Biomedical subjects

J C Barrett

Publications and source records attributed to J C Barrett.

At least 55 records · Page 3Linked to original sources

PubMed2000

Mammalian cell transformation and aneuploidy induced by five bisphenols.

Bisphenol-A (BP-A), a monomer of plastics used in numerous consumer products and a xenoestrogen, induces cellular transformation and aneuploidy in Syrian hamster embryo (SHE) cells. In this study, the abilities of 4 other bisphenols to induce cellular transformation and genetic effects in SHE cells were examined and compared to BP-A. Cellular growth was inhibited by all bisphenols in a concentration-related manner. The growth inhibitory effect of the bisphenols ranked: BP-5 > BP-4 > BP-3 > BP-2 or BP-A. Morphological transformation of SHE cells was induced by BP-A, BP-3, BP-4 and BP-5, and the induced-transformation frequencies were highest with BP-4. None of the bisphenols induced gene mutations at the Na(+)/K(+) ATPase locus or the hprt locus, or chromosomal aberrations in SHE cells. By contrast, aneuploidy induction in the near-diploid range was exhibited by BP-A, BP-3, BP-4 or BP-5, corresponding to the transforming activity of each compound. The results indicate that BP-A, BP-3, BP-4 and BP-5 exhibit transforming activity in SHE cells, while BP-2 does not, and that aneuploidy induction may be a causal mechanism of the transforming activity.

Aneuploidy↗

PubMed2000

Involvement of genotoxic effects in the initiation of estrogen-induced cellular transformation: studies using Syrian hamster embryo cells treated with 17beta-estradiol and eight of its metabolites.

To examine a direct involvement of genotoxic effects of estrogens in the initiation of hormonal carcinogenesis, the abilities of 17beta-estradiol (E2) and 8 of its metabolites to induce cellular transformation and genetic effects were studied using the Syrian hamster embryo (SHE) cell model. Treatment with E2, estrone (E1), 2-hydroxyestrone (2-OHE1), 4-hydroxyestrone (4-OHE1), 2-methoxyestrone (2-MeOE1), 16alpha-hydroxyestrone (16alpha-OHE1), 2-hydroxyestradiol (2-OHE2), 4-hydroxyestradiol (4-OHE2) or estriol (E3) for I to 3 days inhibited SHE cell growth in a concentration-dependent manner. Concentration-dependent increases in the frequency of morphological transformation in SHE cells were exhibited by treatment for 48 hr with each of all estrogens examined, except for E3. The transforming activities of the estrogens, determined by the induced transformation frequencies, were ranked as follows: 4-OHE1 > 2-OHE1 > 4-OHE2 > 2-OHE2 > or = E2 or E1 > 2-MeOE1 or 16alpha-OHE1 > E3. Somatic mutations in SHE cells at the Na+/K+ATPase and /or hprt loci were induced only when the cells were treated with 4-OHE1, 2-MeOE1 or 4-OHE2 for 48 hr. Some estrogen metabolites induced chromosome aberrations in SHE cells following treatment for 24 hr. The rank order of the clastogenic activities of the estrogens that induced chromosome aberrations was 4-OHE1 > 2-OHE1 or 4-OHE2 > 2-OHE2 > E1. Significant increases in the percentage of aneuploid cells in the near diploid range were exhibited in SHE cells treated for 48 hr or 72 hr with each of the estrogens, except for 4-OHE1 and E3. Our results indicate that the transforming activities of all estrogens tested correspond to at least one of the genotoxic effects by each estrogen, i.e., chromosome aberrations, aneuploidy or gene mutations, suggesting the possible involvement of genotoxicity in the initiation of estrogen-induced carcinogenesis.

Aneuploidy↗

PubMed2000

Biomarkers of oxidative stress study: are plasma antioxidants markers of CCl(4) poisoning?

