bluuebunny/crossref_metadata_2025 huggingface dataset:
Influence of DNA repair by ada and ogt alkytransferases on the mutational specificity of alkylating agents
Authors: Roldán‐Arjona Teresa , Luque‐Romero Francisco L. , Ariza Rafael R. , Jurado Juan , Pueyo Carmen
Published: February 2007

We investigated the influence of the alkyltransferases (ATases) encoded by the ada and ogt genes of Escherichia coli on the mutational specificity of alkylating agents. A new mutational assay for selection of supF‐ mutations in shuttle‐vector plasmids was used. Treating plasmid‐bearing bacteria with N‐methyl‐N‐nitrosourea (MNU), N‐ethyl‐N‐nitrosourea (ENU), and ethyl methanesulfonates (EMS) dramatically increased the muttation frequency (from 33‐fold to 789‐fold). The vast majority of mutations (89–100%) were G:C←A:T transitions. This type of mutation increased in ada‐ (MNU) or ogt‐ (ENU) bacteria, suggesting that repair of O6‐methylguanine by ada ATase and repair of O6‐ethylguanine by ogt ATase contribute mainly to the decrease in G:C←A:T transitions. The analysis of neighboring base sequences revealed an overabundance of G:C←A:T transitions at 5′‐GG sequences. The 5′‐PuG bias increased in ATase‐defective cells, suggesting that these sequences were not refractory to repair. G:C←A:T transitions occurred preferentially in the untranscribed strand after in vivo exposure. That this strand specificity was detected even in bacteria devoid of ATase activity (ada‐ ogt‐) and not after in vitro mutagenesis suggests a bias for damage induction rather than for DNA repair. Highly significant differences were found between the in vivo and in vitro incidences of G:C←A:T substitutions at the two major hotspots, positions 123 (5′‐GGG‐3′; antisense strand) and 168 (5′‐GGA‐3′; sense strand). These results are explained by differences in the probability of formation of stem‐loop structures in vivo and in vitro. © 1994 Wiley‐Liss, Inc.
Infrequent loss of heterozygosity and mutation of the p53 gene in immortal and transformed mouse embryo fibroblasts
Authors: Tokumitsu Masayuki , Kadohama Takayuki , Ogawa Katsuhiro
Published: February 2007

Some of the progeny of isolated mouse embryo fibroblasts acquire the ability to grow indefinitely during cultivation, presumably through some mutational events. The relevance of p53 mutations and loss of heterozygosity to the mechanism of such immortal growth capability remains controversial. Since four bases in intron 1 of the p53 gene in C3H/HeJ mice are replaced by 13 different bases in DBA/2J mice, it is possible to distinguish maternal and paternal p53 alleles in the cells of F1 hybrids of these strains (C3D2F1) by electrophoresis of polymerase chain reaction fragments including the region. We established 23 spontaneously immortalized fibroblast cell lines from C3D2F1 mouse embryos and 29 transformed cell lines induced from one of the immortal cell lines, either by treatment with chemical carcinogens or by transfection with the c‐Ha‐ras gene. Of these 52 cell lines, only one, derived from fibroblasts unpassaged for 4 mo, showed p53 gene loss of heterozygosity and a structural alteration in the remaining allele. Our results demonstrated that p53 mutations are not a strict requirement for immortalization and transformation of mouse embryo fibroblasts in Vitro. © 1994 Wiley‐Liss, Inc.
Transcriptlonal activation by viral enhancers: Critical dependence on extracellular matrix‐cell interactions in mammary epithelial cells
Authors: Schmidhauser Christian , Casperson Gerald F. , Bissell Mina J.
Published: February 2007

Extracellular matrix (ECM)‐cell interactions are essential for the regulation of many genes in differentiated cell types. A number of expression vectors that work well in cells cultured on tissue‐culture plastic appear to be inactive or sporadically active in vivo. We reasoned that these responses also may be influenced by the ECM. We therefore examined three commonly used viral enhancers and found that they all responded either positively or negatively to the presence of exogenous ECM. Using mouse mammary epithelial cells, we found that a mouse mammary tumor virus enhancer linked to its own promoter or to a truncated (and by itself inactive) β‐casein promoter drove transcription efficiently only when the cells were in contact with an ECM (more than a 100‐fold induction over tissue‐culture plastic). Similarly, the cytomegalovirus enhancer was more active in cells in contact with ECM. In contrast, the simian virus 40 enhancer, linked to the β‐casein promoter, was 12‐fold more active in cells on tissue‐culture plastic. This activity was strongly reduced when the cells interacted with ECM. Thus, we conclude that different enhancers can respond to ECM by either activating or suppressing transcription. This observation has important implications for understanding the mechanisms of promoter action and for designing expression systems for use in gene therapy. © 1994 Wiley‐Liss, Inc.
Increased expression of and sensitivity to transforming growth factor‐α: A promotive role during rat liver carcinogenesis
Authors: Strömblad Staffan , Eriksson Lennart C. , Andersson Göran
Published: February 2007

