Ribonuclease and Deoxyribonuclease Activities in Experimental and Human Tumors by the Histochemical Substrate Film Method*

R. DAOUSTJANDHARUKOAMANOÕ

(Laboratoires de Recherche, Institut du Cancer de Montréal,Hôpital Notre-Dame et Universitéde Montréal,Montréal,Canada)

SUMMARY The ribonuclease and deoxyribonuclease activities of 65 experimental and human tu mors (32 different types) have been examined by histochemical substrate film methods. A same general pattern was obtained for the distribution of both nucleases in the various types of experimental and human tumors. The connective tissue stroma and the necrotic regions of the tumor masses showed various levels of nuclease activity, whereas the neoplastic cells showed no demonstrable activity. It appears that deficien cies in ribonuclease and deoxyribonuclease activities represent general properties of cancer cells. The possible significance of the losses of nuclease activities in carcinogenesis is dis cussed.

Studies on nucleases by histochemical methods MATERIALS AND METHODS have shown that losses of ribonuclease (RNase) The experimental tumors used in the present and deoxyribonuclease (DNase) activities take study were mostly rat, mouse, and hamster trans- place in rat liver during azo-dye carcinogenesis (1, plantable tumors (see Table 1). The tumor-bearing 6). The loss of RNase activity is progressive and animals were obtained from commercial or private occurs before parenchymal cells become cancerous, sources, and the tumors were used as supplied or whereas the loss of DNase activity is abrupt and closely associated with the neoplastic transforma TABLE1 tion of parenchymal cells. EXPERIMENTALTUMORS If a loss of RNase or DNase activity plays an important role in tumor formation, the lack of SpeciesRat"""MouseHamsterTumorPrimary demonstrable nuclease activity observed in rat hepatomaNovikoff primary hepatomas should also be observed in a hepatomaWalker variety of tumors. Thus, to assess the significance 256Murphy-Sturmsarcoma of these changes with respect to neoplasia, histo lymphosarcomaEhrlich chemical analyses of nucleases in different experi form)Sarcomatumor (solid mental and human tumors have been undertaken. 37Lymphocytic form)MelanomaSarcomaleukemia (solid * This investigation was supported by a grant from the National Cancer Institute of Canada to Dr. A. Cantero, Direc S32Mammary tor of the Research Laboratories. Reports of this work were B16Papillomacarcinoma presented at the Annual Meetings of the American Association (primary)Renal for Cancer Research held in Atlantic City in April, 1961, and carcinomaNumber332333111133 April, 1962. t Research Scientist of the National Cancer Institute of Canada. following transplantation in animals of his labora ÃŽPresent Address: Department of Anatomy, School of tory. The definition, history, and description of Medicine, Okayama University, Okayama, Japan. these tumors, as well as the methods of transplan Received for publication July 14, 1962. tation, are given by Stewart et al. in their atlas of 131

