Ken Ohmine

Characterization of stage progression in chronic myeloid leukemia by DNA microarray with purified hematopoietic stem cells.

Chronic myeloid leukemia (CML) is characterized by the clonal expansion of hematopoietic stem cells (HSCs). Without effective treatment, individuals in the indolent, chronic phase (CP) of CML undergo blast crisis (BC), the prognosis for which is poor. It is therefore important to clarify the mechanism underlying stage progression in CML. DNA microarray is a versatile tool for such a purpose. However, simple comparison of bone marrow mononuclear cells from individuals at different disease stages is likely to result in the identification of pseudo-positive genes whose change in expression only reflects the different proportions of leukemic blasts in bone marrow. We have therefore compared with DNA microarray the expression profiles of 3456 genes in the purified HSC-like fractions that had been isolated from 13 CML patients and healthy volunteers. Interestingly, expression of the gene for PIASy, a potential inhibitor of STAT (signal transducer and activator of transcription) proteins, was down-regulated in association with stage progression in CML. Furthermore, forced expression of PIASy has induced apoptosis in a CML cell line. These data suggest that microarray analysis with background-matched samples is an efficient approach to identify molecular events underlying the stage progression in CML.

Analysis of gene expression profiles in an imatinib-resistant cell line, KCL22/SR

The BCR/ABL tyrosine kinase inhibitor, imatinib, has shown substantial effects in blast crises of chronic myelogenous leukemia. However, most patients relapse after an initial clinical response, indicating that drug resistance is a major problem for patients being treated with imatinib. In this study, we generated a new imatinib‐resistant BCR/ABL‐positive cell line, KCL22/SR. The 50% inhibitory concentration of imatinib was 11‐fold higher in KCL22/SR than in the imatinib‐sensitive parental cell line, KCL22. However, KCL22/SR showed no mutations in the BCR/ABL gene and no increase in the levels of BCR/ABL protein and P‐glycoprotein. Furthermore, the level of phosphorylated BCR/ABL protein was suppressed by imatinib treatment, suggesting that mechanisms independent of BCR/ABL signaling are involved in the imatinib resistance in KCL22/SR cells. DNA microarray analyses demonstrated that the signal transduction‐related molecules, RAS p21 protein activator and RhoA, which could affect Ras signaling, and a surface tumor antigen, L6, were upregulated, while c‐Myb and activin A receptor were downregulated in KCL22/SR cells. Furthermore, imatinib treatment significantly suppressed the level of phosphorylated p44/42 in KCL22 cells but not in KCL22/SR cells, even when BCR/ABL was inhibited by imatinib. These results suggest that various mechanisms, including disturbance of Ras‐mitogen‐activated protein kinase signaling, are involved in imatinib resistance.

Schedule-dependent synergism and antagonism between methotrexate and cytarabine against human leukemia cell lines in vitro.

Methotrexate (MTX) and cytarabine have been widely used for the treatment of acute leukemias and lymphomas for over 30 years. However, the optimal schedule of this combination is yet to be determined and a variety of schedules of the combination has been used. We studied the cytotoxic effects of MTX and cytarabine in combination against human leukemia cell lines at various schedules in vitro. The effects of the combinations at the concentration of drug that produced 80% cell growth inhibition (IC80) were analyzed using the isobologram method of Steel and Peckham. Simultaneous exposure to MTX and cytarabine for 3 days produced antagonistic effects in human T cell leukemia, MOLT-3 and CCRF-CEM, B cell leukemia, BALL-1, Burkitts lymphoma, Daudi, promyelocytic leukemia, HL-60 and Philadelphia chromosome-positive leukemia, K-562 cells. Simultaneous exposure to MTX and cytarabine for 24 h produced antagonistic effects, sequential exposure to MTX for 24 h followed by cytarabine for 24 h produced synergistic effects, and the reverse sequence produced additive effects in both CCRF-CEM and HL-60 cells. Sequential exposure to MTX for 24 h followed by cytarabine for 3 days also produced synergistic effects in MOLT-3 cells. Cell cycle analysis supported these observations. Our findings suggest that the simultaneous administration of MTX and cytarabine is not appropriate and the sequential administration of MTX followed by cytarabine may be the optimal schedule of this combination.

