Apoptosis In CD34 Research Articles

Apoptosis and antiapoptotic mechanisms in the progression of myelodysplastic syndrome

Myelodysplastic syndrome (MDS), previously known as preleukemia, comprises a spectrum of heterogeneous, clonal disorders of hematopoiesis. A patient's life expectancy can range from a few months to more than a decade. Recent studies provide some insight into the pathophysiology of MDS. One mechanism contributing to the constellation of hypercellular marrow and peripheral blood cytopenia is a significant increase in programmed cell death (apoptosis) in hematopoietic cells. Tumor necrosis factor (TNF)-alpha, Fas ligand, TNF-related apoptosis-inducing ligand, and other proapoptotic cytokines are upregulated in early-stage/low-risk MDS, and neutralization of these signals can improve hematopoiesis. TNF-related apoptosis inducing ligand induces apoptosis preferentially in clonal cells, which can contribute to containment of the clone. In a proportion of patients, MDS will eventually evolve to acute leukemia. This progression has been correlated with upregulation of nuclear factor kappaB; altered expression of adaptor molecules, such as Flice inhibitory protein; and enhanced activity of antiapoptotic members of the Bcl-2 and inhibitors of apoptosis protein families. Also, the ratio of TNF receptors 1 and 2 changes in favor of receptor 2. The role of the microenvironment in the pathophysiology and progression of MDS has remained controversial, although there is evidence that stroma and matrix components, and their interactions with clonal cells, play an important role. Microarray gene-expression studies are consistent with dysregulation of apoptosis, but not all data are in agreement.

Effect of homoharringtonine on bone morrow CD34+CD7+ cells in chronic granulocytic leukemia

ObjectiveTo explore the effect of homoharringtonine (HHT) on bone morrow CD34+CD7+ cells in chronic granulocytic leukemia (CGL).MethodsThe changes of bone morrow CD34+CD7+ cells were observed after the treatment of HHT in 23 cases with CGL. The proliferation and apoptosis of CD34+CD7+ cells treated with HHT in vitro were studied.ResultsThe proportion of CD34+CD7+ cells in CGL (0.145±0.021) was higher than that of normal control (0.052±0.013). The proportion of CD34+CD7+ cells in patients who got cytogenetic responses to HHT (0.072±0.020) decreased remarkably, but not in those patients who did not got cytogenetic responses to HHT, (0.137±0.023). the proliferation of CD34+ cells was inhibited and the proportion of CD34+CD7+ cells decreased after cultured with HHT (0.134 in 24 h, 0.126 in 48 h and 0.102 in 72). The apoptosis rate of CD34+CD7+ cells was higher than that in CD34+CD7− cells (35.39%±4.39% versus 24.57%±4.01%, P<0.05) 72 h after culture with HHT.ConclusionThe proportion of CD34+CD7+ cells in CGL was higher than that of normal control and HHT may inhibit the proliferation and induce apoptosis of bone marrow CD34+CD7+ cells.

Inhibition of Bcl-2 Signaling by Small Molecule BH3 Inhhibitor GX15-070 as a Novel Therapeutic Strategy in AML.

GX15-070 is a novel cycloprodigiosin derived small molecule BH3 inhibitor that binds with moderate affinity to all antiapoptotic Bcl-2 family members, including Mcl-1, and is currently undergoing Phase I clinical trials in leukemias. In this study, we investigated the activity of GX15-070 in acute myeloid leukemia (AML) cell lines and primary AML samples. GX15-070 inhibited cell growth of HL-60, U937, OCI-AML3 and KG-1 cell lines at IC50's of 0.1, 0.5, 0.5 and 2.5μM, respectively, at 72 hours. Neither overexpression of Bcl-2 or Bcl-XL nor loss of expression of Bax conferred resistance to GX15-070. GX15-070 inhibited Bim/Bcl-2 heterodimerization and induced association of activated Bak with Bax in OCI-AML3 cells, as demonstrated by co-immunoprecipitation studies using CHAPS buffer. This was associated with cytosolic release of cytochrome c followed by an increase in annexin positivity, caspase activation and a decrease in mitochondrial inner membrane potential. Notably, GX15-070 induced cytochrome c release from isolated mitochondria of leukemic cells. GX15-070 synergized with both AraC and the novel BH3 mimetic ABT-737 to induce apoptosis in OCI-AML3 cells, a notoriously chemoresistant cell line (GX15-070 and ABT-737 average CI value 0.3; GX15-070 and AraC average CI value 0.36). In 6/7 primary AML samples, GX15-070 induced apoptosis in CD34+ progenitor cells at an average IC50 of 3.6±1.2μM at 24 hours. GX15-070 potently inhibited clonogenic ability of AML blasts at sub-micromolar doses (58.5±10.6% CFU-Blast at 0.1μM and 38.1±10.5% at 0.25μM, n=7). In summary, BH3 inhibitor GX15-070 induces apoptosis in AML cells via inhibition of association of pro-survival Bcl-2 family proteins and BH3-only proteins, followed by Bax/Bak activation and initiation of the intrinsic apoptotic pathway. Hence, GX15-070 alone or in combination with chemotherapeutic agents may have utility in AML therapy.

