この論文は国立情報学研究所の学術雑誌公開支援事業により電子化されました。Immunohistochemical distribution of lactate dehydrogenase (LDH) subunits in human lung cancer cells were studied using fluorescent antibody technique. Both in cytological and histological specimen, specific fluorescence of LDH-H and LDH-M were seen in the cytoplasm of cancer cells, and in most cases no remarkable difference could be demonstrated between the distribution of H and M subunit. But in some cases specific fluorescence of M-subunit was stronger than that of H-subunit. The fluorescence of M-subunit was demonstrated as fine granules diffusely in the cytoplasm. On the other hand the fluorescence of H-subunit was rather localized and demonstrated as rather coarse granules. And this observation was discussed

BABA, Mitsuo

FUKUDA, Haruo

HARADA, Takayuki

ITO, Motohiko

MORIKAWA, Shigeru

Kyoto University Research Information Repository

Title<ORIGINAL REPORT>IMMUNOHISTOCHEMICALSTUDIES ON LACTATE DEHYDROGENASE SUBUNITSIN LUNG CANCER CELLSAuthor(s)ITO, Motohiko; FUKUDA, Haruo; BABA, Mitsuo; HARADA,Takayuki; MORIKAWA, ShigeruCitation京都大学結核胸部疾患研究所紀要 (1974), 8(1): 11-18Issue Date1974-12-30URL http://hdl.handle.net/2433/52248RightType Departmental Bulletin PaperTextversionpublisherKyoto UniversityIMMUNOHISTOCHEMICAL STUDIES ON LACTATEDEHYDROGENASE SUBUNITS IN LUNG CANCER CELLSMotohiko ITOl), Haruo FUKUDAl), Mitsuo BABA2),Takayuki HARADA2) and Shigeru MORIKAWA2)Department of Thoracz'c Surgeryl), and Department of Pathology2),Chest Disease Research Instz"tute, Kyoto Uni'versity(Received for publication on September 21, 1974)INTRODUCTIONI t is well established that lactate dehydrogenase is a tetramer composed of Hand M subunitwhich combine randomly to make five isozymes,l ,2,3) and that the isozyme pattern of tumor tissueis quite different from that of normal tissue in which tumor is originated.4 ,5,6)We have already reported the difference of immunohistochemical distribution of LDHsubunits in normal bovine tissue,7) and in normal porcine liverS) using fluorescent antibodytechnique.In this study we examined the immunological cross reactivity between procine LDH andhuman LDH against anti-porcine LDH antisera, and the immunohistochemical distribution ofLDH subunits in human lung cancer cells in histological sections and in cytological smears ofpulmonary adenocarcinoma.MATERIALS AND METHODSPreparation of antiseraLactate dehydrogenase from porcine heart(LDH-4H) and lactate dehydrogenase fromporcine muscle(LDH-4M) obtained from Boehringer and Sohne GmbH(Mannheim, Germany)were used as antigens without further purification.Antisera were prepared in male albino rabbits as reported in a previous paper.7)Specificity of antisera was examined by double gel diffusion method of Ouchterlony and byimmunoelectrophoresis, procedures of which were fully described in our previous papers. 7,S)Immunological cross reactivity between porcine LDH and human LDH against anti-porcine-LDH antisera was also examined by double gel diffusion method of Outcherlony.This study was supported by a Grant-in Aid for Scientific Research from the Ministry of Education.-11--12 - M. ITO, H. FUKUDA, M. BABA, Bull. Chest Dis. Res. Inst., Kyoto Univ.T. HARADA and S. MORIKAWAPreparatz"on of jluorescez"n conjugatesGlobulin fraction of antisera was conjugated with FITC(Baltimore Biological Lab.,Baltimore). Following separation from free fluorescent dyes by gel filtration on Sephadex G-50~the conjugate were purified by fractionation on DEAE-cellulose columns.These procedures are fully described in our previous papers.7,9)Preparatz"on of spedmenCells and tissues from resected cases of pulmonary adenocarcinoma were used in this study.Touch smears from resected adenocarcinoma were dried for 15~30minutes at room tempera-ture, fixed in ether-alcohol-acetone mixture for 15 minutes, washed with phosphate bufferedsaline(PBS) and then stained with FITC-conjugates.Small piece of fresh cancer tissue was put in a test tube and frozen at -70°C immersing thetest tube in an acetone-dryice mixture.Frozen specimen were then cut into 4-5 JL sections in a cryostatt at -20°C, placed on aglass, dried for 15--30 minutes at room temperature, and then fixed with ether-alcohol-acetonemixture for 15 