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Evidence for a Role of TNF-Related Apoptosis-Inducing Ligand (TRAIL) in the Anemia of Myelodysplastic Syndromes
Abstract
Myelodysplastic syndromes (MDS) are characterized by impaired erythropoiesis, possibly caused by proapoptotic cytokines. We focused our study on the cytokine TRAIL (TNF-related apoptosis-inducing ligand), which has been shown to exhibit an anti-differentiation activity on erythroid maturation. Immunocytochemical analysis of bone marrow mononuclear cells (BMMC) showed an increased expression of TRAIL in MDS patients with respect to acute myeloid leukemia (AML) patients and normal BM donors. TRAIL expression was increased predominantly in myeloid precursors of granulocytic lineage and in a subset of monocytes and pro-erythroblasts. Significant levels of soluble TRAIL were released in 21 of 68 BMMC culture supernatants from MDS patients. On the other hand, TRAIL was detected less frequently in the culture supernatants of AML (4 of 33) and normal BMMC (0 of 22). Analysis of peripheral blood parameters revealed significantly lower levels of peripheral red blood cells and hemoglobin in the subset of patients whose BMMC released TRAIL in culture supernatants compared to the subgroup of patients who did not release TRAIL. Moreover, TRAIL-positive BMMC culture supernatants inhibited the differentiation of normal glycophorin A+ erythroblasts generated in serum-free liquid phase. Thus, increased expression and release of TRAIL at the bone marrow level is likely to impair erythropoiesis and to contribute to the degree of anemia, the major clinical feature of MDS.
... 7,40 As extensive intramedullary cell death in patients with MDS is related to TNFα, and the serum TNFα concentrations are elevated in patients with BD, anti-TNFα agents would theoretically be appropriate for treating intestinal BD-MDS. 41,42 However, previous studies showed that 5-ASA or CS and anti-TNFα agents were ineffective in treating intestinal BD-MDS, and only hematological therapy could improve both intestinal lesions and cytopenias. [25][26][27]29,43 Consistently, in our study, 5-ASA and/or CS, immunomodulators, and anti-TNFα agents provided clinical benefits in 4/32, 0/5, and 4/15 patients, respectively, whereas in all four patients who had been resistant to medical treatments, improvement in intestinal ulcers was observed following HSCT. ...
... However, as a causative correlation to IR, some studies have directly implemented CRP in developing IR [30]. Additionally, evidence has been reported on TNF-α implementation in progressing IR and T2DM [31][32][33]. Moreover, growing evidence has also been documented on molecular changes in hepatocytes affected by IL-6 under inflammatory stress that might promote IR [5,15,34]. ...
Background: Extreme alterations in hematological values and inflammatory biomarkers affecting dependent variables in clinical tests are associated with type-2 diabetes mellitus and anemia. Objective: To investigate some hematological and inflammatory risk factors and their relation in developing insulin resistance in anemic type-2 diabetes mellitus. Patients and Methods: This case-control study was conducted in 190 patients and healthy subjects in the hospital of Layla Qasim Center in Erbil city and ethical clearance was obtained from the university ethical review committee. The included subjects were divided into three groups: non-diabetic (control) group, non-anemic type-2 diabetes mellitus, and anemic type-2 diabetes mellitus. Blood samples were analyzed for hematological parameters by Coulter counter analyzer, and biochemical tests included glycated hemoglobin, fasting blood glucose, insulin, and some inflammatory biomarkers by spectrophotometry and standard sandwich enzyme-linked immunoassay. Harvested data were analyzed by the statistical package for social science version 23. Results: The results showed remarkable alterations in glycated hemoglobin, red and white blood corpuscles, and inflammatory parameters in anemic type-2 diabetes mellitus compared to non-anemic type-2 diabetes mellitus. Inflammatory parameters including interleukin-1 and tumor necrosis factor-alpha exhibited a strong positive correlation with anemic diabetes than non-anemic diabetes. Conclusion: We concluded that some hematological and biochemical parameters were positively related to developing anemic type-2 diabetes mellitus and can be helpful as diagnosing tools for predicting the progression of anemic type-2 diabetes mellitus.
... Although there are no data on the outcomes of CO-VID-19 in patients with MDS, those with lower-risk MDS or MDS/MPN, particularly chronic myelomonocytic leukemia (CMML), typically have high baseline inflammation with increased levels of multiple cytokines and chemokines (including IL-1, IL-6, or TNF) [59][60][61][62][63], and therefore they may be at particular risk of experiencing immune overactivation and hyperinflammation from SARS-CoV-2 infection. Consequently, close monitoring of patients with lower-risk MDS or CMML with SARS-CoV-2 infection is warranted even in case of mild disease. ...
The coronavirus disease 2019 (COVID-19) pandemic poses several challenges to the management of patients with leukemia. The biology of each leukemia and its corresponding treatment with conventional intensive chemotherapy, with or without targeted therapies (venetoclax, FLT3 inhibitors, IDH1/2 inhibitors, Bruton's tyrosine kinase inhibitors), introduce additional layers of complexity during COVID-19 high-risk periods. The knowledge about COVID-19 is accumulating rapidly. An important distinction is the prevalence of "exposure" versus "clinical infectivity," which determine the risk versus benefit of modifying potentially highly curative therapies in leukemia. At present, the rate of clinical infection is <1-2% worldwide. With a mortality rate of 1-5% in CO-VID-19 patients in the general population and potentially of >30% in patients with cancer, careful consideration should be given to the risk of COVID-19 in leukemia. Instead of reducing patient access to specialized cancer centers and modifying therapies to ones with unproven curative benefit, there is more rationale for less intensive, yet effective therapies that may require fewer clinic visits or hospitalizations. Here, we offer recommendations on the optimization of leukemia management during high-risk COVID-19 periods.
... Innate immunity signaling mediated through TLRs, inflammatory cytokines (i.e., IL-1, IL-6, and IL-8), and damage associated molecular patterns such as S100A8/9 are observed among most MDS subtypes [10,11]. This inflammatory background, the unclear role of apoptosis in MDS pathogenesis, and the high levels of tumor necrosis factor (TNF) in MDS [12][13][14] suggest alternative and less immune-silent mechanisms of cell death may be responsible for the disease phenotype. Although recent studies have described NLRP3-inflammasome mediated pyroptosis as a mediator of the MDS phenotype [15,16], there are limited data evaluating the potential role of other inflammatory mechanisms of cell death in MDS pathogenesis. ...
Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis and cytopenias due to uncontrolled programmed cell death. The presence of pro-inflammatory cytokines and constitutive activation of innate immunity signals in MDS cells suggest inflammatory cell death, such as necroptosis, may be responsible for disease phenotype. We evaluated 64 bone marrow samples from 55 patients with MDS or chronic myelomonocytic leukemia (CMML) obtained prior to (n = 46) or after (n = 18) therapy with hypomethylating agents (HMAs). RNA from sorted bone marrow CD34+ cells was isolated and subject to amplification and RNA-Seq. Compared with healthy controls, expression levels of MLKL (CMML: 2.09 log2FC, p = 0.0013; MDS: 1.89 log2FC, p = 0.003), but not RIPK1 or RIPK3, were significantly upregulated. Higher expression levels of MLKL were associated with lower hemoglobin levels at diagnosis (−0.19 log2FC per 1 g/dL increase of Hgb, p = 0.03). Significant reduction in MLKL levels was observed after HMA therapy (−1.06 log2FC, p = 0.05) particularly among nonresponders (−2.89 log2FC, p = 0.06). Higher RIPK1 expression was associated with shorter survival (HR 1.92, 95% CI 1.00–3.67, p = 0.049 by Cox proportional hazards). This data provides further support for a role of necroptosis in MDS, and potentially response to HMAs and prognosis. This data also indicate that RIPK1/RIPK3/MLKL are potential therapeutic targets in MDS.
... The cellular and molecular mechanisms of the cytopenias are not well understood.2,3 In all subtypes of MDS, high rates of apoptosis mediated by inflammatory cytokines in the bone marrow contribute to the ineffective hematopoiesis and peripheral cytopenias.121,122 However, apoptosis is not inherited uniformly by all the cell lineages in the marrow, as the cells most sensitive to apoptosis are the ones undergoing premature death in the marrow, whereas those most resistant to it are the cells that make it into the blood. ...
Myelodysplastic syndromes (MDS)s are a myeloid neoplasm with a propensity for natural evolution or transformation to acute leukemias (AL) over time. Mechanisms for MDS transformation to AL remain poorly understood, but are related to genomic instability, which affects the production of the different cell lineages. Genomic instability is also generated by dysfunctional telomeres. Indeed telomeres, the protective ends of chromosomes, are the backbone of genome stability. Nuclear telomere remodelling is an early indicator of nuclear remodelling preceding the onset of genomic instability and MDS. This review aims to revisit the pathogenesis and pathophysiology of MDS from morphology and cytogenetics to molecular and epigenetic mechanisms. Furthermore, this review will highlight and discuss recent breakthroughs in dysfunctional telomeres and nuclear telomere architecture roles in the pathogenesis and physiopathology of MDS in the global context of genomic instability.
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... It has been demonstrated that it specifically affects erythroid development by targeting on immature erythroblasts [24][25][26][27] and acts in a stage of different particular manner, as a negative regulator of normal erythropoiesis [25]. Expanded expression of TRAIL at the bone marrow level is liable to hinder erythropoiesis and add to the level of iron deficiency, that become the major clinical component of Myelodysplastic syndrome (MDS) [28]. ...
Introduction:
Curcumin, traditionally utilized as a flavouring zest as a part of Indian cooking, has been accounted to decrease the proliferation potential of most cancer cells. Apoptosis is a mechanism by which most anticancer therapies including chemotherapy, radiation and antihormonal therapy kill tumour/cancer cells. Novel agents that may sensitize drug-resistant tumour cells for induction of apoptosis by customary treatments could lead to the regression and improved prognosis of the refractory disease. Indeed, chemotherapeutic agents have been shown to sensitize cancer cells to killing by death ligands such as tumour necrosis factor-α.
Aim:
To investigate cytotoxicity and apoptotic effect of curcumin in chronic myeloid leukaemic cell line KCL-22.
