Apoptosis as a Measure of Chemosensitivity to Cisplatin and Taxol Therapy in Ovarian Cancer Cell Lines

GYNECOLOGIC ONCOLOGY ARTICLE NO.

65, 13–22 (1997)

GO974637

Apoptosis as a Measure of Chemosensitivity to Cisplatin and Taxol Therapy in Ovarian Cancer Cell Lines RANDALL K. GIBB, M.D.,* DOUGLAS D. TAYLOR, PH.D.,* TINA WAN, M.S.,* DENNIS M. O’CONNOR, M.D.,*,† DAVID L. DOERING, M.D.,* AND C ¸ IC¸EK GERC¸EL-TAYLOR, PH.D.* *Department of Obstetrics and Gynecology, Division of Gynecological Oncology, and †Department of Pathology, University of Louisville School of Medicine, Louisville Kentucky 40292 Received December 29, 1995

of an aberrantly expressed p53 gene product, while no p53 was detected in the SKOV-3 cells. Conclusions. Our findings indicate that the ability to achieve significant cytotoxicity by cisplatin and Taxol may be directly related to the induction of apoptosis; however, cellular and genetic characteristics determine the eventual outcome of these treatments. q 1997 Academic Press

Objective. Cisplatin- and Taxol-induced apoptosis was studied in four human ovarian cancer cell lines to evaluate apoptosis as a measure of chemosensitivity. Methods. In vitro sensitivities of OVCAR-3, SKOV-3, UL-1, and UL-2 cells to cisplatin or Taxol were determined by the sulforhodamine B assay. Induction of apoptosis was studied by DNA fragmentation following treatment with cisplatin and/or Taxol after 24- and 48-hr exposure. DNA fragmentation was further quantitated by the diphenylamine assay and the proportion of cells in the G1, G2/M, and S phase of the cell cycle was determined by flow cytometry. Presence of the p53 gene product was examined by Western blotting. Results. The four cell lines represent various sensitivities to cisplatin and Taxol (LD50 range for cisplatin, 5–30 mg/ml; Taxol, 30–1000 nM). UL-2 represents a resistant cell line which was 10– 30 times resistant to Taxol and 6 times resistant to cisplatin when compared to the others. Demonstration of apoptosis correlated with the sensitivity of the cell lines to both cisplatin and Taxol for OVCAR-3 and UL-2. DNA fragmentation of OVCAR-3 was uniformly present when treated with cisplatin or Taxol, at 24 or 48 hr. UL-2 demonstrated no apoptosis after 24 or 48 hr of treatment with either cisplatin or Taxol. When sequencing experiments were performed with cisplatin and Taxol, DNA fragmentation correlated with the cytotoxicity assays, except in UL-1 cells where no significant difference was observed in different interactions of cisplatin and Taxol. Pretreatment with Taxol generally resulted in enhanced cytotoxicity in a schedule-dependent manner, and increased fragmentation was demonstrated; cisplatin pretreatment consistently resulted in decreased fragmentation. Quantitation of the fragmented DNA correlated with that seen on gel electrophoresis. OVCAR-3 and UL-1 demonstrated the greatest change from baseline at 24 hr (3.8 and 3.7 times baseline, respectively), whereas UL-2 had little change from the baseline following treatment. G1 arrest occurred more readily in OVCAR-3 and SKOV-3 cells. UL2 cells had very little change in the proportion of cells entering G1 arrest, but had a significant increase in the G2/M proportion. In OVCAR-3, UL-1, and UL-2 cells, we demonstrated the presence

