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Increased Apoptosis as a Mechanism of Ineffective Erythropoiesis in Myelodysplastic Syndromes
Comprehensive Review Increased Apoptosis as a Mechanism of Ineffective Erythropoiesis in Myelodysplastic Syndromes Rosangela Invernizzi, Erica Travaglino Abstract 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. Clinical Leukemia, Vol. 2, No. 2, 113-120, 2008 Key words: Apoptotic pathways, Ineffective hematopoiesis, Mitochondrial ferritin, Sideroblastic anemia
Introduction Apoptosis, or programmed cell death, discovered in 1972 by Kerr et al,1 is a morphologically and biochemically distinct form of cell death, genetically regulated, without inflammatory tissue injury. It is an active process of dying that requires energy and de novo gene expression. It is implicated in biologic processes ranging from embryogenesis to aging, from normal tissue homeostasis to many human diseases. Decreased or inhibited apoptosis is a feature of many malignancies. The molecular mechanisms involved in death signal, genetic regulation, and activation of effectors have been recently identified.2,3 Myelodysplastic syndromes (MDS) are clonal diseases of hematopoiesis characterized by reduced ability of the proliferating clone to differentiate and mature with a propensity to evolve into acute myeloid leukemia (AML). Premature apoptotic cell death within the bone marrow (BM) might explain the apparent paradox in MDS of persistent cytopenia despite normal or increased BM cellularity, and it is commonly considered the underlying cause of ineffective hematopoiesis.4-12 Department of Internal Medicine, University of Pavia, Fondazione IRCCS Policlinico S. Matteo, Pavia, Italy Submitted: October 30, 2007; Revised: December 10, 2007; Accepted: January 3, 2008 Address for correspondence: Rosangela Invernizzi, MD, Clinica Medica 3, Piazzale Golgi, 27100 Pavia, Italy Fax: 39-0382-526223; e-mail: [email protected] Electronic forwarding or copying is a violation of US and International Copyright Laws. Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by CIG Media Group, LP, ISSN #1931-6925, provided the appropriate fee is paid directly to Copyright Clearance Center, 222 Rosewood Drive, Danvers, MA 01923 USA 978-750-8400.
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Increased Apoptosis in Myelodysplastic Syndromes
Table 1
Apoptosis in Myelodysplastic Syndrome: Published Experience
Study Clark et al13 al5
Marrow Sample
Technique
Apoptotic Cells (%)
Biopsy
Morphology
20 Times controls
Biopsy
ISEL
q 75 in half of cases
Lepelley et al14
Aspirate
TUNEL
2.5-5 (Controls: < 2)
Rajapaksa et al15
Aspirate
FC
9 (CD34+) (controls: 2)
Bogdanovic et al16
Aspirate
Morphology (EM)
3 (Controls: 1)
Hellström et al17
Biopsy
TUNEL
56 (Controls: 16)
al18
Aspirate
TUNEL
39 (Controls: 13)
Parker et al19
Aspirate
FC (annexin V)
56.5 (CD34+) (controls: 18.5)
Tsoplou et al20
Aspirate
TUNEL
34% of Cases (controls: 0)
Shimazaki et al21
Biopsy
TUNEL
5.5 (Controls: 0.6)
Novitzky et al22
Aspirate
TUNEL
49.5
Raza et
Bouscary et
al23
Aspirate
FC (annexin V)
> 50 (CD34+) in RA, RARS, RAEB
Albitar et al24
Aspirate
FC (annexin V)
7
al25
Parker et
Biopsy
TUNEL
47 (Controls: 41)
Brada et al26
Biopsy
TUNEL
2.3 (Controls: 4.8)
Michalopoulou et al27
Aspirate
FC (annexin V)
20 (Controls: 7)
Invernizzi et al28
Aspirate
TUNEL
24 (Controls: 13)
Ramos et
Abbreviations: EM = electron microscopy; FC = flow cytometry; ISEL = in situ end labeling
This review will outline the role of the apoptotic pathways in the pathogenesis of MDS and suggest possible molecular mechanisms whereby apoptosis in MDS might be dysregulated.
Increased Apoptosis in Myelodysplastic Syndrome Findings suggestive of excessive apoptosis in MDS include defective in vitro growth pattern of marrow progenitors, high proportion of cells with DNA strand breaks or apoptotic nuclei engulfed by macrophages in marrow biopsies, increased apoptotic CD34+ cells with increased ratio of proapoptotic/antiapoptotic proteins, and increased levels of various apoptosis-related parameters in BM cells. Several works, using different techniques, have shown that MDS is associated with increased apoptosis of marrow cells. However, the percentages of patients with increased apoptosis and the mean percentage of apoptotic cells greatly varied from one study to another (Table 1).5,13-28 These variations can be, at least partially, explained by differences in the sample origin, French-American-British (FAB) subtype, time between sample procurement and analysis, and the sensitivity of the technique.29 With terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) technique, apoptotic cells show intense, diffuse, often dishomogeneous nuclear reactivity (Figure 1). Using this technique, we demonstrated significant higher apoptotic index in low-risk MDS than in AML; the apoptotic index was also higher than in high-risk MDS and than in normal controls, and it was inversely related to blast cell percentage.28-30
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Figure 1
Bone Marrow Aspirate from a Patient with Refractory Anemia
Bone marrow aspirate from a patient with RA, using the TUNEL Technique. Apoptotic cells show strong nuclear reactivity (magnification ×1250).
In summary, the findings of various authors show that excessive apoptosis occurs in all MDS subtypes and affects all marrow lineages and also stromal cells; most reports agree that the highest apoptotic index occurs in refractory anemia (RA), RA with ring sideroblasts (RARS), and RA with excess of blasts (RAEB), there being a progressive decline in apoptosis as the disease evolves toward RAEB in transformation (RAEB-t) and secondary AML (sAML). The question of whether the excessive apoptosis in MDS predominantly involves the subset of progenitor stem cells or of more differentiated cells is still controversial. We used a double-labeling, 2-color technique that combined the TUNEL method and immunocytochemistry to assess the degree of apoptosis in MDS BM and its possible differences from normal marrow in relation to CD34 antigen expression. Our results suggest that in MDS as well as in normal BM, the apoptotic phenomenon predominantly involves the maturing cells.31 Some correlations between apoptosis levels and clinical and hematologic features were observed: inverse correlation with leukocyte count,16,32 International Prognostic Scoring System and cytogenetic risk group,23 prognosis,21 and response to aggressive chemotherapy.24 Patients showing a higher apoptotic rate had a poorer prognosis regardless of the FAB subtype, and patients who did not achieve complete remission after aggressive treatment had higher levels of apoptosis of CD34+ cells.
Molecular Mechanisms of Increased Apoptosis in Myelodysplastic Syndrome Various anomalies might be responsible for increased apoptosis in MDS (Table 2). Apoptosis is caspase dependent, but the question is whether this increased caspase activity is caused by extrinsic or intrinsic apoptotic stimuli.
The Role of the Extrinsic Pathway Details of the death receptor pathway of caspase activation are shown in Figure 2. Supporting the involvement of this pathway, MDS hematopoietic cells overexpress Fas and tumor necrosis factor
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Table 2
Pathogenesis of Increased Apoptosis in Myelodysplastic Syndrome
Figure 2
The Extrinsic Apoptotic Pathway
• Increase in apoptosis-promoting and/or decrease in survivalpromoting cytokines • Activation of the Fas-FasL system
T Cell
• Intrinsic cell-cycle dysfunction • Abnormalities of apoptosis-related genes
Granzyme B
FasL TNF TRAIL
• Mitochondrial abnormalities Ligand Receptor
• Alteration of the hematopoietic microenvironment
(TNF)–α–related apoptosis-inducing ligand (TRAIL) death receptors as well as their ligands, but the source of the stimuli that increases death-receptor signaling is not entirely clear. Microenvironment cells, such as macrophages, stromal elements, or cytotoxic T cells, might produce excess death-receptor stimuli. Alternatively, the increased expression of ligands on the surface of hematopoietic cells might induce autocrine stimulation of the death receptor pathway.
