Histochemical evaluations of human sarcomas such as reticulum cell sarcoma, fibrosarcoma, lymphosarcoma and neurofibrosarcoma, were carried out with five hydrolytic enzymes and eight oxidative enzymes. The activities of acid phosphatase and beta-glucuronidase were slightly positive in the neoplastic cells observed. Beta-esterase activity was also positive but varied according to the kind of sarcomas. Alkaline phosphatase activity was faint or negative in sarcoma cells, though positive in capillary walls. Leucine aminopeptidase activity was negative giving not any appreciable coloration of the cell as far as the method employed is concerned. Among the activities of dehydrogenases, the most intense activity was observed in lactic dehydrogenase. The activities of succinic and beta-hydroxybutyric dehydrogenases were slight. The activities of alpha-glycerophosphate, glutamic and betahydroxybutyric
dehydrogenases were faint or slight. The activities of NADPlinked dehydrogenases, glucose-6-phosphate and isocitric dehydrogenase were all faint or slight in these sarcoma cells.</p

Kawashima, Takao

Mihara, Akemi

Nobuto, Hideo

Ogata, Takuro

Seito, Takashi

Shimatani, Nobuto

Takeuchi, Kisaburo

Okayama University Scientific Achievement Repository

Acta Medica OkayamaVolume 20, Issue 4 1966 Article 3AUGUST 1966A histochemical study on hydrolytic andoxidative enzymes in human sarcomasHideo Nobuto∗ Takao Kawashima† Nobuto Shimatani‡Kisaburo Takeuchi∗∗ Akemi Mihara††Takashi Seito‡‡ Takuro Ogata§∗Okayama University,†Okayama University,‡Okayama University,∗∗Okayama University,††Okayama University,‡‡Okayama University,§Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.A histochemical study on hydrolytic andoxidative enzymes in human sarcomas∗Hideo Nobuto, Takao Kawashima, Nobuto Shimatani, Kisaburo Takeuchi, AkemiMihara, Takashi Seito, and Takuro OgataAbstractHistochemical evaluations of human sarcomas such as reticulum cell sarcoma, fibrosarcoma,lymphosarcoma and neurofibrosarcoma, were carried out with five hydrolytic enzymes and eightoxidative enzymes. The activities of acid phosphatase and beta-glucuronidase were slightly posi-tive in the neoplastic cells observed. Beta-esterase activity was also positive but varied accordingto the kind of sarcomas. Alkaline phosphatase activity was faint or negative in sarcoma cells,though positive in capillary walls. Leucine aminopeptidase activity was negative giving not anyappreciable coloration of the cell as far as the method employed is concerned. Among the ac-tivities of dehydrogenases, the most intense activity was observed in lactic dehydrogenase. Theactivities of succinic and beta-hydroxybutyric dehydrogenases were slight. The activities of alpha-glycerophosphate, glutamic and betahydroxybutyric dehydrogenases were faint or slight. The ac-tivities of NADPlinked dehydrogenases, glucose-6-phosphate and isocitric dehydrogenase wereall faint or slight in these sarcoma cells.∗PMID: 4226976 [PubMed - indexed for MEDLINE] Copyright c©OKAYAMA UNIVERSITYMEDICAL SCHOOLActa Med. Okayama 20, 159-166 (1966)A HISTOCHEMICAL STUDY ON HYDROLYTIC ANDOXIDATIVE ENZYMES IN HUMAN SARCOMASHideo NOBUTO, Takao KAWASHIMA, Nobuto SHIMATANI,Kisaburo TAKEUCHI, Akemi MIHARA, Takashi SEITO,and Takuro OGATADepartment of Surgery, Okayama University Medical School, OkayamaJapan (Director: Prof. S. Tanaka)Received for publication, May 16, 1966Biochemical investigations occasionaly fall in great errors on the estimationof chemical substance or