The influence of the tumor promoter 2‐acetylaminofluorene (2‐AAF) on cell proliferation and on the epidermal growth factor receptor (EGFR) system was assessed in normal and nodular rat livers. DNA replication in vivo was inhibited below the detection level after 8 d of dietary 2‐AAF treatment of previously unexposed rats. The 2‐AAF‐induced growth inhibition was accompanied by downregulation of the number of epidermal growth factor (EGF)‐binding sites and decreased levels of EGFR transcripts, whereas no changes in the transforming growth factor‐α (TGF‐α) mRNA levels were observed. The persistent liver nodules generated by intermittent 2‐AAF feeding had a 30‐ to 35‐fold higher replicating cell fraction than normal liver. Treatment with 2‐AAF in vivo reduced the replicating cell fraction to one third in nodules after 14 d of 2‐AAF treatment. The initial EGFR mRNA levels and number of EGF binding sites in nodules before 2‐AAF administration was about 60% that of control livers and was slightly reduced by 2‐AAF feeding. The levels of EGFR mRNA after 14 d of 2‐AAF feeding were thus similar in the nodules and in the 2‐AAF‐treated control livers, whereas the fraction of proliferating cells in nodules after the 2‐AAF treatment was much larger than in normal liver. The TGF‐α mRNA level in the nodules Was found to be 1.4‐fold and in malignant hepatomas 1.7‐fold the level in normal liver. Primary hepatocytes isolated from control livers were four to five times more sensitive to replicative stimulation with EGF than with TGF‐α, whereas nodular cells responded at lower concentrations than control cells and equally well to both EGF and TGF‐α. We conclude that the decreased amounts of EGFR in the nodular cells with respect to proliferate stimulation could be more than compensated for by elevated synthesis of TGF‐α combined with an increased TGF‐α sensitivity. Collectively, these changes implicate TGF‐α in sustaining cell proliferation during chemically induced rat liver carcinogenesis. © 1994 Wiley‐Liss, Inc.
Preferential repair of ultraviolet light‐induced dna damage in the transcribed strand of the human p53 gene
Authors: Ford James M. , Lommel Lori , Hanawalt Philip C.
Published: February 2007

Mutations in the p53 tumor suppressor gene have been found in most human tumors. Analyses of the spectrum of p53 mutations in certain tumor types have shown a bias for mutations originating from lesions presumed to be in the untranscribed strand of the gene. This implies strand specificity for the formation or repair of DNA damage. We measured the induction and repair of ultraviolet light‐induced cyclobutane pyrimidine dimers (CPD) in each strand of the human p53 gene in a normal human lung fibroblast cell line using quantitative Southern hybridization. We found that the removal of CPD from the transcribed strand was more rapid than that from the untranscribed strand of this gene, although the frequency of CPD induction was similar in both strands. Preferential repair of the transcribed strand of the p53 gene may account for the mutational spectra of this gene in human tumors. © 1994 Wiley‐Liss, Inc.
Cellular control of human papillomavirus oncogene transcription
Authors: Hoppe‐Seyler Felix , Butz Karin
Published: February 2007

Specific types of human papillomaviruses (HPVs) are closely associated with the development of cervical cancer. The transforming ability of these high‐risk HPV types depends on the expression of the viral E6 and E7 oncogenes. It is therefore of particular interest to elucidate the molecular mechanisms that result in the activation of E6/E7 expression during HPV‐associated tumorigenesis. Recently, much progress has been made in characterizing the proteins involved in the regulation of HPV oncogene transcription. This review describes the functional significance of cellular factors involved in the transcriptional control of the E6/E7 promoter for the two most common HPV types associated with cervical cancer, HPV16 and HPV18. In addition, we discuss regulatory pathways that may contribute to the epithelial cell specificity of E6/E7 transcription. The definition of the factors that regulate HPV oncogene transcription could provide new insights into the molecular mechanisms activating viral oncogene expression during cervical carcinogenesis and forms an experimental basis for investigating the specific biochemical pathways that contribute to HPV‐associated malignant cell transformation. © 1994 Wiley‐Liss, Inc.
Non‐Ap‐1 tumor promoter 12‐O‐tetradecanoylphorbol‐13‐acetate‐responsive sequences in the human ornithine decarboxylase gene
Authors: Kim Yong‐Joo , Pan Huichin , Verma Ajit K.
Published: February 2007

To define the mechanisms of 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)‐induced transcription of the ornithine decarboxylase (ODC) gene, we isolated a genomic clone (hODC41 B) of ODC from a human leukocyte genomic DNA library. The restriction endonuclease map, in comparison with the previously published sequences of the human ODC gene, indicated that hODC41B contained a 15.7‐kb sequence that extended from the sixth exon to about 10 kb upstream of the ODC gene. A 2.5‐kb genomic fragment containing the 5′ flanking region and the first exon was subcloned and sequenced. Sequence analysis revealed multiple putative promoter/enhancer elements (a TATA box, a CAAT box, 17 GC boxes, and a cAMP‐responsive element) but no consensus AP‐1 sequences (TGAGTCA) in the 2.5‐kb 5 flanking region. However, three AP‐1 sequences were located in introns 3, 5, and 11. We constructed aseries of chimeric genes containing part of the first exon and increasingly longer 5′ flanking sequences of the ODC gene fused to either bacterial chloramphenicol acetyltransferase (CAT) or luciferase reporter genes. TPA inducibility was determined by transient transfection and measurement of CAT or luciferase expression in HeLa cells. The induction of CAT activity by TPA decreased with decreasing lengths of the 5′ flanking sequences up to nt‐82. The TPA induction from the construct‐72 ODC CAT was threefold to sevenfold, and the TPA inducibility of the same fragment was about ninefold to 30‐fold with the luciferase reporter gene. Further deletion analysis revealed TPA‐responsive sequences in ODC nt‐42 to +54. Gel mobility shift assays using α‐32P‐end labeled ODC nt‐42 to +60 revealed that nt‐42 to +60 specifically bound HeLa cell nuclear proteins. HeLa cell nuclear protein binding to ODC nt‐42 to +60 could not be completely competed by AP‐1‐, AP‐2‐, AP‐3‐, or SP1‐responsive sequences. © 1994 Wiley‐Liss, Inc.
Effects of the genotoxic carcinogen chromium(VI) on basal and hormone‐inducible phosphoenolpyruvate carboxykinase gene expression in vivo: Correlation with glucocorticoid‐and developmentally regulated expression
Authors: McCaffrey Jennifer , Wolf Chad M. , Hamilton Joshua W.
Published: February 2007