DD
DcR
Nucleus
Perforin
Fas-R TNF-R TRAIL-R
cFLIP
FADD SODD
FADD
DED
NIK
IKB
Caspase 10 P
IKB NF-KB
Fas Signaling
BID
Mitochondrial Pathway
The interaction between Fas ligand (FasL) and Fas has been studied extensively in MDS. Increased Fas expression on marrow cells, including CD34+ progenitors, erythroblasts, and myeloid cells, has been reported in approximately 40% of patients,18,33-37 but no correlation between Fas expression and rate of apoptosis could be demonstrated. FasL is expressed on similar hematopoietic populations as Fas, including CD68+ macrophages, and its expression correlates with FAB subtype and anemia and predicts survival.38 Because Fas expression is elevated during all stages of erythroid differentiation/maturation and mononuclear cells from patients with MDS undergo increased apoptosis after treatment with activating antibody, it is likely that MDS erythroid cells exhibit an exacerbation of the physiologic mechanisms of Fas-mediated control of erythropoiesis and a Fas-dependent apoptosis, even in the presence of high erythropoietin levels.39 This suggestion is also supported by the results of in vitro studies demonstrating that lentivirus-mediated introduction of a construct encoding the adapter Fas-associated death domain (FADD) in CD34+ cells from patients with low-grade MDS increases apoptosis of erythroid cells and dramatically reduces burst forming unit erythroid (BFU-E) growth, whereas transduction of a dominant-negative mutant of FADD inhibits caspase-8 activity and apoptosis of erythroid precursors grown in liquid medium and rescues BFU-E growth without abrogating erythroid differentiation.40 Interestingly, Fas-triggered apoptosis in RARS BM cells is significantly improved by granulocyte colony-stimulating factor (G-CSF) treatment.41 The extrinsic Fas-mediated pathway of apoptosis seems important in regulating cell replication and death, especially in trisomy 8 hematopoietic cells.42 The immune system plays an active role in the pathogenesis of trisomy 8 syndrome: Besides upregulation of immune/inflammatory genes and downregulation of apoptosis-inhibiting genes in CD34+ cells,43 T-cell receptor skewing, highly suggestive of an antigen-driven process, has been observed.44 Upregulation of antiapoptotic proteins, such as survivin, c-Myc, and CD1, allows CD34+ cells from patients with trisomy 8 MDS to avoid apoptosis.45
DISC
Caspase 8
Caspase 8
Effector Caspases
Interaction between death ligands and their receptors leads to receptor trimerization, clustering of the adapter protein FADD or TRADD, and creation of a DISC. This recruits and activates initiator procaspase-8 through its DED. Caspase-8 directly activates effector caspases or triggers the mitochondrial pathway through Bid cleavage. Also the T-cell–mediated cell death is an example of extrinsic apoptotic pathway. The death-receptor pathway is negatively regulated by the nuclear transcription factor NF-KB. Abbreviations: DcR = decoy receptor; DD = death domain; DED = death effector domain; DISC = death-inducing signaling complex; FLIP = Fas-associated death domain–like interleukin-1B –converting enzyme-inhibitory protein; IKB = inhibitor of KB; NF-KB = nuclear factor–KB; NIK = NF-inducing kinase; SODD = silencer of death domain; TNF-R = TNF-receptor; TRADD = TNF receptor–associated death domain
TRAIL/TRAILR System There is also evidence that the TRAIL/TRAIL receptors (TRAILRs) system might play a role in the ineffective hemopoiesis of MDS, but it contributes to the myeloid rather than erythroid apoptosis.41 TRAIL and TRAILRs are expressed at increased levels in the BM of patients with MDS. Furthermore, MDS hematopoietic precursors are supranormally sensitive to TRAIL-mediated apoptosis.46,47 However, no mutations in the death domains of TRAILR genes is found.48 Myeloid nuclear differentiation antigen downregulation increases progenitor cell response to TRAILinduced apoptosis.49 With regard to clinical correlations, hemoglobin levels are lower in patients whose BM mononuclear cells release TRAIL in culture supernatants.50
Cytokines Several authors have reported increased levels of TNF-α and other inflammatory cytokines in serum and BM in MDS,33,51-53 and levels of TNF-α and interleukin (IL)–1β correlate with the degree of apoptosis.52,54 These cytokines might upregulate Fas and FasL expression in MDS colony-forming cells and accelerate progenitor death. Moreover, they might activate the p38 mitogen-activated protein kinase pathway to mediate signals for apoptosis.55,56 Tumor necrosis factor–α can induce apoptosis also by oxidation of DNA and proteins.57
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Increased Apoptosis in Myelodysplastic Syndromes
Figure 3
The Intrinsic Apoptotic Pathway Reduced Growth Factor
DNA damage
ROS
BH-3
tBid
Bcl-2
Bax
Bim
Nucleus
IAP
AIF
Omi
IAP
Endo G
smac
IAP
IAP IAP
Cytochrome C Caspase-9 Apaf-1 Cytochrome C Apaf-1
Caspase-3/7
Apoptotic substrates
Apoptosome
Activation of the mitochondrial pathway leads to a loss of mitochondrial membrane potential and release of several apoptogenic factors. A key event is the translocation of cytochrome C from the intermembrane space into cytosol. Released cytochrome C and Apaf-1 associate with procaspase-9 and adenosine triphosphate to form the apoptosome complex. Pro–caspase-9 becomes activated and triggers the proteolytic cascade, leading to apoptosis. Apoptosisinducing factor can directly bind and cleave DNA. Bcl-2 family members are the major regulators of mitochondrial membrane permeabilization and release of apoptogenic factors. Abbreviations: AIF = apoptosis-inducing factor; Apaf-1 = apoptotic protease-activating factor–1; Endo G = endonuclease G; IAP = inhibitor of apoptosis protein; ROS = reactive oxygen species; smac = second mitochondrial activator of caspases
On the other hand, in vivo treatment with TNF-α–blocking agents,58,59 anti–TNF-α antibody,60 and cytokines such as G-CSF and erythropoietin61,62 reduces apoptosis in responding patients. Also anti-inflammatory therapy is effective in patients with MDS with increased apoptosis of BM cells.22 The involvement of inflammatory cytokines in the pathogenesis of MDS is confirmed by the recent finding of Toll-like receptor 4 upregulation in MDS hematopoietic progenitor cells.63
Stromal Microenvironment The precise role of the stromal microenvironment in triggering MDS apoptosis is still largely unknown. Some authors have demonstrated that adherent cells grown from the biopsy of patients with MDS are able to support normal hematopoiesis,64 whereas others have shown that stromal growth of MDS marrow is impaired and is associated with a decreased capacity to support normal and myelodysplastic hematopoiesis65,66; moreover, the adherent layers in cultures grown from patients with MDS are hemopoietically defective and show abnormal IL-1β expression.67 On the other hand, increased numbers of stromal macrophages are the key regulators in the increased production of proapoptotic cytokines. Whether altered integrin-mediated focal contacts between MDS progenitors and marrow stroma are involved in apoptosis is at present unknown.
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The Role of the Intrinsic Pathway There is evidence that, in MDS, in addition to the extrinsic apoptotic pathway, the intrinsic pathway is also activated.70 The hallmark of activation of the intrinsic pathway is the release of apoptogenic factors from mitochondria (Figure 3).
ATP
Caspase-9
With regard to angiogenesis, the overexpression of the angiogenic factor vascular endothelial growth factor along with its receptors has been demonstrated in MDS cells.68 Vascular endothelial growth factor production might stimulate angiogenesis that could favor disease progression by altering the microenvironment to favor unregulated cell growth. On the other hand, we observed by immunocytochemistry an abnormal expression of matrix metalloproteinases (MMP)–2 and –9 in both myeloid and erythroid MDS cells. Vascular endothelial growth factor and MMPs can stimulate endothelial cells and macrophages to produce inflammatory cytokines such as TNF-α that might reinforce FasL expression in erythroid cells and, as a consequence, ineffective erythropoiesis.69 Also, extracellular matrix degradation by MMPs might contribute to decreased erythroid adherence and increased apoptosis.
