HISTOCHEMICAL STUDIES ON DPN DIAPHORASE SYSTEMIN HUMAN TUMOR CELLS
Akira MIZUTANIThe Pathological Division (Chief: Prof. H. Takamatsu) of
the Tuberculosis Research Institute, Kyoto University, Kyoto(Received for publication July 27, 1960)

INTRODUCTION

The biochemical significance of diaphorase or coenzyme factor has beenemphasized since by Adler & Euler l - 6 ) and Green & his coworkers. 7 - 12 )The histochemical studies, however, on the enzyme system have started muchlater. Farber and his coworkers ('54-'56)13,14,15) have originally reported the histochemical methods for the demonstration of DPN~ and TPN-diaphorases based upon
the principle to utilize tetrazolium salts as a hydrogen acceptor. The author has
also studied the histochemical detection of lactic dehydrogenase-DPN diaphorasesystem in the previous report and suggested its marked activity in tumor cells. 16 )The purpose of this paper is to present the interesting histochemical patternsof the enzyme system in gastric cancer and other neoplasms and to describe its
biochemical significance for the metabolic pathway of malignant tumors.
MATERIALS AND METHOD

Most of the tissues were obtained from the surgical operation under the
diagnosis of gastric cancer and pulmonary cancer, and also from the necropsy
cases of various malignant tumors; 43 samples of gastric cancer, 4 pulmonary
cancer, each case of liver metastasis of gastric cancer, hepatoma, breast carcinoma,embryonal carcinoma of the testicle, multiple myeloma and malignant lymphoma
were included. The additional examinations of the chronic gastritis, incident tomost of gastric cancers, and of benign gastric polyp were also carried out.
The fresh frozen sections of these tissues were made in a cryostat after rapid
chilling of the materials by sticking on the out-side wall of a glass vessel inwhich dry ice-acetone or -alcohol was filled, and those were successively fixed incold acetone about a half or an hour. The specimens 10 ~ 15 micron in thickness
were incubated in the freshly prepared reaction mixture at 37°C. Since the reaction mixture required expensive chemicals, a technique employing small amountsof the mixture was utilized. The incubation was carried out in small closed glass
vessels in which slide glasses sticking the specimens were horizontally supported.

Akira MIZUTANI

2

Two or three tenth of one m!. of the reaction mixture preheated to 37°C wasplaced on the sections on each slide glass. A few amounts of water added in
the vessels in order to prevent the drying of the mixture during the incubation.
The incubating mixture contained the following reagents,
0.1 % Neotetrazolium chloride (or Nitro BT)
2 parts
0.1 M Sod. lactate (adjusted to pH 8.5 by NaOH)
2 parts
0.4% DPN..
2 parts
0.1 M KeN
1 part0.1 M AMPD-HCI buffering solution (pH 8.5)
2 parts
Other mixture in which DPNH was substituted for lactate and DPN was
also used.
The incubation period was about 10 to 15 minutes at 37°C, and was shortenedwhen the reaction was strong. The initial color of. the reactive tissues was pink,
and by increasing the incubating period, progressively violet precipitate wasyielded. The additional incubation resulted in the formation of blue color, but
the excessive precipitation of diformazan disturbed the cytological examinations.

RESULTS
1. Chronic gastritis.
At the most cases of gastric cancer, the neighboring non-cancerous mucosa
shows the histological pattern of atrophic or hypertrophic gastritis. With thelactic dehydrogenase-DPN diaphorase procedure, atrophic mucosa exhibited thedecreased activity staining in pale red or pink color; the surface epithelium andthin atrophic layer of gastric glands, containing sparcely scattered remaining
chief cells, stained in light color. The remainder of parietal cells, which normallyshowed the most intense activity and were relatively resistant to atrophic anddegenerative process, still stained intensely in violet color. But the reaction
was also weaker than that of normal glands. Somewhat productive interstitialconnective tissue cells often showed a moderate activity.
On the other hand, at the cases of hypertrophic gastritis, the gland cells of
the lengthend pits, which often showed the intestinal metaplasia containg goblet
and Paneth cells, revealed the moderate reaction except for mucoidal substances.
Sometimes this histochemical pattern resembled that of the relatively differentiatedtype of cancer.
Lymphfolicles which often formed in the deep portion of mucosa associatedwith chronic inflammatory process, showed light or no staining.
2.

