Chem.-BioL Interacticms, 14 (1976) 93-99 0 Elsevier Scientific Publishing Company, Amsterdam
- Printed in The Netherlands
THE SENSITIVITY OF HeLa AND CHINESE HAMSTER (OVARY) CELLS TO METHYLENE DIMETHANE SULPHONATE
A.H.W. NIAS * and B.W. FOX Paterson Laboratories, Christie Hospital and Holt Radium institute, Manchester M20 9BX (Great Britain) (Received (Accepted
December 5th, 1975) January 23rd, 1976)
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-__I-
..- __
.._____~ --
SUMMARY
HeLa and Chinese hamster (ovary) cells were expcsed in vitro to methylene dimethanesulphonate (MDMS) and their survival of colony-forming ability was assayed in monolayer culture. Asynchronous cultures were exposed to the drug for the whole culture period but cell survival was complicated by the toxicity of formaldehyde which is a final breakdown product of the drug. A short treatment schedule of 15 min within the hydrolytic half life of the agent was therefore employed and the response of synchronous cultures of HeLa cells was then assayed throughout the course of the cell cycle. Cells were most sensitive at the beginning of the DNA synthetic phase (early-S) and most resistant at the end (late-S). -.-
_ ..___
_____.~_. _ ~.____._ __.~--.--~~- .--
INTRODUCTION
MDMS is the first member of a series of bifunctional methanesulphonates of straight-chain diols. It exerts an effective anti-tumour action on the transplanted Yoshida lymphosarcoma in the Wistar rat [ 1,2] and the mechanism of this action has been further investigated (31 using Yoshida cells in culture. The responses in vivo and in vitro to MDMS have also been compared [4] using L1210 cells and the mouse. In the present investigation, the pharmacokinetics of MDMS were studied using two established mammalian cell lines, HeLa and Chinese hamster ovary, which grow in monolayer culture. * Present address: Glasgow Institute of Radiotherapeutics and Oncology, BeEvidere HOSpital, Glasgow G31 4PG (Great Britain). Abbreviations: BCNU, 1,3-bis( S-chloroethyl)-l-nitrosourea; IMS, isopropyl methanesulMMS, methyl methanesulphonate; phonate; MDMS, methylene dimethanesulphonate; [ jH]TdR, [ 3H]thymidine.
Also HeLa cells were synchronised with respect to their progress through the cell cycle and the response to the drug was examined throughout the cycle. MATERIALS
AND METHODS
HeLa and Chinese hamster ovary cells were grown in monolayer culture using established techniques [ 51. Cell survival was estimated by the cloneforming ability of single cells, according to the method of Puck and Marcus [6]. For the data shown in Figs. 1 to 3 asynchronous cells were obtained by trypsinising logarithmically growing cultures. For the data shown in Figs. 4 and 5 synchronised cells were obtained by the method of Terasima and Tolmath [‘I] by which cells in mitosis are selected from a logarithmically growing culture. The degree of synchronisation [8] was estimated for each experiment by determining (1) the mitotic index at zero time (2) the average cellular multiplicity at intervals during the first cycle (3) the [3H] TdR labelling index at similar intervals. MDMS was freshly dissolved in physiological saline or culture medium after sterilising by filtration (Millipore 0.22 p). The solution was kept on ice to keep the rate of hydrolysis minimal. Suitable volumes were added to each culture bottle to achieve the desirec$.concentration of the agent. For studies with synchronised cells the comhou::d was dissolved in absolute ethanol to enable aliquots to be administered throughout the cell cycle without any prior hydrolysis. In all cases the compound was added to the bottles in a room at 37°C for the prescribed time interval. The drug-containing medium was then removed by aspiration and replaced by fresh drugfree medium in this room so that the cells were maintained at 37°C throughout. The sub-clone was obtained using the method already described [9] for clones of HeLa cells sensitive to MMS. RESULTS
Continuous exposure