Previous studies have shown that a number of different genotoxic carcinogens that induce different types of DNA damage preferentially alter the expression of inducible genes in vivo. To investigate further the mechanistic basis for these effects, we examined the effects of the human lung carcinogen chromium(VI) on expression of the hormone‐inducible cytosolic phosphoenolpyruvate carboxykinase (PEPCK) gene in chick embryo liver. Chromium(VI) pretreatment had significant effects on both basal and glucocorticoid‐inducible PEPCK expression in 14‐d‐old embryo liver. These effects were principally a result of changes in PEPCK transcription. In contrast, treatment with chromium(VI) 1 h after treatment with glucocorticoid had no effect on PEPCK induction, suggesting that an early event in the induction process is the target for carcinogen effects. In 16‐d‐old liver, in which PEPCK expression is no longer responsive to glucocorticoid induction, both basal and inducible PEPCK expression were also refractory to chromium(VI) effects, indicating that carcinogen responsiveness is a phenotypic rather than an inherent property of inducible genes and is related to their competence for induction. Chromium(VI) had no effect on cAMP induction of PEPCK expression, demonstrating that carcinogens target their effects to specific regulatory pathways. Comparison of the effects of chromium(VI) with those of cycloheximide suggests that chromium(VI) targets its effects to a labile, constitutively expressed repressor involved in PEPCK gene regulation. © 1994 Wiley‐Liss, Inc.
Differences in the regulation of phosphatidylinositol‐specific phospholipase C in normal and neoplastic keratinocytes
Authors: Punnonen Kari , Denning Mitchell F. , Rhee Sue Goo , Yuspa Stuart H.
Published: February 2007

The induction of epidermal differentiation by Ca2+ in vitro is associated with enhanced activity of phosphatidylinositol‐specific phospholipase C (PLC). Neoplastic keratinocyte cell lines expressing a mutant c‐Ha‐ras gene and normal keratinocytes transformed to the neoplastic phenotype by transduction with the v‐Ha‐ras gene (v‐Ha‐ras keratinocytes) have elevated constitutive activity of PLC that increases further in response to Ca2+, but the cells do not differentiate normally. PLC‐γ1 (145 kDa) is the major isoform detected by immuno‐blotting of extracts from control, v‐Ha‐ras, and neoplastic keratinocyte cell lines cultured in 0.05 mM Ca2+ medium. The amount of PLC‐γ1 protein was higher in neoplastic cell lines than in normal and v‐Ha‐ras keratinocytes that had similar PLC‐γ1 protein levels. Thus, higher PLC‐γ1 protein levels cannot account for the elevated constitutive activity PLC in v‐Ha‐ras keratinocytes. After induction of differentiation by Ca2+, the amount of PLC‐γ1 protein increased in all cell types, and PLC‐δ1 (85 kDa), barely detectable in 0.05 mM Ca2+, increased. PLC‐β1 was not detected at any Ca2+ concentration. PLC‐γ1 and PLC‐δ1 mRNA did not increase after elevation of extracellular Ca2+, suggesting that posttranscriptional mechanisms can regulate PLC‐γ1 and PLC‐δ1 protein levels in normal and neoplastic keratinocytes. Activation of protein kinase C by treatment with 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) inhibited the stimulation of inositol phosphate (InsP) formation by Ca2+ but did not alter basal InsP levels in normal keratinocytes. In contrast, TPA treatment reduced both Ca2+‐stimulated and basal InsP formation in neoplastic cells lines and v‐Ha‐ras keratinocytes. In both normal and v‐Ha‐ras keratinocytes labeled with [32P]orthophosphate, antibodies against PLC‐γ1 immunoprecipitated a complex of 32 P‐labeled proteins. The relative labeling of the PLC‐γ1 band was greater in normal than in v‐Ha‐ras keratinocytes. Furthermore, treatment with TPA specifically increased the relative phosphorylation of PLC‐γ1 in v‐Ha‐ras keratinocytes but not in normal keratinocytes. These results suggest that the negative regulation of constitutive activity of PLC by protein kinase C differs in normal and neoplastic keratinocytes and that this could be the mechanism of increased PLC activity produced by an oncogenic ras gene in keratinocytes. © 1994 Wiley‐Liss, Inc.
Use of representational difference analysis for the identification of mdm2 oncogene amplification in diethylstilbestrol‐induced murine uterine adenocarcinomas
Authors: Risinger John I. , Terry Lori A. , Boyd Jeff
Published: February 2007

Exposure in utero to the synthetic estrogen diethylstilbestrol (DES) is associated with the subsequent development of reproductive‐tract malignancies in female offspring. To search for the genetic targets of DES, representational difference analysis was used to compare genomic DNA from DES‐associated mouse uterine adenocarcinoma cells with genomic DNA from normal CD‐1 mouse tissue. Several difference clones were obtained, all of which recognized rearranged and amplified sequences in tumor compared with normal DNA. One of these difference fragments mapped to a region of mouse chromosome 10 that includes the mdm2 oncogene. Amplification and overexpression of mdm2 was found in all three early‐passage cell lines established from independent DES‐associated cancers. These findings demonstrate the potential power of representational difference analysis in cancer research and suggest a genetic mechanism for DES‐induced carcinogenesis. ©1994 Wiley‐Liss, Inc.
Differences in the expression of connexin genes in rat hepatomas in vivo and in vitro
Authors: Neveu Mark J. , Sattler Carol A. , Sattler Gerald L. , Hully Jim R. , Hertzberg Elliot L. , Paul David L. , Nicholson Bruce J. , Pitot Henry C.
Published: February 2007

