ABSTRACT
Centrosome abnormalities are observed in human cancers and have been associated with aneuploidy, a driving force in tumour progression. However, the exact pathways that tend to cause centrosome abnormalities have not been fully elucidated in human tumours. Using a series of 68 non-small-cell lung carcinomas and an array of in vitro experiments, the relationship between centrosome abnormalities, aneuploidy, and the status of key G1 to S-phase transition cell-cycle molecules, involved in the regulation of centrosome duplication, was investigated. Centrosome amplification and structural abnormalities were common (53%), were strongly related to aneuploidy, and, surprisingly, were even seen in adjacent hyperplastic regions, suggesting the possibility that these are early lesions in lung carcinogenesis. Cyclin E and E2F1 overexpression, but not p53 mutation, was observed to correlate with centrosome abnormalities in vivo (p = 0.029 and p = 0.015, respectively). This was further strengthened by the observation that cyclin E was specifically present in the nucleus and/or cytoplasm of the cells that contained centrosome aberrations. The cytoplasmic cyclin E signal may be attributed, in part, to the presence of truncated low-molecular-weight isoforms of cyclin E. In order to isolate the effect of cyclin E on the appearance of centrosome abnormalities, a U2OS tetracycline-repressible cyclin E cell line that has a normal centrosome profile by default was used. With this system, it was confirmed in vitro that persistent cyclin E overexpression is sufficient to cause the appearance of centrosome abnormalities.
Subject(s)
Aneuploidy , Carcinoma, Non-Small-Cell Lung/pathology , Centrosome/ultrastructure , Cyclin E/genetics , Lung Neoplasms/pathology , Aged , Carcinoma, Non-Small-Cell Lung/genetics , Chi-Square Distribution , E2F1 Transcription Factor/genetics , Electrophoresis, Polyacrylamide Gel , Female , Gene Expression , Gene Expression Regulation, Neoplastic , Genes, p53 , Humans , In Situ Hybridization , Lung Neoplasms/genetics , Male , Middle Aged , Statistics, NonparametricABSTRACT
Actin and vinculin are two of the most abundant cytoskeletal proteins, widely expressed in nearly all types of eukaryotic cells. It has been well established that long-term exposure to the tumor promoter phorbol myristate acetate (PMA) affects Sertoli cell morphology, as well as F-actin and vinculin organization in vitro. To analyze in a quantitative manner the F-actin and vinculin content of rat immature Sertoli cells in vitro in response to PMA exposure, cytoskeletal fractions were prepared following extraction with Triton X-100. Analysis of the isolated cytoskeletal fractions by immunoblotting showed that exposure of immature rat Sertoli cells to PMA for 8h has an appreciable effect on the cellular level of both the actin and vinculin. Interestingly, as revealed by using calphostin C, a specific protein kinase C inhibitor, the observed F-actin and vinculin changes are most probably mediated by a mechanism that depends on protein kinase activity. A discussion is made concerning PKC modulation by PMA and the subsequent actin and vinculin quantitative changes and reorganization, phenomena that have been closely related to cell transformation.
Subject(s)
Actins/biosynthesis , Carcinogens/pharmacology , Sertoli Cells/metabolism , Tetradecanoylphorbol Acetate/pharmacology , Vinculin/biosynthesis , Actin Cytoskeleton/metabolism , Actin Cytoskeleton/ultrastructure , Animals , Cells, Cultured , Gene Expression Regulation/drug effects , Male , Rats , Rats, Wistar , Sertoli Cells/drug effects , Sertoli Cells/ultrastructureABSTRACT
The objective of the present study was to examine the effects of the well-known tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) on the morphology of cultured Sertoli cells from immature rats. The cells were cultured for 5 days and the TPA was added at the end of the culture period for 8 h at a concentration of 10-7 M. Viability tests showed that controls as well as TPA-treated cells remained viable during the culture period and no deleterious effects were observed as a result. Application of computerized morphometry at both light and electron microscopic levels revealed that TPA caused important changes in cell morphology in vitro. Statistical analysis of the results indicated that compared to the controls, Sertoli cells treated with TPA exhibited fewer astrocytic-type cytoplasmic extensions and a smaller size. Our results support the conclusion that the tumor promoter TPA, when applied to immature Sertoli cells in vitro, causes significant morphological alterations.
