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Radiosensitivity Patterns of Four Human Ovarian Cancer Cell Linesin Vitro
GYNECOLOGIC ONCOLOGY ARTICLE NO.
64, 490–492 (1997)
GO964574
Radiosensitivity Patterns of Four Human Ovarian Cancer Cell Lines in Vitro E. PETRU,* B. U. SEVIN,†
AND
CH. GOTTLIEB‡
*Department of Obstetrics and Gynecology, School of Medicine, University of Graz, 8036 Graz, Austria, and †Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, and ‡Department of Radiology, School of Medicine, University of Miami, Miami, Florida 33101 Received June 10, 1996
Clinical evidence and the majority of experimental data suggest a cross-resistance between cisplatin and radiation in ovarian cancer. The authors are not aware of any report of a human ovarian cancer cell line for which the dose–response relationships to both radiation and chemotherapy including paclitaxel and cisplatin have been evaluated with the same methodology. The present study investigated the radiosensitivity profiles of four established human ovarian cancer cell lines in vitro using the ATP assay, which measures total cell kill. The CAOV-3 cell line showed the highest degree of radiosensitivity of the four cell lines. SKOV-3 cells were the most resistant. The BG-1 cell line, previously shown to be highly resistant to cisplatin but sensitive to paclitaxel, was distinctly sensitive to radiation. This was particularly true for the lower dosages (2–6 Gy). The four cell lines tested are a good representation of cell lines with different radiosensitivities. The different response patterns to cytotoxic agents and radiation, make the BG-1 cell line in particular an interesting candidate for future studies on mechanisms of resistance and combination effects between radiation and chemotherapy. q 1997 Academic Press
INTRODUCTION
Cisplatin and paclitaxel are currently the most active drugs in the therapy of ovarian cancer [1]. The role of radiotherapy in addition to chemotherapy remains to be defined [2]. Clinical evidence and the majority of experimental data suggest a cross-resistance between cisplatin and radiation in ovarian cancer. Multiple mechanisms including enhanced DNA repair and increased cellular glutathione levels have been suggested to be involved [3–7]. A limited number of experiments have studied the resistance patterns between cisplatin and radiation in vitro [8– 10]. In one study, low doses of paclitaxel were used to sensitize ovarian cancer cells to radiation [11]. The authors are not aware of any report of a human ovarian cancer cell line for which the dose–response relationships to both radiation and chemotherapy including paclitaxel have been evaluated with the same methodology.
Gyn 4574
/
6d17$$$201
MATERIAL AND METHODS
Cells were grown and harvested as previously described [12]. Tubes with 4-ml cell suspensions (20,000 cells/ml) were arranged in concentric circles from the radiation source (60Co irradiator with a dose rate of 151 cGy/min; Gamma Beam 150 Panoramic Irradiator, Atomic Energy of Canada) and irradiated at 0, 2, 4, 6, 8, and 10 Gy as previously described [13]. Cells were then plated, in triplicate, in 24well tissue culture plates. On Day 7, ATP was extracted from the living cells in situ with 4% trichloroacetic acid. ATP luminescence was determined with the luciferin–luciferase reaction [12, 14]. Dose–response curves were obtained from percentage of treated/untreated control ATP. All experiments were repeated twice. The coefficient of variation based on double triplicate in vitro samples (six values) ranged from 0.6 to 8.4% of the mean in untreated controls. In treated groups, the double triplicate samples revealed a standard error of the mean for the survival fraction between 0.6 and 9.2 (mean, 5.1). RESULTS
The sensitivity patterns of the four human ovarian cancer cell lines to radiation in vitro are shown in Fig. 1. The CAOV-3 cell line showed the highest degree of radiosensi-
490
0090-8258/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
AID
The ATP cell viability assay has been developed at the University of Miami to evaluate effects of cytotoxic drugs and radiation in vitro [12–15]. Among four established human ovarian cancer cell lines studied (SKOV-3, BG-1, OVCAR-3, CAOV-3), the BG-1 cell line, derived from an untreated patient, showed the highest degree of resistance to cisplatin (inhibitory concentration Å IC50 ú 12.5 mg/ml) [12]. In contrast, BG-1 cells proved highly sensitive to paclitaxel [15]. In the present study, we investigated the radiosensitivity profiles of the four cell lines in vitro in comparison to their chemotherapy response.
