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Summary The effect of CMC-544, a calicheamicin-conjugated anti-CD22 monoclonal antibody, was analysed in relation to CD22 and P-glycoprotein (P-gp) in B-cell chronic lymphocytic leukaemia (CLL) and non-Hodgkin lymphoma (NHL) in vitro. The cell lines used were CD22-positive parental Daudi and Raji, and their P-gp positive sublines, Daudi/MDR and Raji/MDR. Cells obtained from 19 patients with B-cell CLL or NHL were also used. The effect of CMC-544 was analysed by viable cell count, morphology, annexin-V staining, and cell cycle distribution. A dose-dependent, selective cytotoxic effect of CMC-544 was observed in cell lines that expressed CD22. CMC-544 was not effective on Daudi/MDR and Raji/MDR cells compared with their parental cells. The MDR modifiers, PSC833 and MS209, restored the cytotoxic effect of CMC-544 in P-gp-expressing sublines. In clinical samples, the cytotoxic effect of CMC-544 was inversely related to the amount of P-gp (P = 0·003), and to intracellular rhodamine-123 accumulation (P

We studied the effect of CMC-544, the calicheamicin-conjugated anti-CD22 monoclonal antibody, used alone and in combination with rituximab, analyzing the quantitative alteration of target molecules, that is, CD20, CD22, CD55 and CD59, in Daudi and Raji cells as well as in cells obtained from patients with B-cell malignancies (BCM). Antibody inducing direct antiproliferative and apoptotic effect, complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) were tested separately. In Daudi and Raji cells, the CDC effect of rituximab significantly increased within 12 h following incubation with CMC-544. The levels of CD22 and CD55 were significantly reduced (P

地方病院の輸血部門においては，安全性や経済性を考慮した積極的な取り組みは重要な課題である．本研究では大量輸血への対応体制を構築し，術者，輸血部門そして血液センター（BC）との連絡を緊密にし，輸血製剤の有効利用を目指すとともに術者の安心感を得ようとした．BC側には通常の輸血用血液の時間的，量的な情報以外に緊急性と追加発注の可能性を伝えた．術者には各血液製剤がどのくらいの時間で術者の手元に届くかを明確にした．対象は赤血球で約10単位以上の大量輸血が必要とされる手術例99例で，心臓血管手術が94%を占めた．血液の追加発注は30%に認められ，供給上の問題点を認めなかった．術者の理解とともに，大量輸血対応は順調に増加し，82%の外科医の安心感を向上させた．また輸血の返納血は有意に減少し，製剤単位数から算出された全体廃棄率は1.6%から0.4%にまで低下した．病院内の輸血部門の重要性を再確認し，地方の輸血供給にも配慮した視点に立った体制を構築していく必要性がある．

CEM, MOLT4 and SUP-B15 cells were transduced with lentivirus-mediated siRNA KIS gene. The mRNA expressions of KIS were successfully reduced in all cell lines. On the other hand, the mRNA expressions of p27(Kip1) in CEM, MOLT4 and SUP-B15 cells were not affected by the transduction with siRNA KIS gene. We showed that KIS protein directly interacted with p27(Kip1) protein, and reduction of KIS inhibited the S10 phosphorylation of p27(Kip1) in leukemia cells. On these cells transfected with siRNA KIS, the inhibition of S10 phosphorylation of p27(Kip1) was strongly suppressed cell proliferation in a time-dependent manner. Moreover, the inhibition of S10 phosphorylation of p27(Kip1) increased a significant population in G0/G1 fraction. These data demonstrated that the KIS activity was induced during G0/G1, and it promotes cell cycle progression by phosphorylation of S10 on p27(Kip1). We showed that KIS mRNA expression was increased in primary leukemia specimens (acute myelogenous leukemia (AML); 37, myelodysplastic syndrome (MDS); 72, acute lymphoblastic leukemia (ALL); 23), and the mean ratios of KIS to G3PDH in AML, MDS and ALL specimens were 3.62+/-0.68, 3.27+/-0.73 and 3.17+/-0.58, respectively. Moreover, we found that KIS protein was overexpressed in all 132 adults cases of various leukemias, including 37 AML (8 M1, 12 M2, 2 M3, 7 M4, 8 M5), 72 MDS (42 RAEB-I, 30 REAB-II) and 23 ALL (23 L2). This study demonstrates that the elevated levels of KIS protein in leukemia cells promote the cell cycle progression in leukemia cells.

[Background] Homeobox (Hox) genes are grouped together in 4 clusters, A to D. Recent studies has shown that the Hox proteins are important in the control of differentiation and proliferation in hematopoietic cells. We found that the Abl kinase inhibitors increased the expression of HoxA10 gene in CML cells. In this study, we analyzed the role of HoxA10 in CML cell lines and the hematopoietic progenitor cells derived from CML patients by inhibiting the expression of HoxA10. Moreover, we investigated whether the regulation of HoxA10 eradicate Ph+ hematopoietic stem/progenitor cells, which are the targets for leukemic transformation in CML. [Methods] We used AMN107 and BMS354825 for the Abl kinase inhibitors, LY294002 for a PI3K inhibitor, PP2 for a Src kinase inhibitor, and SB203580 for a p38 MAP kinase inhibitor. For analysis of HoxA10 mRNA and protein, RT-PCR and western blot were performed in K562, Meg-01 and U937 cells, which untreated or treated with AMN107, BMS354825, LY294002, PP2, or SB203580 respectively. We then attempted to localize the intracellular locations of HoxA10 in K562 and Meg01, which untreated or treated with AMN107, BMS354825, or LY294002 by using conforcal fluorescence microscopy. For analysis of proliferation in K562, Meg-01 and U937 transfected with siRNA HoxA10, MTT assays were performed in untransfected or transfected K562, Meg-01 and U937 treated with or without AMN107, BMS354825, or LY294002. Finally, we counted the colony numbers of CFU-GEMM, CFU-GM, and BFU-E in K562 and Meg-01 treated with the Abl kinase inhibitors or LY294002. Results Both K562 and Meg01 cells expressed HoxA10 mRNA and protein at lower level compared to U937 cells. Interestingly, treatment with AMN107, BMS354825, or LY294002 increased the expression of HoxA10 mRNA and protein in both K562 and Meg01 cells. The fluorescence of HoxA10 was more strongly observed in the area corresponding to the cell’s cytoplasm than nucleus, and the treatment with AMN107, BMS354825, or LY294002 increased the fluorescence in nucleus of K562 and Meg01 cells in a time-dependent manner. In K562 and Meg01 cells transfected with the siRNA HoxA10, treatment with AMN107 or BMS354825 slightly inhibited the proliferation compared to K562 and Meg01 transfected with control siRNA. Finally, we showed that the inhibition of HoxA10 expression by siRNA increased the numbers of CFU-GEMM, BFU-E, and CFU-GM when the cells were treated with the combination of BMS354825 and LY294002 compared to control cells. [Conclusions] In this study, we showed that the Abl kinase inhibitors induced the expression of HoxA10, and HoxA10 was regulated by PI3K pathway in CML cells. This finding indicates a new insight in the regulation of cell proliferation via the PI3K signal pathway in CML cells. Moreover, we found the role of HoxA10 in CML cell lines and the hematopoietic progenitor cells derived from CML patients by inhibiting the expression of HoxA10. We showed that the regulation of HoxA10 eradicated Bcr-Abl+ hematopoietic stem/progenitor cells, which are the targets for leukemic transformation in CML.

We describe 2 patients with acute promyelocytic leukemia (APL) in whom torsade de pointes (TdP) developed during treatment with arsenic trioxide. Patient 1 was a 23-year-old woman with second-relapse APL. Ventricular premature beat bigeminy developed on day 27 of treatment, and episodes of TdP developed on day 28. Patient 2 was a 51-year-old woman with second-relapse APL who had cardiomyopathy due to prior anthracycline treatment. TdP developed on day 17 of treatment. Arsenic trioxide is known to cause electrocardiographic abnormalities, such as ventricular tachycardia and prolongation of QT interval. Patient 1 was given fluconazole as a concomitant drug. Patient 2 had cardiomyopathy and hypokalemia. Careful management is needed during arsenic trioxide therapy because this treatment prolongs the QT interval, possibly inducing episodes of TdP.

Acute promyelocytic leukemia (APL) cells express a considerable level of CD33, which is the target of gemtuzumab ozogamicin (GO), and a significantly lower level of P-glycoprotein (P-gp). Therefore, GO is predicted to be a successful treatment for APL. In this article, we report on the GO treatment of 2 patients with APL, who had fully relapsed after induction therapy with all -trans retinoic acid (ATRA) following chemotherapy. Both patients had relapsed 3 times and were resistant to reinduction therapy with ATRA. GO (9 mg/m2) was administered on days 1 and 15. After GO treatment, both patients achieved complete hematologic and molecular remission. GO may be another promising agent for the treatment of ATRA-resistant relapsed APL when given as salvage chemotherapy.

