# Patent

## Title
Polynucleotides comprising a reporter sequence operatively linked to a regulatory element

## Abstract

The invention provides a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of a gene selected from Bscl2, Srxnl, Cbr3, Ephxl, Nope, Cdknla, Perp, Pltp, Cgrefl, Ltb4r1, Btg2, Gpx2, Ltb4r2, Ddit4l, Fosl1, and Egr1, which regulatory element stimulates expression of the reporter sequence in response to a genotoxic agent or to an oxidative stress-inducing agent. The invention also provides a method of detecting a genotoxic or oxidative stress-inducing agent comprising subjecting a cell containing the polynucleotide of the invention to a test agent; and assessing the expression of the reporter sequence. The invention provides a method of detecting a genotoxic or oxidative stress-inducing agent comprising subjecting one or more cells that comprise a reporter sequence operatively linked to a regulatory element of a gene, which regulatory element stimulates expression of the reporter sequence in response to a genotoxic agent or to an oxidative stress-inducing agent, to a test agent, and assessing the expression of the one or more reporter sequences; wherein at least one cell subjected to a test agent comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene.

## Background
This invention relates to methods for detecting agents that are genotoxic or are oxidative stress-inducing, and to molecules and cell lines that may be employed in such methods.
The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.
DNA damage may be induced by a range of agents including ultraviolet light, X-rays, free radicals, methylating agents and other mutagenic compounds. DNA damage can also be caused indirectly either by agents that affect enzymes and proteins which interact with DNA (including polymerases and topoisomerases) or by promutagens (agents that can be metabolised to become mutagenic). Any of these agents may directly or indirectly cause damage to the DNA that comprises the genetic code of an organism and cause mutations in genes. Such agents may be collectively known as genotoxic agents.
Exposure to a range of agents may also directly or indirectly lead to the production of reactive oxygen species that can react with various cellular biomolecules and affect their functionality. These agents are collectively known as oxidative stress-inducing agents.
Industry yearly develops millions of new chemical compounds for a wide range of applications. These compounds may react with various cellular structures and organelles, including DNA, proteins and lipids and affect multiple cellular processes. Reactivity towards cellular DNA of exposed organisms may cause mutations, affect genome stability and may ultimately lead to the development of genetic diseases and cancer. Compound exposure may also result in the formation of reactive oxygen species which have also been implicated in cellular dysfunctioning, the generation of mutations and aging. One regulator in the cellular response to a wide variety of cellular stressors is the p53 tumor suppressor gene [1]. The p53-induced stress responses include activation of DNA repair systems, cell cycle checkpoints, activation of gene transcription and protein expression, and induction of apoptosis or cellular senescence. Activation of these pathways provides protection against the mutagenic and possibly oncogenic consequences of exposure to DNA damaging and oxidative stress-inducing compounds and enhances organism survival.
A worldwide legislation demands rigorous testing of new chemicals for potential genotoxic or other deleterious properties of newly developed chemical compounds. For the chemical and pharmaceutical industries it is important that these tests are cheap, fast and predictive for human health risk. In addition, these tests should preferably be applicable as high-throughput assays and should provide insights into the mode of toxicity of the compounds.
Various in vitro assays for genotoxicity testing are currently in use.
The oldest and widely used test is the Ames test, which is a Salmonella based test for bacterial mutagenicity. However, the Ames test has a relatively low sensitivity and fails to establish genotoxicity of compounds that interact with cellular structures that are specific for eukaryotic cells. Further, due to the low mutation frequencies that are induced by compounds, the Ames test is not suitable as a high-throughput test system.
Other tests for bacterial mutagenicity include those that depend on the bacterial SOS response that is activated upon exposure to genotoxic compounds. The SOS/umu-assay is a Salmonella bacterial test that is based on beta-galactosidase expression under control of the umuC stress response [2]. In the Vitotox assay, a bioluminescent marker is placed under control of the SOS response [3], and the assay was recently developed to increase testing throughput [4].
To establish genotoxicity in a eukaryotic test system, various yeast-based assays have been developed. The DELL assay is based on reversion mutations in modified Saccharomyces cerevisiae strains containing a deletion in the his3 gene [5, 6]. Reversion depends on intrachromosomal recombination and is therefore restricted to specific classes of carcinogens. The Greenscreen (GS) assay uses the enhanced green fluorescent protein (eGFP) coupled to the RAD54 DNA damage repair gene [7]. Validation of the yeast Greenscreen assay showed high specificity and sensitivity in the identification of a large collection of known genotoxic compounds [8].
Development of sensitive mammalian cell based genotoxicity tests, which are more relevant for risk assessment in higher organisms, has proven challenging [9]. The comet assay depends on higher mobility of cells with DNA breaks, in agarose gels and is a widely used test for chromosomal damage. The assay is often used in combination with a chromosomal aberration test or an in vitro micronucleus test. However, these assays have reduced sensitivity, will only score positive when using compounds that induce DNA strand breaks and are not suitable for high throughput screening. Recently there has been efforts to improve the throughput of the comet assay [10].
H2AX is a specific variant of the H2A histone protein family and is rapidly phosphorylated by various kinases, including ATM and ATR, involved in the DNA damage response. It was proposed that phosphorylation of H2AX (γH2AX) could be used as a sensitive marker for detection of DNA damage [11]. Recent evaluation of the γH2AX assay shows that phosphorylation of H2AX can be used to assess genotoxicity [12].
The Greenscreen HC assay uses the human lymphoblastoid TK6 cells and is a validated mammalian in vitro test for genotoxicity [9, 16]. The assay depends on an eGFP reporter linked to the GADD45a (Growth Arrest and DNA Damage) gene. GADD45a is regulated by the p53 tumor suppressor and plays an important role in cell cycle control, DNA repair mechanisms and signal transduction and thereby is required for genome maintenance [17]. The GreenScreen HC GADD45a assay provides a reliable and sensitive genotoxicity test that allows discrimination between genotoxic and non-genotoxic carcinogens [18]. The fluorescent eGFP reporter can be detected using flow cytometry thereby allowing screening in a high throughput setup. However, the GreenScreen HC assay is solely representative for the global p53 response and provides little mechanistic information on the reactivity of genotoxic compounds.
Given the demand for genotoxicity assays, there remains a demand for fast and highly sensitive, animal free test systems to establish the genotoxicity of both known and novel chemical compounds.
Surprisingly and unexpectedly, the inventors have now identified 16 genes in mice that can serve as biomarkers for exposure to genotoxic stress, namely Bscl2 (Bernardinelli-Seip congenital lipodystrophy 2 homolog (human)), Cbr3 (carbonyl reductase 3), Ephx1 (epoxide hydrolase 1, microsomal), Nope (immunoglobulin superfamily, DCC subclass, member 4), Cdkn1a (cyclin-dependent kinase inhibitor 1A (P21)), Perp (TP53 apoptosis effector), Pltp (phospholipid transfer protein), Srxn1 (sulfiredoxin 1 homolog (S. cerevisiae)), Cgref1 (cell growth regulator with EF hand domain 1), Ltb4r1 (leukotriene B4 receptor 1), Btg2 (B-cell translocation gene 2, anti-proliferative), Gpx2 (glutathione peroxidise 2), Ltb4r2 (leukotriene B4 receptor 2), Ddit4l (DNA-damage inducible transcript 4-like), Fosl1 (Fos-like antigen 1), and Egr1 (Early growth response 1). The inventors have developed highly sensitive mouse embryonic stem cell systems that allow establishing genotoxicity at non-cytotoxic concentrations.
Accordingly, a first aspect of the invention provides a polynucleotide comprising a reporter polynucleotide operatively linked to a regulatory element of a gene selected from a group consisting of the genes Bscl2, Cbr3, Ephx1, Nope, Cdkn1a, Perp, Pltp, Srxn1, Cgref1, Ltb4r1, Btg2, Gpx2, Ltb4r2, Ddit4l, Fosl1, and Egr1, which regulatory element stimulates expression of the reporter sequence in response to a genotoxic or oxidative stress-inducing agent.
By ‘reporter sequence’ we include the meaning of a polynucleotide whose expression is detectable by means of a suitable assay. For example, the polynucleotide may be one whose expression can be detected directly, for instance by using RT-PCR according to standard procedures in the art and as described in Example 1. In one embodiment, the reporter sequence is not the naturally occurring polynucleotide of the gene whose regulatory element the reporter sequence is operatively linked to. Alternatively, expression of the polynucleotide can be detected indirectly. For example, the polynucleotide may encode a reporter protein, and expression of the encoded reporter protein assessed. By ‘reporter protein’, we include the meaning of a protein that can be detected by means of an appropriate assay. It will be appreciated that the reporter protein may be one that is directly detected (e.g. a light emitting reporter protein) or one that is indirectly detected (e.g. an enzyme that produces a detectable signal).
In an embodiment, the reporter sequence is one that encodes any of DsRed fluorescent protein, horse radish peroxidise (HRP), Green Fluorescent Protein (GFP) (or an analogue or derivative thereof), luciferase, chloramphenicol acetyl transferase (CAT) or β-galactosidase. However, it may encode any protein whose cellular quantity can be accurately and rapidly assessed.
The reporter sequence may be fatal to the cells, or alternatively may allow cells to survive under otherwise fatal conditions. Cell survival can then be measured, for example using colorimetric assays for mitochondrial activity, such as reduction of WST-1 (Boehringer). WST-1 is a formosan dye that undergoes a change in absorbance on receiving electrons via succinate dehydrogenase.
Several techniques are available in the art to detect and measure expression of a reporter sequence which would be suitable for use in the present invention. Many of these are available in kits both for determining expression in vitro and in vivo.
For example, levels of mRNA transcribed from a reporter sequence can be assayed using RT-PCR (see Example 1). The specific mRNA is reverse transcribed into DNA which is then amplified such that the final DNA concentration is proportional to the initial concentration of target mRNA.
Levels of expression can also be determined by measuring the concentration of a reporter protein encoded by the reporter sequence. Assaying protein levels in a biological sample can occur using any suitable method. For example, protein concentration can be studied by a range of antibody based methods including immunoassays, such as ELISAs and radioimmunoassays. In one such assay, a protein-specific monoclonal antibody can be used both as an immunoadsorbent and as an enzyme-labelled probe to detect and quantify a specific protein. The amount of the protein present in the sample can be calculated by reference to the amount present in a standard preparation using a linear regression computer algorithm. In another ELISA assay, two distinct specific monoclonal antibodies can be used to detect the specific protein. In this assay, one of the antibodies is used as the immunoadsorbent (primary antibody) and the other as the enzyme-labelled probe (secondary antibody).
Suitable enzyme labels include those from the oxidase group, which catalyze the production of hydrogen peroxide by reacting with substrate. Glucose oxidase is particularly preferred as it has good stability and its substrate (glucose) is readily available. Activity of an oxidase label may be assayed by measuring the concentration of hydrogen peroxide formed by the enzyme-labeled antibody/substrate reaction. Besides enzymes, other suitable labels include radioisotopes such as iodine (125I, 121I) carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99mTc), and fluorescent labels such as fluorescein and rhodamine, and biotin.
The concentration of a specific protein expressed by a reporter sequence may also be detected in vivo by imaging, for example when testing an agent in an animal model. For example, the reporter sequence may encode a bio-illuminescent reporter protein, or a fluorescent reporter protein.
Conveniently, the reporter protein is a light emitting protein (e.g. fluorescent) and its concentration is measured by assessing the light emitted, for example by using a fluorometer, or flow cytometry (eg. fluorescence assisted cell sorting (FACS) analysis), or fluorescence microscopy (eg confocal immunofluorescence microscopy) and (automated) live cell imaging.
In a preferred embodiment, the reporter sequence is one that encodes the light emitting reporter protein Discosoma sp red fluorescent protein (DsRed), and light emitting derivatives thereof. DsRed is from the reef coral Discosoma sp. and is readily detectable by virtue of its red light emission. An advantage of using a red fluorescent marker compared to the commonly used GFP is that it allows analysis of compounds that show green autofluorescence.
Derivatives of DsRed include polypeptide analogues, mutants or fragments of DsRed which are able to emit light.
One such derivative is DsRed-Express. DsRed-Express is a rapidly maturing variant of DsRed which contains nine amino acid substitutions that enhance solubility, reduce green emission, and accelerate maturation [43]. Recent development of a particular non-cytotoxic DsRed variant, DsRed-express2 [34], has been shown to allow stable and high expression of red fluorescent proteins in mammalian cells, and so in a particularly preferred embodiment, the reporter sequence is one that encodes DsRed-Express 2. The DsRed-Express2 sequence may be obtained from the pDsRed-express2.1 plasmid (e.g. obtained from Clontech), which is shown in FIG. 5A.
In a further preferred embodiment, the reporter sequence is one that encodes GFP.
Where the reporter sequence encodes an enzyme, determining the expression of a reporter sequence may comprise measuring the activity of the enzyme. Enzyme assays typically measure either the consumption of substrate or production of product over time. It is appreciated that a large range of methods exist for determining the concentrations of substrates and products such that many enzymes can be assayed in several different ways as is well known in the art (e.g. Bergmeyer (1974)).
The reporter sequences can be operatively linked to the specified regulatory element using standard molecular biology techniques.
By ‘operatively linked’ we include the meaning that the reporter sequence and regulatory element are linked such that the regulatory element is able to regulate the expression of the reporter sequence with which it is associated.
By a ‘regulatory element of a gene’ we include the meaning of a polynucleotide sequence that regulates the expression of a gene with which it is associated. Thus, when the regulatory element is operatively linked to a reporter sequence, the regulatory element is one that regulates expression of that reporter sequence. In the context of the invention, the regulatory element is also one that stimulates expression of the reporter sequence in response to a genotoxic or oxidative stress-inducing agent. By ‘stimulates expression’, it is understood that the regulatory element may act to switch on expression of the reporter sequence (i.e. from an undetectable level) in the presence of a genotoxic or oxidative stress-inducing agent, or it may act to increase existing expression of the reporter sequence in the presence of a genotoxic or oxidative stress-inducing agent.
Whether or not a particular regulatory element stimulates expression of a reporter sequence in response to a genotoxic or oxidative stress-inducing agent can be assayed using routine methods, including, for example, those described above and detailed in the Examples. For example, a polynucleotide comprising a candidate regulatory element operatively linked to a reporter sequence may be transfected into a cell, and expression of the reporter sequence assessed in the presence and absence of a genotoxic or oxidative stress-inducing agent.
It is appreciated that the regulatory element generally contains one or more regulatory sequence motifs that are able to bind to particular transcription factors. In this way, expression is regulated by one or more transcription factors binding to the regulatory element in the presence of a genotoxic or oxidative stress-inducing agent.
By the genes Bscl2, Cbr3, Ephx1, Nope, Cdkn1a, Perp, Pltp, Srxn1, Cgref1, Ltb4r1, and Btg2, we include mouse genes Bscl2 (NC_000085; SEQ ID No: 58), Cbr3 (NC_000082; SEQ ID No: 49), Ephx1 (NC_000067; SEQ ID No: 50), Nope (NC_000075; SEQ ID No: 51), Cdkn1a (NC_000083; SEQ ID No: 52), Perp (NC_000076; SEQ ID No: 53), Pltp (NC_000068; SEQ ID No: 54), Srxn1 (NC_000068; SEQ ID No: 55), Cgref1 (NC_000071; SEQ ID No: 56), Ltb4r1 (NC_000080; SEQ ID No: 57), and Btg2 (NC_000067; SEQ ID No: 59), whose polynucleotide sequences (downstream of the promoter) are listed in FIG. 3, and whose expression has been shown to be upregulated in response to genotoxic or oxidative stress.
By the genes Gpx2, Ltb4r2, Ddit4l, Fosl1, and Egr1 we include mouse genes Gpx2 (NC_000078; SEQ ID No: 60), Ltb4r2 (NC_000080; SEQ ID No: 61), Ddit4l (NC_000069; SEQ ID No: 62), Fosl1 (NC_000085; SEQ ID No: 63) and Egr1 (NC_000084; SEQ ID No: 64), whose polynucleotide sequences (downstream of the promoter) are listed in FIG. 4, and whose expression has been shown to be upregulated in response to genotoxic or oxidative stress.
It is appreciated that various types of transcription factors, and therefore regulatory sequences, are highly conserved in mammals. Thus, the inventors believe that if a regulatory element of one of the mouse genes Bscl2, Cbr3, Ephx1, Nope, Cdkn1a, Perp, Pltp, Srxn1, Cgref1, Ltb4r1, Btg2, Gpx2, Ltb4r2, Ddit4l, Fosl1, and Egr1, is conserved with a regulatory element of an orthologue of the respective gene, and the required transcription factors are available (some transcription factors are tissue and cell type specific), the regulatory elements will function in the same way. Accordingly, by the genes Bscl2, Cbr3, Ephx1, Nope, Cdkn1a, Perp, Pltp, Srxn1, Cgref1, Ltb4r1, Btg2, Gpx2, Ltb4r2, Ddit4l, Fosl1, and Egr1, we also include orthologues of the mouse genes Bscl2, Cbr3, Ephx1, Nope, Cdkn1a, Perp, Pltp, Srxn1, Cgref1, Ltb4r1, Btg2, Gpx2, Ltb4r2, Ddit4l, Fosl1, and Egr1, that are present in other species (eg human, rat, monkey, dog and horse). Orthologues of mouse genes Bscl2, Cbr3, Ephx1, Nope, Cdkn1a, Perp, Pltp, Srxn1, Cgref1, Ltb4r1, Btg2, Gpx2, Ltb4r2, Ddit4l, Fosl1, and Egr1, can be readily identified by a person of skill in the art, for example using sequence comparison programmes. Preferably, the orthologue has a polynucleotide sequence with at least 60%, or 65%, or 70%, or 75%, or 80%, or 85% or 90% sequence identity with the coding polynucleotide sequence (eg cDNA sequence) of the corresponding mouse gene, whose sequence (downstream of the promoter) is provided in FIGS. 3 and 4, and more preferably 95% and 99% sequence identity.
Typically, the regulatory element of a gene comprises the promoter of the gene since the promoter immediately upstream of the transcription initiation site is expected to contain most of the regulatory sequence motifs that bind to transcription factors. Thus, the regulatory element may comprise the promoter sequence of a gene selected from a group consisting of the genes Bscl2, Cbr3, Ephx1, Nope, Cdkn1a, Perp, Pltp, Srxn1, Cgref1, Ltb4r1, Btg2, Gpx2, Ltb4r2, Ddit4l, Fosl1, and Egr1.
The regulatory element is conveniently a polynucleotide fragment upstream of the transcription start site of the gene. For example, the regulatory element may comprise at least a 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or 2000 base pair polynucleotide sequence immediately upstream of the transcription start site of the gene. Transcription start sites of the genes Bscl2, Cbr3, Ephx1, Nope, Cdkn1a, Perp, Pltp, Srxn1, Cgref1, Ltb4r1, and Btg2 are indicated in FIG. 1. Transcription start sites of the genes Gpx2, Ltb4r2, Ddit4l, Fosl1, and Egr1 are indicated in FIG. 2.
In a preferred embodiment, the regulatory element comprises at least a 1400 or 1500 base pair polynucleotide sequence immediately upstream of the transcription start site of the gene.
In a preferred embodiment, the regulatory element of a gene selected from a group consisting of the genes Bscl2, Cbr3, Ephx1, Nope, Cdkn1a, Perp, Pltp, Srxn1, Cgref1, Ltb4r1, and Btg2, comprises the respective polynucleotide sequences listed in FIG. 1 (SEQ ID Nos: 1-11).
In a preferred embodiment, the regulatory element of a gene selected from a group consisting of the genes Gpx2, Ltb4r2, Ddit4l, Fosl1, and Egr1 comprises the respective polynucleotide sequences listed in FIG. 2 (SEQ ID Nos: 34-38).
It is appreciated that genes typically have multiple regulatory sequences which may or may not be adjacent to one another. For example, regulatory sequences of genes can reside up to megabase distances away from the transcription initiation site. While regulatory sequences for the common basic transcription factors often reside close to the transcription initiation site of a gene (eg. within a few hundred base pairs), regulatory sequences responsible for responding to particular stimuli (eg. regulating expression in response to genotoxic stimuli) may either reside within a few hundred base pairs of the transcription initiation site, or further away from it. Thus, it is understood that not all regulatory sequences of a gene may be located in the promoter directly upstream of its transcription initiation site.
Accordingly, in one embodiment, the regulatory element corresponds to two or more regulatory sequences which act together to increase expression of a reporter sequence in the presence of a genotoxic or oxidative stress-inducing agent. In other words, the regulatory element may comprise one or more portions of the gene, which portions have been joined together.
In an embodiment, the regulatory element of the gene comprises sequences downstream of the promoter sequence. For example, the regulatory element may comprise polynucleotide sequences that bind transcription factors which promote gene expression following DNA damage, or may comprise a 3′ untranslated region (UTR).
In an embodiment, the regulatory element comprises at least one exon of the respective gene, or at least one intron of the respective gene, or at least one exon and one intron of the respective gene.
In an embodiment, the regulatory element comprises an enhancer sequence of the respective gene, which acts to enhance gene expression in response to a genotoxic or oxidative stress-inducing agent.
In an embodiment, the regulatory element comprises a regulatory sequence motif that is known to bind a transcription factor which serves to increase gene expression in response to a genotoxic or oxidative stress-inducing agent. Thus, the regulatory element may comprise a p53 binding motif.
In a preferred embodiment, the regulatory element of a gene comprises the whole gene, such that the gene sequence encoding the endogenous protein is operatively linked to a reporter sequence. The polynucleotide sequences (downstream of the promoter) of each of the genes: Bscl2, Cbr3, Ephx1, Nope, Cdkn1a, Perp, Pltp, Srxn1, Cgref1, Ltb4r1, and Btg2, are provided in FIG. 3 and the polynucleotide sequences of each of the genes: Gpx2, Ltb4r2, Ddit4l, Fosl1, and Egr1 (downstream of the promoter) are provided in FIG. 4. Without wishing to be bound by any theory, the inventors believe that in this way, all regulatory sequences of the gene are captured in the regulatory element such that expression of the reporter sequence will better mimic expression of the endogenous gene. Methods for linking a whole gene to a reporter sequence are known in the art, and include, for example, BAC TransgeneOmics technology (Poser et al, 2008 Nature Methods, 5: 409-415) described in the Examples.
It is appreciated that the regulatory element may be a functional derivative, or a variant (eg in which one or more nucleotides have been substituted or deleted), or a portion of one of the sequences mentioned above.
For example, the regulatory element may be a variant of any of the sequences listed in FIGS. 1A-K or FIG. 2A-E, having at least, for example, 60%, 65%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with said sequences, provided that the variant is one that stimulates expression of a reporter sequence operatively linked to it in response to a genotoxic or oxidative stress-inducing agent.
Percent sequence identity between two polynucleotides may be determined by any suitable method known in the art, for example by using appropriate computer programs such as WU-BLAST-2 (Altschul et al., Methods in Enzymology 266:460-480 (1996)) and Blast search, MacVector and Vector NTI (eg AlignX program in Vector NTI version 11) (Invitrogen).
In another example, the regulatory element may be a portion of any of the sequences listed in FIGS. 1A-K or FIGS. 2A-E or variants of said sequences, which portions have at least, for example, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of the bases of said sequences, provided that the portion is one that stimulates expression of a reporter sequence operatively linked to it in response to a genotoxic or oxidative stress-inducing agent. Conveniently, the portion is at least 50 bp or 100 bp or 200 bp or 300 bp or 400 bp or 500 bp or 600 bp or 700 bp or 800 bp or 900 bp or 1000 bp or 1100 bp or 1200 bp or 1300 bp or 1400 bp or 1500 bp in length.
Now that the importance of the sixteen genes identified by the inventors in genotoxic and oxidative stress has been realised, it is appreciated that any regulatory element of the genes can be readily identified by assessing whether or not expression of a reporter sequence operatively linked to a putative regulatory element is activated in the presence of a genotoxic or oxidative stress-inducing agent. The regulatory element may also be identified by conducting mutagenesis on a promoter of a gene selected from a group consisting of the genes Bscl2, Cbr3, Ephx1, Nope, Cdkn1a, Perp, Pltp, Srxn1, Cgref1, Ltb4r1, Btg2, Gpx2, Ltb4r2, Ddit4l, Fosl1, and Egr1, when in natural association with the respective gene, and assessing whether or not expression of the gene occurs.
Typically, the regulatory elements activate expression of a reporter sequence in response to a genotoxic agent selected from any of an agent causing base damage, an agent causing bulky DNA adducts, an agent causing single-stranded DNA breaks, an agent causing double-stranded DNA breaks, an agent causing intra-strand crosslinks, or an agent causing inter-strand crosslinks. Thus, the genotoxic agent may be an intercalator, a topoisomerase II poison or a methylating agent or an alkylating agent. It will be appreciated that there are no genotoxic agents that selectively induce only one type of DNA damage. Hence, as well as causing base damage, the genotoxic agent may also cause bulky DNA adducts.
Preferably, the regulatory element stimulates expression of a reporter sequence in response to any of cisplatin, mitomycin C, doxorubicin, etoposide, methylmethane sulphonate, and N-methylnitrosurea, or to any of the genotoxic or oxidative stress-inducing compounds mentioned in the Examples, such as a genotoxin or pro-oxidant as suggested by the European Centre for Validation of Alternative Methods (ECVAM) (see Tables 2 and 3).
The regulatory element may stimulate expression of a reporter polynucleotide in response to oxidative stress-inducing agent such as a pro-oxidant and including any of hydrogen peroxide, t-butyl hydroperoxide, menadione and diethyl maleate.
It will be understood that some agents may induce both genotoxicity and oxidative stress to varying degrees.
Exposure to high concentrations of genotoxic or oxidative stress-inducing compounds rapidly induces apoptosis to protect cells against the mutagenic effects of DNA damage. Thus, it is preferred if the regulatory element stimulates expression of the reporter sequence in response to a non-cytotoxic concentration of a genotoxic or oxidative stress-inducing agent. Preferably, the regulatory element stimulates expression of the reporter sequence in response to a concentration of genotoxic or oxidative stress-inducing agent that induces apoptosis in less than 35% of a population of cells, such as less than 30%, 20% or 10% of a population of cells. Methods for assessing apoptosis are well known in the art and include annexin V and caspase 3 staining assays as described in Example 1.
The polynucleotide may be RNA (eg mRNA) or DNA, although typically it is DNA.
It will be appreciated that the polynucleotide of the first aspect of the invention may be incorporated into a vector and used as an expression cassette. Accordingly, a second aspect of the invention provides a vector comprising a polynucleotide according to the first aspect of the invention.
Suitable vectors are ones which propagate in and/or allow expression of the reporter sequence in prokaryotic (e.g. bacterial) or eukaryotic (e.g. mammalian) cells. For example, the vector may be a plasmid, a cosmid, a phage or a bacterial artificial chromosome (BAC). The polynucleotide sequence of the vector will depend upon the nature of the intended host cell, the manner of the introduction of the polynucleotide of the first aspect of the invention into the host cell, and whether episomal maintenance or integration is desired. Conveniently, the vector comprises at least one selectable marker such as antibiotic resistance (e.g. kanamycin or neomycin).
Vectors are useful to replicate the polynucleotide of the first aspect of the invention, and are also useful to transfect cells with the polynucleotide, and may also promote expression of the reporter sequence.
Typical prokaryotic vector plasmids are: pUC18, pUC19, pBR322 and pBR329 available from Biorad Laboratories (Richmond, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540 and pRIT5 available from Pharmacia (Piscataway, N.J., USA); pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16A, pNH18A, pNH46A available from Stratagene Cloning Systems (La Jolla, Calif. 92037, USA).
A typical mammalian cell vector plasmid is pSVL available from Pharmacia (Piscataway, N.J., USA). This vector uses the SV40 late promoter to drive expression of cloned genes, the highest level of expression being found in T-antigen-producing cells, such as COS-1 cells. Another example is pcDNA3.1 (neo) (Invitrogen) for use in COS-1 or COS-7 cells. An example of an inducible mammalian expression vector is pMSG, also available from Pharmacia (Piscataway, N.J., USA). This vector uses the glucocorticoid-inducible promoter of the mouse mammary tumour virus long terminal repeat to drive expression of the cloned gene.
