Radiation resistant derivatives of L strain mouse cells

Experimental

RADIATION

RESISTANT DERIVATIVES MOUSE CELLS J. F. WHITFIELD

Biology

531

Cell Research 19, 531-538 (1960)

and Health Physics Division

OF L STRAIN

and R. H. RIXON

Atomic, Energy of Canada Ltd., Chalk River, Ont., Canada

ReceivedJune 3, 1959

EXPOSURE of a

tumour to a number of sublethal irradiations frequently results in the acquisition of radioresistance by the tumour [l, 7, 8, 151. Several attempts have been made to produce resistant tumour strains, but these attempts have met with only variable success. For example, Conger and Luippold [ 1 ] and Montgomery and Warren [7] were unable to observe the development of radioresistance in transplantable mouse tumours irradiated with sublethal doses of X-radiation under conditions which precluded development of apparent resistance due to host reactions (such as described by Nice [8]). On the other hand, it would appear that Dittrich et al. [2] have obtained a true resistant, transplantable mouse tumour strain. It should be noted that radioresistant strains of bacteria have been described [16, 171. In the present study we reported the isolation of a colony of radioresistant L strain mouse cells from a culture exposed to a single large dose of X-rays. The resistant and sensitive lines have been compared with respect to postirradiation survival in terms of colony formation and in terms of suspension growth. MATERIAL

AND

METHODS

Cell strain.-In the experiments reported here, strain L mouse cells have been used. The isolation of this strain from mouse connective tissue has been described by Sanford et al. [ll]. Earle el al. [3] showed that cells of this strain could be propagated in suspension and detailed studies of their characteristics while growing in suspension have been reported by Siminovitch et al. [12] and Whitfield and Rixon [13]. The original cultures were obtained from Dr. L. Siminovitch of the Ontario Cancer Institute (Toronto). Medium-Cells were cultivated in medium CMRL-1066 supplemented with horse serum. The final concentration of the two constituents was 80 per cent CMRL-1066 and 20 per cent horse serum. CMRL-1066 is a chemically defined mixture of amino acids, vitamins, coenzymes and other metabolites in a balanced salts solution contalning glucose [6]. In addition, the medium contained penicillin (10 rg per ml) and Experimental

Cell Research 19

J. F. Whitfield and R. H. R&on streptomycin (100 pg per ml). These antibiotics do not affect the growth characteristics of the cells used [13]. Propagation of cells.-Cells, suspended in 30 ml of medium, were grown in 25 mm x 150 mm test-tubes closed with silicone stoppers.’ The tubes were rotated horizontally in a revolving drum at 45 rpm. in incubators at 37°C. The number of cells per ml was determined from the mean number of cells in four chambers of standard haemacytometers (improved Neubauer ruling). The cells were not stained before counting. The medium was partially replenished by centrifuging the 30 ml cultures, removing 15 ml of the supernatant liquid and replacing this with an equal volume of fresh, warm (37°C) medium. Medium was partially replenished every 24 hours when the cell count exceeded 5 x 10” per ml. When the cell count was below this level the medium was replenished at 48 hour intervals. Cultures with cell counts above 2 x 10” per ml were diluted with an equal volume of fresh medium and the subsequent changes in concentration estimated by multiplying by the dilution factor. Due to the strong effect of dilution on multiplication [13], cultures were not diluted in the period from 24 hours before to 2 days after irradiation. Stock cultures (30 ml) were maintained with cell concentrations between 2 and 4 x lo6 cells per ml. The titer was kept in this range by dilution with fresh, warm medium. ~~r~~t~tto~.-A suspension culture (in complete medium) in a 25 mm x 150 mm tube was rotated about its long axis at 60 rpm while in the X-ray beam. Cultures were irradiated in the unfiltered beam of a 2000 kVp X-ray machine operating at 1.5 ma. The dose was measured in air with a parallel plate ionization chamber. All doses were administered at a rate of 14 r per sec., 47 cm from the target. Colony formation.-The techniques used were similar to those employed by Puck et al. [lo] and Gwatkin et al. [5]. No feeder layer was used. A 30 ml (1 x lo5 cells per ml) suspension culture was diluted with fresh, warm (37%) medium to contain 1 x lO* cells per ml. Eight 40 ml samples were taken, 7 of which were irradiated with doses ranging from 50 to 1000 r and one of which served as the control. For each dose, 7 ml of suitably diluted culture were placed in each of 5 Petri dishes (6 cm diameter). Cultures of strain L and the resistant strains were irradiated at the same time. The plates were incubated for 14 days at 37°C in an atmosphere of 5 per cent CO, in air. After incubation, the colonies were washed with Tyrode’s solution {minus glucose) and fixed with methanol-formalin fixative (9: 1). The colonies were then stained with Giemsa’s stain and scored. At high doses (600 to 1000 r) colonies were quite small and only those colonies containing 50 or more normal appearing cells were considered to have survived [9]. RESULTS Initid isolation.-Between 0.4 and 1.0 per cent of strain L cells irradiated with 1000 r of X-rays was able to form large colonies such as those illustrated in Fig. I. One colony was lifted from the glass with a sterile platinum spatula,

