Proteomics Profiling of Pancreatic Cancer and Pancreatitis for Biomarkers Discovery.

Pancreatic cancer is one of the most aggressive malignancies with an increase in incidence predicted, particularly in African Americans. Pancreatic cancer is considered a silent disease with poor prognosis and a lack of early biomarkers for detection. Proteomics has been applied in many diseases for identifying or discovering biomarkers. It has long been suggested that chronic pancreatitis may be a risk factor for developing pancreatic cancer. This study identified proteins that are altered in expression in pancreatic cancer and pancreatitis compared to normal using proteomic technology. Proteins were extracted from laser captured micro-dissected tissues and separated in 2-DPAGE and imaged. The protein profiles of pancreatic cancer and pancreatitis are similar but differed with the protein profile of normal adjacent tissues. Representative proteins, overexpressed in tumor and pancreatitis but not normal tissues, were excised from gels, subjected to in-gel digestion, and analyzed by MALDI-TOF mass spectrometry. Proteins identified included transferrin, ER-60 protein, proapolipoprotein, tropomyosin 1, alpha 1 actin precursor, ACTB protein, and gamma 2 propeptide, aldehyde dehydrogenase 1A1, pancreatic lipase and annexin A1. Several proteins, which were shown in pancreatic cancer, were also observed in pancreatitis samples. Understanding the role of these specific proteins and their mechanistic action will give insights into their involvement in pancreatic cancers.

Canopy nitrogen, carbon assimilation, and albedo in temperate and boreal forests: Functional relations and potential climate feedbacks.

The availability of nitrogen represents a key constraint on carbon cycling in terrestrial ecosystems, and it is largely in this capacity that the role of N in the Earth's climate system has been considered. Despite this, few studies have included continuous variation in plant N status as a driver of broad-scale carbon cycle analyses. This is partly because of uncertainties in how leaf-level physiological relationships scale to whole ecosystems and because methods for regional to continental detection of plant N concentrations have yet to be developed. Here, we show that ecosystem CO(2) uptake capacity in temperate and boreal forests scales directly with whole-canopy N concentrations, mirroring a leaf-level trend that has been observed for woody plants worldwide. We further show that both CO(2) uptake capacity and canopy N concentration are strongly and positively correlated with shortwave surface albedo. These results suggest that N plays an additional, and overlooked, role in the climate system via its influence on vegetation reflectivity and shortwave surface energy exchange. We also demonstrate that much of the spatial variation in canopy N can be detected by using broad-band satellite sensors, offering a means through which these findings can be applied toward improved application of coupled carbon cycle-climate models.

Biogeographic history of an Australian freshwater shrimp, Paratya australiensis (Atyidae): the role life history transition in phylogeographic diversification.

The widespread distribution of the freshwater shrimp Paratya australiensis in eastern Australia suggests that populations of this species have been connected in the past. Amphidromy is ancestral in these shrimps, although many extant populations are known to be restricted to freshwater habitats. In this study, we used a fragment of the cytochrome c oxidase I mitochondrial DNA (mtDNA) gene to examine diversity within P. australiensis and to assess the relative importance of amphidromy in its evolutionary history. We hypothesized that if transitions from an amphidromous to a freshwater life history were important, then we would find a number of divergent lineages restricted to single or groups of nearby drainages. Alternatively, if amphidromy was maintained within the species historically, we expected to find lineages distributed over many drainages. We assumed that the only way for divergence to occur within amphidromous lineages was if dispersal was limited to between nearby estuaries, which, during arid periods in the earth's history, became isolated from one another. We found nine highly divergent mtDNA lineages, estimated to have diverged from one another in the late Miocene/early Pliocene, when the climate was more arid than at present. Despite this, the geographic distribution of lineages and haplotypes within lineages did not support the notion of a stepping-stone model of dispersal between estuaries. We conclude that the extensive divergence has most likely arisen through a number of independent amphidromy-freshwater life history transitions, rather than via historical isolation of amphidromy populations. We also found evidence for extensive movement between coastal and inland drainages, supporting the notion that secondary contact between lineages may have occurred as a result of drainage rearrangements. Finally, our data indicate that P. australiensis is likely a complex of cryptic species, some of which are widely distributed, and others geographically restricted.

Increased levels of NAD(P)H: quinone oxidoreductase 1 (NQO1) in pancreatic tissues from smokers and pancreatic adenocarcinomas: A potential biomarker of early damage in the pancreas.