02-01-97 01:08:36
goas
AP: GYN
RADIOSENSITIVITY OF OVARIAN CANCER CELLS
491
the most sensitive to a variety of cytotoxic agents [12], also demonstrated the highest level of radiosensitivity of the four cell lines. The BG-1 cell line, previously shown highly resistant to cisplatin but sensitive to paclitaxel [12, 15], was distinctly sensitive to radiation (Fig. 1). Based on these data, the four cell lines tested are a good representation of cell lines with different radiosensitivities. The different response patterns to cytotoxic agents and radiation make the BG-1 cell line in particular an interesting candidate for future studies on mechanisms of resistance and combination effects between these two treatment modalities. FIG. 1. Comparative sensitivity of SKOV-3, BG-1, OVCAR-3, and CAOV-3 cells to radiation doses ranging from 2 to 10 Gy.
tivity. We observed a 64% reduction of the surviving fraction at 4 Gy. SKOV-3 cells were the most resistant. Even at 10 Gy, only a 31% reduction of the surviving fraction was observed. The BG-1 cell line was distinctly sensitive to radiation, which was particularly true for the lower dosages (2– 6 Gy). At 4 Gy, a 55% reduction of the surviving fraction was found. Dosages of 8 and 10 Gy only marginally increased cytotoxicity compared with 6 Gy. DISCUSSION
Whether the addition of radiotherapy to chemotherapy may improve the treatment results in ovarian cancer remains unclear [2, 3]. In vitro experiments may contribute to a better understanding of resistance mechanisms involved [7]. Tumor cells high in glutathione peroxidase activity have been found more resistant to activated macrophage-mediated oxidant injury [16]. Neutrophils, also capable of generating free radicals, have been reported to remain resistant to effector agents by maintaining high glutathione and glutathione peroxidase activity [17]. Endothelial cells rich in glutathione peroxidase activity were more resistant to activated neutrophil damage, while cells that were glutathione peroxidase poor were highly susceptible to activated neutrophil-mediated cytotoxicity [18]. In vitro experiments may form the impetus for the design of new clinical trials. Resistance may be circumvented by optimally sequencing radiotherapy and chemotherapy or by administering buthionine sulfoxamine, a specific inhibitor of glutathione synthesis [7]. An experimental model with differential sensitivity to chemotherapy and radiation would seem particularly useful for studies on the interaction between these two treatment modalities. The ATP assay, which measures total cell kill, has previously been used to obtain sensitivity profiles of four established human ovarian cancer cell lines to various cytotoxic drugs in vitro [12, 14, 15]. In the present experiment, the radiosensitivity patterns of these four cell lines were investigated. The CAOV-3 cell line, which had previously been
AID
Gyn 4574
/
6d17$$$202
02-01-97 01:08:36
ACKNOWLEDGMENT This work was supported by the Doris Kleiber Memorial Fellowship, University of Miami.
