Abstract: We evaluated the long-term outcome of very dose-intensive chemotherapy (TCC-NHL-91) for advanced intermediate-grade lymphoma, in which an eight-cycle regimen with 11 drugs was given with granulocyte colony-stimulating factor (G-CSF) support (total 18 weeks). Fifty-nine patients were treated during February 1, 1991 and March 31, 2001 (median age: 48 years). Forty-three patients (73%) were in a high-intermediate risk or high-risk group (HI/H) according to the age-adjusted International Prognostic Index (aa-IPI). Forty-six patients received 7 or 8 cycles of therapy. Ten of 15 patients over age 60 stopped before 7 cycles. Forty-three patients with an initial bulky mass or a residual mass received involved-field radiation. Overall, 56 patients (95%) achieved complete remission (CR). Grade 4 hematotoxicity was observed in all patients. With a median follow-up of 128 months, the 10-year overall survival (OS) and progression-free survival (PFS) rates were 76% and 61%, respectively. Neither aa-IPI risk factors nor the index itself was associated with response, OS, or PFS. One patient died of sepsis during the therapy and one died of secondary leukemia. This retrospective study suggests that the TCC-NHL-91 regimen achieves high CR, OS, and PFS in patients with advanced intermediate-grade lymphoma up to 60 years old and may be a valuable asset in the management of this disease. Further evaluation and prospective studies of the TCC-NHL-91 are warranted.
Abstract: We describe a Japanese family with familial platelet disorder with propensity to develop myeloid malignancies (FPD/MM). Among the three affected individuals, two members developed myeloid malignancies. Sequence studies demonstrate that all affected individuals of the pedigree display a heterozygous single nucleotide deletion in exon 8 of the RUNX1 gene.
Abstract: Hypoxia inducible factor (HIF)-1 is a master transcriptional regulator mediating the cellular adaptation to hypoxia. In addition, HIF-1 is also vital for the development of hematopoietic stem cells (HSCs). In a previous study we found that thrombopoietin (TPO), an important and non-redundant cytokine for HSC maintenance and expansion, induces HIF-1alpha expression in HSCs by enhancing the stability of HIF-1alpha under normoxic conditions. However, the molecular mechanisms of these effects are not yet fully understood. In this study, we explored the mechanisms and found that TPO-induced mitochondrial reactive oxygen species (ROS) played a crucial role in stabilization of HIF-1. Both ROS scavengers and inhibitors of mitochondrial electron transport completely blocked HIF-1alpha induction by TPO in UT-7/TPO cells and in primary immature mouse bone marrow cells. We also found that TPO-induced HIF-1alpha induction was tightly coupled with glucose metabolism. Inhibition of glucose transporter or glycolytic enzyme blocked HIF-1alpha elevation of TPO. These results indicate that TPO induces HIF-1alpha expression in a manner very similar to that of hypoxia.
Abstract: FKHRL1 (also called FOXO3a) is a member of the Forkhead Box, class O (FOXO) subfamily of forkhead transcription factors and functions downstream of Bcr-Abl tyrosine kinase as a phosphorylated inactive form in chronic myelogenous leukemia (CML). The Bcr-Abl tyrosine kinase inhibitor imatinib induces cell cycle arrest and subsequent apoptosis via the conversion of FKHRL1 from the phosphorylated inactive form to the dephosphorylated active form in CML-derived cell lines. In the present study, we examined whether active FKHRL1 can overcome resistance to imatinib. To this end, we generated a 4-hydroxytamoxifen-inducible active FKHRL1 (FKHRL1-TM; a triple mutant of FKHRL1 in which all three Akt phosphorylation sites have been mutated)-estrogen receptor fusion protein expression system in CML-derived imatinib-resistant cell lines. 4-Hydroxytamoxifen inhibited cell growth and cell cycle progression, and subsequently induced apoptosis, accompanied by upregulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Thus, active FKHRL1 antagonized deregulated proliferation and induced apoptosis in these cell lines. In addition, imatinib-resistant cells underwent apoptosis after transfection with full-length TRAIL cDNA. Collectively, our results suggest that active FKHRL1 can overcome imatinib resistance in CML cells, in part via TRAIL production.
Abstract: FMS-like tyrosine kinase-3 (FLT3) is a new therapeutic target for acute myelocytic leukemia (AML), because FLT3 mutations are the most common genetic alterations in AML and are directly related to leukemogenesis. We studied cytotoxic interactions of a FLT3 inhibitor, PKC412, with eight conventional antileukemic agents (cytarabine, doxorubicin, idarubicin, mitoxantrone, etoposide, 4-hydroperoxy-cyclophosphamide, methotrexate and vincristine) using three leukemia cell lines carrying FLT3 mutations (MOLM13, MOLM14 and MV4-11) and five leukemia cell lines without FLT3 mutations (KOPB-26, THP-1, BALL-1, KG-1 and U937). PKC412 showed synergistic effects with all agents studied except methotrexate for FLT3-mutated cell lines in isobologram analysis. In contrast, PKC412 was rather antagonistic to most drugs, except for 4-hydroperoxy-cyclophosphamide and vincristine, in leukemia cell lines without FLT3 mutations. Cell-cycle analysis revealed that PKC412 induced G1 arrest in leukemia cell lines carrying FLT3 mutations, whereas it arrested cells in G2/M phase in the absence of FLT3 mutations, which may underlie the divergent cytotoxic interactions. These results suggest that the simultaneous administration of PKC412 and other agents except methotrexate is clinically effective against FLT3 mutation-positive leukemias, whereas it would be of little benefit for FLT3 mutation-negative leukemias. Our findings may be of help for the design of PKC412-based combination chemotherapy.
Abstract: FKHRL1 is one of the human homologues of DAF-16, which is concerned with longevity in Caenorhabditis elegans. Previously, we demonstrated that FKHRL1 functions downstream of Akt in erythropoietin (EPO) signaling and that it is directly phosphorylated by activated Akt. Because phosphorylated FKHRL1 loses its transcriptional activity and translocates into the cytoplasm, FKHRL1 appears to be nonfunctional in the presence of EPO. Conversely, EPO deprivation leads to FKHRL1 dephosphorylation and its translocation into the nucleus, suggesting that FKHRL1 becomes active as a transcription factor in the absence of EPO. On the basis of these findings, we hypothesized, by analogy with C elegans, that erythroid cells possess self-defense machinery against life-threatening surroundings. We prepared a dominant-negative mutant of FKHRL1 (FKHRL1-DN) lacking the transactivation domain and prepared FKHRL1 small interfering RNA (siRNA), and we used constructs to transfect a human EPO-dependent cell line, UT-7/EPO. In the parental cells, 24-hour EPO deprivation induced transient cell cycle arrest without apoptosis. On the other hand, stable transfectants expressing FKHRL1-DN or FKHRL1 siRNA underwent rapid apoptosis after EPO deprivation in the UT-7/EPO cells. In conclusion, FKHRL1 activation plays an important role in the extension of survival of erythroid cells after EPO deprivation. This phenomenon appears to correspond to dauer formation in C elegans. Thus, the mechanism of lifespan extension may be broadly conserved from C elegans to humans.
