Disruption of the p53-Mdm2 complex by nutlin-3 reveals different cancer cell phenotypes

Introduction: Mdm2 inhibits p53 transactivation by forming a p53-Mdm2 complex on chromatin. Upon DNA damage-induced complex disruption, such latent p53 can be activated, but in cells overexpressingMdm2 because of a homozygous single nucleotide polymorphism at position 309 (T→C) of mdm2, the complex is highly stable and cannot be disrupted by DNA damage, rendering p53 inactive. Methods: To determine whether the p53 response phenotype is influenced differentially in cells with variable mdm2 genotypes, we compared responses to DNA damage and targeted p53-Mdm2 complex disruption by Nutlin-3 in the following wild-type p53 human cancer cell lines: A875 and CCF-STTC-1 (C/C for mdm2 SNP309), SJSA-1 {mdm2 genomic amplification and T/T for mdm2 SNP309), MCF-7 (estrogen-induced Mdm2 overexpres- sion and T/C for mdm2 SNP309), ML-1 and H460 (T/T for mdm2 SNP309), and K562 (p53- null and T/C for mdm2 SNP309). We also examined mdm2 gene-splicing patterns in these lines by cloning and sequencing analyses. Results: While Mdm2-overexpressing C/C cells were resistant to p53 activation by DNA damage, they were sensitive to Nutlin-3. Strikingly, the p53 C1 checkpoint in C/C cells was activated by Nutlin-3 but not by etoposide, whereas in other Mdm2-overexpressing cells, both drugs activated p53 and subsequent C1 arrest or apoptosis. cDNA clones lacking exons 5-9 were generated at a high frequency in cells overexpressing Mdm2. Conclusion: Nutlin-3 and DNA damage distinguish a differential phenotype in human cancer cells with C/C mdm2 SNP309 from other Mdm2 overexpressers. Categorization of the Mdm2 isoforms produced and their influence on p53 activity will help in characterization and treatment development for different cancers.