Radiation Survival Parameters of Antineoplastic Drug-sensitive and -resistant
Human Ovarian Cancer Cell Lines and Their Modification by Buthionine
Sulfoximine
Karen G. Louie, Brent C. Behrens, Timothy J. «insella,Thomas C. Hamilton, Karen R. Grotzinger,Wilma M. McKoy, Margaret A. Winker, and Robert F. Ozols1
Medicine Branch [K. G. L, B. C. B., T. C. H., K. R. G., W. M. M., M. A. W., R. F. O.] and Radiation Oncology Branch [T. J. K.J, Division of Cancer Treatment, NationalCancer Institute, Bethesda, MD 20205
ABSTRACT
The optimum integration of chemotherapy and irradiation is ofpotential clinical significance in the treatment of ovarian cancer.A series of human ovarian cancer cell lines have been developedin which dose-response relationships to standard anticancerdrugs have been determined, and the patterns of cross-resist
ance between these drugs and irradiation have been established.By stepwise incubation with drugs, sublines of A2780, a drug-sensitive cell line, have been made 100-fold, 10-fold, and 10-foldmore resistant to Adriamycin (2780- melphalan (2780ME),andcisplatin (2780CP). Two additional cell lines, NIHiOVCAR-S""1*8*'and NIH:OVCAR-4(A9+), were established from drug-refractorypatients. 2780ME,2780CP,OVCAR-3nu(Afl+>,and OVCAR-4(A9+)are
all cross-resistant to irradiation, with D0s of 146, 187, 143, and203, respectively. However, 2780*° remains sensitive to radia
tion, with a Do of 111, which is similar to that of A2780 (101).Glutathione (GSH) levels are elevated in 2780ME, 2780CP, OV-CAR-S"**9*', and OVCAR-4(A9+>to 4.58, 6.13, 12.10, and 15.14nmol/106 cells as compared to A2780, with 1.89 nmol/106 cells.However, the GSH level in 2780*° is only minimally higher thanthat in A2780 (2.94 nmol/106 cells). Buthionine sulfoximine, a
specific inhibitor of GSH synthesis, significantly increases theradiation sensitivity of 2780ME (changing the D0 from 143 to 95)and 2780CPto a lesser extent, suggesting that intracellular GSH
levels may play an important role in the radiation response ofcertain neoplastia cells. These results suggest that the sequentialuse of irradiation following chemotherapy with melphalan andcisplatin may be less effective than a combined modality approach, which integrates radiation and chemotherapy prior tothe development of drug resistance and cross-resistance to
irradiation.
INTRODUCTION
Most patients with ovarian cancer have advanced disease(FIGO Stage III to IV) at the time of diagnosis (10). Combinationchemotherapy regimens have produced clinical complete responses in 40 to 50% patients with advanced disease, andsecond-look laparotomies have demonstrated that the patholog
ical complete response rate is 20 to 30% (5, 14, 44, 46, 47).Whole abdominal irradiation has also been demonstrated to beeffective therapy in patients with small volume residual disease(11, 12). In an effort to increase the complete response rate,combined modality approaches with radiation and chemotherapy
1To whom requests for reprints should be addressed.
Received 8/20/84; revised 12/13/84; accepted 1/31/85.
are currently under investigation at the National Cancer Instituteand elsewhere (18, 24, 28). The designs of these clinical trialsintegrating irradiation and chemotherapy have been empiricallyderived, and the preliminary results of most studies have failedto demonstrate a marked advantage over the use of combinationchemotherapy alone (24, 28). Experimental studies on patternsof radiation and chemotherapeutic sensitivity in relevant modelsystems of ovarian cancer may be useful in optimizing treatmentschedules.
