Electrophoretic variant of a lactate dehydrogenase isoenzyme and selective promoter methylation of the LDHA gene in a human retinoblastoma cell line.
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.
Maekawa
Masato
M
Department of Laboratory Medicine,Hamamatsu University School of Medicine, Hamamatsu 431-3192, Japan. mmaekawa@hama-med.ac.jp
Inomata
Motoko
M
Sasaki
Masao S
MS
Kaneko
Akihiro
A
Ushiama
Mineko
M
Sugano
Kokichi
K
Takayama
Jun
J
Kanno
Takashi
T
D015152:Blotting, Northern; D015153:Blotting, Western; D018899:CpG Islands; D006801:Humans; D007223:Infant; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D000080864:Lactate Dehydrogenase 5; D008297:Male; D008745:Methylation; D009928:Organ Specificity; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D012175:Retinoblastoma; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013737:Testis / Q000378:metabolism; D014407:Tumor Cells, Cultured
D007527:Isoenzymes; D012333:RNA, Messenger; D007770:L-Lactate Dehydrogenase; D000080864:Lactate Dehydrogenase 5; C409667:lactate dehydrogenase 1