Effects of a green tea extract, Polyphenon E, on systemic biomarkers of growth factor signalling in women with hormone receptor-negative breast cancer.

BACKGROUND: Observational and experimental data support a potential breast cancer chemopreventive effect of green tea. METHODS: We conducted an ancillary study using archived blood/urine from a phase IB randomised, placebo-controlled dose escalation trial of an oral green tea extract, Polyphenon E (Poly E), in breast cancer patients. Using an adaptive trial design, women with stage I-III breast cancer who completed adjuvant treatment were randomised to Poly E 400 mg (n = 16), 600 mg (n = 11) and 800 mg (n = 3) twice daily or matching placebo (n = 10) for 6 months. Blood and urine collection occurred at baseline, and at 2, 4 and 6 months. Biological endpoints included growth factor [serum hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF)], lipid (serum cholesterol, triglycerides), oxidative damage and inflammatory biomarkers. RESULTS: From July 2007-August 2009, 40 women were enrolled and 34 (26 Poly E, eight placebo) were evaluable for biomarker endpoints. At 2 months, the Poly E group (all dose levels combined) compared to placebo had a significant decrease in mean serum HGF levels (-12.7% versus +6.3%, P = 0.04). This trend persisted at 4 and 6 months but was no longer statistically significant. For the Poly E group, serum VEGF decreased by 11.5% at 2 months (P = 0.02) and 13.9% at 4 months (P = 0.05) but did not differ compared to placebo. At 2 months, there was a trend toward a decrease in serum cholesterol with Poly E (P = 0.08). No significant differences were observed for other biomarkers. CONCLUSIONS: Our findings suggest potential mechanistic actions of tea polyphenols in growth factor signalling, angiogenesis and lipid metabolism.

Mechanism of genotoxicity induced by targeted cytoplasmic irradiation.

BACKGROUND: Direct damage to DNA is generally accepted as the main initiator of mutation and cancer induced by environmental carcinogens or ionising radiation. However, there is accumulating evidence suggesting that extracellular/extranuclear targets may also have a key role in mediating the genotoxic effects of ionising radiation. As the possibility of a particle traversal through the cytoplasm is much higher than through the nuclei in environmental radiation exposure, the contribution to genotoxic damage from cytoplasmic irradiation should not be ignored in radiation risk estimation. Although targeted cytoplasmic irradiation has been shown to induce mutations in mammalian cells, the precise mechanism(s) underlying the mutagenic process is largely unknown. METHODS: A microbeam that can target the cytoplasm of cells with high precision was used to study mechanisms involved in mediating the genotoxic effects in irradiated human-hamster hybrid (A(L)) cells. RESULTS: Targeted cytoplasmic irradiation induces oxidative DNA damages and reactive nitrogen species (RNS) in A(L) cells. Lipid peroxidation, as determined by the induction of 4-hydroxynonenal was enhanced in irradiated cells, which could be suppressed by butylated hydroxyl toluene treatment. Moreover, cytoplasmic irradiation of A(L) cells increased expression of cyclooxygenase-2 (COX-2) and activation of extracellular signal-related kinase (ERK) pathway. CONCLUSION: We herein proposed a possible signalling pathway involving reactive oxygen/nitrogen species and COX-2 in the cytoplasmic irradiation-induced genotoxicity effect.

Detection of smoking-related covalent DNA adducts in human placenta.

The presence of covalent DNA chemical addition products (adducts) in human term placentas was investigated by recently developed immunologic and 32P-postlabeling assays. DNA from placental specimens of smokers showed a small but not statistically significant increase in adduct levels when tested by antibodies to DNA modified with a benzo[a]pyrene dihydrodiol epoxide (BPDE-I), the ultimate carcinogenic derivative of benzo[a]pyrene. The postlabeling assay detected several modified nucleotides, one of which (adduct 1) strongly related to maternal smoking during pregnancy. This adduct was present in placental tissue from 16 of 17 smokers, but only 3 of 14 nonsmokers. Among smokers, levels of adduct 1 in general were only weakly related to questionnaire and biochemical measures of the intensity of smoking exposures, which suggests modulation by individual susceptibility factors. The adduct seemed to be derived from an aromatic carcinogen, but it may not result from several of the most intensely studied polycyclic aromatic hydrocarbons or aromatic amines in tobacco smoke. The data show the association of cigarette smoking with covalent damage to human DNA in vivo.

