bluuebunny/crossref_metadata_2025 huggingface dataset:
The molecular progression of plutonium‐239–induced rat lung carcinogenesis: Ki‐ras expression and activation
Authors: Stegelmeier Bryan L. , Gillett Nancy A. , Rebar Alan H. , Kelly Gregory
Published: February 2007

Specific, transforming point mutations of ras and alterations in ras expression have been associated with many neoplastic processes, and their presence may be pivotal in neoplastic transformation. Our objectives were to evaluate the molecular and genetic alterations of Ki‐ras in preneoplastic foci and neoplasms in the lungs of rats that inhaled 239PuO2 aerosols. Histologically classified pulmonary lesions were evaluated by in vitro nucleic acid amplification, oligonucleotide hybridization, and direct nucleic acid sequencing for activating Ki‐ras point mutations. We evaluated ras expression in similar lesions using immunohistochemistry and in situ hybridization. Specific Ki‐ras point mutations were present in 46% of the radiation‐induced malignant neoplasms. Spontaneous pulmonary neoplasms, which are rare in rats, had similar activating mutations and frequencies (40%). We found similar mutation frequencies in radiation‐induced adenomas and foci of alveolar epithelial hyperplasia. No mutations were identified in normal lung tissue. Ras expression in hyperplastic lesions and neoplasms was similar to that observed in normal pulmonary epithelia. These findings suggest that Ki‐ras activation, not alterations in expression, is an early lesion associated with many radiation‐induced, proliferative pulmonary lesions and that this molecular alteration may be an important component of both radiation‐induced and spontaneous pulmonary carcinogenesis in the rat.
An arylamine acetyltransferase (AT‐I) from syrian golden hamster liver: Cloning, complete nucleotide sequence, and expression in mammalian cells
Authors: Abu‐Zeid Medhat , Nagata Kiyoshi , Miyata Masaaki , Ozawa Shogo , Fukuhara Morio , Yamazoe Yasushi , Kato Ryuichi
Published: February 2007

A cDNA clone (designated hamAT101) encoding an arylamine acetyltransferase, AT‐I, was isolated from a hamster liver λgt11 cDNA library using a specific polyclonal antibody raised against AT‐I. The cloned cDNA insert consisted of 1181 nucleotides, including an open reading frame of 870 nucleotides encoding 290 amino acids (Mr 33,503). The isolated cDNA displayed high sequence similarity to those of chicken, rabbit, and human acetyltransferases. In Northern blots, the hamAT101 cDNA probe hybridized to an RNA band of 18S in the livers of both slow and rapid acetylator phenotypes. To confirm that hamAT101 cDNA encodes the monomorphic but not the polymorphic protein, the isolated cDNA was expressed in monkey kidney cells (COS‐1 cells) using the vector p91023(B). A protein with a molecular weight similar to that of AT‐I was detected upon Western blotting in the 9000 × g supernatant from the transfected cells. The activity toward four different substrates of the 9000 × g supernatant was also examined. In agreement with the results of purified AT‐I, the cDNA‐expressed protein exhibited a high capacity for N‐acetylation of 4‐aminoazobenzene and 2‐aminofluorene, and O‐acetylation of 2‐hydroxyamino‐6‐methyldipyrido[1,2‐a:3′,2′‐d]imidazole, whereas no activity was found for the N‐acetylation of p‐aminobenzoic acid. These results, in addition to the RNA blot hybridization, indicate that hamAT101 encodes the hamster acetyltransferase AT‐I.
Analysis of ras gene mutations in rainbow trout liver tumors initiated by aflatoxin B1
Authors: Chang Yung‐Jin , Mathews Catherine , Mangold Kathy , Marien Koenraad , Hendricks Jerry , Bailey George
Published: February 2007

The suspect human hepatocarcinogen aflatoxin B1 (AFB1) is a well‐known potent initiator of hepatic tumors in rainbow trout (Oncorhynchus mykiss). Both hepatocellular carcinomas and mixed hepatocellular/cholangio‐cellular carcinomas are induced by AFB1 in trout, with the mixed form predominating. We previously isolated two c‐ras genes from trout liver cDNA, and in the present study we analyzed DNA from 14 AFB1‐induced trout liver tumors for point mutations in exon 1 of both genes. Using the polymerase chain reaction (PCR) and oligo‐nucleotide hybridization methods, a high proportion (10/14) of the AFB1‐initiated tumor DNAs showed evidence of activating point mutations in the trout c‐Ki‐ras gene. Of the 10 mutant ras genotypes, seven were codon 12 GGA→GTA transversions, two were codon 13 GGT→GTT transversions, and one was a codon 12 GGA→A transition. Nucleotide sequence analysis of cloned PCR products from four of these tumor DNAs provided definitive evidence for two codon 12 GGA→GTA mutations, one codon 12 GGA→A mutation, and one codon 13 GGT→GTT mutation, in complete agreement with the oligonucleotide hybridization results. No mutations were detected in exon 1 of a second trout ras gene also expressed in liver, nor in DNA from control livers. This is the first report of experimentally induced ras gene point mutations in a lower vertebrate fish model. The results indicate that the hepatocarcinogen AFB1 induces c‐Ki‐ras gene mutations in trout similar to those in rat liver tumors.
TGF‐β1 and skin carcinogenesis: Antiproliferative effect in vitro and TGF‐β1 mRNA expression during epidermal hyperproliferation and multistage tumorigenesis
Authors: Krieg Peter , Schnapke Ruben , Fürstenberger Gerhard , Vogt Ingeborg , Marks Friedrich
Published: February 2007

