Abstract
The p73 protein is a p53 homolog and acts on cell cycle and apoptosis regulation. Resistance to apoptosis is a common feature of Acute Myeloid Leukemia (AML), but mutations on the genes p53 and p73 are rare. It is translated in two distinct isoforms: TAp73 and ΔNp73. The later does not possess the N-terminal transactivation domain and exerts a dominant negative action over TAp73 and p53 functions. Theoretically an aberrant high expression of ΔNp73 may lead to a block of p53 and TAp73, thus conferring a proliferative advantage to the leukemic cells. In order to evaluate this issue, we proposed to:
Compare the gene expression levels of TAp73 and ΔNp73 isoforms in the bone marrow from de novo AML patients and normal individuals; Correlate these expression patterns with the presence of the rearrangements PML-RARα, AML1-ETO and CBFβ-MHY11, (previously determined by RT-PCR according the BIOMED-1 protocol).
From 137 AML patients whose samples were evaluated by Real Time PCR, 78 harbored the genetic rearrangements (referred to as RP group): PML-RARα (n = 30), AML1-ETO (n = 16) or CBFβ-MHY11 (n = 32), whereas in the 59 remaining samples these rearrangements were not detected (RN group). Additionally, CD34+ cell samples of 22 normal bone marrow donors were also evaluated. Sample input was normalized by GAPDH expression and the relative expression was calculated using the cell line k562 as reference sample. The mean expression of TAp73 and ΔNp73 was significantly lower on normal CD34+ cell compared to leukemic samples [(TAp73: mean (m) = 0.0162 ± standard deviation = 0.004 vs m = 0.623 ± 0.0845, p = 0,0047); (ΔNp73: m = 0.277 ± 0.09 vs m = 8.09 ± 1.34, p = 0,0215)]. A higher expression of TAp73 and ΔNp73 was observed on RN compared to RP samples [(TAp73: m = 0.992 ± 0.171 vs m = 0.344 ± 0.055, p < 0,0001); (ΔNp73: m = 12.44 ± 2.434 vs m = 4.80 ± 1.382, p = 0,0046)]. There was no difference in the expression of TAp73 between PML-RARα positive samples (m = 0.391 ± 0.095) and the remaining leukemic samples (m = 0.688 ± 0.104, p = 0,1476). However, the expression levels of ΔNp73 were significantly lower in the PML-RARα positive samples (m = 2.656 ± 0.370 vs m = 9.62 ± 1.69, p = 0,0317). No significant difference was observed in ΔNp73 and TAp73 expression between PML-RARα positive samples and the remaining samples with gene rearrengements (TAp73: m = 0.391 ± 0.095 vs m = 0.3144 ± 0.0671, p = 0,4990; ΔNp73: m = 2.656 ± 0.37 vs m = 6.153 ± 2.221, p = 0,2205). When compared to AML1-ETO and CBFβ-MHY11, the RN samples had a higher expression level of TAp73 (m = 0.3144 ± 0.0672 vs m = 0.992 ± 0.1717, p = 0.001), while there was no significant difference on the expression levels of ΔNp73 (m = 6.15 ± 2.22 vs m = 12.44 ± 2.43, p = 0.0642). These findings suggest that both p73 isoforms pathways are involved in the leukemogenic process. Moreover, the lower expression of ΔNp73 in the group with gene rearrangements may contribute to its better prognosis. The distinct pattern of ΔNp73 isoforms expression in AML with PML-RARα rearrangements suggests that it may be associated to a distinct response to apoptotic stimuli and to treatment outcome.
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