Differentiation induction subtraction hybridization (DISH): a strategy forcloning genes displaying differential expression during growth arrest and terminal differentiation

Human cancers often display aberrant patterns of differentiation. By approp riate chemical manipulation, specific human cancers, such as human melanoma , leukemia and neuroblastoma, can be induced to lose growth potential irrev ersibly and terminally differentiate. Treatment of HO-1 human melanoma cell s with a combination of recombinant human fibroblast interferon (IFN-P) and the antileukemic compound mezerein (MEZ) results in irreversible growth ar rest, a suppression in tumorigenic properties and terminal cell differentia tion. A potential mechanism underlying these profound changes in cancer cel l physiology is the activation of genes that can suppress the cancer phenot ype and/or the inactivation of genes that promote the cancer state. To defi ne the repertoire of genes modulated as a consequence of induction of growt h arrest and terminal differentiation in human melanoma cells, we are using a differentiation induction subtraction hybridization (DISH) approach. A s ubtracted cDNA library, differentiation inducer treated cDNAs minus uninduc ed cDNAs, was constructed that uses temporally spaced mRNAs isolated from H O-1 cells treated with IFN-beta+MEZ. Approximately 400 random clones were i solated from the subtracted DISH library and analyzed by reverse Northern a nd Northern blotting approaches. These strategies resulted in the identific ation and cloning of both 30 known and 26 novel cDNAs displaying elevated e xpression in human melanoma cells induced to growth arrest and terminally d ifferentiate by treatment with IFN-beta+MEZ. The DISH scheme and the genes presently identified using this approach should provide a framework for del ineating the molecular basis of growth regulation, expression of the transf ormed phenotype and differentiation in melanoma and other cancers. (C) 1999 Elsevier Science B.V. All rights reserved.