Exisulind induces apoptosis in advanced myelodysplastic syndrome (MDS) and acute myeloid leukaemia/MDS

The influence of Exisulind on the viability and apoptosis of CD34(+) stem cells from patients with advanced myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML)/MDS was investigated. In eight out of 10 patient samples Exisulind reduced the fraction of viable cells by inducing apoptosis. We found evidence that Exisulind-mediated apoptosis depends on c-Jun NH(2)-terminal kinase (JNK) activation. Addition of a specific JNK-inhibitor to Exisulind-treated advanced MDS and AML/MDS cells partly abrogated apoptosis. We propose that Exisulind is tested in clinical phase I/II trials for the treatment of advanced MDS and AML/MDS.

Early apoptosis in CD34+ cells as a potential heterogeneity in quality of cryopreserved umbilical cord blood

The increased use of umbilical cord blood (UCB) raises issues regarding the quality of cryopreserved UCB. This study investigated whether early apoptosis of CD34+ cells is part of the functional heterogeneity of cryopreserved UCB. Annexin V binding of CD34+ PI(-) cells showed wide variations in both fresh and cryopreserved UCBs, with greater variation among units frozen for > 5 years. Xenotransplantation of sorted cells into non-obese diabetic severe combined immunodeficient mice demonstrated that the Annexin V assay identified most repopulating activities in UCB units. Thus, early apoptosis of CD34+ cells could influence the outcome of transplantation using cryopreserved UCB.

Hyaluronic Acid Enhances To Differentiate into Myelo-Megakaryocytic Lineages during Ex-Vivo Expansion of Human Cord Blood(HCB) Using TPO, SCF and FL.

Hyaluronic acid(HA) is the major glycosaminoglycan component of extracellular matrices. CD44 is a ligand for HA and expresses on a wide variety of cell types. CD44 has been implicated in many cell-cell and cell-matrix interactions. In the present study, we studied the effects of HA on proliferation, differentiation and apoptosis of CD34+ cells during HCB ex vivo expansion using TPO, SCF and FL. CD34+ cells from cord blood MNC were cultured for 4 weeks in the combination of TPO, SCF and FL on HA-coated well and CD34+cells were cultured in identical conditions except HA-coated well. At day 4, 7, 14, 21, 18, we counted the number of expanded cells and analyzed the cell surface markers (CD3, CD14, CD19, CD34, CD44, CD61, CD66b) and apoptosis using 7-AAD staining. To analyze the effects of HA on CD34+ cell differentiation, anti-CD44 antibodies (clone A3D8 and 515) were used. The morphology on the expanded cells was examined using wright's staining. In results, cell counts were increased in HA-coated well compared with the control group until D7, but proliferation rate was decreased after D7. Apoptosis portion of CD34+ using 7-AAD staining was increased in HA-coated well and % of CD34+ cells was decreased in HA-coated wells compared with that of the control groups. The expanded cells in HA-coated well were negative for CD3 (pan T cell), CD19 (B cell) but CD14 (monocyte), CD61 (megakaryocyte) and CD66b (neutrophil) were highly expressed than those of control groups. Apoptotic portion was also increased in the HA-coated group. CD66b (neutrophil) expression in HA-coated well was significantly higher than those of control groups at D14 and above findings were confirmed by wright's stain. CD66 expression was reduced by the anti-CD44 antibody clone, A3D8 and however CD66 expression was increased by the clone, 515. Therefore, it might be possible that A3D8 and clone 515 recognize each different epitopes of CD44. In conclusion, CD34+ cell differentiation from HCB into CD66, CD14 and CD61 positive cells was enhanced by the contact of HA during ex vivo expansion using TPO, SCF and FL. This result suggests that the expanded human cord blood graft with HA might be useful as the “post-progenitor” to shorten the severe cytopenia period after myeloablative conditioning for HCB transplantation.

