Overexpression Of HTERT Enhance Proliferation And Anti-apoptosis In Multiple Myeloma Cells And Its Mechanisms

Background Multiple myeloma is a common neoplastic hematologic disorder with feature of bone ma(?)w plasma cells malignant hyperplasia and its incidence (?) of all hematological malignancies. With new drugs such as pro(?)asome and immunomodulatory adents entering into clinic, survival of MM patients was significantly prolonged. Although most untreated MM patients sensitive to st all patients will relapse and ultimately invalid to treatment. So far, MM is considered an incurable disease.The genetic background of MM is complicated, there are several ways participate in the drug resitance mechanism. To date, the known mechanism including drugsuch as P-gp, MRP or LRP, and growth factor such as IL-6, as well as endogenous anti-apoptotic factors such as Bcl-2or NF-kB. In earlier clinical studies,(?)ents such as verapamil, ruinine, or Cyclosporine A was used to reverse drug resistance (?)diated by efflux pump P-gp, but the effect was limited. New drug proteasome, immunomodulators and arsenic can directly or indirectly inhibit anti-apoptotic factors such as IL-6, Bcl-2and NF-kB, although the early treatment effect is obvious, but still can’t cure the disease. So it is still necessary to find new therapy target of MM.Telomeres is the special capping structure at the top and bottom of every chromosome. The telomere becomes shortened following every chromosome replication and cell division and when it’s length comes near critical conditions, the cell turns into senescence procedure. Telomerase can utilize its RNA component as template to synthesizes TTAGGG repeat sequences with its reverse transcription subunit and add them to the bottom of chromosomes, thus maintain telomere’s length and chromosome’s stability. Telomerase is activated in almost all cancer cells, and endow the power of immortalization to the cells. Moreover, the activation of telomerase can regulate tumor cell proliferation and apoptosis through various channels, enhance cell resistance to chemotherapetic drugs, oxidative damage, and ionizing radiation.MM’s resistance is closely related to their proliferation and apoptosis capability. By now, it is not yet clear through which downstream pathway that the activated telomerase regulate the process of MM cells’ proliferation and apoptosis. And whether telomerase itself and its downstream regulatory pathway, is a effective target for MM treatment. So our idea was to use adenovirus vector with which TERT imported into myeloma cells and leading to TERT gene overexpression, then detect proliferation and apoptosis change in myeloma cells under the pressure from drugs, as well as the corresponding changes in cell proliferation and anti-apoptosis signaling pathway. Our research was divided into two parts, the first part for the construction and packaging of TERT-adenovirus vector, and the second part for the changes of proliferation and apoptosis as well as its mechanism in TERT overexpression myeloma cells.Part one Construction and packaging of non-proliferative adenovirus vector load TERT gene.Why choose adenovirus vector Adenovirus expression vector has a unique set of advantages. Adenovirus genome is homologous with the human’s, and thus the target protein expressed in a high level and has a complete function. Furthermore, adenovirus genes are not integrated with the host genome, so it can’t be interference on host genes nonspecifically, nor cause inserted mutation, thus make accurate gene expression. In addition, adenovirus has a high efficiency of infection on human cells, and be contagious either on proliferating cells or non-proliferating cells as well as its genome into the nucleus efficiency up to40%. Moreover, adenovirus vector is relatively safe because of its low pathogenicity and mutagenic force. So, we choose the non-replicating adenovirus expression vector to construct the TERT gene transfection platform.Methods By method of enzyme digestion, the target gene TERT and PDC318was obtained from plasmid PIRES2-EGFP-TERT and vector PDC318, then connected and transferred to DH5a E. coli competent cells reorganization as PDC318-TERT plasmid. The PDC318-TERT plasmid was introduced into HEK-293A cells packing load TERT gene replication-defective adenovirus particles. The virus were identified correct by PCR, then abundance proliferated in HEK-293A cells, and purified by method of HPLC. Finally, the virus titer was determined by the method of TCID50.Results Load TERT gene replication-defective adenovirus expression vectors were obtained, and the virus titer was determined as3.5×109PTU/ml Part two Study of proliferation and apoptosis impacted by TERT gene overexpression in myeloma cells and its mechanism.Object To research the changes of proliferation and apoptosis after TERT gene overexpression in myeloma cells, and the change of major intracellular signaling pathway involved in proliferation and apoptosis, the change of IL-6signaling pathway, and the change of drug-resistance protein expression, as well as the potential targets for myeloma therapy.Methods Transfected the myeloma cell lines, RPMI-8226and U-266, with5-10MOI TERT-adenovirus. Then verified the efficiency of infection of the virus and the TERT expression level after transfection by methods of fluorescence microscopy, PT-PCR, Western Blot as well as TRAP-ELISA. Determined the change of cell proliferation and apoptosis rate under the pressure of different concentrations of epirubicin after TERT overexpression. Determined the expression level of gene of drug-resistance in TERT overexpression myeloma cells by method of RT-PCR. Determined the expression level of IL-6and its receptor by method of ELISA. Determined the expression of apoptosis related protein and expression of ERK1/2 MAPK signaling pathway as well as expression of PI3K/AKT/mTOR signaling pathway by method of Western Blot.Results The infection rate of adenovirus vector to myeloma cells RPMI-8226and U-266was about90%. The TERT mRNA levels increased about200-400times after transfection in TERT-8226cells and TERT-U266cells, and TERT protein level increase about2-7times, and the telomerase activity increased about2-3times. By semi-quantitative RT-PCR, we found that the mRNA level of MDR1/GP170increased about10-15times in TERT-8226cells, but it had no significant change in TERT-U266cells. And the mRNA level of multidrug resistance associated protein and mRNA levelof lung drug resistance related protein had no significant change in the two myeloma cells. The mRNA level of IL-6and its receptor, IL-6R and gp130, had no significant change in TERT overexpression cells. But by method of ELISA, we found that the level of soluble gp130(sgp130) in the supernatant of TERT-8226cells increased, whereas the level of soluble IL-6R (sIL-6R) in the supernatant decreased significantly compared to8226-GFP-vector cells and untransfected8226-control cells. By the Western Blot assay, we found that in TERT overexpression8226cells, the expression of p-AKT and p-mTOR was up-regulated significantly compared with8226wild type cells. On the contrast, either p-ERK or c-MYC was down-regulated significantly. By apoptosis detection with the AV-PI, we found when treated with100nM and200nM epirubicin for24hours, the apoptosis rates of TERT-8226cells decreased by21.73%and21.58%respectively, P value was0.113and0.041respectively, compared with8226wild type cells. Determined by MTT, we found that the48hours IC50value of TERT-8226cells to epirubicin raised by59.31%, P value was0.043, and the48hours IC50value of TERT-U266cells to epirubicin raised by147.12%, p value was0.024, compared with their wild type cells respectively.Conclusion VDC318-TERT adenovirus expression vector can introduce TERT gene into either RPMI-8226cells or U-266myeloma cells efficiently. Overexpression of TERT endow myeloma cells higher capacity of proliferation and anti-apoptosis, increase drug resistance to epirubicin. These enhanced effects maybe directly associated with the phosphorylation of the PI3K/AKT/mTOR signaling pathway. TERT overexpression inhibits ERK1/2MAPK signal, and its downstream transcription factor, c-Myc’s expression. TERT gene overexpression perhaps inhibits the function of IL-6/IL-6R/gp130signaling pathway in RPMI-8226cells, whereas had no significant effect on the expression of common resistance associated genes (such as MDR1, MRP, LRP, etc.) in myeloma cells.