The morbidity of colon cancer reflects the lack of an effective chemotherapeutic strategy for treatment of the disseminated form of the disease. Our long term objective is to devise strategies that permit intervention at the level of the primary regulatory defect that leads to malignant transformation. Our strategy is based upon four observations concerning growth factor regulation of epithelial cell proliferation. l) TGF-beta is a major effector of gut epithelial cell differentiation. 2) Undifferentiated colon adenocarcinoma is unresponsive to TGF-beta, as are intestinal epithelial cells that have been transformed with activated Ras genes. 3) Growth factors, including TGF-beta, regulate the expression of D cyclins, which are important in maintaining the proliferative state. 4) D cylcin genes are oncogenic in many different types of adenocarcinoma. Our immediate objective is to elucidate the role of TGF-beta in regulating gut epithelial cell proliferation and the role of D cyclins in that process. Our initial studies will focus upon IEC-6 intestinal epithelial cells, which, like normal gut epithelial cells, cease to proliferate in the presence of TGF-beta. Our data indicate that the gene encoding cyclin D1 (CcnD1) is the primary focus of TGF-beta inhibition of IEC-6 cells. TGF-beta inhibits transcription of CcnD1 resulting in G0 arrest. Antisense oligonucleotides directed against cyclin D1 mRNA can recapitulate the effects of TGF-beta. Inducible overexpession of cyclin D1 renders the cells resistant to TGF-beta. Two related hypotheses are derived from our initial observations. Hypothesis l: We propose that TGF-beta inhibition of cyclin D1 expression accounts for the normal cessation of proliferation that accompanies differentiation of the gut epithelium. This hypothesis will be tested by experiments described in the companion IRPG proposal, submitted by R.D. Beauchamp, M.D. Hypothesis 2: We propose that TGF-beta inhibition of epithelial cell proliferation is due to inhibition of CcnD1 transcription. The proposal in hand addresses the second hypothesis.
Five specific aims are proposed to test predictions that emanate from the working hypothesis.
Specific Aims l deals with analysis of nuclear run-on transcription and mRNA turnover in IEC-6 cells in the presence and absences of TGF-beta.
Specific Aims 2 A address the cloning and analysis of CcnD7 promoter structure.
Specific Aim 5 addresses the hypothesis that cyclin D1 is both the target of TGF-beta and a component of the TGF-b signal transduction pathway. Our objective is to understand CcnD7 regulation in detail sufficient to the framing of hypotheses concerning means to block cyclin D1 expression in tumor cells.
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| Ko, Tien C; Pan, Fuming; Sheng, Hongmiao et al. (2002) Cyclin D3 is essential for intestinal epithelial cell proliferation. World J Surg 26:812-8 |
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| Ko, T C; Yu, W; Sakai, T et al. (1998) TGF-beta1 effects on proliferation of rat intestinal epithelial cells are due to inhibition of cyclin D1 expression. Oncogene 16:3445-54 |
| Ko, T C; Bresnahan, W A; Thompson, E A (1997) Intestinal cell cycle regulation. Prog Cell Cycle Res 3:43-52 |
| Beauchamp, R D; Sheng, H M; Shao, J Y et al. (1996) Intestinal cell cycle regulations. Interactions of cyclin D1, Cdk4, and p21Cip1. Ann Surg 223:620-7;discussion 627-8 |
