- Email: [email protected]
Radiation response of mammalian cells grown in culture
Experimental
Cell Research 30, 481-491 (1963)
RADIATION III.
481
RESPONSE OF MAMMALIAN GROWN IN CULTURE
CELLS
MODIFICATION OF X-RAY SURVIVAL OF CHINESE CELLS BY 5-BROMODEOXYURIDINE W. C. MOHLER
Clinical PharmacoIogy and Experimental of Physiology, National Cancer Institute,
HAMSTER
and M. M. ELKIND
Therapeutics Service, Medicine Branch and Laboratory National Institutes of Health, Bethesda, Md., U.S.A.
ReceivedJuly 16, 19621
THE thymidine analogue, 5-bromodeoxyuridine (BUDR), is known to increase the X-ray response of a strain of human cells grown in vitro [B]. We have studied this effect in a cell culture originating from a different tissue in a different species and have examined the relationships between varied exposures to BUDR and changes in X-ray survival parameters. We found sensitization occurring after growth in BUDR at concentrations lower than those previously reported [8]. The characteristic alterations found in radiation survival parameters, in cell growth and in viability suggest that in our cell line the cytotoxic effects of BUDR and at least part of its potentiating effect on X-ray response are closely related. MATERIAL
AND METHODS
The techniques employed were similar to those reported previously [6, 121; differences are described below. A subculture of a Chinese hamster cell line derived from female lung tissue designated V79-1 [6] was established September, 1959 and propagated serially. These cells grow attached to glass and appear “fibroblast-like”. Stock cultures were grown in milk dilution bottles with medium HU-15 [6] and were subdivided forty-fold twice weekly. Except where otherwise indicated, cultures for an experiment were grown in medium EM-10 plus 1 x 10-OM thymidine for six days prior to irradiation according to the schedule shown in Table I. Medium EM-IO is the same as HU-15 except that it lacks the supplementary 4 per cent culture medium NCTC-109 and contains only 10 per cent undialyzed fetal calf serum. In our early experiments we found variations in X-ray survival curves after growth in BUDR which we thought might be due to differences in endogenous thymidine production by the cells. Thymidine was added to prevent these variations. Because much data had been accumulated using EM-10 plus thymidine, the addition was 1 Revised 31- 631811
version
received
November
12, 1962. Experimental
Cell Research 30
W. C. Mohler
and M. M. Elkind
continued even after it became apparent that factors other than endogenous thymidine production were probably responsible for the original variations. Cell suspensions used for stock cultures or experiments were prepared by washing glass-attached cells once with a trypsin solution, incubating the cells at 37°C for IO min in another aliquot of the same solution, adding an equal volume of medium, and dispersing the cells by gentle pipetting. The trypsin solution contained 0.05 per cent trypsin (I-300, Nutritional Biochemical Corp.) in phosphate buffered saline (0.15 M NaCl plus 0.005 M potassium phosphate; pH 7.4). TABLE
I. Protocol for Growth
of Experimental
Cultures Prior
to Irradiation.
Culture of V79-1 presumably incorporate BUDR and deplete the medium at a rate proportional to the total cell number, which increases exponentially. Therefore, the medium was replaced frequently toward the end of each three day period. On day 0 and day 3 a sufficient number of cells was planted to give approximately 5 x lo8 cells on day 3 and day 6. Day Day Day Day Day
O-Start cultures from V79-1 stock into 20 ml of medium. a--Replace medium with 25 ml in early morning and late evening. 3-Trypsinize in early morning and replant in 20 ml of medium. B-Replace medium with 25 ml in early morning and late evening. 6-Trypsinize, dilute, attach and irradiate. Medium replaced with for cultures irradiated in afternoon.
25 ml in early morning
For the X-ray studies, cells suspended in EM-IO were allowed to attach for 60 to 100 min at 37°C to the bottoms of 9 cm petri dishes. A pH of 7.4 to 7.6 was mamtained by gassing the incubator with a humidified mixture of air and CO*. Since soft X-rays were used (55 kv, 722 rad/min) the medium was removed after attachment and the cells were irradiated under a humidified CO,-air atmosphere [6]. Appropriate amounts of HU-15 were added to the dishes immediately after irradiation, and stained macroscopic colonies were counted without magnification after the surviving cells had grown for 11 days at 37°C. Unirradiated cells, which otherwise received the same treatment, served as controls for the determination of surviving fraction. The inoculum of cells in plates receiving irradiation was increased to result in approximately the same final number of colonies, i.e. 200, expected on the control plates. BUDR for these studies was obtained from the Cancer Chemotherapy National Service Center of the National Cancer Institute.
Radiation Survival Parameters The survival curve of those observed with other region is followed at higher [6]. Without presupposing model, one can-describe and the dose (Da) which the exponential region. Experimental
Cell Research 30
V79-1 cells not mammalian cell X-ray doses by the applicability such curves by reduces survival
treated with BUDR is similar to lines. In such curves a shoulder an exponential decline in survival of any particular inactivation an extrapolation number (ii) [l] by the fraction l/e (i.e. 0.37) in
X-ray survival modified by BUDR
483
Straight lines were fitted by eye to the exponential portion of survival curves plotted on semi-logarithmic paper and extrapolated to zero dose to obtain ii. fiO was calculated from the dose which reduced survival by (l/e)’ or 0.00091 on the fitted line. The cell suspensions prepared for X-ray survival work always contained more than 90 per cent single cells with a few clumps of 2 or 3 cells. Since V79-1 cells have been shown to survive X-irradiation independently [6], we reduced the observed extrapolation number by the cellular multiplicity (i.e., the average number of cells per potential colony forming unit) in the data to be presented below. This correction is not exact but the uncertainty involved has no effect on the interpretation of our data, since the multiplicity was always less than 1.10. RESULTS
Growth of V79-1 in BUDR.-The
rate of cell growth changes markedly during the first two to three days for V79-1 grown in concentrations of BUDR greater than 1 X 10-e M. We therefore calculated the doubling time (T,) of cells in mass culture from the increase in cell number during the last three days of the standard six day growth period described in Table I. The results are shown as open circles in Fig. 1. For cells grown in the absence of BUDR, T, averaged 11.5 hr. No significant increase in T, was seen for celis growing in concentrations of BUDR less than 2 X 10-7 M. A marked increase in Tz occurred above 1 x 10-e M. In one experiment (closed triangles in Fig. 1). T, was calculated from growth curves obtained by counting the number of cells per colony in 100 colonies on plates which were fixed and stained at daily intervals after inoculation 6Or
’ Fig. l.-Doubling
““,’ 10%
I BUDR IN MEDIUM
‘O-”
time (Ta) of V79-1 grown in BUDR.
