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ABSTRACT: RAD51D, a paralog of the mammalian RAD51 gene, contributes towards maintaining genomic integrity by homologous recombination DNA repair and telomere maintenance. A RAD51D variant, E233G, was initially identified as a potential susceptibility allele in high-risk, site-specific, familial breast cancer. We describe in this report that the Rad51d (E233G) genetic variant confers increased cisplatin resistance and cell growth phenotypes in human breast carcinoma cell lines with a mutant p53 gene (BT20 and T47D) but not with a wild-type p53 gene (MCF-7). Treatment with a p53 specific inhibitor, pifithrin alpha, restored this resistant phenotype in the MCF-7 cell line. Additionally, Rad51d (E233G) conferred increased cisplatin resistance of an MCF7 cell line in which p53 expression was stably knocked down by shRNAp53, indicating that the effect of this variant is dependent upon p53 status. Further study of Rad51d (E233G) will provide mechanistic insight towards the role of RAD51D in cellular response to anticancer agents and as a potential target for cancer therapy.
Molecular Carcinogenesis 05/2009; 48(7):586-91. · 3.16 Impact Factor
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Sonia M Najjar,
Yan Yang,
Mats A Fernström,
Sang-Jun Lee,
Anthony M Deangelis,
George A Abou Rjaily,
Qusai Y Al-Share,
Tong Dai,
Tiffany A Miller,
Shobha Ratnam, Randall J Ruch,
Stuart Smith,
Sue-Hwa Lin,
Nicole Beauchemin,
Ana Maria Oyarce
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ABSTRACT: Insulin is viewed as a positive regulator of fatty acid synthesis by increasing fatty acid synthase (FAS) mRNA transcription. We uncover a new mechanism by which insulin acutely reduces hepatic FAS activity by inducing phosphorylation of the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) and its interaction with FAS. Ceacam1 null mice (Cc1(-/-)) show loss of insulin's ability to acutely decrease hepatic FAS activity. Moreover, adenoviral delivery of wild-type, but not the phosphorylation-defective Ceacam1 mutant, restores the acute effect of insulin on FAS activity in Cc1(-/-) primary hepatocytes. Failure of insulin to acutely reduce hepatic FAS activity in hyperinsulinemic mice, including L-SACC1 transgenics with liver inactivation of CEACAM1, and Ob/Ob obese mice, suggests that the acute effect of insulin on FAS activity depends on the prior insulinemic state. We propose that this mechanism acts to reduce hepatic lipogenesis incurred by insulin pulses during refeeding.
Cell Metabolism 08/2005; 2(1):43-53. · 13.67 Impact Factor
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ABSTRACT: Phosphorylation of the cell adhesion protein CEACAM1 increases insulin sensitivity and decreases insulin-dependent mitogenesis in vivo. Here we show that CEACAM1 is a substrate of the EGFR and that upon being phosphorylated, CEACAM1 reduces EGFR-mediated growth of transfected Cos-7 and MCF-7 cells in response to EGF. Using transgenic mice overexpressing a phosphorylation-defective CEACAM1 mutant in liver (L-SACC1), we show that the effect of CEACAM1 on EGF-dependent cell proliferation is mediated by its ability to bind to and sequester Shc, thus uncoupling EGFR signaling from the ras/MAPK pathway. In L-SACC1 mice, we also show that impaired CEACAM1 phosphorylation leads to ligand-independent increase of EGFR-mediated cell proliferation. This appears to be secondary to visceral obesity and the metabolic syndrome, with increased levels of output of free fatty acids and heparin-binding EGF-like growth factor from the adipose tissue of the mice. Thus, L-SACC1 mice provide a model for the mechanistic link between increased cell proliferation in states of impaired metabolism and visceral obesity.