Acute promyelocytic leukemia (APL) cells express a considerable level of CD33, which is a target of gemtuzumab ozogamicin (GO), and a significantly lower level of P-glycoprotein (P-gp). In this study, we examined whether GO was effective on all-trans retinoic acid (ATRA)- or arsenic trioxide (ATO)-resistant APL cells. Cells used were an APL cell line in which P-gp was undetectable (NB4), ATRA-resistant NB4 (NB4/RA), NB4 and NB4/RA that had been transfected with MDR-1 cDNA (NB4/MDR and NB4/RA/MDR, respectively), ATO-resistant NB4 (NB4/As) and blast cells from eight patients with clinically ATRA-resistant APL including two patients with ATRA- and ATO-resistant APL. The efficacy of GO was analyzed by (3)H-thymidine incorporation, the dye exclusion test and cell cycle distribution. GO suppressed the growth of NB4, NB4/RA and NB4/As cells in a dose-dependent manner. GO increased the percentage of hypodiploid cells significantly in NB4, NB4/RA and NB4/As cells, and by a limited degree in NB4/MDR and NB4/RA/MDR cells. Similar results were obtained using blast cells from the patients with APL. GO is effective against ATRA- or ATO-resistant APL cells that do not express P-gp, and the mechanism of resistance to GO is not related to the mechanism of resistance to ATRA or ATO in APL cells. Leukemia (2005) 19, 1306-1311. doi:10.1038/sj.leu.2403807; published online 26 May 2005.

Adeno-associated virus (AAV) vector system has several useful advantages with regard to in vitro and in vivo gene transfer. However, their usages have been limited by cumbersome and labor-intensive vector production in the traditional method. To overcome limitations in AAV production, in this report, we explored the possibility of generating AAV packaging cell line, 293T R/C.VA.E2A.E4. cells, by using lentivirus-mediated transduction of Rep/Cap gene of AAV-2, VA RNA, E2A, and E4 genes of Ad5 into 293T cells. In packaging cell lines, it is important that supply of the AAV vector can be stably performed for long time. We showed that the 293T R/C.VA.E2A.E4. cells have stably maintained the transduced components after more than 10 passages and yielded high-titer AAV vectors, and the titer of AAV vectors did not decline even if culture of the packaging cells was continued for long time. The Rep/Cap and E4 gene products caused no remarkable cytotoxicity. The 293T R/C.VA.E2A.E4. cells might be able to tolerate the Rep/Cap and E4 gene products, or have less copy numbers of the Rep/Cap and E4 genes than the traditional method. Moreover, we showed that the AAV vectors derived from 293T R/C.VA.E2A.E4. cells infected the primary human CD34+ haematopoietic progenitor cells with high efficiency (50-70%). In the 293T R/C.VA.E2A.E4. cells, the AAV vectors can be generated by the transfection of one AAV vector plasmid, and large-scale AAV production can be easily achieved. It is important that cumbersome, variable, and costly transfection is avoided.

A NUP98 gene translocation occurring with a del(6p23) and an add(11)(p15) was determined in a 61-year-old patient with therapy-related atypical chronic myelocytic leukemia after complete remission from acute promyelocytic leukemia that eventually underwent clonal evolution and transformed to CD56-positive acute myelocytic leukemia (French-American-British classification M0). Precise chromosome analysis by G-banding, spectral karyotyping analysis, and dual-color fluorescence in situ hybridization showed this abnormality as 46,XY,del(6)(p23),add(p15). ish del(6)(NUP98-,D6Z1+),der(7)(NUP98+,D7Z1+),der(11)(NUP98+,D11Z1). A split signal of NUP98 was observed in 68.4% of the 117 cells analyzed, which clearly indicated that the NUP98 partially translocated to chromosome 7. However, the potential fusion partner of the NUP98 was not HOX family or DEK. The fusion gene has not been found by a differential display method. The significance of simultaneously combined del(6)(p23), which also has been reported with secondary leukemogenesis, has not been elucidated. Additional karyotype abnormalities evolved increasingly, and leukocytosis with blasts with more complex karyotypic abnormalities appeared 5 months later. Careful and continuous analysis of karyotype change clarified the process of the clonal evolution after NUP98 translocation. Further investigation of molecular characterization of this NUP98 translocation and interaction with 6p23 abnormalities might be worthwhile for understanding leukemogenesis.

We studied the cytotoxic effect of an organic arsenical compound, phenylarsine oxide (PAO) on an acute promyelocytic leukemia (APL) cell line (NB4) and an As2O3-resistant NB4 subline (NB4/As). Cell growth was inhibited by 50% (IC50) upon 2-day treatment with As2O3 or PAO at 0.54 and 0.06 microM, respectively in NB4 cells (P = 0.025), and 2.80 and 0.08 microM, respectively in NB4/As (P = 0.030). 0.1 microM PAO increased the proportion of hypodiploid cells (50.3%) by a greater degree than the same dose of As2O3 (3.8%) in NB4 cells. In NB4 cells, 0.1 microM PAO reduced the mitochondrial transmembrane potential (20.5% in a PI(negative)-Rhodamine123(low) fraction) by a greater degree than 1 microM As2O3 (7.1%). Western blotting showed that 0.1 microM PAO downregulated the expression of both Bcl-2 and Bcl-X(L) proteins, whereas I microM As2O3 downregulated only Bcl-2 expression. These results suggest that the cytotoxic effect of PAO on an APL cell line and As2O3-resistant subline is significantly higher than that of As2O3. PAO-induced apoptosis seems to be related to the activation of the mitochondrial pathway and downregulation of both Bcl-2 and Bcl-X(L). PAO is a considerable agent for relapsed/refractory APL and for purging APL cells following stem cell transplantation.

Gemtuzumab ozogamicin (CMA-676), a calicheamicin-conjugated humanized anti-CD33 mouse monoclonal antibody, has recently been introduced clinically as a promising drug for the treatment of patients with acute myeloid leukemia (AML), more than 90% of which express CD33 antigen. However, our recent study suggested that CMA-676 was excreted by a multi- drug-resistance (MDR) mechanism in P-glycoprotein (P-gp)-expressing leukemia cell lines. We analyzed the in vitro effects of CMA-676 on leukemia cells from 27 AML patients in relation to the amount of P-gp, MDR-associated protein 1 (MRP1), CD33 and CD34, using a multi-laser-equipped flow cytometer. The cytocidal effect of CMA-676, estimated by the amount of hypodiploid portion on cell cycle, was inversely related to the amount of P-gp estimated by MRK16 monoclonal antibody (P = 0.004), and to the P-gp function assessed by intracellular rhodamine-123 accumulation in the presence of PSC833 or MS209 as a MDR modifier (P = 0.0004 and P = 0.002, respectively). In addition, these MDR modifiers reversed CMA-676 resistance in P-gp-expressing CD33(+) leukemia cells (P = 0.001 with PSC833 and P = 0.0007 with MS209). In CD33(+) AML cells from 13 patients, CMA-676 was less effective on CD33(+)CD34(+) than CD33(+)CD34(-) cells (P = 0.002). PSC833 partially restored the effect of CMA-676 in CD33(+)CD34(+) cells. These results suggest that the combined use of CMA-676 and a MDR modifier will be more effective on CD33(+) AML with P-gp-related MDR.

Recent studies have shown that arsenic trioxide (As(2)O(3)) can induce complete remission in patients with acute promyelocytic leukemia (APL). We tested the efficacy and safety of As(2)O(3) for the treatment of patients with APL who had relapsed from or become refractory to all-trans retinoic acid (ATRA) and conventional chemotherapy in a prospective study. As(2)O(3) at a dose of 0.15 mg/kg was administered until the date of bone marrow remission to a maximum of 60 days. In patients who achieved complete remission (CR), one additional course of As(2)O(3) was administered using the same dose for 25 days. Of 14 patients, 11 (78%) achieved CR. Six of 10 patients who achieved CR showed disappearance of PML-RARalpha transcript by RT-PCR assay. The duration of As(2)O(3)-induced CR ranged from 4 to 22 months (median, 8 months) at a median follow-up of 17 months. Adverse events included 13 electrocardiogram abnormalities (13 QTc prolongation, eight ventricular premature contraction, four nonsustained ventricular tachycardia and two paroxysmal supraventricular tachycardia), seven nausea and vomiting, four pruritus, three peripheral neuropathy, three fluid retention and one APL differentiation syndrome. Four patients received antiarrhythmic agents. Hyperleukocytosis developed in five patients and in three cytotoxic drugs were necessary. Other adverse events were relatively mild. As(2)O(3) treatment is effective and relatively safe in relapsed or refectory patients with APL. Cardiac toxicities in patients with QTc prolongation should be carefully monitored.

