Human Megakaryocytic Leukemia Cell Line Research Articles

Gestodene, a novel positive allosteric modulator of PAR1, enhances PAR1-mediated human platelet aggregation.

Background: Protease-activated receptor 1 (PAR1) is expressed in human platelets and can be activated by low concentrations of thrombin. Vorapaxar, a selective antagonist of PAR1, inhibits thrombin-induced calcium mobilization in human platelet, which is associated with an increased risk of bleeding. Conversely, the administration of a positive allosteric modulator (PAM) of PAR1 may pose a substantial risk of thrombosis due to inducing excessive platelet activation. In this study, we discovered a novel PAM of PAR1 and investigated the effect of enhanced PAR1 activation by PAM of PAR1 on platelet activation. Methods: To find PAMs of PAR1, a cell-based screen was performed in HT29 cells, and finally, gestodene, an oral contraceptive drug (OC), was identified as a novel PAM of PAR1. The mechanism of action of gestodene and its effects on platelet activation were investigated in human megakaryocytic leukemia cell line MEG-01 cells and human platelet. Results: Gestodene enhanced both thrombin- and PAR1-activating peptide (AP)-induced intracellular calcium levels in a dose-dependent manner without altering PAR2 and PAR4 activity. Gestodene significantly increased PAR1-AP-induced internalization of PAR1 and phosphorylation of ERK1/2, and the enhancing effects were significantly blocked by vorapaxar. Furthermore, gestodene potently increased PAR1-AP induced morphological changes in MEG-01 cells. Remarkably, in human blood, gestodene exerted a robust augmentation of PAR1-AP-induced platelet aggregation, and vorapaxar effectively attenuated the gestodene-induced enhancement of platelet aggregation mediated by PAR1. Conclusion: Gestodene is a selective PAM of PAR1 and suggest one possible mechanism for the increased risk of venous thromboembolism associated with OCs containing gestodene.

Apoptosis of Megakaryocytic Leukemia Cell Line Meg-01 Induced by TSP-1 Via CD36/Caspase-3

To investigate the effect of thrombospondin-1 (TSP-1) on apoptosis of human megakaryocytic leukemia cell line Meg-01 and its possible mechanism. The expression of CD36 antigen in Meg-01 cells was detected by flow cytometry and immunocytochemistry. Meg-01 cells were cultured for 48 hours with TSP-1 and CD36 antibody FA6-152 at different concentrations. The early apoptosis and activity of caspase-3 were detected by flow cytometry. The effect of TSP-1 on the growth and differentiation of megakaryocytes was investigated by cell counting and CFU-MK culture. The flow cytometry and immunocytochemistry showed that CD36 antigen was expressed on the surface of Meg-01 cells. TSP-1 (5 μg/ml) inhibited the growth of Meg-01 cells, but had unobvious effect on M-07e cells. After addition of CD36 antibody FA6-152 (5, 10, and 25 μg/ml), the inhibition effect of TSP-1 was significantly reduced. TSP-1 (2.5, 5, and 7.5 μg/ml) increased the positive expression of Annexin V (P<0.01) and caspase-3 activity (P<0.01), which indicated that TSP-1 had a significant effect on inducing apoptosis. After addition of CD36 antibody FA6-152 (25 μg/ml), the apoptosis induced by TSP-1 in Meg-01 cells was significantly reduced. TSP-1 (5, 10, and 25 μg/ml) could significantly inhibit the formation of CFU-MK in mouse bone marrow cells, while β-TG could not. CD36 antibody FA6-152 (25 μg/ml) could significantly reduce the inhibition of TSP-1 on CFU-MK. TSP-1 may induce apoptosis of megakaryocytic leukemia cell line Meg-01 cells via CD36/caspase-3, which provides a potential new drug development and treatment target for clinical treatment of megakaryocytic leukemia.

