Enhanced activation of autophagy in β-thalassemia/Hb E erythroblasts during erythropoiesis

Erythropoiesis in β-thalassemia patients is ineffective, primarily because of death of the erythroid progenitor cells at the polychromatic normoblast stage. While it is known that autophagy plays a critical role during erythropoiesis by removing organelles from erythroid cells during terminal differentiation, its role in erythroid cells whose function is impaired remains to be explored. To investigate this, CD34+ erythroid progenitor cells from normal controls and β-thalassemia/Hb E patients were isolated from peripheral blood and cultured under conditions driving differentiation into an erythroid lineage, and levels of autophagy and apoptosis were analyzed both directly and after biochemical manipulation with l-asparagine. A significantly higher level of autophagy was seen in β-thalassemia/Hb E erythroblasts as compared to normal control erythroblasts during erythropoiesis. Interestingly, this activation was mediated in part by the presence of high levels of Ca²⁺ as modulation of Ca²⁺ levels significantly reduced the level of autophagy in these cells. Inhibition of autophagic flux in normal erythroblasts significantly increased apoptosis in normal erythroblasts, but not in thalassemic erythroblasts, although sensitivity to autophagic flux inhibition was restored by reduction of Ca²⁺ levels. These results suggest that high levels of autophagy in β-thalassemia/HbE erythroblasts may contribute to the increased levels of apoptosis that lead to ineffective erythropoiesis in β-thalassemia/Hb E erythroblasts.     

Expression of microRNA-451 in normal and thalassemic erythropoiesis

MicroRNAs (miRNAs) are negative regulators of gene expression that play an important role in hematopoiesis. Thalassemia, a defective globin synthesis leading to precipitate of excess unbound globins in red blood cell precursors, results in defective erythroid precursors and ineffective erythropoiesis. Expression pattern of miR-451, an erythroid-specific miRNA, was analyzed during differentiation of erythroid progenitors derived from normal and thalassemic peripheral blood CD34-positive cells, after 14 days of culture. A biphasic expression with transient up-regulation of miRNA-451 on day 3 of cultures was observed during thalassemic erythroid differentiation. In contrast, the expression pattern of the miR-451 in erythroid cells obtained from the other extravascular hemolytic anemia, i.e., hereditary spherocytosis patients showed no transient up-regulation of miR-451 on day 3 of cultures. Our results suggest that early erythroid progenitors in β-thalassemia have a dysregulated miRNA-451 expression program, and analysis of microRNA is a relevant approach to determine abnormalities of erythropoiesis.     

Imbalanced globin chain synthesis determines erythroid cell pathology in thalassemic mice

Background

β-thalassemia occurs from the imbalanced globin chain synthesis due to the absence or inadequate β-globin chain production. The excessive unbound α-globin chains precipitate in erythroid precursors and mature red blood cells leading to ineffective erythropoiesis and hemolysis.

Design and Methods

In vitro globin chain synthesis in reticulocytes from different types of thalassemic mice was performed. The effect of imbalanced globin chain synthesis was assessed from changes of red blood cell properties including increased numbers of red blood cells vesicles and apoptotic red blood cells, increased reactive oxygen species and decreased red blood cell survival.

Results

The α/β-globin chain ratio in β^IVSII-654-thalassemic mice, 1.26±0.03, was significantly higher than that of wild type mice, 0.96±0.05. The thalassemic mice show abnormal hematologic data and defective red blood cell properties. These values were improved significantly in doubly heterozygous thalassemic mice harboring 4 copies of human β^E-globin transgene, with a more balanced globin chain synthesis, 0.92±0.05. Moreover, transgenic mice harboring 8 extra copies of the human β^E-globin transgene showed inversely imbalanced α/β-globin synthesis ratio, 0.83±0.01, that resulted in a mild β-thalassemia phenotype due to the excessive β-globin chains. The degree of ineffective erythropoiesis also correlated with the degree of imbalanced globin chain synthesis. Bone marrow and splenic erythroid precursor cells of β^IVSII-654-thalassemic mice showed increased phosphatidylserine exposure in basophilic and polychromatophilic stages, which was restored to the normal level in doubly heterozygous mice.

