| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2004: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2003: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2002: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Research Abstract |
The fact that the genetic alterations of PTEN are frequently found in hormone-dependent cancers such as endometrial, breast, and prostata cancers, might suggest the involvement of PTEN in the hormone dependent cell growth of such tumors. Estrogen and estrogen-induced peptide growth factors promote the cell growth of endometrial glandular cells and would be associated with endometrial carcinogenesis. Endometrial carcinoma cells often lose steroid hormone-responsiveness during the carcinogenesis. We investigated whether PTEN modulates estrogen receptor status, the levels of enzymes of endogenous estrogen synthesis, and responsiveness against peptide growth factors in endometrial carcinoma cells. [Materials and methods] Recombinant adenovirus was produced by Adeno-X expression system. PTEN-null Ishikawa cell, an endometrial carcinoma cell line, was infected with the recombinant adenovirus at 20MOI to express efficiently PTEN protein. In Ishikawa cells expressing PTEN protein (PTEN-IK cell
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s), the followings were analyzed. 1)The expression levels of the enzymes for estrogen synthesis such as estrone sulfatase, estrone sulfotransferase, aromatase, 17 beta-hydroxysteroid dehydrogenases were analyzed by R-PCR. 2)The expression levels of estrogen receptors (alpha and beta) and their phosphorylated forms were analyzed by western blot and/or immunoprecipitation. 3)responsiveness against peptide growth factors including EGF was analyzed by MTT assay and western blot. [Results] (1)In-PTEN-IK cells, the estrone sulfatase and 17beta-hydroxysteroid dehydrogenase type 1 were upregulated when compared with those of parental Ishikawa cells. (2)The expression level of ER-beta was upregulated in PTEN-IK cells, but ER-alpha was not changed regardless of the downregutation of its phosphorylated form. (3)The cell growth of parental IK cells was significantly stimulated by EGF and IGF-1, and PTEN-IK cells were further sensitized the EGF or IGF1-growth stimulation. EFGR antibody could completely compromise the stimulatory effects of EGF in both cell lines. Wortmannin, a PI3K inhibitor, or UO126, a MPAK inhibitor, suppressed EGF-mediated cell growth stimulation partly in both cell lines. EGF augmented the level of phospho-Akt/PKB of PTEN-IK cells more effectively than that of parental IK cells. [Conclusion]PTEN regulates the level of ER-beta, phosphorylation level of ER-alpha, and intracellular estrogen concentration by modulating the levels of estrogen synthetic enzymes. Thus, the deficiency of PTEN protein alters estrogenic environment of the cells. Furthermore, the dysfunction of PTEN reduces responsiveness of EGF or other peptide growth factors of the cells. Endometrial carcinoma cells with deficient PTEN, thus, escape from hormone-dependency. Less
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