Bosong Dai

Autocrine gastrins in colon cancer cells Up-regulate cytochrome c oxidase Vb and down-regulate efflux of cytochrome c and activation of caspase-3.

Suppression of the gastrin gene in human colon cancer cells by stably expressing antisense (AS) gastrin RNA results in significant growth suppression of AS cells. To understand mechanisms mediating the growth effects of autocrine gastrins, differential expression of transcripts by AS and control (C) clones of a representative cell line (HCT-116) was analyzed to identify target genes of autocrine gastrins. Six differentially expressed transcripts were confirmed and sequenced. Of these, the RNA and protein levels of cytochrome c oxidase (COX) Vb were significantly higher in C versus AS cells. The expression of COX Vb by colon cancer cells was proportional to the expression of gastrin. Higher levels of COX Vb coprecipitated with cytochrome c in the mitochondria of C versus AS cells. Treatment of mitochondria with digitonin resulted in a 2-fold higher release of cytochromec from AS versus C mitochondria. As a corollary, the cytosolic levels of cytochrome c were significantly higher in AS versus C cells, which correlated with ∼2- and ∼3-fold higher activation of caspase-9 and -3, respectively, in AS versus C cells in response to camptothecin. Thus, autocrine gastrins may support growth/survival of cells by up-regulating COX Vb, which may decrease the sensitivity of the cancer cells to apoptotic stimuli by increasing retention of cytochrome c in mitochondria.

Smooth muscle uses another promoter to express primarily a form of human Cav1.2 L-type calcium channel different from the principal heart form.

Several different first exons and amino termini have been reported for the cardiac Ca channel known as alpha(1C) or Ca(V)1.2. The aim of this study was to investigate whether the expression of this channel is regulated by different promoters in smooth muscle cells and in heart in humans. Ribonuclease protection assay (RPA) indicates that the longer first exon 1a is found in certain human smooth muscle-containing tissues, notably bladder and fetal aorta, but that it is not expressed to any significant degree in lung or intestine. On the other hand, all four smooth muscle-containing tissues examined strongly express transcripts containing exon 1b, first reported cloned from human fibroblast cells. In addition, primary cultures of human colonic myocytes and coronary artery smooth muscle cells express predominantly transcripts containing exon 1b. The promoter immediately upstream of exon 1b was cloned, and it displays functional promoter activity when luciferase-expressing constructs were transfected into three different cultured smooth muscle cells: primary human coronary artery smooth muscles cells, primary human colonocytes, and the fetal rat aorta-derived A7r5 cell line. These results indicate that expression in smooth muscle is primarily driven by a promoter different from that which drives expression in cardiac myocytes.

A new promoter for α1C subunit of human L-type cardiac calcium channel CaV1.2

The cardiac Ca channel known as a1C or CaV1:2 is shown to express a new longer first exon equivalent to that formerlyreported in rabbit heart or rat aorta. Ribonuclease protection assayindicates that this exon is found in the majorityof Ca V1:2 transcripts in human heart RNA. The presence of this exon also suggests that expression of this transcript is driven bya promoter immediately upstream of this exon and its 5 0 untranslated region. The putative promoter exhibits 69% homologyto its rat counterpart and displays functional promoter activity when transfected into heart cells in culture in luciferase-expressing constructs. 2002 Elsevier

Role of autocrine and endocrine gastrin-like peptides in colonic carcinogenesis.

Colon carcinogenesis is a multistep process that involves deletions, mutations, and changes in expression of genes that regulate growth, differentiation, and apoptosis. Hyperproliferation can initiate dysplastic growth, resulting in accumulation of genetic defects and progression of colon cancer. Although genetic instability, because of inheritance of specific genetic defects, plays a dominant role in familial cancers, in the majority of sporadic cancers hyperproliferation is likely to play a permissive role in initiation and progression of the disease. Thus factors that regulate growth, differentiation, and apoptosis are likely to play an important role in colon carcinogenesis. Autocrine gastrins, insulin-like growth factor-II, transforming growth factor-alpha, and endocrine gastrins have been implicated in the tumorigenic potential of colon cancer cells. In this article we focus on the role of endocrine and autocrine gastrins in colon cancer and review recent advances that suggest a role of processing intermediates of gastrin in colon carcinogenesis.

Identification of a Novel Cis Element Required for Cell Density-dependent Down-regulation of Insulin-like Growth Factor-2 P3 Promoter Activity in CaCo2 Cells