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1963 American Association for Cancer Research. 132 Cancer Research Vol. 23, January 1963 animal tumors (8). Analyses were also carried out strate films (white pattern) correspond to the sites on rat primary hepatomas produced by azo-dye of nuclease activity in the tissue sections. The feeding (1,6) andmouse skin papillomas induced by letter C indicates the reaction given by control rat 9,10-dimethyl-l,2-benzanthracene (2). In total, 27 liver, and T is the part of the film exposed to a experimental tumors, representing twelve different tumor section; d stands for connective tissue and n types, were studied. for necrotic region. The rat liver sections were used The human tumors (see Table 2) were fresh sur in these experiments primarily for controlling the gical specimens obtained within a few hours after film technic. However, since most normal rat tis excision. Thirty-eight human tumors, representing sues (4, 5) as well as normal human tissues (unpub twenty different types, were investigated. lished results) also present appreciable RNase and Samples of animal or human tumors were DNase activities, the rat liver may, in addition, be mounted on the stage of a microtome kept in a considered as representative of various normal tis refrigerated cabinet at —15°to—20°C.A piece of sues. Many reactions for control livers in Figures 1-20 show a poor definition, because the films were TABLE2 overexposed to the tissue sections to increase the HUMANTUMORS chances to detect enzyme activities in the neo plastic tissues. SiteSkin"BreastTongueGumsSalivary RNase activity in experimental tumors.—Results obtained with gelatin-RNA films exposed to sec carcinomaMalignant melanoma (metasta tions of experimental tumors are illustrated in Fig sis in thebreast)CarcinomaEpidermoid ures l^i. Figure 1 shows a positive reaction corre sponding to the connective tissue capsule (ci) of a carcinomaEpidermoid carcinomaMixed mouse Ehrlich tumor (solid form) ; the tumor cells glandsLarynxEpiglottisStomachColonRectumLungKidneyBladderLymphtumorEpidermoid (T7)showed negligible RNase activity. Figure 2 il carcinomaEpidermoid carcinomaCarcinomaAdenocarcinomaAdenocarcinomaEpidermoidlustrates the relative inactivity of a section of mouse Sarcoma 37 (T) as compared with the con trol tissue (C). In Figure 3 is shown a weak reac tion given by a section of rat Murphy-Sturm lym- carcinomaClear-cell carcinomaTransitional phosarcoma (T) and attributed to the connective carcinomaReticulum-cell-cell tissue of the tumor. In Figure 4 is presented a reac node'* sarcomaReticulum-cell **Ovary*•Uterus sarcoma (metas tion obtained with a section of rat Walker sarcoma skin)Seroustasis in the 256 (T) ; the tumor cells show negligible RNase ac cystadenocarcinomaTeratomaAdenocarcinoma tivity except in small necrotic regions (bottom). (Endo-metrium)Eye inthe (metastasis DNase activity in experimental tumors.—Reac omentum)MeningiomaNumber11611221444131111111tions obtained with gelatin-DNA films exposed to (Opticalnerve)TumorBasal-cell sections of experimental tumors are illustrated in Figures 5-8. Positive reactions in Figure 5 corre spond to the control liver tissue (C) and the con normal rat liver, used as control, was placed next nective tissue (ct) of a mouse Ehrlich tumor; the to the tumor samples in each series of experiments. neoplastic cells (T) show no appreciable DNase Sections of frozen tissues were cut at 15 n, and the activity. In mouse Sarcoma 37 (Fig. 6), reactions distributions of RNase and DNase activities in the corresponding to connective tissue bands (upper sections were examined by the substrate film half) and small necrotic regions (bottom left) are methods previously described (4, 5). observed; no appreciable DNase activity is shown by the cancer cells (T). In rat Murphy-Sturm lym- RESULTS phosarcoma (Fig. 7), DNase activity is found in A same general pattern was obtained for the the connective tissue capsule (ct) and necrotic re distribution of RNase and DNase activities in the gion (n); the neoplastic cells (T) are relatively in various types of experimental and human tumors. active. Figure 8 illustrates the inactivity of a sec The connective tissue stroma and the necrotic re tion of rat Walker sarcoma 256 (T) as compared gions of the tumor masses showed various levels of with the control tissue (C). nuclease activity, whereas the neoplastic cells RNase activity in human tumors.—Figures 9-14 were in all cases relatively inactive. are examples of the results obtained with gelatin- Examples of the reactions obtained with various RNA films exposed to sections of human tumors. types of tumors are presented in Figures 1-20. In Figure 9 shows a positive reaction given by control these figures, the unstained regions of the sub liver (C); no appreciable RNase activity is found