Conclusions

Imbalanced α/β-globin chain as a consequence of either reduction or enhancement of β-globin chain synthesis can cause abnormal red blood cell properties in mouse models.     

A dual activation and inhibition role for the paired immunoglobulin-like receptor B in eosinophils

The accumulation of eosinophils in inflammatory foci is a hallmark characteristic of Th2 inflammation. Nevertheless, the expression of inhibitory receptors such as paired immunoglobulin-like receptor B (PIR-B) and their function regulating eosinophil accumulation have received limited attention. We now report that Pirb was up-regulated in an eosinophil-dependent manner in the lungs of allergen-challenged and interleukin (IL)-13–overexpressing mice. Eosinophils expressed high levels of PIR-B, and Pirb^–/– mice displayed increased gastrointestinal eosinophils. Consistent with these findings, PIR-B negatively regulated eotaxin-dependent eosinophil chemotaxis in vivo and in vitro. Surprisingly, Pirb^–/– eosinophils and neutrophils had decreased leukotriene B4 (LTB₄)–dependent chemotactic responses in vitro. Furthermore, eosinophil accumulation was decreased in a chitin-induced model, partially dependent on LTB₄. Mechanistic analysis using a miniphosphoproteomic approach revealed that PIR-B recruits activating kinases after LTB₄ but not eotaxin stimulation. Consequently, eotaxin-activated Pirb^–/– eosinophils displayed markedly increased extracellular signal-related kinase 1 and 2 (ERK1/2) phosphorylation, whereas LTB₄-activated eosinophils had reduced ERK1/2 phosphorylation. We provide multiple lines of evidence supporting a model in which PIR-B displays opposing but potent regulatory functions in granulocyte activation. These data change the conventional wisdom that inhibitory receptors are restricted to inhibitory signals; we therefore propose that a single receptor can have dual functionality in distinct cell types after unique cellular signals.     

Recombinant AAV2-mediated β-globin expression in human fetal hematopoietic cells from the aborted fetuses with β-thalassemia major

Genetic correction of autologous hematopoietic stem cells has been proposed as an attractive treatment method for β-thalassemia. Our previous study has shown that recombinant adeno-associated virus 2 (rAAV2) efficiently transduces human fetal liver hematopoietic cells, and mediates the expression of the human β-globin gene in vivo. In this study, we investigated whether rAAV2 could also mediate the expression of normal β-globin gene in human hematopoietic cells from β-thalassemia patients. Human hematopoietic cells were isolated from aborted β-thalassemia major fetuses, transduced with rAAV2-β-globin, and then transplanted into nude mice. We found that rAAV2-β-globin transduced human fetal hematopoietic cells, as determined by allele-specific PCR analysis. Furthermore, β-globin transgene expression was detected in human hematopoietic cells up to 70 days post-transplantation in the recipient mice. High-pressure liquid chromatography analysis showed that human β-globin expression levels increased significantly compared with control, as indicated by a 1.2–2.8-fold increase in the ratio of β/α-globin chain. These novel data demonstrate that rAAV2 can transduce and mediate the normal β-globin gene expression in fetal hematopoietic cells from β-thalassemia patients. Our findings further support the potential use of rAAV-based gene therapy in the treatment of human β-thalassemia.     

