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Electrophoretic variant of a lactate dehydrogenase isoenzyme and selective promoter methylation of the LDHA gene in a human retinoblastoma cell line.

Lactate dehydrogenase (LD; [8] EC is a tetrameric molecule composed of polypeptide subunits encoded by two structurally distinct genes, LDHA and LDHB (1). The association of the subunits A and B is random and generates five isoenzymes, LD1 to LD5, whose subunit compositions are [B.sub.4], [B.sub.3][A.sub.1], [B.sub.2][A.sub.2], [B.sub.1][A.sub.3], and [A.sub.4], respectively, where [B.sub.4] (LD1) has the highest and [A.sub.4] (LD5) the lowest electrophoretic migration rate toward the anode. Expression of mammalian genes for LDHA and LDHB is regulated developmentally and in a tissue-specific manner (2,3). Consequently, alterations in the serum LD isoenzyme pattern are used to indicate sites of pathologic involvement and cancer development (3). Serum LD1 concentrations are often increased in patients with several types of brain tumor and various types of germinal cell tumors of the ovary, testis, and mediastinum (3). An unusual extra electrophoretic band has occasionally been detected in sera from cancer patients with brain tumors (4, 5), esophageal cancer (6), and neuroblastomas (7) and in erythrocytes (8), first-trimester placenta, trophoblasts, and a choriocarcinoma cell line (9). The electrophoretic mobility of this extra band (designated LD2ex) is often close to that of LD2 and between the mobilities of the LD2 and LD3 isoenzyme bands; the LD2ex band is present in addition to the relatively high-intensity LD1 and relatively low-intensity LD2, LD3, LD4, and LD5 bands. LD activity migrating close to the LD2ex position has also been observed in cases with amino acid alterations attributable to missense mutations at coding exons of the LDHA gene (10). The properties of LD2ex have been examined (8,11), but the molecular nature of tumor-related LD2ex has yet to be elucidated.

In this study, we investigated the molecular origin of the LD2ex variant isoenzyme in a newly established retinoblastoma (Rb) cell line, NCC-RbC-51 (R51). The tumor cells expressed only LD1 and LD2ex, which was reflected in the patient's serum LD isoenzyme pattern. In the tumor cells, the promoter region of the LDHA gene was hypermethylated and the wild-type somatic LDHA mRNA (type 1 transcript) was not transcribed; a testis-specific variant mRNA (type 2 transcript) was transcribed instead. This aberrant transcription of the LDHA gene, attributable to promoter methylation, is suggested as the explanation for the absence of tetrameric molecules other than the LDHB homotetramer (LD1) and a combination of three LDHB subunits with one subunit encoded by a variant transcript of the LDHA gene (LD2ex).

Materials and Methods


A 4-year-old boy was hospitalized for hereditary bilateral Rb that had metastasized to his cervical lymph nodes (12). His serum LD activity was markedly increased. The LD isoenzyme pattern in his serum was abnormal, having an extra band (LD2ex) that migrated between LD2 and LD3, with a relatively large amount of the LD1 fraction and relatively small amount of LD3, -4, and -5. During the hospitalization, the patient was treated with surgery and chemotherapy. As a result, serum LD activity decreased close to the reference interval, and the LD isoenzyme pattern exhibited a reduction of LD1 and the extra band and increases in the LD3, -4, and -5 fractions.


A Rb cell line, R51 (NCC-RBC-51), was established from a surgical specimen resected from the patient described above (12). Two representative Rb cell lines, Y79 and WERI-Rb1, were also studied for comparison. The cells were grown in a 94% air-6% C[O.sub.2] humidified atmosphere at 37[degrees]C in RPMI 1640 supplemented with 90 mL/L heat-inactivated fetal bovine serum and 70 mg/L kanamycin. Karyotype analysis of R51 cells was by a standard G-banding technique (13).


R51 cells (~5 x [10.sup.5]) were cultured with or without 5 or 50 /,mol/L 5-aza-2'-deoxycytidine (5AzaCdR; Sigma) or 5-azacytidine (5AzaCR; Sigma) for 7 days, washed with phosphate-buffered saline, harvested, and stored at -70[degrees]C until required for use.


