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

癌症患者血清中乳酸脱氢酶(LDH)活性较高，有时还会出现异常的LDH同工酶带外。这条额外的条带通常与LDH2相邻，并位于LDH2和LDH3同工酶之间(命名为LDH2 ex)，然而，LDH2 ex的分子性质尚不清楚。我们经历了一例转移性遗传性视网膜母细胞瘤的4岁男孩，他的血清除了5种正常的同工酶外，还显示了LDH2 ex。从手术标本中建立的视网膜母细胞瘤细胞系NCC-RBC-51也显示出一种不寻常的同工酶图谱，仅由两条带组成：正常的LDH1带和LDH2 ex。我们分析了NCC-RBC-51这种异常LDH同工酶图谱的分子机制。以人LDHA基因为探针的Northern印迹分析表明，NCC-RBC-51细胞不表达正常/体细胞LDHA基因，但与SLI…有少量的LDHA基因表达比正常LDHA mRNA的相对分子质量高得多。50μM的5-氮杂脱氧胞苷诱导正常的LDH2、3、4和5同工酶的表达，提示正常的LDHa的转录被启动子甲基化抑制。亚硫酸氢钠处理和PCR分析表明，在LDHA基因中，编码5‘非编码序列(外显子a，Takano和Li，1990)区域的CpG岛完全甲基化。RT-PCR和直接测序表明，NCC-RBC-51表达的RNA具有小鼠睾丸特异变异体的人类对应序列，其5’非编码序列的外显子0(-434至-232)而不是外显子A(-2248至-2172)。综上所述，根据这些结果，我们推测NCC-RBC-51细胞中LDH同工酶图谱异常的机制如下：(1)体细胞LDHA被外显子a附近的启动子高甲基化沉默，只剩下正常同工酶的LDH1(B4)；以及(Ii)LDH2 ex(B3A‘1)由一个睾丸特异剪接变异体LDHA(A’)和三个正常LDHB分子组成。虽然LDH2 ex在肿瘤中的新表达是由于体细胞LDHA转录抑制还是独立事件的结果，我们认为这一机制至少解释了先前报道的LDH2 ex异常同工酶模式的一部分。较少

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