The role of transforming growth factor‐β1 (TGF‐β1) in multistage carcinogenesis in mouse skin was assessed by studying its growth inhibitory effects on nontumorigenic and tumorigenic keratinocytes and by examining its mRNA expression in vitro and during epidermal hyperproliferation and multistage carcinogenesis. While growth of primary basal keratinocytes was inhibited by TGF‐β1 in doses as low as 0.1 ng/mL, the immortal keratinocyte line MCA/3D (“putatively initiated” cells) responded to TGF‐β1 with slightly reduced sensitivity, and the papilloma‐producing keratinocyte line 308 was considerably less sensitive. In contrast, the squamous carcinoma cell line Carc B was completely nonresponsive, and two other tumorigenic cell lines (PDV and PDVC57) were sensitive to growth inhibition by TGF‐β1. Steady‐state levels of TGF‐β1 mRNA were high in all the malignant cell lines and in line 308 papilloma cells, but low in primary basal cells and in the nontumorigenic keratinocyte lines V2, Reb1, and MCA/3D. Our in vivo studies showed that tumor promoters, but not mitogenic or weak hyperplasiogenic agents, were able to induce transient expression of TGF‐β1 mRNA in mouse epidermis. A constitutive overexpression of TGF‐β1 mRNA was observed in malignant carcinomas but not in the benign premalignant lesions, indicating that overexpression may be associated with malignant progression.
Mutational specificities of environmental carcinogens in the lacl gene of Escherichia coli H. V: DNA sequence analysis of mutations in bacteria recovered from the liver of swiss mice exposed to 1,2‐dimethylhydrazine, azoxymethane, and methylazoxymethanolacetate
Authors: Zeilmaker Marco J. , Horsfall Michael J. , van Helten José B. M. , Glickman Barry W. , Mohn Georges R.
Published: February 2007

The host‐mediated assay (HMA) was used to determine the spectra of mutations induced in the lacl gene of Escherichia coli cells recovered from the livers of Swiss mice exposed to the carcinogens 1,2‐dimethylhydrazine (SDMH), azoxymethane (AOM), and methylazoxymethanolacetate (MAMA). These spectra were further compared with changes induced by dimethylnitrosamine (DMNA) in the HMA methodology. A total of 177 independent lacl mutations arising in the HMA following exposure to SDMH, AOM, and MAMA were analyzed. Single‐base substitutions accounted for 97% of all mutations analyzed. The vast majority of the single‐base substitutions consisted of G:C→ A:T transitions (94% of all mutations). The remaining mutations consisted of A:T →G:C transitions (3% of all mutations) while non‐base substitutions accounted for only 3% of the total mutagenesis. The latter mutations consisted of one frameshift mutation and four lacO deletions. The distribution of G:C→ A:T transitions induced by the three chemicals in the first 200 bp of the lacO gene was not random, but rather clustered at sites where a target guanine was flanked at the 5′ site by a purine residue.
Altered Regulation of TGF‐β1 and TGF‐α in Primary Keratinocytes and Papillomas Expressing v‐Ha‐ras
Authors: Glick Adam B. , Sporn Michael B. , Yuspa Stuart H.
Published: February 2007

The influence of an oncogenic v‐Ha‐ras gene on the expression of TGF‐β and TGF‐α by mouse keratinocytes and derived tumors has been investigated. Normal mouse keratinocytes cultured as basal cells in 0.05 mM Ca2+ secreted low levels of TGF‐β2 peptide, and this increased markedly following culture in 1.4 mM Ca2+, retinoic acid, or phorbol esters. In contrast, introduction of a v‐Ha‐ras gene into normal keratinocytes increased basal expression and secretion of TGF‐β1 (rather than TGF‐β2) in response to all three agents. The selective secretion of TGF‐β1 in v‐Ha‐ras keratinocytes in response to 1.4 mM Ca2+ occurred even though the four TGF‐β2 transcripts were induced and the TGF‐β1 transcript decreased, suggesting that the activated v‐Ha‐ras gene product regulates expression of the TGF‐β isoforms at the posttranscriptional level. Immunohistochemical analysis of papillomas formed following skin grafting of v‐Ha‐ras keratinocytes onto nude mice indicated that TGF‐β1 was abundant in the basal and spinous layers, while there was no expression of TGF‐β1 in normal skin. In contrast, both normal and neoplastic tissues expressed TGF‐β2 and TGF‐β3 in the granular layers. Furthermore, TGF‐α mRNA expression was also elevated fivefold in cultured v‐Ha‐ras keratinocytes, and TGF‐α protein was overexpressed in the grafted papillomas, but there was no detectable expression in normal skin. Elevated expression of both TGF‐β1 and TGF‐α in the basal and spinous layers of benign tumors may be important for the high proliferation rate in these tumors as well as for increased proliferation in the suprabasal layer.
Morphological transformation of 10T1/2 mouse embryo cells can be initiated by DNA double‐strand breaks alone
Authors: Borek Carmia , Ong Augustus , Morgan William F. , Cleaver James E.
Published: February 2007

Malignant transformation of mouse fibroblasts was produced by electroporation with restriction enzymes. Similar transformation frequencies were observed with Pstl, Pvull, and Xbal, which cut genomic DNA at similar overall frequencies but have different termini, i.e., a 3′ overhang, a blunt end, and a 5′ overhang, respectively. The dose‐response curve for restriction enzyme transformation shows a marked plateau in frequencies of transformed foci per surviving cell, whereas x‐irradiation of the same cells gives a linear dose‐response curve. Evidently, transformation can be caused by DNA double‐strand breaks alone at a limited number of sites, but the evidence from x rays suggests that other kinds of DNA damage can cause transformation independently.
The use of rabbit polyclonal antibodies for the isolation of carcinogen‐adducted DNA by immunoprecipitation
Authors: Brylawski Bruna P. , Cordeiro‐Stone Marila , Kaufman David G.
Published: February 2007