Abstract: PURPOSE OF REVIEW: Thrombopoietin, the primary regulator of megakaryopoiesis, acts by modulating several nuclear transcription factors. This review focuses on recent studies that have provided new insights into the functional roles of these proteins. RECENT FINDINGS: Among the transcription factors responsible for megakaryopoiesis, important roles for the signal transducer and activator of transcription and Forkhead box proteins have been defined. In addition, in the past several years, two new groups of transcription factors have been found to be affected by thrombopoietin signaling, homeodomain-containing HOX proteins and hypoxia-inducible factor. The HOX transcription factors, which play key roles in body pattern development, are also expressed in adult hematopoietic stem cells and play pivotal roles in their proliferation. Thrombopoietin controls the levels of expression and modifies the function of homeodomain proteins in thrombopoietin-responsive leukemic cells and in primary immature hematopoietic cells. Levels of hypoxia-inducible factor, a master transcription factor required for the adaptation to hypoxic conditions, is also affected by thrombopoietin in these cells. SUMMARY: The discovery that a diverse range of transcription factors are downstream effectors of thrombopoietin helps to explain the molecular mechanisms by which the hormone affects hematopoiesis.
Abstract: Thrombopoietin (TPO), the primary regulator of thrombopoiesis, is also an important, nonredundant mediator of hematopoietic stem cell (HSC) development. For example, following transplantation, HSC expansion is approximately 15-fold more robust in normal than in Tpo(-/-) mice. Vascular endothelial growth factor (VEGF) also plays an important role in HSC development, where it acts in an intracellular autocrine fashion to promote cell survival. Thus, we tested the hypothesis that TPO affects the autocrine production of VEGF to account for its favorable effects on HSCs. We found that VEGF transcripts are reduced in purified sca-1(+)/c-kit(+)/Gr-1(-) marrow cells derived from Tpo(-/-) mice and that TPO induces VEGF transcripts in these primitive hematopoietic cells. Additional studies determined that TPO induces VEGF expression by increasing the level of its primary transcription factor, hypoxia-inducible factor 1alpha (HIF-1alpha), by enhancing its protein stability. Moreover, VEGF expression is important for the TPO effect on primitive hematopoietic cells because blockade of the VEGF receptor with a specific inhibitor substantially blunts TPO-induced growth of single sca-1(+)/c-kit(+)/Gr-1(-) marrow cells in serum-free cultures. Along with previous findings that TPO affects Hox transcription factors that regulate HSC proliferation, these data contribute to our growing understanding of the mechanisms by which a hormone can influence stem cell development.
Abstract: Thrombopoietin (TPO) is a pivotal and nonredundant hematopoietic cytokine, supporting the survival, self-renewal activity and proliferation of hematopoietic stem and progenitor cells, the growth and differentiation of megakaryocytes, and the functional activation of their progeny, blood platelets. TPO exerts these effects through regulating the abundance or subcellular localization of several transcription factors, including the homeodomain proteins HOXB4 and HOXA9. In addition to these effects, TPO helps orchestrate a cytokine-network in the bone marrow microenvironment that supports hematopoietic stem cell (HSC) function. In recent studies we have shown that TPO stimulates production of vascular endothelial cell growth factor (VEGF), another cytokine vital for HSC physiology, promoting their survival and expansion into committed hematopoietic progenitors. Like several other effects of the cytokine, the effect of TPO on VEGF expression is mediated by stabilization and activation of the primary transcription factor responsible for VEGF expression, the oxygen tension responsive hypoxia inducible factor-1 (HIF-1). Together with the observation that bone marrow microenvironment is hypoxic and hypoxia simulates the repopulating activity of HSCs, our observations suggest that TPO mimics hypoxia and controls important genes required for HSC cycling, including VEGF, adding to our understanding of how the hormone contributes to HSC function.
Abstract: Members of the homeobox family of transcription factors are major regulators of hematopoiesis. Overexpression of either HOXB4 or HOXA9 in primitive marrow cells enhances the expansion of hematopoietic stem cells (HSCs). However, little is known of how expression or function of these proteins is regulated during hematopoiesis under physiological conditions. In our previous studies we demonstrated that thrombopoietin (TPO) enhances levels of HOXB4 mRNA in primitive hematopoietic cells (K. Kirito, N. Fox, and K. Kaushansky, Blood 102:3172-3178, 2003). To extend our studies, we investigated the effects of TPO on HOXA9 in this same cell population. Although overall levels of the transcription factor were not affected, we found that TPO induced the nuclear import of HOXA9 both in UT-7/TPO cells and in primitive Sca-1(+)/c-kit(+)/Gr-1(-) hematopoietic cells in a mitogen-activated protein kinase-dependent fashion. TPO also controlled MEIS1 expression at mRNA levels, at least in part due to phosphatidylinositol 3-kinase activation. Collectively, TPO modulates the function of HOXA9 by leading to its nuclear translocation, likely mediated by effects on its partner protein MEIS1, and potentially due to two newly identified nuclear localization signals. Our data suggest that TPO controls HSC development through the regulation of multiple members of the Hox family of transcription factors through multiple mechanisms.