Recently, several ovarian cancer cell lines have been established, both from previously untreated patients and from patientsclinically resistant to chemotherapy (2, 22). NIH:OVCAR-4(A9+)2and NIH:OVCAR-3nu<A9+)were derived from patients who had
become refractory to cyclophosphamide, cisplatin, and Adra-
mycin. In addition, by stepwise incubation of A2780 (a sensitivecell line cloned from an untreated patient) with drugs, variant celllines with resistance to Adriamycin (2780*°),melphalan (2780ME),or cisplatin (2780CP)were established (16, 22). The sublines are
approximately 100-fold, 10-fold, and 10-fold more resistant to
the respective drugs than is the parent line (A2780). These celllines have been used to determine dose-response relationships
to chemotherapy, to study the radiosensitivity of human ovariancancer cells, and to establish the pattern of cross-resistance toirradiation in cell lines with primary resistance induced againstantineoplastic drugs. 2780ME is 3-fold more resistant to Adriamycin than is A2780, and 2780*° is 2.5-fold more resistant to
melphalan than is the parent line (2). In addition, these cell linesmay be useful for evaluating the role of radiation and chemosen-
sitizing agents such as BSO. This drug, a specific inhibitor ofglutathione synthesis, has been shown to markedly depress theintracellular GSH level and enhance both radiation and chemo-sensitivity of many cell types (7,13,19-21, 29,39). The results
of these studies may have direct relevance to the design ofclinical trials aimed at evaluating combined modality therapy inovarian cancer.
MATERIALS AND METHODS
Chemicals and Reagents. RPM11640, fetal bovine serum, penicillin/streptomycin, and glutamine were obtained from Grand Island Biological
2The abbreviations used are: NIHrOVCAR-S™**«*',a variant of National Institutes
of Health Human Ovarian Carcinoma cell line #3 which has been selected forgrowth in nude mice (nu) and substrate independent growth in agarose (Ag+);NlHiOVCAR^"«*', a variant of OVCAR cell line #4 selected for substrate inde
pendent growth in agarose; A2780, a human ovarian cancer cell line kindly providedby Or. S. A. Aaronson, Laboratory of Cellular and Molecular Biology, NationalCancer Institute (16); 2780*°, 2780**, and 2780CP, variants of A2780 selected for
resistance to Adriamycin, melphalan, and cisplatin, respectively; BSO, buthionmesulfoximine; GSH, glutathione; IC»,drug concentration which inhibits 50% colonygrowth; DO,slope of the exponential portion of the curve.
RADIOSENSITIZATION OF HUMAN OVARIAN CANCER CELLS WITH BSD
Company, Chagrin Falls, OH. Insulin (lletin U-100) was from EN Lilly &Company, Indianapolis, IN. D,L-Buthionine^S, fl-sulfoximine was from
Chemalog (Chemical Dynamics Corporation), South PlainfieW, NJ. TypeVII agarose was obtained from Sigma Chemical Company, St. Louis,MO.
Tissue Culture Medium. Cells were passaged and maintained inRPMI 1640 supplemented with 10% (v/v) fetal bovine serum, insulin(0.25 U/ml), penicillin (100 U/ml), streptomycin (100 ^g/ml), and glutamine(0.3 mg/ml) as described previously (32). Cells were incubated at 37°C
in a humidified atmosphere of 5% (v/v) CO2 and medium was changedevery 3 to 4 days.
Clonogenic Assay. Drug sensitivity and radiation survival curves weredetermined using clonogenicity in soft agarose as described previously
(32). In brief, cells were harvested with a trypsin (0.05%, w/v)/EDTA(0.02%, w/v) solution and counted with a Coulter counter (CoulterElectronics model ZBI). Cells in single cell suspension were plated in amixture of 0.3% (w/v) agarose and RPM11640 (including the ingredientslisted above) over a layer of solidified 0.6% (w/v) agarose in 10-sq cm
dishes. A2780 and its variants were plated at a concentration of 10,000to 15,000 cells/dish, yielding cloning efficiencies of 20 to 45%. OVCAR-4(A«+>had a cloning efficiency of 4%, with 20,000 cells/dish, and OVCAR-3nu(*g+)nad a C|on¡ngefficiency of 1%, with 75,000 cells/dish.
