Kv1.3 钾离子通道在卵巢癌细胞株中的表达及活性

目的:研究Kv1.3钾离子通道在SKOV3卵巢癌细胞中的表达及其在细胞增殖和细胞周期中的作用。方法:应用RT-PCR和免疫细胞化学鉴别Kv1.3钾离子通道在SKOV3卵巢癌细胞中的表达。应用MTT和流式细胞技术观察KV1.3钾离子通道对SKOV3卵巢癌细胞增殖及细胞周期的影响。结果:4-氨基吡啶是Kv1.3钾离子通道特异性阻滞剂。不同浓度的4-氨基吡啶可以明显抑制SKOV3细胞的增殖,并且细胞周期也受到影响。G0/G1细胞比例增加,S期和G2/M期细胞比例下降。结论:Kv1.3钾离子通道在SKOV3卵巢癌细胞中表达,并且在细胞增殖及细胞周期变换中扮演着重要的角色。     

Loss of ovarian function and the risk of ovarian cancer

Animal models with premature ovarian failure resulting from the loss or depletion of germ cells consistently develop ovarian surface epithelial cell hyperplasia with invasion into the stroma and the development of ovarian tubular adenomas. In human ovaries, deep epithelial invaginations and inclusion cysts occur at increasing frequency with age and are thought to be the structures from which the majority of ovarian cancers arise. A feature that is common to these animal models and to post-menopausal women is a deficiency in the number of oocytes. The potential consequences of the loss or depletion of female germ cells, naturally or otherwise, include failure of follicle development, significant reductions in oestrogen and progesterone levels and elevation of circulating levels of gonadotropins. This review will consider the way in which these structural and hormonal changes affect ovarian cancer risk. Some lessons may be learned from gonad formation, since notable similarities exist between ovarian tumorigenesis and embryonic gonadogenesis including fragmentation of the basement membrane underlying the coelomic (surface) epithelium, the potential for the migration of epithelial cells into the gonad and the importance of the germ cells for the regulation of ovarian structure and function. Research was supported by grants from the National Cancer Institute of Canada and the Canadian Institutes of Health Research.     

Esterase-like and fibronectin-like polypeptides share similar sex-cell-biased patterns in the gonad of hermaphroditic and gonochoric species of bivalve mollusks

The mechanism of sexualization of the tubular gonad in seawater bivalves is unknown, and no information regarding the genes involved in this process is yet available, except for the identification of esterase (Est)-like "male-associated polypeptide" in the male gonad of Mytilus galloprovincialis. Our present work reveals distinct protein profiles specific for the testicular or ovarian portion of the ovotestis of Pecten maximus. Two proteins exhibiting testis- or ovary-dependent enrichment in the ovotestis have been identified and partially characterized as Est-like and fibronectin (Fn)-like polypeptides, respectively. Immunofluorescence has demonstrated a close association between the localization of these polypeptides and the gonad tubule network and interstitial stroma of the ovotestis of P. maximus. We also present evidence of Est-like and Fn-like protein enrichment, respectively, in testicular and ovarian tissue in hermaphroditic, sex-reversal, and gonochoric species of seawater bivalves. Together, the results (1) strongly suggest that sex-cell-biased expression of Est-like and Fn-like polypeptides in gonad tissue is a widespread phenomenon among bivalve mollusks, despite the high diversification of their sexual patterns, (2) confirm and expand our previous demonstration of sex-biased protein expression in M. galloprovincialis, and (3) indicate a direct link between germ cell differentiation and sexual specialization of the bivalve somatic gonad.     

Novel action of estrone on vascular tissue: regulation of NOS and COX activity

The hypothesis tested in the present work is that estrone non-genomically regulates aortic nitric oxide synthase (NOS) and cyclooxygenase (COX) activities in female rats, and that such regulation depends on ovarian function. We found that physiological concentrations of estrone (E(1)) (0.1-10nM) significantly increased nitric oxide (NO) production (133 and 163% above control). The stimulatory action of E(1) on NOS activity was independent of calcium influx since the increase in NO elicited by the hormone was not affected by EGTA or verapamil. When COX activity was measured, we observed that estrone enhanced thromboxane (TXB(2)) production and prostacyclin (PGI(2)) release, but not prostaglandin (PGF(2), PGD(2), and PGE(2)) synthesis. Finally we demonstrated that the hormonal effect on NOS activity was not detected in rat aortic strips (RAS) isolated from animals deprived of ovarian activity (FR(-)) or ovariectomized rats (OVX). These results suggest that estrone exerts a direct, non-genomic action on rat aortic metabolism, which involves NOS and COX activation and depends on ovarian activity.     

