2458

Alterations in the APC/beta-catenin pathway initiate almost all human colorectal cancers, and c-myc and cyclin D1 were identified as key transcriptional targets of this pathway. Further, c-myc and cyclin D1 are down-regulated by chemopreventive agents such as the short chain fatty acid butyrate, and Vitamin D. Utilizing a novel variation of fluorescence in situ hybridization (FISH) we have shown that increased transcriptional initiation of c-myc induced by butyrate, consistent with our previous report of butyrate elevation of β-catenin/TCF activity, was efficiently abrogated by a block to transcriptional elongation, resulting in decreased c-myc expression (Wilson et al., Cancer Research, 62, 6006, 2002). 1α,25-dihydroxyvitamin D3 also induced this transcriptional blockage. It was important to extend these experiments to cyclin D1 since: it too is regulated by the beta-catenin/TCF signaling pathway; abnormalities in the cell cycle are linked to the majority of human neoplasias; and cyclin D1 over-expression enhances cell transformation and tumorigenesis, and enhances the amplification of other genes. Therefore we investigated if the ascribed down regulation of cyclin D1 mRNA and protein content after incubation with sodium butyrate is also totally or partially caused by blockage to transcriptional initiation or elongation. We again used the novel FISH technique which detects transcription sites in cell nuclei. The rates of transcription initiation and termination could be determined by positioning probes along the cyclin D1 transcription unit. These oligodeoxynucleotide probes were labelled with up to five fluorochromes per molecule. The data demonstrated that butyrate induced a transcriptional block in the cyclin D1 gene, leading to a decrease in steady state levels of the transcript. We are investigating the fate of the truncated transcripts, and, since the short chain fatty acid butyrate is a physiological regulator of cell maturation in the intestinal mucosa, whether the transcriptional block induced by butyrate in vitro can be detected as cells undergo maturation in vivo as they migrate along the crypt-villus axis.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]