Gap‐junctional intercellular communication (GJIC) in normal rat liver cells involves at least three different connexins (Cxs)—Cx32, Cx26, Cx43—depending on the cell type, position in the lobule, or both. Whereas rat hepatocyte primary cultures expressed Cx32 and Cx26 as observed in vivo, cell lines derived from normal rat liver (WB‐F344, Clone 9, RLEC, and BRL) expressed Cx43 and to a lesser extent Cx26. Hepatoma cells propagated in vitro were either deficient in GJIC and Cx expression (7777, 8994, H4IIE‐C3) or communicated via gap junctions composed of Cx43 protein (N1S1‐67, 9618A). Analysis of neoplasms that resulted from injection of hepatoma cells into rat femoral muscle showed differences in Cx expression when compared with cells grown in vitro. Whereas hepatoma cells 7777 and H4IIE‐C3 failed to express Cx mRNAs in culture, these cells transplanted in vivo expressed levels of Cx32 mRNA comparable to those in normal liver. However, detectable Cx32 immunostaining was observed in less than 5% of the neoplastic cells in vivo. These results indicate that Cx32 protein was posttranscriptionally downregulated in 7777 and H4IIE‐C3 tumor cells. Unexpectedly, 9618A cells expressed Cx43 mRNA and protein in cell culture but expressed Cx32 mRNA in vivo. In contrast, N1S1 transplants continued to express Cx43 mRNA and protein in vivo. Unlike the punctate Cx43 staining observed in suspension cultures of N1S1 cells, diffuse intracellular Cx43 staining was observed in N1S1‐derived neoplasms in vivo, although the electrophoretic pattern of Cx43 isolated from N1S1 tumors grown in vivo (43 kDa) was different from that observed in suspension cell cultures (43 and 45 kDa). Thus, the findings reported here demonstrate that Cx expression in hepatoma cells depends on the environment, whether in vivo or in vitro, in which the cells are propagated.©1994 Wiley‐Liss, Inc.
Augmented expression of cytokines in mouse epidermal tumor cells and its possible involvement in the induction of hematopoietic alterations
Authors: Bauluz Cristina , Larcher Fernando , Ballestin Claudio , Grande Teresa , Jorcano José L.
Published: February 2007

Mice with skin tumors induced either by 7,12‐dimethylbenz[a]anthracene complete carcinogenesis or subcutaneous injection of a carcinogenic keratinocyte cell line showed moderate to severe splenomegaly as a result of an increase in splenic granulocyte‐macrophage and erythroid (erythroid burst‐forming unit) progenitors. To test whether the observed alterations involve the release of soluble factors by the epidermal component of skin tumors, we used an in vitro approach. A series of mouse keratinocyte cell lines resembling progressive stages of skin carcinogenesis and carrying either normal or activated Ha‐ras genes were assayed for their ability to produce the factors required for colony growth of hematopoietic‐committed progenitors. Only the conditioned media of keratinocytes harboring activated Ha‐ras genes were able to support the growth of granulocyte‐macrophage colony‐forming units. In addition, preincubation of normal bone‐marrow cells with conditioned media from the transformed epidermal cell lines stimulated in vitro amplification of the hematopoietic granulocyte‐macrophage progenitor compartment. To identify the possible factors responsible for the activities detected in the keratinocyte‐conditioned media, we performed northern blot analysis using the cytokine probes granulocyte colony‐stimulating factor, macrophage colony‐stimulating factor, granulocyte‐macrophage colony‐stimulating factor, stem cell factor, interleukin‐1α, interleukin‐3, and tumor necrosis factor‐α. The cell lines expressed different cytokine mRNA combinations that positively correlated with the colony‐stimulating activity detected in the corresponding conditioned medium. These results suggest that transformed epidermal tumor cells in vivo may alter normal hematopoiesis as a consequence of the production of cytokines that act in autocrine or paracrine loops probably related to tumor growth. ©1994 Wiley‐Liss, Inc.
Brief communications oncogene amplification per se: An independent prognostic factor in human breast cancer
Authors: Champème Marie‐Hélène , Biéche Ivan , Hacéne Kamel , Lidereau Rosette
Published: February 2007

As the prognostic significance of the three most frequently amplified oncogenes in breast cancer (c‐myc, int‐2IFGF3, and c‐erbB‐2lnew) is still unclear, and as the amplification of these genes appears to be mutually exclusive, we investigated the prognostic significance of oncogene amplification per se by multivariate analysis in a group of 112 primary human breast cancer cases. Amplification of at least one gene (c‐myc, int‐2lFGF3, or c‐erbB‐2Ineu), progesterone receptor status, and pathological tumor size were the only independent variables predictive of metastasis‐free survival. Moreover, We constructed prognostic profilles by computing risks associated with the three parameters predictive of poor survival and disciminated high‐,moderate‐, and low‐risk categories. ©1994 Wiley‐Liss, Inc.
Reduced expression of phospholipase c‐δ, a signal‐transducing enzyme, in rat colon neoplasms induced by methylazoxymethanol acetate
Authors: Yoshimi Naoki , Wang Aijin , Makita Hiroki , Suzui Masumi , Mori Hideki , Okano Yukio , Banno Yoshiko , Nozawa Yoshinori
Published: February 2007