Combined effect of all-trans retinoic acid and arsenic trioxide in acute promyelocytic leukemia cells in vitro and in vivo

All-trans retinoic acid (tRA) and arsenic trioxide(As₂O₃) induce non-cross-resistant completeclinical remission in patients with acute promyelocytic leukemia witht(15;17) translocation and target PML-RAR, the leukemogenic protein,by different pathways suggesting a possible therapeutic synergism. Toevaluate this possibility, this study examined the effect ofAs₂O₃ on tRA-induced differentiation and,conversely, the effect of tRA on As₂O₃-induced apoptosis. As₂O₃ at subapoptotic concentrations(0.5 µM) decreased tRA-induced differentiation in NB4 cells butsynergized with atRA to induce differentiation in tRA-resistant NB4subclones MR-2 and R4 cells as measured by nitrobluetetrazolium reduction and tRA-inducible genes(TTGII, RAR, RIG-E). tRA cleaved PML-RAR into distinct fragments in NB4 but not in tRA-resistant MR-2 or R4cells, whereas As₂O₃ completely degradedPML-RAR in all 3 cell lines. As₂O₃-inducedapoptosis was decreased by tRA pretreatment of NB4 cells but not of R4cells and was associated with a strong induction of Bfl-1/A1expression, a Bcl-2 protein family member. Severe combinedimmunodeficient mice bearing NB4 cells showed an additive survivaleffect after sequential treatment, but a toxic effect was observedafter simultaneous treatment with tRA andAs₂O₃. These data suggest that combinedAs₂O₃ and tRA treatment may be more effectivethan single agents in tRA-resistant patients. Although in vitro data donot always translate to in vivo response, toxicity and potential drugantagonism may be diminished by decreasing the concentration ofAs₂O₃ when given at the same time withtherapeutic levels of tRA.     

Red Cells Shrink During Sickling

Rapid transformation of discoid hemoglobin SS erythrocytes into grossly distorted sickled forms suggests alteration ofsurface/volume ratio. We, therefore, examined the effect of deoxygenation on redcell volume. ¹³¹I-labeled albumin wasused as a marker of plasma volume innormal (Hb AA), sickle (Hb SS), andsickle-C (Hb SC) blood. Heparinized bloodwas incubated for 30 min at 37°C in atonometer under deoxy (90% N₂, 10%CO₂) or oxy (90% O₂, 10% CO₂) atmospheres. Whole blood and plasma concentrations of radioactivity were determined.The hematocrits thus obtained with¹³¹I-albumin showed that deoxygenationcaused: (1) Hb AA cells to swell (meanMCV +4.0 cu µ ± 3.0, p < 0.01), as ispredicted by the Bohr effect; (2) Hb SScells to shrink (mean MCV -9.4 cu µ ±2.9, p < 0.001); Hb SC cells to shrink(mean MCV -4.1 cu µ ± 0.8, p < 0.02).We also measured size distributions ofglutaraldehyde-fixed oxy and deoxy cellsusing a Coulter Model B counter. Thismethod confirmed the results of the radioalbumin experiments. These results demonstrate loss of cell volume during sicklingof intact erythrocytes. Possible mechanismsfor this loss include potassium efflux withobligatory water efflux, increased intracellular hydrostatic pressure from deformation of the cell membrane, or loss of intracellular osmotic activity of hemoglobinsecondary to polymerization. This dehydration enables the sickled cell to developmore extreme shape distortion withoutmembrane disruption, may facilitate continued hemoglobin polymerization, andmay play a role in the formation of "irreversibly" sickled cells.

Submitted on May 16, 1974 Accepted on June 24, 1974     

Short-term BMP-4 treatment initiates mesoderm induction in human embryonic stem cells

Human embryonic stem cells (hES cells) have unlimited self-renewal capacity and can differentiate into most, if not all, possible cell types. This unique property makes them valuable not only for investigation of early developmental processes, but also for regenerative medicine. Mesoderm-derived cardiac cells and hematopoietic cells both have the potential for various therapeutic applications. However, efficient induction of hES cell differentiation into mesoderm remains a challenge. Here, we showed that treatment of hES cells with bone morphogenetic protein-4 (BMP-4) exhibited differential effects: long-term treatment results in trophoblast and extra-embryonic endoderm differentiation, whereas short-term treatment can promote early mesoderm induction. The induction of mesoderm in hES cells occurs at a high efficiency as measured using several markers, such as Brachyury, WNT3, and MIXL1 expression. Moreover, these mesoderm progenitor cells can differentiate into cardiac and hematopoietic lineages in vitro. Further analysis showed that the mesoderm-inducing capacity of BMP-4 requires endogenous FGF and TGF-β/Nodal/activin signaling activities. Thus, our results uncover a novel role for BMP-4 in regulation of hES cell differentiation and should provide insights into the mechanism of mesoderm induction in hES cells.     