REFERENCES 1. McGuire W, Hoskins W, Brady M, Kucera P, Patridge E, Look K, Clarke-Pearson D, Davidson M: Cyclophosphamide and cisplatin compared with paclitaxel and cisplatin in patients with stage III and IV ovarian cancer. N Engl J Med 334:1–6, 1996 2. Thomas G: Is there a role for consolidation or salvage radiotherapy after chemotherapy in advanced epithelial ovarian cancer? Gynecol Oncol 51:97–103, 1993 3. Lambert H, Rustin G, Gregory W, Nelstrop A: A randomized trial comparing single-agent carboplatin with carboplatin followed by radiotherapy for advanced ovarian cancer: A North Thames ovary group study. J Clin Oncol 11:440–448, 1993 4. Carmichael J, Hickson I: Mechanisms of cellular resistance to cytotoxic drugs and X-irradiation. Int J Radiat Oncol Biol Phys 20:197–202, 1991 5. Coleman N, Bump E, Kramer R: Chemical modifiers of cancer treatment. J Clin Oncol 6:709–733, 1988 6. Lau D, Lewis A, Ehsan M, Sikic B: Multifactorial mechanisms associated with broad cross-resistance of ovarian carcinoma cells selected by cyanomorpholino doxorubicin. Cancer Res 51:5181–5187, 1991 7. Louie K, Behrens B, Kinsella T, Hamilton T, Grotzinger K, McKoy W, Winkler M, Ozols R: Radiation survival parameters of antineoplastic drug-sensitive and -resistant human ovarian cancer cell lines and their modification by buthionine sulfoximine. Cancer Res 45:2110–2115, 1985 8. Dempke W, Hosking L, Hill B: Expression of collateral sensitivity to cisplatin, methotrexate, and fluorouracil in a human ovarian carcinoma cell line following exposure to fractionated x-irradiation in vitro. Semin Oncol 19(Suppl. 3):66–72, 1992 9. DePooter C, Scalliet P, Elst H, Huybrechts J, Gheuens E, VanOsteroom A, Fichtinger-Schepman A, DeBruijn E: Resistance patterns between cis-diamminedichloroplatinum and ionizing radiation. Cancer Res 51:4523–4527, 1991 10. Gorodetsky R, Mou X, Pfeffer R, Peretz T, Levy-Agababa F, Vexler A: Sub-additive effect of the combination of radiation and cisplatin in cultured murine and human cell lines. Isr J Med Sci 31:95–100, 1995 11. Steren A, Sevin BU, Perras J, Angioli R, Nguyen H, Guerra L, Koechli O, Averette H: Taxol sensitizes human ovarian cancer cells to radiation. Gynecol Oncol 48:252–258, 1993 12. Petru E, Sevin BU, Perras J, Boike G, Ramos R, Nguyen H, Averette
goas
AP: GYN
492
PETRU, SEVIN, AND GOTTLIEB
H: Comparative chemosensitivity profiles in four human ovarian cancer cell lines measuring ATP bioluminescence. Gynecol Oncol 38:155– 160, 1990 13. Rodriguez M, Steren A: Application of the ATP-cell viability assay for assessment of in vitro radiation treatment response in gynecologic cancer cell lines, in Ko¨chli O, Sevin BU, Haller U (eds): Chemosensitivity Testing in Gynecologic Malignancies and Breast Cancer. Contrib. Gynecol. Obstet. Vol. 19. Basel, Karger, 1994, pp 156–165 14. Sevin BU, Perras J, Averette H, Donato D, Penalver M: Chemosensitivity testing in ovarian cancer. Cancer 71:1613–1620, 1993 15. Untch M, Sevin BU: Evaluation of new drugs and combinations in
AID
Gyn 4574
/
6d17$$$202
02-01-97 01:08:36
gynecologic tumors and cancer cell lines with the ATP-cell viability assay, in Ko¨chli O, Sevin BU, Haller U (eds): Chemosensitivity Testing in Gynecologic Malignancies and Breast Cancer. Contrib. Gynecol. Obstet. Vol. 19. Basel, Karger, 1994, pp 145–155 16. Nathan C: Secretion of oxygen intermediates: Role in effector functions of activated macrophages. Fed Proc 41:2206–2211, 1982 17. Wright CD, Mu¨lsch A, Busse R, Osswald H: Generation of nitric oxide by human neutrophils. Biochem Biophys Res Commun 160:813–819, 1989 18. Vercellotti GM, Dobson M, Schorer A, Moldow C: Endothelial cell heterogeneity: Antioxidant profiles determine vulnerability to oxidant injury. Proc Soc Exp Biol Med 187:181–189, 1988
goas
AP: GYN
64, 490–492 (1997)
GO964574
Radiosensitivity Patterns of Four Human Ovarian Cancer Cell Lines in Vitro E. PETRU,* B. U. SEVIN,†
AND
CH. GOTTLIEB‡
*Department of Obstetrics and Gynecology, School of Medicine, University of Graz, 8036 Graz, Austria, and †Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, and ‡Department of Radiology, School of Medicine, University of Miami, Miami, Florida 33101 Received June 10, 1996