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1963 American Association for Cancer Research. DAOUSTANDAMANO—EnzymeActivitiesin Human Tumors 133 in the section of breast carcinoma (T) except in salivary glands and an epidermoid carcinoma of bands of connective tissue and small necrotic re the epiglottis—no definite conclusion could be gions. Positive reactions in Figure 10 correspond drawn because of the complexity of their structure. to the connective tissue (ci) of a reticulum-cell sar coma; no appreciable RNase activity is observed DISCUSSION in the neoplastic cells (T). Similarly, sections of The observation of a lack of demonstrable nu- gastric carcinoma (Fig. 11) show some RNase ac clease activity in rat primary hepatoma (1,6) was tivity in the connective tissue (cf) but none in the extended to a large number of malignant tumors in neoplastic cells (T). Figure 12 illustrates the re the present work. Deficiencies in RNase and sults obtained with a section of gastric adenocar- DNase activities thus appear as general properties cinoma: a nodule of neoplastic cells (T) with low of cancer cells. RNase activity surrounded by highly active con Control studies on the substrate film method nective tissue; the reactions observed within the have been reported (4, 5), and the advantages as nodule correspond to strands of connective tissue well as the limitations of the method have been and necrotic regions. In Figure 13 is shown a film previously discussed (3). It may be pointed out exposed to a section of colon adenocarcinoma. again, however, that the film method, like many RNase activity is found in the bands of connective other methods used in histochemical and biochem tissue (ci) and necrotic regions (n) of the tumor ical studies on enzymes, demonstrates exclusively mass; the neoplastic cells (T) are relatively inac the active form of the enzymes. The fact, there tive. The reactions observed in Figure 14 are given fore, that cancer cells show negligible nuclease ac by the connective tissue (cf) of a malignant mela tivity by the film method does not exclude the noma; negligible RNase activity is found in the possibility that RNase and DNase are present in tumor cells (T). an inactive form in the cells. The observation that DN ase activity in human tumors.—Figures15-20 necrotic regions of tumors give intense reactions are examples of the results obtained with gelatin- could actually be interpreted as indicating the DNA films exposed to sections of human tumors. presence of inhibited nucleases in cancer cells. The In Figure 15 is shown a reaction given by control activity shown by the necrotic tissue could result liver tissue ((7); no appreciable DNase activity from the activation of nucleases in cancer cells or was found in the section of breast carcinoma. Posi originate from the lymphatic cells present in such tive reactions observed in Figure 16 are given by areas. At any rate, the object of the present work the connective tissue (ct) of a reticulum-cell sar by the film method was to study the distribution coma; the neoplastic cells (T) show no DNase ac of the active nucleases; and, in this regard, the tivity. Figure 17 illustrates the relative inactivity conclusion can be drawn that cancer cells, as com of a section of gastric adenocarcinoma (T). In pared with normal tissues, are generally deficient adenocarcinoma of the colon (Fig. 18), DNase ac in RNase and DNase activity. tivity is shown by the connective tissue bands (ct) Biochemical assays have commonly demon and necrotic regions (n); the neoplastic cells (T) strated nuclease activities in homogenates of tu show no demonstrable activity. Sections of malig mor masses. The present histochemical observa nant melanoma (T) are relatively inactive (Fig. tions do not contradict these results but specify 19), but necrotic regions (n) of the same tumor that the RNase and DNase activities are localized (Fig. 20) show high DNase activity. in the connective tissue stroma and the necrotic All malignant tumors listed in Tables 1 and 2 regions of the tumor masses and not in the neo gave results similar to those presented in Figures plastic cells themselves. Biochemical and histo 1-20—i.e., positive reactions corresponding to the chemical results have been compared in studies on connective tissue and necrotic regions of the tu nuclease activities of rat primary hepatomas (1,6, mors and negligible activity in the neoplastic cells. 7) and discussed in more general terms in other ar The distribution of the nucleases in benign ticles (see references in [1]). The conclusion that mouse skin papillomas was similar to that ob the biochemical data on hepatomas represent served in normal skin. The results obtained with mean activities of cell types and extracellular fluids these tumors will be described separately and com with very different enzymatic properties seems, pared with results on various types of normal from the present work, to apply to tumors in stratified squamous epithelia. Nuclease activity general. was also observed in polyps of the colon and the The lack of demonstrable nuclease activity ob rectum, the mucous cells showing both RNase and served in neoplastic cells of various origins sup DNase activities as they do in normal intestine. ports the view that the losses of RNase and DNase With some tumors—i.e., the mixed tumors of the activities observed in rat liver parenchyma during