Useful yeast plasmid vectors are pRS403-406 and pRS413-416 and are generally available from Stratagene Cloning Systems (La Jolla, Calif. 92037, USA). Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (YIps) and incorporate the yeast selectable markers HIS3, TRP1, LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (YCps).
In a preferred embodiment, the vector which comprises the polynucleotide of the first aspect of the invention is pDsRed-expression2.1 plasmid (Clontech; Strack et al, “A noncytotoxic DsRed variant for whole-cell labelling” Nat Methods, 2008, 5(11): 955-7 (FIG. 5)).
Any suitable method known in the art may be used to construct vectors containing the polynucleotide of the first aspect of the invention. One such method involves ligation via homopolymer tails. Homopolymer polydA (or polydC) tails are added to exposed 3′ OH groups on the DNA fragment to be cloned by terminal deoxynucleotidyl transferases. The fragment is then capable of annealing to the polydT (or polydG) tails added to the ends of a linearised plasmid vector. Gaps left following annealing can be filled by DNA polymerase and the free ends joined by DNA ligase.
Another method involves ligation via cohesive ends. Compatible cohesive ends can be generated on the DNA fragment and vector by the action of suitable restriction enzymes. These ends will rapidly anneal through complementary base pairing and remaining nicks can be closed by the action of DNA ligase. For example, using appropriate primers in PCR reactions, the regulatory element of the polynucleotide of the first aspect of the invention may be produced so as to be flanked by desired restriction enzyme sites. The regulatory element may then be cloned into a vector that contains a reporter sequence downstream of where the regulatory element is inserted.
A further method uses synthetic molecules called linkers and adaptors. DNA fragments with blunt ends are generated by bacteriophage T4 DNA polymerase or E. coli DNA polymerase I which remove protruding 3′ termini and fill in recessed 3′ ends. Synthetic linkers, pieces of blunt-ended double-stranded DNA which contain recognition sequences for defined restriction enzymes, can be ligated to blunt-ended DNA fragments by T4 DNA ligase. They are subsequently digested with appropriate restriction enzymes to create cohesive ends and ligated to an expression vector with compatible termini. Adaptors are also chemically synthesised DNA fragments which contain one blunt end used for ligation but which also possess one preformed cohesive end.
Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including International Biotechnologies Inc, New Haven, Conn., USA.
Generating a BAC comprising the polynucleotide of the first aspect of the invention may be done using BAC TransgeneOmics technology as described in Poser et al, 2008 Nature Methods, 5: 409-415.
A third aspect of the invention provides a cell comprising a polynucleotide according to the first aspect of the invention, or a vector according to the second aspect of the invention. Such cells may be used to replicate the polynucleotide of the first aspect of the invention, or may be used in a genotoxic or oxidative stress-inducing screening assay, as discussed in more detail below.
The cell can be either prokaryotic or eukaryotic.
It is appreciated that construction and amplification of the polynucleotide of the first aspect of the invention is conveniently performed in bacterial cells, whereas the use of the polynucleotide for genotoxic or oxidative stress-inducing screening is typically limited to mammalian cells.
Bacterial cells are preferred prokaryotic host cells and typically are a strain of E. coli such as, for example, the E. coli strains DH5 available from Bethesda Research Laboratories Inc., Bethesda, Md., USA, and RR1 available from the American Type Culture Collection (ATCC) of Rockville, Md., USA (No ATCC 31343). Preferred eukaryotic host cells include yeast, insect and mammalian cells or cell lines, preferably vertebrate cells or cell lines such as those from a mouse, rat, monkey or human. The cells may be stem cells (e.g. embryonic stem cells) or they may be immortalised cells (e.g. hTert immortalised primary human fibroblasts), or they may be primary cells such as hepatocytes (e.g. obtained through differentiation of stem cells). Yeast host cells include YPH499, YPH500 and YPH501 which are generally available from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA. Preferred insect cells are Sf9 cells which can be transfected with baculovirus expression vectors.
Cells used for expressing the reporter sequence are ideally stably transfected. However, it is appreciated that non-stable transfectants (eg when using viral expression vectors) may also be used.
Preferably, the cell used for expressing the reporter sequence is one that has a non-compromised DNA damage response. For example, the cell may have a functional DNA repair response and so be capable of one or more of, and preferably all of, inter-strand crosslink repair, base excision repair, homologous recombination, and non-homologous end joining. The cell may have operational cell cycle checkpoints, such as G1-S, intra S, G2 and mitosis checkpoints. The cell may have a functional apoptotic pathway. Methods of determining whether a cell has a non-compromised DNA damage response are routine in the art, and include, for example, sequence analysis of genes, and functional assays of proteins (eg following genotoxic insult), known to be involved in the DNA damage response.
In a particular embodiment, the cell has a functional p53 protein which is required for the functionality of some cell cycle checkpoints and for the induction of apoptosis. Thus, the cell may be an embryonic stem cell or a hTert immortalised primary human fibroblast.
The inventors have found that mouse embryonic stem cells are particularly useful cell lines in the method of the invention. Thus, in a particularly preferred embodiment, the cell is a mouse stem cell, most preferably an embryonic stem cell.
It is appreciated that a non-genotoxic or a non-oxidative stress-inducing agent may be converted into a genotoxic or into an oxidative stress-inducing agent, respectively, within a cell. Thus, in order to identify such agents in a genotoxic or oxidative stress-inducing screening assay, the cell is conveniently a cell that expresses metabolising enzymes, such as an hepatocyte (eg Hep G2 or an hepatocyte derived from an embryonic stem cell), or a STO (mouse embryonic fibroblast) cell, or a lung epithelial cell (eg one grown in 3D cell culture).
Transformation of appropriate cells with a vector is accomplished by well known methods that typically depend on the type of vector used. With regard to transformation of prokaryotic host cells, see, for example, Cohen et al (1972) Proc. Natl. Acad. Sci. USA 69, 2110 and Sambrook et al (2001) Molecular Cloning, A Laboratory Manual, 3rd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Transformation of yeast cells is described in Sherman et al (1986) Methods In Yeast Genetics, A Laboratory Manual, Cold Spring Harbor, N.Y. The method of Beggs (1978) Nature 275, 104-109 is also useful. With regard to vertebrate cells, reagents useful in transfecting such cells, for example calcium phosphate and DEAE-dextran or liposome formulations, are available from Stratagene Cloning Systems, or Life Technologies Inc., Gaithersburg, Md. 20877, USA. Electroporation may also be used as described in Example 1.
It is appreciated that in addition to the transformed host cells themselves, the present invention also contemplates a culture of those cells, preferably a monoclonal (clonally homogeneous) culture, or a culture derived from a monoclonal culture, in a nutrient medium.
It will be understood that the polynucleotide of the first aspect of the invention or vector of the second aspect of the invention may be incorporated into a cell of an organism in vivo, or into the cell of a tissue ex vivo. Thus, the invention also includes an organism or tissue that comprises the polynucleotide of the first aspect of the invention or vector of the second aspect of the invention. For example, it will be appreciated that the invention includes a non-human transgenic mammal that comprises a cell of the third aspect of the invention. Preferably, the mammal is a laboratory animal, such as any of a rodent (eg mouse or rat), a primate, a dog or a cat.
In this way, the polynucleotide of the first aspect of the invention can be used to screen for genotoxic or oxidative stress-inducing agents in vivo or ex vivo. For example, the polynucleotide may be incorporated into a laboratory animal such as a primate, mouse or rat that can be used to screen for genotoxic or oxidative stress-inducing agents.
It will be appreciated that the organism or tissue may comprise more than one of the polynucleotides, vectors or cells of the invention, each one containing a regulatory element of a different gene.
The invention provides an organism or tissue that comprises (i) a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene, and (ii) one or more cells that comprise a reporter sequence operatively linked to a regulatory element of a gene, which regulatory element stimulates expression of the reporter sequence in response to a genotoxic agent or to an oxidative stress-inducing agent. Thus, the organism or tissue may comprise (i) a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene, and (ii) one or more cells that comprise a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of a gene selected from a respective one or more of (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 of) Srxn1, Cbr3, Ephx1, Nope, Cdkn1a, Perp, Pltp, Cgref1, Ltb4r1, Btg2, Gpx2, Ltb4r2, Ddit4l, Fosl1 and Egr1. It is appreciated that the cells comprising the various reporter sequences operatively linked to regulatory elements may be the same or different. In other words, the organism or tissue may comprise a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene, and, either in the same or respectively different cells, one or more polynucleotides that comprise a reporter sequence operatively linked to a regulatory element of a gene, which regulatory element stimulates expression of the reporter sequence in response to a genotoxic agent or to an oxidative stress-inducing agent. Thus, it is also appreciated that the invention provides a cell that comprises more than one polynucleotide or vector of the invention, each one containing a regulatory element of a different gene (eg Bscl2 and Srxn1 genes).
Where the organ or tissue or cell contains a regulatory element from more than one gene, it is appreciated that the different regulatory elements may be responsive to different genotoxic or oxidative stress-inducing agents. For example, the regulatory element of one gene may be responsive specifically to genotoxic agents and the regulatory element of another gene responsive specifically to oxidative stress-inducing agents. Thus, by exposing more than one polynucleotide comprising a regulatory element according to the invention, to an agent, the mode of toxicity of that agent can be assessed. As described in Examples 4 and 5, the inventors have found that Bscl2 reporters are selectively activated following exposure to genotoxic stress, Srxn1 reporters are selectively activated following exposure to oxidative stress, and Btg2 reporters are activated by both genotoxic and oxidative stress. Thus, by exposing different reporters to an agent, it is possible to categorise that agent as a primarily a genotoxic or primarily an oxidative-stress inducing compound, or both a genotoxic and oxidative-stress inducing compound.
In a preferred embodiment, the organ or tissue comprises a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene, and a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Srxn1 gene. It is understood that the cells may be same.
In a further preferred embodiment, the organ or tissue comprises a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene, and a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Srxn1 gene, and a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Btg2 gene. It is understood that the cells may be the same.
In one embodiment, regulatory elements that respond in a specific way (e.g. to genotoxic agent or oxidative stress-inducing agents or classes thereof) are operatively linked to different reporter sequences. For instance, a regulatory element that is responsive to genotoxic agents may be linked to a first reporter sequence and a regulatory element that is responsive to oxidative stress-inducing agents may be linked to a second reporter sequence. In this way, the property of the agent can readily be determined by the readout from the different reporter sequences.
The polynucleotide of the first aspect of the invention or vector of the second aspect of the invention may be stably integrated into the genome of a cell in vivo or ex vivo, using standard methods in the art. For example, the polynucleotide may be integrated by regular transgene methods such as cre-lox. Alternatively, the polynucleotide may be somatically incorporated. For example, somatic incorporation of reporter constructs in rodents (e.g. mice or rats) can be achieved through tail vein injection.
The invention does not provide a process for cloning a human being, nor does it provide a process for modifying the germ line identity of human beings, nor does it provide a use of a human embryo for industrial or commercial purposes, nor does it provide a process for modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal, and also animals resulting from such a process.
As described in Example 1, the inventors have found the polynucleotides of the first aspect of the invention to be particularly useful in genotoxic or oxidative stress-inducing screening assays. Accordingly, a fourth aspect of the invention provides a method of detecting a genotoxic or an oxidative stress-inducing agent comprising subjecting a cell according to the third aspect of the invention to a test agent; and assessing the expression of the reporter sequence.
It is appreciated that cells containing different regulatory elements, may be subjected to a test agent, and so the invention similarly provides a method of detecting a genotoxic or oxidative stress-inducing agent comprising (a) subjecting one or more cells that comprise a reporter sequence operatively linked to a regulatory element of a gene, which regulatory element stimulates expression of the reporter sequence in response to a genotoxic agent or to an oxidative stress-inducing agent, to a test agent, and (b) assessing the expression of the one or more reporter sequences; wherein at least one cell subjected to a test agent in step (a) comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene.
Thus, the method may involve subjecting cells to a test agent, wherein the cells contain regulatory elements of different genes operatively linked to a reporter sequence, provided that at least one cell comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene. By using different regulatory elements, it is believed that more potentially genotoxic and oxidative stress-inducing agents can be detected, and also further insight into the mode of toxicity can be gained. It is appreciated that the different regulatory elements may be operatively linked to the same or different reporter sequences. It is also understood that the polynucleotides that comprise the different regulatory elements operatively linked to a reporter sequence may be present in the same or different cells.
In one embodiment, (i) a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene, and (ii) one or more cells that comprise a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of a gene selected from a respective one or more of (e.g. 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 of) Srxn1, Cbr3, Ephx1, Nope, Cdkn1a, Perp, Pltp, Cgref1, Ltb4r1, Btg2, Gpx2, Ltb4r2, Ddit4l, Fosl1 and Egr1, are subjected to a test agent. The cells of (i) and (ii) may be the same or different. Thus, a cell that comprises a Bscl2-reporter (i.e. a reporter sequence operatively linked to a regulatory element of the Bscl2 gene) and one or more cells that respectively comprise any of a Srxn1-reporter, Cbr3-reporter, Ephx1-reporter, Nope-reporter, Cdkn1a-reporter, Perp-reporter, Pltp-reporter, Cgref1-reporter, Ltb4r1-reporter, Btg2-reporter Gpx2-reporter, Lbt4r2-reporter, Ddit4l-reporter, Fosl1-reporter and Egr1-reporter, may be subjected to a test agent. In a particularly preferred embodiment, a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene, and a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Srxn1 gene, are subjected to a test agent, which cells may or may not be the same. In a further preferred embodiment, a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene, and cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Srxn1 gene, and a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Btg2 gene, are subjected to a test agent, which cells may or may not be the same.
It is appreciated that the test agent may be any physical or chemical agent in the environment to which organisms are exposed. Thus, the method may be used to screen compounds, such as candidate medicaments, food additives, plasticisers or cosmetics to assess whether it is safe to expose an organism (e.g. human) to the compounds. Alternatively, the method may be used to assess contamination of samples with genotoxic or oxidative stress-inducing agents. For instance, the presence of genotoxic or oxidative stress-inducing agents in water supplies or in industrial effluents may be assessed.
Preferred reporter sequences include those described above with respect to the first aspect of the invention. Thus, the method may involve assessing the expression of any one or more reporter proteins such as a DsRed fluorescent protein, horse radish peroxidise (HRP), Green Fluorescent Protein (GFP) (or an analogue or derivative thereof), luciferase, chloramphenicol acetyl transferase (CAT) or β-galactosidase, in the presence of a test agent. Preferably, the method involves assessing the expression of a DsRed fluorescent protein (e.g. DsRed-express2) by FACS or assessing the expression of GFP by fluorescence microscopy (eg confocal immunofluorescence microscopy) or (automated) live cell imaging. It is also appreciated that in this aspect of the invention, the reporter sequence may be the naturally occurring polynucleotide of the gene whose regulatory element the reporter sequence is operatively linked to.
When cells containing different regulatory elements are exposed to the test agent, it is appreciated that the different regulatory elements may be operatively linked to the same or different reporter sequences. As discussed above, it may be desirable to operatively link regulatory elements that respond in a specific way (e.g. to genotoxic agent or oxidative stress-inducing agents or classes thereof or to both genotoxic and oxidative stress-inducing agents) to different reporter sequences, such that the mode of toxicity of a particular agent can be readily determined from the reporter readout.
In one embodiment, the method is performed in vitro. By in vitro we include the meaning of cell-based assays. By cell-based assays we include the meaning of cell cultures in two dimensions, such as on plastic or glass culture plates, as well as cell cultures which are cultured in three dimensional matrices. These matrices may be composed of natural matrix components and contain, for example, collagen, fibronectin, laminin, or matrigel, or they may be composed of artificial matrix components.
Conveniently, the method is performed by growing cells transfected with a vector that comprises a reporter sequence operatively linked to a regulatory element of a gene, which regulatory element stimulates expression of the reporter sequence in response to a genotoxic agent or to an oxidative stress-inducing agent (e.g. one or more cells according to the third aspect of the invention), incubating the cells with a genotoxic or oxidative stress-inducing agent, and assessing the expression of the one or more reporter sequences from a sample of the cells.
It may be desirable to compare the expression of the reporter sequence in the one or more reporter cells used in the method of the invention, to expression in control cells that contain the reporter sequence but which lack the regulatory element of a gene which regulatory element stimulates expression of the reporter sequence in response to a genotoxic agent or to an oxidative stress-inducing agent (e.g. regulatory elements of any of the genes Bscl2, Cbr3, Ephx1, Nope, Cdkn1a, Perp, Pltp, Srxn1, Cgref1, Ltb4r1, Btg2, Gpx2, Ltb4r2, Ddit4l, Fosl1, and Egr1).
To enable high-throughput analysis, the cells may be seeded on a multiwell plate, such as a 96-well plate. For example, as described in Example 1, cells containing a fluorescent reporter protein (eg DsRed) may be seeded on a multiwell plate and expression assessed by flow cytometry.
In another embodiment, the method is performed in vivo or ex vivo. For example, one or more polynucleotides that comprise a reporter sequence operatively linked to a regulatory element of a gene, which regulatory element stimulates expression of the reporter sequence in response to a genotoxic agent or to an oxidative stress-inducing agent (e.g. polynucleotides of the first aspect of the invention), each polynucleotide comprising a different regulatory element may have been incorporated into either the same or respectively different cells of a living organism (e.g. a laboratory animal such as a mouse or rat), and the expression of the one or more reporter sequences in the organism assessed in the presence and absence of a test agent. Conveniently, the method is performed in vivo using transgenic animals (e.g. mice) and expression of reporter sequences assessed using whole-body small animal imaging machines. Alternatively, the one or more polynucleotides that comprise a reporter sequence operatively linked to a regulatory element of a gene, which regulatory element stimulates expression of the reporter sequence in response to a genotoxic agent or to an oxidative stress-inducing agent, each polynucleotide comprising a different regulatory element may have been incorporated into a tissue ex vivo, and the expression of the reporter sequences in the tissue assessed in the presence and absence of a test agent. Performing the method ex vivo typically involves using isolated cells from transgenic animals. For example, following hydrodynamic tail injection of the polynucleotide of the first aspect of the invention into a mouse or rat, reporter activity may be assessed in liver slices ex vivo.
As mentioned above and exemplified in Example 6, the polynucleotide of the first aspect of the invention can be used in a genotoxic or oxidative stress-inducing screening assay to detect non-genotoxic agents or non-oxidative stress-inducing agents that may be converted into genotoxic agents or oxidative stress-inducing agents in a cell. To do so, it is appreciated that either the cells containing polynucleotides such as the polynucleotide of the first aspect of the invention (i.e. that comprise a reporter sequence operatively linked to a regulatory element of a gene, which regulatory element stimulates expression of the reporter sequence in response to a genotoxic agent or to an oxidative stress-inducing agent) are metabolically active, or that metabolising cells are added to the cells (either as feeder cells or as a co-culture), or that a metabolising cell extract (eg. liver extract such as rat S9 mix) is added to the cells.
Expression of the reporter sequence may be assessed at several intervals over time. For example, expression may be assessed at 8 h, 12 h, 16 h, 24 h, 30 h and 48 h after incubation with the test agent.
As mentioned above, the inventors have identified 16 genes whose expression is activated in response to genotoxic or oxidative stress-inducing agents, and which therefore may be used as biomarkers for genotoxic or oxidatively induced stress. It follows that an agent may counteract genotoxic or oxidative stress by reducing or preventing the expression of such genes upon exposure to a genotoxic or oxidative stress-inducing agent. In this way, the reporter sequence of the first aspect of the invention may also be used to identify agents that counteract genotoxic or oxidative stress, for example by assessing the expression of the reporter sequence in the presence of a genotoxic or oxidative stress-inducing agent and by determining what effect a test agent has on expression.
Accordingly, a fifth aspect of the invention provides a method of detecting an agent to counteract genotoxic or oxidative stress, the method comprising:
a) subjecting a cell according to the third aspect of the invention to a genotoxic or oxidative stress-inducing agent, and to a test agent; and
b) assessing the expression of the reporter sequence.
It may be desirable to use cells in the method that comprise a different regulatory element operatively linked to a reporter sequence, either in the same or different cells. It is appreciated that the different regulatory elements may be operatively linked to the same or different reporter sequences. For example, it may be desirable to operatively link regulatory elements that respond in a specific way (e.g. to genotoxic agent or oxidative stress-inducing agents or classes thereof or to both genotoxic and oxidative stress-inducing agents) to different reporter sequences, such that the counteracting effect of an agent on different types of toxicity can be readily determined by the reporter readout from the reporter sequences.
Thus, the invention similarly provides a method of detecting an agent to counteract genotoxic or oxidative stress, the method comprising a) subjecting one or more cells that comprise a reporter sequence operatively linked to a regulatory element of a gene, which regulatory element stimulates expression of the reporter sequence in response to a genotoxic agent or to an oxidative stress-inducing agent, to a genotoxic or oxidative stress-inducing agent, and to a test agent; and
b) assessing the expression of the one or more reporter sequences;
wherein at least one of the cells comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene.
Thus, step (a) may comprise subjecting either the same or respectively different cells that comprise different regulatory element operatively linked to a reporter sequence, to a genotoxic or oxidative stress-inducing agent, and to a test agent. It is appreciated that the different regulatory elements may be operatively linked to the same or different reporter sequences.
In one embodiment, (i) a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene, and (ii) one or more cells that comprise a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of a gene selected from a respective one or more of (e.g. 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 of) Srxn1, Cbr3, Ephx1, Nope, Cdkn1a, Perp, Pltp, Cgref1, Ltb4r1, Btg2, Gpx2, Ltb4r2, Ddit4l, Fosl1, and Egr1, are subjected to a genotoxic or oxidative stress-inducing agent, and to a test agent. It is understood that the cells may be the same or different.
In a particularly preferred embodiment, a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene, and a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Srxn1 gene are subjected to a genotoxic or oxidative stress-inducing agent, and to a test agent.
In a further preferred embodiment, a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene, and a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Srxn1 gene, and a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Btg2 gene, are subjected to a genotoxic or oxidative stress-inducing agent, and to a test agent.
By an agent that ‘counteracts genotoxic or oxidative stress’ we include the meaning of an agent that prevents or reduces DNA damage caused by a genotoxic agent, or the oxidative stress caused by an oxidative stress-inducing agent.
An agent that counteracts genotoxic or oxidative stress is expected to reduce the expression of a reporter sequence relative to the expression of the reporter sequence in the absence of the agent. Conveniently therefore, the method comprises first assessing the expression of the reporter sequence in the presence of a genotoxic or oxidative stress-inducing agent, but in the absence of a test agent, and subsequently comparing the expression with the expression of the reporter sequence in the presence of a genotoxic or oxidative stress-inducing agent, and a test agent.
Accordingly, the invention includes a method of determining whether an agent (e.g. compound) can counteract genotoxic or oxidative stress, the method comprising:
a) subjecting a cell according to the third aspect of the invention to a genotoxic agent or an oxidative stress-inducing agent;
b) determining the effect of the genotoxic agent or oxidative stress-inducing agent on the expression of the reporter sequence;
c) subjecting the cell to a test agent; and
d) determining whether the test agent counteracts the effect of the genotoxic agent or oxidative stress-inducing agent on the expression of the reporter sequence.
Similarly, it is appreciated that the invention includes a method of determining whether an agent (e.g. compound) can counteract genotoxic or oxidative stress, the method comprising:
a) subjecting one or more cells that comprise a reporter sequence operatively linked to a regulatory element of a gene, which regulatory element stimulates expression of the reporter sequence in response to a genotoxic agent or to an oxidative stress-inducing agent, to a genotoxic agent or an oxidative stress-inducing agent;
b) determining the effect of the genotoxic agent or oxidative stress-inducing agent on the expression of the one or more reporter sequences;
c) subjecting the one or more cells to a test agent; and
d) determining whether the test agent counteracts the effect of the genotoxic agent or oxidative stress-inducing agent on the expression of the one or more reporter sequences; wherein at least one of the cells in step (a) comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene. It is appreciated that the cells in step (a) containing different regulatory elements operatively linked to reporter sequences may be the same or different, and that the different regulatory elements may be operatively linked to the same or different reporter sequences.
By “counteracts the effect of the genotoxic agent or oxidative stress-inducing agent on the expression of the reporter sequence” we include the meaning of determining whether the test agent reduces the expression of the reporter sequence, stimulated by a genotoxic or oxidative stress-inducing agent.
It is appreciated, however, that the expression of the reporter sequence in the presence of a genotoxic or oxidative stress-inducing agent alone may already be known, in which case it is only necessary to assess the expression of the reporter sequence in the presence of both the genotoxic or oxidative stress-inducing agent, and test agent.
Preferences for the cell, the genotoxic or oxidative stress-inducing agent and methods for assessing expression of the reporter sequence for this and subsequent aspects of the invention are as mentioned above.
The test agent may be any compound. Examples include any of a polypeptide, a peptide, a nucleic acid, a small molecule (e.g. less than 5000 daltons), or a natural product.
The method of the fifth aspect of the invention may be performed in vitro, in vivo or ex vivo, in the same way as the method of the fourth aspect of the invention.
A sixth aspect of the invention provides a kit of parts comprising:

    
    
        (i) a cell according to the third aspect of the invention; and
        (ii) a means for detecting expression of the reporter sequence.
    
    


The invention similarly provides a kit of parts comprising:

    
    
        (i) one or more cells that comprise a reporter sequence operatively linked to a regulatory element of a gene, which regulatory element stimulates expression of the reporter sequence in response to a genotoxic agent or to an oxidative stress-inducing agent; wherein at least one of the cells comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene; and, optionally,
        (ii) one or more means for detecting expression of the one or more reporter sequences.
    
    


It is appreciated that the cells in part (i) containing different regulatory elements operatively linked to reporter sequences may be the same or different.
Thus, the kit of parts may contain one or more cells that comprise a different regulatory element (e.g. 2, 3, 4 or 5 different regulatory elements) operatively linked to a reporter sequence. The cells may the same or different. It is appreciated that the different regulatory elements may be operatively linked to the same or different reporter sequences.
In one embodiment, the kit of parts comprises (i) a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene, and (ii) one or more cells that comprise a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of a gene selected from a respective one or more of Srxn1, Cbr3, Ephx1, Nope, Cdkn1a, Perp, Pltp, Cgref1, Ltb4r1, Btg2, Gpx2, Ltb4r2, Ddit4l, Fosl1, and Egr1. It is understood that the cells may be the same or different.
In a particularly preferred embodiment, the kit of parts comprises a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene, and a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Srxn1 gene.
In a further preferred embodiment, the kit of parts comprises a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene, and a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Srxn1 gene, and a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Btg2 gene.
The means for detecting expression of the reporter sequence may be any suitable means that can be used to assess expression of a particular reporter sequence. For example, if expression of the reporter sequence is assessed directly by RT-PCR, the means may comprise a primer or probe. Alternatively, if expression of the reporter sequence is assessed by assessing the expression of a reporter protein encoded by the polynucleotide, the means may be an agent that can be used to measure expression of the reporter protein, such as an antibody or an enzyme substrate.
It may be desirable to compare expression to that of a control cell which lacks the regulatory element and/or reporter sequence present in the cell of the kit of parts (e.g. cell of the third aspect of the invention). Thus, the kit may further comprise a cell which does not contain a reporter sequence operatively linked to a regulatory element of a gene which regulatory element stimulates expression of the reporter sequence in response to a genotoxic agent or to an oxidative stress-inducing agent (e.g. a regulatory element from a gene selected from a group consisting of the genes Bscl2, Cbr3, Ephx1, Nope, Cdkn1a, Perp, Pltp, Srxn1, Cgref1, Ltb4r1, Btg2, Gpx2, Ltb4r2, Ddit4l, Fosl1, and Egr1). For example, the kit may further comprise a cell which does not contain a polynucleotide according to the first aspect of the invention, or a vector according to the second aspect of the invention.
A further aspect of the invention provides a method of determining the effect of a genotoxic agent or an oxidative stress-inducing agent, the method comprising subjecting a cell according to the third aspect of the invention to a genotoxic agent or an oxidative stress-inducing agent; and assessing the expression of the reporter sequence. It is believed that by assessing the effect of various genotoxic agents or oxidative stress-inducing agents, agents may be classified by their effect and insights gained into modes of toxicity.
Similarly, the invention provides a method of determining the effect of a genotoxic agent or an oxidative stress-inducing agent, the method comprising the steps of (a) subjecting one or more cells that comprise a reporter sequence operatively linked to a regulatory element of a gene, which regulatory element stimulates expression of the reporter sequence in response to a genotoxic agent or to an oxidative stress-inducing agent, to a genotoxic agent or an oxidative stress-inducing agent; and (b) assessing the expression of the one or more reporter sequences, wherein at least one of the cells comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene.
Thus, step (a) may comprise subjecting one or more cells that comprise a different regulatory element (e.g. 2, 3, 4 or 5 different regulatory elements) operatively linked to a reporter sequence, to a genotoxic or oxidative stress-inducing agent, and to a test agent. The cells may the same or different. It is appreciated that the different regulatory elements may be operatively linked to the same or different reporter sequences.
In one embodiment, (i) a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene, and (ii) one or more cells that comprise a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of a gene selected from a respective one or more of Srxn1, Cbr3, Ephx1, Nope, Cdkn1a, Perp, Pltp, Cgref1, Ltb4r1, Btg2, Gpx2, Ltb4r2, Ddit4l, Fosl1, and Egr1, are subjected to a genotoxic or oxidative stress-inducing agent. It is understood that the cells of (i) and (ii) may be the same or different.
In a particularly preferred embodiment, a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene, and a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Srxn1 gene are subjected to a genotoxic or oxidative stress-inducing agent.
In a further preferred embodiment, a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene, and a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Srxn1 gene, and a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Btg2 gene, are subjected to a genotoxic or oxidative stress-inducing agent.
The method may be performed in vitro, in vivo, or ex vivo as described above.
A yet further aspect of the invention provides a method of selecting an agent (e.g. compound) that reduces expression of the reporter sequence in the polynucleotide of the first aspect of the invention, the method comprising:
a) subjecting a cell according to the third aspect of the invention to a test agent;
b) assessing the effect of the test agent on the expression of the reporter sequence; and
c) selecting a test agent that reduces expression of the reporter sequence.
Similarly, the invention includes a method of selecting an agent that reduces expression of one or more reporter sequences, the method comprising:
a) subjecting one or more cells that comprise a reporter sequence operatively linked to a regulatory element of a gene, which regulatory element stimulates expression of the reporter sequence in response to a genotoxic agent or to an oxidative stress-inducing agent, to a test agent;
b) assessing the effect of the test agent on the expression of the one or more reporter sequences; and
c) selecting a test agent that reduces expression of the one or more reporter sequences;
wherein at least one of the cells subjected to a test agent, comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene.
Thus, step (a) may comprise subjecting cells that comprise a different regulatory element (e.g. 2, 3, 4 or 5 different regulatory elements) operatively linked to a reporter sequence, to a test agent. The cells may the same or different. It is appreciated that the different regulatory elements may be operatively linked to the same or different reporter sequences.
In one embodiment, (i) a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene, and (ii) one or more cells that comprise a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of a gene selected from a respective one or more of Srxn1, Cbr3, Ephx1, Nope, Cdkn1a, Perp, Pltp, Cgref1, Ltb4r1, Btg2, Gpx2, Ltb4r2, Ddit4l, Fosl1, and Egr1 are subjected to a test agent. It is understood that the cells may be the same.
In a particularly preferred embodiment, a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene, and a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Srxn1 gene are subjected to a test agent.
In a further preferred embodiment, a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene, and a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Srxn1 gene, and a cell that comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Btg2 gene, are subjected to a test agent.
As discussed above in relation to other methods of the invention, the method may be performed in vitro, in vivo or ex vivo.
It is appreciated that in this and other screening methods of the invention, the one or more cells may be subjected to a combination of different test agents and the effect of the combination of test agents on the expression of the reporter sequences assessed. Examples of suitable test agents include those listed above.
Suitable methods for assessing the expression of the reporter sequence are as described above. By ‘reduces expression’ it is understood that the expression may be switched off (i.e. to an undetectable level), or that existing expression may be decreased.
It is appreciated that the method of this aspect of the invention may be performed with or without first subjecting the one or more cells to a genotoxic or oxidative stress-inducing agent, depending upon the background level of expression of the one or more reporter sequences in the absence of a genotoxic or oxidative stress-inducing agent.
If the background expression is very low for example, it may be desirable to first subject the one or more cells to a genotoxic or oxidative stress-inducing agent so that a baseline level expression of the one or more reporter sequences can be established to which the expression of the one or more reporter sequences in the presence of a genotoxic or oxidative stress-inducing agent can be compared. Of course, this may not be necessary if the baseline expression of the one or more reporter sequences in the presence of a genotoxic or oxidative stress-inducing agent is known. Alternatively, the background expression of the one or more reporter sequences may be used as the baseline expression to which expression in the presence of a genotoxic or oxidative stress-inducing agent is compared to, in which case it is not necessary to first subject the cells to a genotoxic or oxidative stress-inducing agent.
Preferably, the method is performed on isolated cells. Thus, the invention includes a method of selecting an agent or a combination of agents that reduce expression of the reporter sequence in the polynucleotide of the first aspect of the invention, the method comprising:
a) culturing a cell according to the third aspect of the invention (eg a cell line) in a suitable medium;
b) optionally adding a genotoxic or oxidative stress-inducing agent to the medium;
c) adding a test agent or combination of test agents to said medium;
d) assessing the effect of the test agent or combination of test agents on the expression of the reporter sequence; and
e) selecting a test agent or combination of test agents that reduces expression of the reporter sequence.
Similarly, the invention includes a method of selecting an agent or a combination of agents that reduce expression of one or more reporter sequences, the method comprising:
a) culturing one or more cells (e.g. cell line) that comprise a reporter sequence operatively linked to a regulatory element of a gene, which regulatory element stimulates expression of the reporter sequence in response to a genotoxic agent or to an oxidative stress-inducing agent, in a suitable medium;
b) optionally adding a genotoxic or oxidative stress-inducing agent to the medium;
c) adding a test agent or combination of test agents to said medium;
d) assessing the effect of the test agent or combination of test agents on the expression of the one or more reporter sequences; and
e) selecting a test agent or combination of test agents that reduces expression of the one or more reporter sequences; wherein at least one of the cells comprises a polynucleotide comprising a reporter sequence operatively linked to a regulatory element of the Bscl2 gene.
Preferences for the cell and the genotoxic or oxidative stress-inducing agent include those listed above.