Stimulation of GATA-2 as a mechanism of hydrogen peroxide suppression in hypoxia-induced erythropoietin gene expression

Hydrogen peroxide (H2O2) has previously been shown to inhibit the DNA binding activity of hypoxia inducible factor‐1 (HIF‐1), the accumulation of HIF‐1α protein and erythropoietin (Epo) gene expression. Epo gene expression has been previously shown to be down‐regulated through a GATA binding site at its promoter region. In this study, the effect of H2O2 on Epo gene expression under hypoxic conditions through a GATA transcription factor was investigated. Hypoxic induction was found to be inhibited upon the addition of H2O2, and this effect could be reversed through the addition of catalase. Hypoxic induction was found to be suppressed by co‐transfection with a human GATA‐2 cDNA expression plasmid. Transfection of Hep3B cells with a reporter gene bearing a mutation at the promoter GATA binding site was found to be only mildly affected by the addition of H2O2. Electrophoretic gel mobility shift assays (EMSAs), using the Epo promoter GATA site as a probe and the GATA‐2 protein extracted from Hep3B cells, showed that addition of H2O2 enhanced the binding of GATA‐2 while addition of catalase inhibited this binding. From these results, we conclude that H2O2 increases the binding activity of GATA‐2 in a specific manner, thereby suppressing the activity of the Epo promoter and thus inhibiting Epo gene expression. J. Cell. Physiol. 186:260–267, 2001.

Cancer gene therapy using mesenchymal stem cells

Abstract Cellular and gene therapies represent promising treatment strategies at the frontier of medicine. Hematopoietic stem cells, lymphocytes, and mesenchymal stem cells (MSCs) can all serve as sources of cells for use in such therapies. Strategies for gene therapy are often based on those of cell therapy, and it is anticipated that some examples will be put to practical use in the near future. Given their ability to support hematopoiesis, MSCs may be useful for the enhancement of stem cell engraftment, and the acceleration of hematopoietic reconstitution. Furthermore, MSCs may advance the treatment of severe graft-versus-host disease, based on their immunosuppressive ability. This application is also based on the homing behavior of MSCs to sites of injury and inflammation. Interestingly, MSCs possess tumor-homing ability, opening up the possibility of applications in the targeted delivery of anti-cancer genes to tumors. Many reports have indicated that MSCs can be utilized to target tumors and to deliver anti-cancer molecules locally, as tumors are recognized as non-healing wounds with inflammatory tissue. Here, we review both the potential of MSCs as cellular vehicles for targeted cancer therapy and the molecular mechanisms underlying MSC accumulation at tumor sites.

Rituximab plus 70% cyclophosphamide, doxorubicin, vincristine and prednisone for Japanese patients with diffuse large B-cell lymphoma aged 70 years and older

Abstract In the rituximab era, several large studies have suggested that full-dose rituximab plus cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) might be the best treatment for patients with diffuse large B-cell lymphoma (DLBCL) aged 60 years and older. However, it remains unclear whether this is also the case for those aged 70 years and older. Previously untreated patients with DLBCL aged 70 years and older (elderly) were treated with R-70%CHOP, and patients younger than 70 years (younger) were treated with full-dose R-CHOP every 3 weeks, for a total of 6–8 cycles. Complete remission (CR) rates in elderly versus younger patients were 75 vs. 78% (p = 0.7), respectively. The 3-year overall survival, event-free survival and progression-free survival of elderly versus younger patients were 58 vs. 78% (p < 0.05), 45 vs. 70% (p < 0.05) and 64 vs. 72% (p = 0.43), respectively. Severe adverse events were more frequent in the elderly, even with the dose reduction in that age group. Three-year PFS with R-70%CHOP for patients aged 70 years and older was not significantly worse than that with full-dose R-CHOP for younger patients, suggesting that R-70% CHOP might be a reasonable choice for patients with DLBCL aged 70 years and older, especially for those with comorbidities.

Screening of genes specifically activated in the pancreatic juice ductal cells from the patients with pancreatic ductal carcinoma.

Pancreatic ductal carcinoma (PDC) is one of the most intractable human malignancies. Surgical resection of PDC at curable stages is hampered by a lack of sensitive and reliable detection methods. Given that DNA microarray analysis allows the expression of thousands of genes to be monitored simultaneously, it offers a potentially suitable approach to the identification of molecular markers for the clinical diagnosis of PDC. However, a simple comparison between the transcriptomes of normal and cancerous pancreatic tissue is likely to yield misleading pseudopositive data that reflect mainly the different cellular compositions of the specimens. Indeed, a microarray comparison of normal and cancerous tissue identified the INSULIN gene as one of the genes whose expression was most specific to normal tissue. To eliminate such a “population‐shift” effect, the pancreatic ductal epithelial cells were purified by MUC1‐based affinity chromatography from pancreatic juice isolated from both healthy individuals and PDC patients. Analysis of these background‐matched samples with DNA microarrays representing 3456 human genes resulted in the identification of candidate genes for PDC‐specific markers, including those for AC133 and carcinoembryonic antigen‐related cell adhesion molecule 7 (CEACAM7). Specific expression of these genes in the ductal cells of the patients with PDC was confirmed by quantitative real‐time polymerase chain reaction analysis. Microarray analysis with purified pancreatic ductal cells has thus provided a basis for the development of a sensitive method for the detection of PDC that relies on pancreatic juice, which is routinely obtained in the clinical setting. (Cancer Sci 2003; 94: 263–270)