Abstract: Targeting BCR-ABL tyrosine kinase by treatment with the selective inhibitor imatinib (formerly STI571, Gleevec) has proved to be highly efficient for inhibiting leukemic growth in vitro. In addition, in clinical trials, imatinib has produced high response rates in patients with chronic myeloid leukemia (CML) in chronic phase and blastic crisis. However, episodes of severe cytopenia were also frequently observed, leading to discontinuation of therapy in some cases. Therefore, it is important to examine whether administration of cytokines overcomes the adverse effects of imatinib in in vitro systems. In this study, we examine the effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (EPO) on TF-1/bcr-abl (which was generated by transduction of a bcr-abl fusion gene into the TF-1 cell line) as a model system for CML with blastic crisis. Imatinib induced apoptosis in TF-1/bcr-abl cells but not in the parental TF-1 cells. However, GM-CSF, a survival factor of the parental TF-1 cells, protected TF-1/bcr-abl cells from imatinib-induced apoptosis in a dose-dependent manner. Concomitantly, constitutive phosphorylation of Stat5 and FKHRL1 was significantly inhibited by imatinib, and the inhibition was canceled by the addition of GM-CSF, accompanied by upregulation of Bcl-xL and downregulation of p27/Kip1. In addition, although untreated TF-1/bcr-abl cells had lost responsiveness to both GM-CSF and EPO and showed autonomous growth, GM-CSF enhanced phosphorylation of Stat5 and FKHRL1 in these cells. Importantly, imatinib-treated TF-1/bcr-abl cells differentiated into hemoglobin-positive cells in the presence of EPO, as in the case for the parental TF-1 cells. Taken together, imatinib-treated CML cells may differentiate into mature cells in the presence of differentiation-inducing cytokines such as EPO.
Abstract: A member of the Forkhead transcription factor family, FKHRL1, lies downstream of the phosphatidylinositol 3-kinase-Akt activation pathway in cytokine signaling. Because the phosphatidylinositol 3-kinase-Akt activation pathway is required for BCR-ABL-mediated transformation and survival signaling in chronic myelogenous leukemia (CML), in this study we examined the involvement of FKHRL1 in the BCR-ABL-mediated signaling pathway. FKHRL1 was constitutively phosphorylated in BCR-ABL-expressing cell lines KCL22 and KU812, and its phosphorylation was inhibited by treatment with STI571, a specific inhibitor of BCR-ABL tyrosine kinase. Concomitantly, STI571 induced cell cycle arrest at the G(0)/G(1) phase, accompanied by up-regulation of a cyclin-dependent kinase inhibitor p27/Kip1 in KCL22 cells. In addition, FKHRL1 was constitutively phosphorylated in the TF-1/bcr-abl cell line ectopically expressing BCR-ABL but not in the parent TF-1 cell line. Considering several lines of evidence that phosphorylated FKHRL1 has lost transcriptional activity and that p27/Kip1 expression is positively regulated by dephosphorylated "active" FKHRL1, BCR-ABL may down-regulate p27/Kip1 expression via the loss of FKHRL1 function as a transcription factor. To demonstrate this hypothesis, we generated a tamoxifen-inducible "active FKHRL1" FKHRL1-TM (a triple mutant of FKHRL1, in which all three Akt phosphorylation sites have been mutated), estrogen receptor system in the KCL22 cell line. The addition of tamoxifen inhibited the cell growth indicating that overexpression of FKHRL1 in the nucleus antagonized deregulated proliferation of CML cells. Collectively, FKHRL1 regulates the expression of p27/Kip1 as a downstream molecule of BCR-ABL signaling in CML cells. BCR-ABL-induced loss of FKHRL1 function may be involved in oncogenic transformation of CML partially via the down-regulation of p27/Kip1 proteins.
Abstract: Erythropoietin (EPO) can rescue erythroid cells from apoptosis during erythroid development, leading to red cell production. However, the detailed mechanism of how EPO protects erythroid cells from apoptosis is still open to question. To address this problem, we used a human EPO-dependent leukemia cell line UT-7/EPO and normal erythroid progenitor cells. After deprivation of EPO, UT-7/EPO cells underwent apoptosis, accompanied by down-regulation of the Bcl-xL protein. In addition, the cleaved products of caspase-3, p11 and p21, and a few cleaved forms of inhibitor of caspase-activated DNase (ICAD) were detected in these cells. When the cells were pre-treated with the pancaspase inhibitor Z-VAD-FMK, the ratio of apoptotic cells was significantly reduced, suggesting that EPO protects the UT-7/EPO cells from apoptosis via inhibition of caspase activities. When an MEK 1/2 inhibitor U0126 inhibited activities of extracellular signal-regulated kinases (ERKs), the expression of Bcl-xL protein was down-regulated and subsequently apoptosis was induced. Interestingly, Z-VAD-FMK blocked U0126-induced down-regulation of Bcl-xL protein and apoptosis, strongly suggesting that Bcl-xL expression is regulated by caspases which lies downstream of ERK activation pathway in EPO signaling. Importantly, these findings were also observed in normal erythroid progenitor cells. In conclusion, the activation of ERKs by EPO up-regulates Bcl-xL expression via inhibition of caspase activities, resulting in the protection of erythroid cells from apoptosis.
Abstract: Thrombopoietin (TPO), the primary regulator of platelet production, also plays an important role in hematopoietic stem cell (HSC) biology. In previous studies we demonstrated that the self-renewal and expansion of HSCs is 10 to 20 times less robust in tpo-/- mice than in controls. To explore the molecular basis of this effect, we postulated that Hoxb4 might mediate at least part of the TPO effect on these cells. We first analyzed the effects of TPO on Hoxb4 expression in primitive hematopoietic cell lines; TPO increased expression of the gene 2- to 3-fold in EML and UT-7/TPO cells. We also compared Hoxb4 levels in a candidate HSC population derived from tpo-/- and control mice; Hoxb4 expression was 2- to 5-fold lower in null HSCs. Of the numerous signal transduction molecules induced by TPO, we found that p38 mitogen-activated protein kinase (MAPK) was responsible for the TPO-induced Hoxb4 elevation. We also demonstrated that upstream stimulating factor 1 (USF-1), a transcription factor previously shown to regulate Hoxb4 expression, is also induced by TPO in a p38-dependent manner. Together, these data provide a molecular pathway by which a growth factor can modulate a transcription factor and thereby help direct a critical developmental process.
Abstract: Signal transducers and activators of transcription (Stat) proteins play important roles in the regulation of hematopoiesis as downstream molecules of cytokine signal transduction. Previously, we demonstrated that Stat1 and Stat3 are activated by erythropoietin (EPO) in a human EPO-dependent erythroleukemia cell line UT-7/EPO. We report here that Stat1 and Stat3 are constitutively activated in freshly isolated erythroleukemia cells. In addition, EPO promoted cell growth of these cells, accompanied by enhanced activities of Stat1 and Stat3. Furthermore, mutation in the Statl/Stat3-binding sites of the c-myc gene promoter clearly blocked its promoter activity in EPO-stimulated primary erythroleukemia cells. Thus, Stat1 and Stat3 may support cell growth in part via c-myc gene activation in primary erythroleukemia cells.