Phospholipase C (PLC), Which hydrolyzes phosphoinositides, has been implicated as a key enzyme in signal transduction. We examined the expression of an isozyme of PLC, PLC,‐δ, in rat colon neoplasms induced by methylazoxymethanol (MAM) acetate. Large‐bowel neoplasms were observed in five of 10 rats given MAM acetate (25 mg/kg body weight, by interperitoneal injection at 6 and 7 wk of age) 40 wk after treatment. Expression of PLC‐δ in the neoplasms was not detected by northern blot analysis, and a low level of expression was detected by immunoblot analysis, although PLC‐δ expression was apparent in the non‐neoplastic colon mucosae of MAM acetate‐treated rats as well as in the colon mucosae of control rats. Furthermore, analysis by reverse transcriptase‐polymerase chain reaction revealed that the ratio of the expression of PLC‐δ to that of β‐action in the neoplasms was significantly lower than the ratios in the non‐neoplastic colon mucosae of carcinogen‐treated and control rats (P<0.01) However, the ornithine decarboxylase (ODC) activity in the neoplsms was significantly greater than that of the non‐neoplastic and control mucosae (P<0.001). The differences in the levels of PLC‐δ expression in neoplastic and non‐neoplastic tissues and the inverse correlation of PLC‐δ expression with ODC activity may suggest that PLC‐δ has little effect on the PLC‐mediated mitogenic signaling system, at least in MAM acetate‐induced colon neoplasms in rats.
DNA recombination induced by aflatoxin B1 activated by cytochrome P450 1A enzymes
Authors: Sengstag Christian , Würgler Friedrich E.
Published: February 2007

Mutations in tumor suppressor genes are intricately associated with the etiology of neoplasia. Often, such mutations are followed by the second, functional alleles of tumor suppressor genes, a phenomenon known as loss of heterozygosity. Loss of heterozygosity may occur by different molecular mechanisms, including mitotic recombination, and it is conceivable that these molecular events are influenced by endogenous as well as exogenous factors. To test whether mitotic recombination is induced by certain carcinogens, we genetically engineered a Saccharomyces cerevisiae tester strain so that it metabolized two important classes of carcinogens, polycyclic aromatic hydrocarbons and heterocyclic arylamines. This was accomplished by expressing human cDNA's coding for the cytochrome P450 (CYP) enzymes CYP1A1 or CYP1A2 in combination with NADPH‐CYP oxidoreductase in a strain heterozygous for two mutations in the trp5 gene. Microsomes isolated form the transformed yeast strains activated various xenobiotics to powerful mutagens that were detected in the were detected in the Ames test. Of these, the mycotoxin aflatoxin B1, when activated intracellularly in the strains containing either human CYP enzyme, significantly induced mitotic recombination. These results are discussed in light of possible mechanisms that are involved in aflatoxin B1‐mediated hepatocarcinogenesis. Similarly, benzo[a]pyrene‐trans‐7,8‐dihydrodiol and 3‐amino‐1‐methyl‐5H‐pyrido [4,3‐b]indole were activated to recombinagenic products, whereas benozo[a]pyrene and 2‐amino‐3,8‐dimethylimidazo[4,5‐f] quinoxaline were negative in this assay. Our results argue that the contstructed yeast strains may be a valuable tool for the investigation of drug‐induced mitotic recombination. ©1994 Wiley‐Liss, Inc.
Frequent P53 mutation in mouse tumors induced by repeated β‐irradiation
Authors: Ootsuyama Akira , Makino Hiroshi , Nagao Minako , Ochiai Atsushi , Yamauchi Yuko , Tanooka Hiroshi
Published: February 2007

On examination of cDNA of the p53 gene in 65 murine tumors of the skin and bone produced by repeated exposure to a suprathreshold dose of 1–8 Gy of 90Sr‐90Y β‐radiation per exposure, we found 20 cases of mutation: 11 minute deletions (loss of 1‐24bp), including two cases possibly due to aberrant splicing: three insertions of 4–8 bp; and six base‐pair substitutions, including four at CpG sites, three being identical changes at codon 122 (a p53 hot spot). All but one of these mutations were confined to the central region of the p53 gene. From frameshifts created by deletions and insertions, the minimum size of the mutant p53 proteins found in these tumors was estimated to be 55% of the intact size. Cells of 17 of 19 tested tumors with p53 mutation were positive for immunostaining of p53 proteins accumulated in the nuclei and so were clearly different from nonneoplastic cells. Ha‐ras mutation was absent in these tumors, indicating that repeated β‐irradiation created a cell‐growth stimulating effect similar to that of ras mutation.
Ha‐ras p21‐GTP levels remain constant during primary keratinocyte differentiation
Authors: Betz Natalie A. , Pelling Jill C.
Published: February 2007

Several lines of evidence that indicate that mutation of the Ha‐ras oncogene is the initiating event in mouse skin carcinogenesis. Keratinocytes known to possess a mutated Ha‐ras have been shown to be resistant to differentiation. Thus, overstimulation of the Ha‐ras signaling pathway appears to block normal keratinocyte differentiation, and we hypothesized that for normal keratinocytes to terminally differentiate, the Ha‐ras signaling cascade must be turned off. In the present studies, we measured the level and activity state of Ha‐ras p21 protein in cultured keratinocytes undergoing calcium‐induced differentiation. We have employed Western blot analysis to demonstrate that Ha‐ras p21 protein levels remain constant during primary newborn and adult keratinocyte differentiation. The overall level of Ha‐ras p21 was higher in immortalized, benign, and malignant mouse keratinocyte cell lines than in normal keratinocytes but did not change within each cell type when subjected to differentiating conditions. The percentage of Ha‐ras p21 protein in its active, GTP‐bound form also remained unchanged during primary adult keratinocyte differentiation and in immortalized, benign, and malignant keratinocytes subjected to differentiating conditions. Our results indicate that terminal differentiation of primary adult mouse keratinocytes occurred in the presence of constant levels of Ha‐ras p21‐GTP, suggesting that the Ha‐ras signaling pathway may be blocked at a point distal to a step involving the Ha‐ras p21 protein itself. © 1995 Wiley‐Liss Inc.
Expression of the liver‐enriched transcription factors C/EBPα, C/EBPβ, HNF‐1, and HNF‐4 in preneoplastic nodules and hepatocellular carcinoma in rat liver
Authors: Flodby Per , Liao De‐Zhong , Blanck Agneta , Xanthopoulos Kleanthis G. H. , Hällström Inger Porsch
Published: February 2007

