Dehydrogenase Activity in Human Tumors

MASAHIKO MORI, TETSUHIKO MIYAJI, IWAO

AND MADOKA MURAKAMI

(Department of Oral Surgery and Hi.@tochemwal Laboratory, Osaka University Dental School, Kita/cu, Osaka, Japan)

SUMMARY

A histochemical study was made of a-glycerophosphate dehydrogenase in humantumors of @30cases. Enzymatic activity was generally confined to the cell plasma inneoplastic cells, and stromal reaction was usually low or absent. The stainability fora-glycerophosphate dehydrogenase was constant in normal tissue, whereas that of neoplastic cells was variable. Squamous-cell carcinomas showed varying degrees of enzymatic activity. Adenocarcinoma exhibited a moderate to strong activity, and 47 percent of the stomach cancers seemed to be moderately reactive. Adenomas and fibroadenomas in the breast showed a moderate activity.

Activity and localization for a-glycerophosphate dehydrogenase showed no characteristics, and no correlation was found with the degree of differentiation and malignancy.

Although it has already been reported thata-glycerophosphate dehydrogenase is importantfor neutral fat and phospholipide metabolism andthat it catalyzes the reversible oxidation of ia-aglycerophosphate (5), there are few reports concerning a histochemical detection of a-glycerophosphate dehydrogenase in human neoplasms (1, 7).The biological significance of a-glycerophosphatedehydrogenase in tumor tissue from the biochemical and histochemical viewpoints is still notclarified at the present time. The present expeniments were attempted to determine the localizationof a-glycerophosphate dehydrogenase activity inhuman neoplastic tissue and to investigate apossible relationship between neoplastic cells andhomologous non-neoplastic cells.

MATERIALS AND METHODS

The human tumors consisted of 140 malignantand 90 benign tumors. The blocks were obtainedfrom surgical specimens. All specimens were immediately frozen in dry ice, and fresh frozen sections,1@—18ii thick, were made in a cryostat at —@0°C.with the use of a slide microtome. After being driedat room temperature, these sections were incubated in the following solution at 37°C. for 1 hourfor the histochemical study of a-glycerophosphatedehydrogenase. The incubation solution consisted

Received for publication June 24, 1968.

of 4 ml. of 1 M sodium a-glycerophosphate, 3 ml.of Nitro-BT', @.5mg. of 100 per cent DPN, 11 ml.of 0.1 M phosphate buffer, pH 7.6, to which@ ml.of 0.1 M potassium cyanide was added, and thesolution was adjusted with 0.5 M hydrochlorideto pH 7.6 (i). After incubation, the sections werefixed in 10 per cent neutral formalin for over 10minutes, rinsed in distilled water, and mounted inbalsam. Positive enzymatic activity was revealedby a blue color. All specimens were histopathologically diagnosed on sections stained with hematoxylin and eosin.

RESULTS

The stainability for a-glycerophosphate dehydrogenase activity in tissue sections with Nitro-BTused as an electron acceptor showed ,a fine bluishpigment. The grades of enzymatic intensity werearbitrarily divided into the following groups: 0(negative), ± (trace), +1 (slight), +@ (moderate), +3 (high or strong), and +4 (the highest ormost intense). The degrees of color reaction in bothmalignant and normal tissues were determined bydifferent stainability in the same section.

Oral carcinoma.—Epidermoid cancer with highkeratinization (sixteen cases) exhibited a low to

1 Five mg. of Nitro-BT (2,2'-di-[p-nitrophenylj-5,5'-diphen

yl-8,8'-[S,8'-dimethoxy-4,4'-biphenylene] ditetrazolium chloride) was dissolved in S ml. of distilled water.

168.5

Histochemical Observations of a—Glycerophosphate

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DIAGNOSISTOTALcastsDzozzzs

OFACTIYITT*—±+1+5+3+4Epidermoid

@.,mouthSquamousca.,uterusAdenoca.,stomachUndiff.ca.,stomachPolyp,stomàchAdenoca.,rectumAdenoca., breastAdenomaand fibroadenoma,breast

Adenoca.,thyroidgl.StrumanodosaPleomorphic adenoma,

salivarygl.Adenoca.,pancreasAdenoca.,ovary16

2254156

127

282

21

9280

000000

000

0000

001001

001

0009

6114011

110

10

5118

625

7564

506

8124

10168141

524

1000

000010

000

000

Cancer Research Vol. @3,November 1963I 686

marked enzymatic activity in the malignant epithelium and no activity in the keratinized portion.The stromal elements were usually devoid of thisenzymatic reaction. The normal oral squamousepithelium showed a moderate a-glycerophosphatedehydrogenase activity in all the epithelial layersexcept for the superficial kenatinized layer (Table1).

