Showing papers in "Molecular Carcinogenesis in 1995"
866 citations
TL;DR: The high incidence of early‐onset lymphomas in the p53‐nullizygous mice makes these animals a good lymphoma model, whereas the heterozygous mice may be a useful model for Li‐Fraumeni syndrome, a human inherited cancer predisposition.
Abstract: Mice with disrupted germline p53 alleles have been engineered by us and others and have been shown to have enhanced susceptibility to spontaneous tumors of various types. We monitored a large number of p53-deficient mice (p53+/- and p53-/-) and their wild-type littermates (p53+/+) of two different genetic backgrounds (129/Sv and mixed C57BL/6 x 129/Sv) up to 2 yr of age. p53+/- and p53-/- 129/Sv mice show accelerated tumorigenesis rates compared with their p53-deficient counterparts of mixed C57BL/6 x 129/Sv genetic background. The tumor spectra of the two strains of mice are similar except that almost half of 129/Sv p53-/- males develop malignant teratomas, whereas these tumors are rarely observed in C57BL/6 x 129/Sv mice and never in 129/Sv p53+/- males. In the study reported here, we further characterized the lymphomas that arose in the p53-nullizygous mice and found that over three-quarters of the lymphomas were of thymic origin and contained primarily immature (CD4+/CD8+) T-cells, whereas the remainder originated in the spleen and peripheral lymph nodes and were of B-cell type. The high incidence of early-onset lymphomas in the nullizygous mice makes these animals a good lymphoma model, whereas the heterozygous mice may be a useful model for Li-Fraumeni syndrome, a human inherited cancer predisposition.
268 citations
TL;DR: Data suggest that PGHS‐2 has a critical role in skin carcinogenesis, and the anti–tumor‐promoting effect of the PGHS inhibitor indomethacin is specifically reversed by prostaglandin F2α, indicating that this prostag landin type has a significant role in tumor development.
Abstract: An anti-tumor-promoting effect of indomethacin and related nonsteroidal anti-inflammatory drugs (NSAIDs) as well as the ability of the tumor promoter 12-O-tetradecanoylphorbol-13- acetate (TPA) to increase the level of prostaglandins in murine keratinocytes and mouse epidermis in vivo has been repeatedly documented. Here, the expression of prostaglandin H synthase (PGHS) isozymes, which are major targets of NSAIDs, was investigated in different stages of tumor development in mouse skin. Mouse epidermis in vivo constitutively expressed PGHS-1. PGHS-1 steady-state levels remained unchanged upon induction of acute or chronic epidermal hyperplasia by TPA and in papillomas and carcinomas generated by the initiation-promotion procedure, with 7,12-dimethylbenz[a]anthracene as initiator and TPA as promoter. Thus, the elevated prostaglandin level in the acute hyperplastic epidermis was very likely due to PGHS-2 induction. Repeated applications of TPA resulted in stationary hyperplasia and downregulation of PGHS-2 expression and prostaglandin levels, suggesting that the epidermis had adapted to the TPA stimulus. In papillomas and carcinomas, however, constitutive overexpression of PGHS-2 was found, with a large amount of prostaglandin E2 and prostaglandin F2 alpha. Keratinocyte cell lines corresponding to different stages of tumor development also constitutively over-expressed PGHS-2. Considered with inhibitor studies, these data suggest that PGHS-2 has a critical role in skin carcinogenesis. The anti-tumor-promoting effect of the PGHS inhibitor indomethacin is specifically reversed by prostaglandin F2 alpha, indicating that this prostaglandin type has a significant role in tumor development.
151 citations
TL;DR: The 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.
Abstract: 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.
108 citations
TL;DR: W wild‐type p53 gene transfer has dominant effects over mutant p53 in sensitizing tumor cells to therapy, which supports the potential of p 53 gene therapy to enhance the efficacy of traditional therapy.
Abstract: It is known that transfer of the wild-type p53 gene into p53-negative cells from transgenic mice increases their sensitivity to drug and radiation-induced apoptosis. However, unlike many human tumors, these transgenic cells do not express mutant p53, and it is not known from these earlier studies whether wild-type p53 dominates the effects of mutant p53 with respect to drug and radiation sensitivity. We addressed this question in glioblastoma, a disease characterized by an unusually high level of intrinsic resistance to therapy and poor prognosis: mean survival time from diagnosis is only about 1 yr. We introduced the gene for wild-type p53 into human T98G glioblastoma cells, which express endogenous mutant p53 but not wild-type p53. Stable transfectants that co-expressed mutant and wild-type p53 had enhanced sensitivity to cisplatin and gamma radiation, compared with parental cells, control vector-transduced cells, and transduced cells that had lost expression of wild-type p53. Transient wild-type p53 expression after high-efficiency gene transfer by a p53 adenovirus also sensitized the cells to cisplatin and correlated with the induction of apoptosis. The sensitization effect was also observed in p53 adenovirus-infected H23 small cell lung carcinoma cells, which express endogenous mutant p53. Therefore, wild-type p53 gene transfer has dominant effects over mutant p53 in sensitizing tumor cells to therapy, which supports the potential of p53 gene therapy to enhance the efficacy of traditional therapy. © 1995 Wiley- Liss, Inc.
