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

Abstract

Treatment of human melanoma cells with a combination of recombinant fibroblast interferon (IFN-beta) and the protein kinase C (PKC) activator mezerein (MEZ) causes a rapid and irreversible suppression in growth and terminal cell differentiation. Temporal subtraction hybridization combined with random clone selection, reverse Northern hybridization, high throughput microchip cDNA array screening, and serial cDNA library arrays permit the identification and cloning of genes that are differentially expressed during proliferative arrest and terminal differentiation in human melanoma cells. A specific melanoma differentiation associated (mda) gene, mda-7, exhibits reduced expression as a function of melanoma progression from melanocyte to metastatic melanoma. In contrast, treatment of metastatic melanoma cells with IFN-beta + MEZ results in expression of mda-7 mRNA and protein. To evaluate the mechanism underlying the differential expression of mda-7 as a function of melanoma progression and induction of growth arrest and differentiation in human 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 transcription factors. Employing a heterologous mda-7 luciferase gene reporter system, we demonstrate that ectopic expression of either AP-1/cJun or C/EBP can significantly enhance expression of the mda-7 promoter in melanoma cells. In contrast, a dominant negative mutant of cJun, TAM67, is devoid of promoter-enhancing ability. Western blot analyses reveals that cJun and the C/EBP family member C/EBP-beta are physiologically relevant transcription factors whose expression corresponds with mda-7 mRNA expression. Electrophoretic mobility shift assays (EMSA) performed using nuclear protein extracts from terminally differentiated human melanoma cells document binding to regions of the 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 human melanoma cells.