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Inhibitory activity of l -asparaginase from Mycobcacterium tuberculosis on Yoshida ascites sarcoma in rats
ARCHIVES
OF
BIOCHEMISTRY
Inhibitory
AND
BIOPHYSICS
Activity
of L-Asparaginase
tuberculosis V. V. SUBBA REDDY, Microbiology
133, 262-267 (1969)
on Yoshida
Ascites
H. N. JAYARAM,
and Pharmacology Received
Laborarory, February
from
Sarcoma
M. SIRSI,
Indian
Mycobacterium
Institute
18, 1969; accepted
in Rats
T. RAMAKRISHNAN
AND
of Science, Bangalore
March
12, India
20, 1969
The antitumor activity of tiasparagine amidohydrolases (EC 3.5.1.1) from Mycobacterium tuberculosis H,,R, and Hs?R, strains has been tested on Yoshida ascites sarcoma in rats. The enzyme specific to M. tuberculosis Ha7R, but not to H,,R, has proved to be effective in inhibiting tbe growth of the sarcoma. Comparative studies on the activity of this enzyme with that of similar enzyme from Escherichia coli B, has shown that at the same levels the former is more effective than the latter. Long-lived immunity to this tumor in A/IISc Wistar rats following treatment of tumor bearing animals with M. tuberculosis H37Ra, pH 9.6 Gasparaginase has been observed. Immunity in these rats was demonstrated by tumor rejection and detection of humoral antibodies in the sera to the antigen of the cell-free extract of the tumor. The enzyme was ineffective in inhibiting fibrosarcoma in mice at the dose levels tested.
The antilymphoma factor in guinea pig serum first reported by Kidd (1) has been proved to be the enzyme L-asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) (2-5). The search for other sources of the enzyme has led to the discovery of asparaginases in Escherichia coli B by Mashburn and Wriston (6) and in Serratia marcescens by Rowley and Wriston (7). Recently Wade et al. (8) have reported that Erwinia caroLovora strains are more potent sources of the enzyme than E. c&i. Tests undertaken by many workers for sensitivity of tumors to asparaginase have included Walker Carcinoma, Jensen sarcoma, and a fibrosarcoma in rats and some lymphomas both in rats and mice (9). We have mentioned earlier (10) that L-asparaginase from Mycobackrium tuberculosis Hs7Ra strain but not from Ha,& strain is effective in inhibiting Yoshida ascites sarcoma in rats. In the present paper we have discussed the comparative effectiveness of enzyme preparations from 44. tuberculosis Hs,I& M. tuberculosis H37Ra, and E. coli B on Yoshida ascites sarcoma in rats and the effectiveness of enzyme from
M. tuberculosis H37Ra on fibrosarcoma mice. MATERIALS
AND
in
METHODS
ChemicaZs. Diethylaminoethyl (DEAE) cellulose was obtained from the Sigma Chemical Co., St. Louis, Missouri; L--asparagine was obtained from E. Merck Ag., Darmstadt; tannic acid (light) B.P. was obtained from W. J. Bush & Co. Ltd., London, E.8; the other chemicals were of reagent grade. Calcium phosphate gel was prepared by the method described by Colowick (11). Standard test system was the same as reported earlier (10). Unit activity of the enzyme is expressed as pmoles of ammonia liberated at 37” in 30 min and specific activity is expressed as unit activity per milligram of protein under the same experimental conditions. Protein was estimated by the method of Lowry et al. (12). Growth
of Organism Cell-Free
and Preparation Extract
of
M. tuberculosis H,TR, strain No. 7416 NCTC, London, and M. tuberculosis H,,R, strain No. 7417 NCTC, London, were grown on Youman’s medium (13) for 14 days at 37”. The cells were harvested by filtration and were suspended in phosphate buffer pH 7.5, 0.005 M (5 ml buffer per gram wet weight of the cells) and subjected to
262
INHIBITORY
ACTIVITY
sonic oscillation for 40 min in a lo-KHz Sonic Oscillator. The cell-free extract collected after centrifugation at 13,000g for 40 min was used as crude extract. Enzyme purification procedures were followed as reported (10). In certain experiments, as indicated under Results, the purification was carried out only to the ammonium sulfate stage. Escherichia coli B obtained from Dr. S. P. Champe, Purdue University, Indiana, was grown on nutrient broth for 48 hr at 37” and harvested by centrifugation. The cells thus collected were suspended in phosphate buffer pH 7.5, 0.005 M (10 ml buffer per gram weight of cells) and subjected to sonication for 20 min in a IO-kHz Sonic Oscillator. The cell-free extract was collected after centrifugation at 13,OOOg for 40 min. The purification of the enzyme from E. coli was carried out by taking a 55-100% saturated fraction of ammonium sulfate. This fractionated enzyme, which was nontoxic to rats, was used for the studies. By this step a seven-fold purification could be obtained. Tumor systems. Yoshida ascites sarcoma (YAS) in rats and mouse fibrosarcoma (MFS) have been used in the studies. YAS and MFS are carried in substrains of isogeneic Wistar rats (A/IISc) and Swiss mice (SWR/IISc), respectively. Originally, both the tumor systems and hosts were obtained from Indian Cancer Research Center, Bombay, and are being maintained since then in inbred colonies of rats and mice in our laboratory. Maintenance of YAS (14) and the criteria for the