Core binding factors are necessary for natural killer cell development and cooperate with Notch signaling during T-cell specification

CBFβ is the non-DNA binding subunit of the core binding factors (CBFs). Mice with reduced CBFβ levels display profound, early defects in T-cell but not B-cell development. Here we show that CBFβ is also required at very early stages of natural killer (NK)–cell development. We also demonstrate that T-cell development aborts during specification, as the expression of Gata3 and Tcf7, which encode key regulators of T lineage specification, is substantially reduced, as are functional thymic progenitors. Constitutively active Notch or IL-7 signaling cannot restore T-cell expansion or differentiation of CBFβ insufficient cells, nor can overexpression of Runx1 or CBFβ overcome a lack of Notch signaling. Therefore, the ability of the prethymic cell to respond appropriately to Notch is dependent on CBFβ, and both signals converge to activate the T-cell developmental program.     

CELL CYCLE EFFECTS OF THE ANTI-RHEUMATIC AGENT CPH82

The benzylidated podophyllotoxin glycoside CPH82, a potentiallyuseful drug for treatment of RA, was tested in vitro on ninehuman haematopoietic cell lines for cell kinetic effects. Previousstudies have shown CPH82 to behave like a colchinetype ‘metaphase’blocker.The distribution of cells within different cell cyclecompartments (G₁, S, G₂ and M) was analysed by a novel methodusing dual parameter flow cytometric analysis of stage specificantigens (proliferating cell nuclear antigen and Ki-67). WithCPH82 concentrations chosen to mimic clinical conditions, eightout of nine lines showed an accumulation of cells in the G₂phase of the cell cycle. In many lines a delayed progress throughS seemed to occur. Three lines were blocked in both G₁ and G₂,whereas the major effect on one line (HL-60) was an accumulationof cells in the G₁ phase. Progression of M cells seemed onlyslightly delayed for some cell lines. In comparison with tworelated ‘metaphase’ blocking agents (podophyllotoxinand taxol), CPH82 had a different and dose-dependent patternof cell cycle retardation. It is speculated that the cell kineticaction of CPH82 might give insight into the question why it,unlike other ‘metaphase’ blockers, has proved valuablein the treatment of RA. KEY WORDS: Microtubule antagonist, Cell cycle analysis, Podophyllotoxin, Taxol, Proliferating cell nuclear antigen, Ki-67, Flow cytometry     

Integrin alpha E(CD103)beta 7 influences cellular shape and motility in a ligand-dependent fashion

While the extravasation cascade of lymphocytes is well characterized, data on their intraepithelial positioning and morphology are scant. However, the latter process is presumably crucial for many immune functions. Integrin _E(CD103)β₇ has previously been implicated in epithelial retention of some T cells through binding to E-cadherin. Our current data suggest that _E(CD103)β₇ also determines shape and motility of some lymphocytes. Time-lapse microscopy showed that wild-type _E(CD103)β₇ conferred the ability to form cell protrusions/filopodia and to move in an amoeboid fashion on E-cadherin, an activity that was abrogated by _E(CD103)β₇-directed antibodies or cytochalasin D. The _E-dependent motility was further increased (P < .001) when point-mutated _E(CD103) locked in a constitutively active conformation was expressed. Moreover, different yellow fluorescent protein–coupled _E(CD103) species demonstrated that the number and length of filopodia extended toward purified E-cadherin, cocultured keratinocytes, cryostat-cut skin sections, or epidermal sheets depended on functional _E(CD103). The in vivo relevance of these findings was demonstrated by wild-type dendritic epidermal T cells (DETCs), which showed significantly more dendrites and spanned larger epidermal areas as compared with DETCs of _E(CD103)-deficient mice (P < .001). Thus, integrin _E(CD103)β₇ is not only involved in epithelial retention, but also in shaping and proper intraepithelial morphogenesis of some leukocytes.     