Abstract: Signal transducers and activators of transcription (Stat) proteins play important roles in the regulation of hematopoiesis as downstream molecules of cytokine signal transduction. It was previously demonstrated that erythropoietin (EPO), a major regulator of erythropoiesis, activates 3 different Stat members, Stat1, Stat3, and Stat5, in a human EPO-dependent cell line, UT-7/EPO. To clarify the mechanism by which EPO activates Stat1 and Stat3 via the EPO receptor (EPOR), a series of chimeric receptors was constructed bearing the extracellular domain of the granulocyte colony-stimulating factor receptor linked to the transmembrane domain of EPOR and the full length or several mutants of the cytoplasmic domain of EPOR, and these chimeric receptor complementary DNAs were introduced into UT-7/EPO cells. Tyr432 on human EPOR was important for activation of Stat1 and Stat3 and c-myc gene induction. In addition, Jak2 and Fes tyrosine kinases were involved in EPO-induced activation of Stat1 and Stat3. These results indicate that Stat1 and Stat3 are activated by EPO via distinct mechanisms from Stat5.
Abstract: Phosphorylation of the Fanconi anemia complementation group A (FANCA) protein is thought to be important for the function of the FA pathway. However, the kinase for FANCA (so-called FANCA-PK) remains to be identified. FANCA has a consensus sequence for Akt kinase near serine 1149 (Ser1149), suggesting that Akt can phosphorylate FANCA. We performed in vitro kinase assays using as substrate either a GST-fusion wild-type (WT) FANCA fragment or a GST-fusion FANCA fragment containing a mutation from serine to alanine at 1149 (FANCA-S1149A). These experiments confirmed that FANCA is phosphorylated at Ser 1149, in vitro. However, (32)P-orthophosphate labeling experiments revealed that FANCA-S1149A was more efficiently phosphorylated than WT-FANCA. Furthermore, phosphorylation of wild-type FANCA was blocked by coexpression of a constitutively active (CA)-Akt and enhanced by a dominant-negative (DN) Akt. Our results suggest that Akt is a negative regulator of FANCA phosphorylation.
Abstract: Thrombopoietin (TPO), an essential factor for megakaryopoiesis and thrombopoiesis, works as a survival factor for megakaryocytic lineage cells. However, little is known about the molecular mechanism in detail. We show here that TPO supports the survival of TPO-dependent leukemia cell line UT-7/TPO and normal megakaryocytic progenitors via the induction of Bcl-xL, an anti-apoptotic member of the Bcl-2 family. We further analyzed the signal transduction pathways required for TPO-induced Bcl-xL gene expression. A reporter assay with various lengths of Bcl-x gene promoter revealed that both Stat- and nuclear factor kappa B-binding sites are prerequisites for TPO-induced promoter activity. Consistent with these results, TPO induced the binding of Stat5 and subunits of nuclear factor kappa B, p50, and c-Rel to the Bcl-x gene promoter. AG490, a specific inhibitor for Jak2, and LY294002, a specific inhibitor for phosphatidylinositol (PI) 3-kinase, reduced the protein level of Bcl-xL in UT-7/TPO cells, accompanied by an increase in the ratio of apoptotic cells. Interestingly, LY294002 enhanced the TPO-induced DNA binding activity of Stat5 without affecting the Jak2 activation and tyrosine phosphorylation of Stat5. Concomitantly, confocal microscopy revealed that LY294002 clearly inhibited the nuclear export of Stat5, suggesting that PI 3-kinase regulates the subcellular localization of Stat5. Taken together, our results suggest that both Jak-Stat and PI 3-kinase activation pathways regulate the TPO-induced survival of megakaryocytic cells via Bcl-xL gene expression. In addition, our data suggest possible cross-talk between these two signaling pathways.
Abstract: A 71-year-old man visited our hospital complaining of fever and a bleeding tendency. The peripheral blood WBC count was 10,400/microliter with 90% promyelocytes. The bone marrow was hypercellular with 88% promyelocytes. Disseminated intravascular coagulation was recognized. The patient was diagnosed as having acute promyelocytic leukemia and was treated with daily oral administration of all-trans retionic acid (ATRA) (45 mg/m2/day) and cytarabine (160 mg/day, intravenous drip infusion for the initial five days). The ATRA treatment induced leukemic cells to undergo mature myeloid differentiation. On day 24 after the start of treatment, the WBC count rapidly increased and acute myocardial infarction appeared, with consciousness disturbance and bilateral Babinski reflex appearing three hours later. Magnetic resonance imaging showed a fresh lacunar infarction of the right lenticular nucleus, and serum levels of IL-6 and PAI-1 were found to be elevated at the onset of infarction. Since there was a possibility that the retinoic acid syndrome (RAS) might have helped bring about the infarctions, we stopped the ATRA treatment and started administration of methyl-prednisolone (500 mg/body/day for 3 days) and gabexate mesilate. The WBC count decreased immediately and the consciousness disturbance improved. In this case, ATRA treatment might have initiated the RAS and resulted in some endothelial damage, thus causing the infarctions.
Abstract: The signal transducer and activator of transcription 3 (Stat3), a member of the Stat family of proteins, is commonly activated by thrombopoietic cytokines including thrombopoietin (TPO), interleukin (IL)-6, and interleukin-11. This finding strongly suggested that Stat3 has an important role in megakaryopoiesis and thrombopoiesis. To clarify the functional role of Stat3 in in vivo megakaryopoiesis and thrombopoiesis, we generated transgenic mice overexpressing a dominant-negative Stat3, Stat3F, to suppress the function of endogenous Stat3. To accomplish the selective expression of Stat3F in megakaryocytic lineage cells, we used the regulatory gene region of GATA-1 transcription factor selectively expressed in megakaryocytic and erythroid lineage cells. Two independent transgenic (Tg) mice lines were established. It was confirmed by Western blotting analysis that Stat3F proteins were highly expressed in the platelets from the Tg mice. In addition, it was found that Stat3 activation induced by TPO stimulation was drastically suppressed in these Tg mice compared with littermates. These findings indicate that Stat3F works well in the Tg mice. Platelet counts were within the normal range in steady-state conditions and were recovered normally from transient thrombocytopenia induced by antiplatelet serum injection. Interestingly, the platelet recovery from myelosuppression after 5-fluorouracil treatment was significantly delayed in the Tg mice. Collectively, our results strongly suggest that Stat3 plays an important role in the early stage of megakaryopoiesis, presumably through the expansion of megakaryocytic progenitor cells.