BAG-1 p29 protein prevents drug-induced cell death in the presence of EGF and enhances resistance to anoikis in SKOV3 human ovarian cancer cells

BAG-1 is a multi-functional protein that exists in three major isoforms, BAG-1 p50, p46, and p36. A fourth isoform of 29 kDa also exists but its function remains mostly unknown. To further understand the role of this smaller isoform in ovarian cancer cells, the SKOV3 cell line was transfected with a doxycycline-inducible human BAG-1 p29 isoform or control plasmid. Ovexpression of BAG-1 p29 promotes protection from apoptosis in the presence of EGF as shown by decreased cell death measured by XTT assay and caspase-3 activity. Unexpectedly, however, BAG-1 p29 does not associate with the EGF receptor. When BAG-1 p29 transfectants were incubated in hydrogel-coated plates, BAG-1 p29-expressing SKOV3 cells were significantly more resistant to anoikis as compared to controls, and this correlated with decreased activation of caspase-3. The results of this study implicate BAG-1 p29 in the regulation of both the EGF signaling cascade and the apoptotic cascade induced by loss of anchorage.     

The immunomodulatory protein B7-H4 is overexpressed in breast and ovarian cancers and promotes epithelial cell transformation

B7-H4 protein is expressed on the surface of a variety of immune cells and functions as a negative regulator of T cell responses. We independently identified B7-H4 (DD-O110) through a genomic effort to discover genes upregulated in tumors and here we describe a new functional role for B7-H4 protein in cancer. We show that B7-H4 mRNA and protein are overexpressed in human serous ovarian cancers and breast cancers with relatively little or no expression in normal tissues. B7-H4 protein is extensively glycosylated and displayed on the surface of tumor cells and we provide the first demonstration of a direct role for B7-H4 in promoting malignant transformation of epithelial cells. Overexpression of B7-H4 in a human ovarian cancer cell line with little endogenous B7-H4 expression increased tumor formation in SCID mice. Whereas overexpression of B7-H4 protected epithelial cells from anoikis, siRNA-mediated knockdown of B7-H4 mRNA and protein expression in a breast cancer cell line increased caspase activity and apoptosis. The restricted normal tissue distribution of B7-H4, its overexpression in a majority of breast and ovarian cancers and functional activity in transformation validate this cell surface protein as a new target for therapeutic intervention. A therapeutic antibody strategy aimed at B7-H4 could offer an exciting opportunity to inhibit the growth and progression of human ovarian and breast cancers.     

Follicular epithelial cell apoptosis of atretic follicles within developing ovaries of the mosquito Culex pipiens pallens

Follicular atresia, the degeneration of developing follicles, is always incident to normal oogenesis in both oviparous and viviparous animals. Photo- and electron-microscopic observation of degenerating follicles within developing ovaries taken from blood-fed Culex pipiens pallens mosquitoes showed gradual degradation of the internal structures including yolk granules in the oocyte. The epithelial cells, which sometimes incorporated yolk granules from the oocyte along with the shrinkage of the follicle, gradually lost their uniform columnar shape, while their integrity as a covering layer remained. In situ active caspase analysis detected active enzymes in these epithelial regions. In the latest stages of atresia where either the nurse cells or oocyte were lost, the follicle was mainly comprised of irregularly shaped epithelial cells, and some of these cells' nuclei contained condensed chromatin peripherally, one of the characteristics of apoptotic cells. Also terminaldeoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling treatment indicated that DNA fragmentation occurred in these follicles. It seems likely that in atretic follicles the epithelial cells survive to play key roles in the event, and then finally undergo their own apoptotic cell death so as to give the developmental site to the next follicle in the same ovariole.     