Radiation. Cells growing exponentially were plated in 10-sq cm dishes
as described above. At 4 to 6 h after trypsinization, cells were irradiatedat room temperature using a 15-MeV photon beam from a Vanan 20
linear accelerator. Dishes received graded doses of radiation from 50 to600 rads at a dose rate of 500 rads/min. Dosimetry was carried outusing a Baldwin-Farmer ionization chamber connected to a Keithly elec
trometer system having direct National Bureau of Standards calibration.Full electron equilibrium was insured for all radiation. Dishes were incubated for 1 week at 37°C in a humidified 5% (v/v) CO2 atmosphere.
Colonies measuring greater than 60 /¿mand containing greater than 50cells were counted on a Bausch and Lomb Omnicon FAS II system.Using the linear portion of the curves, a least squares regression analysisof the individual dose data points for each cell line was determined togenerate radiation survival curves. At least 2 experiments were conducted on each cell line. The radiation curves are characterized by ashoulder at low doses, merging into an exponential line at higher doseson a semilogarithmic plot. Conventionally, two parameters have beenused to describe the radiation survival curve: D0, the slope of theexponential portion of the curve (the dose required to reduce survival to
0.37 on any survival level of the exponential portion of the curve) and Dqor n (extrapolation number), which is a measure of the width of theshoulder of the curve and which is calculated by extrapolating the straightportion of the curve back to the surviving fraction axis. Multiplicity at thetime of irradiation was assumed to be unity.
BSD Treatment. A2780 and its variants growing in log phase werepretreated in monolayer culture with 25 ^M BSO for 48 h prior to cloning.BSO 25 iiM was also added to the upper agarose-cell suspension before
plating. Both control and BSO treated plates were fed with growthmedium, without BSO, on Day 4 and scored for colony growth on Day10. NIH:OVCAR-3nu(Ag+)and NIHiOVCAR^'*«*' were treated similarly with
15 /IM BSO. Doses of BSO which were minimally cytotoxic were selectedfor the individual cell lines. For 2780ME and 2780°*",treatment with 25 /¿M
BSO resulted in 70 to 95% survival compared to untreated controls. InOVCAR-S""1*"*' and OVCAR-4(AB+),incubation with 15 ?M BSO produced
survival of 45% compared to untreated controls.Measurement of GSH. Five x 106 cells from each line both with and
without prior BSO treatment in monolayer were centrifugea at 10°C,
300 x g, for 10 min. The pellet was resuspended in 5 ml saline andcentrifugea again as above. Total GSH levels were measured a minimumof 3 times by the method of Tietze after preparation of a protein-free
filtrate (41). GSH levels of all other cell lines were compared to A2780by a 2-tailed f-test.
RESULTS
Characteristics of Ovarian Cancer Cell Lines. By stepwiseincubation with individual drugs, variants of a œil line derivedfrom an untreated ovarian cancer patient (A2780) have beendeveloped with resistance to melphalan, Adriamycin, or cisplatin(22). 2780*° was exposed to gradually increasing doses of
Adriamycin over a 6-month period up to a maximum dose of 2
fiu and then grown in the absence of drug for more than 6months without loss of resistance to the induction drug. 2780MEand 2780CP were treated similarly with maximum drug concen
trations of 10 and 8 tiM, respectively. The doubling times for theparent line, as well as the variants, are approximately 14 h,Cross-resistance patterns to chemotherapeutic agents havebeen described previously (2). 2780MEis partially cross-resistantto Adriamycin, and 2780*° is partially cross-resistant to mel
phalan (2).NIH:OVCAR-4(A9+) and NIHiOVCAR-Sâ„¢1*9*' were obtained
from patients who were clinically refractory to cyclophospha-
mide, Adriamycin, and cisplatin at the time the lines were established (23). The doubling time of OVCAR-4(A9+)is 25 h, and thatof OVCAR-3nu<Ag+>is 63 h. The inherent resistance of OVCAR-3nU(Ag+)and ovCAR-4(A9+) to melphalan is the same order ofmagnitude as 2780ME; however, 2780*° is much more resistantto Adriamycin than either OVCAR-4(A8+)or OVCAR-S""1*9*' (Table
1) (19). The ICso (drug concentration which inhibits 50% colonygrowth) for melphalan is 5.5 UM in 2780ME as compared to 1.5UM in OVCAR-S"**9*»and 1.4 ^M in OVCAR-4(A9+>.The resultsfor Adriamycin show an IC^ of 1.6 UM in 2780*°versus 0.02 UMand 0.06 MMfor OVCAR-S""^' and OVCAR-4(A9+>,respectively.
