APC-dependent regulation of polyamine metabolism and apoptosis in human colon tumor cells

Abstract

Mutation/deletion of the adenomatous polyposis coli (APC) tumor suppressor gene in germline cells of rodents and humans is associated with increased intestinal activity of ornithine decarboxylase (ODC), the first enzyme in polyamine synthesis, and intestinal neoplasia. To study the role of APC in signaling ODC expression, the human colon tumor cell line HT29 (wtAPC -/-) was stably transfected with a zinc-inducible wild-type APC gene. Addition of ZnCl2 to HT29-APC cells increased wild-type APC protein and Mad1 RNA and protein, and decreased levels of c-myc and ODC RNA and protein, relative to these parameters in HT29 cells transfected with the same plasmid containing the beta-galactosidase (betaGal) gene in place of APC. Upon induction of APC expression, ODC promoter activity and RNA levels were suppressed. To examine the role of APC-dependent regulation of ODC, the two sets of E-boxes were analyzed. When the E-box domain in the 5' flanking region of the ODC gene was mutated, ODC promoter activity was unaffected by wild-type APC expression. Antisense, but not missense, c-myc oligonucleotides decreased ODC activity in HT29 cells expressing mutant APC. These results indicate that APC expression can inhibit ODC via the 5' E-box. Using the cell model previously described, APC selectively represses the ODC A allele, apparently through selective binding of Mad1. These results demonstrate that wild-type APC suppresses c-myc and activates Mad1 expression in HT29 colon-derived cells. Treatment of Min mice with the ODC inhibitor, difluoromethylornithine (DFMO), suppresses intestinal polyamine contents and intestinal tumorigenesis. The data presented in this dissertation indicate that ODC is a modifier of APC-dependent signaling in intestinal cells and tissues. Apoptosis is significantly reduced in both the small intestines and colons of Min (multiple intestinal neoplasia) mice when compared to normal littermates. Apoptotic indices can be restored by treating the mice with alpha-difluoromethylornithine (DFMO). DFMO is a specific, irreversible inhibitor of ornithine decarboxylase (ODC), the first enzyme in polyamine biosynthesis. These results indicate that APC induces apoptosis via the mitochondrial pathway rather than through the death receptor pathway. APC also affects a variety of other proteins involved in the regulation of apoptosis including transcription factors (i.e., ets2, FKHR, JunB, etc.) and bcl-2 (i.e., Bcl-xL) family members. The multiple levels at which APC functions suggest a variety of possible targets for the prevention and treatment of colon cancer. (Abstract shortened by UMI.)