Dynamics of Bcl-2, Bax and ACE Expression In CD34+ Cells of Peripheral Blood and Bone Marrow In Patients with Acute Leukemia During Induction Therapy

Abstract

4840 The causes of drug resistance in acute leukemias (AL) have been studied very intensively and the key research was done on Bcl-2 family proteins. Last studies have showed that high level Bcl-2 expression in acute leukemia is really associated with drug resistance andpoor prognosis [Haematologica 2007, U. Testa]. It was demonstrated that lower Bax/Bcl-2 ratio (<0,3) was associated with FAB M0-M1 classes (p=.00001), poor-risk cytogenetics and poor prognosis [Blood 2003, G. Poeta]. But there were no studies on the dynamic evaluation of Bcl2 and Bax expression on CD34+ cells during chemotherapy. Renin-angiotensin system and angiotensin concertin enzyme (ACE) influence on leukogenesis is extensively investigated. It was reported that ACE expression on blast cells is high [Leuk Lymphoma 2006, S. Aksu]. Recent publications indicate that primitive hematopoietic precursors have different characteristics regarding ACE: CD34+ACE+cells transplanted into NOD/SCID mice contribute 10-fold higher numbers of multilineage blood cells than their CD34+ACE- counterparts and contain a significantly higher incidence of SCID-repopulating cells than the unfractionated CD34+ population [Blood 2008, V. Jokubaitis]. But it's still unknown how CD34+ACE+ cells in AL behave on and after chemotherapy. We have studied the dynamics of Bcl-2 and Bax expression by flow cytometry in CD34+ cells of peripheral blood (PB) and bone marrow (BM) in pts with AL. PB and BM samples were collected before treatment, on days +8, +36, only PB - on day + 21. Bcl-2 and Bax were detected on CD34+ cells by flow cytometry using specific monoclonal antibodies: CD34 (8G12, BD), Bcl-2 (100, BD), Bax (2D2, Santa Cruz). ACE (9B9, BD) expression was also evaluated. We calculated 10 000 cells in each sample. 10 pts were included in the study: 4 AML, 6 ALL. The control group comprised 4 healthy donors. At time of diagnosis CD34+ cells number in BM was 38,7%± 9,75, in PB - 38,3%± 8,14 in AL pts, not differing much in AML and ALL, and indicating blast cells population. CD34+ cells numbers in BM and PB of healthy donors were 1,35% and 0,23%, respectively. After induction therapy and WBC recovery (days +36-38) CD34+ cells number in AL pts decreased dramatically in BM to 3,83%±1,51 (p=0,001) and in PB to 0,98%± 0,29 (p=0,0001), indicating the efficacy of chemotherapy. The dynamics of Bcl-2, Bax and ACE expression on CD34+ cells of BM and PB in AL pts are presented in fig.1-6 As seen in the fig.1,2 CD34/Bcl-2 expression in BM is significantly higher (p=0,04) and in PB is similar in AL pts at the diagnosis comparing with donors. It's also worth to note that BM and PB CD34+ cells in donors had different expression characteristics of Bcl-2 demonstrating much higher level of antiapoptotic marker in PB cells. On the contrast CD34+ AL cells in BM and PB had similar characteristics regarding CD34/Bcl-2 expression. This expression level decreased substantially in BM at day +36 comparing with day 0 (p=0,04), but it never reached the donors level remaining extremely high and supposing the persistence of antiapoptotic activity in CD34+ cells in AL pts. It did not change at all during chemotherapy in PB cells, being identical to donors characteristics. The fig.2,3 demonstrate that, CD34/Bax expression in BM is almost 3-times higher (p=0,14) and in PB is twice lower (p=0,02) in AL pts in comparison with donors. It's interesting that CD34/Bax expression in leukemic BM and PB cells looks very similar, when in donors we had very low expression in BM and high - in PB. This fact demonstrates the heterogeneity of donor CD34+cells in BM and PB and points that leukemia CD34+cells in BM and PB are rather similar in Bax expression. Chemotherapy caused the significant augmentation of CD34/Bax expression in PB on day +8 (p=0,01) and near significant on day +21 (p= 0,09) showing the increased level of “dieing” cells in PB after cytostatic influence. The fig. 5,6 show that CD34/ACE coexpression in BM cells of AL pts and donors did not differ much at any time of evaluation. But CD34/ACE expression in PB cells of AL pts was much lower (p=0,02) than in donors and substantially increased at day +36 almost reaching the donor level. We may conclude that Bcl-2, Bax, ACE expression on CD34+ cells in AL pts and donors significantly differs, the dynamics of expression in AL while chemotherapy shows critical changes in CD34/Bcl-2 expression in BM, CD34/Bax and CD34/ACE in PB. Disclosures: No relevant conflicts of interest to declare.

Local hematopoietic renin-angiotensin system in myeloid versus lymphoid hematological neoplastic disorders

“…31 ACE and p53 expressions were detected in the CD34+ cells of patients with acute leukemia during and after induction chemotherapy. 17 ACE -expressing macrophages in lymph nodes of patients with Hodgkin’s disease have been detected. 15 Enhanced ACE activity was also linked to multiple myeloma.…”

Local bone marrow renin–angiotensin system in primitive, definitive and neoplastic haematopoiesis

“…Neoplastic malignant blood cells are derived from leukaemic stem cells within a complex series of pathological proliferative steps. There is preliminary evidence that the local BM RAS could affect neoplastic tumoral blood cell production [34][35][36][40][41][42][43][44][45]. The prominent functions of the local RAS in primitive embryonic haematopoiesis [21][22][23][24] support further the hypothesis that the local autocrine BM RAS could be active in neoplastic haematopoiesis.…”

“…Immunohistochemical studies have shown the possible role of ACE/RAS in BM by evaluating ACE expression in normal BM, several myeloproliferative disorders and myelodysplasia [51]. ACE and p53 expression were detected in CD34 + cells in patients with acute leukaemia during and after the induction of chemotherapy [43]. ACE-expressing macrophages in the lymph nodes of patients Hodgkin's disease have been detected [118].…”

Local Renin-Angiotensin System in Normal Hematopoietic and Multiple Myeloma-Related Progenitor Cells

“…Malignant blood cells, including multiple myeloma (MM) cells, are derived from the clonal neoplastic stem cells within a complex series of pathological proliferative steps. The local BM RAS could affect neoplastic tumoral blood cell production [ 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 ]. The local RAS is effective even in embryonic hematopoiesis [ 23 , 24 , 25 , 26 ].…”