Materials and methods:
In present study, different doses of curcumin (10,25,50,75,100μM) and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) (25,50 μM) alone and combine regimen were exposed to myeloid leukaemic cell KCL-22. The cell viability was monitored by MTT assay, apoptotic activity by binding of Annexin V-FITC using fluorescence microscopy and cell cycle check points by flow cytometry.
Results:
Cytotoxic assay revealed that curcumin and TRAIL induced both dose and time-dependent decrease in cell viability. Significant cell cytotoxicity was seen in combine regimen of both curcumin and TRAIL at 48 h of exposure. Cells treated with curcumin and TRAIL was arrested at the S phase, as revealed by flow cytometric analysis. Subtoxic concentrations of the curcumin-TRAIL combination induced strong apoptotic response in KCL-22 cells as demonstrated by the binding of Annexin V-FITC.
Conclusion:
Our study conclude that curcumin inhibits the cancer cell growth by inducing apoptosis and enhance the therapeutic potential of TRAIL which recommends that both curcumin alone or in combination with TRAIL might be useful for leukaemic prevention and better therapeutic responses.
... Myelodysplastic syndromes (MDSs) are malignant hematopoietic stem cell (HSC) disorders characterized by ineffective hematopoiesis resulting in blood cytopenias and the risk of clonal evolution to acute myelogenous leukemia. 1 In vitro stromal abnormalities in MDS [2][3][4][5][6][7][8][9][10][11] have suggested the possibility that the bone marrow microenvironment (BMME) may contribute to disease progression and may therefore be a valuable therapeutic target. However, the lack of examination of the BMME in a robust in vivo model of MDS has limited progress in understanding the reciprocal interactions between abnormal MDS hematopoietic cells and the BMME. ...
Key Points
An in vivo model of MDS displays time-dependent defects in HSPCs and in microenvironmental populations. Normalization of the marrow microenvironment alters disease progression and transformation and improves hematopoietic function.
... In non--pathologic conditions TRAIL is expressed on human immune effector cells, such as interleukin 12--stimulated natural killer (NK) cells , peripheral blood T and B lymphocytes [Kayagaki et al 1999, Ehrlich et al 2003, interferon--activated monocytes [Griffith et al 1999], LPS-induced monocytes and macrophages [Halaas et al 2000], neutrophils [Tecchio et al 2004] and dendritic cells. Even if its pioneering and several following reports denoted that TRAIL mRNA in contrast to TNFα and FASL mRNA is expressed in many normal human tissues [Wiley et al 1995, Pitti et al 1996 [Campioni et al 2005]) and further degenerating diseases like intervertebral disk degeneration in scoliosis or vertebrae fracture [Bertram et al 2009]. Since TRAIL--stimulated apoptosis requires a functional DR5 and/or DR4--mediated extrinsic apoptosis pathway within the target cell, the origins for each of these conditions are apparently potent sensitisers of (normal) cells to TRAIL--apoptosis. ...
The cytokine TRAIL is a promising cancer therapeutic candidate as it induces apoptosis selectively in transformed cells. TRAIL-induced clustering of its receptors (DR) is essential for the DISC complex formation, which induces cell death. The mechanism for TRAIL’s tumour selective effect is largely unknown. We identified the cytoskeleton proteins non-muscle myosin heavy chain IIa, IIb (NMHCIIa, NMHCIIb), myosin regulatory light chain (MLC2) and ß-actin as novel DR-interactors. An initially weak and TRAIL-induced abrogation of NMHCII/DR interaction correlated with efficient DISC formation in tumour cells. In contrast, a robust NMHCII/DR interaction that was sustained upon TRAIL stimulus was accompanied by incomplete DISC arrangement. Weakening the NMHCII/DR interaction in normal cells using chemical inhibitors enhanced TRAIL-induced apoptosis. Intriguingly, siRNA-mediated NMHCIIa- but not NMHCIIb depletion potently released TRAIL resistance in normal cells and influenced DISC composition. Reduced NMHCII/DR interaction in transformed cells was characterised by diminished MLC2 phosphorylation and altered protein expression of upstream regulatory kinases. Our results suggest that normal cell resistance to TRAIL-apoptosis is based on the interaction of cytoskeleton components with DR that is impaired upon transformation. Since NMHCII function in cell adhesion and migration, it will be interesting to study possible roles of the interaction in cell detachment and altered TRAIL sensitivity; moreover this link may provide clues as to the cause of TRAIL resistance in some cancers.
... (Apo2L), a nível medular altera provavelmente a eritropoiese contribuindo para a anemia, a principal característica da SMD (Campioniet D., 2005 A inactivação deste gene tem vindo a ser associada com o risco de evolução da doença para LMA, conferindo mau prognóstico (Hirai H., 2003). ...
... In spite of its initial characterization in the cancer biology field, our group have provided robust experimental evidence that TRAIL can drive prosurvival and/or proliferative effects and can modulate differentiation in normal cell types such as human intestinal cells [8], bone marrow multipotent stromal cells [9], myeloid cells [10][11][12], and osteoclastic cells [13,14]. These trophic effects of TRAIL are mediated by the activation of the PI3K/Akt and ERK pathways [15,16] and of the transcription factor NF-kB [17,18] in a cell type specific context. ...
TNF-related apoptosis inducing ligand (TRAIL) is an intensively studied cytokine, in particular for its anticancer activity. The discovery that conjunctival sac fluid contains extremely high levels of soluble TRAIL as compared to other body fluids suggested important implications in the context of the immunological surveillance of the eye, in particular of the anterior surface. In this review, we discuss the potential physiopathologic and therapeutic role of the TRAIL/TRAIL receptor system in a variety of ocular cancers. Moreover, since an increasing amount of data has indicated the important biological activities of the TRAIL/TRAIL receptor systems also in a completely different pathologic context such as diabetes mellitus, in the second part of this review we summarize the currently available data on the involvement of TRAIL in the ocular complications of diabetes mellitus as modulator of the inflammatory and angiogenic response in the eye.
... TNFα produced by stromal cells plays a role in inducing apoptosis of maturing hematopoietic progeny. [56][57][58] Vascular endothelial growth factor (VEGF) levels have been found to be increased in the marrow of patients with MDS. 59,60 Keith et al. found increased microvascular density in the marrow of patients with MDS, which correlated with increased VEGF expression. ...
The BM microenvironment and its components regulate hematopoietic stem and progenitor cell (HSC) fate. An abnormality in the BM microenvironment and specific dysfunction of the HSC niche could play a critical role in initiation, disease progression, and response to therapy of BM failure syndromes. Therefore, the identification of changes in the HSC niche in BM failure syndromes should lead to further knowledge of the signals that disrupt the normal microenvironment. In turn, niche disruption may contribute to disease morbidity, resulting in pancytopenia and clonal evolution, and its understanding could suggest new therapeutic targets for these conditions. In this chapter, we briefly review the evidence for the importance of the BM microenvironment as a regulator of normal hematopoiesis, summarize current knowledge regarding the role of dysfunctions in the BM microenvironment in BM failure syndromes, and propose a strategy through which niche stimulation can complement current treatment for myelodysplastic syndrome.
© 2014 by The American Society of Hematology. All rights reserved.
... After 30 minutes of incubation at room temperature, blood samples were centrifuged (4.650 g for 20 min), and the obtained sera were stored at −80 ∘ C until analysis. For the measurement of TRAIL levels in this specimen, analyses were performed in duplicate by using a specific ELISA kit (R&D Systems, Minneapolis, MN) in agreement with the manufacturer's instructions, as previously described [22]. ...
A low chronic inflammation mediated by cytokine release is considered a major pathogenic mechanism accounting for the higher risk of cardiovascular disease in the overweight/obese population. In this context, although the existence of a possible interaction between soluble tumor necrosis factor- (TNF-) related apoptosis inducing ligand (TRAIL) and quantity and localization, of adiposity in the body has been hypothesized, no studies have yet investigated this link by radiologic techniques able to assess directly fat mass (FM) in different body regions. To address this issue, we assessed body fat distribution by dual X-rays absorptiometry (DXA) in a sample of 103 women and investigated the possible association between the derived adiposity measures and serum TRAIL concentration. The level of TRAIL showed a positive and independent correlation with arms FM (P < 0.05), trunk FM (P < 0.001) and trunk FM% (P < 0.05), total FM and total FM% (P < 0.001 for both), and an inverse association with legs FM% (P < 0.05). Only trunk FM retained a significant correlation (P < 0.05) with TRAIL after adjusting for all the other indices of regional adiposity. In conclusion, from our study it emerged a significant and independent association of serum TRAIL levels with overall, and, mainly, central adiposity. Further studies are needed to longitudinally investigate the cause-effect relationship between change in body fat distribution and TRAIL.
... In parallel, the degree of apoptosis was quantified by Annexin V-FITC/propidium iodide (PI) staining (Immunotech, Marseille, France) followed by flow cytometry analysis, as previously described (24,25). To analyze the cell cycle profile, cells were incubated with 50 mM 5-bromodeoxyuridine (BrdU; Sigma Aldrich) at 37˚C for 1 hour, the anti-BrdU antibody (BD Biosciences Pharmingen, Franklin Lakes, NJ) was bound to BrdU, and the complex was detected by an FITC-conjugated secondary antibody (Beckman-Coulter, Marseille, France) (26,27). After additional staining with 50 mg/ml PI (Sigma-Aldrich), the cell samples were analyzed by flow cytometry. ...
It has been shown that SOCS-1 plays an important role in the proper control of cytokine/growth factor responses and acts as a tumor suppressor in acute myeloid leukemias. Therefore, the objective of the present study was to evaluate the in vitro effect of treatment with Nutlin-3, a small molecule inhibitor of the MDM2/p53 interaction, on the expression of the suppressor of cytokine signaling 1 in primary acute myeloid leukemia cells and in myeloid cell lines with differential p53 status.
