| P15INK4b tumor suppressor gene, a new tumor suppressor gene , is located on the chromosome 9P21. P15 is an important factor of the cyclin dependent kinase inhibitors (CKI) concerning the regulation of cell cycle. Inactivation of P15INK4b gene is closedly related to the generation,^development and prognosis of various tumors. Studies show that DNA abnormal methylation is one of the important ways to inactivate P15INK4b gene. And 5' -CpG islands in P15 gene promoter are usually found abnormal hypermethylation. It was reported that about 86% of DNA hypermethylation is detected in acute myelogenous leukemia (AMI). Thus, demethylation therapy aimed at Pl5INK4b tumor suppressor gene becomes a new therapeutic strategy for hematologic malignancies.Current researches about methylation inhibitors mainly focus on 5-aza-2-deoxycyitidine(5-aza-CdR), but the potential mechanisms of demethylation and the possible approach to regulate cell cycle are still not illuminated clearly. In the other hand, that arsenic trioxide is used to treat leukemia as methylation inhibitor is rarely reported. Accordingly in our study acute T-cell lymphoblastic leukemia cell lineMolt-4 was treated by arsenic trioxide in which P15INK4b gene expression was suppressed due to DNA hypermethylation. The purpose is to investigate the demethylating effect on P15INK4b gene in leukemia cell and the relations between demethylation and gene expression, to explore the correlation between arsenic trioxide and DNA methylaion and clarify the possible mechanisms of demethylating treatment, also to provide experimental basis for arsenic trioxide as methylation inhibitor.The subject of the thesis were focused on the following two aspects :1. The impacts on P15INK4b genes in Molt-4 cell by arsenic trioxide: Molt-4 cells were incubated with different concentrations (0μM, 2.5 μM,5.0μM and 10.0μM)of arsenic trioxide for some time. Then restricted endonucleases PCR (REP-PCR) was used to detect the methylation of P15INK4b gene. The expression of P15 gene , DNA methyltransferase-1 (DNMT-1) and DNA demethylases (MBD-2) gene mRNA were determined by RT-PCR . Immunocytochemistry and flow cytometry were used to investigate the expression of Pl5INK4b proteins. Those foregoing means can reflect the connection between DNA methylation and P15INK4b gene expression from genome DNA replication, mRNA transcription and protein translation levels. The major results indicated that after 48 hours treatment, the methylation of P15 gene decreased markedly to disappear. Compared with the untreated group, p15INK4b gene mRNA expressions were recovered partly or completely. And P15 protein positive ratios and intensities went up obviously. As results showed , P15INK4b protein expressions in arsenic trioxide (2. 5μM, 5. 0μM and 10. 0μM) -treated groups increased 33.49%, 56.84% and 69.21% respectively (P<0.05). Moreover we can analyse the correlation between P15INK4b gene methylation and expression of key enzyme in methylation pattern. The results indicated that with P15INK4b gene methylaation decreasing , expression of DNMT-1 went downwhile MBD-2 went up.2. The effects on proliferation energy and cell cycle of Molt-4 cell by arsenic trioxide: Molt-4 cells were incubated with different concentration of arsenic trioxide for a certain time. Then, we observed the clone formation, do cell-counting and MTT assay to analyse the impact of arsenic trioxide on the cell proliferation and energy. Flow cytometry was used to detect the change of cell cycle for Molt-4 cells in order to explore the demethylation effect of arsenic trioxide. Results showed that arsenic trioxide displayed concentration-dependent and time-dependent inhibition of cell proliferation and cell cycle arrest.Conclusions:(1) arsenic trioxide is able to effectively wipe off the methylation of P15INK4b tumor suppressor gene, recover the expression of P15 , block the cell cycle in Go/Gl and ultimately inhibit the growth and proliferation of Molt-4 cell. ?The possible mechanism of demethylation of arsenic trio...
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