Mitochondrial Abnormalities Mitochondrial abnormalities are commonly seen in MDS, including reduced membrane potential in the erythroid subpopulations of RARS71 and in BM cells of all MDS subtypes and cytochrome C-oxidase gene mutations.72-75 A potential role for the mitochondria in MDS was suggested also by analysis of the crimsonless zebrafish mutant that develops an MDS as the mutation of a mitochondrial heat-shock protein (HSPA9B) produces oxidative stress and apoptosis distinctly in blood cells.76 The erythroid apoptosis of low-risk MDS is initiated at a very early stage of stem cells through a spontaneous cytochrome C release from the mitochondrial intermembrane space, with subsequent activation of caspase-9 and effector caspase-3.77 The role of the mitochondrial intrinsic pathway might be relevant especially in RARS, where G-CSF shows an antiapoptotic effect in vitro, being able to inhibit spontaneous release of cytochrome C, drop of mitochondrial membrane potential, caspase activation, and restore erythroid proliferation.78-80 In summary, in low-risk MDS, the increased sensitivity to triggering of death receptors is likely dependent on constitutive mitochondrial signaling. The factors that stimulate the mitochondrial pathway of apoptosis are unclear. In RARS, the mitochondrial pathway might be stimulated by increased iron deposition.
Mitochondrial Ferritin A novel human ferritin, which is encoded by an unusual intronless gene on chromosome 5q23.1 and has limited tissue expression, has been identified.81,82 The encoded protein is a 242–amino acid precursor with a 57–amino acid leader sequence for mitochondrial import. In mitochondria, this protein is processed to a subunit that assembles into functional shells that have a well-known crystallographic structure83 and incorporate iron as efficiently as H ferritin.84 This novel mitochondrial ferritin (FtMt) might play an important role in regulating mitochondrial iron homeostasis and heme synthesis. Mitochondrial ferritin expression might increase with mitochondrial iron loading, possibly through transcriptional mechanisms.85-87
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Rosangela Invernizzi, Erica Travaglino
Figure 4
Bone Marrow Aspirate from a Patient with RARS
A
Figure 5
Cytospin Samples of Erythroid Progenitors from a Patient with RARS
A
B B
CD34+ cells were separated from the BM mononuclear cells and cultured in a liquid medium according to a procedure that allows the expansion of high concentrations of erythroid progenitors in the presence of various cytokines and growth factors. (A) At day 11 of culture, most cells show morphologic features of proerythroblasts or basophilic erythroblasts (MayGrünwald-Giemsa stain). (B) Immunoalkaline phosphatase staining for mitochondrial ferritin shows unequivocal positivity in the same cells (magnification ×1250). (A) Perls staining showing positive granules forming a ring surrounding the nucleus in the erythroblasts. This peculiar stain is caused by mitochondrial iron overload. (B) Immunoalkaline phosphatase staining for mitochondrial ferritin showing its localization in perinuclear granules (magnification ×1250).
The massive increases in mitochondrial iron in conditions of defective heme synthesis led recently to examine the expression of the FtMt in BM erythroblasts from patients with sideroblastic anemia, a group of hereditary or acquired disorders characterized by ineffective erythropoiesis associated with iron accumulation in the mitochondria of immature red cells.88 We demonstrated that most of the iron deposited in perinuclear mitochondria of ring sideroblasts is present in the form of FtMt that can be detected by immunocytochemistry in granules ringing the nucleus (Figure 4). The close relationship between the number of FtMt-positive erythroblasts and that of ring sideroblasts suggests that FtMt might be a specific marker of sideroblastic anemia and that FtMt detection by immunocytochemistry or flow cytometry might be a useful tool in the work-up of patients with MDS.89,90
On the basis of preliminary findings showing that FtMt reduces TNF-α–induced apoptosis in transduced HeLa cells, we hypothesized that, functionally, FtMt might play an important role in regulating iron homeostasis and toxicity; it might protect mitochondria from oxidative damage and increase cell resistance to apoptotic signals.86,91 In an attempt to look for the potential pathogenetic role of FtMt in the ineffective erythropoiesis of RARS, we used an in vitro model to evaluate the distribution of FtMt in RARS erythroid progenitors.92 Mitochondrial ferritin expression occurs very early during RARS erythroid differentiation, in cells that are still CD34+ and without any visible iron accumulation (Figure 5). In very immature erythroblasts in RARS, FtMt expression is closely linked to mitochondrial damage in terms of release of cytochrome C. The strong antiapoptotic effect of G-CSF in RARS seems to be caused by upregulation of compensatory survival mechanisms rather than a direct effect on mitochondrial iron accumulation. We can suppose
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Increased Apoptosis in Myelodysplastic Syndromes
Table 3
Myelodysplastic Syndrome Disease Progression
Table 4
Controversial Issues
• What are the properties of the stem cell that produces MDS? • Telomere shortening that might contribute to genomic instability • Increased angiogenesis
• Why are MDS cells unusually susceptible to apoptosis?
• Defective apoptotic machinery (Fas signaling downregulation, p53 inactivation)
• Is increased apoptosis in MDS related to the molecular pathogenesis of the disease or is it only a consequence of ineffective hematopoiesis caused by genomic damage and microenvironmental changes?
• Upregulation of negative apoptosis regulators (Bcl-2 family members, IAP)
• How many undiscovered genes are involved in death decision and how will we find them?
• Constitutive activation of NF-KB
• Is apoptosis clinically important in MDS?
• Epigenetic changes such as p15INK4b
• Are there possible therapeutic implications?
Abbreviations: IAP = inhibitor of apoptosis protein; INK = kinase inhibitor; NF = nuclear factor
Figure 6
Pathogenetic Model of Myelodysplastic Syndrome Hypercellular marrow
Progenitor cell transformation
Monoclonal hematopoiesis Early event
Cytokines Abnormal microenvironment Immunologic response
Toxin exposure Spontaneous mutation
Apoptosis Variable cytopenia
enigmatic whether increased apoptosis in MDS is related to the molecular pathogenesis of the disease or it is only a consequence of ineffective hematopoiesis caused by genomic damage and microenvironmental changes. Moreover, the properties of the stem cell that produces MDS are still obscure.95,96 Elucidation of this fascinating enigma might be important also from the clinical point of view because it might lead to the development of novel therapeutic strategies.
Acknowledgements Rosangela Invernizzi, MD, is supported by a grant from the Ministry for Education, University and Research, Italy, and Erica Travaglino, MSc, is supported by a grant from Fondazione IRCCS Policlinico S. Matteo, Pavia, Italy.
References that the aberrant expression of FtMt might reflect a defense mechanism of the cell against the oxidative damage caused by excess iron, but further excessive iron accumulation might cause further damage to the mitochondria and trigger apoptosis. The pathogenesis of RA without ring sideroblasts seems to be more heterogeneous, with the involvement of alternative mechanisms not associated with iron overloading, including T-cell–mediated BM failure. Ongoing studies seem to confirm that abnormalities in FtMt expression in RARS progenitors might contribute to alter the balance between cell growth and cell death.
Myelodysplastic Syndrome Progression Early MDS is a highly proliferative disorder, but despite the increased cell proliferation, the marrow does not export sufficient cells into the blood because intramedullary apoptosis mechanism prevails over proliferation.23,28,93,94 Progression to RAEB-t/sAML is associated with a significant fall in proliferation in most cases. Disease progression has been presumed to be the result of chromosomal lesions and genetic mutations, some affecting cell proliferation and others conferring resistance to apoptosis (Table 3).
