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Some properties of radiation resistant derivatives of L strain mouse cells
242 SOME PROPERTIES
OF RADIATION OF L STRAIN
J. F. WHITFIELD Biology
RESISTANT
MOUSE
DERIVATIVES
CELLS
and R. H. RIXON
and Health Physics Division, Atomic Energy of Canada Limited, Chalk River, Ontario, Canada
Received April 5, 1960
STAEZE,radiation
resistant lines of mammalian cells have been rarely produced [2, 5, 11, 151 and little is known of their metabolic properties. In the present communication, we have compared the aerobic glycolysis, respiration and catalase activity of radiosensitive and radiation resistant strains of L mouse cells. We have concentrated on the properties of three strains, L, Ll and R3. Strain R3 is radiation resistant [15] and strains L and Ll are radiosensitive. These strains represent a descending scale of radiosensitivity when arranged in the order Ll, L and R3 [15]. Other strains were used in one part of this study; these were 7K2, 7K9 and 10K. They were obtained from Rhynas and Newcombe whose observations indicate that they have about the same degree of radiation resistance as R3 [ll]. Our method of handling and maintaining suspension cultures of L cells has been described in previous publications [14, 151. For estimates of glucose utilization and lactate production, cells were removed from their medium (80 per cent CMRL1066 and 20 per cent horse serum) by centrifugation and the glucose and lactate contents of the medium determined. Glucose was determined by the method of Somogyi [la] employing Nelson’s color reagent [7] and lactate determined by the method of Barker and Summerson [l]. Oxygen uptake was measured by the direct method of Warburg [13]; 1 x 10’ cells from a growing suspension culture were suspended in 2.7 ml of Krebs-Ringer phosphate solution [13] containing 0.1 per cent glucose (pH 7.35 to 7.40). 0.3 ml of horse serum was added to minimize cell death in the respirometer flask [8]. For determination of the rate of oxygen uptake, the initial cell concentration and the terminal viable cell concentration were averaged; the distinction between viable and dead cells was made on the basis that dead cells can be stained with Erythrosin B [lo]. The catalase activities of homogenates of strain L, Ll and R3 were measured by the sodium perborate method of Feinstein [6]. Cells in suspension cultures were spun down, resuspended in phosphate buffer (pH 6.8) and homogenized in a Raytheon 200 watt, 10 Kc. oscillator (35 min exposure). Lyophilized beef liver catalase (Worthington Biochemical Corp., Freehold, New Jersey) served as a standard. In static cultures (see footnote, Table I), the most radiosensitive strain Ll and the resistant strain R3 invariably consumed glucose at significantly (P -= 0.001) higher rates than strain L, but the difference between Ll and R3 was not significant (Table I). Along with their higher glucose consumption, Ll and R3 consistently produced more lactate than strain L (Table I). Again, the difference between Ll and R3 was not significant. Therefore, an increased aerobic glycolysis was not consistently associated with radiation resistance since it was observed both in a sensitive and a rcsistant strain. Experimental
Cell Research 20
Radiation resistance Cells of resistant strain R3 consumed oxygen at a significantly (P i 0.001) lower rate than did cells of strain L (Table I); the QO, of L was 9.02 and of R3 was 7.01. The respiration rates of the two sensitive strains L and Ll were the same (Table I). A reduced oxygen consumption of R3 cells as compared with cells of strain L was also observed when glucose and horse serum were omitted from the Krebs-Ringer solution; this demonstration of the ability of L cells to respire in the absence of glucose and horse serum contradicts the findings of Phillips and Feldhaus [Cl]. TABLE
Strain
I. Glucose uptake, lactic acid production and oxygen consumption of radiosensitive and resistant lines of L cells. Lactic acid production @g/10’ cells/hr)
Glucose uptake (/q/10’ cells/hr)
oxygen consumption (mm3/ccll/min.)
L
251.9f
1.1010.02
x 10-7
Ll R3
379.4 i 11.8
167.7 + 4.5
1.11 io.02
366.1 i21.6
150.2 i 19.4
0.81~0.02
X 10-7 x 10-7
93.1 iI
6.5
7.2
In the experiments on glucose uptake and lactate production about 1.5 x 10’ cells were suspended in 5 ml of complete medium in a 15 x 120 mm test tube and incubated for 5 hours at 37”C, during which time there was no detectable growth. The values recorded are the means and standard errors of the means of 6 experiments. The difference between the lactate production by L and Ll and L and R3 arc significant; P < 0.001 and 0.02 respectively.