Journal of Clinical Investigation 11/2004; 114(7):944-52. · 15.39 Impact Factor
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ABSTRACT: The inhibition of gap junctional intercellular communication (GJIC) is a common effect of nongenotoxic carcinogens and might be a biomarker for these agents. To further test this relationship, we hypothesized that phenobarbital would inhibit mouse hepatocyte GJIC and this would correlate with strain-specific hepatocarcinogenicity. Phenobarbital is a strong nongenotoxic hepatocarcinogen in B6C3F1 mice, but not in C57BL/6 mice. Hepatocytes were isolated from males of both strains, placed in coculture with rat liver epithelial cells, and treated with phenobarbital for up to 14 days. Male mice were also administered PB by single intraperitoneal injection (0.1 mg/kg), then sacrificed 24 h later, or given phenobarbital in the drinking water (500 ppm) for 14 days before sacrifice. GJIC was assayed in cocultures by fluorescent dye microinjection and in isolated liver tissue by fluorescent dye "cut-loading." Phenobarbital decreased GJIC only in cultured B6C3F1 hepatocytes; this was dose-responsive and temporary, because hepatocyte GJIC returned to control levels within 24 h of phenobarbital exposure. Administration of phenobarbital to mice for 14 days also decreased hepatocyte dye coupling in B6C3F1 liver, but this effect was not seen in C57BL/6 mice or observed after a single administration of the drug. Phenobarbital did not alter connexin32 and connexin26 expression, but increased hepatic Cyp2b1 expression and the liver weight:body weight ratio in both strains. In summary, phenobarbital inhibited mouse hepatocyte GJIC in vivo and in vitro and in correlation with strain-specific hepatocarcinogenicity. These data support the hypothesis that decreased GJIC is a biomarker for nongenotoxic carcinogens and involved in their carcinogenic mechanism.
Toxicological Sciences 03/2003; 71(2):190-7. · 4.65 Impact Factor
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ABSTRACT: Connexin32 (Cx32) encodes the predominant gap junction protein expressed by hepatocytes. We investigated the transcriptional control of Cx32 in expressing and nonexpressing rat liver cell lines and hypothesized that a putative hepatocyte nuclear factor-1 (HNF-1) binding site (centered at mp -187) in the liver-active, P1 promoter is essential for transcription of Cx32. HNF-1alpha was expressed by Cx32-expressing rat liver cell lines and bound the promoter at the -187 site, but was not expressed by non-Cx32-expressing hepatic lines. Stable transfection of non-Cx32-expressing WB-F344 rat liver epithelial cells with HNF-1alpha stimulated a transfected Cx32 promoter element (mp -244 to -33), binding of HNF-1alpha to the -187 site, and expression of endogenous Cx32. Site-directed mutagenesis of this HNF-1 binding site abolished HNF-1alpha binding and proximal promoter activity. Hepatic Cx32 expression was also significantly decreased in HNF-1alpha(-/-) mice. These data indicate that HNF-1alpha is a positive regulator of Cx32 expression in hepatic cells.
Archives of Biochemistry and Biophysics 12/2002; 407(2):160-7. · 2.93 Impact Factor
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Randall J Ruch
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ABSTRACT: The article highlighted in this issue is "Inhibition of Gap Junctional Intercellular Communication by Perfluorinated Compounds in Rat Liver and Dolphin Kidney Epithelial Cell Lines in Vitro and Sprague-Dawley Rats in Vivo" by W. Hu, P. D. Jones, B. L. Upham, J. E. Trosko, C. Lau, and J. P. Giesy (pp. 429-436).
Toxicological Sciences 09/2002; 68(2):265-6. · 4.65 Impact Factor
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ABSTRACT: Gap junctional intercellular communication and expression of gap junction proteins (connexins) are decreased frequently in neoplastic cells including human ovarian carcinoma cells. In order to test the hypothesis that these changes contribute to the neoplastic phenotype of ovarian carcinoma cells, we transfected human ovarian carcinoma SKOV-3 cells with connexin43. Stable, connexin43-expressing transfectants were characterized for cell proliferation in vitro in normal, low-serum, and serum-free culture medium, for tumorigenicity in nude mice, and for sensitivity to adriamycin in vitro. Transfected clones expressed higher levels of connexin43 and gap junctional intercellular communication, reduced proliferation and greater dependence upon serum for growth in vitro, decreased tumor formation, increased sensitivity to adriamycin, and reduced expression of p-glycoprotein. These data suggest that gap junctional intercellular communication and/or connexin43 expression suppresses the neoplastic phenotype of ovarian carcinoma cells and their downregulation is involved in neoplastic transformation of ovarian epithelial cells. The increased sensitivity to adriamycin and elevated expression of p-glycoprotein by the transfected cells also suggest that gap junctional intercellular communication and connexin43 expression are involved in drug sensitivity and might be manipulated to enhance the clinical response.