The activity of the exogenous, full-length insulin-like growth factor-2 (IGF-2) P3 promoter is significantly up-regulated during the logarithmic growth phase but rapidly declines in confluent CaCo2 cells undergoing differentiation. Nuclear run-on assays confirmed cell density-dependent regulation of endogenous P3 promoter. To identify regulatory elements in the P3 promoter that may be required for regulating cell density-dependent transcriptional activity, we used the methods of promoter truncation, electrophoretic mobility shift assay, DNase footprinting, and mutation analysis. The relative activity of the full-length (−1229/+140) and truncated (−1090/+140) promoter was identical, being ∼19, 27, 7, and 3% of pSV-luc activity on days 3, 5, 7, and 9 of cell culture, respectively. However, truncation to −1048 resulted in complete loss of cell density-dependent down-regulation of P3 promoter activity on days 7 and 9, suggesting the presence of regulatory elements between −1091 and −1048 sequence. Further stepwise truncation to −515 did not change promoter activity. Truncation to −138/+140 resulted in complete loss of promoter activity, suggesting that the core promoter was within the −515/−138 segment. A 14-base pair footprint (−1084/−1070) was identified by DNase footprinting within the distal −1091/−1048 segment. Electrophoretic mobility shift assay with wild type and mutant probes confirmed the presence of a novel 7-base pair (CGAGGGC) (−1084/−1078) cis element (P3-D); its mutation abolished binding. Functionality of P3-D cis element was confirmed by measuring the activity of core P3 promoter ligated to distal P3 segment containing either the mutant or wild type P3-D element. We have, therefore, identified a novel cis element, P3-D, that appears to play a critical role in regulating IGF-2 P3 promoter activity in a cell density/differentiation-dependent manner.

Differential activation of IGF-II promoters P3 and P4 in Caco-2 cells during growth and differentiation

BACKGROUND & AIMS Insulin-like growth factor (IGF)-II gene is overexpressed in colon cancers. Transcriptional up-regulation may be the major mechanism contributing to its overexpression. IGF-II messenger RNA (mRNA) levels are up-regulated during proliferation followed by a significant decline during differentiation of Caco-2 cells. Mechanisms underlying transcriptional regulation of the IGF-II gene promoters (P1-P4) have yet to be examined in colon cancers, which was the basis for this study. METHODS Ribonuclease protection assay was used to measure IGF-II mRNA derived from P1-P4. To determine if changes in the IGF-II transcripts reflected differences in promoter activity, transient transfection assays with the full-length P1-P4-luciferase expression vectors were performed. RESULTS Both P3- and P4-derived transcripts were significantly up-regulated during the proliferative phase of the cells (days 3-6 in culture) and declined rapidly in cells undergoing differentiation (days 7-10); conversely, P1- and P2-derived transcripts were not detected. Similarly, transcriptional activity of P3 and P4 promoters reached peak levels by days 4-6 and declined rapidly thereafter. P1 and P2 were relatively inactive on all days. CONCLUSIONS The activity of the P3 and P4 promoters may play a selective role in regulating IGF-II mRNA levels during growth and differentiation of colon cancer cells.

Gastrin gene expression in human colon cancer cells measured by a simple competitive PCR method

Gastrin is mitogenic for several colon cancers and is postulated as an autocrine growth factor for colon cancer cells. In the present study we report the development of a simple competitive polymerase chain reaction (PCR) method for measuring relative abundance of gastrin gene expression in colon cancer cells. Primers flanking exons 2 and 3 of the gastrin gene were utilized for co-amplification of cDNA and genomic DNA. The amplification of genomic DNA was distinguished from that of cDNA by the presence of the 130 bp intron sequence which was resolved by electrophoresis on agarose gels. A standard reaction of competitive PCR, using known concentrations of genomic DNA and cDNA, was first established. The steady state levels of gastrin mRNA were next quantitated in three human colon cancer cell lines (HCT-116, Colo-205 and DLD-1) by competitive PCR. Gastrin mRNA levels in these cell lines ranged from approximately 0.1 to 1.0 fmoles/mg total RNA (approximately 2-25 copies of gastrin mRNA per cell). Thus low to moderate levels of gastrin were expressed by human colon cancer cell lines which may function as autocrine growth factors for colon cancers.

Autocrine gastrins in colon cancer cells up-regulate cytochrome C oxidase VB (COX VB) and down-regulate efflux of cytochrome C in activation of caspase 3

Suppression of the gastrin gene in human colon cancer cells by stably expressing antisense (AS) gastrin RNA results in significant growth suppression of AS cells. To understand mechanisms mediating the growth effects of autocrine gastrins, differential expression of transcripts by AS and control (C) clones of a representative cell line (HCT-116) was analyzed to identify target genes of autocrine gastrins. Six differentially expressed transcripts were confirmed and sequenced. Of these, the RNA and protein levels of cytochrome c oxidase (COX) Vb were significantly higher in C versus AS cells. The expression of COX Vb by colon cancer cells was proportional to the expression of gastrin. Higher levels of COX Vb coprecipitated with cytochrome c in the mitochondria of C versus AS cells. Treatment of mitochondria with digitonin resulted in a 2-fold higher release of cytochrome c from AS versus C mitochondria. As a corollary, the cytosolic levels of cytochrome c were significantly higher in AS versus C cells, which correlated with approximately 2- and approximately 3-fold higher activation of caspase-9 and -3, respectively, in AS versus C cells in response to camptothecin. Thus, autocrine gastrins may support growth/survival of cells by up-regulating COX Vb, which may decrease the sensitivity of the cancer cells to apoptotic stimuli by increasing retention of cytochrome c in mitochondria.