P53 guardian of the genome and target for improved treatment of leukemia
Abstract: P53 acts in several cellular processes as cell cycle checkpoints, DNA repair senescence and the surveillance of genomic integrity. Wt p53 inhibits cancer processes by causing cell cycle arrest and apoptosis. P53 is strongly regulated by the HDM-2 protein, which builds a complex with p53 leading to proteosomal degradation of p53. HDM-2 in turn is regulated by the p14ARF protein. The p53 gene is located on 17p13 and chromosome 17p abnormalities are strongly associated with p53 mutations by loss of one p53 allele and point mutation of the remaining. The gene for p14ARF is encoded on chromosome 9 in 9p21. The objectives were to identify chromosomal aberrations of importance for p53 function in leukemia and to study if small molecules can restore impaired p53 function. Leukemic cells were isolated from 399 adult patients with AML and chromosomal analysis was successful in 336. After incubation with cytostatics and culturing, in vitro sensitivity assay was performed by measuring ATP. Chromosomal analysis showed 39% normal karyotype. Of adverse aberrations, 16% had complex karyotype and 10% abnormal 7. Of abnormalities affecting p53, 7% (24 patients) had abnormal 17 and 13 patients (4%) abnormal chromosome 9. In vitro, patients with abnormal 17 were significantly more resistant against antileukemic cytostatics compared to both normal and complex karyotype. Although abnormal 9p showed higher drug resistance compared to normal karyotype, the difference was not significant. The shortest survival was seen in patients with abnormal 17, mean survival two months and all died within 11 months. p<0.0001 vs normal and <0.05 vs complex karyotype. Also patients with abnormal 9 had significantly shorter survival (mean 5 months) compared to normal karyotype. These findings prompted us to study the effect of two small molecules with a potential to restore the p53 function. PRIMA-1 can restore wild type confirmation and specific DNA binding of mutant p53 and trigger apoptosis in mutant tumor cells. RITA affects wt p53 by inducing intracellular accumulation of p53 and by preventing p53-HDM-2 interaction. Samples from 14 CLL patients incubated with 0.1-2.5ìM PRIMA-1 and 1-2ìM fludarabine. Drug sensitivity was measured with the ATP method and apoptosis by Annexin V. PRIMA induced pronounced apoptosis in the leukemic cells. Samples with mutated p53 were significantly more resistant to fludarabine and when combined with PRIMA-1, synergy was observed. 62 AML samples were incubated with PRIMA.1 5-20 muM. The p53-mutated cells were significantly (p<0.002-0.00001) more sensitive to PRIMA-1 and more resistant to Ara-C (p<0.0002). In contrast, incubation with RITA 0.1-1 muM in AML and 5-10mumM in CLL showed a significantly higher effect in samples without abnormal 17. In both AML and B-CLL cells exposure to RITA and RITA combined with PRIMA-1 resulted in time and dose dependent induction of intracellular p53. The increase was highest in AML cells. Co-incubation with PRIMA-1 and RITA showed a synergistic effect in the majority of AML samples. While combining RITA with fludarabine resulted in 100% synergy in CLL. We conclude that genetic abnormalities related with mutations in the HDM-2-p53 pathway occur in about 11 percent of patients with adult AML and that this is correlated to intracellular drug resistance and poor survival. Small molecules targeting the p53 function appears to be a promising way to improve the outcome of these patients.
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