Polyclonal rabbit antibodies elicited against DNA with high levels of (±) 7r, 8t‐dihydroxy‐9t,10t‐epoxy‐7,8,9,10‐tetrahydrobenzo[a]pyrene (BPDE‐I) adducts were used to isolate DNA fragments modified by this carcinogen. DNA treated in vitro with different concentrations of BPDE‐I was used as substrate in double‐antibody immuno‐precipitation reactions. The IgG fraction from immune rabbit serum (primary antibody) was reacted with single‐stranded plasmid DNA bearing BPDE‐I adducts, and the complexes were immunoprecipitated using goat antirabbit‐IgG as secondary antibody. DNA was isolated from the immunoprecipitated pellet, blotted onto nitrocellulose or nylon, and hybridized with 32P‐labeled sequences homologous to a fragment of the plasmid DNA used in the assay. The recovery of both DNA and adducts in the immunoprecipitated pellet increased with the level of carcinogen adduction of the DNA. The immunoprecipitation reaction appeared to be more efficient for fragments of DNA containing a high number of adducts. The amount of 32P‐hybridizing material recovered by immunoprecipitation was virtually identical to the amount added to the reaction in DNA samples that contained three adducts per 103 nucleotides.
Low‐level β1 protein kinase C expression in cloned rat embryo fibroblast cells enhances transformation induced by the adenovirus type 5 E1A gene
Authors: Su Zao‐Zhong , Duigou Gregory J. , Fisher Paul B.
Published: February 2007

Expression of the E1A gene of adenovirus type 5 (Ad5) in a cloned rat embryo fibroblast (CREF) cell line results in morphological transformation. The efficiency of E1A‐mediated transformation of CREF cells is increased if a wild‐type Ad5 E1A gene is cotransfected with a rat β1 protein kinase C (β1 PKC) gene. A direct demonstration of complementation between a functional‐transforming Ad5 E1A gene and β1 in inducing transformation was demonstrated using Ad5 E1A cold‐sensitive mutant (E1ACS) genes. The E1Acs gene enhanced transformation only at the transformation‐permissive temperature of 37° C and not at the nonpermissive transforming temperature of 32° C. CREF cells constitutively expressing low levels of β1, PKC mRNA were transformed at a higher frequency than parental CREF cells after transfection with an Ad5 E1A gene or infection with wild‐type Ad5 or the Ad5 host‐range cold‐sensitive mutant H5hr1. There was no enhancement of transformation in low‐level β1 PKC‐expressing CREF cells when cultures were grown continuously in the presence of the PKC‐inhibitor 1‐(5‐isoquinolynsulfonyl)‐2‐methylpiperazine dihydrochloride. Transfected CREF cells expressing low levels of β1 PKC mRNA displayed CREF‐like morphology and did not form colonies when grown in agar. In contrast, retroviral vector‐transformed CREF cells expressing high levels of β1 PKC mRNA and β1 PKC enzyme activity were morphologically transformed and grew efficiently in agar. These findings indicate that the β1, PKC gene, when expressed at low levels, can cooperate with the Ad5 E1A gene in the initiation of viral oncogene‐mediated transformation.
Chromatin and cell surface receptors mediate melanoma cell growth response to nerve growth factor
Authors: Rakowicz‐Szulczynska Ewa M. , Reddy Usha , Vorbrodt Andrzej , Herlyn Dorothee , Koprowski Hilary
Published: February 2007

Growth response to nerve growth factor (NGF) was tested in the primary melanoma cell line WM 164, which expressed a low level of NGF cell‐surface receptor, and in WM 164 cells transfected with cDNA for the cell‐surface receptor (TrWM 164 cells), which expressed a higher level of the cell‐surface receptor. Neither cell line expressed the chromatin receptor for NGF or internalized NGF. Both cell lines were stimulated to growth by NGF. After 10 d of exposure to NGF, a 230 000 Mr chromatin protein (receptor) was induced in both cell lines; as a result, NGF bound to the chromatin, and ribosomal RNA synthesis and cell proliferation were inhibited. We suggest that the cell‐surface and chromatin receptors each mediate a different function of NGF.
Spectrum of cis‐diamminedichloroplatinum(II)‐induced mutations in a shuttle vector propagated in human cells
Authors: Bubley Glenn J. , Ashburner Brian P. , Teicher Beverly A.
Published: February 2007

The supF gene of the shuttle vector pZ189 was used as a target for the study of mutations induced by cis‐diamminedichloroplatinum(II) (cis‐DDP). Normal human repair‐proficient fibroblasts and cis‐DDP repair‐deficient xeroderma pigmentosum (XP) cells were used as host cells to study the effect of cis‐DDP on the inhibition of shuttle vector replication and mutagenesis. Transfection of cis‐DDP‐treated pZ189 into normal and XP cell lines resulted in a marked increase in the mutation frequency and a decrease in the replication efficiency of the vector. However, these effects were much greater for the plasmid propagated in XP cells. Atomic absorption spectroscopy showed that six to eight Pt‐DNA adducts per plasmid were necessary to inhibit plasmid replication by 50% in normal cells. In contrast, only one to two Pt‐DNA adducts were necessary to inhibit replication of the plasmid by 50% in XP cells. Analysis of mutation sites demonstrated that cis‐DDP treatment resulted primarily in single and double mutations separated by one base and limited to a few locations within the 85‐bp mature tRNA. Propagation of the cis‐DDP‐treated vector in either normal or XP cells led to predominantly transversion mutations at AGA, AGG, and GAG sites and a cis‐DDP‐associated deletion of 174 bp. Although mutations occurred at target sites for cis‐DDP adduct formation, there was no correlation between sites of mutation and the most frequent sites of adduct formation.
Cytochrome P4501A2 constitutively expressed from transduced DNA mediates metabolic activation and DNA‐adduct formation of aromatic amine carcinogens in NIH 3T3 cells
Authors: Battula Narayana , Schut Herman A. J. , Thorgeirsson Snorri S.
Published: February 2007

