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Clinical Chemistry 48: 1938-1945, 2002;
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(Clinical Chemistry. 2002;48:1938-1945.)
© 2002 American Association for Clinical Chemistry, Inc.

Electrophoretic Variant of a Lactate Dehydrogenase Isoenzyme and Selective Promoter Methylation of the LDHA Gene in a Human Retinoblastoma Cell Line

Masato Maekawa1a, Motoko Inomata2, Masao S. Sasaki3, Akihiro Kaneko4, Mineko Ushiama5, Kokichi Sugano6, Jun Takayama7 and Takashi Kanno1

1 Department of Laboratory Medicine,Hamamatsu University School of Medicine, Hamamatsu 431-3192, Japan.

2 Pharmacology Division, National Cancer Center Research Institute

4 Department of Ophthalmology

5 Clinical Laboratory

7 Department of Pediatrics, National Cancer Center Hospital, Tokyo 104-0045, Japan.

3 Radiation Biology Center, Kyoto University, Kyoto 606-8501, Japan.

6 Oncogene Research Unit/Cancer Prevention Unit, Tochigi Cancer Center Research Institute, Utsunomiya 320-0834, Japan.

aAuthor for correspondence. Fax 81-53-435-2794; e-mail mmaekawa{at}hama-med.ac.jp.

Background: Lactate dehydrogenase (LD), a tetrameric product of the genes LDHA and LDHB, may be increased in sera of cancer patients. A variant isoenzyme with electrophoretic mobility between LD2 and LD3 (LD2ex) has been described in patients, but its molecular nature is largely unknown.

Methods: A newly established retinoblastoma cell line, NCC-RbC-51 (R51), showed an isoenzyme pattern with only two bands, LD1 and LD2ex. We investigated the isoenzymes by Northern blot, Western blot, and methylation analysis and PCR.

Results: Northern blot analysis revealed that R51 cells expressed no wild-type/somatic LDHA mRNA, but did express a small amount of LDHA-related mRNA with a slightly higher molecular mass. Western blot analysis confirmed the anti-LDHA-reactive protein with a 3-kDa higher molecular mass. Treatment of R51 cells with the demethylating agent 5-aza-2'-deoxycytidine restored the expression of the LD2, -3, -4, and -5 isoenzymes. PCR analysis of sodium bisulfite-treated genomic DNA revealed that the CpG island in the promoter region around exon a of the LDHA gene was completely methylated. Reverse transcription-PCR analysis and direct sequencing revealed that R51 cells expressed a RNA with the sequence of the human homolog of a murine testis-specific variant that has exon 0 as the 5' noncoding sequence. LDHB was expressed normally in R51 cells.

Conclusions: The somatic LDHA in R51 cells is transcriptionally silenced by promoter hypermethylation around exon a, leaving only LDHB to be expressed normally and a testis-specific variant transcript of LDHA containing exon 0. LD2ex possibly results from tetramerization of three wild-type LDHB molecules and one variant LDHA product.




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