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1963 American Association for Cancer Research. 134 Cancer Research Vol. 23, January 1963

azo-dye feeding (1, 6) may represent important debted to Dr. Gilles Tremblay for his help in the classification steps of the carcinogenic process. The progressive of human tumors. We wish to thank also Miss Pierrette Laver loss of RNase activity occurring before parenchy- dure for preparing histological sections of the tumors and Mr. Jean Lafond for the photographs. mal cells become cancerous may be an essential feature of preneoplasia, whereas the sudden loss of REFERENCES DNase activity associated with the neoplastic 1. AMANO,H., and DAOCST,R. The Distribution of Ribo- nuclease Activity in Rat Liver during Azo-dye Carcinogene- transformation may represent a change more im sis. J. Histochem. Cytochem., 9:161-64, 1961. mediately related to tumor formation. How these 2. BOHUM,K.The Role of the Mouse Hair Cycle in Epidermal changes could alter the nucleic acid metabolism Carcinogenesis. Acta Pathol. Microbiol. Scandinav., 34: and result in tumor formation is difficult to an 542-53, 1954. swer, since the intracellular function(s) of the nu- 3. DAODST,R. Localization of Deoxyribonuclease Activity by the Substrate Film Method. In: J. F. DANJELU(ed.), Gen cleases is still poorly understood. A plausible hy eral Cytochemical Methods, 2:153-70. New York: Academ pothesis, however, is the following: Intracellular ic Press, Inc., 1961. nucleases might constitute a defense mechanism 4. DAOTJST,R., and AMANO,H. The Localization of Ribo- against foreign or abnormal nucleic acids; in the nuclease Activity in Tissue Sections. J. Histochem. Cyto event of a loss of nuclease activity, foreign nucleic chem., 8:131-34, 1960. acids (viral or other) could become part of the 5. . Improved DNA Film Method for Localizing DNAase Activity in Tissue Sections. Exp. Cell Res., 24: genetic material, alter the properties of the cells, 559-64, 1961. and possibly induce metaplastic and neoplastic 6. DAOUST,R., and CANTERO,A.The Distribution of Deoxy transformations. This briefly outlined working ribonuclease in Normal, Cirrhotic and Neoplastic Rat hypothesis is the object of further investigation. Livers. J. Histochem. Cytochem., 7:139-i3, 1959. 7. LAMIEANDE,G.DE;DAOUST,R.; and CANTERO,A.Enzyme ACKNOWLEDGMENTS Studies of Tumors—Evaluation and Perspectives. Canadian We wish to thank Dr. Alex B. Novikoff from the Albert Cancer Conference, 4:43-56. New York: Academic Press, Einstein College of Medicine, New York, and Dr. Peter Inc., 1961. Scholefield from the McGill-Montreal General Hospital Re 8. STEWART,H. L.; SNELL, K. C.; DUNHAM,L. J.; and search Institute for supplying animals with transplanted tu SCHLTEN,S. M. Atlas of Tumor Pathology, Section XII— mors. We are grateful to the members of the Departments of Fascicle 40, Transplantable and Transmissible Tumors of Surgery and Pathology of this Hospital for their collaboration Animals. Armed Forces Institute of Pathology, Washing in obtaining samples of human tumors. We are especially in ton, 1959.