Antioxidants in the blood plasma of rats were measured as part of a comprehensive, multilaboratory validation study searching for noninvasive biomarkers of oxidative stress. For this initial study an animal model of CCl(4) poisoning was studied. The time (2, 7, and 16 h) and dose (120 and 1200 mg/kg, intraperitoneally)-dependent effects of CCl(4) on plasma levels of alpha-tocopherol, coenzyme Q (CoQ), ascorbic acid, glutathione (GSH and GSSG), uric acid, and total antioxidant capacity were investigated to determine whether the oxidative effects of CCl(4) would result in losses of antioxidants from plasma. Concentrations of alpha-tocopherol and CoQ were decreased in CCl(4)-treated rats. Because of concomitant decreases in cholesterol and triglycerides, it was impossible to dissociate oxidation of alpha-tocopherol and the loss of CoQ from generalized lipid changes, due to liver damage. Ascorbic acid levels were higher with treatment at the earliest time point; the ratio of GSH to GSSG generally declined, and uric acid remained unchanged. Total antioxidant capacity showed no significant change except for 16 h after the high dose, when it was increased. These results suggest that plasma changes caused by liver malfunction and rupture of liver cells together with a decrease in plasma lipids do not permit an unambiguous interpretation of the results and impede detection of any potential changes in the antioxidant status of the plasma.

Animals↗

PubMed2000

Increased cell survival by inhibition of BRCA1 using an antisense approach in an estrogen responsive ovarian carcinoma cell line.

STATEMENT OF FINDINGS: We tested the hypothesis that BRCA1 may play a role in the regulation of ovarian tumor cell death as well as the inhibition of ovarian cell proliferation. Introduction of BRCA1 antisense retroviral constructs into BG-1 estrogen-dependent ovarian adenocarcinoma cells resulted in reduced BRCA1 expression. BRCA1 antisense pooled populations and derived subclones were able to proliferate in monolayer culture without estrogen, whereas control cells began to die after 10 days of estrogen deprivation. In addition, both populations and subclones of BRCA1 antisense infected cells demonstrated a growth advantage in monolayer culture in the presence of estrogen and were able to proliferate in monolayer culture without estrogen, while control cells did not. Furthermore, clonal studies demonstrated that reduced levels of BRCA1 protein correlated with growth in soft agar and greater tumor formation in nude mice in the absence of estrogen. These data suggest that reduction of BRCA1 protein in BG-1 ovarian adenocarcinoma cells may have an effect on cell survival during estrogen deprivation both in vitro and in vivo.

Adenocarcinoma↗

PubMed2000

The insulin-like growth factor-1 elevates urokinase-type plasminogen activator-1 in human breast cancer cells: a new avenue for breast cancer therapy.

Tumor recurrence is a common problem in the treatment of breast cancer. In breast cancer, the expression of high protein levels of the insulin-like growth factor-1 receptor (IGF-1R) and urokinase-type plasminogen activator-1 (uPA) is strongly associated with breast cancer recurrence and decreased survival. The expression of uPA by tumors is thought to not only stimulate tumor invasion but also facilitate angiogenesis. In this study, our goal was to address whether IGF-1R could influence the expression of the extracell ular matrix proteases, matrix metalloproteinase (MMP), or uPA thus allowing a selective advantage for tumor invasion and concomitant neovascularization. Initially, we determined whether or not insulin-like growth factor (IGF)-1 regulated the production MMP or uPA in the human breast cancer MDA-MB-231 cells. There was no increase in MMP activity when the cells were treated with IGF-1 (10 ng/mL) for 24 h. In contrast, uPA mRNA and protein were induced in a time-dependent manner. Furthermore, clones expressi ng a dominant negative inhibitor of IGF-1R termed 486stop had less uPA mRNA, and the clones were less invasive through Matrigel. Taken together, these data illustrate that IGF-1R stimulates uPA production. Hence, these two prognostic indicators may be interrelated, suggesting they may function in a synergistic manner to facilitate local tumor invasion as well as angiogenesis. Our data suggest that disruption of IGF-1 signaling in breast cancer may lead to breast cancer prevention and intervention by decreasing uPA expression.

Blotting, Northern↗

PubMed2000

Establishment and characterization of a breast cell strain containing a BRCA1 185delAG mutation.