The expression of the suppressor of cytokine signaling 1 was quantitatively analyzed by real-time PCR in myeloid p53wild-type (OCI and MOLM) and p53null HL-60, leukemic cell lines, in patient-derived acute myeloid leukemia blasts, and in primary normal cell types, such as macrophages, endothelial cells, and bone marrow mesenchymal stem cells. The p53-dependence of the suppressor of cytokine signaling 1 upregulation that is induced by Nutlin-3 was analyzed in experiments performed using siRNA for p53, while the functional upregulation of the suppressor of cytokine signaling 1 was analyzed by assessing the levels of phosphorylated STAT-3.
Nutlin-3 significantly upregulated the transcription of the suppressor of cytokine signaling 1 in p53wild-type OCI and MOLM but not in p53deleted p53null HL60, myeloid leukemic cell lines, as well as in primary acute myeloid leukemia blasts. Conversely, and somewhat unexpectedly, Nutlin-3 did not modulate the suppressor of cytokine signaling 1 expression in primary normal macrophages, endothelial cells, and bone marrow mesenchymal stem cells. The p53-dependent upregulation of the suppressor of cytokine signaling 1 by Nutlin-3 was associated with the downregulation of phosphorylated STAT-3, a major molecular target of the suppressor of cytokine signaling 1.
Overall, our data suggest a potential role for the suppressor of cytokine signaling 1 as a therapeutic target of Nutlin-3 in p53 wild-type acute myeloid leukemias.
... For the measurement of TRAIL levels in plasma, serum, and cell lysates, analyses were performed in duplicate by using a specific ELISA kit (R&D Systems), in agreement with the manufacturer's instructions, as previously described (18). The levels of TRAIL in cell lysates were normalized to the milligrams of total proteins, determined by a bicinchoninic assay protein assay (Thermo Scientific). ...
Context:
The regulation of the circulating levels of TNF-related apoptosis-inducing ligand (TRAIL), a cytokine of the TNF family, playing a key role in the immune surveillance against cancer, is incompletely understood.
Objective:
The objective of the study was to investigate the potential link between TRAIL and 17β-estradiol.
Design, setting, and participants:
Circulating TRAIL levels were measured by an ELISA in plasma samples (n = 246) of healthy, age-matched (range 30-70 y) men and women and in the sera (n = 180) of females belonging to different physiopathological conditions (childhood, pregnancy, under gonadotropin treatment, menopause) characterized by different levels of circulating 17β-estradiol.
Results:
TRAIL plasma levels in women with aged younger than 50 years were significantly lower compared with age-matched men, whereas in woman 50 years old or older, TRAIL levels were significantly higher compared with the age-matched men and with the younger women. Moreover, an analysis of women with different conditions revealed a significant inverse correlation between the serum levels of TRAIL and 17β-estradiol, with the lowest levels of TRAIL being observed during pregnancy and the highest in childhood and in postmenopausal women. Moreover, gonadotropin treatment in women undergoing assisted reproduction was accompanied by an acute decrease of serum TRAIL levels. Finally, in vitro treatment with 17β-estradiol decreased the TRAIL expression levels in peripheral blood mononuclear cells.
Conclusions:
Our data suggest that 17β-estradiol plays a role in regulating TRAIL circulating levels. The demonstration that postmenopausal women exhibit the highest TRAIL levels is of particular interest in light of a previous large study population showing that TRAIL is positively correlated to the overall survival.
... Macrophages, stromal elements or cytotoxic T cells might be implicated as sources of the stimuli that increase death-receptor signaling in MDS [12]. Alternatively, the increased levels of TNF-a and CD95 ligand on the surface of hematopoietic cells from patients with MDS might induce autocrine stimulation of the death-receptor pathway [13]. ...
Several classification and scoring systems have been developed in myelodysplastic syndromes (MDS to predict the risk of progression to acute myeloid leukemia and survival. These prognostication models have been also used to inform therapeutic decision-making in a risk-adapted fashion. Patient-related factors such as age, comorbidities, and functional status have to be considered as well. Here we review a risk-guided therapeutic approach for the management of MDS patients. It is anticipated that the improved understanding of the complex pathogenesis of MDS and the recent discovery of important molecular lesions will be translated into novel therapeutic approaches. Additionally, some prognostic aberrations are expected to be incorporated into the prognostic tools with the goal of improving their prognostic precision and therefore allow for a more informed therapeutic decision-making based on the individual's risk profile.
... ICA antibodies were measured with an indirect immunofluorescence assay using cryosections of freshfrozen human blood group O pancreas as substrate[21]; GADA, IA2A, and IAA antibodies were detected by radioimmunoassay (RIA) commercial kits (Biochem Immuno-Systems, Italy for GADA and IA-2A; CIS Italy for IAA)[22]; ZnT8A antibodies were assessed by ELISA commercial kits (RSR limited, United Kingdom). Serum TRAIL was measured in duplicate by using a commercially available ELISA kit (R&D Systems, Minneapolis , MN, USA) following the manufacturer's instructions, as previously described[23,24]. Selected samples were run in each ELISA plate as internal controls, confirming the reproducibility of determinations over time. ...
Experimental evidence in animal models suggests that TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily, might play an important role in type 1 diabetes (T1D). We have performed a retrospective study by analyzing the sera of a cohort of pediatric subjects (age ≤18 years; n = 507) consisting of (1) patients diagnosed with T1D (n = 387), (2) healthy individuals (n = 98, considered as controls), and (3) healthy autoantibody-positive subjects (n = 22). Patients with T1D exhibited significantly decreased levels of circulating TRAIL with respect to the control healthy subjects, as well as to the healthy autoantibody-positive subjects. Within the T1D group, no differences in the levels of circulating TRAIL were observed between patients with or without other concomitant autoimmune pathologies. Of note, the levels of TRAIL were significantly lower in the T1D patients analyzed at onset, although reduction in TRAIL levels persisted also in patients analyzed after disease onset (>1 year from diagnosis). In particular, T1D patients who exhibited ketoacidosis at onset showed significantly lower levels of circulating TRAIL with respect to patients without ketoacidosis at onset. Moreover, the levels of TRAIL at diagnosis correlated inversely with the insulin requirement up to 21 months of follow-up. This is the first study demonstrating that the levels of circulating TRAIL are significantly decreased in T1D, with the lowest levels of TRAIL being observed in patients with ketoacidosis at the onset and with the highest insulin requirement.
... Furthermore, loss of FANCC results in constitutively phosphorylation and increased binding affinity for double-stranded RNA (dsRNA) kinase PKR, a key effector of apoptosis to various extra and intracellular cues [71], suggesting another function of FANCC in suppressing cytokine-induced apoptosis through modulating the activity of a growth inhibitory kinase [80]. In addition, elevated expression/release of TNF-related apoptosis-inducing ligand (TRAIL) at the bone marrow level has also been found in FA, which may also implicate in the pathogenesis of FA [90] and MDS [91]. FANCD2 was shown to be a target for apoptosis mediated by caspase(s) but not proteasome-mediated pathway following DNA damage [92]. ...
... 30,31 TRAIL by itself is able to inhibit erythropoietic differentiation. 32 Interestingly, apoptosis can be modulated by interfering with known downstream factors including FADD 33 and FLIP, 34,35 the latter being underexpressed in MDS stem cells. TNF-a upregulates FAS on CD34+ cells. ...
Allogeneic hematopoietic cell transplantation (HCT) is a curative treatment option for patients with myelodysplastic syndromes (MDS). Although improvements in donor selection, supportive therapy and post-grafting immunosuppression have been achieved throughout the last two decades, allogeneic HCT after standard conditioning remains restricted to a small minority of patients. The long-term success of allogeneic HCT depends on several disease and patient specific risk factors, leading to probabilities of disease-free survival after three years between 20% and 65%. Early transplantation seems to be warranted especially in patients with intermediate-2 and high-risk IPSS scores as defined by marrow blasts and cytogenetics. The outcome of grafts from matched unrelated donors is comparable to that from matched sibling donors. Many investigators favour the use of peripheral blood stem cells instead of marrow grafts. Given that most patients with MDS are older than 60 years, the development of less toxic conditioning regimens has allowed patients with a higher comorbidity score to be transplanted with similar results as with standard conditioning in younger cohorts. Still, a reduced-intensity of the preparative regimen increases the risk of relapse. Therefore current clinical trials focus on the relevance of pretransplant induction therapy and the direct comparison of standard-intensity with reduced-intensity conditioning. Additional efforts will be made to integrate new pharmacological strategies in order to reduce the risk of relapse. Further improvements are needed before allogeneic HCT will become the standard therapy for high-risk MDS.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
Hematologic malignancies were the first human cancers to be studied in depth at the molecular level, and recent years have seen important advances in treatment. This comprehensive reference book covers the full range of hematologic malignancies, including all subtypes of leukemias, lymphomas, and plasma cell dyscrasias. Authored by internationally known experts, each chapter emphasizes diagnostic work-up, staging, and therapeutic approaches. Up-to-date hematopathology, treatment, and outcomes data are presented in a way which is directly applicable to patient care. Highly illustrated with color images, graphs, flowcharts and treatment algorithms, the book is perfect for quick clinical reference as well as providing detailed reference lists for further study. With its authoritative and practical focus and visually stimulating presentation, this is a key text for hematology and oncology fellows, physicians, oncology nurses, physician assistants and other healthcare workers in the field of oncology.
The myelodysplastic syndromes (MDS) include a group of clonal hematopoietic stem cell disorders defined by marrow failure, dysplastic cell morphology, and a risk of clonal evolution including to acute myeloid leukemia (AML). Most cases arise de novo, but 10% to 20% are a consequence of prior cytotoxic chemotherapy or radiation therapy, and familial MDS is increasingly recognized. More than 40 genes are recurrently mutated in association with MDS, including genes encoding factors important in epigenetic patterning, chromatin remodeling, mRNA splicing, signal transduction, transcription, and DNA repair. More than half of patients have cytogenetic abnormalities in marrow cells, usually aneuploidy such as monosomy 7 or trisomy 8, or a deletion of the long arm of chromosomes 5 or 20. The most commonly used MDS classification system is the 2016 World Health Organization criteria. Because the natural history of MDS is highly variable, risk stratification using tools such as the 2012 Revised International Prognostic Scoring system, supplemented by molecular genetic information, is important for counseling patients and choosing appropriate therapy. Only allogeneic hematopoietic cell transplant offers the possibility of cure, but most patients with MDS are not transplant candidates due to advanced age and comorbid conditions. Approved disease-modifying therapies include azacitidine, decitabine, and lenalidomide. Supportive care with erythropoiesis stimulating agents, transfusions, and iron chelation is important for some patients. Clinical trial enrollment is essential given the poor outcomes for patients, especially those who have been failed by hypomethylating agents.