Effect of Thrombospondin-1 on Apoptosis of Human Megakaryocytic Leukemia Cells

Background: Thrombospondin 1 (TSP-1) is an extracellular matrix protein that interacts with a wide array of ligands including cell receptors, growth factors, cytokines, and proteases to regulate various physiological and pathological processes. TSP-1 induces apoptosis of endothelial and cancer cells via its receptor CD36. This study was to investigate the effect of TSP-1 on apoptosis of human megakaryocytic leukemia cell line Meg-01 and its possible mechanism.Methods: The expression of CD36 antigen in Meg-01 cells was detected by flow cytometry and immunocytochemistry. Meg-01 cells were cultured for 48 hours with TSP-1 and CD36 antibody FA6-152 at different concentrations, to investigate the effect of TSP-1 on the growth of megakaryocytes. The early apoptosis and the activity of the apoptotic protease caspase-3 were detected by flow cytometry. Bone marrow cells of mice were cultured for CFU-MK and stained with acetylcholine to detect the differentiation of megakaryocytes.Results: CD36 antigen was detected on the surface of Meg-01 cells by flow cytometry and immunocytochemistry. TSP-1 (5 μg/mL) inhibited the proliferation of Meg-01 cells, but not M-07e cells (CD36 -). After the addition of CD36 antibody FA6-152 (5, 10 and 25 μg/mL), the inhibitory effect of TSP-1 was significantly reduced. TSP-1 (2.5, 5 and 7.5 μg/mL) exerted a pro-apoptotic effect by increasing the expression of Annexin V (P<0.01) and caspase-3 activation (P<0.01). Addition of FA6-152 (25 μg/mL) can significantly reduce the apoptosis induced by TSP-1 in Meg-01 cells. In addition, TSP-1 (5, 10 and 25 μg/mL) repress the formation of CFU-MK in mouse bone marrow cells, while β-TG did not. This repression was relieved efficiently by FA6-152 (25 μg/mL).Conclusion: TSP-1 could inhibit the proliferation of Megakaryocyte cell line Meg-01 cells, and induce it's apoptosis. TSP-1 may induce apoptosis of Meg-01 cells via CD36 or caspase-3, which provides a potential new drug choice for clinical treatment of megakaryocytic leukemia. DisclosuresNo relevant conflicts of interest to declare.

Enhanced Functional Response to CXCL12/SDF-1 through Retroviral Overexpression of CXCR4: Implications for Hematopoietic Stem & Progenitor Cell Homing and Engraftment.