enzyme activity of neoplastic tissues because of theunavoidable inclusion of non-neoplastic tissues during homogenating process,while histochemical investigations have great advantage to analyse the localiza-tion of enzyme activity in the unit of cellular or tissue structure. In this decade,there appeared many histochemical observations on tumor cells and several obser-vations on human sarcomas such as those reported by MONIS et al.2,9,l1,12, SELIGMANet al.s ,1,1O, MORl et al. Is and KABAT et a[,l. They observed various tumorshistochemically but only a few of human tumors, and only a poor informationon the enzyme activities of human sarcomas. Therefore, the authors observedthe enzyme activities of human sarcoma cells by histochemical methods for thepurpose to disclose some possible difference in enzyme activity between normaland tumor cells. The present paper deals with the enzyme activities of ninecases of human sarcomas. The enzyme activities observed were those of hydro-lytic and oxidative ones, such as alkaline phosphatase, acid phosphatase, beta-estrase, leucine aminopepidase, j9-glucuronidase, succinic dehydrogenase, lacticdehydrogenase, malic dehydrogenase, a-glycerophosphate dehydrogenase, gluta-mic dehydrogenase, ;9-hydroxybutyric dehydrogenase, glucose-6-phosphate de-hydrogenase and iso-citric dehydrogenase.MATERIALS AND METHODSNine cases of sarcomas were studied. They were·5 cases of reticulosarcoma,2 cases of fibrosarcoma, 1 case of lymphosarcoma and 1 case of neurofibro-sarcoma. The blocks for the experiment were obtained exclusively fromsurgical specimens and frozen in the deep freezer at - 30°C. Frozen blockswere sliced 20f.L thick in a -20°C cryostat, with a sliding microtome. Parallelsections were stained with hematoxylin and eosin for morphological orientation.1591Nobuto et al.: A histochemical study on hydrolytic and oxidative enzymes inProduced by The Berkeley Electronic Press, 1966160 H. NOBUTO and othersThe sliced sections were processed for the histochemical procedures. For thehistochemical demonstration of hydrolytic enzymes, the sections were fixed in10 per cent neutral fromalin for 10 minutes.Each reaction mixture was composed as followsAlkaline phosphatase (ALP): Sections on slides, fixed in 10 per centformalin for 10 minutes, were incubated for 1 hour at 20°C in the solutioncontaining 10 mg of sodium a-naphthyl phosphate, 20 mg of Clarks and Lubs'buffer at pH 9.2, and 20 mg of Diazo blue B.Acid phosphatase (A CP): The same procedures were taken as those foralkaline phosphatase except that 0.1 M acetate buffer was used at pH 5.8.ft-Esterase (ES T): The formalin fixed sections were incubated for 30minutes at 20°C in the reaction mixture; 10 mg of ft-naphthyl acetate dissolvedin 1 ml of acetone, 20 ml of O. 1 M Michaelis buffer at pH 7. 2, and 20 mg ofDiazo blue B. The stained sections were mounted in glycerin.(i-Glucuronidase (ftGL): The post-azo coupling method was employed.Formalin fixed sections were incubated for 6 hours at 37°C in the followingmixture; 15 mg of 6-bromo-2-naphthyl-(i-D-glucuronide dissolved in 2.5 ml ofmethanol, 10 ml phospho-citric buffer at pH 4.95 and 37.5 ml of distilledwater. Therafter these sections were rinsed in water and immersed in 50 ml of0.02 M phosphate buffer at pH 7.5 containing 50 mg of Diazo blue B.Leucine Aminopeptidase (LAP): The sections, fixed in 10 per centneutral formalin for 10 minutes, were incubated for 30 minutes to one hour at37°C in the following solution; 8 mg of L-Ieucyl-(i-naphthylamine, 