Patent Publication Number: US-9429566-B2

Title: Assay for inhibitors of CIP/KIP protein degradation

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a National Entry Application of PCT application no PCT/CA2012/050682 filed on Sep. 28, 2012 and published in English under PCT Article 21(2), which itself claims benefit of U.S. provisional application Ser. No. 61/540,151, filed on Sep. 28, 2011. All documents above are incorporated herein by reference in their entirety. 
    
    
     TECHNICAL FIELD 
     The present generally concerns assays, and more particularly to screening assays and systems for the identification of inhibitors of p21 degradation. 
     SEQUENCE LISTING 
     Pursuant to 37 C.F.R. 1.821(c), a sequence listing is submitted herewith as an ASCII compliant text file named “15691_47- sequence listing_ST25.txt”, created on Sep. 28, 2012 and having a size of ˜42 kilobytes. The content of the aforementioned file is hereby incorporated by reference in its entirety. 
     BACKGROUND ART 
     The Cell Cycle as a Therapeutic Target for Cancer 
     Progression through the cell division cycle is controlled by oscillating waves of Cdk activity (1). These kinases are regulated positively by association with cyclin subunits and negatively by binding to Cdk inhibitors (2, 3). The Ubiquitin-Proteasome System (UPS) ( FIG. 1 ) plays a key role in controlling cell cycle progression by promoting the periodic degradation of cyclins and Cdk inhibitors (4, 5). 
     Deregulation of cell cycle progression is a hallmark of human cancer (6). Although Cdks are rarely mutated in cancer, their activity is universally deregulated owing to hyperactivation of upstream signaling pathways (Ras-MAP kinase, PI 3-kinase), amplification of Cdk or cyclin genes, genetic/epigenetic inactivation of Ink4 Cdk inhibitors, or downregulation of p21 and p27 Cdk inhibitors (7-9). For example, cyclin D1 is overexpressed in several tumors as a result of transcriptional activation, gene amplification, or translocation. p16 Ink4a  a is frequently inactivated by gene deletion, point mutation or epigenetic silencing, resulting in activation of cyclin D-dependent kinases. Aberrant activation of Cdk2 and Cdk1 is observed in various malignancies. Other protein kinases such as Aurora A/B and Plk1, which are involved in centrosome duplication and mitosis execution, are overexpressed in a wide range of tumor types (10, 11). In addition to cell cycle kinases, deregulation of the mechanisms that control protein stability has been shown to contribute to tumorigenesis. Overexpression of oncogenic E3 ligases (such as Skp2), which target negative regulators of the cell cycle, or inactivation of tumor suppressor E3 ligases like Fbxw7 is observed in many human tumors (4, 5, 12). 
     Since it was established that aberrant cell cycle control is a hallmark of cancer, development of agents targeting the cell cycle has been viewed as a promising strategy for cancer therapy. For more than a decade, there has been an intensive search for small molecules that target Cdks, but no Cdk inhibitor drug has yet been approved for clinical use (7, 13, 14). More recent efforts have focused on the development of inhibitors for Aurora and Polo kinases (15-17). However, further investigation is necessary to assess the clinical potential of these targets. On the other hand, the FDA approval of the proteasome inhibitor bortezomib (Velcade; Millenium) for the treatment of multiple myeloma in 2003 (18) has heralded an entirely new class of cancer drugs and validated the therapeutic potential of the UPS (12, 19-22). 
     The Cip/Kip Family of Cdk Inhibitors 
     The activity of Cdks is negatively regulated by Cdk inhibitors. In human, 7 Cdk inhibitors have been identified and classified into two families, according to structural and functional similarities (1, 23). The Ink4 proteins, which include p16 Ink4A , p15 Ink4B , p18 Ink4C  and p19 INK4D  contain multiple ankyrin repeats and interact specifically with Cdk4 and Cdk6 to inactivate cyclin D-Cdk complexes. Members of the Cip/Kip family, which is composed of p21, p27 and p57, inhibit all cyclin-Cdk complexes and are not specific to a particular cell cycle phase. Structurally, the three Cip/Kip proteins share a conserved domain at their N-terminus, consisting of two separable subdomains for binding to cyclin and Cdk subunits ( FIG. 2 ). They also have a nuclear localization signal (NLS) near the C-terminus. Notably, p21 also contains a proliferating cell nuclear antigen (PCNA) binding domain. 
     Biochemical and genetic analyses indicate that p21, p27 and p57 have both overlapping and specific cellular functions. p21 is a transcriptional target of p53 and is believed to be one of the main effectors of p53-mediated cell cycle arrest (24). The p21 protein is expressed ubiquitously in adult tissues. In the developing embryo, the expression of p21 correlates with terminal differentiation of a variety of tissues such as skeletal and heart muscle, cartilage and skin (25, 26). These observations implicated p21 in the regulation of cell cycle withdrawal during terminal differentiation. p27 is expressed ubiquitously and act as a negative regulator of cell proliferation in a variety of cell types (26). Accordingly, the expression of p27 is high in quiescent cells and in cells exposed to anti-proliferative signals, and declines in response to mitogenic factor stimulation (27-29). p57 is highly expressed in the developing embryo, but its expression declines in adults (26). 
