ALBERT

Description: Abstract 4861 In cancer cells, glucose uptake is elevated and glycolysis persists even under aerobic conditions (Warburg effect). Glutamine metabolism is another target for alteration in cancer development. Glutaminolysis (catabolism of glutamine to generate ATP) is known to increase in tumors. We examined the dependency of the leukemia cells (Kasumi-1, THP-1, HL-60 and NB4) on glucose or glutamine by measuring the growth (MTS count) in glucose- or glutamine-deprived condition. Glucose withdrawal greatly suppressed the growth of all 4 cell lines. However, glutamine withdrawal showed different growth suppressive effects among the cell lines (Kasumi-1: 55% of control, THP-1: 60%, HL-60: 39%, NB4: 70%). HL-60 was most sensitive to glutamine deprivation. The growth suppression of HL-60 due to glutamine withdrawal was partially rescued by oxaloacetate (OAA), a TCA cycle metabolite, while the growth of other cell lines was not rescued by OAA. In the course of glutamine catabolism, ammonia is liberated. Although basal level of the ammonia concentration was not so different among each cell line, glycolysis inhibitor (2-deoxyglucose) treatment enhanced the ammonia generation in HL-60 (Kasumi-1: 2.8% increased, THP-1: 1.7%, HL-60: 6.1%, NB4: 2.8%). Glutaminase, an enzyme converting glutamine to glutamate, is most abundantly expressed in HL-60 in western blot analysis. In addition, HL-60 was most sensitive to the treatment with aminooxyacetate, an inhibitor of glutamate-dependent transaminases that convert glutamate into a-ketoglutarate in the glutaminolytic pathway (Kasumi-1: 86% of control, THP-1: 97%, HL-60: 79%, NB4: 83%). Taken together, HL-60 was considered as glutamine dependent cell line. Therapies targeting glutamine metabolism, such as glutamine depletion or use of inhibitor of glutaminolytic pathway, might be effective against some leukemia. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4991 Bortezomib improved the survival in patients with multiple myeloma (MM), however, cannot cure this disease even when it is combined with autologous stem cell transplant(s) and other new drugs. Myeloma cells cannot be excluded completely from a patient due to drug refractoriness and/or inefficiency of drug delivery in extramedullary sites such as cerebrospinal fluid (CSF). Therefore, it is important to clarify the mechanisms of bortezomib resistance and invasion to central nerve system (CNS) of myeloma for the progress of myeloma therapy. In this study, we established a novel human myeloma cell line from a myeloma patient involved with CNS and analyzed its characters including sensitivity to bortezomib. The patient had been treated with bortezomib for 1.5 years when her CNS was involved with myeloma. The cells from patient's CSF were cultured in RPMI 1640 supplemented with 20% heat-inactivated fetal bovine serum (FBS) and rhIL-6. After 3 months of culture, cell proliferation became continuous with 10% FBS and without rhIL-6. The cell line, named AMU-MM1 was established and negative for EBV. A doubling time of AMU-MM1 cells was about 48 hours. AMU-MM1 cells were positive for CD38, CD54, CD138 and cytoplasmic kappa chain and negative for CD19, CD20, CD33 and CD56 by flow cytometry analysis while those in patient's bone marrow were positive for CD56. AMU-MM1 showed hypo- and pseudodiploid karyotypes with t(4;14), t(8;13), t(1;19), del13q, amp1q21 and others but without del17p by cytogenetic analyses including FISH. The G322A mutation in the proteosome beta 5 subunit (PSMB5) gene, which is reported as a mutation found in bortezomib-resistant cell lines induced via repeated drug selection, was not detected in AMU-MM1 by direct sequencing. Apoptosis analysis using Annexin V/PI assay indicated that AMU-MM1 was sensitive to bortezomib. Our data suggest that AMU-MM1 was derived from the cells that invaded to CSF in which bortezomib concentration was very low and not resistant to bortezomib, and the downregulation of CD56 might play a role in the pathogenesis of CNS involvement as reported before. In addition, we are now focusing on new chimera or dysregulated genes in t(8;13), t(1;19) and other sites as well. In conclusion AMU-MM1 is a useful cell line for analysis of mechanisms of CNS involvement and also possibly the acquired resistance to bortezomib. Disclosures: No relevant conflicts of interest to declare.

