(CANCER RESEARCH 48. 4484-4488, August 15, 1988]

Mechanism of cw-Diamminedichloroplatinum(II)-induced Cytotoxicity: Role of G2Arrest and DNA Double-Strand Breaks1

Christine M. Sorenson and Alan Eastman2

Eppley Institute for Research in Cancer, University of Nebraska Medical Center, Omaha, Nebraska 68105

ABSTRACT

DNA has been implicated as the critical target for cis-diamminedi-chloroplatinum(II) (m-DDP)-induced cytotoxicity. In vitro, DNA-plati-IIIIMIadducts inhibit DNA synthesis. An assessment of the inhibition ofDNA synthesis in murine leukemia 1.121(1 cells demonstrated that,although cell division was halted, DNA replication continued for a periodof time. The DNA underwent almost a complete doubling even in cellsthat did not divide. Flow cytometric analysis demonstrated a slowedsynthetic phase which progressed to a block in the <•_•phase of the cellcycle. The duration of the G2 block was proportional to the concentrationof ciî-DDP.Low concentrations of c/ï-DDP caused the cells to betransiently blocked in the G2 phase for 24 to 48 h. Higher concentrationsof c/ï-DDPresulted in a G2 arrest that was not reversed by 96 h. Afterthis time, the arrested cells appeared to disintegrate, rather than recover.Cell survival and trypan blue exclusion studies indicated that, at low drugconcentrations, cells which had transiently arrested in the (..•phasesurvived, while at higher concentrations only a limited number of survivors were responsible for the observed recovery of growth. Analysis ofDNA double-strand breaks showed that significant numbers of breaksonly occurred at concentrations of cis-DDP that subsequently led todebris detectable on the flow cytometer and to loss of trypan blueexclusion. The formation of these breaks appeared to be the first detectable change that was indicative of cell death. It is proposed that cellsarrest in the G2 phase because they are unable to transcribe damagedDNA and make mRNA essential for passage into mitosis. DNA repairprobably overcomes this arrest. Cell death may therefore be a consequence of the inability to adequately recover transcription.

INTRODUCTION

In 1969, Rosenberg et al.(\) demonstrated that the platinumcoordination compound m-DDP3 had potent antitumor activity in a murine leukemia LI210 cell line. c/5-DDP was subsequently shown to be an effective therapy for such humandisorders as testicular, ovarian, head and neck, bladder, prostate, and lung cancers (2).

DNA has been implicated as the critical target for c/s-DDP-induced cytotoxicity (3). The major damage is DNA-intrastrandcross-links; DNA-protein and DNA-interstrand cross-links represent less than 1% of the total DNA platination (reviewed inRef. 4). Evidence from this and other laboratories has demonstrated that DNA polymerases are halted by a's-DDP adducts

in DNA in vitro (5, 6). However, the lack of integrity of thesein vitro systems precludes the possibility of bypass mechanismsthat might be present in intact cells. Inhibition of DNA synthesis has been shown to be a consequence of platination of humanAV3 cells in culture (7) and Ehrlich ascites cells in vivo (8).However, an attempt to correlate the extent of the inhibitionof DNA synthesis with toxicity showed that much higher levels

Received 8/18/87; revised 3/7/88, 5/4/88; accepted 5/11/88.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' This work was supported by National Cancer Institute Research GrantsCA36039 and CA00906 and by Cancer Center Support Grant CA36727. C. M.S. was supported by a fellowship from the Elizabeth Bruce and Parents MemorialEndowment.

2To whom requests for reprints should be addressed.'The abbreviations used are: c/s-DDP, c/s-diamminedichloroplai ¡muniII),

LCso. 50% lethal concentration.

of drug were required to inhibit DNA synthesis than wereneeded to inhibit cell growth (9). This experiment only measured DNA synthesis for 90 min immediately after drug treatment. This type of experiment has several potential drawbacks:were the criteria for toxicity and DNA synthesis inhibitionlegitimate? For example, it is possible that DNA synthesis isnot immediately inhibited by the drug.

In the present studies we investigated inhibition of DNAsynthesis in murine leukemia LI210 cells at extended timeperiods and noted a progressive inhibition of DNA synthesis.This led to an investigation of the cell cycle distribution ofplatinated cells, and we report here on the G-i arrest that isinduced by this drug. In addition, we have attempted to relatethese observations to various indices of toxicity, i.e., clonalgrowth in soft agar, growth curves, trypan blue exclusion, andDNA breakage.

