CHARACTERIZATION OF A NOVEL MELANOMA DIFFERENTIATION-ASSOCIATED GENE,MDA-9, THAT IS DOWN-REGULATED DURING TERMINAL CELL-DIFFERENTIATION

Terminal differentiation in human melanoma cells correlates with tempo ral changes in the expression of specific target genes. To define thos e genes that may be critical for this process we have used a subtracti on hybridization approach. cDNA libraries were constructed from active ly proliferating H0-1 human melanoma cells (driver cDNA library) and c ultures treated for various time periods with the combination of recom binant human fibroblast interferon (IFN-beta) and mezerein (MEZ) (temp orally spaced tester cDNA library) that induces terminal differentiati on (Jiang and Fisher, 1993). From these two cDNA libraries, an HO-I IF N-beta + MEZ temporally spaced subtracted (TSS) cDNA library was const ructed. Random screening of this TSS cDNA library identifies cDNAs tha t display differential expression as a function of induction of growth arrest and terminal differentiation, called melanoma differentiation- associated (mda) genes. In the present study we analyzed the propertie s of the novel mda-9 gene. This cDNA encodes a unique protein of 298 a mino acids with a predicted size of similar to 34 kDa. Southern blotti ng analysis indicates that mda-9 is an evolutionary conserved gene. Ti ssue distribution analysis documents comparable expression in 50 human tissues, with slightly elevated expression in brain (putamen) and spl een (adult and fetal). Treatment of H0-1 human melanoma cells with IFN -beta + MEZ results in a biphasic induction of mda-9 with maximum expr ession 8 and 12 h posttreatment and reduced expression at 24 h. In ter minally differentiated and irreversibly growth arrested human melanoma cells, the level of mda-9 mRNA is reduced. The suppression in mda-9 e xpression is not simply a function of growth inhibition, because treat ment of H0-1 cells with interferons, including IFN-beta, leukocyte int erferon (IFN-alpha), or immune interferon (IFN-gamma), elevates mda-9 expression even though they suppress growth. These studies demonstrate that subtraction hybridization using temporally spaced RNA samples, r esulting in a TSS cDNA library, can identify genes, such as mda-9, tha t are down-regulated during terminal cell differentiation in human mel anoma cells. Further studies are necessary to define the precise role of mda-9 in the process of terminal differentiation.