     Regulation of p21 Expression in Normal and Cancer Cells 
     The regulation of p21 protein is exerted at multiple levels. The amount of p21 is controlled mainly at the levels of transcription and protein turnover (30). p21 was originally identified as the product of a gene activated by p53 (31). Since then, a variety of cellular and viral factors have been shown to induce or repress p21 transcription by p53-independent mechanisms (30, 32). In cancer cells, repression of p21 gene transcription is associated either with loss of function of activators (p53) or upregulation or gain of function mutations of transcriptional repressors. For example, the Myc oncogene is a potent repressor of p21 transcription (33). Importantly, p21 is a very unstable protein that is degraded by the proteasome ( FIG. 3 ). Four E3 ubiquitin ligase complexes, SCF skp2  (34), CRL4 cdt2  (35-37), APC/C Cdc20  (38) and MKRN1 (39) have been shown to promote the degradation of p21 at specific stages of the cell cycle. Several proteins involved in the ubiquitin-dependent proteolysis of p21 are upregulated in a variety of human tumours, indicating that p21 downregulation may account for the oncogenic properties of these proteins. For example, Skp2, the substrate binding subunit of the SCF skp2  E3 ligase, is frequently upregulated in human cancers and displays oncogenic properties (4). Similarly, Cdt2 and Cul4a, two subunits of the CRL4 cdt2  E3 ligase are overexpressed in breast and advanced liver cancers (40-43). 
     p21 is a Potent Tumor Suppressor 
     Mouse genetic studies and human clinical investigations have provided compelling evidence that p21 is a bona fide tumor suppressor. Mice deficient in p21 develop tumours of hematopoietic, endothelial and epithelial origin with late onset (44). Furthermore, p21 deficiency accelerates the development of chemically induced tumors in mice (45-47) and cooperates with oncogenes to promote tumorigenesis (48). Importantly, two recent studies have shown that knock-in mice expressing the p53 R172P mutant, that is deficient for apoptosis but maintains its ability to induce p21 and cell cycle arrest, are able to suppress tumorigenesis in different cancer models (49, 50). Tumor suppression by this p53 mutant was modulated by p21, which induced senescence and preserved chromosomal stability. p21 is not a classical tumor suppressor gene as it is very rarely mutated in human tumors. However, p21 levels are frequently downregulated in human cancers (including carcinomas, gliomas and hematological malignancies) and this is usually associated with a poor prognosis (30, 51). As mentioned above, downregulation of p21 is most often associated with increased turnover of the protein. 
     Accumulating evidence suggest that p21 exerts its tumor suppressor activity through multiple mechanisms. In addition to its ability to inhibit cyclin-Cdks and induce cell cycle arrest, microarray-based studies indicate that p21 expression is associated with the suppression of genes important for cell cycle progression and the induction of senescence genes (52). Interestingly, recent work suggests that tumor regression can be achieved through the reactivation of senescence, by restoring p53 function (53) or by inactivation of Myc in tumors with functional p53 (54). Reactivation of p53 and Myc inactivation both leads to p21 upregulation. p21 can compete for PCNA binding with several PCNA-reliant proteins involved in DNA repair processes (55). Finally, p21 has been reported to either inhibit or promote apoptosis depending on the cellular context (30). Interestingly, a recent study showed that p21 promotes apoptosis of intestinal stem/progenitor cells in response to gamma irradiation, suggesting that increasing p21 expression may be a viable approach to selectively target colon cancer stem cells (56). 
     There is thus a need for the development of novel strategies to inhibit p21 degradation, such as novel methods and assays to identify inhibitors of p21 degradation. 
     The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety. 
     SUMMARY OF THE INVENTION 
     In a first aspect, the present invention provides a high throughput Screening (HTS)-compatible method for determining whether a test compound may be useful for treating cancer, said method comprising (a) contacting said test compound with a cell expressing a fusion protein in the presence of a protein synthesis inhibitor, said fusion protein comprising (i) a Cip/Kip polypeptide; and (i) a reporter protein linked to the C-terminal of said Cip/Kip polypeptide, wherein said fusion protein has a half-life that is similar to that of said Cip/Kip polypeptide, and (b) measuring a readout signal from the reporter protein, wherein a higher readout signal from the reporter protein in the presence of said test compound, relative to the readout signal in the absence of said test compound, is indicative that said test compound may be useful for treating cancer. 
     In another aspect, the present invention provides a high throughput Screening (HTS)-compatible system for determining whether a test compound may be useful for treating cancer, said system comprising:
         a cell expressing a fusion protein, said fusion protein comprising (i) a Cip/Kip polypeptide; and (i) a reporter protein linked to the C-terminal of said Cip/Kip polypeptide, wherein said fusion protein has a half-life that is similar to that of said Cip/Kip polypeptide;   a protein synthesis inhibitor; and   a detection system to measure the readout signal from the reporter protein.       

