Growth of xenotransplanted leukemia cells is influenced by diet nutrients and is attenuated with 2-deoxyglucose.

We examined the effects of diet nutrients on xenotransplanted leukemia cells, THP-1 or NB4. THP-1 tumors showed more growth when fed with high fat diet, while NB4 tumors grew more with high carbohydrate diet. Then, administration of 2-deoxyglucose (a glycolysis inhibitor) showed a significant antitumor effect on both tumors: NB4 tumor showed large necrotic areas, while THP-1 tumor did not, but had augmented expression of enzymes for fatty acid oxidation. 2-Deoxyglucose inhibited the growth of NB4 by cell death because main energy producing pathway (glycolysis) was abolished, while 2-deoxyglucose slowed the growth of THP-1 by shifting energy metabolism to fatty acid ß-oxidation.

Leukemia cells demonstrate a different metabolic perturbation provoked by 2-deoxyglucose.

The shift in energy metabolism from oxidative phosphorylation to glycolysis can serve as a target for the inhibition of cancer growth. Here, we examined the metabolic changes induced by 2-deoxyglucose (2-DG), a glycolysis inhibitor, in leukemia cells by metabolome analysis. NB4 cells mainly utilized glucose as an energy source by glycolysis and oxidative phosphorylation in mitochondria, since metabolites in the glycolytic pathway and in the tricarboxylic acid (TCA) cycle were significantly decreased by 2-DG. In THP-1 cells, metabolites in the TCA cycle were not decreased to the same extent by 2-DG as in NB4 cells, which indicates that THP-1 utilizes energy sources other than glucose. TCA cycle metabolites in THP-1 cells may be derived from acetyl-CoA by fatty acid ß-oxidation, which was supported by abundant detection of carnitine and acetylcarnitine in THP-1 cells. 2-DG treatment increased the levels of pentose phosphate pathway (PPP) metabolites and augmented the generation of NADPH by glucose-6-phosphate dehydrogenase. An increase in NADPH and upregulation of glutathione synthetase expression resulted in the increase in the reduced form of glutathione by 2-DG in NB4 cells. We demonstrated that a combination of 2-DG and inhibition of PPP by dehydroepiandrosterone (DHEA) effectively suppressed the growth of NB4 cells. The replenishment of the TCA cycle by fatty acid oxidation by carnitine palmitoyltransferase in THP-1 cells, treated by 2-DG, might be regulated by AMPK, as the combination of 2-DG and inhibition of AMPK by compound C potently suppressed the growth of THP-1 cells. Although 2-DG has been effective in preclinical and clinical studies, this treatment has not been fully explored due to concerns related to potential toxicities such as brain toxicity at high doses. We demonstrated that a combination of 2-DG and DHEA or compound C at a relatively low concentration effectively inhibits the growth of NB4 and THP-1 cells, respectively. These observations may aid in the identification of appropriate combinations of metabolic inhibitors at low concentrations which do not cause toxicities.

Establishment of a novel human myeloid leukemia cell line, AMU-AML1, carrying t(12;22)(p13;q11) without chimeric MN1-TEL and with high expression of MN1.

In this study, we established and analyzed a novel human myeloid leukemia cell line, AMU-AML1, from a patient with acute myeloid leukemia with multilineage dysplasia before the initiation of chemotherapy. AMU-AML1 cells were positive for CD13, CD33, CD117, and HLA-DR by flow cytometry analysis and showed a single chromosomal abnormality, 46, XY, t(12;22)(p13;q11.2), by G-banding and spectral karyotyping. Fluorescent in situ hybridization analysis indicated that the chromosomal breakpoint in band 12p13 was in the sequence from the 5' untranslated region to intron 1 of TEL and that the chromosomal breakpoint in band 22q11 was in the 3' untranslated region of MN1. The chimeric transcript and protein of MN1-TEL could not be detected by reverse-transcriptase polymerase chain reaction or Western blot analysis. However, the MN1 gene was amplified to three copies detected by array comparative genomic hybridization analysis, and the expression levels of the MN1 transcript and protein were high in AMU-AML1 cells when compared with other cell lines with t(12;22)(p13;q11-12). Our data showed that AMU-AML1 cells contain t(12;22)(p13;q11.2) without chimeric fusion of MN1 and TEL. The AMU-AML1 cells gained MN1 copies and had high expression levels of MN1. Thus, the AMU-AML1 cell line is useful for studying the biological consequences of t(12;22)(p13;q11.2) lacking chimeric MN1-TEL.

