非何杰金淋巴瘤P15基因甲基化和去甲基化再表达的研究
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摘要
P_(15)基因是肿瘤抑制基因的候选基因，受细胞生长抑制蛋白FGF-β
的诱导，编码周期素依赖激酶4/6（CDK4/6）抑制因子，对细胞周期起负调
控作用，P_(15)基因操纵区5’-CpG岛的甲基化被认为是基因缺失之外的又
一失活机制。P_(15)基因5’-CpG岛的异常甲基化导致该基因转录的抑制，5-
杂氮脱氧胞嘧啶（5-Aza-2cdR）通过共价俘获DNA甲基转移酶抑制DNA
的甲基化，使因甲基化失活的生长调控基因重新激活并表达。本文用甲
基化特异性PCR（MSP）的方法以亚硫酸氢钠修饰后的DNA为模扳，
检测了32例非杰何金淋巴瘤石蜡标本P_(15)基因甲基化，用5-Aza-2cdR诱
导非何杰金淋巴瘤raji细胞株P_(15)基因去甲基化，结合RT-PCR方法测定
诱导前后P_(15)基因的表达，结果提示：非何杰金淋巴瘤P_(15)基因操纵区甲
基化的发生率为18.75％（6/32），高度恶性比低度恶性更易发生甲基化，
其发生率分别为31.6％和0％。在5-Aza-2cdR10~(-7)-10~(-6)mol/L浓度时P_(15)基
因可去甲基化再表达。并且raji细胞生长显著抑制，停留G0/G1期细胞
数升高近2倍。结果表明：5-Aza-2cdR可通过P_(15)基因去甲基化再表达
抑制raji细胞的生长，为恶性血液病去甲基化临床治疗提供实验依据。
P15
gene is a candidate as tumor suppressor gene. As cell cycle negative regulator, it is a inhibitor of cyclin-dependent kinase CDK4 and CDK6,whose expression is induced by transforming growth factor(TGF-β)which is a potent inhibitor of cell growth . Abberrant methylation of 5’-CpG islands of P15 gene promoter has been recently described as an alternative mechanism of gene inactivation besides gene deletion. 5’-Aza-2cdR, which can inhibit DNA methylation by covalently traping DNA methylases as their target site in DNA, plays a role in demethylation and reactivate growth-regulator gene inactivated by demethylation. Using a recently found method that methylation-specific ploymerose chain reaction (MSP) is preceded by bisulfite modification of DNA, we investigated the methylation status of 32 NHL cases and raji cell lines of non-Hodgkin’s lymphoma (NHL) induced by 5-Aza-2cdR, and investigated the P15 gene reexpression of raji cell lines before and after induced by 5-Aza-2cdR by RT-PCR.The results have showed that 18.75% (6/32) in NHL is methylation in P15 gene,and methylation was much more frequently in high grade malignant NHL patients 31.6% than in low grade malignant patients 0%. P15 gene can be demethylated and reexpressed with 5-Aza-2cdR of 10-7-10-6 mollL concentration so that inhibited the growth of rajicell lines and resulted in about 2-fold increase in the proportion of cells in G1\G0.The results have also suggested that 5-Aza-2cdR can inhibit the raji cell growth by inducing the reexpression of P15 gene.The method of P15 gene of demethylation provides an experimental foundation for hematologic malignant clincal
therapy.
的诱导，编码周期素依赖激酶4/6（CDK4/6）抑制因子，对细胞周期起负调
控作用，P_(15)基因操纵区5’-CpG岛的甲基化被认为是基因缺失之外的又
一失活机制。P_(15)基因5’-CpG岛的异常甲基化导致该基因转录的抑制，5-
杂氮脱氧胞嘧啶（5-Aza-2cdR）通过共价俘获DNA甲基转移酶抑制DNA
的甲基化，使因甲基化失活的生长调控基因重新激活并表达。本文用甲
基化特异性PCR（MSP）的方法以亚硫酸氢钠修饰后的DNA为模扳，
检测了32例非杰何金淋巴瘤石蜡标本P_(15)基因甲基化，用5-Aza-2cdR诱
导非何杰金淋巴瘤raji细胞株P_(15)基因去甲基化，结合RT-PCR方法测定
诱导前后P_(15)基因的表达，结果提示：非何杰金淋巴瘤P_(15)基因操纵区甲
基化的发生率为18.75％（6/32），高度恶性比低度恶性更易发生甲基化，
其发生率分别为31.6％和0％。在5-Aza-2cdR10~(-7)-10~(-6)mol/L浓度时P_(15)基
因可去甲基化再表达。并且raji细胞生长显著抑制，停留G0/G1期细胞
数升高近2倍。结果表明：5-Aza-2cdR可通过P_(15)基因去甲基化再表达
抑制raji细胞的生长，为恶性血液病去甲基化临床治疗提供实验依据。
P15
gene is a candidate as tumor suppressor gene. As cell cycle negative regulator, it is a inhibitor of cyclin-dependent kinase CDK4 and CDK6,whose expression is induced by transforming growth factor(TGF-β)which is a potent inhibitor of cell growth . Abberrant methylation of 5’-CpG islands of P15 gene promoter has been recently described as an alternative mechanism of gene inactivation besides gene deletion. 5’-Aza-2cdR, which can inhibit DNA methylation by covalently traping DNA methylases as their target site in DNA, plays a role in demethylation and reactivate growth-regulator gene inactivated by demethylation. Using a recently found method that methylation-specific ploymerose chain reaction (MSP) is preceded by bisulfite modification of DNA, we investigated the methylation status of 32 NHL cases and raji cell lines of non-Hodgkin’s lymphoma (NHL) induced by 5-Aza-2cdR, and investigated the P15 gene reexpression of raji cell lines before and after induced by 5-Aza-2cdR by RT-PCR.The results have showed that 18.75% (6/32) in NHL is methylation in P15 gene,and methylation was much more frequently in high grade malignant NHL patients 31.6% than in low grade malignant patients 0%. P15 gene can be demethylated and reexpressed with 5-Aza-2cdR of 10-7-10-6 mollL concentration so that inhibited the growth of rajicell lines and resulted in about 2-fold increase in the proportion of cells in G1\G0.The results have also suggested that 5-Aza-2cdR can inhibit the raji cell growth by inducing the reexpression of P15 gene.The method of P15 gene of demethylation provides an experimental foundation for hematologic malignant clincal
therapy.
引文
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2． Ehrlich M, Wang RYH. 5-methylcytosine in eukaryotic DNA Science, 1981,212:1350
3． LiE, Bestor TH, Jaenisch R Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 1992; 69: 915-926
4． Rideout. W. M.. III. Coetzee. G.A. Olumi, F., and Jones, P.A.5-Methylcytosine in eukaryotic DNA Science, 1981,212:1350
5． Fearon. E. R. and Jones. P.A. Progessing toward a molecular description of colorectal cancer development. FASEB J. 6:2783-2790,1992
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7． Peter A. Jones. DNA Methylation Errors and Cancer: Research 56,2463-2467． June 1． 1996
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