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

Abstract

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 overexpressing Mdm2 because of a homozygous single nucleotide polymorphism at position 309 (T --> G) 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-STTG-1 (G/G for mdm2 SNP309), SJSA-1 (mdm2 genomic amplification and T/T for mdm2 SNP309), MCF-7 (estrogen-induced Mdm2 overexpression and T/G for mdm2 SNP309), ML-1 and H460 (T/T for mdm2 SNP309), and K562 (p53-null and T/G for mdm2 SNP309). We also examined mdm2 gene-splicing patterns in these lines by cloning and sequencing analyses.

Results: While Mdm2-overexpressing G/G cells were resistant to p53 activation by DNA damage, they were sensitive to Nutlin-3. Strikingly, the p53 G1 checkpoint in G/G cells was activated by Nutlin-3 but not by etoposide, whereas in other Mdm2-overexpressing cells, both drugs activated p53 and subsequent G1 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 G/G 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.

Fig 1

mdm2 SNP309 cells have functionally compromised p53 due to a p53-Mdm2 complex. A) Table depicting the different cell lines used in the project, their origins, and mdm2 and p53 status. B) Model demonstrating that in cells homozygous for G/G mdm2 SNP309, p53 is induced after DNA damage and does not dissociate from the excessively expressed Mdm2 protein. This association does not interfere with the ability of p53 to bind to DNA but does impair its transcriptional activity. This model has been slightly modified from one published previously.

Fig 2

Nutlin-3 can reactivate p53 in cancer cell lines with overexpressed Mdm2 when activation by etoposide is compromised. Quantitative reverse transcription–polymerase chain reaction was used to detect fold induction of the mdm2, waf1, gadd45, puma, and pig3 transcripts in K562 (panel A), A875 and CCF-STTG-1 (panel B), SJSA-1 and MCF-7 (panel C), and H460 and ML-1 (panel D) cells. Exponentially growing cells were either left untreated (white bars), treated with 8 µM etoposide (50 µM for MCF-7 cells) (black bars), or 10 µM Nutlin-3 (gray bars), for 24 hours. All results were normalized to untreated samples and gapdh values. Results are representative of three independent experiments, and error bars indicate standard deviation.

Fig 3

Cell cycle analysis of human cancer cells treated with etoposide or Nutlin-3. Fluorescence-activated cell sorting (FACS) analysis of K562 (panel A), A875 and CCF-STTG-1 (panel B), SJSA-1 and MCF-7 (panel C), and H460 and ML-1 (panel D) cells. Exponentially growing cells were either left untreated (left panel), treated with 8 µM etoposide (50 µM for MCF-7 cells) (center panel), or treated with 10 µM Nutlin-3 (right panel) for 48 hours. Cells were harvested, fixed in 30% ethanol, and cellular DNA was stained with propidium iodide before FACS analysis. Cell cycle phase is indicated on the graphs in the bottom panel.

Fig 4

Spliced variants have the potential to produce in-frame truncated Mdm2 products. cDNA was prepared by using reverse transcription–polymerase chain reaction (RT-PCR). Using specific primers flanking exons 2 and 12 of mdm2, PCR products were generated, cloned, and sequenced. The resulting nucleotide sequences were translated by using the translation tool found at www.ExPASy.org. The output amino acid sequences were compared to the Mdm2 full-length protein as depicted above, where letters represent amino acids.

Fig 5

Nutlin-3 can release the p53-Mdm2 complex in mdm2 SNP309 cells. A model illustrating that in cells homozygous for G/G mdm2 SNP309, p53 transcriptional activity is blocked through a chromatin-bound inhibitory complex that might contain an alternative Mdm2 isoform. DNA damage is unable to release p53 from this complex; however, the p53-Mdm2 antagonist, Nutlin-3, activates p53 function.