The Role And Regulating Mechanisms For β Subunit Of Mitochondrial F₁-f₀ Atp Synthase In Acute Leukemia With Multidrug Resistance

BackgroundAcute leukemia(AL) is one of the most common malignant tumors in hematological system. Multidrug resistance(MDR) to chemotherapy is the main reason for treatment failure in patients with AL. The mechanisms of MDR have been extensively studied in the past 20 years. And some new chemo-resistant reversal treatments have been under clinical practice. However, the specific molecular mechanism of MDR remains to be further elucidated. Besides, the therapeutic effect of current chemo-resistant reversal treatments have been proved to be very limited. Therefore, it is of great clinical importance to investigate new links and mechanisms of MDR in patients with AL and explore novel molecular targets for drug-resistant reversal.Mitochondria plays a pivotal role in harnessing cellular energetic metabolism, maintaining cellular calcium homeostasis and regulating apoptosis. Mitochondria F1F0-ATP synthase is the enzyme complexes of the inner mitochondrial membrane which carries out the synthesis of ATP, theβsubunit (β-F1-ATPase) of which is a rate-limiting component of the activity of oxidative phosphorylation. Down-regulation ofβ-F1-ATPase has been found in many different types of human carcinomas. A defect in mitochondrial ATP synthase is not only a bioenergetic feature of many cancer types, but may also be a link to chemotherapy and radiotherapy resistance, because the overall oxidative phosphorylation capability of the cell is diminished and, thus, the apoptotic potential of the cancer cell was hampered. A recent study showed that down-regulation of mitochondial F1F0-ATP synthase in human colon cancer cells was associated with induced 5-fluorouracil resistance, but the mechanisms explaining drug-resistance remained to be determined. It has been evidenced in many recent researches that intrinsic connection exists between abnormal methylation status of CpG sites at gene promoter region and the repressed transcriptional activity, as well as the induced MDR in malignant tumor cells. Our previous study showed that down-regulation of mitochondrial ATPase was associated with induced adriamycin(ADR) resistance in chronic myeloid leukemia(CML) cells. And our further study showed that DNA methylation regulation was associated with drug-resistance to ADR in CMLIn this study, on basis of previous researches, for a purpose of exploring novel mechanisms for MDR and locating novel molecular targets for MDR reversal in patients with AL, we aim to learn the relationship betweenβ-F1- ATPase expression in primary cells of patients with AL and the clinical effects as well as in vitro sensitivitity to ADR; to investigate the effect of DNA methylation on MDR in patients with AL. PartⅠExpression ofβsubunit of mitochondria F1F0-ATP synthase in primary cells of patients with acute leukemia and the relationship between its expression and clinical effects as well as in vitro drug sensitivityObjective:To investigate the role of abnomal expression ofβ-F1-ATPase in the development of MDR in patients with ALMethods:Bone marrow samples were isolated from 82 patients with AL (51 AML,31 ALL) and 11 patients with benign hematological deseases(for control). According to clinical drug-resistance diagnostic criteria, AL patients were divided into clinical chemosensitive group and clinical chemoresistant group. Bone marrow mononuclear cells(BMMCs) were isolated and cultured in primary. Expression levels ofβ-F1-ATPase mRNA and protein in BMMCs of patients with AL were detected by real-time quantitative RT-PCR(RQ-PCR) and Western blot respectively.β-F1-ATPase mRNA and protein levels were compared between chemosensitive and chemoresistant group. And consecutive changes ofβ-F1-ATPase mRNA levels of 6 patients with AL were observed when initially diagnosed and when their deseases relapsed for the first time. In vitro sensitivity of BMMCs to ADR in chemosensitive and chemoresistant group patients with AL was detected by MTT assay. Linear correlation relationship between expressions ofβ-F1-ATPase mRNA and protein and in vitro drug sensitivity in the two groups was analysed respectively.Results:1. Different levels ofβ-F1-ATPase mRNA and protein expression were detected in all patients with AL and benign hematological deseases. Compared with control group, expression levels ofβ-F1-ATPase mRNA and protein in BMMCs decreased significantly in patients with AL (P<0.01)2. Compared with chemosensitive group, expression levels ofβ-F1-ATPase mRNA and protein in BMMCs decreased significantly in patients with