105 citations
TL;DR: It is demonstrated that TGC tumors fail to be selected for p53 mutations but nonetheless frequently expressed high levels of wild‐type p53 protein in the cell nucleus, which produces the excellent response to radiation and chemotherapy of these tumors, which generally have a good prognosis.
Abstract: Mutations in the p53 gene are common in many cancers. They have been documented to occur in about 55% of all cancers of 51 different cell and tissue types. These mutations are accompanied by overexpression of the p53 protein in the nucleus of the cell, and this protein has lost its tumor suppressor function. In this study, 25 testicular germ-cell (TGC) tumors were tested for p53 mutations and the level of p53 protein expression. While 67% of the tumors overproduced the p53 protein in the nucleus of 10-60% of their cells, in all cases the DNA sequence of exons 4-9 of the p53 gene was wild type. In this tumor type, there was apparently no selection pressure for p53 mutations. The mdm-2 gene resides on chromosome 12 (12q13-q14), a chromosome often altered in TGC tumors. mdm-2 gene amplification (2.5- to 10-fold) was detected in three (12%) of these TGC tumors. These three tumors, and eight additional TGC tumors, overexpressed mdm-2 mRNA. There was a good correlation between overexpression of p53 protein and overexpression of mdm-2 mRNA (P = 0.01). This may well result from the fact that the level of mdm-2 mRNA is regulated by the p53 level. These studies demonstrate that TGC tumors fail to be selected for p53 mutations but nonetheless frequently expressed high levels of wild-type p53 protein in the cell nucleus. Perhpas this produces the excellent response to radiation and chemotherapy of these tumors, which generally have a good prognosis. Wild-type p53 may mediate apoptosis in these cells in response to the DNA damage. © 1995 Wiley-Liss Inc.
95 citations
TL;DR: The 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.
Abstract: 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.
79 citations
TL;DR: It is proposed that these transgenic mice will be a model for studying the process of multistage melanoma carcinogenesis and a system for evaluating potential chemopreventive agents.
Abstract: The tyrosinase promoter has been used to target expression of the mutated human T24 Ha-ras oncogene in pigment-producing cells of transgenic mice. Two independent founder mice carrying the transgene survived and showed the same distinct phenotype of mutated coat color, deeply pigmented skin with multiple nevi, and twirling behavior. The offspring of one of these founders were developed into a line that stably expressed the same phenotype. Histopathological analysis of the tissues revealed hyperpigmentation and/or melanocytic hyperplasia in the skin, eyes, inner ear, and meningeal membranes in the brain. Reverse transcriptase-polymerase chain reaction analysis revealed expression of the transgene in skin, brain, and spleen. We propose that these transgenic mice will be a model for studying the process of multistage melanoma carcinogenesis and a system for evaluating potential chemopreventive agents.
79 citations
TL;DR: The pattern and frequency of LOH at the predisposing locus in 77 primary tumors and cell lines were examined to gain an understanding of the role of Tsc2 allelic loss in the pathogenesis of Eker‐derived tumors and confirmed the inactivation of both alleles plays an important role in renal and uterine tumor development.
Abstract: Somatic events leading to the inactivation of tumor suppressor genes often involve chromosomal alterations that can be detected as loss of heterozygosity (LOH). In the Eker rat, spontaneous tumors of the kidney, uterus, and spleen develop as a result of a germline mutation of the tuberous sclerosis 2 (Tsc2) gene. We examined the pattern and frequency of LOH at the predisposing locus in 77 primary tumors and cell lines to gain an understanding of the role of Tsc2 allelic loss in the pathogenesis of Eker-derived tumors. Although most renal and uterine tumors (primary and cell lines) displayed LOH, splenic hemangiosarcomas did not. Although the presence of normal tissue may account for some of this difference, the possibility exists that an alternative mechanism, such as subtle mutation or gene dosage effects, may be involved during splenic tumorigenesis. Northern analysis confirmed that LOH resulted in loss of the wild-type transcripts for the Tsc2 gene. Thus, the inactivation of both alleles plays an important role in renal and uterine tumor development, in keeping with Knudson's two-hit hypothesis. In addition, renal tumors that retained the wild-type allele also did not express the normal transcript, suggesting that the remaining Tsc2 alleles had acquired subtle mutations resulting in loss of gene function.
73 citations
TL;DR: The 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.
