Apoptosis and Cell Cycle Regulatory Gene Expression in Adult De Novo Myelodysplastic Syndromes (MDS)

Introduction: MDS constitute a heterogeneous group of clonal haematopoietic disorders characterized by ineffective haematopoiesis and an increased risk of transformation to acute myeloid leukaemia. Increased apoptosis has been associated with ineffective progenitor and maturing haematopoietic cell survival and associated cytopenias early in the disease whereas the progression of MDS to AML occurs concomitant with decreased apoptosis and an increased degree of neoplastic cell survival. The aim of the present study was to identify molecular alterations of cell cycle and apoptosis regulatory genes in the bone marrow cells of adult patients with de novo MDS and to correlate these with clinical characteristics.

Patients and methods: A total of 60 cases of MDS, 40 males and 20 females, classified according to FAB classification as follows: 17 RA, 5 RARS, 19 RAEB, 9 RAEBT and 10 CMML, were included in our study. Twelve non-Hodgkin’s lymphoma patients without bone marrow involvement were used as normal controls. BM aspirates were obtained at diagnosis from the patients as well as the control group. We used RNAse Protection Assay to detect alterations of expression at the mRNA level of cell cycle regulatory genes, particularly CDK1, CDK2, CDK3, CDK4, p27, p21, PISSLRE, p16, cyclins A, B, C, D1, D2, D3, A1 as well as apoptosis regulatory genes, particularly caspases 1,2,3,5,6,7,8,9, Granzyme B, bclxL, bclxS, bfl1, bik, bak, bax, bcl2, mcl1. Moreover our study included estimation of apoptosis using the Annexin V affinity assay, as well as analysis of cell cycle by determining the percentage of cells in S phase using flow cytometry.

Results: The median value of apoptosis for all MDS cases was 2,79% (range 0–34,9). A positive correlation was found between caspases 3, 5, 9, CDKs 1, 2 and 4, as well as p21 expression and the level of apoptosis. The median value of cells in S phase of the cell cycle was 10,6% (range 0,19–27,4). A positive correlation was found between caspase 3 expression levels and S phase. Patients of the IPSS ≥1 score group were associated with higher values of mcl1 gene expression. Cases with bone marrow blasts ≥5% showed higher bclw, mcl1 and bfl1 gene expression values. Regarding FAB classification, CMML correlated with higher cyclin D1 gene expression. bclxL and mcl1 gene median values were found higher in patients with MDS, compared to normal controls. On the contrary CDK3 and p21 gene median values were lower in patients compared to healthy individuals. Multivariate analysis revealed that combined expression of caspases 8, 3, 6, 5, 2, 7 and Granzyme B was lower in MDS patients compared to normals, as well as that combined expression of cyclins B, C, D1 and D2 was higher in patients with abnormal karyotype compatible with a higher proliferation rate and a higher probability of revealing karyotypic abnormalities.

Conclusions: Our study demonstrated that the expression of the anti-apoptotic mcl1 was higher in MDS patients with IPSS>1 and the anti-apoptotic genes bclw, mcl1 and bfl1 had higher expression in cases with ³5% blasts in the bone marrow. In the myeloproliferative CMML category a significantly higher cyclin D1 expression was found. Taking into consideration the multifactorial pathogenetic features of MDS, we consider the developing understanding of apoptosis and cell cycle function essential, with the view to proceed to molecularly targeted treatment and improved clinical outcome.