Benign adenomatous polyp.

The columnar tumor cells of well differentiated adenomatous polyp stained
only in light red or pink color. However, it was merely one case and so willneed further examinations.

Histochemical Studies on DPN-Diaphorase System in Human Tumor Cells

3.

3

Gastric cancer

The strict histological classification of gastric cancer is not easy and, as amatter of fact, most of gastric cancers are adenocarcinomas. The author has
conveniently devided it in 4 types upon the basis of histological and histochemicalpatterns, although with some difficulties such as containing histological findings
to belong partly to type 1 and partly to type 2 or 3 in the same case. This
classification may be suitable not to the summarization of each case but rather
to the description of respective pattern of each specimen.
The classification of the present cases are as follows;
Type 1
Type 2Type 3
Type 4

relatively differentiated typerelatively undifferentiated typedegenerating typeundifferentiated type

8 cases
16
"
10
"
8
"

One squamous cell carcinoma of esophagus origin was also examined.
a)Relatively well differentiated adenocarcinoma (Type 1).
Well differentiated columnar or cuboid epithelial cells with round or elongatednuclei in basal portion proliferated at the mucosa with regular or irregular glandformation and infiltrated into submucosal layer beyond muscularis mucosae.
Two malignant polyps were also included in this type.
With the histochemical procedure for the eyzyme system, the cancer cells
moderately stained in violet color with crude formazan sedimentation but the
precipitation was not so fine as of normal parietal cells.
The invading margins of the tumor clusters more intensely stained and thesuperficial and central areas were rather lighter although degenerative tendency
was not prominent in this type. These intracellular stainings did not work on
the cytoplasmic vacuoles and nuclei.
b)Relatively undifferatiated adenocarcinoma and medullar type (Type 2).
The relatively undifferatiated type showed various histological features;
secreting or nonsecreting flat epithelial cells proliferated with irregular glandformation such as cords, highly atypical glands, and almost medullar solid clumps,
some of which were accompanied with stromal hyperplasia but not so marked
as "Scirrhus".
With the enzyme procedure, the undifferentiated tumor cells stained in violet
color with somewhat crude or fine formazan sedimentation and this staining
reaction was most intense at the invading margins or infiltrating solid cells of
submucosa or muscle layer. The formazan precipitated on the small cell aggregates was mostly fine and dense. On the other hand, at the central portion of
medullar proliferation, the reactions were relatively weak. The cytoplasmic
vacuoles containing mucus and nuclei exactly did not stain. The atypical glands
sometimes showed the histochemical patterns of intense periluminal staining incontrast with weak reaction of basal portion.

Akira MIZUrANI

4

The poorly differentiated cancer often illustrated the variations, for example,
showing side by side partly differentiated glands and smaller rounded or polygonalcells without any gland formation. These anaplastic cells showed most intense
reactions or almost negative. The latter was degenerative or necrotic tumor cells.
As the exceptional findings, 3 cases did not intensely stained.
c)

Degenerative type (Type 3).
The rapidly growing tumor cells often tend to degeneration and necrosis,
especially in fungating type. A typical type of degeneration is so-called colloid,mucoid or gelatinous carcinoma. The intense formation of mucus results in the
floating of degenerative cancer cells in it.
In this type, the lactic dehydrogenase-DPN diaphorase procedure revealed
various activities. The proliferating solid masses and nondegenerative cells
stained in intense color, and otherwise, the floating degenerative cells were weakor no color with some exceptive cells to show marked activity. Generally, any
cancer cell in the necrotic regio n did not stain. The so-called "signet ring cells"
showed merely weak or no staining reaction.
d)Undifferentiated type (Type 4).
In eight cases, the tumors were composed of completely undifferentiatedcells almost without any gland formation. The anaplastic cancer cells withmarked anisocytosis and anisokaryosis proliferated and infiltrated in cluster eveninto the muscle bundles. The isolated anaplastic cells were also scattered in theloose or dense connective tissues.
Most of this type were included in so-called Carcinoma simplex. In somecases, however, atypical small glands were observed and there were signet ringcells in the scattering cell groups.
For the histochemical procedure, the anaplastic crude cells stained mostintensely with dense precipitation of fine diformazan. The small anaplastic cancer
cells were easily distinguished from noncancerous migrating or histiocytic cellsby the marked enzyme reaction. However, signet ring cells, as already described,
yielded light or no staining.
Thus, the histochemical characteristics of lactic dehydrogenase-DPN diaphorase at gastric cancers were summarized as follows; the most cancer cells
had a strong activity, especially the poorly differentiated and peripherally invading
cells were markedly positive, and otherwise, the degenerative and necrotic cells
such as signet ring form weak or negative.
The substitution of DPNH for lactate and DPN in the reaction medium gave
the identical results.
4.