Abstract: Carcinogenic polycyclic aromatic hydrocarbons (PAHs) form DNA adducts via a complex metabolic activation pathway that includes cytochrome P450 (CYP) 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 (WBCs) and should reflect the net effect of competing activation and detoxification pathways and DNA repair as well as exposure. We have previously shown that WBC PAH-DNA adducts measured by an enzyme-linked immunosorbent assay (ELISA) 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/microgram DNA vs 0.07 fmol/microgram 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 CYP1A1 exon 7 polymorphism are not associated with increased susceptibility for PAH-DNA adduct formation in peripheral WBCs measured by ELISA in nonsmoking populations.
61 citations
TL;DR: It is suggested that cofactors provide a template that is required for productive interaction of PKC and the inhibitor, and the significance of the proposed proximity effect of calphostin C action is discussed.
Abstract: Protein kinase C (PKC) undergoes specific inactivation by nanomolar concentrations of calphostin C. Both PKC-α (a Ca2+-dependent conventional isoform) and PKC-α (a Ca2+-independent novel isoform) are similarly inactivated by calphostin C (75–100 nM produced 50% inhibition), suggesting that inactivation requires a site common to both classes of PKC. We therefore performed studies to identify a critical region in the regulatory domain of PKC-α required for inactivation by calphostin C. A series of N-terminal–truncation mutants of bovine PKC-α expressed in Saccharomyces cerevisiae was tested with 500 nM calphostin C, a concentration sufficient to inactivate wild-type PKC-α by 80–90%. This concentration was as effective with mutant proteins containing deletions of up to 91 amino acid (aa) residues from the amino terminus (ND91), whereas a mutant protein truncated by 140 aa (ND 140) was inactivated by only 20%. These findings imply that the aa sequence 92–140 is a structural determinant of PKC-α inactivation by calphostin C. This sequence contains one of the phorbol ester-binding sites (aa 102–144), which is highly conserved among most PKC isoforms including PKC-α. In addition to aa 92–140, PKC-stimulating cofactors (phosphatidylserine, phorbol ester, and Ca2+) are required for inactivation by calphostin C even in the case of PKC mutants that do not require these cofactors for enzymatic activity. These results suggest that cofactors provide a template that is required for productive interaction of PKC and the inhibitor. The significance of the proposed proximity effect of calphostin C action is discussed. © 1995 Wiley-Liss Inc.
TL;DR: The observation of different mutant frequencies and spectra in treated and control mice demonstrates the utility of this approach in the study of mutagenesis in vivo, and is consistent with the types of DNA damage induced by DMBA.
Abstract: Transgenic mice carrying the bacterial lacl gene in a lambda shuttle vector were used to isolate and characterize background and 7,12-dimethylbenz[a]anthracene (DMBA)-induced mutations in skin Adult male mice were treated once topically with either DMBA or acetone or were left untreated Seven days later, DMBA treatment had significantly increased the mutant frequency in the skin (mean +/- SEM, 36 +/- 3 x 10(-5)) versus in vehicle-treated (64 +/- 12 x 10(-5)) and untreated mice (71 x 10 x 10(-5)) At least 10 mutants from each of three DMBA-treated and three untreated mice were selected for DNA sequence analysis In each case, the entire 1080-bp target gene was sequenced Base-pair substitutions predominated (86 of 96 mutations), although frameshift and deletion mutations were also detected Twelve percent of the mutants carried more than one mutation In controls, the mutations were predominantly GC-->AT transitions (26 of 42), and no AT-->TA transversions were recovered In contrast, in the DMBA-treated mice, AT-->TA transversions represented 42% of the mutations (23 of 54) and GC-->AT transitions accounted for only 11% The AT-->TA transversions occurred mostly at 5'-CA sites This class of mutation has been recovered frequently in ras genes from DMBA-treated mice and probably represents an early event in carcinogenesis (Nelson MA et al, Proc Natl Acad Sci USA 89:6398-6402, 1992) Our present results are consistent with the types of DNA damage induced by DMBA The observation of different mutant frequencies and spectra in treated and control mice demonstrates the utility of this approach in the study of mutagenesis in vivo
TL;DR: A role for GJIC in the establishment of contact inhibition of in vitro cell growth is supported and is correlated with altered cellular growth, tumor promotion, and neoplastic transformation.
Abstract: Many studies have correlated reductions in gap junctional intercellular communication (GJIC) with altered cellular growth, tumor promotion, and neoplastic transformation. To test directly whether reduced GJIC affects cellular growth, GJIC was inhibited in murine BALB/c 3T3 fibroblasts by treatment with a phosphorothioate-modified antisense oligonucleotide targeted against the connexin43 translation start codon, and in vitro cell growth was monitored. The cells were incubated with the oligonucleotide (0.1-0.5 microM) in liposomes in serumless culture medium for 16 h; washed and refed with serum-containing medium; and analyzed for dye-coupling, connexin43 protein and mRNA levels, and cell growth over the next 5 d. The antisense oligonucleotide inhibited dye-coupling and reduced connexin43 protein levels in a concentration-dependent manner but had no effect on connexin43 mRNA levels. Cell growth rate was not affected, but saturation density was increased approximately threefold by the oligonucleotide. These data support a role for GJIC in the establishment of contact inhibition of in vitro cell growth.
