SHORT NOTE Cathepsin D Stimulates DNA Synthesis and Mitosis in Mouse Liver in vivo MIZUE
and HIROSHI TERAYAMA
Zoological Institute, Faculty of Science, University of Tokyo, Hongo, Tokyo 113, Japan Effects of a single intraperitoneal injection of cathepsin D (CatD) on DNA synthesis and mitosis in the mouse liver and kidney were investigated. Twenty ug of catD induced a significant stimulation of DNA synthesis in the liver, but not in the kidney, in a dosedependent fashion and with a peak activity at 38 h after the injection. CatD also stimulated liver mitosis, with a peak value at 44 h after the injection.
Although various effecters are known to stimulate or initiate hepatocellular proliferation [l], little is known about the regulatory mechanisms underlying the proliferation of liver cells in vivo. In a preceding paper  we reported that intraperitoneal injection of leupeptin and/or pepstatin in partially hepatectomized rats inhibits or retards the sequential events of liver regeneration, such as the activation of RNA synthesis, DNA synthesis and mitosis. Moreover, we found that the temporary disappearance of cell coat glycosaminoglycans in the early stages of liver regeneration  was also retarded by the administration of these protease inhibitors after partial hepatectomy . These previous findings suggest that some endogenous proteases, such as cathepsins, may be somehow involved in the process of cell proliferation. In the present study, we investigated the effect of an intraperitoneal injection of cathepsin D (catD) on the DNA syntheses and mitoses in the mouse liver and kidney. Materials and Methods Animals. Male dd-strain mice weighing 2C~25g were used throughout the present study. Labeled compounds, enzymes and chemicals. [MethyL3H]thymidine (5 Ci/mmole) was purchased from the Radiochemical Centre, Amersham, UK. Cathepsin D (catD) (bovine spleen, 12.5 unit/mg protein) and bovine serum albumin (BSA) (Fr. V) were the products of Sigma Chem. Co., USA, and papain (36 units/mg protein) was from Worthington Biochem. Corp., USA. Inactivated catD was prepared by heating catD solution (20 ug/ml saline) at 100°Cfor 30 min. In vivo incorporation of a labeled precursor into DNA of liver and kidnq. For the assay of the DNA synthetic activity in the mouse liver and kidney in vivo, [3Hlthymidine (10 yCi/25 g body wt) was injected intraperitoneally 1.5 h prior to sacrifice. The excised tissues were homogenized in 2 vol of cold de-ionized water in a glass homogenizer. An aliquot of the homogenate was mixed with an equal volume of cold 10% trichloroacetic acid (TCA) and centrifugated at 1000 g for 10 min. The pellets were washed 4 times with the acid, once ethanol and ethanol-ether (3 : l), and then hydrolysed overnight at 37°C with 0.4 N KOH. The DNA was precipitated by adding an equal volume of 10% TCA. The pelleted DNA was suspended in 5 % perchloric acid (RCA) and heated at 80°C for 15 min. The radioactivity incorporated into DNA fraction was measured by a liquid scintillation system and the amount of DNA was determined by the method of Dische [41with calf thymus DNA as a standard. Mitotic count. Colchicine (0.1 mg/25 g body wt) was injected 2 h prior to killing. Excised livers were fixed in the Bouin’s fixatives and the paraffin wax sections (5 urn thickness) were stained with Copyright @ 1984 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/84 $03.00
274 Short notes
Hours after injection
Fig. 1. Kinetic changes in DNA synthetic activities in mouse liver and kidney after catD administration. To determine the DNA synthetic activity, each mouse received an intraperitoneal injection of 20 pg of catD, papain or heat-inactivated catD, and then [3H]thymidine (10 uCi/25 g body wt) was injected intraperitoneally to each mouse 1.5 h prior to sacrifice. DNA synthesis activities in the liver (fig. 1 a) and kidney (fig. 1 b) were expressed as the value relative to the control injected with saline 1.5 h prior to killing. The amounts of r3H]thymidine incorporated into DNA in the liver and kidney of control mice were 1336+70 dpm/mg DNA and 1439+ 17 dpm/mg DNA, respectively. Each point in the figure represents the average of 2-3 experiments + SD. O- - -0, Saline (as a control); @[email protected]
, catD; A---A, papain; W---m, heat-inactivated catD.
Mayer’s hematoxylin-eosin. The mitotic figures were counted in the 30 arbitrary microscopic fields, each containing about 200 parenchymal cells. The mitotic index (MI) was expressed in terms of the number of mitotic nuclei per 1000 hepatic nuclei.