Efficacy of the Shinki bioclean room for preventing infection in neutropenic patients Aim of the study. To investigate the effectiveness of a new type of bioclean room named Shinki bioclean room (SBCR) for the prevention of infection during neutropenia after intensive chemotherapy in comparison with a standard laminar air flow room (LAFR). Background. Recently, a new industrial technology, wherein a dust-free and aseptic environment is created by circulating air containing nanometre order ultra fine water droplets with abundant negative air ions, has been developed in Japan. Methods. The air cleanliness of SBCR was examined by measuring airborne particles and microorganisms. Bacteriological samples for environment culture were taken by means of exposed settle-plates. In addition, the frequency of pneumonia and fever higher than 38°C were examined in 34 patients with acute leukaemia who received intensive chemotherapy in SBCR or LAFR. Results. The number of airborne particles (≥0·5 μm) was 70 particles/ft3, and that of airborne microorganisms was 0·00 colony forming unit/ft3 in SBCR, and neither bacteria nor fungi were detected. The numbers of colonies of bacteria and fungi on air settle-plates were fewer in the SBCR than in the LAFR regardless of the presence of patients or the nurse entering. The frequency of pneumonia during chemotherapy for acute leukaemia was lower in the SBCR group (0%, 0/19 cases) than in the LAFR group (27%, 4/15 cases) (P=0·0294) and the frequency of fever higher than 38°C also tended to be lower in the SBCR group (53%, 10/19 cases) than in the LAFR group (80%, 12/15 cases) (P=0·0973). Conclusion. The SBCR is equal or superior to LAFR in preventing infection during neutropenia. Other advantages for SBCR are a low level of noise (40 dB), easy control of temperature and humidity, and efficient removal of odour. In addition to the quiet and comfortable atmosphere, expected favourable effects of negative air ions may give higher quality of life for patients in SBCR than those in LAFR. Further studies will be needed to examine the safety, benefits and effects of the negative ion exposure.

Erythropoietin (Epo) is one of the main regulators of growth and differentiation of hematopoietic cells. In normal bone marrow cells, the amount of erythropoietin receptor (EpoR) was highest in the CD34+ CD38- subset, and decreased on lineage committed progenitor cells expressing CD38 antigens. Among the erythroid cells expressing GpA antigens, CD34-positive fractions expressed more EpoR than CD34-negative fractions. Although the amounts of EpoR of bone marrow cells from patients with refractory anemia (RA) were less than those of normal bone marrow cells in all phenotypes examined, there was no statistical significant difference. EpoR was detected on leukemia cells from 60% of acute myeloblastic leukemia (AML) cases and 29% of acute lymphoblastic leukemia (ALL) cases, and distributed widely among all FAB-subtypes. In spite of the presence of EpoR, in vitro proliferative response to Epo was not observed in a large proportion of AML. And there was no correlation between the amount of EpoR and the in vitro response to EPO. Patients with both EpoR expression and in vitro response to Epo had shorter remission duration than those without EpoR.

Using a non-isotopic ligand binding assay and immunofluorescence, we examined the amount of mpl, glycoprotein IIb/IIIa (gpIIb/IIIa) and glycoprotein Ib (gpIb) on platelets from healthy volunteers and patients with refractory anemia (RA). For the analysis of mpl expression, we applied both a non-isotopic ligand binding assay and immunofluorescence using anti-mpl monoclonal antibody, and compared the results from both methods. The non-isotopic ligand binding assay has been developed in our laboratory and is suitable for the quantitative analysis of a small amount of cytokine receptors such as mpl on platelets. In platelets from patients with RA, the amount of mpl expressed by the D value was 0.05+/-0.03 (mean+/-standard deviation), and was significantly lower than that in healthy volunteers (0.15+/-0.05, p

Calicheamicin-conjugated humanized anti-CD33 mouse monoclonal antibody, CMA-676, has recently been introduced to clinics as a promising drug to treat patients with acute myeloid leukemia (AML) in relapse. However, the mechanism of action of CMA-676 has not been well elucidated. The cytotoxic effect of CMA-676 on HL60, NOMO-1, NB4, NKM-1, K562, Daudi, and the multidrug-resistant sublines, NOMO-1/ADR and NB4/MDR, was investigated by cell cycle distribution and morphology. These studies were done by a video-microscopic system, DNA fragmentation, dye exclusion and 3H-thymidine uptake after analysis of CD33, CD34, P-glycoprotein (P-gp), multidrug resistance (MDR)-associated protein and lung-related protein on these cells. A dose-dependent, selective cytotoxic effect of CMA-676 was observed in cell lines that expressed CD33, and was dependent on the amount of CD33 and the proliferative speed of the cells. Sensitive cells were temporally arrested at the G2/M phase before undergoing morphological changes. CMA-676 is not effective on P-gp-expressing multidrug-resistant sublines compared with parental cell lines. MDR modifiers, MS209 and PSC833, restored the cytotoxic effect of CMA-676 in P-gp-expressing sublines. CMA-676 is a promising agent in the treatment of patients with AML that expresses CD33. The combined use of CMA-676 and MDR modifiers may increase the selective cytotoxic effect in multidrug-resistant AML.

Spicamycin is a potent inducer of differentiation of human myeloid leukemia cells (HL-60) and murine myeloid leukemia cells (M1). One of the spicamycin derivatives, KRN5500, shows a broad spectrum of antitumor activity against human tumor xenografts in nude mice. In this study, we first investigated the differentiation efficacy of spicamycin and KRN5500 in HL-60 and acute promyelocytic leukemia cell line, NB4, and found that low concentrations of both compounds induced differentiation to a small extent in both cell lines, but markedly induced apoptosis in NB4 cells. Further investigation in a myeloid leukemia cell line, NKM-1, a lymphoma cell line, Daudi, and a multiple myeloma cell line, NOP-1, showed that high concentrations of both compounds also induced apoptosis in these cells. The 50% inhibitory concentration (IC(50)) determined by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that myeloid cells were more sensitive to both compounds than lymphoid cells, and spicamycin was more potent than KRN5500. Western blot analysis of Bcl-2, Bcl-xL and Bax expression and immunofluorescence analysis of promyelocytic leukemia (PML) protein indicated that apoptosis induced by spicamycin and KRN5500 was associated with down-regulation of Bcl-2 expression and modulation of PML protein. Thus, spicamycin and KRN5500 may be useful for the treatment of myeloid and lymphoid neoplasms.

Myelodysplastic syndromes (MDS) are clonal disorders in which the proper differentiation of hematopoietic stem cells is impaired. There is no effective treatment for this stem cell disorder at present. In an attempt to find a new strategy that promotes the differentiation of MDS blast cells, we tried retroviral transduction of granulocyte colony-stimulating factor receptor (G-CSFR) into an interleukin-3-dependent MDS cell line, MDS-L, since expression of G-CSFR is known to be essential for the differentiation of myeloid progenitor cells and this expression is impaired in most MDS cells. Ectopic expression of human G-CSFR cDNA in MDS-L cells gave rise to granulocytic differentiation by G-CSF stimulation. G-CSF caused the transformants expressing G-CSFR to display a morphological characteristic of mature granulocytes, upregulated CD11b on the cell surface, and improved NBT reduction activity. These results demonstrate that MDS-L cells ecopically expressing G-CSFR are induced to granulocytic differentiation upon exposure to G-CSF, and shed light on the molecular mechanisms of maturation arrest in MDS cells.

We studied serum thrombopoietin (TPO) levels and circulating numbers of platelet during five courses of myelosuppressive post-remission chemotherapy in three patients with acute leukemia in complete remission. Serum TPO levels were measured by a newly developed and sensitive sandwich enzyme-linked immunosorbent assay. In all courses, serum TPO levels changed reciprocally with the platelet counts. When platelets were transfused into patients near the time of platelet nadir, the TPO levels dropped temporarily, while platelet counts temporarily increased. In addition, platelets obtained after transfusion in a thrombocytopenic patient showed lower binding to biotinylated TPO than donor platelets prior to the transfusion. The finding indicated that the TPO receptors were saturated with endogenous TPO of the patient with a high serum TPO level. These results suggest that the platelet mass directly regulates serum TPO levels by receptor-mediated absorption and is one of the major regulators of serum TPO levels in humans.

