CELL BIOCHEMISTRY AND FUNCTION VOL.
8: 91-97 (1990)
Suppressive Effect of Adrenalectomy on Growth of L1210 Leukemic Cells in Ascites KOTOHIKO KIMURA' AND YUKIYA SAKAMOTO Division of Biochemistry, Department of Oncology, BiomedicaI Research Center, Osaka University Medical School, I-I-SO, Fukushirna, Fukushima-ku, Osaka 553, Japan
This study was designed to evaluate the effect of adrenalectomy on growth of L1210 leukemic cells in ascites of BDF, mice. Varying doses of 1.5 x lo4, 5.0 x lo5, and 1-5 x lo6 viable tumour cells were inoculated intraperitoneally into groups of either adrenalectomized or sham-operated mice. At days 4 to 7 after the inoculation, adrenalectomized mice inoculated with 1.5 x lo4 or 5.0 x lo5 tumour cells had a smaller number of tumour cells in ascites than sham-operated controls. However, after inoculation of 1-5 x lo6 cells, no significant differences were found at days 2 to 4 between adrenalectomized and sham-operated mice. The growth retardation by adrenalectomy was not observed in adrenalectomized mice supplemented with 4 or 6 pg dexamethasone per day per mouse. It suggested that the ablation of glucocorticoids was at least partially responsible for the growth retardation observed in adrenalectomized mice. Cell kinetic analysis revealed that the difference in a potential doubling time could not explain these results. Tumour retention in the peritoneal cavity was measured using ['ZSI]-iododeoxyuridine-labelled tumour cells as a tracer. At days 4 to 6 after inoculation of 5.0 x lo5 labelled cells, radioactivity in the peritoneal cavity in adrenalectomized mice was about 70 per cent of that in sham-operated mice. This ratio was almost equivalent to the ratio of the number of cells in ascites of adrenalectomized mice to that of sham-operated ones. Consequently, growth retardation observed in adrenalectomized mice resulted from an increase in tumour cell migration and/or in tumour cell death, but not from an increase in doubling time. KEY WORDS-
Adrenalectomy; L1210; growth; iododeoxyuridine.
INTRODUCTION Mouse lymphocyte leukemia L1210 was first de- However the effect of an adrenalectomy pretreatscribed by Law et al.' This cell line arose in female ment of growth kinetics of L1210 leukemic cells has no. 234 of subline 212 of the DBA strain, at 8 not been investigated in detail despite their fremonths of age, after skin paintings with 0-2 per cent quent use. The present study was designed to methylcholanthrene dissolved in diethyl ether. It evaluate growth of L1210 cells in mice adrenalectohas since been maintained in DBA/2 or BDF, mice mized before tumour challenge and also to reassess by subcutaneous, intraperitoneal, or intramuscular contradictory data about the effect of adrenalecinoculation. These cells have been utilized in vivo tomy on tumour growth. for biochemical studies and for assays of chemotherapeutic Intensive studies of growth kinetics have been made for this cell line because of MATERIALS AND METHODS its importance in evaluating the results of experiAnimuls and Cell Line mental screening of cytotoxic Tumour growth is affected by many intrinsic Male BDF, mice, 4 weeks of age, weighing about factors in the host animals. Among these factors, 20 g, were purchased from Shizuoka Laboratory adrenal steroids have been studied most because of Animals Center, Shizuoka, Japan. All of the experitheir use as anticancer drugs.13 Some authors re- ments were performed with mice at 6 to 7 weeks of ported that adrenalectomy enhanced graft age. Either bilateral adrenalectomy or sham-operarejecti~n'~.'and suppressed tumour tion was performed by the retroperitoneal apOn the other hand, other studies showed enhancing proach under ether anesthesia. Care was taken not effects of adrenal ablation on tumour g r o ~ t h . ~ ~ - to ' ~ rupture the adrenal capsule and to completely 0263-6484/90/02009 1 -07$05.00 0 1990 by John Wiley & Sons, Ltd.
