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Human cytochrome P4501A2 (CYP1A2) is involved in the metabolism of a large number of common drugs and is responsible for the metabolic activation of numerous promutagens and procarcinogens. Large interindividual differences exist in the expression of this enzyme. This variability has important implications for drug efficacy and cancer susceptibility. In this sudy, the methylation status of the CCGG site (bp -2759) located adjacent to an AP-1 site in the 5'-flanking region of the CYP1A2 gene was assessed in liver samples from a pool of 55 human donors. DNA methylation is an important epigenetic mechanism controlling gene expression and may be one of the molecular mechanisms underlying the interindividual variation. Analysis was conducted using Hpa II digestion and PCR. Results showed that individual samples varied in the methylation status at this site. The site was found to be hypermethylated in approximately 60% of the samples. To compare methylation status with level of CYP1A2 expression, results were analyzed by median test. In 44% of the samples with expression levels above the median the CCGG site was hypermethylated. However, for those samples with levels below the median hypermethylation of the site was found in 73% of the samples. The difference was statistically significant (p<0.05). These findings indicate that CpG methylation may be involved in controlling the expression of CYP1A2, with hypermethylation reducing expression. Moreover, this approach can be useful in assessing the role of site-specific DNA methylation in interindividual variation of CYP1A2. Analysis of other CpG sites in potentially important regulatory elements of the CYP1A2 gene will continue.

Cytochrome P4501B1 (CYP1B1) is involved in the activation of many carcinogens and in the metabolism of steroid hormones, including 17beta-oestradiol (E2) and testosterone. We report a significant difference in the allele frequencies of two point mutations in the coding region of the CYP1B1 gene among Caucasian (n = 189), African-American (n = 52) and Chinese (Linxian) (n = 109) populations. A (C to G) transversion at position 1666 in exon 3, which results in an amino acid substitution of Leu432 to Val, was present in African-Americans with an allele frequency for Va1432 of 0.75, in Caucasians of 0.43, and in Chinese of 0.17. A (C to T) transition at position 1719 in exon 3, with no amino acid change (Asp449), appeared to be closely linked with the Val432 variant. Results using human lung microsomal preparations from individuals with the CYP1B1Val/Val and CYP1B1Leu/Leu genotypes indicate that Val432 variant may be a high activity allele and thus may contribute to the interindividual differences in CYP1B1 activity. Because CYP1B1 is involved in hormone and carcinogen metabolism, and given the disparate rates of prostate cancer among ethnic groups, we also evaluated the association of the CYP1B1 Leu432Val polymorphism with prostate cancer risk in a pilot case-control study. Among Caucasians, 34% of men with cancer (n = 50) were homozygous for the Val432 polymorphism, while only 12% of matched control subjects (n = 50) had this genotype. These preliminary data indicate that genetic polymorphisms in CYP1B1 might play an important role in human prostate carcinogenesis.

Diet has been implicated as a possible link to the etiology, promotion and/or progression of many diseases, including cancer. Recently, interest has been focused on the cancer-protective role of several of the hormone-like diphenolic phytoestrogens, lignans, and isoflavonoids. This study examined the chemoprotective effects of genistein, biochanin A, equol, and coumestrol on human pancreatic adenocarcinoma cells in vitro. Two human adenocarcinoma cell lines, HPAF-11 from a male and Su 86.86 from a female, were used. HPAF-11 cells were exposed for 24 h to these agents at concentrations of 1 and 10 microM. Su 86.86 cells were exposed for 24 h at a concentration of 1 microM. Coumestrol and equol at higher concentrations were toxic to the Su 86.86 cells. These agents displayed marked differences between cell lines in inhibition of growth. Equol and coumestrol inhibited the growth of the female pancreatic tumor cells by 95%; however, these agents stimulated the growth of pancreatic tumor cells from the male. Genistein also stimulated growth in the male pancreatic tumor cells, but had little effect on pancreatic tumor cells from the female. Biochanin A inhibited growth of both male and female tumor cells, but to a lesser extent than other agents. This study also indicated a difference in K-ras expression in pancreatic tumors cells treated with these agents. Equol and coumestrol decreased K-ras expression in the female tumor cell line. Genistein increased expression of K-ras in both male and female pancreatic tumor cells. Genistein also increased expressions of the multidrug resistant (mdr-1) gene in the male tumor-cell line, while coumestrol and biochanin A decreased expression. Equol had no effect on mdr-1 expression. Whether the chemoprotective potential of equol and coumestrol against pancreatic cancer is greater in females than males is being further studied.

