MGSA/GRO-mediated melanocyte transformation involves induction of Ras expression.
The MGSA/GRO protein is endogenously expressed in almost 70% of the melanoma cell lines and tumors, but not in normal melanocytes. We have previously demonstrated that over-expression of human MGSA/GROalpha, beta or gamma in immortalized murine melanocytes (melan-a cells) enables these cells to form tumors in SCID and nude mice. To examine the possibility that the MGSA/GRO effect on melanocyte transformation requires expression of other genes, differential display was performed. One of the mRNA's identified in the screen as overexpressed in MGSA/GRO transformed melan-a clones was the newly described M-Ras or R-Ras3 gene, a member of the Ras gene superfamily. Over-expression of MGSA/GRO upregulates M-Ras expression at both the mRNA and protein levels, and this induction requires an intact glutamine-leucine-arginine (ELR)-motif in the MGSA/GRO protein. Western blot examination of Ras expression revealed that K- and N-Ras proteins are also elevated in MGSA/GRO-expressing melan-a clones, leading to an overall increase in the amount of activated Ras. MGSA/GRO-expressing melan-a clones exhibited enhanced AP-1 activity. The effects of MGSA/GRO on AP-1 activation could be mimicked by over-expression of wild-type M-Ras or a constitutively activated M-Ras mutant in control melan-a cells as monitored by an AP-1-luciferase reporter, while expression of a dominant negative M-Ras blocked AP-1-luciferase activity in MGSA/GRO-transformed melan-a clones. In the in vitro transformation assay, over-expression of M-Ras mimicked the effects of MGSA/GRO by inducing cellular transformation in control melan-a cells, while over-expression of dominant negative M-Ras in MGSA/GROalpha-expressing melan-a-6 cells blocked transformation. These data suggest that MGSA/GRO-mediated transformation requires Ras activation in melanocytes.
Wang
D
D
Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Yang
W
W
Du
J
J
Devalaraja
M N
MN
Liang
P
P
Matsumoto
K
K
Tsubakimoto
K
K
Endo
T
T
Richmond
A
A
D000818:Animals; D019898:Autocrine Communication / Q000502:physiology; D002461:Cell Line, Transformed / Q000378:metabolism; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D054360:Chemokine CXCL1; D019743:Chemokines, CXC; D002630:Chemotactic Factors / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D002999:Clone Cells; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D017930:Genes, Reporter; D011905:Genes, ras; D006133:Growth Substances / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D020935:MAP Kinase Signaling System; D008544:Melanocytes / Q000473:pathology*; D008545:Melanoma / Q000235:genetics / Q000473:pathology; D051379:Mice; D020559:Monomeric GTP-Binding Proteins / Q000096:biosynthesis; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics; D020033:Protein Isoforms / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D012334:RNA, Neoplasm / Q000096:biosynthesis; D018121:Receptors, Cytokine / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000502:physiology; D015398:Signal Transduction; D013382:Subtraction Technique; D018808:Transcription Factor AP-1 / Q000502:physiology; D014162:Transfection; D018631:ras Proteins
C475829:CXCL1 protein, human; D054360:Chemokine CXCL1; D019743:Chemokines, CXC; D002630:Chemotactic Factors; C475895:Cxcl1 protein, mouse; D006133:Growth Substances; D036341:Intercellular Signaling Peptides and Proteins; D009363:Neoplasm Proteins; D020033:Protein Isoforms; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D018121:Receptors, Cytokine; D011993:Recombinant Fusion Proteins; D018808:Transcription Factor AP-1; C078862:melanoma growth stimulating activity receptor; C109418:Mras protein, mouse; D020559:Monomeric GTP-Binding Proteins; D018631:ras Proteins