MATERIALS AND METHODS

Cell Culture Methods. L1210/0 cells were maintained in an exponential suspension culture at 37°Cin a humidified atmosphere of 5% CO2-95% air in McCoy's medium 5a (modified) (Gibco, Grand Island, NY),supplemented with 15% calf serum, penicillin, streptomycin, and Fun-gizone. In all experiments, 112100 cells were incubated in variousconcentrations of c/s-DDP (Bristol Laboratories, Syracuse, NY) for 2h at 37°C.To measure growth inhibition, the cells were then centri-

fuged, washed once, resuspended in fresh medium at 30,000 to 50,000cells/ml, and incubated for various time periods. Cell numbers weredetermined on a Coulter Counter. At the same time intervals, an aliquotof cells was diluted with an equal volume of 0.4% trypan blue. Viabilitywas recorded as the percentage of cells that had excluded the dye. Cellsincubated with drug as above were also diluted into 0.1% agar andallowed to grow for 2 wk at which time colonies were counted (10).

Measurement of DNA Synthesis. LI210/0 cells were grown in thepresence of 0.04 ^Ci/ml of [3H]thymidine (3.9 Ci/mmol; Amersham

Corporation, Arlington Heights, IL) for 48 h after which they wereincubated with varying concentrations of cis-DDP for 2 h. The cellswere then washed, resuspended in fresh medium, and harvested up to3 days later, at which time the cell growth was determined on a CoulterCounter. DNA was purified from the cells by a modification of thetechnique of hydroxyapatite column chromatography (11). The purifiedDNA was redissolved in 0.1 N HC1, and absorbance (A26o)and radioactivity were assessed. Total DNA synthesis occurring during thisperiod was calculated from the reduction in the DNA specific radioactivity.

Flow Cytometry. Cell cycle analysis was performed by a modificationof the method of Darzynkiewicz et al. (12). Briefly, the LI210 cellswere rinsed once with phosphate-buffered saline containing 5 IHMMgCl2 and fixed in 80% ethanohacetone (1:1) for at least 18 h at 4'C.

The fixed cells were centrifuged, rinsed once in phosphate-bufferedsaline, and resuspended in 50 ¿igof propidium iodide/ml of phosphate-buffered saline, and 1000 units of RNase A (Sigma Chemical Co., St.Louis, MO) were added. This was incubated for 30 min at 37°C,and

the cells were analyzed on a Becton-Dickinson flow cytometer.Neutral Elution. The detection of double-strand breaks was accom

plished by the method of Long et al. (13). LI 210/0 cells were incubatedwith 0.01 fiCi/ml of [MC]thymidine (61 mCi/mmol; Amersham Cor

poration) for 48 h, washed, incubated with various concentrations ofc/'s-DDP for 2 h, washed, and incubated with 0.03 ¿iCi/mlof [3H]

thymidine for 6 h. This provided different radiolabels in the parent

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MECHANISM OF c/j-DDP-INDUCED CYTOTOXICITY

strand and the posttreatment, daughter strand. Approximately IO6cellswere harvested at 24 to 96 h after removal of [3H]thymidine, resus-

pended in 75 mM NaCl-24 mM disodium EDTA, pH 7.0, and layeredon 25-mm-diameter, 2-Mm-pore-size polycarbonate filters (NucleoporeCorp., Pleasanton, CA). The cells were lysed at room temperature inthe dark by pumping 10 ml of 2% sodium laurylsarcosine-2 M NaCl-

40 mM disodium EDTA, pH 9.6, containing 100 vg/ml of proteinaseK through the filters at 10 ml/h. The filters were rinsed by pumping 10ml of 25 mM disodium EDTA, pH 9.6, containing 100 jig/ml ofproteinase K through the filter at the same flow rate. The DNA waseluted in a solution consisting of 20 mM tetrahydrogen EDTA andenough tetrapropylammonium hydroxide (Eastman Kodak Co., Rochester, NY) to yield a pH of 9.6. Fractions, 2 ml each, were collected ata rate of 2 ml/h for 16 h and neutralized, and the radioactivity wasdetermined using a Beckman scintillation counter programmed fordouble-labeled samples.