We transduced mouse cytochrome P4501A2 DNA into NIH 3T3 cells by retrovirus‐mediated gene transfer. The capacity of the transduced cytochrome P4501A2 for metabolic activation and DNA‐carcinogen adduct formation of aromatic amine carcinogens was investigated. Clones of NIH 3T3 cells that constitutively express cytochrome P4501A2 and controls were exposed to a prototype food‐derived carcinogenic heterocyclic amine, 2‐amino‐3‐methylimidazo[4,5‐f]quinoline (IQ), and an aromatic amine, 2‐acetylaminofluorene (AAF), and their genomic DNAs were analyzed for adducts by 32P‐postlabeling assays. Kinetic analysis of DNA‐carcinogen adducts indicated that adduct formation was dependent on the level of the enzyme, the dose of carcinogen, and the duration of exposure. Addition of 7,8‐benzoflavone, an inhibitor of P4501A2, blocked both the enzyme activity and DNA‐adduct formation, indicating the specific role of P4501A2 in metabolic activation and adduct formation. Three specific IQ‐DNA adducts were detected in cells expressing P4501A2. Fingerprints of the in situ DNA adducts were similar to those of the in vivo adducts in rodent hepatic DNA after the administration of IQ. A single AAF‐DNA adduct was observed in cells exposed to AAF, but other minor adducts were also detected in vivo. These results show that cells expressing constitutive levels of single cytochrome P450s provide an excellent in situ model system for analyzing the catalytic specificity, metabolic activation, and genotoxicity of putative toxic, mutagenic, and carcinogenic substances.
Activation of Ki‐ras gene by point mutation in human liver angiosarcoma associated with vinyl chloride exposure
Authors: Marion Marie‐Jeanne , Froment Olivier , Trépo Christian
Published: February 2007

Point mutations of c‐ras genes were investigated in human angiosarcomas of the liver associated with occupational exposure to vinyl chloride. DNA prepared from either frozen or paraffin‐embedded tissues was amplified by the polymerase chain reaction, and putative point mutations at codons 12, 13, and 61 of c‐Ha‐ras, c‐Ki‐ras, and N‐ras were analyzed by dot‐blot hybridization with allele‐specific oligonucleotides. A G·C→A·T transition in the second nucleotide at codon 13 of the c‐Ki‐ras‐2 gene was detected in 5 of 6 tumors. This mutation is likely a consequence of vinyl chloride‐DNA adduct formation. It leads to the substitution of glycine by aspartic acid in the resulting p21 protein, a consistent amino acid substitution found so far in all types of human cancer exhibiting a codon 13‐mutated Ki‐ras gene.
Involvement of fos in spontaneous and ultraviolet light‐induced genetic changes
Authors: van den Berg Susanne , Kaina Bernd , Rahmsdorf Hans J. , Ponta Helmut , Herrlich Peter
Published: February 2007

Transient overexpression of ras, mos, or fos transcribed from various inducible promoters in NIH 3T3 cells causes significant increases in the frequency of chromosomal aberrations and, as shown for fos, in gene mutations. Under the experimental conditions of exponential growth and full serum supply, overexpression of the oncogenes does not increase the proliferation rate of cells. The generation of ras‐ and mos‐induced chromosomal aberrations was suppressed in cells that had been deprived of fos protein by antisense c‐fos oligodeoxynucleotides. The induction of chromosomal aberrations by ultraviolet irradiation is also suppressed by antisense c‐fos oligodeoxynucleotides. The data suggest that fos protein alone, or a transcription factor that contains fos protein as a subunit, activates or induces the synthesis of one or several mutator functions. Oncogenedriven mutagenesis could account for the accumulation of additional mutations after the activation of an oncogene, which may furnish a mechanistic basis for tumor promotion and tumor progression.
Expression of mouse cytochrome P4501A1 cDNA in repair‐deficient and repair‐proficient CHO cells
Authors: Trinidad Alma C. , Wu Rebekah W. , Thompson Larry H. , Felton James S.
Published: February 2007

Recombinant DNA techniques have been used to develop Chinese hamster ovary cell lines for studying chemically induced genotoxicity. These cell lines express a specific cytochrome P450 isozyme responsible for the metabolism of polycyclic aromatic hydrocarbons and exhibit defined differences in DNA repair capacity. A bacterial gene (neo) conferring resistance to gentamicin was inserted into the pcD expression vector containing the mouse cytochrome P1450 (P450IA1) cDNA to facilitate the selection of transformed cells. This plasmid was introduced into the nucleotide‐excision‐repair‐deficient UV5 cell line by electroporation. Transformed clonal isolates expressing the P1450 cDNA were identified by differential cytotoxicity assays using benzo[a]pyrene (B[a]P). One such clone, termed UV5P1, was mutagenized with ethyl methanesulfonate and selected for resistance to killing by UV radiation to derive a repair‐competent clone that expresses similar P1450 activity to that of the parental cell line. Two repair‐competent clones were selected and called 5P1R1 and 5P1R3. The resulting cell lines (UV5P1, 5P1R1, and 5P1R3) expressed arylhydrocarbon hydroxylase activity. UV5P1 and 5P1R3 were compared in terms of cytotoxicity and mutagenicity after exposure to B[a]P. Induced mutations were measured at the adenine phosphoribosyltransferase (aprt) and hypoxanthine guanine phosphoribosyltransferase (hprt) loci. Repair‐deficient UV5P1 cells were killed by B[a]P at concentrations below 0.1 μM, while the repair‐proficient 5P1R3 cells showed no toxicity up to 60 μM. Mutation induction at both loci was also much more efficient in UV5P1 cells. These new cell lines provide a more sensitive system that can be used in a battery of assays to evaluate the genotoxicity of chemicals requiring P450IA1 metabolic activation and to assess the role of DNA repair in modulating the biological effects of DNA damage.
Effect of serum growth factors and phorbol ester on growth and survival of human thyroid epithelial cells expressing mutant ras
Authors: Bond Jane , Dawson Tim , Lemoine Nick , Wynford‐Thomas David
Published: February 2007