The invention will now be described in more detail with the aid of the following Figures and Examples.
FIG. 1: Polynucleotide sequences of PCR fragments containing regulatory elements of genes (A) Bscl2, SEQ ID No: 1; (B) Ephx1, SEQ ID No: 2; (C) Nope, SEQ ID No: 3; (D) Cdkn1a, SEQ ID No: 4; (E) Perp, SEQ ID No: 5; (F) Pltp, SEQ ID No: 6; (G) Srxn1, SEQ ID No: 7; (H) Cgref1, SEQ ID No: 8; (I) Ltb4r1, SEQ ID No: 9; (J) Cbr3, SEQ ID No: 10; (K) Btg2, SEQ ID No: 11. Forward and reverse primers are indicated below the sequence of each PCR fragment. Transcription start sites are indicated in a box in each sequence.
(A) Forward, SEQ ID No: 12; Reverse, SEQ ID No: 13; (B) Forward, SEQ ID No: 14; Reverse, SEQ ID No: 15; (C) Forward, SEQ ID No: 16; Reverse, SEQ ID No: 17; Reverse 2, SEQ ID No: 89 (D) Forward (2900 bp), SEQ ID No: 18; Reverse, SEQ ID No: 19; Forward (2364 bp), SEQ ID No: 90 (E) Forward, SEQ ID No: 20; Reverse, SEQ ID No: 21; (F) Forward, SEQ ID No 22; Reverse, SEQ ID No: 23; (G) Forward, SEQ ID No: 24; Reverse, SEQ ID No: 25; (H) Forward, SEQ ID No 26; Reverse, SEQ ID No: 27; (I) Forward, SEQ ID No: 28; Reverse, SEQ ID No: 29; (J) Forward, SEQ ID No: 30; Reverse, SEQ ID No: 31; (K) Forward, SEQ ID No: 32; Reverse, SEQ ID No: 33.
FIG. 2: Polynucleotide sequences of PCR fragments containing regulatory elements of genes (A) Gpx2, SEQ ID No: 34; (B) Ltb4r2, SEQ ID No: 35; (C) Ddit4l, SEQ ID No: 36; (D) Fosl1, SEQ ID No: 37; (E) Egr1, SEQ ID No: 38. Forward and reverse primers are indicated below the sequence of each PCR fragment. Transcription start sites are indicated in a box in each sequence.
(A) Forward, SEQ ID No: 39; Reverse, SEQ ID No: 40; (B) Forward, SEQ ID No: 41; Reverse, SEQ ID No: 42; (C) Forward, SEQ ID No: 43; Reverse, SEQ ID No: 44; (D) Forward, SEQ ID No: 45; Reverse, SEQ ID No: 46; (E) Forward, SEQ ID No: 47; Reverse, SEQ ID No: 48.
FIG. 3: Polynucleotide sequences of genes (downstream of promoter).
(A) Bscl2; NC_000085; SEQ ID No: 58; (B) Ephx1; NC_000067; SEQ ID No: 50; (C) Nope; NC_000075; SEQ ID No: 51; (D) Cdkn1a; NC_000083; SEQ ID No: 52; (E) Perp; NC_000076; SEQ ID No: 53; (F) Pltp; NC_000068; SEQ ID No: 54; (G) Srxn1; NC_000068; SEQ ID No: 55; (H) Cgref1; NC_000071; SEQ ID No: 56; (I) Ltb4r1; NC_000080; SEQ ID No: 57; (J) Cbr3; NC_000082; SEQ ID No: 49; (K) Btg2; NC 000067; SEQ ID No: 59.
FIG. 4: Polynucleotide sequences of genes (downstream of promoter).
(A) Gpx2; NC_000078; SEQ ID No: 60; (B) Ltb4r2; NC_000080; SEQ ID No: 61; (C) Ddit4l; NC_000069; SEQ ID No: 62; (D) Fosl1; NC_000085; SEQ ID No: 63; (E) Egr1; NC 000084; SEQ ID No: 64.
FIG. 5: Cloning of promoter region by cloning a  ̃1500 bp fragment upstream of transcription start site that is believed to contain most of the regulatory promoter sequences. Also shown is a diagram of the pDsRed-Express 2.1 vector.
FIG. 6: Compound class specificity of Ephx1, Btg2, Perp and Cbr3 genes. Changes in gene expression of the endogenous biomarker genes were established by quantitative RT-PCR following a 16 hrs treatment of ES cells with indicated compounds. Depicted values are the average of tree independent experiments and error bars represent the standard deviation.
FIG. 7: Sensitivity of mouse ES cells to different genotoxic agents was established by determining the apoptotic response after exposure. (A) Apoptosis as percentage of subG1 cells using flow cytometry and (B) by using a Caspase3 activity assay. Error bars indicate the standard deviation of three independent experiments. (C) Percentage of apoptotic cells by Annexin-V staining at different times after exposure to increasing concentrations of Cisplatin.
FIG. 8: DsRed reporter cell lines for genotoxicity and oxidative stress assessment. (A) Flow cytometry analysis of DsRed expression in monoclonal mouse ES cell lines containing a DsRed fluorescent reporter driven by a putative biomarker gene promoter. As controls a promoterless and CMV-driven DsRed reporter cell line were used. Cell lines were exposed to 10 μM Cisplatin (Ephx1, Btg2, Perp, CMV, Promotorless) or 250 μM DEM (Cbr3) for 16 hrs. (B) Clonal survival of wild type mouse ES cells and the DsRed reporter cell lines after treatment with Cisplatin and Etoposide. Shown data is the average of three independent experiments. Error bars represent the standard deviation.
FIG. 9: Expression kinetics of the Ephx1-DsRed reporter mimics the expression changes of the endogenous gene. (A) Expression levels of the DsRed reporter and the endogenous Ephx1 gene as determined by quantitative RT-PCR. (B) Ratio between DsRed and Ephx1 expression. Background expression of Ephx1-DsRed is higher than that of the endogenous Ephx1 gene, while the response to CisPt is comparable. Shown data are the average of four experiments.
FIG. 10: Differential response of the DsRed reporter cell lines to genotoxic and oxidative stress-inducing agents. (A) DsRed reporter cell lines were exposed to 0.5 μM Etoposide or 5 μM Cisplatin and total DsRed fluorescence was determined using flow cytometry after different times of exposure. (B) DsRed reporter cell lines were exposed to increasing concentrations of the DNA damage-inducing agents Cisplatin, Etoposide, MMS and the oxidative stress-inducer DEM. The increase in total DsRed fluorescence was determined after 30 hrs. of exposure using flow cytometry. (C) Ratio of fold changes in DsRed expression of Btg2 vs. Cbr3 DsRed reporter cell lines after exposure to different agents. Exposure to the DNA damaging agents (Cisplatin, Etoposide) resulted in a ratio of approximately 1.2. while induction of oxidative stress (DEM, MMS) resulted in a ratio of approximately 0.8. All data shown are the average of four independent experiments. Error bars indicate the standard error of the mean.
FIG. 11: Compound class specificity of the reporter cell lines. (A) At each dose of compounds used the ratio of fold change (=relative reactivity) were calculated for each combination of two reporter cell lines. The relative reactivity of every pair of reporter cell lines was used to determine which combination of cell lines discriminated best between exposure to genotoxic and oxidative stress-inducing compounds. Values were calculated from at least four independent experiments and error bars represent standard error of the mean. (B) Fold change ratio between Btg2-DsRed and Cbr3-DsRed after exposure to the DNA damaging agents doxorubicin and mitomycin C (MMC) and the pro-oxidant CuSO4.
FIG. 12: DsRed reporters are not activated by general cytotoxic stress. (A) DsRed reporter cell lines were exposed to increasing concentrations of Cisplatin (CisPt), Wyeth-14,643 and Cyclosporin-A (CsA) and cell viability was determined by Alamar blue staining. (B) DsRed reporter cell lines were exposed to increasing concentration of CisPt, CsA, Wyeth-14,643 and DES. Total DsRed fluorescence was determined 24, 30 and 48 hrs. after exposure using flow cytometry. Depicted values are the average of three independent experiments and error bars indicated the standard error of the mean.
FIG. 13: Differential response of the GFP reporter cell lines generated by BAC TransgeneOmics to genotoxic and oxidative stress-inducing agents. GFP reporter cell lines were exposed to increasing concentrations of the DNA damage-inducing agents Cisplatin, Doxorubicin, MMC, and the oxidative stress-inducer DEM and CuSO4. The increase in total GFP fluorescence was determined after 30 hrs of exposure using flow cytometry. All data shown are the average of 3 independent experiments. Error bars indicate the standard deviation.
FIG. 14: DsRed reporter cell lines for genotoxicity and oxidative stress assessment. Flow cytometry analysis of DsRed expression in monoclonal mouse ES cell lines containing a DsRed fluorescent reporter driven by five different putative biomarker gene promoters. Cell lines were exposed to 10 μM Cisplatin for 16 hrs.
FIG. 15: Genes being upregulated upon exposure to different genotoxic agents. We performed genome-wide expression profiling of mouse ES cells that were exposed to a wide variety of genotoxic compounds. (A) Genes that showed the highest change in expression upon exposure (+) compared to untreated controls (−), were grouped in three gene sets based on fold change and p-value. Gene set A contains genes that are induced by DNA damaging agents, gene set B contains genes that show specificity for oxidative stress. Gene set D contains genes that are activated by compounds that induce DNA damage, oxidative stress and the cell cycle inhibitor flavopiridol. (B) Responsive genes show a dose dependent increase in gene expression following exposure to the compounds tested. Data in the heatmaps is the average of three independent treatments and array hybridizations. Cells were exposed to low (L), medium (M) and high (H) concentrations of compounds that induce >10%, 10-30% or 30-50% apoptosis respectively.
FIG. 16: GFP-based mES reporter cells for genotoxicity and oxidative stress. (A) Two putative biomarker genes, selected after genome-wide transcription profiling of mES cells, show a selective response to DNA damaging agents (Bscl2) or pro-oxidants (Srxn1). The red intensity in the heatmap represents for every treatment the expression level under non-treated and treated conditions. (B) Selected genes were fused to a GFP fluorescent reporter using BAC transgenomics. (C) Fluorescence microscopy analysis of Bscl2-GFP and Srxn1-GFP reporter cells following exposure to cisplatin (CisPt) or DEM. (D) Expression of the GFP reporters was compared to expression of the endogenous biomarker genes after exposure to CisPt and DEM by quantitative RT-PCR. (E) Expression of the GFP reporters was compared to expression of the endogenous biomarker genes after 8 or 16 h exposure to CisPt and DEM by quantitative RT-PCR.
FIG. 17: The Bscl2-GFP and Srxn1-GFP mES reporter cells show specificity for genotoxic compounds or pro-oxidants respectively. (A) GFP reporter cells were exposed to CisPt and DEM and the induction in total GFP fluorescence of intact cells was determined by flow cytometry. (B) Toxicity of CisPt and DEM is comparable in the reporter cell lines. Survival of the Bscl2-GFP and Srxn1-GFP reporter cells was determined as the fraction of intact cells after 24 h treatment by flow cytometry. (C) Specific activation of the Bscl2-GFP reporter by the DNA damaging agents mitomycin C (MMC), doxorubicin and etoposide and of Srxn1-GFP after exposure to the oxidative stress-inducing agents sodium arsenite (NaAsO2), methyl methanesulphonate (MMS) and cadmium chloride (CdCl2). (D) GFP reporter cells were exposed to the DNA damaging agents CisPt, etoposide, doxorubicin and mitomycin C (MMC) or to the oxidative stress-inducing agents DEM, sodium arsenite (NaAsO2), cadmium chloride (CdCl2) and methyl methanesulphonate (MMS) for 24 h and the induction in total GFP fluorescence of intact cells was determined by flow cytometry. (E) Kinetics of Bscl2- and Srxn1-GFP reporter induction upon exposure to genotoxins or pro-oxidants. Bscl2-GFP and Srxn1-GFP reporter cells were exposed to 5 μM CisPt or 100 μM DEM and GFP expression was determined by live cell imaging up to 24 h exposure.
FIG. 18: Activation of the Srxn1-GFP reporter for oxidative stress depends on ROS production. (A) Bscl2-GFP and Srxn1-GFP mES reporter cells were treated with different concentration of the pro-oxidant DEM. The cells were simultaneously incubated with increasing concentrations of the ROS-scavenger n-acetyl cysteine (NAC). (B) Srxn1-GFP reporter cells were exposed to the pro-oxidants DEM, NaAsO2, CuSO4 and CdCl2. Increasing concentrations of NAC were added to the cells to reduce ROS levels.
FIG. 19: Activation of the Bscl2-GFP DNA damage reporter is associated with inhibition of DNA replication. (A) Bscl2-GFP and Srxn1-GFP mES reporter cells were treated with increasing concentrations of hydroxyurea and aphidicolin. (B) Cell viability of Bscl2-GFP and Srxn1-GFP reporter cell after exposure to hydroxyurea and aphidicolin.
FIG. 20: Activation of the Bscl2-GFP reporter depends on the ATR DNA damage signaling pathway. The Bscl2-GFP and Srxn1-GFP mES reporter cell lines were treated with the DNA damaging agent CisPt or the DNA replication inhibitor aphidicolin in the presence of specific inhibitors for ATM (ku55933), ATR (schisandrin B) or Chk1/Chk2 signaling (UCN01).
FIG. 21: Activation of the Bscl2-GFP DNA damage reporter is independent of p53 and expression of Srxn1-GFP oxidative stress reporter is controlled by the Nrf2 antioxidant pathway. siRNA knockdown of p53 and Nrf2 in the Bscl2-GFP and Srxn1-GFP reporter cells followed by exposure to the genotoxic compounds CisPt or etoposide and to the pro-oxidants DEM, MMS and NaAsO2.
FIG. 22: Sensitivity and specificity of the GFP reporter assays using ECVAM class 1 compounds. Bscl2-GFP and Srxn1-GFP reporter cells were exposed to increasing concentrations of ECVAM-recommended carcinogens that should be positive in an in vitro genotoxicity assay. Induction of the GFP reporters was determined after 24 h exposure by flow cytometry.
FIG. 23: Sensitivity and specificity of the GFP reporter assays using ECVAM class 2 compounds. Bscl2-GFP and Srxn1-GFP reporter cells were exposed to increasing concentrations of ECVAM-recommended non-carcinogens that should be negative in an in vitro genotoxicity assay. Induction of the GFP reporters was determined after 24 h exposure by flow cytometry.
FIG. 24: Sensitivity and specificity of the GFP reporter assays using ECVAM class 3 compounds. Bscl2-GFP and Srxn1-GFP reporter cells were exposed to increasing concentrations of ECVAM-recommended compounds that are non-carcinogens or non-genotoxic carcinogens and which scored positive in one in vitrolin vivo genotoxicity test. Induction of the GFP reporters was determined after 24 h exposure by flow cytometry.
FIG. 25: Sensitivity and specificity of the GFP reporter assays using additional (geno)toxic compounds. Bscl2-GFP and Srxn1-GFP reporter cells were exposed to increasing concentrations of (geno)toxic compounds with different reactive properties. These compounds were not specifically recommended by ECVAM for validation of in vitro genotoxicity testing. Induction of the GFP reporters was determined after 24 h exposure by flow cytometry.
FIG. 26: Bscl2-GFP reporter induction by progenotoxins that require metabolic activation. (A) Bscl2-GFP reporter cells were treated with increasing concentrations of aflatoxin B1 (AFB1), benzo [a] pyrene (B[a]P), cyclophosphamide and dimethylbenz(a)anthracene (DMBA) either in the presence of S9 rat liver extract plus the required cofactors or in the presence of the cofactors only. After 3 h incubation, S9 mix and genotoxins were removed by washing and culturing of cells was continued for 24 h in regular culture medium. GFP reporter activity was established after 24 h by flow cytometry. (B) Relative cell survival was determined by the fraction of intact cells following exposure by flow cytometry.
FIG. 27: Activation of the Srxn1-GFP reporter depends on ROS production and is controlled by the Nrf2 pathway. (A) Bscl2-GFP and Srxn1-GFP mES reporter cells were treated with different concentration of the pro-oxidants DEM, CuSO4, NaAsO2, CdCl2 or MMS and simultaneously incubated with increasing concentrations of the ROS-scavenger N-acetyl cysteine (NAC). GFP reporter induction was determined after 24 h incubation by flow cytometry. (B) Western blot analysis of Bscl2-GFP and Srxn1-GFP reporter cells that had been transfected with siRNAs against Nrf2. Nrf2 protein level was determined 4 days after transfection. Hprt protein level was used as loading control. (C) Bscl2-GFP and Srxn1-GFP reporter cells were exposed to 10 μM CisPt, 1.5 μM etoposide, 150 μM DEM, 0.5 mM MMS or 10 μM NaAsO2 after knockdown of Nrf2 by siRNA transfection. GFP reporter activation was determined after 24 h exposure by flow cytometry.
FIG. 28: Activation of the Bscl2-GFP DNA damage reporter is associated with inhibition of DNA replication. (A) Bscl2-GFP and Srxn1-GFP mES reporter cells were treated with increasing concentrations of hydroxyurea and aphidicolin. GFP reporter activation was determined after 24 h exposure by flow cytometry. Survival of Bscl2-GFP and Srxn1-GFP reporter cell after exposure to hydroxyurea and aphidicolin was determined by the fraction of intact cells by flow cytometry. (B) Western blot analysis of cells that were exposed to 10 μM CisPt or 1.5 μM Aph in the presence of ATR (schisandrin B) or ATM (ku55933) inhibitors. Phosphorylation of the ATM target Kap-1, ATR target Chk1 and p53 was investigated. Detection of Hprt was used as control for equal protein loading. (C) Bscl2-GFP and Srxn1-GFP mES reporter activation by the DNA damaging agent CisPt or the DNA replication inhibitor Aph in the presence of specific inhibitors for ATM, ATR or Chk1/Chk2 signaling (UCN-01) was determined after 24 h by flow cytometry.
FIG. 29: Activation of the Bscl2-GFP and Srxn1-GFP reporters is independent of p53. (A) Western blot analysis of p53 expression in the Bscl2-GFP and Srxn1-GFP reporter cells after transfection with siRNAs against p53. (B) Induction of the Bscl2-GFP and Srxn1-GFP reporters upon exposure to 10 μM CisPt, 15 μM etoposide, 150 μM DEM, 0.5 mM MMS or 10 μM NaAsO2 after p53 knockdown by siRNA transfection. (C) Induction of the Btg2-GFP reporter by several (geno)toxic compounds after transfection with siRNAs against p53, Nrf2 or a scrambled siRNA pool as control.



