ALBERT

Description: In a search for key molecules that prevent murine M1 leukemia cells from undergoing interleukin (IL)-6–induced differentiation into macrophages, we isolated an antisense complementary DNA (cDNA) that encodes full-length mouse MgcRac-GTPase-activating protein (GAP) through functional cloning. Forced expression of this antisense cDNA profoundly inhibited IL-6–induced differentiation of M1 cells into macrophage lineages. We also isolated a full-length human MgcRacGAP cDNA, which encodes an additional N-terminal polypeptide of 105 amino acid residues compared with the previously published human MgcRacGAP. In human HL-60 leukemic cells, overexpression of the full-length form of human MgcRacGAP alone induced growth suppression and macrophage differentiation associated with hypervacuolization and de novo expression of the myelomonocytic marker CD14. Analyses using a GAP-inactive mutant and 2 deletion mutants of MgcRacGAP indicated that the GAP activity was dispensable, but the myosin-like domain and the cysteine-rich domain were indispensable for growth suppression and macrophage differentiation. The present results indicated that MgcRacGAP plays key roles in controlling growth and differentiation of hematopoietic cells through mechanisms other than regulating Rac GTPase activity.

Description: Platelet transfusions are widely used for patients with severe thrombocytopenia. There are, however, practical problems in the current donor-dependent platelet transfusions, such as the limited supply and risk of serious immune reactions. Thus, the development of new strategies for generating platelets for transfusion is crucial. Platelets have been differentiated from hematopoietic stem cells, fetal liver cells, embryonic stem cells, induced pluripotent stem cells, NF-E2-transduced fibroblasts, and preadipocytes. Here, among these cells preadipocytes, especially in the subcutaneous adipose tissue, could be ideal candidate cells for manufacturing megakaryocytes (MKs) and platelets, because (1) they are relatively easy to obtain large quantities and have ability to proliferate in vitro, (2) their differentiation does not require gene transfer, as they possess genes in relation to megakaryopoiesis and thrombopoiesis, such as p45NF-E2 and c-mpl, and (3) they differentiate into MKs and platelets using an endogenous thrombopoietin. Thus, to clarify the usefulness of preadipocytes as a donor-independent source for platelet transfusion, we compared both number and function between platelets derived from mouse subcutaneous preadipocytes and those from bone marrow mononuclear cells (BMMNCs), the established cell source for manufacturing platelets. First, BMMNCs were not feasible for their expansion in vitro and therefore the cells were directly seeded in MK lineage induction media. In contrast, preadipocytes were to be passaged 6 times without any morphological changes, and then cultured in MK lineage induction media for their differentiation into platelets. Thus, as assessed by CD41-positive platelet-sized cells, 106.2±5.0 ×105 or 3.9±1.0 ×105 platelets were obtained from 106 preadipocytes or 106 BMMNCs, respectively (p

Description: Cutaneous T-cell lymphoma (CTCL) is a disease entity characterized by a primary sporadic T-cell proliferation in the skin. Human T-lymphotropic virus type 1 (HTLV-1) is a retrovirus that causes adult T-cell leukemia/lymphoma. Recently, several authors have detected the HTLV-1 genome in genomic DNA from patients with CTCL and proposed a causal relation of HTLV-1 to CTCL. However, it remains controversial because these studies contain some problems in materials used to detect HTLV-1. We investigated both fresh and cultured T lymphocytes (128 specimens) derived from 50 Japanese patients with CTCL, where HTLV-1 is endemic, by using polymerase chain reaction with four sets of primers including gag,pol,env, and pX regions of HTLV-1 to elucidate the relationship between HTLV-1 and CTCL in Japan. However, none of the 128 DNA specimens revealed positive for HTLV-1 in contrast to the previous studies. We conclude that CTCL, which does not include HTLV-1, is present although the pathogenesis of CTCL may be different by areas or races. © 1997 by The American Society of Hematology.