OBJECTIVE: The aim of this study was to examine whether cells containing the heterozygous form of a BRCA1 185delAG mutation would exhibit abnormal growth or an altered response to DNA damage. METHODS: A primary culture of human mammary epithelial cells (90P) was obtained from the nontumor breast tissue of a 35-year-old patient who had undergone a mastectomy for removal of a breast tumor. These cells were immortalized (90PE6E7) following retroviral infection with HPV-16 viral E6/E7. genes. Both the 90P cell strain and the cell line were characterized for their ability to grow in culture, form colonies in soft agar, and produce tumors in athymic nude mice compared to normal breast epithelial cells containing wild-type BRCA1. 90P cells were also analyzed for cellular response to gamma radiation and H(2)O(2). RESULTS: These cells were confirmed to contain a frameshift mutation, 185delAG, of the BRCA1 gene. Despite being heterozygous for wild-type BRCA1, the 220-kDa full-size BRCA1 protein was abundantly expressed. 90P and 90PE6E7 cells grew at a similar rate and were anchorage dependent. 90PE6E7 also failed to form tumors in athymic nude mice. Finally, 90P cells exhibited a survival response similar to that of normal mammary epithelial cells to radiation damage and exposure to oxidative stress. CONCLUSION: To our knowledge the 90P cells and the 90PE6E7 cells are the first characterized, non-tumor-derived breast epithelial cells that are heterozygous for the BRCA1 germline mutation 185delAG. Our conclusion is that these BRCA1 mutant cells appear to have growth and stress response characteristics similar to those of normal human breast cells, which is consistent with the hypothesis that loss of heterozygosity must occur to impair putative BRCA1 function.

Adult↗

PubMed2000

Regulation of p53 stability and activity in response to genotoxic stress.

The p53 tumor suppressor is a universal sensor of genotoxic stress that regulates the transcription of genes required for cell-cycle arrest and apoptosis. In response to DNA damage, the p53 protein is phosphorylated at its amino-terminus and becomes stabilized upon disruption of an interaction with its negative regulator, MDM2. Subsequent phosphorylation and acetylation of p53 promote different interactions with other proteins and with target gene regulatory elements to facilitate cell-cycle arrest, apoptosis, or adaptation in response to DNA damage. Downstream of p53, p21 is responsible for growth arrest in G1, but other p53 target genes are responsible for G2 cell-cycle arrest. In response to genotoxic insult, p53-induced apoptosis results from overlapping downstream pathways that both suppress mitogenic and survival signaling and promote pro-apoptotic signaling. Adaptation to DNA damage is manifested by p53-mediated expression of its negative regulator, MDM2. The frequency of observed mutations in p53 predicts that its inactivation is a requisite step in tumorigenesis, as p53 is mutated in approximately 50% of human tumors. Thus, it is likely that in the remaining tumors, genetic aberrations will occur in pathways that regulate p53 or in pathways directly downstream of p53. The advances in the understanding of p53 signaling over the past few years point to many potential overlapping signaling pathways, where mutations may occur as alternative modes to p53 mutation.

DNA Damage↗

PubMed2000

Apoptotic response to growth factor deprivation involves cooperative interactions between c-Fos and p300.

Two preneoplastic cell lines have been utilized to study changes in the regulation of apoptosis during neoplastic progression [sup+I (stage I) and sup-II (stage II)]. Sup+I cells are prone to undergo apoptosis, while sup-II cells are relatively resistant. We report that induction of apoptosis in sup+I cells is tightly correlated with the formation of c-Fos/p300 complexes, which were not present in the non-apoptotic sup-II cells under the same conditions. When apoptosis was induced in the sup-II cells by over-expression of c-Fos, concomitant c-Fos:p300 complexes were detected. Over-expression of p300 resulted in apoptosis in sup-II cells and also in p53wt human tumor cells, but not in p53mutant human tumor cells. Over-expression of the C-terminal fragment of p300, which contains the c-Fos binding site, enhanced apoptosis, suggesting that the c-Fos:p300 complex is actively involved in apoptosis. We propose that p300 could function as a general mediator of transcription factor-induced apoptosis.

Animals↗

PubMed2000

Suppressed tumorigenicity of human endometrial cancer cells by the restored expression of the DCC gene.

To obtain functional evidence for DCC as a tumour suppressor associated with endometrial cancer, the human DCC cDNA encoding a complete open reading frame (ORF) was transfected into highly tumorigenic human endometrial carcinoma cells, HHUA and Ishikawa in which DCC expression was completely deleted. Reconstituted expression of DCC in HHUA had little effect on in vitro growth, but suppressed tumour formation in mice completely. The clones from Ishikawa had abundant DCC expression similar to that in normal endometrium. Their growth in vitro was suppressed and showed apoptotic phenotype. Lower levels of DCC expression in the prolonged passaged clones did not induce apoptosis, but still had the potential to suppress tumorigenicity. These observations imply a role of DCC in regulation of normal endometrial cell growth, and categorize DCC as the tumour suppressor gene for endometrial cancer.

