The present study was performed to identify biomarkers for exposure of benzene in blood cells and hematopoietic tissues. Peripheral mononuclear cells, hematopoietic stem cells, and leukemia cell lines were cultured in RPMI 1640 media with the addition of 0, 1, and 10 mM of benzene. Hydrogen peroxide was measured using an enzyme immunoassay. Mitochondrial mass, membrane potential, and mitochondrial DNA (mtDNA) copy number were measured using MitoTracker Green/Red probes, and real-time polymerase chain reaction. In addition, two-dimensional gel electrophoresis and mass spectrometry matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) technology were performed to identify protein markers. The mitochondrial contents and membrane potentials were dramatically increased after three weeks of direct benzene exposure. The hydrogen peroxide level increased significantly after two weeks of treatment with benzene (4.4 ± 1.9 µM/mg protein) compared to the non-benzene treatment group (1.2 ± 1.0; p = 0.001). The mtDNA copy number gradually increased after exposure to benzene. Numerous protein markers showed significant aberrant expression after exposure to benzene. Among them, the heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 was markedly decreased after exposure to benzene. Thus, increased mitochondrial mass, mtDNA copy number, and the hnRNP A2/B1 protein were biomarkers for benzene-related toxicity and hematotoxicity.
4426 Background Exposure to benzene and its metabolites increases risk of marrow failure disorders, leukemia and other hematological diseases. However, biomarkers of benzene toxicity have not been comprehensively studied in hematopoietic cells and leukemia cells. We previously reported that benzene metabolites may impair electron chain transport and mitochondrial function (3rd WHO Conference on Children's Health and the Environment, 7-10 June 2009). Therefore, we hypothesized that alterations in mitochondrial mass and mitochondrial DNA (mtDNA) may occur in bone marrow cells and leukemia cells after benzene exposure to compensate for damaged mitochondria. Materials and Methods Total bone marrow cells from healthy individuals and leukemia cell lines (THP-1, Kasumi-1, K562, Molt-4 and HL-60) were cultured in RPMI media containing 10% fetal bovine serum for 5 days. Benzene was added in cell culture media with 0, 1 and 10mM concentration at 24 hour interval. Cell count was performed using an automated blood cell analyzer (ADVIA120, Siemens, Germany). Viability and apoptosis were assessed by tryptophan blue dye exclusion test and flowcytometry based annexin V staining protocol. Hydrogen peroxide content is measured using the commercial kit (Bioxytech® H2O2-560TM, OXIS International) according to the manufacturer's instructions. Mitochondrial mass, membrane potential and mtDNA copy number were measured using MitoTracker Green, MitoTracker Red probes (Invitrogen), and real time PCR using the QuantiTect SYBR Green PCR kit (Qiagen) and Rotor-Gene 3000 (Corbett Research), respectively. Results The number of cells were gradually increased regardless of concentration of benzene in day 3, and then steadily maintained during 3 weeks culture. Interestingly, the growth of K562 cells showed no growth inhibition effect (three fold increase) after 5-day exposure to benzene. Overall viability of five leukemia cell lines disclosed significant decrease after two week treatment of benzene (about 60% of viability was observed in 3- week suspension culture). The proportion of apoptosis was increased in time and dose dependent manner after 2-week treatment of benzene. Interestingly, mitochondrial contents and membrane potentials were dramatically increased in 3-week suspension culture after benzene exposure at dose dependent manner. The level of hydrogen peroxide significantly elevated after two week treatment of benzene (4.4 ± 1.9 μM/mg protein) compared with non-benzene treatment group (1.2 ± 1.0 μM/mg protein; P = 0.001). The average mtDNA copy number was gradually increased after exposure to benzene. Conclusions Benzene exposure caused increased mitochondrial mass and mtDNA copy number in response to oxidative stress induced by benzene. So, these mitochondrial changes can be used for biomarkers of benzene toxicity in hematopoietic tissue and leukemia cell. Disclosures: No relevant conflicts of interest to declare.