P1eOmOrphW adenonui of salivary glands.—Ninecases of mixed tumor of salivary glands and pleomorphic adenoma in oral mucosae were examined.In general, the majority of adenomatous cells ofpleomorphic adenoma showed a low enzymatic reaction—e.g., a low reaction in five of the nine casesand a moderate reaction in three cases. The normalgland in subepithelial tissue of the palate and the

epithelium (Figs. 7, 8, 14), and in its undifferentiated type the activity was variable in the neoplastic cells (Figs. 11, 1@). Forty-seven per cent ofthe cases of stomach cancer (3@of 69 cases) showeda moderate activity for a-glycerophosphate dehydrogenase. The surface epithelium of the stomachpolyp was stained slightly for a-glycerophosphatedehydrogenase reaction (Fig. 10).

Rectal cancer.—Ten cases of rectal adenocarcinoma and one case each of sigmoid cancer andmalignant polyp were examined (Table 1) Malignant epithelia of rectal cancer were usually moderately reactive (Fig. 18).

Pancreatic cancer.—Two cases of adenocarcinoma in the pancreas showed a low and moderateenzymatic activity in the neoplastic epithelium.

TABLE 1

a-GLYCEROPHOSPHATE DEHYDROGENASE ACTIVITY IN TUMOR CELLS

S Degrees of activity: —, negative; ±, trace; +1, slight; +2, moderate; +8. strong; +4,most intense.

buccal region revealed a marked enzymatic activity in duct cells and a moderate to low activityin acini.

Ameloblastoma.—Seven cases of ameloblastomawere studied. A slight reaction was observed inthree cases, and a moderate and high activity intwo cases. The neoplastic epithelium of this tumorwas positive for a-glycerophosphate dehydrogenase and generally slightly positive in the stroma.

Stomach tumor.—Sixty-nine cases of gastric cancer consisted of 54 cases of adenocarcinoma andfifteen cases of undifferentiated cancer; six cases ofgastric polyp and four cases of gastritis were alsoexamined (Table 1). Most cases of differentiatedadenocarcinoma of the stomach showed a moderate activity for a-glycerophosphate dehydrogenase. In well differentiated adenocarcinoma theenzymatic activity was confined to the neoplastic

Tumors of female genital organs.—Twenty-twocases of squamous-cell cancer in cervix uteri, onecase of chorioepithelioma, three cases of ovarianadenocarcinoma, and one case of Kruckenberg'stumor were studied (Table 1). All the squamouscell carcinomas contained a low to marked enzymatic reaction in the neoplastic epithelium; however, the surrounding necrotic areas in cancer focihad a somewhat higher activity than did peripheralcells. The activity of chorioepithelioma was low,that of ovarian cystoadenocarcinoma low, that oftwo cases of papillary adenocarcinoma high, andthat of Kruckenberg's tumor moderate (Figs. 3, 4)The stromal elements of squamous-cell cancer incervix uteri contained a trace and low enzymaticactivity. The homologous squamous epithelium ofthe cervix exhibited a moderate enzymatic activityin the basal-cell layer to the parakeratotic layer, as

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Morn et al.—a-Glycerophosphate Dehydrogenase in Human Tumors 1687

observed in the normal oral epithelium. The distribution of a-glycerophosphate dehydrogenase insquamous-cell cancer was similar to the homo!•ogousnon-neoplastic epithelium.

Breast tumors.—Seven cases of adenocarcinomaand @3cases of the so-called mastopathy werestudied (Table 1). a-Glycerophosphate dehydro

@enase was significantly reduced in the neoplasticepithelium of both adenocarcinoma and adenomaof the breast (Fig. 1). It was moderate in amountin eleven out of @8cases of adenosis and fibroadenosis. The stroma of breast tumors generallyshowed a low enzymatic activity in fibrous components in periductal and periacinous regions(Fig. @).

Thyroid tunwrs.—Two cases of thyroid cancerand @1cases of struma nodosa were studied (Table1). Thyroid cancer was markedly reactive fora-glycerophosphate dehydrogenase in the malignant epithelium, 48 per cent of @1cases of strumanodosa showed a low enzymatic activity, andmany cases of goiter showed a slight to moderateactivity in their epithelia.

