Epidemiological studies and animal experiments have shown carcinogenic properties of estrogen. Studies to clarify the molecular mechanisms of carcinogenesis by estrogen suggest that estrogen causes carcinogenic effects by combined genotoxicity and stimulation of cell proliferation. [1][2][3] Estrogen causes DNA damage by estrogen-derived oxidants, 4,5) DNA adducts formed by estrogen metabolites 5,6) and formation of micronuclei. 7,8) Recent studies strongly suggest that DNA damage induced by estrogen is dependent on estrogen receptors (ER): the ER antagonist tamoxifen inhibits E2 effects in ER-positive MCF-7 cells, but not in ERnegative MDA-MB-231 cells. 4,9) Detoxifying enzyme activity markedly decreases by treatment with 17b-estradiol (E2) in MCF-7 cells, leading to increased susceptibility of cells to DNA damage, but E2 has no effect on detoxifying enzyme activity in MDA-MB-231 cells. 4)Bisphenol A (BPA) was first shown to be estrogenic in 1938 in ovariectomized rats 10) and later in MCF-7 human breast cancer cell culture assay. 11) BPA is an endocrine-disrupting chemical and has a weak affinity for ER, estimated at about 1/1000 of E2, 12) and its additional estrogenic effects on the hormonal homeostatic system has recently received much attention.We also studied proteins involved in the repair of DNA damage induced by E2 and BPA. Histone H2AX (H2AFX) is responsible for maintaining genomic stability by recognizing DNA double-strand breaks.13) At the sites of stalled replication forks, H2AX is phosphorylated to gH2AX, which forms foci 14) that appear immediately after DNA damage and recruit proteins responsible for repair of DNA damage, including Bloom helicase (BLM), 14,15) the product of BLM that is the causative gene of Bloom syndrome, an autosomal recessive genetic disorder. 16,17) Clinical features of patients having Bloom syndrome include growth retardation, immunodeficiency, male infertility but not female infertility, and a high incidence of cancers. Cells from Bloom syndrome patients show a high frequency of sister chromatid exchange. 18,19) BLM responds to DNA damage and accumulates at the site of DNA double-strand breaks and physically interacts with gH2AX.15) In this study we investigated the effects of E2 and BPA on DNA damage in ER-positive MCF-7 and ER-negative MDA-MB-231 cells, both of which are derived from adenocarcinomas; these effects were assessed by alkaline single cell electrophoresis (Comet assay). We also investigated colocalization of gH2AX and BLM at sites of DNA damage. Science and Technology, Japan Science and Technology Agency; Kawaguchi Center Building, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan: and c Department of Urology, St Marianna University School of Medicine; 2-16-1 Sugao, Miyamae, Kawasaki 216-8511, Japan. Received September 15, 2005; accepted November 15, 2005 Evidence exists that raises concern about genotoxic effects induced by estrogen: oxidative stress caused by estrogen-derived oxidants, DNA adducts formed by estrogen metabolites and estrogen-induce...

Differentiation of ST-13 preadipocytes into adipocytes was inhibited almost completely by addition of rat plasma fibronectin (FN) (approximately 100 micrograms/mL), but was reversed by GRGDSP cell recognition peptide (1.5 mM) and anti-alpha 5 beta 1. On the contrary, the thermolysin digest of FN stimulated adipocyte differentiation in a dose-dependent manner, in which remarkable increases in the values of the differentiation indexes, the number of adipocytes (8-fold above the control), glycerophosphate dehydrogenase (GPD) activity (12-fold), and triacylglycerol content (5-fold), were observed by inclusion of the thermolysin digest (100 micrograms/mL). The increase in GPD activity by the thermolysin digest was inhibited remarkably (about 70% inhibition) by an antibody directed to the amino-terminal fibrin-binding (Fib 1) domain of FN and slightly (about 15%) by an antibody directed to the central cell-binding (Cell) domain, but not by anti-gelatin-binding domain and anti-carboxy-terminal fibrin-binding domain. Treatment of ST-13 cells by a purified 24K fragment (100 micrograms/mL) derived from the Fib 1 domain caused an over 20-fold augmentation of the GPD activity, accounting for a major part of the differentiation stimulatory activity of the thermolysin digest. The differentiation stimulatory effect of the 24K Fib 1 fragment was not affected by either GRGDSP peptide or anti-alpha 5 beta 1. Thus, FN can regulate adipose development of ST-13 cells by its two antipodal, inhibitory and stimulatory, activities, the latter of which is expressed only upon fragmentation. Proteolytic cleavage of FN may play an important role in controlling the action of FN on adipocyte differentiation.