RESULTS

DNA Synthesis. DNA synthesis and cell division were assessed in untreated and ds-DDP-treated L1210/0 cells. Theuntreated cells doubled both their cell number and DNA contentevery 24 h (Fig. 1). Cells incubated with c/s-DDP displayed adose-dependent inhibition of cell growth. At the highest concentrations used, the cells did not divide during the 3-dayexperiment (Fig. \A). In cells incubated with cis-DDP, the DNAdoublings were inhibited much less than cell growth (Fig. IB).Even in cells that did not divide during the 3-day incubation,the DNA appeared to have almost completely doubled. Inhibi-

0 Ma/ml

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1 ug/ml

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4jug/ml8pg/ml

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Days of Growth

Fig. 1. Comparison of the inhibition of cell growth (A) and DNA synthesis(B) in LI210/0 cells. The cells were incubated for 2 h with the indicatedconcentrations of cis-DDP, and the cell doublings (A) and DNA doublings (B)were determined at 1 to 3 days.

tion of DNA synthesis therefore did not correlate with inhibition of cell growth.

Flow Cytometry. Cell cycle analysis was performed to determine the cause of the increased DNA content in these cells.Control LI 210/0 cells had approximately 30% of their cells inthe G2 + M phases of the cell cycle. In initial experiments,drug-treated cells were followed for 4 days (Fig. 2). After 24 h,a large proportion of the cells had become arrested in the G2phase of the cell cycle. At intermediate concentrations of cis-DDP, the G2 arrest was maximum at 48 h, and an extended Sphase was apparent. The G2 arrest was transient at low concentrations, being reversed by 72 to 96 h. At higher concentrationsthe G2 arrest persisted throughout the 4-day experiment. Insubsequent experiments, cells treated at the higher concentrations of cis-DDP were followed for up to 10 days (Fig. 2). Asignificant increase occurred in the amount of cellular debrisdetected (peak prior to d phase). The cell cycle analysis demonstrated that cells were able to bypass the G2 arrest. It wastherefore necessary to determine whether these cells survived.

Long-Term Survival Curves. Rather than terminating theexperiment at 3 days as previously (Fig. 1), cells were incubatedfor up to 11 days after drug treatment (Fig. 3). It was evidentthat cells that did not divide during the first 3 days eventuallyregained their ability to divide. The time at which growthresumed was dependent on the drug concentration. At thehighest concentration of cis-DDP, the cells did not regain theirability to divide during the experiment.

Trypan Blue Exclusion. Trypan blue exclusion was used todetermine the membrane integrity of LI210/0 cells as an indicator of cell viability (Fig. 4). The percentage of viability remained above 90% for the first 3 days. At later time points,differences in viability became evident. LI210/0 cells incubatedwith low concentrations of c/s-DDP remained viable throughoutthe entire experiment. In contrast, cells incubated with highdrug concentrations demonstrated a continual decrease in viability up to 8 days after incubation. After this time, the viabilityof the population increased dramatically. This illustrated that,at low concentrations, cis-DDP induced minimal cell death.However, at higher drug concentrations, cell death was quitepronounced with only 4% viability at the highest concentration.By 10 days after incubation, these few survivors had begun to

DRUGCONCENTRATION(jjg/ml>

l|0 OS 1 1.5 2 4 8

1<Jay A\ A\ A\ rA2 days

3 days IU\ IU\.AA

4 days

6 days

8 days

10 days

Fig. 2. Cell cycle distribution of LI210/0 cellsindicated concentrations of m-DDP, posttreatmentfixed, stained, and analyzed by flow cytometry.

incubated for 2incubated for 1

h with theto 10 days,

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MECHANISM OF cís-DDP-INDUCED CYTOTOXICITY

100

p4ug/ml

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,'2 ,4/6' 8 10 12

Incubation Time (days)

-8J

Fig. 3. Assessment of the long-term growth of L1210/0 cells after a 2-hincubation with the indicated concentrations of cis-DDP. The back extrapolationof these growth cunes was used as a parameter of cell survival presented in Fig.5.