EXAMPLE 1
Novel Highly Sensitive Reporter Cell System for Genotoxicity and Oxidative Stress Risk Assessment
Introduction
Exposure of organisms to genotoxic or oxidative stress-inducing agents can result in cell death and development of various genetic diseases, including cancer. Existing test systems for genotoxicity display low specificity and often require animal testing. Therefore, there is a strong demand for fast and highly sensitive, animal-free test systems to establish the genotoxicity or oxidative stress-inducing capacity of both known and novel chemical compounds.
We have identified genes that can serve as biomarkers for exposure to genotoxic or oxidative stress. Promoters of selected genes were fused to a DsRed reporter gene and stably integrated in mouse ES cells. Established reporter cell lines displayed significant DsRed expression upon exposure to genotoxic agents already at concentrations that did not induce apoptosis. Exposure of DsRed reporter cell lines to various known non-genotoxic carcinogenic compounds did not induce DsRed expression. By QPCR we confirmed that changes in expression of the DsRed biomarkers after exposure to genotoxic stress are comparable to that of the respective endogenous genes.
Mouse embryonic stem (ES) cells are undifferentiated pluripotent cells that have the unique capability to divide unlimited. ES cells have an intact DNA damage response including the p53 pathway [20, 21] and are highly sensitive to various DNA damaging agents [22]. Therefore, mouse ES cells are highly suitable to use as an in vitro mammalian cell based assay for genotoxicity testing.
In conclusion, we generated highly sensitive mouse ES cell systems that allow establishing genotoxicity or oxidative stress-inducing capacity of compounds at non-cytotoxic concentrations. In addition, the fluorescent DsRed markers provide an easy and sensitive tool to study the DNA damage response in mammalian stem cells.
Materials and Methods
ES Cell Culture and Treatments
C57/Bl6 B4418 wild type ES cells were cultured as described previously [23]. Prior to exposure, cells were cultured on gelatin-coated plates in the absence of MEF feeders. Sub-confluent ES cells were exposed to different concentrations of genotoxic and non-genotoxic agents for 8 or 24 hours. Used concentrations: Cisplatin (CDDP: 10 μM), mitomycin C (MMC: 0.1-0.5-1.5 μM), N-methylnitrosurea (MNU: 0.1-0.5-2 μM), methyl methanesulfonate (MMS: 0.1-0.2-0.5 mM), etoposide (Etop: 0.1-0.5-2 μM), doxorubicin (Dox: 0.01-0.05-0.2 μM), four pro-oxidant agents: hydrogen peroxide (H2O2: 25-50-200 μM), t-butyl hydroperoxide (t-BHP: 25-50-100 μM), menadione (MEN: 25-50-100 μM), diethyl maleate (DEM: 25-100-250 μM), and three non-genotoxic agents: diethylstilbestrol (DES: 0.5-2.5-10 μM), Wyeth14,643 (1-50-250 μM), and Cyclosporine-A (CsA: 0.5-2.5-10 μM). All compounds were prepared freshly in DMSO, PBS or Medium and added directly to the culture medium.
Generation of DsRed Reporter Cell Lines
1.5 Kb PCR fragments, containing the promoter sequence and transcription start site (TSS) of the putative biomarker genes (Sharov A A, Dudekula D B, Ko. M S. 2006. CisView: a browser and database of cis-regulatory modules predicted in the mouse genome. DNA Res. 2006 Jun. 30; 13(3):123-34.) were inserted into the cloning site of pDsRed-express2.1 plasmid (Clontech). Primer sequences are summarized in Table 1. The DsRed reporter plasmids were stably transfected into wild type mouse ES cells using electroporation and various stable monoclonal cell lines were established. Mouse ES cells containing a promoterless DsRed or a CMV-driven DsRed reporter were generated as control cell lines. Sensitivity of the DsRed reporter cell lines to genotoxic compounds was determined using a clonal survival assay as described previously [22].
Detection of DsRed Expression
DsRed expression in the reporter cell lines was determined using flow cytometry (BD FACSCanto II). Cells were seeded on gelatin-coated 96 wells plates 24 prior to exposure and subsequently exposed to various genotoxic agents. Cells were washed with PBS, trypsinized and resuspended into PBS+2% serum for FACS analysis.
Induction of the DsRed reporters was compared to expression of the endogenous genes using quantitative real time (qRT-PCR). Cells were exposed to various genotoxic agents and total RNA was isolated after 6, 16 and 24 hours using the RNeasy mini kit (Qiagen). cDNA was synthesized using oligo(dT)12-18 primers and SuperscriptIII reverse transcriptase (invitrogen) according to the manufacturer's instructions. Expression of DsRed and Ephx1 was determined using FastStart SYBR Green Master (Rox) QPCR mix on a 7900HT Fast Real-Time PCR System (Applied Biosystems). Relative expression was normalized using expression of the YWHAD and HPRT genes. See Table 1 for primer sequences.







TABLE 1







Sequences of oligonucleotides used in this study









Gene
Forward primer (5′-3′)
Reverse primer (5′-3′)










Amplification biomarker promoter region









Ephx1
CGGAATTCACACACCAGAAGAGGGCATC
GGGGTACCGTCGCTCTCGGGTTCCTACT



(SEQ ID No: 65)
(SEQ ID No: 69)


 


Cbr3
CGGAATTCCATTGCTGCCTTGATCACATAAC
ACGCGTCGACGACACACGGACCACCTGATG



(SEQ ID No: 66)
(SEQ ID No: 70)


 


Btg2
CGGGAGATCTGATGAAATGTCTTGCTG
GATAAGTCGACACCACTCGGGGAG



(SEQ ID No: 67)
(SEQ ID No: 71)


 


Perp
CGGAATTCTTTGAGCCGACAGAACCAAT
ACGCGTCGACGCGGAGCGGAGGAACGCCGG



(SEQ ID No: 68)
(SEQ ID No: 72)


 







qRT-PCR









YWHAD-1
CTGGTGATGACAAGAAAGGAATTG
GGTGTGTCGGCTGCATCTC



(SEQ ID No: 73)
(SEQ ID No: 80)


 


YWHAD-2
GCCGACACACCCCATCAG
GCAATGGCTTCATCGAAAGC



(SEQ ID No: 74)
(SEQ ID No: 81)


 


DsRed-1
AGTACGGCTCCAAGGTGTACGT
CATCACGCGCTCCCACTT



(SEQ ID No: 75)
(SEQ ID No: 82)


 


DsRed-2
GTGAAGCTGCCCGGCTACT
GTACTGCTCCACCACGGTGTAGT



(SEQ ID No: 76)
(SEQ ID No: 83)


 


Ephx1-1
CAAAGCCATCAGCCAAAGAAG
AACCTATCTATCCTCTGGTGCAAGTC



(SEQ ID No: 77)
(SEQ ID No: 84)


 


Ephx1-2
TGTGGGCTGTGCTCTGAATG
AGGCCTCCATCCTCCAGTTC



(SEQ ID No: 78)
(SEQ ID No: 85)


 


Hprt
TTGCTCGAGATGTCATGAAGGA
AGCAGGTCAGCAAAGAACTTATAG



(SEQ ID No: 79)
(SEQ ID No: 86)









Apoptosis Assay

DsRed reporter cell lines were seeded on gelatin-coated 6 well plates and treated as described above. After 24 hr., adherent cells were trypsinized and combined with the detached cells in the culture medium. Cell pellets were washed with PBS and resuspended in 0.5 ml Annexin V staining buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 5 mM KCL, 1.8 mM CaCl2 and 1 mM MgCl2) containing freshly added 1 ul/ml FITC-conjugated AnnexinV (Home made). Cells were incubated for 15 min. at room temp in the dark and analyzed by flow cytometry. Alternatively, apoptosis was determined using a Ac-DEVD-AMC-based Caspase3 activity detection assay as described in Kruse et al (Mutat Res, 2007. 617(1-2): p. 58-70).
Results
Expression of Ephx1, Cbr3, Btg2 and Perp Genes is Increased Upon Exposure to Genotoxic and Oxidative Stree Inducing Agents
Changes in expression upon exposure to different genotoxic and oxidative-stress inducing agents were assessed by quantitative RT-PCR (FIG. 6). Ephx1, of which expression in induced upon exposure to DNA damaging agents, is an epoxide hydrolase that is located at the membrane of the endoplasmic reticulum where it is important in the biotransformation of aromatic compounds [27]. The oxidative stress-induced Cbr3 gene encodes an NADPH-dependent carbonyl reductase that is involved in the reduction of a large number of biological and pharmacological compounds [28]. The Btg2 and Perp genes respond to a broader spectrum of DNA damage and stress-inducing compounds and expression of both genes has been suggested to depend on the p53 tumor suppressor gene [29, 30]. Btg2 is involved in regulation of the G1 to S phase transition of the cell cycle, while Perp is a membrane protein involved in cell-cell adhesion [31-33].
For each compound a low concentration that induced less than 10% apoptosis, a medium concentration that induced between 10-30% apoptosis and a high concentration that resulted in 30-50% apoptosis, was used. Apoptosis measurements were performed by determining the percentage of sub-G1 cells using flow cytometry and by measuring caspase 3 activity. FIG. 7A-B shows the results for Etoposide, doxorubicin, MNU and MMC (see Table 2 for a complete list of compounds used). In addition, we determined the level of apoptosis in time after exposure to different concentrations of CisPt by Annexin-V staining (FIG. 7C).







TABLE 2







Genotoxic compounds used in study










Compound
Abbreviation
Concentrations
Mode of action














Cis-Platin
CisPt
1-10
uM
Intra- and interstrand crosslinks. Lesions on G or A.


Doxorubicin
Dox
0.001-0.2
uM
Intercalating agent. Inhibits progression of topoisomerase-II.


Etoposide
Etop
0.1-2
uM
Topoisomerase-II inhibitor.


Mitomycin-C
MMC
0.1-1.5
ug/ml
DNA crosslinking agent. Interstrand and intrastrand crosslinks and mono aducts at guanine.


Methyl Methane
MMS
0.05-0.5
mM
Alkylating agent, methylates DNA on N7-deoxyguanine and N3-deoxyadenine.


Sulphonate






Diethyl Malonate
DEM
25-250
uM
Glutathion depletion, results in increased levels of oxigen radicals.


Menadione
MEN
5-100
uM
Vitamin K. Prooxidant.


Hydrogen Peroxide
H2O2
25-200
uM
Produces oxigen radicals resulting in protein and DNA damage


Tert-butyl Hydroperoxide
tBHP
25-100
uM
Prooxidant. Depletion of cellular stores of GSH and oxidation of






functionally important SH groups on mitochondrial enzymes


Antimycin A1
Ant-A1
0.5-10
uM
Mitochondrial toxin. Respiratory chain inhibitor


Potassium Cyanide
KCN
0.5-5
uM
Mitochondrial toxin. Respiratory chain inihbitor


Rotenone
Rot
1-10
uM
Mitochondrial toxin. Respiratory chain inhibitor


2-Acetylamino Fluorene
2-AAF
10-100
uM
CYP450 substrate. AF and AAF adducts at C(8) and N(2)






position of guanine.


Cytosine arabinoside
AraC
0.05-5
uM
Incorporated into DNA. Chain terminator.


Vincristine
Vin
1-100
uM
Microtubule disruption. Blocks cells in mitosis


Flavopiridol
Flavo
10-100
uM
Cyclin-dependent kinase (cdk) inhibitor. Downregulation of Bcl-2. Causes cell cycle