BACKGROUND

Ovarian cancer continues to present a challenge despite many advances in our knowledge over the past 20 years in understanding its pathophysiology and treatment, with most women at diagnosis presenting with advanced-stage disease and a poor prognosis. Our understanding of the etiological factors involved in the pathogenesis of ovarian cancer, its lack of a durable response to chemotherapy, and the development of resistance to initially responsive chemotherapy are poorly understood. Regimens containing the combination of cisplatin and Taxol are the most effective against this tumor, but they are rarely curative. Remissions that result from treatment are often short with frequent relapses, and patients are usually resistant to subsequent chemotherapy. In the past 5 years, increasing evidence has suggested that the characteristics of tumor cell death may be the most important determinant for successful chemotherapy. Apoptosis is defined by morphological and biochemical changes resulting in cell loss and has been found to be relevant to a wide spectrum of biological processes, including neoplasia and cancer chemotherapy [1–6]. Multiple studies have concluded that both cisplatin and Taxol arrest the cell cycle at G1 or G2/M, with subsequent double-stranded DNA breaks consistent with apoptosis and correlate with cell death from these agents [7–10]. Recent evidence has supported these findings and confirmed the presence of chemotherapy-induced apoptosis in epithelial ovarian cancer following treatment with cisplatin and Taxol [11, 12]. Concentration on the mechanisms of action of chemotherapeutic agents has

Presented at the 27th Annual Meeting of the Society of Gynecologic Oncologists, New Orleans, LA, February 10–14, 1996. 13

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0090-8258/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.

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allowed us to establish that most of these agents exert their biological effects by triggering apoptotic cell death. The ability of these agents to induce apoptosis in tumor cells has now become a rationale for therapeutic approaches and entertains the possibility that apoptosis may be enhanced in tumors for therapeutic benefit. Although available data demonstrate the induction of apoptosis in tumor cells, the information is limited on the correlation of apoptotic cell death to success of chemotherapy and resistance. This is especially true for ovarian cancer where failure of chemotherapy is extremely common. Since cisplatin and Taxol are used alone and in combination [13] in the treatment of ovarian cancer, and a schedule-dependent interaction between the two drugs is reported [14, 15], we also studied the induction of apoptosis in sequencing experiments with these agents. In this study, we are presenting data on apoptosis induced by cisplatin and Taxol in four ovarian cancer cell lines, which include two recent clinical isolates, and represent different levels of sensitivities to cisplatin and Taxol. Our study also includes the interaction of these chemotherapeutic agents on ovarian cancer cell lines with respect to the apoptosis, and cytotoxicity assays as a quantitative measure of the efficiency of killing. The data are presented to correlate the role of apoptosis to their apparent in vitro behavior following cytotoxic insult by cisplatin or Taxol. MATERIALS AND METHODS

Cell lines and culture conditions. Human ovarian cancer cell lines, OVCAR-3 and SKOV-3, were obtained from American Type Culture Collection. UL-1 and UL-2 are ovarian cancer cell lines recently established in our laboratory from the ascites of two different ovarian cancer patients [16]. SKOV-3 and UL-1 were grown in DMEM,1 while OVCAR3 and UL-2 were grown in RPMI 1640. Both media were supplemented with 0.1 mM nonessential amino acids, 20 mM L-glutamine, 100 mg/ml streptomycin, 100 IU/ml penicillin, and 10% fetal bovine serum in a humidified 5% CO2 atmosphere. Cytotoxicity assays. All cytotoxicity experiments were performed twice in quadruplicate samples. cis-DDP (Sigma Chemical Co., St. Louis, MO) was made as 1 mg/ml stock in ddH2O. Taxol (Sigma Chemical Co.) was made as 10 mM stock in DMSO. Cell preparations from cultures in log-phase 1 Abbreviations used: ATP, adenosine triphosphate; cis-DDP, cisplatin; ddH2O, double-distilled water; DMEM, Dulbecco’s modification of Eagle’s media; DMSO, dimethyl sulfoxide; ECL, enhanced chemiluminescence; EDTA, ethylenediaminetetraacetic acid; F(ab)2 , antigen binding fragment derived from pepsin digestion; PAGE, polyacrylamide gel electrophoresis; PBS, 10 mM sodium phosphate-buffered saline at pH 7.4; PMSF, phenylmethylsulfonyl fluoride; RPMI, Roswell Park Memorial Institute; SDS, sodium dodecyl sulfate; Tax, Taxol; TBE, Tris–borate–EDTA; TBS, Trisbuffered saline; TCA, trichloroacetic acid; TE, Tris–EDTA.