Description: Because of the difficulty in obtaining the sufficient amounts of hematopoietic stem cells from bone marrow, peripheral blood, and cord blood cells, studies of an in vitro experimental system that allows the production of abundant number of megakaryocytes (MKs) and platelets are currently the focus of research. Here, we present a novel system where human subcutaneous adipocytes were successfully differentiated into MK lineages in an in vitro liquid culture system. We also show that subcutaneous preadipocytes could be successfully transfected with vectors, to obtain modified MKs. Primary human subcutaneous preadipocytes (Cambrex Bio Science Walkersvile, Inc. Walkersville, MD, USA) were cultured in conditioned media to differentiate into mature adipocytes. Cells were cultured in serum-free media containing thrombopoietin for differentiation into MK lineages. The MKs or platelets were counted by flow cytometric analysis on day 14 using the relative value of CD41(+)/propidium iodide(+) cells or platelet size CD41(+) cells, respectively, versus 107 subcutaneous preadipocytes on day 0. The MK and platelet cell count was approximately 9600/ 107 and 2200/ 107, respectively. Morphological analysis with electron microscopy demonstrated that MKs, which had typical organelles such as granules, demarcation membrane, and nuclei, and platelets, which had typical contents such as granules, mitochondria, and open canalicular system, were successfully obtained from subcutaneous preadipocytes. We then attempted to transfect subcutaneous preadipocytes with vectors to obtain transformed MKs. Two glycoprotein (GP) Ib alpha polymorphisms, 145Thr/Met and 1–4 repeats of variable number tandem repeat of 13 amino-acid sequences, were used as the marker of gene transfer, which were detected by PCR-restricited fragment length polymorphism (RFLP) and Western blot analysis, respectively. Subcutaneous preadipocytes with the 145Thr/Thr and 1 repeat sequences were transfected with the expression vector carrying GPIb alpha with the 145Met and 4 repeats sequences. PCR-RFLP analysis with gel electrophoresis was performed on each RNA sample from the expression vector-transfected and non-transfected cells. Non-transfected cell sample had bands corresponding to the 145Thr sequence position, while the expression vector-transfected cell sample had bands corresponding to both the 145Thr and Met sequences. Western blot analysis with an anti-GPIb alpha monoclonal antibody, LJ-Ib alpha1 (a generous gift from Dr. ZM Ruggeri, The Scripps Research Institute, La Jolla, CA, USA), showed bands of the expected size corresponding to 1R and 1R/4R. In summary, we established an in vitro culture system to produce MKs and platelets from subcutaneous adipocytes. We were also able to obtain transfected MKs from subcutaneous preadipocytes.

Description: CD47 belongs to immunoglobulin superfamily, and is expressed as a 50 kDa cell surface antigen in wide variety of tissues. CD47 associates with integrins of β1, β2, and β3, and it serves as a receptor for thrombospondin (TSP) and also as a ligand for transmembrane signal regulatory protein SIRP-α. CD47 has a number of different functions such as platelet activation, cell motility, leukocyte adhesion and migration. Recently, it was reported that the ligation of CD47 induces cell death in T-cells and chronic lymphocytic leukemic B cells (B-CLL) in a caspase-independent manner. B-CLL is the most common hematological malignancy in Western countries. Although new chemotherapeutic agents including fludarabine and 2-chlorodeoxyadenosine have been introduced into the clinic, B-CLL is not curable, and therefore, there is a strong need of new effective drugs. In an attempt to establish a novel therapeutic agent for B-CLL, we generated a murine monoclonal antibody against an extracellular domain of human CD47 (designated MABL). Soluble MABL (10 μg/ml) induced apoptosis in CD47-positive CCRF-CEM and MOLT-4 cells but not in CD47-negative cells as judged by annexin V staining. Caspase-3 was not activated by MABL, confirming that the cell death mediated by CD47 was caspase independent. In addition, administration of the F(ab’)2 of MABL (200 μg/mouse twice a day on day 3, 4 and 5) significantly prolonged the survival of the SCID mice inoculated CCRF-CEM (〉150 days in MABL-treated group vs.