1
IO-%
See text for details. Experimental
Cell Research 30
484
u’. C. Mohler
and M. M. Elkind
with 1000 cells. The results agree rather well with Tz obtained from mass culture measurements. Viability of cells.-As a measure of the viability of cells after six days growth in BUDR, we calculated the plating efficiency (P.E.), i.e., the percentage of the potential colony forming units in a trypsinized suspension which grew into macroscopic colonies. For control cultures grown in the
. - -.-,-_r . . .
‘\ .
a\
T
\
‘. \
0 -’ c!+ BUDR IN MEDIUM
Fig. 2.-Plating
efficiency of cells after growth for six days in BUDR. See text for details.
absence of BUDR, the P.E. averaged 88 per cent with a range from 80 per cent to 95 per cent. Fig. 2 shows the P.E. of cells grown in BUDR plotted as the per cent of the P.E. for control cells in each experiment. No significant change was seen at BUDR concentrations less than 2 x 10-7 M, A marked decrease in viability was noted above 1 X 10-G M. Effect of growth in BUDR on X-ray survival parameters.-Cells grown in BtJDR showed an increased response to the lethal effects of X-rays. Fig. 3 illustrates the progression in response with increasing concentrations of BUDR. There is a decrease in both ii and 8, as the BUDR concentration increases. The radiation response of V79-1 cells is stable after approximately the first two days of growth in a given concentration of BUDR. This is illustrated in Fig. 4 for cells grown in 1 x 10-e M (closed symbols) and 5 x lo-6 M (open symbols). The X-ray survival parameters remained unchanged even though the percentage of viable cells in the culture decreased. For example, the P.E. for cells grown in 1 X 10-e M BUDR dropped from 80 per cent of control at 60 hr to 65 per cent at 108 hr and the P.E. for cells grown in 5 X 10-e M BUDR decreased from 28 per cent of control at 46 hr to 8 per cent at 94 hr. The variation in go with BUDR concentration is shown in Fig. 5. fiO is a Ezperimenfal
Cell Resenrch 30
X-ray survival modified by BUDR smooth function of BUDR concentration even up to 5 x 10-S M, a concentration at which less than 10 per cent of the cells were viable after six days growth. There is a decrease in fiO even at BUDR levels below 2 X 10-7 M, which do not detectably alter the growth rate or viability. This can be seen
Fig. J.-X-ray survival curves for V79 after growth in BUDR for 6 days. The specific ISUDR concentration is indicated for each curve. Standard errors, estimated by the method of Mantel [13], are shown when larger than the symbols.
-
6
-
5
-
4;
: x2 -3 -2
50 HOURS IN EUDR PRIOR TO IRRADIATION
Fig. I.-Change in X-ray survival parameters during growth in BUDR..Closed symbols for cells grown in 1 x lo-‘M BUDR. Open symbols for cells grown in 5 x 10-8M BUDR. Arrow indicates time of trypsinization and replanting, explained in Table I. Experimental
Cell Research 30
W. C. Mohler and M. M. Elkind
486
more clearly in Fig. 6 where g,, for each culture grown in BUDR is plotted as a percentage of that for the control (no BUDR) culture in the same experiment. (For 17 cultures grown without BUDR, b,, averaged 144.5 rad with a standard deviation of 6.2 rad or 4.3 per cent). 150 v 1
v A
. 8 .
p ‘;I00 ZD
. . .
I
BUDR IN MEDIUM
Fig. 5.-Change
0 t h-J 0
in 0, as a function
,(I”,’
of BUDR
I lo-%/
IO-‘M
concentration.
_u_wJ
IO-M
BUDR IN MEDIUM
Fig. 6.-Change in 0, compared with 0, of control cultures which were grown under identical conditions in the absence of BUDR. This presentation emphasizes the change in 0: which occurs at low BUDR concentration where no other alterations in growth, viability or extrapolation number can be detected.
The extrapolation number behaved differently, as shown in Fig. 7. The value for control cultures varied from experiment to experiment. In seventeen cultures grown without BUDR, fi averaged 5.70 with a standard deviation of 1.44 or 25 per cent. In spite of this variability, cells grown in BUDR concentrations of 1 x 10-7 M or less did not give X-ray survival curves with ii less than the ii of the control culture in the same experiment. (Compare values for individual experiments represented by different symbols in Fig. 7). At 2 X 10-T M and above, fi was always less than corresponding control value Experimental
Cell Research 30
X-ray survival modified by BUDR
487
and at 2 x 10-e M and above, survival curves were exponential and hence ii=l. Radiation response after BUDR is removed.-A subculture of V79-1 was grown in medium HU-15 (which contains 1.6 X 10-S M thymidine) plus
I
,,,,I,’
I
I IO-%
lOA BUDR IN MEDIUM’“-‘M
Fig. 7.-Change
TABLE
II.
