Modulation of the Expression of Bloom Helicase by Estrogenic Agents

Abstract

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 ...

Effects of Estrogenic Agents 17.BETA.-Estradiol (E2) and Bisphenol A on the Expression of RecQ DNA Helicases in Mammary Tumor MCF-7 Cells

“…This study supported our previous conclusions 7) that estrogenic agents stimulate cell proliferation by interaction with ER and also stimulate the expression of BLM helicase participating in DNA repair during the cell cycle. However, whether this stimulation is the direct cosequence of E2 signalling affecting BLM transcription or due to an indirect effect by DNA damage induced by E2-based oxygen radicals remains to be studied.…”

“…5,6) E2 upregulates cell cycle-associated genes, especially associated with DNA synthesis, and downregulates the expression of antiproliferative and proapoptotic genes, 5) resulting collectively in the formation of mammary tumor cells and sustained proliferation. A similar effect is also observed for bisphenol A (BPA), 7) an endocrine-disrupting chemical, widely used in material for polycarbonate plastic and epoxy resin. 8) BPA has a weak estrogenic activity to stimulate cell proliferation of estrogen receptor (ER)-positive mammary tumor cells, and so is attracting considerable attention.…”

“…8) We showed that transcription of not only BLM, but also cell-cycle-associated genes, such as MCM5, MCM2, Myt1, PCNA and AuroraA, were stimulated under the same conditions. 7) In this study, we examined mRNA expression of BLM, WRN, RTS, RecQ5 and RecQL1 (Fig. 1A).…”

Mutagenicity and DNA Damage of Bisphenol a and its Structural Analogues in Hepg2 Cells

“…Iso et al (53) reported that BPA induced DNA strand breaks in ER-positive MCF-7 cells (at 1 μmol L -1 and 100 μmol L -1 ) and that its genotoxicity was ER-dependent, as evidenced by much lower effect in ER-negative MDA-MB-231 cells. The increase in DNA strand breaks was signifi cant as soon as after three hours of exposure and increased even further up to hour 24, which is in agreement with our study.…”

Candidate SNP analyses integrated with mRNA expression and hormone levels reveal influence on mammographic density and breast cancer risk

“…Such women likely have elevated estrogen levels. Estradiol(E2) has previously been found to up-regulate MCM5, suggesting participation in DNA stability during increased cell proliferation by E2 [51]. To our knowledge CDC20 has not been associated with hormones previously but could share the same qualities as MCM5.…”