TL;DR: The expression patterns of liver-enriched transcription factors CCAAT/enhancer-binding protein (C/EBP) alpha and beta 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.
Abstract: The expression patterns of the liver-enriched transcription factors CCAAT/enhancer-binding protein (C/EBP) alpha and beta 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/EBP alpha 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/EBP alpha 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/EBP beta, HNF-1, and HNF-4. A slight decrease in C/EBP beta 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/EBP alpha expression, regarded as a negative growth regulator, could contribute to the proliferative advantage of nodules during liver carcinogenesis.
TL;DR: While ras mutations can be detected with increasing frequency in azoxymethane‐induced adenomas and carcinomas, they are reportedly absent in IQ‐induced colon tumors, thus, for IQ and related compounds additional factors (possibly increased cell proliferation) may be important in the later stages of colorectal tumorigenesis.
Abstract: Aberrant crypt foci (ACF) are putative peneoplastic lesions that develop after treatment of animals with colon carcinogens, including cooked-meat heterocyclic amines such as 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). Male F344 rats given IQ by gavage on alternating days for 2 wk (130 mg/kg body weight) and killed 12 wk after the final carcinogen dose had an average of 4.4 ACF/colon and an average of 3.2 crypts/focus. The DNA from these ACF was amplified by the polymerase chain reaction and analyzed by 3′-primer mismatch and direct sequencing methods for mutations in the Ki-ras proto-oncogene. Of the 37 IQ-induced ACF screened, three contained a GGT→GAT mutation in codon 12 and one contained a GGC→GCC mutation in codon 13. The approximately 11% frequency of mutation in IQ-induced ACF is within the range of previous ACF studies of azoxymethane, which reported a 7–37% incidence of Ki-ras mutaion. These findings suggest that for both compounds, ras mutations occur during early stages of colorectal tumorigenesis. However, while ras mutations can be detected with increasing frequency in azoxymethane-induced adenomas and carcinomas, they are reportedly absent in IQ-induced colon tumors. Thus, for IQ and related compounds additional factors (possibly increased cell proliferation) may be important in the later stages of colorectal tumorigenesis. © 1995 Wiley-Liss Inc.
TL;DR: Germline p53 deficiency does not enhance the rate of development of diethylnitrosamine‐induced hepatocellular adenoma or carcinoma but may instead favor development of hepatic hemangiosarcoma.
Abstract: To determine whether a constitutive p53 deficiency would enhance the rate of development of chemically induced hepatocellular carcinoma, we treated groups of wild-type, p53-heterozygous (+/-), and null (-/-) male mice with a single dose of diethylnitrosamine at 12 d of age. Although the null mice had to be killed very early, at 15 wk of age because of the development of nonliver tumors, hemangosarcoma of the liver had already developed in two of seven mice. More detailed analysis of the wild-type and heterozygous mice showed no difference in the number, size, or growth rate of early microscopic lesions or in the number or apparent malignancy of hepatocellular adenomas or carcinomas at later time points. Thus, germline p53 deficiency does not enhance the rate of development of diethylnitrosamine-induced hepatocellular adenoma or carcinoma but may instead favor development of hepatic hemangiosarcoma. © 1995 Wiley-Liss Inc.
TL;DR: The results suggest that an enzyme or enzymes similar to mouse liver Cyp2a‐5, one of which may be CYP2A3, is expressed at high levels in rat nasal epithelium but not in the liver and that its expression is increased by coumarin, an odorant and a substrate of Cyp2 a‐5.
Abstract: In this study, we found that rat nasal coumarin-7-hydroxylase (COH) activity was two orders of magnitude higher than rat hepatic COH activity and could be induced by adding coumarin to the rats' drinking water. In western blot analysis, an anti-cytochrome P450 (Cyp) 2a-5 (mouse liver COH) antibody recognized a sharp band in the microsomal fraction of rat nasal epithelium but not of the liver; the band comigrated with Cyp2a-5. The intensity of the band was increased by the coumarin treatment. Similarly, in northern blot analysis, a cDNA probe specific for Cyp2a-5 recognized an mRNA in the nasal epithelium having the same size as mouse liver Cyp2a-5 mRNA; however, no hybridizable mRNA was recognized in liver preparations. Unlike the protein level, the level of the mRNA was not increased by coumarin. When northern blot analyses were performed with two oligoprobes specific for rat lung CYP2A3, an mRNA of similar size to Cyp2a-5 mRNA was recognized. In immunoinhibition analysis, anti-Cyp2a-5 antibody inhibited rat nasal COH activity and aflatoxin B1 (AFB1) metabolism completely. It inhibited N-nitrosodiethylamine (NDEA) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) metabolism by 80-90%. In contrast, the hepatic metabolism of the four compounds was not affected by the antibody. When coumarin instead of anti-Cyp2a-5 antibody was used, a strong but variable inhibition of the nasal metabolism of AFB1, NDEA, and NNK was seen. The results suggest that an enzyme or enzymes similar to mouse liver Cyp2a-5, one of which may be CYP2A3, is expressed at high levels in rat nasal epithelium but not in the liver and that its expression is increased by coumarin, an odorant and a substrate of Cyp2a-5. The increase probably occurs by protein stabilization or stimulation of translation. The results also show that the enzyme has a key role in the nasal metabolism of three well-known carcinogens, AFB1, NDEA, and NNK and may therefore be an important contributing factor in nasal carcinogenesis.