Fig. 1 shows survival curves for asynchronous HeLa cells of the standard line and also for a sub-clone selected from survivors of a previous treatment with MDMS. The drug was added to the culture medium at the time the cells were inoculated into bottles and this medium plus drug was left undisturbed for the whole lOday period of culture. The survival curves show a broad “shoulder” before following a steep exponential shape. The sub-clone is more sensitive to second treatment with MDMS than its parent cell line. The survival curve for Chinese hamster ovary cells is also shown (Fig. 1) after a similar treatment with MDMS except that in this case the culture period was halved to 5 days because the cells divide twice as frequently as HeLa cells (i.e. the doubling time of Chinese hemster ovary cells is 12 h, that of HeLa cells 24 h). A similar shape is seen in the survival curve with a broad shoulder followed by a steep exponential slope. 94
Fig. 1. Effect of MDMS on asynchronous cells. Dose-response curves for celh eu MOMS for the whole period of culture. (The points show the mean and stani.ard error of the mean of data pooled from 5 experiments with Chinese hamster ovgry &HO) cells, 11 with HeLa cells, and 2 with the HeLa sub-clone.) Fig. 2 Rate of inactivation of MDMS in Eagle’s medium (with 10% human serum) expressed in terms of increased survival of HeLa cells treated with aliquots of drugcontaining medium incubated at 37%. (Initial drug concentration 8 yglml.)
Pharmacokinetics The rate of inactivation of MDMS in culture medium at 37°C is shown in Fig. 2. HeLa cell survival increases with time but does not reach 100%. Rather, a plateau level of 40% survival is attained. MDMS is known [ 101 to hydrolyse to formaldehyde and 8 c(g MDMS will produce 1 JI dehyde. With 1 pg/ml formaldehyde the survival level was 39% to the final level in Fig. 3 after the hydrolysis of MDMS at 8 pg 0.5 I.cg/ml, the survival was 85%. The half-life of hydrolysis of MDMS [l] in water at 37°C is 22 min. A comparison of the data in Figs. 1 and 2 suggest that the biolo half-life does not conform with this. however; an inactivati min being the lowest estimate that can be derived. A simil tions was made with Chinese hamster ovary cells which activation time considerably in excess of 22 min. Cle sidual formaldehyde reduces the value of observations like tho where cells have been exposed continuously to MDMS and its b~~kdown products. 95
-0’0
0.040 MDMS uglmlll5minr
ot 37’C)
2
4 HOURS
6
8
IO
II
I4
t6--ik-%-k-%%
alter COLLECTION0f MIT~TIC
CELLS
Fig. 3. Dose-response curges for cells exposed to MDMS for 15 min at 37’C. (Data from 7 HeLa cell and 2 CH0 cell experiments.) -‘-ed HeLa cells exposed to MDMS (50 I.cg/ml for 15 min at Fig. 4. Survival of synchro..,a 37OC) at different times aT.*r mitotic selection. (Data from 2 experiments.)
Short treatment of asyrlchronous cells Fig. 3 shows survival curves for HeLa and Chinese hamster ovary cells exposed to MDMS for only 15 min before the drug-containing medium was removed and fresh medium substituted for the remainder of the culture period. Again, each curve has an initial shoulder but the subsequent exponential is much less steep.
Spchronised
HeLa celis
Fig. 4 shows the response of aliquots of synchronised populations of HeLa cells to the same concentration of MDMS (50 pg/ml, for 15 min) at different times during progress through the first cell cycle after mitotic selection. There is a small decrease in survival as the cells traverse the G, phase of the cycle (O-10 h) and then as the cells enter the DNA synthetic (S) period they 2 3 most sensitive. Towards the end of the S period sensitivity to MDMS is decreased reaching a minimum at the end of S. Then a small reduction in survival occurs as the cells pass through the G2 period (22-26 h). Thus in the HeLa cell. early-S is the most sensitive period and late-S is the most resistant to MDMS. Dose-response cwws were obtained at these two periods (Fig. 5). The exponential slope is the same for each period in the cell cycle; the sensitivity of cells in early-S, however, is shown as a diminution in the size of the initial shoulder of the survival curve.