Carcinogenesis by 17b-estradiol (E2) is believed to be associated with its genotoxicity and stimulating activity of cell proliferation.1,2) Studies using laboratory animals 3,4) and cultured cells [5][6][7][8] showed that E2 induces tumors. E2 suppresses detoxifying enzymes, such as catalase and glutathione-S-transferase, leading to an increase in genotoxicity by E2. 9) We showed previously that E2 causes DNA damage in mammary tumor MCF-7 cells assessed by single cell gel electrophoresis, the so-called Comet assay, and that the estrogen receptor (ER) is responsible for DNA damage.10) A similar effect was also observed for bisphenol A (BPA), an endocrine-disrupting chemical, 11) widely used as material for polycarbonate plastic and epoxy resin. BPA has a weak estrogenic activity to stimulate cell proliferation of ER-positive mammary tumor cells, and so is attracting considerable attention. 12,13) We showed previously the genotoxic effect of BPA as assessed by Comet assay, 10) but the BPA effect was much less than the effect of E2.DNA microarray technology identifies profiles of estrogen responsive genes.14,15) Cell cycle-associated genes whose expression is up-regulated by E2, particularly those having a role in DNA synthesis 14) have been studied. E2 down regulates antiproliferative and proapototic genes, 14) resulting in cell proliferation and cell survival in mammary tumor cells. During active proliferation cells need genome-maintaining enzymes, such as RecQ helicases, 16) to keep DNA fidelity in replictation. In this study, we focused on the expression of Bloom helicase (BLM), a RecQ helicase, in MCF-7 cells stimulated by E2 to study the possible roles of BLM in E2-treated cells. In additional experiments, pre-treatment with ICI182780 was done for 1 h and then by E2 or BPA treatments. The chemical agents were solubilized in ethanol and the final concentration of ethanol in the culture was adjusted to 0.1%. Control culture cells were exposed to a culture medium containing 0.1% ethanol. MATERIALS AND METHODS ChemicalsGene Expression Reverse transcriptase-polymerase chain reaction (RT-PCR) based on the TaqMan method with ABI prism 7000 (Applied Biosystems, Foster City, CA, U.S.A.) was used to quantify BLM mRNA. A One-Step RT-PCR Master Mix Reagents Kit and TaqMan MGB probes specific for BLM was purchased from Applied Biosystems. Total RNA was extracted by using an RNeasy Mini kit (Qiagen, Valencia, CA, U.S.A.) according to the manufacturer's protocol. All RNA samples were assayed in triplicate. The number of changes in expression was calculated by using a comparative C T method (ABI PRISM user bulletin #2) with human b-glucronidase expression as an internal control.Antibodies and Immunoblotting Immunoblot analysis of BLM was done as described previously.17) Briefly, after treatment with E2 or BPA, cells were solubilized in RIPA buffer containing 20 mM Tris-HCl (pH 7.4), 0.1% sodium dodecyl sulfate (SDS), 1% TritonX-100, 1% sodium deoxy- Kawasaki, Kanagawa 216-8511, Japan. Received September 19, 2006; accepted ...

Nateglinide is an oral antidiabetic medication that acts through rapid, short-term stimulation of insulin production. This study was undertaken to identify the incidence and nature of adverse effects of nateglinide and to assess its efficacy in clinical practice. Patients (n = 3254) were recruited from 606 centers in Japan with a 12-week observation period. Pretreatment and posttreatment values were obtained for fasting blood glucose, postprandial blood glucose, hemoglobin A1c (HbA1c), triglycerides, cholesterol, and body mass index. All adverse events were reported, along with standard laboratory blood variables. The incidence of adverse events was 7.40%; hypoglycemia, including hypoglycemic symptoms, was reported as the most prevalent (1.62%). Adverse events were observed more frequently in patients with hepatic or renal dysfunction; no significant findings were noted in the remaining patient population. The efficacy rating determined by the treating physicians was 76.40%. HbA1c decreased by 0.81% from 7.70+/-1.53% to 6.89+/-1.22%, postprandial glucose decreased by 54.05 mg/dL from 228.91+/-73.69 mg/dL to 174.86+/-62.86 mg/dL, and fasting glucose decreased by 23.73 mg/dL from 164.15+/-51.42 mg/dL to 140.43+/-42.63 mg/dL. These effects were most marked in patients who were previously medication naïve or who had been diagnosed with diabetes for a short period. Mean body mass index decreased, and nateglinide was equally effective in obese patients. Nateglinide showed good therapeutic effect when used as the first choice in patients with a short duration of diabetes, and in those with no history of previous treatment. Moreover, nateglinide seemed to be useful for the treatment of elderly patients and obese patients.