The expression patterns of the liver‐enriched transcription factors CCAAT/enhancer‐binding protein (C/EBP) α and β and hepatocyte nuclear factor (HNF)‐1 and HNF‐4 were studied in liver nodules and hepatocellular carcinomas from male rats treated according to the resistant hepatocyte (RH) model. C/EBPα expression was lower at the transcriptional, mRNA, and protein levels in persistent nodules than in the respective surrounding livers. Expression was further decreased in the tumors. Transcriptional downregulation of C/EBPα gene expression was observed already in very early nodules, isolated 3 wk after partial hepatectomy in the RH model. However, no detectable changes were observed in preneoplastic nodules in the transcription or in steady‐state mRNA levels of C/EBPβ, HNF‐1, and HNF‐4. A slight decrease in C/EBPβ protein and a more pronounced attenuation of HNF‐1 and HNF‐4 levels was observed in nodules, being 67%, 37%, and 46% of the levels in the corresponding surrounding livers, respectively. In conclusion, differential regulation of several transcription factors that are associated with the maintenance of the differentiated state of the hepatocytes was observed in preneoplastic and neoplastic liver lesions. This could have an impact on the regulation of a wide array of genes during liver carcinogenesis. Furthermore, the attenuation of C/EBPα expression, regarded as a negative growth regulator, could contribute to the proliferative advantage of nodules during liver carcinogenesis. © 1995 Wiley‐Liss Inc.
ogt alkyltransferase enhances dibromoalkane mutagenicity in excision repair–deficient escherichia coli K‐12
Authors: Abril Nieves , Luque‐Romero Francisco L. , Prieto‐Alamo María‐josé , Margison Geoffrey P. , Pueyo Carmen
Published: February 2007

We examined the role of the O6‐alkylguanine‐DNA alkyltransferase encoded by ogt gene in the sensitivity of Escherichia coli to the mutagenic effects of the dibromoalkanes, dibromoethane and dibromomethane, by comparing responses in ogt− bacteria to those in their isogenic ogt+ parental counterparts. The effects of the uvrABC excision‐repair system, the adaptive response, mucAB and umuDC mutagenic processing, and glutathione bioactivation on the differential responses of ogt− and ogt+ bacteria were also studied. Mutation induction was monitored by measuring the frequency of forward mutations to L‐arabinose resistance. Induced mutations occurred only in excision repair–defective strains and were totally (with dibromomethane) or substantially (with dibromoethane) dependent on the alkyltransferase (ATase) encoded by the ogt gene. An increased mutagenic response to both dibromoalkanes was also seen in ogt− bacteria that overexpressed the ogt protein from a multicopy plasmid, indicating that the differences in mutability between ogt+ and ogt− bacteria were not dependent on the ogt− null allele carried by the defective strain. The ATase encoded by the constitutive ogt gene was more effective in promoting dibromoalkane mutagenicity than the ada ATase induced by exposure to low doses of a methylating agent. The mutagenicity promoted by the ogt ATase was dependent on both glutathione bioactivation and SOS mutagenic processing. To our knowledge, this paper presents for the first time evidence that DNA ATases, in particular the ATase encoded by the ogt gene, can increase the mutagenic effects of a DNA‐damaging agent. The mechanism of this effect has yet to be established. © 1995 Wiley‐Liss Inc.
p53 status in spontaneous and dimethylnitrosamine—induced renal cell tumors from rats
Authors: Horesovsky Greg , Recio Leslie , Everitt Jeff , Goldsworthy Thomas , Wolf Douglas C. , Walker Cheryl
Published: February 2007

Rats carrying the Eker tumor–susceptibility mutation (Eker rats) are predisposed to developing renal cell carcinoma. Rats heterozygous for the Eker mutation develop spontaneous multiple bilateral renal cell tumors by the age of 1 yr. In a previous study, Eker‐mutation carrier and noncarrier rats were exposed to the renal carcinogen dimethylnitrosamine (DMN), and male rats carrying the Eker mutation exhibited a 70‐fold increase in the induction of renal adenomas and carcinomas when compared with noncarrier rats. In this study, spontaneous and DMN‐induced rat renal cell tumors (adenomas and carcinomas) were analyzed for mutations of the p53 gene by direct sequencing of cDNA polymerase chain reaction products. There were no mutations in p53 cDNA derived from renal tumors from six untreated rats. Mutations were found in one of 15 of the DMN‐induced tumors: a transition at codon 140, CCT → CTT, in a renal adenoma. Additionally, seven cell lines derived from spontaneous renal cell tumors did not contain mutations in p53. The low frequency of p53 mutations (one of 21 renal cell tumors and none of seven cell lines derived from renal cell tumors) indicates that the development of both spontaneous and carcinogen‐induced renal tumors involved a non–p53‐dependent pathway. As p53 is infrequently mutated in human renal cell carcinomas and in rat renal mesenchymal tumors, it is likely that a tumor suppressor gene or genes other than p53 are involved in the development of renal cancer. © 1995 Wiley‐Liss Inc.
N‐methylnitrosourea—lnduced Ki‐ras codon 12 mutations: Early events in mouse thymic lymphomas
Authors: Newcomb Elizabeth W. , Bayona William , Pisharody Sobha
Published: February 2007