arrest and apoptosis


Cyclosporin A
CsA
0.5-10
uM
Non-genotoxic carcinogen. Immuno suppressor


Wyeth-14,643
Wyeth
1-250
uM
Non-genotoxic carcinogen. Peroxisome proliferator









Generation of Fluorescent Reporter Cell Lines

A 1500 bp DNA fragment upstream of the transcription start site of each of mouse genes Cbr3, Ephx1, Nope, Cdkn1a, Perp, Pltp, Srxn1, Cgref1, Ltb4r1, Bscl2, and Btg2 was fused to a recently described highly stable DsRed fluorescent protein reporter gene [34].
These promoter fragments were cloned upstream of the DsRed-express2 reporter [34] and transfected into mouse ES cells. In addition, control cell lines were generated containing either a promoterless or a CMV promoter driven DsRed reporter gene. Multiple stable clones for each construct were analyzed for DsRed expression upon exposure to genotoxic or oxidative stress-inducing agents using flow cytometry (FIG. 8A and data not shown). For every biomarker gene a DsRed reporter cell line was selected based on low background DsRed expression and a strong increase in DsRed expression after exposure to different genotoxic compounds (FIG. 8A).
Promoterless DsRed cells did not show any DsRed expression irrespective of exposure to genotoxic agents, while the CMV-driven reporter displayed constitutive high DsRed expression when untreated but appeared to be somewhat responsive to genotoxic treatment, in agreement with previous reports [35, 36].
Cytotoxicity of the reporter cell lines for the tested compounds was similar compared to wild type ES cells (FIG. 8B), indicating that both DsRed expression and integration of the reporter gene in the cellular genome did not affect cellular sensitivity.
DsRed Reporter Vs. Endogenous Gene Activation
We tested to what extent the changes in DsRed mRNA expression of our reporter genes upon genotoxic stress resembled the transcriptional activation of the endogenous biomarker genes. mRNA expression levels of the Ephx1-DsRed reporter and the endogenous Ephx1 gene were determined at different times after exposure to the DNA crosslinking agent Cisplatin by quantitative RT-PCR. Both genes were transcriptionally activated by Cisplatin (FIG. 9A). The transcriptional activation of the Ephx1-DsRed reporter and the endogenous Ephx1 gene was enhanced with increasing dose of Cisplatin and continued to increase even after 24 h of exposure. This suggests that the Ephx1 and DsRed messenger RNAs are stable and accumulate in the cells. Direct comparison of the increase in mRNA for Ephx1 and DsRed shows that the transcription activation by Cisplatin is similar for both genes but that the DsRed expression levels in untreated cells are higher than of Ephx1 (FIG. 9B). High background expression of the DsRed reporter is possibly caused by integration of the gene in a transcriptionally active region in the genome. However, the DsRed expression in untreated cells apparently does not affect transcriptional induction upon exposure to genotoxic agents.
Highly Sensitive and Selective Biomarkers
We next determined the time-dependent increase in DsRed expression in the reporter cell lines after exposure to Cisplatin and the topoisomerase II poison Etoposide. DsRed reporter cell lines were grown in 96-wells microplates, exposed to these two compounds and DsRed fluorescence was monitored using high-throughput flow cytometry at various time points. In all four reporter cell lines, both genotoxic agents induced a strong, up to 12-fold increase in total DsRed fluorescence (FIG. 10A). DsRed expression gradually increased in time, even at 48 hours after treatment, but specifically at higher genotoxicant concentrations, high levels of apoptotic cells negatively influenced the reliability of the assay. These findings indicate that the DsRed protein is rather stable and accumulates in the cells, increasing the sensitivity of the reporter cell systems.
To determine the sensitivity and specificity of the reporter cell lines for detection of either genotoxic or oxidative stress, cells were exposed to increasing concentrations of different classes of genotoxic agents: Cisplatin, Etoposide, the methylating agent methylmethane sulphonate (MMS) and the oxidative stress inducer diethyl malonate (DEM). DsRed expression was determined at 30 hours after treatment using flow cytometry. In all reporter cell lines a clear induction of DsRed expression upon exposure to the different agents could be observed (FIG. 10B). However, the extent of induction was clearly different for some of the reporter cell lines. Btg2, Ephx1 and Perp were relatively more responsive to DNA damaging agents, while Cbr3 responded more strongly to the oxidative stress inducer DEM.
To determine the differential response of the reporter cell lines to these two classes of compounds, we calculated for every combination of two reporter cell lines the ratio of fold changes of reporter expression at each dose of the compounds used. The average ratio between two reporter cell lines was used to determine which combination of reporter cell lines allows best discrimination between genotoxic and oxidative stress-inducing compounds (FIG. 11A). The fold induction ratio of Btg2-DsRed and Cbr3-DsRed proved to be most informative. While the ratios for the DNA damaging agents tested, i.e. Cisplatin and Etoposide were all around 1.2, the ratio for MMS and DEM was 0.8. Although the alkylating agent MMS is genotoxic, various reports show that exposure to MMS results in high levels of oxidative stress due to GSH depletion [37-39]. We verified the use of these cell lines for the detection of genotoxicity and oxidative stress by calculation the fold change ratio between Btg2-DsRed and Cbr3-DsRed following exposure to the genotoxic compounds doxorubicin and mitomycin C and to the oxidative stress inducer copper sulphate (FIG. 11A). Together these results indicate that the generated DsRed reporter cell lines can be used to identify genotoxic activity of compounds and in addition can help to discriminate between direct acting DNA damaging agents and oxidative damage-inducing compounds.
Reporter Activity Specific for Genotoxic Agents
To investigate whether the reporter cell lines were selectively reactive for genotoxic carcinogens or oxidative stress, cells were exposed to a number of non-genotoxic carcinogenic compounds i.e. the immunosuppressant Cyclosporin A (CsA), the peroxisome proliferator Wyeth-14,643 and the synthetic hormone diethylstilbestrol (DES) [40] at concentrations that clearly affected cell viability (FIG. 12A). While exposure of the reporter cell lines to Cisplatin resulted in a clear induction of DsRed expression, exposure to these non-genotoxic carcinogens did not induce any reporter activation, not even at the highest concentrations (FIG. 12B). These results indicate that activation of the DsRed reporter genes is not part of a general cellular stress response. The reporter cell lines therefore may provide a highly sensitive system for assessment of genotoxicity or oxidative stress.
Discussion
Recently, visualization of the activation of cellular stress pathways using fluorescent or chemi-illuminescent reporter cell lines has become an attractive approach to identify potential (cyto)toxic properties of chemical agents [16, 19]. Various assays, like the Vitotox and Greenscreen HC tests, show that the use of luminescent or fluorescent markers for the DNA damage response provides a reliable tool for genotoxicity prediction. However, these systems provide only limited information about the mode of toxicity of the tested compounds. Depending on the reactivity of compounds, various cellular pathways are activated upon exposure. Reporters that would be able to discriminate between the different DNA damage response and cellular stress pathways would not only predict (geno)toxicity of compounds but would also provide insight in the mode of toxicity.
In this study, we have identified biomarker genes which have low background expression and a robust induction upon agent exposure. To monitor biomarker induction, promoter regions of the identified genes were placed upstream of the DsRed Express-2 fluorescent marker gene. This DsRed variant is non-cytotoxic allowing stable and high expression of red fluorescent protein in mammalian cells [34]. Indeed, expression of DsRed Express-2 reporters in mouse ES cells did not affect cellular growth or sensitivity to various genotoxic compounds (FIG. 8B). An advantage of using a red fluorescent marker, compared to the commonly used eGFP, is that it allows analysis of compounds that show green autofluorescence [41]. For four of the identified biomarker genes, a stably integrated fluorescent reporter mES cell line was selected that fulfilled the criteria of displaying low basal background expression and a robust induction upon cytotoxicant exposure.
In the four selected ES cell lines, expression of the DsRed reporters that is driven by the selected biomarker promoters closely followed the induction of the endogenous genes upon exposure to genotoxic stress (FIG. 9). Basal DsRed biomarker expression in untreated cells was higher compared to the endogenous genes possibly because of positional effects of the integration site in the genome on gene expression. However, the higher basal expression of the reporters did not influence their ability to sensitively detect DNA damage-induced expression. This indicates that the strategy cloning of promoter regions upstream of a fluorescent reporter gene and expression in mammalian cells allows development of stable and highly specific in vitro systems for genotoxicity testing.
Other genotoxicity test systems have been described previously that rely in fluorescent or luminescent reporter activation in response to cellular exposure to DNA damaging agents. The bacterial Vitotox screen depends on a luciferase gene that is under control of the bacterial DNA damage SOS signaling system and the yeast Radarscreen is based on a β-galactosidase reporter that is controlled by the promoter of the Rad54 DNA repair gene. Validation of these genotoxicity assays using the ECVAM compound list indicated that both systems show a low number of false positives and false negatives [42]. Both assays showed a strong correlation (80-90%) with the Ames mutagenicity and in vitro clastogenicity tests. The mammalian Greenscreen HC assay that depends on expression of a GFP-tagged GADD45a gene that is activated by the p53-dependent DNA damage response. Also validation of this genotoxicity test system indicated a low percentage of false positives and negatives and a good correlation with mutation induction [18].
These data confirm that monitoring the activation of the DNA damage response can be used as a reliable tool to predict genotoxicity of mutagenicity of compounds. Preliminary data indicate that also the DsRed-based reporter cell lines provide a reliable test system to assess genotoxicity after exposure to a selection of ECVAM recommended list of compounds (data not shown). In contrast to the majority of current genotoxicity assays, the DsRed reporter cell systems provides additional information about the mode of toxicity of compounds.
The sensitivity of the bacterial Vitotox, yeast RadarScreen and the mammalian Greenscreen HC assays is relatively low compared to our mES cell baser reporter system.
The use of stem cells as a basis for a (geno)toxicity test system contributes to the sensitivity of the assay. mES cells are proficient in all the major DNA damage response pathways, including the p53-dependent signalling pathway. The use of ES cells as reporter system allows detection of genotoxicity of compounds at 10 to 100-fold lower concentrations compared to the Vitotox and Greenscreen HC assays (FIG. 10 and [16, 42]). Although for many compounds the concentration used for testing might not be a relevant issue, it becomes important when compounds are poorly soluble in water or show autofluorescence. Indeed, our DsRed reporter cell lines show increased DsRed expression upon exposure to various genotoxic agents already low cytotoxic concentrations (FIG. 10). In addition, due to its high stability the DsRed proteins will accumulate in the cells which will further increase the sensitivity of the cell system.
In conclusion, we generated a mES cell base fluorescent reporter system that can be used for genotoxicity assessment of compounds. We have created four independent cell lines that express reporter genes with different substrate specificity. We showed that these reporter cell lines are able to discriminate between genotoxic compounds and pro-oxidants.
EXAMPLE 2
Performance of Srxn1, Bscl2 and Btg2 as Biomarkers for Genotoxic and Oxidative Stress-Inducing Agents
FIG. 13 demonstrates activated expression of GFP in reporter cell lines generated by BAC TransgeneOmics. Each of genes Srxn1, Bscl2 and Btg2 were fused to GFP via BAC recombineering. GFP expression in response to the genotoxic agents Cisplatin, Doxorubicin and MMC, and the oxidative stress-inducing agents DEM and CuSO4 was increased. The results confirm the use of these genes as biomarkers for genotoxic and oxidative stress-inducing agents.
EXAMPLE 3
Performance of Ltb4ra, Cgref1, Cdkn1a, Pltp, and Nope as Biomarkers for Genotoxic and Oxidative Stress-Inducing Agents
FIG. 14 demonstrates activated expression of DsRed in mouse ES cells driven by the promoter regions of each of Ltb4ra, Cgref1, Cdkn1a, Pltp, and Nope. Reporter cell lines were generated as described in Example 1. The results confirm the use of these genes as biomarkers for genotoxic and oxidative stress-inducing agents.
EXAMPLE 4
Identification of Five Genes (Gpx2, Ltb4r2, Ddit4L, Fosl1 and Egr1) as Biomarkers for Genotoxic or Oxidative Stress
We have identified genes that could serve as potential biomarkers for exposure to genotoxic stress by exposing mouse embryonic stem (ES) cells to various genotoxic compounds and changes in gene expression were determined.
Materials and Methods
ES Cell Culture and Treatments (See Example 1)
Transcription Profiling
Total RNA was isolated using the RNeasy mini kit (Qiagen, Valencia, Calif.) according to the manufacturer's protocol. RNA quality and integrity was assessed with the Agilent 2100 Bioanalyzer system (Agilent technologies, Palo Alto, Calif.). RNA sample labeling and hybridization on Affymetrix arrays (Genechip Mouse Genome 430A arrays) was performed according to the manufacturer's protocols (Affymetrix). The data were analyzed using Rosetta Resolver (Rosetta Biosoftware, Seattle, Wash., USA). Upon importing the Affymetrix Genechip data (CEL files), data pre-processing including background correction and normalization was performed. Triplicate hybridizations were combined and the data points were compared to control treatments to create log ratios and associated p-values for the significance of differential expression. Genes with a false discovery rate (FDR) of 10% were identified as differentially expressed. In order to identify biomarker candidates, we divided our compounds into 2 classes: 1-DNA damage agents (CDDP, Dox, Etop, MMC, MMS, MNU), 2-pro-oxidant agents (H2O2, t-BUP, Dem, Men), and selected only genes with pvalues<0.01 and fold change>2 for all the treatments belonging to a class of compounds. To select the most significantly expressed genes, we combine the multiple treatments points for each class, we summed the logarithms of the multiple testing corrected p-values (log(pvalues)) and the fold change. A number of biomarker candidates either responsive to DNA damage or to prooxidants were identified with the lowest summed log(pvalues) and the highest summed fold change.
Results
Identification of Putative Biomarkers
Five genes have been identified that can serve as biomarkers for genotoxicity or oxidative stress. To establish the cellular response to genotoxic stress, we exposed mouse embryonic (ES) cells to various genotoxic agents. ES cells are p53 proficient and are capable of indefinite cell division without transformation, in contrast to the majority of established mammalian cell lines.
We first established cytotoxicity of various DNA damaging agents in mouse ES cells by measuring apoptosis (see Example 1 and FIG. 7).







TABLE 4







Genotoxic compounds used in study










Compound
Abbreviation
Concentrations
Mode of action














Cis-Platin
CisPt
1-10
uM
Intra- and interstrand crosslinks. Lesions on G or A.


Doxorubicin
Dox
0.001-0.2
uM
Intercalating agent. Inhibits progression of topoisomerase-II.


Etoposide
Etop
0.1-2
uM
Topoisomerase-II inhibitor.


Mitomycin-C
MMC
0.1-1.5
ug/ml
DNA crosslinking agent. Interstrand and intrastrand crosslinks and mono aducts at guanine.


Methyl Methane
MMS
0.05-0.5
mM
Alkylating agent, methylates DNA on N7-deoxyguanine and N3-deoxyadenine.


Sulphonate






Diethyl Malonate
DEM
25-250
uM
Glutathion depletion, results in increased levels of oxigen radicals.


Menadione
MEN
5-100
uM
Vitamin K. Prooxidant.


Hydrogen Peroxide
H2O2
25-200
uM
Produces oxigen radicals resulting in protein and DNA damage


Tert-butyl Hydroperoxide
tBHP
25-100
uM
Prooxidant. Depletion of cellular stores of GSH and oxidation of






functionally important SH groups on mitochondrial enzymes


Antimycin A1
Ant-A1
0.5-10
uM
Mitochondrial toxin. Respiratory chain inhibitor


Potassium Cyanide
KCN
0.5-5
uM
Mitochondrial toxin. Respiratory chain inihbitor


Rotenone
Rot
1-10
uM
Mitochondrial toxin. Respiratory chain inhibitor


2-Acetylamino Fluorene
2-AAF
10-100
uM
CYP450 substrate. AF and AAF adducts at C(8) and N(2) position of guanine.


Cytosine arabinoside
AraC
0.05-5
uM
Incorporated into DNA. Chain terminator.


Vincristine
Vin
1-100
uM
Microtubule disruption. Blocks cells in mitosis


Flavopiridol
Flavo
10-100
uM
Cyclin-dependent kinase (cdk) inhibitor. Downregulation of Bcl-2. Causes