Relationship of Erythropoietin Effectiveness to the Generative Cycle of Erythroid Precursor Cell

Hydroxyurea, a cytotoxic agent that kills cells in DNA synthesis, was usedto study the relationship between erythropoietin and the generative cycle of theimmediate erythroid precursor cell. When OHU and EP were administeredsimultaneously to hypertransfused mice, the resultant erythroid response wasdiminished relative to EP treated controls. OHU given at intervals after EPresulted in a progressively greater diminution of erythroid response.

From these studies, then, we would suggest that in the suppressed animal thecommitted stem cell compartment is in cycle but with a prolonged G₁.After EP there is a shortening of the generation time and an increase in therate of turnover of the committed stem cells. The data also indicate that cellsin cycle are differentiated into the pronormoblast compartment. It furthermay be suggested that erythropoietin is effective throughout the bulk of thegenerative cycle although it seems unlikely that differentiation is accomplishedduring the mitotic phase. Whether erythropoietin must be present in both G₁and S as suggested by Kretchmar cannot be answered by the present studies.The data also indicate that cells of the pluripotential compartment are normallyin G₀ or perhaps a prolonged G₁. Damage to the committed compartment appears to be in part repaired by the influx of cells from the pluripotentialcompartment.

Submitted on July 9, 1969 Accepted on September 8, 1969     

Prevalence of thrombotic risk factors among β-thalassemia patients from Western Iran

Background There is evidence for increased risk of thrombosis in patients with β-thalassemia intermedia and β-thalassemia major. The present study investigated the prevalence of thromboembolic risk factors of prothrombin G20210A, factor V Leiden G1691A and methylentetrahydrofolate reductase (MTHFR) C677T, as well as the hematological and clinical profiles in β-thalassemia major and intermedia patients from Western Iran. Methods Patients consisted of 158 β-thalassemia patients, 151 β-thalassemia major and 7 β-thalassemia intermedia patients, including 82 males and 76 females aged 13.6 ± 6.3 years. The control group were 180 healthy blood donors and school students, consisting of 103 males and 77 females aged 16.8 ± 2.1. Genotyping was done by PCR-RFLP using Mnl I, Hind III and Hinf I for factor V Leiden and prothrombin G20210A and MTHFR, respectively. Results The prevalence of prothrombin G20210A variant in patients and healthy individuals were 1.3 and 3.3%, respectively. Factor V Leiden G1691A was insignificantly higher in β-thalassemia patients (prevalence 5.7% and allele frequency 3.2%) compared to healthy individuals (2.8%). This mutation was found in eight β-thalassemia major (5.3%) and one β-thalassemia intermedia (14.3%) patients. The prevalence of MTHFR C677T polymorphism was slightly higher in patients (50%) compared to healthy individuals (48.3%). Around 71% of β-thalassemia intermedia and 38.4% of β-thalassemia major patients had undergone splenectomy. In β-thalassemia major patients, 5.3% had insulin dependent diabetes mellitus (IDDM) and 6.6% had HCV antibodies. All patients with IDDM were splenectomized and in one of them the prothrombin G20210A variant was found. Two patients, a 7-year-old boy with β-thalassemia intermedia receiving regularly blood transfusion and a β-thalassemia major patient (a 22-year-old splenectomized female), were found to be homozygous for MTHFR 677TT and heterozygous for factor V Leiden G1691A. Double heterozygosity for factor V Leiden G1691A and MTHFR C677T and also homozygous factor V Leiden 1691AA were found in two β-thalassemia major patients. No thromboembolic event has been recorded in the files of patients. Conclusions The results of present study establish the prevalence of biological risk factors of thrombosis in β-thalassemia patients from Western Iran. It seems that thrombophilic mutations may not be associated with thrombotic events in thalassemic patients, which needs to be confirmed by the study of larger sample sizes.     