1 West Co., Phoenixville, Pa. Experimental

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Radiation resistance

Fig. I.-An illustration of surviving colonies from cultures of strains L and Rl irradiated 1000 r. 3.81 x lOa L cells and 2.42 x 10e Rl cells were plated. These plates (6 cm.-diameter) been incubated at 37°C in an atmosphere of 5 per cent CO, for 19 days.

with had

placed in 10 ml of medium in a 16 X 150 mm test tube, and rotated at 45 rpm. When a normally growing suspension culture had been established, the survival of the colony-forming ability of the newly isolated strain, Rl, was compared with that of strain L after irradiation with 500 and 1000 r. Table I and Fig. 1 show that the Rl strain retained its colony-forming ability to a much greater extent than did cells from the original L strain. A culture of Rl was then irradiated with an additional 3000 r. given as 3 irradiations with 1000 r at intervals of 15 cell generations. The resulting culture was designated R3 and was then subjected to detailed testing for resistance. TABLE

I. Comparison of colony formation

by irradiated

cells of strains L and Rl.

Dose (r)

Original No. of cells

Colonies formed

Per cent survival

L Rl

500 500

1300 972

173 472

13.3 48.5

L Rl

1000 1000

2286 1944

20 104

0.9 5.3

Strain

A suspension culture of each of strains L and Rl containing 1 x 10s cells per ml was irradiated at the same time. The plates were incubated together in the same incubator for 14 days. 35 - 603703

Experimental

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534

J. F. Whitfield and R. H. Rixon

Colony formation.-With increasing dose between 0 and 400 r a stepwise decline in colony forming ability has been observed in a total of five experiments on cultures of strain L, but the decline was less in experiments on cultures of R3 (compare curves A and B of Fig. 2). The L survival curve resembles the survival curves of X-irradiated cultures of haploid strains of Saccharomgces cerevisiae [4]. Such a curve could arise from a heterogeneity of the population with respect to radiosensitivity [4]. The survival of R3 was higher than that of L at all doses, but the difference was greatest at 1000 r (Fig. 2). The average number of colonies formed by strain L was 1.0 per cent and by R3 was 4.2 per cent of the control numbers. For comparison, a new resistant strain (R2) was obtained from a surviving colony of Rl which had been irradiated with a further dose of 1000 r (accumulated dose 2000 r). The number of colonies formed from a culture of R2, after irradiation with 1000 r, was 4.4 per cent of the control (the survival in an L culture irradiated at the same time was 1.0 per cent). This is the same as R3. Therefore, further selection by irradiation has not resulted in a significant change in resistance compared with the initial isolate (Table I). Suspension culfures.-Both strain L and strain R3 multiplied loga~.ithmically in suspension (Fig. 3A). The mean generation time of a culture of strain L was 23 hours, while that of an R3 culture was 27 to 28 hours. After irradiation with 1000 r, the pattern of multiplication of strain L differed considerably from that of R3. Multiplication was reduced and was not logarithmic in L cultures during the first 6 days, but it accelerated and became logarithmic after day 6 (Fig. 3B). The average generation time (3 cultures) during this second multiplication phase was 31.9 hours. R3 cultures continued to multiply logarithmically after irradiation, but at a reduced rate; the average generation time of three cultures was 66.2 hours. The rate of multiplication sharply accelerated at day 7 and the average generation time of the cultures decreased to 34.1 hours. During the course of this work, we obtained suspension cultures of L cells (Ll) maintained in this laboratory by Mr. P. Rhynas. The overall changes with time in the titre of these cultures after irradiation with 1000 r were similar to those described above, but the phase of rapid multiplication did not appear until day 10, four days later than it did in cultures of I.,. A postirradiation growth curve of this strain is included in Fig. 3B for comparison with the L strain as well as with R3. A greater difference was observed between the changes in cell concentration of L and R3 cultures irradiated with 2000 r. Four 30 ml cultures of L Experimental