The chemokine CXCL12 (stromal cell-derived factor 1/SDF-1) induces the migration of hematopoietic stem and progenitor cells (HSC/HPC) through the corresponding chemokine receptor, CXCR4. CXCL12 is thought to be important for proper homing and engraftment of HSC/HPC to the bone marrow (BM) and mobilization of HSC/HPC out of the BM. Previous studies have suggested that breaking the CXCL12-CXCR4 interaction mobilizes both human and mouse HPC (Liles WC, et al. Blood. 2003 102(8): 2728-30.) (Broxmeyer HE, et al. ASH 44th Annual Meeting. 2002 #2397). Other studies suggest that blocking CXCR4 inhibits homing (Peled A, et al. Science. 1999 283(5403): 845-8) and CXCR4 overexpression by lentiviral vector increases repopulation (Kahn J, et al. Blood. 2004 103(8): 2942-9) of CD34+ cells to the BM of NOD/SCID recipient mice. Expression of CXCR4 on the surface of HSC/HPC appears to be variable, depending highly on the cytokine and growth factor composition of the environment. This may partially explain the reduction in homing efficiency observed during the expansion of cord blood (CB). The efficiency of engraftment therefore appears to be dependent on the response of HSC/HPC to CXCL12 which is in turn dependent upon levels of CXCR4 expressed on HSC/HPC. In order to study the functional cellular response of HSC/HPC independent of variable levels of CXCR4 expression on the surface of cells we utilized the MSCV-based bicistronic (EGFP) retroviral vector, MIEG3, to overexpress human CXCR4 in M07e cells. The human megakaryocytic leukemia cell line, M07e, (a CD34+, c-Kit+, growth-factor-dependent human cell line) has been established as a model for human HPC. Cells infected with the MIEG3 empty vector or the MIEG3-CXCR4 construct were sorted based on their expression of GFP and CXCR4 expression was measured by flow cytometric analysis. MIEG3-CXCR4 M07e cells (GFP+) and MIEG3-CXCR4 bright M07e cells (GFP+++) express significantly higher levels of CXCR4 than M07e cells, MIEG3 M07e cells (GFP+), or MIEG3 bright M07e cells (GFP+++). Migratory response of cells to CXCL12 was assessed by chemotaxis assay. Increased CXCL12 induced chemotaxis was observed in MIEG3-CXCR4 M07e cells (p<0.05) and MIEG3-CXCR4 bright M07e cells (p<0.01). Overexpression of CXCR4 also resulted in a significant increase in CXCL12 induced cell survival during growth factor withdrawal, most markedly at low doses of CXCL12 (10 and 1.0ng/ml) (p<0.05). Thus, retroviral overexpression of CXCR4 enhances both the migratory response and survival of the human HPC line, M07e, to CXCL12. Most importantly cells respond to levels of CXCL12 that are normally too low. This data validates the ability of the MIEG3-CXCR4 construct to efficiently overexpress CXCR4 and validates the future use of MIEG3-CXCR4 M07e cells for further study of CXCR4 in HPC. This is important given the previously established ability of MIEG3 to infect CD34+ CB cells (Tao W et al. Gene Ther. 2004 11(1): 61-9). This information also suggests that previously reported increases in engraftment resulting from CXCR4 overexpression is a function of both increased HSC/HPC settlement and increased HSC/HPC survival in the transplant recipient's BM. This information may have potential therapeutic application for improvements in overall transplant efficiency.

Ectopic Expression of Protein-tyrosine Kinase Bcr-Abl Suppresses Tumor Necrosis Factor (TNF)-induced NF-κB Activation and IκBα Phosphorylation: RELATIONSHIP WITH DOWN-REGULATION OF TNF RECEPTORS

Bcr-Abl, the product of the protooncogene bcr-abl, is a constitutively active protein-tyrosine kinase that is highly expressed in chronic myelogenous leukemia and in acute myeloid leukemia cells. Because Bcr-Abl is known to provide mitogenic signals through suppression of apoptosis, we investigated the effect of this oncogene product on signaling by tumor necrosis factor (TNF), a proapoptotic cytokine. We used a bcr-abl-deficient human megakaryocytic leukemia cell line MO7E and an isogenic MBA cell line stably transfected with bcr-abl. Electrophoretic mobility shift assay revealed that TNF activated the nuclear transcription factor NF-kappaB in MO7E cells but not in MBA cells. The impaired NF-kappaB activation in Bcr-Abl-expressing cells was not due to absence of the NF-kappaB proteins p65, p50, or p100 or of IkappaBalpha or IkappaBbeta. Okadaic acid-induced NF-kappaB activation was unaffected by Bcr-Abl expression. TNF induced IkappaBalpha phosphorylation and degradation in MO7E cells but not in MBA cells. The suppression of TNF-induced NF-kappaB activation by Bcr-Abl was not restricted to MBA cells, because ectopic expression of Bcr-Abl in human acute myeloid leukemia HL-60 cells also blocked TNF-induced NF-kappaB activation. When examined for the TNF receptors by the radioreceptor assay, flow cytometry, or Western blot analysis, we found that Bcr-Abl expression down-regulated the expression of the TNF receptors. The RNase protection assay and Northern blot analysis revealed the transcriptional down-regulation of the TNF receptor by Bcr-Abl protein. Overall, these results indicate that ectopic expression of Bcr-Abl interferes with the TNF signaling pathway through the down-regulation of TNF receptors.

