p53 inactivation by point mutations and splice site mutations in human and mouse tumors

Sammanfattning: The p53 tumor suppressor gene is frequently mutated in human tumors. p53 induces cell cycle arrest and/or apoptosis in response to cellular stress, such as DNA damage, hypoxia and certain activated oncogenes like c-myc. The status of p53 in Burkitt's Iymphoma (BL) cell lines was investigated. The majority of BL lines expressed mutated p53 protein. Functional reconstitution with exogenous wild type (wt) p53 induced apoptosis in a BL line that carried a mutated p53 gene. These results substantiate the findings that c-myc induced apoptosis is p53 mediated, since all BLs carry activated c-myc by a c- myc/Ig translocation. Established ascites tumor cells grow under highly crowded, virtually anoxic conditions. Hypoxia is a powerful inducer of p53-dependent apoptosis. Would conversion of relatively well-vascularized solid mouse tumors into freely growing ascitic cell variants favor cells with mutated or deleted p53? PCR and sequence analysis showed that all solid tumors tested carried exclusively wild type pS3 while most of the ascites tumors carried mutant p53. The naturally occurring alternatively spliced p53 (p53as) mRNA was detected in all solid tumors, but was only present in few ascites tumors, in a mutated form. Our findings indicate that conversion of solid into ascites tumors favors the selection of cells with nonfimctional p53 or p53as. Furthermore, in three separate studies unusual point mutations in the p53 gene, which alter expression and function of the p53 protein are described. In a mouse sarcoma cell, a mutation in a splice donor site activates a cryptic donor site further downstream in the same intron and gives rise to an insertion of 5 extra amino acids to the p53 protein. Human skin fibroblasts derived from a patient with Bloom's syndrome (BS), lack p53 mRNA and protein. A highly sensitive RT-PCR and sequence analysis of p53 revealed that exon 6 was missing as a result of an unusual base substitution at a splice acceptor site of intron 5. These results thus suggest that the lack of p53 expression is due to aberrant splicing that destabilizes p53 mRNA. Analysis of a human tonsil tumor that expressed elevated levels of p53 showed a deletion of the entire intron 7 in one of the p53 alleles, leaving the coding sequence intact. The p53 protein is a key tumor-suppressor, as evidenced by its frequent inactivation in human cancers and understanding the mechanism behind the functional inactivation of p53 is essential for developing new therapeutic strategies. Akademitryck AB, Edsbruk. Printed on environmental-friendly nonhleached paper ©Kristinn P Magnússon, Stockholm 1998