     In an embodiment, the above-mentioned half-life is about 1 hour or less, in a further embodiment the half-life is from 30 minutes to about 1 hour. 
     In an embodiment, the above-mentioned protein synthesis inhibitor is cycloheximide (CHX). 
     In an embodiment, the above-mentioned said reporter protein is a luciferase, in a further embodiment  Renilla  luciferase. In a further embodiment, the  Renilla  luciferase is a polypeptide comprising the amino acid sequence of SEQ ID NO:4, or a functional variant or fragment thereof having  Renilla  luciferase activity. In yet a further embodiment, the  Renilla  luciferase is a polypeptide comprising the amino acid sequence of SEQ ID NO:4. 
     In an embodiment, the above-mentioned readout signal from the reporter protein is bioluminescence in the presence of a luciferase substrate. In a further embodiment, the luciferase substrate is coelenterazine or an analog thereof. 
     In an embodiment, the above-mentioned the Cip/Kip polypeptide is a p21 polypeptide, in a further embodiment a polypeptide comprising the amino acid sequence of SEQ ID NO:2, or a functional variant or fragment thereof having p21 activity. In a further embodiment, the p21 polypeptide is a polypeptide comprising the amino acid sequence of SEQ ID NO:2. 
     In an embodiment, the above-mentioned cell further comprises an inducible expression system for inducible expression of the fusion protein. In a further embodiment, the above-mentioned inducible expression system is a tetracycline-controlled expression system. 
     In an embodiment, the nucleic acid encoding said fusion protein is operably linked to tetracycline-responsive elements (TREs). 
     In an embodiment, the above-mentioned cell further expresses a reverse tetracycline-responsive transcriptional activator (rtTA). 
     In an embodiment, the above-mentioned method further comprises contacting said cell with tetracycline (Tc), or an analog thereof, in a further embodiment the Tc analog is doxycycline (Dox). 
     In an embodiment, the above-mentioned cell is a fibroblast, in a further embodiment a Rat1 cell. 
     Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
       In the appended drawings: 
         FIG. 1  shows an overview of the Ubiquitin-Proteasome System (UPS); 
         FIG. 2  shows a schematic representation of the human Cip/Kip family of Cdk inhibitors; 
         FIG. 3  shows that p21 is an unstable protein degraded by the UPS. ts20 cells, which bear a temperature-sensitive mutation in the E1 enzyme, were incubated at the permissive (34° C., E1 active) or non-permissive (39° C., E1 inactive) temperature and treated with the protein synthesis inhibitor cycloheximide (CHX) for different times. Expression of p21 was measured by immunoblotting 
         FIG. 4  shows the underlying principle of the p21 degradation assay. p21 is an unstable protein with an half-life of about 30-60 minutes. Upon addition of CHX to block protein synthesis, the p21 protein is rapidly degraded. Addition of a small molecule inhibitor of p21 degradation is predicted to stabilize p21 leading to its accumulation in the cells; 
         FIGS. 5A-C  show the design and basis of the p21 degradation reporter assay.  FIG. 5A  shows a schematic representation of the p21-Renilla luciferase (Rluc) reporter construct.  FIG. 5B  shows an immunoblot analysis of the degradation rate of Rluc and p21-Rluc fusion protein upon addition of CHX in the presence or absence of the proteasome inhibitor MG 132. A specific antibody to Rluc was used for detection.  FIG. 5C  shows a quantification of the data in  FIG. 5B  expressed as relative abundance; 
         FIG. 6  shows a schematic representation of the HTS assay in 384-well plates used to screen a library of small molecule compounds using the p21-Rluc reporter assay described herein; 
         FIG. 7  shows (A) the results expressed as fold stabilization values for one HTS run representing 9,984 small molecule compounds. (B) Distribution of the fold stabilization data for the 112,900 compounds tested in the primary screen using the p21-Rluc reporter assay described herein; 
         FIG. 8  shows a summary of the screen and decision tree showing the different assays implemented and the corresponding statistical methods applied for hits selection. The number of compounds tested at each step is indicated; 
         FIG. 9  shows dose-response curves of selected compounds identified from the primary screen using the p21-Rluc reporter assay. The proteasome inhibitor MG132 was used as control. 
         FIGS. 10A and 10B  shows the validation of the p21-Rluc reporter assay by ELISA.  FIG. 10A  shows a dose-response curve of the effect of the proteasome inhibitor MG132 and an inactive compound X in the p21-Rluc assay. Luciferase values are normalized to the control DMSO (set to 1).  FIG. 10B  shows a dose-response curve of MG132 and compound X using a p21 ELISA assay to measure the expression of endogenous p21 protein. ELISA values are normalized to the control DMSO. 
         FIG. 11A  shows the nucleotide sequence of human p21 mRNA (transcript variant 1, NCBI Reference Sequence: NM_000389.4, SEQ ID NO:1), with the coding sequence in italics (nucleotides 126-620); 
         FIG. 11B  shows the amino acid sequence of human p21 protein (NCBI Reference Sequence: NP_000380.1, SEQ ID NO:2); 
         FIG. 12A  shows the nucleotide sequence of  Renilla reniformis  luciferase mRNA (GenBank: M63501.1, SEQ ID NO:3), with the coding sequence in italics (nucleotides 10-945); 
         FIG. 12B  shows the amino acid sequence of  Renilla reniformis  luciferase (GenBank: AAA29804.1, SEQ ID NO:4); 
         FIG. 13  shows the nucleotide (SEQ ID NO:5) and amino acid (SEQ ID NO:6) sequences of the p21-Rluc fusion construct used in the experiments described herein. The construct comprises a “linker” (highlighted in grey) corresponding to a KpnI restriction site (used to prepare the fusion construct) between p21 and Rluc, which results in the presence of 2 amino acids (Gly and Thr) between the two proteins in the fusion; 
         FIG. 14A  shows the nucleotide sequence of human p27 mRNA (NCBI Reference Sequence: NM_004064.3, SEQ ID NO:7), with the coding sequence in italics; 
         FIG. 14B  shows the amino acid sequence of human p27 protein (NCBI Reference Sequence: NP_004055.1, SEQ ID NO:8); 
         FIG. 15A  shows the nucleotide sequence of human p57 mRNA (NCBI Reference Sequence: NM_000076.2, SEQ ID NO:9), with the coding sequence in italics; 
         FIG. 15B  shows the amino acid sequence of human p57 protein (NCBI Reference Sequence: NP_000067.1, SEQ ID NO:10). 
     
