TPA-INDUCED DIFFERENTIATION OF HUMAN RHABDOMYOSARCOMA CELLS INVOLVES DEPHOSPHORYLATION AND NUCLEAR ACCUMULATION OF MUTANT P53

Previous studies have shown that human rhabdomyosarcoma cells are indu ced to differentiate by TPA, in the absence of appreciable alterations of the muscle regulatory genes and their products (1). The question w as addressed whether the tumor suppressor p53 could be a target of TPA action in these cells. Genomic analysis by a Polymerase Chain Reactio n/Single-Strand Conformation Polymorphism (PCR/SSCP) and direct sequen cing indicate the presence of a mutation in exon VII at codon 248 (C t o T transition) and a loss of heterozygosity of p53 gene in human rhab domyosarcoma cell line (RD). It is here shown that transcription of p5 3 mRNA strongly decreases in RD cells induced to growth arrest and dif ferentiate by TPA treatment. In these cells immunoprecipitation and im munoblot analysis show that both synthesis and total cellular concentr ation of the protein are also reduced by TPA. Nevertheless nuclear p53 accumulation is at much higher extent, whereas (32) P-orthophosphate labelling, followed by immunoprecipitation, demonstrates a decrease of phosphorylation of both cytoplasmic and nuclear p53. These results in dicate that TPA causes a number of alterations of mutant p53, likely m ediated through a protein kinase C dependent mechanism, which might im pair the transforming ability of mutant p53 in growth-arrested and dif ferentiating RD cells. (C) 1994 Academic Press, Inc.