Macromolecular adducts: biomarkers for toxicity and carcinogenesis.
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Animals↗
Biomedical subjects
L L Hsieh
Publications and source records attributed to L L Hsieh.
Urinary aflatoxin levels, hepatitis-B virus infection and hepatocellular carcinoma in Taiwan.
Using a urinary immunoassay to measure aflatoxin metabolites, we examined the associations between exposure to aflatoxin, chronic infection with the hepatitis-B virus (HBV) and background rates of hepatocellular carcinoma (HCC) mortality in a cross-sectional survey of 250 residents from 8 areas of Taiwan with a 4-fold variation in age-adjusted HCC mortality. Specimens of fasting blood and overnight urines were used to determine HBV carrier status and excretion of aflatoxin in the subjects surveyed. While the prevalence of hepatitis-B virus carriers showed moderate variability, there was a 500-fold range in urinary aflatoxin levels. Mean log-transformed levels of aflatoxin metabolites were similar in males and females and in HBV carriers and non-carriers. In the 8 townships, HCC mortality correlated positively with both area HBV carrier prevalence and mean aflatoxin levels. The primary analyses, however, were conducted at the individual level. Each subject's aflatoxin level was treated as the response variable in a multiple regression model, and the corresponding sex-specific area HCC rate was included as a predictor along with the individual's carrier status, age and sex; alcohol consumption and cigarette smoking were also considered. In these analyses, a significant association was again observed between the marker of aflatoxin exposure and the background rate of HCC mortality. In females, the slope of the regression line was somewhat steeper in HBV carriers, but this pattern was not seen in males and formal testing yielded no statistically significant evidence of an interaction. Our findings are consistent with the hypothesis that aflatoxin plays an independent role in hepatocellular carcinoma in Taiwan.
Adult↗
Detection of aflatoxin B1-DNA adducts in human placenta and cord blood.
Human placenta and cord blood are readily available specimens that respond to maternal environmental insult and are being used to investigate metabolism, bioactivation, and transplacental transfer of procarcinogens. Enzyme-linked immunosorbent assay was used to quantitate 120 placentas and 56 cord bloods from term, uncomplicated pregnancies at Taipei Chang Gung Memorial Hospital, Taiwan, for the presence of the imidazole ring-opened form of aflatoxin B1-DNA (AFB1-DNA) adducts. Of the 120 samples of placentas, 69 (57.5%) contained AFB1-DNA adducts in levels from 0.6 to 6.3 mumol/mol DNA. Of the 56 samples of cord bloods, 5 (8.9%) contained AFB1-DNA adducts in levels from 1.4 to 2.7 mumol/mol DNA. A higher positive rate was found in samples collected in the summer than in the winter. These results indicate that a significant number of individuals in an area of high liver cancer risk have been exposed to AFB1, and it is possible to transfer AFB1 and its metabolites to the progeny through the transplacental unit. Thus, monitoring adduct levels in human specimens may provide information not only on carcinogen exposure but also on the relationship among infection with hepatitis B/C virus, dietary exposure to AFB1, and liver cancer.
Aflatoxin B1↗
Secular trend and geographical variation in hepatitis A infection and hepatitis B carrier rate among adolescents in Taiwan: an island-wide survey.
During the last two decades an economic boom has occurred in Taiwan, a region where the prevalence of both hepatitis A and B virus infection was formerly very high. To examine the impact of socioeconomic developments on the secular trend and geographical variation in hepatitis A and B virus infection, 875 adolescents selected randomly from 20 junior high schools were studied. Serum samples collected from the subjects were tested for hepatitis A antibody (anti-HAV) and hepatitis B surface antigen (HBsAg) by enzyme immunoassay using commercial reagents. The anti-HAV prevalence increased from northern through central to southern Taiwan; the prevalence was highest in aboriginal townships and lowest in metropolitan precincts. This striking variation in anti-HAV prevalence in different geographical locations and at different urbanization levels remained significant in multiple logistic regression analysis. The HBsAg prevalence was significantly higher in aboriginal townships than in rural and urban townships and metropolitan precincts. In addition, HBsAg prevalence was related inversely to the number of physicians per 1,000 population. The prevalence of both anti-HAV and HBsAg declined significantly during the last decade in Taipei City and County. The decrease in anti-HAV prevalence may be due to improvements in environmental hygiene, water supply, and food sanitation, while the decline in the HBsAg carrier rate may result from the use of disposable needles and syringes as well as screening for HBsAg in blood banks.
