The uncontrolled growth which is characteristic of transformed cells is often attributed to the absence of appropriate cellular responses to environmental stimuli such as autocrine growth regulators. Understanding the mechanisms which account for the inappropriate control of growth by natural growth regulators in transformed cells is critical to identifying novel, non-cytotoxic means of preventing cancer progression. In order to test potential means of restoring altered control of growth by polypeptide growth factors in transformed cells, defined model systems in serum-free culture, displaying both normal and altered responsiveness to exogenous growth factors are necessary. The overall goal of this proposal is to utilize two previously established model culture systems for altered growth factor control in human colon carcinoma and intestinal epithelial cells to investigate the role of the ras oncogene in the observed growth factor independence. Activated ras genes are found in 50% of colon carcinomas. Moreover, overexpression of ras alters growth factor production, as is observed for transforming growth factor alpha (TGF-alpha) in the poorly-differentiated (PD) colon carcinoma cells. Further, the ras oncogene is capable of uncoupling mitogenic responses from cellular stimulation by polypeptide growth factors. Areas of investigation include: (1) Characterization of ras expression levels and gene mutations in relation to the growth factor autonomy of the cells. K-ras mutations at codons 12, 13, and 61 will be analyzed by amplification of tumor cell DNA using polymerase chain reaction, followed by direct sequencing. Immunoprecipitation of p21ras using mutation-specific antibodies will reveal alterations in expression levels. (2) Analysis of alterations in the activation state of ras. GTPase activities and guanine nucleotide exchange rates will be measured in permeabilized cells. The activity of GTPase activating protein (GAP), a potential effector or negative regulator of ras action, which greatly accelerates the intrinsic GTPase activity of ras, will be analyzed in cell lysates. (3) Antisense agents directed against mutations of p21ras will be used to demonstrate the ras dependence of transformation-related properties of colon carcinoma cells. (4) Inhibitors of post-translational processing of ras will be examined for their ability to selectively reverse transformation-related alterations associated with overexpression of p21ras in colon carcinoma cells.
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