Description: Plasma cell myeloma (PCM) is a mature B cell malignancy characterized by monoclonal gammopathy and destructive bone lesions. Although recent development of therapeutic drugs including thalidomide or proteasome inhibitor may prolong survival time, PCM is still an incurable disease. To improve the treatment outcome of PCM, the development of novel, pathophysiology-based therapies are needed. Histone acetylation plays a role in transcriptional regulation by altering the chromatin structure, which allows the transcription factors to access the DNA. Histone acetyltransferases (HATs) are involved in tumor suppression and inhibition of histone deacetylases (HDACs) results in antitumor effect. HDAC inhibitors have also reported to induce apoptosis or differentiation of tumor cells. Trichostatin A (TSA) is one of the HDAC inhibitors, and it has shown to be effective in vitro against leukemic cells with characteristic chromosomal translocations. Although none of the disease-specific genetic alterations involving aberrant transcription factors in PCM have been reported, previous studies have demonstrated the antitumor effect of HDAC inhibitors on PCM. Accordingly, to address the detailed molecular mechanisms of HDAC inhibitor-induced apoptosis, we examined the in vitro effects of TSA on PCM cell lines. By using flow cytometric analyses, we found that cell cycle arrest at G1 phase and apoptosis were induced in various PCM cell lines, RPMI8226, IM9, and U266 treated with 100nM of TSA for 24 hours. Especially, the viability of TSA-treated RPMI8226 cells was strongly suppressed to approximately 30% compared to the cells without TSA. Nuclear factor (NF) -κB is thought to be a key molecule of cellular growth in PCM; therefore, we studied the functional alterations of NF-κB in PCM cell lines stimulated with TSA. Immunoblot and electrophoretic mobility shift assays demonstrated that TSA augmented the nuclear localization of NF-κB, which resulted in the increased DNA binding activity and upregulation of downstream target gene expression such as ICAM-1. Increased phosphorylation of Iκ-Bα by TSA was observed simultaneously. Transient transfection assays showed that the transcriptional activity of NF-κB was upregulated more than 4-fold by TSA, and the synergistic effects of TSA with TNF-α or PMA were observed. Interestingly, an NF-κB-specific inhibitor, SN50, induced apoptosis of PCM cell lines, and TSA showed a synergism with SN50. These data may provide a notion that increased activity of NF-κB by TSA could be a compensatory response against proapoptotic signals induced by TSA. Additionally, we investigated other molecules responsible for TSA-stimulated apoptosis, and found the downregulation of Mcl-1, Bcl-XL, XIAP proteins, as well as dephosphorylation of Akt and cleavage of Bid proteins in a time-dependent manner. These results suggest that either PI 3-K/Akt pathway or JNK-dependent pathway could be involved in the TSA-induced apoptosis. In conclusion, HDAC inhibitors will be promising agents to improve survival of patients with PCM, and the combination of HDAC inhibitor and NF-κB inhibitor may provide a novel therapeutic strategy against PCM.

Description: Human interferon (IFN)-α is a standard treatment for the patients with chronic hepatitis C. It is well known that chronic hepatitis C can transform into liver cirrhosis and hepatocellular carcinoma, and the risk of progression to liver cirrhosis is around 40% in 5 years. Thus, it is important for chronic hepatitis C patients to receive IFN-α treatment to prevent its malignant transformation. Thrombocytopenia is one of the major adverse effects of IFN-α and often leads to a dose reduction or discontinuation of IFN-α therapy. However, there is little information how IFN-α inhibits human megakaryopoiesis. In this study, we demonstrated that IFN-α does not inhibit colony formation of megakaryocytes (CFU-MK) from human CD34-positive hematopoietic stem cells. IFN-α also does not inhibit endomitosis (DNA duplication without cytokinesis), but inhibits cytoplasmic maturation of megakaryocytes and platelet production in vitro. The inhibitory effects of IFN-α on the development of demarcation membrane in human megakaryocytes were visualized by staining primary megakaryocytes with di-8-ANEPPS dye under a confocal laser microscope. Loss of platelet production by IFN-α was analyzed by measuring proplatelet formation (PPF) and counting the human platelets produced in the supernatant of human primary megakaryocytes by flow cytometry. The expression of transcription factors regulating late stage megakaryopoiesis such as GATA-1, p45 NF-E2 and MafG was suppressed by IFN-α. To confirm these in vitro observations in vivo system, we transplanted human CD34-positive hematopoietic cells into the immunodeficient NOD/Shi-scid/IL-2Rγnull (NOG) mice (hu-NOG) after 2.4 Gy irradiation. Recombinant human(rh) IFN-α significantly reduced the number of human platelets but did not decrease the number of human megakaryocytes in hu-NOG mice. rhIFN-α did not change murine platelet counts in hu-NOG mice, as IFN-α has species specificity. The DNA ploidy of human megakaryocytes in the bone marrow of hu-NOG mice was not altered after treatement with rhIFN-α. We also confirmed the life time of human platelets was not shortend by rhIFN-α in hu-NOG mice, indicating IFN-α does not promote the clearance of human platelets. We have also confirmed that a novel thrombopoietin mimetics, NIP-004, prevented rhIFN-α-induced thrombotytopenia and increased the number of polyploid human megakaryocytes in hu-NOG mice, suggesting NIP-004 might be useful for the patients with hepatitis C to avoid IFN-α-induced thrombocytopenia. In conclusion, these results clarified that IFN-α induces thrombocytopenia by inhibiting the cytoplasmic maturation of megakaryocytes but not proliferation and endomitosis in human megakaryocytes.