OF
evaluation of antitumor activity in these tumor systems were the same as described previously (15). Tumor transplantations were performed as follows : YAS. Cells aspirated from the peritoneum of rats bearing 5-day-old tumors were used for transplantation. This was done by injecting intraperitoneally (ip) 107YAS cells into A/IISc Wistar rats, 2 months old, weighing 110-130 g. MFS. Tumors from mice implanted 15 days earlier were used to transplant into the experimental animals. This was carried out by subcutaneous (SC) trocar implantation of 2-4-mm tumor pieces (one each) into SWR/IISc mice, 2 months old weighing 20-25 g. Enzyme therapy. Treatment of experimental animals was started a day after transplantation unless otherwise stated. This consists of ip injection of the enzyme preparations from the sources described above. Control animals were injected with same volume of 0.005 M phosphate buffer of pH 7.5. The general behavior and weights of all the animals were regularly recorded. Autopsy studies for tumor manifestations were carried out in all the animals after death or sacrifice, accordingly. The tumor-free rats, after treatment with 45, 90, and 135 units of asparaginase (Table I) from M. tuberculosis H37Ra, were challenged with 5 X 10’ YAS cells thrice, once each at 3, 5, and 10 months. The sera from these rats were used for
TABLE THE
EFFECT
OF L-ASPARAGINASES
B, Enzyme sowce
activity
Eu7Jlll.2’ injected (units)
Buffer controlsc M
tuberculosis H&v
M
tuberculosis H&a
(pH 9.0 and pH 9.6 enzyme) E coli B
I tuberculosis HarR,, Ha7R,,
FROM Mycobacterium ON YOSHIDA ASCITES
sp4ic
SARCOMA
Survivors - Treated
O/B 6.4
90
l/5
6.4 12.5
135 45
2/5
12.5 12.5
90 135 45 90 135 500
5/5 5/5 O/5 O/5 O/5 4/5
43.3 43.3 43.3 43.3
263
L-ASPARAGINASE
O/5
-
AND Escherichia
coli
IN RATS Survival” period of tumor-bearing rats (Days)
Day of sacrifice of tumor-free survivors
8.2rk0.37 7.5f0.28
320 (1 Rat)
8.6f0.24 8.6f0.33
320 (2 Rats)
320 (5 Rats) 320 (5 Rats) 8.8f0.20 20.8f2.72 32.4f2.85 63
0 Administered by intraperitoneal route in a single dose, 24 hr after transplantation stated “Mean f SE. c 0.005 M phosphate buffer, pH 7.5.
320 (4 Rats)
unless otherwise
REDDY
264
the detection of antibodies for YAS cell free extract at the termination of the experiment. Antigen preparation. Rats with prominent ascites, 5 days after transplantation of tumor, were selected. The animals were killed by decapitation. Ascitic fluid was aspirated into flask containing sufficient anticoagulant (aqueous 10% potassium oxalate) in the cold. Subsequent operations were performed at O-4”. The tumor cells were collected by centrifugation and washed by suspending in medium containing 0.14 M NaCl, 0.02 M glucose and 0.04 M Tris buffer at pH 8.5 followed by centrifugation. This procedure was repeated until the supernate was clear. In order to lyse the contaminating red blood cells (RBC), the packed cells were suspended in 10 vol of distilled water. After immediate centrifugation at 6009 the packed cells were suspended in 10 vol (using original cell volume) of the above medium and were homogenized for 30 min with washed glass powder (1:2 ratio) in a glass mortar. The homogenate was centrifuged for 20 min at 10,OOOgand the supernatant fluid was aspirated carefully and dialyzed for 12 hr against distilled water. This dialyzate was centrifuged and the supernate was used for the studies. Passive hemagglutination technique. The highly sensitive technique of passive hemagglutination as described by Campbell et al. (16) was used to detect the presence of antibody in the sera of YAS resistant rats. Freshly drawn sheep blood in Alsever’s solution was used. Diluted YAS cellfree extract consisting of 0.032yo protein served as antigen. Sheep RBC treated with dilute tannic acid (l/26,000 dilution) for 10 min at 37” were coated with the above antigen. These antigen coated RBC were suspended in tubes containing dilute, inactivated, and absorbed normal rat serum and serially diluted immune rat serum treated similarly. Necessary controls were kept. These tubes were incubated for 60 min at room temperature and were kept overnight in the cold. The pattern of RBC sedimentation and microscopic observation of cells were taken into consideration for the evaluation of the agglutination titer. RESULTS
The data presented in Table I show the effect of L-asparaginase from different sources on Yoshida ascites sarcoma in rats. A 55-100% ammonium sulfate fraction (as given under Methods) of E. coli B was used as a source of n-asparaginase from this organism, as the lower fractions which contained some enzyme activity were found to be toxic to rats. A 30-60% ammonium sul-
ET AL.