Recombinant human parvovirus B19 vectors: erythroid cell-specific delivery and expression of transduced genes.

A novel packaging strategy combining the salient features of two human parvoviruses, namely the pathogenic parvovirus B19 and the nonpathogenic adeno-associated virus type 2 (AAV), was developed to achieve erythroid cell-specific delivery as well as expression of the transduced gene. The development of such a chimeric vector system was accomplished by packaging heterologous DNA sequences cloned within the inverted terminal repeats of AAV and subsequently packaging the DNA inside the capsid structure of B19 virus. Recombinant B19 virus particles were assembled, as evidenced by electron microscopy as well as DNA slot blot analyses. The hybrid vector failed to transduce nonerythroid human cells, such as 293 cells, as expected. However, MB-02 cells, a human megakaryocytic leukemia cell line which can be infected by B19 virus following erythroid differentiation with erythropoietin (N. C. Munshi, S. Z. Zhou, M. J. Woody, D. A. Morgan, and A. Srivastava, J. Virol. 67:562-566, 1993) but lacks the putative receptor for AAV (S. Ponnazhagan, X.-S. Wang, M. J. Woody, F. Luo, L. Y. Kang, M. L. Nallari, N. C. Munshi, S. Z. Zhou, and A. Srivastava, J. Gen. Virol. 77:1111-1122, 1996), were readily transduced by this vector. The hybrid vector was also found to specifically target the erythroid population in primary human bone marrow cells as well as more immature hematopoietic progenitor cells following erythroid differentiation, as evidenced by selective expression of the transduced gene in these target cells. Preincubation with anticapsid antibodies against B19 virus, but not anticapsid antibodies against AAV, inhibited transduction of primary human erythroid cells. The efficiency of transduction of primary human erythroid cells by the recombinant B19 virus vector was significantly higher than that by the recombinant AAV vector. Further development of the AAV-B19 virus hybrid vector system should prove beneficial in gene therapy protocols aimed at the correction of inherited and acquired human diseases affecting cells of erythroid lineage.

Polyploidization and Functional Maturation Are Two Distinct Processes During Megakaryocytic Differentiation: Involvement of Cyclin-Dependent Kinase Inhibitor p21 in Polyploidization

AbstractThe mechanism of megakaryocytic differentiation was investigated using human megakaryocytic leukemia cell line UT-7. Polyploidization of UT-7 cells was induced by the microtubule-depolymerizing agent, nocodazole, and 12-O-tetradecanoylphorbol-13-acetate (TPA), but the effect was much more striking with nocodazole. By contrast, induction of cytoplasmic maturation, as judged by β-thromboglobulin production and platelet factor 4 expression, was more prominent in TPA-treated cells than in nocodazole-treated cells. Nocodazole and TPA could act synergistically to increase ploidy and to enhance the expression of mature phenotypes. Human thrombopoietin induced functional maturation but not polyploidization in UT-7 cells and also acts synergistically with nocodazole. Cyclin-dependent kinase inhibitor p21 was upregulated at the early stage of megakaryocytic differentiation, and overexpression of p21 resulted in an increase in ploidy of UT-7 cells. This suggests that p21 is implicated in polyploidization via suppression of CDC2 activity at mitosis. UT-7 but not HL-60 cells could incorporate [3H]thymidine in the presence of TPA, indicating the presence of megakaryocyte-specific licensing factor to allow DNA replication during differentiation. Taking these data together, we propose that megakaryocytic differentiation consists of two distinct processes, polyploidization and functional maturation, and that these two processes are independently regulated.

Association between Lyn protein tyrosine kinase (p53/56lyn) and the beta subunit of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors in a GM-CSF-dependent human megakaryocytic leukemia cell line (M-07e).