Animals↗

PubMed2000

Molecular and environmental causes of cancer.

The multistep, clonal evolution model of cancer predicts fundamental mechanisms by which chemicals may influence the cancer process. These are illustrated by studies of the mechanisms of induction of carcinogenesis by estrogens and the inhibition of carcinogenesis by caloric restriction.

Carcinogens, Environmental↗

PubMed2000

Induction of mammalian cell transformation and genotoxicity by 2-methoxyestradiol, an endogenous metabolite of estrogen.

2-methoxyestradiol (2-MeOE(2)) is an endogenous metabolite of 17beta-estradiol and a proposed inhibitor of tumor growth and angiogenesis. However, 2-MeOE(2) is also an inhibitor of microtubule assembly and other microtubule inhibitors, e.g. colcemid and diethylstilbestrol, induce aneuploidy and cell transformation in cultured mammalian cells. To assess the in vitro carcinogenicity and related activity of 2-MeOE(2), the abilities of this metabolite to induce cell transformation and genetic effects were studied simultaneously using Syrian hamster embryo (SHE) fibroblasts. Growth of these cells was reduced by treatment with 2-MeOE(2) at 0.1-1.0 microg/ml in a concentration-dependent manner. Treatment of SHE cells with 2-MeOE(2) at 0.3 or 1.0 microg/ml for 2-48 h also resulted in a concentration- and treatment time-related increase in the mitotic index and the percentage of multinucleated cells. Treatment with 2-MeOE(2) at 0.1-1.0 microg/ml for 48 h induced a statistically significant increase in the frequencies of morphological transformation of SHE cells in a concentration-dependent manner. A statistically significant increase in the frequencies of somatic mutations at the Na(+)/K(+) ATPase or hprt locus was also observed in cells treated with 2-MeOE(2) for 48 h at 0.1 or 0.3 microg/ml, respectively. Treatment of SHE cells with 2-MeOE(2) at 0.3 or 1.0 microg/ml for 24 h induced chromosome aberrations, mainly breaks, exchanges and chromosome pulverization. The incidence of chromosome aberrations was not affected by co-treatment with alpha-naphthoflavone, an inhibitor of 2-hydroxylase that inhibits oxidative conversion of 2-MeOE(2) to 2-hydroxyestradiol, but the incidence was slightly increased by co-treatment with L-ascorbic acid. Numerical chromosomal changes in the near diploid range and in the tetraploid and near tetraploid ranges were also detected in 2-MeOE(2)-treated cells. These findings indicate that 2-MeOE(2) has cell transforming and genotoxic activities in cultured mammalian cells and potential carcinogenic activity.

2-Methoxyestradiol↗

PubMed2000

Hypermethylation of the p16 (Ink4a) promoter in B6C3F1 mouse primary lung adenocarcinomas and mouse lung cell lines.

Primary lung tumors from B6C3F1 mice and mouse lung cell lines were examined to investigate the role of transcriptional silencing of the p16 (Ink4a) tumor suppressor gene by DNA hypermethylation during mouse lung carcinogenesis. Hypermethylation (>/=50% methylation at two or more of the CpG sites examined) of the p16 (Ink4a) promoter region was detected in DNA from 12 of 17 (70%) of the B6C3F1 primary mouse lung adenocarcinomas examined, whereas hypermethylation was not detected in normal B6C3F1, C57BL/6 and C3H/He mouse lung tissues. Immunohistochemistry performed on the B6C3F1 lung adenocarcinomas revealed heterogeneous expression of the p16 protein within and among the tumors. Laser capture microdissection was employed to collect cells from immunostained sections of four tumors displaying areas of relatively high and low p16 expression. The methylation status of the microdissected samples was assessed by sodium bisulfite genomic sequencing. The pattern of p16 expression correlated inversely with the DNA methylation pattern at promoter CpG sites in nine of 11 (82%) of the microdissected areas displaying variable p16 expression. To provide further evidence that hypermethylation is involved in the loss of p16 (Ink4a) gene expression, three mouse lung tumor cell lines (C10, sp6c and CMT64) displaying complete methylation at seven promoter CpG sites and no p16 (Ink4a) expression were treated with the demethylating agent, 5-aza-2'-deoxycytidine. Re-expression of p16 (Ink4a) and partial demethylation of the p16 (Ink4a) promoter were observed in two cell lines (C10 and sp6c) following treatment. These are the first reported studies to provide strong evidence that DNA methylation is a mechanism for p16 inactivation in mouse lung tumors.

