- Email: [email protected]
A heritable change in radiation resistance of strain L mouse cells
Experimental
326
A HERITABLE
CHANGE OF STRAIN
P. 0. W. RHYNAS Biology
Cell Research 21, 326-331
(1960)
IN RADIATION RESISTANCE L MOUSE CELLS and H. B. NEWCOMBE
and Health Physics Division, Atomic Energy of Canada Ltd., Chalk River, Ontario, Canada Received October 27. 1959
I-~CKI..~SE:I~ :radiation
resistance has been frequently observetl in turnours following repeated exposure to ionizin g radiation [7]. In an ascites turnour this resistance has been directly related to a change in the tumour tissue itself, rather than to a change in the host tissue or in the relationship between the tumour and its host [l]. resistant strains have been derived from In the present study, radiation in vitro suspension cultures of’ Earle’s strain I, mouse cells. The development of a number of resistant cell lines was studied in replicate cultures, ancl the chromosome complements of cells of the resistant lines were comparetl with those of unirradiatetl control line cells. MATERIALS
AND
METHODS
Cells crnd medium-Strain L mouse fibroblasts [5] were grown in suspension using CMRL 1066 [2] supplemented with 20 per cent horse serum together with penicillin (1 mg/ml) and dihydrostreptomycin (100 mg/ml). Cultures had a mean generation time of about 30 hours when grown in 1 by 6 inch test tubes rotated horizontally around their long axes at 45 rpm and maintained at 37” [6]. Suspension cultures of L cells were exposed to hard (2 Mev.) X-rays at a rate of 500 r per minute during the logarithmic phase of growth. The medium was changed (by centrifugation and re-suspension), and cell counts were made (using samples of unstained cells in a haemocytomcter) at least every second day after irradiation until the cultures had resumed their pre-irradiation growth rate, and at less frequent intervals thereafter. Densities of the cultures were then maintained between 5 ,: 10” and 1 x lo6 cells per ml by dilution with pre-warmed medium. The amount of growth achieved by a culture could be calculated by multiplying the cell count at any time by all previous dilution factors. Cytology.PChromosome counts were made of air-dried cells in metaphase from suspensions grown 1X-24 hours in colchicine using the method of Rothfels and Siminovitch [4]. Intact cells were selected for chromosome counts under “low power” ( x 100). Camera lucida tracings were made of each cell at x 1250 and then checked by direct microscopic examination at the same magnification, a green filter (Wratten X2) being used throughout. A series of ten cultures of L cells Development of radiation resistant cultures. Experimental
Cell Research 21
Radiation
327
resistance in strain L mouse cells
was exposed to a total dose of 5000 r distributed over a period of five months (three exposures to 1000 r and a final exposure to 2000 r) (Table I). Post-irradiation growth in these ten cultures was compared with that in ten control cultures (from the unirradiated parent line) following a 2000 r test exposure to both groups. ‘TABLE
Cycle 1 2 3 -I ’ Calculated cvclc.
I. Radiation
histories of the ten radiation
Dose (r) 1000 1000 1000 2000 from initial
pre-irradiation
Total no. cells irrad. (in ten 30 ml cultures) 1.0 1.2 0.8 0.7
x 108 Y 108 x lo* x 10”
resistant strains.
No. of days to next irradiation
Av. no. gencrations before next irrad.’
77 22 32 42
31 11 21 21
cell counts to the pre-irradiation
cdl counts of the next
RESULTS Hesistance.-All of the ten lines which had accumulated an exposure of .5000 r proved more resistant to the test exposure than did any of the ten control (parental line) cultures (Fig. 1). The initial growth in the resistant lines was depressed to a lesser extent, and the period of declining cell numbers \\fhich followed showed a less severe efTect than in the control cultures. Further, the resumption of a normal growth rate occurred much earlier than in the controls. Altertrtions in the chromosome numbers of’ reistnnt cclltures.--Another resistant culture, also a derivative of the unirradiated parent strain of L cells, and which had received a total of 10,000 r over an eight month period, was csamined in order to determine whether repeated irradiation had altered its chromosome number. Chromosome counts were made on thirty cells from the resistant lint and thirty from the normal parental line on days 43 and 54 folio\\-ing the final irradiation of the resistant line. Cells of the resistant line had a modal chromosome number of 57, five fewer than the parent line \vhich had a modal value of 62 chromosomes. The lower modal number of chromosomes in the resistant line \\-as also associated with a wider distribution of chromosome numbers (Fig. 2). In this and subsequent experiments occasional polpploid cells, naturally occurring or produced by the colchicine treatment, were not counted. There \yas no apparent dill-erencc in the numbers of these cells occurring in either the resistant or control lines. Experimenlai
Cell Kesearch 21
328
P. 0. W. Rhynas and H. B. Newcombe
Following this earlier observation, the chromosomes of 10 cells of each of the eight tested resistant lines were counted, and t\vo of the lines \vere sampled twice to make a total of 100 cells in all (on days 45, 46, 17, 53, 65, 67, and 68 following the test irradiation). These counts \vere compared with
I I , 1 I’
jr
4 RESISTANT J
rl
I 4
I
I
(
16 12 DAYS AFTER IRRADIATION 8
I
I
c
20
Fig. 1.--A comparison of the average growth of 10 pre-irradiated cultures and 10 previously unirradiated control cultures of strain L cells following a test exposure of 2000 r. The average of the initial cell counts in both resistant and control cultures was 2.2 x lo6 cells/ml. Short vertical lines at top indicate days on which medium was replaced. For comparative purposes, the increase or decrease factor was calculated from individual culture cell counts on days 0, 2, 4, 6, . . . 22 after irradiation. These values were averaged to give the points on the curve. Two resistant cultures were accidentally destroyed on day 16.