92 remove the adrenal gland and the surrounding fat. The operation was carried out at least four days before tumour transplantation. The adrenalectomized mice were provided with 0-9 per cent saline ad libitum. Ascitic leukemic cells, strain L1210, were a gift from the Research Institute for Tuberculosis and Cancer, Tohoku University, Sendai, Japan, and were maintained in our laboratory by intraperitoneal implantation in BDF, mice. Preparation and Inoculation of Tumour Cells
After tumour-bearing mice had been killed by ether anesthesia, their ascites were collected by aseptic methods. This ascitic fluid was centrifuged at 400g for 5 min at 4°C. The pellet was resuspended in 5 ml ice-cold buffer (17 mM Tris-HC1, 140 mM NH,Cl, pH 7-2).The suspended pellet was incubated for 15 min at 4"C, a time sufficient to destroy almost all red blood cells contained in the ascites. The cells were washed twice with 0.9 per cent saline. Cell concentration was properly adjusted by dilution with 0.9 per cent saline. About 0.1 ml of the diluted ascites, containing a particular number of viable L1210 leukemic cells, was inoculated intraperitoneally into mice. Viable cell counts were determined by the trypan blue exclusion technique. Determination of the Number of Cells in Ascites
After tumour-bearing mice had been killed by ether anesthesia, their tumour cells were completely harvested by flushing the peritoneal cavity with 0-9 per cent saline. The number of cells was determined with a Turk-Burger hemocytometer. At day 7 after inoculation of 1.5 x lo4 tumour cells, the mean weight of adrenalectomized mice was 25.2 k 2.0 g and that of sham-operated mice was 24.1 1.9 g. No significant differences were found between them by means of Student's t-test. Administration of Dexamethasone
Dexamethasone (Sigma Chemical Co., St. Louis, U.S.A.) was suspended in 0.05 ml of vehicle (0.5 per cent carboxymethylcellulose) at given concentrations. This suspension was injected subcutaneously into the hindlimb of mice. For the control, mice received injections of 0-05ml vehicle only. Measurement of Mitotic Rate
Groups of mice inoculated with L1210 leukemic cells were injected intraperitoneally with different
K. KIMURA AND Y. SAKAMOTO
doses of demecolcine (Wako Pure Chemical Industries Ltd., Osaka, Japan) and killed after 3 h. Smears of the cells were made, fixed in 99 per cent ethanol and stained with hematoxylin for quantification of metaphase. The lowest dose giving the highest number of cells accumulated in metaphase within 3 h was 25 to 75 pg demecolcine per mouse. Then a time/response experiment was made at 50 pg per mouse. A linear increase was observed in the cells in metaphase up to 3 h after injection with the line of best fit going through the origin. Thus, the numbers of the cells accumulated in metaphase during a 3 h period were valid estimates of the mitotic rate of the cell line. Calculations of Doubling Times
During exponential growth the potential doubling time ( T ) is: T, = ln2/R, where R, is the mitotic rate. 2 8 DNA Flow Cytometry
Cells were harvested and washed by the method described above. The samples for DNA analysis were prepared according to Vindelerv et a1.27 Determination of Tumour Retention in the Peritoneal Cavity
The method of Hofer and Hofer" was applied with small modifications. A group of intact mice was inoculated intraperitoneally with 1-5 x lo4 L1210 leukemic cells. Five days after the inoculation, the L1210 cells were labelled by two intraperitoneal injections of 10 pCi ['251]-iododeoxyuridine ('2sIUDR) per mouse. The two injections were given 6 h apart to ensure complete labelling of the tumour population. Seven days after the inoculation of cells, the mice were killed in an ether chamber and the leukemic cells were harvested under sterile conditions by flushing the peritoneal cavity with 0.9 per cent saline repeatedly. Tumour cells were pooled and washed by the method described above. In the present study radioactivity per lo6 cells was kept below 0-015 $3, since, in the case of L1210 cell line, [1251]-incorporation must be kept below 0.03 pCi per lo6 labelled cells to avoid adverse effects of ['2SI]-irradiation on the viability of the tumour cells."To study tumour retention in the peritoneal cavity, 5 x lo5 [1251UDR]-labelled L1210 cells were inoculated intraperitoneally into either adrenalectomized or sham-operated mice. For the control, groups of
93
EFFECT OF ADRENALECTOMY O N L1210 CELLS