Aliquots containing ~5 x [10.sup.6] cultured cells were washed once with phosphate-buffered saline, pelleted by centrifugation, and sonicated with a Sonifier 450. The sonicated extract and patient's serum samples were frozen and stored at -70[degrees]C until required for use, when they were subjected to zymogram analysis with use of a LD staining reagent set (Helena Laboratory), according to the manufacturer's instructions.


DNA was extracted by a previously described method (14), and total RNA was extracted by use of Isogene (Nippon Gene). Reverse transcription (20-[micro]L reaction mixture) was carried out with ~0.5-1.0 [micro]g of total RNA as templates with random hexamers as primers and Super-Script RNase-free reverse transcriptase (Invitrogen). One-twentieth of the resulting cDNA was amplified using several pairs of LDHA primers (Table 1). To examine for possible differential expression of the LDHA gene, PCR analysis was performed with specific primers for amplifying exons a and 0 (RT-F4 and RT-F6, respectively; see Table 1) on the cDNAs of the Rb cell lines, which were compared with a cDNA panel containing cDNAs prepared from healthy and cancer tissues (Clontech).


RNA (10 /xg) was electrophoresed on a 1.2% agarose gel in 160 mL/L formalin buffer and blotted on a nylon transfer membrane (Hybond-N; Amersham Pharmacia Biotech.). The RNA was cross-linked to the membrane by ultraviolet irradiation for 1 min, prehybridized for 6 h, and then hybridized for 17 h at 42[degrees]C with [sup.32]P-labeled human LDHA cDNA (15). The membrane was washed twice with SSPE buffer (150 mmol/L sodium chloride, 10 mmol/L sodium biphosphate,1 mmol/L EDTA) containing 10 g/L sodium dodecyl sulfate at 55[degrees]C and autoradiographed for 6 days at -70'C using Hyper film MP (Amersham Pharmacia Biotech.) and an intensifying screen. The filter was then rehybridized with a [sup.32]P-labeled human [beta]-actin probe (Nippon Gene) as an internal control.


The PCR products were treated with shrimp alkaline phosphatase and exonuclease III to remove excess PCR primers and nucleotides (Amersham Pharmacia Biotech.) and then directly sequenced by the dideoxysequencing procedure using a BigDye Terminator Cycle Sequencing FS Ready Reaction Kit and a PRISM 310 Genetic Analyzer (PE Applied Biosystems), according to the manufacturers' instructions.


Each supernatant was treated with sodium dodecyl sulfate and subjected to electrophoresis on a 10-20% polyacrylamide gradient gel, after which the proteins were transferred to a polyvinylidene fluoride membrane (Millipore) and Western blot analysis was performed. Briefly, the polyvinylidene fluoride membrane was incubated with a 2000-fold diluted mouse monoclonal antibody against human LD5 ([A.sub.4]; a gift from Professor T. Komoda, Saitama Medical School, Saitama, Japan) for 1 h, and then with a 2500-fold diluted horseradish peroxidase-conjugated goat polyclonal antibody against mouse IgG (Biosource International) for 1 h at room temperature. Finally, the labeled peroxidase was detected by use of Chemiluminescent Reagent Plus (NEN Life Science Products Inc.).


Methylation analysis was performed using bisulfite sequencing and the bisulfite-PCR-single-strand conformation polymorphism (BiPS) procedure (16). Briefly, bisulfite treatment was carried out with the reagents provided in a CpGenome DNA Modification Kit (Intergen). Sssl methylase (New England Biolab) was used to prepare control methylated DNA from genomic DNA derived from peripheral blood cells of healthy volunteers. The PCR primers were designed to be complementary to the converted DNA with no CpG sequences in the corresponding region of the original DNA (Table 2). The PCR was performed with AmpliTaq Gold (PE Applied Biosystems) and the hot start procedure, and the PCR products were directly sequenced or subjected to single-strand conformation polymorphism analysis using 10% nondenaturing polyacrylamide gels and silver staining detection (Daiichi Pure Chemicals).