Hint1 is a haplo-insufficient tumor suppressor in mice.

The HINT1 protein, a member of the histidine triad (HIT) family, is highly conserved in diverse species and ubiquitously expressed in mammalian tissues. However, its precise function in mammalian cells is not known. As a result of its structural similarity to the tumor-suppressor protein FHIT, we used homozygous-deleted Hint1 mice to study its role in tumorigenesis. We discovered that after 2 to 3 years of age the spontaneous tumor incidence in Hint1 -/- mice was significantly greater than that in wild-type Hint1 +/+ mice (P < 0.05). Using a well-established mouse model of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary carcinogenesis we found a marked and significant (P < 0.05) increase in the incidence of mammary and ovarian tumors in both, Hint1 -/- and +/- mice versus +/+ mice. The Hint1 -/- and +/- mice had similar tumor incidence and similar tumor histologies. Therefore, deletion of Hint1 in mice enhances both spontaneous tumor development and susceptibility to tumor induction by DMBA. In addition, since the Hint1 +/- tumors retained expression of the unmutated wild-type allele, Hint1 is haplo-insufficient with respect to tumor suppression in this model system.

Organ, species, and compound specificity in the metabolic activation of primary aromatic amines.

For the evaluation of the role of target tissue activation in the induction of bladder cancer, microsome-mediated N-hydroxylation of the bladder carcinogen 4-biphenylamine (4-BA) was studied in bovine and canine bladder mucosae, relative to the activity in liver. Bovine bladder microsomes mediated the N-hydroxylation of 4-BA at an exceptionally high rate, whereas no detectable activity was found with bovine liver microsomes. Dog bladder microsomes were 40-100 times less active than bovine bladder microsomes and contained approximately one-third the amount of cytochrome P450. Dog liver microsomes were as active as dog bladder microsomes per nanomole P450 and an order of magnitude more active when normalized to microsomal protein. Rat liver microsomes contained the highest level of P450 of all the preparations studied, and N-hydroxylase activity was approximately twice the rate of that of dog liver. The rate of N-hydroxylation of 2-naphthylamine (2-NA) and 1-naphthylamine (1-NA) was compared in bovine bladder mucosa and was found to correlate well with the relative potency of these compounds as bladder carcinogens (4-BA greater than 2-NA greater than 1-NA). Such a comparison could not be made with dog bladder mucosa because of its low N-hydroxylation activity. In addition, bovine bladder mucosa S-9 mutagenic activation of 4-BA, 2-NA, and 1-NA was investigated in Salmonella typhimurium and found to parallel the carcinogenic potency of these compounds. These results demonstrate considerable tissue, species, and compound specificity for the metabolic activation of aromatic amines and provide further evidence in support of bladder activation as a mechanism of aromatic amine-induced bladder cancer.

Quantitative associations between DNA damage in human placenta and maternal smoking and birth weight.

Specimens of human placental DNA were tested for chemical addition products (adducts) by recently developed 32P-postlabeling and immunologic assays, and results were compared with data concerning maternal exposures and birth weight. A total of 7 different adducts were detected in the 53 specimens of human placental tissue examined by the 32P-postlabeling assay. Three of these adducts were found almost exclusively in smokers. Among smokers there were positive dose-response relationships between levels of the smoking-related adducts and biochemical estimates of doses of maternal exposure to cigarette smoke during pregnancy. Levels of 1 adduct found only in smokers appeared to relate directly to amounts of caffeine consumption by the mother. In addition to these relationships with maternal exposures, levels of smoking-related adducts were inversely associated with the birth weight of offspring. Results from this study suggest that even at their current formative stage of development, assays for DNA adducts may help identify determinants of DNA damage to human tissues and improve our ability to demonstrate dose-response relationships for the effects of environmental exposures to potentially carcinogenic agents.