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Radiafion resistance

535

and four of R3 were diluted with fresh medium to contain 7 to 8 x lo4 cells per ml and incubated until the cell concentrations had reached 1.5 to 1.6 x 105 cells per ml (c. 24 hours). The cultures in each group were pooled, divided into 30 ml aliquots and then irradiated. The results are presented in Fig. 4: the points on the curves are averages of the cell counts in the four cultures of each group. lO0.U

,

,

1

’

,

,

1

1

,

,

1

’

,

,

1

’

,

6.0 -

0.8 0.8

i

0

200

400 OOSE

600

so0

ROEWTOENS)

1 IWO

0

2

4 DAYS

6

60

2 4 DAYS

6 6 AFTER

IO I 4 IRRAOIATION

Fig. 2. Fig. 3. Fig. 2.-Survival of colony formation in irradiated cultures of (A) strain L, (B) strain R3. Each point on the curves is the mean of two similar experiments. Experiments on both strains were performed at the same time and were stored in the same incubator. The average plating efficiencies of L and R3 were 75 and 69 per cent respectively. Fig. 3.-A. Multiplication in unirradiated cultures of strain L ( - 0 -) and strain R3 ( - 0 -). B. Multiplication in irradiated (1000 r) cultures of strain L ( - 0 - ), strain Ll ( - x -) and strain R3(-0

-).

During the first 3 days after irradiation, cell multiplication in cultures of R3 (Fig. 4, curve B) was more rapid than in cultures of L (Fig. 4, curve A): the average generation time of L was 218 hours and of R3 was 73 hours. Increase of cell count in both groups ceased after day 3 and was replaced by a decline, which was more rapid and greater in L cultures than in R3 cultures. At the lowest point on the L curve, the average titre was 19.2 per Experimental

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J. F. WhitfieEd md R. R. Rixon

536

cent of the highest level attained shortly after irradiation whereas in the R3 cultures it was 40 per cent of the highest level attained. The average ceI1 count of two cultures of Ll irradiated with 2000 r dropped steadily during the period of observation (21 days}. At the end of this period, the average count was only 3.8 per cent of the highest level attained during the first three days after irradiation.

DAYS

AFTER

IRRADIATION

Fig. 4.

Fig. 5, Fig. 4.--Muftiplkation in cultures of strain L (A) and strain R3 (B) irfadiated with 2000 r. Each point on both curves is the mean titre of four cultures. The cultures within each group were identical in their response to irradiation. Fig. 5.---Multiplication in cultures of strain 1; (A) and strain R3 (B) irradiated with 3000 r. Each point on both curves is the mean titre of two cultures. The cultures within each group were identical in their response to irradiation.

Recovery growth began in all four L cultures between days 17 and 18, but in R3 cultures it started between days 13 and 15. The beginning of rapid multiplication was heralded by the appearance of normal size cells among the larger cells commonly observed in cultures irradiated with large doses P, 141. A comparison was also made between suspension cultures irradiated with

537 3000 r (Fig. 5). The experiment was carried out as described above, but only two cultures of each strain were studied. Again, cell multiplication was more rapid in cultures of R3 during the first three days after irradiation than it was in L cultures. The average generation time of the L cultures was 216 hours and 137 hours for R3 cultures. After day 3, the cell count began to fall more rapidly in L cultures than in cultures of R3 (compare curves A and B of Fig. 5). There was no evidence of recovery during the period of observation (31 days). StabiZify.-The resistant strains have so far proven to be relatively stable. For example, the experiments on suspension growth of R3 following irradiation with 2000 r were performed 20 days after a preliminary experiment using the same dose. The results of the preliminary experiment were exactly the same as the ones reported here. During the interval, the stock culture had gone through about 20 generations. In addition, no decrease in resistance of R3 has been found after over two months of continuous cultivation.

