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

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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