PIP: The use of P-postlabeling assay to find the determinants of DNA damage demonstrates an innovative approach for measuring toxic end points. This approach begins with estimates of biochemical or molecular damage to tissue and then figures in exposure or susceptibility factors that are associated with the damage. Using this method, researchers studied smoking-related DNA adducts (chemical addition products) and found 7 different DNA adducts in human placental tissue specimens. Maternal smoking during pregnancy was related to 3 of the adducts observed. Levels of adducts were associated with caffeine use, age, race and education of the mother. The intensity of the smoking was determined by three methods: a questionnaire; a biological approach that scored smoking exposure and combined data from questionnaires and assays and a molecular approach that was related to levels of DNA adducts. There was a clear association between birth weight and birth length and levels of adduct that demonstrated that cigarette smoke does cause low birth weight and birth length. Little correlation between levels of tar, nicotine or carbon monoxide and adduct levels was found. The information and study was at an inchoate stage; therefore, further comparison and interpretation are needed to assess the findings.

DNA adducts, protein adducts, and sister chromatid exchange in cigarette smokers and nonsmokers.

In order to validate markers of internal dose and biologically effective dose of carcinogens, a battery of measurements was made on blood samples from 22 smokers and 24 nonsmokers. The markers included immunoreactivity in an enzyme-linked immunosorbent assay (ELISA) quantified in white blood cells with the use of a polyclonal anti-benzo[a]pyrene diol epoxide-I-DNA antibody, 4-aminobiphenyl hemoglobin (4-ABP-Hb) adducts measured by negative chemical ionization mass spectrometry, sister chromatid exchange (SCE) in cultured lymphocytes, and cotinine in plasma measured by radioimmunoassay. Several blood samples were drawn from each subject. In blood samples 1 and 3 having detectable levels of DNA adducts, mean femtomole-per-microgram levels were consistently higher among smokers compared to nonsmokers. The borderline significance of this difference may be attributable to the small numbers of subjects. Consistently higher adduct levels were seen in females compared to males. In sample 3, adduct levels were significantly correlated with measurements of active smoking in smokers and with passive smoking in nonsmokers. By contrast to the ELISA data, which may reflect cumulative exposure from multiple background sources, the 4-ABP-Hb assay was able to distinguish clearly between smokers and nonsmokers. SCEs were significantly elevated in the smokers compared to nonsmokers. Also observed were significant correlations between 4-ABP-Hb and both cotinine and SCEs, as well as a positive correlation between the 4-ABP-Hb and DNA adduct levels (sample 3) that was highly significant. The correlation between DNA and 4-ABP-Hb adducts was significant in smokers but not nonsmokers (sample 3). These results support the need for batteries of markers to detect and to quantify the carcinogenic dose to humans resulting from both specific and "background" environmental exposures.

Biomarkers of environmental tobacco smoke in preschool children and their mothers.

BACKGROUND: Adverse health effects attributable to environmental tobacco smoke (ETS) include respiratory illness and lung cancer in nonsmokers. There is accumulating evidence that children may be at heightened risk of cancer later in life as a result of exposure to carcinogens during their early development. It is of concern that as many as 9 million American children under the age of 5 years may be exposed to ETS. PURPOSE: Our goal was to assess whether levels of cotinine and polycyclic aromatic hydrocarbon-albumin (PAH-albumin) are associated with ETS exposure in children and in women of reproductive age, after accounting for background exposures to PAHs in the diet, workplace, and the home environment. METHODS: The study cohort was composed of 87 Hispanic and African-American mothers and 87 of their preschool children (2-5 years of age). Plasma cotinine was analyzed by gas chromatography; PAH-albumin adducts in peripheral blood were analyzed by enzyme-linked immunosorbent assay. Exposure data were obtained by interview-administered questionnaires. RESULTS: Both cotinine and PAH-albumin were significantly higher in the children whose mothers smoked than in the children of nonsmoking mothers (P < .001 and P < .05, respectively). Among the children of nonsmoking mothers, cotinine levels were also significantly higher in those who had ETS exposure from others in the household compared with the unexposed children. By regression analysis, after adjustment for ethnicity, there was a significant dose-response relationship between cotinine and the number of cigarettes smoked per day by the mother, both in the children (partial r2 = .23; P = .01) and in the mothers (partial r2 = .22; P = .01). Among the nonsmoking mothers, regression of biomarkers against total passive smoking exposure also showed a significant association with cotinine (r2 = .25; P = .04). PAH-albumin did not show the same dose-related response with the smoking variables. Mothers' cotinine levels were significantly correlated with those of their children (r = .76; P < .001) as were PAH-albumin adducts (r = .27; P = .014). CONCLUSION: ETS exposure of young children via their mothers' smoking is associated with increases not only in the internal dose of ETS (cotinine), which has been previously reported, but also in the biologically effective dose of the carcinogenic (PAH) components of ETS (PAH-albumin adducts). This observation underscores the carcinogenic and public health hazard of ETS. IMPLICATIONS: Given the relatively low level of ETS exposure in this study, these results reinforce the need for effective programs aimed at smoking prevention and cessation among women, particularly women of reproductive age and minorities.