We previously demonstrated a high incidence of ras mutation in thyroid follicular (epithelial) cell neoplasms and showed that expression of mutant ras is a potent mitogenic stimulus for normal human follicular cells in culture. Here we show that induction of cell proliferation in primary follicular cells by a mutant human Ha‐ras (val 12) expressed from a retroviral vector was absolutely dependent on the presence of serum growth factors. Induction of DNA synthesis showed partial dependence. Mutant ras‐induced growth was also inhibited by exposure to phorbol ester at concentrations sufficient to downregulate protein kinase C. More importantly, we observed an unexpected toxic effect of phorbol ester in this system that was specific to cells expressing mutant ras. This has potential significance both for elucidating the basic mechanism of ras action in epithelial cells and also as a pointer to a novel therapeutic strategy. © 1992 wiley‐list, Inc.
Isolation and partial characterization of murine O6‐alkyiguanine‐DNA‐alkyltransferase: Comparative sequence and structural properties
Authors: Santibanez‐Koref Mauro , Elder Rhoderick H. , Fan Chun‐Yang , Cawkwell Lynn , McKie James H. , Douglas Kenneth T. , Margison Geoffrey P. , Rafferty Joseph A.
Published: February 2007

A cDNA encoding murine O6‐alkylguanine‐DNA‐alkyltransferase (ATase) has been sequenced after isolation from total liver RNA by the polymerase chain reaction using oligonucleotide primers derived from the rat ATase cDNA sequence. Functionally active murine ATase protein has been expressed in Escherichia coli at high levels (about 2% of total protein) and purified to apparent homogeneity (molecular mass 26 kDa). In liquid hybridization experiments, anti‐human ATase polyclonal antibodies inhibited human but not rat or mouse ATase, whereas anti‐rat polyclonal antibodies inhibited rat and mouse but not human ATase. Both antibodies detected all mammalian ATases tested by western analysis so far. These results indicate some common epitopes and at least one unique human epitope. We compared the amino‐acid sequence of the murine ATase with those of other mammalian and bacterial ATases. The proteins of this family all have a large domain (approximately 70 amino acids) of highly conserved residues flanking the sequence PCHRV, which contains the alkyl‐accepting cysteine residue of the active site. No evidence was found in the sequences for helix‐turn‐helix, leucine‐zipper, or zinc‐finger motifs for DNA recognition and binding. Nuclear localization signals (basic‐residue‐rich regions) could not be uniquely identified in the mammalian members of the family. Outside of the conserved PCHRV region, there were major differences between prokaryotic and eukaryotic proteins at the primary structure level: there was a series of proline‐rich motifs, but these also varied between sequences. © 1992 wiley‐Liss, Inc.
Effects of N‐Methyl‐N‐nitrosourea on transcriptionally active and inactive nucleosomes: Macromolecular damage and DNA repair
Authors: Boffa Lidia C. , Mariani Maria R. , Carpaneto Elisabetta M.
Published: February 2007

We previously reported a separation, on an organomercurial column, of transcriptionally inactive nucleosomes (class 1) from those containing active gene sequences (classes 2 and 3). In this paper, we analyzed nucleosomal damage caused by exposure of HeLa S3 cells in suspension culture to the directly alkylating carcinogen N‐methyl‐N‐nitrosourea (MNU). The extent and site of methylation induced by the compound in nucleosomal DNA and RNA were determined by cell incubation in the presence of [3H]MNU. The highest amount of damage was detected in DNA of class 3 nucleosomes, while RNA alkylation was comparable in all nucleosomal classes. Cellular capacity for repair of MNU‐induced DNA strand breaks (estimated after a short pulse with [3H]thymidine) was found to be higher in active nucleosomal fractions (classes 2 and 3) than in the inactive fraction (class 1). Our data support the postulate that chromatin primary structure plays a role in modulating carcinogen damage to chromosomal macromolecules and in DNA strand breakage and repair mechanisms. Some of these initial steps are believed to be critical in the process of carcinogenesis. © 1992 Wiley‐Liss, Inc.
Genetic linkage between copper accumulation and hepatitis/hepatoma development in LEC rats
Authors: Sone Hideko , Maeda Mitsuaki , Gotoh Masahiro , Wakabayashi Keiji , Ono Takao , Yoshida Michihiro C. , Takeichi Noritoshi , Mori Michio , Hirohashi Setsuo , Sugimura Takashi , Nagao Minako
Published: February 2007

The concentration of copper in the livers of Long‐Evans rats with cinnamon‐like coat color (LEC), in which hepatitis and then hepatomas develop spontaneously, was recently found to be abnormally high. Therefore, we examined the copper concentrations in the livers of LEC F1 backcrosses (LEC F1 × LEC) to determine the linkage of copper accumulation with development of hepatitis. Consistent with a previously reported ratio of rats with hepatitis to rats without hepatitis of about 1:1, hepatitis developed in 14 of 30 F1 backcrosses. The copper concentrations in the livers of all LEC F1 backcrosses with hepatitis were abnormally high and comparable to those of LEC rats. In contrast, the concentrations in all backcrosses without hepatitis were similar to those in normal Long‐Evans with agouti coat color or Brown‐Norway rats. Copper accumulation was shown to be closely linked with the development of hepatitis in LEC rats and appeared to be a possible cause of hepatitis. The concentrations of copper in the livers of Fischer 344 rats after carbon tetrachloride treatment were in the range for normal liver, indicating that a high copper concentration in the liver is specific to LEC rats and not a specific characteristic of hepatitis. Furthermore, we found that the size and level of ceruloplasmin mRNA in the livers of LEC rats were the same as those in LEA rats and that the size and level of ceruloplasmin polypeptide in their livers and plasma were almost the same as those in LEA rats. Therefore, these results suggest that the copper accumulation is not due to alteration of expression or to gross alteration of the ceruloplasmin gene. © 1992 Wiley‐Liss, Inc.
Elevated expression of the ribosomal protein S2 gene in human tumors
Authors: Chiao Paul J. , Shin Dong M. , Sacks Peter G. , Hong Waun K. , Tainsky Michael A.
Published: February 2007