Abstract: A 19-year-old male with de novo acute myeloid leukemia (AML) in complete remission received a bone marrow transplant from a HLA-matched donor. Because of major incompatibility for ABO blood type, bone marrow mononuclear cells of the donor were infused after conditioning including total body irradiation (TBI). Engraftment was confirmed on day +23. On day +91, recipient ABO blood genotype was detected in burst forming-unit erythroid (BFU-E) using polymerase chain reaction. Thereafter, myelodysplastic syndrome (MDS) of recipient origin rapidly developed and progressed into a chronic myelomonocytic leukemia-like disorder. An association between MDS and TBI is suggested.
Abstract: FKHRL1, a member of the Forkhead transcription factor family, is one of the downstream molecules of phosphatidylinositol 3-kinase-Akt. This molecule is a mammalian homolog of DAF-16, which plays an important role in the longevity of Caenorhabditis elegans. In this study we found that Akt and FKHRL1 proteins were detectable in highly purified normal human megakaryocytes and that these molecules were actually phosphorylated by thrombopoietin (TPO). To clarify the functional role of FKHRL1 in TPO signaling, we established a tetracycline-inducible system in the human TPO-dependent leukemia cell line UT-7/TPO. Induced expression of active FKHRL1 led to cell cycle arrest at G0/G1 phase in this cell line. These results suggest that FKHRL1 plays an important role in the cell cycle of megakaryocytic cells as one of the downstream target molecules of phosphatidylinositol 3-kinase-Akt, presumably mediated through the activation or inactivation of cell cycle-associated gene(s).
Abstract: The mitogen-activated protein (MAP) kinase cascade is a key regulator of mammalian cell proliferation and differentiation. In this study, we examined the roles of 2 members of the MAP kinase family, extracellular signal-regulated kinase 1 (Erk1) and Erk2, in erythropoietin (EPO)-induced erythroid differentiation and thrombopoietin (TPO)-induced megakaryocytic differentiation. UT-7/GM was used as a model system because this cell line is an erythroid/megakaryocytic bipotent cell line that can be induced to differentiate into the erythroid and megakaryocytic lineages by EPO and TPO, respectively. The kinetics of activation of Erk1 and Erk2 were examined during erythroid and megakaryocytic differentiation of UT-7/GM cells. EPO induced a transient activation of these kinases, peaking after 1 minute of stimulation and then declining quickly almost to the basal level. In contrast, TPO-induced activation of the kinases peaked at 10 minutes and persisted for up to 60 minutes, similar to the activation by granulocyte-macrophage colony-stimulating factor. The percentage of EPO-induced hemoglobin-positive cells was elevated by the addition of PD98059, a specific inhibitor of MEK1 (MAP kinase/ERK kinase 1). In contrast, PD98059 clearly reduced the amount of glycoprotein IIb/IIIa antigens induced by TPO on UT-7/GM cells. Thus, inactivation of Erk1 and Erk2 kinases promoted EPO-induced erythroid differentiation and suppressed TPO-induced megakaryocytic differentiation of UT-7/GM cells. In conclusion, the activation of Erk1 and Erk2 kinases may be a critical event in the determination of cell fate and the differentiation processes of the erythroid and megakaryocytic lineages.
Abstract: Chronic myeloid leukemia (CML) is characterized by the clonal expansion of hematopoietic stem cells (HSCs). Without effective treatment, individuals in the indolent, chronic phase (CP) of CML undergo blast crisis (BC), the prognosis for which is poor. It is therefore important to clarify the mechanism underlying stage progression in CML. DNA microarray is a versatile tool for such a purpose. However, simple comparison of bone marrow mononuclear cells from individuals at different disease stages is likely to result in the identification of pseudo-positive genes whose change in expression only reflects the different proportions of leukemic blasts in bone marrow. We have therefore compared with DNA microarray the expression profiles of 3456 genes in the purified HSC-like fractions that had been isolated from 13 CML patients and healthy volunteers. Interestingly, expression of the gene for PIASy, a potential inhibitor of STAT (signal transducer and activator of transcription) proteins, was down-regulated in association with stage progression in CML. Furthermore, forced expression of PIASy has induced apoptosis in a CML cell line. These data suggest that microarray analysis with background-matched samples is an efficient approach to identify molecular events underlying the stage progression in CML.
Abstract: Myelodysplastic syndrome (MDS) is a slowly progressing hematologic malignancy associated with a poor outcome. Despite the relatively high incidence of MDS in the elderly, differentiation of MDS from de novo acute myeloid leukemia (AML) still remains problematic. Identification of genes expressed in an MDS-specific manner would allow the molecular diagnosis of MDS. Toward this goal, AC133 surface marker-positive hematopoietic stem cell (HSC)-like fractions have been collected from a variety of leukemias in a large-scale and long-term genomics project, referred to as "Blast Bank," and transcriptome of these purified blasts from the patients with MDS were then compared with those from AML through the use of oligonucleotide microarrays. A number of genes were shown to be expressed in a disease-specific manner either to MDS or AML. Among the former found was the gene encoding the protein Delta-like (Dlk) that is distantly related to the Delta-Notch family of signaling proteins. Because overexpression of Dlk may play a role in the pathogenesis of MDS, the disease specificity of Dlk expression was tested by a quantitative "real-time" polymerase chain reaction analysis. Examination of the Blast Bank samples from 22 patients with MDS, 31 with AML, and 8 with chronic myeloid leukemia confirmed the highly selective expression of the Dlk gene in the individuals with MDS. Dlk could be the first candidate molecule to differentiate MDS from AML. The proposal is made that microarray analysis with the Blast Bank samples is an efficient approach to extract transcriptome data of clinical relevance for a wide range of hematologic disorders.
Abstract: The phosphatidylinositol 3-kinase (PI3K) signaling pathway is important for the regulation of a number of cellular responses. Serine/threonine kinase Akt (protein kinase B; PKB) is downstream of PI3K and activated by growth factors. This study found that erythropoietin (EPO) induced tyrosine phosphorylation of Akt in a time- and dose-dependent manner in EPO-dependent human leukemia cell line UT-7/EPO. In vitro kinase assay using histone H2B and glucose synthase kinase as substrates demonstrated that Akt was actually activated by EPO. EPO-induced phosphorylation of Akt was completely blocked by a PI3K-specific inhibitor, LY294002, at 10 micromol/L, indicating that activation of Akt by EPO is dependent on PI3K activity. In addition, overexpression of the constitutively active form of Akt on UT-7/EPO cells partially blocked apoptosis induced by withdrawal of EPO from the culture medium. This finding suggested that the PI3K-Akt activation pathway plays some role in the antiapoptotic effect of EPO. EPO induced phosphorylation of a member of the trancription factor Forkhead family, FKHRL1, at threonine 32 and serine 253 in a dose- and time-dependent manner in UT-7/EPO cells. Moreover, results showed that Akt kinase activated by EPO directly phosphorylated FKHRL1 protein and that FKHRL1 phosphorylation was completely dependent on PI3K activity as is the case for Akt. In conjunction with the evidence that FKHRL1 is expressed in normal human erythroid progenitor cells and erythroblasts, the results suggest that FKHRL1 plays an important role in erythropoiesis as one of the downstream target molecules of PI3K-Akt.