Adolescent↗
Immunohistochemical detection of aflatoxin B1-DNA adducts and hepatitis B virus antigens in hepatocellular carcinoma and nontumorous liver tissue.
Monoclonal antibodies recognizing the stable imidazole ring-opened form of the major N7-guanine aflatoxin B1-DNA adduct have been used in competitive enzyme-linked immunosorbent assays (ELISA) and indirect immunofluorescence assays to quantitate adduct levels in liver tissue. Methods were developed in AFB1-treated animals, then applied to paired tumor and nontumor liver tissues of hepatocellular carcinoma patients from Taiwan. An avidin-biotin complex staining method was also used for of the detection of hepatitis B surface (HBsAg) and X (HBxAg) antigens in liver sections. A total of 8 (30%) hepatocellular carcinoma (HCC) samples and 7 (26%) adjacent nontumor liver tissue samples from Taiwan were positive for AFB1-DNA adducts. For HBsAg, 10 (37%) HCC samples and 22 (81%) adjacent nontumorous liver samples were positive, and 9 (33%) HCC samples and 11 (41%) adjacent nontumor liver samples were HBxAg positive. No association with AFB1-DNA adducts was observed for HBsAg and HBxAg. These methods should be useful in determining the role of exposure in the induction of HCC in Taiwan.
Aflatoxin B1↗
Genetic monitoring of human polymorphic cancer susceptibility genes by polymerase chain reaction: application to glutathione transferase mu.
Several genes involved in the metabolism of carcinogens have been found to be polymorphic in human populations and are associated with increased risk of cancer at some sites. This study focuses on the polymorphic enzyme glutathione transferase mu (GT mu). Smokers with low lymphocyte GT mu activity are at an approximately 2-fold higher risk for lung cancer and an approximately 3-fold higher risk for stomach and colon adenocarcinomas. Recent cloning and sequencing of the GST1 gene has allowed the development of convenient genotyping methods based on restriction fragment length polymorphisms (RFLP) or the polymerase chain reaction (PCR). The GST1 polymorphism has been shown to be a deletion of the gene locus. To detect the presence or absence of the gene we amplified exons 4-5 and/or exons 6-7 of the GST1 gene by PCR. PCR amplification produced bands of 215-bp or 273-bp from individuals with one or two copies of the GST1 allele and no band if the individual was homozygously deleted (0/0). In the exon 6-7 PCR, we co-amplified a 268-bp portion of the beta-globin gene as an internal reference standard for quantitative analysis of product yield. This allowed homozygote individuals (+/+) to be distinguished from heterozygotes (+/0). We have compared the GST1 genotype to lymphocyte GT mu activity measured on trans-stilbene oxide (TSO) in the lymphocytes of 45 individuals. Low GT mu activity (< 67 pmole/min/10(7) cells) was strongly associated (24/24) with the GST1 0/0 genotype. With the exception of one individual, activities greater than 67 pmole/min/10(7) were associated with the presence of the GST1 allele (20/21). Individuals with the highest GT-TSO activity were found to be homozygous for GST1. (+/+), while heterozygotes (+/0) generally had lower activity, suggesting a gene dosage effect in lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)
Gene Deletion↗
Quantitation of aflatoxin B1-DNA adducts in woodchuck hepatocytes and rat liver tissue by indirect immunofluorescence analysis.
A quantitative indirect immunofluorescence technique was developed utilizing a monoclonal antibody (6A10) recognizing the imidazole ring-opened form of the major N-7 guanine adduct of aflatoxin B1 (AFB1). This method was used to investigate adduct formation in woodchuck hepatocytes treated in culture and in liver tissue of rats treated i.p. with AFB1. Fluorescein isothiocyanate-labeled secondary antiserum was used for adduct localization in conjunction with 4',6-diamidino-2-phenylindole dihydrochloride staining to localize nuclei. Quantitation of AFB1-DNA adducts was carried out by densitometric analysis of photographic slides. Specific nuclear staining was observed in both woodchuck hepatocytes and rat liver tissue. There was a dose-response relationship between fluorescence intensity and AFB1 dose in treated animals. Turnover of adducts could also be followed in animals over 48 h with this method. DNA was isolated from liver tissue of treated animals and adduct levels were quantitated by competitive enzyme-linked immunosorbent assay with antibody 6A10 and by fluorescence spectroscopy. There was a significant correlation of the quantitative immunofluorescence intensity with levels of AFB1 adducts detected by enzyme-linked immunosorbent assay (r = 0.61, P less than 0.05) and spectrofluorescence (r = 0.78, P less than 0.01). This immunohistochemical method should be applicable to the detection of adducts in liver tissues of humans exposed to high levels of dietary AFB1.