0
I, 20
I I I a I. 40
60
so
I I I I loo
120
140
MINUTES
l:ig. l.-The effect of addition of glucose on the oxygen consumption of cells of strain R3 suspended in Krebs-Ringer solution plus horse serum. Before addition of glucose the rate of oxygen consumption was 1.08 x lo-’ mm3/cell/min. After addition of glucose the rate was lowered to 0.75 x lo-’ mm3/cell/min. The viable cell count at the end of the experiment was 98 per cent of the initial count. This is a typical experiment out of seven showing the effect.
Experimenfal Cell Research 20
244
.J. F. Whitfield and R. H. Rixon
Other radioresistant strains did not show the same relationship between respiration rate and radioresistance as did R3. Two of them (7K2 and 7K9) had higher rates of oxygen consumption (1.42 and 1.32 “ lo-’ mm3 per cell per min, respectively) than the sensitive strains while one (101~) showed only a slight reduction in respiration rate (0.94 x IO-’ mm3 per cell per min). Therefore, there was no consistent association between the rates of oxygen consumption and the degree of radiosensitivity. It was also noted that the rates of oxygen consumption by cells of strain L and R3 were reduced (Fig. 1) by the addition of glucose (the Crabtree effect [4]), the reduction of the rate of oxygen consumption of R3 being greater than that of L. \Vhen glucose was added (0.1 per cent) the rate of oxygen consumption by R3 cells was reduced to X2 per cent of the rate without glucose (mean of 7 experiments). Strain L cells respired in the presence of glucose at 91 per cent of the rate without added glucose (mean of 5 experiments). Decreased sensitivity to x-rays could be due to a higher level of catalase activity in the resistant cells (radiation resistance in the classical strain Escherichia coli B/r is accompanied by an increased catalase activity [3]). However, homogenates of the most radiosensitive strain, Ll, consistently had the highest catalase activity (1.48 times L; mean of 4 determinations) while the activity of R3 was invariably intermediate between the activities of L and Ll (1.17 times L; mean of 4 experiments). In summary, no evidence has been found to relate radiation resistance in L cells to increased catalase activity, glycolysis or rate of oxygen consumption. Only two cell properties have hitherto been observed to be consistently associated with radiation reistance in these mouse cells. These are the absence of a long metacentric chromosome and a lower modal chromosome number in the resistant derivatives of the sensitive lines 1111. The authors gratefully acknowledge the technical T. Youdale in carrying out the experiments reported
assistance here.
of K. M. Baird
and
REFERENCES 1. BARKER, S. B. and SUMMERSON, W. H., J. Riot. Chem. 138, 535 (1941). 2. RETZ, E. H., LELIEYRE, P. and Booz, G., Proc. 2nd. conf. Peaceful Uses Atomic Energy,
Vol. 22, p 171. United Nations, Geneva, 1958. 3. CLARK, J. B., J. 13acterioZ. 64, 527 (1952). 4. CRABTREE, H. G., Hiochem. .J. 23, 53ti (1929). 5. DITTRICII, W., HOHNE, G. and SCHUBERT, G., in Progress in Radiobiology, p 381. (J. S. Wtchell, B. E. Holmes, and G. L. Smith, Eds.) Oliver and Boyd, Edinburgh, 1956. R. N., J. BioZ. Chem. 180, 1197 (1949). 6. FEINSTEIN, 7. NELSON. X.. .J. Riot. Chem. 153, 375 (1944). 8. PHILLIPS, g. .J. and ANDREWS, R. W:, Expt/. Cell Research 16, 678 (1959). 9. PHILLIPS. H. J. and FELDHAUS, R. J., I’roc. Sot. Exptl. Biol. Med. 92, 478 (1956). .J. ‘E., Exptl. Cett’Research 13, 341 (1957). 10. PHILLIPS; H. J. and TERRYBE&, 11. RHYNAS, P. 0. W. and NEWCOZVIBE.H. B., Exptt. Cell Research. In press. 12. SOYOGYI, M., J. Biol. Chem. 160, 62 (1945). 13. UMBREIT, W. W., BURRIS, R. K. alld STAUFFER, J. F., Manometric Techniques and Related Methods for the Study of Tissue Metabolism. Burgess Pub. Co. Minneapolis, 1949. 14. WHITFIELD, J. I>. and RIXON, R. H., Exptl. Cetl Research 18, 126 (1959). ibid. 19, 531 (1960). 15. ~
Experimental
Cell Research 20
OF RADIATION OF L STRAIN
J. F. WHITFIELD Biology
RESISTANT
MOUSE
DERIVATIVES
CELLS
and R. H. RIXON
and Health Physics Division, Atomic Energy of Canada Limited, Chalk River, Ontario, Canada
Received April 5, 1960
STAEZE,radiation
