Abstract

Acute promyelocytic leukemia (APL) is associated with the t(15;17) which generates the PML-RARa fusion gene. The encoded PML-RARα oncoprotein physically interacts with native PML impairing its function. PML is a potent inhibitor of proliferation and apoptosis. In addition, mouse embryonic fibroblasts in which PML was inactivated (PML−/− MEFs) exhibit a significant increase in the percentage (%) of cells in S phase accompanied by the decrease in the G0/G1 subpopulation. Transgenic mice (TM) hCG-PML-RARα develop a form of leukemia similar to human APL after a long period of latency, suggesting that PML-RARα expression is necessary but not sufficient to leukemogenesis. Leukemic cells of the TM model present increased proliferation associated with resistance of apoptosis. Nevertheless, it is not known whether these changes are present from birth and thus related to exclusively to PML/RARα expression, or appear late in life and are associated with additional mutagenic events. To address this issue, we have analyzed TM of different ages without hematological abnormalities. We characterized the distribution of cells in the phases of cell cycle, cell proliferation and resistance to apoptosis in vivo. Cell cycle was analyzed in bone marrow (BM) cells stained with propidium iodate (PI) and analyzed by flow cytometry (FC). In TM younger than 9m (n=12), there was no significant difference in cell cycle distribution compared to wild-type (WT) controls. In contrast, in TM older than 9m (n=15) the % of BM cells in S phase was significantly lower (TM=14,84 ± 3,39%; WT=18,26 ± 3,55%; p=0,005) and was associated with increase in the % of cells in G0/G1 (TM=81,69 ± 3,79%; WT=78,14 ± 3,70%; p=0,001). The proliferation was tested in vivo by injecting bromodeoxyuridine i.p. and assessing its incorporation by BM cells after 2h. Compared to WT controls, there was a significantly lower % of proliferating cells in TM older than 9m (19,48 ± 7,81 versus 23,20 ± 10,80% in WT; p=0,06). Apoptosis was induced by γ irradiation, and after 24h BM cells were obtained and stained with PI. The % of apoptotic cells was determined by quantifying sub-G0 peak by FC. BM cells from TM older than 9m, but not from younger mice, were resistant to apoptosis. In this age group, γ-irradiation induced a 2,24 ± 0,81-fold increase in the % of apoptotic cells, whereas in WT controls this increase was of 4,06 ± 3,01-fold (p=0,018). Finally, in order to analyze the transcriptional mechanisms subjacent to the resistance, we measured the expression of two candidate genes involved in cell cycle and apoptosis regulation: p21Waf1/Cip1 and CDKN2A. The analysis was restricted to myeloid precursors by isolating CD117+ cells through an immunomagnetic technique. We found that both p21Waf1/Cip1 and CDKN2A are up-regulated in TM older than 9m. Moreover, this up-regulation was detected in both irradiated and unirradiated TM. Our results showed that resistance to apoptosis is associated with a block of the transition G1/S in the pre-leukemic phase of hCG-PML-RARα TM model, in contrast with the previously demonstrated increase in proliferation and resistance to apoptosis in the leukemic phase. Therefore deregulation of cell cycle is a late event during APL genesis and may be associated with additional mutagenic events.

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