Pancreatic and prostate cancers pose serious problems to human health. To determine the potential for chemopreventive intervention against pancreatic and prostate cancers, black and green tea extracts and components of these extracts were examined in vitro for their effect on tumor cell growth. Components included a mixture of polyphenols from green tea (GTP), mixtures of polyphenols (BTP) and of theaflavins (MF) from black tea, and the purified components epicatechin-3-gallate (ECG) and epigallocatechin-3-gallate (EGCG). Two human cell lines, pancreatic adenocarcinoma (HPAC) and prostate tumor (LNCaP), were exposed to these agents for 24 hours. Results showed inhibition (approx 90%) of cell growth in pancreatic tumor cells by black and green tea extracts (0.02%). GTP (10 micrograms/ml) and MF (100 micrograms/ml) significantly inhibited growth (approx 90%); ECG and EGCG inhibited growth as well (approx 95%). Black and green tea extracts, GTP, and EGCG decreased the expression of the K-ras gene, as determined by reverse transcription-polymerase chain reaction. Green and black tea extracts decreased the multidrug-resistant gene (mdr-1), although GTP and EGCG increased expression. Similar data were obtained in the prostate cell line LNCaP. All agents significantly inhibited growth. These agents increased expression of the mdr-1 gene. This study suggests that components from black and green tea extracts can modulate the expression of genes known to play a role in the carcinogenesis process and, therefore, may be potential agents for chemoprevention against pancreatic cancer.

The DNA methyltransferase enzyme (DNA MTase) catalyzes DNA methylation at cytosines in CpG dinucleotides. 5-Methylcytosine modification of DNA is important in gene regulation, DNA replication, chromatin organization and disease. Increased levels of DNA MTase have been associated with the initiation and promotion of cancer. This study was conducted to assess whether cigarette smoking and other factors, such as age and gender, influence DNA MTase expression in nontumorous tissue. DNA MTase was significantly (p<0.05) higher in samples from cigarette smokers; the mean level of DNA MTase mRNA was almost 2-fold higher in these samples than in those from nonsmokers. Levels of DNA MTase mRNA were higher in samples from females than in those from males, but the difference was not statistically significant. Age was not associated with DNA MTase levels. Increased levels of DNA MTase in individuals who smoke may indicate a greater susceptibility to the risk of cancer since increased levels of this enzyme are found in cancer cell lines and human tumors. The results of this study suggest that further investigations of increased expression of this enzyme as a predisposing factor for cancer susceptibility are needed.

The resiliency of rats during early post-natal development to CCl4 or to an interactive hepatotoxicity of chlordecone (CD) + CCl4 has been shown to be due to an efficient stimulation of tissue repair. The objective of the current study was to investigate if this is due to efficient expression of transforming growth factor-alpha (TGF-alpha) and proto-oncogenes. Postnatally developing (20 day old) and adult (60 day old) male Sprague-Dawley rats were challenged with a single low dose of CCl4 (100 microL/kg, ip) or corn oil. Liver samples were collected during a time course (0-96 h) after the administration of CCl4 and used to examine TGF-alpha and early (c-fos) and late (H-ras and K-ras) proto-oncogenes mRNA expressions. Significant increases in TGF-alpha, H-ras, and K-ras gene expressions were evident as early as 12 hours after CCl4 and peaked between 24 and 48 hours in an age-dependent manner as detected by slot-blot analysis. Results of the study revealed three- and twofold increases in TGF-alpha gene expression in 20 and 60 day old rats, respectively, after CCl4. There were 3.5- and 2.5-fold increases in H-ras and 4.4- and 3.4-fold increases in K-ras in 20 and 60 day old rats, respectively. In contrast, a 10-fold increase in c-fos mRNA expression was evident in 20 day old rats 1 hour after CCl4 treatment, returning to the baseline value by 3 hours, whereas in 60 day old rats, this increase was less than twofold. The overall findings of this study indicate that TGF-alpha and the early and late proto-oncogene mRNA expressions were enhanced in an age- and time-dependent manner in response to a low dose of CCl4. These results further strengthen the view that the remarkable resiliency of rats to hepatotoxicants during early postnatal development is due to substantial increases in stimulation of hepatocellular regeneration and tissue repair mechanisms, leading to regression of liver injury and recovery.