    
    
     DISCLOSURE OF INVENTION 
     An assay compatible with high-throughput screening (HTS) that is capable of identifying inhibitors, such as small-molecule inhibitors, of the degradation of the Cdk inhibitor of the Cip/Kip family (e.g., p21), was designed. Inhibitors identified by this assay may be useful to inhibit the proliferation of tumor cells, and thus for the treatment of cancers. Accordingly, in a first aspect, the present invention provides a high throughput screening (HTS)-compatible method for determining whether a test compound may be useful for treating cancer, said method comprising 
     (a) contacting said test compound with a cell expressing a fusion protein in the presence of a protein synthesis inhibitor, said fusion protein comprising a reporter protein fused to the C-terminal end of a Cip/Kip polypeptide, wherein said fusion protein has a half-life that is similar to that of said Cip/Kip polypeptide, and 
     (b) determining a readout signal from the reporter protein, 
     wherein a higher readout signal from the reporter protein in the presence of said test compound, relative to the readout signal in the absence of said test compound, is indicative that said test compound may be useful for treating cancer. 
     In another aspect, the present invention provides a high throughput screening (HTS)-compatible method for determining whether a test compound may be useful for (i) inhibiting (e.g., preventing, decreasing) Cip/Kip protein degradation, (ii) stabilizing Cip/Kip protein expression, and/or (iii) inducing the cellular accumulation of Cip/Kip protein, said method comprising 
     (a) contacting said test compound with a cell expressing a fusion protein in the presence of a protein synthesis inhibitor, said fusion protein comprising a reporter protein fused to the C-terminal end of a Cip/Kip polypeptide, wherein said fusion protein has a half-life that is similar to that of said Cip/Kip polypeptide, and 
     (b) determining a readout signal from the reporter protein, wherein a higher readout signal from the reporter protein in the presence of said test compound, relative to the readout signal in the absence of said test compound, is indicative that said test compound may be useful for inhibiting (e.g., preventing, decreasing) Cip/Kip degradation (or stabilization of Cip/Kip expression). 
     In another aspect, the present invention provides a high throughput screening (HTS)-compatible method for determining whether a test compound may be useful for inhibiting cell growth arrest and/or cell cycle progression, said method comprising 
     (a) contacting said test compound with a cell expressing a fusion protein in the presence of a protein synthesis inhibitor, said fusion protein comprising a reporter protein fused to the C-terminal end of a Cip/Kip polypeptide, wherein said fusion protein has a half-life that is similar to that of said Cip/Kip polypeptide, and 
     (b) determining a readout signal from the reporter protein, wherein a higher readout signal from the reporter protein in the presence of said test compound, relative to the readout signal in the absence of said test compound, is indicative that said test compound may be useful for inhibiting cell growth arrest, and/or cell cycle progression. 
     In another aspect, the present invention provides a high throughput screening (HTS)-compatible system for determining whether a test compound may be useful for treating cancer, said system comprising: 
     a cell expressing a fusion protein, said fusion protein comprising (i) a Cip/Kip polypeptide; and (i) a reporter protein linked to the C-terminal of said Cip/Kip polypeptide, wherein said fusion protein has a half-life that is similar to that of said Cip/Kip polypeptide; 
     a protein synthesis inhibitor; 
     a detection system to measure the readout signal from the reporter protein. 
     In another aspect, the present invention provides a high throughput screening (HTS)-compatible system for determining whether a test compound may be useful for (i) inhibiting (e.g., preventing, decreasing) Cip/Kip protein degradation, (ii) stabilizing Cip/Kip protein expression, and/or (iii) inducing the cellular accumulation of Cip/Kip protein, said system comprising: 
     a cell expressing a fusion protein, said fusion protein comprising (i) a Cip/Kip polypeptide; and (i) a reporter protein linked to the C-terminal of said Cip/Kip polypeptide, wherein said fusion protein has a half-life that is similar to that of said Cip/Kip polypeptide; 
     a protein synthesis inhibitor; 
     a detection system to measure the readout signal from the reporter protein. 
     In another aspect, the present invention provides a high throughput screening (HTS)-compatible system for determining whether a test compound may be useful for inhibiting cell growth arrest and/or cell cycle progression, said system comprising: 
     a cell expressing a fusion protein, said fusion protein comprising (i) a Cip/Kip polypeptide; and (i) a reporter protein linked to the C-terminal of said Cip/Kip polypeptide, wherein said fusion protein has a half-life that is similar to that of said Cip/Kip polypeptide; 
     a protein synthesis inhibitor; 
     a detection system to measure the readout signal from the reporter protein. 
     The term “high-throughput screening” (HTS) as used herein refers to a method that allow screening rapidly and in parallel large numbers of compounds (hundreds, thousands) for binding activity or biological activity against target molecules. Such HTS methods are typically performed in microtiter plates having several wells, for example 384, 1536, or 3456 wells. For HTS, it is important that the readout signal be detected with high sensitivity, accuracy and reproducibility. 
     The above-mentioned fusion protein has a half-life that is similar to that of said Cip/Kip (e.g., p21) polypeptide. In an embodiment, the half-life is the half-life within a cell, for example a cell cultured in vitro, in petri culture dishes. “Similar” as used in that context means that the difference between the half-life of the fusion protein and a Cip/Kip (e.g., p21) polypeptide (alone, not in the fusion protein), under the same conditions (e.g., same cells, same culture conditions) is less than 25%, in further embodiments less than 20, 15 or 10%. In an embodiment, the half-life of said fusion protein is about 1 hour or less, in a further embodiment between about 30 minutes to about 1 hour. Methods to measure the half-life of proteins are well known in the art. In embodiments, the half-life of the fusion protein may be measured using the cycloheximide chase and p21 immunoblotting analysis described below. 