Tumors of the nervous system.—Seven cases ofmeningioma, four cases of glioma, one case eachof glioblastoma, neurinoma of the skin, astrocytoma, and ganglioma, and two cases of pinealomawere examined. In cases of meningioma, a-glycerophosphate dehydrogenase was slight in three cases,moderate in three cases, and strong in one case.Meningiofibroma showed a somewhat lower activity than multiform type. Of the four cases ofglioma studied the enzymatic intensity was lowand moderate in two cases. The enzymatic activityof astrocytoma was the highest; two cases of pinealoma and a case of ganglioma had moderate, acase of neuninoma had low, and a case of glioblastoma had a trace of activity (Fig. 6).

Others.—One case each of epithelial tumor, basalioma, and melanoma, and one case each of fibrosarcoma, liposarcoma, lymphosarcoma, and osteosarcoma were studied, as well as one case of adrenocortical adenoma. The grade of enzymatic intensityof basalioma was moderate and that of melanomalow. The enzymatic intensity in mesenchymal malignant tumors was moderate in liposarcoma (Fig..5), and in other sarcomas it was low.

DISCUSSIONa-Glycerophosphate dehydrogenase catalyzes

the oxidation of r@-a-glycerophosphate to dihydroxyacetone phosphate, which constitutes an important intermediate for neutral fats and phospholipide synthesis (4). However, the biological significance of a-glycerophosphate dehydrogenase in

tumor tissue has not included reference to histochemical studies of the enzymatic activity.

The data reported in the present study indicatedthat the malignant proliferating epithelium usuallystained moderately for a-glycerophosphate dehydrogenase, with a suggestion that it would show anoncharactenistic distribution in tumor tissue. Insquamous-cell carcinoma the enzymatic intensityshowed little difference between high-keratinizedepidermoid cancer developed in the oral cavity andnon- or low-keratinized squamous-cell cancer incervix uteri. Besides, the distribution pattern ofthe neoplastic epithelium of squamous-cell cancerwas similar to that of the homologous non-neoplastic epithelium. It was assumed on the basis ofthe present report, as well as previous data (3),that the intensity and localization of a-glycerophosphate dehydrogenase in human squamous-cellcancer were nonspecific.

A histochemica! demonstration of a-glycerophosphate dehydrogenase activity in the gastrointestinal tract was carried out by previous investigator (@) and that in carcinoma of the largeintestine in man was reported by Wattenberg (6).Wattenberg reported that the enzymatic reactivityin normal colon mucosae exhibited a moderateintensity, that of the cells at the base of cryptsbeing the highest and that of the surface epitheliumslight. Both poorly and well differentiated carcinoma cells in the colon showed a weak activity forthis enzyme, and a considerably higher proportionof poorly differentiated neoplastic cells exhibitedan intense staining. In the present study theenzymatic activity of both well differentiatedadenocarcinoma and undifferentiated cancer in thehuman stomach was moderate in 31 cases of 67cases; ten cases of twelve rectum cancers showed amoderate to high enzymatic activity. The enzymatic intensity for all gastrointestinal cancer caseswas moderate in the malignant and polyp epithelia.However, the highest staining in the neoplasticepithelium was observed in a few cases in stomachand rectum specimens. The significance of thehighest staining in gastrointestinal adenocarcinoma has not been clarified. Furthermore, it mustbe admitted that those cases with the most intenseactivity were not always so advanced in malignancy. Possibly, those differences indicated in theenzymatic intensity may be related to quite different primary cells in the gastrointestinal tract.

Studies on normal salivary glands indicated thatmost of the oxidative and reducing enzymes werepresent in the duct system, with a high to thehighest staining reaction (s). In the present study,pleomorphic adenoma which developed from minorsalivary glands in oral mucosae showed a varying

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1688 Cancer Research

intensity for a-glycerophosphate dehydrogenase.In general, the minor salivary gland present in theconnective tissue under the oral epithelium wasthe so-called mucous gland, and a dehydrogenasewas usually localized in the basal layer in aciniwith a relatively low grade of activity. Furtherstudies for a-glycerophosphate dehydrogenase andother oxidative enzymes in mixed tumors of thesalivary glands seem to be necessary.