Controversial Issues Whatever the cause or mechanism leading to apoptosis, it is now generally accepted that increased intramedullary apoptosis is responsible for the ineffective hematopoiesis in MDS (Figure 6). However, some controversial issues remain (Table 4). In particular, it is
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Leuk Lymph 2004; 45:2229-37. Choi JW. Apoptotic rate varies with detection methods in myelodysplastic syndromes: impact of method selection. Eur J Haematol 2002; 69:62-3. Invernizzi R, Pecci A, Bellotti L, et al. Expression of p53, Bcl-2 and Ras oncoproteins and apoptosis levels in acute leukaemias and myelodysplastic syndromes. Leuk Lymph 2001; 42:481-9. Pecci A, Travaglino E, Klersy C, et al. Apoptosis in relation to CD34 antigen expression in normal and myelodysplastic bone marrow. Acta Haematol 2003; 109:29-34. Reza S, Dar S, Andric T, et al. Biologic characteristics of 164 patients with myelodysplastic syndromes. Leuk Lymph 1999; 33:281-7. Gersuk GM, Beckham C, Loken MR, et al. A role for tumour necrosis factor-alpha, Fas and Fas ligand in marrow failure associated with myelodysplastic syndrome. Br J Haematol 1998; 103:176-88. Kitagawa M, Yamaguchi S, Takahashi M, et al. Localization of Fas and Fas ligand in bone marrow cells demonstrating myelodysplasia. Leukemia 1998; 12:486-92. Lepelley P, Poulain S, Grardel N, et al. Expression of lung resistance protein and correlation with other drug resistance proteins and outcome in myelodysplastic syndromes. Leuk Lymph 1998; 29:547-51. Fontenay-Roupie M, Bouscary D, Guesnu M, et al. Ineffective erythropoiesis in myelodysplastic syndromes: correlation with Fas expression but not with lack of erythropoietin receptor signal transduction. Br J Haematol 1999; 106:464-73. Ribeiro E, Lima CS, Metze K, et al. Flow cytometric analysis of the expression of Fas/Fasl in bone marrow CD34+ cells in myelodysplastic syndromes: relation to disease progression. Leuk Lymph 2004; 45:309-13. Gupta P, Niehans GA, LeRoy SC, et al. Fas ligand expression in the bone marrow in myelodysplastic syndromes correlates with FAB subtype and anemia, and predicts survival. Leukemia 1999; 13:44-53. Claessens YE, Bouscary D, Dupont JM, et al. In vitro proliferation and differentiation of erythroid progenitors from patients with myelodysplastic syndromes: evidence for Fas-dependent apoptosis. Blood 2002; 99:1594-601. Claessens YE, Park S, Dubart-Kupperschmitt A, et al. Rescue of early-stage myelodysplastic syndrome-deriving erythroid precursors by the ectopic expression of a dominant-negative form of FADD. Blood 2005; 105:4035-42. Schmidt-Mende J, Tehranchi R, Forsblom AM, et al. Granulocyte colony-stimulating factor inhibits Fas-triggered apoptosis in bone marrow cells isolated from patients with refractory anemia with ringed sideroblasts. Leukemia 2001; 15:74251. Sloand EM, Kim S, Fuhrer M, et al. Fas-mediated apoptosis is important in regulating cell replication and death in trisomy 8 hematopoietic cells but not in cells with other cytogenetic abnormalities. Blood 2002; 100:4427-32. Chen G, Zeng W, Miyazato A, et al. Distinctive gene expression profiles of CD34 cells from patients with myelodysplastic syndrome characterized by specific chromosomal abnormalities. Blood 2004; 104:4210-8. Sloand EM, Mainwaring L, Fuhrer M, et al. Preferential suppression of trisomy 8 compared with normal hematopoietic cell growth by autologous lymphocytes in patients with trisomy 8 myelodysplastic syndrome. Blood 2005; 106:841-51. Sloand EM, Pfannes L, Chen G, et al. CD34 cells from patients with trisomy 8 myelodysplastic syndrome (MDS) express early apoptotic markers but avoid programmed cell death by up-regulation of antiapoptotic proteins. Blood 2007; 109:2399-405. Zang DY, Goodwin RG, Loken MR, et al. Expression of tumor necrosis factorrelated apoptosis-inducing ligand, Apo2L, and its receptors in myelodysplastic syndrome: effects on in vitro hemopoiesis. Blood 2001; 98:3058-65. Allampallam K, Shetty V, Mundle S, et al. Biological significance of proliferation, apoptosis, cytokines, and monocyte/macrophage cells in bone marrow biopsies of 145 patients with myelodysplastic syndrome. Int J Hematol 2002; 75:289-97.
48. Liu LG, Tanaka H, Ito K, et al. Absence of gene mutation in TRAIL receptor 1 (TRAIL-R1) and TRAIL receptor 2 (TRAIL-R2) in chronic myelogenous leukemia and myelodysplastic syndrome, and analysis of mRNA expression of TRAIL and TRAIL-related genes in chronic myelogenous leukemia. Acta Haematol 2005; 113:113-23. 49. Briggs RC, Shults KE, Flye LA, et al. Dysregulated human myeloid nuclear differentiation antigen expression in myelodysplastic syndromes: evidence for a role in apoptosis. Cancer Res 2006; 66:4645-51. 50. Campioni D, Secchiero P, Corallini F, et al. Evidence for a role of TNF-related apoptosis-inducing ligand (TRAIL) in the anemia of myelodysplastic syndromes. Am J Pathol 2005; 166:557-63. 51. Verhoef GE, De Schouwer P, Ceuppens JL, et al. Measurement of serum cytokine levels in patients with myelodysplastic syndromes. Leukemia 1992; 12:1268-72. 52. Shetty V, Mundle A, Alvi S, et al. Measurement of apoptosis, proliferation and three cytokines in 46 patients with myelodysplastic syndromes. Leuk Res 1996; 20:891-900. 53. Stifter G, Heiss S, Gastl G, et al. Over-expression of tumor necrosis factor-alpha in bone marrow biopsies from patients with myelodysplastic syndromes: relationship to anemia and prognosis. Eur J Haematol 2005; 75:485-91. 54. Mundle SD, Venugopal P, Cartlidge JD, et al. Indication of an involvement of interleukin-1β converting enzyme-like protease in intramedullary apoptotic cell death in the bone marrow of patients with myelodysplastic syndromes. Blood 1996; 88:2640-7. 55. Katsoulidis E, Li Y, Yoon P, et al. Role of the p38 mitogen-activated protein kinase pathway in cytokine-mediated hematopoietic suppression in myelodysplastic syndromes. Cancer Res 2005: 65:9029-37. 56. Navas TA, Mohindru M, Estes M, et al. Inhibition of overactivated p38 MAPK can restore hematopoiesis in myelodysplastic syndrome progenitors. Blood 2006; 108:4170-7. 57. Peddie CM, Wolf CR, McLellan LI, et al. Oxidative DNA damage in CD34+ myelodysplastic cells is associated with intracellular redox changes and elevated plasma tumor necrosis factor-alpha concentration. Br J Haematol 1997; 99:62531. 58. Raza A. Anti-TNF therapies in rheumatoid arthritis, Crohn’s disease, sepsis, and myelodysplastic syndromes. Microsc Res Tech 2000; 60:229-35. 59. Maciejewski J, Ristiano AM, Sloand EM, et al. A pilot study of the recombinant soluble human tumour necrosis factor receptor (p75)-Fc fusion protein in patients with myelodysplastic syndrome. Br J Haematol 2002; 117:119-26. 60. Boula A, Voulgarelis M, Giannouli S, et al. Effect of cA2 anti-tumor necrosis factor therapy on hematopoiesis of patients with myelodysplastic syndromes. Clin Cancer Res 2006; 12:3099-108. 61. Hellström Lindberg E, Negrin R, Stein R, et al. Erythroid response to treatment with G-CSF plus erythropoietin for the anaemia of patients with myelodysplastic syndromes: proposal for a predictive model. Br J Haematol 1997; 99:344-51. 