DISCUSSION

Among the possible mechanisms which have been proposed to explain the origin of radioresistance in tumours [l, 7,151, is the selection by radiation of resistant mutants from among the tumour cell population. These mutants would exist either prior to irradiation or be induced by radiation. Clear demonstrations of a tumour change have seldom been obtained [2]. In most cases of experimentally induced radiation resistance of tumours, the change has been at~ibuted to alterations in the tumour environment rather than to repopulation of the tumour itself by genetically radioresistant cells [7,8]. Irradiation causes vascular damage, fibrosis and chronic inflammation of the tumour bed with a resulting alteration of the nutrition and oxygen supply of the surviving tumour cells. In the present study, there can be no question of the intervention of environmental or nutritional factors in the origin of resistant strain Rl and its derivatives. The evidence presented here strongly indicates that the cell population in a suspension culture of strain L is heterogeneous with respect to radiosensitivity. A highly resistant strain was obtained following adminis~ation of a single Zarge dose of jrra~~afio~ (1000 r> by a method which is commonly used in microbiology to isolate mutant strains of bacteria, yeast and bacterial viruses. In preliminary experiments we have observed that the resistant strains Rl and R3 multiply to a higher level in an atmosphere of nitrogen than does Experimenfal

Ceil Research 19

538

J. F, W~i~~iel~ and R. N. Rixon

strain L (and Ll). Therefore, there seems to be a metabolic difference between the resistant and sensitive strains. Other radioresistant cultures of strain L have also been obtained in this laboratory by Rhynas and Newcombe (in preparation) using other methods. SUMMARY

A radiation resistant colony has been isolated from the survivors of strain L mouse cells after irradiation with a single dose of 1000 r. Further selection by additional exposures to 1000 r did not result in significant changes in radioresistance. Cells from suspension cultures of this strain (Rl) and its derivatives (R2 and R3) retained their colony forming ability to a greater extent than cells of strain L after irradiation with doses ranging from 50 to 1000 r; the survival of the resistant lines was 4 to 5 times that of strain L after irradiation with 1000 r. Initial growth in suspension cultures of R3 was much less affected by irradiation with 1000 r than it was in cultures of strain L. In addition, decline in cell numbers in cultures of R3 irradiated with 2000 and 3000 r was less marked than in cultures of strain L. The authors gratefully acknowledge the technical assistance of K. M. Baird and m 1. Youdale in carrying out the experiments reported here.

REFERENCES 1. CONGER, A. D. and LUIPPOLD, H. J., Cancer Research 17, 897 (1957). p. 381. J. S. Mit2. DITTRICH, W., HBHNE, G. and SCHUBERT, G., in Progress in Ra~ob~olo~, chell, B. E. Hohnes, and C. L. Smith, eds. Oliver and Boyd, Edinburgh, 1956. 3. EARLE, W. R., SCHILLING, E. L., BRYANT, J. C. and EVANS, V. J., J. Nati. Cancer Inst. 14, 1159 (1954). 4. ELKIND, M. M. and SUTTON, H., Science 128,1082 (1958). 5. GWATKIN, R. B. L., TILL, J. E., WHITMORE, G. F., SIMINOVITCW, L. and GRAHAM, A. F., Proc. Natl. Acad. Sci. (Wash.) 43, 451 (1957). 6. HEALY, G. M., FISHER, D. C. and PARKER, R. C., Proc. Sot. Exptl. Biol. Med. 89, 71 (1955). MONTGOMERY, P. O’B. and WARREN, S., Radiology 60, 421 (1953). i: NICE. C. M.. Am. J. Roentaenol. Radium Theranu Nuclear Med. 78. 831 (1957). 9. Pu&, T. T,and MARCUS, b. I., J. Exptl. Med.‘fO3, 653 (1956). ’ ’ ’ 10. PUCK, T. T., MARCUS, P. I. and CIECIURA, S. J., J. Exptt. Med. 103, 273 (1955). 11. SANFORD, K. K., EARLE, W. R. and LIKELY, G. D., J. AWL Cancer Inst. 9,229 (1948). 12. SIMINOVITCH, L., GRAHAM, A. F., LESLEY, S. M. and NEVILL, A., Exptl. Ceil Research 12, . 299 (1957): 13. WHITFIELD, J. F. and RIXON, R. H., Exptl. Cell Research 18, 126 (1959). 14. WHITMORE, G. F., TILL, J. E., GWATKIN, R. B. L., SIMINOVITCH, L. and GRAHAM, A. F., Biochim. et Biophys. Acta 30, 583 (1958). 15. WINDHOLZ, F., Radiology 48, 398 (1947). 16. WITKIN, E. M., Genetics 32, 221 (1947). 17. ZELLE, M. R. and OGG, J. IL, J. Bacterial. 74, 485 (1957). Experimental

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