Inhibition of Bcl-2 Signaling by Small Molecule BH3 Inhibitor GX15-070 as a Novel Therapeutic Strategy in AML.

GX15-070 is a novel cycloprodigiosin derived small molecule BH3 inhibitor that binds with moderate affinity to all antiapoptotic Bcl-2 family members, including Mcl-1, and is currently undergoing Phase I clinical trials in CLL. In this study, we investigated anti-leukemic activity of GX15-070 in acute myeloid leukemia (AML) cell lines and primary AML samples. GX15-070 inhibited cell growth of HL-60, U937, OCI-AML3 and KG-1 at IC50's of 0.1, 0.5, 0.5 and 2.5μM, respectively as determined by trypan blue exclusion method at 72 hours. Overexpression of Bcl-2 or Bcl-XL did not confer resistance to GX15-070 (IC50's of HL-60/neo, HL-60/Bcl-2 and HL-60/Bcl-XL cells 0.25μM). MDR-overexpressing HL-60/Dox cells were similarly sensitive compared to their non-MDR counterpart, and specific MDR inhibitor (PSC 833) did not enhance apoptosis caused by GX15-070. GX15-070 (250nM) inhibited Mcl-1/Bak heterodimerization in HL-60 cells starting at 3 hours as evidenced by IP/Western blot analysis, however it did not affect Bax/Bcl-2 heterodimerization. This was associated with cytosolic release of cytochrome c followed by an increase in annexin positivity (21±2.5% annexin + cells with 250nM) and a decrease in mitochondrial inner membrane potential with 64±4.8% of cells losing membrane potential at 72 hours. GX15-070 did not affect cell cycle distribution of HL-60 cells. In 6/7 primary AML samples, GX15-070 induced apoptosis in CD34+ progenitor cells at an average IC50 of 3.6±1.2μM at 24 hours. In conclusion, the small molecule BH3 inhibitor GX15-070 potently induces apoptosis in myeloid leukemia cells via disruption of Mcl-1/Bak dimerization and activation of the intrinsic apoptotic cascade. Our findings provide rationale for the further development of this BH3 inhibitor as a novel therapeutic strategy for AML.

Myelotoxicity of trichothecenes and apoptosis: An in vitro study on human cord blood CD34 + hematopoietic progenitor

Previous studies have revealed that hematological disorders associated with trichothecenes intoxication in humans could result from hematopoiesis inhibition. The most frequent and potent trichothecene mycotoxins are T-2 toxin and deoxynivalenol (DON), respectively. Apoptosis induction by these two toxins was investigated in vitro on human hematopoietic progenitors (CD34 + cells). Hoechst coloration, DNA fragmentation and annexin-V/PI labeling in flow cytometry showed that T-2 toxin, in contrast to DON, induced apoptosis in CD34 + cells. T-2 toxin effect was dose- and time-dependent with a significant increase of apoptotic cells as early as 3 h after incubation at 10 −7 M and a maximum reached at 12 h. This observation evidenced the high sensitivity of hematopoietic progenitors to T-2 toxin. The inhibition of T-2 toxin-induced apoptosis by a pan-caspase inhibitor (Z-VAD-fmk) suggested the involvement of caspases. The proportional increase of caspase-3 specific activity (DEVDase) with T-2 toxin concentration confirmed its role in the process. After incubation of CD34 + cells with T-2 toxin, in conditions that induced apoptosis, clonal expansion of granulo-monocytes, erythrocytes and megakaryocytes precursors was dose-dependently inhibited. The hematological effects observed in T-2 toxin mycotoxicosis could then be assigned to hematopoiesis inhibition by apoptosis. Different mechanisms that need to be further elucidated are involved in DON myelotoxicity.