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growth were removed from the culture flask by trypsinization. Cells were added to 96-well tissue culture plates in 100 ml of medium at a concentration of 1.0 1 104 cells per well. One hundred microliters of each drug concentration was administered in complete medium and the plates were incubated for 2 hr at 377C. Cisplatin concentrations ranged from 0.05 to 50 mg/ml, while Taxol concentrations ranged from 0.05 to 1000 nM. The supernatant was then aspirated and the cells were refed with complete medium. The assay was terminated after 96 hr. When sequencing experiments were performed, cells were treated simultaneously or in sequence (24 hr apart) for 2 hr with cisplatin and Taxol at the appropriate concentrations (1 1 LD50 ) unless otherwise stated. In vitro determinations of the effect of cisplatin and Taxol were performed by the sulforhodamine B cytotoxicity assay and read at 540 nm. Cell culture treatment conditions. For quantitation and analysis of DNA fragmentation, and cell cycle analysis experiments, all cultures were treated in a similar fashion with 2 hr of drug exposure at 5 1 LD50 doses as determined by the individual cytotoxicity assays. In all of these experiments both attached cells and those in the supernatant were combined for analysis. In sequencing and simultaneous exposure experiments, cells were treated with various drug doses, but generally at the 1 1 LD50 concentration. The schedules consisted of 2-hr treatments separated with 24 hr, or 2-hr concurrent treatments, and all cytotoxicity assays were terminated 96 hr following the initial treatment. In the cell lines tested, Taxol pretreatment experiments in which 2-hr exposure was followed 24 hr later with concurrent exposure with cisplatin gave more consistent results in cytotoxicity determinations, and was therefore used in the experiments presented in this article. Cell cultures which were not treated with drugs were used as controls. DNA extraction and gel electrophoresis. Cell cultures following no treatment, or treatment at 5 1 LD50 or 1 1 LD50 as determined by the cytotoxicity assays, were removed from the culture flask by gentle scraping after either 24 or 48 hr. In sequencing and simultaneous exposure experiments, cells were harvested 48 hr following the initial treatment whether it was cisplatin or Taxol pretreatment or simultaneous treatment with both agents. Cells were pelleted at 300g and the medium was removed by aspiration. Cell pellets were resuspended in lysis buffer (5 mM Tris at pH 7.5, 0.5% Triton X-100, and 20 mM EDTA) and kept on ice for 20 min. Ribonuclease A (DNase free, Sigma Chemical Co.) was added to a final concentration of 15 mg/ml and incubated for 20 min at 377C, followed by the addition of NaCl to 1 M, with further incubation at 47C for 2 hr. The sample was centrifuged at 13,000g for 30 min at 47C and the DNA was extracted with phenol–chloroform and precipitated with 2 vol of ethanol at 0207C for 24 hr. The precipitated DNA was spun at 13,000g for 30 min at 47C and allowed to air

