The MGSA/GRO gene was initially described as an autocrine growth factor for melanoma cells and as a growth related early response gene in fibroblasts. Subsequently, a family of these cytokines was identified including MGSA/GRO alpha, beta, gamma, and delta. the homology of the cDNAs for these four MGSA/GRO genes range from 82-93%. Many tumor cells over-express MGSA/GRO and there is evidence for tissue specific over- expression of the various subtypes of MGSA/GRO. The degradation rate of the various sub-forms appear to differ in tissues. The mRNA levels for these genes is increased when cells are treated with TPA, LPS, TNFalpha, IL-1, EGF, cycloheximide, serum, thrombin, and a number of other factors. MGSA/GRO mRNA levels are suppressed by TGFbeta in some cells. The regulation of mRNA levels by these agents appears to be the result of transcriptional effects as well as increased/decreased mRNA stability. The Hs294T malignant melanoma cell line over-expresses MGSA/GROalpha but not MGSA/GRObeta, gamma, or delta. Mutations of the NF-kappaB element markedly reduces basal transcription of a 5' MGSA/GRO CAT construct in melanoma tumor cells. In order to determine the mechanism for the elevated expression of MGSA/GRO, we will compare the basal and cytokine regulated expression of MGSA/GRO genes in melanoma to that in normal melanocytes. The goal of this study is to test the hypothesis that altered expression of MGSA/GROalpha in melanoma cells results from a combination of alterations in the level of NF-kappaB transactivation and altered mRNA stabilization. Specifically, we plan: (1) to compare the basal levels of transcription of MGSA/GRO alpha, beta, gamma, delta genes in normal and transformed melanocytes; (2) to compare the transcriptional regulation of the MGSA/GRO alpha, beta, gamma, delta genes by the cytokines IL-1, TNFalpha, TGFbeta, and by cycloheximide in these cell types, and to characterize alterations in NF-kappaB regulation of basal transcription in transformed melanocytes as compared to non-transformed melanocytes; (3) to examine the role for basal and cytokine effected MGSA/GRO in mRNA stabilization in normal melanocytes as compared to malignant melanoma cells, and (4) to examine the expression of MGSA/GRO alpha, beta, gamma, delta genes in non-malignant and malignant melanocytic lesions by in situ hybridization.
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