4771 Background: Leukemic stem cell (LSC) has been accused to play a pivotal role in pathogenesis of hematological malignancy such as acute myeloblastic leukemia (AML). AML stem cell (ASC) is known as CD34+CD38– leukemic cell population. Yet, despite their critical importance, much remains to be learned about selectively targeting ASC. Recently metformin selectively targets breast cancer stem cells, and acts together with chemotherapy to block tumor growth. Thus, the current study investigated whether metformin selectively inhibits AML cells and AML stem cells directly or indirectly. Materials and Methods: ASC showing CD34+CD38– phenotype were selectively sorted from primary bulk AML cells and KG-1 AML cell lines using single cell sorter (BD FACS Aria cell sorter, BD Biosciences, USA). ASC from primary AML samples and cell lines were cultured in serum media with each 100 ng/mL of stem cell factor, Flt-3, thrombopoietin and G-CSF. Metformin (1,1-dimethylbiguanide hydrochloride) and idarubicin (IDA) were dissolved and diluted with high purity distilled water in various concentrations (0.1 mmol/L to 30 mmol/L). Morphological change, cell count, viability and apoptosis using annexin V flowcytometry were checked after treatment of metformin and IDA. Phosphorylation of AMPK was assayed by western blotting and mitochondrial membrane potential was measured using MitoTracker green dye. Results: The frequency and proportion of ASC varied according to FAB subtypes. The mean proportion of ASC from AML M2, M4 and M3 showed 25.1±22.5% (mean±SD), 15.1±16.6% and 6.5±3.3%, respectively. The mean proportion of ASC from KG-1 cell lines was 78.6±6.2%. Total cell count of ASC was gradually decreased after treatment of metformin in dose-time dependent manner. Overall viability of AML cell lines and ASC disclosed significant decrease after treatment of metformin. The proportion of apoptosis was increased in time and dose dependent manner after 24-hr treatment of metformin. Mitochondrial membrane potentials were increased after metformin exposure at dose dependent manner. Moreover, when combined with lower concentration of IDA, more profound selective killing effect on AML cell lines and ASC was observed. Western blot analysis showed that the treatment of AML cell lines and ASC with 30 mmol/L of metformin resulting in the increased phosphorylation of AMP-activated protein kinase (AMPK) at approximately 1.5-fold over total AMPK. Conclusion: Metformin selectively kills AML cells as well as AML stem cells, and acts together with chemotherapeutic drugs to eradicate leukemic cells and their stem cells. Disclosures: No relevant conflicts of interest to declare.
3002 Background: Prohibitin (PHB) is localized to the mitochondria where it might have a role in the maintenance of mitochondrial function. The diverse function of PHB, together with the emerging evidence that its function can be modulated specifically in certain diseases, implies that PHB would be a potential target for new therapeutics. Materials and Methods: We analyzed mitochondrial proteins and develop new anti-proliferative agents targeting multiple myeloma (MM) cells. Mitochondria were isolated from primary leukemia and MM cell lines (RPMI8226, ARH77, U266 and IM9) by density-gradient ultracentrifugation using swelling buffer and sucrose buffer. Dysregulated mitochondrial protein was identified using 2-DE and mass spectrometry (MALDI-TOF/TOF technology). Results: Out of 38 remarkable deregulated mitochondrial proteins in MM cell lines, prohibitin (PHB) (gi4505773) was highly expressed in primary MM and leukemia cells, which was confirmed by Western blot, immunohistochemistry and immunofluorecenct study in the primary bone marrow cells and sections. Potent chemical substances that can alkylate PHB, two molecules of phenyl-chloroethyl urea family such as cyclohexylphenyl-chloroethyl urea (CCEU) and iodophenyl-chloroethyl urea (ICEU), were synthesized independently from the reaction with 2-ethylisocyanate and 4-cyclohexylaniline and 4-iodoaniline, respectively. They were characterized by 1H NMR and 13C NMR. Time and dose dependent manner of proliferation suppression when treated with CCEU and ICEU was observed in MM and leukemia cells. Moreover, notable morphological transformation of MM cells was observed when treated with 10 – 100 umol of CCEU and ICEU for 24 hours. The half of maximal inhibitory concentration (IC50) was 25umol of most MM cell lines. Cell cycle analysis of CCEU-and-ICEU-treated- MM cells showed a remarkable increase of the sub-G1 phase. Immunoblotting experiment revealed the change of cytoplasmic and nucleoplasmic PHB. Expression of cleaved caspase3 and poly ADP-ribose polymerases were also observed to have increased in MM cell lines. Conclusions: By analyzing mitochondrial protein in leukemia and MM cell, we discovered a new molecular marker, PHB, characteristically overexpressed in leukemia and MM cells and developed new anti-cancer agents such as CCEU and ICEU that target against PHB in MM cells. Disclosures: No relevant conflicts of interest to declare.
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