The role of the lyn product (p53/p56lyn), a membrane-associated protein tyrosine kinase in the signaling pathway used by granulocyte macrophage-CSFR (GM-CSFR) was investigated by using the GM-CSF-dependent human megakaryoblastic leukemia cell line M-07e. M-07e cells express GM-CSFR and are dependent on GM-CSF for survival and proliferation in vitro. Treatment with anti-lyn Abs coimmunoprecipitated, along with lyn product, the beta subunit of GM-CSFR and a phosphoprotein with a molecular mass of 120 kDa (p120) in the lysates of M-07e cells but not in the lysates of human monocyte-derived macrophages (HMDM) or human lymphoid leukemia cells. That the 120-kDa phosphoprotein coimmunoprecipitated by anti-lyn Abs is the beta subunit of GM-CSFR was confirmed in the immunoprecipitates (IP) of M-07e cells with the use of an agarose-conjugated anti-p-tyr mAb. The formation of GM-CSF/GM-CSFR/lyn signaling complexes was verified in an autoradiographic study with anti-lyn IP of M-07e cells that had been bound with 125I-labeled recombinant human (rh)GM-CSF. The p120 protein (beta subunit) was not detected in the IP of M-07e cells with anti-fyn or anti-PI3 Abs. A direct association of Lyn kinase with the beta subunit of GM-CSFR was illustrated with a reversed approach showing the recovery of Lyn protein in anti-beta (CRS1) but not anti-alpha IP of M-07e cells that had been starved for a prolonged period. Finally, the interaction of Lyn kinase with the GM-CSFR complexes was further corroborated using anti-GM-CSF (G133) mAb, which coimmunoprecipitated both the p120 beta subunit and lyn product in the lysates of M-07e cells that had been bound with rhGM-CSF before cell lysis. Removal of rhGM-CSF from culture medium for 10 to 12 h resulted in a marked decrease in lyn-associated kinase activity but not the beta subunit/lyn kinase complex formation. Taken together, our results showed that, in M-07e cells, Lyn protein tyrosine kinase (p53/p56lyn) is stably associated with a constitutively phosphorylated beta subunit of the GM-CSFR in a manner that seems to be independent of lyn kinase activity.

Tyrosine Phosphorylation and Activation of JAK Family Tyrosine Kinases by Interleukin-9 in MO7E Cells

Interleukin-9 (IL-9) is a T-cell-derived multifunctional cytokine that can stimulate the proliferation of a human megakaryocytic leukemia cell line, MO7E. Previous studies suggested that protein tyrosine phosphorylation may be involved in IL-9 signaling pathways. However, tyrosine kinases activated by IL-9 have not been identified. In this report we show that IL-9 induces tyrosine phosphorylation and activation of the JAK family tyrosine kinases including JAK1, JAK3, and Tyk2. The kinetic studies indicate that tyrosine phosphorylation and activation of JAK kinases induced by IL-9 occurred within 1 minute, peaked by 5 to 10 minutes, and persisted at least for 45 minutes. Furthermore, we show that signal transducers and activators of transcription (Stat) 91 or related protein and an 88-kD Stat 91-associated protein are rapidly tyrosine phosphorylated following IL-9 treatment. Gel shift assays confirm that nuclear extracts from MO7E cells stimulated with IL-9 specifically interact with a DNA element termed gamma activated site. These results suggest that actions of IL-9 may, in part, be mediated through JAK kinase-Stat signaling cascades.

Evidence for Separate Calcium-Signaling P2t and P2U Purinoceptors in Human Megakaryocyte Dami Cells