     The term “reporter protein” refers to a protein that can be detected (e.g., by fluorescence, spectroscopy, luminometry, etc.) easily and that is not present normally (endogenously) in the system used. Commonly used reporter proteins include enzymes such as β-galactosidase (encoded by the bacterial gene IacZ), luciferase, chloramphenyl acetyltransferase (CAT; from bacteria), GUS (β-glucuronidase), bioluminescent proteins and fluorescent proteins. In the context of the present invention, the reporter protein is selected so as to not significantly affect the half-life of Cip/Kip (e.g., p21), i.e. so that the Cip/Kip-reporter protein fusion has a half-life that is similar to that of the Cip/Kip (e.g., p21) polypeptide alone. The skilled person would be able to easily determine the suitable reporter proteins for the above-noted methods/systems by measuring the half-life of a fusion protein comprising Cip/Kip (e.g., p21) and the reporter protein, and comparing it to the half-like of Cip/Kip (e.g., p21). In an embodiment, the reporter protein is a luciferase. The term luciferase refers to a class of oxidative enzymes used in bioluminescence. Many luciferases are known in the art, for example firefly luciferase (for example from the firefly  Photinus pyralis ),  Renilla  luciferase ( Renilla reniformis ),  Metridia  luciferase (MetLuc, derived from the marine copepod Metridia longa),  Aequorea  luciferase, Dinoflagellate luciferase, or  Gaussia  luciferase (Gluc). In an embodiment, the luciferase is a  Renilla  luciferase. In an embodiment, the  Renilla  luciferase is a polypeptide comprising the amino acid sequence of SEQ ID NO:4 ( FIG. 12B ), or a functional variant or fragment thereof having  Renilla  luciferase activity.  Renilla  Luciferase activity as used herein refers to the ability to metabolize the substrate coelenterazine (6-(4-hydroxyphenyl)-2-[(4-hydroxphenylmethyl]-8-(phenylmethyl)-7H-imidazo[3,2-a]pyrazin-3-one). In an embodiment, the functional variant or fragment comprises a sequence having at least 70% identity with the sequence of SEQ ID NO:4 ( FIG. 12B ). In further embodiments, the functional variant or fragment comprises a sequence having at least 75, 80, 85, 90, 95, 96, 97, 98 or 99% identity with the sequence of SEQ ID NO:4 ( FIG. 12B ). In an embodiment, when an enzyme is used as the reporter protein, the above-mentioned method further comprises contacting the cell with a substrate of the enzyme so as to induce the production of a detectable metabolite. In an embodiment, when the reporter protein is a  Renilla  luciferase, the above-mentioned method further comprises contacting the cell with coelenterazine or an analog thereof, which catalyzes coelenterazine oxidation by oxygen to produce light. Coelenterazine and several coelenterazine analogs (coelenterazine cp, f, h, hcp, fcp, i, ip, n, 400a, methyl Coelenterazine) are commercially available from Life Technologies™, Molecular Probes™ and Biotium™, for example (see also, e.g., Zhao et al.,  Mol Imaging,  2004 3(1):43-54). In a further embodiment, the just-noted contacting the cell with coelenterazine or an analog thereof is for a period of about 1 to about 10 minutes, for example about 3 to about 7 minutes, more specifically about 5 minutes. 
     The method to determine the readout signal from the reporter protein depends from the nature of the reporter protein. For example, for fluorescent reporter proteins, the readout signal corresponds to the intensity of the fluorescent signal. The readout signal may be measured using spectroscopy-, fluorometry-, photometry-, and/or luminometry-based methods and detection systems, for example. Such methods and detection systems are well known in the art. 
     The term “Cip/Kip polypeptide” refers to a cyclin-dependent kinase (CDK) inhibitors of the Cip/Kip family and includes the protein p21, p27 and p57. The nucleotide and amino acid sequences of p21, p27 and p57 are depicted in  FIGS. 11A-11B, 14A-14B and 15A-15B , respectively. In an embodiment, the Cip/Kip polypeptide is a polypeptide comprising the amino acid sequence of SEQ ID NO:2, 8 or 10 ( FIG. 11B, 14B or 15B ), or a functional variant or fragment thereof having the activity of native p21, p27 or p57 (e.g., inhibition of CDK, regulation of cell cycle progression). In an embodiment, the functional variant or fragment comprises a sequence having at least 70% identity with the sequence of SEQ ID NO:2, 8 or 10 ( FIG. 11B, 14B or 15B ). In further embodiments, the functional variant or fragment comprises a sequence having at least 75, 80, 85, 90, 95, 96, 97, 98 or 99% identity with the sequence of SEQ ID NO:2, 8 or 10 ( FIG. 11B, 14B or 15B ). 
     In an embodiment, the Cip/Kip polypeptide is a p21 polypeptide. The term “p21 polypeptide” refers to a polypeptide that inhibits cyclin-dependent kinase (CDK) and regulates cell cycle progression. The sequences of p21 polypeptides from various organisms and species are known in the art, for example mouse: NCBI Reference Sequence NP_001104569.1; Rat: GenBank AAC52221.1; cow: NCBI Reference Sequence NP_001092428.1; human: NCBI Reference Sequence NP_000380.1, SEQ ID NO:2 ( FIG. 11B ). In an embodiment, the p21 polypeptide is a polypeptide comprising the amino acid sequence of SEQ ID NO:2 ( FIG. 11B ), or a functional variant or fragment thereof having the activity of native p21 (e.g., inhibition of CDK, regulation of cell cycle progression). In an embodiment, the functional variant or fragment comprises a sequence having at least 70% identity with the sequence of SEQ ID NO:2 ( FIG. 11B ). In further embodiments, the functional variant or fragment comprises a sequence having at least 75, 80, 85, 90, 95, 96, 97, 98 or 99% identity with the sequence of SEQ ID NO:2 ( FIG. 11B ). 
     The term “protein synthesis inhibitor” refers to an agent that blocks/inhibits the processes that lead to the generation of new proteins. Such agents usually act at the ribosome level. In an embodiment, the protein synthesis inhibitor is a eukaryotic protein synthesis inhibitor. Examples of eukaryotic protein synthesis inhibitors include cycloheximide (CHX), puromycin, isomigrastatin, lactimidomycin (LTM), Actinomycin D, Anisomycin, emetine, and analogs thereof. In an embodiment, the protein synthesis inhibitor is cycloheximide (CHX). 
     In embodiments, the Cip/Kip (e.g., p21) polypeptide may be covalently linked to the reporter protein either directly (e.g., through a peptide bond) or via a suitable linker moiety, e.g., a linker of one or more amino acids (e.g., a polyglycine linker) or another type of chemical linker (e.g., a carbohydrate linker, a lipid linker, a fatty acid linker, a polyether linker, PEG, etc. (see, e.g., Hermanson (1996) Bioconjugate techniques). In an embodiment, the Cip/Kip (e.g., p21) polypeptide and the reporter protein are covalently linked through a peptide bond. In an embodiment, the p21 polypeptide and the reporter protein are covalently linked through a linker, in a further embodiment a 2-amino acid linker. In a further embodiment, the linker comprises a glycine residue and a threonine residue. In a further embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO:6 ( FIG. 13 ). 
     In an embodiment, the above-mentioned reporter protein is under inducible expression. Accordingly, in another embodiment, the cell further comprises an inducible expression system. 
     In a further embodiment, the inducible expression system is a tetracycline-controlled/regulated expression system. Inducible expression systems, such as tetracycline-controlled/regulated expression systems, are well known in the art and are commercially available. Examples of such systems include the RheoSwitch® Mammalian Inducible Expression System from New England BioLabs Inc., Tet-Express™ Inducible Expression Systems from Clontech, and the T-REx™ System from Life Technologies. 