Flow cytometric analysis of 8-methoxypsoralen-DNA photoadducts in human keratinocytes.

Flow cytometric methods have been developed for the analysis of 8-methoxypsoralen-DNA adducts in SV40-transformed human keratinocytes (svk-14). Monoclonal antibodies which specifically recognize these adducts were used in conjunction with fluorescein isothiocyanate-labeled second antibodies and propidium iodide staining to simultaneously determine 8-methoxypsoralen-DNA adduct levels and DNA content, respectively. The sensitivity of the method was determined by quantitation of adduct levels in DNA isolated from treated cells by enzyme-linked immunosorbent assay. Adduct formation during various stages of the cell cycle was also investigated. Two separate cell populations with a DNA content indicative of cells in G1 but with different levels of fluorescein isothiocyanate labeling were observed. Pretreatment of cells with aphidicolin decreased the number of cells in S phase, and only one fluorescein isothiocyanate-staining G1 population was observed. There is an indication of greater adduct formation in S-phase cells when immunofluorescence is corrected for DNA content. These results suggest that adduct formation is not simply linear with respect to DNA content.

Immunological detection and visualization of 8-methoxypsoralen-DNA photoadducts.

Monoclonal antibodies specific for DNA damaged by 8-methoxypsoralen (8-MOP) plus ultraviolet A (UVA) light were used to study adduct formation in human keratinocytes and mouse and rat skin in vivo. This antibody does not cross-react with nonmodified DNA or free 8-MOP. Sensitive competitive enzyme-linked immunosorbent assays with color or fluorescence endpoints were used to quantitate adducts on DNA isolated from treated keratinocytes or skin samples. Localization of 8-MOP-DNA adducts was studied by indirect immunofluorescence with fluorescein-conjugated anti-mouse-IgG antibodies. When cultured keratinocytes were treated with 8-MOP and UVA, immunofluorescence was localized in the nucleus. There was no fluorescence in untreated control cells or treated cells incubated with nonspecific serum. Comparison of intensity of immunofluorescence staining with quantitation of adduct levels by enzyme-linked immunosorbent assay indicated that the limit of sensitivity of the immunofluorescence technique is 9.0 fmol adduct/micrograms DNA or 2.9 adducts/10(6) nucleotides.

Immunological detection of aflatoxin B1-DNA adducts formed in vivo.

Two monoclonal antibodies (6A10 and 12F5) were obtained after fusion of mouse P3X63-AG.8.653 myeloma cells with spleen cells isolated from BALB/c mice immunized with imidazole ring-opened aflatoxin B1 (AFB1)-DNA and characterized by competitive enzyme-linked immunosorbent assays. Both antibodies are highly specific for imidazole ring opened AFB1-DNA and show some cross-reactivity with AFB1-DNA and no cross-reactivity with 8,9-dihydro-8-(7-guanyl)-9-hydroxy-AFB1, AFB1 conjugated with bovine serum albumin, aflatoxin M1 conjugated with bovine serum albumin, AFB1, or aflatoxin G1. Antibody 6A10 was further characterized and showed no cross-reactivity with DNA modified by several other carcinogens. It could detect adducts with 4-fold higher sensitivity in highly modified DNA (2.5 adducts/100 nucleotides) than in low modified DNA (4 adducts/10(5) nucleotides). With low modified DNA the limit of sensitivity is 5 adducts/10(7) nucleotides. Antibody 6A10 reliably detected adducts formed in vivo in rats and mice treated with AFB1. In a pilot study, AFB1 adducts were detected in liver tissues from individuals living in areas with suspected exposure to AFB1. Monitoring adduct levels in human tissue may provide information not only on carcinogen exposure but also on the relationship among infection with hepatitis B virus, dietary exposure to aflatoxin B1, and liver cancer.