resistant lines of mammalian cells have been rarely produced [2, 5, 11, 151 and little is known of their metabolic properties. In the present communication, we have compared the aerobic glycolysis, respiration and catalase activity of radiosensitive and radiation resistant strains of L mouse cells. We have concentrated on the properties of three strains, L, Ll and R3. Strain R3 is radiation resistant [15] and strains L and Ll are radiosensitive. These strains represent a descending scale of radiosensitivity when arranged in the order Ll, L and R3 [15]. Other strains were used in one part of this study; these were 7K2, 7K9 and 10K. They were obtained from Rhynas and Newcombe whose observations indicate that they have about the same degree of radiation resistance as R3 [ll]. Our method of handling and maintaining suspension cultures of L cells has been described in previous publications [14, 151. For estimates of glucose utilization and lactate production, cells were removed from their medium (80 per cent CMRL1066 and 20 per cent horse serum) by centrifugation and the glucose and lactate contents of the medium determined. Glucose was determined by the method of Somogyi [la] employing Nelson’s color reagent [7] and lactate determined by the method of Barker and Summerson [l]. Oxygen uptake was measured by the direct method of Warburg [13]; 1 x 10’ cells from a growing suspension culture were suspended in 2.7 ml of Krebs-Ringer phosphate solution [13] containing 0.1 per cent glucose (pH 7.35 to 7.40). 0.3 ml of horse serum was added to minimize cell death in the respirometer flask [8]. For determination of the rate of oxygen uptake, the initial cell concentration and the terminal viable cell concentration were averaged; the distinction between viable and dead cells was made on the basis that dead cells can be stained with Erythrosin B [lo]. The catalase activities of homogenates of strain L, Ll and R3 were measured by the sodium perborate method of Feinstein [6]. Cells in suspension cultures were spun down, resuspended in phosphate buffer (pH 6.8) and homogenized in a Raytheon 200 watt, 10 Kc. oscillator (35 min exposure). Lyophilized beef liver catalase (Worthington Biochemical Corp., Freehold, New Jersey) served as a standard. In static cultures (see footnote, Table I), the most radiosensitive strain Ll and the resistant strain R3 invariably consumed glucose at significantly (P -= 0.001) higher rates than strain L, but the difference between Ll and R3 was not significant (Table I). Along with their higher glucose consumption, Ll and R3 consistently produced more lactate than strain L (Table I). Again, the difference between Ll and R3 was not significant. Therefore, an increased aerobic glycolysis was not consistently associated with radiation resistance since it was observed both in a sensitive and a rcsistant strain. Experimental
Cell Research 20
Radiation resistance Cells of resistant strain R3 consumed oxygen at a significantly (P i 0.001) lower rate than did cells of strain L (Table I); the QO, of L was 9.02 and of R3 was 7.01. The respiration rates of the two sensitive strains L and Ll were the same (Table I). A reduced oxygen consumption of R3 cells as compared with cells of strain L was also observed when glucose and horse serum were omitted from the Krebs-Ringer solution; this demonstration of the ability of L cells to respire in the absence of glucose and horse serum contradicts the findings of Phillips and Feldhaus [Cl]. TABLE
Strain
I. Glucose uptake, lactic acid production and oxygen consumption of radiosensitive and resistant lines of L cells. Lactic acid production @g/10’ cells/hr)
Glucose uptake (/q/10’ cells/hr)
oxygen consumption (mm3/ccll/min.)
L
251.9f
1.1010.02
x 10-7
Ll R3
379.4 i 11.8
167.7 + 4.5
1.11 io.02
366.1 i21.6
150.2 i 19.4
0.81~0.02
X 10-7 x 10-7
93.1 iI
6.5
7.2
In the experiments on glucose uptake and lactate production about 1.5 x 10’ cells were suspended in 5 ml of complete medium in a 15 x 120 mm test tube and incubated for 5 hours at 37”C, during which time there was no detectable growth. The values recorded are the means and standard errors of the means of 6 experiments. The difference between the lactate production by L and Ll and L and R3 arc significant; P < 0.001 and 0.02 respectively.