Using a non-isotopic ligand binding assay, we quantitatively examined the amount of human thrombopoietin (TPO) receptor (TPO-R) on leukaemia cells from 128 patients with acute myeloid leukaemia (AML) or acute lymphoblastic leukaemia (ALL). The TPO-R was expressed in 53 (47%) of 114 AML cases, and an in vitro treatment with TPO led to proliferation (stimulation index >1 5) of leukaemia cells in 13 (20%) of 66 AML cases examined. The TPO levels had no relation to the FAB classification except for FAB-M7 AML. All five FAB-M7 cases expressed TPO-R, and one of three FAB-M7 examined showed in vitro proliferative response to TPO. Although there was no significant correlation (r = 0.3125) between the amount of TPO-R and the proliferative response, all of the AML cases which showed the in vitro response had TPO-R expression. There was no relationship between TPO-R amount and CD phenotypes, or the amount of granulocyte-colony stimulating factor (G-CSF) receptor. TPO-R was also expressed in two (14%) of 14 cases of ALL, and only these two cases had in vitro proliferative response to TPO. One had only lymphoid antigens, and the other had both lymphoid and myeloid antigens. Our results suggest that some leukaemia cells express functionally active TPO-R.

Using a non-isotopic ligand binding assay using multi-colour flow cytometry, we quantitatively examined the amount of mpl in megakaryocyte-platelet lineage cells. Firstly, we quantified the amount of mpl on cell lines. Mpl gene-transfected BaF3 cells expressed a large amount of mpl, whereas original BaF3, K562, HL-60 and NOMO-1 cells showed no mpl. In bone marrow cells from healthy volunteers, mpl was expressed on CD34 + cells from the very early stage of differentiation when they had no CD38 antigen. The amount of mpl increased with differentiation to CV34 + CD41 + cells, but decreased with further differentiation to CD34 + CD41 + cells. In CD34 + CD41 + cells the amount of mpl varied according to cell size: abundant in large cells, moderate in medium-size cells and a little in small cells. In bone marrow cells from patients with refractory anaemia (RA), the amount of mpl was decreased compared with that in bone marrow cells from healthy volunteers. When analysed by the same CD phenotype and same cell size, the amount of mpl was less in RA patients compared with that in healthy volunteers in all phenotypes and sizes tested. The proportion of large CD34 + CD41 + cells was less in RA patients than in normal volunteers.

In order to develop a non-isotopic quantitative assay of erythropoietin (Epo) receptor (EpoR) on human cells, we devised a flow-cytometric assay using cells stained with biotin-labelled and a streptavidine-RED670 conjugate. For quantification, we applied the Kolmogorov-Smirnov test and calculated the D value. The D value was evaluated from the degree of shift in two profiles according to the increase of fluorescence intensity due to the specific binding of biotin-labelled Epo to EpoR. A good correlation was observed between the number of EpoR calculated by 125I-Epo binding assay and the D value. Then, EpoR expression on bone marrow cells from normal individuals was studied by three-colour flow cytometry. In normal bone marrow, the number of EpoR on cells was highest in CD34+CD38 cells (approximately 1600 sites/cell), and decreased in the following order: CD34+CD38- cells > CD34+CD38+ cells > CD34-CD38+ cells. Glycophorin A (GpA) positive erythroid cells also expressed EpoR, and their CD34+ fraction expressed more EpoR than their CD34- fraction. However, the expression levels of EpoR of these fractions were lower than CD34+CD38- cells. These results indicated that EpoR was highly expressed on CD34+ haemopoietic progenitors from very early stages of differentiation without expression of CD38 antigen, and that the level of expression decreased with erythroid differentiation as well as with various lineage commitment in human bone marrow cells.

To evaluate the expression of multidrug resistance (MDR) on normal and leukemia cells, we examined P‐glycoprotein (P‐gp) by a newly devised flow cytometric method, utilizing a biotinylated monoclonal antibody (mAb) against P‐gp (MRK16), a streptavidin‐RED670 conjugate (SA‐RED670) and appropriate emission filters. The combination of biotinylated MRK16 (b‐MRK16) and SA‐RED670 resulted in higher sensitivity as compared with standard methods such as the use of streptavidin‐phycoerythrin (SA‐PE) conjugate. The sensitivity was examined in K562, K562/ADR, NOMO‐1, NOMO‐1/ADR and HL60 cells, and compared with the data obtained from reverse transcription polymerase chain reaction (RT‐PCR) of mdr‐1 gene. P‐gp positively on flow cytometry was 10.4%, 99.9%, 1.4%, 90.4% and 0%, respectively. Mdr‐1 mRNA was well expressed in K562/ ADR and NOMO‐1/ADR cells, hut not in NOMO‐1 and HL60 cells. In K562 cells, mdr‐1 was found after 40 cycles of PCR, but not 25 cycles. These data are well correlated with those from the flow cytometry. We then studied the P‐gp expression on normal peripheral blood cells and acute leukemia cells. P‐gp was little expressed on peripheral lymphocytes, monocytes and granulocytes. It was also little expressed on blast cells from 5 patients with acute promyelocytic leukemia (APL) at diagnosis, ranging from 0.2 to 10.6% (4.6±3.9%). Ten other acute myeloid leukemia (AMD and 5 acute lymphocytic leukemia (ALL) expressed P‐gp at diagnosis, ranging from 8.5% to 34.5% (16.9± 11.8%) and from 2.3% to 45.6% (24.0±17.8%), respectively. All 9 relapsed or refractory cases expressed P‐gp, ranging from 21.1% to 99.8% (52.2±29.9%). Significant differences were found in APL, CD34‐positive and relapse and refractory cases (P=0.0006, 0.0007 and 0.0088, respectively). These results indicate that this flow cytometric analysis is useful for the evaluation of clinical MDR status and can identify a group of patients with resistant leukemia.

P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1) are induced by arsenic trioxide (As 2 O 3 ), but are not the main mechanism of As 2 O 3 -resistance in acute promyelocytic leukemia cells

There are several important biological markers in myeloid leukemia. In particular, WT1, FLT3, P-glycoprotein (P-gp) and surface antigens are important biologic markers. When we monitor the treatment of leukemia, WT1 is considered to be an important marker, and has been applied to immune therapy. The abnormality of the genes of FLT3 and expression of P-gp are poor prognostic factors, and their inhibitors are developed in progress. The surface antigen analysis of blast cells is also used in the classification and the treatment strategy decision. Recently, as for the treatment of leukemia, stratification by various kinds of factors has been adopted in many prospective studies. The results of these biological markers are utilized for the stratification.

To investigate the pharmacokinetics of arsenic species in Japanese patients with relapsed or refractory acute promyelocytic leukemia (APL) treated with arsenic trioxide (ATO) at a daily dose of 0.15 mg/kg.Inorganic arsenic (AsIII and AsV) and the major metabolites monomethylarsonic acid (MAA(V)) and dimethylarsinic acid (DMAA(V)) in plasma and urine collected from 12 Japanese patients were quantified by HPLC/ICP-MS.The plasma concentrations of AsIII and AsV on day 1 reached the similar Cmax (12.4 +/- 8.4 and 10.2 +/- 3.9 ng/ml) immediately after completion of administration followed by a biphasic elimination. The AUC(0-infinity) of AsV was about twice that of AsIII. The appearance of methylated metabolites in the blood was delayed. During the repeated administration, the plasma concentrations of inorganic arsenic reached the steady state. In contrast, the MAA(V) and DMAA(V) concentrations increased in relation to increased administration frequency. The mean total arsenic excretion rate including inorganic arsenic and methylated arsenic was about 20% of daily dose on day 1 and remained at about 60% of daily dose during week 1-4.This study demonstrates that ATO is metabolized when administered intravenously to APL patients and methylated metabolites are promptly eliminated from the blood and excreted into urine after completion of administration, indicating no measurable accumulation of ATO in the blood.

Recently, arsenic trioxide (ATO) has been proved to induce complete remission (CR) at a high rate in patients with acute promyelocytic leukemia (APL). We prospectively investigated the safety and efficacy of ATO therapy in patients with relapsed and refractory APL and examined the duration of CR and the postremission therapies. Initially, 0.15 mg/kg ATO was administered until bone marrow remission to a maximum of 60 days. After the patient achieved CR, 1 additional ATO course at the same dosage was administered for 25 days. Of 34 patients, 31 (91%) achieved CR. PML-RAR3 messenger RNA was not detected in the bone marrow of 18 (72%) of the 25 patients evaluated by reverse transcriptase-polymerase chain reaction analysis. At a median follow-up of 30 months, the estimated 2-year overall survival rate was 56%, and the estimated 2-year event-free survival rate was 17%. During the ATO therapy, QTc prolongation was observed in most cases. Fifteen patients developed ventricular tachycardia, and 1 of them showed torsades de pointes. Other adverse events were nausea, water retention, APL differentiation syndrome, skin eruption, liver dysfunction, and peripheral neuropathy, all of which were quite tolerable. ATO therapy was remarkably effective for relapsed APL; however, postremission therapies were necessary to maintain a durable remission.