Clinical evidence and the majority of experimental data suggest a cross-resistance between cisplatin and radiation in ovarian cancer. The authors are not aware of any report of a human ovarian cancer cell line for which the dose–response relationships to both radiation and chemotherapy including paclitaxel and cisplatin have been evaluated with the same methodology. The present study investigated the radiosensitivity profiles of four established human ovarian cancer cell lines in vitro using the ATP assay, which measures total cell kill. The CAOV-3 cell line showed the highest degree of radiosensitivity of the four cell lines. SKOV-3 cells were the most resistant. The BG-1 cell line, previously shown to be highly resistant to cisplatin but sensitive to paclitaxel, was distinctly sensitive to radiation. This was particularly true for the lower dosages (2–6 Gy). The four cell lines tested are a good representation of cell lines with different radiosensitivities. The different response patterns to cytotoxic agents and radiation, make the BG-1 cell line in particular an interesting candidate for future studies on mechanisms of resistance and combination effects between radiation and chemotherapy. q 1997 Academic Press
INTRODUCTION
Cisplatin and paclitaxel are currently the most active drugs in the therapy of ovarian cancer [1]. The role of radiotherapy in addition to chemotherapy remains to be defined [2]. Clinical evidence and the majority of experimental data suggest a cross-resistance between cisplatin and radiation in ovarian cancer. Multiple mechanisms including enhanced DNA repair and increased cellular glutathione levels have been suggested to be involved [3–7]. A limited number of experiments have studied the resistance patterns between cisplatin and radiation in vitro [8– 10]. In one study, low doses of paclitaxel were used to sensitize ovarian cancer cells to radiation [11]. The authors are not aware of any report of a human ovarian cancer cell line for which the dose–response relationships to both radiation and chemotherapy including paclitaxel have been evaluated with the same methodology.
Gyn 4574
/
6d17$$$201
MATERIAL AND METHODS
Cells were grown and harvested as previously described [12]. Tubes with 4-ml cell suspensions (20,000 cells/ml) were arranged in concentric circles from the radiation source (60Co irradiator with a dose rate of 151 cGy/min; Gamma Beam 150 Panoramic Irradiator, Atomic Energy of Canada) and irradiated at 0, 2, 4, 6, 8, and 10 Gy as previously described [13]. Cells were then plated, in triplicate, in 24well tissue culture plates. On Day 7, ATP was extracted from the living cells in situ with 4% trichloroacetic acid. ATP luminescence was determined with the luciferin–luciferase reaction [12, 14]. Dose–response curves were obtained from percentage of treated/untreated control ATP. All experiments were repeated twice. The coefficient of variation based on double triplicate in vitro samples (six values) ranged from 0.6 to 8.4% of the mean in untreated controls. In treated groups, the double triplicate samples revealed a standard error of the mean for the survival fraction between 0.6 and 9.2 (mean, 5.1). RESULTS
The sensitivity patterns of the four human ovarian cancer cell lines to radiation in vitro are shown in Fig. 1. The CAOV-3 cell line showed the highest degree of radiosensi-
490
0090-8258/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
AID
The ATP cell viability assay has been developed at the University of Miami to evaluate effects of cytotoxic drugs and radiation in vitro [12–15]. Among four established human ovarian cancer cell lines studied (SKOV-3, BG-1, OVCAR-3, CAOV-3), the BG-1 cell line, derived from an untreated patient, showed the highest degree of resistance to cisplatin (inhibitory concentration Å IC50 ú 12.5 mg/ml) [12]. In contrast, BG-1 cells proved highly sensitive to paclitaxel [15]. In the present study, we investigated the radiosensitivity profiles of the four cell lines in vitro in comparison to their chemotherapy response.