Fetal hemoglobin chemical inducers for treatment of hemoglobinopathies

The switch from fetal (^Gγ and ^Aγ) to adult (β and δ) globin gene expression occurs at birth, leading to the gradual replacement of HbF with HbA. Genetic regulation of this switch has been studied for decades, and the molecular mechanisms underlying this developmental change in gene expression have been in part elucidated. The understanding of the developmental regulation of γ-globin gene expression was paralleled by the identification of a series of chemical compounds able to reactivate HbF synthesis in vitro and in vivo in adult erythroid cells. Reactivation of HbF expression is an important therapeutic option in patients with hemoglobin disorders, such as sickle cell anemia and β-thalassemia. These HbF inducers can be grouped in several classes based on their chemical structures and mechanisms of action. Clinical studies with some of these agents have shown that they were effective, in a part of patients, in ameliorating the clinical condition. The increase in HbF in response to these drugs varies among patients with β-thalassemia and sickle cell disease due to individual genetic determinants.     

Cul4A is required for hematopoietic cell viability and its deficiency leads to apoptosis

In vitro studies indicate that Cul4A ubiquitin ligases target for ubiquitin-mediated proteolysis regulators of cell-cycle progression, apoptosis, development, and DNA repair. In hematopoietic cell lines, studies by our group and others showed that Cul4A ligases regulate proliferation and differentiation in maturing myeloid and erythroid cells. In vivo, Cul4A-deficient embryos die in utero. Cul4A haploinsufficient mice are viable but have fewer erythroid and primitive myeloid progenitors. Yet, little more is known about Cul4A function in vivo. To examine Cul4A function in adults, we generated mice with interferon-inducible deletion of Cul4A. Cul4A deficiency resulted in DNA damage and apoptosis of rapidly dividing cells, and mutant mice died within 3 to 10 days after induction with dramatic atrophy of the intestinal villi, bone marrow, and spleen, and with hematopoietic failure. Cul4A deletion in vivo specifically increased cellular levels of the Cul4A ligase targets Cdt1 and p27^Kip1 but not other known targets. Bone marrow transplantation studies with Cul4A deletion in engrafted cells specifically isolated analysis of Cul4A function to hematopoietic cells and resulted in hematopoietic failure. These recipients died within 9 to 11 days, demonstrating that in hematopoietic cells, Cul4A is essential for survival.     

TG101209, a novel JAK2 inhibitor,has significant in vitro activity in multiple myeloma and displays preferential cytotoxicity for CD45+ myeloma cells

Interaction of myeloma cells with the bone marrow microenvironment is mediated in large part through different cytokines, especially VEGF and IL6. These cytokines, especially IL6, leads to upregulation of the JAK/STAT pathway in myeloma cell, contributing to increased proliferation, decreased apoptosis, and acquired drug resistance. Here, we examined the preclinical activity of a novel JAK2 inhibitor TG101209. TG101209 induced dose‐ and time‐dependent cytotoxicity in a variety of multiple myeloma (MM) cell lines. The induction of cytotoxicity was associated with inhibition of cell cycle progression and induction of apoptosis in myeloma cell lines and patient‐derived plasma cells. Evaluation of U266 cell lines and patient cells, which have a mix of CD45 positive and negative cells, demonstrated more profound cytotoxicity and antiproliferative activity of the drug on the CD45+ population relative to the CD45? cells. Exploring the mechanism of action of TG101209 indicated downregulation of pJak2, pStat3, and Bcl‐xl levels with upregulation of pErk and pAkt levels indicating cross talk between signaling pathways. TG101209, when used in combination with the PI3K inhibitor LY294002, demonstrated synergistic cytotoxicity against myeloma cells. Our results provide the rationale for clinical evaluation of TG101209 alone or in combination with PI3K/Akt inhibitors in MM. Am. J. Hematol., 2010. © 2010 Wiley‐Liss, Inc.     