96
SUHVIVING FRACTION
Fig. 5. Dose--response curves of synchronised HeLa cells exposed :o MDMS (far 15 min at 37’C) at 10 h (early S) and 22 h (late S) aftw mitotic selection. (Data from 2 experiments ’
DISCUSSION
Previouly [ 111 the reponse of HeLa cells to the monofunctional analogue MMS in the relatively long hydrolysis time (about 9 h) of ihe alkylating agent had to be taken into account. A short exposure time of 15 min was employed for studies where a pulse treatment was required for the purposes of the experiment, e.g. “split-dose” studies [ 121 or studies of the phase seirsitivity with synchronised cells [9]. With IMS on the other hand the short ilydsolysis time of 13.6 min enabled all such studies to be performed [ 131 without the need to change the culture medium. In both cases, the final hydrolysis products were relatively non-toxic compared with the unhydrolysed drug. An experiment similar to that described for in Fig. 2, when undertaken with MMS, showed cell survival rising t rather than 40% as in the present work. With MDMS a major product of hydrolysis is formaldehyde which is toxic :it the concentration levels expected by hydrolysis. It would be expected . that in vivo, the formaldehyde formed would be rapidly removed . fied, but in vitro, the accumulation of this product would be e nificantly to affect the cell survival. The extent of action with time may be judged from Fig. 2 which in effect suggests that MDMS is approximately eight-fold more toxic than formaldehyde at the same molar concentration, but that this toxicity would only be manifest by treatment within a short time interval after putting in solution. A compromise was adopted in which the cells were subjected to a 15.min “pulse” treatment with the the survival curves for this treatment are shown in Fig. 3 for both HeLa and Chinese hamster celi lines. Fig. 2 shows that the major contribution to cell 97
death from this short exposure must be from the drug and not from the formaldehycie formed. The Do for Chinese hamster ovary cells is 10 pg/ml with D, of ;j2 pg/ml(n), whereas the HeLa cells had a Do of 11.5 pg/ml and a D, of 26.5 pg/ml. The less steep slope of the pulse-treated lines, together with the larger D, values suggest that cell recovery is markedly improved by the pulse treatment. Furthermore the agent has a strong cumulative action in contrast to the big differences in continuous and pulse-treated regimes. The comparative survival of HeLa cells after a 15-min pulse treatment of synchrc nised HeLa cells is shown in Fig. 4. A slow decrease in survival results from treatment in G1 prior to the commencement of S phase. During S phase, an initial phase of 2-3 b (lo-13 h) is most sensitive followed by an increasing resistance through S, rising to least sensitivity during early G2 phase. Thus the greatest sensitivity in early S phase resembles the action of RCNU on Chinese hamster cells [ 141 in strain L mouse fibroblasts [ 153 and to uracil mustard [ 161. A similar pattern of sensitivity may be observed in synchronised HeLa cells, as cells selected in early S were more sensitive (Do = 15 pg/ml, D, = 16.3 I_cg/ml) than those in late S phase (Do = 15 pg/ml, D,= 49 yg/ml), the main difference being due to the extrapolation value and suggesting an increased potential for recovery within this phase. The pattern of sensitivity also closely resembles that reported for X-rays [ 171. Although the mode of action of this alkylating agent is not yet understood it is known that differences in sensitivity between sensitive and resistant lines of Yoshida cells are associated [IS] with a difference in their capacity to repair damaged DNA. The agent is considered 1191 to associate with GCrich regions of DNA producing a region of conformational instability and increasing the number of single-stranded regions. The present data suggest that the early stages of DNA synthesis may be more susceptible to faulty replication sequences resulting from such conformational damage than the later stages when replication is almost complete. This implies that +,he replicating fork itself could be a principal target for action. Thus when precautions are taken to exclude the action of the formaldehyde produced the synchronised HeLa cell line shows maximum sensitivity to cell killing at the end of G, and beginning of S and the least sensitivity at the end of S and during Gz. ACKNOWLEDGEMENT
The authors are grateful for helpful discussions with Dr. Margaret Fox and for the technical assistance of Mrs. Elisabeth Moore. REFERENCES 1 B.W. Fox and H. Jackson, In vivo effects of methylene dimethane sulphonate on proliferating cell systems, Brit. J. Pharmacol. Chemother., 24 (1965) 24. 2 B.W. Fox, The sensitivity of a Yoshida sarcoma to methylene dimethane sulphonate, Jnt. J. Cancer, 4 (1969) 54.
