AP-1 and C/EBP transcription factors contribute to mda-7 gene promoter activity during human melanoma differentiation

Treatment of human melanoma cells with a combination of recombinant fibrobl ast interferon (IFN-P) and the protein kinase C (PKC) activator mezerein (M EZ) causes a rapid and irreversible suppression in growth and terminal cell differentiation. Temporal subtraction hybridization combined with random c lone selection, reverse Northern hybridization, high throughput microchip c DNA array screening, and serial cDNA library arrays permit the identificati on and cloning of genes that are differentially expressed during proliferat ive arrest and terminal differentiation in human melanoma cells. A specific melanoma differentiation associated (mda) gene, mda-7, exhibits reduced ex pression as a function of melanoma progression from melanocyte to metastati c melanoma. in contrast, treatment of metastatic melanoma cells with IFN-be ta + MEZ results in expression of mda-7 mRNA and protein. To evaluate the m echanism underlying the differential expression of mda-7 as a function of m elanoma progression and induction of growth arrest and differentiation in h uman melanoma cells the promoter region of this gene has been isolated from a human placental genomic library and characterized. Sequence analysis by GCG identifies multiple recognition sites for the AP-1 and C/EBP transcript ion factors. Employing a heterologous mda-7 luciferase gene reporter system , we demonstrate that ectopic expression of either AP-1/cJun or C/EBP can s ignificantly enhance expression of the mda-7 promoter in melanoma cells. In contrast a dominant negative mutant of dun, TAM67, is devoid of promoter-e nhancing ability. Western blot analyses reveals that dun and the C/EBP fami ly member C/EBP-beta are physiologically relevant transcription factors who se expression corresponds with mda-7 mRNA expression. Electrophoretic mobil ity shift assays (EMSA) performed using nuclear protein extracts from termi nally differentiated human melanoma cells document binding to regions of th e mda-7 promoter that correspond to consensus binding sites for AP-1 and C/ EBP. These results provide further mechanistic insights into the regulation of the mda-7 gene during induction of terminal cell differentiation in hum an melanoma cells. (C) 2000 Wiley-Liss. Inc.