mice received an inoculum of labelled cells that had been killed by heating in an 80°C water bath for 15 min. Two days before the mice were injected with ['2sIUDR]-labelled tumour cells, their drinking water was supplemented with 0.1 per cent sodium iodine to depress subsequent accumulation of I2'I in the thyroid. At days 4 to 6, after the mice had been killed in an ether chamber, the peritoneal tumour cells were harvested by flushing the peritoneal cavity repeatedly with 0-9 per cent saline, and the liver was dissected out from the mice. Radioactivity in the rest of the body was counted with an Auto-Gamma 5000 series gamma counter (Packard, Downers Grove, IL, U.S.A.), which detected 80.1 f 7.7 per cent of the disintegrations of l Z 5 I . Radioactivity in the DNA of the peritoneal tumour cells and the liver was measured with the gamma counter after homogenization of the organ, followed by precipitation of the DNA with 10 per cent trichloroacetic acid to remove "'1 activity which was not incorporated into the DNA. The whole body I2'J radioactivity was defined as the sum of the counts of the peritoneal tumour cells, the liver, and the rest of the body. RESULTS Efect of Adrenalectomy on Tumour Growth Rate in Ascites Following Varying Doses of Tumour Inoculat ion
Both adrenalectomized and sham-operated mice were divided respectively into three groups receiving an inoculum of either 1.5 x lo4, 5-0 x lo5, or 1.5 x lo6 tumour cells. The growth curve is shown for each group in Figure 1. When the cell count was below 1-0 x lo7, it was not possible to quantitate the number of L1210 leukemic cells, because of too few L1210 cells and relatively high numbers of coexisting leukocytes. The number of cells was significantly less in adrenalectomized mice than in sham-operated mice, both in the 1.5 x lo4 group at days 5 to 7, and in the 5-0 x 10' group at days 4 and 5. In the 1.5 x lo6 group, there was no significant differences in the number of cells between adrenalectomized and sham-operated mice throughout the period examined. Eflect of Administration of Dexarnethasone on Tumour Growth in Ascites of Adrenalectomized Mice
Varying doses of dexamethasone were given to groups of adrenalectomized mice once a day at days 0 to 6 after inoculation of 1.5 x lo4 tumour
Days Figure 1. Apparent tumour growth in the peritonel cavity following varying doses of tumour inoculum. Each point represents the average of four to nine mice and vertical bars represent S.D. Closed squares, sham-operated mice inoculated with 1.5 x lo6 cells; open squares, adrenalectomized mice inoculated with 1-5 x lo6 cells; closed triangles, sham-operated mice inoculated with 5.0 x lo5 cells; open triangles, adrenalectomized mice inoculated with 5.0 x lo5 cells; closed circles, sham-operated mice inoculated with 1.5 x lo4 cells; open circles, adrenalectomized mice inoculated with 1.5 x lo4 cells. Single and double asterisks show significant differences at P < 0.05 and P < 0-01, respectively.
cells. The number of cells at day 7 is shown in Figure 2. At doses of 4 and 6 pg per day per mouse, the number of cells was significantly greater than that without administration of dexamethasone, and even exceeded the number attained in sham-operated mice. However, at a dose of more than 8 pg per day per mouse, the number of cells decreased dosedependently. EHect of Adrenalectomy on a Potential Doubling Time of L1210 Cells
Both groups of adrenalectomized and shamoperated mice were inoculated intraperitoneally with 1.5 x lo4 L1210 cells, and their mitotic rate was determined at day 5 after tumour inoculation, when the growth of tumour cells was still in the exponential phase. The mitotic rate was 5.8 & 0.4 per cent h-' in six sham-operated mice, and 5.6 k 0.3 per cent h-' in six adrenalectomized mice. No significant differences were observed between two
K. KIMURA AND Y. SAKAMOTO
adrenalectomized mice, respectively. There were no significant differences between both groups. Eflect of Adrenalectomy on Tumour Retention in the Peritoneal Cavity
4
1
2
4
6 8 1 0 1 5
50
100
Doses of dexamethasone (pg/day/mouse)
Figure 2. Apparent tumour growth in the peritoneal cavity of adrenalectomized mice supplemented with varying doses of dexamethasone. The number of cells was determined at day 7 after inoculation of 1.5 x lo4 tumour cells. Each point represents the average of four to nine mice. The shaded area shows mean & S.D. The point with a vertical bar on the right side of the figure represents number of cells (mean f S.D.) in ascites of sham-operated mice at day 7 after inoculation of 1.5 x LO4 tumour cells. A significant difference is found between each of the groups administered with 4 or 6 pg dexamethasone per day per mouse and the group with only vehicles at P < 0.01.
