Cell surface overexpression of alphavbeta5 integrin impedes alphavbeta3-mediated migration of the human ovarian adenocarcinoma cell line IGROV1.

Human ovarian surface epithelium and epithelial tumors express integrin alphavbeta5, which can interact with vitronectin. In addition, in vitro acquisition of cisplatin resistance by alphavbeta3-expressing IGROV1 cells is accompanied by cell-surface expression of integrin alphavbeta5. To further explore the role of alphavbeta5 in ovarian carcinoma cells, IGROV1 cells were stably transfected with a human beta5 integrin cDNA construct, and three beta5 transfectant clones were selected for the expression of alphavbeta5 integrin at their cell surface. Despite a delayed entry in the exponential phase of growth, beta5-transfectant cells kept a proliferation ability similar to that of parental cells, while their growth rate was hindered in the presence of an anti-alphavbeta5 blocking antibody. Only simultaneous blockade of alphavbeta3 and alphavbeta5 by specific antibodies impeded the adhesion to vitronectin of beta5 transfectants and of the beta5-expressing cisplatin-resistant variant IGROV1-R10, suggesting that the two heterodimers cooperated in the regulation of this process. Cell surface expression of alphavbeta5 resulted in an attenuation of alphavbeta3-mediated migration on vitronectin. Alphavbeta5 participated to migration events in the absence of exogenous growth factors only in one transfectant clone and in IGROV1-R10 cells. Finally, the response to cisplatin was not significantly modified in beta5 transfectants when compared to IGROV1 parental cells.

Effect of 2-deoxy-D-glucose on various malignant cell lines in vitro.

BACKGROUND: 2-Deoxy-D-glucose (2-DG) is an analog of glucose that is preferentially captured by tumors and is accumulated in transformed cells, because the phosphorylated molecule (2-DG-6P) cannot be metabolized or diffused outside the cells. Targeted with a fluorine atom, 18F-DG is currently used to visualize malignant tumors (PET scan). Although cancer cells have been reported to be strongly dependent on glycolysis (Warburg effect), very few reports have studied the inhibitory effects of 2-DG on cancer. MATERIALS AND METHODS: Our objective was to study, in a large panel of human malignant cells of various origins (ovarian, squamous, cerebral, hepatic, colonic and mesothelial), if the inhibitory activity of 2DG against tumor growth could be considered a general phenomenon and to determine its effect on the cell cycle. RESULTS: Four types of response in the different cell lines were observed when cells were cultured in the presence of 2-DG (5 mM) continuous exposure: proliferation slow down; proliferation arrest without signs of apoptosis; strong cell cycle arrest accompanied by moderate apoptosis induction; massive apoptosis. CONCLUSION: 2-DG appears as an interesting new therapeutic agent against cancer in vitro, and should be tested in in vivo studies.

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.

Urine mutagenicity of farmers occupationally exposed during a 1-day use of chlorothalonil and insecticides.

OBJECTIVES: A study was conducted to evaluate the genotoxic impregnation consecutive to a 1-day open-field spraying of pesticides. METHODS: From 14 farmers (five smokers and nine non-smokers), three urine samples were collected at the end of the spraying season: the morning (S1) of the day of spraying, the evening (S2) and the morning (S3) of the following day. A fourth sample (S0) was obtained before the pesticide-handling period. Mutagenicity of urine extracts was evaluated with the Ames test, using strains TA97a, TA98, TA100 and TA102, with and without S9 mix. RESULTS: The ratio of induced vs spontaneous revertants (induction ratio) was > or =2 in five farmers (including three smokers), with only one strain responding in each. Applying the SALM software proposed by Kim and Margolin in combination with the ANOVA-Dunnett test on crude data (number of revertants), urine extracts were found to be mutagenic on at least one Salmonella strain in 57% and 96% of non-smokers and smokers, respectively. The proportion of mutagenic responses tended to increase from S1 to S3 (not statistically significant) in non-smokers only. Finally, there were no relationships between the relative changes in the number of revertants (adjusted for urine concentration) and any exposure parameters available: area sprayed, number of tanks prepared and time free of exposure to any pesticide. CONCLUSIONS: The lack of significant relationships between urine mutagenicity and exposure data argues against a direct role of the pesticides sprayed, on this impregnation. This result should be considered with caution since the number of farmers involved may limit the significance of the study.

Detection of topoisomerase inhibitor-induced DNA strand breaks and apoptosis by the alkaline comet assay.

The alkaline comet assay is able to identify in individual cells DNA strand breaks associated with different processes. Topoisomerase inhibitors, some of which are used as chemotherapeutic agents, stabilise topoisomerase-DNA cleavable complexes by stimulating DNA strand cleavage and inhibiting religation. This can result in the activation of stress-associated signalling pathways, inducing cell cycle arrest and activation of the biochemical cascade of apoptosis. The aim of our study was to assess the ability of the comet assay to detect stabilisation of cleavable complexes and induction of apoptosis by two topoisomerase II inhibitors, etoposide and ellipticine, and two topoisomerase I inhibitors, camptothecin and topotecan. The study was carried out on Chinese hamster ovary (CHO) cells, DC3F cells and DC3F/C-10, its camptothecin-resistant counterpart. The comet assay was able to identify stabilised cleavable complexes through the presence of DNA strand breaks after 1h treatment that disappeared within 24h after drug removal. Kinetics studies allowed to discriminate between these early DNA damages and DNA fragmentation related to apoptosis characterised by reappearance of DNA strand breaks 48h after treatment.

Biomonitoring of urine mutagenicity with the Ames test: improvement of the extraction/concentration method.

Comparative extraction efficiency of the pre-packed Bakerbond-spe-SDB-1 resin and of Amberlite-AD2 (XAD-2) resin, for the preparation of urine extracts in biomonitoring studies. Urine extracts were prepared in parallel with the Bakerbond column and with the classical XAD-2 resin from urines (1) spiked with mutagenic chemicals, (2) collected from patients after chemotherapy, and (3) from smokers. Mutagenic activities were evaluated on Salmonella typhimurium tester strains TA97a, TA98, TA100 and TA102 with and without S9 mix. Mutagenic activities obtained with Bakerbond extracts were almost always higher or at least equivalent to those prepared on XAD-2 resin. Similar results were observed for the three urine sample groups. When fully validated, the use of the pre-packed columns will be more convenient and time-saving for large population studies.