chromosome counts of a corresponding number of cells from the parent line (i.e. the unirradiated parental line was sampled at five intervals coinciding with days 1, 12, 51, 59, and 67, in relation to the irradiated series). All ten samples from the eight resistant lines showed a lower average chromosome number than any one of the samples from the parental line. The modal chromosome number for the parental line was 63, and for the eight resistant lines 60 (Fig. 3). Experimental
Cell Research 21
Radiation resistance in sfrain L mouse cells
329
Another resistant derivative of strain L, R3 of Whitfield and Rixon [Xl (originally a colony grown from the survivors of a 1000 r exposure) had an average chromosome number of 61.4, which was lower than the lowest average value in the control strain (footnote to Fig. S). 25-
ii, ,coNyL, ,A, , 8
35
40
45
50
55
60
65
50
55
60
65
70
75
70
75
k 5
15-
g1
IO-
RESISTANT
5I 35
I
Lm 40
DISTRIBUTION
45
OF CHROMOSOME Fig.
2.
NUMBER
I
PER CELL
DISTRIBUTION
OF CHROMOSOME
NUMBER
PER CELL
Fig. 3.
l:ig. 2..--Prequcncy distribution of chromosome numbers in 30 cells of strain 1, (black) cells of a resistant strain which had received an accumulated dose of 10,000 r (white).
and 30
Fig. 3--Frequency distribution of chromosome numbers in cells of strain 1. and of eight indcpcndently derived resistant strains (combined). Each value below represents the average of IO cells counted. Strain L (mean -63.5); 63.3, 62.9, 63.9, 63.7, 63.X,63.6, 64.3, 63.1. 63.3, 63.3. Resistant strains (mean -59.2); 56.7, 60.0, 60.9, 58.7, 55.8, 58.5, 61.5, 61.5, 5X.6, 59.3. (Values italicized are repeats on the preceding culture).
A search for other distinguishable tiill’crences in the chromosome complements of resistant and control cultures sho\vetl that cells of the resistant lines almost always lacked a large metacentric chromosome which is the most easily recognized member of the parent cell complement (see Fig. 4 and, for a previous description, Hsu and Klatt [37). The 100 rcsistanl cells originally cxamincd for chromosome number \vcrc re-examined for presence or absence of this large chromosome; only t\Yo cells appeared to contain it (and even these \vere doubtful), as against 98 out of 100 cells examined of Ihe parent line. It was possible therefore to distinguish between normal and resistant lines on the basis of the presence or absence of this chromosome by examining as few as 10 cells of a given line. Experimenfal
Cell Research 21
P. 0. W. Rhynas and H. B. Newcombe
Fig. 4.-The
large metacentric
chromosome
of a typical
strain L mouse cell.
x 4000.