Reversal
in extrapolation
number
as a function
of BUDR
concentration.
of Radiation Response After Culture Medium.
BUDR
is Removed
From
V79-1 was grown in HU-15 plus 2.6 x 10-eM BUDR for 79 days before BUDR was omitted from cultures. See text for details. The growth factor is the increase in number of cells relative to the replicate culture at time 0. P. E. is the plating efficiency, defined under Results. Replicate
#l #2 #3
Hr after Omitting
0 23 47
BUDR
b
ii
90 rad 143 u 188 I)
1.1 1.9 2.5
P. E.
37 37 33
Growth
Factor
1 1.6 4.7
1.6 X 10-6 M BUDR. The subculture was carried by transferring l/20 to l/40 of the cells to fresh medium every 3 to 4 days. At the end of 79 days, an X-ray survival curve was determined with one of three replicate cultures and the other two were refed with HU-15 containing no BUDR. X-ray survival curves were determined with the second replicate culture after 23 hr and with the third after 47 hr of growth in medium lacking BUDR. Table II summarizes the results. The increase in 6, and ii indicates that these cells Experimental
Cell Research 30
488
W. C. Mohler and M. M. Elkind
returned toward a “normal” radiation response during the first few generations after the removal of BUDR. The relatively low ii at 47 hr is compatible with some population heterogeneity. The high fi,, at 47 hr is unexplained and may be related to a change induced by prolonged growth in BUDR since the subline had a hyperploid chromosome number (greater than 35) instead of the near-diploid number of 23 characteristic of the stock strain examined at the same time [14]. Also, it should be noted that in control experiments we have observed over a long period of time that variations in ii are usually accompanied by inverse variations in 5, [6]. In any case, the rapid change back toward an unaltered X-ray survival response is evidence supporting the view that BUDR does not modify radiation response by selecting a pre-existing moiety of V79-1 cells which has an inherently low ii and fiO. A similar rapid change back toward “normal” has been reported for another cell strain in which BUDR increased the response to the lethal effects of ultraviolet light [3].
IHSCUSSION
Our findings with this Chinese hamster cell line, V79-1, can be compared with the results of Erickson and Szybalski [S], who studied the effects of halogenated pyrimidines upon the X-ray response of a human sternal bone marrow cell line, D98/AG. They exposed their cells to BUDR for four days, which was probably equivalent to about 4 doubling times for this slower growing cell line [3]. While it is not clear that this exposure produced maximum changes in n” and & their results differ in at least two respects from ours. First, even though they used 5fluorodeoxyuridine to inhibit thymidylate synthetase and thus increase the amount of thymidine replaced by BUDR in cellular deoxyribonucleic acid (DNA), they required higher concentrations of BUDR to produce equivalent percentage changes in & Their data indicate that about 2 X 10-S M BUDR was required to achieve a 50 per cent reduction in 5, from their control fi,, of 180r. Second, their data suggest that n” either remained the same or increased even at concentrations of BUDR where fiO was reduced to half the control value, a fi,, reduction at which we found fi to have dropped to 1.0. These differences appear even more striking if one assumes from other data [3] that their cells which exhibit about 50 per cent reduction in E,, had about 40 per cent of DNA thymidine replaced by BUDR. Preliminary studies indicate that V79-1 cells which had a comparable reduction in fi,, had less than 10 per cent replacement of thymidine by BUDR [14]. These discrepancies may be due in part to physical factors since Erickson Experimental
Cell Research 30
X-ray survival modified by BUDR and Szybalski used 140 kv X-rays and we used 55 kv X-rays. The relative difference between the X-ray absorption cross sections of the bromine in BUDR and the analogous methyl group in thymidine will be more pronounced for the softer X-ray spectrum due to a relatively larger photoelectric effect. More energy should be absorbed locally with softer X-rays for the same average exposure in both cases. The answer to questions of this type must await definitive studies of the relative biological effectiveness of different qualities of radiation as a function of BUDR incorporation. It might well be noted also that other mammalian cells have exhibited differences in their ability to tolerate exposure to BUDR or its incorporation into their DNA [lo]. Before considering the possible modes of action of BUDR it is first necessary to question whether or not the potentiating effects of BUDR are only apparent. Recent results suggest that single cell survival curves which may appear to represent a homogeneous population can, in fact, be the sum of survival curves of two or more subpopulations which survive differently [7, 171. Hence BUDR might be killing or inhibiting the growth of some cells and thereby selecting for more X-ray responsive cells. Selection seems unlikely at BUDR concentrations less than 2 x 10-7 M since no toxicity was manifest. Yet Do, which is the parameter least responsive to slight modification in the proportions of subpopulations, is significantly and progressively altered at these low concentrations. The alterations in ii seen at BUDR concentrations greater than 2 x 10-7 M might be due to selective killing. However, an analysis of the conditions under which this would occur does not favor a dominant selective influence. First, from the kinds of analyses dealing with population heterogeneity which were considered in an earlier paper [5], it can be shown that our results preclude selection by BUDR if in the absence of drug all moieties had the same fi. In such a case the observed extrapolation number of the remaining population would have to increase, not decrease, as the more X-ray resistant moieties were killed off by the cytotoxic action of BUDR. The general type of population heterogeneity consistent with our results would require that the X-ray survival parameters of subpopulations of V79-1 (not exposed to BUDR) must have small ii’s correlated with small D,‘s and large ii’s with large &‘s. In the second place, the experiments summarized in Fig. 5 show that X-ray survival parameters remained constant after 35 to 45 hr growth in BUDR in spite of a continued cytotoxic action of the drug such that in experiment # 245 (1 x 10-B M BUDR) the P.E. dropped from N 80 per cent at 60 hr # 319 (5 x 10-S M BUDR) it to -65 per cent at 108 hr and in experiment dropped from - 30 per cent at 46 hr to w 8 per cent at 94 hr. Finally, the Experimental