02-01-97 01:08:36
goas
AP: GYN
RADIOSENSITIVITY OF OVARIAN CANCER CELLS
491
the most sensitive to a variety of cytotoxic agents [12], also demonstrated the highest level of radiosensitivity of the four cell lines. The BG-1 cell line, previously shown highly resistant to cisplatin but sensitive to paclitaxel [12, 15], was distinctly sensitive to radiation (Fig. 1). Based on these data, the four cell lines tested are a good representation of cell lines with different radiosensitivities. The different response patterns to cytotoxic agents and radiation make the BG-1 cell line in particular an interesting candidate for future studies on mechanisms of resistance and combination effects between these two treatment modalities. FIG. 1. Comparative sensitivity of SKOV-3, BG-1, OVCAR-3, and CAOV-3 cells to radiation doses ranging from 2 to 10 Gy.
tivity. We observed a 64% reduction of the surviving fraction at 4 Gy. SKOV-3 cells were the most resistant. Even at 10 Gy, only a 31% reduction of the surviving fraction was observed. The BG-1 cell line was distinctly sensitive to radiation, which was particularly true for the lower dosages (2– 6 Gy). At 4 Gy, a 55% reduction of the surviving fraction was found. Dosages of 8 and 10 Gy only marginally increased cytotoxicity compared with 6 Gy. DISCUSSION
Whether the addition of radiotherapy to chemotherapy may improve the treatment results in ovarian cancer remains unclear [2, 3]. In vitro experiments may contribute to a better understanding of resistance mechanisms involved [7]. Tumor cells high in glutathione peroxidase activity have been found more resistant to activated macrophage-mediated oxidant injury [16]. Neutrophils, also capable of generating free radicals, have been reported to remain resistant to effector agents by maintaining high glutathione and glutathione peroxidase activity [17]. Endothelial cells rich in glutathione peroxidase activity were more resistant to activated neutrophil damage, while cells that were glutathione peroxidase poor were highly susceptible to activated neutrophil-mediated cytotoxicity [18]. In vitro experiments may form the impetus for the design of new clinical trials. Resistance may be circumvented by optimally sequencing radiotherapy and chemotherapy or by administering buthionine sulfoxamine, a specific inhibitor of glutathione synthesis [7]. An experimental model with differential sensitivity to chemotherapy and radiation would seem particularly useful for studies on the interaction between these two treatment modalities. The ATP assay, which measures total cell kill, has previously been used to obtain sensitivity profiles of four established human ovarian cancer cell lines to various cytotoxic drugs in vitro [12, 14, 15]. In the present experiment, the radiosensitivity patterns of these four cell lines were investigated. The CAOV-3 cell line, which had previously been
AID
Gyn 4574
/
6d17$$$202
02-01-97 01:08:36
ACKNOWLEDGMENT This work was supported by the Doris Kleiber Memorial Fellowship, University of Miami.