Pancreatic acinar cells from rats treated in vitro with 5-azacytidine and/or transfected with an activated c-H-ras demonstrated transformation and tumorigenic phenotypes. DT-diaphorase (NAD(P)H:quinone oxidoreductase) activity was determined in these non-tumorigenic (3AP) and tumorigenic cells (T3AP and T5AM). T5AM cells were those treated with 5-azacytidine and further treated with N'-methyl-N'-nitro-nitrosoguanidine. Higher levels of enzyme activity were found in transformed cells when compared to that in control cells (> 15-fold, 3AP cells; > 40-fold, T3AP cells; > 20-fold T5AM cells). In contrast, NADPH-cytochrome c reductase activity was decreased in transformed cells (> 10-fold, 3AP cells; > 20-fold, T3AP cells; > 10-fold, T5AM cells). These studies demonstrated that pancreatic acinar cells are capable of undergoing alterations in enzyme activity patterns when transformed and that DT-diaphorase may be a good marker for malignant transformation.

Specific gene hypermethylation has been shown in DNA from neonatal rats exposed to the phytoestrogens, coumestrol, and equol. The pancreas is an organ in which estrogen receptors have been shown to be present. Studies have correlated the development of acute pancreatitis with rising levels of human estrogen binding proteins. Neonatal rats were dosed with 10 or 100 micrograms of coumestrol or equol on postnatal day (PND) 1-10. The animals were sacrificed at Day 15. The pancreas was excised and pancreatic acinar cells isolated for molecular analysis. DNA was isolated from the cells by lysis in TEN-9 buffer supplemented with proteinase K and 0.1% SDS. High molecular weight (HMW) DNA was digested with the methylated DNA specific restriction enzymes, Hpa II and Msp I, for determination of methylation profiles. Both coumestrol and equol at high doses caused hypermethylation of the c-H-ras proto-oncogene. No hypermethylation or hypomethylation was observed in the proto-oncogenes, c-myc or c-fos. Methylation is thought to be an epigenetic mechanism involved in the activation (hypomethylation) or inactivation (hypermethylation) of cellular genes which are known to play a role in carcinogenesis. Epidemiology studies have shown that equol may have anti-carcinogenic effects on some hormone-dependent cancers. Additional studies are needed to further understand the role of phytoestrogens and methylation in relation to pancreatic disorders.

Rat hepatic aryl sulfotransferase IV (AST IV), which catalyses sulfuric acid esterification of N-hydroxy-2-acetylaminofluorene to its ultimate carcinogenic form, is differentially expressed during multistep 2-acetylaminofluorene (AAF) hepatocarcinogenesis. Two molecular mechanisms associated with this effect involve modulation of mRNA translational capacity at the early stages, and gene transcription at the late stages of the carcinogenic process. To characterize further the molecular mechanisms that may be involved in the transient regulation of the enzyme expression, an AST IV cDNA was used to assess the change in methylation profile and restriction fragment length polymorphism (RFLP) in the gene domain of genomic DNA derived from rats at different stages of carcinogenesis. The onset of hypomethylation of the AST IV gene domain and amplification of a 5.3-kb DNA sequence was found to correlate with the stage in AAF hepatocarcinogenesis, where rats begin to exhibit irreversible loss in hepatic enzyme expression and the liver becomes committed to hepatoma formation. This represents the first observation of both altered methylation status of AST IV gene domain and amplification of a DNA sequence whose expression may play a role in the genesis and/or progression of neoplastic transformation of initiated cells during AAF hepatocarcinogenesis.

