Reversion of monochromosome-mediated suppression of tumorigenicity in malignant melanoma by retroviral transduction.
We have developed a general strategy to reverse monochromosome suppression of the malignant phenotypes by retroviral transduction. Our approach involved the introduction of a retroviral expression vector-carried cDNA library into a chromosome 6-suppressed melanoma subline UACC-903(+6) [J. M. Trent et al., Science (Washington DC), 247: 568-571, 1990]. The cDNA library was constructed from polyadenylated RNA isolated from the suppressed UACC-903(+6) cells, packaged into high-titer amphotropic retrovirus particles, and transduced into UACC-903(+6) cells. Revertant his(R) transductants were selected by isolating colony-forming cells in soft agar. A total of 121 large (> 150 microm) colonies was picked from soft agar culture with 18 of 121 (15%) established as permanent sublines. The revertant sublines demonstrated 7-58% cloning efficiency upon plating in agar, in contrast to <0.05% for the UACC-903(+6) subline. All 18 revertant sublines, termed SRS1-SRS18 (for "selection of revertants for suppression"), displayed a reduced population-doubling time, with 9 of 18 showing focus formation in monolayer similar to the parental (nonsuppressed) cell line. Preliminary evidence for reversion of the suppressed phenotype by injection of cells into athymic nude mice has been completed for one revertant subline. Southern analysis has demonstrated integration of the retroviral vector sequence in all 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.
Su
Y A
YA
Laboratory of Cancer Genetics, National Center for Human Genome Research, NIH, Bethesda, Maryland 20892-4470, USA.
Ray
M E
ME
Lin
T
T
Seidel
N E
NE
Bodine
D M
DM
Meltzer
P S
PS
Trent
J M
JM
D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D004273:DNA, Neoplasm / Q000235:genetics; D016147:Genes, Tumor Suppressor; D006801:Humans; D008545:Melanoma / Q000235:genetics*; D008969:Molecular Sequence Data; D009006:Monosomy; D012878:Skin Neoplasms / Q000235:genetics*; D014161:Transduction, Genetic; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D018076:DNA, Complementary; D004273:DNA, Neoplasm