An enzymatic histochemical study of dehydrogenase in the normal nervous system was carriedout in detail by Thomas and Pearse (5). They reported that nerve cells exhibited slight enzymaticactivity but that the surrounding gray mattershowed a high activity, especially in glial cells. Anastrocytoma group showed a slightly higher activity for a-glycerophosphate dehydrogenase thandid a fibrous type in brain tumor. The finding thatastrocytic cells showed more activity than othercells such as fibrous elements is assumed to be related to structural elements in nerve cells. At anyrate, the significance of oxidative enzymes has notbeen fully clarified in normal nerve cells or in

FIG. 1.—Adenoma of the breast. Intense enzymatic activity

was observed in the neoplastic epithelium. Xl00.FIG. 2.—Adenofibroma of the breast. Note the slight a-glyc

erophosphate dehydrogenase activity in fibrous componentsin periductal and periacinous areas. X100.

Fio. 8.—Cystoadenocarcinoma of the ovary. The enzymatic activity was limited to the malignant neoplastic epithehum. Little difference in activity was observed between the

brain tumor cells. It should be approached by biological patterns in the normal nervous system inreference to tumors of this system.

REFERENCES1. BRAUNSTEIN,H.; Fazmwi, D. G.; THOMAS,W., Ja.; and

GALL,E. A. A Histochemical Study of the Enzymatic Activity of Lymph Nodes. III. Granulomatous and PrimaryNeoplastic Conditions of Lymphoid Tissues. Cancer, 15:189—52,1962.

2. thus, R.; Sc4umpzui, D. G.; and Pwmaz, A. G. E. TheCytochemical Localization of Oxidative Enzymes. IL Pyridine Nucleotide-linked Dehydrogenase. J. Biophys. Biochem. CytoL, 4:758-60, 1958.

S. KAwAK.ATSU,K.; Morn, M.; and NAGASUNA,H. Histochemical Study of Hydrolytic Enzymes and Dehydrogenases inInvading Carcinoma of Oral Mucosae. J. Osaka Univ.DentalSchool,3:21—80,1968.

4. Scnzux, F. In: J. B. SusuER and K. MynuXca (eds.), TheEnzymes, 2:802-8. New York: Academic Press, Inc., 1951.

5. THOMAS,E., and Pnsimsz, A. G. E. The Fine Localizationof Dehydrogenase in the Nervous System. Histochemie.,2:266—82,1961.

6. WArrENBERO,L W. A Histochemical Study of Five Oxidetive Enzymes in Carcinoma of the Large Intestine in Man.Am. J. Pathol., 35: 118—87,1959.

basal side and the luminal aspect. X100.Fio. 4.—Ovarian adenocarcinoma. Neoplastic tissue was

intensely reactive for a-glycerophosphate dehydrogenase.x100.

FIG. 5.—Liposarcoma. Sarcoma cells revealed a fine forma

zan deposition. X200.Fio. 6.—Glioblastoma multiforme. Large astrocytes showed

fine formazan pigmentation in their cell plasma. X400.

Vol. @8,November 1968

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FIG. 7.—Adenocarcinoma of the stomach. In the lowerpart tumor cells showed the highest activity, whereas in theupper part the normal glands were slightly stained. X40.

FIG. 8.—Higher magnification of Figure 7. Stromal elements reacted weakly for a-glycerophosphate dehydrogenase.X400.

FIG. 9.—Adenocarcinoma of the stomach. Neoplastic epithelium exhibited low enzymatic activity in contrast withFigure 7. X40.

FIG. 10.—Stomach polyp. Weak activity was found in the

epithelium of polyp. X200.

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FIG. 1 1 .—Undifferentiated carcinoma of the stomach. Lowenzymatic activity was found in neoplastic celLs, while largecells scattered among them revealed a comparatively strongactivity.X400.

FIG. 12.—Undifferentiated adenocarcinoma of the stomach.Malignant tumor cells showed intense activity. X400.

FIG. 13.—Adenocarcinoma of the rectum. a-Glycerophospliate dehydrogenase activity in the neopiastic epithelium wasweaker than that of the homologous normal tissue. X40.

FIG. 14.—a-Glycerophosphate dehydrogenase activity incystoadenocarcinoma of the stomach. X200.

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1963;23:1685-1688. Cancer Res   Masahiko Mori, Tetsuhiko Miyaji, Iwao Murata, et al.   Dehydrogenase Activity in Human Tumors

-GlycerophosphateαHistochemical Observations of

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