Two of the resistant lines have been tested again for resistance after about 25 and 35 cell generations of growth respectively. No loss of resistance occurred. DISCUSSION
It is clear that the observed increase in resistance is heritable over many cell generations. However, the time of occurrence of the initial change, and its nature, need to be considered further. The exposures to radiation may have acted solely to select out resistant Experimental
Cell Research 21
Radiation resistance in strain L mouse cells
331
cells which had resulted from prior changes in the absence of’irradiation, or alternatively the exposures may have produced the changes as well as selecting the products of the change. The former possibility seems much more likely in view of the ease with which resistant lines have been obtained repeatedly in the present experiments. Also, since resistant lines can be obtained following only two exposures (and in Whitfield and Rixon’s experiment [8] following a single exposure), it seems unlikely that the irradiation is responsible for the production as well as the selection of resistant lines of descent. The consistency with which the resistant lines exhibit lolver average chromosome numbers than the parent strain strongly suggests a chromosomal basis for the increased resistance. It is most unlikely that similar changes in number would have occurred fortuitously in all of the eight lines examined. l’olyploidy seems to have played no part in the development of radiation resistance in this material. Polyploid cells occurred in both the resistant and parent lines, but were relatively infrequent throughout and showed no apparent increase in the resistant strains. SUMMARY
Repeated exposure of mass cultures of L strain mouse cells to high doses of ionizing radiation resulted in new strains which had an increased resistance to S-rays. After a cumulative dose of 5000 r spread over five months (80-90 cell generations), growth of these cell lines following a test exposure to 2000 r \vas compared with that of cultures of the control strain. After the test exposure, growth of the pre-irradiated lines was resumed much sooner than in the controls. The resistant cell lines all had a lower average chromosome number with a wider spread of numbers than had the parental control line. The authors wish to thank Dr. L. Siminovitch, of the Ontario Cancer Institute, who kindly supplied the strain of cells used throughout these experiments. REFERENCES 1. 2. 3. 4. 5. 6.
DITTRICH, W., HOHNE, G. and SCHUBERT, G., Progr. in Radiobiol. p. 381 (1956). HEALY, G. M., FISHER, D. C. and PARKER, R. C., Proc. Sot. Exptl. Biot. Med. 89, 71 (1955). Hsu, T. C. and KLATT, O., J. Natl. Cancer Inst. 21, 437 (1958). ROTHFELS, K. H. and SIMINOVITCH, L., Stain Technot. 33, 73 (1958). SANFORD, K. K., EARLE, W. R. and LIKELY, G. D., J. Nat.!. Cancer Inst. 9, 229 (1948). SIMINOVITCH, L., GRAHAM, A. F., LESLEY, S. M. and NEvILL, A., Exptt. Cell Research 12, 299
(1957). 7. UPTON, A. C., Federation 8. WIIITFIELD,
Proc. 17, 698 (1958). R. H., Exptl. Cell Research 19, 531 (1960).
J. F. and RIXOX,
Experimental
Cell Research 21
326
A HERITABLE
CHANGE OF STRAIN
P. 0. W. RHYNAS Biology
Cell Research 21, 326-331
(1960)
IN RADIATION RESISTANCE L MOUSE CELLS and H. B. NEWCOMBE
and Health Physics Division, Atomic Energy of Canada Ltd., Chalk River, Ontario, Canada Received October 27. 1959
I-~CKI..~SE:I~ :radiation
resistance has been frequently observetl in turnours following repeated exposure to ionizin g radiation [7]. In an ascites turnour this resistance has been directly related to a change in the tumour tissue itself, rather than to a change in the host tissue or in the relationship between the tumour and its host [l]. resistant strains have been derived from In the present study, radiation in vitro suspension cultures of’ Earle’s strain I, mouse cells. The development of a number of resistant cell lines was studied in replicate cultures, ancl the chromosome complements of cells of the resistant lines were comparetl with those of unirradiatetl control line cells. MATERIALS
AND
METHODS
Cells crnd medium-Strain L mouse fibroblasts [5] were grown in suspension using CMRL 1066 [2] supplemented with 20 per cent horse serum together with penicillin (1 mg/ml) and dihydrostreptomycin (100 mg/ml). Cultures had a mean generation time of about 30 hours when grown in 1 by 6 inch test tubes rotated horizontally around their long axes at 45 rpm and maintained at 37” [6]. Suspension cultures of L cells were exposed to hard (2 Mev.) X-rays at a rate of 500 r per minute during the logarithmic phase of growth. The medium was changed (by centrifugation and re-suspension), and cell counts were made (using samples of unstained cells in a haemocytomcter) at least every second day after irradiation until the cultures had resumed their pre-irradiation growth rate, and at less frequent intervals thereafter. Densities of the cultures were then maintained between 5 ,: 10” and 1 x lo6 cells per ml by dilution with pre-warmed medium. The amount of growth achieved by a culture could be calculated by multiplying the cell count at any time by all previous dilution factors. Cytology.PChromosome counts were made of air-dried cells in metaphase from suspensions grown 1X-24 hours in colchicine using the method of Rothfels and Siminovitch [4]. Intact cells were selected for chromosome counts under “low power” ( x 100). Camera lucida tracings were made of each cell at x 1250 and then checked by direct microscopic examination at the same magnification, a green filter (Wratten X2) being used throughout. A series of ten cultures of L cells Development of radiation resistant cultures. Experimental
Cell Research 21
Radiation
327
resistance in strain L mouse cells
was exposed to a total dose of 5000 r distributed over a period of five months (three exposures to 1000 r and a final exposure to 2000 r) (Table I). Post-irradiation growth in these ten cultures was compared with that in ten control cultures (from the unirradiated parent line) following a 2000 r test exposure to both groups. ‘TABLE
Cycle 1 2 3 -I ’ Calculated cvclc.