Cell Research 30
W. C. Mohler
and M. M. Elkind
data in Table II indicated that even after a prolonged exposure to BUDR, V79-1 cells quickly returned toward a radiation response resembling that of cells never exposed to BUDR, in spite of evidence that the prolonged growth in BUDR had exerted a selective influence. It is reasonable that the radiation modifying effect of BUDR should be sought at an intracellular level since, as a thymidine analogue, BUDR is incorporated into DNA thereby altering the cell’s genome. The work of Opara-Kubinska and Szybalski [15] on ultraviolet (U.V) damage strongly support this thesis. Their experiments show that transforming principle DNA from Bacillus subtilis is rendered considerably more sensitive as a result of incorporation of the bromine analogue. Szybalski and coworkers [S, 15, 161 have proposed that the potentiating effects of halogenated pyrimidines in general indicate that DNA is the most radiosensitive cell component and therefore the principal target in the lethal action of both U.V. and X-rays. It seems to us that this conclusion must be viewed with caution particularly when one proposes a mechanism of action for ionizing radiation in cells which have not been exposed to pyrimidine analogs. In mammalian cells the vast majority of primary ionizations registered in DNA molecules must be innocuous. For example, in V79-1 cells not exposed to BUDR about lo3 DNA molecules per cell will receive primary ionization per h,, dose. Therefore it seems reasonable to expect that most of the primary DNA damage is ordinarily repaired, by-passed, or registered in sites unrelated to reproductive integrity. The action of BUDR might be either to make sensitive some sites of incorporation which otherwise would not have contributed significantly to X-ray sensitivity in the normal cell or to impede the repair or by-passing of damaged sites. Support for both possibilities exist since it is known that brominated uracil can produce “hot spots” of mutation in DNA [a], and the recent results of Howard-Flanders and Boyce [9] suggest that BUDR may enhance U.V. killing of T, phage by inhibiting repair processes which would otherwise occur after bacterial infection. Drugs like BUDR have been discussed as possible adjuncts in the radiotherapy of tumors. We do not feel able from our studies to comment on the relative merits of “unifilar” or “bifilar” labelling of DNA [3, 111. It is clear, however, from experiments like those shown in Fig. 5 that to be most effective in altering the radiation response of V79-1 BUDR must be maintained in relatively cytotoxic concentrations for several doubling times. In terms of conventional therapeutic fractionation protocols, it appears that the important change induced by BUDR might be a relatively large reduction of fi and reduced recovery between exposures [4, 111. Experimental
Cell Research 30
X-ray survival modified by BUDR
491
SUMMARY
Chinese hamster cells (strain V79-1) cultured in vitro show an increased response to the lethal effects of X-rays when they are grown prior to irradiation in 5-bromodeoxyuridine (BUDR). These cells appear to be more sensitive than the human cell line, D98/AG, studied by Erickson and Szybalski to the radiation modifying as well as the cytotoxic effects of BUDR. BUDR produces a striking change in the extrapolation number of X-ray survival curves of V79-1 as well as the change in slope reported in D98/AG. Both of these changes appear to be the result of alterations in cellular radiation response and not the result of population heterogeneity. When cells having an increased X-ray response induced by BUDR are removed from contact with BUDR, they reassume X-ray survival parameters similar to normal cells within one or two generations. The authors gratefully acknowledge the assistance of Miss Bernadine Bianchi, Mrs. Carolyn Kimler and Mrs. Ovella Ayers during many portions of these experiments. REFERENCES T., GILLIES, N. E. and ELKIND, M. M., Nature 186, 1062 (1960). BENZE~, s., Proc. Natl. Acad. Sci. USi 47, 403 (1961). ’ DJORDJEVIC. B. and SZYBALSKI, W., J. Es&Z. Med. 112, 509 (1960). . , ELKIND, M. M., Radiology 74, 5i9 (iSSO). . __ Brookhaven Symp. in Mot. 14, 220 (1961). ELKIND, M. M. and SUTTON, H., Radiation Res. 13, 556 (1960). ELKIND, M. M., SUTTON, H. and MOSES, W. B., J. Cellular Comp. Physiot. 58, Suppl.
1. ALPER,
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
1, 113 (1961). ERICKSON. R. L. and SZYBALSKI. W., Biochem. Biouhvs. Res. Commun. 4, 258 (1961). HOWARD-FLANDERS, P. and BOYCE, k. P., Radiation kes. 16, 563 (1962): ’ Hsu, T. C. and SOMERS, E. E., Exptl. Cell Res. 26, 404 (1962). KAPLAN, H. S., SMITH, K. C. and TOMLIN, P. A., Radiation Res. 16, 98 (1962). LOCKART, JR., R. Z., ELKIND, M. M. and MOSES, W. B., J. Natl. Cancer Inst. 27,1393 (1961). MANTEL, N., Am. Statistician 5, 26 (1951). MOHLER, W. C. and ELKIND, M. M., Unpublished results. OPARA-KUBINSKA, Z., LoRK;EwIcz,.~~~~SYBALSKI, W., Biochem. Biophys. Res. Commun. 4,
288 (1961). SZYBALSK;, Natl. 17. TERASIMA, 16.
W.; in Research in Radiotherapy, p. 162. KALLMAN, Acad. Sci. Washington, D.C., 1961. T. and TOLMACH, L. J., Nature 190, 1210 (1961).