100

2 4 6 8 10Incubation Time (days)

Fig. 4. Exclusion of trypan blue by LI210/0 cells at various times after a 2-hincubation with m-DDP at concentrations of 0 (•),0.5 (A), 1 (•),2 (A), 4 (O),and 8 (D) „uml

grow and represented the predominant cells in the population.Comparison of Parameters of Toxicity. Previous studies have

routinely analyzed toxicity by the loss of ability of drug-treatedcells to form colonies in soft agar. We therefore repeated suchan experiment (Fig. 5) and obtained an LC5o value of 0.2 ^g/ml. This is consistent with other reports for these cells (14).However, this concentration is considerably lower than would

246

cjs - DDP CONCENTRATION ()jg/ml)

Fig. 5. Percentage of "survival" of L12IO/0 cells calculated from growth insoft agar (•),growth in microtiter wells (•),back extrapolation of long-termgrowth curves (Fig. 3; A), inhibition of cell growth over 3 days (Fig. 1; D), ormaximal loss of trypan blue exclusion (Fig. 4; O).

be predicted from flow cytometry and trypan blue exclusionexperiments. Different methods of evaluating toxicity weretherefore compared. An alternate colony-forming assay wasperformed in microtiter wells to avoid any potential contributions from agar. This survival curve (Fig. 5) gave an LC;o of0.4 ng/m\. Other authors have also used a method of backextrapolation of growth curves such as in Fig. 3 (15, 16). Thisalso gave an LC5oof about 0.4 pg/ml.

In this laboratory and in many others routinely performingdrug testing in LI210 cells, toxicity has been reported as theconcentration of drug that inhibits cell growth by 50% asmeasured over 3 days (as shown in Fig. 1). The 50% inhibitoryconcentration in this experiment was 0.7 /¿g/rnl.The finalsurvival curve shown in Fig. 5 was obtained from the maximumpercentage of cells that lost the ability to exclude trypan blue(Fig. 4). In this case the LC50 was calculated to be about 1.8Mg/ml. In the flow cytometry analysis (Fig. 2), it also requiredabout 1.5 Mg/ml of cis-DDP before about 50% of the cells couldbe considered debris. This maximum occurred 8 days after drugtreatment.

DNA Breakage. Neutral elution was used to detect the formation of DNA double-strand breaks in cells incubated withcis-DDP. The DNA in both the [3H]thymidine-labeled daughterstrand and the "(labeled parent strand eluted at the same rate

in the LI210/0 cell line (Fig. 6). In cells incubated with lowconcentrations of drug, no double-strand breaks were detected.As the concentration of cis-DDP increased, the amount of DNAeluting from the filter increased in a dose-dependent fashion.The double-strand breaks persisted throughout the 4-day experiment.

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MECHANISM OF rii-DDP-INDUCED CYTOTOXICITY

50

40

30

20

10

LUCO< 40LUOCO

Z 30

20

10

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2 ug/ml

1 jjg/ml

0.5 ug/ml

[14CJTHYMIDINE (PARENT)

ug/ml

0.5 ug/ml

24 48 72

INCUBATION TIME (H)

Fig. 6. The formation of double-strand breaks in the DNA of LI 210/0 cellsafter a 2-h incubation with the indicated concentrations of ciî-DDP.DNA breakswere measured by the technique of neutral elution, and values are expressed asthe amount of DNA eluted from the filters during the 16-h elution. Top, DNAlabeled with [3H]thymidine after the incubation with c/i-DDP; bottom, ['4C]-thymidine-labeled "parent" strand.

DISCUSSION

This study was stimulated by a report that inhibition of DNAsynthesis did not correlate with toxicity (9). However, the twoevents were measured at different time points, i.e., DNA synthesis was measured during the first 90 min after platination,while toxicity was the result of events occurring over a muchlonger time period. It seemed likely that inhibition of DNAsynthesis could be a progressive event. In measuring the amountof DNA synthesized, it became evident that this was indeed thecase. Although cell division could be completely inhibited for 3days, DNA replication continued. The outcome was that cellshad up to twice the normal complement of DNA.

Flow cytometry was used to gain an understanding of thediscrepancy between the cell and DNA doublings. At low concentrations of cii-DDP, a slowed synthetic phase and transientG2 arrest were observed, while at higher concentrations thisarrest persisted throughout the 4-day experiment. Many chem-otherapeutic agents preferentially block cells in the G2 phase ofthe cell cycle. The duration and magnitude of the arrest aredependent on the length of treatment time and concentrationof the drug. As the treatment period or concentration of thedrug is increased, the delay becomes more pronounced, and anirreversible block may occur (17). Irreversible drug-induced G2arrest has been associated with extensive chromosome damage(18).

