A TRANSFORMING P53 MUTANT, WHICH BINDS DNA, TRANSACTIVATES AND INDUCES APOPTOSIS REVEALS A NUCLEAR-CYTOPLASMIC SHUTTLING DEFECT

The DG75 Burkitt lymphoma-derived human B cell line is heterozygous fo r p53, carrying wild type (WT) and mutant (Arg283His) alleles, The cel ls constitutively express high levels of both p53 proteins and also Md m2, Arg283His transactivates the p21(Waf1), Mdm2, bar, cyclin G and IG F-BP3 promoters in transient transfection assays equally as well as, i f not better than WT p53. It also suppresses the outgrowth of SAOS-2 c ells and specifically binds DNA like wild type protein, However, in pr imary rodent fibroblasts Arg283His fails to suppress transformation by HPV16-E7 and (Ha-)ras and even has modest transforming activity when transfected alone with (Ha-)ras, When Arg283His is transiently transfe cted into SAOS-2 cells it efficiently induces apoptosis, so - unlike m utants such as Arg175Pro - its behaviour in transformation assays does not clearly correlate with loss of the apoptosis function, Immunofluo rescence staining of both REF transformants and transiently transfecte d SAOS-2 revealed that this unusual mutant becomes excluded from the n ucleus and produces striking cytoplasmic fluorescence, The best correl ation with transformation, therefore, appears to be the lack of nuclea r retention of Arg283His, Since this mutation does not map to any know n nuclear localization signal and its presence seems to result in aber rant exclusion from the nucleus, then it may prove very useful in expl oring mechanisms involved in the nuclear:cytoplasmic shuttling of p53.