fate fraction of M. tuberculosis H3,R. and H&. was used as a source of n-asparaginase from these organisms since it contained the maximum enzyme activity and was not toxic to the animals. At doses of 90 and 135 units, the partially purified enzyme from M. tuberculosis H&L had no inhibitory effect on the development of YAS. A similar fraction from M. tuberculosis HJ!L (which consists of two L-asparaginases, one showing maximum activity at pH 9.0 and the other at pH 9.6) is effective at both these dosages and produced complete regression of YAS in all the treated animals. At a dosage of 45 units tumor regression was observed in two of the five animals treated, the survival period of the remainder being the same as that of the untreated animals. The partially purified enzyme from E. coli B proved to be effective only at a dosage of 500 units resulting in four tumor-free survivals out of five animals treated, the remaining one animal having its survival period extended to 63 days, as compared to 8 days in the untreated controls. At 90- and 135-unit dosages this enzyme extended the survival period of the treated animals to the extent of 250 and 400%, respectively. On the other hand, at 45-unit dosage the E. coli B enzyme has no effect on the growth of YAS. The comparative effect of the enzyme preparations (at 135-unit dosage) from above sources on the growth of YAS has been presented in Fig. 1. Identical patterns of weight curve and survival period were seen with the enzyme from M. tuberculosis H&L and buffer controls. The weight curve of rats treated with the enzyme from M. tuberculosis H3rRa showed a gradual increase as in healthy controls and the animals did not show any signs of tumor development. Rats treated with the enzyme from E. coli B succumbed to tumor by 32 days on the average, with an initial loss in weight. The pH 9.0 and 9.6 n-asparaginases from M. tuberculosis Hs,R, were separated and purified as described earlier (10) and tested separately on YAS in other set of animals. The pH 9.6 enzyme produced complete regression of YAS in all treated animals at as low a dosage as 60 units while the pH 9.0
INHIBITORY
,:*:A 0
2
4
6
8 IO AVERAGE
ACTIVITY
265
OF L-ASPARAGINASE
12 14 SURVIVAL
20 22 16 I8 PERIOD (DAY S )
24
26
32
34
FIG. 1. Average body weight and survival an indication of the effect of period as M. tuberculosis HarRv, H,,R,, and E. coli B n-aspiraginsses, on the growth of Yoshida ascites sarcoma in A/IISc Wistar rats. Rats were treated ip with a single dose of 135 units of asparaginase from above sources 1 day after transplantation. 0, Healthv controls: A. buffer controls; A, treated with asparaginase from c. tubercuZo&‘H~~R,; 6, H3rRs; and l , E. coli B.
enzyme (which resembles the single Lasparaginase in M. tuberculosis HP,RV) was ineffective even at 200 units. In one set of experiments, the pH 9.6 enzyme at a dosage of 400 units was administered to the animals 48 hr after tumor transplantation and was found to be effective. The pH 9.6 enzyme from M. tuberculosis H37Ra which was found effective on YAS, did not influence the growth of mouse fibrosarcoma, the t/c value being of 0.95. Treatment consisted of three successive enzyme doses of 50 units each. No tumors developed as a result of subsequent challenges with YAS cells on tumorfree rats after L-asparaginase treatment from M. tuberculosis H3,Ra. Tumor cells could not be observed in these rats killed at the time specified in Table I. When the sera of these rats were tested for the presence of humoral antibodies, agglutination of the antigencoated erythrocytes could be detected up to a titer of l/1024 (Table II). DISCUSSION
The data presented in this paper show that L-asparaginase present in M. tuberculosis H3,Ra is highly effective in inhibiting the
TABLE
II
AGGLUTINATIONTITEROFTANNEDERYTHROCYTES COATED WITH YOSHIDA ASCITES SARCOMA CELL ANTIGENS Dilution of se~a Sera
Antiseraa
Normal
sera*
w
I/32
$6
I/:0228
I/2% l/2048
l/4096
$24
c-
d-
e-
f-
f-
f-
f-
f-
f-
f-
a Sera obtained from rats in which tumors had regressed after treatment with n-asparaginase and further transplants had been rejected. * Sera obtained from isogeneic non-infected rats. c Compact granular agglutination covering the bottom of the tubes. d Diffuse film of agglutinated cells covering the bottom of the tube; edges of the film ragged. 0 Narrow ring of cells surrounding a diffuse film of agglutinated cells. f Heavy ring of cells or discreet smooth button of cells in center of tubes.
