The goal of this project is to investigate cell cycle control alterations during neoplastic transformation of mammalian cells. In one set of studies, we are investigating cell cycle control in v-mos-transformed NIH3T3 cells. After serum withdrawal, normal NIH3T3 cells arrest in quiescence while v-mos-transformed cells arrest in early G1. In contrast to quiescent NIH3T3 cells, serum starved v-mos-transformed cells are unable to fully down regulate expression and/or associated kinase activity of p34cdc2, p33cdk2, cyclin A, cyclin D1, and cyclin E. At least part of this defect is a consequence of defective transcriptional control of gene expression, with both AP1 and E2F transcriptional complexes inappropriately regulated. We also found that these cells are unable to appropriately regulate MAP kinase activity in response to serum stimulation. Since mos can signal through the ras/MEK/MAP kinase pathway, we have extended our studies to investigate the consequences of metallothionein-inducible expression of activated ras, mos, and MEK on cell cycle control. In other studies, the molecular mechanisms of the G2 delay induced by ionizing radiation (IR) are being analyzed in normal diploid human fibroblasts and in fibroblasts that lack wild type function of the ataxia telangiectasia (AT), p53, or RB cancer susceptibility gene products. Exposure of normal human fibroblasts to IR results in a rapid reduction in the percentage of cells in mitosis and in p34cdc2 kinase activity. AT fibroblasts exposed to IR displayed little reduction in the mitotic index or p34cdc2 kinase activity. To address the role of p53 in the G2 checkpoint, human fibroblasts expressing the HPV type16 E6 oncoprotein (that functionally inactivates p53) as well as cells from embryos of p53-deficient mice were analyzed for G2 checkpoint response to IR. We found that neither wild type p53 nor the cyclin-dependent kinase inhibitor p21 is required for normal G2 checkpoint function. However we found that lack of p53 function results in cells that are chromosomally unstable and show a progressive attenuation of their G2 checkpoint control that accompanies chromosomal abnormalities. Studies are underway to biochemically characterize the mitotically active p34cdc2/cyclin B1 protein kinase complexes and the inactive G2 complexes following IR.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Intramural Research (Z01)
Project #
1Z01ES021157-05
Application #
5202120
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
1995
Total Cost
Indirect Cost
City
State
Country
United States
Zip Code
Ferrucio, Bianca; Tiago, Manoela; Fannin, Richard D et al. (2017) Molecular effects of 1-naphthyl-methylcarbamate and solar radiation exposures on human melanocytes. Toxicol In Vitro 38:67-76
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Innes, Cynthia L; Heinloth, Alexandra N; Flores, Kristina G et al. (2006) ATM requirement in gene expression responses to ionizing radiation in human lymphoblasts and fibroblasts. Mol Cancer Res 4:197-207

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