Kentaro Kinjo

Arsenic trioxide (As2O3)-induced apoptosis and differentiation in retinoic acid-resistant acute promyelocytic leukemia model in hGM-CSF-producing transgenic SCID mice.

Recent clinical studies in China and USA showed that arsenic trioxide (As2O3) is an effective treatment of acute promyelocytic leukemia (APL) patients refractory to all-trans retinoic acid (RA). We here investigate the effects of As2O3on RA-resistant APL in vivo and in vitro using our RA-resistant APL model system. As2O3 can induce inhibition of cellular growth of both RA-sensitive NB4 and RA-resistant UF-1 APL cells via induction of apoptosis in vitro. The expression of BCL-2 protein decreased in a dose- and time-dependent manner in NB4 cells. Interestingly, the levels of BCL-2 protein were not modulated by As2O3, but it did upregulate BAX protein in UF-1 cells. UF-1 cells (1 × 107) were transplanted into hGM-CSF-producing transgenic SCID mice and successfully formed subcutaneous tumors. After 40 days of implantation, mice were treated with As2O3, all-trans RA and PBS for 21 days. In all-trans RA- and PBS-treated mice, tumors grew rapidly, with a 4.5-fold increase in volume at day 21 compared to the initial size. In marked contrast, tumor size was decreased to half of the initial size by the treatment of As2O3, which resulted in cells with the typical appearance of apoptosis. Interestingly, one of the As2O3-treated mice showed mature granulocytes in the diminished tumor, suggesting that As2O3 had dual effects on RA-resistant APL cells in vivo: both inducing apoptosis and differentiation of the leukemic cells. We conclude that our RA-resistant APL model will be useful for evaluating novel therapeutic approaches to patients with RA-resistant APL, and for further investigation of the metabolism of As2O3 in vivo.

Two distinct stem cell lineages in murine bone marrow.

Mesenchymal stem cells (MSC), a distinct type of adult stem cell, are easy to isolate, culture, and manipulate in ex vivo culture. These cells have great plasticity and potential for therapeutic application, but their properties are poorly understood because of their low frequency and the lack of knowledge on cell surface markers and their location of origin. The present study was designed to address the undefined lineage relationship of hematopoietic and mesenchymal stem cells. Genetically marked, highly purified hematopoietic stem cells (HSCs) were transplanted into wild‐type animals and, after bone marrow repopulation, the progeny were rigorously investigated for differentiation potential into mesenchymal tissues by analyzing in vitro differentiation into mesenchymal tissues. None/very little of the hematopoietic cells contributed to colony‐forming units fibroblast activity and mesenchymal cell differentiation; however, unfractionated bone marrow cells resulted in extensive replacement of not only hematopoietic cells but also mesenchymal cells, including MSCs. As a result, we concluded that purified HSCs have no significant potency to differentiate into mesenchymal lineage. The data strongly suggest that hematopoietic cells and mesenchymal lineage cells are derived from individual lineage‐specific stem cells. In addition, we succeeded in visualizing mesenchymal lineage cells using in vivo microimaging and immunohistochemistry. Flow cytometric analysis revealed CD140b (PDGFRβ) could be a specific marker for mesenchymal lineage cells. The results may reinforce the urgent need for a more comprehensive view of the mesenchymal stem cell identity and characteristics.

The Role of CREB as a Proto-oncogene in Hematopoiesis

Cyclic-AMP response element binding protein (CREB) is a transcription factor that functions in glucose homeostasis, growth-factor- dependent cell survival, proliferation and memory. Signaling by hematopoietic growth factors, such as GM-CSF, results in activation of CREB and upregulation of CREB target genes. Data from our laboratory shows that a majority of patients with acute lymphoid and myeloid leukemiaoverexpress CREB in the bone marrow. CREB overexpression is associated with poor initial outcome of clinical disease in AML patients. To study its role in hematopoiesis, we overexpressed CREB in leukemia cell lines and in mice. CREB overexpression resulted in increased survival and proliferation of myeloid cells and blast-transformation of bone marrow progenitor cells from transgenic mice expressing CREB in the myeloid lineage. CREB transgenic mice also develop myeloproliferative disease after one year. Thus, CREB acts as a proto-oncogene to regulate hematopoiesis and contributes to the leukemia phenotype. Our results suggest that CREB-dependent pathways may serve as targets for directed therapies in leukemia in the future.

Novel mutation in the PML/RARα chimeric gene exhibits dramatically decreased ligand-binding activity and confers acquired resistance to retinoic acid in acute promyelocytic leukemia

OBJECTIVE All-trans retinoic acid (RA) resistance in acute promyelocytic leukemia (APL) has been a serious clinical problem in differentiation-inducing therapy. However, the mechanisms underlying acquired RA resistance in APL patients are not well understood. MATERIALS AND METHODS We recently established a spontaneous RA-resistant APL cell line (UF-1) from a patient and used this cell line as an excellent in vitro model for RA-resistant clinical situations. We investigated the structural and functional abnormalities of chimeric PML/RARalpha gene in UF-1 cells and preserved materials from the original patient. RESULTS A novel point mutation was detected in the ligand-binding (E) domain of the RARalpha portion of the PML/RARalpha gene in UF-1 cells. This mutation resulted in amino acid substitution of Arg611 (CGG) for Trp611 (TGG) in the short-form PML/RARalpha protein, which corresponded to Arg276 in wild-type RARalpha. Importantly, the same mutation was also detected in the preserved materials from the original patient. COS-1 cells were transiently transfected with cDNA encoding wild-type and mutant PML/RARalpha constructed by site-directed mutagenesis and performed RA-binding assay. Interestingly, RA-binding activity was dramatically decreased in the mutant PML/RARalpha compared with that of the wild-type chimeric protein, suggesting that this single amino acid substitution is critical for RA binding. CONCLUSIONS These results strongly suggest that a novel point mutation in the ligand-binding domain of the RARalpha portion (Arg611) of the chimeric PML/RARalpha gene decreased sensitivity to all-trans RA. We conclude that acquisition of the PML/RARalpha mutation is one possible mechanism for development of RA resistance in patients with APL in vivo.