Formation of a G2 arrest after incubation with c/s-DDP hasbeen shown by several investigators (19, 20). Mouse mammarycarcinoma cells formed a G2 arrest which was still apparent

after 30 h, the maximum time period in the study (19). Ehrlichascites tumor cells incubated with c/i-DDP demonstrated a G2block which was transient at low concentrations, but at higherconcentrations was reportedly irreversible, even though the cellswere only followed for 48 h. At the highest concentrations, thecells became "frozen" in the cell cycle (20). In the present study

we extended incubation times even further. The G2 arrest wastransient at low concentrations while at higher concentrations,only a portion of the population may eventually bypass thisarrest. An important question therefore is whether such cellssurvive.

The podophy Ilotoxin derivative VP-16-213 has been shownto transiently arrest human lymphoid cells in the G2 phase. Thecells subsequently divided, but the progeny only retained alimited reproductive potential, and the amplitude and durationof the G2 block did not correlate with cell survival (21). In thecase of c/s-DDP in LI210 cells, we observed that, after a periodof no cell division, the cells began to grow. Therefore, cellswhich are transiently arrested in the G2 phase regain theirgrowth potential after the G2 arrest is bypassed. This thenrepresents a slowed population of cells. However, at higherconcentrations, the G2 arrest is persistent, and there is a definiteloss of viability after 4 days. This suggests that cells may remainin the G2 phase of the cell cycle for a limited period of time,after which they either reenter the cell cycle or die. After aperiod of approximately 8 days, repopulation occurs by survivors which accounts for the resumption of growth demonstratedby the long-term survival curves. These types of growth curves

have frequently been used to calculate the number of survivorsin a population (15, 16). This number is obtained by extrapolating the growth curves back to the ordinate. Such an extrapolation assumes that all survivors continue to grow at anunaltered rate, while any cell whose growth is slowed wouldhave to be considered as dead. This is obviously not the case.For example, at 1 ng/m\ of c/'s-DDP, cells maximally arrested

in the G2 phase at 2 days. After 4 days they regained normalgrowth. The trypan blue experiments suggested that less than10% of the cells died. Survival of the cells is therefore 90%,whereas extrapolation of the line suggested only 3% survival(Fig. 5). At higher drug concentrations, significant numbers ofcells lost membrane integrity as assayed by trypan blue exclusion. Although these cells are obviously dying, extrapolation ofthe growth curves would still produce an erroneously low valuefor the number of surviving cells. This type of extrapolationcan therefore only provide a minimum value for LCso. Theclonogenic survival assays gave LC5o values comparable to thisextrapolation value (Fig. 5) in agreement with previous work(15). This suggests that clonogenic assays may only representthe growth of undamaged cells, not cells that are temporarilyarrested. Therefore, longer incubation times after drug treatment might be expected to produce more colonies. We havebeen unable to demonstrate this. At present, we cannot explainwhy transiently damaged cells will not produce colonies,whereas they will grow under normal culture conditions. Thejustification for use of clonogenic assays is that, "in cancer

research, cell death often means reproductive death, the failureof proliferation, not loss of viability" ( 15). This is a justifiable

sentiment, but the assays to measure reproductive death do nottake into consideration that a nonreproductive but viable cellcan regain the capacity to reproduce. This has a considerablesignificance if one considers dormant tumor cells that can bestimulated to divide and perhaps lead to patient relapse. Ourresults show two fates for a drug-damaged cell: recovery of

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MECHANISM OF cij-DDP-INDUCED CYTOTOXICITY

reproductive potential or death. This does not support theconcept of viable but nonreproductive cells.