98
3 M. F*BX and B.W. FOX, The establishment of cloned cell lines from Yoshida sarcomas having differential sensitivities to methylene dimethane sulphonate in vivo and their cross sensitivity to X-rays and UV and other alkylating agents, Chem.-Biol. Interact., 4 (1971/i 2) 363. 4 D. Andtrson and A.H.W. Nias, Resistance to methylene dimethane sulphonate (MDMS) in L1210 cells in vitro, Eur. J. Cancer, 8 (1972) 659. 5 A.H.W. Nias, Clone size analysis: A parameter in the study of cell population kinetics, Cell Tissue Kinetics, 1 (1968) 153. 6 T.T. Puck and P.I. Marcus, Action of X-rays on mammalian cells, J. Exp. Med., 103 (1956) 653. 7 T. Terasima and L.J. Tolmach, X-Ray sensitivity and DNA synthesis in Fynchronous popuhtions of HeLa cells, Science, 140 (1963) 490. 8 A.H.U Nia; and M. Fox, Synchronisation of mammalian cells with respect to the mitotic cycle, Cell Tissue Kinetics, 4 (1971) 375. 9 M. FOX and A.H.W. Niaa, A modification of the sensitivity of mammalian cells surviving treatment with methyl meihanesulphonate, Eur. J. Cancer, 4 (1968) 325. 10 K. Edwards, H. Jackson and A. Jones, Studies with alkylating esters, III. The metabolism and fate of methylene dimethanesulphonate, Biochem. Pharmacol., 19 (1970) 1791. 11 J.J. Roberts, J.M. Pascoe, J.E. Plant, J.E. Sturrock and A.R. Crathorn, Quantitative aspects of the repair of alkylated DNA in cultured mammalian cells, I. The effect on HeLa and Chinese hamster cell survival of alkylation of cellular macromolecules, Chem.-Biol. Interact., 3 (1971) 29. 12 M. Fox, C.W. Gilbert, L.G. La_tha and A.H.W. Nias, The interpretation of “split-dose” experiments in mammalian cells after treatment with alkylating agents, Chem.-Biol. Interact., 1 (1969/70) 241. 13 D. Petrovic and A.H.W. Nias, A comparison of the nffects upon HeLa cells of isopropyl methanesulphonate and X-rays during different phases of the cell cycle, Eur. J. Cancer, 3 (1967) 329. 14 S.C. Barranco and R.M. Humphrey, The effects of 1,3-bis(2chloroethyl)-l-nitrosourea on survival and cell progression in Chinese hamster cells, Cancer Res., 31 (1971) 1218. 15 I.G. Walker and C.W. Helleiner, The sensitivity of cultured mammalian cells in different stages of the division cycle to nitrogen and sulfur mustard, Cancer Res., 23 (1963) 734. 16 F. Mauro and H. MadocJones, Age-response of cultured mammalian cells to cytotoxic drugs, Cancer Res., 30 (1970) 1397. 17 W.K. Sinclair, Cyclic X-ray responses in mammalian cells in vitro, Radiation Res., 33 (1968) 620. 18 M. Fox and B.W. Fox, Repair replication and unscheduled DNA synthesis in mammalian cell lines showing differential sensitivity to alkylating agents, Mutation Res., 19 (1973) 119. 19 D.G. Poppitt and B.W. Fox, The effect of mcthylene dimethanesulphonate (MDMS) on the conformation of DNA ant1 its dependence on base composition, Chem.-Biol. Interact., 11 (1975) 163.