Adenocarcinoma↗

PubMed2000

Elevated ceramide is downstream of altered calcium homeostasis in low serum-induced apoptosis.

Two immortalized cell lines, sup (+) and sup (-), derived from mutagenized Syrian hamster embryo cells, were used to study the relationship and temporal order between calcium and ceramide signals during apoptosis. The early preneoplastic cells, termed sup (+), suppress tumorigenicity when hybridized with tumor cells, whereas later-stage sup (-) cells do not. In reduced serum conditions, sup (+) cells cease proliferating and undergo apoptosis; in contrast, sup (-) cells continue slow growth and undergo necrosis. In sup (+) cells, decreased endoplasmic reticulum (ER) calcium occurs 4 h after low serum treatment and precedes apoptosis. Significant elevations in ceramide are observed 16 h after reduced serum treatment of sup (+) cells but are not found in sup (-) cells. Inhibiting ER calcium depletion in low serum-treated sup (+) cells by treating with high levels of calcium prevents both ceramide generation and apoptosis. Conversely, inducing ER calcium depletion in sup (-) cells by treating with low serum plus thapsigargin results in elevated ceramide levels and apoptosis. Furthermore, C(6)-ceramide treatment induced apoptosis of sup (-) cells in low serum, a condition that does not normally cause apoptosis. C(6)-ceramide treatment did not induce apoptosis in either sup (+) or sup (-) cells in 10% serum but did cause G(2)/M arrest. These studies show that ceramide production is downstream of ER calcium release.

Animals↗

PubMed2000

Genomic imprinting and environmental disease susceptibility.

Genomic imprinting is one of the most intriguing subtleties of modern genetics. The term "imprinting" refers to parent-of-origin-dependent gene expression. The presence of imprinted genes can cause cells with a full parental complement of functional autosomal genes to specifically express one allele but not the other, resulting in monoallelic expression of the imprinted loci. Genomic imprinting plays a critical role in fetal growth and behavioral development, and it is regulated by DNA methylation and chromatin structure. This paper summarizes the Genomic Imprinting and Environmental Disease Susceptibility Conference held 8-10 October 1998 at Duke University, Durham, North Carolina. The conference focused on the importance of genomic imprinting in determining susceptibility to environmentally induced diseases. Conference topics included rationales for imprinting: parental antagonism and speciation; methods for imprinted gene identification: allelic message display and monochromosomal mouse/human hybrids; properties of the imprinted gene cluster human 11p15.5 and mouse distal 7; the epigenetics of X-chromosome inactivation; variability in imprinting: imprint erasure, non-Mendelian inheritance ratios, and polymorphic imprinting; imprinting and behavior: genetics of bipolar disorder, imprinting in Turner syndrome, and imprinting in brain development and social behavior; and aberrant methylation: methylation and chromatin structure, methylation and estrogen exposure, methylation of tumor-suppressor genes, and cancer susceptibility. Environmental factors are capable of causing epigenetic changes in DNA that can potentially alter imprint gene expression and that can result in genetic diseases including cancer and behavioral disorders. Understanding the contribution of imprinting to the regulation of gene expression will be an important step in evaluating environmental influences on human health and disease.

Animals↗

PubMed2000

The environmental genome project: ethical, legal, and social implications.

The National Institute of Environmental Health Sciences is supporting a multiyear research initiative examining genetic influences on environmental response. Proponents of this new initiative, known as the Environmental Genome Project, hope that the information learned will improve our understanding of environmentally associated diseases and allow clinicians and public health officials to target disease-prevention strategies to those who are at increased risk. Despite these potential benefits, the project presents several ethical and social challenges. Of immediate concern is the protection of individual research participants. Other ethical issues relate to the application of research results and how study findings could affect social priorities. Clarifying these emerging areas of concern, many of which have not received adequate attention in the existing bioethics literature, is an important step toward minimizing potential research-related risks and defining research needs.