Abstract: Flow cytometric, immunochemical and molecular studies were performed on leukemic blasts from a patient with minimally differentiated erythroleukemia (AML-M6v). The blasts expressed CD36 and CD71 but not lymphoid antigens, myeloid antigens, CD41 or glycophorin A. Analysis of carbohydrate antigens showed that the blasts expressed the sialyl-Tn antigen. Immunochemistry revealed that the blasts had neuron-specific enolase (NSE). Serum sialyl-Tn and NSE levels were markedly increased. Finally, an erythroid lineage was confirmed in the presence of alpha-globin messages in the blasts. Sialyl-Tn and NSE expression in leukemic blasts may be useful to identify AML-M6v.
Abstract: A retrospective analysis was performed on 76 consecutive elderly patients with acute leukemia aged 60 years or more (48 men, 28 women). Forty patients were 60-69 years old, 28 were 70-79 years old and 8 were > or = 80 years old. There were 55 patients with acute myelogenous leukemia (AML), 13 acute lymphoblastic leukemia (ALL) and 8 AML from myelodysplastic syndrome (MDS/AML). Patients were treated with the JALSG protocol, CAG regimen, or low-dose Ara-C regimen for AML, and DVP/M-CHOP protocol for ALL. The complete remission (CR) rates were 52.7% (29 of 55) in AML, 61.5% (8 of 13) in ALL, and 0% in MDS/AML. The median CR durations were 226, 85, 0 days, and the median survivals were 204, 177, 99 days, respectively. CR rates were 65.3% for the JALSG protocol, 62.5% for the CAG regimen and 25.0% for low-dose Ara-C regimen. According to age, CR was obtained 62.5% in patients aged 60-69 years and 33.3% in patients over 70 years old. Our results indicated that patients aged 60-69 years should be treated with intensive chemotherapy.
Abstract: UT-7 is a human megakaryoblastic leukemia cell line with absolute dependence on interleukin-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), or erythropoietin (EPO) for growth and survival. Among its sublines, UT-7/GM, which remains undifferentiated in the presence of GM-CSF, has a bipotency showing differentiation into erythroid or megakaryocytic cell lineages in the presence of EPO or thrombopoietin (TPO), respectively. To investigate the mechanism underlying determination of cell differentiation, we investigated the role of signal transducers and activators of transcription (STAT) in EPO-induced erythroid differentiation. UT-7 cells grow in response to GM-CSF and EPO but do not differentiate into mature cells. UT-7/GM cells grow in response to GM-CSF and differentiate into erythroid cells by EPO. In UT-7 cells, both GM-CSF and EPO induced the activation of Stat1 alpha, Stat3 and Stat5. In UT-7/GM cells, EPO activated Stat5 alone, although the activation of Stat1 alpha, Stat3, and Stat5 was induced by GM-CSF or TPO. In addition, GM-CSF inhibited EPO-induced erythroid differentiation and concomitantly activated Stat1 alpha and Stat3 in UT-7/GM cells even in the presence of EPO. Further we transfected Stat1 alpha, Stat3 cDNA or both into UT-7/GM cells. Hemoglobin-positive cells were decreased in the stable transfectants stimulated with EPO. These results indicate that Stat1 alpha and Stat3 have an inhibitory effect on the EPO-induced erythroid differentiation, and more complicated combination of transcription factors may play an important role in the decision of cell differentiation.
Abstract: Thrombopoietin (TPO) acts on megakaryopoiesis and erythropoiesis in vitro and in vivo. We isolated a novel subline, UT-7/GMT, from the human leukemia cell line UT-7/GM (N. Komatsu, et al., Blood, 89: 4021-4033, 1997). A small population of UT-7/GM cells positively stained for hemoglobin (Hb) after a 7-day exposure to TPO. More than 50% of TPO-treated UT-7/GMT cells positively stained for Hb. Using UT-7/GMT cells, we examined how TPO promotes hemoglobinization. TPO induced tyrosine phosphorylation of the TPO receptor but not the erythropoietin (EPO) receptor. There was no competition between TPO and EPO for binding to EPO receptor. These findings suggest that TPO has a direct effect on hemoglobinization via a specific receptor on UT-7/GMT cells. Isoelectric focusing demonstrated that TPO induced fetal and adult Hb synthesis, whereas EPO induced embryonic, fetal, and adult Hb synthesis. Thus, our data suggest that TPO has a distinct action on erythropoiesis.
Abstract: We recently determined that erythropoietin (EPO) activates 3 members of the signal transducer and activator of transcription (STAT) family, Stat1alpha, Stat3, and Stat5, in the human EPO-dependent cell lines, UT-7 and UT-7/EPO (Kirito et al, J Biol Chem 272:16507, 1997). In addition, we have shown that Stat1alpha, but not Stat3, is involved in EPO-induced cellular proliferation. In this study, we examined the roles of Stat1alpha and Stat3 in EPO-induced erythroid differentiation. UT-7/GM was used as a model system, because this cell line can differentiate into erythroid-lineage cells with EPO treatment (Komatsu et al, Blood 89:4021, 1997). We found that EPO did not activate Stat1alpha or Stat3 in UT-7/GM cells. Transfection experiments showed that both Stat1alpha and Stat3 inhibited the induction by EPO of gamma-globin and erythroid-specific 5-aminolevulinate synthetase transcripts, resulting in a reduction of the percentage of hemoglobin-positive cells. Dominant negative forms of Stat1alpha or Stat3 promoted the EPO-induced erythroid differentiation of UT-7/GM cells, even in the presence of granulocyte-macrophage colony-stimulating factor, although this cytokine never induced erythroid differentiation of the parent UT-7/GM cells with or without EPO. A cell cycle analysis showed that the constitutive activation of Stat1alpha, but not Stat3, shortened the period of G0/G1 prolongation caused by EPO stimulation. Taken together, our data suggest that Stat1alpha and Stat3 act as negative regulators in EPO-induced erythroid differentiation. Specifically, Stat1alpha may activate a cell cycle-associated gene(s), leading to the entry of cells into the cell cycle.
