title: Opposing roles of the extracellular signal-regulated kinase and p38 mitogen-activated protein kinase cascades in Ras-mediated downregulation of tropomyosin.
abstract: We showed previously that activated Ras, but not Raf, causes transformation of RIE-1 epithelial cells, demonstrating the importance of Raf-independent pathways in mediating Ras transformation. To assess the mechanism by which Raf-independent effector signaling pathways contribute to Ras-mediated transformation, we recently utilized representational difference analysis to identify genes expressed in a deregulated fashion by activated Ras but not Raf. One gene identified in these analyses encodes for alpha-tropomyosin. Therefore, we evaluated the mechanism by which Ras causes the downregulation of tropomyosin expression. By using RIE-1 cells that harbor inducible expression of activated H-Ras(12V), we determined that the downregulation of tropomyosin expression correlated with the onset of morphological transformation. We found that the reversal of Ras transformation caused by inhibition of extracellular signal-regulated kinase activation corresponded to a restoration of tropomyosin expression. Inhibition of p38 activity in Raf-expressing RIE-1 cells caused both morphological transformation and loss of tropomyosin expression. Thus, a reduction in tropomyosin expression correlated strictly with morphological transformation of RIE-1 cells. However, forced overexpression of tropomyosin in Ras-transformed cells did not reverse morphological or growth transformation, a finding consistent with the possibility that multiple changes in gene expression contribute to Ras transformation. We also determined that tropomyosin expression was low in two human tumor cell lines, DLD-1 and HT1080, that harbor endogenous mutated alleles of ras, but high in transformation-impaired, derivative cell lines in which the mutant ras allele has been genetically deleted. Finally, treatment with azadeoxycytidine restored tropomyosin expression in Ras-transformed RIE-1, HT1080, and DLD-1 cells, suggesting a role for DNA methylation in downregulating tropomyosin expression.
authors.affiliation: Department of Pharmacology, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, 27599, USA. shieldsj@med.unc.edu
authors.forename: Janiel M
authors.initials: JM
authors.lastname: Shields
authors.forename: Heena
authors.initials: H
authors.lastname: Mehta
authors.forename: Kevin
authors.initials: K
authors.lastname: Pruitt
authors.forename: Channing J
authors.initials: CJ
authors.lastname: Der
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002455:Cell Division; D002460:Cell Line; D002471:Cell Transformation, Neoplastic / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D019175:DNA Methylation / Q000187:drug effects; D003841:Deoxycytidine / Q000031:analogs & derivatives / Q000494:pharmacology; D015536:Down-Regulation; D005455:Fluorescent Antibody Technique; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015689:Oncogene Protein p21(ras) / Q000235:genetics / Q000378:metabolism; D027384:Oncogene Proteins v-raf / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction; D014335:Tropomyosin / Q000235:genetics / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*; D020741:rho GTP-Binding Proteins / Q000378:metabolism
chemical_list: D012333:RNA, Messenger; D014335:Tropomyosin; D003841:Deoxycytidine; D027384:Oncogene Proteins v-raf; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D015689:Oncogene Protein p21(ras); D018631:ras Proteins; D020741:rho GTP-Binding Proteins