PRELIMINARY COMMUNICATION
/ . Biochem., 79, 451-454 (1976)
Changes in Omithine Decarboxylase Activity in Normal Tissues of Tumor-bearing Mice during Tumor Growth1 Tamio NOGUCHI, Atunori KASHIWAGI, and Takehiko TANAKA Department of Nutrition and Physiological Chemistry, Osaka University, Medical School, Kita-ku, Osaka, Osaka 530 Received for publication, November 17, 1975
The omithine decarboxylase [EC 4.1.1.17] activities in the liver and spleen of tumorbearing mice increased remarkably, reaching a peak 4 to 6 days after inoculation of tumor cells. On the contrary, the enzyme activity in the kidney decreased during tumor growth and had almost disappeared on day 6 after tumor inoculation. Injection of cell-free tumor homogenate also raised the enzyme activities in the liver and spleen, but did not change the activity in the kidney. No increase in enzyme activity in the liver of mice was observed on injection of homogenates of normal tissues, such as liver, spleen, kidney, and muscld
Numerous studies ( / ) have shown that omithine decarboxylase [EC 4.1.1.17], the enzyme responsible for putrescine formation, is rapidly induced in eukaryotic tissues which are undergoing rapid proliferation in response to various stimuli. It has been suggested that this rise in enzyme activity is a necessary event for tissue growth (2). Several investigators ( 3 - 5 ) have also reported an increase in DNA synthesis in normal tissues of tumor-bearing animals. Based on these reports, we examined the changes in omithine decarboxylase activity in the liver, spleen, and kidney of tumor-bearing mice during tumor growth. Male ICR mice were housed in a constanttemperature room with lighting from 9 a.m. to 9 p.m. and were given laboratory chow and tap 1
This study was supported in part by grants from the Ministry of Education of Japan. Vol. 79, No. 2, 197"
451
water ad libitum. Ehrlich ascites tumor cells were maintained in the peritoneal cavity of mice by weekly transfers. The mice were starved for 4 to 6 hr before sacrifice to avoid variation in enzyme activities due to intake of dietary protein (6). The animals were killed by decapitation between 2 and 4 p.m. and the tissues were rapidly excised, washed with cold 0.9% saline, blotted, and homogenized in 0.25 M sucrose containing 1 mM dithiothreitol using a Dounce homogenizer. The tumor cells collected from each mouse were counted, and then washed, separated from blood cells by low speed centrifugation in 0.9% saline, and disrupted by sonication (20 kHz, 150 W) for 1 min. The supernatant fraction obtained by centrifugation at 100,000 xg for 40 min was used for estimation of enzyme activity.
452
PRELIMINARY COMMUNICATION
Ornithine decarboxylase activity was determined by measuring the formation of u COj from DL-{1-14C) ornithine. The reaction mixture consisted of 40 mM Tris-HCl buffer, pH 7.4, 20 fiM pyridoxal 5-phosphate, 1.4 mM dithiothreitol, 0.4 mM L-ornithine, 0.5 fid DL-(l-l4C) ornithine and 0.2 to 0.5 ml of tissue extract in a final volume of 1.25 ml. The mixture was incubated at 37° for 1 hr with shaking in an air-tight, 25 ml Erlenmeyer flask, with a center well containing filter paper (2x2.5 cm) and 0.05 ml of 10% KOH. The reaction was stopped by adding 0.5 ml of 6 N HC1 and then the mixture was incubated for an additional 20 min. Then the filter paper was transferred to a scintillation vial, and 10 ml of scintillation fluid were added. Radioactivity was measured in an Aloka liquid scintillation counter. All enzyme activities were corrected for the value of the blank without enzyme. Protein concentration was measured by the Biuret method ( 7 ) or the method of Lowry et al. (8), with bovine serum albumin as a standard. Figure 1 shows changes in ornithine decarboxylase activity in tumor cells and normal tissues of tumor-bearing mice during tumor growth. The enzyme