Immunological quantitation of levels of tissue inhibitor of metalloproteinase-1 in human colon cancer.

Treatment of rodent cells with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate activates protein kinase C, leading to increased expression of several genes, including a gene originally designated TPA-S1 or phorbin (M. D. Johnson, G. M. Housey, P. T. Kirschmeier, and I. B. Weinstein, Mol. Cell Biol., 7: 2821-2829, 1987). Sequence analysis of this cloned gene indicated homology with human erythroid-potentiating activity and tissue inhibitor of metalloproteinase (TIMP-1). Elevated levels of phorbin mRNA have been observed in human colon tumors (J. G. Guillem, M. F. Levey, L. L. Hsieh, M. D. Johnson, P. LoGerfo, K. A. Forde, and I. B. Weinstein, Mol. Carcinogen., 3: 68-74, 1990) and this increase correlated with the extent of invasion. To further investigate this phenomenon at the protein level, monoclonal antibodies were developed against the recombinant form of TIMP-1. A competitive enzyme-linked immunosorbent assay was developed for quantitation of the TIMP-1 protein in tissue extracts. Elevated levels of TIMP-1 protein were found in 31 human colon tumors, compared to paired samples of adjacent normal mucosa. In a subset of samples, previously analyzed for phorbin mRNA levels (n = 25), there was a good correlation between the abundance of TIMP-1 protein and phorbin mRNA. Immunoaffinity column purification of tumor extracts followed by Western blot analysis was used to confirm the enzyme-linked immunosorbent assay data. These results provide evidence that phorbin and TIMP-1 represent the same gene. In addition, the immunoassays we have developed may be useful in further studies on the role of TIMP-1 in human colon cancer.

Role of oxyradicals in mutagenicity and DNA damage induced by crocidolite asbestos in mammalian cells.

Crocidolite, one of the most carcinogenic forms of asbestos, is mutagenic in cultured mammalian cells when assayed using a system that can detect multilocus deletions. In the present study, we examined the effect of buthionine sulfoximine (BSO) on mutation frequency and the formation of 8-hydroxydeoxyguanosine (8-OHdG) in human-hamster hybrid (A(L)) cells induced by crocidolite fibers in an attempt to determine the role of oxyradicals in mediating fiber mutagenesis. BSO, a competitive inhibitor of the enzyme gamma-glutamyl cysteine synthetase, depleted nonprotein sulfhydryls to <5% of control within 24 h at a nonmutagenic dose of 25 microM. In cells pretreated with BSO for 24 h, the mutation yield at the CD59 locus induced by a 4 microg/cm2 dose of crocidolite fibers was increased by more than 3-fold (P < 0.05). Using immunoperoxidase staining with a monoclonal antibody specific for 8-OHdG, we demonstrated that crocidolite fibers induced a dose-dependent increase in oxidative DNA damage in A(L) cells. Furthermore, addition of DMSO, a well-established hydroxyl radical (OH*) scavenger, dramatically suppressed 8-OHdG induction (P < 0.005). Our results definitely demonstrate that reactive oxygen species mediate fiber-induced DNA damage mutagenesis in A(L) cells in a concentration-dependent manner.

Immunoperoxidase detection of 8-hydroxydeoxyguanosine in aflatoxin B1-treated rat liver and human oral mucosal cells.

An immunoperoxidase method using a monoclonal antiserum that recognizes 8-hydroxydeoxyguanosine has been developed for detection and quantitation of oxidative damage in single cells. The method was initially applied to cultured cells treated with H2O2 or aflatoxin B1 and then to cryostat liver sections of rats treated with aflatoxin B1. To demonstrate that the method has sufficient sensitivity for detection of damage in human samples, oral mucosal cells from a total of 12 pairs of smokers and nonsmokers were analyzed. Mean staining intensity of oral cells of smokers was 1.6-fold higher than in nonsmokers. The immunoperoxidase method, requiring a small number of cells and eliminating the need for isolation of DNA, will be useful for evaluation of oxidative damage in a wide range of biological samples.

Detection of polycyclic aromatic hydrocarbon-DNA adducts in white blood cells of foundry workers.

