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

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