Novel variant isoform of G-CSF receptor involved in induction of proliferation of FDCP-2 cells: relevance to the pathogenesis of myelodysplastic syndrome.

Recent studies have shown that point mutations in granulocyte colony‐stimulating factor receptor (G‐CSFR) are involved in the pathogenesis of severe congenital neutropenia (SCN) and in the transformation of SCN to acute myelogenous leukemia (AML). It is reasonably speculated that the abnormalities in the signal transduction pathways for G‐CSF could be partly responsible for the pathogenesis and the development to AML in patients with myelodysplastic syndromes (MDS). Therefore, we investigated the structural and functional abnormalities of the G‐CSFR in 14 patients with MDS and 10 normal subjects. In in vitro colony forming assay, MDS samples showed reduced response to growth factors. However, G‐CSF, but not GM‐CSF and IL‐3, enhanced clonal growth in three cases of high risk patients with MDS (RAEB, RAEB‐t, and MDS having progressed to acute myeloid leukemia (AML)) and one low risk patient (RA). Eight out of 14 patients including above 4 patients demonstrated a common deletion of the G‐CSFR cDNA; a deletion of three nucleotides (2128–2130) in the juxtamembrane domain of the G‐CSFR, which resulted in a conversion of Asn630Arg631 to Lys630. To assess the functional activities of this deletion in the G‐CSFR isoform, a mutant with the same three‐nucleotide deletion was constructed by site‐directed mutagenesis. FDCP‐2 cells expressing the G‐CSFR isoform responded to G‐CSF, and exhibited proliferative responses than did those cells having wild‐type G‐CSFR. Moreover, these isoforms showed prolonged activation of STAT3 in response to G‐CSF than did the wild‐type. These results suggest that the deletion in the juxtamembrane domain of the G‐CSFR gives a growth advantage to abnormal MDS clones and may contribute to the pathogenesis of MDS.

Serum thrombopoietin and erythropoietin levels in patients with acute promyelocytic leukaemia during all-trans retinoic acid treatment

Endogenous serum thrombopoietin (TPO) and various cytokines including erythropoietin (EPO), interleukin (IL)‐3, IL‐6, IL‐11, granulocyte‐colony stimulating factor (G‐CSF), granulocyte‐macrophage‐colony stimulating factor (GM‐CSF) and stem cell factor (SCF) levels were measured in five patients with acute promyelocytic leukaemia (APL) during all‐trans retinoic acid (RA) treatment. During differentiation‐inducing therapy, platelet counts slowly increased and reached a peak between days 29 and 46 (median day 35). Serum TPO levels increased parallel to the increasing platelet counts and reached a maximum level during the first 10–20 d of all‐trans RA treatment. The circulating TPO levels then decreased in inverse correlation to the platelet counts. These unique changes in serum TPO levels revealed that TPO levels were not regulated by platelet or megakaryocyte mass in patients with APL during differentiation‐inducing therapy, and it would appear that TPO levels are directly regulated by all‐trans RA during the first 10–20 d of treatment. In addition, the change in circulating EPO levels and reticulocyte counts were similar to that of the TPO levels and platelet counts during all‐trans RA treatment, suggesting a close relationship between TPO and EPO signalling.

155 THE CYCLIC ADENOSINE MONOPHOSPHATE RESPONSE ELEMENT BINDING PROTEIN REGULATES HEMATOPOIETIC PROGENITOR CELL PROLIFERATION AND MYELOID ENGRAFTMENT

Purpose The cAMP response element binding protein (CREB) is a transcription factor that regulates gene expression in a variety of cell types and promotes cell proliferation and survival. We previously reported that more than 60% of AML patients overexpressed CREB in the bone marrow. To understand the role of CREB in myelopoiesis, we generated transgenic mice in which CREB is overexpressed in myeloid cells. We analyzed the hematopoietic progenitor cells from CREB transgenic mice in methylcellulose colony and bone marrow transplantation assays. Methods Bone marrow cells obtained from hMRP8-CREB transgenic mice were plated in methylcellulose containing IL-3, IL-6, and SCF. After 14 days the colonies were counted and analyzed using FACs and cytospin preparations. Bone marrow cells (4 ×106) from CREB transgenic mice were transplanted into lethally irradiated wild type C57/BL6 recipient mice. Peripheral blood counts were obtained every 4 weeks and FACs analysis was performed. Results CREB transgenic mice showed evidence of monocytosis, compared to age-matched littermate controls. Bone marrow cells from CREB transgenic mice formed robust colonies earlier and had increased numbers of colony forming units (CFU-GM). Bone marrow from CREB transgenic mice also had evidence of more immature myeloid cells compared to controls. We observed a 10-fold increase in the numbers of bone marrow progenitor cells from CREB transgenic mice (two different founder lines) compared to controls when cultured in the absence of cytokines. In bone marrow transplant experiments, mice transplanted with CREB transgenic mouse bone marrow had signs of earlier myeloid engraftment at 6 weeks following transplantation compared to controls. Recipients of CREB transgenic bone marrow showed increased monocytes and neutrophils in the peripheral blood with a corresponding increase in Mac-1+, Gr-1+ cell populations. The lymphocyte count was significantly lower in mice transplanted with CREB transgenic bone marrow compared to controls. Conclusions Our results suggest that CREB plays a critical role in the regulation of normal myelopoiesis and hematopoietic progenitor cell proliferation.