     In an embodiment, the nucleic acid sequence encoding the above-mentioned fusion protein is operably linked to inducible transcriptional regulatory element sequence(s). A nucleic acid sequence is “operably-linked” with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid. For instance, a promoter is operably-linked to a coding sequence if the promoter affects the transcription or expression of the coding sequences. Generally, operably-linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in reading frame. However, since, for example, enhancers generally function when separated from the promoters by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably-linked but not contiguous. “Transcriptional regulatory element sequence(s)” is a generic term that refers to DNA sequences, such as initiation and termination signals, enhancers, and promoters, splicing signals, polyadenylation signals which induce or control transcription of protein coding sequences with which they are operably-linked. In an embodiment, the transcriptional regulatory element sequences are tetracycline-responsive elements (TREs). The tetracycline response elements consist of 7 repeats of the 19 bp bacterial tet-o sequence separated by spacer sequences. 
     In an embodiment, the above-mentioned cell further expresses a tetracycline-responsive transcriptional activator (tTA, Tet-Off expression system), or a reverse tetracycline-responsive transcriptional activator (rtTA, Tet-On expression system). 
     A tetracycline transactivator (tTA) protein is a fusion of the TetR (tetracycline repressor), found in  Escherichia coli  bacteria with another protein, VP16, produced by Herpes Simplex Virus (HPV). In the absence of tetracycline (Tc) or an analog thereof (doxycycline, Dox), tTA binds to the TRE and activates transcription of the target gene. In the presence of Tc or Dox, which binds tTA, tTA is not capable of binding to TRE sequences, thereby preventing transactivation of target genes (the nucleic acid encoding the fusion protein). 
     A reverse tetracycline-responsive transcriptional activator (rtTA) is also a fusion protein comprised of the TetR repressor and the VP16 transactivation domain; however, a four amino acid change in the tetR DNA binding moiety alters rtTA&#39;s binding characteristics such that it can only recognize the tetO sequences in the TRE of the target transgene in the presence of tetracycline or an analog thereof (doxycycline, Dox). Thus, in such as a system, transcription of the TRE-regulated target gene is stimulated by rtTA only in the presence of tetracycline or an analog thereof. 
     In an embodiment, the above-mentioned cell further expresses a reverse tetracycline-responsive transcriptional activator (rtTA, Tet-On expression system). In an embodiment, the method further comprises culturing the cell in the presence of tetracycline (Tc), or an analog thereof, to induce the expression of the fusion protein by the cell. In a further embodiment, the tetracycline (Tc) derivative is doxycycline (Dox). 
     In another embodiment, the above-mentioned method comprises: 
     (a1) contacting the cell expressing the fusion protein with tetracycline or a tetracycline analog to induce the expression of the fusion protein; 
     (b1) contacting the test compound with the cell of (a) in the presence of a protein synthesis inhibitor; and 
     (c1) determining a readout signal from the reporter protein. 
     In an embodiment, the above contacting at step (a1) is for a period of from about 8 to about 30 hours, for example from about 12 to about 24 hours, more specifically about 18 hours. 
     In an embodiment, the above contacting at step (b1) is for a period of from about 2 to about 10 hours, for example from about 4 to about 8 hours, more specifically about 6 hours. 
     Any cell capable of expressing the fusion protein may be used in the method/system of the invention. In an embodiment, the above-mentioned cell is a mammalian cell (e.g., animal cell, mouse cell, rat cell, human cell). In a further embodiment, the cell is a cell line, in a further embodiment a fibroblast cell line, in yet a further embodiment a rat cell line. In yet a further embodiment, the cell is a Rat1 cell. 
     The cell may be prepared by introducing a nucleic acid encoding the above-mentioned fusion protein (by any transfection, transduction or transformation method), such as the nucleic acid comprising the sequence of SEQ ID NO:6, and providing conditions suitable for the expression of the fusion protein. Methods and systems for introducing a nucleic acid into a cell are well known in the art, and include for example chemical-based transfection (using calcium phosphate, liposomes, cationic polymers such as DEAE-dextran or polyethylenimine), electroporation, gene gun, viral transduction. Kits for introducing a nucleic acid into a cell are commercially available. 
     In an embodiment, the above-mentioned cancer is a cancer associated with a decrease expression, or downregulated levels, of p21, p27 and/or p57 (reviewed in references 30, 51 and 67, for example). In a further embodiment, the above-mentioned cancer is a cancer associated with a decrease expression, or downregulated levels, of p21. In an embodiment, the above-mentioned cancer is a human cancer, in further embodiments a carcinoma, glioma or hematological malignancy (e.g., leukemia). In an embodiment, the cancer is a breast, gastrointestinal (e.g., gastric, colon), liver, tonsillar ovarian, cervical, pancreatic, laryngeal or oral cancer. p57(Kip2) protein is frequently downregulated in different types of human epithelial and nonepithelial cancers as a consequence of genetic and epigenetic events (67). Accordingly, in another embodiment, the cancer is an epithelial or nonepithelial cancer. 
     Test compounds (drug candidates) that may be screened by the method/system of the invention may be obtained from any number of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. 
     In another aspect, the present invention provides a fusion protein as defined above, or a nucleic acid encoding such fusion protein, or a cell comprising the fusion protein or nucleic acid encoding same. 
     In another aspect, the present invention provides a kit comprising the fusion protein defined above, or a nucleic acid encoding such fusion protein, or a cell comprising the fusion protein or nucleic acid encoding same. In embodiments, the kit further comprises one or more of the components of the system defined above, as well as instructions for performing the HTS-compatible method defined above. 
     MODE(S) FOR CARRYING OUT THE INVENTION 
     The present invention is illustrated in further details by the following non-limiting examples. 
     EXAMPLE 1 
     Materials and Methods 
     Generation of p21-Rluc Protein Fusion 
     The human p21Cip1 (p21) and  Renilla  luciferase (Rluc) genes were amplified by polymerase chain reaction (PCR) from plasmids pRmHa-5 HA-p21Cip1 and pcDNA3.1-Rluc respectively. PCR products were digested and ligated into a modified version of pRevTRE vector (Clonetech) previously digested with BamHI and NotI restriction enzymes. The final recombinant molecules (pRevTRE Rluc and pRevTRE-p21-Rluc) were sequenced to ensure the integrity of DNA. 
     Generation of Rat1 rtTA Stable Cell Line 
     Human Embryonic Kidney 293T cells were transfected with pCL-Eco and pRevTet-On vectors in order to produce retroviral particle bearing the reverse tet-transactivator transgene (rtTA). Rat1 cells were infected with these retroviruses and selected with G418 for 2 weeks to generate the Rat1 rtTA cell line. 
     Generation of Rat-1 rtTA Inducible Rluc/p21-Rluc Stable Cell Lines 