To investigate the 5' terminus of the abnormal LDHA cDNA, we used rapid amplification of cDNA ends (RACE) technology. Total RNA treated with DNase was analyzed with use of a FirstChoice RLM-RACE reagent set (Ambion), according to the manufacturer's instructions.



A representative karyotype of R51 cells can be seen in Fig. 1 of the data supplement that accompanies the online version of this article (available at http://www.clinchem. org/content/vo148/issuell/). The chromosomal constitution was 59, XY, del(13)(g14), +del (13)(814), t(2;19)(g13; q13), t(5;11)(gll;p15), t(6;14)(p11;g32), +1, +7, +8, +10, +11, +14, +14, +15, +19, +19, +20, +21, according to the ISCN 1985 nomenclature (17). The status of the RB1 genes of the patient and R51 cells have been studied previously [RB134 family in Ref. (18)]. The mutation in the RB1 gene was attributable to an intragenic deletion of ~1.6 kbp, including exon 24, which produced a premature termination codon in exon 25. The patient from whose specimen the R51 cell line was derived was heterozygous for RB1 genes: one wild-type allele was inherited from his unaffected mother and the other, mutated, allele derived from his unilaterally affected father. The R51 cells had lost the maternal allele as a result of a large deletion involving the RB1 gene, identified by a del(13)(gl4) (shown in Fig. 1 in the data supplement).


The patient's serum LD catalytic activity was markedly increased, and the LD isoenzyme pattern mostly comprised two bands: a strong band of LD1 and a weak wild-type LD2 band and slower LD2-like abnormal band (Fig. 1, patient's serum after 79 days). The slower LD2-like band was defined as LD2ex because of its characteristic mobility between LD2 and LD3. After the patient had received high-dose chemotherapy, the intensities of the LD1 and LD2ex bands clearly diminished and LD2, LD3, LD4, and LD5 bands appeared concomitantly (Fig. 1, after 153 days). The LD isoenzyme pattern of R51 cells comprised two bands, a strong LD1 band with normal mobility and a faint LD2ex band. This abnormal pattern indicated that R51 cells lacked isoenzymes that contain the LDHA component, suggesting that the wildtype LDHB, but not the wild-type LDHA subunit was expressed, whereas the patterns of the representative Rb cell lines, Y79 and WERI-Rb1, showed the five isoenzymes with normal mobilities.


Northern blot analysis using human LDHA cDNA as a probe showed that R51 cells did not express wild-type LDHA mRNA but did express a small amount of abnormal mRNA with a molecular mass slightly higher than that of wild-type LDHA (Fig. 2), whereas Y79 and WERI-Rb1 cells expressed wild-type LDHA mRNA.


Because Northern blotting detected no wild-type LDHA mRNA, the occurrence of some mutations in the LDHA gene was suspected. We therefore used intron-intron primers to amplify all the protein-coding exons and exon-intron boundary regions (10) and determined their sequences. Contrary to our expectations, we detected no missense mutations in the LDHA gene of R51 cells (data not shown).




We next suspected that the deficiency of the wild-type LDHA subunit was associated with DNA methylation because many genes in other types of tumors have been reported to be silenced by CpG hypermethylation and the LDHA promoter contains several CpG dinucleotides [Fig. 3A; Ref. (19); GenBank accession no. U13679]. BiPS analysis revealed that CpG sites in the DNA fragments amplified with the primer pairs F11 and R11 and F12 and R12 were methylated. In particular, CpG islands on a region of the LDHA gene encoding the 5' noncoding sequence (designated exon a) (19) were completely methylated in R51 cells (Fig. 3B). However, the downstream regions of the LDHA promoter, including exon 0, amplified with primer pairs F15 and R17, F13 and R13, and F18 and R18 were unmethylated. None of the regions of the Y79 cells analyzed were methylated. BiPS analysis of the products amplified with primer pairs F11 and R11, F15 and R17, F13 and R13, and F18 and R18 (Fig. 4C in the data supplement that accompanies the online version of this article; available at content/vo148/issuell/) was also carried out, but the products amplified by F12 and R12 were so long that we did not subject them to this analysis.

