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.
Xu
Z
Z
Department of Anatomy & Neurosciences, University of Texas Medical Branch, Galveston 77555.
Dai
B
B
Dhruva
B
B
Singh
P
P
D001483:Base Sequence; D003110:Colonic Neoplasms / Q000235:genetics* / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000032:analysis; D004273:DNA, Neoplasm / Q000032:analysis; D005755:Gastrins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000032:analysis; D012334:RNA, Neoplasm / Q000032:analysis*; D012194:RNA-Directed DNA Polymerase; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D018076:DNA, Complementary; D004273:DNA, Neoplasm; D005755:Gastrins; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D012194:RNA-Directed DNA Polymerase