99
- Printed in The Netherlands
THE SENSITIVITY OF HeLa AND CHINESE HAMSTER (OVARY) CELLS TO METHYLENE DIMETHANE SULPHONATE
A.H.W. NIAS * and B.W. FOX Paterson Laboratories, Christie Hospital and Holt Radium institute, Manchester M20 9BX (Great Britain) (Received (Accepted
December 5th, 1975) January 23rd, 1976)
----
--
-__I-
..- __
.._____~ --
SUMMARY
HeLa and Chinese hamster (ovary) cells were expcsed in vitro to methylene dimethanesulphonate (MDMS) and their survival of colony-forming ability was assayed in monolayer culture. Asynchronous cultures were exposed to the drug for the whole culture period but cell survival was complicated by the toxicity of formaldehyde which is a final breakdown product of the drug. A short treatment schedule of 15 min within the hydrolytic half life of the agent was therefore employed and the response of synchronous cultures of HeLa cells was then assayed throughout the course of the cell cycle. Cells were most sensitive at the beginning of the DNA synthetic phase (early-S) and most resistant at the end (late-S). -.-
_ ..___
_____.~_. _ ~.____._ __.~--.--~~- .--
INTRODUCTION
MDMS is the first member of a series of bifunctional methanesulphonates of straight-chain diols. It exerts an effective anti-tumour action on the transplanted Yoshida lymphosarcoma in the Wistar rat [ 1,2] and the mechanism of this action has been further investigated (31 using Yoshida cells in culture. The responses in vivo and in vitro to MDMS have also been compared [4] using L1210 cells and the mouse. In the present investigation, the pharmacokinetics of MDMS were studied using two established mammalian cell lines, HeLa and Chinese hamster ovary, which grow in monolayer culture. * Present address: Glasgow Institute of Radiotherapeutics and Oncology, BeEvidere HOSpital, Glasgow G31 4PG (Great Britain). Abbreviations: BCNU, 1,3-bis( S-chloroethyl)-l-nitrosourea; IMS, isopropyl methanesulMMS, methyl methanesulphonate; phonate; MDMS, methylene dimethanesulphonate; [ jH]TdR, [ 3H]thymidine.
Also HeLa cells were synchronised with respect to their progress through the cell cycle and the response to the drug was examined throughout the cycle. MATERIALS
AND METHODS
HeLa and Chinese hamster ovary cells were grown in monolayer culture using established techniques [ 51. Cell survival was estimated by the cloneforming ability of single cells, according to the method of Puck and Marcus [6]. For the data shown in Figs. 1 to 3 asynchronous cells were obtained by trypsinising logarithmically growing cultures. For the data shown in Figs. 4 and 5 synchronised cells were obtained by the method of Terasima and Tolmath [‘I] by which cells in mitosis are selected from a logarithmically growing culture. The degree of synchronisation [8] was estimated for each experiment by determining (1) the mitotic index at zero time (2) the average cellular multiplicity at intervals during the first cycle (3) the [3H] TdR labelling index at similar intervals. MDMS was freshly dissolved in physiological saline or culture medium after sterilising by filtration (Millipore 0.22 p). The solution was kept on ice to keep the rate of hydrolysis minimal. Suitable volumes were added to each culture bottle to achieve the desirec$.concentration of the agent. For studies with synchronised cells the comhou::d was dissolved in absolute ethanol to enable aliquots to be administered throughout the cell cycle without any prior hydrolysis. In all cases the compound was added to the bottles in a room at 37°C for the prescribed time interval. The drug-containing medium was then removed by aspiration and replaced by fresh drugfree medium in this room so that the cells were maintained at 37°C throughout. The sub-clone was obtained using the method already described [9] for clones of HeLa cells sensitive to MMS. RESULTS
Continuous exposure