Other tumors.

a)Esophagus cancer.
This was a case of squamous cell carcinoma which originated from esophagu'>
and continuously invaded into the cardiac portion.

Histochemical Studies on DPN-Diaphorase System in Human Tumor Cells

5

With the enzyme reaction, the normal squamous epithelium stained intensely
at the basal layer and weakened in parallel with the cell differentiation to thesuperficial layer.
The cancer cells which invaded into the cardiac submucosa stained in intense
violet color. Although it was relatively differentiated type with keratinizationand pearl formation, each layer from basal to central part uniformly stained withabrupt cease of the reaction at the keratin layer.
b)Pulmonary cancer.
Four pulmonary cancers secured at surgical operation were examined.
The first was bronchogenic papillary adenocarcinoma. The tortuous epithelium yielded the purple formazan sedimentation. The staining was completely
absent at the necrotic regions.
The second was also adenocarcinoma partially of medullar type. The partiallylimited histological pattern resembled that. of metastatic carcinoid. The tumor
cell clusters also moderately stained with the enzyme procedure.
The third was typical squamous cell carcinoma with pearl formation. The
histochemical pattern for the enzyme system was identical with that of esophagus
cancer already described.
In additional one case (bronchogenic carcinoma), the tumor cells lightly
stained with merely loose deposition of crude diformazan and homogeneous light
red coloring.
c)Hepatoma.
Two hepatomas from autopsy were examined.
The aggregations of polygonal carcinoma cells resembling hepatic cells withsinusoidal stroma showed the marked activity. One case revealed more intense
reaction than normal hepatic cells and the other weaker.
d)Others.
The autopsied specimens of breast cancer (mammary duct carcinoma), testicular cancer (embryonal carcinoma), multiple myeloma and malignant lymphoma
were also examined. The both carcinoma cells gave the similar results to gastriccancer for the histochemical staining.
The enzyme reactions at the latter two nonepithelial tumors also yielded thediformazan formation but the intensity in each tumor cells was not uniform.
Further examinations about sarcoma cells will be reported successively.
DISCUSSION

The utilization of tetrazolium salts or tellurite for hydrogen acceptor has
made it possible to study a number of important oxidative enzymes in histologic
setions. As a principle, the formation of water insoluble and intensely coloredformazans by reduction of tetrazoles are also available for the histochemical
demonstration of lactic dehydrogenase--DPN diaphorase system. Hydrogen trans-

Akira MIZUTANI

6

port, as well known, at the lactate dehydration in vitro is shown as follows.
lactate ~ (pyruvate)
H

-j-

\

lactic dehydrogenase~

Co I (DPN) ~ (DPNH)

1 DPN diaphorasecytochrome system
1 cytochrome oxidase
O2
This lactate-pyruvate reaction is reversible according to their quantitative
ratio.
At the histochemical procedure, however, the actual reduction of tetrazolium
salts in incubation mixture was not brought about by their interaction with thedehydrogenase itself, but at the second process of reduced DPN oxidation by
DPN-diaphorase and successively at cytochrome oxidation. The cytochromeoxidation may be obstructed by the addition of cyanide in the reaction mixture.
Farber et a1. 13 - 15 ) have reported about the specificity of the staining reactionsin their successful histochemical studies on DPN- and TPN-diaphorases. They
described that the stains which had been developed, even though the reactions
might be produced by the activity of single dehydrogenase systems in the tissues,
were, in strict sense, specific only for DPN diaphorase and TPN diaphorase
activity. At the reaction mixture of the present study, which contains lactate
as substrate, it is true that the formation of formazan is strictly brought about
by diaphorase activity, but the reaction may not occur without the precedingoxidation of substrate by dehydrogenase, in other words, reduction of DPN.
Thus it is preferable to name "lactic dehydrogenase-DPN diaphorase system"
for the present studies, and it may be rather reasonable to use reduced DPN(DPNH) itself for the "DPN diaphorase" procedure as the present additional
studies and the researches by W attenbergl7) without any other substrates suchas lactate and malate.
Then, as to cyanide, the classical biochemists had considered its necessity asa keton-fixative to maintain the lactic dehydrygenase activity. But Huennekens
and Green (1950)18) have reported by their investigations of cyclophorase preparation that the tests for the dissociated apolactic oxidase were carried out at
pH 8.5-9.5 with cyanide and, otherwise, for the conjugated oxidase no fixative
had to be employed. The previous studies by the present author 16 ) have also
discussed the strict necessity of cyanide for the histochemical examinations.
However, the addition of cyanide, as already described, may be reasonable torestrict the reactions to the dehydrogenase and diaphorase activities inhibiting
cytochrome oxidase action.
And then, as to pH of the reaction mixture, Farber et al. 13 - 15 ) have usedHistochemical Studies on DPN-Diaphorase System in Human Tumor Cells