N‐Methylnitrosourea (NMU)—induced codon 12 Ki‐ras mutations were analyzed in premalignant thymic lymphomas from C57BL/6J mice by using a selective polymerase chain reaction amplification strategy. The frequency of codon 12 Ki‐ras mutations was 67% (16 of 24) in NMU‐treated animals with premalignant stage I disease. Previously, animals with different stages of disease had been analyzed for cytogenetic changes and for mutations in the p53 tumor suppressor gene. The genetic changes observed were early‐activating codon 12 G35→A transition mutations of the Ki‐ras gene, followed closely by trisomy 15 and infrequent mutation of the p53 gene late in tumor development. The consistent and early detection of Ki‐ras mutations in NMU‐treated animals but not in untreated controls suggests that the mutations result from direct carcinogen exposure. Alternate pathways of NMU‐induced thymic lymphomagenesis were implicated. One pathway involved putative NMU‐induced mutations in other, non‐ras oncogenes that cooperate with trisomy 15 to produce similar T‐cell tumors. The frequency of p53 gene mutations in human and murine T‐cell tumors is similar but low. © 1995 Wiley‐Liss, Inc.
Analysis of loss of heterozygosity in murine hepatocellular tumors
Authors: Manenti Giacomo , De Gregorio Laura , Gariboldi Manuela , Dragani Tommaso A. , Pierotti Marco A.
Published: February 2007

Because allelotype analysis of tumors has been important in the identification of new tumor suppressor genes, here we studied loss of heterozygosity (LOH) in a well‐defined animal experimental system. We analyzed spontaneous liver tumors from C3HHc × C57BL/61 (B6C3F1) mice and urethane‐induced hepatocellular tumors from (C3H/He × Mus spretus) × C57BL/6JBy (HSB) interspecific mice. A total of 95 different genetic markers were tested: 13 in 24 B6C3F1 tumors, 76 in 58 HSB tumors, and six in both groups. Minisatellite fingerprinting analysis detected one case of LOH and less than 1% genomic rearrangements in polymorphic and nonpolymorphic bands, respectively. There were no changes at hepatocellular susceptibility loci or at markers homologous to loci frequently lost in human hepatocellular carcinomas. Therefore, our results suggest that LOH and genomic rearrangements are uncommon in mouse hepatocellular tumors. © 1995 Wiley‐Liss, Inc.
Suppression of the tumorigenic phenotype of a rat liver epithelial tumor cell line by the p11.2–p12 region of human chromosome 11
Authors: Coleman William B. , McCullough Karen D. , Esch Gwyn L. , Civalier Chris J. , Livanos Elizabeth , Weissman Bernard E. , Grisham Joe W. , Smith Gary J.
Published: February 2007

Comparative chromosomal mapping studies and investigations of tumor‐associated chromosomal abnormalities suggest that the development of hepatic tumors in humans and rats may share a common molecular mechanism that involves inactivation of the same tumor suppressor genes or common genetic loci. We investigated the potential of human chromosomes 2 and 11 to suppress the tumorigenic phenotype of rat liver epithelial tumor cell lines. These tumor cell lines (GN6TF and GP7TB) display elevated saturation densities in culture, efficiently form colonies in soft agar, and produce subcutaneous tumors in 100% of syngeneic rat hosts with short latency periods. Introduction of human chromosome 11 by microcell fusion markedly altered the tumorigenicity and the transformed phenotype of GN6TF cells. In contrast, the tumorigenic potential and phenotype of GP7TB cells was unaffected by the introduction of human chromosome 11, indicating that not all rat liver tumor cell lines can be suppressed by loci carried on this chromosome. Introduction of human chromosome 2 had little or no effect on the tumorigenicity or cellular phenotype of either tumor cell line, suggesting the involvement of chromosome 11–specific loci in the suppression of the GN6TF tumor cell line. The GN6TF‐11neo microcell hybrid cell lines displayed significantly reduced saturation densities in monolayer cultures, and their ability to grow in soft agar was completely inhibited. Although GN6TF‐11neo cells ultimately formed tumors in 80–100% of syngeneic rat hosts, the latency period for tumor formation was much longer. Molecular characterization of GN6TF‐11neo microcell hybrid cell lines indicated that some of the clonal lines had spontaneously lost significant portions of the introduced human chromosome, partially delineating the chromosomal location of the putative tumor suppressor locus to the region between the centromere and 11p12. Molecular examination of microcell hybrid–derived tumor cell lines further defined the minimal portion of human chromosome 11 capable of tumor suppression in this model system to the region 11p11.2‐p12. © 1995 Wiley‐Liss, Inc.
Cellular immortality: A late event in the progression of human squamous cell carcinoma of the head and neck associated with p53 alteration and a high frequency of allele loss
Authors: Edington Kirsten G. , Loughran Oonagh P. , Berry Isabella J. , Parkinson E. Kenneth
Published: February 2007

Many human tumors contain variant cells that, unlike their normal counterparts, possess indefinite proliferative potential in vitro. However, little is known of the relevance of these immortal cells to human carcinomas in vivo. To investigate immortality in a human tumor system, we established cultures from different stages of head and neck squamous carcinoma (SCC‐HN). All the neoplastic cultures were transformed because they showed very low cornification in surface or suspension culture and were partially or completely resistant to suspension‐induced death. Immortal variants were not detected in premalignant erythroplakia cultures, but their frequency increased with tumor progression, indicating that immortality is a late event in carcinogenesis. Some late‐stage carcinomas still produced senescent cultures, but, significantly, all recurrent tumors were immortal. Immortal but not senescent carcinoma cultures were associated with p53 dysfunction and a high frequency of allele loss, indicative of tumor suppressor gene inactivation. These results show that there are at least two classes of human SCC‐HN that are phenotypically and genotypically distinct and that the pathological stage of a given tumor is not necessarily indicative of the kind of cells it contains. © 1995 Wiley‐Liss, Inc.
The impact of glutathione s‐transferase M1 and cytochrome P450 1A1 genotypes on white‐blood‐cell polycyclic aromatic hydrocarbon‐dna adduct levels in humans
Authors: Rothman Nathaniel , Shields Peter G. , Poirier Miriam C. , Harrington Anita M. , Ford D. Patrick , Strickland Paul T.
Published: February 2007

