IDENTIFICATION AND EVALUATION OF THE ROLE OF ENDOGENOUS TYROSINE KINASES IN AZOXYMETHANE INDUCTION OF PROLIFERATIVE PROCESSES IN THE COLONIC MUCOSA OF RATS

Although tyrosine kinases (Tyr-k) are known to play a role in regulati ng proliferation of normal, preneoplastic and neoplastic cells, little is known about the identity of different species of Tyr-k involved in this process. Utilizing a non-denaturing polyacrylamide gel electroph oresis system, in which the separated proteins from tissue extracts ar e assayed directly for Tyr-k, we attempted to identify the species of Tyr-k that may be involved in azoxymethane (AOM) induction of colonic mucosal ornithine decarboxylase (ODC) activity, an enzyme whose activi ty is known to rise in rapidly proliferating cells. We have observed t hat 5 days after a single injection of the colonic carcinogen AOM (20 mg/kg body wt.) to 3-4-month old rats, a significant 230% rise in colo nic mucosal proliferative activity (as evidenced by 5-bromo-2'-deoxyur idine (BrdU) immunoreactivity) was also accompanied by a 550% increase in ODC activity. This was also associated with a marked rise (140-240 %) in the relative activity of Tyr-k of three mucosal proteins with M( r) of 165, 145 and 125 kDa. Since the molecular mass of one of the Tyr -k (165 kDa) corresponded to that of EGF-receptor (EGF-R), this led us to examine the role of EGF-R Tyr-k in AOM induction of colonic mucosa l ODC. We observed that a 320% increase in mucosal ODC activity, 5 day s after AOM injection, was accompanied by over 200% rise in Tyr-k acti vity of EGF-R. Daily injection of tyrphostin (300 mu g/kg body wt.), a Tyr-k inhibitor with a higher specificity for EGF-R Tyr-k, significan tly attenuated AOM-induced stimulation of both ODC and Tyr-k activity of EGF-R. Administration of AOM also stimulated the rate of synthesis and secretion of TGF-alpha in isolated colonocytes. In addition, the l evels of TGF-alpha and its mRNA in the colonic mucosa were also found to be 100% and 250% higher, respectively, in AOM-treated rats when com pared with the controls. We suggest that (a) activation of intrinsic T yr-k of EGF-R is an important event in AOM induction of colonic mucosa l proliferative processes, and (b) this activation is thought to be me diated by TGF-alpha through an autocrine mechanism.