Analysis of Apoptosis Regulatory Genes Expression in the Bone Marrow of Adult De Novo Myelodysplastic Syndromes (MDS).

Background: MDS cover a range of clonal stem cell disorders characterized by ineffective hematopoiesis associated by excessive intramedullary apoptosis of hematopoietic cells. The bcl2 family of proteins constitutes an essential component of the apoptotic machinery at the level of mitochondria and is involved in the acitvation of caspases a family of cytosolic proteases, the effector molecules of apoptosis

Aim: The aim of the present study was to examine caspases, granzyme B and bcl2 family mRNA expression and the degree of apoptosis in the bone marrow (BM) of adult de novo MDS and to correlate our findings with clinical parameters and prognosis.

Methods. We studied 46 cases of MDS including 14 RA, 21 RAEB, 6 CMML and 5 RARS according to FAB criteria. The degree of apoptosis was determined by flow cytometry using the Annexin method and propidium iodine labelling (PI) on BM mononuclear cells. The expression of caspases 1, 2, 3,5,6,7,8 and 9 and Granzyme-B as well as of bclw, bclxL, bclxS, BFL1, BID, Bik, Bak, Bax, bcl2, Mcl1was determined using a multiprobe RNase Protection Assay System (Riboquant, BD Biosciences). A pool of RNA from normal bone marrow mononuclear cells was used as a normal control. The expression of each gene was compared to that of two housekeeping genes (GAPDH and L32) using the Image Master analysis Software. The level of each gene expression was compared to that obtained from the normal pool RNA. The expression of the genes and the degree of apoptosis were analyzed, taking into consideration haematological parameters, the FAB classification and the IPSS value.

Results: The median value of apoptosis for all MDS cases was 4,07 (0.04–16.9) Apoptosis in the low risk group was higher but not significantly different from the high risk group (8.9 in the RA-RARS vs 4.3 in the RAEB, and CMML). A positive correlation was found between caspases 8, 5, 3, 2, 1 expression and the level ofapoptosis. Moreover a positive correlation was found between all caspases level of expression with the exception of caspase 7 that did not correlate with caspases 8 and 5. The same phenomenon was observed for the bcl2 family members with the exception of BFL1 that did not correlate with bclw, bclxS and bcl2. The level of expression as well as the percentage of positive cases for all caspases and granzyme B was not significantly different between different FAB subgroups and different IPSS risk categories. BFL1 and Mcl1 levels were significantly higher in patients with BM blasts >5%. Cases with ratio of BID gene expression >1 compared to normal pool were associated with IPSS values <=1. The epxression level of all other bcl2 family members and the percentage of positive cases were not significanlty different between different FAB and IPSS risk categories.

Conclusion. The expression level of caspases 1,2,3,5,6,8,9 and granzyme B did not correlate with the FAB, calssification and the IPSS risk groups in patients with MDS. The antiapoptotic genes BFL1 and Mcl1 were significanlty higher in cases with BM blasts >5% while cases overexpressing the proapoptotic gene BID were mostly represented by the low risk IPSS subgroups. Larger number of cases need to be examined to draw definite conclusion about the role of these apoptosis regulatory genes in the pathogenesis and prognosis of MDS.