AL in chemoresistant group (P<0.05)3. Compared with chemosensitive group, expression levels ofβ-F1-ATPase mRNA and protein in BMMCs decreased significantly in patients with acute myeloid leukemia(AML) in chemoresistant group (P <0.05). In patients with acute lymphoblastic leukemia(ALL), expression levels ofβ-F1-ATPase mRNA and protein in BMMCs showed a decreasing trend in chemoresistant group when compared to chemosensitive group, but no statistical difference was observed between the two groups (P>0.05)4. Compared with initial diagnosis, expression levels ofβ-F1-ATPase mRNA in BMMCs of 6 patients with AL who achieved complete remission(CR) after initial induction therapy significantly decreased when their deseases relapsed for the first time (P<0.05)5. Average 50% concentration of inhibition(IC50) of ADR to primary BMMCs of patients with AL in chemosensitive group and chemoresistant group were 0.94μg/mL and 7.39μg/mL respectively. The extent of drug-resistance to ADR in chemoresistant group was 7.9 times higher than that in chemosensitive group.6. Expression levels ofβ-F1-ATPase mRNA and protein significantly and inversely correlated with IC50 of ADR to primary BMMCs in chemoresistant group patients with AL (r value were-0.467 and-0.628 respectively, both P<0.05)Conclusion:A significant decrease ofβ-F1-ATPase mRNA and protein expression can be observed in bone marrow primary cells in chemoresistant group patients with AL. And this decrease significantly correlates with resistance of primary cells in vitro to ADR, suggesting that chemoresistance in AL is associated with down-regulation ofβ-F1-ATPase activity. PartⅡRelationship between methylation regulation of CpG sites at gene promoter region ofβsubunit of mitochondria F1F0-ATP synthase and clinical effects in acute leukemiaObjective:To investigate the relationship between methylation regulation of CpG sites at gene promoter region ofβ-F1-ATPase and clinical drug-resistance in patients with AL.Methods:Bone marrow samples were isolated from 52 patients (34 AML,18 ALL) with AL and 8 patients with benign hematological deseases(for control). According to clinical drug-resistance diagnostic criteria, AL patients were divided into clinical chemosensitive group and clinical chemoresistant group. According to gene promoter methylation status, patients with AL were divided into methylation group and unmethylation group. BMMCs were isolated and cultured in primary. Methylation status of CpG sites at gene promoter region ofβ-F1-ATPase and expression level of mRNA in BMMCs in chemosensitive and chemoresistant group of patients with AL were detected using methylation specific PCR(MSP) and RQ-PCR respectively. Methylation frequency ofβ-F1-ATPase between chemosensitive group and chemoresistant group was compared. Expression levels ofβ-F1-ATPase mRNA were compared between methylation group and unmethylation group.11 patients with AL in chemoresistant group who showed a complete methylation status inβ-F1-ATPase promoter region were chosen. The reversal effects of BMMCs of these 11 patients to ADR resistance were observed after using methylation inhibitor 5-azacytidine(5-Aza)Results:1. No methylation of CpG sites ofβ-F1-ATPase promoter region was detected in BMMCs of 8 patients with benign hematological deseases, with 0 methylation frequency. Methylation frequency ofβ-F1-ATPase in BMMCs of 52 patients with AL was 55.8%.2. Methylation frequency in BMMCs of 25 patients with AL in chemoresistant group was 76%, which was significantly higher than that in 27 patients with AL in chemosensitive group(37%) (P<0.05)3. Methylation frequency ofβ-F1-ATPase in BMMCs of 16 patients with AML in chemoresistant group was 81.3%, which was significantly higher than that in 18 patients with AML in chemosensitive group(38.9%) (P<0.05)4. Compared with a single use of ADR, IC50 of ADR to BMMCs of 11 patients with AL significantly decreased after 5-Aza treatment (P< 0.01). And a significant increase could be observed in expression levels ofβ-F1-ATPase mRNA in BMMCs of these 11 patients after 5-Aza treatment (P<0.05)Conclusion:1. Significant difference of methylation status ofβ-F1-ATPase promoter region in BMMCs of patients with AL can be observed between chemosensitive group and chemoresistant group. Hypermethylation ofβ-F1-ATPase promoter region exists in chemoresistant group, and influentsβ-F1-ATPase mRNA expression.2.5-Aza, a methylation inhibitor, can enhance the sensitivity of drug-resistant leukemia cells to ADR, and up-regulateβ-F1-ATPase mRNA expression. So it is expected for 5-Aza to become a new intervention method for reversal of drug-resistance in AL...