Regulation of mda-7 gene expression during human melanoma differentiation

]Induction of irreversible growth arrest and terminal differentiation in hu man melanoma cells following treatment with recombinant human fibroblast in terferon (IFN-beta) and mezerein (MEZ) results in elevated expression of a specific melanoma differentiation associated gene, mda-7. Experiments were conducted to define the mechanism involved in the regulation of mda-7 expre ssion in differentiating human melanoma cells. The mda-7 gene is actively t ranscribed in uninduced HO-1 human melanoma cells and the rate of transcrip tion of nInn-7 is not significantly enhanced by treatment with IFN-beta, ME Z or IFN-beta+MEZ. The high basal activity of the mda-7 promoter in uninduc ed melanoma cells and the absence of enhancing effect upon treatment with d ifferentiation inducers is corroborated by transfection studies using the p romoter region of mda-7 linked to a luciferase reporter gene containing the SV40 polyadenylation signal sequence. RT-PCR analysis detects the presence of low levels of mda-7 transcripts in uninduced and concomitant increases in differentiation inducer treated HO-1 cells. However, steady-state mda-7 mRNA is detected only in IFN-beta+MEZ and to a lesser degree in MEZ, treate d cells, We show that induction of terminal differentiation of HO-1 cells w ith IFN-beta + MEZ dramatically increases the half-life of mcCn-7 mRNA whil e treatment with cycloheximide results in detectable mda-7 mRNA in control and inducer treated cells. These observations confirm constitative activity of the mda-7 promoter in HO-1 cells irrespective of differentiation status suggesting posttranscriptional processes as important determinants of mda- 7 expression during terminal differentiation, The 3' UTR region of mda-7 co ntains AU-rich elements (ARE) that contribute to rapid mda-7 mRNA turnover during proliferation and reversible differentiation, a process controlled b y a labile protein factor(s), Substitution of the SV40 polyadenylation sign al sequence in the luciferase reporter plasmid with the mda-7-ARE-3'-UTR re nders the Luciferase message unstable,when expressed in proliferating and r eversibly differentiated melanoma cells. In contrast, the luciferase messag e is stabilized when the mda-7-ARE-3'-UTR construct is expressed in termina lly differentiated HO-1 cells. These results provide compelling evidence th at mda-7 expression during terminal differentiation in human melanoma cells is regulated predominantly at a posttranscriptional level.