Differential screening of a cDNA library was used to isolate genes differentially expressed in a nontumorigenic clone and a ras‐transformed variant of the human teratocarcinoma cell line PA‐1. The RNA transcript for one of the cDNA clones that we identified was expressed at a 25‐fold higher level in the ras‐transformed PA‐1 cells than in the nontumorigenic PA‐1 cells. DNA sequence analysis of this clone showed that it had 86% nucleic acid homology to the mouse LLRep3 gene and only differed at a single amino acid codon (codon 198), which is changed from serine in LLRep3 to threonine in this cDNA clone. The rat ribosomal S2 protein is closely related to the yeast omnipotent informational suppressor SUP44, which encodes the yeast ribosomal protein 54; to the mouse protein LLRep3; and to the human cDNA clone we describe in this report. We therefore concluded that this clone codes for the human ribosomal S2 protein. In situ hybridization experiments revealed that expression of this gene was elevated in cultured human head and neck squamous cell carcinomas compared with normal keratinocytes. In situ hybridization experiments also demonstrated that expression of this gene was elevated in histological sections of human premalignant leukoplakia, head and neck squamous cell carcinomas, and colon and breast cancers compared with the adjacent normal tissues. S2 expression may be a useful diagnostic or prognostic marker for grading human tumors. © 1992 Wiley‐Liss, Inc.
p53 gene mutations in human endometrial carcinoma
Authors: Risinger John I. , Dent Georgette A. , Ignar‐Trowbridge Diane , McLachlan John A. , Tsao Ming‐Sound , Senterman Mary , Boyd Jeff
Published: February 2007

Although carcinoma of the uterine endometrium is the most frequently diagnosed malignancy of the female reproductive tract, the molecular genetic features of this tumor have yet to be described in significant detail. Since mutations of the p53 tumor suppressor gene are the single most common genetic alteration found in human malignancies, we examined the hypothesis that p53 mutations occur in human endometrial carcinoma. Sequencing analysis of exons 5–8 revealed point mutations in 3 of 21 (14%) tumors; one mutation was an unusual single‐base insertion at codons 176–177, resulting in a premature stop codon, whereas the other two were CGG→TGG transitions at codon 248. Two of these tumors showed reduction to homozygosity at the p53 allele, but one tumor apparently retained heterozygosity. These data indicate that p53 mutations occur in human endometrial carcinoma, although relatively infrequently, and that loss of the normal p53 allele does not necessarily occur with point mutation of the p53 gene in this tumor type. © 1992 Wiley‐Liss, Inc.
Activating mutation of the Ha‐ras gene in chemically induced tumors of the hamster cheek pouch
Authors: Gimenez‐Conti Irma B. , Bianchi Albert B. , Stockman Sharon L. , Conti Claudio J. , Slaga Thomas J.
Published: February 2007

The presence of an activating mutation in the Ha‐ras gene in hamster cheek pouch tumors induced by 7,12‐dimethylbenz[a]anthracene (DMBA) complete carcinogenesis was investigated. The normal sequence of a fragment of genomic DNA encompassing codon 61 of the Ha‐ras gene was amplified by the polymerase chain reaction using primers designed for a highly conserved region of the mouse Ha‐ras‐1 gene. The sequence of the amplified fragment was determined by a direct sequencing technique and exhibited 83.3% and 87.5% homology with the corresponding human and mouse sequences, respectively. At the amino acid level, the sequence was identical among the three species. Paraffin sections of 11 squamous cell carcinomas of the cheek pouch were used to detect mutated Ha‐ras alleles. DNA sequencing of the tumors showed that six of 11 tumors presented an A→T transversion in the second position of codon 61, resulting in an amino acid change from glycine to leucine. As has been demonstrated in other systems, we have shown a specific mutation of the Ha‐ras gene in chemically induced tumors of the hamster cheek pouch, further supporting the role of this oncogene in chemical carcinogenesis. © 1992 Wiley‐Liss, Inc.
Differential retention of tumor‐ and differentiation‐suppressor functions in cells derived from a human squamous cell carcinoma
Authors: Jaffe Deborah R. , Montero‐Puerner Yvonne , Beckett Michael A. , Cowan Janet M. , Weichselbaum Ralph R. , Diamond Alan M.
Published: February 2007

Three morphologically distinct cell lines—F.2a, V, and B.2—were isolated from a single human squamous cell carcinoma. Although all three cell lines can grow indefinitely in culture, they differ in a number of important transformation‐related phenotypes. Only B.2 is strongly tumorigenic when injected into the flanks of nude mice, and only V can efficiently grow in semisolid media. The dominance of these traits was investigated by generating somatic cell hybrids among the three cell lines. F.2a was able to suppress the tumorigenicity of B.2 cells, whereas B.2 inhibited the capacity for anchorage‐independent growth of V, the latter trait being a function of the ability of these epithelial cells to differentiate when deprived of support. The influence of exogenously added growth factors was also evaluated. This study indicates that the particular tumor we examined consisted of a heterogeneous population of cells with distinct growth and differentiation capacities. © 1992 Wiley‐Liss, Inc.
Differential effect of tumor necrosis factor on proliferation of primary human keratinocytes and cell lines containing human papillomavirus types 16 and 18
Authors: Villa Luisa Lina , Vieira Katia Brito Lins , Pei Xu‐Fang , Schlegel Richard
Published: February 2007

Keratinocytes immortalized by human papillomaviruses (HPV) 16 and 18 are partially resistant to the inhibition of proliferation exerted by transforming growth factor‐β (TGF‐β). To determine if this finding reflects a generalized resistance to inhibitory cytokines, we studied the effect of tumor necrosis factor‐α (TNF‐α) on subconfluent cultures of both normal and HPV‐immortalized human foreskin keratinocytes. Whereas primary and HPV‐16‐immortalized keratinocytes were sensitive to TNF‐α, HPV‐18‐immortalized keratinocytes (and those immortalized by simian virus 40) were resistant to the inhibitory effects of this cytokine. The ability of HPV‐18 to induce a more resistant phenotype correlated with its more potent in vitro transforming activity and its apparent association with more aggressive tumors. Interestingly, the state of TNF‐induced growth inhibition in normal or HPV‐16—immortalized keratinocytes was not accompanied by a reduction in the expression of c‐myc RNA or protein. This contrasts sharply with the ability of TGF‐β to inhibit c‐myc RNA expression in normal cells. Evidently, the resistance of HPV‐immortalized keratinocytes to TNF‐β and TGF‐β proceeds along different regulatory pathways. © 1992 Wiley‐Liss, Inc.
Activation of the Ki‐ras gene in spontaneous and chemically induced lung tumors in CD‐1 mice
Authors: Manam Sujata , Storer Richard D. , Prahalada Srinivasa , Leander Karen R. , Kraynak Andrew R. , Hammermeister Christine L. , Joslyn Dennis J. , Ledwith Brian J. , Van Zwieten Matthew J. , Bradley Matthews O. , Nichols Warren W.
Published: February 2007