62. Tehranchi R. Impact of growth factors in the regulation of apoptosis in low-risk myelodysplastic syndromes. Medical Oncology 2006; 23:37-49. 63. Maratheftis CI, Andreakos E, Moutsopoulos HM, et al. Toll-like receptor-4 is upregulated in hematopoietic progenitor cells and contributes to increased apoptosis in myelodysplastic syndromes. Clin Cancer Res 2007; 13:1154-60. 64. Alvi S, Shaher A, Shetty V, et al. Successful establishment of long-term bone marrow cultures in 103 patients with myelodysplastic syndromes. Leuk Res 2001; 25:941-54. 65. Aizawa S, Nakano M, Iwase O, et al. Bone marrow stroma from refractory anemia of myelodysplastic syndrome is defective in its ability to support normal CD34positive cell proliferation and differentiation in vitro. Leuk Res 1999; 23:239-46. 66. Flores-Figueroa E, Gutierrez-Espindola G, Montesions JJ, et al. In vitro characterization of hematopoietic microenvironment cells from patients with myelodysplastic syndrome. Leuk Res 2002; 26:677-86. 67. Tennant GB, Walsh V, Truran LN, et al. Abnormalities of adherent layers grown from bone marrow of patients with myelodysplasia. Br J Haematol 2000; 111:85362. 68. Bellamy WT, Richter L, Sirjani D, et al. Vascular endothelial cell growth factor is an autocrine promoter of abnormal localized immature myeloid precursors and leukaemia progenitor formation in myelodysplastic syndromes. Blood 2001; 97:1427-34. 69. Stasi R, Amadori S, Newland AC, et al. Infliximab chimeric antitumor necrosis factor-α monoclonal antibody as potential treatment for myelodysplastic syndromes. Leuk Lymph 2005; 46:509-16. 70. Fontenay M, Cathelin S, Amiot M, et al. Mitochondria in hematopoiesis and haematological diseases. Oncogene 2006; 25:4757-67. 71. Matthes TW, Meyer G, Samii K, et al. Increased apoptosis in acquired sideroblastic anaemia. Br J Haematol 2000; 111:843-52. 72. Gattermann N, Retzlaff S, Wang YL, et al. Heteroplasmic point mutations of mitochondrial DNA affecting subunit 1 of cytochrome c oxidase in two patients with acquired idiopathic sideroblastic anemia. Blood 1997; 90:4961-72. 73. Gattermann N. From sideroblastic anemia to the role of mitochondrial DNA mutations in myelodysplastic syndromes. Leuk Res 2000; 24:141-51. 74. Reddy PL, Shetty VT, Dutt D, et al. Increased incidence of mitochondrial cytochrome c-oxidase gene mutations in patients with myelodysplastic syndromes. Br J Haematol 2002; 116:564-75. 75. Shin MG, Kajigaya S, Levin BC, et al. Mitochondrial DNA mutations in patients with myelodysplastic syndromes. Blood 2003; 101:3118-25. 76. Craven SE, French D, Ye W, et al. Loss of Hspa9b zebrafish recapitulates the ineffective hematopoiesis of the myelodysplastic syndrome. Blood 2005; 105:3528-
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Increased Apoptosis in Myelodysplastic Syndromes 34. 77. Hellström Lindberg E, Schmidt-Mende J, Forsblom AM, et al. Apoptosis in refractory anaemia with ring sideroblasts is initiated at the stem cell level and associated with increased activation of caspases. Br J Haematol 2001; 112:714-26. 78. Tehranchi R, Fadeel B, Forsblom AM, et al. Granulocyte colony-stimulating factor inhibits spontaneous cytochrome c release and mitochondria-dependent apoptosis of myelodysplastic syndrome hematopoietic progenitors. Blood 2003; 101:1080-6. 79. Hellström Lindberg E. Strategies for biology- and molecular-based treatment of myelodysplastic syndromes. Current Drug Targets 2005; 6:713-25. 80. Tehranchi R, Fadeel B, Schmidt-Mende J, et al. Antiapoptotic role of growth factors in the myelodysplastic syndromes: concordance between in vitro and in vivo observations. Clin Cancer Res 2005; 11:6291-9. 81. Levi S, Corsi B, Bosisio M, et al. A human mitochondrial ferritin encoded by an intronless gene. J Biol Chem 2001; 276:24437-40. 82. Levi S, Arosio P. Mitochondrial ferritin. Int J Biochem Cell Biol 2004; 36:1887-9. 83. Langlois d’Estaintot B, Santambrogio P, Granier T, et al. Crystal structure and biochemical properties of the human mitochondrial ferritin and its mutant Ser144Ala. J Mol Biol 2004; 340:277-93. 84. Corsi B, Cozzi A, Arosio P, et al. Human mitochondrial ferritin expressed in HeLa cells incorporates iron and affects cellular iron metabolism. J Biol Chem 2002; 277:22430-7. 85. Drysdale J, Arosio P, Invernizzi R, et al. Mitochondrial ferritin: a new player in iron metabolism. Blood Cells Mol Dis 2002; 29:376-83. 86. Napier I, Ponka P, Richardson DR. Iron trafficking in the mitochondrion: novel pathways revealed by disease. Blood 2005; 105:1867-74.
87. Nie G, Sheftel AD, Kim SF, et al. Overexpression of mitochondrial ferritin causes cytosolic iron depletion and changes cellular iron homeostasis. Blood 2005; 105:2161-7. 88. Alcindor T, Bridges KR. Sideroblastic anaemias. Br J Haematol 2002; 116:733-43. 89. Cazzola M, Invernizzi R, Bergamaschi G, et al. Mitochondrial ferritin expression in erythroid cells from patients with sideroblastic anemia. Blood 2003; 101:19962000. 90. Della Porta MG, Malcovati L, Invernizzi R, et al. Flow cytometry evaluation of erythroid dysplasia in patients with myelodysplastic syndrome. Leukemia 2006; 20:549-55. 91. Campanella A, Isaya G, O’Neill HA, et al. The expression of human mitochondrial ferritin rescues respiratory function in frataxin-deficient yeast. Hum Mol Genet 2004; 13:2279-88. 92. Tehranchi R, Invernizzi R, Grandien A, et al. Aberrant mitochondrial iron distribution and maturation arrest characterize early erythroid precursors in low-risk myelodysplastic syndromes. Blood 2005; 106:247-53. 93. Raza A, Alvi S, Borok RZ, et al. Excessive proliferation matched by excessive apoptosis in myelodysplastic syndromes: the cause-effect relationship. Leuk Lymph 1997; 27:111-8. 94. Lin CW, Manshouri T, Jilani I, et al. Proliferation and apoptosis in acute and chronic leukemias and myelodysplastic syndrome. Leuk Res 2002; 26:551-9. 95. Liesveld JL, Jordan CT, Phillips GL. The hematopoietic stem cell in myelodysplasia. Stem Cells 2004; 22:590-9. 96. Corey SJ, Minden MD, Barber DL, et al. Myelodysplastic syndromes: the complexity of stem-cell diseases. Nat Rev Cancer 2007; 7:118-29.
Bella Center Copenhagen, Denmark
13th CONGRESS of the
European Hematology Association (EHA) JUNE 12 - 15, 2008
The EHA Annual Congress is organized every June in a major European city. Over the years the congress has become the meeting place for hematologists in all fields of the specialty and now attracts more than 6,000 participants. The congress program includes sessions on clinical and laboratory hematology and covers all the major hematologic sub-specialities, including hemato-oncology, red cell disorders, hemostasis, thrombosis, pediatric hematology, and transfusion medicine. The topics range from stem cell physiology and development to leukemia, lymphoma and myeloma, myeloproliferative disorders and myelodysplasia, thrombocytopenias, thrombosis and bleeding disorders, hemoglobinopathies, transfusion, and stem cell transplantation. The program includes basic and translational science, clinical trials, diagnostic techniques, a major education program, and ethical and regulatory issues.