Radiation Studies. Radiation survival curves for A2780 andthe variant drug resistant cell lines are shown in Chart 1. The D0for A2780 is 101, while the rt is 1.40. These results are similarto those of other tumor cell lines, both nonhuman and human,reported in the literature (17, 45). 2780ME has a D0 of 146 andan n of 2.12. 2780CP has a D0 of 187 and an ñof 1.62. These
two lines demonstrate a statistically significant increase in resistance to irradiation. In contrast, 2780*°has a D0 of 111 with
an n of 1.48, values which are not statistically different fromthose of the parent line (A2780).
The radiation survival curves for OVCAR-4(Afl+) and OVCAR-3n,xAg+)are snown jn chart 2. The D0s of 203 and 143 and rts of1.08 and 1.53 demonstrate radiation responses similar to 2780MEand 2780CP.
GSH Levels and the Effect of Buthionine Sulfoximine. Totalintracellular GSH levels in A2780 and its variants, as well as inOVCAR-Sâ„¢^' and OVCAR-4(Afl+>,were determined at the time
of irradiation. The results are shown in Table 2. Of A2780 and
Table 1
Resistance to melphalan and Adriamycin
ICsoS(drug concentration which inhibits 50% of colony growth) for Adriamycinand melphalan were determined by 24-h monolayer exposure. Each numberrepresents the mean of at least two experiments, each with triplicate dishes.
RADIOSENSITIZATION OF HUMAN OVARIAN CANCER CELLS WITH BSD
guoc
te.CO 0.01-
RADIATION DOSE (rads)Chart 1. Radiation survival curves for A2780 and its resistant variants: (O)
A2780; (A) 2780*°; (•)2780"*; and (•)2780°".Cells were plated in 0.3% agarose
and radiated at doses of 50 to 600 rads. Colonies larger than 60 urn (or 50 cells)were scored on Day 7. Each point represents the mean of at least 4 experiments.Curves were generated using linear regression by least squares analysis. ByStudent's r test, 2780"* and 2780CP are statistically different from A2780 (P <0.01). 2780*°does not differ significantly from A2780. Bars, SE.
its variants, 2780ME and 2780CP have the highest levels of GSH,with 4.58 and 6.13 nmol/106 cells, respectively. In contrast,2780*° has a level of 2.94 nmol GSH/106 cells, which is only50% higher than that of the parent line (1.89 nmol/106 cells).OVCAR-S"1^' and OVCAR-4(A9+)have the highest intracellularGSH levels, with 12.10 and 15.14 nmol/106 cells, respectively.
Preincubation (48 h) with 25 MMBSO in A2780 and its variants(2780CP, 2780*°,and 2780ME) resulted in a decrease of intracellular GSH to less than 0.75 nmol/106 cells (Table 2). The 48-hpreincubation with 15 MM BSO in OVCAR-S"**9"4 and OVCAR-
4<Ag+>resulted in intracellular GSH levels which were greater than2.0 nmol/106 cells (Table 2).
Radiation Sensitizaron by BSO. Depletion of intracellularGSH by 48-h monolayer exposure to BSO followed by maintenance of the GSH-depleted state by the presence of BSO in the
cloning medium, i.e., during irradiation and for 4 days thereafter,caused a marked sensitization in 2780ME,with the D0decreasingfrom 143 to 95 (Chart 3). Similar treatment of 2780CPresulted in
less sensitization to radiation, with the D0 changing from 183 to134 (Table 3). However, no significant change in ñvalues wasseen for these two lines.