cell cycle arrest and apoptosis


Cyclosporin A
CsA
0.5-10
uM
Non-genotoxic carcinogen. Immuno suppressor


Wyeth-14,643
Wyeth
1-250
uM
Non-genotoxic carcinogen. Peroxisome proliferator









These concentrations were used to determine the changes in gene expression using microarray analysis of the complete cellular transcriptome upon exposure to various genotoxic agents. RNA was isolated from cells, 8 h. after exposure to increasing concentrations of various genotoxic agents and subsequently hybridized on Affymetrix arrays. After normalization and background correction, genes that displayed the highest change in expression after treatment were identified, based on both fold change and p-value (FIGS. 15A and 15B). Data analysis showed that Gpx2, Ltb4r2, Ddit4l, Fosl1, and Egr1 are highly upregulated, confirming a specific response to DNA damage. Based on this, we have identified five genes as putative biomarkers for genotoxic stress.
Generation of DsRed Reporter Cell Lines
Regulatory elements of the selected genes are fused to a DsRed reporter gene and stably integrated into mouse ES cells. The fluorescent DsRed markers provide an easy and sensitive tool to study the DNA damage response in mammalian stem cells.
A panel of highly sensitive mouse ES cells that allow assessment of genotoxicity is developed. The assay is based on expression of a DsRed fluorescent marker gene under control of different promoters that are specifically activated upon exposure to DNA damaging agents. Genotoxicity can be tested at non-cytotoxic concentrations and detection of DsRed fluorescence using flow cytometry allows usage of these cell lines as a high throughput assay.
The cell lines are generated, DsRed expression detected, and apoptosis assayed by, for example, methods described in Example 1.
Sensitivity
Sensitivity of the reporter cell lines for detection of genotoxic stress is assessed by exposing the cells to increasing concentrations of the genotoxic agents cisplatin, etoposide, the methylating agent methylmethane sulphonate (MMS) and the indirect oxidative stress inducer diethyl malonate (DEM). At different times after treatment DsRed expression is determined using flow cytometry. Used concentrations of the different compounds is based on the level of apoptosis induction in wild type ES cells (FIG. 7).
Not only exposure to genotoxic agents has been shown to induce cancer but also non-genotoxic compounds have been implicated in carcinogenesis although their mode of action is largely unknown. To investigate whether the reporter cell lines can be used to discriminate between genotoxic and non-genotoxic carcinogens, cells are exposed to cyclosporine A (CsA), Wyeth-14643 and diethylstilbestrol (DES). The concentrations of the non-genotoxic compounds that are used for the exposures are based on their effect on cell proliferation (data not shown). DsRed expression is determined by flow cytometry.
Exposure to high concentrations of genotoxic compounds rapidly induces apoptosis to protect cells against the mutagenic effects of DNA damage. Apoptosis can be easily determined by annexin V or caspase 3 staining assays, for example as described above. To further establish the sensitivity of the reporter cell lines, DsRed expression in the reporter cells is compared to apoptosis induction after exposure to increasing doses of genotoxic agents.
The genes that are identified as putative biomarkers for genotoxicity are selected based on strong transcriptional activation upon exposure to genotoxic agents. The promoter regions of these genes are fused to a fluorescent reporter gene and stable integrated in mouse ES cells. Ideally, the changes in DsRed expression upon genotoxic stress would closely resemble the transcriptional activation of the endogenous biomarker genes. Expression levels of the reporter genes and the endogenous genes are determined at different times after exposure to a genotoxic agent by quantitative RT-PCR.
EXAMPLE 5
GFP Reporter Cell Systems for DNA Replication Inhibition and Oxidative Stress
Summary
Industry yearly develops a large number of new chemical compounds for a wide range of applications that benefit society. Following environmental, occupational, accidental or medical exposure of humans, these compounds may react or interact with various cellular structures and organelles, including DNA, proteins and lipids with possible (geno)toxic consequences. Current approaches for evaluating the safety of chemicals and products are resource- and time-intensive, making it difficult to meet the demands of evaluating the safety of an ever-increasing number of chemicals. Monitoring activation of specific cellular signalling pathways upon exposure to various classes of (geno)toxic carcinogens will on one hand allow assessment of potential (geno)toxic properties of chemicals, and on the other hand can provide more insight into the primary mode of toxicity of compounds.
We have constructed two reporters encoding C terminal green fluorescent GFP-tagged fusion proteins. The Bscl2-GFP reporter is selectively activated after exposure to DNA damaging agents. Induction of the reporter is associated with inhibition of DNA replication and activation of the ATR DNA damage signaling pathway. The Srxn1-GFP reporter is specifically induced upon oxidative stress and is part of the Nrf2 antioxidant response pathway. Employment of these mES reporter cell lines can provide insight into the primary reactive properties of known and unknown chemicals.
Results
FIG. 16 shows GFP fluorescence-based mES reporter cell systems can be used to detect genotoxicity and oxidative stress.
FIG. 17 demonstrates that GFP reporters are specific for genotoxic and oxidative stress.
FIG. 18 shows Srxn1-GFP induction in response to ROS production.
FIG. 19 shows Bscl2-GFP reporter activation by DNA replication inhibition.
FIG. 20 shows that DNA damage reporter activation depends on ATR signalling.
FIG. 21 focuses on the interaction between p53 and Nrf2 signalling pathways and GFP reporter activation.
Conclusions
Mouse embryonic stem cell systems can detect genotoxic or oxidative stress-inducing properties of chemicals.
Bscl2-GFP reporter for DNA damage is activated upon DNA replication inhibition
Bscl2-GFP reporter expression depends on the ATR damage signalling pathway but is p53 independent.
Srxn1-GFP reporter for oxidative stress is activated in response to ROS production and is controlled by the Nrf2 pathway.
EXAMPLE 6
The ToxTracker Assay: GFP Reporter Systems That Provide Mechanistic Insight Into the Genotoxic Properties of Chemicals
Summary
Here we describe the generation of a novel GFP-based (geno)toxicity assay, ToxTracker, consisting of different mES reporter cell lines that are preferentially responsive to genotoxic compounds or to agents that induce oxidative stress. The Bscl2-GFP genotoxicity reporter is activated upon replication inhibition and depends on the ATR-Chk1 signaling pathway. However, Bscl2-GFP expression is not regulated by the p53 tumor suppressor. The Srxn1-GFP reporter is activated upon increased levels of oxidative stress and is controlled by the Nrf2 anti-oxidant pathway. A third GFP reporter cell line based on the p53-responsive Btg2 gene is activated upon exposure to a broad spectrum of (geno)toxic compounds. The ToxTracker assay provides a powerful tool for (geno)toxic risk assessment of novel chemicals while providing mechanistic information on the genotoxic and/or oxidative properties of a compound.
Materials and Methods
ES Cell Culture and Treatments
C57/B16 B4418 wild type mouse ES (mES) cells were cultured in ES knockout medium (Gibco) containing 10% FCS, 2 mM glutamax, 1 mM sodium pyruvate, 100 μM β-mercaptoethanol and leukemia inhibitory factor (LIF) as previously described (Hendriks et al, 2011). Mouse ES cells were propagated on irradiated primary mouse embryonic fibroblasts as feeders according to established protocols. Cells were seeded 24 h prior to chemical exposure on gelatin-coated plates in BRL-conditioned ES cell medium in the absence of feeder cells. For analysis of compounds that require metabolic activation, cells were exposed for 3 h in the presence of 1% S9 rat liver extract in 3.2 mM KCl, 0.8 mM MgCl2, 0.5 mM glucose-6-phosphate and 0.4 mM NADP. After 3 h cells were washed with PBS and cultured for 24 h in BRL-conditioned medium without the tested compounds. In all other treatments cells were continuously exposed for 24 h before GFP reporter analysis. For the inhibition of ATM, ATR and Chk1/Chk2 signaling in response to replication stress, cells were seeded 24 h prior to exposure in gelatin-coated 96-wells plates. Cells were exposed to 10 μM cisplatin (CisPt) or 1.5 μM aphidicolin (Aph) for 24 h in the presence of Ku55933 ATM inhibitor (0, 2, 5, 10 μM), schisandrin B ATR inhibitor (0, 6, 15, 30 μM) or UCN-01 Chk1/Chk2 inhibitor (0, 100, 200 nM).
Generation of GFP Reporter Cell Lines
The GFP reporters were generated by BAC recombineering as described above (Poser et al, 2008). Bacterial strains with a BAC containing the biomarker gene were selected using mouse BAC finder and ordered from BACPAC. The putative biomarker genes on the BAC were modified with a C-terminal GFP green fluorescent marker (Poser et al, 2008) using the Quick & Easy BAC modification Kit (Gene Bridges). Electrocompetent bacterial BAC strains were first transformed with the pRed/ET plasmid that contains the RecE and RecT recombination enzymes. PCR fragments encoding a GFP-ires-neomycin/kanamycin reporter cassette were generated using primers that each contain 50 nucleotide additional sequence homologous to the 3′ sequence of the biomarker gene on the BAC. These homologous sequences on both the 5′ and 3′ ends of the PCR fragment allow RecE/T-mediated site-specific recombination of the GFP-ires-Neo selection cassette at the 3′ end of the biomarker gene on the BAC. BAC strains that contain pRed/ET were grown at 37° C. for 30 minutes in the presence of L-arabinose to induce expression of the recombination enzymes. Subsequently, BAC strains were transformed with the GFP-ires-Neo PCR fragment by electroporation, incubated at 37° C. for 2 h to allow recombination of the PCR fragment with the BAC and plated on kanamycin selection plates. Individual clones were analyzed for proper integration of the GFP cassette by PCR. Modified BACs were isolated using the Nucleobond PC100 DNA isolation kit (Macherey Nagel).
Mouse ES cells were seeded on gelatin-coated culture dishes 24 h prior to transfection. Modified BACs were transfected into the mES cells using Lipofectamine 2000 (Invitrogen) according as described previously (Poser et al, 2008). Monoclonal mES cell lines were selected based on the level of induction of the GFP reporter after exposure to genotoxic compounds or pro-oxidants. GFP expression was determined by flow cytometry.
siRNA Transfection
The GFP reporter cells were transfected with SMARTpools of four individual siRNAs against Nrf2 or p53 (Dharmacon). A scrambled non-targeting siRNA pool was used as negative control. A siRNA against kif11, an essential gene that encodes a kinesin-like protein, was used to determine transfection efficiency. Kif11 knockdown is lethal for mES cells. siRNA transfections were performed in gelatin-coated 96-wells cell culture plates. 1 μM siRNA was mixed with 0.1 μl Dharmafect 1 transfection reagent in 20 μl serum-free medium per transfection. The siRNA mix was transferred to the 96-wells plate and subsequently 11.000 mES reporter cells were seeded in each well. Cells were washed after 16 h with PBS and cultured in fresh BRL-conditioned ES cell medium. After 48 h cells were treated with various genotoxic and oxidative stress inducing compounds. After 24 h incubation, induction of the GFP reporters was determined by flow cytometry.
Detection of GFP Expression
Cells were seeded on gelatin-coated 96 wells plates and 24 hours later subsequently exposed to various genotoxic agents. All tested compounds were dissolved in DMSO or PBS and diluted in fresh BRL-condition ES cell medium just before incubation with the cells. After 24 h exposure, cells were washed with PBS, trypsinized and resuspended into PBS+2% serum, immediately followed by flow cytometry analysis (Guava easyCyte 6HT, Millipore).
GFP reporter expression in mES cells was visualized by confocal immunofluorescence microscopy and live cell imaging. Cells were seeded at low density on fibronectin-coated glass cover slips. For confocal microscopy analysis, the cells were exposed to 5 μM CisPt or 150 μM DEM for 24 h and subsequently fixed with 2% paraformaldehyde in PBS. GFP reporter expression was visualized using a Leica TCS SP2 confocal microscope. For live cell imaging, cells were plated on glass bottom 96 well culture plates (Greiner) and exposed to 5 μM CisPt or 100 μM DEM. GFP reporter activation was determined using a Nikon TiE2000 microscope equipped with a Perfect Focus System and an automated microscope stage at 37° C. with 5% CO2 delivery to the sample plate location. Images were acquired with a 20× (NA 0.75) dry Plan Apochromat objective and the image acquisition was controlled by EZ-C1 software (Nikon). In each well, an image from the same position was acquired every 15 minutes for a period of 24 hours. Automated image analysis of individual images was performed using Image-Pro Plus software (MediaCybernetics) to calculate the induction of overall cellular fluorescence.
Quantitative RT-PCR Analysis of Reporter Expression
Induction of the GFP reporters was compared with the expression of the endogenous gene using quantitative real time PCR (qRT-PCR). Cells were exposed to the genotoxic agent CisPt or the pro-oxidant DEM and total RNA was isolated after 8 or 16 hours using the RNeasy mini kit (Qiagen). cDNA was synthesized using oligo(dT)12-18 primers and SuperscriptIII reverse transcriptase (Invitrogen) according to the manufacturer's protocol. Expression of the GFP reporter and the endogenous biomarker gene was determined using specific primers against the 3′-UTR of either gene with the FastStart SYBR Green Master (Roche) QPCR mix on a 7900HT Fast Real-Time PCR System (Applied Biosystems). Relative expression was normalized using expression of the YWHAD and Hprt genes.
Western Blot Analysis
Activation of the ATM and ATR signaling pathways in response to CisPt and Aph was determined by Western blot analysis. Cells were lysed in Laemmli protein sample buffer after 24 h exposure and subjected to SDS-PAGE. Proteins were transferred to PVDF membrane (Millipore) and detected using antibodies against phospho-Kap1 (Bethyl laboratories), phospho-Chk1 (Bethyl laboratories), phospho-p53 (Cell signaling) or Hprt (Santa Cruz) as protein loading control. Proteins were visualized using enhanced chemoluminescence (ECL).
Validation of GFP Reporter Cell Lines
The mES GFP reporter cells were exposed to at least five different concentrations of 50 genotoxic and non-genotoxic compounds. The selection of compounds was largely based on the ECVAM suggested list of chemicals for validation of in vitro genotoxicity test assays (Kirkland et al, 2008). Compound concentrations that were used for the validation were based on cytotoxicity, where the highest concentration induced significant cell death (10-25% viable cells after 24 h treatment). Cell viability was determined by flow cytometry as the fraction of intact cells after 24 h of treatment compared to untreated cells. For compounds that did not affect cell viability, a maximum concentration of 10 mM was used. Induction of GFP fluorescence in the reporter cells was determined after 24 h exposure by flow cytometry.
Testing Criteria
Activation of a reporter cell line was considered positive when exposure to at least two different concentrations of a compound resulted in >1.5 fold induction of GFP expression which is at least 5 times higher than the standard deviation in background fluorescence. To determine if a reporter gene was activated by a compound, only concentrations that resulted in >25% cell survival were taken into account. At higher cell killing levels GFP induction would often not increase anymore with dose. All presented data are the summary of at least three independent experiments. All shown error bars represent standard deviations.
Results
GFP-Based Reporters for Genotoxicity and Oxidative Stress
We have found two putative biomarker genes to be responsive to either DNA damaging chemicals or to oxidative stress (see Examples 2 and 5, and FIG. 16). The Bscl2 gene was shown to be selectively responsive to genotoxic compounds. The Bscl2 gene is defective in patients suffering from Berardinelli-Seip congenital lipodystrophy and encodes the Seipin protein (Magre et al, 2001)(Szymanski et al, 2007). So far, the Bscl2 gene has not been implicated in the cellular DNA damage response. The Srxn1 gene encodes the sulfiredoxin-1 protein that reduces oxidized cysteines in peroxiredoxins (Prxs) in the peroxisomes. Srxn1 plays an important role in the defense against cellular oxidative stress (Chang et al, 2004).
To allow physiological regulation of gene expression we used BAC recombineering (Poser et al, 2008) (FIG. 16B) to generate Bscl2- and Srxn1-based green fluorescent reporters. Following transfection multiple mES cell lines containing either the Bscl2-GFP or Srxn1-GFP reporter were evaluated for their responsiveness to either the DNA damaging agent cisplatin (CisPt) or the indirect pro-oxidant diethyl maleate (DEM) and a single clonal cell line for either construct was selected for further studies. Bscl2-GFP correctly localized to the endoplasmic reticulum, while Srxn1-GFP was located in both the nucleus and cytoplasm as reported previously, indicating that the GFP tag did not affect their localization (FIG. 16C). By quantitative real-time PCR (qRT-PCR), we compared the responsiveness of the GFP reporters with the corresponding endogenous genes. The Bscl2-GFP reporter was selectively induced upon exposure to CisPt but not to DEM (FIGS. 16D and E). Both the basal level and the kinetics of induction of the GFP reporter were comparable to that of the endogenous Bscl2 gene. The Srxn1-GFP reporter was somewhat responsive to CisPt but highly induced after exposure to DEM. The specificity of the Srxn1-GFP reporter was comparable to endogenous Srxn1 although the extent of induction of the GFP reporter appears to be slightly higher compared to the endogenous gene.
Sensitivity and Specificity of the mES GFP Reporter Cell Lines
To investigate the sensitivity and specificity of the mES Bscl2-GFP and Srxn1-GFP reporter cell lines, cells were exposed to increasing concentrations of the genotoxic compounds CisPt, etoposide, doxorubicin and mitomycin C (MMC) or the oxidative stress-inducing agents DEM, sodium arsenite (NaAsO2), cadmium chloride (CdCl2) and methyl methanesulphonate (MMS). Although MMS is a DNA alkylating agent, we and others have previously shown that the primary toxic response of cells after exposure to MMS is strongly correlated with oxidative stress induction, likely due to the direct reaction of MMS with gluthatione and other proteins (Hendriks et al, 2011)(Wilhelm et al, 1997)(Ashino et al, 2003). The Bscl2-GFP reporter was significantly induced after exposure to all tested genotoxic compounds in a concentration-dependent fashion but hardly responded to either of the pro-oxidants or the alkylating agent MMS (FIG. 17D). In contrast, the Srxn1-GFP reporter cells was highly responsive to all oxidative stress-inducing agents including MMS, but was also induced by the genotoxic compounds, albeit to a lesser extent than the Bscl2-GFP reporter. Cytotoxicity of CisPt and DEM was comparable in the Bscl2-GFP and Srxn1-GFP reporter cells, indicating that induction of the GFP reporters is not correlated with general cellular stress (FIG. 17B). In addition, both GFP reporter cell lines were equally sensitive to CisPt and DEM indicating that expression of the GFP reporters is not cytotoxic.
We evaluated the dynamic response of the GFP reporter cell lines by using time-lapse live cell imaging confocal microscopy and quantitative image analysis (FIG. 17E). The Srxn1-GFP reporter was readily induced upon exposure to DEM but was hardly responsive to CisPt along the entire time period. Expression of the Srxn1-GFP reporter was clearly detectable after 8 h exposure to DEM and reached a plateau after 24 h exposure. Reversely, the Bscl2-GFP reporter was preferentially induced upon exposure to CisPt, in agreement with the flow cytometry analysis (FIG. 17D). Bscl2-GFP expression became visible as early as 12 h and steadily increased up to 24 h after start of treatment.
Validation of the GFP Reporters
To further establish the sensitivity and specificity of the Bscl2-GFP and Srxn1-GFP reporters, they were exposed to a wide variety of carcinogenic and non-carcinogenic compounds, as suggested by the European Center for Validation of Alternative Methods (ECVAM) (Kirkland et al, 2008). ECVAM Class 1 compounds consists of in vivo carcinogens that are either positive or negative in the Ames bacterial mutagenicity test or that should score positive in an in vitro genotoxicity assay. ECVAM Class 2 compounds are Ames negative, non-genotoxic carcinogens or non-carcinogens that should be negative in in vitro genotoxicity tests and ECVAM Class 3 compounds are Ames negative but score equivocal or positive in an in vitro genotoxicity assay. In total exposures to 50 different mainly ECVAM-suggested genotoxins, pro-oxidants and non-genotoxins were performed (Table 3 and FIGS. 22-25). All ECVAM class 1 compounds scored positive in one or both GFP reporter cell lines, except p-chloroaniline. Although this compound is an in vivo carcinogen, it was also not identified as genotoxin in other in vitro genotoxicity assays (Birrell et al, 2010)(Westerink et al, 2010). All tested ECVAM class 2 compounds failed to induce the GFP reporters. The ECVAM class 3 compounds that were previously identified as genotoxin in other in vitro genotoxicity assays also induced the GFP reporters. Interestingly, nearly all of the ECVAM class 3 compounds that scored positive, selectively induced expression of the Srxn1-GFP reporter, suggesting that the primary toxic properties of these compounds are associated with the induction of oxidative stress. An additional collection of non-ECVAM suggested compounds were all correctly identified as genotoxic or oxidative stress-inducing chemicals. Together these data show that the Bscl2-GFP and Srxn1-GFP reporter cells, referred to us as the ToxTracker assay, provide a sensitive and selective test to establish potential toxic activities of chemicals. In addition, by integrated evaluation of results from both reporters, the ToxTracker assay can provide insight in the primary toxic properties of compounds.







TABLE 3





Sensitivity and specificity of the Bscl2-GFP and Srxn1-GFP reporters in detection of genotixic and oxidative properties of chemicals.


















Genotoxicity profile











Bscl2-GFP/Srxn1-GFP

















CAS
Ames
In vivo
In vitro
mES DsRed
GFP
Prim. toxic



Chemical
number
test1
genotoxicity1
genotoxicity1
reporters2
Induction
properties
Information










ECVAM class 1















1. Ames-positive in vivo










genotoxins.


Cisplatin
15663-27-1
Pos.
Pos.
Pos.
Pos.
Pos.
Genotoxin
DNA crosslinking agent.










Used in cancer treatment.


MMS
66-27-3
Pos.
Pos.
Pos.
Pos.
Pos.
Pro-oxidant
Alkylating agent. Strong










mutagen.


Cadmium Chloride
10108-64-2
Pos.
Pos.
Pos.
Pos.
Pos.
Pro-oxidant
Inorganic carcinogen


p-chloroaniline
106-47-8
Pos.
Pos.
Equivocal
Neg.
Neg.

Non-carcinogenic. Used in










herbicides and pesticides


2. In vivo genotoxins, neg. or


equivocol in Ames.


Sodium arsenite
7784-46-5
Neg.
Pos.
Pos.
Pos.
Pos.
Pro-oxidant
Inorganic carcinogen.










Oxidant.


Taxol
33069-62-4
Neg.
Pos.
Pos.
Pos.
Pos.
Genotoxin
Aneugen. Used in cancer










treatment.







ECVAM class 2















1. Non-carcinogens, neg. in in










vivo genotoxicity tests.


n-butyl chloride
109-69-3
Neg.
nd
Neg.
Neg.
Neg.


Phenformin HCl
834-28-6
Neg.
nd
Neg.
Neg.
Neg.


(2-chloroethyl)trimethyl-
999-81-5
Neg.
nd
Neg.
Neg.
Neg.


ammonium chloride


N,N-dicyclohexyl thiourea
1212-29-9
Neg.
nd
Neg.
Neg.
Neg.


Cyclohexanone
108-94-1
Neg.
nd
Neg.
Neg.
Neg.


Erythromycin stearate
643-22-1
Neg.
nd
Neg.
Neg.
Neg.


Fluometron
2164-17-2
Neg.
nd
Neg.
Neg.
Neg.


2. Non-genotoxic carcinogens.


D-limonene
5989-27-5
Neg.
nd
Neg.
Neg.
Neg.


Amitrole
61-82-5
Neg.
Neg.
Neg.
Neg.
Neg.


Tert-butyl alcohol
75-65-0
Neg.
Neg.
Neg.
Neg.
Neg.


Diethanolamine
111-42-2
Neg.
Neg.
Neg.
Neg.
Neg.


Hexachloroethane
67-72-1
Neg.
Neg.
Neg.
Neg.
Neg.


Methyl carbamate
598-55-0
Neg.
Neg.
Neg.
Neg.
Neg.


Pyrldlne
110-86-1
Neg.
Neg.
Neg.
Neg.
Neg.


Tris(2-ethylhexyl)phosphate
78-42-2
Neg.
Neg.
Neg.
Neg.
Neg.













Genotoxicity profile
















CAS
Ames
In vivo
In vitro
mES DsRed
Bscl2-GFP/



Chemical
number
test1
genotoxicity1
genotoxicity1
reporters2
Srxn1-GFP
Information










ECVAM class 3















1. Non-carcinogens,










neg. or equivocal in


in vivo genotoxicity tests.


Tert-butylhydroquinone
1948-33-0
Neg.
Neg.
Pos.
Pos.
Pos.
Pro-oxidant


o-anthranilic acid
118-92-3
Neg.
Neg.
Pos.
Pos.
Neg.


1,3-Dihydroxybenzene
108-46-3
Neg.
Neg.
Pos.
Pos.
Pos.
Equivocal


(Resorcinol)


Sulfisoxazole
127-69-5
Neg.
Neg.
Equivocal
Neg.
Pos.
Pro-oxidant


2. Non-carcinogens,


no in vivo


genotoxicity data.


Ethionamide
536-33-4
Neg.
nd
Weak Pos.
Neg.
Neg.


Curcumln
458-37-7
Neg.
nd
Pos.
Neg.
Neg.


Benzyl alcohol
100-51-6
Neg.
nd
Weak Pos.
Neg.
Neg.


Urea
57-13-6
Neg.
nd
Pos.
Neg.
Neg.


3. non-genotoxic


carcinogens,


carcinogen by


irrelevant mechanism.


Sodium saccharin
128-44-9
Neg.
Neg.
Equivocal
Neg.
Neg.


4. Supplementary


list, in vitro


genotoxicity unclear.


p-Nitrophenol
100-02-7
Neg.
nd
Equivocal
Neg.
Pos.
Pro-oxidant


2,4-dichlorophenol
120-83-2
Neg.
Weak Pos.
Pos.
Pos.
Pos.
Pro-oxidant


Eugenol
97-53-0
Neg.
Equivocal
Pos.
Neg.
Neg.


Ethyl acrylate
140-88-5
Neg.
Weak Pos.
Pos.
Neg.
Neg.


Isobutyraldehyde
78-84-2
Neg.
Equivocal/Pos.
Pos.
Neg.
Neg.


Propyl gallate
121-79-9
Neg.
Equivocal
Pos.
Pos.
Pos.
Equivocal


Additional


Doxorubicin
23214-92-8
Pos.
Pos.
Pos.
Pos.
Pos.
Genotoxin
DNA intercalating










chemical.










Topoisomerase










II inhibitor.


Mitomycin C
50-07-7
Pos.
Pos.
Pos.
Pos.
Pos.
Genotoxin
DNA crosslinking










agent. Used in










cancer treatment.


Etoposide
33419-42-0
Neg.
Pos.
Pos.
Pos.
Pos.
Genotoxin
Topoisomerase










II poison.


Diethyl maleate
141-05-9
nd
nd
nd
Pos.
Pos.
Pro-oxidant
Pro-oxidant.


Tert-butyl
75-91-2
Pos.
Neg.
Neg.
Neg.
Pos.
Pro-oxidant
Pro-oxidant.


hydroperoxide


Hydrogen peroxide
7722-84-1
Pos.
Neg.
Pos.
Neg.
Pos.
Pro-oxidant
Pro-oxidant.


Flavopiridol
146426-40-6
nd
nd
nd
Pos.
Neg.

Cdk2 inhibitor.










Cell cycle










blocking agent.


Copper sulfate
7758-98-7
nd
nd
nd
nd
Pos.
Pro-oxidant
Used as herbicide,










fungicide and










pesticide.


Potassium bromate
7758-01-2
Pos.
Pos.
Pos.
nd
Pos.
Pro-oxidant
Pro-oxidant.










Used as flour










additive.


4-Nitroquinelone-
56-57-5
Pos.
Pos.
Pos.
nd
Pos.
Genotoxin
UV-mimetic agent.


1-oxide


4-Hydroxy-2-nonenal
75899-68-2
Neg.
Neg.
Pos.
nd
Pos.
Pro-oxidant
Lipid peroxi-










dation product.


Cytarabine
147-94-4
Neg.
nd
Pos.
nd
Pos.
Genotoxin
DNA chain










terminator. Used










in chemotherapy.


Camptothecin
7689-03-4
Neg.
Neg.
Pos.
nd
Pos.
Genotoxin
Topoisomerase










I Inhibitor


Bleomycin
11056-06-7
Pos.
Pos.
Pos.
nd
Pos.
Genotoxin
Radiomimetetic agent.





1Data extracted from Kirkland et al., Mutat. Res. 653 (2008), 99-108.


2Hendriks et al., Mutat Res., 709-710 (2011), 49-59.