Caspase-8L expression protects CD34+ hematopoietic progenitor cells and leukemic cells from CD95-mediated apoptosis

Regulation of sensitivity or resistance for apoptosis by death receptor ligand systems is a key control mechanism in the hematopoietic system. Dysfunctional or deregulated apoptosis can potentially contribute to the development of immune deficiencies, autoimmune diseases, and leukemia. Control of homeostasis starts at the level of hematopoietic stem cells (HSC). To this end, we found that CD34+ hematopoietic progenitor cells are constitutively resistant to CD95-mediated apoptosis and cannot be sensitized during short-term culture to death receptor-mediated apoptosis by cytokines. Detailed analysis of the death machinery revealed that CD34+ cells do not express caspase-8a/b, a crucial constituent of the death-inducing signaling complex (DISC) of death receptors. Instead, we found a smaller splice variant termed caspase-8L to be present in HSC. Forced expression of caspase-8L using a recombinant lentiviral vector was able to protect hematopoietic cells from death receptor-induced apoptosis even in the presence of caspase-8a/b. Furthermore, we found that caspase-8L is recruited to the DISC after CD95 triggering, thereby preventing CD95 from connecting to the caspase cascade. These results demonstrate an antiapoptotic function of caspase-8L and suggest a critical role as apoptosis regulator in HSC. Similar to CD34+ HSC, stem cell-derived leukemic blasts from AML(M0) patients only expressed caspase-8L. Additionally we found, caspase-8L expression in several AML and ALL samples. Thus, caspase-8L expression might explain constitutive resistance to CD95-mediated apoptosis in CD34+ progenitor cells and might participate in the development of stem cell-derived and other leukemias by providing protection from regulatory apoptosis.

P-85 Myelodysplastic syndrome with bone marrow hypoplasia is associated with much higher frequency of apoptosis in CD34+ cells

P-85 Myelodysplastic syndrome with bone marrow hypoplasia is associated with much higher frequency of apoptosis in CD34+ cells

Myelodysplastic Syndrome with Bone Marrow Hypoplasia Is Associated with Much Higher Frequency of Apoptosis in CD 34+ Cells.

Myelodysplastic Syndrome (MDS) is a clonal disorder characterized by dysplastic changes and ineffective hematopoiesis. The ineffective hematopoiesis is considered as the results of excessive apoptosis of bone marrow (BM) hematopoietic cells. Recently, immunosuppressive therapy is effective in some hypoplastic MDS patients, resulting in the dramatic improvement of complete blood counts. To elucidate the difference between MDS patients with hypoplasia and normo/hyperplasia in BM, we measured the frequency of apoptosis in each lineage CD34+ BM cells in 35 MDS (12 RA patients with hypoplastic BM, 9 RA, 7 RAEB, and 7 RAEB-t patients with normo/hyperplastic BM) at diagnosis by three color flow cytometric analysis. Apototic cells were analyzed by PE labeled AnnexinV. The lineage cell population was identified as CD34+/GlycophorineA+, CD34+/CD33+, CD34+/CD41+, CD34−/GlycophorineA+, CD34−/CD33+ and CD34−/CD41+. In this study, the higher frequency of apoptosis was observed in each lineage CD34+ cells in all MDS patients (n=35, median: 32.2% (range: 6.3–80.5%) in erythroid, 38.0% (8.8–93.3%) in myeloid, 41.4% (10.2–78.4%) in megakaryocytic lineage, p<0.05, respectively), compared to that in normal controls (n=10, 8.5% (1.5–9.9%) in erythroid, 8.5% (2.2–8.8%) in myeloid, 7.7% (4.4–9.3%) in megakaryocytic lineage, respectively). While much higher frequency of apoptosis was observed in each lineage CD34+ cells in hypoplastic MDS patients (n=12, 49.1% (31.2–80.5%) in erythroid, 66.0% (37.8–93.3%) in myeloid, 68.5% (43.4–78.4%) in megakaryocytic lineage, p<0.05, respectively), compared to that in normo/hyperplastic MDS patients (n=23, 28.7% (6.3–69.4%) in erythroid, 30.0% (8.8–61.7%) in myeloid, 29.8% (10.2–58.1%) in megakaryocytic lineage, respectively). The increased frequency of apoptosis in each lineage CD34+ cells decreased after immunosuppressive therapy (n=5, 25.3% (19.8–36.4%) in erythroid, 35.2% (20.1–42.0%) in myeloid, 38.5% (30.6–47.9%) in megakaryocytic lineage, respectively). Our findings suggested that the excessive apoptosis occurred mainly in CD34+ cells in hypoplastic MDS as well as in non-hypoplastic MDS. Much more increased frequency of excessive apoptosis in CD34+ cells resulted in BM hypoplasia in hypoplastic MDS patients. This method is useful to evaluate quantitatively the ineffective hematopoiesis in BM hematopoietic cells.