Fig. 1 shows survival curves for asynchronous HeLa cells of the standard line and also for a sub-clone selected from survivors of a previous treatment with MDMS. The drug was added to the culture medium at the time the cells were inoculated into bottles and this medium plus drug was left undisturbed for the whole lOday period of culture. The survival curves show a broad “shoulder” before following a steep exponential shape. The sub-clone is more sensitive to second treatment with MDMS than its parent cell line. The survival curve for Chinese hamster ovary cells is also shown (Fig. 1) after a similar treatment with MDMS except that in this case the culture period was halved to 5 days because the cells divide twice as frequently as HeLa cells (i.e. the doubling time of Chinese hemster ovary cells is 12 h, that of HeLa cells 24 h). A similar shape is seen in the survival curve with a broad shoulder followed by a steep exponential slope. 94
Fig. 1. Effect of MDMS on asynchronous cells. Dose-response curves for celh eu MOMS for the whole period of culture. (The points show the mean and stani.ard error of the mean of data pooled from 5 experiments with Chinese hamster ovgry &HO) cells, 11 with HeLa cells, and 2 with the HeLa sub-clone.) Fig. 2 Rate of inactivation of MDMS in Eagle’s medium (with 10% human serum) expressed in terms of increased survival of HeLa cells treated with aliquots of drugcontaining medium incubated at 37%. (Initial drug concentration 8 yglml.)
Pharmacokinetics The rate of inactivation of MDMS in culture medium at 37°C is shown in Fig. 2. HeLa cell survival increases with time but does not reach 100%. Rather, a plateau level of 40% survival is attained. MDMS is known [ 101 to hydrolyse to formaldehyde and 8 c(g MDMS will produce 1 JI dehyde. With 1 pg/ml formaldehyde the survival level was 39% to the final level in Fig. 3 after the hydrolysis of MDMS at 8 pg 0.5 I.cg/ml, the survival was 85%. The half-life of hydrolysis of MDMS [l] in water at 37°C is 22 min. A comparison of the data in Figs. 1 and 2 suggest that the biolo half-life does not conform with this. however; an inactivati min being the lowest estimate that can be derived. A simil tions was made with Chinese hamster ovary cells which activation time considerably in excess of 22 min. Cle sidual formaldehyde reduces the value of observations like tho where cells have been exposed continuously to MDMS and its b~~kdown products. 95
-0’0
0.040 MDMS uglmlll5minr
ot 37’C)
2
4 HOURS
6
8
IO
II
I4
t6--ik-%-k-%%
alter COLLECTION0f MIT~TIC
CELLS
Fig. 3. Dose-response curges for cells exposed to MDMS for 15 min at 37’C. (Data from 7 HeLa cell and 2 CH0 cell experiments.) -‘-ed HeLa cells exposed to MDMS (50 I.cg/ml for 15 min at Fig. 4. Survival of synchro..,a 37OC) at different times aT.*r mitotic selection. (Data from 2 experiments.)
Short treatment of asyrlchronous cells Fig. 3 shows survival curves for HeLa and Chinese hamster ovary cells exposed to MDMS for only 15 min before the drug-containing medium was removed and fresh medium substituted for the remainder of the culture period. Again, each curve has an initial shoulder but the subsequent exponential is much less steep.
Spchronised
HeLa celis
Fig. 4 shows the response of aliquots of synchronised populations of HeLa cells to the same concentration of MDMS (50 pg/ml, for 15 min) at different times during progress through the first cell cycle after mitotic selection. There is a small decrease in survival as the cells traverse the G, phase of the cycle (O-10 h) and then as the cells enter the DNA synthetic (S) period they 2 3 most sensitive. Towards the end of the S period sensitivity to MDMS is decreased reaching a minimum at the end of S. Then a small reduction in survival occurs as the cells pass through the G2 period (22-26 h). Thus in the HeLa cell. early-S is the most sensitive period and late-S is the most resistant to MDMS. Dose-response cwws were obtained at these two periods (Fig. 5). The exponential slope is the same for each period in the cell cycle; the sensitivity of cells in early-S, however, is shown as a diminution in the size of the initial shoulder of the survival curve.