R. F. (ed). Pnblication
Experimental
888,
Cell Research 30
Cell Research 30, 481-491 (1963)
RADIATION III.
481
RESPONSE OF MAMMALIAN GROWN IN CULTURE
CELLS
MODIFICATION OF X-RAY SURVIVAL OF CHINESE CELLS BY 5-BROMODEOXYURIDINE W. C. MOHLER
Clinical PharmacoIogy and Experimental of Physiology, National Cancer Institute,
HAMSTER
and M. M. ELKIND
Therapeutics Service, Medicine Branch and Laboratory National Institutes of Health, Bethesda, Md., U.S.A.
ReceivedJuly 16, 19621
THE thymidine analogue, 5-bromodeoxyuridine (BUDR), is known to increase the X-ray response of a strain of human cells grown in vitro [B]. We have studied this effect in a cell culture originating from a different tissue in a different species and have examined the relationships between varied exposures to BUDR and changes in X-ray survival parameters. We found sensitization occurring after growth in BUDR at concentrations lower than those previously reported [8]. The characteristic alterations found in radiation survival parameters, in cell growth and in viability suggest that in our cell line the cytotoxic effects of BUDR and at least part of its potentiating effect on X-ray response are closely related. MATERIAL
AND METHODS
The techniques employed were similar to those reported previously [6, 121; differences are described below. A subculture of a Chinese hamster cell line derived from female lung tissue designated V79-1 [6] was established September, 1959 and propagated serially. These cells grow attached to glass and appear “fibroblast-like”. Stock cultures were grown in milk dilution bottles with medium HU-15 [6] and were subdivided forty-fold twice weekly. Except where otherwise indicated, cultures for an experiment were grown in medium EM-10 plus 1 x 10-OM thymidine for six days prior to irradiation according to the schedule shown in Table I. Medium EM-IO is the same as HU-15 except that it lacks the supplementary 4 per cent culture medium NCTC-109 and contains only 10 per cent undialyzed fetal calf serum. In our early experiments we found variations in X-ray survival curves after growth in BUDR which we thought might be due to differences in endogenous thymidine production by the cells. Thymidine was added to prevent these variations. Because much data had been accumulated using EM-10 plus thymidine, the addition was 1 Revised 31- 631811
version
received
November
12, 1962. Experimental
Cell Research 30
W. C. Mohler
and M. M. Elkind
continued even after it became apparent that factors other than endogenous thymidine production were probably responsible for the original variations. Cell suspensions used for stock cultures or experiments were prepared by washing glass-attached cells once with a trypsin solution, incubating the cells at 37°C for IO min in another aliquot of the same solution, adding an equal volume of medium, and dispersing the cells by gentle pipetting. The trypsin solution contained 0.05 per cent trypsin (I-300, Nutritional Biochemical Corp.) in phosphate buffered saline (0.15 M NaCl plus 0.005 M potassium phosphate; pH 7.4). TABLE
I. Protocol for Growth
of Experimental
Cultures Prior
to Irradiation.
Culture of V79-1 presumably incorporate BUDR and deplete the medium at a rate proportional to the total cell number, which increases exponentially. Therefore, the medium was replaced frequently toward the end of each three day period. On day 0 and day 3 a sufficient number of cells was planted to give approximately 5 x lo8 cells on day 3 and day 6. Day Day Day Day Day
O-Start cultures from V79-1 stock into 20 ml of medium. a--Replace medium with 25 ml in early morning and late evening. 3-Trypsinize in early morning and replant in 20 ml of medium. B-Replace medium with 25 ml in early morning and late evening. 6-Trypsinize, dilute, attach and irradiate. Medium replaced with for cultures irradiated in afternoon.
25 ml in early morning
For the X-ray studies, cells suspended in EM-IO were allowed to attach for 60 to 100 min at 37°C to the bottoms of 9 cm petri dishes. A pH of 7.4 to 7.6 was mamtained by gassing the incubator with a humidified mixture of air and CO*. Since soft X-rays were used (55 kv, 722 rad/min) the medium was removed after attachment and the cells were irradiated under a humidified CO,-air atmosphere [6]. Appropriate amounts of HU-15 were added to the dishes immediately after irradiation, and stained macroscopic colonies were counted without magnification after the surviving cells had grown for 11 days at 37°C. Unirradiated cells, which otherwise received the same treatment, served as controls for the determination of surviving fraction. The inoculum of cells in plates receiving irradiation was increased to result in approximately the same final number of colonies, i.e. 200, expected on the control plates. BUDR for these studies was obtained from the Cancer Chemotherapy National Service Center of the National Cancer Institute.
Radiation Survival Parameters The survival curve of those observed with other region is followed at higher [6]. Without presupposing model, one can-describe and the dose (Da) which the exponential region. Experimental
Cell Research 30
V79-1 cells not mammalian cell X-ray doses by the applicability such curves by reduces survival
treated with BUDR is similar to lines. In such curves a shoulder an exponential decline in survival of any particular inactivation an extrapolation number (ii) [l] by the fraction l/e (i.e. 0.37) in
X-ray survival modified by BUDR
483
Straight lines were fitted by eye to the exponential portion of survival curves plotted on semi-logarithmic paper and extrapolated to zero dose to obtain ii. fiO was calculated from the dose which reduced survival by (l/e)’ or 0.00091 on the fitted line. The cell suspensions prepared for X-ray survival work always contained more than 90 per cent single cells with a few clumps of 2 or 3 cells. Since V79-1 cells have been shown to survive X-irradiation independently [6], we reduced the observed extrapolation number by the cellular multiplicity (i.e., the average number of cells per potential colony forming unit) in the data to be presented below. This correction is not exact but the uncertainty involved has no effect on the interpretation of our data, since the multiplicity was always less than 1.10. RESULTS
Growth of V79-1 in BUDR.-The
rate of cell growth changes markedly during the first two to three days for V79-1 grown in concentrations of BUDR greater than 1 X 10-e M. We therefore calculated the doubling time (T,) of cells in mass culture from the increase in cell number during the last three days of the standard six day growth period described in Table I. The results are shown as open circles in Fig. 1. For cells grown in the absence of BUDR, T, averaged 11.5 hr. No significant increase in T, was seen for celis growing in concentrations of BUDR less than 2 X 10-7 M. A marked increase in Tz occurred above 1 x 10-e M. In one experiment (closed triangles in Fig. 1). T, was calculated from growth curves obtained by counting the number of cells per colony in 100 colonies on plates which were fixed and stained at daily intervals after inoculation 6Or
’ Fig. l.-Doubling
““,’ 10%
I BUDR IN MEDIUM
‘O-”
time (Ta) of V79-1 grown in BUDR.