GATA Suppresses Erythropoietin Gene Expression through GATA Site in Mouse Erythropoietin Gene Promoter

The promoter and enhancer elements of the mouse erythropoietin (mEpo) gene, which have high homology with those of the human erythropoietin (hEpo) gene, were fused withluciferase. The construct was transfected into erythropoietin-producing hepatoma cell line (Hep3B) cells by lipofectin with lacZ as an internal standard. The wild type (TGATA) showed a 39.5-fold increase in induction by hypoxia. Mouse GATA-2 inhibited the hypoxic induction of the wild-type (m3), promoter-luciferase construct but not the hypoxic induction of the mutant (m4, 5) promoter-luciferase constructs. NG-monomethyl L-arginine (L-NMMA) inhibited the hypoxic induction of the m3 promoter-luciferase construct, but this inhibition was recovered by L-arginine. H2O2 also inhibited the hypoxic induction of the m3 promoter-luciferase construct, but this inhibition was recovered by catalase. Gel shift assays performed on nuclear extracts of 293 cells overexpressing mGATA-1, -2, and -3 revealed that mGATA-1, -2, and -3 bind to the TGATA element of the mEpo promoter. These results indicate that mGATA binds to the TGATA site of the mEpo promoter and negatively regulates mEpo gene expression. Negative regulation of mEpo gene by GATA transcriptional factors is discussed.

Hemin reduces cellular sensitivity to imatinib and anthracyclins via Nrf2.

Heme plays an important biomodulating role in various cell functions. In this study, we examined the effects of hemin on cellular sensitivity to imatinib and other anti‐leukemia reagents. Hemin treatment of human BCR/ABL‐positive KCL22 leukemia cells increased IC50 values of imatinib, that is, the drug resistance, in a dose‐dependent manner without any change in the BCR/ABL kinase activity. Imatinib‐induced apoptosis was also suppressed by hemin treatment in KCL22 cells. Hemin treatment increased the activity of γ‐glutamylcysteine synthetase (γ‐GCS) light subunit gene promoter, which contains a Maf recognition element (MARE). Protein levels of γ‐GCS and heme oxygenase‐1 (HO‐1), two MARE‐containing genes, were also increased after hemin treatment. Knockdown of Nrf2 expression by RNA interference largely abolished the effect of hemin on imatinib‐treated cells, suggesting that Nrf2 recognition of MARE is essential for the hemin‐mediated protective effect. Similar to hemin, treatment of cells with δ‐aminolevulinic acid (δ‐ALA), the obligatory heme precursor, also increased IC50 values of imatinib. In contrast, inhibition of cellular heme synthesis by succinylacetone increased the sensitivity of cells to imatinib in two imatinib‐resistant cell lines, KCL22/SR and KU812/SR. Hemin treatment also decreased the sensitivity of cells to four anthracyclins, daunorubicin, idarubicin, doxorubicin, and mitoxantrone, in BCR/ABL‐negative leukemia U937 and THP‐1 cells, as well as in KCL22 cells. These findings thus indicate that cellular heme level plays an important role in determining the sensitivity of cells to imatinib and certain other anti‐leukemia drugs and that the effect of heme may be mediated via its ability to upregulate Nrf2 activity. J. Cell. Biochem. 104: 680–691, 2008.

Mechanisms of resistance to azacitidine in human leukemia cell lines

The DNA methylation inhibitor azacitidine (5-azacytidine) is used against myelodysplastic syndrome and acute myeloid leukemia, but drug resistance is an ongoing, intractable problem. To investigate resistance mechanisms, we generated two azacitidine-resistant cell lines, THP-1/AR and HL60/AR, and studied genetic disparities between them and their corresponding parental lines. In cells treated with azacitidine, significant mitotic variations were noted in parental cells which were absent in resistant cells, suggesting that resistance arises from negating azacitidine-mediated activation of apoptosis signaling and reestablishing G2/M checkpoint. Importantly, both resistant cell lines have common point mutations in the uridine-cytidine kinase 2 (UCK2) gene, which encodes the rate-limiting enzyme of the azacitidine activation pathway. Forced expression of mutated UCK2 in parental THP-1 cells abrogated azacitidine-induced apoptosis, whereas overexpression of wild type UCK2 in resistant THP-1/AR cells restored sensitivity to azacitidine, implying that UCK2 gene mutations perturb azacitidine activation and advance azacitidine resistance. Our study provides new insights into azacitidine resistance and establishes models useful in developing effective strategies to overcome it.