REFERENCES 1. McGuire W, Hoskins W, Brady M, Kucera P, Patridge E, Look K, Clarke-Pearson D, Davidson M: Cyclophosphamide and cisplatin compared with paclitaxel and cisplatin in patients with stage III and IV ovarian cancer. N Engl J Med 334:1–6, 1996 2. Thomas G: Is there a role for consolidation or salvage radiotherapy after chemotherapy in advanced epithelial ovarian cancer? Gynecol Oncol 51:97–103, 1993 3. Lambert H, Rustin G, Gregory W, Nelstrop A: A randomized trial comparing single-agent carboplatin with carboplatin followed by radiotherapy for advanced ovarian cancer: A North Thames ovary group study. J Clin Oncol 11:440–448, 1993 4. Carmichael J, Hickson I: Mechanisms of cellular resistance to cytotoxic drugs and X-irradiation. Int J Radiat Oncol Biol Phys 20:197–202, 1991 5. Coleman N, Bump E, Kramer R: Chemical modifiers of cancer treatment. J Clin Oncol 6:709–733, 1988 6. Lau D, Lewis A, Ehsan M, Sikic B: Multifactorial mechanisms associated with broad cross-resistance of ovarian carcinoma cells selected by cyanomorpholino doxorubicin. Cancer Res 51:5181–5187, 1991 7. Louie K, Behrens B, Kinsella T, Hamilton T, Grotzinger K, McKoy W, Winkler M, Ozols R: Radiation survival parameters of antineoplastic drug-sensitive and -resistant human ovarian cancer cell lines and their modification by buthionine sulfoximine. Cancer Res 45:2110–2115, 1985 8. Dempke W, Hosking L, Hill B: Expression of collateral sensitivity to cisplatin, methotrexate, and fluorouracil in a human ovarian carcinoma cell line following exposure to fractionated x-irradiation in vitro. Semin Oncol 19(Suppl. 3):66–72, 1992 9. DePooter C, Scalliet P, Elst H, Huybrechts J, Gheuens E, VanOsteroom A, Fichtinger-Schepman A, DeBruijn E: Resistance patterns between cis-diamminedichloroplatinum and ionizing radiation. Cancer Res 51:4523–4527, 1991 10. Gorodetsky R, Mou X, Pfeffer R, Peretz T, Levy-Agababa F, Vexler A: Sub-additive effect of the combination of radiation and cisplatin in cultured murine and human cell lines. Isr J Med Sci 31:95–100, 1995 11. Steren A, Sevin BU, Perras J, Angioli R, Nguyen H, Guerra L, Koechli O, Averette H: Taxol sensitizes human ovarian cancer cells to radiation. Gynecol Oncol 48:252–258, 1993 12. Petru E, Sevin BU, Perras J, Boike G, Ramos R, Nguyen H, Averette
goas
AP: GYN
492
PETRU, SEVIN, AND GOTTLIEB
H: Comparative chemosensitivity profiles in four human ovarian cancer cell lines measuring ATP bioluminescence. Gynecol Oncol 38:155– 160, 1990 13. Rodriguez M, Steren A: Application of the ATP-cell viability assay for assessment of in vitro radiation treatment response in gynecologic cancer cell lines, in Ko¨chli O, Sevin BU, Haller U (eds): Chemosensitivity Testing in Gynecologic Malignancies and Breast Cancer. Contrib. Gynecol. Obstet. Vol. 19. Basel, Karger, 1994, pp 156–165 14. Sevin BU, Perras J, Averette H, Donato D, Penalver M: Chemosensitivity testing in ovarian cancer. Cancer 71:1613–1620, 1993 15. Untch M, Sevin BU: Evaluation of new drugs and combinations in
AID
Gyn 4574
/
6d17$$$202
02-01-97 01:08:36
gynecologic tumors and cancer cell lines with the ATP-cell viability assay, in Ko¨chli O, Sevin BU, Haller U (eds): Chemosensitivity Testing in Gynecologic Malignancies and Breast Cancer. Contrib. Gynecol. Obstet. Vol. 19. Basel, Karger, 1994, pp 145–155 16. Nathan C: Secretion of oxygen intermediates: Role in effector functions of activated macrophages. Fed Proc 41:2206–2211, 1982 17. Wright CD, Mu¨lsch A, Busse R, Osswald H: Generation of nitric oxide by human neutrophils. Biochem Biophys Res Commun 160:813–819, 1989 18. Vercellotti GM, Dobson M, Schorer A, Moldow C: Endothelial cell heterogeneity: Antioxidant profiles determine vulnerability to oxidant injury. Proc Soc Exp Biol Med 187:181–189, 1988
goas
AP: GYN