Experimental and Molecular Pathology 09/2002; 73(1):54-60. · 2.42 Impact Factor
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ABSTRACT: CEACAM1, a tumor suppressor (previously known as pp120), is a plasma membrane protein that undergoes phosphorylation on Tyr(488) in its cytoplasmic tail by the insulin receptor tyrosine kinase. Co-expression of CEACAM1 with insulin receptors decreased cell growth in response to insulin. Co-immunoprecipitation experiments in intact NIH 3T3 cells and glutathione S-transferase pull-down assays revealed that phosphorylated Tyr(488) in CEACAM1 binds to the SH2 domain of Shc, another substrate of the insulin receptor. Overexpressing Shc SH2 domain relieved endogenous Shc from binding to CEACAM1 and restored MAP kinase activity, growth of cells in response to insulin, and their colonization in soft agar. Thus, by binding to Shc, CEACAM1 sequesters this major coupler of Grb2 to the insulin receptor and down-regulates the Ras/MAP kinase mitogenesis pathway. Additionally, CEACAM1 binding to Shc enhances its ability to compete with IRS-1 for phosphorylation by the insulin receptor. This leads to a decrease in IRS-1 binding to phosphoinositide 3'-kinase and to the down-regulation of the phosphoinositide 3'-kinase/Akt pathway that mediates cell proliferation and survival. Thus, binding to Shc appears to constitute a major mechanism for the down-regulatory effect of CEACAM1 on cell proliferation.
Journal of Biological Chemistry 02/2002; 277(2):1076-84. · 4.77 Impact Factor
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ABSTRACT: Gap junctional intercellular communication (GJIC) and the expression of gap junction proteins (connexins) may be involved in growth regulation and neoplastic transformation. The mechanisms of connexin gene regulation in normal and neoplastic tissues are poorly understood. In this study, the glucocorticoids, dexamethasone and hydrocortisone, enhanced fluorescent dye-coupling in primary cultured rat hepatocytes and MH 1 C 1 rat hepatoma cells. Other types of steroids (β-estradiol, testosterone, aldosterone and progesterone) had no effect. Northern blot, Western blot, nuclear run-on and immunohistochemical analyses showed that glucocorticoids enhanced the expression of connexin32 in these cells in a dose- and time-dependent fashion. Connexin26 expression was also enhanced slightly by dexamethasone in hepatocytes, but not MH 1 C 1 cells. Connexin43 expression in these cells was not affected by steroids. In WB-F344 rat liver epithelial cells, which were highly coupled and expressed high levels of connexin43 and no detectable connexin32 or connexin26, dexamethasone had no effect on coupling or connexin expression. These results indicate that dye-coupling and the expression of connexin32 and connexin26, but not connexin43, were upregulated by glucocorticoids in a cell-specific manner. These effects on GJIC and connexin expression may be involved in the induction of hepatic differentiation and inhibition of growth.
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ABSTRACT: Overview summary The transfer of HSV-TK into tumor cells and the subsequent sensitization to GCV have resulted in successful antitumor effects both in vitro and in vivo for a variety of cancers. This study focuses on evaluating and comparing two colon carcinoma cell lines for their ability to metabolize GCV and transfer phosphorylated metabolites to neighboring non-HSV-TK-expressing cells (bystander effect). Here we demonstrate differences in HSV-TK expression, GCV triphosphate accumulation, and incorporation into DNA and their effect on cytotoxicity. We also provide evidence of the transfer of phosphorylated GCV to bystander cells in a cell line deficient in gap junctional intercellular communication. Peer Reviewed http://deepblue.lib.umich.edu/bitstream/2027.42/63405/1/hum.1998.9.6-801.pdf