While the life-extending and disease-modulating effects of caloric restriction (CR) are well documented in whole animal studies and in correlative experiments using cells taken from CR animals, very few studies have used cells in culture after their removal from the CR-fed animal. In using this in vivo-->in vitro approach we have attempted to examine the proposition that the effects of CR can be transferred to individual cells by analyzing the cellular functions of proliferation and transformation, the activation of oncogenes, and the methylation of DNA as a function only of diet. Pancreatic acinar cells excised from CR-fed Brown-Norway rats and placed in rich medium showed different responses compared to cells from ad libitum (AL)-fed controls. CR had the effect of slowing growth rate and protecting against spontaneous and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced transformation over 14 passages of cells in culture. At the molecular level, cells from the CR animals showed reduced c-Ha-ras oncogene expression and mutation as well as reduced mutation of the p53 suppressor gene. CR also increased genomic methylation of ras DNA. We conclude that the effects of CR treatment of the animal are transferred to individual cells and note that these responses (decreased proliferation and transformation; depressed oncogene expression and mutation and decreased suppressor gene mutation; and increased oncogene methylation) are cellular and molecular analogs of in vivo weight loss, life extension, and carcinogenesis modulation, which are hallmarks of CR in the whole animal. The fact that these responses are seen generations after the cells are removed from the CR-treated animal indicates that CR causes a permanent predisposition of pancreatic acinar cells to these modulated responses and shows the value of the in vivo-->in vitro protocol in studies that relate diet to cellular and molecular function.

Testing of compounds for carcinogenic potential in vivo involves various experimental designs. A few of these techniques are directed to demonstrate the genotoxicity and mutagenicity of the compound by histopathology. These changes shown by histochemical means include monoclonal antibody directed cellular markers. Development of the polymerase chain reaction technique (PCR) for amplification of DNA has facilitated the investigation of molecular events related to the formation of malignant neoplasms. We describe here a method for screening tissues for mutations of the H-ras gene using monoclonal antibodies directed toward normal and mutant p21 proteins. Formalin-fixed, paraffin-embedded tissue sections are used to subsequently confirm the gene mutation by PCR amplification of the H-ras gene. The results indicated a successful application of this technique to demonstrate the presence of p21 oncoprotein in the tissues tested.

We have developed a system for serum-free culture of separated uterine epithelium and stroma from 11-day-old rats recombined on extracellular matrix extracted from Englebreth-Holm-Swarm tumors. Epithelium grew and, after 2 days in culture, developed into luminal epithelial spheres (LES) surrounding a fluid-filled lumen. Individual LES cells maintained epithelial cell characteristics such as basally located nuclei, apical microvilli (oriented toward the lumen), lateral membranes with interdigitations and desmosomes, secretory Golgi complexes, and abundant mitochondria and rough endoplasmic reticulum. Secretory vesicles were ubiquitous throughout the luminal fluid. Addition of 17 beta-estradiol to the growth medium increased the number and longevity of the LES. Prior exposure of uteri to tamoxifen via s.c. injection in vivo on postnatal Days 1 to 5 reduced or completely inhibited formation of LES in vitro. These effects occurred regardless of whether the stromal or epithelial component of the recombinant tissue was exposed to tamoxifen. These data suggest a directive property of neonatal stroma in culture resulting in the formation of highly secretory spherical epithelial structures completely enclosing a lumen. LES formation is responsive to both estrogen (positive response) and antiestrogen (negative response).