After continuous exposure to 5 or 50 /[micro]mol/L 5AzaCdR or 5AzaCR for 7 days, R51 cells showed the wild-type LD isoenzyme pattern in addition to the original LD2ex pattern, which suggests that the loss of wild-type LDHA mRNA and protein expression was attributable to DNA methylation (Fig. 4). The demethylation activity of 5AzaCdR was higher than that of 5AzaCR.



The Northern blot results (Fig. 2) suggested that R51 cells did not produce the wild-type LDHA transcript, but did express a LDHA-like transcript that could be detected by hybridization with LDHA cDNA. A human counterpart of a murine testis-specific LDHA variant was predicted by Takano and Li (19) based on comparisons of sequences in their database. The predicted variant has a 5' noncoding exon (exon 0), which is distinct from that of the somatic transcript (exon a) but has the same 24-nucleotide sequence at the 5' position of the translation initiation codon, a protein-coding sequence of 996 nucleotides, a 3' noncoding region of 488 nucleotides, and a poly(A) tail (Fig. 5A). The predicted size of this mRNA is 87 by longer than wild-type LDHA, which is comparable to the size of the mRNA expressed by R51 cells (Fig. 2). We therefore hypothesized that this testicular mRNA is related to the LD2ex extra band and tried to confirm this hypothesis by use of reverse transcription-PCR (RT-PCR) analysis. The RT-PCR strategy and primer locations are presented in Fig. 5A, and the primer design is shown in Table 1. RT-PCR and direct sequencing revealed that R51 cells did not express the wild-type LDHA transcript starting from exon a (designated type 1), but did express a unique LDHA transcript starting from exon 0 without exon a (designated type 2; Fig. 5B). However, after 5AzaCdR treatment, R51 cells expressed the type 1 LDHA transcript. A small amount of the type 2 transcript was also detected in Y79 cells and peripheral blood leukocytes, and trace bands with the same migration properties were observed when the genomic DNAs of R51 and Y79 cells and leukocytes were amplified by PCR with primers F1 and R2. Sequence analysis revealed that these trace bands derived from a novel processed pseudogene, because any nucleotide alterations, including alterations at the translation start codon, were observed in the open reading frame (GenBank accession no. AF461457).



To study the relationship between the type 2 transcript and LD2ex, we performed Western blot analysis using an anti-LDHA monoclonal antibody. Y79 cells yielded two bands, a stronger one at 38 kDa, which corresponds to the molecular mass of somatic LDHA, and a fainter band at 41 kDa, whereas R51 cells yielded only one faint band at 41 kDa. This means that both R51 and Y79 cells possess an unusual cross-reacting material with a molecular mass 3 kDa higher than that of somatic LDHA (Fig. 6).

After 5'-RACE using an inner primer (from the supplied reagent set) and R3, as shown in Table 1 and Fig. 5A, the longest cDNA contained an amplified product of ~240 bp. We determined the nucleotide sequence of this amplified product by direct sequencing, which showed that it contained exon 0, a homolog of the mouse LDHA exon 0 (19, 20), and that its 5' terminus was at position -434 from the translation start site of somatic LDHA.


To quantify the expression of these two types of transcripts in other tissues, we analyzed tissue and cancer cDNA panels by PCR with two primer pairs, F4 and R3 and F6 and R3 (Fig. 7). PCR products of the type 1 transcript starting from exon a were detected in most tissues and cancer tissues after 33 PCR cycles, whereas the type 2 transcript starting from exon 0 was strongly expressed in the testis and weakly expressed in the pancreas, kidney, and placenta. It was also expressed in cancer tissues, and the expression was particularly high in prostatic and colonic adenocarcinomas.