groups. The potential doubling time (Tp)calculated from the mitotic rate was 12.2 h in the mean value. The cell cycle distributions were measured using DNA flow cytometry at day 5 after inoculation of 1.5 x lo4 cells. The cell cycle fraction in the GI, S, and G,/M phases was as follows; 42-2 k 6.9 per cent, 51.2 f 5.8 per cent and 6.8 f 1.5 per cent in five sham-operated mice, and 39.3 ? 2.9 per cent, 54.2 f 4.5 per cent and 6.5 2.0 per cent in six
Tumour retention was measured using [I2'I UDRI-labelled cells as a tracer. The effect of adrenalectomy in retarding growth of tumour cells in ascites was observed more clearly in the experiment using 1.5 x lo4 cells than 5 x lo' cells (Figure 1). But, in this experiment we used 5 x lo' labelled cells as a tracer instead of 1.5 x lo4 labelled cells, because, in the experiment using 1-5 x lo4 labelled cells, the radioactivity of 12'1 retained in the peritoneal cavity was not high enough to be measured precisely. The results are shown in Table 1. At days 4 to 6, the radiactivity retained in the peritoneal cavity was only about two per cent of all the radioactivity of 5 x lo' ['251UDR]-labelled cells inoculated into the mice. The radioactivity in the peritoneal cavity in adrenalectomized mice was about 70 per cent of that in sham-operated mice. This ratio is almost equivalent to the ratio of the number of cells in ascites of adrenalectomized mice to that of shamoperated mice, as shown in Figure 1. The radioactivity detected in the peritoneal cavity was probably due to ''1 incorporated into DNA of viable tumour cells, because the radioactivity was not detected in the peritoneal cavity in mice inoculated with dead labelled cells instead of viable ones. There were no significant differences in the radioactivity retained in the whole body between adren-
Table 1. Comparison between adrenalectomized and sham-operated mice in peritoneal '''I 4 to 6 days after i.p. injecion of 5.0 x lo5 living or heat-killed labelled L1210 cells. Sham-operated Living Dead Day 4
Day 5
Day 6
Whole body Peritoneal L1210 Number of mice
20.7 k 1.7* 2.6 k 0.3* 5
10.9 k 3.3
Whole body Peritoneal L 12 10 Number of mice
8.5 f 0.5 2.5 k 0.2*
Whole body Peritoneal L1210 Number of mice
retention at
Adrenalectomized Living Dead 9.2 k 3.8
8: 91-97 (1990)
Suppressive Effect of Adrenalectomy on Growth of L1210 Leukemic Cells in Ascites KOTOHIKO KIMURA' AND YUKIYA SAKAMOTO Division of Biochemistry, Department of Oncology, BiomedicaI Research Center, Osaka University Medical School, I-I-SO, Fukushirna, Fukushima-ku, Osaka 553, Japan
This study was designed to evaluate the effect of adrenalectomy on growth of L1210 leukemic cells in ascites of BDF, mice. Varying doses of 1.5 x lo4, 5.0 x lo5, and 1-5 x lo6 viable tumour cells were inoculated intraperitoneally into groups of either adrenalectomized or sham-operated mice. At days 4 to 7 after the inoculation, adrenalectomized mice inoculated with 1.5 x lo4 or 5.0 x lo5 tumour cells had a smaller number of tumour cells in ascites than sham-operated controls. However, after inoculation of 1-5 x lo6 cells, no significant differences were found at days 2 to 4 between adrenalectomized and sham-operated mice. The growth retardation by adrenalectomy was not observed in adrenalectomized mice supplemented with 4 or 6 pg dexamethasone per day per mouse. It suggested that the ablation of glucocorticoids was at least partially responsible for the growth retardation observed in adrenalectomized mice. Cell kinetic analysis revealed that the difference in a potential doubling time could not explain these results. Tumour retention in the peritoneal cavity was measured using ['ZSI]-iododeoxyuridine-labelled tumour cells as a tracer. At days 4 to 6 after inoculation of 5.0 x lo5 labelled cells, radioactivity in the peritoneal cavity in adrenalectomized mice was about 70 per cent of that in sham-operated mice. This ratio was almost equivalent to the ratio of the number of cells in ascites of adrenalectomized mice to that of sham-operated ones. Consequently, growth retardation observed in adrenalectomized mice resulted from an increase in tumour cell migration and/or in tumour cell death, but not from an increase in doubling time. KEY WORDS-
Adrenalectomy; L1210; growth; iododeoxyuridine.