I. Radiation
histories of the ten radiation
Dose (r) 1000 1000 1000 2000 from initial
pre-irradiation
Total no. cells irrad. (in ten 30 ml cultures) 1.0 1.2 0.8 0.7
x 108 Y 108 x lo* x 10”
resistant strains.
No. of days to next irradiation
Av. no. gencrations before next irrad.’
77 22 32 42
31 11 21 21
cell counts to the pre-irradiation
cdl counts of the next
RESULTS Hesistance.-All of the ten lines which had accumulated an exposure of .5000 r proved more resistant to the test exposure than did any of the ten control (parental line) cultures (Fig. 1). The initial growth in the resistant lines was depressed to a lesser extent, and the period of declining cell numbers \\fhich followed showed a less severe efTect than in the control cultures. Further, the resumption of a normal growth rate occurred much earlier than in the controls. Altertrtions in the chromosome numbers of’ reistnnt cclltures.--Another resistant culture, also a derivative of the unirradiated parent strain of L cells, and which had received a total of 10,000 r over an eight month period, was csamined in order to determine whether repeated irradiation had altered its chromosome number. Chromosome counts were made on thirty cells from the resistant lint and thirty from the normal parental line on days 43 and 54 folio\\-ing the final irradiation of the resistant line. Cells of the resistant line had a modal chromosome number of 57, five fewer than the parent line \vhich had a modal value of 62 chromosomes. The lower modal number of chromosomes in the resistant line \\-as also associated with a wider distribution of chromosome numbers (Fig. 2). In this and subsequent experiments occasional polpploid cells, naturally occurring or produced by the colchicine treatment, were not counted. There \yas no apparent dill-erencc in the numbers of these cells occurring in either the resistant or control lines. Experimenlai
Cell Kesearch 21
328
P. 0. W. Rhynas and H. B. Newcombe
Following this earlier observation, the chromosomes of 10 cells of each of the eight tested resistant lines were counted, and t\vo of the lines \vere sampled twice to make a total of 100 cells in all (on days 45, 46, 17, 53, 65, 67, and 68 following the test irradiation). These counts \vere compared with
I I , 1 I’
jr
4 RESISTANT J
rl
I 4
I
I
(
16 12 DAYS AFTER IRRADIATION 8
I
I
c
20
Fig. 1.--A comparison of the average growth of 10 pre-irradiated cultures and 10 previously unirradiated control cultures of strain L cells following a test exposure of 2000 r. The average of the initial cell counts in both resistant and control cultures was 2.2 x lo6 cells/ml. Short vertical lines at top indicate days on which medium was replaced. For comparative purposes, the increase or decrease factor was calculated from individual culture cell counts on days 0, 2, 4, 6, . . . 22 after irradiation. These values were averaged to give the points on the curve. Two resistant cultures were accidentally destroyed on day 16.
chromosome counts of a corresponding number of cells from the parent line (i.e. the unirradiated parental line was sampled at five intervals coinciding with days 1, 12, 51, 59, and 67, in relation to the irradiated series). All ten samples from the eight resistant lines showed a lower average chromosome number than any one of the samples from the parental line. The modal chromosome number for the parental line was 63, and for the eight resistant lines 60 (Fig. 3). Experimental
Cell Research 21
Radiation resistance in sfrain L mouse cells
329
Another resistant derivative of strain L, R3 of Whitfield and Rixon [Xl (originally a colony grown from the survivors of a 1000 r exposure) had an average chromosome number of 61.4, which was lower than the lowest average value in the control strain (footnote to Fig. S). 25-
ii, ,coNyL, ,A, , 8
35
40
45
50
55
60
65
50
55
60
65
70
75
70
75
k 5
15-
g1
IO-
RESISTANT
5I 35
I
Lm 40
DISTRIBUTION
45
OF CHROMOSOME Fig.
2.