     Human Embryonic Kidney 293T cells were transfected with pCL-Eco in combination with either pRevTRE Rluc of pRevTRE p21-Rluc to produce retroviral particles. Rat1 rtTA cells were infected with these retroviruses in the presence of 10 ug/ml polybrene. Cells were selected with hygromycin B and G418 for 5 days to generate Rat1 rtTA Rluc and Rat1 rtTA p21-Rluc cell lines. 
     High Throughput p21 Degradation Assay 
     Stable cell lines from frozen vials were thawed and resuspended in phenol red-free DMEM (Wisent) supplemented with 10% NBCS (day 0). Two days after, cells were trypsinized and seeded at 2500 cells/well into white 384-well plates (BD Bioscience). Doxycyclin was added at 1 μg/ml into the culture medium in order to induce the expression of Rluc and/or p21-Rluc (day 2). Cells were incubated at 37° C. for 18 h. On day 3, 5 μl of cycloheximide was added into each well to reach a final concentration of 50 μg/ml. The proteasome inhibitor MG132 was added into few wells on each plate as positive control at a final concentration of 25 μM. Dimethyl sulfoxide (DMSO) was added into few wells on each plate as negative control. Compounds were pre-diluted in water and 5 μl of the diluted solutions was added at a final concentration of 10 μM. The final volume in each well was 50 μl and the final concentration of DMSO through the whole screen was 0.5%. Plates were incubated at 37° C. for 6 h. Culture medium was then aspirated and 50 μl of a solution containing the  Renilla  luciferase substrate coelenterazine was added at a final concentration of 5 μM. The reaction was allowed to proceed for 5 minutes and luminescence was monitored using EnVision™ plate reader (Perkin Elmer) set to “Enhanced luminescence” mode. 
     EXAMPLE 2 
     High-throughput Screening (HTS)-compatible Cell-based Assay 
     To identify small molecules that lead to an increase in the expression levels of p21, a highly robust HTS-compatible cell-based assay using a reporter protein made of a fusion between the unstable p21 protein and  Renilla luciferase  (p21-Rluc) was designed. The assay relies on the generation of a fusion protein between p21 and a reporter protein that is quantifiable in a high throughput format. The genetically engineered chimeric protein should behave like the wild type p21 protein, such that the readout signal from the reporter moiety will reflect the regulation of p21. Two fusions proteins were initially constructed: a fusion between p21 and the  Renilla  luciferase (p21-Rluc) and a fusion between p21 and the GFP protein (p21-GFP) ( FIG. 5A ) 
     Luciferase activity is detected by measuring bioluminescence after addition of coelenterazine to intact cells, whereas GFP expression is measured by fluorescence spectroscopy. The two fusion constructs were stably expressed in a fibroblast cell line using an inducible Tet-On retroviral expression system. Since p21 is a negative regulator of the cell cycle, the use of an inducible vector permits to repress its expression and allows the amplification and maintenance of the transduced cell lines. 
     To validate the assay, expression of the p21 fusion protein was induced with the tetracycline derivative doxycycline and the protein synthesis inhibitor cycloheximide was added to stop new protein synthesis. The rate of degradation of the p21 fusion was then measured by cycloheximide chase and immunoblotting analysis with a  Renilla  luciferase-specific antibody (US Biological, Catalog #L6003-20). The proteasome inhibitor MG-132 was used as control to confirm that the degradation was proteasome-dependent. The fusion of GFP to p21 was found to artificially stabilize the p21 protein and this strategy was not pursued further. In contrast, the p21-Rluc protein was found to be highly unstable with a half-life of less than 1 hour, comparable to that of the wild type p21 protein ( FIG. 5B ). However, the Rluc-p21 fusion protein (i.e. in which the Rluc is N-terminal relative to p21) was found to artificially stabilize the p21 protein. To ascertain that the degradation rate of p21-Rluc reflects the half-life of p21, the same assay was used to monitor the degradation of Rluc alone. No degradation of Rluc was observed under these conditions, consistent with the reported stability of the  Renilla  luciferase protein ( FIG. 5C ). From these results, it may be concluded that the stability of the p21-Rluc fusion protein is a true reflection of the stability of p21 and that the construct can be used in a cell-based assay for screening purposes. 
     The p21-Rluc degradation assay was next transposed to a HTS-compatible format in 384-well plates and used to screen the Institut de Recherche en Immunologie et Cancerologie&#39;s (IRIC&#39;s) collection of 112,900 compounds ( FIG. 6 ) derived from the Chembridge DIVERset™ screening library, the Maybridge Hitfinder™ screening library, the Specs screening library, the Microsource SPECTRUM™ collection, the Biomol/Enzo Life Sciences Screen-Well™ library, the Prestwick Chemical Library™ library and the Sigma LOPAC 1280 ™. The potent proteasome inhibitor MG-132 was used as positive control. The mean increase of p21-Rluc signal by all positive controls across the screen was 3.062 ( FIG. 6 ). This value was set at 100% stabilization and used as comparison reference for test compounds. From the primary screen, 686 compounds that increase the p21-Rluc luminescence signal by at least 1.7-fold and 4 SDs above baseline (DMSO control) were identified ( FIG. 5 ). These compounds were re-tested in a reconfirmation experiment using the same assay conditions. A subset of 104 molecules was confirmed to be active by applying the same statistical criteria. Confirmed hits were then tested in a secondary assay using Rluc alone to eliminate compounds that increase luciferase enzymatic activity or boost the luminescence signal. From this assay, 72 molecules were selected for further evaluation. These molecules were tested in secondary screens using p27-Rluc and ERK3-Rluc fusion proteins to determine if they specifically inhibit p21 degradation or if they also block the degradation of p27 and the unrelated protein kinase ERK3, which would suggest that the molecules target the proteasome. Dose-response curves were generated for all compounds to estimate IC 50  values.  FIG. 9  shows a representative example of dose-response curves for a subset of active hit compounds identified in the assay. Interestingly, from the 72 molecules selected, 14 were found to inhibit the degradation of both p21 and p27 by more than 60% compared to the reference MG-132. Another 4 compounds inhibited p21 degradation by more than 60% but had less than 25% inhibitory effect on p27 proteolysis. None of these molecules had a significant effect on ERK3 degradation. Ten molecules had ED 50  values in the low μM range. The screening data for these molecules are summarized in Table 1A and 1B. These hit compounds were re-synthesized and their biological activity was confirmed in the p21 degradation assay. To validate that the increase in luciferase activity of the p21-Rluc fusion protein truly reflects an increase in the expression of the endogenous p21 protein, we have developed a p21 ELISA to measure its abundance. As shown in  FIG. 10  for the MG132 control and an inactive molecule, the increase in luciferase activity reflected an increase in the intracellular expression of the endogenous protein. The same correlation was observed for the positive hits identified in the screen. 
     