7phosphate buffer adjusted to pH 7.4, and Wattenbergl7), 8.0. The reactions became
more intense by elevation of pH, and phosphates are almost powerless as abuffering solution at the higher pH than 8.0. In the previous studies, the author
has used 2-amino-2-methyl-1, 3-propandiol (AMPD)-HCI solution buffered toabout pH 9.0. But, as Scarpelli and Pearse have reported 19 ), alkaline media (pH 10)
resulted in mitochondrial swelling and enzyme diffuse. The author has used
AMPD-HCI buffer solution adjusted to pH 8.3-8.5 in the present studies.
The histochemical findings of lactic dehydrogenase~DPN diaphorase and
DPN diaphorase itself in gastric cancer and other tumors are summarized asfollows; most cancer cells have intense activities, especially undifferentiatedanaplastic and invading cells are found to be markedly positive, whereas thedegenerative or necrotic cells such as signet ring cells are weak or negative.
This is an interesting characteristics. Wattenberg17 ) has reported the similar
results in colon carcinoma.
The intense staining at malignant lymphoma cells, in spite of the weakreaction at normal lymphoid tissues, suggests the independence of the activities
upon the original mother cells. Further studies about these problems will be
carried out.
Generally, in malignant neoplasms, various enzyme activities are found to
be diminished with some exceptions (Greenstein20 ), Novikoff21 ), Wattenberg17 ,22),
Rutenburg23 )). On the other hand, cancer cells have been repeatedly shown topossess a high glycolytic activity 24,25), in spite of the low activities of cytochromes25 ,26\ This suggests the active reoxidation of DPNH during the formationof lactate from pyruvate in anerobic metabolism. In fact, the accumulation oflactic acid in tumor cells has been reported by Greenstein20 ). Moreover, theintense activity of DPN diaphorase may correspond to the anerobic metabolism
for high energy source. The vigorous growth of malignant neoplasms theoretically demands immense energies. In the anerobic pathway, dehydration of intermediate 1, 3-diphosphoglyceraldehyde introduced from 3-phosphoglyceraldehyde
yields 1,3-diphosphoglyceric acid under the influence of triosephosphate dehydrogenase, and it results in DPN reduction.
The results of the present studies may be suggestive of the role of probableanerobic efficient metabolism including active DPN reduction and oxidation fora immense energy source of malignant tumors.
SUMMARY

The histochemical procedures of lactic dehydrogenase~DPN diaphorase andof DPN diaphorase itself were employed in the tissues of gastric cancer andrelated gastric changes. Other various neoplasms such as pulmonary cancer,
hepatoma etc. were also examined.
As a characteristic reaction, most of the gastric cancer cells showed distinctive