Stem cells are an essential to repair damaged tissues. Their functions, proliferation and differentiation need to be tightly controlled, as impairments can lead to various diseases including cancer. Induction of apoptosis is one way to control the number of stem cells and to eliminate rogue and/or precancerous cells. One way of triggering apoptosis, and probably the physiologically most important one, is via binding of death ligand to their cognate receptors. The death receptor–ligand family encompasses five pairs: FAS/FASL; TNF-R1/TNF; DR3/TL1A and TWEAK; DR4 and DR5/TRAIL; and DR6/unknown ligand. Of these, FASL and TRAIL and to a lesser extent TNF are strong inducers of apoptosis, whereas the others possess relative weak cell-death-inducing activity. Interestingly, these death receptors and ligands also have non-canonical functions and in specific cellular and molecular contexts can regulate cell proliferation, differentiation, chemokine production and inflammatory responses. Some of these non-apoptotic functions have been shown to be of relevance in stem and progenitor cells.
Stem cells have also been used as part of cell therapies in connection with delivery of death ligands to target their respective receptors, in particular in experimental anti-cancer therapies. Stem cells, at least some types, are attractive in these approaches because they are capable to infiltrate certain tissues including tumours to deliver their therapeutic payload. This way of cellular delivery can be more efficacious and specific compared to recombinant proteins or direct gene therapy.
This chapter summarises our current understanding of stem cell regulation by death receptor–ligand signalling and in the second part how certain types of stem cells have been used to deliver death-ligand gene therapies in the laboratory and increasingly in clinical trials.
Purpose of review This review will focus on the emerging role of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/TRAIL-receptors in the pathophysiology of hematopoiesis and on the potential therapeutic applications of either recombinant TRAIL or anti-TRAIL-R1/-R2 agonistic antibodies for the treatment of hematological malignancies. Recent findings While CD34(+) stem/progenitor cells do not express TRAIL-receptors and are protected from TRAIL-induced apoptosis, accumulating evidence points to a role for elevated expression/release of TRAIL at the bone marrow level in the pathophysiology of aplastic anemia, Fanconi anemia, and myelodysplastic syndromes. In-vitro data show promising synergistic effects of recombinant TRAIL in association with proteasome or histone deacetylase inhibitors, natural compounds or small molecules in the therapy of myeloid and lymphoid malignancies. Moreover, although both recombinant TRAIL and anti-TRAIL-R1/-R2 antibodies are well tolerated in vivo, anti-TRAL-R1/-R2 agonistic antibodies show the potential advantage of avoiding the neutralizing activity of the soluble receptor osteoprotegerin. Summary While a chronic pathological elevation of TRAIL at the bone marrow level might contribute to the impairment of normal hematopoiesis, the use of recombinant TRAIL and anti-TRAIL-R1/-R2 agonistic antibodies appears particularly promising for the treatment of hematological malignancies in particular, of multiple myeloma, especially if used in association with innovative therapeutic compounds.
Myelodysplastic syndromes (MDS) are regarded as clonal disorders of haematopoietic stem cells (HSC). Recent evidence demonstrates that stromal microenvironment, in addition to HSC defects, plays a particular role via its direct contact with haematopoietic precursor cells (HPC). This thesis aims at evaluating the putative growth deficiencies of mesenchymal stromal cells (MSC) from MDS individuals compared with normal controls, exploring their adhesion profile, assessing the adhesion process-involved molecular substrates, and establishing correlations with their growth patterns and HPC dysfunctions. Functional assays revealed that MSC from MDS are intrinsically pathological, show a continuous decline of proliferation over a 14-day culture and a reduced clonogenic capacity in the absence of signals from HPC. MSC growth defects significantly correlate with decreased CD44 and CD49e expression. Moreover, stroma-dependent adhesion mechanisms control HPC clonogenic potential and CD49e might be one of the molecules involved in this process. Qualitative and quantitative abnormalities of focal adhesion (FA) proteins paxillin and pFAK [Y397] and of two regulatory proteins, HSP90αβ and p130CAS were identified via immunofluorescence analysis. Paxillin, pFAK [Y397] and HSP90αβ increased expression, besides its stronger nuclear colocalization in MSC from RAEB correlates with a consistent proliferative advantage and has a negative impact on HPC clonogenic capacity. These results open interesting opportunities, e.g. HPC-to-MSC interactions involve FA proteins signalling, and, as FAK is an HSP90αβ-client protein, it may enhance the utility of HSP90αβ inhibitors as adjuvant therapy in MDS
Purpose: Myelodysplastic syndromes (MDS) are a group of disorders characterized by cytopenias, with a propensity for evolution into acute myeloid leukemias (AML). This transformation is driven by genomic instability, but mechanisms remain unknown. Telomere dysfunction might generate genomic instability leading to cytopenias and disease progression. Experimental Design: We undertook a pilot study of 94 patients with MDS (56 patients) and AML (38 patients). The MDS cohort consisted of refractory cytopenia with multilineage dysplasia (32 cases), refractory anemia (12 cases), refractory anemia with excess of blasts (RAEB)1 (8 cases), RAEB2 (1 case), refractory anemia with ring sideroblasts (2 cases), and MDS with isolated del(5q) (1 case). The AML cohort was composed of AML-M4 (12 cases), AML-M2 (10 cases), AML-M5 (5 cases), AML-M0 (5 cases), AML-M1 (2 cases), AML-M4eo (1 case), and AML with multidysplasia-related changes (1 case). Three-dimensional quantitative FISH of telomeres was carried out on nuclei from bone marrow samples and analyzed using TeloView. Results: We defined three-dimensional nuclear telomeric profiles on the basis of telomere numbers, telomeric aggregates, telomere signal intensities, nuclear volumes, and nuclear telomere distribution. Using these parameters, we blindly subdivided the MDS patients into nine subgroups and the AML patients into six subgroups. Each of the parameters showed significant differences between MDS and AML. Combining all parameters revealed significant differences between all subgroups. Three-dimensional telomeric profiles are linked to the evolution of telomere dysfunction, defining a model of progression from MDS to AML. Conclusions: Our results show distinct three-dimensional telomeric profiles specific to patients with MDS and AML that help subgroup patients based on the severity of telomere dysfunction highlighted in the profiles.
Behçet's disease (BD) is a systemic vasculitis, while myelodysplastic syndrome (MDS) is a heterogeneous group of clonal hematologic disorders characterized by ineffective hematopoiesis. Some studies suggest a relationship between MDS and BD, especially intestinal BD, and trisomy 8 seems to play an important role in both diseases. There are several reports on patients with BD comorbid with MDS involving trisomy 8 that frequently have intestinal lesions refractory to conventional medical therapies. Tumor necrosis factor (TNF)-α is strongly involved in the pathophysiology of several autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease, and BD. In addition, TNF-α plays an important role in the pathophysiology of MDS by inhibiting normal hematopoiesis and inducing the programmed cell death of normal total bone marrow cells and normal CD34+ cells. Recent clinical reports demonstrate the favorable effect of TNF-α antagonists in patients with refractory intestinal BD and in those with MDS. We present the case of a patient with intestinal BD and MDS involving trisomy 8 who was successfully treated with adalimumab.
Accumulating evidence indicates that bone marrow microenvironment plays an important role in the pathogenesis of some myeloid and lymphoid hematological malignancies (HM). Among different environmental associated parameters, those related to functional, cytogenetic and immunological integrity of mesenchymal stromal cells (MSC) are particularly relevant. Functional alterations and immunophenotypic abnormalities have been described in MSC obtained from HM patients. These data seem to confirm the defective biological pattern of MSC especially in myeloid diseases, while MSC cytogenetic profile in HM is still an open question, because it is not clear whether BM stromal cells are "culprit or bystander" displaying or not an abnormal karyotype. Contradictory findings were reported in different HM but the functional implications of altered MSC karyotype need to be further addressed also in light of a clinical use of MSC. A "pathological" in vivo supportive function of endogenous MSC, which provide important survival and drug resistance signals to leukemic cells especially in lymphoproliferative disorders, is suggested. Thus, the mechanisms underlying these protective versus cytotoxic effects exerted by MSC on leukemic cells need further investigations.
Cancerous inhibitor of PP2A (protein phosphatase 2A) (CIP2A) is an inhibitor of PP2A, a phosphatase and tumor suppressor that regulates cell proliferation, differentiation, and survival. The aim of this study was to investigate whether CIP2A plays a role in the progression of myelodysplastic syndromes (MDS). Immunohistochemical analysis revealed that a fraction patients having refractory anemia with excess blasts (RAEB)-1 (4 out of 12) and RAEB-2 (10 out of 14) exhibited significant expression of CIP2A in bone marrow hematopoietic cells, while all patients with refractory cytopenia with unilineage or multilineage dysplasia (RCUD/RCMD) (0 out of 18) and the control group (0 out of 17) were negative. CIP2A was mainly expressed by the MPO-positive myeloid series of cells and partly by the CD34-positive cells in association with the expression of phosphorylated c-MYC (p-c-MYC) protein and the cell cycle-related proteins Ki-67, MCM2, and geminin. The percentage of p-c-MYC-positive cells in the bone marrow of CIP2A-positive MDS cases was significantly higher than that in CIP2A-negative MDS cases (P < 0.01). The expression levels of mRNA for CIP2A and PP2A exhibited positive correlation in MDS/control bone marrow. These results suggest that up-regulated expression of CIP2A might play a role in the proliferation of blasts in the MDS bone marrow and in disease progression in at least some cases.