Objective To examine laboratory data including total blood cell count, leukocyte morphology and coagulation parameters during treatment for acute promyelocytic leukemia (APL) at a single institute, and compare the precise differences between all-trans retinoic acid (ATRA) and arsenic trioxide (As2O3) treatment.Patients and Methods Sixteen patients with APL who were treated with ATRA or As2O3 alone and achieved complete remission (CR) were analyzed. ATRA 45 mg/m2/day was given orally until CR. As2O3 0.15 mg/kg/day was given intravenously until leukemic blasts and promyelocytes were eliminated from the bone marrow.Results All 7 patients in the ATRA-treated group were primary cases and all 9 patients in the As2O3-treated group were relapsed cases after the achievement of CR with the ATRA. There was no difference in the data before treatment between these two groups. The duration of leukocytopenia and neutropenia during As2O3 treatment was significantly longer than those of ATRA treatment. The nadir of leukocyte and neutrophil counts was observed later in the As2O3-treated group. Terminal neutrophil differentiation was observed more obviously in the ATRA-treated group. The red blood cell count and hemoglobin concentration decreased significantly at the end of As2O3 treatment and were lower than those of ATRA treatment. Platelets recovered earlier in the ATRA-treated group. Coagulation parameters were not significantly changed between the two groups.Conclusion In comparison with ATRA treatment, the recovery of several components in the peripheral blood cells was delayed in As2O3 treatment. Therefore we should pay more and longer attention in As2O3 treatment.

Objectives Several studies including ours have suggested that lack of CD56 in multiple myeloma (MM) defines a unique patient subset with poorer prognosis. However, the mechanism underlying this aggressive behavior of CD56 − MM has not been well elucidated. Interleukin-6 (IL-6) or insulin-like growth factor I (IGF-I) induce proliferation of MM cells. In this study, we report about the relationship between CD56 expression and responsiveness to these cytokines. Methods We sorted out both CD56 − and CD56 + fractions from MM cell lines such as KMS-21-BM and U-266, and investigated their different responsiveness to IL-6 or IGF-I. Furthermore, we compared the effects of these cytokines on the regulation of cell-cycle distribution between CD56 − and CD56 + cells. Results Although CD56 − cells in both KMS-21-BM and U-266 cells responded significantly to IL-6, CD56 + cells did not. Ki-67 + cells in the CD56 − cells were significantly increased by IL-6. Western blotting showed that IL-6 phosphorylated Akt, and upregulated and downregulated the level of cyclin D1 and p27 protein in the CD56 − KMS-21-BM cells, respectively. LY-294002 completely blocked these effects of IL-6. On the other hand, Ki-67 + cells in the CD56 + cells did not respond to IL-6. Anti-IGF-I mAb significantly reduced Ki-67 + cells only in the CD56 + cells. IGF-I phosphorylated Akt and upregulated cyclin D1 in the CD56 + KMS-21-BM cells, which was completely blocked by LY294002. Conclusions These results suggest that CD56 − and CD56 + MM cells could be stimulated by IL-6 and IGF-I, respectively, via PI3-K/Akt pathway, and provide useful information for anticytokine therapies.

Cyclooxygenase-2 (COX-2) is reported to be an important cellular target for therapy in malignancies. The growth inhibitory effects of COX-2 inhibitors on malignancies have been demonstrated to be through not only COX-2 dependent, but also independent mechanisms. In this study, we showed that etodolac, COX-2 inhibitor, induced apoptosis via COX-2 independent pathway, and investigated the molecular details of etodolac-induced apoptosis in Burkitt's lymphoma cells. In Daudi and Raji Burkitt's lymphoma cell lines, which expressed no COX-2 enzyme, etodolac more strongly induced apoptosis compared to meloxicam. Moreover, etodolac did not induce apoptosis to normal B-lymphocytes. For the pathway of etodolac-induced apoptosis, reduction of anti-apoptotic bcl-2 mRNA and Bcl-2 protein, activation of Caspase-9 and -3, down-regulation of caspase inhibitors, c-IAP-1 and Survivin were involved. Moreover, EBER-1 and -2 expression in Epstein–Barr virus positive Daudi and Raji cells were reduced to result in down-regulation of Bcl-2 by treatment with etodolac. It has been reported that etodolac has stereoisomers, R- and S-etodolac. We found that racemate of etodolac more strongly induced apoptosis in Daudi and Raji cells compared to R- or S-etodolac. In conclusion, our findings indicated etodolac inhibited EBERs expression and induced apoptosis via a Bcl-2-regulated pathway. Moreover, racemate of etodolac more effectively induced apoptosis than R- and/or S-etodolac. Therefore, these activities of etodolac potentially extend to the treatment of patients with Burkitt's lymphoma resistant to chemotherapy.

Adeno-associated virus (AAV) vector system has several useful advantages with regard to in vitro and in vivo gene transfer. However, their usages have been limited by cumbersome and labor-intensive vector production in the traditional method. To overcome limitations in AAV production, in this report, we explored the possibility of generating AAV packaging cell line, 293T R/C.VA.E2A.E4. cells, by using lentivirus-mediated transduction of Rep/Cap gene of AAV-2, VA RNA, E2A, and E4 genes of Ad5 into 293T cells. In packaging cell lines, it is important that supply of the AAV vector can be stably performed for long time. We showed that the 293T R/C.VA.E2A.E4. cells have stably maintained the transduced components after more than 10 passages and yielded high-titer AAV vectors, and the titer of AAV vectors did not decline even if culture of the packaging cells was continued for long time. The Rep/Cap and E4 gene products caused no remarkable cytotoxicity. The 293T R/C.VA.E2A.E4. cells might be able to tolerate the Rep/Cap and E4 gene products, or have less copy numbers of the Rep/Cap and E4 genes than the traditional method. Moreover, we showed that the AAV vectors derived from 293T R/C.VA.E2A.E4. cells infected the primary human CD34+ haematopoietic progenitor cells with high efficiency (50-70%). In the 293T R/C.VA.E2A.E4. cells, the AAV vectors can be generated by the transfection of one AAV vector plasmid, and large-scale AAV production can be easily achieved. It is important that cumbersome, variable, and costly transfection is avoided.

A 43-year-old woman was hospitalized with skin eruption and liver dysfunction complicated with disseminated intravascular coagulation (DIC) after taking Proporis, a so-called health food. Her clinical course was well correlated with discontinuation and the retaking of Proporis. No other reason for the development of DIC was detected except for allergic reaction to Proporis, as demonstrated by a patch skin test. DIC subsided after the discontinuation of Proporis and treatment with gabexate mesilate and heparin. The consumption of health foods with various ingredients is recently increasing. Thus, attention should be paid to any possible serious allergic reaction related to such foods.

CREB-binding protein (CBP) and highly related p300 protein are transcriptional co-activators that play an essential role in chromatin remodeling through histone acetyltransferase activity and interaction with other transcriptional regulators. In this study, various hematological malignancies, including nine cell lines and 45 clinical samples (32 acute myeloid leukemias (AML), nine acute lymphoblastic leukemias (ALL), two cases of myelodysplastic syndrome (MDS), one multiple myeloma, and one chronic myelogenous leukemia in blast crisis), were examined to ask whether mutation of the CBP and p300 genes could be involved in leukemogenesis. The answer was approached by employing the reverse transcription-polymerase chain reaction and single-strand conformation polymorphism (RT-PCR/SSCP) technique and subsequent sequence analysis. A T-lymphoblastic cell line, CEM had an in-frame 21-base-pair deletion within the bromodomain of its p300 cDNA. Genomic DNA analysis revealed aberrant splicing caused by mutation of the acceptor site of intron 17 from ag to gg, which should interfere with catalytic step II of the pre-mRNA splicing reaction. In 1 MDS patient, a missense mutation was detected, which caused a replacement from Ser to Gly at codon 507 of p300. This is the first report of CBP/p300 mutations in leukemias, which might be relatively rare but nonetheless contribute to pathogenesis in some fraction of cases.