AbstractRecently (J Pharmacol Exp Ther 261:580, 1992), we have shown that K562 leukemia cells express a calcium-signaling purinoceptor with characteristics of the P2T receptor subtype for adenosine diphosphate (ADP) previously found only in platelets. Because these results suggested that the P2T receptor may be an early marker for megakaryocytic differentiation, we studied whether this calcium-signaling receptor is also expressed in Dami cells, a human megakaryocytic leukemia cell line. Here we report evidence that Dami cells express a P2T receptor for ADP. The calcium response EC50 values for ADP, 2-methylthioadenosine diphosphate (2-MeS-ADP), and adenosine 5-0-(2-thiodi-phosphate) (ADPβS) in Dami cells are 0.4 µmol/L, 0.04 µmol/L, and 2 µmol/L, respectively, which approximate the potencies of these agonists in K562 cells and in platelets. The platelet P2T receptor antagonists 2-methylthioadenosine triphosphate (2-MeS-ATP), and 2-chloroadenosine triphosphate (2-CI-ATP) were surprisingly potent agonists at the P2T receptor in both Dami and K562 cells. Dami cells, unlike K562 cells and platelets, also respond to adenosine triphosphate (ATP) and uridine triphosphate (UTP) with an increase in intracellular calcium. Adenosine monophosphate (AMP) is an effective antagonist of the response to ADP, 2-MeS-ADP. ADPβS, 2-MeS-ATP, and 2-CI-ATP, but not to ATP and UTP. The responses to maximal concentrations of UTP in combination with either ADP, 2-MeS-ADP, ADPβS, or 2-MeS-ATP are additive. In contrast, ADP in combination with either 2-MeS-ADP, ADPβS, 2-MeS-ATP, or 2-CI-ATP are not additive. UTP desensitized Dami cells to ATP but not to ADP, 2-MeS-ADP, ADPβS, or 2-MeS-ATP. Addition of ATP after UTP desensitization antagonized subsequent responsiveness to ADP. The data suggest that the receptor for ADP may be a unique P2T subtype, and the receptor for ATP and UTP is distinct from that of ADP and is most characteristic of the P2U (nucleotide) receptor subtype. Activation of either the P2T or P2U receptor causes a rapid generation of inositol trisphosphate in Dami cells.

Evidence for separate calcium-signaling P2T and P2U purinoceptors in human megakaryocytic Dami cells

Recently (J Pharmacol Exp Ther 261:580, 1992), we have shown that K562 leukemia cells express a calcium-signaling purinoceptor with characteristics of the P2T receptor subtype for adenosine diphosphate (ADP) previously found only in platelets. Because these results suggested that the P2T receptor may be an early marker for megakaryocytic differentiation, we studied whether this calcium- signaling receptor is also expressed in Dami cells, a human megakaryocytic leukemia cell line. Here we report evidence that Dami cells express a P2T receptor for ADP. The calcium response EC50 values for ADP, 2-methylthioadenosine diphosphate (2-MeS-ADP), and adenosine 5′-O-(2-thiodiphosphate) (ADP beta S) in Dami cells are 0.4 mumol/L, 0.04 mumol/L, and 2 mumol/L, respectively, which approximate the potencies of these agonists in K562 cells and in platelets. The platelet P2T receptor antagonists 2-methylthioadenosine triphosphate (2- MeS-ATP), and 2-chloroadenosine triphosphate (2-Cl-ATP) were surprisingly potent agonists at the P2T receptor in both Dami and K562 cells. Dami cells, unlike K562 cells and platelets, also respond to adenosine triphosphate (ATP) and uridine triphosphate (UTP) with an increase in intracellular calcium. Adenosine monophosphate (AMP) is an effective antagonist of the response to ADP, 2-MeS-ADP, ADP beta S, 2- MeS-ATP, and 2-Cl-ATP, but not to ATP and UTP. The responses to maximal concentrations of UTP in combination with either ADP, 2-MeS-ADP, ADP beta S, or 2-MeS-ATP are additive. In contrast, ADP in combination with either 2-MeS-ADP, ADP beta S, 2-MeS-ATP, or 2-Cl-ATP are not additive. UTP desensitized Dami cells to ATP but not to ADP, 2-MeS-ADP, ADP beta S, or 2-MeS-ATP. Addition of ATP after UTP desensitization antagonized subsequent responsiveness to ADP. The data suggest that the receptor for ADP may be a unique P2T subtype, and the receptor for ATP and UTP is distinct from that of ADP and is most characteristic of the P2U (nucleotide) receptor subtype. Activation of either the P2T or P2U receptor causes a rapid generation of inositol trisphosphate in Dami cells.

