RESUMO
To identify new molecular markers for differentiation of normal and neoplastic colon epithelium, we have studied changes in gene expression during the in vitro differentiation of the HT29-D4 colon carcinoma cell line. Using a modified differential display procedure, we cloned a novel cDNA, designated differentiation-related gene 1 (Drg1). Drg1 mRNA has a length of approximately 3 kb and is induced approximately 20-fold during in vitro differentiation of the colon carcinoma cell lines HT29-D4 and Caco-2. The absence of Drg1 induction in growth-inhibited A431 epidermoid carcinoma cells indicates that Drg1 up-regulation in colon carcinoma cells is not a result of decreased proliferation. The Drg1 cDNA contains an open-reading frame of 1182 bp that encodes a protein with a predicted molecular weight of 43 kd. Drg1 mRNA is expressed most prominently in placental membranes and prostate, kidney, small intestine, and ovary tissues. Compared to normal colon mucosa, Drg1 mRNA expression is decreased in colon adenomas and adenocarcinomas. An antiserum raised against recombinant Drg1 protein detected a band of the expected size in Western blots. Immunohistochemistry showed that in normal colon Drg1 protein is expressed in the cytoplasm and basolateral membranes of surface epithelial cells that border the gut lumen, indicating that Drg1 protein is expressed late during differentiation, just before apoptosis and shedding of cells into the colon lumen.
Assuntos
Proteínas de Ciclo Celular , Colo/citologia , Neoplasias Colorretais/genética , Regulação para Baixo , Mucosa Intestinal/citologia , Proteínas/genética , Regulação para Cima , Sequência de Aminoácidos , Animais , Sequência de Bases , Diferenciação Celular , Colo/metabolismo , Humanos , Soros Imunes , Peptídeos e Proteínas de Sinalização Intracelular , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Ratos , Células Tumorais CultivadasRESUMO
To investigate the working hypotheses that stem cells or their early descendants are prime targets for neoplastic transformation, and that the degree to which a neoplasm retains the immature phenotype is an important determinant of tumor aggressiveness, we have identified several mRNAs that are downregulated during the in vitro differentiation of HT29-D4 colon carcinoma cells. These genes include heat-shock cognate protein Hsc70, adenylosuccinate lyase, B23/nucleophosmin, alpha-tubulin, and a novel gene designated DS-1. The DS-1 mRNA has a length of approximately 0.9 kb and is downregulated 4.7-fold upon differentiation. From the DS-1 cDNA, a protein of 206 amino acids with a molecular mass of 24 kDa and an isoelectric point of 10.9 can be deduced. An antiserum directed against a synthetic peptide detected a minor band of the expected size in Western blots, as well as a major band of lower size that may represent a processed form of the protein.