NAD(P)H:quinone oxidoreductase 1 (NQO1) is elevated in several human tumors. This study was conducted to determine whether increased levels of NQO1 expression also occur in human pancreatic tumor tissue, and to compare expression levels in nontumorous tissue from smokers with those in nonsmokers. The expression of NQO1 was examined in pancreatic tissue samples from 82 human donors. These samples included normal (n = 20), smokers (n = 25), pancreatitis (n = 7), and adenocarcinomas of the pancreas (n = 30). Genotyping for the C609T polymorphism in NQO1 by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was also performed. Polymorphic variants were confirmed by automatic sequencing. Higher levels of NQO1 expression were demonstrated in pancreatic adenocarcinomas (0.831 +/- 0.021) compared to those in nontumorous tissues from nonsmokers (0.139 +/- 0.024). These high levels were also found in smokers (0.729 +/- 0.167) and in pancreatitis tissues (0.923 +/- 0.184). NQO1 activity was also higher in smokers (2.43 +/- 0.61 nmol/min per mg protein) compared to nonsmokers (0.44 +/- 0.05 nmol/min per mg protein; p < 0.05). No differences were found in genotype distribution and frequencies of the variant alleles between normal and cancer tissues in this relatively small sample pool. Seventy-five percent of the normal pancreatic tissues showed 609(C/C) and 25% 609(C/T). In pancreatic adenocarcinomas the frequency distribution was 65% C/C, 30% C/T and 5% T/T. The increased expression in noncancer pancreatic tissue from smokers and the fact that smoking is a moderate risk factor for pancreatic cancer suggest that NQO1 expression may be a good candidate as a biomarker for pancreatic cancer, especially in risk groups such as smokers.

Component and whole-system respiration fluxes in northern deciduous forests.

We measured component and whole-system respiration fluxes in northern hardwood (Acer saccharum Marsh., Tilia americana L., Fraxinus pennsylvanica Marsh.) and aspen (Populus tremuloides Michx.) forest stands in Price County, northern Wisconsin from 1999 through 2002. Measurements of soil, leaf and stem respiration, stem biomass, leaf area and biomass, and vertical profiles of leaf area were combined with biometric measurements to create site-specific respiration models and to estimate component and whole-system respiration fluxes. Hourly estimates of component respiration were based on site measurements of air, soil and stem temperature, leaf mass, sapwood volume and species composition. We also measured whole-system respiration from an above-canopy eddy flux tower. Measured soil respiration rates varied significantly among sites, but not consistently among dominant species (P < 0.05 and P > 0.1). Annual soil respiration ranged from 8.09 to 11.94 Mg C ha(-1) year(-1). Soil respiration varied linearly with temperature (P < 0.05), but not with soil water content (P > 0.1). Stem respiration rates per unit volume and per unit area differed significantly among species (P < 0.05). Stem respiration per unit volume of sapwood was highest in F. pennsylvanica (up to 300 micro mol m(3) s(-1)) and lowest in T. americana (22 micro mol m(3) s(-1)) when measured at peak summer temperatures (27 to 29 degrees C). In northern hardwood stands, south-side stem temperatures were higher and more variable than north-side temperatures during leaf-off periods, but were not different statistically during leaf-on periods. Cumulative annual stem respiration varied by year and species (P < 0.05) and averaged 1.59 Mg C ha(-1) year(-1). Leaf respiration rates varied significantly among species (P < 0.05). Respiration rates per unit leaf mass measured at 30 degrees C were highest for P. tremuloides (38.8 nmol g(-1) s(-1)), lowest for Ulmus rubra Muhlenb. (13.1 nmol g(-1) s(-1)) and intermediate and similar (30.2 nmol g(-1) s(-1)) for T. americana, F. pennsylvanica and Q. rubra. During the growing season, component respiration estimates were dominated by soil respiration, followed by leaf and then stem respiration. Summed component respiration averaged 11.86 Mg C ha(-1) year(-1). We found strong covariance between whole-ecosystem and summed component respiration measurements, but absolute rates and annual sums differed greatly.

Specific site methylation in the 5'-flanking region of CYP1A2 interindividual differences in human livers.

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.

Human CYP1B1 Leu432Val gene polymorphism: ethnic distribution in African-Americans, Caucasians and Chinese; oestradiol hydroxylase activity; and distribution in prostate cancer cases and controls.

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.

The effects of phytoestrogens on human pancreatic tumor cells in vitro.

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.

Increased expression of hepatic DNA methyltransferase in smokers.

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.

Chemopreventive effects of tea extracts and various components on human pancreatic and prostate tumor cells in vitro.

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.

Age-related differences in TGF-alpha and proto-oncogenes expression in rat liver after a low dose of carbon tetrachloride.

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.

Increased DT-diaphorase activity in transformed and tumorigenic pancreatic acinar cells.

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.

Methylation profile and amplification of proto-oncogenes in rat pancreas induced with phytoestrogens.

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.

Hypomethylation of the rat aryl sulfotransferase IV gene and amplification of a DNA sequence during multistage 2-acetylaminofluorene hepatocarcinogenesis.

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.

Effects of caloric restriction in animals on cellular function, oncogene expression, and DNA methylation in vitro.

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.

Retrieval and amplification of DNA from unstained histopathological sections.

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.

Growth of separated and recombined neonatal rat uterine luminal epithelium and stroma on extracellular matrix: effects of in vivo tamoxifen exposure.

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).

Conditional immortalization of human endometrial stromal cells with a temperature-sensitive simian virus 40.

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'.

An in vitro pancreas acinar cell model for testing the modulating effects of caloric restriction and ageing on cellular proliferation and transformation.

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.

The onset of oncogene hypomethylation in the livers of rats fed methyl-deficient, amino acid-defined diets.

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.