growth of Yoshida ascites sarcoma in rats. In comparative studies made with the widely known enzyme from E. coli B, the M. tuberculosis H37Ra enzyme was found to be
266
REDDY
active at much lower concentrations than the former. Unlike the L-asparaginase from E. coli the mycobacterial enzyme is free from glutaminase activity (10) and does not appear to be associated with any toxic factor. L-asparaginases from various sources differ in their effectiveness against transplantable mouse and rat tumors in vivo. The enzymes from normal guinea pig serum (2, 3), from E. coli (6), and from S. marcestens (7) possess antitumor activity but those from some other microbial sources have been found to be ineffective (6). Even in E. coli there are present two L-asparaginases, only one of which has antilymphoma activity (17). M. tuberculosis H&L also possesses two L-asparaginases and only one of them shows antitumor activity. The properties of this enzyme are different from the corresponding one in E. coli B. M. tuberculosis H3,Rv possesses only one L-asparaginase which is inactive against tumor and appears to be identical to the inactive enzyme in the HnR, strain. A number of factors might be influencing the effectiveness of asparaginases in inhibiting tumor growth. The two L-asparaginases differ from each other in pH optima, heat inactivation, Michaelis constant, and effect of inhibitors. However, it is difficult to attribute to any of these properties the difference in their activities against tumors. Though the optimum pH of the effective enzyme in M. tuberculosis H37Ra and of guinea pig serum L-asparaginase is pH 9.6, that of E. coli B is active over the range from pH 6.0 to 8.4 (17). Stability to elevated temperatures seems to be a common property of the n-asparaginases from guinea pig serum (18)) E. coZi (19)) and M. tuberculosis H3,Ra while instability to such temperatures is a common property of the ineffective Lasparaginases of E. coli and M. tuberculosis. However, this will not explain why at the same concentration the mycobacterial enzyme is 5-6 times more effective than E. coli enzyme. Since the properties of these two classes of L-asparaginases are different, it is reasonable to assume that the amino acid sequences are also different, and it is possible that the ineffective L-asparaginases have groupings at the active site which are readily
ET AL.
cleared by the proteolytic enzymes present in the host. It is proposed to test this hypothesis on the two L-asparaginases present in M. tuberculosis H3,Ra. It has now been established that while cellular immunity plays a dominant role in tumor rejections, circulating antibodies may also play a part (20, 21). While classical precipitation reactions may not reveal these antibodies, they have been detected by passive hemagglutination tests (21). Utilizing this technique with homogenized YAS cells as antigen, it is now shown that the sera of rats with completely regressed tumors exhibit an appreciable titer of antibody against antigens of YAS. The specificity of these antibodies to tumor antigens and the extent to which they participate in the mechanisms involved in the rejection of the transplants needs further investigation. Among the L-asparaginase susceptible tumors which are lethal to the host, established immunity has been reported only in C3H and C3H/HE mice to the 6 C3HED lymphosarcoma (20). In our study we report here for the first time a long-lived immunity in A/IISc Wistar rats to a new L-asparaginase susceptible tumor-Yoshida ascites sarcoma. ACKNOWLEDGMENT
One of the authors (V.V.S.R.) thanks the authorities of the Council of Scientific and Industrial Research, India, for awarding a Fellowship. REFERENCES 1. KIDD, J. G., J. Exptl. Med. 98,565, 583 (1953). 2. BROOME, J. D., Nature 191, 1114 (1961). 3. BROOME, J. D., J. Exptl. Med. 118, 99, 121 (1963). 4. HIRAMOTO, R., TATE, C., AND HAMLIN, M., Proc. Sot. Exptl. Biol. Med. 121, 597 (1966). 5. SULD, H. M., AND HERBUT, P. A., J. Biol. Chem. 240,2234 (1965). 6. MASHBURN, L. T., AND WRISTON, J. C., Arch. Biochem. Biophys. 106, 450 (1964). 7. ROWLEY, B., AND WRISTON, J. C., JR., Biothem. Biophys. Res. Commun. 28,160 (1967). 8. WADE, H. E., ELSWORTH, R., HERBERT, D., KEPPIE, J., AND SARGEANT, K., Lancet II, 776 (1968). 9. BROOME, J. D., Trans. N. Y. Acad. Sci. 30. 690 (1968).
INHIBITORY
ACTIVITY
10. JAYARaM, H. N., RAMAKRISHNAN, T., AND VAIDYANATHAN, C. S., Arch. Biochem. Biophys. 126, 165 (1968). 11. COLOWICK, S. P., Methods Enzymol. I, 90 (1955). 12. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). 13. YOUMANS, G. P., AND KARLSON, A. G., Bm. Rev. Tuberc. 66,529 (1947). 14. REDDE-, V. T'. S., KAO, 1’. S., AND SIRSI, M., Current Sci. 36, 143 (1967). 15. REDDY, 1'. V. S., AND SIRSI, >I., Cancer Res. (accepted for publication). 16. CBMPBELL, D. H., GAR~EY, J. S., CREMFX, N.
OF L-ASPARAGINASE
17.
18. 19 20. 21.
267
E., AND SUSSDORF, D. H., “Methods in Immunology,” p. 161, Benjamin, New York (1963). CAMPBELL, H. A., MASHBURN, L. T., BOYSE, E. A., AND OLD. L. J., Biochemistry 6, 721 (1967). TOWER, D. B., PETERS, E. L., AND CURTIS, W. C., J. Biol. Chem. 238, 983 (1963). SCHWARTZ, J.H., REAVES, J.Y., AND BROOME, J. D., Proc. Natl. Acad. Sci. 66, 1516 (1966). PRAGER,)~. D., ROBERTS, J., ANDBACHYNSKY, N., J. Immunol. 98,1045 (1967). MILGROM, F., Cancer Res. 21, 862 (1961).