TL;DR: These studies provide the first evidence that not only does individual variation in mdr induction by AH exist but that AHs regulate mdr in humans by a novel mechanism distinguishable from the classical Ah receptor pathway.
Abstract: To determine whether human liver responds to treatment with aromatic hydrocarbons (AHs) with induction of the multidrug resistance (mdr) gene product P-glycoprotein and whether AH induction of mdr involves the Ah receptor, we compared induction of mdr mRNA with induction of cytochrome P450 (CYP)1A1 mRNA in AH-treated cultures of primary human hepatocytes. Hepatocytes from all 15 individuals tested responded to treatment with 3-methylcholanthrene (MC) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) with induction of CYP1A1 mRNA. However, only 62% and 55% of the preparations responded to treatment with MC and TCDD, respectively, with induction of mdr mRNA. Indeed, in some individuals mdr mRNA was suppressed by MC and TCDD despite robust CYP1A1 induction. These studies provide the first evidence that not only does individual variation in mdr induction by AH exist but that AHs regulate mdr in humans by a novel mechanism distinguishable from the classical Ah receptor pathway. The dramatic variability in AH induction of mdr may be a predictive risk factor that will help to identify an individual's risk of AH-associated toxicities.
TL;DR: It is demonstrated that BCNU resistance is multifactorial and that MGMT makes a modest contribution to resistance in the authors' lines, indicating that another mechanism or mechanisms is operating to limit cytotoxicity.
Abstract: To assess the possible role of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) in resistance of brain neoplasms to the clinically important chloroethylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), we quantitated MGMT activity, BCNU survival, and the effect of ablating MGMT activity on the sensitivity of 14 human medulloblastoma- and glioma-derived cell lines. BCNU resistance, measured as 10% survival dose (LD10), differed eightfold among the lines. Elimination of measurable MGMT activity with the substrate analogue inhibitor O6-benzylguanine (O6-BG) revealed a variable but limited contribution of MGMT to survival. In no case did O6-BG reduce LD10 by more than 3.4-fold. In contrast, 06-BG reduced the LD10 for N-methyl-N′-nitro-N-nitrosoguanidine up to 31-fold in the same cell lines (Bobola MS, Blank A, Berger MS, Silber JR, Mol Carcinog 13:70–80, 1995). Variability in BCNU survival, manifested as a sevenfold range of LD10, persists after measurable MGMT was eliminated, indicating that another mechanism or mechanisms is operating to limit cytotoxicity. Cells alkylated while suspended in growth medium are more resistant to BCNU and display less dependence on MGMT than cells treated while proliferating on a plastic substratum. When alkylated in suspension, most of the lines are either unresponsive to O6-BG or contain a subpopulation that did not respond to O6-BG. Our results demonstrate that BCNU resistance is multifactorial and that MGMT makes a modest contribution to resistance in our lines. © 1995 Wiley-Liss, Inc.
TL;DR: In fibroblasts, β‐carotene and canthaxanthin at concentrations between 10−5 and 3 × 10−6 M were found to strongly enhance GJC in a dose‐ and time‐dependent manner, and the lack of response of keratinocytes suggests differences in regulation of Cx43 expression or in carotenoid processing.