10 ml of0.1 M acetate buffer at pH 6.5, 8 ml of 0.85 per cent sodium chloride, 1 ml of0.02 M sodium cyamide, and 10 mg of Diazo blue B. These incubated sectionswere washed in saline and transferred to a solution of 0.1 M copper sulfate fora few minutes.On dehydrogenases, sections were incubated at 37°C for 30 to 60 minutesin each mixture and then fixed in 10 per cent formalin solution for 10 minutes.Succinic dehydrogenase (SDH): M/5 sodium succinate 5 ml, M/IOphosphate buffer pH 7.6 5 ml, Nitro BT 5 mg/3ml 6 ml, aq. 10 ml.Lactic dehydrogenase (LDH): M/2 sodium lactate, 4 ml; M/10 phos-phate buffer pH 7.4, 10 ml; Nitro BT 5 mg/3 ml, 3 ml; 100% NAD, 2.5 mg;M/I0 KCN, 2 ml; and M/2 HCI, 2 drops.Malic d ehydrogenase (MDH) : 1 M sodium malate, 5 ml; M /10 phosphatebuffer pH 7. 4, 10 ml; Nitro BT 5 mg/3 ml, 3 ml ; 100% NAD, 2.5 mg; M/IOOMgC12' 2 ml; M/2 Hel, 5 drops.Glutamic dehydrogenase (GD H): 1 M sodium glutamate, 4 ml; M/IOphosphate buffer pH 7.6, 11 ml; Nitro BT 5 mg/3 ml, 3 ml; 100% NAD, 2.52Acta Medica Okayama, Vol. 20 [1966], Iss. 4, Art. 3http://escholarship.lib.okayama-u.ac.jp/amo/vol20/iss4/3Histochemical Study in Human Sarcomas 161mg; M/IO KCN, 2ml; M/2 HCN, 5 drops.a-Glycerophosphate dehydrogenase (a:GDH): 1 M sodium a-glycero-phoaphate, 4 ml; M/10 phosphate buffer, 11 ml; Nitro BT 5 mg/3 ml, 3 ml ~10096 NAD, 2.5mg; M/10 KeN, 2ml; M/2 HCl, 5 drops.11-Hydroxybutyric dehydrogenase (j3HyDH): 1 M sodium ,q·hydroxy-butyrate1 4 ml; M/lO phosphate buffer pH 7.9, 11 ml; Nitro BT 5 mg/3 ml;.3ml; 1"0096 NAD, 2.5mg; M/10 KeN, 2ml; M/2 HCI; 5 drops.Glucose-6·phosphate dehydrogenase (G6 PDH): M/200 glucose-6-phosphate,6ml; Veronal buffer pH 7.4, 17ml; Nitro BT 5mg/3ml, 4.5ml;10096 NADP, 7 mg; M/100 MgCl2, 3 ml; M/2 MnCI2, 3 ml.Isocitric dehydrogenase (ICDH): M/10 sodium isocitrate, 4 ml; Vero-nalbufferpH7.4, 11ml; NitroBT5mg/3ml,3ml; 10096NADP,2.5mg;M/100 MgCl2, 2 ml; M/2 MnCI2, 2 ml.RESULTSAL P: Enzymatic reaction was faint or negative m all the sarcomas ex-amined in the present study. Only capillary wall showed a strong enzymeactivity.A CP: Reticulum cell sarcoma had a weak activity, but other sarcomas.showed faint or no activity. The azo-dye pigments were deposited in granularTable 1 Distribution of Enzymatic Activities in Human Sarcoma CellsLo::ationINo. oflI cases IDiagnosisReticulum cell I I~ 74 1-1 + + I-J-l-' +I1 Caecum + _L - + -- + + +-'-sarcoma+1+:-1 +IReticulum cell 2 Retroperitoneum '0 621-1± -\- -'- - + + .J- + ±sarcoma - -'-Reticulum cell 3 Side of neck ~ 34 + + +I± - +1,+ ± + -!- ±I+sarcoma -'-IReticulum cell 4 Side of neck ~ 24 + + + + - ± +:-1+:-1+ + -!- -'- -sarcoma171=-'-Reticulum cell 5 Tonsil '0 + - + - + -I-l-I-II-i + -'- --'- - -sarcoma"I"Lymphosarcoma 1 Inguinal ~ 42 - - +J- - -1- ± -:- '1-1- - -Llymph node -'- - - -I+:-1 +Fibrosarcoma 2 Retroperi toneum 0 12 - -I- + + - + +Fibrosarcoma 1 Fibula '" 38 - - + + - ± -1-]- +:- -I- + +:- + +0Neurofibrosarcoma 1 Inguinal region ~ 1 48 - - -i± - +1- -r-:- +1- -I- +I± +1- -I-i Inegative, ±: faint, +: slight, +1-: moderate, ·11+: intense3Nobuto et al.: A histochemical study on hydrolytic and oxidative enzymes inProduced by The Berkeley Electronic Press, 1966162 O and othersH. NOBUT4Acta Medica Okayama, Vol. 20 [1966], Iss. 4, Art. 3http://escholarship.lib.okayama-u.ac.jp/amo/vol20/iss4/3Histochemical Study in Human Sarcomas 163form in the cytopplasm.LAP: Leucine aminopeptidase activity was absent in all cases of sarcomas.