As part of an evaluation of the effectiveness of using ras mutation analysis for distinguishing carcinogen‐induced from spontaneous tumors, we examined the profile of ras gene point mutations in spontaneous, 7,12‐dimethylbenz[a]anthracene (DMBA)‐induced, and N‐nitrosodiethylamine (DEN)‐induced lung tumors from Crl:CD‐1(ICR)BR (CD‐1) mice. Although all of the lung tumors were assayed for mutations in the Ha‐ras, Ki‐ras, and N‐ras genes (codons 12, 13, and 61), only Ki‐ras mutations were found, which is consistent with other studies that have noted a strong preference for Ki‐ras gene activation in mouse, rat, and human lung tumors. We found that spontaneous CD‐1 mouse lung tumors had a very high frequency of Ki‐ras gene activation (17 of 20 tumors; 85%), distributed among codons 12 (5 of 20), 13 (1 of 20), and 61(11 of 20). DMBA‐induced lung tumors had a slightly higher frequency of Ki‐ras gene mutations (16 of 16; 100%), again distributed among codons 12(5 of 16), 13 (2 of 16), and 61(9 of 16). However, seven of the DMBA tumors had mutations qualitatively different from those found in spontaneous tumors. In contrast to DMBA‐induced tumors, DEN‐induced tumors had a lower frequency of Ki‐ras mutations (36%) when compared with spontaneous lung tumors, suggesting that DEN primarily induces lung carcinogenesis by a mechanism other than ras gene activation. Thus, although spontaneous and induced CD‐1 mouse lung tumors have a strong tissue‐specific preference for carrying an activated Ki‐ras gene, the nature of the initiating carcinogen can influence the frequency or profile of Ki‐ras mutations. © 1992 wiley‐Liss, inc.
Regulation of growth and gene expression in human papillomavirus‐transformed keratinocytes by transforming growth factor‐β: Implications for the control of papillomavirus infection
Authors: Braun Lundy , Dürst Matthias , Mikumo Ryoko , Crowley Annette , Robinson Mava
Published: February 2007

Cervical carcinogenesis is a multistep process that appears to be initiated by infection of squamous epithelial cells in the cervix with one of a limited number of human papillomavirus (HPV) types. However, the mechanisms involved in the evolution of benign, HPV‐induced lesions to malignancy have not yet been fully elucidated. Transforming growth factor‐β (TGF‐β), a multifunctional growth factor produced by cells in the skin, inhibits the proliferation of foreskin and cervical keratinocytes in vitro. We examined the effects of TGF‐β on growth and virus early‐gene expression in cell lines immortalized by two HPV types associated with cervical carcinogenesis as well as the expression of TGF‐β1 mRNA transcripts in normal and HPV‐positive cells in vivo and in vitro. We found that normal and HPV‐positive cells expressed similar levels of TGF‐β1 mRNAs and exhibited similar patterns of responsiveness to three isoforms of TGF‐β in both monolayer and modified organotypic cultures. Of particular interest is our finding that the expression of the E6 and E7 early viral transforming regions of both HPV16 and HPV18 was reversibly and rapidly inhibited by TGF‐β. In one HPV16‐positive cell line examined in detail, inhibition of HPV expression required protein synthesis and occurred at the level of transcription. HPV‐immortalized cells selected for resistance to in vitro differentiation signals remained sensitive to TGF‐β‐mediated growth inhibition. These results, showing that both growth and virus gene expression in HPV‐transformed cells were responsive to TGF‐β, suggest that endogenous growth factors produced by different cell types in squamous epithelium may play a role in the progression of cervical neoplasia. © 1992 Wiley‐Liss, Inc.
Aberrant expression of the simple epithelial type II keratin 8 by mouse skin carcinomas but not papillomas
Authors: Larcher Fernando , Bauluz Cristina , Diaz‐Guerra Margarita , Quintanilla Miguel , Conti Claudio J. , Ballestin Claudio , Jorcano José L.
Published: February 2007

Keratins have been demonstrated to be suitable markers of changes taking place during epithelial neoplasia. Therefore, we analyzed 18 mouse skin tumors (nine papillomas and nine squamous cell carcinomas), induced either by two‐stage carcinogenesis with 7,12‐dimethylbenz[a]anthracene(DMBA)/12‐O‐tetradecanoylphorbol‐13‐acetate or complete carcinogenesis with DMBA, by immunofluorescence with a monoclonal antibody to keratin (K) 8 (TROMA‐1). Immunoperoxidase staining and immunoblotting were also used on selected tumor samples to further explore for the presence of K8. All of the papillomas tested were negative for the presence of K8, whereas the carcinomas were positive. The level of K8 expression in carcinomas showed a positive correlation with the degree of malignancy. Northern blot analysis using a K8 cDNA probe suggested that control of K8 expression in mouse skin tumors occurs at the transcriptional level. Double‐label immunofluorescence staining using TROMA‐1 and RK13 antibodies demonstrated that K8 did not generally colocalize with K13, a keratin normally found in internal stratified epithelia but aberrantly expressed in mouse epidermal tumors. Furthermore, tumors expressing high levels of K8 showed a reduced expression of K13. Histological examination of immunoperoxidase‐stained tumors demonstrated that K8‐positive cells were mainly found in anaplastic areas, whereas K13 foci were restricted to well‐differentiated regions. Our results demonstrate that K8 expression is a marker of late stages of carcinoma progression in the mouse skin carcinogenesis model. © 1992 Wiley‐Liss, Inc.
Stepwise transformation of primary thyroid epithelial cells by a mutant Ha‐ras oncogene: An in vitro model of tumor progression
Authors: Burns Jorge S. , Blaydes Jeremy P. , Wright Pat A. , Lemoine Louise , Bond Jane A. , Dillwyn Williams E. , Wynford‐Thomas D.
Published: February 2007

