TPA-induced differentiation of human rhabdomyosarcoma cells involves dephosphorylation and nuclear accumulation of mutant P53.
Previous studies have shown that human rhabdomyosarcoma cells are induced to differentiate by TPA, in the absence of appreciable alterations of the muscle regulatory genes and their products (1). The question was addressed whether the tumor suppressor p53 could be a target of TPA action in these cells. Genomic analysis by a Polymerase Chain Reaction/Single-Strand Conformation Polymorphism (PCR/SSCP) and direct sequencing indicate the presence of a mutation in exon VII at codon 248 (C to T transition) and a loss of heterozygosity of p53 gene in human rhabdomyosarcoma cell line (RD). It is here shown that transcription of p53 mRNA strongly decreases in RD cells induced to growth arrest and differentiate by TPA treatment. In these cells immunoprecipitation and immunoblot analysis show that both synthesis and total cellular concentration of the protein are also reduced by TPA. Nevertheless nuclear p53 accumulation is at much higher extent, whereas 32P-orthophosphate labelling, followed by immunoprecipitation, demonstrates a decrease of phosphorylation of both cytoplasmic and nuclear p53. These results indicate that TPA causes a number of alterations of mutant p53, likely mediated through a protein kinase C dependent mechanism, which might impair the transforming ability of mutant p53 in growth-arrested and differentiating RD cells.
Germani
A
A
Department of Experimental Medicine, University of L'Aquila, Italy.
Fusco
C
C
Martinotti
S
S
Musarò
A
A
Molinaro
M
M
Zani
B M
BM
D001483:Base Sequence; D015152:Blotting, Northern; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D017931:DNA Primers; D005091:Exons; D015870:Gene Expression / Q000187:drug effects; D016158:Genes, p53; D006801:Humans; D008969:Molecular Sequence Data; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis; D012208:Rhabdomyosarcoma; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis* / Q000235:genetics
D017931:DNA Primers; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; D013755:Tetradecanoylphorbol Acetate