OF
BIOCHEMISTRY
Inhibitory
AND
BIOPHYSICS
Activity
of L-Asparaginase
tuberculosis V. V. SUBBA REDDY, Microbiology
133, 262-267 (1969)
on Yoshida
Ascites
H. N. JAYARAM,
and Pharmacology Received
Laborarory, February
from
Sarcoma
M. SIRSI,
Indian
Mycobacterium
Institute
18, 1969; accepted
in Rats
T. RAMAKRISHNAN
AND
of Science, Bangalore
March
12, India
20, 1969
The antitumor activity of tiasparagine amidohydrolases (EC 3.5.1.1) from Mycobacterium tuberculosis H,,R, and Hs?R, strains has been tested on Yoshida ascites sarcoma in rats. The enzyme specific to M. tuberculosis Ha7R, but not to H,,R, has proved to be effective in inhibiting tbe growth of the sarcoma. Comparative studies on the activity of this enzyme with that of similar enzyme from Escherichia coli B, has shown that at the same levels the former is more effective than the latter. Long-lived immunity to this tumor in A/IISc Wistar rats following treatment of tumor bearing animals with M. tuberculosis H37Ra, pH 9.6 Gasparaginase has been observed. Immunity in these rats was demonstrated by tumor rejection and detection of humoral antibodies in the sera to the antigen of the cell-free extract of the tumor. The enzyme was ineffective in inhibiting fibrosarcoma in mice at the dose levels tested.
The antilymphoma factor in guinea pig serum first reported by Kidd (1) has been proved to be the enzyme L-asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) (2-5). The search for other sources of the enzyme has led to the discovery of asparaginases in Escherichia coli B by Mashburn and Wriston (6) and in Serratia marcescens by Rowley and Wriston (7). Recently Wade et al. (8) have reported that Erwinia caroLovora strains are more potent sources of the enzyme than E. c&i. Tests undertaken by many workers for sensitivity of tumors to asparaginase have included Walker Carcinoma, Jensen sarcoma, and a fibrosarcoma in rats and some lymphomas both in rats and mice (9). We have mentioned earlier (10) that L-asparaginase from Mycobackrium tuberculosis Hs7Ra strain but not from Ha,& strain is effective in inhibiting Yoshida ascites sarcoma in rats. In the present paper we have discussed the comparative effectiveness of enzyme preparations from 44. tuberculosis Hs,I& M. tuberculosis H37Ra, and E. coli B on Yoshida ascites sarcoma in rats and the effectiveness of enzyme from
M. tuberculosis H37Ra on fibrosarcoma mice. MATERIALS
AND
in
METHODS
ChemicaZs. Diethylaminoethyl (DEAE) cellulose was obtained from the Sigma Chemical Co., St. Louis, Missouri; L--asparagine was obtained from E. Merck Ag., Darmstadt; tannic acid (light) B.P. was obtained from W. J. Bush & Co. Ltd., London, E.8; the other chemicals were of reagent grade. Calcium phosphate gel was prepared by the method described by Colowick (11). Standard test system was the same as reported earlier (10). Unit activity of the enzyme is expressed as pmoles of ammonia liberated at 37” in 30 min and specific activity is expressed as unit activity per milligram of protein under the same experimental conditions. Protein was estimated by the method of Lowry et al. (12). Growth
of Organism Cell-Free
and Preparation Extract
of
M. tuberculosis H,TR, strain No. 7416 NCTC, London, and M. tuberculosis H,,R, strain No. 7417 NCTC, London, were grown on Youman’s medium (13) for 14 days at 37”. The cells were harvested by filtration and were suspended in phosphate buffer pH 7.5, 0.005 M (5 ml buffer per gram wet weight of the cells) and subjected to
262
INHIBITORY
ACTIVITY
sonic oscillation for 40 min in a lo-KHz Sonic Oscillator. The cell-free extract collected after centrifugation at 13,000g for 40 min was used as crude extract. Enzyme purification procedures were followed as reported (10). In certain experiments, as indicated under Results, the purification was carried out only to the ammonium sulfate stage. Escherichia coli B obtained from Dr. S. P. Champe, Purdue University, Indiana, was grown on nutrient broth for 48 hr at 37” and harvested by centrifugation. The cells thus collected were suspended in phosphate buffer pH 7.5, 0.005 M (10 ml buffer per gram weight of cells) and subjected to sonication for 20 min in a IO-kHz Sonic Oscillator. The cell-free extract was collected after centrifugation at 13,OOOg for 40 min. The purification of the enzyme from E. coli was carried out by taking a 55-100% saturated fraction of ammonium sulfate. This fractionated enzyme, which was nontoxic to rats, was used for the studies. By this step a seven-fold purification could be obtained. Tumor systems. Yoshida ascites sarcoma (YAS) in rats and mouse fibrosarcoma (MFS) have been used in the studies. YAS and MFS are carried in substrains of isogeneic Wistar rats (A/IISc) and Swiss mice (SWR/IISc), respectively. Originally, both the tumor systems and hosts were obtained from Indian Cancer Research Center, Bombay, and are being maintained since then in inbred colonies of rats and mice in our laboratory. Maintenance of YAS (14) and the criteria for the