Here the relationship between all-trans retinoic acid (ATRA)-resistance and P-glycoprotein (P-gp)-associated multidrug resistance (MDR) is discussed in acute promyelocytic leukemia (APL). First, the remission rates of ATRA therapy are similar in relapsed/refractory APL to the preceding chemotherapy given and in newly diagnosed APL. Second, MDR1 cDNA-transduced NB4 (NB4/MDR) cells accumulate less Rhodamine-123 (Rh123) than NB4 cells, but there is no difference in the intracellular ATRA concentration between them. PSC833 or MS209. MDR modifiers, increases the intracellular accumulation of Rh123 in NB4/MDR and APL cells expressing P-gp, but not of ATRA. Third, the expression of CD11b, the NBT reduction activity, the proportion of apoptotic cells and the morphology are not different between NB4/MDR and NB4 cells, and between APL cells expressing P-gp and not. APL cells express little P-gp, and mainly express CD33 but no CD34. Despite previous reports that ATRA-resistant APL cells express more P-gp than ATRA-sensitive ones, P-gp and ATRA-resistance seems to exist independently.

Interleukin-12 (IL-12) has potent antitumor activities. We examined whether IL-12 enhanced the cytotoxicity of peripheral blood mononuclear cells (PBMNC) and decreased leukemia cells in 30 patients with leukemia or myelodysplastic syndromes (MDS): 12 acute myeloid leukemia (AML) (five in complete remission (CR) and seven in non-CR); six chronic myeloid leukemia (CML); and 12 MDS (three refractory anemia (RA), eight RA with excess of blasts and one chronic myelomonocytic leukemia). PBMNC from patients and five healthy volunteers were cultured at 5 x 10(5)/ml parallel with or without 100 units/ml of IL-12 for 3 days. Cytotoxicity of PBMNC against K562 cells was assessed by flow cytometry. To quantify the amount of leukemia cells, WT1 mRNA was measured by competitive reverse transcription polymerase chain reaction (RT-PCR), since WT1 mRNA is considered as a marker of minimal residual disease (MRD) in leukemia or MDS. The cytotoxicity of non-IL-12-treated PBMNC of 30 patients was 13.4+/-9.3% at the effector to target (E:T) ratio of 20:1, and significantly lower than that of normal subjects (25.7+/-8.4%). The cytotoxicity increased to 30.6+/-17.9% in the IL-12-treated PBMNC. WT1 mRNA in PBMNC of five healthy volunteers was less than 10(3) copies/microg of total RNA. Following the 3-day IL-12 treatment, mean WT1 mRNA of PBMNC was reduced from 10(4.8) to 10(4.2) copies/microg of total RNA in six CML patients, from 10(5.4) to 10(4.8) copies/microg in 12 MDS patients and from 10(5.0) to 10(4.2) copies/microg in five AML patients in CR, but not reduced in five of seven AML in non-CR. These results showed that IL-12 significantly enhanced PBMNC cytotoxicity and decreased the quantity of leukemia cells in PBMNC of most patients with MDS, CML and AML in CR. IL-12 might be of considerable benefit in the elimination of MRD in patients with hematological malignancies.

The regulation mechanism of circulating thrombopoietin (TPO) level in human newborns remains unknown. In the present study, we examined whether the TPO concentrations in cord blood were influenced by the difference in the delivery method and the presence or absence of maternal/fetal complications. Cortisol levels were simultaneously measured to assess the adrenal response of fetuses. Both the TPO level and the cortisol level were substantially greater in the neonates delivered vaginally with and without the complications than in those delivered by cesarean section without the complications. The binding assay showed that the incubation of mpl+/BaF3 cells with cortisol gave rise to a significant decrease in the binding sites of TPO. These results suggest that the stress to the fetuses near the time of delivery affects the cord blood TPO levels, which may be mediated in part by the action of cortisol on the TPO-mpl binding system.

We analysed the relationship between all-trans retinoic acid (ATRA) resistance and P-glycoprotein (P-gp)-associated multidrug resistance (MDR) in acute promyelocytic leukaemia (APL). There was no difference in the intracellular ATRA accumulation between NB4 cells and an MDR1 cDNA-transduced NB4 subline and between ATRA-resistant NB4 cells (NB4/RA) and an MDR1 cDNA-transduced NB4/RA subline. PSC833, a MDR modifier, did not increase the intracellular accumulation of ATRA or affect the expression of CD11b, the nitroblue tetrazolium (NBT) reduction activity, the proportion of apoptotic cells or the morphology of these four ATRA-treated cell lines. Similar results were obtained in the analysis of APL cells from five patients relapsed after ATRA-induced complete remission.

Expression of erythropoietin (EPO) receptor (EPO-R) was analysed in leukaemia cells from 150 patients with acute myeloid leukaemia (AML) or acute lymphoblastic leukaemia (ALL). EPO-R was expressed in 81 (60%) out of 136 AML, and in vitro treatment with EPO led to proliferation of leukaemia cells in 13 (16%) out of 81 AML examined. EPO-R expression and in vitro response to EPO were observed in all subtypes of AML according to the French-American-British (FAB) classification. All eight patients with FAB-M6 expressed EPO-R, and one out of four showed an in vitro response to EPO. Although there was no significant correlation (r = 0.2522) between the amount of EPO-R and the in vitro response to EPO, all of the AML patients who showed in vitro response expressed EPO-R. Stem cell factor significantly enhanced both EPO-R expression and in vitro response to EPO. Interleukin-3 tended to increase in vitro response to EPO. CD phenotypes, the amount of granulocyte colony-stimulating factor (G-CSF) receptors and the amount of TPO receptors had no significant relationship with the amount of EPO-R. Patients with both EPO-R expression and in vitro response to EPO had shorter duration of complete remission than those without EPO-R (P = 0.0053). EPO-R was expressed in four (29%) out of 14 ALL, and none out of five ALL showed in vitro response to EPO.

Granulocyte colony-stimulating factor (G-CSF) is the most important cytokine in granulopoiesis, and induces the proliferation and differentiation of normal bone marrow granulocytic precursors. The physiologic effect of G-CSF is mediated through binding to specific cell surface receptors for G-CSF. Using a newly-devised quantitative flow-cytometric assay, we analyzed the expression of G-CSF receptors on normal and leukemic hematopoietic cells. In normal donors, G-CSF receptors are widely expressed from CD34-positive immature bone marrow cells to mature peripheral granulocytes. The highest expression of G-CSF receptors is observed in peripheral granulocytes. In the bone marrow, the level of G-CSF receptors expression increases in the following order; CD34+ CD33- cellsCD34+ CD33+ cellsCD34- CD33+ cells, indicating that G-CSF receptors are expressed on myeloid cells from a very early stage of differentiation and that the level of expression increases with the progress of cell maturation. G-CSF receptors are also detected on blast cells of patients with acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). The number of cell surface receptors varies from patient to patient, and no clear correlation is observed between the expression of receptors and the leukemia subtype or the cell surface markers. In this respect, the clinical use of G-CSF should be carefully controlled in ALL as well as AML patients.

We examined the multidrug resistant P-glycoprotein (P-gp) on normal bone marrow (BM) cells and acute myeloid leukaemia (AML) cells, using newly devised flow cytometric multi-parameter analysis with CD33, CD34 and MRK16 monoclonal antibodies. In both normal BM cells and AML cells, CD34 + CD33 - cells expressed P-gp strongly, CD34 + CD33 + cells moderately, and CD34 - CD33 + cells weakly. Acute promyelocytic leukaemia, mainly expressing CD34 - CD33 + but not CD34 + CD33 - at diagnosis, expressed less P-gp. P-gp expression of AML cells at diagnosis was increased as compared with normal cells of the same phenotype. P-gp expression was more increased in relapsed cases, especially in immature subpopulations.

Impairment of heart rate variability control during arsenic trioxide treatment for acute promyelocytic leukemia

The constitutive activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway commonly occurs in cancers and is a crucial event in tumorigenesis. Chronic myelogenous leukemia (CML) is characterized by a reciprocal chromosomal translocation (9;22) that generates the Bcr-Abl fusion gene. The PI3K/Akt pathway is activated by Bcr-Abl chimera protein and mediates the leukemogenesis in CML. However, the mechanism by which Bcr-Abl activates the PI3K/Akt pathway is not completely understood. In the present study, we found that pleckstrin homology domain leucine-rich repeat protein phosphatases 1 and 2 (PHLPP1 and PHLPP2) were depleted in CML cells. We investigated the interaction between PHLPPs and Bcr-Abl in CML cell lines and Bcr-Abl+ progenitor cells from CML patients. The Abl kinase inhibitors and depletion of Bcr-Abl induced the expression of PHLPP1 and PHLPP2, which dephosphorylated Ser-473 on Akt1, -2, and -3, resulting in inhibited proliferation of CML cells. The reduction of PHLPP1 and PHLPP2 expression by short interfering RNA in CML cells weakened the Abl kinase inhibitor-mediated inhibition of proliferation. In colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte; colony-forming unit-granulocyte, macrophage; and burst-forming unit-erythroid, treatment with the Abl kinase inhibitors and depletion of Bcr-Abl induced PHLPP1 and PHLPP2 expression and inhibited colony formation of Bcr-Abl+ progenitor cells, whereas depletion of PHLPP1 and PHLPP2 weakened the inhibition of colony formation activity by the Abl kinase inhibitors in Bcr-Abl+ progenitor cells. Thus, Bcr-Abl represses the expression of PHLPP1 and PHLPP2 and continuously activates Akt1, -2, and -3 via phosphorylation on Ser-473, resulting in the proliferation of CML cells.