[3-EST: [3-Esterase activity was negative or faint in all "five reticulum cellsarcomas and a neurofibrsarcoma. In the cytoplasm of fibrosarcoma and lym-phosarcoma, the reaction was faint to moderate. Necrotic area of most tumorsshowed a moderate or intense activity.[3- Gl: A staining reaction for [3-glucuronidase was slightly positive in thecytoplasm of all th~ cases of sarcomas.SDH: The activity of succinic dehydrogenase in tumor cells was ratherslight or weak in lymphosarcoma and reticulum cell sarcomas but rather strongin neurofibrosarcoma. The formazan pigment formed a particularly delicatepattern suggestive of mitochondria.LDH: As compared with other dehydrogenases, the activity of thisenzyme was most intense and accentuated, especially at the periphery of tumorcells. Reticulum cells of reticulum cell sarcomas showed a moderate activityin the cytoplasm and reticulum.MDH: Although the staining of malic dehydrogenase was somewhatweaker than that of lactic dehydrogenase, patterns of its distribution weresimilar to those of lactic dehydrogenase, patterns of its distribution were similarto those of lactic dehydrogenase.Other NAD-linked dehydrogenases: a-Glycerophosohate, glutamic andp-hydroxybutyric dehydrogenases were weak in most cases of sarcomas, espe-cialy in lymphosarcoma. Their deposition patterns of formazan granules weresimilar to those of succinic dehydrogenase.In both of NADP-linked dehydrogenases such as glucose-6-phosphate andisocitric dehydrogenase their activity was in general faint or slight in sarcomacells. Occasionally, a weak activity was observed in the small arterial wall.Fig. 1 Reticulum cell sarcoma. Hematoxylin-Eosin stain. x400Fig. 2 Reticulum cell sarcoma. (same specimen as in Fig. 1). Silver stain. X 400Fig. 3 Reticulum cell sarcoma. (same specimen as in Fig. 1). Acid phosphatase. Enzymaticactivity is observed moderately in scattered histocytes or fibrocytes, while there is weak activityin the tumor cells and connective tissne. X 40Fig. 4 Reticulum cell sarcoma. Lactic dehydrogenase. (same specimen as in Fig. 1). Tumorcells show a moderate or intense activity. X 40Fig. 5 Reticulum cell sarcoma. p-Hydroxybutiric dehydrogenase. (same specimen as in Fig.1) Tumor cells show a weak activity. X 40Fig. 6 Neurofibrosarcoma. Alkaline phosphatase. Small capillary wall shows a strong activity.Tumor cells are not stained. X 40Fig. 7 Neurofibrosarcoma. Glutamic dehydrogenase. Moderate activity is observed. X 40Fig. 8 Lymphosarcoma. Glucose-6-phosphate dehydrogenase. Formazan granules are depo-sited in the cytoplasma and peripher)- of tumor cells. X 4005Nobuto et al.: A histochemical study on hydrolytic and oxidative enzymes inProduced by The Berkeley Electronic Press, 1966164 H. NOBuTo and othersDISCUSSIONAlthough several studies on sarcomas have been reported, histochemicalevaluations of them are not sufficient. With respect to the alkaline phosphataseactivity, KABAT et at.l examined various animal sarcomas induced by virus andmethylcholanthrene and found no reaction in these tumors. MaNIS et at. ob-served that only fibromyosarcomas of 12 sarcomas showed a weak reaction inthe malignant cells. MANHEIMER et at.s demonstrated that only the blood vesselswere stained in a fibrosarcoma of the breast and a uterine myoma. FANGER etat. t observed the absence of reaction in a fibrosarcoma of the breast and in aleiomyosarcoma of the uterus, but SCHAJOWICZ et at.