0
I, 20
I I I a I. 40
60
so
I I I I loo
120
140
MINUTES
l:ig. l.-The effect of addition of glucose on the oxygen consumption of cells of strain R3 suspended in Krebs-Ringer solution plus horse serum. Before addition of glucose the rate of oxygen consumption was 1.08 x lo-’ mm3/cell/min. After addition of glucose the rate was lowered to 0.75 x lo-’ mm3/cell/min. The viable cell count at the end of the experiment was 98 per cent of the initial count. This is a typical experiment out of seven showing the effect.
Experimenfal Cell Research 20
244
.J. F. Whitfield and R. H. Rixon
Other radioresistant strains did not show the same relationship between respiration rate and radioresistance as did R3. Two of them (7K2 and 7K9) had higher rates of oxygen consumption (1.42 and 1.32 “ lo-’ mm3 per cell per min, respectively) than the sensitive strains while one (101~) showed only a slight reduction in respiration rate (0.94 x IO-’ mm3 per cell per min). Therefore, there was no consistent association between the rates of oxygen consumption and the degree of radiosensitivity. It was also noted that the rates of oxygen consumption by cells of strain L and R3 were reduced (Fig. 1) by the addition of glucose (the Crabtree effect [4]), the reduction of the rate of oxygen consumption of R3 being greater than that of L. \Vhen glucose was added (0.1 per cent) the rate of oxygen consumption by R3 cells was reduced to X2 per cent of the rate without glucose (mean of 7 experiments). Strain L cells respired in the presence of glucose at 91 per cent of the rate without added glucose (mean of 5 experiments). Decreased sensitivity to x-rays could be due to a higher level of catalase activity in the resistant cells (radiation resistance in the classical strain Escherichia coli B/r is accompanied by an increased catalase activity [3]). However, homogenates of the most radiosensitive strain, Ll, consistently had the highest catalase activity (1.48 times L; mean of 4 determinations) while the activity of R3 was invariably intermediate between the activities of L and Ll (1.17 times L; mean of 4 experiments). In summary, no evidence has been found to relate radiation resistance in L cells to increased catalase activity, glycolysis or rate of oxygen consumption. Only two cell properties have hitherto been observed to be consistently associated with radiation reistance in these mouse cells. These are the absence of a long metacentric chromosome and a lower modal chromosome number in the resistant derivatives of the sensitive lines 1111. The authors gratefully acknowledge the technical T. Youdale in carrying out the experiments reported
assistance here.
of K. M. Baird
and
REFERENCES 1. BARKER, S. B. and SUMMERSON, W. H., J. Riot. Chem. 138, 535 (1941). 2. RETZ, E. H., LELIEYRE, P. and Booz, G., Proc. 2nd. conf. Peaceful Uses Atomic Energy,
Vol. 22, p 171. United Nations, Geneva, 1958. 3. CLARK, J. B., J. 13acterioZ. 64, 527 (1952). 4. CRABTREE, H. G., Hiochem. .J. 23, 53ti (1929). 5. DITTRICII, W., HOHNE, G. and SCHUBERT, G., in Progress in Radiobiology, p 381. (J. S. Wtchell, B. E. Holmes, and G. L. Smith, Eds.) Oliver and Boyd, Edinburgh, 1956. R. N., J. BioZ. Chem. 180, 1197 (1949). 6. FEINSTEIN, 7. NELSON. X.. .J. Riot. Chem. 153, 375 (1944). 8. PHILLIPS, g. .J. and ANDREWS, R. W:, Expt/. Cell Research 16, 678 (1959). 9. PHILLIPS. H. J. and FELDHAUS, R. J., I’roc. Sot. Exptl. Biol. Med. 92, 478 (1956). .J. ‘E., Exptl. Cett’Research 13, 341 (1957). 10. PHILLIPS; H. J. and TERRYBE&, 11. RHYNAS, P. 0. W. and NEWCOZVIBE.H. B., Exptt. Cell Research. In press. 12. SOYOGYI, M., J. Biol. Chem. 160, 62 (1945). 13. UMBREIT, W. W., BURRIS, R. K. alld STAUFFER, J. F., Manometric Techniques and Related Methods for the Study of Tissue Metabolism. Burgess Pub. Co. Minneapolis, 1949. 14. WHITFIELD, J. I>. and RIXON, R. H., Exptl. Cetl Research 18, 126 (1959). ibid. 19, 531 (1960). 15. ~
Experimental
Cell Research 20