Environmental Health↗

PubMed2000

Pregnancy, parturition, and prostaglandins: defining uterine leiomyomas.

Leiomyomas, benign smooth muscle tumors of the uterus, are the most common gynecologic neoplasm in women. Studies with surgically resected human tissues and primary cultures have revealed that several genes are differentially expressed in leiomyomas compared to matched normal myometrium. An estrogen-driven pattern of gene expression in leiomyomas, similar to that seen in normal myometrium during pregnancy and parturition, is associated with a persistent inappropriate response of neoplastic myometrial smooth muscle cells to ovarian hormones. This is possibly due to aberrant expression levels or signaling via estrogen and progesterone receptors. We propose the hypothesis that uterine leiomyomas mimic a differentiated myometrial cell at pregnancy and exhibit a hypersensitivity to sex steroid hormones that prevents the cells from responding to normal apoptotic or dedifferentiation signals and from returning to a nongravid phenotype. Support of this hypothesis is derived from experimental studies in female Eker rats that develop uterine leiomyomas with many similarities to the human disease. Our hypothesis accounts for the benign nature of these tumors and their high incidence in women during the reproductive years. By identifying the factors that participate in parturition and involution of the pregnant myometrium, we may better define uterine leiomyomas and thus identify novel targets for therapeutic strategies to treat these tumors.

Animals↗

PubMed2000

Molecular cloning of human Hic-5, a potential regulator involved in signal transduction and cellular senescence.

By using differential display, we cloned the human counterpart of the murine gene Hic-5 from senescent human keratinocytes. The full-length cDNA contained a short GC-stretch proceeding a consensus Kozak sequence followed by a single open reading frame of 1338 bp encoding a 461-amino acid protein with a predicted molecular weight of 50 kDa. The expression of this gene was prominent in cells of epithelial origin but low or absent in lymphoid tissues and hematopoietic cells. The deduced protein contained four LIM domains at the carboxyl-terminal end and four LD motifs at the amino-terminal half, sharing high similarities with the focal adhesion protein paxillin. Hic-5 may therefore function, like paxillin, as a potential adapter for the recruitment of structural and signaling molecules to certain subcellular sites or in focal adhesions. Isolation of the genomic sequence revealed that the gene covered a segment of 6 kb and spanned 11 exons from the translation initiation site ATG to the termination signal TGA. Fluorescent in situ hybridization by using a human Hic-5 specific probe localized the gene to human chromosome 16p11.

Amino Acid Sequence↗

PubMed2000

Racial disparity in the frequency of PTEN mutations, but not microsatellite instability, in advanced endometrial cancers.

Survival of African Americans with endometrial cancer is significantly worse than that of whites. Mutation of the PTEN tumor suppressor gene and microsatellite instability occur in some endometrial cancers, and they are associated with favorable prognostic features. The aim of this study was to determine whether there is a racial disparity in the frequency of these molecular alterations that contributes to differences in outcome in advanced endometrial cancer. We screened 140 stage III/IV endometrial adenocarcinomas (78 Caucasian, 62 African American) for mutations in the PTEN gene. Paired DNA samples were available in 100 cases and were analyzed for microsatellite instability using three polymorphic markers. African-American women had cancers with significantly higher stage and grade that were more often nonendometrioid. In addition, median survival of African Americans (1.0 years) was worse than that of whites (2.5 years; P = 0.02). PTEN mutation was seen in 20 of 140 (14%) cancers and was associated with endometrioid histology and more favorable survival. The frequency of PTEN mutations was significantly higher in whites (17 of 78; 22%) than in African Americans (3 of 62; 5%; P = 0.006). Microsatellite instability was found in 15% of cancers, exclusively in endometrioid cases, and was associated with favorable survival (P = 0.01). There was no racial difference in the frequency of microsatellite instability. We conclude that mutation of the PTEN tumor suppressor gene is associated with favorable survival in advanced endometrial cancer and is 4-fold more frequent in Caucasians relative to African Americans. This suggests that differences in the frequency of PTEN mutations contribute to the racial disparity in endometrial cancer survival.

Adenocarcinoma↗