| Objective: The acute leukemia is one of the most common hematopoietic malignanances, there are abnormal DNA methylation in leukemia cell lines and leukemia patients, which may led to the pathogenesis and development of leukemia. DNA methylation modification are catalayzed by DNA methyltyansferase in the context of the sequence 5'-CG-3',which is also referred to as a CpG dinucleotide. It is the most common eukaryotic DNA modification, which can alter the gene expression without a change in nucleotide sequence. Therefore the levels of the target genes transcription are changed by DNMTs genes , which altered the status of genes CpG land (promotor regions which richly contain CpG dinucleotide) methylation. In recent years, DNMTs genes played a major role in the regulation of gene expression, mammalian development, genome imprinting, and X-chromosome inactivation, and are closely associated with many tumor diseases. In tumor cells, the levels of general genome DNA methylayion are lower than normal cells, but with the increase methylation levels of CpG land. Both the demethylation of oncogenes and hypermethylationof the tumor suppressor genes can affect the occurrence and development of cancer. The overseas study that a number of genes were abnormally methylated concurrently in leukemia cells, including either the oncogene widespread demethylation or the tumor suppressor gene promotor hypermethylation, was confirmed. Special the later was the one of important factors causing leukemia pathogenesis. The study was to investigate the relationship between the expression of DNMTs and clinical prognosis in patients with acult leukemia, meanwhile further understood the influence and the mechanism of methylation inhibitor inhibiting leukemia cells growth, so that provide the assistance for the leukemia chemotherapy. Methods: The mRNA expressions of DNMTs, p15, mdr1 and PCNA were measured in 72 patients with AL and 20 normal controls by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR); The ratio of P15 CpG land methylation was measured in 56 patients with AL and 14 normal controls by methylation-special PCR (MSP-PCR). K562,HL-60 cells and primary cells of leukemias were cultured in RPMI 1640 and treated with different concentrations of 5-aza-CdR, the status of cells proliferate were observed with microscop, the cells viability were observed by MTT test and drawed cells growth curves, the differentiation of HL-60 cells were examined by NBT reduction reaction, cells were stained with Wrightï¼Giemsafor general morphology, cells apoptosis was measured by DNA agrose gels electrophoresis. The mRNA expressions of DNMTs, p15, P53 and Bcl-2 in trial teams and control teams were determined by RT-PCR. The status of P15 gene methylation in trail teams and control teams was measured by MSP-PCR. Results 1. All three DNMTs mRNA expression in AL patients were significant higher than nomal controls (all P<0.01), When the internal contrast was changed into proliferation cell nuclear antigen (PCNA), a kind of cell proliferation marker gene, The difference still remain statistic value. It showed that the expressions of DNMTs are not related to cell proliferation. 2. All three DNMTs genes expression were significantly positively correlated in AL patients, showing cooperatively high expression, and there was a negative correlation between the levels of P15, mdr1 gene expression and DNMTs', but P15 and mdr1 gene expression was not correlated. 3. The complete remission (CR) rate was 74.5% in AL patients with all DNMTs genes positive expression, 23.8% in AL patients with DNMTs genes partly positive or negative expression, the two CR rates was significantly different (p<0.01). 4. In 56 AL patients , the P15INK4B was completely methylated in 55.4% (31 of 56), partly methylated in21.4%(12 of 56) and all 14 cases of normal controls were not methylated. 5. K562,HL-60 cells and primary cells of leukemia were significantly inhibited after treated with different concentra... |
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