Ineffective hematopoiesis, with premature death of marrow myeloid precursors, is a hallmark of myelodysplastic syndromes (MDS), with the apparent paradox of peripheral cytopenia associated with hypercellular bone marrow (BM). Excessive apoptosis appears relevant especially in low-risk MDS. Apoptosis, triggered by the BM microenvironment and/or intrinsic cellular defects, is regulated at different levels by numerous factors, such as oncogenes and their protein products, hematopoietic growth factors, immunologic factors, cell-cell or cellstromal interactions, critical adhesion receptors, and various cytokines. Deregulation of both the intrinsic and the extrinsic pathways have been reported in MDS cells. Many studies provide evidence that the activation of the Fas/Fas-ligand system might represent an important pathogenetic mechanism. Recently, it has been demonstrated that the erythroid apoptosis of low-risk MDS is initiated at a very early stage of stem cells and associated with mitochondrial dysfunction. There is a constitutive triggering to apoptosis via cytochrome C release from the mitochondrial intermembrane space, with subsequent activation of effector caspases and increased sensitivity to death ligands triggering the extrinsic apoptotic pathway. The role of the mitochondrial pathway might be relevant especially in refractory anemia with ring sideroblasts, where abnormalities in mitochondrial ferritin expression might directly influence iron homeostasis and contribute to alter the balance between cell growth and death. The pathogenesis of refractory anemia without ring sideroblasts seems to be more heterogeneous, with the involvement of alternative mechanisms, including T-cell–mediated BM failure. Elucidation of these pathogenetic mechanisms might lead to the development of novel therapeutic strategies.
Cell-cycle kinetics were measured in situ after infusions of iododeoxyuridine and/or bormodeoxyuridine in 50 patients with myelodysplastic syndromes (MDS) and the median labeling index in bone marrow (BM) biopsy samples was 28.6%. Unfortunately, 26 of 50 patients showed that > or = 75% of hematopoietic cells of all three lineages were undergoing programmed cell death (PCD) in their biopsy samples as shown by the in situ end labeling (ISEL) technique. Ten patients had 1/3 and eight had 2/3 ISEL+ cells. Stromal cells were frequently ISEL+ and often S-phase cells were also found to be simultaneously ISEL+. Nucleosomal DNA fragments as a ladder in agarose gel were present in BM aspirates of four patients who showed high ISEL and were absent in two who had no ISEL staining in biopsy samples, but only when DNA was extracted after a 4-hour in vitro incubation in complete medium. Therefore, laddering data confirmed the ISEL findings that the majority of hematopoietic cells in MDS are in early stages of PCD. We conclude that extensive intramedullary cell death may explain the paradox of pancytopenia despite hypercellular marrows in MDS patients. Investigating approaches that protect against PCD in some MDS subsets would be of interest.
Cytokines in the tumor necrosis factor (TNF) family regulate development and function of the immune system. We have isolated a new member of this family, designated Apo-2 ligand (Apo-2L), via an expressed sequence tag. Apo-2L is a 281-amino acid protein, related most closely to Fas/Apo-1 ligand. Transfected Apo-2L is expressed at the cell surface with its C terminus exposed, indicating a type II transmembrane protein topology. Like Fas/Apo-1 ligand and TNF, the C-terminal extracellular region of Apo-2L (amino acids 114-281) exhibits a homotrimeric subunit structure. Soluble Apo-2L induces extensive apoptosis in lymphoid as well as non-lymphoid tumor cell lines. The effect of Apo-2L is not inhibited by soluble Fas/Apo-1 and TNF receptors; moreover, expression of human Fas/Apo-1 in mouse fibroblasts, which confers sensitivity to induction of apoptosis by agonistic anti-Fas/Apo-1 antibody, does not confer sensitivity to Apo-2L. Hence, Apo-2L acts via a receptor which is distinct from Fas/Apo-1 and TNF receptors. These results suggest that, along with other family members such as Fas/Apo-1 ligand and TNF, Apo-2L may serve as an extracellular signal that triggers programmed cell death.
Apoptosis of hematopoietic progenitor cells is increased in myelodysplastic syndromes (MDS). We have studied Fas (CD95/Apo-1) antigen expression in 27 MDS patients (RARS 4, RA 3, RAEB 13; RAEB-t 3, CMML 4) and three AML secondary to MDS. We found that the Fas antigen was not expressed on normal bone marrow (BM) CD34+, CD14+, or glycophorin+ cells, and only slightly on CD33+ cells. Patients with MDS had upregulation of Fas expression on total bone marrow nuclear cells (BMMC) (t-test, P = 0.04), CD34+ (P = 0.013), CD33+ (P = 0.04), and glycophorin+ (P = 0.032) BM cells compared to controls. Fas expression did not correlate to the FAB subtype, the Bournemouth score, or to peripheral cytopenias. However, Fas expression intensity on CD34+ cells negatively correlated to the BM blasts number (Spearman, P = 0.01) suggesting that leukemic blasts cells lose Fas antigen expression with progression of myelodysplasia. Using both proliferation assays in liquid cultures and clonogenic progenitor assays in the presence of an agonist anti-Fas MoAb (CH11), we showed that the Fas protein was functional in some patients. Dose-dependent inhibition of DNA synthesis was observed in three out of seven patients studied. CFU-GM and BFU-E colonies suppression in some patients suggested that Fas can induce apoptosis in myeloid and erythroid BM progenitors of MDS patients. The TUNEL technique on BM smears gave a mean of 12.6% +/- 2.5 of bone marrow apoptotic cells in five controls. Patients with MDS had increased bone marrow apoptosis (mean 39% +/- 5.7, t-test, P = 0.012). Four out of 15 (26%) patients studied with a sensitive radiolabeled DNA ladder technique had typical DNA ladders indicative of advanced stages of apoptosis. Massive BM suicide was observed in patients with RA (2/2) and RAEB (8/11), whereas apoptosis rates were normal or low in patients with RAEB-t (3/3) or secondary AMLs (3/3). Moreover, high rates of apoptosis correlated to low Bournemouth score (Spearman, P = 0.01). No statistical correlation could be found between Fas expression and apoptosis rates. Our results confirm the importance of programmed cell death in MDS. The Fas antigen is clearly upregulated on BM cells, but its role in the pathophysiology of apoptosis in myelodysplasia is still unclear, indicating that many factors positively or negatively interfere with the Fas-mediated pathway of apoptosis in vivo and in vitro.
To evaluate the utility of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as a cancer therapeutic, we created leucine zipper (LZ) forms of human (hu) and murine (mu) TRAIL to promote and stabilize the formation of trimers. Both were biologically active, inducing apoptosis of both human and murine target cells in vitro with similar specific activities. In contrast to the fulminant hepatotoxicity of LZ-huCD95L in vivo, administration of either LZ-huTRAIL or LZ-muTRAIL did not seem toxic to normal tissues of mice. Finally, repeated treatments with LZ-huTRAIL actively suppressed growth of the TRAIL-sensitive human mammary adenocarcinoma cell line MDA-231 in CB.17 (SCID) mice, and histologic examination of tumors from SCID mice treated with LZ-huTRAIL demonstrated clear areas of apoptotic necrosis within 9-12 hours of injection.
TNF and Fas ligand induce apoptosis in tumor cells; however, their severe toxicity toward normal tissues hampers their application to cancer therapy. Apo2 ligand (Apo2L, or TRAIL) is a related molecule that triggers tumor cell apoptosis. Apo2L mRNA is expressed in many tissues, suggesting that the ligand may be nontoxic to normal cells. To investigate Apo2L's therapeutic potential, we generated in bacteria a potently active soluble version of the native human protein. Several normal cell types were resistant in vitro to apoptosis induction by Apo2L. Repeated intravenous injections of Apo2L in nonhuman primates did not cause detectable toxicity to tissues and organs examined. Apo2L exerted cytostatic or cytotoxic effects in vitro on 32 of 39 cell lines from colon, lung, breast, kidney, brain, and skin cancer. Treatment of athymic mice with Apo2L shortly after tumor xenograft injection markedly reduced tumor incidence. Apo2L treatment of mice bearing solid tumors induced tumor cell apoptosis, suppressed tumor progression, and improved survival. Apo2L cooperated synergistically with the chemotherapeutic drugs 5-fluorouracil or CPT-11, causing substantial tumor regression or complete tumor ablation. Thus, Apo2L may have potent anticancer activity without significant toxicity toward normal tissues.
