ISOLATION AND CHARACTERIZATION OF GENES ASSOCIATED WITH CHROMOSOME-6 MEDIATED TUMOR SUPPRESSION IN HUMAN-MALIGNANT MELANOMA

Melanocytic transformation is thought to occur by the sequential accum ulation of genetic alterations, Evidence implicating human chromosome- 6 as a site for a gene(s) involved in melanoma suppression comes from studies of LOH [loss of heterozygosity], cytogenetics and biologic rev ersion of tumorigenicity following the introduction of a normal chromo some 6 by microcell-mediated chromosome transfer (Trent et al., 1990), Using a tumorigenic melanoma cell line (UACC 903) and a chromosome-6 suppressed melanoma subline [UACC 903 (+6)], we have isolated a series of genes uniquely expressed in the suppressed subline, A modified PCR -based cDNA subtraction technique was used to generate subtracted cDNA sublibraries for both the parental and (+6) suppressed cells, A total of 32 randomly selected clones from the suppressed sublibrary were is olated and examined, with 24 detecting a transcript by Northern analys is, Of these 24 clones, 21 (88%) demonstrated elevated expressed by No rthern analysis in the suppressed subline relative to the tumorigenic parental cell line, In 6/21 differentially expressed clones (29%), exp ression was exclusive to the suppressed subline, Partial sequence anal ysis and database searching of these clones indicated that 5/6 were no vel with one representing a previously characterized gene, Chromosomal localization of the five novel clones was performed following PCR amp lification of a human/rodent somatic cell hybrid mapping panel or fluo rescent in situ hybridization, One cDNA (termed AIM1) was localized to a band-region of chromosome 6 frequently deleted in melanomas (6q21), This novel approach should facilitate the identification of genes who se expression is causally related to the suppressed phenotype.