GENETIC AND BIOCHEMICAL ANALYSIS OF CELL BEHAVIOR

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We are utilizing the Chinese hamster ovary (CHO) fibroblast to study the genetics and biochemistry of some aspects of the behavior of cultured cells. Our work has emphasized morphology and its relationship to growth control, and response to cyclic AMP. We have isolated a variety of different mutants with altered microtubules which express mutated Alpha or Beta-tubulin subunits and used these mutants to study spindle formation and the mechanism of anti-microtubule drug action. These mutants are defective in spindle formation because the mutant tubulins are present in spindles and interfere with their normal function. One Beta-tubulin mutation has been transferred by DNA mediated gene transfer to wild-type cells where it is amplified, overexpressed and renders the cells dependent on Colcemid for growth at 37 degrees. We have also established two cell systems for examining the ways in which AMP can positively and negatively regulate cell growth. CHO cell growth is inhibited by cAMP; mutants selected for resistance to growth inhibition have defective cAMP dependent protein kinases. We have used DNA from cells carrying dominant cAMP-resistant defects to transfer the cAMP-resistance phenotype to sensitive cells and are cloning these genes from cosmid libraries prepared from our mutant cell lines. CHO cells malignantly transformed by RSV are also cAMP-resistant. Formation of tumors by CHO-RSV cells and human melanoma calls is dependent on prior treatment with cholera toxin which raises cAMP levels within the cells. This increased tumorigenicity is an example of positive regulation of cell growth by cAMP.