P53 TRANS-DOMINANTLY SUPPRESSES TUMOR-FORMATION OF HUMAN BREAST-CANCER CELLS MEDIATED BY RETROVIRAL BULK INFECTION WITHOUT MARKER GENE SELECTION - AN EXPEDITIOUS IN-VITRO PROTOCOL WITH IMPLICATIONS TOWARDS GENE-THERAPY

Studies on the molecular basis of human breast cancer have demonstrate d that mutational inactivation of the p53 tumor supressor gene may be an essential step in the development of this cancer, We and others hav e previously shown that transfer of the wild-type p53 gene into cultur ed breast cancer cells reduced their malignant potential, We report he re on a p53 gene transfer protocol based on a replication-incompetent retrovirus to efficiently inhibit tumor formation of cancer cells with endogenous mutant p53, The susceptibility of the cells to retroviral infection was determined with LZRNL transducing the lacZ reporter gene , A multiplicity of infection (moi) of 2 resulted in 90% of the expose d cell population in cytochemically detectable beta-galactosidase acti vity. Using the p53 vector Lhp53RNL with a moi of 2 was sufficient to completely supress tumor formation by the highly tumorigenic MDAMB231 breast cancer cells carrying a point missense mutation in codon 280. E ven after 12 weeks, no vital tumors were histologically detectable, Fo r comparison, established protocols were used to infect MDAMB231 cells with low moi with the p53 virus. Clones were expanded in G418-selecti ve media for few weeks, pooled and injected into nude mice, Tumor form ation occurred already after 1 week from G418-selected cells,long-term expression of the p53 transgene was more stable in retrovirally bulk- infected and nonselected cells resulting in an efficient suppression o f tumor formation, This approach may facilitate future studies on othe r growth suppressive genes that potentially qualify for in vivo gene t herapy.