The impact of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) on normal hematopoietic development was investigated using adult peripheral blood CD34(+) hematopoietic progenitor cells, induced to differentiate along the erythroid, megakaryocytic, granulocytic, and monocytic lineages by the addition of specific cytokine cocktails. TRAIL selectively reduced the number of erythroblasts, showing intermediate levels of glycophorin A (glycophorin A(interm)) surface expression, which appeared in liquid cultures supplemented with stem cell factor + interleukin 3 + erythropoietin at days 7-10. However, neither immature (day 4) glycophorin A(dim) erythroid cells nor mature (day 14) glycophorin A(bright) erythroblasts were sensitive to TRAIL-mediated apoptosis. Moreover, pre-exposure to TRAIL significantly decreased the number and size of erythroid colonies in semisolid assays. These adverse effects of TRAIL were selective for erythropoiesis, as TRAIL did not significantly influence the survival of cells differentiating along the megakaryocytic, granulocytic, or monocytic lineages. Furthermore, TRAIL was detected by Western blot analysis in lysates obtained from normal bone marrow mononuclear cells. These findings indicate that TRAIL acts in a lineage- and stage of differentiation-specific manner, as a negative regulator of normal erythropoiesis. (Blood. 2000;95:3716-3724)
Bone marrow CD34(+) cell apoptosis (annexin V), proliferation (Ki-67), and Bcl-2-related protein expression was evaluated by flow cytometry in 102 patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia secondary to MDS (MDS-AML) and in 30 normal donors (NBM). Apoptosis was significantly increased in refractory anemia (RA)/RA with ringed sideroblasts (RARS) (56.9% [20.4%-93.6%]) and refractory anemia with excess blasts (RAEB) (51.2% [25.2%-76. 6%]) compared with NBM (16.7% [3.4%-35.3%], P <.0001). In RA/RARS, apoptosis always exceeded proliferation (Ki-67-positivity, 26.1% [9.5%-47.8%]; apoptosis:proliferation ratio 2.08 [1.15-3.63]); whereas in RAEB, this ratio equalized (1.14 [0.93-2.08]) due to increased proliferation (40.4% [22%-69.5%]). Progression to RAEB in transformation (RAEB-t)/MDS-AML was associated with a significant reduction in apoptosis (22.3% [2.1%-53.2%]; P <.0001) and proliferation (16.8% [1.9%-75.8%); P =.04; ratio 1.69 [0.16-12.21]). Pro-apoptotic (Bax/Bad) versus anti-apoptotic (Bcl-2/Bcl-X) Bcl-2-related protein ratios were increased in RA/RARS compared with NBM (2.57 [1.93-9.42] versus 1.89 [0.65-4.1]; P =.06), whereas disease progression was associated with significantly reduced ratios (1.16 [0.06-3.32]; P <.0001) due primarily to increased Bcl-2 expression. Apoptosis and Bax/Bad:Bcl-2/Bcl-X ratio were inversely correlated with both International Prognostic Scoring System score and cytogenetic risk group; highest levels observed in patients with low score and/or good risk cytogenetics. There was a trend toward an association between Bcl-2-related protein expression and apoptosis (P =.07). This study indicates that MDS progression arises through multiple hits that alter levels of CD34(+) cell apoptosis and proliferation. Early disease is associated with excessive apoptosis and elevated ratio of apoptosis to proliferation. Increased proliferative rates are observed in RAEB, whereas leukemic transformation arises through inhibition of apoptosis rather than excessive cell growth. Although disease progression is accompanied by a fall in pro-apoptotic versus anti-apoptotic Bcl-2-related protein ratios, heterogeneity in patterns of protein expression indicates that factors additional to Bcl-2 family members play a role in the deregulated apoptosis in MDS. (Blood. 2000;96:3932-3938)
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL, APO2L) has been shown to induce apoptosis in a number of tumor cell lines as well as in some primary tumors whereas cells from most normal tissues are highly resistant to TRAIL-induced apoptosis. We have studied the susceptibility of primary malignant and normal bone marrow hematopoietic progenitors to TRAIL-induced apoptosis. Extracellular domain of human TRAIL with N-terminal His(6) tag (His-TRAIL, amino acids 95-281) was produced in E. coli and its apoptosis-inducing ability was compared with the leucine-zipper containing TRAIL, LZ-TRAIL. Both variants of TRAIL had the same apoptosis-inducing ability. Clonogenic progenitor assays showed that His-TRAIL significantly reduced the number of myeloid colonies (CFU-GM) and clusters from patients with acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and myelodysplastic syndromes (MDS). His-TRAIL had no negative effect on the number of CFU-GM colonies and clusters derived from bone marrow cells of AML patients in complete remission, and lymphoma patients without bone marrow involvement, as well as those derived from normal cord blood cells. Moreover, we found that normal human stem cells treated with high doses of His-TRAIL maintain a repopulating potential when transplanted into NOD/SCID mice. To conclude, our data document that TRAIL does not affect normal human hematopoiesis but suppresses the growth of early primary leukemia and myelodysplasia progenitors.
Erythropoiesis results from the proliferation and differentiation of pluripotent stem cells into immature erythroid progenitors (ie, erythroid burst-forming units (BFU-Es), whose growth, survival, and terminal differentiation depends on erythropoietin (Epo). Ineffective erythropoiesis is a common feature of myelodysplastic syndromes (MDS). We used a 2-step liquid-culture procedure to study erythropoiesis in MDS. CD34(+) cells from the marrow of patients with MDS were cultured for 10 days in serum-containing medium with Epo, stem cell factor, insulin-like growth factor 1, and steroid hormones until they reached the proerythroblast stage. The cells were then placed in medium containing Epo and insulin for terminal erythroid differentiation. Numbers of both MDS and normal control cells increased 10(3) fold by day 15. However, in semisolid culture, cells from patients with refractory anemia (RA) with ringed sideroblasts and RA or RA with excess of blasts produced significantly fewer BFU-Es than cells from controls. Fluorescence in situ hybridization analysis of interphase nuclei from patients with chromosomal defects indicated that abnormal clones were expanded in vitro. Epo-signaling pathways (STAT5, Akt, and ERK 1/2) were normally activated in MDS erythroid progenitors. In contrast, apoptosis was significantly increased in MDS cells once they differentiated, whereas it remained low in normal cells. Fas was overexpressed on freshly isolated MDS CD34(+) cells and on MDS erythroid cells throughout the culture. Apoptosis coincided with overproduction of Fas ligand during the differentiation stage and was inhibited by Fas-Fc chimeric protein. Thus, MDS CD34(+)-derived erythroid progenitors proliferated normally in our 2-step liquid culture with Epo but underwent abnormal Fas-dependent apoptosis during differentiation that could be responsible for the impaired erythropoiesis.
The potential contribution of abnormal marrow stromal function to the ineffective haemopoiesis seen in myelodysplastic syndromes (MDS) is unclear. We have examined modulation of apoptosis in F36P cells co-cultured with stromal layers from normal (n=7) and MDS (n=9) marrow mononuclear cells. Co-cultures for 36 hours in the absence of exogenous GM-CSF/IL-3 were either in direct contact with stroma or separated by transwell inserts. Apoptosis quantitated by flow-cytometry of F-36P cells stained with 7-aminoactinomycin D was 12.4% after co-culture in contact with normal stroma (range 6.8-18.5%, n=6). On MDS stroma 6/9 co-cultures showed apoptosis in the normal range, but 3 showed a marked increase (range 28.029.1%). These 3 all contained more monocytes/macrophages (13.519.5% of stromal cells), than the remaining MDS layers (1-4.5%) or normal stroma (0.3-6.7%). There was no correlation between FAB type or BFU-E content of marrow and stroma-related apoptosis, no relationship to TNF-a levels ((indétectable in the supernatants from 6 MDS co-cultures, compared with a mean of 1.7, range 0-4 pg/ml in 4 normal co-cultures), and no evidence of involvement of the Fas/lCE pathway (<1% Fas/Apo-1 expression in F-36P cells exposed to normal or MDS stroma). Apoptosis was generally more in cells grown in transwell inserts, with a mean of 22.6% (range 19.9-24.5%, n=4) on normal stroma, but differences with MDS were no longer seen. These results suggest abnormal stromal function in a subset of MDS patients may contribute to increased apoptosis within the marrow microenvironment. The effect appears to be dependent on close cellular contact, rather than the release of soluble factors, but the exact mechanism remains unclear.
Apo2 ligand or tumour necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) is one of the several members of the tumour necrosis factor (TNF) gene superfamily that induce apoptosis through engagement of death receptors (DRs). Apo2L/TRAIL interacts with an unusually complex receptor system of two DRs and three decoys. This protein has garnered intense interest as a potential candidate for cancer therapy because as a trimer it selectively induces apoptosis in many transformed cells but not in normal cells. While much of the early characterisation of Apo2L/TRAIL and its receptors relied on overexpression studies, recent work using untransfected cells has clarified how endogenous proteins transmit apoptotic signals from this ligand. In this review, we focus on the apoptotic signalling pathways stimulated by Apo2L/TRAIL and summarise what is known about its physiological role.
In order to investigate the biologic activity of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on human erythropoiesis, glycophorin A (GPA)+ erythroid cells were generated in serum-free liquid phase from human cord blood (CB) CD34+ progenitor cells. The surface expression of TRAIL-R1 was weakly detectable in the early-intermediate phase of erythroid differentiation (days 4-6; dim-intermediate GPA expression), whereas a clear-cut expression of TRAIL-R2 was observed through the entire course of erythroid differentiation (up to days 12-14; bright GPA expression). On the other hand, surface TRAIL-R3 and -R4 were not detected at any culture time. Besides inducing a rapid but small increase of apoptotic cell death, which was abrogated by the pan-caspase inhibitor z-VAD-fmk, the addition of recombinant TRAIL at day 6 of culture inhibited the generation of morphologically mature erythroblasts. Among the intracellular pathways investigated, TRAIL significantly stimulated the extracellular signal-regulated kinase 1/2 (ERK1/2) but not the p38/mitogen-activated protein kinase (MAPK) or the c-Jun NH2-terminal kinase (JNK) pathway. Consistently with a key role of ERK1/2 in mediating the negative effects of TRAIL on erythroid maturation, PD98059, a pharmacologic inhibitor of the ERK pathway, but not z-VAD-fmk or SB203580, a pharmacologic inhibitor of p38/MAPK, reverted the antidifferentiative effect of TRAIL on CB-derived erythroblasts.
Aneuploidy, especially monosomy 7 and trisomy 8, is a frequent cytogenetic abnormality in the myelodysplastic syndromes (MDSs). Patients with monosomy 7 and trisomy 8 have distinctly different clinical courses, responses to therapy, and survival probabilities. To determine disease-specific molecular characteristics, we analyzed the gene expression pattern in purified CD34 hematopoietic progenitor cells obtained from MDS patients with monosomy 7 and trisomy 8 using Affymetrix GeneChips. Two methods were employed: standard hybridization and a small-sample RNA amplification protocol for the limited amounts of RNA available from individual cases; results were comparable between these 2 techniques. Microarray data were confirmed by gene amplification and flow cytometry using individual patient samples. Genes related to hematopoietic progenitor cell proliferation and blood cell function were dysregulated in CD34 cells of both monosomy 7 and trisomy 8 MDS. In trisomy 8, up-regulated genes were primarily involved in immune and inflammatory responses, and down-regulated genes have been implicated in apoptosis inhibition. CD34 cells in monosomy 7 showed up-regulation of genes inducing leukemia transformation and tumorigenesis and apoptosis and down-regulation of genes controlling cell growth and differentiation. These results imply distinct molecular mechanisms for monosomy 7 and trisomy 8 MDS and implicate specific pathogenic pathways.
