alexa Identification of p53 gene mutations in breast cancers and their effects on transcriptional activation function.


Journal of Cancer Science & Therapy

Author(s): Oh SJ, Jung JY, Shim SS, Im MY, Kim HD,

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Abstract Somatic mutations in the p53 tumor suppressor gene are the most common genetic alterations found in human malignancies. In the present study, we studied 36 primary human breast carcinomas, using a polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and sequencing analysis of exons 2 through 9 for the presence of p53 gene mutations. Six of 36 (17\%) breast cancers contained mutations within the core domain of the p53 protein responsible for sequence-specific DNA binding (codons 102-292); all 5 missense mutations clustered between codons 240 and 291 (codons 240, 243, 250, 285, and 291), whereas one nonsense mutation occurred at codon 199. By using recombinant PCR in vitro mutagenesis, we introduced point mutations at codons 199 from Gly to stop (gly199stop), 240 from Ser to Ile (ser240Ile), 250 from Pro to Ala (pro250ala), 285 from Glu to Lys (glu285lys), and 291 from Lys to Asn (lys291asn), and all the p53 sequences were subcloned into the CMVneoBam vector under the control of the cytomegalovirus (CMV) promoter. To test whether the mutants p53 were functionally wild-type (wt) or mutant, we transfected them to p53-null Saos-2 cells with a reporter plasmid containing a p53-responsive element, and performed chloramphenicol acetyltransferase (CAT) assay. Transient CAT assay for transcriptional activation revealed that one group, including gly199stop, ser240ile, glu285lys, and lys291asn, abolished the transcriptional activity, whereas the other group, including pro250ala, retained stronger transcriptional transactivation activity than that of wt p53.
This article was published in Mol Cells and referenced in Journal of Cancer Science & Therapy

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