activity in the tumor cells increased markedly, reaching a peak 4 days after tumor inoculation, when proliferation was most rapid, and then quickly decreased. A rise in ornithine decarboxylase activity like that in tumor cells was also observed in the liver and spleen of tumor-bearing mice, although the times of peak activity in these tissues were slightly later. The kidney of normal mice showed the highest enzyme activity of all the tissues examined. This very high activity in the kidney decreased significantly 4 days after inoculation of the tumor cells and had almost disappeared on day 6, remaining very low until the time of death. This unexpected, and marked decrease in the activity in the kidney of tumor-bearing mice, in contrast to the activities in liver and spleen, is very interesting. It is unknown why it occurred, but it suggests that ornithine decarboxylase in the kidney is regulated by a different mechanism from the enzyme in other tissues, such as tumor cells, spleen, and liver. Neish and Key ( 9 ) and Andersson and
0 5 10 QWS AFTER INOCULATION
Fig. 1. Ornithine decarboxylase activities in tumor cells and normal tissues of tumor-bearing mice during tumor growth. Mice were inoculated intraperitoneally with 2.9 xlO7 Ehrlich ascites tumor cells and killed at intervals. Enzyme activity was determined as described in the text. Points and bars are means and standard deviations of values in 4 mice. A: Ornithine decarboxylase activity in the liver ( • , xlO), kidney (O, X10"1), and spleen ( a ) of tumor-bearing mice. B : Cell number (O) and ornithine decarboxylase activity ( • ) in Ehrlich ascites tumor cells.
Heby (10), independently, reported increase in spermidine concentration in the liver of tumorbearing animals. Heby and Russell (77) also observed increases of the enzymes involved in polyamine biosynthesis and in the polyamine concentrations in the spleen and liver of tumorbearing mice. However, in the latter case the increases were due to tumor cell infiltration. In the present study histological examination of the liver, spleen and kidney of tumor-bearing mice 8 days after tumor inoculation showed that these tissues were normal with no tumor cell infiltration or metastasis. Recently, Yanagi and Potter also observed increase in ornithine decarboxylase activity in normal liver tissue of tumor-bearing animals (personal communication). Suda et al. (12,13) observed a remarkable deviation in the pattern of key / . Biochtm.
453
ORNITHINE DECARBOXYLASE IN TUMOR-BEARING MICE
TABLE I. Effects of cell-free homogenates of tumor cells and various normal tissues on ornithine decarboxylase activities in the liver, spleen, and kidney of normal mice. Tumor cells collected 7 days after inoculation, and tissues from normal 5-week-old mice were kept frozen at —20° before use. In experiment I, the tumor cells were homogenized in 10 mM Tris-HCl buffer (pH 7.6) containing 1 mM MgSO4, 3.3 mM CaClt, and 50 mM NaCl using a Waring blendor, and then sonicated for 5 min to disrupt them completely. No viable tumor cells were present in the homogenate, because no growth of tumor cells was observed for at least 45 days after injection of the homogenate. In experiment II, the tumor cells and normal tissues were homogenized in 10 mM phosphate buffer (pH 7.4) containing 0.9% NaCl with a Waring blendor or a Dounce homogenizer, and then sonicated until all the nuclei were broken, as monitored by microscopy. Cell-free homogenates of tumor cells and normal tissues, and bovine serum albumin, Fr. V, in the solution used for homogenization were injected intraperitoneally at a dose of 10 mg protein. Control mice were injected with the solution used for homogenization only. All mice were killed 24 hr after the injection. Enzyme activity was determined as described in the text. Data are expressed as mean values±S.D.
Exp. No.