Introduction Apoptosis, or programmed cell death, discovered in 1972 by Kerr et al,1 is a morphologically and biochemically distinct form of cell death, genetically regulated, without inflammatory tissue injury. It is an active process of dying that requires energy and de novo gene expression. It is implicated in biologic processes ranging from embryogenesis to aging, from normal tissue homeostasis to many human diseases. Decreased or inhibited apoptosis is a feature of many malignancies. The molecular mechanisms involved in death signal, genetic regulation, and activation of effectors have been recently identified.2,3 Myelodysplastic syndromes (MDS) are clonal diseases of hematopoiesis characterized by reduced ability of the proliferating clone to differentiate and mature with a propensity to evolve into acute myeloid leukemia (AML). Premature apoptotic cell death within the bone marrow (BM) might explain the apparent paradox in MDS of persistent cytopenia despite normal or increased BM cellularity, and it is commonly considered the underlying cause of ineffective hematopoiesis.4-12 Department of Internal Medicine, University of Pavia, Fondazione IRCCS Policlinico S. Matteo, Pavia, Italy Submitted: October 30, 2007; Revised: December 10, 2007; Accepted: January 3, 2008 Address for correspondence: Rosangela Invernizzi, MD, Clinica Medica 3, Piazzale Golgi, 27100 Pavia, Italy Fax: 39-0382-526223; e-mail: [email protected] Electronic forwarding or copying is a violation of US and International Copyright Laws. Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by CIG Media Group, LP, ISSN #1931-6925, provided the appropriate fee is paid directly to Copyright Clearance Center, 222 Rosewood Drive, Danvers, MA 01923 USA 978-750-8400.
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Table 1
Apoptosis in Myelodysplastic Syndrome: Published Experience
Study Clark et al13 al5
Marrow Sample
Technique
Apoptotic Cells (%)
Biopsy
Morphology
20 Times controls
Biopsy
ISEL
q 75 in half of cases
Lepelley et al14
Aspirate
TUNEL
2.5-5 (Controls: < 2)
Rajapaksa et al15
Aspirate
FC
9 (CD34+) (controls: 2)
Bogdanovic et al16
Aspirate
Morphology (EM)
3 (Controls: 1)
Hellström et al17
Biopsy
TUNEL
56 (Controls: 16)
al18
Aspirate
TUNEL
39 (Controls: 13)
Parker et al19
Aspirate
FC (annexin V)
56.5 (CD34+) (controls: 18.5)
Tsoplou et al20
Aspirate
TUNEL
34% of Cases (controls: 0)
Shimazaki et al21
Biopsy
TUNEL
5.5 (Controls: 0.6)
Novitzky et al22
Aspirate
TUNEL
49.5
Raza et
Bouscary et
al23
Aspirate
FC (annexin V)
> 50 (CD34+) in RA, RARS, RAEB
Albitar et al24
Aspirate
FC (annexin V)
7
al25
Parker et
Biopsy
TUNEL
47 (Controls: 41)
Brada et al26
Biopsy
TUNEL
2.3 (Controls: 4.8)
Michalopoulou et al27
Aspirate
FC (annexin V)
20 (Controls: 7)
Invernizzi et al28
Aspirate
TUNEL
24 (Controls: 13)
Ramos et
Abbreviations: EM = electron microscopy; FC = flow cytometry; ISEL = in situ end labeling
This review will outline the role of the apoptotic pathways in the pathogenesis of MDS and suggest possible molecular mechanisms whereby apoptosis in MDS might be dysregulated.
Increased Apoptosis in Myelodysplastic Syndrome Findings suggestive of excessive apoptosis in MDS include defective in vitro growth pattern of marrow progenitors, high proportion of cells with DNA strand breaks or apoptotic nuclei engulfed by macrophages in marrow biopsies, increased apoptotic CD34+ cells with increased ratio of proapoptotic/antiapoptotic proteins, and increased levels of various apoptosis-related parameters in BM cells. Several works, using different techniques, have shown that MDS is associated with increased apoptosis of marrow cells. However, the percentages of patients with increased apoptosis and the mean percentage of apoptotic cells greatly varied from one study to another (Table 1).5,13-28 These variations can be, at least partially, explained by differences in the sample origin, French-American-British (FAB) subtype, time between sample procurement and analysis, and the sensitivity of the technique.29 With terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) technique, apoptotic cells show intense, diffuse, often dishomogeneous nuclear reactivity (Figure 1). Using this technique, we demonstrated significant higher apoptotic index in low-risk MDS than in AML; the apoptotic index was also higher than in high-risk MDS and than in normal controls, and it was inversely related to blast cell percentage.28-30
114
Figure 1
Bone Marrow Aspirate from a Patient with Refractory Anemia
Bone marrow aspirate from a patient with RA, using the TUNEL Technique. Apoptotic cells show strong nuclear reactivity (magnification ×1250).
In summary, the findings of various authors show that excessive apoptosis occurs in all MDS subtypes and affects all marrow lineages and also stromal cells; most reports agree that the highest apoptotic index occurs in refractory anemia (RA), RA with ring sideroblasts (RARS), and RA with excess of blasts (RAEB), there being a progressive decline in apoptosis as the disease evolves toward RAEB in transformation (RAEB-t) and secondary AML (sAML). The question of whether the excessive apoptosis in MDS predominantly involves the subset of progenitor stem cells or of more differentiated cells is still controversial. We used a double-labeling, 2-color technique that combined the TUNEL method and immunocytochemistry to assess the degree of apoptosis in MDS BM and its possible differences from normal marrow in relation to CD34 antigen expression. Our results suggest that in MDS as well as in normal BM, the apoptotic phenomenon predominantly involves the maturing cells.31 Some correlations between apoptosis levels and clinical and hematologic features were observed: inverse correlation with leukocyte count,16,32 International Prognostic Scoring System and cytogenetic risk group,23 prognosis,21 and response to aggressive chemotherapy.24 Patients showing a higher apoptotic rate had a poorer prognosis regardless of the FAB subtype, and patients who did not achieve complete remission after aggressive treatment had higher levels of apoptosis of CD34+ cells.
Molecular Mechanisms of Increased Apoptosis in Myelodysplastic Syndrome Various anomalies might be responsible for increased apoptosis in MDS (Table 2). Apoptosis is caspase dependent, but the question is whether this increased caspase activity is caused by extrinsic or intrinsic apoptotic stimuli.
The Role of the Extrinsic Pathway Details of the death receptor pathway of caspase activation are shown in Figure 2. Supporting the involvement of this pathway, MDS hematopoietic cells overexpress Fas and tumor necrosis factor
Clinical Leukemia • May 2008
Rosangela Invernizzi, Erica Travaglino
Table 2
Pathogenesis of Increased Apoptosis in Myelodysplastic Syndrome
Figure 2
The Extrinsic Apoptotic Pathway
• Increase in apoptosis-promoting and/or decrease in survivalpromoting cytokines • Activation of the Fas-FasL system
T Cell
• Intrinsic cell-cycle dysfunction • Abnormalities of apoptosis-related genes
Granzyme B
FasL TNF TRAIL
• Mitochondrial abnormalities Ligand Receptor
• Alteration of the hematopoietic microenvironment
(TNF)–α–related apoptosis-inducing ligand (TRAIL) death receptors as well as their ligands, but the source of the stimuli that increases death-receptor signaling is not entirely clear. Microenvironment cells, such as macrophages, stromal elements, or cytotoxic T cells, might produce excess death-receptor stimuli. Alternatively, the increased expression of ligands on the surface of hematopoietic cells might induce autocrine stimulation of the death receptor pathway.