The questions posed by these observations relate to why cellsarrest in the G2 phase, how they overcome this block to surviveand, if they fail, how cell death results. We tested the hypothesisthat the G2 period was required for postreplication repair, thatis, the repair of damage caused by the process of DNA synthesison damaged DNA. Blocks to DNA synthesis are thought tocause gaps in the newly synthesized daughter strand. Thesemay be Tilled by a gap-filling or recombination mechanism; thelatter mechanism is reportedly associated with formation andrepair of DNA double-strand breaks in E. coli (22). A similarreport pertains to eukaryotic cells (23), but in that study it wastranslated as repair even though greater than 99% of the cellswere unable to produce colonies. Therefore, we investigated theformation of single- and double-strand breaks in the DNA ofLI210/0 cells. The single-strand breaks were analyzed by thetechnique of alkaline elution (not shown) but demonstrated noadditional breaks than detected by neutral elution for double-strand breaks. Significant numbers of double-strand breaks onlyoccurred at concentrations of c/s-DDP that subsequently led todebris detectable on the flow cytometer and to loss of trypanblue exclusion. Therefore, the formation of double-strandbreaks appeared to be the first change detected in cells that aredestined to die. We could not provide evidence for postreplication repair occurring during the (i.- phase. It is assumed thatthis process occurs during, and is probably responsible for, theextended S phase. These experiments have been repeated in anLI210 subline 100-fold resistant to c/s-DDP (24), and similarresults were obtained at equitoxic concentrations of c/s-DDPwhen the cells were analyzed for flow cytometry, long-termgrowth curves, and DNA double-strand breaks (not shown).

A reason for the G2 arrest is still needed. Tobey has suggestedthat a surveillance mechanism operates during the G2 phase,which eliminates cells with unrepaired DNA while cells withrepaired DNA are returned to the cycle (25). Such a mechanismcould work as follows. It is known that transcription is requiredfor passage into mitosis (18). In other studies, we have demonstrated that every c/s-DDP adduct in a bacterial gene trans-fected into LI 210/0 cells is capable of eliminating gene expression (26). Cells may therefore arrest in G2 because they areunable to make essential transcripts. In DNA repair-competentcells, the arrest would be overcome after the critical adductshave been repaired.

These results can be summarized in the following hypothesis:inhibition of transcription is more critical to toxicity thaninhibition of DNA synthesis. Cells are obviously capable ofreplicating past lesions in DNA by processes collectively knownas postreplication repair, although the mechanisms are unknown. However, there is no known pathway whereby transcription can bypass lesions. Presumably at high concentrations ofdrug, the repair processes required for recovery of transcriptionare themselves overwhelmed, endonucleases are aberrantly expressed, DNA is degraded, and the cells die.

ACKNOWLEDGMENTS

The authors wish to thank Dr. Robert Briggs, Vanderbilt University,for advice in performing flow cytometric analysis and Dr. William

Vaughan, University of Nebraska Medical Center, for making his flowcytometer available for these studies.

REFERENCES

1. Rosenberg, B., Van Camp, L., Trosko, J. E.. and Mansour, V. H. Platinumcompounds: a new class of potent antitumor agents. Nature (Lond.), 222:385-386, 1969.

2. Loehrer, P. J., and Einhorn, L. H. Cisplatin. Ann. Intern. Med., 700: 704-713, 1984.

3. Roberts, J. J.. and Thomson, A. J. The mechanism of action of antitumorplatinum compounds. Prog. Nucí.Acid Res. Mol. Biol., 22: 71-133, 1979.

4. Eastman, A. The formation, isolation, and characterization of DNA adductsproduced by anticancer platinum complexes. Pharmacol. Ther., 34; 155-166.1987.

5. Eastman, A. Characterization of the interaction of ru-diamminedichloropla-tinum(II) with DNA. In: M. P. Hacker, E. B. Douple, and I. H. Krakoff(eds.). Platinum Coordination Complexes in Cancer Chemotherapy, p. 56.Boston: Martinus Nijhoff Publishing, 1984.

6. Pinto, A. L., and Lippard, S. J. Sequence-dependent termination of in vitroDNA synthesis by CM-and fra/u-diamminedichloroplatinum(II). Proc. Nati.Acad. Sci. USA, «2:4616-4619, 1985.

7. Harder, H. C., and Rosenberg, B. Inhibitory effects of anti-tumor platinumcompound on DNA, RNA, and protein synthesis in mammalian cells in vitro.Int. J. Cancer, 6: 207-216, 1970.

8. Howie, J. A., and Gale, G. R. cú-Dichlorodiammineplatinum(II) persistentand selective inhibition of deoxyribonucleic acid synthesis in vivo. Biochem.Pharmacol., 19: 2757-2762, 1970.

