Maryse Dupouy

Flt3-ligand induces adhesion of haematopoietic progenitor cells via a very late antigen (VLA)-4- and VLA-5-dependent mechanism.

Summary. The adhesion of haematopoietic progenitor cells (HPC) to the bone marrow microenvironment is a process regulated by cytokines. In this study, we have shown that flt3‐ligand (FL), a growth factor that controls early haematopoiesis, regulated the function and expression of the beta‐1 integrins, very late antigen (VLA)‐4 and VLA‐5 on HPC. The modulation of the adhesiveness of HPC by FL was studied by adhesion assays on umbilical vein endothelial cells (HUVEC). Stimulation by FL induced two peaks of increased adhesiveness of HPC. The first peak was at around 30 min and was mechanistically related to an activation of the beta‐1 integrins, mainly VLA‐4 and VLA‐5. The second peak was at around 12 h and was related to increased expression of VLA‐4 and VLA‐5. The control of HPC adhesiveness by FL is a previously unreported property of FL that may be important for the homing and the retention of flt3‐expressing HPC within the bone marrow microenvironment.

α-Defensin 1-3 And α-Defensin 4 as Predictive Markers of Imatinib Resistance and Relapse in CML Patients

Objective: Imatinib mesylate is a tyrosine kinase inhibitor used as first line treatment in chronic myeloid leukaemia. Despite a remarkable effectiveness, treatment failure cases have been reported in 20 percent of CML patients. The identification of biomarkers which can predict the response to imatinib is our point of interest. Methods: Gene expression profiling microarray was carried out on secondary imatinib resistant patients. Longitudinal studies were performed on imatinib treated responder/resistant patients. Then, Q-RT/PCR studies were realized on patients prior imatinib initiation. Results: For imatinib responder patients, we observed a strong and lasting decrease of α-defensin 1-3 and α-defensin 4 expression. For relapse patients, we observed a dramatic increase of α-defensin 1-3 and α-defensin 4 expression before BCR-ABL transcript increase. Moreover, before imatinib initiation, α-defensin 1-3 and α-defensin 4 expression was significantly lower in the resistant group than in the responder group. Conclusion The variation of expression of α-defensin 1-3 and α-defensin 4 in peripheral blood is associated with imatinib resistance and may reflect an adequate immune control of the disease. Monitoring of α-defensin 1-3 and α-defensin 4 could be helpful to predict the patients who are not going to respond to the treatment.

Expansion and differentiation of haemopoietic progenitor cells on endothelialized hydroxyapatite under static conditions.

We have analysed the potential of an endothelialized hydroxyapatite matrix (HAP), a synthetic bone substitute, as a cellularized support for the expansion of haemopoietic progenitor cells. Scanning electron microscopy (SEM) showed that the endothelial cells (EC) tended to form a monolayer fitting closely to the matrix, and that the progenitors adhered to the EC layer. Endothelialized HAP supported the proliferation and differentiation of the progenitors with the addition of the exogenous cytokines IL‐1, and IL‐3. The expanded cell population essentially belonged to the myeloid and monocytic lineages, with a smaller percentage of the megakaryocyte lineage. In comparative experiments CD34+ progenitors were expanded on endothelialized tissue culture flasks, and a significant higher viability of the expanded cells was found with the endothelialized HAP. A high percentage (approx. 40%) of mature granulocytes was generated in accordance with the presence of differentiating cytokines such as G‐CSF and GM‐CSF at high concentrations in the coculture medium. Other cytokines that could be detected were IL‐6, M‐CSF, SCF, flt3‐ligand. More than 50% of the expanded cell population was able to phagocytose bacteria and to generate an oxydative burst. These data indicate that cellularized HAP may be a useful matrix for stromal cell‐based expansion systems.

Effect of stem cell factor on leukemic progenitor cell growth and sensitivity to cytosine-arabinoside

Recruitment of quiescent, clonogenic blasts from patients with acute myeloid leukemia (AML) by hematopoietic growth factors (HGFs) may improve the cytotoxic effects of cell-cycle-specific drugs like cytosine-arabinoside (Ara-C). Using the culture methods described by Nara and McCulloch and making a distinction between self-renewing and post-deterministic mitoses, we analyzed the effects of stem cell factor (SCF), a growth factor acting on early hematopoietic progenitor and stem cells. First, we demonstrated that SCF, used in combination with other HGFs included in fetal calf serum (FCS) and/or in 5637 cell line supernatant (5637-CM), stimulated both colony formation and self-renewal of blast progenitors from 10 patients, unlike SCF alone. We tested the effects of SCF on the recruitment of cells in the S-phase by using a bromodeoxyuridine/DNA (BrdUrd/DNA) staining method in flow cytometry (FCM). We showed that SCF stimulated proliferation of AML cells significantly in 9/18 patients with AML. Second, we tested the influence of SCF on the sensitivity to Ara-C of self-renewing leukemic cells from 18 patients with AML. We showed that SCF was efficient in increasing the toxicity of Ara-C on the self-renewing blast progenitors, especially with high concentrations of Ara-C. However, a large patient-to-patient heterogeneity was found and the activity of SCF was not correlated with its effect on the cell cycle. These data indicate that SCF can enhance sensitivity to Ara-C of some leukemic cells with self-renewing capacity.