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dry. The DNA was then resuspended in 20 ml of TE buffer (10 mM Tris at pH 7.5, and 10 mM EDTA) and quantified by absorbance at 260 nm. Ten micrograms of DNA was applied to a 1.5% agarose gel in TBE buffer (89 mM Tris base, 89 mM boric acid, and 2 mM EDTA) and resolved at 3 V/cm constant current, and the DNA was visualized by 0.5 mg/ml ethidium bromide staining. Quantitation of DNA fragmentation. Quantitation of DNA fragmentation was determined via the colorimetric diphenylamine assay as described by Burton [17, 18]. Approximately 5 1 106 cells in each experimental group was gently scraped after 24 hr following no treatment or treatment and centrifuged at 300g at 47C for 10 min to pellet the cells. The cell pellet was resuspended in 0.8 ml of 0.01 M PBS at pH 7.4, and 0.7 ml of ice-cold lysis buffer was added containing 5 mM Tris; 20 mM EDTA, 0.5% Triton X-100 at pH 8.0. The suspension was incubated on ice for 15 min and centrifuged at 13,000g at 47C. The entire supernatant containing fragmented DNA was transferred to a 5-ml glass tube, while the pellet that contained intact DNA was resuspended in 1.5 ml of TE buffer containing 10 mM Tris and 1 mM EDTA at pH 8.0. Following this, 1.5 ml of 10% TCA was added to each tube and incubated at room temperature for 10 min, followed by centrifugation of the TCA precipitates at 500g at 47C for 15 min. The supernatant was discarded and the precipitate was resuspended in 0.7 ml of 5% TCA, boiled at 1007C for 15 min, and allowed to cool to room temperature. The samples were then centrifuged at 300g at 47C for 15 min after which 0.5 ml of the supernatant was transferred to a new glass tube. One milliliter of the reagent containing 1.5 g diphenylamine in 100 ml acetic acid and 1.5 ml H2SO4 with acetaldehyde at a final concentration of 16 mg/ml was added and then incubated overnight a 307C. The amount of DNA was determined colorimetrically by the absorbance at 600 nm of the supernatant and the pellet. The absorbance of low molecular weight DNA versus total DNA in each sample was expressed as a relative ratio. Cell cycle analysis. In order to analyze the entire population of cells (attached and detached), the cells were gently scraped directly into the medium. After aspirating the medium, the cells were centrifuged and then washed with cold PBS. The cells were counted manually and equal aliquots were fixed in 70% cold ethanol overnight. The fixed cells were incubated for 30 min at 377C in PBS containing 1 mg/ ml ribonuclease A and 100 mg/ml propidium iodide [19]. An average of 25,000 events was subsequently analyzed for DNA content in a Coulter Corp. Elite flow cytometer using the Multicycle DNA Analysis program by Phoenix Flow Systems. Proportions of cells in G1, G2/M, and S were analyzed and reported. SDS–electrophoresis and Western blotting. In order to study the expression of p53, cells in log-phase growth were removed from the culture flask by gentle scraping. The cells

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FIG. 1. Cytotoxicity results of the four ovarian cancer cell lines following cisplatin treatment (0.05–50 mg/ml) assayed at 96 hr. The ovarian cancer cell lines studied were as follows: ,, OVCAR-3; l, SKOV-3; s, UL-1; ., UL-2.

were centrifuged and the cell pellet was resuspended in lysis buffer containing 5% SDS. The suspension was centrifuged at 10,000g for 10 min. Prior to electrophoresis the level of protein was determined by the Bio-Rad DC protein assay kit and was adjusted to a 30-mg protein concentration per well. The proteins were separated by SDS–PAGE with a 12.5% acrylamide separating gel. Molecular weight determinations were based on simultaneous electrophoresis of prestained molecular weight standards (Bio-Rad, Richmond, CA). Following the electrophoretic separation, the proteins were transferred to nitrocellulose paper and blocked using 5% nonfat dry milk (Bio-Rad). The presence of the oncogene product was detected using primary mouse monoclonal antibody (Oncogene Science, Ab-2) incubated at room temperature for 60 min. This antibody detects both wild-type and mutant forms of p53. The primary antibody incubation was followed by three washes with 0.2% Tween 20 in TBS and a 45-min incubation with secondary peroxidase-labeled F(ab)2 of rabbit anti-mouse immunoglobulins. The blots were then visualized by staining for peroxidase with ECL (Amersham). RESULTS

Cytotoxicity assays were performed on the four cell lines and the results indicate that they represent various sensitivities to cisplatin (Fig. 1) and Taxol (Fig. 2). LD50’s as determined by the sulforhodamine B assay were as follows: cisplatin (OVCAR-3, 5 mg/ml; SKOV-3, 5 mg/ml; UL-1, 7 mg/

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FIG. 3. The induction of apoptosis by cisplatin and Taxol in ovarian cancer cell lines SKOV-3 and OVCAR-3. These cell lines were treated at 5 1 LD50 concentrations as outlined previously in the text.