INTRODUCTION Mouse lymphocyte leukemia L1210 was first de- However the effect of an adrenalectomy pretreatscribed by Law et al.' This cell line arose in female ment of growth kinetics of L1210 leukemic cells has no. 234 of subline 212 of the DBA strain, at 8 not been investigated in detail despite their fremonths of age, after skin paintings with 0-2 per cent quent use. The present study was designed to methylcholanthrene dissolved in diethyl ether. It evaluate growth of L1210 cells in mice adrenalectohas since been maintained in DBA/2 or BDF, mice mized before tumour challenge and also to reassess by subcutaneous, intraperitoneal, or intramuscular contradictory data about the effect of adrenalecinoculation. These cells have been utilized in vivo tomy on tumour growth. for biochemical studies and for assays of chemotherapeutic Intensive studies of growth kinetics have been made for this cell line because of MATERIALS AND METHODS its importance in evaluating the results of experiAnimuls and Cell Line mental screening of cytotoxic Tumour growth is affected by many intrinsic Male BDF, mice, 4 weeks of age, weighing about factors in the host animals. Among these factors, 20 g, were purchased from Shizuoka Laboratory adrenal steroids have been studied most because of Animals Center, Shizuoka, Japan. All of the experitheir use as anticancer drugs.13 Some authors re- ments were performed with mice at 6 to 7 weeks of ported that adrenalectomy enhanced graft age. Either bilateral adrenalectomy or sham-operarejecti~n'~.'and suppressed tumour tion was performed by the retroperitoneal apOn the other hand, other studies showed enhancing proach under ether anesthesia. Care was taken not effects of adrenal ablation on tumour g r o ~ t h . ~ ~ - to ' ~ rupture the adrenal capsule and to completely 0263-6484/90/02009 1 -07$05.00 0 1990 by John Wiley & Sons, Ltd.
92 remove the adrenal gland and the surrounding fat. The operation was carried out at least four days before tumour transplantation. The adrenalectomized mice were provided with 0-9 per cent saline ad libitum. Ascitic leukemic cells, strain L1210, were a gift from the Research Institute for Tuberculosis and Cancer, Tohoku University, Sendai, Japan, and were maintained in our laboratory by intraperitoneal implantation in BDF, mice. Preparation and Inoculation of Tumour Cells
After tumour-bearing mice had been killed by ether anesthesia, their ascites were collected by aseptic methods. This ascitic fluid was centrifuged at 400g for 5 min at 4°C. The pellet was resuspended in 5 ml ice-cold buffer (17 mM Tris-HC1, 140 mM NH,Cl, pH 7-2).The suspended pellet was incubated for 15 min at 4"C, a time sufficient to destroy almost all red blood cells contained in the ascites. The cells were washed twice with 0.9 per cent saline. Cell concentration was properly adjusted by dilution with 0.9 per cent saline. About 0.1 ml of the diluted ascites, containing a particular number of viable L1210 leukemic cells, was inoculated intraperitoneally into mice. Viable cell counts were determined by the trypan blue exclusion technique. Determination of the Number of Cells in Ascites
After tumour-bearing mice had been killed by ether anesthesia, their tumour cells were completely harvested by flushing the peritoneal cavity with 0-9 per cent saline. The number of cells was determined with a Turk-Burger hemocytometer. At day 7 after inoculation of 1.5 x lo4 tumour cells, the mean weight of adrenalectomized mice was 25.2 k 2.0 g and that of sham-operated mice was 24.1 1.9 g. No significant differences were found between them by means of Student's t-test. Administration of Dexamethasone
Dexamethasone (Sigma Chemical Co., St. Louis, U.S.A.) was suspended in 0.05 ml of vehicle (0.5 per cent carboxymethylcellulose) at given concentrations. This suspension was injected subcutaneously into the hindlimb of mice. For the control, mice received injections of 0-05ml vehicle only. Measurement of Mitotic Rate
Groups of mice inoculated with L1210 leukemic cells were injected intraperitoneally with different
K. KIMURA AND Y. SAKAMOTO
doses of demecolcine (Wako Pure Chemical Industries Ltd., Osaka, Japan) and killed after 3 h. Smears of the cells were made, fixed in 99 per cent ethanol and stained with hematoxylin for quantification of metaphase. The lowest dose giving the highest number of cells accumulated in metaphase within 3 h was 25 to 75 pg demecolcine per mouse. Then a time/response experiment was made at 50 pg per mouse. A linear increase was observed in the cells in metaphase up to 3 h after injection with the line of best fit going through the origin. Thus, the numbers of the cells accumulated in metaphase during a 3 h period were valid estimates of the mitotic rate of the cell line. Calculations of Doubling Times