Distinct roles for long-term hematopoietic stem cells and erythroid precursor cells in a murine model of Jak2V617F-mediated polycythemia vera

In the current model of the pathogenesis of polycythemia vera (PV), the JAK2V617F mutation arises in hematopoietic stem cells (HSCs) that maintain the disease, while erythroid precursor populations expand, resulting in excessive red blood cell production. We examined the role of these specific cell populations using a conditional Jak2V617F knockin murine model. We demonstrate that the most immature long-term (LT) HSCs are solely responsible for initiating and maintaining the disease in vivo and that Jak2V617F mutant LT-HSCs dominate hematopoiesis over time. When we induced Jak2V617F expression in erythropoietin receptor expressing precursor cells, the mice developed elevated hematocrit, expanded erythroid precursors, and suppressed erythropoietin levels. However, the disease phenotype was significantly attenuated compared with mice expressing Jak2V617F in LT-HSCs. In addition to developing a PV phenotype, all mice transplanted with Jak2V617F LT-HSCs underwent myelofibrotic transformation over time. These findings recapitulate the development of post-PV myelofibrosis in human myeloproliferative neoplasms. In aggregate, these results demonstrate the distinct roles of LT-HSCs and erythroid precursors in the pathogenesis of PV.     

Constitutive expression of IL-12R beta 2 on human multiple myeloma cells delineates a novel therapeutic target

The interleukin-12 (IL-12) receptor (R) B2 gene acts as tumor suppressor in human acute and chronic B-cell leukemias/lymphomas and IL-12rb2–deficient mice develop spontaneously localized plasmacytomas. With this background, we investigated the role of IL-12Rβ2 in multiple myeloma (MM) pathogenesis. Here we show the following: (1) IL-12Rβ2 was expressed in primary MM cells but down-regulated compared with normal polyclonal plasmablastic cells and plasma cells (PCs). IL-6 dampened IL-12Rβ2 expression on polyclonal plasmablastic cells and MM cells. (2) IL-12 reduced the proangiogenic activity of primary MM cells in vitro and decreased significantly (P = .001) the tumorigenicity of the NCI-H929 cell line in SCID/NOD mice by inhibiting cell proliferation and angiogenesis. The latter phenomenon was found to depend on abolished expression of a wide panel of proangiogenic genes and up-regulated expression of the antiangiogenic genes IFN-, IFN-, platelet factor-4, and TIMP-2. Inhibition of the angiogenic potential of primary MM cells was related to down-regulated expression of the proangiogenic genes CCL11, vascular endothelial-cadherin, CD13, and AKT and to up-regulation of an IFN-–related antiangiogenic pathway. Thus, IL-12Rβ2 directly restrains MM cell growth, and targeting of IL-12 to tumor cells holds promise as new therapeutic strategy.     

Increase in γ-globin mRNA content in human erythroid cells treated with angelicin analogs

The aim of the present study was to identify molecular analogs of angelicin (ANG) able to increase erythroid differentiation of K562 cells and expression of γ-globin genes in human erythroid precursor cells, with low effects on apoptosis. ANG-like molecules are well-known photosensitizers largely used for their antiproliferative activity in the treatment of different skin diseases (i.e., psoriasis, vitiligo, eczema, and mycosis fungoides). To verify the activity of these derivatives, we employed three experimental cell systems: (1) the human leukemic K562 cell line, (2) K562 cell clones stably transfected with a pCCL construct carrying green-EGFP under the γ-globin gene promoter, and (3) the two-phase liquid culture of human erythroid progenitors isolated from normal donors and β-thalassemia patients. The results of our study suggest that trimethyl ANG is a powerful inducer of erythroid differentiation, compared with known inducers, such as ANG, cytosine arabinoside, mithramycin, and cisplatin. These data could have practical relevance, because pharmacologically mediated regulation of human γ-globin gene expression, with the consequent induction of fetal hemoglobin, is considered a potential therapeutic approach in hematological disorders including β-thalassemia and sickle cell anemia.