Aflatoxin B1↗
Changes in expression of cellular oncogenes and endogenous retrovirus-like sequences during hepatocarcinogenesis induced by a peroxisome proliferator.
Previous studies have demonstrated that BR-931, a hepatic peroxisome proliferator, can induce liver tumours in mice and rats. Since alterations in gene expression may play a critical role in multistage hepatocarcinogenesis, the present studies examined the expression of the c-myc, c-H-ras, epidermal growth factor (EGF) receptor and ODC (ornithine decarboxylase) genes, as well as endogenous retrovirus-like sequences, in F344 rat liver during the first 8 weeks of feeding a 0.16% Br931 diet and in liver tumours induced by chronic feeding of this diet. Northern blot analysis of poly A + liver RNA samples showed an increase in the level of RNAs homologous to rat leukaemia virus (RaLV) but no significant change in the level of 30S-retrovirus related RNAs in the liver RNA samples obtained from rats during the first 8 weeks of feeding the diet containing BR931. An increase in the levels of c-myc, c-H-ras and ODC transcripts was also seen in the liver RNA samples from the treated rats. Of particular interest was a decrease in the abundance of EGF receptor transcripts in the liver RNA samples from rats fed the BR931 diet. Increased levels of RaLV, c-myc, and ODC RNAs were also seen in the tumours induced by BR931, but this was not the case for 30S and c-H-ras. The liver tumour samples also showed a decrease in EGF receptor RNA. These changes in cellular levels of specific RNAs resemble, in several respect, those we previously described in rodent liver during regeneration and tumour promotion, and also those seen in rodent hepatomas induced by other agents. Therefore, they may reflect a common profile of gene expression relevant to liver proliferation and carcinogenesis.
Animals↗
Protein kinase C heterogeneity in GH4C1 rat pituitary cells. Characterization of a Ca2(+)-independent phorbol ester receptor.
Clonal GH4C1 rat pituitary cells are heterogeneous with respect to phorbol dibutyrate receptors (PDBu-R) and protein kinase C (PKC) content. GH cell PDBu-Rs can be separated into two categories based on Ca2(+)-modulation of receptor affinity. Approximately 70% of the cytosolic PDBu-Rs demonstrate Ca2(+)-sensitive receptor affinity and redistribute from the soluble to the particulate fraction in the presence of excess Ca2+. The other 30% of the receptors remain in the cytosol in the presence of excess Ca2+. Their receptor affinity is Ca2(+)-independent. Northern blot hybridization and immunoblot analysis showed that GH4C1 cells express Ca2(+)-independent epsilon-PKC as well as Ca2(+)-dependent alpha- and beta-PKCs. Cell lysis in Ca2+ caused the redistribution of greater than 95% of alpha- and beta-PKC to the particulate fraction, whereas approximately 90% of the epsilon-PKC remained in the cytosol. In contrast, brief treatment of GH cell cultures with PDBu or thyrotropin-releasing hormone caused redistribution of all three isozymes. Prolonged treatment with PDBu down-modulated all three isozymes but at different rates and to different extents. In contrast, prolonged thyrotropin-releasing hormone treatment selectively down-modulated epsilon-PKC. These results demonstrate that GH cells have both Ca2(+)-sensitive and -insensitive PKCs and PDBu-Rs and that both populations are regulated by agonists that control prolactin synthesis and secretion by these cells.
Animals↗
Isolation of a complementary DNA encoding the catalytic subunit of protein kinase A and studies on the expression of this sequence in rat hepatomas and regenerating liver.