Description: Multiple myeloma is a plasma cell malignancy that remains incurable despite the use of conventional and high dose chemotherapy with hematopoietic stem cell transplantation, and so novel therapeutic approaches are urgently required in the clinical settings. Recent understanding of the biology of myeloma has led to the development of biological treatments such as thalidomide and bortezomib, which target the myeloma cell and the bone marrow microenvironment. These agents have shown remarkable activity against refractory multiple myeloma in the early clinical trials, but prolonged drug exposures may result in the development of de novo drug resistance in some cases. Therefore, identification and validation of more novel targeted therapies to overcome drug resistance and improve patient outcome of multiple myeloma will be needed. Recently, green tea attracted much attention due to its beneficial health effects; the polyphenolic compound, (−)-epigallocatechin-3-gallate (EGCG), has potent chemopreventive effects against various tumors. EGCG has been shown to inhibit cellular proliferation and induce apoptosis of various cancer cells. The aim of this study was to investigate the possibility of EGCG as a novel therapeutic agent for the patients with multiple myeloma. EGCG rapidly induced apoptotic cell death in various myeloma cell lines (U266, IM9, RPMI 8226, and HS-Sultan) and fresh myeloma cells from patients in a dose (0–100 μM)- and time (0–72 h)-dependent manner. IM9 cells were most sensitive to EGCG with an IC50 of 17 μM, and cell growth was suppressed as early as 6 h and the typical morphological appearance of apoptosis was observed. Treatment with EGCG (20 μM) increased the population of cells in the G0/G1 phase with the reduction of S phase followed by the appearance of a sub-G1 DNA contents, indicating that EGCG led to cell cycle arrest at G1 phase followed by apoptosis. EGCG-induced apoptosis in myeloma cells was in association with the loss of mitochondrial transmembrane potentials (Δψm), the release of cytochrome c, Smac/DIABLO and AIF from mitochondria into the cytosol, and the activation of caspase-3 and -9. Treatment with 20 μM EGCG for 1 h in IM9 cells as well as fresh myeloma cells from patients showed rapid elevation of intracellular reactive oxygen species (ROS) production. Antioxidant, catalase and Mn-SOD completely blocked ROS generation, the loss of Δψm, caspase-3 activation and consequently inhibited EGCG-induced apoptosis in IM9 cells, suggesting that ROS plays a key role in EGCG-induced apoptosis in myeloma cells. Recently, arsenic trioxide (AS) was reported to inhibit the proliferation of myeloma cells by induction of apoptosis via intracellular production of ROS. Therefore, we further tested the possibility of using an ROS-producing agent, EGCG, to enhance the activity of AS. The combination with AS and EGCG significantly enhanced induction of apoptosis compared to AS or EGCG alone via decreased intracellular GSH levels and increased production of ROS in all myeloma cells, suggesting that EGCG potentiated AS-induced cytotoxicity. In conclusion, EGCG has potential as a novel therapeutic agent for patients with multiple myeloma via induction of apoptosis mediated by modulation of the molecules of the redox system. Furthermore, the combination of EGCG and ROS-producing agents such as AS may provide a new strategy to enhance therapeutic activity for the patients with multiple myeloma.