During exponential growth the potential doubling time ( T ) is: T, = ln2/R, where R, is the mitotic rate. 2 8 DNA Flow Cytometry
Cells were harvested and washed by the method described above. The samples for DNA analysis were prepared according to Vindelerv et a1.27 Determination of Tumour Retention in the Peritoneal Cavity
The method of Hofer and Hofer" was applied with small modifications. A group of intact mice was inoculated intraperitoneally with 1-5 x lo4 L1210 leukemic cells. Five days after the inoculation, the L1210 cells were labelled by two intraperitoneal injections of 10 pCi ['251]-iododeoxyuridine ('2sIUDR) per mouse. The two injections were given 6 h apart to ensure complete labelling of the tumour population. Seven days after the inoculation of cells, the mice were killed in an ether chamber and the leukemic cells were harvested under sterile conditions by flushing the peritoneal cavity with 0.9 per cent saline repeatedly. Tumour cells were pooled and washed by the method described above. In the present study radioactivity per lo6 cells was kept below 0-015 $3, since, in the case of L1210 cell line, [1251]-incorporation must be kept below 0.03 pCi per lo6 labelled cells to avoid adverse effects of ['2SI]-irradiation on the viability of the tumour cells."To study tumour retention in the peritoneal cavity, 5 x lo5 [1251UDR]-labelled L1210 cells were inoculated intraperitoneally into either adrenalectomized or sham-operated mice. For the control, groups of
93
EFFECT OF ADRENALECTOMY O N L1210 CELLS
mice received an inoculum of labelled cells that had been killed by heating in an 80°C water bath for 15 min. Two days before the mice were injected with ['2sIUDR]-labelled tumour cells, their drinking water was supplemented with 0.1 per cent sodium iodine to depress subsequent accumulation of I2'I in the thyroid. At days 4 to 6, after the mice had been killed in an ether chamber, the peritoneal tumour cells were harvested by flushing the peritoneal cavity repeatedly with 0-9 per cent saline, and the liver was dissected out from the mice. Radioactivity in the rest of the body was counted with an Auto-Gamma 5000 series gamma counter (Packard, Downers Grove, IL, U.S.A.), which detected 80.1 f 7.7 per cent of the disintegrations of l Z 5 I . Radioactivity in the DNA of the peritoneal tumour cells and the liver was measured with the gamma counter after homogenization of the organ, followed by precipitation of the DNA with 10 per cent trichloroacetic acid to remove "'1 activity which was not incorporated into the DNA. The whole body I2'J radioactivity was defined as the sum of the counts of the peritoneal tumour cells, the liver, and the rest of the body. RESULTS Efect of Adrenalectomy on Tumour Growth Rate in Ascites Following Varying Doses of Tumour Inoculat ion
Both adrenalectomized and sham-operated mice were divided respectively into three groups receiving an inoculum of either 1.5 x lo4, 5-0 x lo5, or 1.5 x lo6 tumour cells. The growth curve is shown for each group in Figure 1. When the cell count was below 1-0 x lo7, it was not possible to quantitate the number of L1210 leukemic cells, because of too few L1210 cells and relatively high numbers of coexisting leukocytes. The number of cells was significantly less in adrenalectomized mice than in sham-operated mice, both in the 1.5 x lo4 group at days 5 to 7, and in the 5-0 x 10' group at days 4 and 5. In the 1.5 x lo6 group, there was no significant differences in the number of cells between adrenalectomized and sham-operated mice throughout the period examined. Eflect of Administration of Dexarnethasone on Tumour Growth in Ascites of Adrenalectomized Mice
Varying doses of dexamethasone were given to groups of adrenalectomized mice once a day at days 0 to 6 after inoculation of 1.5 x lo4 tumour
Days Figure 1. Apparent tumour growth in the peritonel cavity following varying doses of tumour inoculum. Each point represents the average of four to nine mice and vertical bars represent S.D. Closed squares, sham-operated mice inoculated with 1.5 x lo6 cells; open squares, adrenalectomized mice inoculated with 1-5 x lo6 cells; closed triangles, sham-operated mice inoculated with 5.0 x lo5 cells; open triangles, adrenalectomized mice inoculated with 5.0 x lo5 cells; closed circles, sham-operated mice inoculated with 1.5 x lo4 cells; open circles, adrenalectomized mice inoculated with 1.5 x lo4 cells. Single and double asterisks show significant differences at P < 0.05 and P < 0-01, respectively.