1
IO-%
See text for details. Experimental
Cell Research 30
484
u’. C. Mohler
and M. M. Elkind
with 1000 cells. The results agree rather well with Tz obtained from mass culture measurements. Viability of cells.-As a measure of the viability of cells after six days growth in BUDR, we calculated the plating efficiency (P.E.), i.e., the percentage of the potential colony forming units in a trypsinized suspension which grew into macroscopic colonies. For control cultures grown in the
. - -.-,-_r . . .
‘\ .
a\
T
\
‘. \
0 -’ c!+ BUDR IN MEDIUM
Fig. 2.-Plating
efficiency of cells after growth for six days in BUDR. See text for details.
absence of BUDR, the P.E. averaged 88 per cent with a range from 80 per cent to 95 per cent. Fig. 2 shows the P.E. of cells grown in BUDR plotted as the per cent of the P.E. for control cells in each experiment. No significant change was seen at BUDR concentrations less than 2 x 10-7 M, A marked decrease in viability was noted above 1 X 10-G M. Effect of growth in BUDR on X-ray survival parameters.-Cells grown in BtJDR showed an increased response to the lethal effects of X-rays. Fig. 3 illustrates the progression in response with increasing concentrations of BUDR. There is a decrease in both ii and 8, as the BUDR concentration increases. The radiation response of V79-1 cells is stable after approximately the first two days of growth in a given concentration of BUDR. This is illustrated in Fig. 4 for cells grown in 1 x 10-e M (closed symbols) and 5 x lo-6 M (open symbols). The X-ray survival parameters remained unchanged even though the percentage of viable cells in the culture decreased. For example, the P.E. for cells grown in 1 X 10-e M BUDR dropped from 80 per cent of control at 60 hr to 65 per cent at 108 hr and the P.E. for cells grown in 5 X 10-e M BUDR decreased from 28 per cent of control at 46 hr to 8 per cent at 94 hr. The variation in go with BUDR concentration is shown in Fig. 5. fiO is a Ezperimenfal
Cell Resenrch 30
X-ray survival modified by BUDR smooth function of BUDR concentration even up to 5 x 10-S M, a concentration at which less than 10 per cent of the cells were viable after six days growth. There is a decrease in fiO even at BUDR levels below 2 X 10-7 M, which do not detectably alter the growth rate or viability. This can be seen
Fig. J.-X-ray survival curves for V79 after growth in BUDR for 6 days. The specific ISUDR concentration is indicated for each curve. Standard errors, estimated by the method of Mantel [13], are shown when larger than the symbols.
-
6
-
5
-
4;
: x2 -3 -2
50 HOURS IN EUDR PRIOR TO IRRADIATION
Fig. I.-Change in X-ray survival parameters during growth in BUDR..Closed symbols for cells grown in 1 x lo-‘M BUDR. Open symbols for cells grown in 5 x 10-8M BUDR. Arrow indicates time of trypsinization and replanting, explained in Table I. Experimental
Cell Research 30
W. C. Mohler and M. M. Elkind
486
more clearly in Fig. 6 where g,, for each culture grown in BUDR is plotted as a percentage of that for the control (no BUDR) culture in the same experiment. (For 17 cultures grown without BUDR, b,, averaged 144.5 rad with a standard deviation of 6.2 rad or 4.3 per cent). 150 v 1
v A
. 8 .
p ‘;I00 ZD
. . .
I
BUDR IN MEDIUM
Fig. 5.-Change
0 t h-J 0
in 0, as a function
,(I”,’
of BUDR
I lo-%/
IO-‘M
concentration.
_u_wJ
IO-M
BUDR IN MEDIUM
Fig. 6.-Change in 0, compared with 0, of control cultures which were grown under identical conditions in the absence of BUDR. This presentation emphasizes the change in 0: which occurs at low BUDR concentration where no other alterations in growth, viability or extrapolation number can be detected.
The extrapolation number behaved differently, as shown in Fig. 7. The value for control cultures varied from experiment to experiment. In seventeen cultures grown without BUDR, fi averaged 5.70 with a standard deviation of 1.44 or 25 per cent. In spite of this variability, cells grown in BUDR concentrations of 1 x 10-7 M or less did not give X-ray survival curves with ii less than the ii of the control culture in the same experiment. (Compare values for individual experiments represented by different symbols in Fig. 7). At 2 X 10-T M and above, fi was always less than corresponding control value Experimental
Cell Research 30
X-ray survival modified by BUDR
487
and at 2 x 10-e M and above, survival curves were exponential and hence ii=l. Radiation response after BUDR is removed.-A subculture of V79-1 was grown in medium HU-15 (which contains 1.6 X 10-S M thymidine) plus
I
,,,,I,’
I
I IO-%
lOA BUDR IN MEDIUM’“-‘M
Fig. 7.-Change
TABLE
II.
