Abstract

The tumor suppressor gene p53 is the most commonly mutated gene in solid tumors. Although less common in hematologic malignancies, about 10–20% of CLL cases carry a p53 mutation. Recently, the compound RITA (Reactivation of p53 and Induction of tumor cells Apoptosis) has been shown to able to bound to p53 and induce its accumulation in tumor cells, its seems to have a cytotoxic effects in tumors with wild type p53.

Peripheral blood or bone marrow was collected from 17 patients with B-CLL, 6 samples wit deleted p53 and 11 without. The cryopreserved samples were incubated in 2 and 3 days with RITA and fludarabine alone or RITA and fludarabine in combination. The cells (3.0x105 cells/ml) were incubated in a medium consisting of RPMI 1640, glutamax, 10% PBS (Phosphate Buffered Saline). Cell viability was assessed after incubation by a bioluminescence method measuring cellular ATP. RITA induced dose and time dependent cell toxic effects in all B-CLL cells but was most effective in those with wild type p53. The combination of 10μM RITA and 1μM of fludarabine resulted in a very high significant synergistic effect. RITA induced dose dependent cell toxic effects in both B-CLL cells with p53 mutation and those without. In the non mutated cells, the main viability compared to unexposed controls after two days of incubation was 54 % (range 20–88 %) at 10μM, 42 % (13–83 %) at 20μM and 31 % (6–870 %) at 50μM. In the patient with a confirmed p53 mutation, the cell viability values were 70 %, 56 % and 30 %, respectively. The combination of 10μM RITA and 1μM of fludarabine decreased the cell viability with an average of 56 percentage points compared to fludarabine alone. We conclude that RITA induces cytotoxic effect in B-CLL and that CLL cells without p53 deletion are more sensitive in vitro. (Preliminary results suggest that RITA cause accumulation of p53 and cause massive apoptosis) Determination of p53 status and apoptosis in the tested patients before and after exposure to RITA is currently performed and the results will be presented at the meeting.

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