[Objective] PHLPP1 and 2 (pleckstrin homology (PH) domain leucine-rich repeat protein phosphatase 1 and 2) is identified as the dephosphorylation enzyme of Akt, the same as that of PP2A of the dephosphorylation enzyme of Akt, and regulate the cell-growth signal of Akt through a dephosphorylation of the phosphorylated Akt (p-Akt). Previously, we reported the expression of PHLPP1 and 2 were suppressed in CML cell lines. In this study, we investigated the the CML and its progenitor cell proliferation through the phosphorylation of Akt by depletion of PHLPP1 and 2. [Method] CML cell lines (K562, Meg01, SHG3) and the CML clinical specimen (n= 10) were used for this research. The changes in the expression of PHLPP1 and 2 by treatment with Abl kinase inhibitors (STI571, ANM107 and BMS354825) or knock down with Bcr-Abl gene were evaluated by RT-PCR method. The influence on the expression of PHLPP1 and 2 in AML cell line transfected with Bcr-Abl also evaluated. CML progenitor cells derived from CML patients separated by ALDH activity, and the expression of PHLPP1 and 2 were investigated by quantitative RT-PCR method. p-Ser473 Akt1, 2 and 3 by Abl kinase inhibitor or knock down with PHLPP1 and 2 were measured, and the influence on the effect of cell growth inhibition by MTT assay. The expression of PHLPP1 and 2 and colony formation in colony forming unit-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM), colony forming unit-granulocyte, macrophage (CFU-GM) and burst forming unit-erythroid (BFU-E) derived from Bcr-Abl positive hematopoietic progenitor cells also evaluated. [Result] The expression of mRNA and protein of PHLPP1 and 2 were increased by treatment with Abl kinase inhibitor or Bcr-Abl knock down by siRNA in CML cell lines and AML cell line trasfected with Bcr-Abl gene. Moreover, p-Ser473 Akt 2 and 3 were decreased according to the expression of PHLPP1, and also p-Ser473 Akt1 and 3 according to the expression of PHLPP2. The Abl kinase inhibitors induced the expression of PHLPP1, 2 and the reduction of p-Ser473 Akt isoforms. The Abl kinase inhibitors inhibited the CML cell proliferation via the depletion of PHLPP1 and 2. In Bcr-Abl positive progenitor cells, the expression of PHLPP1 and 2 was increased, and colony formation was suppressed by Abl kinase inhibitor and by Bcr-Abl siRNA. The colony formation in progenitor cells knocked down PHLPP1 and 2 were decreased when treated with Abl kinase inhibitors. [Conclusion] These results suggest that Bcr-Abl might promote CML cell proliferarion through continuous phosphorylation of p-Ser473 Akt1, 2 and 3 by suppression of PHLPP1 and 2, and that the induction of PHLPP1 and 2 may be effective to regulate the cell proliferation in CML cells. It may be also that the induction of expression of PHLPPs has the potential possibility as the targets on the regulation of cell proliferation in Bcr-Abl positive progenitor cells. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5053.

Abstract 2182 Poster Board II-159 Objective PHLPP1 and 2 (pleckstrin homology (PH) domain leucine-rich repeat protein phosphatase 1 and 2) is identified as the dephosphorylation enzyme of Akt, the same as that of PP2A of the dephosphorylation enzyme of Akt, and regulate the cell-growth signal of Akt through a dephosphorylation of the phosphorylated Akt (p-Akt). Previously, we reported the expression of PHLPP1 and 2 were suppressed in CML cell lines. In this study, we investigated the the CML and its progenitor cell proliferation through the phosphorylation of Akt by depletion of PHLPP1 and 2. Method CML cell lines (K562, Meg01, SHG3) and the CML clinical specimen (n= 10) were used for this research. The changes in the expression of PHLPP1 and 2 by treatment with Abl kinase inhibitors (STI571, AMN107 and BMS354825) or knock down with Bcr-Abl gene were evaluated by RT-PCR method. The influence on the expression of PHLPP1 and 2 in AML cell line transfected with Bcr-Abl also evaluated. CML progenitor cells derived from CML patients separated by ALDH activity, and the expression of PHLPP1 and 2 were investigated by quantitative RT-PCR method. p-Ser473 Akt1, 2 and 3 by Abl kinase inhibitor or knock down with PHLPP1 and 2 were measured, and the influence on the effect of cell growth inhibition by MTT assay. The expression of PHLPP1 and 2 and colony formation in colony forming unit-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM), colony forming unit-granulocyte, macrophage (CFU-GM) and burst forming unit-erythroid (BFU-E) derived from Bcr-Abl positive hematopoietic progenitor cells also evaluated. Result The expression of mRNA and protein of PHLPP1 and 2 were increased by treatment with Abl kinase inhibitor or Bcr-Abl knock down by siRNA in CML cell lines and AML cell line trasfected with Bcr-Abl gene. Moreover, p-Ser473 Akt 2 and 3 were decreased according to the expression of PHLPP1, and also p-Ser473 Akt1 and 3 according to the expression of PHLPP2. The Abl kinase inhibitors induced the expression of PHLPP1, 2 and the reduction of p-Ser473 Akt isoforms. The Abl kinase inhibitors inhibited the CML cell proliferation via the depletion of PHLPP1 and 2. In Bcr-Abl positive progenitor cells, the expression of PHLPP1 and 2 was increased, and colony formation was suppressed by Abl kinase inhibitor and by Bcr-Abl siRNA. The colony formation in progenitor cells knocked down PHLPP1 and 2 were decreased when treated with Abl kinase inhibitors. Conclusion These results suggest that Bcr-Abl might promote CML cell proliferarion through continuous phosphorylation of p-Ser473 Akt1, 2 and 3 by suppression of PHLPP1 and 2, and that the induction of PHLPP1 and 2 may be effective to regulate the cell proliferation in CML cells. It may be also that the induction of expression of PHLPPs has the potential possibility as the targets on the regulation of cell proliferation in Bcr-Abl positive progenitor cells. Disclosures: No relevant conflicts of interest to declare.

Abstract 4814 Background and Aims We synthesized two phospha sugar derivatives, 2,3,4-tribromo-3-methyl-1-phenylphospholane 1-oxide (TMPP) by reacting 3-methyl-1-phenyl-2-phospholene 1-oxide with bromine, and we has reported their potential as an antileukemic agent in cell lines. In this study, we have investigated that TMPP have antileukemic effects as shown in various leukemia cell lines as well as primary AML patient specimens. Moreover, we have identified the target molecules of TMPP by using docking simulation. Methods In human leukemia cell lines (HL60, NB4, U937, NOMO-1, CEM, MOLT4, SUP-B15, and YRK2 cells), the anti-leukemic effects were elevated by cell proliferation assay, Cell cycle analysis, Western blotting. In AML cells derived from AML patients and normal hematopoietic progenitor cells derived from healthy volunteers, the changes of viability by treatment with TMPP were evaluated. By using docking simulation, the target molecules were identified in leukemia cells treated with TMPP. Results TMPP showed inhibitory effects on leukemia cell proliferation, with mean IC50 values of 10.2 nmol/L for TMPP, indicating that inhibition appeared to be dependent on the number of bromine atoms in the structure. Further, TMPP at 8.4 nmol/L induced G2/M cell cycle block in leukemia cells, and TMPP at 17.3 nmol/L induced apoptosis in these cells. TMPP treatment reduced cell cycle progression signals (FoxM1, KIS, Cdc25B, Cyclin D1, Cyclin A, and Aurora-B) and tumor cell survival (Bcl-2, p27Kip1 and p21Cip1). Further, treatment with TMPP significantly reduced the viability of AML specimens derived from AML patients, but only slightly reduced the viability of normal ALDHhi progenitor cells. We also found that TMPP showed the strong activity against Aurora kinase B and Bcl-2 by using docking simulation. Conclusion We demonstrate that TMPP inhibits Aurora kinase B and Bcl-2 and shows a dominant Aurora B kinase inhibition-related cell cycle arrest and the induction of mitochondrial potential catastrophe-related apoptosis in acute leukemia cells. Moreover, we identified Aurora kinase B and Bcl-2 as the target molecules of TMPP. We conclude that TMPP has great therapeutic potential in anticancer therapy in a wide range of cancers. Disclosures: No relevant conflicts of interest to declare.