FIG. 2. Cytotoxicity results of the four ovarian cancer cell lines following Taxol treatment (0.05–1000 nM) assayed at 96 hr. The ovarian cancer cell lines studied were as follows: ,, OVCAR-3; l, SKOV-3; s, UL-1; ., UL-2.

ml; and UL-2, 30 mg/ml) and Taxol (UL-1, 30 nM; OVCAR3, 32 nM; SKOV-3, 100 nM; and UL-2, 1000 nM). The dosages tested to effect cell kill between LD50 and LD90 for OVCAR-3, SKOV-3, and UL-1 fell within reported achievable serum levels found with these two chemotherapeutic agents for epithelial ovarian cancer; cisplatin 3–5 mg/ml, and Taxol 0.5–1.5 mM. UL-2 represents a resistant cell line when compared to the others and is 10 to 30 times more resistant to Taxol and 6 times more resistant to cisplatin. Due to this resistance, the cytotoxicity assay was extended in the UL-2 cell line to cover a 90% log kill resulting in doses of 250 mg/ml for cisplatin and 10,000 nM for Taxol. The induction of apoptosis was studied by DNA fragmentation studies following treatment with cisplatin or Taxol. DNA fragmentation was analyzed after 24- and 48-hr periods from the time of initial exposure. Untreated controls failed to exhibit sufficient apoptosis to yield any laddering (Figs. 3 and 4). OVCAR-3, which represents the most chemosensitive cell line studied, demonstrated apoptosis more readily at 24 hr with cisplatin or Taxol than the other cell lines and the DNA fragmentation of OVCAR-3 was uniformly present when treated with cisplatin or Taxol, at 24 and 48 hr. UL2 cells did not exhibit DNA fragmentation consistent with apoptosis after treatment with either cisplatin or Taxol, for 24 or 48 hr. However, both SKOV-3 and UL-1 demonstrated increased DNA fragmentation at 48 hr, when compared to 24 hr with either agent used. When comparing the two chemotherapeutic agents tested, Taxol was more likely to cause laddering at 48 hr, when compared to its 24-hr counterpart.

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In order to demonstrate the significance of DNA fragmentation in the cytotoxic outcome of ovarian cancer cells, sequencing experiments were performed; simultaneous treatment with cisplatin and Taxol was compared to pretreatment by either agent at various dose and time combinations. The results presented are at 1 1 LD50 doses unless otherwise shown (Fig. 5). The interaction of cisplatin and Taxol appears to be variable depending on time of initial treatment, period between treatments, and the final treatment with the second agent. We obtained consistent results showing significantly increased cytotoxicity when 2-hr Taxol pretreatment was followed by a 2-hr concurrent treatment with cisplatin and Taxol at a 24-hr interval (Fig. 5). With the UL1 cells, we did not demonstrate a significant change in the level of cytotoxicity even when different sequencing schedules were used. Significant increase in cytotoxic ability was demonstrated in OVCAR-3, SKOV-3, and UL-2 with Taxol pretreatment followed by cisplatin with the schedule outlined above. We compared the cytotoxicity data to DNA fragmen-

FIG. 4. The induction of apoptosis by cisplatin and Taxol in ovarian cancer cell lines UL-1 and UL-2. These cell lines were treated at 5 1 LD50 concentrations as outlined previously in the text.

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FIG. 5. Cytotoxicity results from sequencing experiments with cisplatin and Taxol. Pretreatment with cisplatin or Taxol (PreC / T, PreT / C) was performed by a 2-hr exposure 24 hr prior to 2-hr concurrent exposure with the second agent. Simultaneous cisplatin and Taxol treatments (C / T) were for 2 hr.

tation, and the results are presented in Fig. 6. Increased DNA fragmentation consistent with cytotoxicity was demonstrated in OVCAR-3 and SKOV-3 cells, while with UL-1 cells slight increase of DNA fragmentation was observed with Taxol pretreatment compared to simultaneous exposure of the two agents. As with single-agent treatment, no fragmentation was seen in UL-2 cells in sequencing experiments (data not shown). Further studies were conducted to quantitate the amount of DNA fragmentation, to determine if cell cycle arrest occurs, and to determine if the p53 gene product was present and may be related to the apoptotic response. DNA fragmentation studies were undertaken to quantitate the proportion of cellular DNA exhibiting low-molecular-weight fragmentation which is consistent with the laddering evident on gel electrophoresis. These results are depicted as the relative level of low-molecular-weight DNA compared to the baseline untreated controls following treatment with cisplatin or Taxol at 24 and 48 hr (Fig. 7). In the four cell lines the baseline percentage of DNA fragmentation in the controls was given a relative level of one and is demonstrated by the hatched line. Following treatment with cisplatin at 24 hr, both OVCAR-3 and UL-1 demonstrated the greatest increase in the relative level of DNA fragmentation at 3.8 and 3.7 times the baseline, respectively. When examined at 48 hr, the fragmentation in SKOV-3 increases to 4.6 times the baseline (from 2.6 at 24 hr), while the amount of fragmentation in OVCAR-3 and UL-1 are 3.4 and 2.5 times the baseline. This may explain the observed DNA fragmentation at 24 hr following cisplatin treatment in OVCAR-3 and UL-1 and the apparent lag in SKOV-3 cells until 48 hr. The amount