minutes. Fixed specimen were then washed with PBS and stained with FITClabelled antisera.Staining with FITC conjugate and microscopyAfter washing with PBS, specimen were stained with FITC labelled conjugates in a moistchamber at room temperature overnight.These slides were then washed with PBS, mounted in glycerol containing 100/0 PBS andexamined under a Carl-Zeiss fluorescence microscope.RESULTSSpecijidty of antz"seraThe antisera were tested for the specificity by immunodiffusion and immunoelectrophoresisas shown in Fig. 1 and Fig. 2. No contamination was observed between antigen and antisera inour immune system.Cross reaetivz'ty between antz'-porcz"ne LDH antisera and human LDHThe results of Ouchterlony's immunodifiusion between the anti-porcine LDH antisera andhuman LDH is shown in Fig. 3. Cross reactivity is seen between the antisera and human lactatedehydrogenase.Immunohistochemical d£stributz"on of LDH subunits in lung cancer cellsGenerally specific fluorescence of LDH Hand LDH M in the cytological smears is con-centrated in the perinuclear area in the cytoplasm of cancer cells. Diffuse fine fluorescence ofLDH in the cytoplasm is stronger in LDH M than in LDH H. But no remarkable differenceof distribution between both subunits can be seen in the cytological smears of adenocarcinoma.These findings are demonstrated in Fig. 4 and Fig. 5.In cryostatt-sectioned specimen, the fluorescence of LDH M seems to be fine and diffusecomparing with that of LDH H in some cases as shown in Fig. 6 and Fig. 7. But in most casesno difference of distribution between both subunits can be seen in the cryostatt sectioned specimen.Vol. 8 No.1 December, 1974 Immunohistochemical Studies on Lactate DehydrogenaseFig. 1 Double gel diffusion study of LDH subunit and antisera. A precipitationband was observed between LDH 4H and anti-LDH H, and betweenLDH 4M and anti-LDH M.DISCUSSION- 13 --The difference of the distribution of LDH Hand LDH M subunits in normal cells and tissueshave been reported from the electrophoretical studyl) and the enzyme histochemical studies.lO,ll)Recently, the distribution of lactate dehydrogenase subunits were studied immunohistochemicallyin our laboratory.7,8)We extended the immunohistochemical study of the subunits in neoplastic tissues, for wehave not seen any report on the distribution of both subunits in cancer cells or tissues.In this paper we tried to demonstrate the distribution of Hand M subunits of lactate de-hydrogenase in lung cancer cells.The isozyme pattern of tumor tissue generally shows the predominant M -component of thelactate dehydrogenase comparing with that of normal tissue. 4 ,5,6) So some differences may beexpected in the distribution of LDH Hand LDH M subnnits.Antisera prepared from porcine lactate dehydrogenase reported in previous papers7,8) wasrevealed to cross-react with human lactate dehydrogenase in our immune system as some authorsreported.l2,l3) So our immunofluorescent conjugates might be able to locate the distribution ofLDH subunits in human cancer cells.The tissue from the case of squamous cell carcinoma contain many necrotic masses, andthere appear many non-specific fluorescence. Small cell undifferentiated carcinoma cells havepoor cytoplasm. Both of them are not adequate for studying the intracytoplasmic distributionof lactate dehydrogenase subunits.So we examined the distribution of lactate dehydrogenase subunits in the cytological smears-14 - M. ITO, H. FUKUDA, M. BABA,T. HARADA and S. MORIKAWA Bull. Chest Dis. Res. Inst., Kyoto Univ.Fig.2 Immunoelectrophoretic study of LDH subunit.Fig.3 Double gel diffusion study of human LDH and porcine LDH.(S. :Human serum)Vol. 8 No.1 December, 1974 Immunohistochemical Studies on Lactate Dehydrogenase - 15-Fig. 4 The immunofluorescence of LDH H in the cytological smear.Fig. 5 The immunofluorescence of LDH M in the cytological smear.-16 - M. ITO, H. FUKUDA, M. BABA, Bull. Chest Dis. Res. Inst., Kyoto Univ.T. HARADA and S. MORIKAWAFig. 