Scanning electron microscopic study of the adhesion of a human megakaryocytic leukemia cell line (CMK11-5)

To characterize the CMK11-5 subclone of a human megakaryocytic leukemia cell line (CMK), an ultrastructural study on adhesion was performed. The CMK11-5 cells showed irreversible attachment to rabbit aortic subendothelium accompanied by several morphological changes such as flattening and the spreading out of pseudopodia. These morphological changes are similar to those observed in normal platelets during attachment. The attachment of CMK11-5 cells to rabbit aortic subendothelium indicates that human megakaryocytes may possess an adhesion function like that of platelets. The CMK cell line and its CMK11-5 subclone with megakaryocytic properties appear to be useful for studying the function of megakaryocytes.

Successful replication of parvovirus B19 in the human megakaryocytic leukemia cell line MB-02

The pathogenic human parvovirus B19 has been shown to undergo productive replication in the erythroid lineage in primary normal human hematopoietic progenitor cells. However, none of the established erythroleukemia cell lines has allowed B19 virus replication in vitro. The remarkable erythroid tissue tropism of B19 virus was evaluated with a human megakaryocytic leukemia cell line, MB-02, which is dependent on the growth factor granulocyte-macrophage colony-stimulating factor but can be induced to undergo erythroid differentiation following treatment with erythropoietin (Epo). Whereas these cells did not support B19 virus DNA replication in the presence of granulocyte-macrophage colony-stimulating factor alone, active viral DNA replication was observed if the cells were exposed to Epo for 5 to 10 days prior to B19 virus infection, as detected by the presence of the characteristic B19 virus DNA replicative intermediates on Southern blots. No replication occurred if the cells were treated with Epo for 3 days or less. In addition, complete expression of the B19 virus genome also occurred in Epo-treated MB-02 cells, as detected by Northern blot analysis. B19 progeny virions were released into culture supernatants that were biologically active in secondary infection of normal human bone marrow cells. The availability of the only homogeneous permanent cell line in which induction of erythroid differentiation leads to a permissive state for B19 virus replication in vitro promises to yield new and useful information on the molecular basis of the erythroid tissue tropism as well as parvovirus B19-induced pathogenesis.

Modulation of GM-CSF receptor beta-subunit and interleukin-6 receptor mRNA expression in a human megakaryocytic leukemia cell line.

Our present study was designed to clarify the mechanism by which the same megakaryocyte progenitor cells respond to various cytokines at different stages of megakaryocyte development. We examined the changes in mRNA expression of granulocyte macrophage colony-stimulating factor receptor beta-subunit (GM-CSFR beta-subunit), which was a common subunit of a high-affinity interleukin-3 receptor (IL-3R) and a high-affinity GM-CSFR, and interleukin-6 receptor (IL-6R) during megakaryocyte development in a human megakaryocytic leukemia cell line (CMK) which could proliferate and/or differentiate in the presence of 12-O-tetradecanoylphorbol 13-acetate (TPA), IL-3, GM-CSF, and IL-6. We found that GM-CSFR beta-subunit mRNA was expressed constitutively in CMK cells and was transiently down-regulated by TPA and IL-6, while the expression of IL-6R mRNA was increased by TPA in association with the differentiation of megakaryocytes. Furthermore, the TPA-induced down-regulation of GM-CSFR beta-subunit mRNA expression and its recovery were blocked by cycloheximide (CHX), a protein synthesis inhibitor, suggesting that these modulations required de novo protein synthesis. These findings imply that multi-lineage cytokines such as GM-CSF and IL-3 may contribute preferentially to the regulation of the earlier development of megakaryocyte progenitor cells with high densities of multi-lineage cytokine receptors, while IL-6 may be limited in its action to supporting the maturation of more differentiated megakaryocyte progenitor cells.(ABSTRACT TRUNCATED AT 250 WORDS)