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of DNA fragmentation following Taxol treatment increases from 24 to 48 hr in each of the three cell lines as previously demonstrated by gel electrophoresis: OVCAR-3, 2.7 r 5.2; SKOV-3, 1.4 r 4.0; and UL-1, 1.8 r 3.6 times the baseline. The UL-2 cell line, despite treatment with either cisplatin or Taxol, did not show any significant change in the relative level of DNA fragmentation when compared to the baseline control. This is consistent with the absence of DNA laddering in these treated cells. Since recent studies have concluded that both cisplatin and Taxol arrest the cell cycle at G1 or G2/M followed by doublestranded DNA breaks consistent with apoptosis, the proportion of cells in G1, G2/M, and S following treatment with cisplatin or Taxol after 24 hr were measured. A DNA histogram is depicted for the OVCAR-3 cell line comparing the untreated cells to those following treatment with either cisplatin or Taxol (Fig. 8). A normal DNA histogram is achieved in the untreated cells with a majority of the cells in G1 (Fig. 8A). In comparison, cells treated with cisplatin or Taxol resulted in a shift of the proportion of cells into G1 arrest (Figs. 8B and 8C). The OVCAR-3-treated histogram also exhibited a second pronounced peak to the left of the G1 population. This peak is consistent with an apoptotic population of cells with hypodiploid nuclei as previously demonstrated by others [19, 20]. This corresponds with the findings of extensive DNA fragmentation, laddering, and apoptosis seen in the OVCAR-3 cell line compared to the others. The cell cycle profiles of SKOV-3, UL-1, and UL-2 were consistent with the other biochemical markers of apoptosis (Fig. 9). The proportion of each cell population is graphically represented as a percentage of total cells in each cycle (Fig. 10). In SKOV-3 and OVCAR-3, the addition of cisplatin or Taxol increased the proportion of cells accumulating in G1. When OVCAR-3 and SKOV-3 were treated with Taxol, there was an absence of cells in the G2/M phase, suggesting accumulation in G1 arrest followed by apoptosis. In the UL-1 and UL2 cell lines the observations were less defined. The UL-1 cell line in response to cisplatin increased the proportion of G1

FIG. 6. The induction of apoptosis as a result of simultaneous or sequencing experiments with cisplatin and Taxol. DNA fragmentation induced by the respective treatments 48 hr following the initial treatment of the cells by one or both of the agents. 1, cisplatin and Tax; 2, pre-cisplatin followed by Taxol; 3, pre-Tax followed by cisplatin according to the schedule outlined under Materials and Methods.

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FIG. 7. Relative level of apoptosis quantified by the diphenylamine assay in the individual ovarian cancer cell lines following treatment with 5 1 LD50 cisplatin or Taxol. Data are expressed as a relative level to the untreated control (---).

slightly but also at the expense of the proportion in G2/M. However, when treated with Taxol, the proportion of cells entering G2/M increased significantly. The UL-2 cell line, following treatment with either cisplatin or Taxol, exhibited little change in the proportion of cells in G1 compared to the control, but there was a significant increase in the proportion of cells in G2/M arrest. Few cells were demonstrated in synthesis (S) following treatment with either agent and this finding appears unique to this specific cell line. Since p53 has been implicated extensively in tumor development and plays a crucial role in the execution of some forms of apoptosis, the expression of p53 in the four cell lines was assessed. Three of the four cell lines exhibited an abnormal accumulation of p53 (Fig. 11). SKOV-3 is the only cell line which did not exhibit abnormal accumulation. A more intense band representing p53 was observed in OVCAR-3 and UL-1 compared to UL-2. CONCLUSIONS We demonstrate that the apoptotic response correlates with cisplatin and Taxol sensitivity in ovarian cancer cell lines.