DD
DcR
Nucleus
Perforin
Fas-R TNF-R TRAIL-R
cFLIP
FADD SODD
FADD
DED
NIK
IKB
Caspase 10 P
IKB NF-KB
Fas Signaling
BID
Mitochondrial Pathway
The interaction between Fas ligand (FasL) and Fas has been studied extensively in MDS. Increased Fas expression on marrow cells, including CD34+ progenitors, erythroblasts, and myeloid cells, has been reported in approximately 40% of patients,18,33-37 but no correlation between Fas expression and rate of apoptosis could be demonstrated. FasL is expressed on similar hematopoietic populations as Fas, including CD68+ macrophages, and its expression correlates with FAB subtype and anemia and predicts survival.38 Because Fas expression is elevated during all stages of erythroid differentiation/maturation and mononuclear cells from patients with MDS undergo increased apoptosis after treatment with activating antibody, it is likely that MDS erythroid cells exhibit an exacerbation of the physiologic mechanisms of Fas-mediated control of erythropoiesis and a Fas-dependent apoptosis, even in the presence of high erythropoietin levels.39 This suggestion is also supported by the results of in vitro studies demonstrating that lentivirus-mediated introduction of a construct encoding the adapter Fas-associated death domain (FADD) in CD34+ cells from patients with low-grade MDS increases apoptosis of erythroid cells and dramatically reduces burst forming unit erythroid (BFU-E) growth, whereas transduction of a dominant-negative mutant of FADD inhibits caspase-8 activity and apoptosis of erythroid precursors grown in liquid medium and rescues BFU-E growth without abrogating erythroid differentiation.40 Interestingly, Fas-triggered apoptosis in RARS BM cells is significantly improved by granulocyte colony-stimulating factor (G-CSF) treatment.41 The extrinsic Fas-mediated pathway of apoptosis seems important in regulating cell replication and death, especially in trisomy 8 hematopoietic cells.42 The immune system plays an active role in the pathogenesis of trisomy 8 syndrome: Besides upregulation of immune/inflammatory genes and downregulation of apoptosis-inhibiting genes in CD34+ cells,43 T-cell receptor skewing, highly suggestive of an antigen-driven process, has been observed.44 Upregulation of antiapoptotic proteins, such as survivin, c-Myc, and CD1, allows CD34+ cells from patients with trisomy 8 MDS to avoid apoptosis.45
DISC
Caspase 8
Caspase 8
Effector Caspases
Interaction between death ligands and their receptors leads to receptor trimerization, clustering of the adapter protein FADD or TRADD, and creation of a DISC. This recruits and activates initiator procaspase-8 through its DED. Caspase-8 directly activates effector caspases or triggers the mitochondrial pathway through Bid cleavage. Also the T-cell–mediated cell death is an example of extrinsic apoptotic pathway. The death-receptor pathway is negatively regulated by the nuclear transcription factor NF-KB. Abbreviations: DcR = decoy receptor; DD = death domain; DED = death effector domain; DISC = death-inducing signaling complex; FLIP = Fas-associated death domain–like interleukin-1B –converting enzyme-inhibitory protein; IKB = inhibitor of KB; NF-KB = nuclear factor–KB; NIK = NF-inducing kinase; SODD = silencer of death domain; TNF-R = TNF-receptor; TRADD = TNF receptor–associated death domain
TRAIL/TRAILR System There is also evidence that the TRAIL/TRAIL receptors (TRAILRs) system might play a role in the ineffective hemopoiesis of MDS, but it contributes to the myeloid rather than erythroid apoptosis.41 TRAIL and TRAILRs are expressed at increased levels in the BM of patients with MDS. Furthermore, MDS hematopoietic precursors are supranormally sensitive to TRAIL-mediated apoptosis.46,47 However, no mutations in the death domains of TRAILR genes is found.48 Myeloid nuclear differentiation antigen downregulation increases progenitor cell response to TRAILinduced apoptosis.49 With regard to clinical correlations, hemoglobin levels are lower in patients whose BM mononuclear cells release TRAIL in culture supernatants.50
Cytokines Several authors have reported increased levels of TNF-α and other inflammatory cytokines in serum and BM in MDS,33,51-53 and levels of TNF-α and interleukin (IL)–1β correlate with the degree of apoptosis.52,54 These cytokines might upregulate Fas and FasL expression in MDS colony-forming cells and accelerate progenitor death. Moreover, they might activate the p38 mitogen-activated protein kinase pathway to mediate signals for apoptosis.55,56 Tumor necrosis factor–α can induce apoptosis also by oxidation of DNA and proteins.57
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Increased Apoptosis in Myelodysplastic Syndromes
Figure 3
The Intrinsic Apoptotic Pathway Reduced Growth Factor
DNA damage
ROS
BH-3
tBid
Bcl-2
Bax
Bim
Nucleus
IAP
AIF
Omi
IAP
Endo G
smac
IAP
IAP IAP
Cytochrome C Caspase-9 Apaf-1 Cytochrome C Apaf-1
Caspase-3/7
Apoptotic substrates
Apoptosome
Activation of the mitochondrial pathway leads to a loss of mitochondrial membrane potential and release of several apoptogenic factors. A key event is the translocation of cytochrome C from the intermembrane space into cytosol. Released cytochrome C and Apaf-1 associate with procaspase-9 and adenosine triphosphate to form the apoptosome complex. Pro–caspase-9 becomes activated and triggers the proteolytic cascade, leading to apoptosis. Apoptosisinducing factor can directly bind and cleave DNA. Bcl-2 family members are the major regulators of mitochondrial membrane permeabilization and release of apoptogenic factors. Abbreviations: AIF = apoptosis-inducing factor; Apaf-1 = apoptotic protease-activating factor–1; Endo G = endonuclease G; IAP = inhibitor of apoptosis protein; ROS = reactive oxygen species; smac = second mitochondrial activator of caspases
On the other hand, in vivo treatment with TNF-α–blocking agents,58,59 anti–TNF-α antibody,60 and cytokines such as G-CSF and erythropoietin61,62 reduces apoptosis in responding patients. Also anti-inflammatory therapy is effective in patients with MDS with increased apoptosis of BM cells.22 The involvement of inflammatory cytokines in the pathogenesis of MDS is confirmed by the recent finding of Toll-like receptor 4 upregulation in MDS hematopoietic progenitor cells.63
Stromal Microenvironment The precise role of the stromal microenvironment in triggering MDS apoptosis is still largely unknown. Some authors have demonstrated that adherent cells grown from the biopsy of patients with MDS are able to support normal hematopoiesis,64 whereas others have shown that stromal growth of MDS marrow is impaired and is associated with a decreased capacity to support normal and myelodysplastic hematopoiesis65,66; moreover, the adherent layers in cultures grown from patients with MDS are hemopoietically defective and show abnormal IL-1β expression.67 On the other hand, increased numbers of stromal macrophages are the key regulators in the increased production of proapoptotic cytokines. Whether altered integrin-mediated focal contacts between MDS progenitors and marrow stroma are involved in apoptosis is at present unknown.
116
The Role of the Intrinsic Pathway There is evidence that, in MDS, in addition to the extrinsic apoptotic pathway, the intrinsic pathway is also activated.70 The hallmark of activation of the intrinsic pathway is the release of apoptogenic factors from mitochondria (Figure 3).
ATP
Caspase-9
With regard to angiogenesis, the overexpression of the angiogenic factor vascular endothelial growth factor along with its receptors has been demonstrated in MDS cells.68 Vascular endothelial growth factor production might stimulate angiogenesis that could favor disease progression by altering the microenvironment to favor unregulated cell growth. On the other hand, we observed by immunocytochemistry an abnormal expression of matrix metalloproteinases (MMP)–2 and –9 in both myeloid and erythroid MDS cells. Vascular endothelial growth factor and MMPs can stimulate endothelial cells and macrophages to produce inflammatory cytokines such as TNF-α that might reinforce FasL expression in erythroid cells and, as a consequence, ineffective erythropoiesis.69 Also, extracellular matrix degradation by MMPs might contribute to decreased erythroid adherence and increased apoptosis.
Mitochondrial Abnormalities Mitochondrial abnormalities are commonly seen in MDS, including reduced membrane potential in the erythroid subpopulations of RARS71 and in BM cells of all MDS subtypes and cytochrome C-oxidase gene mutations.72-75 A potential role for the mitochondria in MDS was suggested also by analysis of the crimsonless zebrafish mutant that develops an MDS as the mutation of a mitochondrial heat-shock protein (HSPA9B) produces oxidative stress and apoptosis distinctly in blood cells.76 The erythroid apoptosis of low-risk MDS is initiated at a very early stage of stem cells through a spontaneous cytochrome C release from the mitochondrial intermembrane space, with subsequent activation of caspase-9 and effector caspase-3.77 The role of the mitochondrial intrinsic pathway might be relevant especially in RARS, where G-CSF shows an antiapoptotic effect in vitro, being able to inhibit spontaneous release of cytochrome C, drop of mitochondrial membrane potential, caspase activation, and restore erythroid proliferation.78-80 In summary, in low-risk MDS, the increased sensitivity to triggering of death receptors is likely dependent on constitutive mitochondrial signaling. The factors that stimulate the mitochondrial pathway of apoptosis are unclear. In RARS, the mitochondrial pathway might be stimulated by increased iron deposition.