The study of immortalization and other alterations associated with neoplastic transformation of endometrial stromal cells is important to understanding the development of uterine sarcomas and mixed tumors. Because stromal cells are important regulators of associated epithelial cells, alterations in the regulation of stromal cell proliferation that influence epithelial cells may also contribute to the development of endometrial carcinomas. To study immortalization and associated phenotypic and genetic alterations of human endometrial stromal cells, cultures were transfected with a plasmid containing an ori-, temperature-sensitive mutant SV40, A209 (tsSV40). Morphologically transformed colonies were selected and propagated at the permissive temperature until they entered 'crisis'. In contrast to human fibroblasts, every clone tested was immortalization competent. The frequency of immortalization was approximately 1 x 10(-6). One uncloned and six cloned cell lines escaped from crisis and appear to be immortal. Two clones, M4 and B10T1, were selected for further study. Immortalization is conditional; proliferative arrest occurs at the restrictive temperature for large T antigen function. Furthermore, withdrawal of the large T antigen results in expression of the senescent phenotype of enlarged, flattened cells. Colony-forming efficiency at the restrictive temperature was undetectable. Immortalization is also associated with several genetic alterations. The DNA content of tsSV40 transfected cells was either diploid or tetraploid in the precrisis stage of proliferation, but became aneuploid upon immortalization. Several structural rearrangements of chromosomes were detected in the immortalized stromal cells which differ from those found in SV40 immortalized fibroblasts. Although their capacity for anchorage-independent proliferation (AIP) is variable, tsSV40-immortalized endometrial stromal cells have a higher capacity for AIP than their tsSV40-transfected progenitor cells in the period of proliferation prior to 'crisis'.

Pancreatic acinar cells were isolated for culture from a young (Y) and an old (O) Brown-Norway or Fischer 344 rat fed an ad libitum (AL) or calorically restricted (CR) diet. The cells were cultured and cellular growth rates were determined as a function of passage number. An overall increase in cellular growth rate and transformation frequency with age and/or AL diet relative to youth as well as a decrease with CR diet were concordant with reported responses in vivo. Transformation frequency was measured in Brown-Norway cells and followed the same pattern as the growth response: AL/O > AL/Y = CR/Y > CR/O. The cellular model is shown to fit the general multistage requirements of the carcinogenic process as well as general age and diet characteristics of pancreatic cancer. This pancreatic acinar cell age-diet approach may prove to be a valuable tool for determining mechanisms of exocrine pancreatic carcinogenesis as well as other disease states; it may also be of utility in in vitro gerontological nutritional and pharmacological studies since some of the age and diet determinants of biological effects appear to be segregable. Propensity of cells from an old and/or AL diet animal for faster growth and for cellular transformation are programmed into the cells by the time of their excision from the animal (as late as 14 months), indicating a heritable component in the model or a mechanism that is dependent upon elements that control gene expression.

This study examines proto-oncogene hypomethylation in rat livers during the early stages of hepatocarcinogenesis by dietary methyl deprivation in the presence and absence of initiation by diethylnitrosamine (DEN). Male weanling F344 rats were fed a complete diet, or a diet deficient in methionine and choline (MDD). Half the animals in each dietary group were given a single initiating dose of DEN (20 mg/kg). Animals from each of the treatment groups were killed at 1, 3, 8, 16 and 32 weeks, and hepatic DNA was isolated. This DNA was digested with the restriction enzymes MspI and HpaII to determine the extent of methylation of the CCGG sequences in c-Ha-ras, c-Ki-ras and c-fos proto-oncogenes. The results indicate that the administration of the MDD produced hypomethylation of these proto-oncogenes at all times investigated, independent of DEN initiation. The methylation changes in the c-Ha-ras gene increased in intensity throughout the experiment until at 32 weeks they were similar to the patterns seen in both neoplastic and preneoplastic livers of rats fed the deficient diet for 18 months. These results demonstrate that early, selective hypomethylation of some, but not all, CCGG sites occurs in rats undergoing hepatocarcinogenesis by dietary methyl deprivation.

