Methylation of LDHA Gene Promoter Region and Regulation of LDHA Expression in Cancer Cells

LDHA基因启动子区甲基化及癌细胞中LDHA表达的调控

• 批准号: 13470518
• 负责人: MAEKAWA Masato
• 金额: $ 1.54万
• 依托单位: Hamamatsu University School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13470518/
• 关键词: LDH; Promoter; Methylation; PCR-SSCP; Cancer; Expression regulation; LDHB; cancer-testis antigen

In cancer patients high lactate dehydrogenase (LDH) activity has been often observed in their sera, and unusual extra band of LDH isoenzyme has been sometimes observed in cancer patients. The extra band is often adjacent to LDH2 and between LDH2 and LDH3 isoenzyme bands (designated as LDH2ex), however, molecular nature of the LDH2ex has not been clarified yet. We experienced a 4-year-old boy with metastasized hereditary retinoblastoma, whose serum showed the LDH2ex in addition to the 5 normal isoenzymes. A retinoblastoma cell line, NCC-RbC-51 established from the surgical specimen obtained from this patient showed also an unusual isoenzyme pattern composed of only 2 bands : the normal LDH1 band and the LDH2ex. We analyzed molecular mechanism underlying this aberrant LDH isoenzyme pattern of the NCC-RbC-51. Northern blot analysis using human LDHA cDNA as the probe revealed that the NCC-RbC-51 line did not express normal/somatic LDHA mRNA but a small amount of LDHA relative mRNA with sli … More ghtly higher molecular weight than that of the normal LDHA mRNA. Treatment with 50 μM of 5-azadeoxycytidine induced the expression of the normal LDH2, 3, 4 and 5 isoenzymes in the NCC-RbC-51, suggesting that the transcription of the normal LDHA was silenced by the promoter methylation. The sodium bisulfite treatment and PCR analysis revealed that in the LDHA gene, the CpG island in the region encoding the 5' non-coding sequence (exon a, Takano and Li, 1990) was completely methylated .The RT-PCR and direct sequencing revealed that the NCC-RbC-51 expressed an RNA having the sequence of the human counterpart of the murine testis-specific variant that has exon 0 (-434 to -232) instead of exon a (-2248 to -2172) for the 5' non-coding sequence. Taken together, from these results the mechanism underlying the abnormal LDH isoenzyme pattern in the NCC-RbC-51 line was presumed as follows : I) somatic LDHA was silenced by the promoter hypermethylation around exon a, leaving only LDH1 (B4) as for the normal isoenzyme ; and ii) the LDH2ex (B3A'1) was constituted with one molecule of the testis-specific splicing variant of the LDHA (A') and three molecules of the normal LDHB. Although it remains to determine whether the novel expression of the LDH2ex in the tumor occurred as a result of the suppression of somatic LDHA transcription or independent event, we think this mechanism explains at least, a part of LDH2ex-involved abnormal isoenzyme patterns reported previously. Less

在癌症患者的血清中经常观察到高乳酸脱氢酶（LDH）活性，并且在癌症患者中有时观察到异常的LDH同工酶带。这条额外的条带通常位于LDH2附近以及LDH2和LDH3同工酶带之间（称为LDH2ex），但LDH2ex的分子性质尚未明确。我们经历了一个4岁的男孩转移遗传性视网膜母细胞瘤，他的血清显示LDH2ex除了5正常同工酶。从该患者手术标本中建立的视网膜母细胞瘤细胞系nc - rbc -51也显示出异常的同工酶模式，仅由2个条带组成：正常的LDH1条带和LDH2ex条带。我们分析了NCC-RbC-51这种异常LDH同工酶模式的分子机制。以人LDHA cDNA为探针的Northern blot分析显示，NCC-RbC-51细胞系不表达正常/体细胞LDHA mRNA，只表达少量LDHA相对mRNA，分子量略高于正常LDHA mRNA。50 μM的5-azadeoxycytidine诱导NCC-RbC-51中正常LDH2、3、4和5同工酶的表达，表明正常LDHA的转录被启动子甲基化沉默。亚硫酸氢钠处理和PCR分析显示，在LDHA基因中，编码5'非编码序列（外显子a， Takano and Li, 1990）区域的CpG岛完全甲基化。RT-PCR和直接测序结果显示，nc - rbc -51表达的RNA序列与小鼠睾丸特异性变异的人类对应序列相同，其5'非编码序列具有外显子0（-434至-232）而不是外显子a（-2248至-2172）。综上所述，根据这些结果，NCC-RbC-51细胞系中LDH同工酶异常模式的机制可以推测如下：1)体细胞LDHA被外显子a周围的启动子超甲基化沉默，只留下LDH1 （B4）作为正常同工酶；ii) LDH2ex （B3A'1）由LDHA的睾丸特异性剪接变体（A'）和正常LDHB的三个分子组成。尽管LDH2ex在肿瘤中的新表达是由于抑制体细胞LDHA转录还是独立事件的结果仍有待确定，但我们认为这一机制至少解释了先前报道的LDH2ex参与异常同工酶模式的一部分。少

项目成果

Maekawa M: "Cancer Biomarker"Jpn J Clin Pathol. Suppl.124. 15-20 (2003)

前川 M：“癌症生物标志物”Jpn J Clin Pathol。

前川真人: "Electrophoretic variant of lactate dehydrogenase isoenzyme and selective promoter methylation of the LDHA gene in a human retinoblastoma cell line"Clinical Chemistry. 48・11. 1938-1945 (2002)

Masato Maekawa：“人视网膜母细胞瘤细胞系中乳酸脱氢酶同工酶的电泳变体和选择性启动子甲基化”临床化学48·11（2002）。

Maekawa M, Inomata M, Sasaki MS, Kaneko A, Ushiama M, Sugano K, Takayama J, Kanno T: "Electrophoretic variant of a lactate dehydrogenase isoenzyme and selective promoter methylation of the LDHA gene in a human retinoblastoma cell line"Clin Chem. 48. 1938-

Maekawa M、Inomata M、Sasaki MS、Kaneko A、Ushiama M、Sugano K、Takayama J、Kanno T：“人视网膜母细胞瘤细胞系中乳酸脱氢酶同工酶的电泳变体和 LDHA 基因的选择性启动子甲基化”Clin Chem。

Maekawa M: "Lactate Dehydrogenase (LD)"Jpn J Clin Pathol. Suppl.116. 81-89 (2001)

Maekawa M：“乳酸脱氢酶（LD）”Jpn J Clin Pathol。