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 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 a (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 date5/1/934/30/99


  • National Cancer Institute


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