The Effect of Telomerase Antisense on the Differentiation of CD34+ Cells from Bone Marrow to Blood Cells.

Background: This study was designed to evaluate the effect of adenovirus-mediated telomerase antisense (TA) expression on cellular proliferation and apoptosis of CD34+ cells in the ex-vivo culture condition collected from normal bone marrow donors.Methods: We used adenovirus vector containing recombinant antisense telomerase template RNA. Mononuclear cells were obtained from 21 bone marrow donors with normal marrow profile. Each samples were divided into two groups, consisted of the study group treated with telomerase antisense oligonucleotides(TA) and control group without treatment of TA. And then, CD34+ cells were purified from each sample with MACS method and were cultured with LTC-IC method. We evaluated the total and viable cell number, apoptosis degree and blood cell morphology at 2 weeks after LTC-IC culture, and compared between control and experimental groups.Results: Total cell number of study group (2.3–3.3(median:2.6)X104) was not significantly different from that of control group(2.1–3.5(median:2.7)X104). Viable cell percentage of control and study groups were 71–83(median:76)% and 69–84(median:77)%, respectively, without significant difference. Apoptosis degree of study group (25–38%, median:32%) was significantly higher than that of control (13–27%, median 18%), Blood cell morphology of study group was more differentiated without blast cells (0%) than that of control group showing blast cells (4–9%, median 7%) at 2 weeks of culture.Conclusion: We found that adenovirus-mediated antisense expression of telomerase RNA might accelerate the differentiation of hematopoietic stem cells with significant effect on the apoptosis degree. Further studies will be warranted to confirm these results and verify the mechanism of these phenomena.

Triterpenoid Methyl-CDDO Is a Potent Inducer of Apoptosis in CD34+ AML Progenitor Cells Via Activation of SAPK Pathways and Inhibition of MAPK Cascades.

Outcome results in AML are a continued challenge for the development of novel therapeutic strategies. C-28 methyl ester of 2-cyano-3,12-dioxoolen-1,9-dien-28-oic acid, CDDO-Me, a novel triterpenoid, induces apoptosis in myeloid and lymphoid leukemic cell lines and in primary AML samples in sub-micromolar concentrations. We reported previously that CDDO-Me inhibits the activation of ERK1/2, blocks Bcl-2 phosphorylation, and promoted apoptosis in AML-derived U937 cells (Blood 2002, 99(1):326–35). Here, we examined the effects of CDDO-Me on CD34+ AML progenitor cells in vitro. Exposure to 1μM CDDO-Me induced apoptosis in all but one AML sample (46±4% annexin(+) CD34+ cells, n=20). To assess the anti-leukemia activity of CDDO-Me in vivo, scid mice intravenously injected with U937 cells were treated with liposomally-delivered CDDO-Me (20mg/kg/day IV every other day, starting at day 7, for a total of 9 injections). While CDDO-Me was not toxic to the mice, pathological and flow cytometry analysis revealed reduced (55–86%) leukemia burden in the bone marrow, liver, and spleen of mice. Since we had shown that CDDO-Me inhibits phosphorylation of pERK in U937 cells, a further goal of this study was to assess the role of ERK in CDDO-Me-induced cell death in primary AML samples. ERK was expressed and phosphorylated in all (n=15) patients' samples studied. CDDO-Me inhibited ERK phosphorylation in 9 of 15 patient samples and promoted apoptosis. However, CDDO-Me still induced apoptosis in 5/6 samples that displayed no significant changes in pERK levels in response to the drug. This finding suggests that ERK is not the sole target of the compound. The stress-activated protein kinases JNK and p38 are related to ERK but in general activate pathways that are opposed to ERK. By Western blot analysis, CDDO-Me induced early (30 min) phosphorylation of JNK and p38, which preceded induction of cell death. Pre-treatment of U937 cells with JNK and p38 inhibitors SP600125 and SB203580 partially abrogated induction of apoptosis, while MEK inhibitor PD-98059 significantly enhanced cytotoxicity. CDDO-Me induced p38 phosphorylation in 5 of 6 primary AML samples tested. Collectively, these finding indicate that CDDO-Me markedly shifts signaling toward the JNK and p38 MAPK stress-related pathways and away from the cytoprotective MAPK cascade. In summary, the triterpenoid CDDO-Me is a potent inducer of apoptosis in primary AML including CD34+ AML progenitor cells. Induction of apoptosis is in part mediated via inhibition of ERK signaling combined with JNK/p38 activation. These studies in primary AML samples and the anti-leukemia activity of the compound in vivo suggest potential utility of CDDO-Me for the treatment of AML patients.