Carcinogenic polycyclic aromatic hydrocarbons(a) form DNA adducts via a complex metabolic activation pathway that includes cytochrome P450(a) 1A1, whereas intermediate metabolites can be detoxified by conjugation through pathways including glutathione s‐transferase M1 (GSTM1). PAH‐DNA adducts can be measured in peripheral white blood cells (a) and should reflect the net effect of competing activation and detoxification pathways and ONA repair as well as exposure. We have previously shown that WBC PAH‐DNA adducts measured by an enzyme‐linked immunosorbent assay (a) were associated with recent, frequent consumption of charbroiled food among 47 nonsmoking wildland fire‐fighters who provided two blood samples 8 wk apart. In the investigation reported here, which was performed in the same population, we measured the association between the GSTM1 null genotype, which results in loss of enzyme activity, and PAH‐DNA adduct levels, hypothesizing that subjects with this genotype would have higher levels of DNA adducts because of their decreased ability to detoxify PAH metabolites. However, PAH‐DNA adduct levels were nonsignificantly lower in subjects with the GSTM1 null genotype (n = 28) compared with other subjects (n=19) (median 0.04 fmol/μg DNA vs 0.07 fmol/μg DNA, respectively, P = 0.45, Wilcoxon rank‐sum test). Adduct levels were also lower in the nine subjects heterozygous or homozygous for the CYP1A1 exon 7 polymorphism (which codes for a valine rather than isoleucine and is thought to be associated with greater CYP1A1 activity) compared with the 38 wild‐type subjects (P = 0.12). In the entire group, there was a positive association between consuming charbroiled food and PAH‐DNA adduct formation (r = 0.24, P = 0.02, Spearman rank‐order correlation). This association was weaker in the subgroup of subjects with the GSTM1 null genotype (r = 0.03, P = 0.84) and stronger among the remaining subjects (r = 0.57, P = 0.0002). These results suggest that the GSTM1 null genotype and CYPT1A1 exon 7 polymorphism are not associated with increased susceptibility for PAH‐DNA adduct formation in peripheral WBCs measured by ELISA in nonsmoking populations.© 1995 Wiley‐Liss, Inc
Promoter demethylation in MMTV/N‐rasN transgenic mice required for transgene expression and tumorigenesis
Authors: Mangues Ramon , Schwartz Simo , Seidman Irving , Pellicer Angel
Published: February 2007

We studied demethylation within the transgene promoter in transgenic mice carrying the N‐ras proto‐onco‐gene driven by the mouse mammary tumor long terminal repeat (MMTV/N‐rasN) and the relationship of demethy!ation to transgene overexpression and tumorigenesis. Demethylation at Fspl or Clal sites correlated with age of the animal and transgene expression in nontumorous mammary gland. Demethylation preceded expression in this tissue. In lymphomas and mammary tumors, the promoter Fspl and Clal sites were significantly more demethylated than in nontumorous control tissues. The Aval, Cfol, and Hpall sites were also found to be undermethylated in older animals and showed differences between tumor and control tissues. Two additional sites (Eagl and Narl) remained fully methylated in all tissues. In contrast with normal tissue, demethylation at the Fspl and Clal sites and expression were not correlated in tumor tissue. An increase in expression in normal tissue initially occurred and was correlated with the level of promoter demethylation; this increase was followed by a further increment in transgene expression when tumors developed. Thus, promoter demethylation leading to transgene overexpression was associated with long‐latency tumorigenesis in MMTV/N‐rasN transgenic mice. Demethylation of proto‐oncogene promoters may therefore be a mechanism of carcinogenesis that requires further investigation in human tumors.
12‐lipoxygenase isoenzymes in mouse skin tumor development
Authors: Krieg Peter , Kinzig Andreas , Ress‐Löschke Marion , Vogel Sonja , Vanlandingham Ben , Stephan Michael , Lehmann Wolf‐Dieter , Marks Friedrich , Fürstenberger Gerhard
Published: February 2007

12‐lipoxygenase‐catalyzed arachidonic acid metabolism in normal and neoplastic mouse epidermis was assessed by cDNA cloning of the epidermal 12‐lipoxygenases and by studying their expression patterns, enzyme activities, and product levels. Papillomas and squamous cell carcinomas induced by the initiation/promotion protocol contained 50‐to 60‐fold more 12‐hydroxy‐5,8,10,14‐eicosatetraenoic acid (a) than normal epidermis. The ratio of S to R enantiomers was 9:1. This indicates that most of this eicosanoid was of enzymatic origin. Accordingly, cell‐free preparations of the tumors exhibited about fivefold elevated 12‐lipoxygenase activities. A papilloma‐derived cDNA library was screened with human platelet‐type 12‐lipoxygenase cDNA probes. Two cDNA clones encoding the platelet‐type and the leukocyte‐type isoforms of murine 12‐lipoxygenase were isolated, demonstrating the coexpression of the isoenzymes in the same tissue and species. When expressed in COS‐7 cells, the recombinant enzymes showed the characteristic substrate selectivity and product profile, with the leukocyte‐type enzyme metabolizing linoleic and arachidonic acid to 13‐hydroxy‐9,11‐octadecadienoic acid and to 12‐ and 15‐HETE, respectively, and the platelet‐type enzyme oxygenating exclusively arachidonic acid to 12‐HETE. In epidermis in vivo and in keratinocytes in culture, only the platelet‐type 12‐lipoxygenase (mRNA and protein) was detectable. In mouse epidermis both isoenzymes were induced transiently by phorbol esters. Most tumors showed constitutive overexpression of platelet‐type mRNA, whereas leukocyte‐type specific transcripts were detectable only in a few tumors. These data suggest that the platelet‐type enzyme is the 12‐lipoxygenase isoform of keratinocytes that is responsible for the generation of most of the 12‐HETE found in neoplastic epidermis. ©1995 Wiley‐Liss, Inc.