~ reported a strikingly strongreaction in a liposarcoma of bone. BIESEL et at.6 examined tissue cultures fromCROCKER mouse sarcoma 180 and observed only a faint nuclear reaction. Theyalso studied rat sarcoma 339 which showed a strong reaction while in RON'Ssarcoma of the chicken showed only a weak activity.In the present study likewise, the enzyme activity of alkaline phosphatasein human sarcomas very weak or negative except for capillary walls.With respect to acid phosphatase activity, REINEH et at,7 studied fibrosar-coma and polymorph cell sarcoma and found a moderate activity in malignantmesenchymal tumors which showed stronger coloration than benign ones.FANGRE et at. revealed a positive rcaction in a leiomyosarcoma of the uterus anda fibrosarcomas of the breast. TAKEUCHI et at.s observed a positive reaction inYOSHIDA sarcoma. In the present study acid phosphatase activity was found inthe cyotplasm of sarcomas with slight activity and in the histocytes withmoderate activity.On aminopeptidase, MaNIs et at.9 found a strong activity in a humanfibrosarcoma while entirely negative in a spindle cell sarcoma. In the presentstudy, aminopeptidase activity was negative in all the sarcomas. CHOEN et al,1°could not find esterase activity in sarcomas of soft tissues.;9-Glucuronidase activity in seven sarcomas was faint or none by the studyof MaNIs et at,ll. The activity in the present study was slightly positive in thecytoplasm and was increased in the necrotic foci of tumors. The reticulum cellsof sarcomas showed a faint or weak activity.There are very few studies of dehydrogenases on sarcoma. However, itwas found that lactic dehydrogenase activity was most intense as compared withthe other dehydrogenases studied. MaNIS et at.12 observed a malignant lym-phoma (lymphocytic), a fibrosarcoma and a spindle cell sarcoma and demon-strated an intense to moderate activity in malic dehydrogenases and a weakactivity in the other dehydrogenases such as succinic and ;9-hydroxybutiric dehy-drogenases. In the present study, the simillar intensity of coloration was6Acta Medica Okayama, Vol. 20 [1966], Iss. 4, Art. 3http://escholarship.lib.okayama-u.ac.jp/amo/vol20/iss4/3Histochemical Study in Human Sarcomas 165perceived. MORI et at.13 reported that reticular cell sarcoma and Hodgkin'ssarcoma showed a low activity of iso-citric dehydrogenase, and liposarcoma andlymphosarcoma exhibited varying stainability in glucose-6-phosphate dehydro-genase.In general, the human sarcomas examined in this study showed a loweractivity of hydrolytic and oxidative enzymes than in those of the cancer ofstomach, intestines, breast and thyroid glands, as were reported in the previouspapersIO - 18•SUMMARYHistochemical evaluations of human sarcomas such as reticulum cell sarcoma,fibrosarcoma, lymphosarcoma and neurofibrosarcoma, were carried out withfive hydrolytic enzymes and eight oxidative enzymes.The activities of acid phosphatase and beta-glucuronidase were slightlypositive in the neoplastic cells observed. Beta-esterase activity was also positivebut varied according to the kind of sarcomas. Alkaline phosphatase activitywas faint or negative in sarcoma cells, though positive in capillary walls.Leucine aminopeptidase activity was negative giving not any appreciable colora-tion of the cell as far as the method employed is concerned. Among the acti-vities of dehydrogenases, the most intense activity was observed in lactic dehy-drogenase. The activities of succinic and beta-hydroxybutyric dehydrogenaseswere slight. The activities of alpha-glycerophosphate, glutamic and beta-hydroxybutyric dehydrogenases were faint or slight. The activities