9. Salles. B., Butour, J.-L., Lesea, C., and Macquet, J.-P. cii-Pt(NH3)¡CI¡and/rans-Pt(NHj)2Cl2 inhibit DNA synthesis in cultured L12IO leukemia cells.Biochem. Biophys. Res. Commun., II2: 555-563, 1983.

10. Chu, M.-Y..and Fischer, G. A. The incorporation of [3H]cytosine arabinosideand its effect on murine leukemic cells (L5178Y). Biochem. Pharmacol., 17:753-767, 1968.

11. Eastman. A., and Schulte, N. Enhanced DNA repair as a mechanism ofresistance to ci5-diamminedichloroplatinum(II). Biochemistry, in press,1988.

12. Darzynkiewicz, Z. Discrimination of Go, Gì,S, G2, and M phases bycytofluorographic analysis. In: Ortho Instruments Protocol No. 25. West-wood, MA: Ortho Instruments, 1978.

13. Long, B. H., Musial. S. T., and Braltain, M. G. Single- and double-strandDNA breakage and repair in human lung adenocarcinoma cells exposed toetoposide and teniposide. Cancer Res., 45: 3106-3112, 1985.

14. Micetich, K., Zwelling, L. A., and Kohn, K. W. Quenching of DNA:platinum(II) monoadducts as a possible mechanism of resistance to cis-diamminedichloroplatinum(II) in 11210 cells. Cancer Res.. 43: 3609-3613,1983.

15. Ducore, J. M., and Barth, E. Quantitative cell survival from growth curvesfollowing anti-neoplastic drug treatment. Mutât.Res., 122: 391-396, 1983.

16. Sladek, N. E., Low, J. E., and Landkamer, G. J. Collateral sensitivity tocross-linking agents exhibited by cultured LI210 cells resistant to oxaza-phosphorines. Cancer Res.. 45:625-629, 1985.

17. Barlogie, B., and Drewinko, B. Cell cycle perturbation effects. In: B. W. Foxand M. Fox (eds.), Antitumor Drug Resistance, pp. 101-141. New York:Springer-Verlag, 1984.

18. Rao, P. N. The molecular basis of drug-induced G: arrest in mammaliancells. Molec. Cell. Biochem., 29:47-57, 1980.

19. Ranno, S., Hyodo, M., Suzuki, K., and Ohkido, M. Effect of DNA-damagingagents on DNA replication and cell cycle progression of cultured mousemammary carcinoma cells. Jpn. J. Cancer Res., 76: 289-296, 1985.

20. Kopf-Maier, P., Wagner, W., and Liss, E. Induction of cell arrest at G,/Sand in <¡•after treatment of Ehrlich ascites tumor cells with metallocenedichlorides and m-platinum in vitro. J. Cancer Res. Clin. Oncol., 106: 44-52, 1983.

21. Drewinko, B.. and Barlogie, B. Survival and cycle-progression delay of humanlymphoma cells in virro exposed to VP-16-213. Cancer Treat. Rep., 60:1295-1305, 1976.

22. Wang, T. V., and Smith, K. C. Postreplicational formation and repair ofDNA double-strand breaks in UV-irradiated Escherichia coli uvr B cells.Mutât.Res., 165: 39-44, 1986.

23. Wang, T. V., and Smith, K. C. Postreplication repair in ultraviolet-irradiatedhuman fibroblasts: formation and repair of DNA double-strand breaks.Carcinogenesis (Lond.). 7: 389-392, 1986.

24. Richon, V. M., Schulte, N., and Eastman, A. Multiple mechanisms of cellularresistance to platinum coordination complexes. Cancer Res., 47:2056-2061,1987.

25. Tobey. R. A. Different drugs arrest cells at a number of distinct stages in G:.Nature (Lond.), 254: 245-247, 1975.

26. Eastman, A., Schulte, N., Sheibani, N., and Sorenson, C. M. Mechanisms ofresistance to platinum drugs. /;/.•M. Nicolini (ed.). Platinum and Other MetalCoordination Compounds in Cancer Chemotherapy, pp. 178-196. Boston:Martinus Nijhoff Publishing, 1988.

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1988;48:4484-4488. Cancer Res   Christine M. Sorenson and Alan Eastman 

Arrest and DNA Double-Strand Breaks2Cytotoxicity: Role of G-Diamminedichloroplatinum(II)-inducedcisMechanism of

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