Description: Bone morphogenetic proteins (BMPs), members of the transforming growth factor (TGF)–β superfamily, are a group of related proteins that are capable of inducing the formation of cartilage and bone but are now regarded as multifunctional cytokines. We show in this report a novel function of BMPs in hematopoietic cells: BMP-2 induces apoptosis not only in human myeloma cell lines (U266, RPMI 8226, HS-Sultan, IM-9, OPM-2, and KMS-12 cells), but also in primary samples from patients with multiple myeloma. The mechanism of BMP-2–induced apoptosis was investigated with the use of U266 cells, which are dependent on the interleukin-6 autocrine loop. We showed that BMP-2 caused cell-cycle arrest in the G1 phase and the subsequent apoptosis of myeloma cells. BMP-2 up-regulated the expression of cyclin-dependent kinase inhibitors (p21CIP1/WAF1 and p27KIP1) and caused hypophosphorylation of retinoblastoma (Rb) protein. In studies of apoptosis-associated proteins, BMP-2 was seen to down-regulate the expression of Bcl-xL; however, BMP-2 had no effects on the expression of Bcl-2, Bax, or Bad. Therefore, BMP-2 induces apoptosis in various human myeloma cells by means of the down-regulation of Bcl-xL and by cell-cycle arrest through the up-regulation of p21CIP1/WAF1 and p27KIP1 and by the hypophosphorylation of Rb. Further analysis showed that the signal transducer and activator of transcription 3 (STAT3) was inactivated immediately after BMP-2 treatment. We conclude that BMP-2 would be useful as a novel therapeutic agent in the treatment of multiple myeloma both by means of its antitumor effect of inducing apoptotis and through its original bone-inducing activity, because bone lesions are frequently seen in myeloma patients.

Description: Multiple myeloma is an incurable hematological malignancy of plasma cells despite advances in conventional chemotherapy or high dose chemotherapy with stem cell transplantation. Recent advances in the biological treatment such as thalidomide and bortezomib will offer great promise to improve the outcome of refractory or relapsed patients with multiple myeloma; therefore, more novel biologically based therapies targeting both myeloma cells and its bone marrow microenvironment are urgently required. Shikonin, a Chinese herb derived from purple gromwell, Boraginaceae Lithospermum erythrorhizon, is previously known to have anti-inflammatory, anti-tumor, and anti-pyretic properties. Several studies have shown that Shikonin induces p53-mediated cell cycle arrest and apoptosis in human malignant melanoma cells, and induces apoptosis in human myeloid leukemic HL60 cells. However, none of the studies have concerned in the effects of Shikonin on myeloma cells. In this study, we investigated the possibilities to develop Shikonin as a novel promising agent for multiple myeloma. Shikonin suppressed the cellular growth of RPMI8226 and IM9 myeloma cells, via induction of apoptosis in a dose (0–1 μM)- and time (0–24 h)-dependent manner. Apoptotic cell death was induced in both RPMI8226 and IM9 cells within 3 h for treatment with only 0.5 μM of Shikonin, and finally 78% of RPMI8226 cells and 91% of IM9 cells were dead by apoptosis within18 h. Treatment with 0.5 μM Shikonin rapidly increased the population of cells in the G0/G1 phase with reduction of cells in the S phase, and then induction of apoptosis was confirmed by the appearance of cells in the sub-G1 fraction. Shikonin-induced apoptosis was in association with the loss of mitochondrial transmembrane potentials, and activation of caspase-3. Shikonin-induced apoptosis was completely blocked by the treatment with pan-caspase inhibitor (20 μM, Z-VAD), suggesting that caspase plays an important role in Shikonin-induced cell death in myeloma cells. Expression of p53 and Bax proteins was increased with down-regulation of Mcl-1 protein, but no changes were confirmed of the expression of Bcl-2 and Bcl-XL. Recent investigations have shown that cytokines such as IL-6, insulin growth factor (IGF), VEGF, and TNF-α mediate myeloma cell growth, survival and migration. IGF-1 increases survival of myeloma cells by activating various down-stream signal transduction molecules. Recently, inhibitors of IGF-1 receptor demonstrate promising anti-multiple myeloma activity in preclinical studies. Shikonin has reported to be an inhibitor of protein tyrosine kinase such as EGFR, v-Src, and KDR/Flk-1. To address the mechanism of Shikonin-induced apoptosis in myeloma cells, we thus examined the effects of Shikonin on the phosphorylation of IGF-1 receptor. IGF-1 (100 ng/ml) stimulated the proliferation of both RPMI8226 and IM9 cells. Interestingly, Shikonin (0.5 μM) overcame IGF-1-induced cell proliferation, and inhibited proliferation of myeloma cells via induction of apoptosis. Shikonin inhibited phosphorylation of IGF-1 receptor as early as 30 min with inhibition of PI3K/Akt signaling. These results suggest that Shikonin-induced apoptosis in myeloma cells was mediated by inhibiting phosphorylation of IGF-1 receptor and modulating its down-stream signaling pathway. In conclusion, Shikonin inhibited cellular growth by inhibiting IGF-1 receptor signaling in myeloma cells, and may have a potential as a novel biologically based therapeutic agent for the patients with multiple myeloma..