Wild-type LD has five isoenzymes (LD1 to LD5) with five possible combinations of two independently expressed subunits, A and B, arranged in tetrameric molecules, [B.sub.4], [B.sub.3][A.sub.1], [B.sub.2][A.sub.2], [B.sub.1][A.sub.3], and [A.sub.4]. In this study, a zymogram of the newly established Rb cell line R51 showed only two bands, wild-type LD1 ([B.sub.4]) and abnormal LD2ex. The original patient's serum yielded the same abnormal LD2ex band in addition to the five wild-type isoenzymes with an increased LD1 concentration. The patient's abnormal serum pattern changed to the typical five-isoenzyme pattern after treatment with high-dose chemotherapy. This finding indicates that the abnormal LD2ex in R51 cells was expressed in the original tumor and released into the patient's serum and was not the result of mutations that occurred during establishment of the cell line. It is evident from the LD isoenzyme pattern that R51 cells do not express wild-type LDHA. Indeed Northern blot analysis using LDHA cDNA as the probe revealed only a faint signal with a molecular mass slightly higher than that of wild-type LDHA. Sequence analysis of all the coding regions of LDHA revealed no mutation analogous to the previously reported electrophoretic variant of LDHA (10). However, we found that expression of the wild-type somatic type LDHA gene (type 1 transcript) was silenced by methylation of CpG islands in the 5' noncoding exon (exon a), and a human homolog of a mouse testis-specific LDHA variant (type 2 transcript) that included exon 0 was expressed instead. Therefore, LD2ex in R51 cells is likely to comprise three wild-type LDHB molecules and one LDHA type 2 product.



Promoter methylation is considered to play an important role in carcinogenesis by regulation of gene expression (21,22). In this respect it should be noted that the LDHA gene has been mapped to the p15 region of human chromosome 11, which is a hotspot for hypermethylation in human neoplasia (23, 24). As shown in Fig. 1 in the online data supplement, R51 cells contained three copies of chromosome 11, two of which were apparently normal, whereas the third was a derivative resulting from a reciprocal translocation, t(5;11)(g11;p15). However, it is unlikely that the translocation split the LDHA gene or influenced the methylation status, because only full-length mRNA was recovered and the methylation status of the CpG dinucleotides in the promoter regions was not heterogeneous.

Hiraoka and Li (20) reported that LDHA mRNA from mouse testis differed from somatic LDHA mRNA in that it possessed a 5' noncoding exon (exon 0) instead of exon a. Takano and Li (19) carried out computerized sequence comparison analysis and predicted the existence of a human analog of this murine testicular LDHA. However, actual expression of this variant by human cells had not been confirmed. We are, we believe, the first to demonstrate expression of this testicular LDHA (LDHA type 2 transcript) by human Rb cells. A partial nucleotide sequence of this type 2 mRNA has been reported and can be seen in the EST bank of human cDNA clones originating from choriocarcinoma, neuroblastoma, T-cell lymphoma (Jurkat), placental, colonic, and testicular tissues (25). In our study, we found that the human LDHA type 2 transcript was also expressed predominantly in the testis, although low-level expression was observed in leukocytes, pancreas, spleen, thymus, placenta, and liver. Whether the two different LDHA mRNAs are transcribed by differential usage of two promoters or differential splicing of the mRNA precursor (19) remains to be elucidated. However, the presence of the LDHA type 2 transcript despite methylation of the upstream region (-2460 to -1651) supports the former mechanism.

Our Western blot analysis showed that the molecular mass of the anti-LDHA-reactive protein in the R51 cells was higher than that of somatic LDHA. Exon 0 has three start codons, at -272, -216, and -87, and the one at -87 seems the most likely to become an actual start codon in the type 2 transcript because its sequence connected with the exact open reading frame and the sequence surrounding the start codon (TAAACCGCGATGG) approximates to the consensus sequence (GCCGCCG/ACCAUGG) derived by Kozak (26) more closely than do the corresponding sequences of the former two start codons. Furthermore, the former two start codons formed only a short open reading frame that was independent of the normal transcript. This would lead to the generation of a novel LDHA subunit with 29 additional amino acids (Fig. 8). In fact, the results of the Western blot analysis are compatible with such a prediction. The tetrameric LD isoenzymes of vertebrates possess an additional 20-amino acid N[H.sub.2] terminus compared with dimeric LD isoenzymes from bacteria (27). These 20 N-terminal residues of vertebrate LD isoenzymes are variable, and their postulated primary function is to stabilize the quaternary structure of tetrameric LD isoenzymes through their interaction with the C-terminal tails of other subunits (28). Because the LDHA type 2 transcript possesses the same 3' end sequence as somatic LDHA, it is reasonable to assume that the extra band, LD2ex, is a consequence of tetrameric formation with three wild-type LDHB molecules and one LDHA type 2 molecule with an additional 29 amino acid residues.