A complementary DNA (cDNA) clone (B4) encoding the catalytic subunit of a cAMP-dependent protein kinase (PKAc) was isolated from a lambda gt10 rat brain cDNA library, using a synthetic oligonucleotide probe whose sequence was based on the known amino acid sequence of a bovine cardiac PKAc. Sequence analysis of this clone revealed a region of 1002 nucleotides which encodes a protein that is 92% homologous to amino acids 17-350 of the bovine cardiac PKAc protein. This clone lacks coding sequences for amino acids 1-16 of the latter protein. Nevertheless, it provided a useful probe to analyze expression of the related gene in a variety of systems. Northern blot analyses using a 32P-labeled probe prepared from a 0.6-kilobase PstI fragment of clone B4 revealed an abundant 4.6-kilobase band in rat brain RNA and lesser amounts of this 4.6-kilobase RNA in rat heart and liver. A 4.6-kilobase RNA was also detected in RNA samples obtained from mouse fibroblasts. This probe also detected homologous RNA in a variety of nonrodent species. In subsequent experiments, this cDNA was used as a probe to elucidate the role of PKAc in post-surgical hepatic regeneration and diethylnitrosamine-induced hepatomas in the rat. These experiments revealed that, following partial hepatectomy, PKAc mRNA is decreased 3-fold by 12 h, returning to normal by 72 h; hepatomas showed no consistent pattern of change in PKAc mRNA levels as compared to controls. Our results indicate that this cDNA encodes an isoform of PKAc which is distinct from PKAc-alpha isolated by Uhler et al. (Proc. Natl. Acad. Sci. USA, 83: 1300-1304, 1986) but highly homologous to PKAc-beta isolated by Showers and Maurer (J. Biol. Chem., 261: 16288-16291, 1986), that depression of cAMP-dependent protein phosphorylation may be an important mechanism in the regeneration of mature rat liver but is not a consistent alteration in chemically induced hepatoma, and that this cDNA is useful as a probe for the study of the role of PKAc gene expression in growth control, particularly in rodent species.
Amino Acid Sequence↗
Increased levels of phorbin, c-myc, and ornithine decarboxylase RNAs in human colon cancer.
Our previous work on protein kinase C (PKC) and colon cancer has shown altered levels of PKC activity in human colon tumors, as well as activation of PKC by colon tumor promoters such as bile acids. To understand further the role of PKC in colon carcinogenesis, we analyzed the expression of phorbin, a gene induced by PKC activation, in a series of different stages of human colon tumors. As shown by northern blot analyses of poly (A)+ RNA, higher levels of phorbin RNA were seen in 26 colon tumor samples than in their adjacent normal colonic mucosa. There also appeared to be a correlation between the abundance of phorbin RNA in the tumors and the extent of invasion (tumor-to-normal tissue phorbin RNA ratio = 4.2, 8.0, and 11.9 for Dukes' A, B, and C, respectively). Phorbin RNA was also abundant in a human colon cancer line (HT29). We also examined the expression of other mitogen-responsive genes (c-myc, ODC, and beta-actin) in a set of 19 colon tumor samples. All tumors displayed significant (mean 3.8-fold) increases in the level of c-myc RNA compared with their adjacent normal colonic mucosa. About 47% and 16% of these tumor samples also showed increased levels of ODC (mean 3.1-fold) and beta-actin (mean 1.6-fold) RNA, respectively. The increased levels of c-myc, ODC, and beta-actin RNA did not correlate with the extent of tumor invasion. Taken together, these results demonstrate that human colon tumors usually display increased levels of both phorbin and c-myc RNAs. The marked increases in phorbin RNA suggest that this could serve as a useful biomarker in studies on human colon cancer.
Actins↗
Factors influencing the expression of endogenous retrovirus-like sequences in Rat 6 cells.
Previous studies from this laboratory have demonstrated increased levels of expression of endogenous rat leukemia virus (RaLV) and 30S retrovirus-like sequences in liver and colon tumors induced by chemical carcinogens in rats. During the process of normal liver regeneration, the levels of RaLV RNA were dramatically increased, whereas the levels of 30S RNA did not change. The present study examined several factors that might influence the expression of these sequences in the Rat 6 embryo fibroblast cell line. Rat 6 cells in either log-phase or confluent cultures were treated with cycloheximide, the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), or the activated carcinogen benzo[a]pyrene diol epoxide (BPDE) for various periods of time up to 48 h. Northern blot analyses of total RNA indicated that cells in untreated cultures in log phase had higher levels of RaLV and 30S RNAs than did confluent cells. Within 10 h cycloheximide (2 or 10 micrograms/mL) markedly increased the levels of RaLV and 30S RNAs in both log-phase and confluent cells. BPDE (100 ng/mL) induced a marked increase in the levels of RaLV RNA at 4 to 10 h, which returned to the basal level by 24 h in the log-phase cells; but no significant change was seen in the confluent cells. The level of 30S RNA also increased moderately in the BPDE-treated log-phase cells and was maximal at 24 h; but no change was seen in confluent cells. Treatment with TPA (100 ng/mL) induced no significant increase in either RaLV or 30S RNA levels in the log-phase or confluent cells. The exposure of Rat 6 cells to 5-azacytidine (3 microM for 24 h) led to a marked increase in the levels of both RaLV and 30S RNAs, which persisted during at least 15 subsequent passages in the absence of the drug. Thus, inhibition of protein synthesis, DNA damage, or hypomethylation can increase the expression of certain endogenous retrovirus-like sequences, but an activator of protein kinase C does not.