Incubation of OVCAR-S""**9*' with 15 ^M BSO resulted in only
zou
Ite.W 0.01-
RADIATION DOSE (rads)Chart 2. Radiationsurvivalcurves for OVCAR-4"*»*1(O)and OVCAR-S™**9*'(•).
Cells were plated in 0.3% agarose and radiated at doses of 50 to 600 rads.Colonies greater than 60 urn in size were scored on Days 14 and 21 for OVCAR-4<*<ff)and OVCAR-S™**"*',respectively. Each poinf represents the mean of 3experiments for OVCAR^"«*1and of triplicate dishes from a single experiment forOVCAR-3"**9*'.Curves were generated using linear regression analysis.Bars, SE.
Table 2Jota! intracellular GSH
GSH levels were determined with and without a 48-h monolayer incubation with15 to 25 ,iM BSO. GSH levels of all other cell lines were compared to A2780 by atwo-tailed f-test.
a small change in D0 but a significant change in n. In contrast,BSO treatment of OVCAR-4(A9+)did not result in radiosensitiza-
tion (Table 3).
DISCUSSION
The development of resistance and cross-resistance to chem
otherapy and irradiation is a major clinical problem ¡nthe treatment of ovarian cancer. Dose-response relationships and patterns of cross-resistance in established ovarian cancer cell lines
RADIOSENSITIZATION OF HUMAN OVARIAN CANCER CELLS WITH BSO
RADIATION DOSE (rads)Chart 3. Radiation survival curves for 2780** both without (O) and with (•)
BSO 25 MMtreatment for 48 h prior to and during irradiation.Eachpo/nf representsthe mean of at least 3 experiments. Linear regression analysiswas used. The D0sare statistically different at P < 0.02 by a two-tailed Mest. Bars, SE.
Table3Modulation of radiation survival parametersby BSO
For each cell line, D„sand fis with and without BSO treatment were comparedby a two-tailed f-test.
Cellline2780"*
2780e"OVCAR-3™"8*'OVCAR-4'*»*1-BSO143
183143222D„+BSO95
134133232P<0.02
<0.01NSNS-BSO1.76
1.601.531.01n+BSO1.95
1.601.211.01PNS"
NS<0.01NS
' NS, not significant.
and their resistant variants may provide clinically important insights into the optimal integration of irradiation and chemotherapy, even though the exact mechanisms of resistance remain tobe defined. Since the relationship between in vitro cytotoxicityand clinical response is also undefined, the term resistance maybe defined operationally as a change in survival parameters.
The results in Chart 1 demonstrate that cross-resistance toirradiation develops in both the cisplatin and melphalan-resistantovarian cancer cell lines, whereas no cross-resistance developsin the Adriamycin-resistant cell line. Ionizing radiation is thought
to induce formation of free radicals on DNA either directly or viaradiolytic products and eventually cause DNA strand breaks (4).Melphalan directly affects DNA by causing formation of inter-strand cross-links (8). The mechanism(s) of melphalan resistance
have not been fully evaluated. However, possibilities includealtered drug transport, changes in the binding or cross-linking to
DNA, changes in the repair of DNA, and metabolism to less toxicproducts (9,15,33,35,37,40,50). While less is known regardingthe action of cisplatin, it is thought to form interstrand crosslinks with DNA in a manner similar to melphalan (48-50). Thus,
melphalan and cisplatin have direct effects on cellular DNA,causing interstrand cross-links, and resistance to these drugsleads to cross-resistance with irradiation.
In contrast, the mechanism of action of Adriamycin is leastunderstood. Adriamycin-resistant cells in some experimental sys
tems have been found to accumulate less drug, which appearsto be caused either by an increased rate of active drug efflux orby changes in the lipid structural order of the cell membrane (25,26, 31, 34, 36, 38, 42, 43). It is felt that the cardiac toxicity ofAdriamycin is mediated via free radical damage; however, it isnot known whether tumor cell cytotoxicity is similarly mediated(1, 30). It is of considerable interest therefore that Adriamycin-resistant cells do not develp cross-resistance to irradiation.
