Different p53 mutations produce distinct effects on the ability of colon carcinoma cells to become blocked at the G1/S boundary after irradiation.

ABSTRACT

The LoVo colon carcinoma cell line that presents two wild type p53 alleles was used as the recipient for a series of transfections with p53 expression vectors coding for wild-type or three different mutants (143ala, 175his or 273his). The parental cell line as well as all clones that had rearranged the plasmid with consequent loss of p53 c-DNA were readily blocked at the G1/S boundary following 10 Gy of irradiation. For each mutation two clones with different levels of mutant protein expression were selected. Confirmation of the integration of the exogenous sequence was obtained by the expression of the mutant m-RNA, established by reverse transcription and DGGE or Southern blot. Flow cytometric measurements of 5-bromodeoxyuridine incorporation revealed a total G1/S block of the 143ala transfectants, similarly to the parental and control transfectant cells, but little or no cell cycle block for the 175his and 273his clones. Although it has been shown in vitro that all three mutations interfere with transcriptional activation by the wild-type protein, not only did we observe p53 protein induction and nuclear accumulation following irradiation, but WAF-1/CIP-1 m-RNA was increased in some of the clones for which the G1/S block was abolished. Our results show that mutant p53 proteins are to some extent submitted to the control of the cellular environment in cancer cells with wild type p53 alleles, but with an efficacy that depends on the mutation.