7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
Immunologic methods for the detection of carcinogen adducts in humans.
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Antibodies, Monoclonal↗
Metabolic activation of nitropyrenes and diesel particulate extracts.
The aim of this research was to investigate the possible risks of genotoxicity associated with human exposure to diesel engine emissions. We sought to identify and evaluate the critical components of such emissions by using a variety of short-term biological systems. Adducts formed between benzo[a]pyrene and DNA in several short-term test systems have been thoroughly investigated. Although benzo[a]pyrene has long been used as an index of the potential carcinogenicity of polycyclic aromatic hydrocarbon mixtures and is present in diesel engine emissions, it may not be the best measure of the carcinogenicity of these emissions if, indeed, they are confirmed to exert such an effect in humans. Certain nitroarenes, known to be present in diesel particulate extracts, are very potent mutagens in the Ames assay. The major adducts formed in Salmonella typhimurium with 1-nitropyrene (Howard and Beland 1982) and 1,8-dinitropyrene (Andrews et al. 1986; Fifer et al. 1986) have been identified. We undertook, therefore, a comparison of the DNA adducts formed between 1-nitropyrene, 1,3-dinitropyrene, 1,6-dinitropyrene, and 1,8-dinitropyrene and cellular DNA in various systems, including human bronchial segments, in rabbit lung and trachea, and mouse embryo fibroblast C3H/10T1/2 cells, with those reported to be formed in S. typhimurium. In these studies, we administered radiolabeled nitropyrenes, analogous to the treatments previously employed for testing benzo[a]pyrene, and isolated and digested the modified DNA. We then compared elution times, by high-pressure liquid chromatography, of the radioactive adducts with synthetic standards. Not all combinations of exposures were undertaken, since the direction of the investigations changed to include studies on adducts formed in animals exposed to diesel engine emissions themselves. Of the samples studied, metabolism of 1-nitropyrene was most evident in the human bronchial tissue. Little metabolism was evident in duodenal and colonic samples. Mouse C3H/10T1/2 embryo fibroblasts showed no detectable metabolism of, DNA adduct formation with, or transformation by 1-nitropyrene. DNA adducts were detected in cells exposed to 1,8-dinitropyrene and traces of adducts were formed with either 1,3-dinitropyrene or 1,6-dinitropyrene. Measurements of DNA adducts formed after treatment with [4,5,9,10-3H]-1-nitropyrene in rabbit tracheal samples showed that only about 2 to 15 percent of the associated radiolabel could be accounted for as the previously identified C-8 adduct. To facilitate the quantification of adduct levels that might occur in humans and laboratory animals exposed to the nitroarenes in diesel engine emissions, antisera were prepared against DNA modified with 1-nitrosopyrene.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Immunologic methods for the detection of carcinogen adducts in humans.
Monoclonal antibodies have been developed which recognize a number of carcinogen-DNA and protein adducts. These antibodies can be used in highly sensitive competitive enzyme linked immunosorbent assays (ELISA) to detect femtomole levels of adducts in human samples. With the most sensitive antibodies, DNA adducts in the range of 1/10(8) nucleotides can be measured. In addition, antibodies to DNA adducts can be used to investigate immunohistochemically the localization of adducts in specific cell and tissue types. Antibodies recognizing benzo(a)pyrene diol epoxide-DNA have been used to monitor adducts in white blood cell DNA of foundry workers and placental and white blood cell DNA of smokers and nonsmokers. Because of antibody crossreactivity with structurally related adducts of other polycyclic aromatic hydrocarbons, this assay is not specific for benzo(a)pyrene adducts. Antibodies to the stable guanine imidazole ring opened aflatoxin-B1-DNA adduct have been used to detect elevated levels of adducts in liver tissue from Taiwanese hepatocellular cancer patients. Monoclonal antibodies against 8-methoxypsoralen-DNA have been used to monitor adducts in psoriasis and cancer patients treated with psoralen plus UVA light. These patients have also served as model systems for the development of immunofluorescence methods for adduct detection. Immunohistochemical staining of skin biopsies from psoriasis patients demonstrated specific staining of epidermal cells. With further increases in sensitivity, this method should be applicable to the detection of adducts in other human tissues. Adduct detection in humans is now established as a viable method for determination of exposure to certain chemical carcinogens. However, the relationship of adduct measurements to individual risk requires further investigation.