Iron foundry workers, exposed to high levels of polycyclic aromatic hydrocarbons (PAHs), silica, and metal fumes and dusts, are at elevated risk of lung cancer. Benzo(a)pyrene and a number of structurally related PAHs are metabolically activated to diol epoxides (e.g., 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a) pyrene) which are mutagenic, carcinogenic in experimental animals, and form covalent adducts with DNA. The levels of these adducts were measured in an enzyme-linked immunosorbent assay using a polyclonal anti-benzo(a)pyrene diol epoxide-I-DNA antibody which cross-reacts with DNA modified by diol epoxides of structurally related PAHs. DNA was analyzed from peripheral blood cells of 35 Finnish foundry workers and 10 controls. Workers were classified as having low (less than 0.05 micrograms/m3), medium (0.05-0.2 micrograms/m3), or high (greater than 0.2 micrograms/m3) exposure to benzo(a)pyrene (as an indicator of PAH). When adjustment was made for cigarette smoking and time since vacation, benzo(a)pyrene exposure was significantly related to adduct levels (P = 0.0001). Each of the three exposure groups had significantly elevated adduct levels compared to controls. Among the exposed workers, the low group differed significantly from the high and medium categories. This study supports the usefulness of monitoring adduct formation in a population occupationally exposed to carcinogens.

Immunohistochemical quantitation of polycyclic aromatic hydrocarbon-DNA adducts in human lymphocytes.

The formation of polycyclic aromatic hydrocarbon-DNA adducts was studied in peripheral blood lymphocytes obtained from men with occupational and environmental exposure. Subjects included coke factory workers, residents from the vicinity of the cokery, and rural region inhabitants (16 individuals in each exposure group). Adducts were determined by immunohistochemical analysis using a polyclonal antiserum recognizing benzo(a)pyrene and related polycyclic aromatic hydrocarbon diol epoxide-DNA adducts, a biotinylated secondary antiserum, and streptavidin-conjugated FITC. Propidium iodide was used to quantitate nuclear DNA. Dual fluorescence intensities were simultaneously measured with a Zeiss Axiovert microscope and a Bio-Rad MRC-600 argon laser scanning confocal attachment. Adducts were significantly elevated (P < 0.001) in both occupational and environmental groups, as compared to the rural control group by Mann-Whitney U test. The distribution of the data indicated the existence of cells with relatively higher adduct levels. The percentages of these so called "higher adduct-level cells" were 13.6, 11.5, and 3.7 in cokery workers, environmentally exposed individuals, and rural controls, respectively. The immunohistochemical method allows visualization and relative quantitation of polycyclic aromatic hydrocarbon-DNA adducts in individual lymphocytes. It requires a much smaller amount of blood than the previously used 32P-postlabeling and ELISA methods, which used isolated bulk DNA. It can also be used for adduct quantitation in biopsy material. The results of this pilot study indicate that this technique is a promising addition to biomonitoring studies.

p53 mutations, chronic hepatitis B virus infection, and aflatoxin exposure in hepatocellular carcinoma in Taiwan.

Recent studies have implicated aflatoxin B1 (AFB1) exposure as an etiological agent in hepatocellular carcinoma (HCC) and suggested an interaction with chronic hepatitis B virus (HBV) infection. Worldwide AFB1 exposure correlates with a specific mutation at codon 249 in the p53 tumor suppressor gene in liver tumors. This study investigated the roles of HBV and AFB1 in the HCC carcinogenic pathway involving p53 mutations. In cases and controls, chronic HBV infection was assessed by serum hepatitis B surface antigen (HBsAg) and AFB1 exposure by immunohistochemical detection of AFB1-DNA adduct in liver tissue. p53 protein mutations in tumor tissues of HCC cases were identified by immunohistochemistry and DNA mutations by single-stranded conformational polymorphism and sequencing analysis. Both chronic HBsAg carrier status and liver AFB1-DNA adducts were significantly higher in cases than in controls with odds ratios (OR) of 8.4 and 3.9, respectively (P < 0.01). Moreover, HCC risk was greatest in individuals with both AFB1-DNA adducts and HBsAg, suggesting a viral-chemical interaction. Mutant p53 protein, mutations in the p53 gene, and specific codon 249 mutations were detected in 37, 29, and 13%, respectively, of the HCC cases. Most of the DNA mutations were transversions, and the only major clustering site for mutations was codon 249. AFB1-DNA adducts were associated with p53 protein (OR = 2.9, P = 0.054) and DNA mutations (OR = 2.9, P = 0.082) but with borderline significance. All of the codon 249 mutations (n = 12) occurred in HBsAg-seropositive carriers, resulting in an OR of 10.0 (P < 0.05), suggesting that HBV may be involved in the selection of these mutations. The ORs between HBsAg and p53 DNA and protein mutations were 2.6 (P = 0.077) and 1.8 (P > 0.05), respectively. Both p53 DNA and protein mutations were related to tumor stage, suggesting that they are late events. These studies provided further support for the role of aflatoxin exposure in HCC in Taiwan and insight into viral-chemical interactions and molecular pathogenesis.