8

Akira MIZUTANI

activity by formazan formation. This staining pattern was not uniformly present
in all components of the neoplasms. The enzyme activities were most intensein the cells at the invading clusters and isolated cell groups. Generally, theundifferentiated anaplastic cells stained in deep violet color. Whereas, thedegenerative cells always decreased or diminished the staining reaction.
The distinctive reaction pattern in other various tumors was also suggested
to be identical with gastric cancer.
The significance of these findings for metabolic pathway of tumor cells was
discussed.
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E. Adler, H. v. Euler & H. Hellstrom: Arkif for Kemi, 12B; Nr 38, 1937.
E. Adler, H. v. Euler & GUnther: Ibid. 12B; Nr 54, 1938.
E. Adler, H. v. Euler & W. HUghes: Z. physioi. Chern., 252; 1-23, 1938.
H. v. Euler & H. Hellstrom: Ibid., 252; 31-40, 1938.
E. Adler, H. Hellstrom, G. GUnther & H. v. Euler: Ibid. 255; 14-26, 1938.
H. v. Euler & K. Hasse: Naturwiss., 26; 187, 1938.
D. E. Green & ]. G. Dewan: Biochem. ]., 31; 1069-1073, 1937.
]. G. Dewan & D. E. Green: Nature, 140; 1097-1098, 1937.
D. E. Green, D. M. Needham & ]. G. Dewan: Biochem. J" 31; 2327-2352, 1937.
H. S. Corran & D. E. Green: Nature, 142; 149-150, 1938.
]. G. Dewan & D. E. Green: Biochem. ]., 32; 626-639, 1938.
H. S. Corran & D. E. Green: Ibid., 32; 2231-2240, 1938.
E. Farber, W. H. Sternberg & C. E. Dunlap: Proc. Soc. Exp. BioI. & Med., 86; 534-537, 1954.
E. Farber, W. H. Sternberg & C. E. Dunlap: Am.]. Path., 30; 616-619, 1954.
E. Farber et al: ]. Histochem. & Cytochem., 4; 254-265, 266-283, 283-294, 347-356, &357-362, 1956.
A. Mizutani: Rep. Tuberc. Res. lnst., Kyoto Univ. (in ]apanease) 8-2; 9-50, 1959.
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A. B. Novikoff: Cancer Res., 17; 1010-1027, 1957.
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A. M. Rutenburg, R. Gofstein and A. M. Seligman: Ibid. 10; 113-121, 1950.
N. Warburg: Science, 123; 309-314, 1956.
]. P. Greenstein: Cancer Res., 16; 641-653, 1956.
W. C. Schneider and V. T. Potter: Ibid., 3; 353-357, 1943.

Histochemical Studies on DPN-Diaphorase System in Human Tumor Cells

Explanation of the plates
Fig.

1.

Atrophic gastritis. Parietal cells remaining in the atrophic thinmucosa still intesely stained.
X 100

Fig.

2.

Metaplasic gastritis. The metaplasic intestinal mucosa of thepylorus portion moderately stained. Intracellular mucus did
not stained.
X 100

Fig.

3.

Gastric polyp.
stained.

Fig.

4.

Gastric cancer. Relatively differentiated cancer cells with glandformation intensely stained.
X 100

Fig.

5.

Gastric cancer.
uniform.

Fig.

6.

Gastric cancer. The invading adencarcinoma cells proved theintense staining reaction. The dilated glands of right lower
portion showed the marked periluminal localization of theactivity.
X 100

Fig.

7.

Gastric cancer.

Fig.

8.

Gastric cancer. The relative undifferentiated cells with irregulargland formation intensely stained.
X 100

Fig.

9.

Gastric cancer. Degenerating adenocarcinoma.
reaction proved to be intense or negative.

The well differentiated colummar cells lightly
X 100

The reaction in the irregular glands are not
X 100

Relatively undifferentiated type.

X 100

The staining
X 100

Fig. 10.

Gastric cancer. Gelatinous carcinoma. The floating cancer cells
showed the various enzymatic activities.
X 100

Fig. 11.

Gastric cancer. The anaplastic cell clusters invading throughmuscle bundles stained most intensely. The muscle bundles
also somewhat stained.
X 100

Fig. 12.

Squamous cell carcinoma which originated from esophagus
mucosa and invaded into cardiac submucosa. The cancer cells
markedly stained but the keratinized central portions did notx100
stained.

Fig. 13.

Hepatoma.

Fig. 14.

Pulmonary cancer. Papillary adencarcinoma.
carcinoma tissues stained intensely.

Fig. 15.

Pulmonary cancer.
noticed.

The tumor cells showed marked activity.

x100

The tortuousx100

The marked peripheral staining is to be
X 100

9

10

Fig. 2

Fig. 1

Fig. 3Fig. 4

Fig. 6

Fig. 5

11

Fig. 7

Fig. 8Fig. 9Fig. 10

Fig. 11

Fig. 1212Fig. 13

Fig. 15