6 Immunohistochemical findings of LDH H in the cryostatt-sectioned specimen.Fig. 7 Immunohistochemical findings of LDH M in the cryostatt-sectioned specimen.Vol. 8 No.1 December, 1974 Immunohistochemical Studies on Lactate Dehydrogenase - 17-or cryostatt-sectioned specimen from the case of pulmonary adenocarcinoma.Both in cytological smears and in cryostatt-sectioned specimen, intracytoplasmic distributionof LDH subunits are almost same. But in some cases specific fluorescence of LDH-M was ratherstronger and diffuser comparing with the fluorescence of LDH-H which was somewhat localizedand granular in the cytoplasm.Diffuse distribution of LDH-M fluorescence may be due to the fact that M-subunit is mainlycontained in soluble fraction by cytochemical analysis. On the other hand, H-subunit is observedto be combined with subcellular organellas.In order to clarify these problems, ultramicroscopical immunohistochemistry must be per-formed in future.SUMMARYImmunohistochemical distribution of lactate dehydrogenase(LDH) subunits in human lungcancer cells were studied using fluorescent antibody technique.Both in cytological and histological specimen, specific fluorescence of LDH-H and LDH-Mwere seen in the cytoplasm of cancer cells, and in most cases no remarkable difference could bedemonstrated between the distribution of Hand M subunit.But in some cases specific fluorescence of M-subunit was stronger than that of H-subunit.The fluorescence of M-subunit was demonstrated as fine granules diffusely in the cytoplasm.On the other hand the fluorescence of H-subunit was rather localized and demonstrated as rathercoarse granules.And this observation was discussed.REFERENCES1) Kaplan, N. 0.: Lactate dehydrogenase--structure and function. Brookhaven Symp. Bio!., 17: 131, 1964.2) Markert, C. L. and Apella, E.: Immunochemical properties of lactate dehydrogenase isozymes. Ann.N. Y. Acad. Sci., 103: 915,1963.3) Cahn, R. D., Kaplan, N. 0., Levine, L. and Zwilling, E.: The nature and development oflactic dehydro-genases. The two major types of this enzyme form molecular hybrids which change in make up duringdevelopment. Science, 136: 962, 1962.4) Goldmann, R. D. and Kaplan, N. 0.: Alterations of tissue lactate dehydrogenase in human neoplasm.Biochim. Biophys. Acta, 77: 515, 1963.5) Wieland, T.: Uber die Verscheidenheit der Milchsaure-dehydrogenasen. Biochem. Z., 331: 103, 1959.6) Wieland, T. and PReider, G.: Chemical differences between multiple forms of lactic acid dehydrogenases.Ann. N. Y. Acad. Sci., 94: 691, 1961.7) Ito, M., Harada, T., Baba, M. and Morikawa, S.: Immunohistochemical studies on lactate dehydrogenasesubunits in normal bovine tissues. Acta Histochem. et Cytochem., 6: 183, 1973.8) Ito, M., Fukuda, H., Matsushita, T., Baba, M., Harada, T. and Morikawa, S.: Immunohistochemicallocalization of lactate dehydrogenase sutunits in porcine liver. Acta Histochem. et Cytochem., in press.9) Morikawa, S.: Studies on alkaline and acid ribonuc1eases in mammalian tissues: Immunohistochemicallocalization and immunochemical properties. J. Histochem. Cytochem., 15: 662, 1967.10) Brody, I. A.: Isozyme histochemistry: A new method for display of selective lactate dehydrogenase isozymeson an electrophoretic pattern. Nature, 201: 685,1964.11) Brody, 1. A. and Engell, W. K.: Isozyme histochemistry: the display of selective lactate dehydrogenaseisozymes in sections of skeletal muscle. J. Histochem. Cytochem., 12: 687, 1964.18 - M. ITO, H. FUKUDA, M. BABA, B 11 Ch t D' R I t K t U .T. HARADA and S. MORIKAWA u. es IS. es. us., yo 0 mv.12) Kaplan, N. O. and White, S.: Immunologic characteristics of dehydrogenases. Ann. N. Y. Acad. Sci.,103: 835, 1963.13) Nisselbaum, J. S. and Bodansky, O. :,Reactions of human tissue lactic dehydrogenases with antisera tohuman heart and liver lactic dehydrogenases. J. BioI. Chern., 236: 401, 1961.14) Agostoni, A., Vergani, C. and Villa, L.: Intracellular distribution of the different forms of lactic dehydro-genases. Nature, 209: 1024,1966.15) Nemchinskaya, V. L., Ganeriar, L. Sh. and Braun, A. D.: Differences in isozyme patterns of nucleic andcytoplasmic lactate dehydrogenase of rat liver cell. Nature, 217: 251, 1968.

<ORIGINAL REPORT>IMMUNOHISTOCHEMICAL STUDIES ON LACTATE DEHYDROGENASE SUBUNITS IN LUNG CANCER CELLS

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<ORIGINAL REPORT>IMMUNOHISTOCHEMICAL STUDIES ON LACTATE DEHYDROGENASE SUBUNITS IN LUNG CANCER CELLS

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