Expression of Proteins P16INK 4a, P53 and Bmi-1 in Hematopoietic Stem Cells of Patients with MDS. the Role of Cellular Senescence.

The myelodysplastic syndromes (MDS) comprise a spectrum of heterogeneous clonal stem/progenitor cell disorders characterized by marrow failure. One potential reason, apart from apoptosis, for hematopoietic stem cells (HSCs) functional failure is cellular senescence, which is believed to have evolved as a tumor suppressor mechanism capable of arresting growth to reduce risk of malignancy. It can be activated by both telomere-dependent (telomere shortening) and telomere-independent pathways (DNA damage, oncogenic or oxidative stress). There are two widely recognized major tumor suppressor pathways p16/pRb and p53-p14/ARF that regulate cellular senescence. Activation of either pathway is profoundly associated with cellular senescence. Bmi-1, on the other hand, is a transcriptional repressor that plays an essential role in the self-renewal of HSCs and leukemic stem cells as well. One of its major targets is the INK/ARF locus, which encodes p16 and p53 independently.

To study the role of cellular senescence in the pathogenesis of MDS through the expression of p16, p53, phospho p53 (activated form of p53, phosphorylated at Ser-15) and bmi-1.

We examined the expression level of p16INK4a, Bmi-1, p53 and phospho p53 by flow cytometry (direct and indirect staining) in CD34+ bone marrow mononuclear cells (BMMNCs). Furthermore, we analyzed the relative telomere length (RTL) in BMMNCs by quantitative fluorescence in situ hybridization assay using flow cytometry (flow-FISH).We verified the presence of p16 by SDS PAGE western blotting. We studied 36 samples of MDS patients (11 RA, 6 RARS, 3 RCMD, 4 5q- and 12 RAEB I-II), 17 samples of age-matched healthy controls after informed consent and 8 cord blood samples. All MDS diagnoses were histologically confirmed by bone marrow examination and categorized into low and high risk according to the international prognostic scoring system (IPSS).

Expression of p16 is significantly increased in low risk MDS (mean± SD=8,7±12) when compared to healthy controls (p=0,02) and high risk patients(p=0,004). Patients with 5q- syndrome express lower level of p16 in comparison with the other low risk group patients. Increased level of bmi-1 expression is noticed in high risk group (mean± SD=30,3 ±33,9) when compared to low risk group (mean± SD=25±24,6) and healthy controls (mean± SD=18 ±16,4), while 5q- syndrome patients appear to express higher level of bmi-1 than any other risk group. Significantly increased expression of p53 (p=0,03) is noticed in high risk group (mean ±SD=64,3±27) when compared to low risk (mean±SD=34,2±28,8). Patients with 5q- syndrome express increased level of p53 (mean± SD=57,4±26,3) similar to that of high risk group. Expression of phospho p53 seams increased (p=0,055) in low risk group(mean± SD=7,1± 13,9) in comparison to high risk group (mean± SD=0,09± 0,18) and healthy controls (mean± SD=0,19± 0,63). Phospho p53 is expressed in lower level in 5q- syndrome patients. Regarding the RTL there is a significant difference (p=0,0018) between MDS (mean± SD=9,7± 2,7) and cord blood samples (mean± SD=13,8± 1,9).

Increased levels of p16 and phospho p53 in low risk MDS suggest that cellular senescence may contribute to the ineffective hematopoiesis of MDS and probably in a telomere independent way. Low expression level of of phospho p53 in high risk MDS raises the question of genetic integrity of p53 in this group of patients and when combined with high levels of bmi-1 and low expression of p16 may play a role in the disease progression to AML. Further investigation of bmi-1, especially in the 5q- group - and other markers of cellular senescence (SA- b galactosidase, SAHF) is needed in order to clarify the impact of cellular senescence in different risk groups of MDS.

No relevant conflicts of interest to declare.