Project: Research project

Project Details


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

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.
Effective start/end date4/1/924/30/01