NUMBER
I
PER CELL
DISTRIBUTION
OF CHROMOSOME
NUMBER
PER CELL
Fig. 3.
l:ig. 2..--Prequcncy distribution of chromosome numbers in 30 cells of strain 1, (black) cells of a resistant strain which had received an accumulated dose of 10,000 r (white).
and 30
Fig. 3--Frequency distribution of chromosome numbers in cells of strain 1. and of eight indcpcndently derived resistant strains (combined). Each value below represents the average of IO cells counted. Strain L (mean -63.5); 63.3, 62.9, 63.9, 63.7, 63.X,63.6, 64.3, 63.1. 63.3, 63.3. Resistant strains (mean -59.2); 56.7, 60.0, 60.9, 58.7, 55.8, 58.5, 61.5, 61.5, 5X.6, 59.3. (Values italicized are repeats on the preceding culture).
A search for other distinguishable tiill’crences in the chromosome complements of resistant and control cultures sho\vetl that cells of the resistant lines almost always lacked a large metacentric chromosome which is the most easily recognized member of the parent cell complement (see Fig. 4 and, for a previous description, Hsu and Klatt [37). The 100 rcsistanl cells originally cxamincd for chromosome number \vcrc re-examined for presence or absence of this large chromosome; only t\Yo cells appeared to contain it (and even these \vere doubtful), as against 98 out of 100 cells examined of Ihe parent line. It was possible therefore to distinguish between normal and resistant lines on the basis of the presence or absence of this chromosome by examining as few as 10 cells of a given line. Experimenfal
Cell Research 21
P. 0. W. Rhynas and H. B. Newcombe
Fig. 4.-The
large metacentric
chromosome
of a typical
strain L mouse cell.
x 4000.
Two of the resistant lines have been tested again for resistance after about 25 and 35 cell generations of growth respectively. No loss of resistance occurred. DISCUSSION
It is clear that the observed increase in resistance is heritable over many cell generations. However, the time of occurrence of the initial change, and its nature, need to be considered further. The exposures to radiation may have acted solely to select out resistant Experimental
Cell Research 21
Radiation resistance in strain L mouse cells
331
cells which had resulted from prior changes in the absence of’irradiation, or alternatively the exposures may have produced the changes as well as selecting the products of the change. The former possibility seems much more likely in view of the ease with which resistant lines have been obtained repeatedly in the present experiments. Also, since resistant lines can be obtained following only two exposures (and in Whitfield and Rixon’s experiment [8] following a single exposure), it seems unlikely that the irradiation is responsible for the production as well as the selection of resistant lines of descent. The consistency with which the resistant lines exhibit lolver average chromosome numbers than the parent strain strongly suggests a chromosomal basis for the increased resistance. It is most unlikely that similar changes in number would have occurred fortuitously in all of the eight lines examined. l’olyploidy seems to have played no part in the development of radiation resistance in this material. Polyploid cells occurred in both the resistant and parent lines, but were relatively infrequent throughout and showed no apparent increase in the resistant strains. SUMMARY
Repeated exposure of mass cultures of L strain mouse cells to high doses of ionizing radiation resulted in new strains which had an increased resistance to S-rays. After a cumulative dose of 5000 r spread over five months (80-90 cell generations), growth of these cell lines following a test exposure to 2000 r \vas compared with that of cultures of the control strain. After the test exposure, growth of the pre-irradiated lines was resumed much sooner than in the controls. The resistant cell lines all had a lower average chromosome number with a wider spread of numbers than had the parental control line. The authors wish to thank Dr. L. Siminovitch, of the Ontario Cancer Institute, who kindly supplied the strain of cells used throughout these experiments. REFERENCES 1. 2. 3. 4. 5. 6.
DITTRICH, W., HOHNE, G. and SCHUBERT, G., Progr. in Radiobiol. p. 381 (1956). HEALY, G. M., FISHER, D. C. and PARKER, R. C., Proc. Sot. Exptl. Biot. Med. 89, 71 (1955). Hsu, T. C. and KLATT, O., J. Natl. Cancer Inst. 21, 437 (1958). ROTHFELS, K. H. and SIMINOVITCH, L., Stain Technot. 33, 73 (1958). SANFORD, K. K., EARLE, W. R. and LIKELY, G. D., J. Nat.!. Cancer Inst. 9, 229 (1948). SIMINOVITCH, L., GRAHAM, A. F., LESLEY, S. M. and NEvILL, A., Exptt. Cell Research 12, 299
(1957). 7. UPTON, A. C., Federation 8. WIIITFIELD,
Proc. 17, 698 (1958). R. H., Exptl. Cell Research 19, 531 (1960).
J. F. and RIXOX,
Experimental
Cell Research 21