Expression And Significance Of HTERTmRNA In Myelodysplastic Syndrome And The Effect Of HTERT In Apoptosis Of MUTZ-1 Cells Induced By Arsenic Trioxide

Myelodysplastic syndrome (MDS) is a group of monoclonal hematopoietic stem cell disorders characterized by cytopenias, transfusion-dependence and progression to AML. No uniformly effective treatment exists for patients with MDS, there is a tremendous need for novel approaches in the management of MDS. During the last decade, the efficacy of AS₂O₃ in both newly diagnosed and relapsed patients with APL has been established. The impact of AS₂O₃ on multiple tumor pathways has spurned its investigation in other hematologic malignancies, including MDS.Human telomerase reverse transcriptase (hTERT) stabilizes chromosomal ends by extending the telomeric repeat sequences and plays a critical role in cellular survival, development and functions of multiple tissues and organs. hTERT activity is repressed in normal somatic tissues, but both hTERT expression and activity are elevated in most human tumors. It is a key molecular in tumorigenesis.To determine the expression and significance of hTERT in MDS, We detectedthe expression of hTERTmRNA in the bone marrow cells of 41 patients with MDS by RT-PCR. Our results showed that the positive rate of hTERT mRNA was 73.2%. There were 66.7%(10/15), 33.3%(2/6), 83.3%(10/12) and 100%(8/8) in RA, RAS, RAEB and RAEB-t groups respectively. Positive rate of hTERT mRNA in high-risk MDS(RAEB+RAEB-t) was higher than that in low-risk MDS(RA+RAS). According to IPSS, positive rates of hTERT were 100.0%, 57.14% and 64.29% in high-risk, intermediate and low-risk MDS respectively. It was higher in high-risk MDS than that in intermediate and low-risk MDS too.Meanwhile we detected the expression of WTl mRNA in MDS, which was reported as the transcription inhibitor of hTERT. We found that it was high expressed in patients with MDS. The positive rate of WTl mRNA was 61%. There were 29.4%(4/15), 16.7%(l/6), 100%(12/12) and 100%(8/8) in RA, RAS, RAEB and RAEB-t groups respectively. Positive rate of WTl mRNA in high-risk MDS(RAEB+ RAEB-t) was higher than that in low-risk MDS(RA+RAS). According to IPSS, positive rates of WTl were 100.0%, 71.43% and 14.29% in high-risk, intermediate and low-risk MDS respectively. It increased with the risk grade of MDS patients. So we concluded that hTERT and WTl were important factors in the tμMorigenesis of MDS patients and may be new targets in the treatment of MDS.To study the effect of As₂O₃ on MDS and investigate the molecular mechanism of As₂O₃-induced apoptosis, MDS cell line MUTZ-1 cells were cultured with AS2O3. We first evaluated the effect of AS₂O₃ on the growth of MUTZ-1 cells using MTT assays. In our study, dose- and time-related inhibitions of cell growth were observed.Growth inhibition (5-52% of the control) was induced in MUTZ-1 cells after it was exposured at the concentration of 1 -8μM AS₂O₃ for 24h, and there were 28-78% and 52-89% for 48h and 72h respectively. The respective IC50s of As₂O₃ on MUTZ-1 cells for 24h, 48h and 72h were 6.32μM, 1.94μM and 0.59μM. We further detected the apoptosis of MUTZ-1 cells incubated with variousconcentration of As₂O₃ by morphologic changes, apoptotic DNA ladder and PI-AnnxineV staining. The results showed that As₂O₃ induced apoptosis on MUTZ-1 cells in a dose-related manner. Typical DNA ladder was observed when MUTZ-1 incubated with 4μM As₂O₃ for 24h and 2μM As₂O₃ for 48h. Rates of apoptotic cells were 5.89%±1.56%, 29.62%±9.95%, 43.88%+15.08% and 74.8% ±7.97% when cells were incubated with 0, 2, 4, 8μM As₂O₃ by flow cytometry repectively.To characterize the apoptotic pathway triggered by As₂O₃ in MUTZ-1 cells, we measured caspase 3, caspase 8 and PARP activation in MUTZ-1 cells incubated with 0-8μM As₂O₃ for 24h. The 20kDa and 42 kDa active forms of caspase3 and caspase8 were observed by Western blotting. To confirm the involvement of caspase in As₂O₃ induced apoptosis, we examined the specific cleavage of the general substrate (PARP) by caspase3 during apoptosis. Both the uncleaved 116kDa proform and the cleaved 89kDa fragments of PARP were observed by Western blot a...