OF
evaluation of antitumor activity in these tumor systems were the same as described previously (15). Tumor transplantations were performed as follows : YAS. Cells aspirated from the peritoneum of rats bearing 5-day-old tumors were used for transplantation. This was done by injecting intraperitoneally (ip) 107YAS cells into A/IISc Wistar rats, 2 months old, weighing 110-130 g. MFS. Tumors from mice implanted 15 days earlier were used to transplant into the experimental animals. This was carried out by subcutaneous (SC) trocar implantation of 2-4-mm tumor pieces (one each) into SWR/IISc mice, 2 months old weighing 20-25 g. Enzyme therapy. Treatment of experimental animals was started a day after transplantation unless otherwise stated. This consists of ip injection of the enzyme preparations from the sources described above. Control animals were injected with same volume of 0.005 M phosphate buffer of pH 7.5. The general behavior and weights of all the animals were regularly recorded. Autopsy studies for tumor manifestations were carried out in all the animals after death or sacrifice, accordingly. The tumor-free rats, after treatment with 45, 90, and 135 units of asparaginase (Table I) from M. tuberculosis H37Ra, were challenged with 5 X 10’ YAS cells thrice, once each at 3, 5, and 10 months. The sera from these rats were used for
TABLE THE
EFFECT
OF L-ASPARAGINASES
B, Enzyme sowce
activity
Eu7Jlll.2’ injected (units)
Buffer controlsc M
tuberculosis H&v
M
tuberculosis H&a
(pH 9.0 and pH 9.6 enzyme) E coli B
I tuberculosis HarR,, Ha7R,,
FROM Mycobacterium ON YOSHIDA ASCITES
sp4ic
SARCOMA
Survivors - Treated
O/B 6.4
90
l/5
6.4 12.5
135 45
2/5
12.5 12.5
90 135 45 90 135 500
5/5 5/5 O/5 O/5 O/5 4/5
43.3 43.3 43.3 43.3
263
L-ASPARAGINASE
O/5
-
AND Escherichia
coli
IN RATS Survival” period of tumor-bearing rats (Days)
Day of sacrifice of tumor-free survivors
8.2rk0.37 7.5f0.28
320 (1 Rat)
8.6f0.24 8.6f0.33
320 (2 Rats)
320 (5 Rats) 320 (5 Rats) 8.8f0.20 20.8f2.72 32.4f2.85 63
0 Administered by intraperitoneal route in a single dose, 24 hr after transplantation stated “Mean f SE. c 0.005 M phosphate buffer, pH 7.5.
320 (4 Rats)
unless otherwise
REDDY
264
the detection of antibodies for YAS cell free extract at the termination of the experiment. Antigen preparation. Rats with prominent ascites, 5 days after transplantation of tumor, were selected. The animals were killed by decapitation. Ascitic fluid was aspirated into flask containing sufficient anticoagulant (aqueous 10% potassium oxalate) in the cold. Subsequent operations were performed at O-4”. The tumor cells were collected by centrifugation and washed by suspending in medium containing 0.14 M NaCl, 0.02 M glucose and 0.04 M Tris buffer at pH 8.5 followed by centrifugation. This procedure was repeated until the supernate was clear. In order to lyse the contaminating red blood cells (RBC), the packed cells were suspended in 10 vol of distilled water. After immediate centrifugation at 6009 the packed cells were suspended in 10 vol (using original cell volume) of the above medium and were homogenized for 30 min with washed glass powder (1:2 ratio) in a glass mortar. The homogenate was centrifuged for 20 min at 10,OOOgand the supernatant fluid was aspirated carefully and dialyzed for 12 hr against distilled water. This dialyzate was centrifuged and the supernate was used for the studies. Passive hemagglutination technique. The highly sensitive technique of passive hemagglutination as described by Campbell et al. (16) was used to detect the presence of antibody in the sera of YAS resistant rats. Freshly drawn sheep blood in Alsever’s solution was used. Diluted YAS cellfree extract consisting of 0.032yo protein served as antigen. Sheep RBC treated with dilute tannic acid (l/26,000 dilution) for 10 min at 37” were coated with the above antigen. These antigen coated RBC were suspended in tubes containing dilute, inactivated, and absorbed normal rat serum and serially diluted immune rat serum treated similarly. Necessary controls were kept. These tubes were incubated for 60 min at room temperature and were kept overnight in the cold. The pattern of RBC sedimentation and microscopic observation of cells were taken into consideration for the evaluation of the agglutination titer. RESULTS
The data presented in Table I show the effect of L-asparaginase from different sources on Yoshida ascites sarcoma in rats. A 55-100% ammonium sulfate fraction (as given under Methods) of E. coli B was used as a source of n-asparaginase from this organism, as the lower fractions which contained some enzyme activity were found to be toxic to rats. A 30-60% ammonium sul-
ET AL.