Abstract: Consumption of dietary carotenoids has been statistically associated with decreased risk of cancer at several anatomic sites. In a model murine system of carcinogenesis (the 10T1/2 assay), we have previously shown that carotenoids can inhibit chemically and physically induced neoplastic transformation. This action is strongly correlated with the ability of carotenoids to increase gap-junctional communication (GJC) by induction of connexin43 (Cx43) gene expression. Here we extend these studies to human foreskin-derived dermal fibroblasts and keratinocytes. In fibroblasts, β-carotene and canthaxanthin at concentrations between 10−5 and 3 × 10−6 M were found to strongly enhance GJC in a dose- and time-dependent manner. This was accompanied by an increase in the number of immunofluorescent junctional plaques recognized by an anti-Cx43 antibody and by an increase in Cx43 protein level as determined by western blot analysis. No decrease in proliferation rates was detected by [H3]thymidine labeling. Human keratinocytes grown in monolayer culture did not respond to carotenoids in terms of GJC as measured by dye transfer, immunofluorescent analysis of Cx43 distribution, or Cx43 levels as measured by western blotting. Both cell types accumulated high levels of carotenoids. Because canthaxanthin, which has no known provitamin A activity in mammals, is as active in fibroblasts as is β-carotene, the carotenoid with the highest provitamin A activity, the induction of GJC and Cx43 expression by carotenoids in human dermal fibroblasts seems unrelated to their provitamin A status. The lack of response of keratinocytes suggests differences in regulation of Cx43 expression or in carotenoid processing. © 1995 Wiley-Liss Inc.
TL;DR: Using reverse transcription‐polymerase chain reaction and Southern blot analysis, no evidence for loss of DCC expression in pancreatic cancer cell lines, and allele loss only rarely in tumor biopsies suggest that abnormalities of RB1 and DCC are unlikely to play a major role in pancreatIC carcinogenesis.
Abstract: Evidence of RB1 allele loss was found in only 6% of pancreatic cancers, and we found no significant sequence abnormalities nor loss of RB protein expression in a panel of tumors and cell lines. Using reverse transcription-polymerase chain reaction and Southern blot analysis, we found no evidence for loss of DCC expression in pancreatic cancer cell lines, and allele loss only rarely in tumor biopsies. These findings suggest that abnormalities of RB1 and DCC are unlikely to play a major role in pancreatic carcinogenesis. © 1995 Wiley-Liss, Inc.
TL;DR: It is shown that variability in resistance, manifested as a 20‐fold range in LD10, persists after measurable MGMT is eliminated, disclosing differential contributions of other resistance mechanisms to survival.
Abstract: The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) has been implicated in resistance of human brain tumors to alkylating agents. We observed that 14 human medulloblastoma- and glioma-derived cell lines differ in sensitivity to the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), as shown by their 28-fold range in 10% survival dose (LD10). By using the substrate analogue inhibitor O6-benzylguanine (O6-BG), we showed that the contribution of MGMT to resistance varies widely, as evidenced by 3- to 30-fold reductions in LD10 among the lines, and varies up to 20-fold among subpopulations of individual lines. Importantly, variability in resistance, manifested as a 20-fold range in LD10, persists after measurable MGMT is eliminated, disclosing differential contributions of other resistance mechanisms to survival. Cells exposed to MNNG while suspended in growth medium are more resistant than cells alkylated as subconfluent monolayers, and MGMT accounts for a smaller proportion of their resistance. Notably, the MGMT content of the lines is not statistically correlated with MNNG resistance or with potentiation of killing by O6-BG, even though MGMT is a biochemically demonstrated determinant of resistance. In contrast, the same lines vary less in resistance to the ethylating agent N-ethylnitrosourea (ENU), and MGMT makes only a small contribution to resistance. Our results strongly indicate that resistance to both MNNG and ENU is multifactorial.
TL;DR: It is demonstrated that relatively undifferentiated osteosarcomas commonly display c‐myc amplification, p53 and RBI mutation, and autocrine growth‐factor production, all of which may play a role in osteOSarcomagenesis.
Abstract: Human osteosarcoma and fibrosarcoma cell lines were investigated for alterations in oncogenes, tumor suppressor genes, and growth factors, all of which have been implicated in tumor formation. Characterization of oncogenes that are involved in osteosarcoma formation, including the c-fos and c-myc oncogenes, indicated that all six osteosarcoma cell lines examined had 5- to 20-fold amplification of the c-myc oncogene, whereas neither of two fibrosarcoma cell lines c-myc amplification. Interestingly, only three of six osteosarcoma cell lines displayed altered c-myc immediate-early gene function. c-fos was found to be normal, both at the gene and functional levels, in all six osteosarcoma and both fibrosarcoma cell lines tested. Characterization of two tumor suppressor genes, p53 and RB1, that have been implicated in osteosarcoma formation indicated that p53 was altered in five of six osteosarcoma cell lines, whereas RB1 was altered in only two or six of these cell lines. Neither RB1 nor p53 was found to be altered in the fibrosarcoma cell lines tested. An additional transformation marker, autocrine growth-factor production, was observed in all six osteosarcoma cell lines and both fibrosarcoma cell lines examined. Finally, the differentiation state of the osteosarcoma cell lines was investigated via the bone differentiation markers alkaline phosphates and osteocalcin. Alkaline phosphatase activity was observed in four of six osteosarcoma cell lines but not in the two fibrosarcoma cell lines examined. The alkaline phosphatase activity was a result of the expression of the bone/liver/kidney alkaline phosphatase isoform. High-level osteocalcin expression was observed in one of the osteosarcoma cell lines but not in the two fibrosarcoma cell lines examined, although all cell lines demonstrated low-level osteocalcin expression. Together, these data demonstrate that relatively undifferentiated osteosarcomas commonly display c-myc amplification, p53 and RB1 mutation, and autocrine growth-factor production, all of which may play a role in osteosarcomagenesis.