96
SUHVIVING FRACTION
Fig. 5. Dose--response curves of synchronised HeLa cells exposed :o MDMS (far 15 min at 37’C) at 10 h (early S) and 22 h (late S) aftw mitotic selection. (Data from 2 experiments ’
DISCUSSION
Previouly [ 111 the reponse of HeLa cells to the monofunctional analogue MMS in the relatively long hydrolysis time (about 9 h) of ihe alkylating agent had to be taken into account. A short exposure time of 15 min was employed for studies where a pulse treatment was required for the purposes of the experiment, e.g. “split-dose” studies [ 121 or studies of the phase seirsitivity with synchronised cells [9]. With IMS on the other hand the short ilydsolysis time of 13.6 min enabled all such studies to be performed [ 131 without the need to change the culture medium. In both cases, the final hydrolysis products were relatively non-toxic compared with the unhydrolysed drug. An experiment similar to that described for in Fig. 2, when undertaken with MMS, showed cell survival rising t rather than 40% as in the present work. With MDMS a major product of hydrolysis is formaldehyde which is toxic :it the concentration levels expected by hydrolysis. It would be expected . that in vivo, the formaldehyde formed would be rapidly removed . fied, but in vitro, the accumulation of this product would be e nificantly to affect the cell survival. The extent of action with time may be judged from Fig. 2 which in effect suggests that MDMS is approximately eight-fold more toxic than formaldehyde at the same molar concentration, but that this toxicity would only be manifest by treatment within a short time interval after putting in solution. A compromise was adopted in which the cells were subjected to a 15.min “pulse” treatment with the the survival curves for this treatment are shown in Fig. 3 for both HeLa and Chinese hamster celi lines. Fig. 2 shows that the major contribution to cell 97
death from this short exposure must be from the drug and not from the formaldehycie formed. The Do for Chinese hamster ovary cells is 10 pg/ml with D, of ;j2 pg/ml(n), whereas the HeLa cells had a Do of 11.5 pg/ml and a D, of 26.5 pg/ml. The less steep slope of the pulse-treated lines, together with the larger D, values suggest that cell recovery is markedly improved by the pulse treatment. Furthermore the agent has a strong cumulative action in contrast to the big differences in continuous and pulse-treated regimes. The comparative survival of HeLa cells after a 15-min pulse treatment of synchrc nised HeLa cells is shown in Fig. 4. A slow decrease in survival results from treatment in G1 prior to the commencement of S phase. During S phase, an initial phase of 2-3 b (lo-13 h) is most sensitive followed by an increasing resistance through S, rising to least sensitivity during early G2 phase. Thus the greatest sensitivity in early S phase resembles the action of RCNU on Chinese hamster cells [ 141 in strain L mouse fibroblasts [ 153 and to uracil mustard [ 161. A similar pattern of sensitivity may be observed in synchronised HeLa cells, as cells selected in early S were more sensitive (Do = 15 pg/ml, D, = 16.3 I_cg/ml) than those in late S phase (Do = 15 pg/ml, D,= 49 yg/ml), the main difference being due to the extrapolation value and suggesting an increased potential for recovery within this phase. The pattern of sensitivity also closely resembles that reported for X-rays [ 171. Although the mode of action of this alkylating agent is not yet understood it is known that differences in sensitivity between sensitive and resistant lines of Yoshida cells are associated [IS] with a difference in their capacity to repair damaged DNA. The agent is considered 1191 to associate with GCrich regions of DNA producing a region of conformational instability and increasing the number of single-stranded regions. The present data suggest that the early stages of DNA synthesis may be more susceptible to faulty replication sequences resulting from such conformational damage than the later stages when replication is almost complete. This implies that +,he replicating fork itself could be a principal target for action. Thus when precautions are taken to exclude the action of the formaldehyde produced the synchronised HeLa cell line shows maximum sensitivity to cell killing at the end of G, and beginning of S and the least sensitivity at the end of S and during Gz. ACKNOWLEDGEMENT
The authors are grateful for helpful discussions with Dr. Margaret Fox and for the technical assistance of Mrs. Elisabeth Moore. REFERENCES 1 B.W. Fox and H. Jackson, In vivo effects of methylene dimethane sulphonate on proliferating cell systems, Brit. J. Pharmacol. Chemother., 24 (1965) 24. 2 B.W. Fox, The sensitivity of a Yoshida sarcoma to methylene dimethane sulphonate, Jnt. J. Cancer, 4 (1969) 54.