Reversal
in extrapolation
number
as a function
of BUDR
concentration.
of Radiation Response After Culture Medium.
BUDR
is Removed
From
V79-1 was grown in HU-15 plus 2.6 x 10-eM BUDR for 79 days before BUDR was omitted from cultures. See text for details. The growth factor is the increase in number of cells relative to the replicate culture at time 0. P. E. is the plating efficiency, defined under Results. Replicate
#l #2 #3
Hr after Omitting
0 23 47
BUDR
b
ii
90 rad 143 u 188 I)
1.1 1.9 2.5
P. E.
37 37 33
Growth
Factor
1 1.6 4.7
1.6 X 10-6 M BUDR. The subculture was carried by transferring l/20 to l/40 of the cells to fresh medium every 3 to 4 days. At the end of 79 days, an X-ray survival curve was determined with one of three replicate cultures and the other two were refed with HU-15 containing no BUDR. X-ray survival curves were determined with the second replicate culture after 23 hr and with the third after 47 hr of growth in medium lacking BUDR. Table II summarizes the results. The increase in 6, and ii indicates that these cells Experimental
Cell Research 30
488
W. C. Mohler and M. M. Elkind
returned toward a “normal” radiation response during the first few generations after the removal of BUDR. The relatively low ii at 47 hr is compatible with some population heterogeneity. The high fi,, at 47 hr is unexplained and may be related to a change induced by prolonged growth in BUDR since the subline had a hyperploid chromosome number (greater than 35) instead of the near-diploid number of 23 characteristic of the stock strain examined at the same time [14]. Also, it should be noted that in control experiments we have observed over a long period of time that variations in ii are usually accompanied by inverse variations in 5, [6]. In any case, the rapid change back toward an unaltered X-ray survival response is evidence supporting the view that BUDR does not modify radiation response by selecting a pre-existing moiety of V79-1 cells which has an inherently low ii and fiO. A similar rapid change back toward “normal” has been reported for another cell strain in which BUDR increased the response to the lethal effects of ultraviolet light [3].
IHSCUSSION
Our findings with this Chinese hamster cell line, V79-1, can be compared with the results of Erickson and Szybalski [S], who studied the effects of halogenated pyrimidines upon the X-ray response of a human sternal bone marrow cell line, D98/AG. They exposed their cells to BUDR for four days, which was probably equivalent to about 4 doubling times for this slower growing cell line [3]. While it is not clear that this exposure produced maximum changes in n” and & their results differ in at least two respects from ours. First, even though they used 5fluorodeoxyuridine to inhibit thymidylate synthetase and thus increase the amount of thymidine replaced by BUDR in cellular deoxyribonucleic acid (DNA), they required higher concentrations of BUDR to produce equivalent percentage changes in & Their data indicate that about 2 X 10-S M BUDR was required to achieve a 50 per cent reduction in 5, from their control fi,, of 180r. Second, their data suggest that n” either remained the same or increased even at concentrations of BUDR where fiO was reduced to half the control value, a fi,, reduction at which we found fi to have dropped to 1.0. These differences appear even more striking if one assumes from other data [3] that their cells which exhibit about 50 per cent reduction in E,, had about 40 per cent of DNA thymidine replaced by BUDR. Preliminary studies indicate that V79-1 cells which had a comparable reduction in fi,, had less than 10 per cent replacement of thymidine by BUDR [14]. These discrepancies may be due in part to physical factors since Erickson Experimental
Cell Research 30
X-ray survival modified by BUDR and Szybalski used 140 kv X-rays and we used 55 kv X-rays. The relative difference between the X-ray absorption cross sections of the bromine in BUDR and the analogous methyl group in thymidine will be more pronounced for the softer X-ray spectrum due to a relatively larger photoelectric effect. More energy should be absorbed locally with softer X-rays for the same average exposure in both cases. The answer to questions of this type must await definitive studies of the relative biological effectiveness of different qualities of radiation as a function of BUDR incorporation. It might well be noted also that other mammalian cells have exhibited differences in their ability to tolerate exposure to BUDR or its incorporation into their DNA [lo]. Before considering the possible modes of action of BUDR it is first necessary to question whether or not the potentiating effects of BUDR are only apparent. Recent results suggest that single cell survival curves which may appear to represent a homogeneous population can, in fact, be the sum of survival curves of two or more subpopulations which survive differently [7, 171. Hence BUDR might be killing or inhibiting the growth of some cells and thereby selecting for more X-ray responsive cells. Selection seems unlikely at BUDR concentrations less than 2 x 10-7 M since no toxicity was manifest. Yet Do, which is the parameter least responsive to slight modification in the proportions of subpopulations, is significantly and progressively altered at these low concentrations. The alterations in ii seen at BUDR concentrations greater than 2 x 10-7 M might be due to selective killing. However, an analysis of the conditions under which this would occur does not favor a dominant selective influence. First, from the kinds of analyses dealing with population heterogeneity which were considered in an earlier paper [5], it can be shown that our results preclude selection by BUDR if in the absence of drug all moieties had the same fi. In such a case the observed extrapolation number of the remaining population would have to increase, not decrease, as the more X-ray resistant moieties were killed off by the cytotoxic action of BUDR. The general type of population heterogeneity consistent with our results would require that the X-ray survival parameters of subpopulations of V79-1 (not exposed to BUDR) must have small ii’s correlated with small D,‘s and large ii’s with large &‘s. In the second place, the experiments summarized in Fig. 5 show that X-ray survival parameters remained constant after 35 to 45 hr growth in BUDR in spite of a continued cytotoxic action of the drug such that in experiment # 245 (1 x 10-B M BUDR) the P.E. dropped from N 80 per cent at 60 hr # 319 (5 x 10-S M BUDR) it to -65 per cent at 108 hr and in experiment dropped from - 30 per cent at 46 hr to w 8 per cent at 94 hr. Finally, the Experimental