Description: Cancer cells are more dependent on glycolysis than oxidative phosphorylation in the mitochondria for generation of ATP as energy source. By using 2-deoxy-D-glucose (2-DG: glycolysis inhibitor) and oligomycin (inhibitor of oxidative phosphorylation), we examined the energy metabolism of various leukemia cell lines. The growth of the cell lines was measured by MTS assay, which detects viable cells in proliferation. 2-DG suppressed the growth of all leukemia cell lines examined in dose-dependent manners. The IC50 of each cell line was as follows: Kasumi-1 0.5±0.1mM, KG-1a 1.8±0.6mM, HL-60 3.3±0.1mM, NB4 3.8±0.4mM, and THP-1 23.1±3.8mM. The concentration of lactic acid (the final product of glycolytic pathway) in the culture supernatant was greatly reduced by the treatment with 0.2mM 2-DG for 24 hours in Kasumi-1 (54.5% of the control), compared with THP-1 (92.2%). It is suggested that the growth of Kasumi-1 was strongly suppressed by 2-DG through inhibition of glycolysis, which is supposed to be a main metabolic pathway in this cell line. On the other hand, treatment with oligomycin (1μg/ml) for 48 hours potently suppressed the growth of THP-1 (44.7%), then Kasumi-1 (72.1%). The growth of NB4, KG-1a and HL-60 was minimally suppressed (more than 90%) by oligomycin. Cell cycle was analyzed after 24 hours treatment with 2-DG or oligomycin. Sub-G1 fraction (apoptosis) was greatly increased by 2-DG (5mM) in Kasumi-1 (56.5%) and NB4 (30.6%), compared with THP-1 (7.6%). The apoptosis inducing effect was confirmed by annexinV staining. Oligomycin treatment (1μg/ml) increased apoptosis (subG1) in THP-1 (35.8%), then Kasumi-1 (16.6%) and NB4 (12.2%). Oligomycin treatment also increased G1 population (G1 arrest) in THP-1 (35.9% to 69.4%). AMP-activated protein kinase (AMPK) is activated by an elevated AMP/ATP ratio, which means the energy-deprived status of the cell. Western blot analysis using phospho-AMPK α (Thr172) antibody revealed that treatment with 2-DG or oligomycin induced prompt (30 min) phosphorylation of AMPK in leukemia cell lines. The extent of AMPK phosphorylation was almost proportional to the suppression of the growth. Collectively, it is suggested that leukemia cells are dependent almost exclusively on either glycolysis or oxidative phosphorylation in the mitochondria for energy production. Then, inhibition of glycolysis by 2-DG or oxidative phosphorylation by oligomycin results in growth suppression by inducing apoptosis and/or cell cycle arrest through activation of AMPK. Our data clarified the characteristics of the energy metabolism of each leukemia cell, and showed the key to produce novel therapeutic approach targeting metabolic pathway.

Description: Abstract 4375 Leukemia cell lines are ubiquitous powerful research tools that are available to many investigators. In balanced chromosomal aberration in leukemia, a chimeric fusion gene formed by genes existing on breakpoints is frequently related to leukemogenesis. Cytogenetic abnormalities of chromosome band 12p13 are detected non-randomly in various hematological malignancies and usually involved TEL, which encodes a protein of the ETS transcription factor family. Chromosome band 22q11-12 is one of partners of translocation 12p13 and t(12;22)(p13;q11-12) results in fusion of TEL and MN1 or in just the partial inactivation of TEL. It is important to analyze precisely the breakpoint in a non-random translocation such as t(12;22)(p13;q11-12) and in addition it contributes to the better understanding of the molecular pathogenesis of leukemogenesis. In this study, we established a novel human myeloid leukemia cell line, AMU-AML1, having t(12;22) from a patient with acute myeloid leukemia with multilineage dysplasia and analyzed its characters. Mononuclear cells were isolated by Ficoll-Hypaque sedimentation from patient's bone marrow before initiation of chemotherapy and cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS). After 3 months, cell proliferation became continuous. The cell line, named AMU-AML1, was established. In AMU-AML1, the following pathogens were negative for EBV, CMV, HBV, HCV, HIV-1, HTLV-1 and mycoplasma. A doubling time of AMU-AML1 cells was about 96 hours. Proliferation of the cells was stimulated by rhG-CSF (10 ng/ml), rhGM-CSF (10 ng/ml), M-CSF (50 ng/ml), rhIL-3 (10 ng/ml) and rhSCF (100 ng/ml) but not by IL-5 (10 ng/ml), rhIL-6 (10 ng/ml), and rhEPO (5 U/ml). AMU-AML1 was positive for CD13, CD33, CD117 and HLA-DR, negative for CD3, CD4, CD8 and CD56 by flow cytometry analysis. G-banding combined with SKY analysis of AMU-AML1 cells showed single structural abnormality; 46, XY, t(12;22)(p13;q11.2). Double-color FISH using PAC/BAC clones listed in NCBI website and array CGH analyses indicated that the breakpoint in 12p13 was within TEL or telomeric to TEL and it of 22q11 was centromeric to MN1. A chimeric MN1-TEL transcript and fusion protein of MN1-TEL could not be detected by RT-PCR and western blot analysis. The wild type of MN1 protein was strongly expressed in AMU-AML1 compared with other leukemic cell lines with t(12;22), MUTZ-3 and UCSD/AML1. Our data suggest that AMU-AML1 had a t(12;22)(p13;q11.2) without fusion of MN1-TEL and the expression level of MN1 protein was relatively high, which might have some effects on leukemogenesis. In conclusion, AMU-AML1 is a useful cell line to analyze the biological consequences of the leukemic cells with t(12;22)(p13;q11.2) but no fusion of MN1-TEL. Disclosures: No relevant conflicts of interest to declare.