Results Stimulation of liver DNA synthesis in mice after an intraperitoneal injection of catD. Twenty ug of catD dissolved in 0.1 ml of saline was intraperitoneally injected into mice, and DNA syntheses in the mouse liver and kidney at 18, 32, 38,44 and 48 h after the protease injection were determined. As illustrated in fig. 1 a, a remarkable stimulation of liver DNA synthesis was observed in the mice treated with catD. The stimulation of liver DNA synthesis started by 25 h after the injection, peaked at 38 h, and then declined gradually. Fig. 1 a also shows that the injection of the same amount (20 ug) of papain and heat-inactivated catD failed to stimulate DNA synthesis in liver. In contrast to the liver DNA synthesis, DNA synthesis in the kidney was not affected by the catD injection, as shown in fig. 1 b, suggesting that catD-mediated stimulation of DNA synthesis may be specific to liver cells. Nor had papain any effect on the DNA synthesis in kidney. The dose-dependency of catD-mediated stimulation of liver DNA synthesis was studied by intraperitoneal injection of various amounts of the protease into mice and then determining the amount of [3H]thymidine incorporated into liver Exp Cd
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Bovine serum dose
Hours after injection
Fig. 2. Dose-dependency of catD effect on liver DNA synthesis. Various amounts of catD of bovine serum albumin were injected intraperitoneally to each mouse 38 h before sacrifice and [3H]thymidine (10 @i/25 g body wt) was injected intraperitoneally in each mouse 1.5 h prior to sacrifice. DNA synthesis activity is expressed as the control value relative to the control injected with saline. The amount of [‘Hlthymidine incorporated into DNA in the liver of control mice was 1095+7 dpm/mg DNA. Each point in the figure represents the average of 2-3 experiments + SD. O-0, CatD; O---O, BSA. Fig. 3. Kinetic change in mitotic activity in mouse liver after catD administration. To determine the mitotic activity, mice received an intraperitoneal injection of 80 ug of catD, and the mitotic figures were counted, as described in the text. Each point represents the average + SD. W, catD; 0- - -0, saline.
DNA. The mice were killed 38 h after the protease injection when the liver DNA synthesis was maximally stimulated, as shown in fig. 1 a. The degree of stimulation of liver DNA synthesis was dependent on the amount of catD injected, being as high as seven-fold in the mice injected with 200 pg of catD (fig. 2). The injection of bovine serum albumin (BSA) instead of catD had no effect on liver DNA synthesis. Stimulation of mitotic activity in mouse liver after an intraperitoneal injection ofcatD. As illustrated in fig. 3, the MI in the liver of mice that received a single intraperitoneal injection of 80 ug of catD increased significantly after the administration of the protease, reaching a peak value 44 h after the injection, i.e., 6 h behind the time of the peak activity of DNA synthesis. At this time, 16.2 mitotic figures were found per 1000 hepatic nuclei, while in the liver of the control mice the mitotic figures were only 1.5 per 1000 hepatic nuclei. The mitotic figures distributed evenly within the liver lobules. Necrosis was not detectable in any part of the hepatic tissues examined. Discussion In the present study, we have shown that a single intraperitoneal injection of a small amount of catD (20 ug) into mice induced a significant stimulation of DNA synthesis in the liver, but not in the kidney. The stimulation of Iiver DNA synthesis was followed by an increase in mitosis. Exp Cell Res 151 (1984)
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These results are consistent with our earlier findings that the administration of leupeptin and/or pepstatin immediately after partial hepatectomy blocks the synthesis of RNA and DNA, as well as mitosis in regenerating liver of rat. Previously, it was reported from this laboratory that the injection of a large amount of mitogenic papain in normal rats causes alterations in the function of plasma membranes (activation of adenylate cyclase ) and in the structure of the cell surface (disappearance of cell glycosaminoglycans ), as well as the stimulation of DNA synthesis . All these events are also known to occur during rat liver regeneration; moreover, the administration of the protease inhibitors to partially hepatectomized animals brought about retardation or disappearance of cell coat glycosaminoglycans . In view of these previous findings and the present results, it is conceivable that certain endogenous proteases are somehow involved in the process of the hepatocellular proliferation. A study of the stimulatory mechanisms of DNA synthesis and mitosis by catD is now in progress at our laboratory. M. M. wishes to thank Dr H Shimada at our laboratory for reading the manuscript and for helpful discussion.
References 1. 2. 3. 4. 4. 6.
Leffert, H L, Koch, S K, Moran, T & Rubalcava, B, Gastroenterology 76 (1973) 1470. Miyamoto, M & Terayama, H, Biochem biophys res commun 55 (1973) 84. Ohnishi, T, Yamamoto, K & Terayama, H, Histochemie 36 (1973) 15. Dische, Z, Microchemie 8 (1930) 4. Koji, T & Terayama, H, Biochim biophys acta 633 (1980) 10. Yamamoto, K, Omata, S, Ohnishi, T & Terayama, H, Cancer res 33 (1973) 567.
Received July 18, 1983 Revised version received October 5, 1983
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