REVERSION OF MONOCHROMOSOME-MEDIATED SUPPRESSION OF TUMORIGENICITY INMALIGNANT-MELANOMA BY RETROVIRAL TRANSDUCTION

We have developed a general strategy to reverse monochromosome suppres sion of the malignant phenotypes by retroviral transduction. Our appro ach involved the introduction of a retroviral expression vector-carrie d cDNA library into a chromosome 6-suppressed melanoma subline UACC-90 3(+6) [J. M. Trent et al., Science (Washington DC), 247: 568-571, 1990 ]. The cDNA library was constructed from polyadenylated RNA isolated f rom the suppressed UACC-903(+6) cells, packaged into high-titer amphot ropic retrovirus particles, and transduced into UACC-903(+6) cells. Re vertant his(R) transductants were selected by isolating colony-forming cells in soft agar. A total of 121 large (>150 mu m) colonies was pic ked from soft agar culture with 18 of 121 (15%) established as permane nt sublines. The revertant sublines demonstrated 7-58% cloning efficie ncy upon plating in agar, in contrast to <0.05% for the UACC-903(+6) s ubline. All 18 revertant sublines, termed SRS1-SRS18 (for ''selection of revertants for suppression''), displayed a reduced population-doubl ing time, with 9 of 18 showing focus formation in monolayer similar to the parental (nonsuppressed) cell line. Preliminary evidence for reve rsion of the suppressed phenotype by injection of cells into athymic n ude mice has been completed for one revertant subline. Southern analys is has demonstrated integration of the retroviral vector sequence in a ll 18 sublines. This approach should facilitate the identification of genes involved in the tumorigenic phenotype of malignant melanoma, and is readily adaptable to other model systems.