       
         
           
               
             
               
                 TABLE 1A 
               
             
            
               
                   
               
               
                 List of potential inhibitors of p21 and p27 degradation 
               
               
                 Threshold p21 &gt; 60% 
               
               
                 Threshold p27 &gt; 60% 
               
            
           
           
               
               
               
            
               
                   
                 Secondary screen 
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Primary screen 
                   
                 Secondary 
                 Secondary 
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 Primary 
                 Primary 
                 Confirmation 
                 Rluc 
                 Anisomycine 
                 screen 
                 screen 
                   
               
               
                   
                 screen (Fold 
                 screen 
                 (Fold 
                 (Fold 
                 (Fold 
                 p21-Rluc 
                 p27-Rluc 
                 IC 50   
               
               
                   
                 stabilization) 
                 (SSMD) 
                 stabilization) 
                 stabilization) 
                 stabilization) 
                 (% stabilization) 
                 (% stabilization) 
                 (μM) 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 UM1 
                 2.24 
                 6.85 
                 2.27 
                 1.04 
                 2.69 
                 84.83 
                 83.61 
                 6.30 
               
               
                 UM2 
                 2.28 
                 9.83 
                 2.36 
                 0.84 
                 2.71 
                 93.86 
                 89.88 
                 3.04 
               
               
                 UM3 
                 2.00 
                 6.57 
                 2.23 
                 0.84 
                 2.06 
                 71.27 
                 74.62 
                 4.61 
               
               
                 UM4 
                 2.26 
                 7.70 
                 2.52 
                 1.05 
                 1.98 
                 68.41 
                 72.74 
                 &gt;20.00 
               
               
                 UM5 
                 2.20 
                 9.47 
                 2.46 
                 1.19 
                 2.80 
                 97.53 
                 66.88 
                 1.11 
               
               
                 UM6 
                 1.93 
                 5.62 
                 2.49 
                 1.18 
                 3.10 
                 98.35 
                 68.60 
                 1.04 
               
               
                 UM7 
                 2.10 
                 5.66 
                 2.19 
                 0.86 
                 2.10 
                 68.99 
                 89.22 
                 4.84 
               
               
                 UM8 
                 2.28 
                 7.11 
                 2.09 
                 0.98 
                 2.68 
                 70.88 
                 75.45 
                 &gt;20.00 
               
               
                 UM9 
                 2.32 
                 9.85 
                 2.24 
                 1.02 
                 2.50 
                 87.13 
                 89.74 
                 12.02 
               
               
                 UM10 
                 1.84 
                 4.91 
                 2.10 
                 1.12 
                 2.79 
                 74.28 
                 82.27 
                 0.83 
               
               
                 UM11 
                 2.54 
                 7.70 
                 2.83 
                 0.78 
                 2.10 
                 78.06 
                 74.91 
                 2.07 
               
               
                 UM12 
                 2.60 
                 10.89 
                 2.18 
                 1.01 
                 2.87 
                 78.16 
                 73.30 
                 0.76 
               
               
                 UM13 
                 2.15 
                 8.05 
                 2.54 
                 1.19 
                 2.57 
                 68.59 
                 61.23 
                 1.83 
               
               
                 UM14 
                 1.83 
                 5.01 
                 2.16 
                 1.11 
                 2.26 
                 63.67 
                 61.44 
                 8.37 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 1B 
               
             
            
               
                   
               
               
                 List of potential specific inhibitors of p21 degradation 
               
               
                 Threshold p21 &gt; 60% 
               
               
                 Threshold p27 &lt; 25% 
               
            
           
           
               
               
               
            
               
                   
                 Secondary screen 
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Primary screen 
                   
                 Secondary 
                 Secondary 
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 Primary 
                 Primary 
                 Confirmation 
                 Rluc 
                 Anisomycine 
                 screen 
                 screen 
                   
               
               
                   
                 screen (Fold 
                 screen 
                 (Fold 
                 (Fold 
                 (Fold 
                 p21-Rluc 
                 p27-Rluc 
                 IC 50   
               
               
                   
                 stabilization) 
                 (SSMD) 
                 stabilization) 
                 stabilization) 
                 stabilization) 
                 (% stabilization) 
                 (% stabilization) 
                 (μM) 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 UM15 
                 2.10 
                 10.04 
                 2.20 
                 0.96 
                 2.70 
                 62.31 
                 18.35 
                 3.41 
               
               
                 UM16 
                 1.87 
                 6.80 
                 2.07 
                 1.06 
                 2.56 
                 62.61 
                 4.18 
                 &gt;20.00 
               
               
                 UM17 
                 1.68 
                 5.62 
                 2.41 
                 1.08 
                 2.64 
                 60.94 
                 13.14 
                 &gt;20.00 
               
               
                 UM18 
                 1.89 
                 5.27 
                 2.20 
                 1.03 
                 2.53 
                 67.32 
                 23.44 
                 1.89 
               
               
                   
               
            
           
         
       
     
     Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims. In the claims, the word “comprising” is used as an open-ended term, substantially equivalent to the phrase “including, but not limited to”. The singular forms “a”, an and the include corresponding plural references unless the context clearly dictates otherwise. 
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