TL;DR: These findings provide the first evidence for amplification of the Ki‐ras gene in human esophageal cancer, which is restricted to adenocarcinomas.
Abstract: Mutated ras genes have been found to be conspicuously absent from primary tumors of the esophagus, although high expression of ras p21 oncoprotein in some esophageal squamous cell carcinomas and mutations of the Ki- and Ha-ras genes in esophageal carcinoma cell lines have been reported. In this study, we found amplification of the Ki-ras gene in four of 10 esophageal adenocarcinomas (40%). No such amplification was observed among 61 squamous cell carcinomas, one pseudosarcomatous carcinoma, and eight esophageal cell lines, nor in six adenocarcinomas of the stomach. In two samples on which immunohistochemical analysis could be performed, we found overexpression of Ki-ras proteins when compared with normal samples. This Ki-ras amplification in esophageal tumors did not correlate with any pathological feature of the tumors, with the survival of the patients, or with the presence of other genetic alterations. These findings provide the first evidence for amplification of the Ki-ras gene in human esophageal cancer, which is restricted to adenocarcinomas. We also found that six of eight adenocarcinomas had point mutations in the p53 gene; this is a considerably higher prevalence than that reported for esophageal squamous cell carcinomas. These results strongly suggest that esophageal adenocarcinomas differ from squamous cell carcinomas in their molecular genetic characteristics. © 1995 Wiley- Liss, Inc.
TL;DR: Data from 28 tumor and normal pairs at five microsatellite markers suggest that homozygous deletion is a common mechanism of loss of tumor suppressor gene function in this region of chromosome 9p21, which is involved in bladder carcinogenesis.
Abstract: Chromosome 9p21 appears to harbor a tumor suppressor gene, as evidenced by deletions in this region in a variety of human primary tumors and cell lines. To map the deletion at 9p21 in bladder tumors, we analyzed DNA from 28 tumor and normal pairs at five microsatellite markers that flank the region occupied by the putative tumor suppressor genes p16 and p15. Loss of heterozygosity (a) at the markers human interferon (a) α and D9S171, which are adjacent to the p15 and p16 loci, was detected in 41% and 33%, respectively, of informative cases of bladder tumors. No sequence mutations were detected in exons 1 or 2 of either p15 or p16 in any of the bladder tumors. Three sequence-tagged site markers in the region bordered by HIFNα and D9S171 were used to further map the deleted region by multiplex polymerase chain reaction with the HIFNγ marker (on chromosome 12) as a control for amplification. Six of 11 tumors with LOH at surrounding markers had homozygous deletions of the marker c5.1, which is located within the p16 gene; and two tumors appeared to have homozygous deletions within p15(RN1.1) but not p16 (c5.1). A recently identified microsatellite marker, p16-CA-1, located 16 kb distal to p16, proved valuable in defining the minimal deletion involved in these bladder tumors. Five tumors exhibited homozygous deletions of this marker but not HIFNα and two tumors showed LOH at this marker and homozygous deletion of p16. Although these data could not be used to identify p16 or p15 as the definitive tumor suppressor gene in this region that is involved in bladder carcinogenesis, they suggest that homozygous deletion is a common mechanism of loss of tumor suppressor gene function in this region. © 1995 Wiley- Liss, Inc.
TL;DR: Results indicate that alterations in the p53‐regulated pathway are important in bladder carcinogenesis.
Abstract: To investigate the importance of oncogenes and tumor suppressor genes in bladder carcinogenesis, we determined the status of the expression of the MDM-2 and p53 genes and genetic alterations in the p53 gene in five bladder carcinoma cell lines and one kidney urothelial carcinoma cell line. Overexpression of MDM-2 mRNA was observed in three bladder carcinoma cell lines, J82, SCaBER, and BFTC-905. Amplification of the MDM-2 gene was not detected in any of the six cell lines by Southern analysis. The deletion in the p53 gene was observed in J82, and point mutation was detected in J82 and BFTC-909, the kidney urothelial carcinoma cell line. In contrast, no mutations were found in codons 12, 13, and 61 in the Ha-ras and Ki-ras genes in these six cell lines. These results indicate that alterations in the p53-regulated pathway are important in bladder carcinogenesis. © 1995 Wiley-Liss, Inc.
TL;DR: Results indicate that mechanistically diverse tumor promoter stimuli Elevated levels of TGFα mRNA and protein in SENCAR mouse epidermis may play an essential role in mitogenic stimulation during tumor promotion by diverse promoting stimuli.