We have developed a tissue culture system using an extract of basement membrane (extracellular matrix) which promotes the in vitro growth and development of uterine luminal epithelium from the 5-day-old rat. Uterine luminal epithelium, free of stroma, was obtained as short tubes by trypsinization of uterine segments followed by mechanical separation. Epithelial segments were grown in a serum-free medium on culture dishes coated with an extracellular matrix. After 2 days, rapid cell growth resulted in monolayer cultures, which subsequently formed organoid structures similar to differentiated uterine glands present in uterine tissue taken from older rats. Electron microscopy of cultures revealed columnar cells with basally located nuclei, apical microvilli, lateral membranes with interdigitations, desmosomes, and secretory Golgi complexes, all features found in functioning uterine epithelium in vivo. This model will allow the in vitro investigation of the development of uterine epithelium-specific functions free of the influence of stromal cell factors.

Stromal cells isolated from normal human endometrium were cotransfected in primary culture with pSVc-myc, a plasmid containing a truncated c-myc gene regulated by simian virus 40 promoter, and pRSV neo, a plasmid containing a neomycin resistance gene regulated by Rous sarcoma virus (RSV) promoter. These cells demonstrated properties of transformed cells in vitro, including altered morphology, focus formation, anchorage-independent growth, chromosomal alterations, and tumor formation in athymic mice. When these cells were treated subsequently with a direct-acting carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine, they demonstrated higher colony-forming efficiency in soft agar and reduced tumor latency. Cells transfected with pRSV neo alone exhibited some properties associated with neoplastic transformation, including altered morphology and formation of colonies in soft agar. It is presumed that in normal cells transfected with pRSV neo, RSV long terminal repeats activated cellular genes that normally regulate growth of human endometrial stromal cells.

Various regulators of protein kinase activities were tested for their effects on the in vitro transfer of phosphate from [gamma-32P]ATP to four proteins of rat brain synaptic particulate preparations. One protein, of apparent molecular weight 44,000, accepted 32P in the presence of 8 mM EDTA and no added Mg2+. It was the major phosphoprotein of brain mitochondria. Its phosphorylation was inhibited by pyruvate and stimulated by K+, and it comigrated in electrophoretic gels with authentic alpha-subunit of pyruvate: lipoamide oxidoreductase (decarboxylating) (EC 1.2.4.1) from bovine heart. The major kinase acting on three proteins of apparent molecular weights 24,000, 21,000, and 19,000 was stimulated by Ca2+, by preincubation with phospholipase C, and by 12-tetradecanoyl 4-beta-phorbol 13-acetate. Phosphorylation of these lower-molecular-weight proteins was inhibited by ACTH1-24, by cyclic 3',5'-adenosine monophosphate, and by 50 microM trifluoperazine. The stimulatory effect of Ca2+ was antagonized by calmodulin. The kinase in question appears to be B-50 protein kinase or protein kinase C.

In vitro transfer of 32P from [gamma-32P]-ATP into proteins of particulate fractions from osmotically shocked preparations enriched in rat brain synaptosomes was studied. Phosphate incorporation into protein bands of apparent molecular weights (MW) 44,000, 24,000, 21,000, and 19,000 was affected by the prior experiences of the rats from which the particulate fractions were prepared. Incorporation into all four proteins was increased in particulate fraction from previously naive rats that received active avoidance training. Handling of the subjects prior to training prevented the response of the 24,000 MW protein to training. Phosphate incorporation into 24,000 and 19,000 MW proteins was increased in preparations from previously naive rats that underwent a yoked experience, while incorporation into the 21,000 MW protein was slightly decreased. The yoked experience did not affect in vitro phosphate incorporation into any of these proteins in particulate fractions from previously handled rats.

We have developed methods for the culture of human endometrial glandular epithelia in vitro. The culture medium is serum-free and is used in combination with Matrigel, an extracellular matrix material applied as a coating on cell culture plates. Cell growth begins as a monolayer, but the cells subsequently form glandular or organoid structures. The glands are composed of polar columnar cells facing a central lumen, which is enclosed by the apical surfaces of cells displaying numerous microvilli and sealed by tight junction complexes. The ability to study in vitro the complex process of glandular morphogenesis represents an important new tool in cell biology which may be used to investigate growth regulation, hormone production and dependency, and cellular recognition and interactions. Ultimately, these characteristics may be applied to study the alterations of glandular epithelia associated with neoplasia.