Mitochondrial Ferritin A novel human ferritin, which is encoded by an unusual intronless gene on chromosome 5q23.1 and has limited tissue expression, has been identified.81,82 The encoded protein is a 242–amino acid precursor with a 57–amino acid leader sequence for mitochondrial import. In mitochondria, this protein is processed to a subunit that assembles into functional shells that have a well-known crystallographic structure83 and incorporate iron as efficiently as H ferritin.84 This novel mitochondrial ferritin (FtMt) might play an important role in regulating mitochondrial iron homeostasis and heme synthesis. Mitochondrial ferritin expression might increase with mitochondrial iron loading, possibly through transcriptional mechanisms.85-87
Clinical Leukemia • May 2008
Rosangela Invernizzi, Erica Travaglino
Figure 4
Bone Marrow Aspirate from a Patient with RARS
A
Figure 5
Cytospin Samples of Erythroid Progenitors from a Patient with RARS
A
B B
CD34+ cells were separated from the BM mononuclear cells and cultured in a liquid medium according to a procedure that allows the expansion of high concentrations of erythroid progenitors in the presence of various cytokines and growth factors. (A) At day 11 of culture, most cells show morphologic features of proerythroblasts or basophilic erythroblasts (MayGrünwald-Giemsa stain). (B) Immunoalkaline phosphatase staining for mitochondrial ferritin shows unequivocal positivity in the same cells (magnification ×1250). (A) Perls staining showing positive granules forming a ring surrounding the nucleus in the erythroblasts. This peculiar stain is caused by mitochondrial iron overload. (B) Immunoalkaline phosphatase staining for mitochondrial ferritin showing its localization in perinuclear granules (magnification ×1250).
The massive increases in mitochondrial iron in conditions of defective heme synthesis led recently to examine the expression of the FtMt in BM erythroblasts from patients with sideroblastic anemia, a group of hereditary or acquired disorders characterized by ineffective erythropoiesis associated with iron accumulation in the mitochondria of immature red cells.88 We demonstrated that most of the iron deposited in perinuclear mitochondria of ring sideroblasts is present in the form of FtMt that can be detected by immunocytochemistry in granules ringing the nucleus (Figure 4). The close relationship between the number of FtMt-positive erythroblasts and that of ring sideroblasts suggests that FtMt might be a specific marker of sideroblastic anemia and that FtMt detection by immunocytochemistry or flow cytometry might be a useful tool in the work-up of patients with MDS.89,90
On the basis of preliminary findings showing that FtMt reduces TNF-α–induced apoptosis in transduced HeLa cells, we hypothesized that, functionally, FtMt might play an important role in regulating iron homeostasis and toxicity; it might protect mitochondria from oxidative damage and increase cell resistance to apoptotic signals.86,91 In an attempt to look for the potential pathogenetic role of FtMt in the ineffective erythropoiesis of RARS, we used an in vitro model to evaluate the distribution of FtMt in RARS erythroid progenitors.92 Mitochondrial ferritin expression occurs very early during RARS erythroid differentiation, in cells that are still CD34+ and without any visible iron accumulation (Figure 5). In very immature erythroblasts in RARS, FtMt expression is closely linked to mitochondrial damage in terms of release of cytochrome C. The strong antiapoptotic effect of G-CSF in RARS seems to be caused by upregulation of compensatory survival mechanisms rather than a direct effect on mitochondrial iron accumulation. We can suppose
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Increased Apoptosis in Myelodysplastic Syndromes
Table 3
Myelodysplastic Syndrome Disease Progression
Table 4
Controversial Issues
• What are the properties of the stem cell that produces MDS? • Telomere shortening that might contribute to genomic instability • Increased angiogenesis
• Why are MDS cells unusually susceptible to apoptosis?
• Defective apoptotic machinery (Fas signaling downregulation, p53 inactivation)
• Is increased apoptosis in MDS related to the molecular pathogenesis of the disease or is it only a consequence of ineffective hematopoiesis caused by genomic damage and microenvironmental changes?
• Upregulation of negative apoptosis regulators (Bcl-2 family members, IAP)
• How many undiscovered genes are involved in death decision and how will we find them?
• Constitutive activation of NF-KB
• Is apoptosis clinically important in MDS?
• Epigenetic changes such as p15INK4b
• Are there possible therapeutic implications?
Abbreviations: IAP = inhibitor of apoptosis protein; INK = kinase inhibitor; NF = nuclear factor
Figure 6
Pathogenetic Model of Myelodysplastic Syndrome Hypercellular marrow
Progenitor cell transformation
Monoclonal hematopoiesis Early event
Cytokines Abnormal microenvironment Immunologic response
Toxin exposure Spontaneous mutation
Apoptosis Variable cytopenia
enigmatic whether increased apoptosis in MDS is related to the molecular pathogenesis of the disease or it is only a consequence of ineffective hematopoiesis caused by genomic damage and microenvironmental changes. Moreover, the properties of the stem cell that produces MDS are still obscure.95,96 Elucidation of this fascinating enigma might be important also from the clinical point of view because it might lead to the development of novel therapeutic strategies.
Acknowledgements Rosangela Invernizzi, MD, is supported by a grant from the Ministry for Education, University and Research, Italy, and Erica Travaglino, MSc, is supported by a grant from Fondazione IRCCS Policlinico S. Matteo, Pavia, Italy.
References that the aberrant expression of FtMt might reflect a defense mechanism of the cell against the oxidative damage caused by excess iron, but further excessive iron accumulation might cause further damage to the mitochondria and trigger apoptosis. The pathogenesis of RA without ring sideroblasts seems to be more heterogeneous, with the involvement of alternative mechanisms not associated with iron overloading, including T-cell–mediated BM failure. Ongoing studies seem to confirm that abnormalities in FtMt expression in RARS progenitors might contribute to alter the balance between cell growth and cell death.
Myelodysplastic Syndrome Progression Early MDS is a highly proliferative disorder, but despite the increased cell proliferation, the marrow does not export sufficient cells into the blood because intramedullary apoptosis mechanism prevails over proliferation.23,28,93,94 Progression to RAEB-t/sAML is associated with a significant fall in proliferation in most cases. Disease progression has been presumed to be the result of chromosomal lesions and genetic mutations, some affecting cell proliferation and others conferring resistance to apoptosis (Table 3).
Controversial Issues Whatever the cause or mechanism leading to apoptosis, it is now generally accepted that increased intramedullary apoptosis is responsible for the ineffective hematopoiesis in MDS (Figure 6). However, some controversial issues remain (Table 4). In particular, it is
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Bella Center Copenhagen, Denmark
13th CONGRESS of the
European Hematology Association (EHA) JUNE 12 - 15, 2008
The EHA Annual Congress is organized every June in a major European city. Over the years the congress has become the meeting place for hematologists in all fields of the specialty and now attracts more than 6,000 participants. The congress program includes sessions on clinical and laboratory hematology and covers all the major hematologic sub-specialities, including hemato-oncology, red cell disorders, hemostasis, thrombosis, pediatric hematology, and transfusion medicine. The topics range from stem cell physiology and development to leukemia, lymphoma and myeloma, myeloproliferative disorders and myelodysplasia, thrombocytopenias, thrombosis and bleeding disorders, hemoglobinopathies, transfusion, and stem cell transplantation. The program includes basic and translational science, clinical trials, diagnostic techniques, a major education program, and ethical and regulatory issues.