fate fraction of M. tuberculosis H3,R. and H&. was used as a source of n-asparaginase from these organisms since it contained the maximum enzyme activity and was not toxic to the animals. At doses of 90 and 135 units, the partially purified enzyme from M. tuberculosis H&L had no inhibitory effect on the development of YAS. A similar fraction from M. tuberculosis HJ!L (which consists of two L-asparaginases, one showing maximum activity at pH 9.0 and the other at pH 9.6) is effective at both these dosages and produced complete regression of YAS in all the treated animals. At a dosage of 45 units tumor regression was observed in two of the five animals treated, the survival period of the remainder being the same as that of the untreated animals. The partially purified enzyme from E. coli B proved to be effective only at a dosage of 500 units resulting in four tumor-free survivals out of five animals treated, the remaining one animal having its survival period extended to 63 days, as compared to 8 days in the untreated controls. At 90- and 135-unit dosages this enzyme extended the survival period of the treated animals to the extent of 250 and 400%, respectively. On the other hand, at 45-unit dosage the E. coli B enzyme has no effect on the growth of YAS. The comparative effect of the enzyme preparations (at 135-unit dosage) from above sources on the growth of YAS has been presented in Fig. 1. Identical patterns of weight curve and survival period were seen with the enzyme from M. tuberculosis H&L and buffer controls. The weight curve of rats treated with the enzyme from M. tuberculosis H3rRa showed a gradual increase as in healthy controls and the animals did not show any signs of tumor development. Rats treated with the enzyme from E. coli B succumbed to tumor by 32 days on the average, with an initial loss in weight. The pH 9.0 and 9.6 n-asparaginases from M. tuberculosis Hs,R, were separated and purified as described earlier (10) and tested separately on YAS in other set of animals. The pH 9.6 enzyme produced complete regression of YAS in all treated animals at as low a dosage as 60 units while the pH 9.0
INHIBITORY
,:*:A 0
2
4
6
8 IO AVERAGE
ACTIVITY
265
OF L-ASPARAGINASE
12 14 SURVIVAL
20 22 16 I8 PERIOD (DAY S )
24
26
32
34
FIG. 1. Average body weight and survival an indication of the effect of period as M. tuberculosis HarRv, H,,R,, and E. coli B n-aspiraginsses, on the growth of Yoshida ascites sarcoma in A/IISc Wistar rats. Rats were treated ip with a single dose of 135 units of asparaginase from above sources 1 day after transplantation. 0, Healthv controls: A. buffer controls; A, treated with asparaginase from c. tubercuZo&‘H~~R,; 6, H3rRs; and l , E. coli B.
enzyme (which resembles the single Lasparaginase in M. tuberculosis HP,RV) was ineffective even at 200 units. In one set of experiments, the pH 9.6 enzyme at a dosage of 400 units was administered to the animals 48 hr after tumor transplantation and was found to be effective. The pH 9.6 enzyme from M. tuberculosis H37Ra which was found effective on YAS, did not influence the growth of mouse fibrosarcoma, the t/c value being of 0.95. Treatment consisted of three successive enzyme doses of 50 units each. No tumors developed as a result of subsequent challenges with YAS cells on tumorfree rats after L-asparaginase treatment from M. tuberculosis H3,Ra. Tumor cells could not be observed in these rats killed at the time specified in Table I. When the sera of these rats were tested for the presence of humoral antibodies, agglutination of the antigencoated erythrocytes could be detected up to a titer of l/1024 (Table II). DISCUSSION
The data presented in this paper show that L-asparaginase present in M. tuberculosis H3,Ra is highly effective in inhibiting the
TABLE
II
AGGLUTINATIONTITEROFTANNEDERYTHROCYTES COATED WITH YOSHIDA ASCITES SARCOMA CELL ANTIGENS Dilution of se~a Sera
Antiseraa
Normal
sera*
w
I/32
$6
I/:0228
I/2% l/2048
l/4096
$24
c-
d-
e-
f-
f-
f-
f-
f-
f-
f-
a Sera obtained from rats in which tumors had regressed after treatment with n-asparaginase and further transplants had been rejected. * Sera obtained from isogeneic non-infected rats. c Compact granular agglutination covering the bottom of the tubes. d Diffuse film of agglutinated cells covering the bottom of the tube; edges of the film ragged. 0 Narrow ring of cells surrounding a diffuse film of agglutinated cells. f Heavy ring of cells or discreet smooth button of cells in center of tubes.