Cell Research 30
W. C. Mohler
and M. M. Elkind
data in Table II indicated that even after a prolonged exposure to BUDR, V79-1 cells quickly returned toward a radiation response resembling that of cells never exposed to BUDR, in spite of evidence that the prolonged growth in BUDR had exerted a selective influence. It is reasonable that the radiation modifying effect of BUDR should be sought at an intracellular level since, as a thymidine analogue, BUDR is incorporated into DNA thereby altering the cell’s genome. The work of Opara-Kubinska and Szybalski [15] on ultraviolet (U.V) damage strongly support this thesis. Their experiments show that transforming principle DNA from Bacillus subtilis is rendered considerably more sensitive as a result of incorporation of the bromine analogue. Szybalski and coworkers [S, 15, 161 have proposed that the potentiating effects of halogenated pyrimidines in general indicate that DNA is the most radiosensitive cell component and therefore the principal target in the lethal action of both U.V. and X-rays. It seems to us that this conclusion must be viewed with caution particularly when one proposes a mechanism of action for ionizing radiation in cells which have not been exposed to pyrimidine analogs. In mammalian cells the vast majority of primary ionizations registered in DNA molecules must be innocuous. For example, in V79-1 cells not exposed to BUDR about lo3 DNA molecules per cell will receive primary ionization per h,, dose. Therefore it seems reasonable to expect that most of the primary DNA damage is ordinarily repaired, by-passed, or registered in sites unrelated to reproductive integrity. The action of BUDR might be either to make sensitive some sites of incorporation which otherwise would not have contributed significantly to X-ray sensitivity in the normal cell or to impede the repair or by-passing of damaged sites. Support for both possibilities exist since it is known that brominated uracil can produce “hot spots” of mutation in DNA [a], and the recent results of Howard-Flanders and Boyce [9] suggest that BUDR may enhance U.V. killing of T, phage by inhibiting repair processes which would otherwise occur after bacterial infection. Drugs like BUDR have been discussed as possible adjuncts in the radiotherapy of tumors. We do not feel able from our studies to comment on the relative merits of “unifilar” or “bifilar” labelling of DNA [3, 111. It is clear, however, from experiments like those shown in Fig. 5 that to be most effective in altering the radiation response of V79-1 BUDR must be maintained in relatively cytotoxic concentrations for several doubling times. In terms of conventional therapeutic fractionation protocols, it appears that the important change induced by BUDR might be a relatively large reduction of fi and reduced recovery between exposures [4, 111. Experimental
Cell Research 30
X-ray survival modified by BUDR
491
SUMMARY
Chinese hamster cells (strain V79-1) cultured in vitro show an increased response to the lethal effects of X-rays when they are grown prior to irradiation in 5-bromodeoxyuridine (BUDR). These cells appear to be more sensitive than the human cell line, D98/AG, studied by Erickson and Szybalski to the radiation modifying as well as the cytotoxic effects of BUDR. BUDR produces a striking change in the extrapolation number of X-ray survival curves of V79-1 as well as the change in slope reported in D98/AG. Both of these changes appear to be the result of alterations in cellular radiation response and not the result of population heterogeneity. When cells having an increased X-ray response induced by BUDR are removed from contact with BUDR, they reassume X-ray survival parameters similar to normal cells within one or two generations. The authors gratefully acknowledge the assistance of Miss Bernadine Bianchi, Mrs. Carolyn Kimler and Mrs. Ovella Ayers during many portions of these experiments. REFERENCES T., GILLIES, N. E. and ELKIND, M. M., Nature 186, 1062 (1960). BENZE~, s., Proc. Natl. Acad. Sci. USi 47, 403 (1961). ’ DJORDJEVIC. B. and SZYBALSKI, W., J. Es&Z. Med. 112, 509 (1960). . , ELKIND, M. M., Radiology 74, 5i9 (iSSO). . __ Brookhaven Symp. in Mot. 14, 220 (1961). ELKIND, M. M. and SUTTON, H., Radiation Res. 13, 556 (1960). ELKIND, M. M., SUTTON, H. and MOSES, W. B., J. Cellular Comp. Physiot. 58, Suppl.
1. ALPER,
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
1, 113 (1961). ERICKSON. R. L. and SZYBALSKI. W., Biochem. Biouhvs. Res. Commun. 4, 258 (1961). HOWARD-FLANDERS, P. and BOYCE, k. P., Radiation kes. 16, 563 (1962): ’ Hsu, T. C. and SOMERS, E. E., Exptl. Cell Res. 26, 404 (1962). KAPLAN, H. S., SMITH, K. C. and TOMLIN, P. A., Radiation Res. 16, 98 (1962). LOCKART, JR., R. Z., ELKIND, M. M. and MOSES, W. B., J. Natl. Cancer Inst. 27,1393 (1961). MANTEL, N., Am. Statistician 5, 26 (1951). MOHLER, W. C. and ELKIND, M. M., Unpublished results. OPARA-KUBINSKA, Z., LoRK;EwIcz,.~~~~SYBALSKI, W., Biochem. Biophys. Res. Commun. 4,
288 (1961). SZYBALSK;, Natl. 17. TERASIMA, 16.
W.; in Research in Radiotherapy, p. 162. KALLMAN, Acad. Sci. Washington, D.C., 1961. T. and TOLMACH, L. J., Nature 190, 1210 (1961).
R. F. (ed). Pnblication
Experimental
888,
Cell Research 30