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.

Correlations of chronic hepatitis B virus infection and cigarette smoking with elevated expression of neu oncoprotein in the development of hepatocellular carcinoma.

To investigate the potential role of neu oncogene expression in hepatocarcinogenesis, a nested case-control study was conducted within a cohort of 9691 male adults in Taiwan. Blood samples of study subjects were collected during 1984-1986 and frozen at -30 degrees C until subsequent analysis. The neu oncoprotein level in the stored serum was measured by an enzyme-linked immunosorbent assay for 27 cases of newly developed hepatocellular carcinoma (HCC), 12 liver cirrhosis cases, and 40 healthy controls. The mean level of neu oncoprotein was significantly higher in HCC and liver cirrhosis cases than in controls. The risk of HCC increased significantly with increasing serum level of neu oncoprotein (trend test, P = 0.02). The proportion of subjects having an elevated serum level of neu oncoprotein, defined as a level greater than the mean level of all controls, was significantly higher among asymptomatic HBsAg carriers than noncarriers (P = 0.05), showing a multivariate-adjusted odds ratio of 4.0. Among HCC cases, a strong association was observed between cigarette smoking and elevated prediagnostic serum level of neu oncoprotein. The association remained highly significant (P = 0.017) even when adjustment was made for potential confounders. The multivariate-adjusted odds ratio of having an elevated serum level of neu oncoprotein, defined as a level greater than the mean plus 1 SD of control levels, for HCC cases who smoked more than 10 cigarettes a day was as high as 386.5 compared with the cases who smoked less than 10 cigarettes a day or nonsmoking cases. The results suggest that both HBsAg carrier status and cigarette smoking are related to the increased expression of neu oncogene, and cigarette smoking seems to play a significant role in the latter stages of hepatocarcinogenesis. There was no association between alcohol drinking and serum neu oncoprotein level.

Overexpression of p27Kip1 inhibits the growth of both normal and transformed human mammary epithelial cells.

We previously reported increased expression of p27Kip1 in a series of human breast cancer cell lines, as compared to cell lines established from normal mammary epithelial cells. These data were surprising because this protein exerts a growth-inhibitory function in normal cells, and p27Kip1 has been proposed as a candidate tumor suppressor gene. A possible explanation for the paradoxical increase in p27Kip1 in the breast cancer cell lines is that they had become refractory to the inhibitory effects of this protein. To address this question, here, we transfected the MCF7 breast cancer cell line and the MCF10F nontumorigenic mammary epithelial cell line with a vector containing the p27Kip1 cDNA to obtain derivatives that express increased levels of p27Kip1. The increased expression of p27Kip1 in both of these cell lines was associated with lengthening of the G1 phase, an increase in the doubling time, a decreased saturation density, and a decreased plating efficiency. In the MCF7 cells, anchorage-independent growth and in vivo tumorigenicity were also suppressed. These effects were associated with decreased cyclin E-associated in vitro kinase activity in both cell lines. The increased expression of p27Kip1 was associated with a decreased level of expression of cyclin D1 in the MCF10F cells but an increased level of the cyclin D1 protein in the MCF7 cell line. Both derivatives expressed slightly increased levels of the cyclin E protein. Thus, breast cancer cells are still responsive to p27Kip1-mediated inhibition of cell growth despite the high basal level of this protein. These results suggest that therapeutic strategies that further increase the level of expression of p27Kip1 or mimic its activity might be useful in cancer therapy.