Energy metabolism of leukemia cells: glycolysis versus oxidative phosphorylation.

For generation of energy, cancer cells utilize glycolysis more vigorously than oxidative phosphorylation in mitochondria (Warburg effect). We examined the energy metabolism of four leukemia cell lines by using glycolysis inhibitor, 2-deoxy-d-glucose (2-DG) and inhibitor of oxidative phosphorylation, oligomycin. NB4 was relatively sensitive to 2-DG (IC(50): 5.75 mM), consumed more glucose and produced more lactate (waste product of glycolysis) than the three other cell lines. Consequently, NB4 was considered as a "glycolytic" leukemia cell line. Dependency on glycolysis in NB4 was confirmed by the fact that glucose (+) FCS (-) medium showed more growth and survival than glucose (-) FCS (+) medium. Alternatively, THP-1, most resistant to 2-DG (IC(50): 16.14 mM), was most sensitive to oligomycin. Thus, THP-1 was recognized to be dependent on oxidative phosphorylation. In THP-1, glucose (-) FCS (+) medium showed more growth and survival than glucose (+) FCS (-) medium. The dependency of THP-1 on FCS was explained, at least partly, by fatty acid oxidation because inhibitor of fatty acid ß-oxidation, etomoxir, augmented the growth suppression of THP-1 by 2-DG. We also examined the mechanisms by which THP-1 was resistant to, and NB4 was sensitive to 2-DG treatment. In THP-1, AMP kinase (AMPK), which is activated when ATP becomes limiting, was rapidly phosphorylated by 2-DG, and expression of Bcl-2 was augmented, which might result in resistance to 2-DG. On the other hand, AMPK phosphorylation and augmentation of Bcl-2 expression by 2-DG were not observed in NB4, which is 2-DG sensitive. These results will facilitate the future leukemia therapy targeting metabolic pathways.

Growth inhibition of AML cells with specific chromosome abnormalities by monoclonal antibodies to receptors for vascular endothelial growth factor.

By using neutralizing monoclonal antibodies to vascular endothelial growth factor receptor type 1 (VEGFR1) and VEGFR2, we have shown that acute myelogenous leukemia (AML) cells with specific chromosome abnormalities are dependent on VEGF/VEGFR system. AML with t(8;21) is the most dependent subtype on VEGF with both VEGFR1 and VEGFR2. t(15;17)AML cells depend on VEGF with VEGFR1. AML cells with 11q23 abnormalities showed variable dependence on VEGF. The growth of t(11;19)AML cells are most extensively inhibited by anti-VEGFR1 antibody. Then, the growth of Kasumi-1, a t(8;21) cell line was suppressed by either anti-VEGFR1 antibody (p=0.0022) or anti-VEGFR2 antibody (p=0.0029) in a dose-dependent manner. The growth of NB4, a t(15;17) cell line was more potently suppressed by anti-VEGFR1 antibody (p=0.0111) than by anti-VEGFR2 antibody (p=0.0477). These results are quite concordant with the results of clinical samples with t(8;21) or t(15;17). In addition, anti-VEGFR2 monoclonal antibody significantly potentiated the growth inhibitory effect of idarubicin for Kasumi-1. As for downstream signals, we have shown that VEGFR2 transduce growth and survival signals through phosphorylation of Akt and MEK in leukemia cells (Kasumi-1). However, VEGFR1 transduce growth and survival signals through pathways other than MEK and Akt (NB4), although Akt phosphorylation may account for some of the VEGFR1 signals (Kasumi-1). Finally, our data suggested that autocrine pathway of VEGF and VEGFRs observed in AML cells with specific chromosomal translocations have contributed to leukemogenesis as activated signaling of receptor tyrosine kinase.

Autocrine pathway of angiopoietins-Tie2 system in AML cells: association with phosphatidyl-inositol 3 kinase.