growth of Yoshida ascites sarcoma in rats. In comparative studies made with the widely known enzyme from E. coli B, the M. tuberculosis H37Ra enzyme was found to be
266
REDDY
active at much lower concentrations than the former. Unlike the L-asparaginase from E. coli the mycobacterial enzyme is free from glutaminase activity (10) and does not appear to be associated with any toxic factor. L-asparaginases from various sources differ in their effectiveness against transplantable mouse and rat tumors in vivo. The enzymes from normal guinea pig serum (2, 3), from E. coli (6), and from S. marcestens (7) possess antitumor activity but those from some other microbial sources have been found to be ineffective (6). Even in E. coli there are present two L-asparaginases, only one of which has antilymphoma activity (17). M. tuberculosis H&L also possesses two L-asparaginases and only one of them shows antitumor activity. The properties of this enzyme are different from the corresponding one in E. coli B. M. tuberculosis H3,Rv possesses only one L-asparaginase which is inactive against tumor and appears to be identical to the inactive enzyme in the HnR, strain. A number of factors might be influencing the effectiveness of asparaginases in inhibiting tumor growth. The two L-asparaginases differ from each other in pH optima, heat inactivation, Michaelis constant, and effect of inhibitors. However, it is difficult to attribute to any of these properties the difference in their activities against tumors. Though the optimum pH of the effective enzyme in M. tuberculosis H37Ra and of guinea pig serum L-asparaginase is pH 9.6, that of E. coli B is active over the range from pH 6.0 to 8.4 (17). Stability to elevated temperatures seems to be a common property of the n-asparaginases from guinea pig serum (18)) E. coZi (19)) and M. tuberculosis H3,Ra while instability to such temperatures is a common property of the ineffective Lasparaginases of E. coli and M. tuberculosis. However, this will not explain why at the same concentration the mycobacterial enzyme is 5-6 times more effective than E. coli enzyme. Since the properties of these two classes of L-asparaginases are different, it is reasonable to assume that the amino acid sequences are also different, and it is possible that the ineffective L-asparaginases have groupings at the active site which are readily
ET AL.
cleared by the proteolytic enzymes present in the host. It is proposed to test this hypothesis on the two L-asparaginases present in M. tuberculosis H3,Ra. It has now been established that while cellular immunity plays a dominant role in tumor rejections, circulating antibodies may also play a part (20, 21). While classical precipitation reactions may not reveal these antibodies, they have been detected by passive hemagglutination tests (21). Utilizing this technique with homogenized YAS cells as antigen, it is now shown that the sera of rats with completely regressed tumors exhibit an appreciable titer of antibody against antigens of YAS. The specificity of these antibodies to tumor antigens and the extent to which they participate in the mechanisms involved in the rejection of the transplants needs further investigation. Among the L-asparaginase susceptible tumors which are lethal to the host, established immunity has been reported only in C3H and C3H/HE mice to the 6 C3HED lymphosarcoma (20). In our study we report here for the first time a long-lived immunity in A/IISc Wistar rats to a new L-asparaginase susceptible tumor-Yoshida ascites sarcoma. ACKNOWLEDGMENT
One of the authors (V.V.S.R.) thanks the authorities of the Council of Scientific and Industrial Research, India, for awarding a Fellowship. REFERENCES 1. KIDD, J. G., J. Exptl. Med. 98,565, 583 (1953). 2. BROOME, J. D., Nature 191, 1114 (1961). 3. BROOME, J. D., J. Exptl. Med. 118, 99, 121 (1963). 4. HIRAMOTO, R., TATE, C., AND HAMLIN, M., Proc. Sot. Exptl. Biol. Med. 121, 597 (1966). 5. SULD, H. M., AND HERBUT, P. A., J. Biol. Chem. 240,2234 (1965). 6. MASHBURN, L. T., AND WRISTON, J. C., Arch. Biochem. Biophys. 106, 450 (1964). 7. ROWLEY, B., AND WRISTON, J. C., JR., Biothem. Biophys. Res. Commun. 28,160 (1967). 8. WADE, H. E., ELSWORTH, R., HERBERT, D., KEPPIE, J., AND SARGEANT, K., Lancet II, 776 (1968). 9. BROOME, J. D., Trans. N. Y. Acad. Sci. 30. 690 (1968).
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ACTIVITY
10. JAYARaM, H. N., RAMAKRISHNAN, T., AND VAIDYANATHAN, C. S., Arch. Biochem. Biophys. 126, 165 (1968). 11. COLOWICK, S. P., Methods Enzymol. I, 90 (1955). 12. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). 13. YOUMANS, G. P., AND KARLSON, A. G., Bm. Rev. Tuberc. 66,529 (1947). 14. REDDE-, V. T'. S., KAO, 1’. S., AND SIRSI, M., Current Sci. 36, 143 (1967). 15. REDDY, 1'. V. S., AND SIRSI, >I., Cancer Res. (accepted for publication). 16. CBMPBELL, D. H., GAR~EY, J. S., CREMFX, N.
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E., AND SUSSDORF, D. H., “Methods in Immunology,” p. 161, Benjamin, New York (1963). CAMPBELL, H. A., MASHBURN, L. T., BOYSE, E. A., AND OLD. L. J., Biochemistry 6, 721 (1967). TOWER, D. B., PETERS, E. L., AND CURTIS, W. C., J. Biol. Chem. 238, 983 (1963). SCHWARTZ, J.H., REAVES, J.Y., AND BROOME, J. D., Proc. Natl. Acad. Sci. 66, 1516 (1966). PRAGER,)~. D., ROBERTS, J., ANDBACHYNSKY, N., J. Immunol. 98,1045 (1967). MILGROM, F., Cancer Res. 21, 862 (1961).