Cell CAM 105, herein referred to as C-CAM, is a rat liver cell-cell adhesion molecule (CAM) and member of the Ig supergene family. Based on extensive localization studies in normal and developing liver, we hypothesize that it plays an important role in membrane morphogenesis and specifically, in the formation of the bile canalicular domain. Studies by our laboratory and others have shown that the expression of C-CAM or its human and mouse homologues is greatly reduced in liver and colorectal carcinomas. The proposed aims are directed at defining the consequences of C-CAM loss during rat liver carcinogenesis.
In aim 1, hepatocytes and transplantable hepatoma lines (THC) will be transduced with retroviral expression vectors carrying cDNAs for the L and S isoforms (C-CAM 1 and C-CAM 2) or an antisense oligomer corresponding to a sequence common to both isoforms. Transduced cells will be evaluated for changes in histotypic interactions, expression of E-cadherin, growth and invasion of collagen gels in vitro and tumorgenicity in nude mice.
In aim 2, interactions of hepatocytes or THC transduced with double expression vectors carrying cDNAs for C-CAM and dipeptidyl peptidase IV (DPPIV), a cell surface exoprotease, will be characterized following transplantation into the livers of syngeneic host rats carrying a mutated gene encoding an inactive form of DPPIV (D-rats). Transplanted cells will be recognized by a histochemical staining protocol or by immunocytochemical labeling with monoclonal antibodies specific for the enzymatically active form of DPPIV.
Aim 3 will determine the role of C-CAM during the initial stages of carcinogenesis. """"""""Transgenic livers"""""""" will be produced in D-rats by liver transplantation of DPPIV positive (D+) hepatocytes initiated in vivo with diethylnitrosamine and transduced in vivo or in vitro by infection with DPPIV/C-CAM retroviruses. The effect of transduction on the frequency, growth, persistence, histological organization and phenotypic properties of preneoplastic foci and nodules (PN) induced in transgenic livers by the """"""""resistant hepatocyte"""""""" selection protocol will be evaluated from changes in the levels of DPPIV activity in serum, and tissue extracts and by microscopic quantitation of D+ foci and nodules. To gain further insight into changes in C-CAM expression during the initial stages of carcinogenesis, C-CAM expression in individual PN will be examined at the protein and RNA levels by immunoblot, Northern blot and other appropriate methods.
The final aim will center on confirming the specific nature of associations between C-CAM and proteins co-eluted from immunoaffinity columns. Initial efforts will focus on a pair of proteins with molecular masses of 75kDa and 80 kDa. The proposed studies will provide new insights into the role of C-CAM in the interactions of normal hepatocytes and the consequences of the loss of C-CAM during liver carcinogenesis.
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