Recently, a unique category of tumor antigens, which was first revealed by the analysis of T-cell-recognized epitopes, has been referred to as cancer-testis antigens, which are expressed by a variable proportion of a wide range of different human tumor types (29). In view of its expression site, the LDHA type 2 transcript might be a cancer-testis antigen, although its autoimmunogenicity has not been examined. Although the mechanism underlying the aberrant expression of cancer-testis antigens in cancer cells is not fully understood, dysregulation of DNA methylation has been implicitly presumed (30). The R51 cell line might offer an example of such a mechanism.

In the R51 cells, LD1 concentrations were relatively increased. Increased LD1 concentrations might be a result of reduced expression of LDHA and/or increased expression of LDHB in several types of tumors, such as neuronal (4, 7, 31) and germ-cell (9) tumors. Von Eyben et al. (32) reported that serum LD1 seems to be a useful marker for testicular germ cell tumors. Huang et al. (33) used cDNA microarrays and observed that LDHA expression was repressed in 9 of 11 tumor specimens from patients with low-grade diffuse astrocytomas. Whether such down-regulation of LDHA in these neoplastic cells is attributable to silencing by DNA methylation is unknown. However, it is interesting to note that most of the LD2ex reported in the literature has been found in neuronal tumors.

It remains to be determined whether the novel alternative expression of LDHA type 2 occurs to compensate for the down-regulation of wild-type somatic LDHA (type 1) or is an independent phenomenon. We consider it likely that most of the LD2ex bands reported in the literature can be explained by expression of this LDHA type 2.

This research was supported in part by Grants-in-Aid for Cancer Research (9-13, 13-5) and for the Second Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health, Labour and Welfare, Japan, and by a Grant-in-Aid for Scientific Research (13470518) from the Ministry of Education, Science, Sports, Culture and Technology, Japan. We thank Prof. T. Komoda of Saitama Medical College for supplying the mouse monoclonal antibody against human LDHA protein, Prof. K. Sudo for reading the manuscript, and T. Taniguchi and C. Tatebayashi for technical assistance.

Received June 20, 2002; accepted August 5, 2002.


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[8] Nonstandard abbreviations: LD, lactate dehydrogenase; Rb, retinoblastoma; 5AzaCdR, 5-aza-2'-deoxycytidine; 5AzaCR, 5-azacytidine; RACE, rapid amplification of cDNA ends of 5' terminus; BiPS, bisulfite PCR-single-strand conformation polymorphism; and RT-PCR, reverse transcription-PCR.


[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, 'Clinical Laboratory, and [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.

* Author for correspondence. Fax 81-53-435-2794; e-mail mmaekawa@
Table 1. PCR primer sets for RT-PCR. (a)

Primers Sequence

(a) Annealing temperature and [Mg.sup.2+] concentration for RT-PCR
are 58[degrees]C and 1.5 mM, respectively.

Table 2. Summary of primer sets and PCR conditions for
methylation analysis.

 Primer temperature,
 pairs Sequence [degrees]C


 Length of
 [Mg.sup.2+] PCR
 Primer concentration, product,
 pairs mm by

 F11 1.5 350
 F12 1.5 477
 F15 1.5 205
 F13 1.5 346
 F18 2.5 234
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Title Annotation:Proteomics and Protein Markers
Author:Maekawa, Masato; Inomata, Motoko; Sasaki, Masao S.; Kaneko, Akihiro; Ushiama, Mineko; Sugano, Kokich
Publication:Clinical Chemistry
Date:Nov 1, 2002
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