98
3 M. F*BX and B.W. FOX, The establishment of cloned cell lines from Yoshida sarcomas having differential sensitivities to methylene dimethane sulphonate in vivo and their cross sensitivity to X-rays and UV and other alkylating agents, Chem.-Biol. Interact., 4 (1971/i 2) 363. 4 D. Andtrson and A.H.W. Nias, Resistance to methylene dimethane sulphonate (MDMS) in L1210 cells in vitro, Eur. J. Cancer, 8 (1972) 659. 5 A.H.W. Nias, Clone size analysis: A parameter in the study of cell population kinetics, Cell Tissue Kinetics, 1 (1968) 153. 6 T.T. Puck and P.I. Marcus, Action of X-rays on mammalian cells, J. Exp. Med., 103 (1956) 653. 7 T. Terasima and L.J. Tolmach, X-Ray sensitivity and DNA synthesis in Fynchronous popuhtions of HeLa cells, Science, 140 (1963) 490. 8 A.H.U Nia; and M. Fox, Synchronisation of mammalian cells with respect to the mitotic cycle, Cell Tissue Kinetics, 4 (1971) 375. 9 M. FOX and A.H.W. Niaa, A modification of the sensitivity of mammalian cells surviving treatment with methyl meihanesulphonate, Eur. J. Cancer, 4 (1968) 325. 10 K. Edwards, H. Jackson and A. Jones, Studies with alkylating esters, III. The metabolism and fate of methylene dimethanesulphonate, Biochem. Pharmacol., 19 (1970) 1791. 11 J.J. Roberts, J.M. Pascoe, J.E. Plant, J.E. Sturrock and A.R. Crathorn, Quantitative aspects of the repair of alkylated DNA in cultured mammalian cells, I. The effect on HeLa and Chinese hamster cell survival of alkylation of cellular macromolecules, Chem.-Biol. Interact., 3 (1971) 29. 12 M. Fox, C.W. Gilbert, L.G. La_tha and A.H.W. Nias, The interpretation of “split-dose” experiments in mammalian cells after treatment with alkylating agents, Chem.-Biol. Interact., 1 (1969/70) 241. 13 D. Petrovic and A.H.W. Nias, A comparison of the nffects upon HeLa cells of isopropyl methanesulphonate and X-rays during different phases of the cell cycle, Eur. J. Cancer, 3 (1967) 329. 14 S.C. Barranco and R.M. Humphrey, The effects of 1,3-bis(2chloroethyl)-l-nitrosourea on survival and cell progression in Chinese hamster cells, Cancer Res., 31 (1971) 1218. 15 I.G. Walker and C.W. Helleiner, The sensitivity of cultured mammalian cells in different stages of the division cycle to nitrogen and sulfur mustard, Cancer Res., 23 (1963) 734. 16 F. Mauro and H. MadocJones, Age-response of cultured mammalian cells to cytotoxic drugs, Cancer Res., 30 (1970) 1397. 17 W.K. Sinclair, Cyclic X-ray responses in mammalian cells in vitro, Radiation Res., 33 (1968) 620. 18 M. Fox and B.W. Fox, Repair replication and unscheduled DNA synthesis in mammalian cell lines showing differential sensitivity to alkylating agents, Mutation Res., 19 (1973) 119. 19 D.G. Poppitt and B.W. Fox, The effect of mcthylene dimethanesulphonate (MDMS) on the conformation of DNA ant1 its dependence on base composition, Chem.-Biol. Interact., 11 (1975) 163.
99
