The continuously renewing intestinal and colonic epithelia are dependent upon a well-regulated system of proliferation and differentiation. A small proliferative compartment is located in the intestinal and colonic crypts; as the cells migrate from this compartment they differentiate. The mechanisms directing the cessation of proliferation and induction of differentiation are unknown. The overall objective of this proposal is the study of one possible mechanism coordinating these events. Expression of the intestinal enterocyte cell phenotype is dependent upon the induction of intestine-specific genes. The caudal-related genes, CDX1 and CDX2, are known to be required for the correct developmental expression of several intestine-specific genes, including sucrase isomaltase (SI), lactase-phlorizin hydrolase, and intestinal phospholipase A/lyso-phospholipase (IPAL). In addition to this role in differentiation, there is mounting evidence that the CDX1 and CDX2 genes can regulate proliferation of intestinal epithelia. Our preliminary data will establish that induction of CDX1 expression in IEC6 cells (non-transformed intestinal cell line) and DLD1 cells (human colon cancer cell line) leads to a G0/G1 block. We will also demonstrate that this block is associated with hypophosphorylation of the retinoblastoma protein, and down-regulation of cyclin D1 and D2 protein. We propose to further explore this effect by testing the following hypothesis: (1) CDX1 specifically interacts with the cell-cycle regulatory machinery to induce a G0/G1 block by an inhibition in G1 cdk kinase activity, and (2) This effect is mediated primarily by down-regulation of cyclins D1 and D2.
The Specific Aims of this proposal are: (1) Evaluate the effects of CDX1 on G1 CDK-kinase activity; (2) Examine the effects of CDX1 expression on proliferation of human colon cancer cells; (3) Further characterize the quiescent G0/G1 state induced by forced CDX1 expression; (4) Initiate studies on the mechanism of CDX1- induced inhibition of proliferation. Forced expression of CDX1, and CDX1 mutants in IEC6 and colon cancer cell lines will serve as our model system. Adenovirus and retrovirus vector systems will deliver the gene into the cells for expression. Proliferative rates, DNA synthetic rates, and flow cytometry measurements will be obtained as needed. G1 cyclin-dependent kinase (cdk) activity will be assayed, and E2F EMSA and trans- activating activity will be characterized. Our study aims and hypotheses are directed to identify a mechanism for the CDX1 induced G0/G1 arrest. Understanding this mechanism would greatly improve our knowledge of the events occurring as cells migrate from intestinal and colonic crypts. It will also enhance our understanding of the events leading to dysregulation of proliferation and human colon cancers.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Clinical Investigator Award (CIA) (K08)
Project #
1K08DK002695-01
Application #
2883893
Study Section
Special Emphasis Panel (ZDK1-GRB-2 (M2))
Program Officer
Podskalny, Judith M,
Project Start
2000-03-15
Project End
2004-11-30
Budget Start
2000-03-15
Budget End
2000-11-30
Support Year
1
Fiscal Year
2000
Total Cost
$125,847
Indirect Cost
Name
University of Pennsylvania
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Ezaki, Toshihiko; Guo, Rong-Jun; Li, Hong et al. (2007) The homeodomain transcription factors Cdx1 and Cdx2 induce E-cadherin adhesion activity by reducing beta- and p120-catenin tyrosine phosphorylation. Am J Physiol Gastrointest Liver Physiol 293:G54-65
Keller, Matthew S; Ezaki, Toshihiko; Guo, Rong-Jun et al. (2004) Cdx1 or Cdx2 expression activates E-cadherin-mediated cell-cell adhesion and compaction in human COLO 205 cells. Am J Physiol Gastrointest Liver Physiol 287:G104-14
Guo, Rong-Jun; Huang, Edward; Ezaki, Toshihiko et al. (2004) Cdx1 inhibits human colon cancer cell proliferation by reducing beta-catenin/T-cell factor transcriptional activity. J Biol Chem 279:36865-75
Lynch, John; Keller, Matthew; Guo, Rong-Jun et al. (2003) Cdx1 inhibits the proliferation of human colon cancer cells by reducing cyclin D1 gene expression. Oncogene 22:6395-407