Clinical trials combining combination chemotherapy and subsequent radiation are currently undergoing evaluation (24, 28).Preliminary results suggest that irradiation confers no additionalbenefit over combination chemotherapy alone. The results of ourstudy demonstrate that cross-resistance to radiation develops inmelphalan- and cisplatin-resistant variant cell lines as well as inlines established from clinically drug-refractory patients. These
findings may provide an explanation for the failure of combinedmodality regimens that utilize radiation after combination chemotherapy. The optimum regimen might not be chemotherapy(with cisplatin and alkylating agents) followed by irradiation, butperhaps simultaneous radiation and chemotherapy.
Intracellular thiols, such as glutathione, are thought to beinvolved either in protection from radiation-induced damage or in
the immediate reduction of free radicals formed by irradiation (4,6, 7, 29). Agents which lower the level of intracellular GSH havealso been shown to increase the radiation sensitivity of somecell lines but not others (3,7,13,24). However, ataxia telangiec-tasia cell lines which have a markedly increased sensitivity tokilling by X-rays have normal GSH levels (27). Elevated intracellular GSH levels have been found both in melphalan-resistantL1210 leukemia cells and in melphalan-resistant lines of humanovarian cancer (19,39). 2780MEand 2780CPboth have 2.5 to 3.5
times the level of intracellular GSH found in A2780.2780*°, on the other hand, had a smaller GSH elevation
(approximately 1.5-fold). As expected, the two sublines withhigher GSH levels (2780ME + 2780CP) are more radioresistantthan those sublines with lower GSH (A2780 + 2780*°).Additionally, OVCAR-3nu(A9+)and OVCAR-4(A9+),from patients refractory
to cyclophosphamide, Adriamycin and cisplatin, also have veryhigh GSH levels and are quite radioresistant. These findings arein agreement with the theory that intracellular thiols such as GSHare intimately involved in protection from radiation damage (7,29).
BSO is a specific inhibitor of the enzyme 7-glutamyl cysteine
synthetase, which is required for GSH synthesis (20, 21). Melphalan-resistant L1210 leukemia cells (which have elevated base
line GSH levels), when treated with BSO, developed markedlydecreased levels of GSH and increased sensitivity to melphalan(39). In human ovarian cancer cell lines, BSO has also beenshown to completely reverse melphalan resistance (19). In this
RADIOSENSITIZATION OF HUMAN OVARIAN CANCER CELLS WITH BSO
study, we have now demonstrated that decreasing the level ofintracellular GSH with BSO can completely restore radiationsensitivity to 2780ME. Despite GSH levels which were decreasedto the same extent as in 2780ME (<0.75 nmol/106 cells), BSOtreatment in 2780CPcaused less sensitization to irradiation. This
suggests that the elevation of intracellular GSH may not be theonly mechanism involved in the development of cross-resistance
to irradiation in cells with primary resistance induced to cisplatin,in contrast to the apparent situation in which primary resistanceis induced with melphalan.
Because of the increased cytotoxicity of BSO in the OVCARcell lines, OVCAR-3nu(Afl+)and OVCAR-4(A9+) were treated with
15 /¿MBSO. At this dose level, total GSH could not be depressedbelow 2.0 nmol/106 cells (a level higher than the inherent level of
A2780). The observation that BSO treatment had no effect onthe radiation survival parameters of OVCAR-Sâ„¢"*9*'and OVCAR-4<Ag+>suggests that there may be a threshold below which GSH
levels must be depressed in order to produce radiation sensitization.
While it is clear that cells with elevated intracellular GSH levelsare more resistant to ionizing radiation and that reduction ofGSH levels (via specific enzyme inhibition) can lead to reversalof that resistance, the molecular mechanisms involved have notbeen established. Possible explanations for GSH protection fromradiation-induced damage include the immediate reduction offree radicals both on DNA itself and on other radiolytic products,as well as modifications of DNA repair enzymes or their environment to enhance activity. Studies to evaluate these possibilitiesas they relate to both radiation and chemotherapeutic drugresistance are in progress.
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