Fios. 1-4.—Gelatin-RNA films exposed to sections of ex FIGS. 5-8.—Gelatine-DNA films exposed to sections of ex perimental tumors and stained with toluidine blue, X30. The perimental tumors and stained with toluidine blue, X30. unstained regions of the films (white pattern) correspond to FIG. 5.—Mouse, Ehrlich tumor (10 min.). DNase activity the sites of RNase activity in the tumor masses. shown by control liver tissue (C) and connective tissue of tu FIG. 1.—Mouse, Ehrlich tumor (20 min.). Positive reac mor (ct); inactivity of tumor cells (T). tions given by control liver tissue (C) and connective tissue Fio. 6.—Mouse, Sarcoma 37 (5 min.). Positive reaction capsule (rf) of tumor (T); negligible RNase activity in neo given by control (C); no DNase activity observed in tumor (T) plastic cells. FIG. 2.—Mouse, Sarcoma 37 (5 min.). Positive reaction except in strands of connective tissue (adjacent to control) and given by control (C) ; relative inactivity of RNase in tumor (7"). small necrotic regions (bottom left). FIG. 3.—Rat, Murphy-Sturm lymphosarcoma (5 min.). FIG. 7.—Rat, Murphy-Sturm lymphosarcoma (5 min.). RNase activity shown by control tissue (C); weak activity cor Positive reactions given by connective tissue capsule (ci) and responding to connective tissue in tumor (T). necrotic region (n) of tumor section but negligible DNase ac FIG. 4.—Rat, Walker Sarcoma 256 (40 min.). Intense reac tivity in tumor cells (T). tion given by control section (C); negligible RNase activity in FIG. 8.—Rat, Walker Sarcoma 256 (20 min.). High DNase tumor (T), but positive reactions given by small necrotic re activity in control tissue (C); no reaction given by tumor (T). gions (bottom).

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1963 American Association for Cancer Research. Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1963 American Association for Cancer Research. FIGS. 9-14.—Gelatin-RNA films exposed to sections of hu man tumors and stained with toluidine blue, X30. FIG. 9.—Breast carcinoma (10 min.). Intense reaction given by control liver tissue (C) ; no RNase activity observed in tumor (T) except in bands of connective tissue and small necrotic regions. FIG. 10.—Lymph node, reticulum-cell sarcoma (10 min.). Positive reactions given by control (C) and connective tissue of tumor (ct); the neoplastic cells (T) do not show any appre ciable RXase activity. FIG. 11.—Stomach carcinoma (10 min.). RNase activity shown by control section (C) and connective tissue of tumor (ct); no reaction given by tumor cells (T). FIG. la.—Stomach adenocarcinoma (aO min.). Nodule of neoplastic tissue (7") with low RNase activity surrounded by highly active connective tissue (ct). The reactions observed within the nodule correspond to strands of connective tissue and necrotic regions. FIG. 13.—Colonadenocarcinoma (10 min.). Positive reac tions given by the connective tissue bands (ct) and the necrotic regions (n) of the tumor mass; the neoplastic cells do not show RNase activity (T). FIG. 14.—Skin, malignant melanoma, metastasis in the breast (20 min.). RNase activity observed in connective tissue (ct), no appreciable activity shown by tumor cells (T).

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1963 American Association for Cancer Research. Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1963 American Association for Cancer Research. FIGS. 15-¿0.—Gelatin-DNAfilms exposed to sections of liiiinan tumors ami stained with toluidine blue, X30. FIG. 15.—Breast carcinoma (5 rain.). Positive reaction given by control tissue (Ci, no appreciable DNase activity ob served in tumor (T). FIG. 16.—Lymph node, reticulum-cell sarcoma (10 min.). DNase activity in control (Ci and connective tissue of tumor (Hi; the tumor cells are inactive (T). FIG. 17.—Stomach carcinoma (¿Omin.). Intense reaction given by control tissue (Ci, relative inactivity of tumor (T). FIG. 18.—Colonadenocarcinoma (10 min.). Positive reac tions given by connective tissue bands (<•<)andnecrotic region (n) of tumor section; negligible DXase activity of tumor cells (T). FIG. 19.—Skin, malignant melanoma, metastasis in the breast (5 min.). DNase activity shown by control tissue (C), negative reaction given by tumor section (T). FIG. ¿0.—Anotherpartof the same tumor: high DNase ac tivity observed in necrotic region (n) of tumor (T).

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1963 American Association for Cancer Research. Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1963 American Association for Cancer Research. Ribonuclease and Deoxyribonuclease Activities in Experimental and Human Tumors by the Histochemical Substrate Film Method

R. Daoust and Haruko Amano

Cancer Res 1963;23:131-134.

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