Activating mutations of the ras oncogene family occur at high frequency in all stages of thyroid tumorigenesis, both human and experimental. To test the causal nature of this association, and to investigate the biological role of ras mutation, we introduced a mutant c‐Ha‐ras gene into normal rat thyroid follicular cells using an ecotropic retroviral vector. The major immediate effect was to greatly extend the proliferative lifespan of these cells in culture from less than 3 to more than 15 doublings, without any observable loss of growth‐factor dependence or differentiated functions. This in vitro phenotype strongly supports an initiating role for ras mutation in the genesis of benign thyroid tumors (adenomas) in vivo. Spontaneous transformation was observed at low frequency on continuous culture of mutant ras‐expressing cells, giving rise to fully immortalized, growth factor‐independent, highly tumorigenic lines. Transformation was associated with (i) loss of responsiveness to the growth inhibitor TGF‐β1, and (ii) greatly increased nuclear levels of p53 protein, which unexpectedly was not due to point mutation in the conserved regions of the p53‐coding sequence. We postulate that these two phenomena are causally related to each other and to the transformed phenotype. © 1992 Wiley‐Liss, Inc.
Mutational analysis of a dominant oncogene (c‐Ki‐ras‐2) and a tumor suppressor gene (p53) in hamster lung tumorigenesis
Authors: Oreffo Victor I. C. , Lin Hsiu‐Wei , Gumerlock Paul H. , Kraegel Susan A. , Witschi Hanspeter
Published: February 2007

In human lung cancers, alterations of both a dominant oncogene (ras) and a tumor suppressor gene (p53) have been identified. Polymerase chain reaction (PCR) analysis of mRNA was used to amplify the c‐Ki‐ras‐2 andp53genes from Syrian golden hamsters. The PCR products were confirmed by predicted‐size analysis, probing with nonradioactive (biotin‐labeled) oligonucleotides, and direct sequencing. Lung tumors were produced in hamsters by repeated injections of 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK). Of six tumors examined, three (50%) had mutations in codon 12 of Ki‐ras. Examination of the conserved regions ofp53revealed no mutations. We conclude that NNK‐induced carcinogenesis in the hamster results in characteristic alterations of Ki‐rasbut may not necessarily involve thep53gene. © 1992 Wiley‐Liss, Inc.
Sequence analysis of the int‐2/fgf‐3 gene in aggressive human breast carcinomas
Authors: Meyers Shari L. , Dudley Jaquelin P.
Published: February 2007

A number of primary human breast carcinomas exhibit amplification of the chromosome 11 region containing the int‐2/fgf‐3 proto‐oncogene, and progression of breast cancer has been correlated with int‐2 amplification or with certain restriction fragment length polymorphisms (RFLPs) of the int‐2 gene. Using the polymerase chain reaction (PCR), we obtained the int‐2 coding sequences from six primary tumors, four of which exhibited amplification of the int‐2 gene and one of which exhibited amplification of the neu gene. The majority of these tumors (five of six) were aggressive, as judged by their early recurrence, metastasis, or both. Nucleotide sequencing of PCR products revealed that previously described BamHl and Pstl RFLPs of the int‐2 gene, as well as a new polymorphism at position 9154, were located within the intron between the second and third exons. A seventh tumor was used to localize one of the Pstl RFLPs 5 bp from the splice‐acceptor site of the third exon. However, none of the tumor DNAs analyzed showed differences in the int‐2 protein coding regions when compared with normal placenta DNA. These results imply that aggressive human breast cancers encode an unaltered form of the int‐2 protein. © 1992 Wiley‐Liss, Inc.
Cholangiocarcinomas in japanese and thai patients: Difference in etiology and incidence of point mutation of the c‐KI‐ras proto‐oncogene
Authors: Tsuda Hitoshi , Satarug Soisungwan , Bhudhisawasdi Vajarabhongsa , Kihana Toshimasa , Sugimura Takashi , Hirohashi Setsuo
Published: February 2007

Point‐mutational activation of the c‐Ki‐ras proto‐oncogene has been shown to be rare in human hepatocellular carcinoma, the most common primary liver cancer and one usually associated with chronic viral infection. To reveal the association of c‐Ki‐ras activation with cholangiocarcinogenesis under different etiological backgrounds, the incidence of point mutation at codons 12 and 13 of the c‐Ki‐ras proto‐oncogene was examined in three groups of human liver cancers with differentiation to biliary epithelial cells: Group 1, cholangiocellular carcinoma in Japanese with normal livers; Group 2, cholangiocellular carcinoma in Thais who had lived in an area where the liver fluke Opisthorchis viverrini is endemic; and Group 3, combined hepatocellular‐cholangiocellular carcinoma, a rare type showing features of both hepatocellular and biliary epithelial differentiation, in Japanese with chronic viral hepatitis with or without cirrhosis. The polymerase chain reaction and direct sequencing of its product were used to detect the mutation. Point mutation at codon 12 of the c‐Ki‐ras gene was detected in five (56%) of nine cases in Group 1. In contrast, the mutation was not detected in any of the cases in Groups 2 and 3. Therefore, point‐mutational activation of c‐Ki‐ras did not seem to be involved in the development of primary liver cancers associated with apparent chronic irritation of liver cells or biliary epithelial cells caused by exogenous liver‐fluke or viral infection. On the other hand, point‐mutational activation of the c‐Ki‐ras proto‐oncogene may be involved in cholangiocarcinogenesis in liver without preexisting liver‐fluke or viral infection. © 1992 Wiley‐Liss, Inc.