[Background] The proto-oncogene Akt/PKB is activated in many human cancers including leukemias, and regulates the cell survival and apoptosis. Akt/PKB is activated by growth factors or the phosphatidylinositol-3,4,5-triphosphate via PI3K, phosphorylates many substrates for cell survival or proliferation. The tumor suppressor PTEN prevents Akt/ PKB phosphorylation and activation. On the other hand, the PH domain leucine-rich repeat protein phosphatase (PHLPP) dephosphorylates Akt/PKB, induces apoptosis, and suppresses tumor growth. It has been reported that the expression levels of PHLPP are significantly reduced in several colon cancer and glioblastoma cell lines, and Akt/PKB phosphorylation is elevated. However, its function is little known in CML. We found that the expression of PHLPP gene and protein was suppressed in CML cells. In this study, we have investigated the expression and the function of the PHLPP in CML cells. [Methods] The cells used in this study were human CML cell lines, K562 and Meg01 cells. Primary CML (CP; n=10) cells were obtained from the peripheral blood or bone marrow. Human normal mononuclear cells (MNCs) were isolated from peripheral blood or bone marrow of healthy volunteers after obtaining informed consents. For analysis of PHLPP mRNA expression, quantitative RTPCR was performed in all cell lines treated with Abl kinase inhibitors (STI571, AMN107, and BMS354825). For proliferation analysis and the levels of Akt phosphorylation in CML cells, MTT assays and western blot were performed in all cell lines transfected with Bcr-Abl or PHLPP siRNA, respectively. For colony analysis, the colonies of CFU-GEMM, CFUGM, and BFU-E were counted in CML stem/progenitor cells transfected with PHLPP siRNA or treated with Abl kinase inhibitors. [Results] In CML cell lines, the expressions of PHLPP mRNA and protein were significantly increased more than normal MNCs by treatment with Abl kinase inhibitors or transfection with Bcr-Abl siRNA. On the other hands, in CML cells transfected with the PHLPP siRNA, it is shown that the phosphorylation levels of Akt were markedly increased compared to the untransfected cells. In CML stem/progenitor cells obtained from patients with CML, the expression of PHLPP gene was suppressed in 10/10 (100 %) of CML. This higher expression was not associated to the counts of blasts of PB (P < 0.001). The transfection with Bcr-Abl siRNA or treatment with Abl kinase inhibitors increased the expression of PHLPP mRNA and protein, and decreased the counts of CFU-GEMM, CFU-GM and BFU-E. [Conclusions] Our results suggest that the Bcr- Abl suppressed the expression of PHLPP mRNA and protein, and the phosphorylation of Akt in CML cells to induce the proliferation of CML cells. Moreover, induction of PHLPP expression might regulate the proliferation of CML stem/progenitor cells.

[Background] Sugar derivatives whose oxygen atom in the hemiacetal ring is replaced by a carbon, nitrogen, sulfur atom, and etc., are celled as pseudo sugars. The synthesis and bioassay of these pseudo sugars are well investigated and are known as potential bioactive materials. Phospha sugar, which is one of pseudo sugars, having a phosphorus atom instead of the oxygen atom in the hemiacetal ring, is little known. The synthesis and bioassay of phospha sugars are not well studied in detail. We synthesized the 2, 3, 4-tribromo-3-methyl-1-phenyl phospholane 1-oxide (TMPP) and 2, 3-dibromo-3-methyl-1-phenyl phospholane 1-oxide (DMPP) by the nucleophilic substitution reactions, and found their anti-leukemic activities. [Purpose] The aims of the present study were to evaluate the inhibition of proliferation and induction of apoptosis in leukemia cell treated with TMPP or DMPP, and define the target molecules for TMPP in leukemia cells. [Methods] The cells used in this study were human leukemia cell lines, K562, U937, and YRK2 cells. For proliferation analysis, MTT assays were performed in leukemia cells treated with TMPP. For cell cycle analysis, flow cytometory analysis was performed in leukemia cells treated with TMPP or DMPP by PI staining. For analysis of mitotic regulatory proteins (p27, p21, Skp2, Cdc25B, Cyclin D1, Survivin, and Aurora kinase B), Western blotting was performed in leukemia cells treated with TMPP. [Results] In leukemia cell lines, DMPP and TMPP significantly inhibited the cell proliferation, and TMPP more strongly inhibited the cell proliferation than DMPP. 10 μM TMPP significantly induced G2/M phase arrest, and 25 μM TMPP induced apoptosis in leukemia cells. In leukemia cells, 10 μM TMPP reduced protein of Aurora kinase B, Survivin, Cyclin D1, Skp2 KIS, and FoxM1, while increased protein expression of p21and p27 by western blot analysis. Moreover, 25 μM TMPP activated caspase-3 and caspase-9, cleaved PARP, and reduced Bcl-2. [Conclusions] In this study, we report in the first time the possibility of phospha sugar derivatives as anti-leukemic agents in therapy for leukemias, and analysis of their characterizations. DMPP significantly inhibited the proliferation, and induced apoptosis of leukemia cells. These results demonstrated that the FoxM1, which is an essential transcription factor for cell growth and regulates expression of the mitotic regulators, is one of the targets for DMPP. Therefore, DMPP or TMPP has possibility as new agents for FoxM1 in leukemia therapy.

[Background] FoxM1, a member of the Fox transcription factor family, plays an important cell cycle regulator of both the transition from G1 to S phase and progression to mitosis. FoxM1 expression was also found to be up-regulated in some solid tumors (basal cell carcinomas, hepatocellular carcinoma, and primary breast cancer). These results suggested that FoxM1 plays a role in the oncogenesis of malignancies. However, it is unknown whether FoxM1 expression contributes to the development or progression of leukemia cells. Therefore, we investigated how FoxM1 regulated the cell cycle of leukemia cells and the expression analysis of the FoxM1 gene in patients with acute leukemias. [Methods] The cells used in this study were human acute leukemia cell lines, U937 and YRK2 cells. Primary acute myeloblastic (25 AML (4 M1, 11 M2, 6 M4, 4 M5)) cells were obtained from the peripheral blood. Human normal mononuclear cells (MNCs) were isolated from peripheral blood (PB) of healthy volunteers after obtaining informed consents. For analysis of proliferation and mitotic regulatory proteins (p27, p21, Skp2, Cdc25B, Cyclin D1, Survivin, Aurora kinase B, and KIS) in leukemia cells, MTT assays and western blot were performed in all cell lines, which untransfected or transfected with siRNA FoxM1, respectively. For cell cycle analysis, flow cytometory analysis was performed in leukemia cells untransfected or transfected with siRNAFoxM1 by PI staining. For analysis of FoxM1 mRNA, quantitative RT-PCR was performed in all cell lines and clinical samples. [Results] In all leukemia cell lines, the expression of FoxM1B mRNA were significantly higher than normal MNCs. When transfected with the siRNA FoxM1 in leukemia cells, suppression of FoxM1 caused a mean 71% (range 62 to 80%) reduction in S phase cells and a mean 4.4-fold (range 3.2 to 5.6-fold) increase in G2/M phase cells compared to controls. MTT assay demonstrated that the proliferation of the siRNA FoxM1 transfected cells was inhibited compared to the untransfected cells. Moreover, FoxM1 knockdown by siRNA in leukemia cells reduced protein and mRNA expression of Aurora kinase B, Survivin, Cyclin D1, Skp2 and Cdc25B, while increased protein expression of p21and p27. In the clinical samples obtained from patients with acute leukemias, the FoxM1B gene was overexpressed in 22/25 (88%). The relative folds of FoxM1B gene expression were for AML: 2.83 compared to normal MNCs. [Conclusions] In this study, we report in the first time that FoxM1 is overexpressed in myeloid leukemia cells. These results demonstrated that expression of FoxM1 is an essential transcription factor for growth of leukemia cells, and regulate expression of the mitotic regulators. Moreover, we showed that FoxM1 induced the expression of KIS protein. Therefore, FoxM1 might be one of moleculer targets of therapy for acute leukemias.