Regulation of survival, expansion, and differentiation of erythroid progenitors requires the well-controlled activity of signaling pathways induced by erythropoietin (Epo) and stem cell factor (SCF). In addition to qualitative regulation of signaling pathways, quantitative control may be essential to control appropriate cell numbers in peripheral blood. We demonstrate that Bruton's tyrosine kinase (Btk) is able to associate with the Epo receptor (EpoR) and Jak2, and is a substrate of Jak2. Deficiency of Btk results in reduced and delayed phosphorylation of the EpoR, Jak2, and downstream signaling molecules such as Stat5 and PLCgamma1 as well as in decreased responsiveness to Epo. As a result, expansion of erythroid progenitors lacking Btk is impaired at limiting concentrations of Epo and SCF. In addition, we show that SCF induces Btk to interact with TNF-related apoptosis-inducing ligand (TRAIL)-receptor 1 and that lack of Btk results in increased sensitivity to TRAIL-induced apoptosis. Together, our results indicate that Btk is a novel, quantitative regulator of Epo/SCF-dependent expansion and survival in erythropoiesis.
A novel tumor necrosis factor (TNF) family member has been cloned and characterized. This protein, designated TNF-related apoptosis-inducing ligand (TRAIL), consists of 281 and 291 aa in the human and murine forms, respectively, which share 65% aa identity. TRAIL is a type II membrane protein, whose C-terminal extracellular domain shows clear homology to other TNF family members. TRAIL transcripts are detected in a variety of human tissues, most predominantly in spleen, lung, and prostate. The TRAIL gene is located on chromosome 3 at position 3q26, which is not close to any other known TNF ligand family members. Both full-length cell surface expressed TRAIL and picomolar concentrations of soluble TRAIL rapidly induce apoptosis in a wide variety of transformed cell lines of diverse origin.
Apoptosis of haemopoietic cells in the marrow of patients with myelodysplastic syndrome (MDS) has been suggested as a mechanism for peripheral cytopenias. We determined the expression of Fas (CD95), Fas-Ligand (Fas-L) and TNF-alpha factors known to be involved in apoptosis, in the marrow of 44 patients with MDS and characterized their functional relevance in in vitro assays of haemopoiesis. Multidimensional flow cytometry revealed phenotypically aberrant blasts as defined by orthogonal light scatter and CD45 expression in the marrow of 24/44 patients. Among those blasts Fas expression was increased on CD34-positive cells and on cells co-expressing HLA-DR. In addition, Fas-L was expressed on some CD34+ cells of MDS patients but was never detected on CD34+ cells in normal marrow. Fas and Fas-L mRNAs as well as mRNA for TNF-alpha, known to increase Fas expression in normal marrow, were up-regulated in patients with MDS. TNF-alpha protein and sTNF-R1 levels in marrow plasma were higher in MDS patients than in controls (P<0.002 and <0.003, respectively). However, results were dependent upon disease category: TNF-alpha levels were significantly higher in patients with refractory anaemia (RA) than in patients with RA with excess blasts (RAEB) or RAEB in transformation (RAEB-T) (P=0.043). Conversely, the proportion of Fas-L-positive cells was lowest in patients with RA (P=0.037). In marrow cultures, Fas-Ig, rhuTNFR:Fc or anti-TNF-alpha antibody, by blocking Fas or TNF mediated signals, respectively, significantly increased the numbers of haemopoietic colonies compared to untreated cells (P<0.001, P<0.003, P<0.001, respectively). These results show significant dysregulation in the expression of TNF-alpha, Fas and Fas-L in the marrow from MDS patients. Altered expression of these molecules appears to be of functional relevance in the dysregulation of haemopoiesis in MDS and may be amenable to therapeutic interventions.
The paradox of peripheral cytopenias despite cellular bone marrow (BM) observed in myelodysplastic syndromes (MDS) has been associated with excessive intramedullary apoptosis of hematopoietic cells. Since MDS is regarded as a stem cell disorder, the present studies were undertaken to examine the relative susceptibility and propensity of early progenitor CD34+ cells to undergo apoptosis as compared to more maturing/matured CD34- cells. Five serial studies were performed on 4 independent groups of 36 newly diagnosed MDS patients. First, in 2 separate groups of 16 and 8 patients each, measurement of the extent of apoptosis in CD34+ and CD34- fractions of the BM aspirate mononuclear cells was carried out using independent biparametric flow cytometry methods, CD34 labeling/terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) (n = 16), and CD34 labeling/reduced uptake of nucleic acid staining dye LDS751 (n = 8). The difference in the median degrees of apoptosis in CD34+ vs. CD34- cells was not statistically significant by either technique (P = 0.583 and P = 0.674 for TUNEL and LDS751, respectively). In the next group of 4 MDS patients, a double-labeling was performed on plastic embedded marrow biopsy sections, to detect CD34 antigen with specific monoclonal antibody and apoptosis by in situ end labeling (ISEL) of fragmented DNA. Despite high overall apoptosis (56.2% +/- 18.4%), only an occasional CD34+ cell was found to be simultaneously labeled with ISEL. Finally, in the last group of 8 MDS patients, CD34+ cells were separated from CD34- cells on affinity column and cultured in serum containing medium for 4 hours. At 0- and 4-hour time points, ISEL was carried out to label apoptotic cells. In addition, a fluorometric assay was employed to estimate the activity of a proapoptotic enzyme, Caspase 3. Both the net increase in % ISEL labeled cells (apoptotic index or AI) and Caspase-3 activity were significantly lower in CD34+ cells as compared to CD34- cells (AI, 0.87% +/- 0.5% vs. 3.97% +/- 1.4%, n = 6, P = 0.028 and Caspase-3 Units/mg protein, 46.9 +/- 25.0 vs. 71.7 +/- 23.03, n = 5, P = 0.042, respectively). We conclude that when estimated in a total population of mononuclear cells, CD34+ cells and CD34- cells show comparable degrees of apoptosis. However, once separated the CD34+ fraction demonstrates lower propensity to undergo apoptosis, thereby suggesting the CD34- fraction as being a possible source for proapoptotic signaling.
DidierBouscary, LaurenceLegros, MichelineTulliez, StephanieDubois, BeatriceMahe, OdileBeyne-Rauzy, Marie C.Quarre, DominiqueVassilief, BrunoVaret, AchilleAouba, MartineGardembas, StephaneGiraudier, AgnesGuerci, PhilippeRousselot, FannyGaillard, AnneMoreau, Marie C.Rousselet, NorbertIfrah, PierreFenaux, FrancoisDreyfus, . (2005) A non-randomised dose-escalating phase II study of thalidomide for the treatment of patients with low-risk myelodysplastic syndromes: the Thal-SMD-2000 trial of the Groupe Francais des Myelodysplasies. British Journal of Haematology 131:10.1111/bjh.2005.131.issue-5, 609-618
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The production of red blood cells follows the sequential formation of proerythroblasts and basophilic, polychromatophilic and orthochromatic erythroblasts, and is promoted by the hormone erythropoietin (Epo) in response to tissue hypoxia. However, little is known about the negative regulation of this process. Death receptors are a family of surface molecules that trigger caspase activation and apoptosis in a variety of cell types. Here we show that immature erythroid cells express several death receptors whose ligands are produced by mature erythroblasts. Exposure of erythroid progenitors to mature erythroblasts or death-receptor ligands resulted in caspase-mediated degradation of the transcription factor GATA-1, which is associated with impaired erythroblast development. Expression of a caspase-resistant GATA-1 mutant, but not of the wild-type gene, completely restored erythroid expansion and differentiation following the triggering of death receptors, indicating that there is regulatory feedback between mature and immature erythroblasts through caspase-mediated cleavage of GATA-1. Similarly, erythropoiesis blockade following Epo deprivation was largely prevented by the expression of caspase-inhibitory proteins or caspase-resistant GATA-1 in erythroid progenitors. Caspase-mediated cleavage of GATA-1 may therefore represent an important negative control mechanism in erythropoiesis.
The mechanism that leads to hemopoietic failure in patients with myelodysplastic syndrome (MDS) is not well understood. There is evidence, however, that regulatory molecules such as tumor necrosis factor (TNF)-alpha, Fas (CD95), and Fas-ligand, which negatively affect hemopoiesis by way of apoptosis are upregulated. Here we analyzed marrow samples from 80 patients with MDS in regard to TNF-alpha and Fas-ligand levels and a possible correlation with various disease parameters and risk factors. TNF-alpha levels were elevated in comparison to samples from normal marrow donors, however, no significant correlation with FAB subtype, cytogenetic risk group or score by the International Prognostic Scoring System (IPSS) was observed. However, there was an inverse correlation between the cytogenetic risk category (low, intermediate, high) and levels of soluble Fas-ligand. The major source of TNF-alpha were mononuclear (non-stromal) cells which appeared to produce TNF-alpha at maximum levels. Limiting dilution analysis of CD34+ precursor cells showed that individually assayed cells, removed from companion cells that presumably provided negative signals such as TNF-alpha or Fas-ligand, were able to generate progressively increasing numbers of colonies. Stromal layers derived from MDS marrow did not have an inhibitory effect. In fact, higher colony numbers were obtained from both normal and MDS marrow derived hemopoietic precursors propagated on irradiated stromal layers from MDS marrow than on stromal layers from normal marrow. These results show that substantial numbers of normal hemopoietic precursors persist in MDS marrow. However, differentiation into mature cells is inhibited by negative signals originating from accessory or abnormal hemopoietic precursors in the non-adherent marrow fraction.