GFP Reporter Induction by Compounds that Require Metabolic Activation

To test the performance of the ToxTracker assay in the detection of genotoxic chemicals that require biotransformation we exposed Bscl2-GFP reporter cells to various pro-genotoxic compounds in the presence of S9 rat liver extract. All four tested carcinogenic compounds (aflatoxin B1 (AFB1), benzo[a]pyrene (B[a]P, cyclophosphamide and dimethylbenz(a)antracene (DMBA)) induced expression of the Bscl2-GFP reporter only when incubated in the presence of rat S9 mix (FIG. 26). In agreement, S9-dependent activation of these pro-genotoxins resulted in reduced survival of the reporter cells.
Srxn1-GFP Reporter is Induced by ROS and Controlled by Nrf2 Signaling
To investigate whether induction of the Srxn1-GFP reporter upon exposure to oxidative stress-inducing compounds was directly related to ROS production, we treated Bscl2-GFP or Srxn1-GFP reporter cells with the pro-oxidants DEM, CuSO4, NaAsO2, CdCl2 and the DNA alkylating agent MMS, in the presence of the ROS scavenger N-acetyl cysteine (NAC). Incubation of cells with NAC did not affect cell viability or reporter activity (data not shown). Exposure of Srxn1-GFP reporter cells to the various pro-oxidants, including MMS, resulted in a strong, concentration-dependent increase in expression of the GFP reporter (FIG. 27A). Addition of the ROS scavenger completely inhibited activation of the reporter, indicating that induction of the Srxn1-GFP reporter directly depends on the production of oxygen radicals. In line with our previous results, the Bscl2-GFP reporter was not induced by pro-oxidant exposure.
Nrf2 is a key regulator of the induced expression of anti-oxidative enzymes in response to oxidative stress (Hayes and McLellan, 1999). Also Srxn1 has previously been identified as a potential Nrf2 target gene (Singh et al, 2009). To investigate whether the Nrf2 pathway controls expression of the Srxn1-GFP reporter, we exposed reporter cells to various genotoxic and oxidative stress-inducing agents following siRNA mediated knock down of Nrf2 (FIG. 27B). Srxn1-GFP was preferentially induced by the pro-oxidants DEM, MMS and NaAsO2 while the Bscl2-GFP reporter was selectively activated by the genotoxic agents CisPt and etoposide. The response of both GFP reporter cell lines to genotoxic agents was not affected by Nrf2 knockdown. In contrast, induction of the Srxn1-GFP reporter by pro-oxidants was strongly decreased after knockdown of Nrf2, indicating that Srxn1-GFP is under control of the Nrf2 anti-oxidant response (FIG. 27C).
Bscl2-GFP Reporter is Activated by DNA Replication Stress
The Bscl2-GFP reporter is activated by a wide variety of genotoxic chemical compounds with different reactive properties (FIGS. 16A and 17E and Table 3). CisPt and MMC are DNA crosslinking agents, etoposide and doxorubicin induce DNA double strand breaks during DNA replication by inhibition of topoisomerase II, while MNU methylates DNA. We assessed which mechanism exposure to these compounds with different chemical reactive properties resulted in induction of the Bscl2 gene. A common property of genotoxic compounds is that they cause DNA damage that can interfere with transcription and DNA replication. To investigate whether activation of the Bscl2-GFP reporter is correlated with DNA replication stress, we exposed the reporter cell lines to different DNA replication inhibitors. Hydroxyurea (HU) inhibits the enzyme ribonucleotide reductase, thereby depleting the pool of free ribonucleotides in the cells, while aphidicolin (Aph) directly inhibits DNA polymerases. Both drugs inhibit DNA replication without inflicting DNA damage. Exposure of Bscl2-GFP cells to HU or Aph resulted in a significant induction of GFP expression, indicating that activation of the Bscl2-GFP reporter by various genotoxic agents is related to DNA replication stress (FIG. 28A). Exposure of Srxn1-GFP cells to HU or Aph resulted in slight reporter activation, in agreement with its limited activation by various genotoxic agents (FIGS. 28A and 17E).
Bscl2-GFP Reporter Activation by ATR-Dependent DNA Damage Signaling
To investigate the involvement of various DNA damage response kinases in activation of the GFP reporters, we treated cells with the DNA crosslinking agent CisPt or the DNA replication inhibitor Aph in the presence of inhibitors of ATM (ku55933), ATR (schisandrin B) (Hickson et al, 2004)(Nishida et al, 2009) or Chk1 and Chk2 (UCN-01) (Yu et al, 2002). ATR inhibition diminished Chk1 and p53 phosphorylation, while inhibition of ATM prevented phosphorylation of Kap1 and p53 in response to CisPt and Aph (FIG. 28B). Bscl2-GFP reporter activation could almost completely be repressed by either the ATR or the Chk1/Chk2 inhibitor, but was unaffected by the ATM inhibitor (FIG. 28C). These data indicate that the Bscl2-GFP reporter is activated by the ATR-Chk1 signaling pathway in response to stalled DNA replication forks. As previously, while the Srxn1-GFP reporter is also slightly activated upon exposure to CisPt or Aph, its activation is not dependent on the ATM or ATR DNA damage signaling pathways.
Bscl2-GFP Reporter Activation is p53-Independent
The p53 tumor suppressor plays a central role in the DNA damage response (Meek, 2009). p53 is activated by various cellular signaling pathways, including the ATM-Chk2 and ATR-Chk1 kinases, (see also FIG. 28B), and controls the activity of DNA repair systems, cell cycle checkpoints and apoptosis. Interestingly, induction of the Nrf2 pathway also appears to trigger p53 activation (Wakabayashi et al, 2010). Meanwhile, p53 also affects expression of Nrf2 target genes (Wakabayashi et al, 2010). To investigate whether the Bscl2-GFP and Srxn1-GFP reporters were under control of p53, we transiently knocked down p53 expression by siRNAs (FIG. 29A), exposed cells to various genotoxic and oxidative stress-inducing compounds and analyzed GFP reporter activation. Activation of neither the Bscl2-GFP nor the Srxn1-GFP reporter by any of the tested compounds was affected by p53 knockdown (FIG. 29B). To confirm that the extent of p53 knockdown was sufficient to prevent activation of p53 target genes, we employed a Btg2-GFP reporter. Btg2 is a known p53 target gene that is transcriptionally activated upon exposure to genotoxic and oxidative stress (Rouault et al, 1996). Exposure of Btg2-GFP reporter cells to genotoxic agents as well as pro-oxidants resulted in increased expression of the reporter (FIG. 29C). Knockdown of p53 resulted in a strongly reduced induction of the Btg2-GFP reporter after exposure to all tested compounds. Interestingly, also knockdown of Nrf2 resulted in decreased Btg2-GFP reporter activation, specifically after exposure to oxidative stress-inducing compounds.
Discussion
Here we describe the generation of the ToxTracker assay, which consists of different GFP fluorescence mES reporter cell lines that are preferentially responsive to either genotoxic or oxidative stress-inducing compounds. Expression of the Bscl2-GFP reporter is specifically induced when mES cells are exposed to genotoxic compounds but is not elevated by chemicals that induce oxidative stress even though oxygen radicals might also damage DNA (FIG. 17). We provide evidence that Bscl2-GFP reporter activation correlates with inhibition of DNA replication progression (FIG. 28). Most types of oxidative DNA lesions do not provide strong blocks for the DNA replication machinery and are therefore unlikely to induce expression of the Bscl2-GFP reporter (Tolentino et al, 2008). The observation that reporter activation depends on ATR and Chk1 signaling (FIG. 29B) provides further evidence that DNA damage-induced replication blocks induce expression of the Bscl2-GFP reporter. Both kinases are activated upon persistent stalling of DNA replication forks (Paulsen and Cimprich, 2007). Interestingly, Bscl2 expression appears to be independent of p53, although p53 is phosphorylated by Chk1 upon replication stress to halt progression of the cell cycle. Expression of the Srxn1-GFP reporter is induced after exposure of cells to chemicals that result in increased levels of cellular oxidative stress (FIG. 17). Although the extent of activation is much stronger by oxidative stress, the Srxn1-GFP reporter is also somewhat responsive to genotoxic compounds. This suggests that either Srxn1 gene expression is directly induced upon DNA damage, although expression of Srxn1 is independent of the ATR-Chk1 DNA damage signaling (FIG. 28C), or that exposure of cells to genotoxic agents also results in increased ROS levels. Indeed, exposure of cells to the DNA crosslinking agent CisPt was shown to result in increased levels of ROS, caused by impaired mitochondrial function and during apoptosis (Jing et al, 2007). In agreement, exposure of the Srxn1-GFP reporter cells to CisPt in the presence of the ROS scavenger NAC significantly reduced reporter activation (data not shown). Additionally, exposure of cells to genotoxic agents results in activation of p53 that can induce expression of Nrf2. We provide evidence that expression of the Srxn1-GFP reporter is part of the Nrf2 anti-oxidant pathway (FIG. 27) being in line with recent reports that Srxn1 expression is directly controlled by Nrf2 via various ARE elements in the Srxn1 promoter (Singh et al, 2009). Results obtained with the Btg2-GFP reporter were not extensively discussed since its sensitivity and specificity is comparable to the previously described Btg2-DsRed reporter cell line (Hendriks et al, 2011). The Btg2-GFP reporter is activated by p53 upon genotoxic stress and by both p53 and Nrf2 after exposure to oxidative stress-inducing agents. Therefore, activation of the Btg2-GFP reporter displays no selectivity for either genotoxins or pro-oxidants (FIG. 29C).
The Bscl2 gene has originally been identified in patients that suffer from Berardinelli-Seip congenital lipodystrophy, a rare autosomal recessive disease that is characterized by an almost complete absence of adipocytes (Magre et al, 2001). The Bscl2 gene encodes a protein called Seipin that is located to the membrane of the endoplasmic reticulum (Szymanski et al, 2007). In addition, mutations in the Bscl2 gene have also been associated with autosomal-dominant disorders of motoneurons that results in severe atrophy and wasting of distal limb muscles (Agarwal and Garg, 2004). Although Seipin is involved in adipocyte differentiation, it is mainly expressed in the nervous system and testis (Magre et al, 2001). The function of Seipin is largely unknown, although it has been implicated in cytosolic lipid droplet morphology and in intracellular transport of lipids and proteins. So far, there is no implication of Bscl2 in the DNA damage response. Our data show that expression of Bscl2 in mES cells is strongly induced upon exposure to various DNA damaging agents. Basal Bscl2 expression level is low in mES cells. Since Bscl2 expression has been associated with adipocyte differentiation, it is attractive to hypothesize that also in mES cells Bscl2 expression is correlated with the induction of cell differentiation after exposure to genotoxic agents. It is well established that cell differentiation is induced upon DNA damage to maintain genome stability and to prevent malignant transformation of stem cells (Sherman et al, 2011). In agreement, exposure to various genotoxic chemicals did not result in increased expression of the Bscl2 gene in primary liver cells (M. Schaap, personal communication) and only a marginal induction in HepG2 liver carcinoma cells (Magkoufopoulou et al. Submitted). We are currently investigating the role of Bscl2 in the DNA damage response in mES cells.
Various assays are currently used to establish the genotoxic potential of novel chemicals. Although each of these assays has limitations in prediction of genotoxicity, a combination of these tests has proven to reliably predict the genotoxic risk of chemicals (Kirkland et al, 2011). However, many of these genotoxicity tests are based on biological endpoints such as mutations or chromosomal damage and therefore provide limited information on the reactive properties of compounds or the cellular responses activated after exposure. More recently, various genotoxicity assays have been described, including a HepG2 cell luciferase reporter system and the GreenScreen HC assay, that use altered transcriptional activities of specific genes to genotoxic chemicals as basis for their reporter system (Hastwell et al, 2006)(Westerink et al, 2010). Most of the genes selected depend on transcriptional activation by p53. The GreenScreen HC test employs the p53 target gene Gadd45a. Various validation studies show that the GreenScreen HC assay provides a highly sensitive and selective assay to identify (geno)toxic carcinogens (Knight et al, 2009)(Olaharski et al, 2009). However, the use of p53 target genes does not allow discrimination between different classes of (geno)toxic compounds, as exemplified by our Btg2-GFP reporter cells (FIG. 29C). We previously described four DsRed-based reporters including the p53-dependent Btg2-Dsred, which were all responsive to DNA damaging agents and pro-oxidants (Hendriks et al, 2011). By using combination of different DsRed reporter cell lines, we were able to discriminate between compounds that primarily induce genotoxic or oxidative stress. The novel ToxTracker assay that we describe here has a higher degree of specificity. The Bscl2-GFP reporter is exclusively induced by compounds that affect ongoing DNA replication, while the Srxn1-GFP reporter is preferentially induced by oxidative stress. In conclusion, the ToxTracker assay that consists of three independent GFP-based reporter cell lines is a novel, highly sensitive and specific genotoxicity test that can provide insights in the relative toxicity potential of chemicals.
REFERENCES FOR EXAMPLE 1


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FURTHER REFERENCES FROM EXAMPLE 6


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    Birrell, L., Cahill, P., Hughes, C., Tate, M., and Walmsley, R. M. (2010). GADD45a-GFP GreenScreen HC assay results for the ECVAM recommended lists of genotoxic and non-genotoxic chemicals for assessment of new genotoxicity tests. Mutation research. 1-2, 87-95.
    Chang, T.-S., Jeong, W., Woo, H. A., Lee, S. M., Park, S., and Rhee, S. G. (2004). Characterization of mammalian sulfiredoxin and its reactivation of hyperoxidized peroxiredoxin through reduction of cysteine sulfinic acid in the active site to cysteine. J Biochem. 49, 50994-51001.
    Hayes, J. D. and McLellan, L. I. (1999). Glutathione and glutathione-dependent enzymes represent a coordinately regulated defence against oxidative stress. Free radical research. 4, 273-300.
    Hendriks, G., Atallah, M., Raamsman, M., Morolli, B., van der Putten, H., Jaadar, H., Tijdens, I., Lange, R. E.-V., Mullenders, L., van de Water, B., and Vrieling, H. (2011). Sensitive DsRed fluorescence-based reporter cell systems for genotoxicity and oxidative stress assessment. Mutation research. 
    Hickson, I., Zhao, Y., Richardson, C. J., Green, S. J., Martin, N. M. B., Orr, A. I., Reaper, P. M., Jackson, S. P., Curtin, N. J., and Smith, G. C. M. (2004). Identification and characterization of a novel and specific inhibitor of the ataxia-telangiectasia mutated kinase ATM. Cancer Res. 24, 9152-9159.
    Jing, X.-B., Cai, X.-B., Hu, H., Chen, S.-Z., Chen, B.-M., and Cai, J.-Y. (2007). Reactive oxygen species and mitochondrial membrane potential are modulated during CDDP-induced apoptosis in EC-109 cells. Biochemistry and cell biology=Biochimie et biologie cellulaire. 2, 265-271.
    Kaspar, J. W., Niture, S. K., and Jaiswal, A. K. (2009). Nrf2:INrf2 (Keap1) signaling in oxidative stress. Free radical biology & medicine. 9, 1304-1309.
    Kirkland, D., Kasper, P., Muller, L., Corvi, R., and Speit, G. (2008). Recommended lists of genotoxic and non-genotoxic chemicals for assessment of the performance of new or improved genotoxicity tests: a follow-up to an ECVAM workshop. Mutation research. 1-2, 99-108.
    Kirkland, D., Reeve, L., Gatehouse, D., and Vanparys, P. (2011). A core in vitro genotoxicity battery comprising the Ames test plus the in vitro micronucleus test is sufficient to detect rodent carcinogens and in vivo genotoxins. Mutation research. 
    Magre, J., Delépine, M., Khallouf, E., Gedde-Dahl, T., Van Maldergem, L., Sobel, E., Papp, J., Meier, M., Mégarbané, A., Bachy, A., Verloes, A., d'Abronzo, F. H., Seemanova, E., Assan, R., Baudic, N., Bourut, C., Czernichow, P., Huet, F., Grigorescu, F., de Kerdanet, M., Lacombe, D., Labrune, P., Lanza, M., Loret, H., Matsuda, F., Navarro, J., Nivelon-Chevalier, A., Polak, M., Robert, J. J., Tric, P., Tubiana-Rufi, N., Vigouroux, C., Weissenbach, J., Savasta, S., Maassen, J. A., Trygstad, O., Bogalho, P., Freitas, P., Medina, J. L., Bonnicci, F., Joffe, B. I., Loyson, G., Panz, V. R., Raal, F. J., O'Rahilly, S., Stephenson, T., Kahn, C. R., Lathrop, M., Capeau, J., BSCL Working Group (2001). Identification of the gene altered in Berardinelli-Seip congenital lipodystrophy on chromosome 11q13. Nat Genet. 4, 365-370.
    Meek, D. W. (2009). Tumour suppression by p53: a role for the DNA damage response? Nat Rev Cancer. 10, 714-723.
    Nishida, H., Tatewaki, N., Nakajima, Y., Magara, T., Ko, K. M., Hamamori, Y., and Konishi, T. (2009). Inhibition of ATR protein kinase activity by schisandrin B in DNA damage response. Nucleic Acids Res. 17, 5678-5689.
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## Claims

1. A polynucleotide comprising a reporter sequence operatively linked to a regulatory element of a gene wherein the gene is Bscl2, wherein the regulatory element stimulates expression of the reporter sequence in response to a genotoxic agent or to an oxidative stress-inducing agent, wherein the polynucleotide does not encode the naturally occurring polypeptide of the gene whose regulatory element the reporter sequence is operatively linked to, and the reporter sequence is not the naturally occurring polynucleotide of the gene whose regulatory element the reporter sequence is operatively linked to.

2. The polynucleotide of claim 1, wherein the reporter sequence comprises a gene encoding DsRed fluorescent protein, horse radish peroxidise (HRP), Green Fluorescent Protein (GFP), luciferase, chloramphenicol acetyl transferase (CAT), or P-galactosidase.

3. The polynucleotide of claim 1, wherein the regulatory element comprises a promoter of the gene.

4. The polynucleotide of claim 1, wherein the regulatory element comprises a transcription start site of the gene.

5. The polynucleotide of claim 1, wherein the regulatory element comprises an at least 400 base pair polynucleotide sequence immediately upstream of the transcription start site of the gene.

6. The polynucleotide of claim 1, wherein the regulatory element comprises a polynucleotide sequence of SEQ ID NO: 1.

7. The polynucleotide of claim 1, wherein the regulatory element stimulates expression of the reporter sequence in response to a genotoxic agent that causes any of base damage, bulky DNA adducts, single-stranded DNA breaks, double-stranded DNA breaks, intra-strand crosslinks, or inter-strand crosslinks.

8. The polynucleotide of claim 7, wherein the regulatory element stimulates expression of the reporter sequence in response to any of an intercalator, a topoisomerase II poison or a methylating agent.

9. The polynucleotide of claim 1, wherein the genotoxic or oxidative stress-inducing agent comprises cisplatin, mitomycin C, doxorubicin, etoposide, methylmethane sulphonate, N-methylnitrosurea, hydrogen peroxide, t-butyl hydroperoxide, menadione, or diethyl maleate.

10. The polynucleotide of claim 1, further defined as comprised in a vector.

11. The polynucleotide of claim 10, wherein the vector is a pDsRed-expression2.1 plasmid.

12. The polynucleotide of claim 1, further defined as comprised in a cell.

13. The polynucleotide of claim 12, wherein the cell is a stem cell, an embryonic stem cell, a mammalian cell, has a functional p53 protein, and/or is a mouse embryonic stem cell.

14. A polynucleotide comprising a reporter sequence operatively linked to a regulatory element of a gene wherein the gene is Bscl2, wherein the regulatory element stimulates expression of the reporter sequence in response to a genotoxic agent or to an oxidative stress-inducing agent wherein the reporter sequence is not the naturally occurring polynucleotide of the gene whose regulatory element the reporter sequence is operatively linked to and wherein the 3′ end of the regulatory element is defined as the transcription start site or a region upstream thereof of Bscl2.

15. A polynucleotide comprising a reporter sequence operatively linked to a regulatory element of a gene defined as the upstream or downstream noncoding region of Bscl2, wherein the regulatory element stimulates expression of the reporter sequence in response to a genotoxic agent or to an oxidative stress-inducing agent wherein the reporter sequence is not the naturally occurring polynucleotide of the gene whose regulatory element the reporter sequence is operatively linked to.

16. A polynucleotide comprising a reporter sequence operatively linked to a regulatory element of a gene wherein the gene is Bscl2, wherein the regulatory element stimulates expression of the reporter sequence in response to a genotoxic agent or to an oxidative stress-inducing agent, wherein the polynucleotide does not contain the coding sequence of the gene whose regulatory element the reporter sequence is operatively linked to, and the reporter sequence is not the naturally occurring polynucleotide of the gene whose regulatory element the reporter sequence is operatively linked to.