Téma: Novel therapeutics, targeted therapies and gene therapy
Číslo abstraktu: P992
The tumour suppressor protein p53 is a transcription factor that has an essential role in guarding the cell from genotoxic stress. By contrast, mutated p53 can be extremely dangerous as it may gain new oncogenic properties and thus enhance tumorigenesis. Mutated p53 frequently accumulates in cancer cells and it has been shown that subtle molecular manipulation can lead in some cases (in some mutations) to p53 reactivation to its wild-type conformation. Although p53 mutations are frequent in various types of tumors, prognosis of affected patients is especially poor in hematological malignancies.
To assess a feasibility of mutated p53 reactivation in B-cell lines using comercially available small molecules.
For p53 reactivation, the following cell lines were used: RAMOS (Burkitt lymphoma; mutation I245D and 17p-); RAJI (Burkitt lymphoma; mutations R213 and Y234H); and SU-DHL-4 (DLBCL; mutation R273C and 17p-). All cell lines expressed high level of mutated p53 protein. Small molecules PRIMA-1 (Cayman Chemical Company) and ellipticine (Calbiochem) were used as reactivation agents. Cell lines were cultured under standard conditions (37°C, 5% CO2); Since RAJI cell line harbors temperature-sensitive p53 mutation (Y234H), these cells were also tested for reactivation at 30°C. The reactivation was tested at the p53 protein level by western blot (WB) and at p53 downstream pathway by quantitative real-time PCR.
In all three cell lines, both PRIMA-1 and ellipticine afftected cellular viability in a concentration-dependent manner (50; 25; 12.5 and 6.25 µM and 10; 5; 2.5 and 1.25 µM, respectively). Time-dependent effect (24, 48, and 72 h) was substantially more apparent in the case of ellipticine. The same concentration range was subsequently applied for testing of p53 reactivation by WB. In RAMOS cells, PRIMA-1 reduced mutated p53 level at two highest concentrations and already after 24 h. In RAJI cells the reduction of mutated p53 protein level was obvious only at 30°C; under these conditions the two highest concentrations again led to disappearance of mutated p53, prominently after 72 h. Situation with SU-DHL-4 cells was similar, as the two highest concentrations led once again to disappearance of mutated p53. For ellipticine, WB was performed after 6, 12, and 24 h. In RAMOS cells, the most pronounced effect was observed at highest concentration after 6 and 12 h cultivation; the cells cultured 24 h exhibited already massive apoptosis. In RAJI cells, the reduction of mutated p53 was only very subtle at both 37°C and 30°C. Analysis of p53 downstream target genes induction was performed after 24, 48, and 72 h. Concerning PRIMA-1, RAMOS cell line was inert to any reactivation. In RAJI cell line, we observed a subtle induction (up to 350% compared to untreated control set at 100%) of p21 and PUMA genes. In SU-DHL-4 there was a clear induction (620%) of p21 gene, but only at a single time interval. Regarding ellipticine, we observed subtle induction of BAX in RAJI cells and no induction of the studied genes in RAMOS cells.
Summary / Conclusion:
Our results show that the small targeted molecules can manipulate with mutated p53 protein in cancer B-cells. These molecules can lead to mutated p53 reduction (clinically highly desirable due to predicted mutated p53 oncogenic gain-of-function) and to induction of cell cycle regulatory and proapoptotic genes. However, further experiments are necessary to more deeply understand exact mechanisms standing behind these effects. Supported by grant NT/13519-4/2012 by IGA MH CR and by project MUNI/A/0723/2012.
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Keywords: Cell line, Lymphoma, p53
Datum přednesení příspěvku: 15. 6. 2013