Description: Most human leukemia cell lines are dependent on serum supplementation (usually fetal calf serum (FCS)), although the extent of serum dependency differs among each cell line. Kasumi-1, a t(8;21) AML cell line is one of the most serum-dependent cell lines. Since growth and survival of many leukemia cell lines are associated with phosphorylation of Akt, we examined the Akt phosphorylation by FCS treatment. In Kasumi-1, Akt was phosphorylated by culture with FCS in a dose-dependent manner, although no such Akt phosphorylation was observed in NB-4, a t(15;17) cell line. By FCS stimulation, Akt (Thr308, Ser473) was phosphorylated from 0.5 hr and the phosphorylation sustained until 48 hours in Kasumi-1. Then, we tested the effect of VEGF/VEGFR signaling in phosphorylation of Akt by FCS. The addition of VEGFR1/Fc and VEGFR2/Fc (which bind external VEGF and abrogate its function) inhibited the Akt phosphorylation from 2 hours until 10 hours, although the growth of Kasumi-1 was not inhibited. The addition of VEGFR2 kinase inhibitor (which inhibits internal VEGF signal) inhibited the Akt phosphorylation from 0.5 hr until 2 hours, and the growth of Kasumi-1 was greatly inhibited. Taken together, it is suggested that serum dependency of Kasumi-1 is at least in part attributed to VEGF/VEGFR pathway. Then, both external and internal VEGF/VEGFR pathways work in Kasumi-1, which in turn phosphorylate Akt. However, blockade of only internal VEGF signal (by VEGFR2 kinase inhibitor) inhibit the early Akt phosphorylation (0.5 hr), which resulted in growth inhibition, indicating the importance of early Akt phosphorylation.

Description: [Background and Objectives] One of the causes of death in patients with myelodysplastic syndrome (MDS) is leukemic transformation. After the indolent phase, which can be treated with chemotherapy, leukemic MDS will progress to a phase of aggressive blast proliferation, which cannot be controlled by chemotherapy. In this study, we would like to know the molecular mechanisms of the disease progression. [Materials and Methods] Bone Marrow samples were collected from both indolent (at initial diagnosis) and aggressive (after chemotherapies) phases from 4 MDS-leukemia patients. We extracted RNA from each sample and then amplified the RNA, reverse transcribed the amplified RNA to cDNA, and fluorescently labeled before hybridization for the microarray. [Results and Discussion] Compared to the expression level at indolent phase, several genes including MEK kinase 5 (ASK1), Rho-associated protein kinase 1 (ROCK1), LIM domain kinase 2 (LIMK2) and GTP binding protein Rheb, were commonly up-regulated more than 2-fold at aggressive phase of 4 cases. These results were confirmed by real time RT-PCR. Since multidrug resistance protein was up-regulated at the ratio of around 1.5, more than 2-fold up-regulation in all 4 examined cases was considered to be significant. These molecules can be candidate markers to predict transition from indolent to aggressive phase of the disease, and also can be targets for novel therapy.