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

在癌症患者的血清中经常观察到高乳酸脱氢酶（LDH）活性，并且有时在癌症患者中观察到不寻常的额外LDH同工酶带。额外的条带通常与LDH2相邻并且介于LDH2和LDH3同工酶条带之间（称为LDH2ex），然而，LDH2ex的分子性质尚未阐明。我们经历了一名患有转移性遗传性视网膜母细胞瘤的 4 岁男孩，其血清除了 5 种正常同工酶外还显示 L​​DH2ex。从该患者的手术标本中建立的视网膜母细胞瘤细胞系 NCC-RbC-51 也显示出仅由 2 个条带组成的不寻常同工酶模式：正常 LDH1 带和 LDH2ex。我们分析了 NCC-RbC-51 异常 LDH 同工酶模式背后的分子机制。使用人LDHA cDNA作为探针的Northern印迹分析表明，NCC-RbC-51系不表达正常/体细胞LDHA mRNA，但表达少量LDHA相对mRNA，其分子量比正常LDHA mRNA明显更高。用 50 μM 5-氮杂脱氧胞苷处理诱导 NCC-RbC-51 中正常 LDH2、3、4 和 5 同工酶的表达，表明正常 LDHA 的转录因启动子甲基化而沉默。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）的区域中的CpG岛完全甲基化。RT-PCR和直接测序表明，NCC-RbC-51表达的RNA具有与具有外显子0（-434至-232）而非外显子a的鼠睾丸特异性变体的人类对应物的序列（-2248 至 -2172）表示 5' 非编码序列。综上所述，根据这些结果，NCC-RbC-51系中异常LDH同工酶模式的机制推测如下：I)体细胞LDHA被外显子a周围的启动子高甲基化沉默，仅留下LDH1(B4)作为正常同工酶； ii)LDH2ex(B3A'1)由LDHA(A')的睾丸特异性剪接变体的一分子和正常LDHB的三分子构成。尽管尚待确定LDH2ex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而发生，但我们认为这种机制至少解释了之前报道的LDH2ex涉及的异常同工酶模式的一部分。较少的

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