II
Material injected
No. of mice
Ornithine decarboxylase activity (nmoles "COi/hr/mg protein) Liver
Spleen
Kidney
Tumor homogenate Bovine serum albumin Buffer
4 3 3
0.301±0.088 0.012±0.011 0.005±0.001
1.305±0.781 0.180±0.061 0.310±0.152
66.05±14.45 42.30 ±18.92 81.04±37.20
Tumor homogenate Kidney homogenate Liver homogenate Muscle homogenate Spleen homogenate Buffer
4 4 4 4 4 4
0.108±0.066 0.011±0.006
/ . Biochem., 79, 451-454 (1976)
Changes in Omithine Decarboxylase Activity in Normal Tissues of Tumor-bearing Mice during Tumor Growth1 Tamio NOGUCHI, Atunori KASHIWAGI, and Takehiko TANAKA Department of Nutrition and Physiological Chemistry, Osaka University, Medical School, Kita-ku, Osaka, Osaka 530 Received for publication, November 17, 1975
The omithine decarboxylase [EC 4.1.1.17] activities in the liver and spleen of tumorbearing mice increased remarkably, reaching a peak 4 to 6 days after inoculation of tumor cells. On the contrary, the enzyme activity in the kidney decreased during tumor growth and had almost disappeared on day 6 after tumor inoculation. Injection of cell-free tumor homogenate also raised the enzyme activities in the liver and spleen, but did not change the activity in the kidney. No increase in enzyme activity in the liver of mice was observed on injection of homogenates of normal tissues, such as liver, spleen, kidney, and muscld
Numerous studies ( / ) have shown that omithine decarboxylase [EC 4.1.1.17], the enzyme responsible for putrescine formation, is rapidly induced in eukaryotic tissues which are undergoing rapid proliferation in response to various stimuli. It has been suggested that this rise in enzyme activity is a necessary event for tissue growth (2). Several investigators ( 3 - 5 ) have also reported an increase in DNA synthesis in normal tissues of tumor-bearing animals. Based on these reports, we examined the changes in omithine decarboxylase activity in the liver, spleen, and kidney of tumor-bearing mice during tumor growth. Male ICR mice were housed in a constanttemperature room with lighting from 9 a.m. to 9 p.m. and were given laboratory chow and tap 1
This study was supported in part by grants from the Ministry of Education of Japan. Vol. 79, No. 2, 197"
451
water ad libitum. Ehrlich ascites tumor cells were maintained in the peritoneal cavity of mice by weekly transfers. The mice were starved for 4 to 6 hr before sacrifice to avoid variation in enzyme activities due to intake of dietary protein (6). The animals were killed by decapitation between 2 and 4 p.m. and the tissues were rapidly excised, washed with cold 0.9% saline, blotted, and homogenized in 0.25 M sucrose containing 1 mM dithiothreitol using a Dounce homogenizer. The tumor cells collected from each mouse were counted, and then washed, separated from blood cells by low speed centrifugation in 0.9% saline, and disrupted by sonication (20 kHz, 150 W) for 1 min. The supernatant fraction obtained by centrifugation at 100,000 xg for 40 min was used for estimation of enzyme activity.
452
PRELIMINARY COMMUNICATION
Ornithine decarboxylase activity was determined by measuring the formation of u COj from DL-{1-14C) ornithine. The reaction mixture consisted of 40 mM Tris-HCl buffer, pH 7.4, 20 fiM pyridoxal 5-phosphate, 1.4 mM dithiothreitol, 0.4 mM L-ornithine, 0.5 fid DL-(l-l4C) ornithine and 0.2 to 0.5 ml of tissue extract in a final volume of 1.25 ml. The mixture was incubated at 37° for 1 hr with shaking in an air-tight, 25 ml Erlenmeyer flask, with a center well containing filter paper (2x2.5 cm) and 0.05 ml of 10% KOH. The reaction was stopped by adding 0.5 ml of 6 N HC1 and then the mixture was incubated for an additional 20 min. Then the filter paper was transferred to a scintillation vial, and 10 ml of scintillation fluid were added. Radioactivity was measured in an Aloka liquid scintillation counter. All enzyme activities were corrected for the value of the blank without enzyme. Protein concentration was measured by the Biuret method ( 7 ) or the method of Lowry et al. (8), with bovine serum albumin as a standard. Figure 1 shows changes in ornithine decarboxylase activity in tumor cells and normal tissues of tumor-bearing mice during tumor growth. The enzyme activity in