The goal of this project is to gain insight into aberrations of cell cycle control mechanisms in neoplastically transformed mammalian cells. An integral part of the carcinogenesis process is the loss of normal control of cellular proliferation which can result, at least in part, from gain-of-function mutations (such as activations of oncogenes), loss-of-function mutations (such as inactivations of normal tumor suppressor gene functions), and both heritable and non-heritable alterations in the normal pattern of gene expression and function. We are studying alterations in growth control signal transduction pathways in transformed mammalian cells in culture as compared with their non-transformed counterparts. Specifically, we are investigating cell cycle related parameters in mouse fibroblasts that constitutive overexpress specific oncogene products. We have been carefully defining the cell division cycle kinetics of clonal populations of the parental NIH3T3 cells or NIH3T3 cells overexpressing mos(mu), mos(Xe) , H-ras(va112), tpr-met, v-src, and c-src(mu). These studies involve optimizing synchrony protocols through the use of various combinations of either serum deprivation, mitotic shake-off, hydroxyurea treatment, or aphidicolin treatment. The degree of synchrony is being monitored by such parameters as incorporation of (3)H-thymidine or BrdU into newly synthesized DNA, mitotic indices, and population doublings. Using tightly synchronized populations and immunohistochemical techniques, we are monitoring cells in early G2 phase, midole G2, late G2, mitotic prophase, metaphase, anaphase, telophase, and cytokinesis for such events as the reorganization of interphase microtubules to mitotic spindles, the phosphorylation status of tubulin, and phosphorylation events associated with the activation of microtubule organizing centers (MTOCs) and maturation promoting factor (MPF) activity. Additional studies will follow the phosphorylation status of specific gene products (such as p34(cdc2) cyclin proteins, and p53) as transformed and nontransformed cells progress through their division cycle. The goal of these studies is to monitor alterations in cell cycle control signal transduction pathways following exposure to genotoxic and non-genotoxic carcinogens.
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