Abstract: The study presented here was designed to further investigate the role of transforming growth factor-alpha (TGF alpha) in skin tumor promotion by examining the ability of 12-O-tetradecanoylphorbol-13-acetate (TPA) and several non-phorbol ester promoters to alter TGF alpha mRNA and protein levels in mouse epidermis. Total RNA was isolated from SENCAR mouse epidermis at various times after single topical treatments with TPA (3.4 nmol), chrysarobin (220 nmol), okadaic acid (2.5 nmol), and thapsigargin (8.5 nmol). Northern analyses of these isolated RNA samples revealed that all four tumor promoters transiently elevated TGF alpha mRNA levels. Whereas TPA, okadaic acid, and thapsigarin elevated TGF alpha mRNA levels over similar time courses (peak at 4-8 h), chrysarobin elevated TGF alpha mRNA levels with a markedly delayed time course (peak at 24-48 h). More detailed studies with TPA also revealed that multiple treatments (four over a 2-wk period) transiently elevated TGF alpha mRNA in both the epidermis and the dermis. The time courses for changes in TGF alpha mRNA after multiple TPA treatments were similar for both tissues. To facilitate studies of altered TGF alpha mRNA expression in mouse epidermis and possibly other mouse tissues, a semiquantitative reverse transcriptase-polymerase chain reaction method was developed. This method faithfully revealed changes in TGF alpha mRNA levels with all four tumor-promoting agents similar to those determined by northern blot analyses. Immunofluorescence analysis of frozen sections from promoter-treated skin revealed elevated TGF alpha protein levels in both epidermis and dermis, although staining was most intense in the epidermal layer. Immunofluorescence analysis of epidermal hyperplasia adjacent to a full-thickness wound also demonstrated significant epidermal TGF alpha staining. Collectively, these results indicate that mechanistically diverse tumor promoter stimuli elevate TGF alpha mRNA and protein in SENCAR mouse epidermis. Elevated levels of TGF alpha may play an essential role in mitogenic stimulation during tumor promotion by diverse promoting stimuli.
TL;DR: The data suggest that the carcinogen induction of mdr1b expression is mediated through sequences that overlap or that are identical to the basal promoter element.
Abstract: In this report we characterized the transcriptional regulation of the rat mdr1b gene by xenobiotics. The expression of this gene was increased in primary rat hepatocytes and in the H4-II-E hepatoma cell line by exposure to carcinogens such as aflatoxin B1, N-acetoxy-2-acetylaminofluorene, and methyl methanesulfonate. Nuclear run-on experiments indicated that the higher steady-state levels of mdr1b mRNA were due to an increase in transcription. The 5′-flanking region of the mdr1b gene was isolated, sequenced, and functionally characterized in transient and stable transfection assays. A single transcription start site was identified for this gene; no alternate start sites were used after induction with aflatoxin B1. Deletion analysis of this promoter demonstrated that the sequence between nt −214 and −178 was critical for basal promoter activity. This region did not contain any consensus-binding sites for previously identified transcription factors. A negative regulatory region was also identified between nt −940 and −250. No specific carcinogen-responsive element was identified; the xenobiotic response required a large part of the promoter. These data suggest that the carcinogen induction of mdr1b expression is mediated through sequences that overlap or that are identical to the basal promoter element. © 1995 Wiley-Liss, Inc.
TL;DR: Evidence that colon carcinomas display altered expression of individual isoforms of PLCs is provided and increased expression of P LCγ1 may play an important role in colon carcinogenesis is suggested.
Abstract: The levels of expression of phosphoinositide-specific phospholipase Cs (PLCs) were examined in a series of primary human colon carcinomas and in eight colon carcinoma cell lines by using monoclonal antibodies and cDNA probes for PLC gamma 1, PLC beta 1, and PLC delta 1. Western and northern blot analyses of PLC gamma 1 revealed elevated expression of this isozyme at both the protein and mRNA levels in most tumors when compared with paired adjacent normal mucosa samples (in 11 of 13 pairs in the western blots and 8 of 9 pairs in the northern blots). On the other hand, decreased levels of the PLC delta 1 protein were seen in most colon carcinomas (12 of 13 paired samples). The levels of PLC beta 1 protein were too low to detect possible differences between the carcinoma and normal mucosa samples. Relatively high expression of PLC gamma 1 was found in almost all of the eight human colon carcinoma cell lines at both the protein and mRNA levels. Only weak expression of PLC beta 1 was detected in these cell lines, by both western and northern blot analyses, and PLC delta 1 protein was not detected in any of the carcinoma cell lines. These findings provide evidence that colon carcinomas display altered expression of individual isoforms of PLCs and suggest that increased expression of PLC gamma 1 may play an important role in colon carcinogenesis.