Darbepoetin Efficacy in Myelodysplastic Syndrome.

Recombinant human epoetin (rHu-EPO) is an effective treatment of anemia in myelodysplastic syndrome (MDS) in up to 40% of patients, mainly in low-risk MDS not yet requiring transfusions. Darbepoetin alpha is an rHuEPO analogue with an approximately 3-fold longer half-life than epoetin alfa, which leads to greater biological activity. We evaluated its effects on anemia in a pilot group of low and intermediate-1 risk MDS patients. The primary objective was to evaluate the efficacy of darbepoetin in terms of response/no response. Secondary objectives were to evaluate: 1) drug safety; 2) variations of Hb and the number of monthly transfusions; 3) changes in quality of life (QoL) of patients; and 4) changes in apoptosis of CD34+ cells. Twelve patients with Hb<11 g/dL were included in the 6-month study to receive an initial weekly dose of darbepoetin 150 mcg s.c. to be increased to 300 mcg in non-responders. Response criteria were defined as follows: complete response if an Hb increase of at least 2 g/dL or Hb = 12.0 g/dL, and no transfusions in transfusion-dependent patients; partial response if an Hb increase of 1 to <2 g/dL, or a 50% or greater (but not complete) reduction of transfusion requirement; no response if variations of Hb levels and of transfusion requirement were not included in the definitions of partial and complete response. At baseline and at 3 months, flow cytometric assays gating the CD34+ cells to observe the CD34+/Annexin V + events were performed on bone marrow aspirates. QOL measures were obtained by the QOL-E© questionnaire. Mean age was 76 (range 63–91) years. Serum erythropoetin levels were median 91 (range 24 – 421) IU/mL. Seven patients required 1 to 4 monthly transfusions and 5 patients were transfusion-free with baseline Hb values ranging from 8.0 to 10.9 g/dL. At the 150 mcg dose, 2 of the 7 transfusion-dependent patients became transfusion-free after 2 months, reaching stable Hb levels of 10.3 and 10.8 g/L, respectively, and 2 patients obtained a partial response. Of these latter 2 patients, one lost the response and one became transfusion-free after 2 months at the same dose. After dosage increase, the remaining 3 non-responders did not obtain a response up to 6 months follow-up. Of the transfusion-free patients, 3 were complete responders after 1 month at a dosage of 150 mcg, 2 of whom had to stop therapy for more than 2 months for Hb>13.0 g/dL (baseline Hb 10.3 and 10.9 g/dL, respectively); 1 became a complete responder after dosage increase. Overall 7 out of 12 patients were complete responders to darbepoetin treatment. At flow cytometric analysis, there was a trend in a reduction in apoptotic cells (p=0.064) associated with treatment response. At univariate ANOVA analysis, response to treatment was associated with increases in QOL-E© functional (p=0.036) and social (p=0.013) scores. Two responsive patients died during study period for unrelated adverse events. No side effects were observed. In conclusion, darbepoetin is safe in patients with MDS. A reduction in apoptotic cells is observed during treatment. Therapeutic response is associated with improvements in QOL. This pilot study suggests that darbepoetin is effective for the treatment of moderate-severe anemia of MDS, though a larger trial is required to evaluate predictive factors for response and QOL scores.