Abstract: We examined expression of the erythroid-associated genes GATA-1 and erythropoietin receptor (EPOR) in primary leukemia using the reverse transcriptase-polymerase chain reaction (RT-PCR). GATA-1 and EPOR mRNAs were detectable in all cases of erythroleukemia (French-American-British classification: M6) or early erythroblastic leukemia. In all other leukemia cases, including M2 through M5, stem cell leukemia, and adult T-cell leukemia, these gene transcripts were undetectable. GATA-2 was detectable in all the cases of primary leukemias examined in this study, except one case of M5. In one case, the phenotype switched from myeloid (M2) to erythroid (M6) and then back to myeloid. Northern blotting and RT-PCR revealed that GATA-1 and EPOR mRNAs were significantly upregulated at the M6 stage compared with the M2 stage. GATA-1 may be involved in the expression of an erythroid phenotype in acute leukemia. We generated HL-60 transfectants exogenously expressing GATA-1. The majority of HL-60 cells expressing GATA-1 lacked azurophilic granules, and electron microscopic analysis revealed that myeloperoxidase activity was negative. Platelet peroxidase activity, which was detectable in both megakaryoblasts and erythroid progenitors, was positive. However, EPOR and glycophorin A mRNAs were undetectable by RT-PCR. These findings suggest that besides GATA-1, a third factor may be required for the expression of mature erythroid phenotypes. In addition, our results indicate that GATA-1 is involved in inactivation of myeloperoxidase and activation of the platelet peroxidase.
Abstract: To understand the regulatory mechanism of erythropoietin (EPO) receptor (EPOR) gene expression, the effect of EPO on the steady-state level of EPOR mRNA was examined using the human EPO-dependent cell line UT-7 as a model system. We found that the treatment of UT-7 cells with EPO resulted in a transient decrease of the EPOR mRNA level. This transient downregulation was also induced by stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF), another stimulator of UT-7 cell growth. These results raised the possibility that EPOR gene expression is in part related to cell growth. Moreover, it was found that EPO-induced downregulation of EPOR mRNA level was preceded by a transient downregulation of GATA-1 mRNA. To examine the relationship between the expression of EPOR, GATA-1, and GATA-2 mRNA levels and the cell cycle, logarithmically growing UT-7 cells were centrifugically fractionated according to the cell-cycle phase. Both EPOR and GATA-1 mRNA levels, but not the GATA-2 mRNA level, concomitantly decreased at the G0/G1 phase and increased at the S and G2/M phases. An electrophoretic mobility shift assay (EMSA) showed that in EPO-stimulated UT-7 cells, the dynamic changes in EPOR gene expression paralleled the GATA-1 DNA-binding activity to the oligonucleotide probe containing a GATA-binding site located at the promoter region of the EPOR gene. These findings suggest that the regulation of EPOR mRNA level is mainly associated with GATA-1 gene expression in UT-7 cells undergoing proliferation, and that these serial events are under the control of, or related to, the cell cycle.
Abstract: The Janus kinase (JAK)-signal transducers and activators of transcription (STAT) pathway is an important signaling pathway of interferons and cytokines. We examined the activation of STAT proteins induced by interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or erythropoietin (EPO) using the human leukemia cell line, UT-7, which requires these cytokines for growth. IL-3, GM-CSF, and EPO induced DNA-binding activity to the oligonucleotides corresponding to the sis-inducible elements (SIE) of c-fos, in addition to the beta-casein promoter (beta-CAP), SIE- and beta-CAP-binding proteins were identical to Stat1alpha and Stat3 complex and to Stat5 protein, respectively. This indicates that IL-3, GM-CSF, and EPO commonly activated Stat1alpha, Stat3, and Stat5 proteins in UT-7. However, EPO hardly activated Stat1alpha and Stat3 in UT-7/GM, which is a subline of UT-7 that grows slightly in response to EPO. Transfection studies revealed that UT-7/GM cells constitutively expressing Stat1alpha, but not Stat3, can grow as well in response to EPO as GM-CSF, suggesting that Stat1alpha is involved in the EPO-induced proliferation of UT-7. Thus, although Stat1alpha, Stat3, and Stat5 proteins are activated by GM-CSF, IL-3, and EPO, our data suggest that each STAT protein has a distinctive role in the actions of cytokines.
Abstract: UT-7 is a human megakaryoblastic leukemia cell line with absolute dependence on interleukin-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), or erythropoietin (EPO) for growth and survival. We isolated a novel subline, UT-7/GM after long-term culture of UT-7 with GM-CSF. The hemoglobin concentration and gamma-globin and EPO-receptor mRNA levels were significantly higher in EPO-treated UT-7/GM cells than in untreated cells. In contrast, the platelet factor 4 and glycoprotein IIb mRNA levels were much higher in thrombopoietin (TPO)-treated UT-7/GM cells than in untreated cells. Some TPO-treated cells had morphologically mature megakaryocytic characteristics such as a developed demarcation membrane in the cytoplasm and multilobular nuclei. These findings indicate that UT-7/GM is a bipotential cell line that can be induced to differentiate into erythroid and megakaryocytic lineages by EPO and TPO, respectively. Moreover, a minority of UT-7/GM cells acquired a high hemoglobin concentration by treatment with TPO, suggesting that TPO in part induced the erythroid differentiation of the UT-7/GM cells. Interestingly, GM-CSF inhibited the EPO- or TPO-induced erythroid differentiation and the TPO-induced megakaryocytic differentiation of UT-7/GM cells. These results support the hypothesis that cytokines influence the programming of gene expression required for lineage commitment or differentiation.
Abstract: The mechanism of interleukin-3 (IL-3) independent cell growth and of IL-3-associated ganglioside expression was analysed using the IL-3 dependent murine myelogenous leukaemia cell line NFS60-I7 and IL-3 gene-transfected sublines. The transfected cell lines showed autonomous cell growth, tumorigenicity, and IL-3 associated ganglioside GD1a expression in spite of their IL-3 production. While the parental NFS60-I7 cells did not express significant amounts of GD1a, exogenous recombinant IL-3 (rIL-3) was demonstrated to induce IL-3-associated ganglioside GD1a expression in NFS60-I7 cells. Furthermore, the growth potential of the transfected cells was not blocked by anti-IL-3 antibody and expression of GD1a was not affected by anti-IL-3 antibody. These findings suggest that IL-3 expressed intracellularly by gene transfection might act independently of the normal IL-3 receptor on autonomous cell growth and on IL-3-associated GD1a expression in murine myelogenous leukaemia NFS60 cells.
