The BH3-Only Protein Puma Plays An Essential Role in p53-Mediated Apoptosis of Chronic Lymphocytic Leukemia Cells

Chronic lymphocytic leukemia (CLL) is a heterogeneous malignant hematologic disease; the response to chemotherapy partly determines the prognosis of patients. Fludarabine, which is frequently used in the treatment of CLL, induces CLL cell apoptosis by activating p53 and then down stream BH3-only family members, consequent to the induction of DNA damage. To identify which is the key factor in this apoptosis pathway, we incubated CLL cells with an active metabolite of fludarabine, 9-β-D-arabinosyl-2-fluoroadenine (F-ara-A), in vitro and then analyzed the role of Puma, Noxa and Bim in p53-mediated apoptosis of CLL cells.

CLL cells from 15 CLL patients were incubated in medium with 3.5 mM fludarabine. Isometric cells were incubated with medium only, as blank control. CLL cells were harvested after 48-hour incubation. The apoptosis of cells was measured by staining with annexin V-FITC/propidium iodide (PI). The mRNA levels of Puma, Noxa, Bim and Bcl-2 genes were quantified using real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) with SYBR Green by ABI LightCycler. Fluorescence in situ hybridization (FISH) analysis was used to detect ATM and p53 gene deletions. CLL cells were evaluated for immunoglobulin heavy chain variable region (IgVH) gene and p53 gene mutational status by PCR and sequencing. The function of p53 gene was detected by flow cytometry.

1. The percentage of apoptosis at the beginning of incubation was 7.41% (4.11%-10.71%), and the percentages of spontaneous and fludarabine-induced apoptosis after 48 h incubation were 31.40% (18.14%-44.66%) and 59.6% (43.72%-75.48%), respectively. When CLL cells were cultured in vitro, the apoptosis was raised obviously (P=0.0002). Mean fludarabine-induced apoptosis was higher than the mean spontaneous apoptosis of CLL cells (P=0.0019).

2. After 48 h incubation without fludarabine, the expression level of Puma was significantly higher in CLL cells (P=0.0317), but the expression levels Noxa (P=0.5404), Bim (P=0.3067) and Bcl-2 (P=0.1747) were not changed. Treatment of CLL cells with fludarabine further enhanced Puma expression in all samples compared to cells in medium only (P=0.0359), but the expression levels of Noxa (P=0.8063), Bim (P=0.5703) and Bcl-2 (P=0.5322) were not changed.

3. In this analysis, 4 patients carried p53 abnormality and 3 patients carried heterozygous ATM gene deletion. The level of p21 in cytoplasm was not raised in CLL cells from patients with p53 abnormality after incubated with fludarabine. Elevation of p21 was detected in CLL cells carried heterozygous ATM gene deletion, after fludarabine induction.

4. It was appeared that apoptosis level (P=0.1704) and Puma expression level (P=0.5334) of CLL cells cultured in medium only were indepented of p53 status, but apoptosis level (P=0.0109) and Puma expression level (P=0.0430) were up-regulated higher by fludarabine in CLL cells with functional p53 than in ones with dysfunctional p53. Fludarabine-induced apoptosis level (P=0.228) and Puma expression level (P=0.164) in CLL cells with heterozygous ATM gene deletion were similar to p53 wild type ones.

5. Fludarabine-induced apoptosis and the expression level of Puma seemed to be higher in CLL cells with mutated IgVH than those with ummutated IgVH, but the difference was not significant.

Functional p53 was necessary in fludarabine-inducd apoptosis. CLL cells with p53 gene deletion and/or p53 mutation showed dysfunction of p53. CLL cells with heterozygous ATM gene deletion carried functional p53. Fludarabine induced CLL cells apoptosis via activating p53 and then up-regulating its downstream effector Puma. Only Puma was the essential factor for p53-mediated apoptosis, and Noxa and Bim seemed to play a more restricted role.

No relevant conflicts of interest to declare.