Classifications of ovarian cancer tissues by proteomic patterns

Ovarian cancer is a morphologically and biologically heterogeneous disease. The identification of type-specific protein markers for ovarian cancer would provide the basis for more tailored treatments, as well as clues for understanding the molecular mechanisms governing cancer progression. In the present study, we used a novel approach to classify 24 ovarian cancer tissue samples based on the proteomic pattern of each sample. The method involved fractionation according to pI using chromatofocusing with analytical columns in the first dimension followed by separation of the proteins in each pI fraction using nonporous RP HPLC, which was coupled to an ESI-TOF mass analyzer for molecular weight (MW) analysis. A 2-D mass map of the protein content of each type of ovarian cancer tissue samples based upon pI versus intact protein MW was generated. Using this method, the clear cell and serous ovarian carcinoma samples were histologically distinguished by principal component analysis and clustering analysis based on their protein expression profiles and subtype-specific biomarker candidates of ovarian cancers were identified, which could be further investigated for future clinical study.     

Comparative proteomic analysis of cisplatin sensitive IGROV1 ovarian carcinoma cell line and its resistant counterpart IGROV1-R10

Ovarian cancer is one of the leading causes of mortality due to gynaecological cancer. Despite a good response to surgery and initial chemotherapy essentially based on cisplatin (cis-diamino-dichloro-platinum(II) (CDDP)) compounds, late tumour detection and frequent recurrences with chemoresistance acquisition are responsible for poor prognosis. Several mechanisms have been implicated in CDDP resistance but they are not sufficient to exhaustively explain this resistance emergence. We applied a proteomic approach based on 2-DE coupled with MS to identify proteins associated with the chemoresistance process. We first established a proteomic pattern of the CDDP sensitive ovarian cell line IGROV1 using MALDI-TOF-MS and PMF. We then compared this 2-D pattern with that of the CDDP-resistant counterpart IGROV1-R10. Among the 40 proteins identified, cytokeratins 8 and 18 and aldehyde dehydrogenase 1 were overexpressed in IGROV1-R10, whereas annexin IV was down-regulated. These observations have been confirmed by Western blotting. The characterization of such variations could lead to the development of new protein markers or to the establishment of new therapeutic strategies. Moreover, the identification of proteins involved in CDDP resistance in ovarian tumours would be useful in completing our understanding on this complex mechanism.     

Estradiol does not influence strategy choice but place strategy choice is associated with increased cell proliferation in the hippocampus of female rats

Adult neurogenesis occurs in the hippocampus of most mammals. While the function of adult hippocampal neurogenesis is not known, there is a relationship between neurogenesis and hippocampus-dependent learning and memory. Ovarian hormones can influence learning and memory and strategy choice. In competitive memory tasks, higher levels of estradiol shift female rats towards the use of the place strategy. Previous studies using a cue-competition paradigm find that 36% of male rats will use a hippocampus-dependent place strategy and place strategy users had lower levels of cell proliferation in the hippocampus. Here, we used the same paradigm to test whether endogenous or exogenous ovarian hormones influence strategy choice in the cue-competition paradigm and whether cell proliferation was related to strategy choice. We tested ovariectomized estradiol-treated (10 μg of estradiol benzoate) or sham-operated female rats on alternating blocks of hippocampus-dependent and hippocampus-independent versions of the Morris water task. Rats were then given a probe session with the platform visible and in a novel location. Preferred strategy was classified as place strategy (hippocampus-dependent) if they swam to the old platform location or cue strategy (hippocampus-independent) if they swam to the visible platform. All groups showed a preference for the cue strategy. However, proestrous rats were more likely to be place strategy users than rats not in proestrus. Female place strategy users had increased cell proliferation in the dentate gyrus compared to cue strategy users. Our study suggests that 78% of female rats chose the cue strategy instead of the place strategy. In summary the present results suggest that estradiol does not shift strategy use in this paradigm and that cell proliferation is related to strategy use with greater cell proliferation seen in place strategy users in female rats.