The chemopreventive agent alpha-difluoromethylornithine blocks Ki-ras-dependent tumor formation and specific gene expression in Caco-2 cells.
Mutation of the Kirsten-ras (Ki-ras) proto-oncogene occurs frequently in colorectal cancers. alpha-Difluoromethylornithine (DFMO), an irreversible inhibitor of the polyamine biosynthetic enzyme, ornithine decarboxylase (ODC), inhibits Ki-ras transformation and colon tumorigenesis in carcinogen-treated animal models by mechanisms yet to be elucidated. Caco-2 cells transfected with an activated Ki-ras, but not parental cells, formed tumors in severe combined immunodeficient (SCID) mice. DFMO treatment (2% in drinking water) prevented tumor growth. Gene expression profiling was performed to identify Ki-ras-and DFMO-dependent patterns of gene expression. Microarray results were validated with real-time or semi-quantitative RT-PCR and/or Western blot analysis. Genes upregulated in Caco-2 cells expressing an activated Ki-ras encoded cytoskeletal-, transport-, protease-, and gap junction-associated proteins. These genes are important for normal development and maintenance of colonic epithelial tissue. Caco-2 cells transfected with an activated Ki-ras displayed increased expression of the integrin alpha 1 (INGA1) and enhanced cell migration on laminin. These parameters were unaffected by DFMO, but Ki-ras-dependent migration was inhibited by INGA1 antibodies. Other Ki-ras-dependent, but DFMO-independent, genes included transglutaminase (TGase) and kallikrein 6 (KLK6). Ki-ras-transfected cells also expressed increased levels of connexin43 (Cx43) (RNA and protein), tight junction protein, and endothelin 1. DFMO reversed these increases. The results indicated that the Ki-ras oncogene caused changes in experimental cell migration and cell-cell communication genes and that some of these changes could be reversed by DFMO.
Ignatenko
Natalia A
NA
Department of Cell Biology and Anatomy, Arizona Cancer Center, The University of Arizona, Tucson, Arizona, USA.
Zhang
Hui
H
Watts
George S
GS
Skovan
Bethany A
BA
Stringer
David E
DE
Gerner
Eugene W
EW
D000818:Animals; D000970:Antineoplastic Agents / Q000627:therapeutic use*; D018938:Caco-2 Cells; D002450:Cell Communication; D002465:Cell Movement; D003110:Colonic Neoplasms / Q000235:genetics / Q000378:metabolism / Q000517:prevention & control*; D000518:Eflornithine / Q000627:therapeutic use*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects*; D011905:Genes, ras / Q000187:drug effects*; D006801:Humans; D007797:Laminin / Q000378:metabolism; D051379:Mice; D016513:Mice, SCID; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D009955:Ornithine Decarboxylase / Q000378:metabolism; D065108:Ornithine Decarboxylase Inhibitors; D000090063:Proto-Oncogene Mas; D014162:Transfection
D000970:Antineoplastic Agents; D007797:Laminin; C000715470:MAS1 protein, human; D009363:Neoplasm Proteins; D065108:Ornithine Decarboxylase Inhibitors; D000090063:Proto-Oncogene Mas; D009955:Ornithine Decarboxylase; D000518:Eflornithine