Abstract: Murine interleukin-3 (IL-3)-associated expression of gangliosides has been investigated using a gene transfection technique. A murine IL-3 cDNA was introduced into the parental NFS60-17 cells that was exclusively dependent on IL-3. We analyzed the glycosphingolipids from the parental cells and the transfected cells by fast atom bombardment mass spectrometry analyses and/or immunostaining techniques using specific antibodies. Two major gangliosides, IV3NeuAc-GgOse4Cer (GM1b) and IV3-NeuAc,III6NeuAc-GgOse4Cer (GD1 alpha), were expressed, in the parental cells. By contrast, in the IL-3 gene-transfected cells, a ganglioside IV3NeuAc,II3NeuAc-GgOse4Cer (GD1a) was strikingly expressed, in addition to GM1b and GD1 alpha that were already present in the parental cells. In spite of various IL-3-secreting capabilities, all transfectants investigated have exhibited the same ganglioside patterns and expressed GD1a. Furthermore, the appearance of GD1a was a consequence of the up-regulation of a single glycosyltransferase, CMP-NeuAc:lactosylceramide alpha 2-->3-sialytransferase (GM3 synthase). Activities of the other downstream glycosyltransferases that were involved in GD1a synthesis were not significantly different between the parental and the transfected cells. According to these data, the progression of tumor stage by the acquisition of autonomous cell growth ability after IL-3 gene transfection resulted in dramatic changes in cell surface gangliosides and their biosynthetic pathways. GD1a could be considered as an IL-3-associated ganglioside and was expressed in a tight connection with a single glycosyltransferase (GM3 synthase) up-regulation and with IL-3 expression in murine myelogenous leukemia cells.
Abstract: We described a case of adult T cell leukemia (ATL) not associated with human T-cell leukemia virus type I (HTLV-I), a clinical entity that was first reported by Shimoyama et al. A 79-year-old male was admitted with anorexia and fever in October, 1989. Physical examination revealed marked hepatosplenomegaly and superficial lymphadenopathies. Hematological examination revealed marked leukocytosis (136,300/microliters) with abnormal lymphoid cells showing highly lobulated nuclei. Hypercalcemia (11.2 mg/dl) and elevation of lactic dehydrogenase were also recognized. Surface marker analysis showed that the abnormal lymphoid cells in the peripheral blood were positive for CD2 and CD4 but negative for CD8. Southern blot analysis of the DNA from peripheral blood leukemic cells revealed monoclonal rearrangement of T-cell receptor beta-chain gene. The clinical and hematological findings of the patient were compatible with those of acute type ATL, however, serum anti-HTLV-I antibody was negative and HTLV-I proviral DNA was not detected in the leukemic cells by Southern blot analysis. Furthermore, the polymerase chain reaction showed no integration of the HTLV-I proviral DNA in the leukemic cells.
Abstract: A 65-year-old man, who had been treated for multiple myeloma (MM) since 1986, was admitted because of loss of consciousness in September 1989. An electrocardiogram taken just before admission showed a sinus arrest, junctional escaped rhythm, and marked bradycardia. The diagnosis of sick sinus syndrome (SSS) was made. Soon a temporary pacemaker was inserted, and the dyspnea ameliorated. However on the second day in the hospital, he had a high fever and Staphylococcus aureus was detected in the cultured blood. A diagnosis of septicemia was made, and the pacemaker was removed. He was then treated with beta-stimulants, but died in November 1989. Necropsy revealed cardiomegaly and microscopic examination showed amyloid deposits in the sinoatrial node, and the walls of the ventricles and coronary arteries. Although amyloidosis is often a complication of MM and the heart is frequently affected, SSS caused by amyloidosis associated with MM is quite unusual. In such patients, the use of a pacemaker is controversial, because amyloid deposits are occasionally accelerated by insertion of a pacemaker and for patients with hematological disorders, septicemia associated with pacemaker insertion may prove fatal.
Abstract: The role of acidic glycosphingolipids in cell growth and differentiation was investigated using the multipotent leukemia cell line K562. When GM3 was added to cell culture media, the growth of K562 cells was remarkably inhibited and the cells were shown to have megakaryocytoid morphology. Ultrastructural study demonstrated that K562 cells treated with GM3 had platelet peroxidase-positive structures, which were considered to be the specific marker of megakaryocyte. Furthermore, AP-3 directed against an epitope present on membrane glycoprotein IIIa reacted with the GM3-treated cells. Free N-acetylneuraminic acid, GM1, GM2, GD1a, and a mixture of bovine brain gangliosides containing GD1a and GT1b did not affect growth of K562 cells or show morphological changes. According to chemical analyses, GM3 content increased in megakaryocytoid differentiation induced by tetradecanoylphorbol-13-acetate, whereas GM3 decreased in erythroid differentiation induced by hemin. Enzymatic analysis showed that the GM3 increase during megakaryocytoid differentiation was a result of the sialyltransferase activation. These results indicated that exogenous GM3 induced differentiation of K562 cells into a "GM3-rich" lineage, i.e., mainly megakaryocytoid lineage, and that GM3 accumulation in the GM3-rich lineage was the result of the activation of GM3 synthase. These findings strongly suggested that GM3 ganglioside, a minor membrane component, has a crucial role in not only the differentiation induction but also the determination of the differentiation direction in pluripotent K562 cells.
Abstract: Neolacto-series gangliosides having linear poly-N-acetyl-lactosaminyl oligosaccharide structure have been demonstrated to be increased characteristically during granulocytic differentiation of human promyelocytic leukemia cell line HL-60 cells induced by dimethyl sulfoxide or retinoic acid (Nojiri, H., Takaku, F., Tetsuka, T., Motoyoshi, K., Miura, Y., and Saito, M. (1984) Blood 64, 534-541). When HL-60 cells were cultured in the presence of neolacto-series gangliosides prepared from mature granulocytes, the cells were found to be differentiated into mature granulocytes on the basis of the changes of morphology, surface membrane antigens, nonspecific esterase activity, and the activity of phagocytosis and respiratory burst. The differentiation of cells was dependent on the concentration of gangliosides and accompanied with inhibition of cell growth. These findings suggest that the particular ganglioside molecules play an important role in regulation of cell differentiation and that the appearance of neolacto-series gangliosides on cell surface membrane not only triggers the differentiation but also determines the direction of differentiation in HL-60 cells.