Flow cytometric detection of accelerated telomere shortening in myelodysplastic syndromes: correlations with aetiological and clinical-biological findings.

Using quantitative fluorescence in situ hybridisation and flow cytometry (flow-FISH), we investigated the biological and clinical relevance of telomere length in 55 patients affected by myelodysplastic syndromes (MDS) compared with 55 sex- and age-matched controls. We found that telomere fluorescence in MDS granulocytes, and CD34+ cells did not decline with age as in normal controls and that MDS granulocytes and CD34+ cells had significantly shorter telomeres than healthy controls. A significant higher incidence of cases with intermediate-unfavourable cytogenetics and International Prognostic Scoring System (IPSS) int-2/high-risk group was observed among patients with lower telomere fluorescence. We also found that apoptosis in CD34+ cells was significantly higher in IPSS int-1 low-risk patients when compared with IPSS int-2 high-risk cases and healthy controls and that CD34+ cell telomere fluorescence directly correlated with CD34+ cell apoptosis. Reduced telomere fluorescence was associated with a history of occupational exposure to toxic agents and with worse survival in univariate and multivariate analyses. Our results suggest that flow-cytometry assessment of telomere dynamics may represent a valuable tool in the biological and clinical-prognostic characterisation of MDS disorders.

A perforin/granzyme-positive MDS-derived T cell line, K2-MDS, induces apoptosis in CD34+ cells through the fractalkine–CX3CR1 system

Fractalkine (CX3CL1) and its receptor CX3CR1 play an important role in natural killer (NK) cell- and cytotoxic T cell-mediated endothelium damage. Here we describe the cytotoxicity of myelodysplastic syndrome (MDS)-derived T cell line, K2-MDS, through the fractalkine–CX3CR1 system. K2-MDS cells induced apoptosis against CD34+ cells from normal bone marrow (BM) in a direct cell contact manner. K2-MDS cells expressed perforin and granzyme B, but they lacked Fas ligand expression. A specific inhibitor for perforin, concanamycin A, blocked K2-MDS-dependent cytotoxicity. Furthermore, a CX3C-chemokine, fractalkine, was expressed in CD34+ cells, and its receptor, CX3CR1, was expressed on K2-MDS cells. The neutralizing monoclonal antibody (MoAb) for fractalkine and soluble fractalkine significantly inhibited K2-MDS-dependent cytotoxicity. K2-MDS cells also induced the cytotoxicity against human umbilical cord endothelial cells (HUVECs) expressing fractalkine. These data indicate that K2-MDS may be a perforin–granzyme-positive T cell line that exerts a cytotoxic effect on CD34+ cells mediated through the fractalkine–CX3CR1 system.

Apoptosis Susceptibility and Cell-Cycle Distribution in Cells from Myelodysplastic Syndrome Patients: Modulatory In-Vitro Effects of G-CSF and Interferon-α

Susceptibility to apoptosis varies in different forms of myelodysplastic syndromes (MDS). Our in vitro study aimed at better defining the cell kinetic profile by investigating whether G-CSF and interferon-alpha (IFNalpha) were capable of controling apoptotic/proliferative mechanisms in RAEB as well as in RAEB-t forms. Apoptosis and cell-cycle distribution were measured in mononuclear and in CD34+ cells from bone marrow samples of 27 MDS patients with RAEB (n = 15) and RAEB-t (n = 12). In selected samples, the in vitro influence of G-CSF and lymphoblastoid (Ly)-IFNalpha on the apoptotic susceptibility and on the cell kinetics of the above MDS populations was evaluated. RAEB samples showed a significantly greater apoptosis than RAEB-t ones, both in mononuclear cells (14.76%+/-8.73 vs. 5.95%+/-3.88, P= 0.0058) and in CD34+ cells (24.66%+/-16.08 vs. 3.96%+/-2.57, P = 0.0007). Short-term cell culture in the presence of G-CSF reduced apoptosis in CD34+ cells in all 4 RAEB samples tested (39.1%+/-40.7 vs. 21.0%+/-23.5, P = n.s.); the percentage of cells in S-phase significantly increased in 3/4 samples (19.90%+/-4.40 vs. 32.40%+/-7.85, P = 0.03). Ly-IFNalpha protected CD34+ cells from apoptosis in 3/4 RAEB samples (25.7%+/-8.06 vs. 10.9%+/-8.8, P = n.s.), but did not modulate cell-cycle distribution. G-CSF and Ly-IFNalpha failed to affect apoptosis and proliferation in RAEB-t. These observations indicate that in RAEB forms increased apoptosis can be efficiently counteracted in most of the samples by both G-CSF and Ly-IFNalpha, suggesting that only in these forms a retained regulatory mechanism on the apoptotic/ proliferative balance may allow therapeutic intervention with apoptotic regulators.