of NADP-linked dehydrogenases, glucose-6-phosphate and isocitric dehydrogenase wereall faint or slight in these sarcoma cells.REFERENCES1. KABAT, K. aud FUTH, ].: A histochemical study of the distribution of alkaline phosphatasein various normal and neoplastic tissues. Amer. ]. Path. 17, 303, 19412. MONIS, B. and RUTENBERG, A. M.: Alkaline phosphatase activity in neoplastic and inflam-matory tissues of man. Cancer 13, 538, 19603. MANHEIMAR, L. H. and SELIGMAN, A. M.: Improvement in the methcd for the histo-chemical demonstration of alkaline phosphatase and its use in a study of normal and neoplastictissues. ]. Nat Cancer Inst. 9, 181, 19484. FANGER, H. and BACKER, B.: Capillaries of normal and diseased breasts. A. M. A Arch.Path. 69, 67, 19605. SCHAJOWICZ, F. and CARBRINI, R. L.: Histochemical studies of bone in normal and patho-logical conditions. ]. Bone Surg. 36-B, 474, 19546. BIESEJ,.E, ].]. and WILSON, A. Y.: Alkaline phosphatase substrate specificities in culturednormal and malignant cells of mouse, rat and fowl. Cancer Reserch 11, 174, 19517. REINER, L., RUTENBERG, A. M. and SELIGMAN, A. M.: Acid phosphatase activity in humanneoplasm. Cancer 10, 563, 19577Nobuto et al.: A histochemical study on hydrolytic and oxidative enzymes inProduced by The Berkeley Electronic Press, 1966166 H. NOBUTO and others8. TAKEUCHI, T. and TANOUE, M.: Histochemical studies on alkaline and acid phosphatasein Yoshida sarcoma. Cann 42, 217, 19519. MONIS, B.: Study of leucine aminopeptidase in neoplastic and inflamatopry tissues with anew histochemical method. Cancer, 12, 601, 195910. COEHN, R. B., NACHLE M. M. and SELIGMAN, AM.: Histochemical demonstration of esterasein malignant tumors. Cancer Reserch Res. I L 709, 195111. MONIS, B., BANKS, M. and RUTENBURG, AM.: ,a-Glucuronidase activity in malignantneoplasm of man. Cancer 13, 386, 196012. MONIS, B., NACHLAS, M. M. and SELIGMAN, AM.: Histochemical study of 3 dehydro-genase systems in human tumors. Cancer, 12, 1238, 195913. MORI, M., MATSUMURA, T., SUGIMURA, M. and KAWASHIMA, H.: Histochemical studieson the localization of iso-citric dehydrogenase in human tumors. Cann 55, 117, 196414. TANAKA, S., SHIMIZU, }., OGATA, T. NISHIYAMA, A and NOBUTO, H.: A histochemicalstudy on human alimentary canal. Part 1. On esophagus and stomach. japanese journalof Gastroenterology 62, 7, 816, 1965 (ill Japanese)15. TANAKA, S., SHIMIZU, J., OGATA, T., SEITO, T., KAWASHIMA, T. and NOBUTO, H.:A histochemical study on human alimentary canal. Part II. On intestine. japanese journalof Gastroenterology 62. 7 835, 1965 (in Japanese)16. SEITO, T., KAWASHIMA, T., NOBUTO, H. and OGATA, H. and OGATA, T.: Histochemicalstudies of hydrolytic and oxidative enzymes in human intestinal tumors. Acta MedicinaeOkayama in press17. NOBUTO, H.: Histochemical studies on human breast tumors. Okayama-Igakkaizasshi 74,251, 1965 (in Japanese)18. NOBUTO, H, TAKEUCHI, K., MIHARA, A, SEITO, T .• KAWASHIMA, T. and OGATA, T.:Histochemical studies on human thyroid gland. Acta Medicinae Okayama in press8Acta Medica Okayama, Vol. 20 [1966], Iss. 4, Art. 3http://escholarship.lib.okayama-u.ac.jp/amo/vol20/iss4/3

A histochemical study on hydrolytic and oxidative enzymes in human sarcomas

Okayama University Digital Information Repository

A histochemical study on hydrolytic and oxidative enzymes in human sarcomas

Abstract

Similar works

Full text

Available Versions

Okayama University Scientific Achievement Repository

Okayama University Digital Information Repository