cells. The number of cells at day 7 is shown in Figure 2. At doses of 4 and 6 pg per day per mouse, the number of cells was significantly greater than that without administration of dexamethasone, and even exceeded the number attained in sham-operated mice. However, at a dose of more than 8 pg per day per mouse, the number of cells decreased dosedependently. EHect of Adrenalectomy on a Potential Doubling Time of L1210 Cells
Both groups of adrenalectomized and shamoperated mice were inoculated intraperitoneally with 1.5 x lo4 L1210 cells, and their mitotic rate was determined at day 5 after tumour inoculation, when the growth of tumour cells was still in the exponential phase. The mitotic rate was 5.8 & 0.4 per cent h-' in six sham-operated mice, and 5.6 k 0.3 per cent h-' in six adrenalectomized mice. No significant differences were observed between two
K. KIMURA AND Y. SAKAMOTO
adrenalectomized mice, respectively. There were no significant differences between both groups. Eflect of Adrenalectomy on Tumour Retention in the Peritoneal Cavity
4
1
2
4
6 8 1 0 1 5
50
100
Doses of dexamethasone (pg/day/mouse)
Figure 2. Apparent tumour growth in the peritoneal cavity of adrenalectomized mice supplemented with varying doses of dexamethasone. The number of cells was determined at day 7 after inoculation of 1.5 x lo4 tumour cells. Each point represents the average of four to nine mice. The shaded area shows mean & S.D. The point with a vertical bar on the right side of the figure represents number of cells (mean f S.D.) in ascites of sham-operated mice at day 7 after inoculation of 1.5 x LO4 tumour cells. A significant difference is found between each of the groups administered with 4 or 6 pg dexamethasone per day per mouse and the group with only vehicles at P < 0.01.
groups. The potential doubling time (Tp)calculated from the mitotic rate was 12.2 h in the mean value. The cell cycle distributions were measured using DNA flow cytometry at day 5 after inoculation of 1.5 x lo4 cells. The cell cycle fraction in the GI, S, and G,/M phases was as follows; 42-2 k 6.9 per cent, 51.2 f 5.8 per cent and 6.8 f 1.5 per cent in five sham-operated mice, and 39.3 ? 2.9 per cent, 54.2 f 4.5 per cent and 6.5 2.0 per cent in six
Tumour retention was measured using [I2'I UDRI-labelled cells as a tracer. The effect of adrenalectomy in retarding growth of tumour cells in ascites was observed more clearly in the experiment using 1.5 x lo4 cells than 5 x lo' cells (Figure 1). But, in this experiment we used 5 x lo' labelled cells as a tracer instead of 1.5 x lo4 labelled cells, because, in the experiment using 1-5 x lo4 labelled cells, the radioactivity of 12'1 retained in the peritoneal cavity was not high enough to be measured precisely. The results are shown in Table 1. At days 4 to 6, the radiactivity retained in the peritoneal cavity was only about two per cent of all the radioactivity of 5 x lo' ['251UDR]-labelled cells inoculated into the mice. The radioactivity in the peritoneal cavity in adrenalectomized mice was about 70 per cent of that in sham-operated mice. This ratio is almost equivalent to the ratio of the number of cells in ascites of adrenalectomized mice to that of shamoperated mice, as shown in Figure 1. The radioactivity detected in the peritoneal cavity was probably due to ''1 incorporated into DNA of viable tumour cells, because the radioactivity was not detected in the peritoneal cavity in mice inoculated with dead labelled cells instead of viable ones. There were no significant differences in the radioactivity retained in the whole body between adren-
Table 1. Comparison between adrenalectomized and sham-operated mice in peritoneal '''I 4 to 6 days after i.p. injecion of 5.0 x lo5 living or heat-killed labelled L1210 cells. Sham-operated Living Dead Day 4
Day 5
Day 6
Whole body Peritoneal L1210 Number of mice
20.7 k 1.7* 2.6 k 0.3* 5
10.9 k 3.3
Whole body Peritoneal L 12 10 Number of mice
8.5 f 0.5 2.5 k 0.2*
Whole body Peritoneal L1210 Number of mice
retention at
Adrenalectomized Living Dead 9.2 k 3.8