Decreased apoptosis of bone marrow progenitor cells in HIV-1-infected patients during highly active antiretroviral therapy.

Impaired haematopoiesis during HIV-1 infection may be caused by the overproduction of inflammatory cytokines by immune cells at the bone marrow level inducing Fas-mediated apoptosis of stem progenitors. In this study, we evaluated the effects of highly active antiretroviral therapy on apoptosis of CD34+ stem cells derived from the bone marrow of HIV-1-infected patients, and observed decreased Fas expression on progenitor cells, in parallel with the diminution of TNF-alpha levels and the amelioration of clonogenic parameters.

Differential apoptosis and Fas expression on GPI-negative and GPI-positive stem cells: a mechanism for the evolution of paroxysmal nocturnal haemoglobinuria.

Paroxysmal nocturnal haemoglobinuria (PNH) has a dual pathogenesis. PIG-A mutations generate clones of haemopoietic stem cells (HSC) lacking glycosylphosphatidylinositol (GPI)-anchored proteins and, secondly, these clones expand because of a selective advantage related to bone marrow failure. The first aspect has been elucidated in detail, but the mechanisms leading to clonal expansion are not well understood. We have previously shown that apoptosis and Fas expression in HSC play a role in bone marrow failure during aplastic anaemia. We have now investigated apoptosis in PNH. Ten patients were studied. Apoptosis, measured by flow cytometry, was significantly higher among CD34+ cells from patients compared with healthy controls. Fas expression was also increased. Cells that were stained for CD34, CD59 and apoptosis showed a significantly lower apoptosis in CD34+/CD59- compared with CD34+/CD59+ cells from the same patient. In three patients, staining for CD34, CD59 and Fas revealed lower Fas expression on CD34+/CD59- cells. Differential apoptosis of CD34+/CD59- HSC may be sufficient in itself to explain the expansion of PNH clones in the context of aplastic anaemia. In addition to demonstrating a basic mechanism underlying PNH clonal expansion, these results suggest further hypotheses for the evolution of PNH, based on the direct or indirect resistance of GPI-negative HSC to pro-inflammatory cytokines.

Transendothelial migration leads to protection from starvation-induced apoptosis in CD34+CD14+circulating precursors: evidence for PECAM-1 involvement through Akt/PKB activation

AbstractIn the present paper we show that transendothelial migration of a subset of CD14+ circulating leukocytes, coexpressing the CD34 precursor marker, leads to protection from the apoptosis that follows growth factor(s) withdrawal. The resistance of this cell subset to starvation-induced programmed cell death, lasting from 48 to 96 hours, is accompanied by a rise of mitochondrial adenosine triphosphate (ATP), a high nicotinamide adenine dinucleotide (NAD)/reduced nicotinamide adenine dinucleotide (NADH) ratio, and by the up-regulation of expression of the antiapoptotic proteins Bcl-2 and Bcl-X, together with an increase in the cytoplasmic, inactive, form of Bax. This suggests that protection from apoptosis is due to the preservation of mitochondrial function(s). Interestingly, ligation of the platelet endothelial cell adhesion molecule-1 (PECAM-1), which drives CD14+CD34+ transendothelial migration, leads to an increase in Bcl-2 A1 and Bcl-X intracellular content, and to protection from starvation-induced apoptosis. This event is dependent on the engagement of phosphatidylinositol-3 kinase and activation of Akt/PKB that is known to contribute to Bcl-2 and Bcl-X induction. These data point to a critical role of endothelium in preventing the apoptotic program triggered by starvation, possibly inducing a prolonged survival of antigen presenting cell precursors, in order to allow recirculation of these cells and localization to the site of priming of T lymphocytes.