A higher percentage of apoptotic cells (apoptotic index or AI) is consistently found in bone marrow (BM) biopsies compared to BM aspirates of patients with myelodysplastic syndrome (MDS). Most studies have only investigated the low-density fraction (LDF) mononuclear cells from BM aspirates following density separation for AI determination. In the present study, both LDF and high-density fraction (HDF) cells for AI were examined by electron microscopy (EM) in 10 MDS patients and 4 healthy donors. Matched BM biopsies were subjected to AI detection by in situ end labeling (ISEL) of fragmented DNA. The results indicate that in LDF and HDF cells, AI is consistently higher in MDS patients (8.5% vs 1.5%, respectively; P =.039) compared to healthy donors (27% vs 4%, respectively; P =. 004). The BM biopsy AI was also higher in MDS patients than in healthy donors (3+ vs 0+, respectively; P =.036). In addition, in MDS patients, more apoptotic cells were found in HDF cells than in LDF cells (27% vs 8.5%, respectively;P =.0001). All stages of maturation, ranging from blasts to terminally mature cells belonging to all 3 lineages, were represented in the dying cells in both compartments. Using EM, typical Pelger-Huett-type cells appeared to be apoptotic granulocytes. Both LDF and HDF cells should be examined for an accurate estimation of apoptotic cells because AI would be underestimated if only the LDF cells were studied. Ultrastructural studies consistently show a higher AI in BM biopsies compared to BM aspirates despite the correction factor of HDF cells provided by AI. This may represent the actual extant state, which could conceivably be due to a higher concentration of proapoptotic signals in the biopsies. (Blood. 2000;96:1388-1392)
To determine the relation of apoptosis and clonal proliferation in the bone marrow (BM) to the effectiveness of a therapeutic protocol described to downmodulate monokine activity in patients with myelodysplastic syndromes (MDS). Prior to protocol therapy, BM stroma was cultivated and selected CD34(+) cells were studied in stroma and cytokine-dependent clonogenic assays. The TUNEL assay was used to establish the degree of apoptosis occurring in the marrow and CD34(+) population. The effectiveness of oral ciproloxacin 500 mg b. i.d., pentoxifylline 800 mg t.i.d., and dexamathasone 4 mg t.i.d. (CPD) antiinflammatory therapy was correlated with the intensity of cell apoptosis and proliferation of BM progenitor cells. Seventeen patients were studied. Twelve patients (10 transfusion dependent) received therapy for a median of 99 days (range 49-284). Toxicity caused four patients to discontinue the drug combination. Six patients fulfilled response criteria. Four patients became transfusion independent, and 50% reduction in the need for blood transfusions was noted in one patient. Blood parameters of one untransfused patient increased by >30%. Blood count remained unsupported in three patients, even at a median of 12 months after trial discontinuation. Apoptosis of marrow cells and selected CD34(+) progenitors was detected in a median of 49.5% (range 3. 6%-90%) and 10.6% (range 3.6%-100%; p < 0.01), respectively. In patients who responded to therapy, the median apoptosis rate in the bone marrow population was 71%, in contrast to the nonresponder's rate of 13% (p = 0.002). Overall clonogenic growth of selected precursors corresponded significantly with response to CPD protocol (p = 0.004). In some patients with MDS, ineffective hematopoiesis is related to high apoptotic index despite proliferation of the CD34(+) precursors. These patients seem to benefit from CPD cytokine modulatory therapeutic strategy.
Under the auspices of the European Association of Hematopathologists and the Society for Hematopathology, 100 hematopathologists and clinicians have met together and in subcommittees over a 5-year period. The model used was that developed by the International Lymphoma Study Group, which used morphologic, immunologic, and genetic features. It resulted in the revised European-American Lymphoma classification. The newly proposed leukemia classification uses a similar format and eliminates purely morphologic subtypes that have no current clinical relevance. As with all consensus proposals, these classifications must withstand criticism and debate by potential users.
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of the tumor necrosis factor (TNF) family, binds to several cell-surface receptors with distinct functions (agonistic receptors 1 and 2 [TRAIL-R1, TRAIL-R2]; decoy receptors 3 and 4 [TRAIL-R3, TRAIL-R4]). Expression and function was characterized in patients with myelodysplastic syndromes (MDSs). While normal marrow showed negligible expression of TRAIL and receptors (except TRAIL-R3), TRAIL and all receptors were constitutively expressed in MDS marrow. Following TRAIL exposure, MDS marrow showed significant increases in apoptosis, whereas normal marrow, except for a subset of CD34+ precursors, did not (P =.012). Marrow from 21 patients with MDS was then propagated in long-term cultures in the presence or absence of TRAIL. While in advanced MDS (refractory anemia with excess blasts in transformation [RAEB-T] and tAML [MDS transformed into AML]), colony numbers decreased in the presence of TRAIL (63.0% +/- 10.4% of untreated group [100%]), numbers increased in patients with RA or RAEB (160.2% +/- 90.5% of untreated group). TRAIL eliminated preferentially clonally abnormal cells as identified by chromosomal markers. Thus, TRAIL and receptor expression differed significantly between normal and MDS marrow, and TRAIL modulated in vitro hemopoiesis in MDS dependent upon disease stage but not, to a detectable extent, in normal marrow.
The aim of this study was to evaluate which CD34(+) cell subset contained in leukapheresis products could be regarded as the most predictive of long-term hematopoietic recovery after autologous peripheral blood stem cell transplantation (auto-PBSCT).
Based on data from 34 patients with hematologic malignancies, doses of CD34(+) cells and CD34(+) cell subsets, defined by the expression of HLA-DR, CD38, CD117 (c-kit/R), CD123 (alpha subunit of IL-3/R), CD133 (AC133), and CD90 (Thy-1) antigens, were correlated with the number of short-term (i.e., colony-forming cells [CFC]) and long-term culture CFC (LTC-CFC) (generated at week 5 of culture) and with the kinetics of hematopoietic engraftment following auto-PBSCT. The capacity of autologous stroma (AS), normal human bone marrow stroma, and M2-10B4 murine cell line to sustain CD34(+) cell growth was comparatively evaluated in the LTC assay.
Our data demonstrated that some of the most primitive progenitor subsets (CD34(+)CD117(-)HLA-DR(-), and CD34(+)CD38(+)HLA-DR(-)) showed the strongest correlation with LTC-CFC numbers generated within the AS, whereas no significant correlation was noted using normal bone marrow stroma. Multivariate analysis showed that the only CD34 cell subset independently associated with long-term (3 to 6 months) platelet engraftment after auto-bone marrow transplantation was the CD34(+)CD117(-)HLA-DR(-) phenotype; long-term erythrocyte engraftment was correlated with CD34(+)CD38(+)HLA-DR(-) cell content. The latter further influenced platelet engraftment in the first 3 months after auto-PBSCT. The most predictive parameters for neutrophil engraftment were CD34(+)CD38(+)HLA-DR(-) cell subtype and the total LTC-CFC quantity infused.
These data further support the hypothesis that the type of stromal feeders influences the frequency of LTC-CFC, possibly because they differ in their ability to interact with distinct subsets of hematopoietic stem cells. Furthermore, as the use of AS in LTC assay can mimic in vitro the human bone marrow microenvironment, it can be speculated that this culture system could be a useful means to study the kinetics of recovery of bone marrow stroma following chemotherapy and PBSCT. From these results, it can be concluded that some CD34(+) cell subsets appear to be more reliable predictors of long-term hematopoietic recovery rates than total CD34(+) cell quantity.
Laboratory observations suggest that, in some myelodysplastic syndromes (MDS), immune mechanisms may contribute to the impaired blood cell production. Tumor necrosis factor alpha (TNF-alpha), a potent inhibitor of haematopoiesis, has been hypothesized to mediate suppressive effects in MDS: TNF-alpha levels are elevated and correlated with marrow apoptosis and cytopenia. Inhibition of TNF-alpha production using the soluble TNF receptor (Enbrel) has been successful in rheumatoid arthritis, and we have now applied the same principle to MDS. We determined spontaneous TNF-alpha production by marrow cells in MDS; TNF-alpha production was elevated (> mean + 2 x SD of controls) in > 1/3 of patients, but did not correlate with clinical parameters. Sixteen patients participated in a 3-month pilot study of Enbrel. The drug was well tolerated and 15 patients were evaluable. Of these, one became temporarily (14 weeks) transfusion independent. In another patient, absolute neutrophil count (ANC) rose from 0.5 x 10(9)/l to 0.84 x 10(9)/l. Serious infections were seen in two out of six neutropenic patients. Progression to refractory anaemia with excess blasts in transformation (RAEBt) or leukaemia was observed in three patients. When the effects of Enbrel on haematopoietic colony formation were studied, no significant increase was seen in MDS and there was no correlation with TNF-alpha levels. Although anti-TNF therapy with Enbrel was well tolerated at the dosages used in MDS, its efficacy as a single agent appears low.
Labeling index (LI), apoptosis, levels of 2 pro-apoptotic cytokines tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta(TGF-beta), and the number of monocyte/macrophage cells that are the likely source of the cytokines were simultaneously measured in plastic-embedded bone marrow (BM) biopsy sections of 145 patients with myelodysplastic syndromes (MDS). TNF-alpha was correlated with TGF-beta (P = .001) and with monocyte/macrophage cells (P = .003). Patients with excess blasts in their marrows had a higher TGF-beta level (P = .01) and monocyte/macrophage number (P = .05). In a linear regression model,TGF-beta emerged as the most significant biological difference between patients who have excess of blasts and those who do not (P = .01). We conclude that in addition to TNF-alpha, TGF-beta also plays a significant role in the initiation and pathogenesis of MDS, and that a more precise definition of its role will likely identify better preventive and therapeutic strategies.
Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF/nerve growth factor superfamily that, apart from inducing cell death in susceptible cells, displays immunoregulatory functions influencing, for instance, T cell proliferation. It can be found in two forms: membrane-bound and soluble protein. The regulation of these is still not fully understood. In this study, we have analyzed the regulation of TRAIL surface expression and secretion in human T cells, B cells, and monocytes in response to specific stimuli. T cells, B cells, and monocytes were cultured in the presence of phytohemagglutinin (PHA)+interleukin (IL-2), anti-CD40+IL-4, and lipopolysaccharide (LPS), respectively. In particular, not only PHA+IL-2 but also LPS were able to induce secretion of soluble TRAIL, but did not enhance the expression of surface-bound TRAIL. Simultaneously, we investigated the effect of the pleiotropic stimulus interferon (IFN)-beta, known to target all leukocyte subsets, on TRAIL. Predominantly, monocytes were affected by IFN-beta, causing both release of soluble TRAIL and upregulation of the surface-bound form. IFN-beta, however, did not cause any upregulation of TRAIL in T cells. Our data serve as a basis to better understand the complex regulation of TRAIL in human peripheral immune cells and might help to clarify the role of the TRAIL system in immunopathology.
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