Abstract
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/GROα, β or γ 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/GROα-expressing melan-a-6 cells blocked transformation. These data suggest that MGSA/GRO-mediated transformation requires Ras activation in melanocytes.
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Abbreviations
- CXC chemokine:
-
chemokine with the first two conserved cysteine residues separated by an intervening amino acid
- CMV:
-
cytomegalovirus
- DMEM:
-
Dulbecco's modified Eagle's medium
- IL-8:
-
interleukin-8
- MGSA/GRO:
-
melanoma growth-stimulatory activity/growth-regulated protein
- AP-1:
-
activation protein-1
- RT-PCR:
-
reverse transcription-polymerase chain reaction
- SV-40:
-
simian virus-40
- EMSA:
-
electrophoresis mobility shift assay
- and MMLV:
-
moloney murine virus
- ELR motif:
-
glutamine-leucine-arginine tripeptide sequence near the N-terminus of MGSA/GRO proteins
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Acknowledgements
We are indebted to the NIH for support: CA56704 (A Richmond); Vanderbilt Cancer Center CA 68485, Department of Veterans Affairs Merit Award and Career Scientist Award (A Richmond), and to James Owen and Eddy Balentien for developing the MGSA/GRO expressing melan-a clones. We are also indebted to Ben Johnston and Amy Pruitt for excellent technical assistance.
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Wang, D., Yang, W., Du, J. et al. MGSA/GRO-mediated melanocyte transformation involves induction of Ras expression. Oncogene 19, 4647–4659 (2000). https://doi.org/10.1038/sj.onc.1203820
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DOI: https://doi.org/10.1038/sj.onc.1203820
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