Aflatoxins↗
Transformation by a ras oncogene causes increased expression of protein kinase C-alpha and decreased expression of protein kinase C-epsilon.
Rat embryo fibroblasts and liver epithelial cell lines normally express two isoforms of protein kinase C (PKC), PKC alpha and PKC epsilon. Derivatives of these cells transformed by an activated human c-H-ras oncogene display a several-fold increase in expression of PKC alpha and a concomitant decrease in PKC epsilon, at both the protein and mRNA levels. Similar changes are seen when the transformed phenotype is induced by Zn2+ in cells carrying the activated ras oncogene under the control of a metallothionein promoter. Studies using cell lines that express very high levels of PKC beta 1, studies using a specific inhibitor of PKC (CGP 41251), and studies in which PKC activity is down-regulated by treatment with a phorbol ester tumor promoter provide evidence that the effects of the ras oncogene on the expression of PKC alpha and PKC epsilon are mediated mainly through a PKC-independent pathway. The present results provide the first evidence that transformation of cells by an oncogene can alter the relative expression of specific isoforms of PKC. It is possible that these changes contribute to the malignant phenotype of these cells.
Animals↗
Altered expression of retrovirus-like sequences and cellular oncogenes in mice fed methyl-deficient diets.
Methyl-deficient (lipotrope-deficient) diets enhance liver carcinogenesis in rodents. Although the mechanisms responsible for the cancer-promoting activity of such diets have not been identified, they have been observed to cause impaired immune response, alterations in methylation of liver RNA and DNA, and enhanced susceptibility to oxidative damage. Since alterations in gene expression may also play a critical role, the present studies examined the expression of the c-myc, c-H-ras, epidermal growth factor receptor, and ornithine decarboxylase genes, as well as endogenous retrovirus-like sequences, in C57BL/6J x C3H/HeJ F1 mouse liver during the first 2 weeks of feeding of a methyl-deficient diet. The kinetics of liver cell proliferation was investigated in parallel. Increased [3H]thymidine incorporation into liver DNA was found at day 4 and reached a maximum at days 7-11 after commencement of the methyl-deficient diet, when compared to age-matched mice fed a complete diet. Northern blot analysis of polyadenylated liver RNA samples indicated an increase in the levels of RNA homologous to Moloney murine leukemia virus and intracisternal A particle sequences but no significant change in the level of VL30 retrovirus-related RNA in the samples from mice fed methyl-deficient diets. A marked increase in the levels of c-myc and a slight increase in the levels of ornithine decarboxylase and c-H-ras transcripts were seen in the liver RNA samples from the treated mice. Of particular interest was a decrease in the abundance of epidermal growth factor receptor transcripts in the liver RNA samples from the treated mice. These changes in cellular levels of specific RNA resemble, in several respects, those we have previously described in rodent liver during regeneration and tumor promotion and also those seen in rodent hepatomas. They may reflect, therefore, a common profile of gene expression relevant to cell proliferation.
Animals↗
Phenotypic effects of overexpression of PKC beta 1 in rat liver epithelial cells.
We have used a previously described retroviral expression vector pMV7-PKC beta 1 to develop derivatives of two rat liver epithelial cell lines, K16 and K22, that stably express about tenfold-higher PKC activity than control cells. Despite these high levels of PKC, these cells did not exhibit gross morphologic changes, anchorage-independent growth, or tumorigenicity. K16PKC-4 and K22PKC-2, two lines with the highest PKC enzyme activity, were studied further in terms of several responses to the phorbol ester tumor promoter TPA. When treated with 100 ng/ml of TPA, the control K16MV7 and K22MV7 cells displayed a slight change in morphology, whereas the K16PKC-4 and K22PKC-2 cells displayed a marked change in morphology. Northern blot analyses demonstrated that TPA induced increased levels of fos, myc, phorbin, and ODC RNAs in control K16MV7 and K22MV7 cells, with maximum induction occurring at about 0.5, 1, 8, and 8 h, respectively. In K16PKC-4 and K22PKC-2 cells, TPA induction of phorbin and ODC RNAs was markedly enhanced, but this was not the case for myc and fos RNAs. In addition, the levels of myc RNA were constitutively higher in both K16PKC-4 and K22PKC-2 cells than in the control cells. Taken together, these results provide direct evidence that PKC plays a critical role in modulating the expression of myc, phorbin, and ODC RNAs. On the other hand, overexpression of PKC beta 1 is not itself sufficient to cause cell transformation.
Animals↗