Hematopoietic cells and endothelial cells are mutually correlated in their development and growth. Various angiogenic factors, such as vascular endothelial growth factor (VEGF) and angiopoietins (Angs), are thought to be associated with leukemia cell growth. In this study, we examined if the Angs-Tie2 autocrine pathway works in primary AML cells or not by using soluble Tie2-Fc, which inhibits Angs from binding to Tie2 receptor. After 48 h of culture with Tie2-Fc, nine AML cells from 19 examined samples were not influenced by Tie2-Fc (group A), while AML cells from remaining 10 patients demonstrated remarkable reduction of cell number by Tie2-Fc treatment (group B). Tie2 receptor, upon binding to Angs, are known to activate phosphatidyl-inositol 3 kinase (PI3 kinase). Then, we examined the effect of LY294002, a potent PI3 kinase inhibitor, on primary AML cells. Cell number reduction effect by the treatment of LY294002 was much more prominent in cells of group B than of group A. In addition, extent of cell number reduction by Tie2-Fc and LY294002 was quite well correlated. These observations demonstrated that cells from a part of AML were dependent on autocrine Angs-Tie2 pathway. This notion was further supported by the study of two AML cell lines, KG-1 and HL-60: the growth of KG-1 was suppressed by Tie2-Fc, and also by anti-Tie2 antibody, which inhibits receptor-ligand interaction, while that of HL-60 was not suppressed by Tie2-Fc or anti-Tie2 antibody. Our results will help to explore the angiogenesis-oriented or endothelial cell-mediated therapy for leukemia.

Acute promyelocytic leukemia with drug-induced hypersensitivity syndrome associated with Epstein-Barr virus infection.

We report a case of acute promyelocytic leukemia (APL) with drug-induced hypersensitivity syndrome associated with Epstein-Barr virus (EBV) infection. A 33-year-old woman was admitted because of APL. After complete remission was obtained with the use of all-trans retinoic acid (ATRA), intensive chemotherapy was administered. She developed high grade fever and severe systemic erythematous eruptions followed by cervical lymphoadenopathy, hepatosplenomegaly, hepatitis and hypotension in a state of myelosuppression during consolidation chemotherapy. Systemic corticosteroids alleviated the symptoms. Since an anti-EB VCA IgM antibody titer was continuously positive, persistent infection of EBV was suspected. In this case, EBV infection may have contributed to the development of drug-induced hypersensitivity syndrome.

Augmented expression of P-gp/multi-drug resistance gene by all-trans retinoic acid in monocytic leukemic cells.

P-glycoprotein (P-gp)/multi-drug resistance 1 (MDR1) gene is recognized to be, at least in part, responsible for the refractoriness to chemotherapy of leukemia. The transcriptional mechanism of MDR1 gene is largely unknown. However, recent reports have clarified that early growth response 1 gene (Egr1) positively regulates MDR1 transcription, while Wilms' tumor suppressor gene (WT1) does negative regulation of MDR1 gene expression in 12-O-tetradecanoylphorbol-13-acetate treated K562 cells. In addition, Egr1 and WT1 are structurally related transcription factors and bind to quite similar DNA sequences. Our study of mRNA expression profile of Egr1, WT1 and MDR1 in fresh AML samples demonstrated that there are disease-specific patterns. Egr1 mRNA was frequently and strongly expressed in monocytic leukemia cells, especially in AML M4 cells. WT1 mRNA was undetectable in t(8;21) AML cells. mRNA expression of MDR1 was frequent in AML M1 and t(8;21) AML cells, in which the expression level was highest in AML M1 and was low in monocytic leukemia (M4 and M5). Then, expression level of MDR1 was inversely correlated with Egr1. By liquid culture of leukemia cell lines and fresh AML cells with the addition of all-trans retinoic acid (ATRA), modulation of P-gp/MDR1 and Egr1 was observed and the pattern of modulation was divided into four groups: (1) blastic AML type, in which distinct expression of P-gp/MDR1 and CD34 was not influenced by ATRA; (2) t(8;21)AML type, in which P-gp/MDR1 expression was augmented by ATRA, while CD34 was kept high; (3) AML M3 type, in which P-gp/MDR1 expression was reduced with granulocytic differentiation by ATRA; (4) monocytic AML type, in which P-gp/MDR1 expression was augmented by ATRA, while CD34 expression decreased, and strong Egr1 expression was downregulated just prior to the augmentation of P-gp/MDR1 expression. WT1 expression was not influenced by the addition of ATRA in each group. Previous reports have suggested that P-gp/MDR1 plays an important role in resistance to chemotherapy, and is recognized as one of the stem cell marker. However, P-gp/MDR1 expression augmented by ATRA, which was observed in monocytic AML, was recognized as a functional molecule of mature monocyte/macrophage, because CD34 expression decreased and CD13 expression increased by ATRA. Finally, expression of P-gp/MDR1 in monocytic leukemia, which was functionally confirmed by Rh123 efflux study, was thought to be closely related to the characteristic modulation of Egr1 expression by ATRA.