the tumor cells increased markedly, reaching a peak 4 days after tumor inoculation, when proliferation was most rapid, and then quickly decreased. A rise in ornithine decarboxylase activity like that in tumor cells was also observed in the liver and spleen of tumor-bearing mice, although the times of peak activity in these tissues were slightly later. The kidney of normal mice showed the highest enzyme activity of all the tissues examined. This very high activity in the kidney decreased significantly 4 days after inoculation of the tumor cells and had almost disappeared on day 6, remaining very low until the time of death. This unexpected, and marked decrease in the activity in the kidney of tumor-bearing mice, in contrast to the activities in liver and spleen, is very interesting. It is unknown why it occurred, but it suggests that ornithine decarboxylase in the kidney is regulated by a different mechanism from the enzyme in other tissues, such as tumor cells, spleen, and liver. Neish and Key ( 9 ) and Andersson and
0 5 10 QWS AFTER INOCULATION
Fig. 1. Ornithine decarboxylase activities in tumor cells and normal tissues of tumor-bearing mice during tumor growth. Mice were inoculated intraperitoneally with 2.9 xlO7 Ehrlich ascites tumor cells and killed at intervals. Enzyme activity was determined as described in the text. Points and bars are means and standard deviations of values in 4 mice. A: Ornithine decarboxylase activity in the liver ( • , xlO), kidney (O, X10"1), and spleen ( a ) of tumor-bearing mice. B : Cell number (O) and ornithine decarboxylase activity ( • ) in Ehrlich ascites tumor cells.
Heby (10), independently, reported increase in spermidine concentration in the liver of tumorbearing animals. Heby and Russell (77) also observed increases of the enzymes involved in polyamine biosynthesis and in the polyamine concentrations in the spleen and liver of tumorbearing mice. However, in the latter case the increases were due to tumor cell infiltration. In the present study histological examination of the liver, spleen and kidney of tumor-bearing mice 8 days after tumor inoculation showed that these tissues were normal with no tumor cell infiltration or metastasis. Recently, Yanagi and Potter also observed increase in ornithine decarboxylase activity in normal liver tissue of tumor-bearing animals (personal communication). Suda et al. (12,13) observed a remarkable deviation in the pattern of key / . Biochtm.
453
ORNITHINE DECARBOXYLASE IN TUMOR-BEARING MICE
TABLE I. Effects of cell-free homogenates of tumor cells and various normal tissues on ornithine decarboxylase activities in the liver, spleen, and kidney of normal mice. Tumor cells collected 7 days after inoculation, and tissues from normal 5-week-old mice were kept frozen at —20° before use. In experiment I, the tumor cells were homogenized in 10 mM Tris-HCl buffer (pH 7.6) containing 1 mM MgSO4, 3.3 mM CaClt, and 50 mM NaCl using a Waring blendor, and then sonicated for 5 min to disrupt them completely. No viable tumor cells were present in the homogenate, because no growth of tumor cells was observed for at least 45 days after injection of the homogenate. In experiment II, the tumor cells and normal tissues were homogenized in 10 mM phosphate buffer (pH 7.4) containing 0.9% NaCl with a Waring blendor or a Dounce homogenizer, and then sonicated until all the nuclei were broken, as monitored by microscopy. Cell-free homogenates of tumor cells and normal tissues, and bovine serum albumin, Fr. V, in the solution used for homogenization were injected intraperitoneally at a dose of 10 mg protein. Control mice were injected with the solution used for homogenization only. All mice were killed 24 hr after the injection. Enzyme activity was determined as described in the text. Data are expressed as mean values±S.D.
Exp. No.
II
Material injected
No. of mice
Ornithine decarboxylase activity (nmoles "COi/hr/mg protein) Liver
Spleen
Kidney
Tumor homogenate Bovine serum albumin Buffer
4 3 3
0.301±0.088 0.012±0.011 0.005±0.001
1.305±0.781 0.180±0.061 0.310±0.152
66.05±14.45 42.30 ±18.92 81.04±37.20
Tumor homogenate Kidney homogenate Liver homogenate Muscle homogenate Spleen homogenate Buffer
4 4 4 4 4 4
0.108±0.066 0.011±0.006