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OVCAR-3 was the most chemosensitive cell line to treatment with cisplatin or Taxol followed by UL-1 and SKOV3. UL-1 was more resistant to cisplatin when compared to OVCAR-3 and SKOV-3, while SKOV-3 was more resistant to Taxol when compared to OVCAR-3 and UL-1. UL-2 represents a resistant cell line with 5 1 LD50 values outside the clinically achievable serum levels for these two agents. On gel electrophoresis, variable amounts of DNA fragmentation consistent with apoptosis was seen in these cell lines. The differences reflect the ability of the individual cell lines to undergo apoptosis upon insult by cisplatin or Taxol. The delay associated with Taxol treatment in three of the four cell lines studied correlates with our ability to detect DNA fragmentation better at 48 hr following Taxol treatment. In addition, the most resistant cell line (UL-2) to cisplatin and Taxol was associated with inability to activate the apoptotic pathway. Our data demonstrate that while rapid, uniform apoptosis relates to sensitivity of cells to chemotherapy with the agents studied, lack of apoptosis can constitute a mechanism for resistance. Our sequencing experiments as well as

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FIG. 8. DNA cell cycle histogram of OVCAR-3 following treatment with 5 1 LD50 cisplatin (B) or Taxol (C) after 24 hr compared to untreated control (A). Peak A, G1; peak B, S; and peak C, G2/M. The large peak to the left of the G1 population in B and C represents hypodiploid nuclei consistent with apoptosis.

FIG. 9. Cell cycle FACS results following treatment with 5 1 LD50 cisplatin or Taxol after 24 hr compared to untreated controls in SKOV-3, UL1, and UL-2 cells.

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FIG. 10. Individual cell cycle populations from the FACS data expressed as a percentage of total cells following treatment with 5 1 LD50 cisplatin or Taxol after 24 hr compared to untreated controls. The cell lines tested were as follows: A, SKOV-3; B, OVCAR-3; C, UL-1; D, UL-2. Periods of the cell cycle are represented as follows: G1, ; G2/M, j; S, .

others [14, 15] clearly demonstrate that the interaction beBoth UL-1 and U t w e e n c i s p l a t i n a n d T a x o l i s s c h e d u l ef r do emp ep na dt ie en nt t as n w d ivt ah r ei ep si t h e l i a l o v a r i a n c a n c b e t w e e n c e l l l i n e s . H o w e v e r , c o n s i s t iennst t ii nt uc tr ie oa ns e[ 1i n6 ]c. yCt o tmo px a- r i s o n o f t h e s e t w o n i c i t y w a s o b s e r v e d w i t h p r e t r e a t m e n tc eo lf l Tl ai n x eo sl ft o l al ol rwe ea dd yb ye s t a b l i s h e d c a n c e r c e c i s p l a t i n i n O V C A R - 3 a n d U L - 2 c e l l s ,OaVn Cd At oR a- 3l ea sn sde Sr K d eOgVr -e 3e , p r o v i d e s a d d i t i o n a l i n S K O V - 3 c e l l s . T h i s i n t e r a c t i o n i s fianl ds io n dg es mc o nm spt ar ra et de dt ob oy t h e r p u b l i s h e d d a t a [ i n c r e a s e d D N A f r a g m e n t a t i o n , e x c e p 2t ic ne lUl Ll -i 2n ec ewl al s . oTb ht ua is n, e d f r o m a 2 7 - y e a r - o e x a m i n a t i o n o f a p o p t o t i c c e l l d e a t h bS yt aDg N e AI I fI rCa gp m a pe inl tl aa tri yo ns e r o u s o v a r i a n a d e n o a l s o c o r r e l a t e s w i t h i n c r e a s e d c y t o t ooxvi ac r eyf fiw chi oe nf ca yi l oe fd ci on m i tbi ia-l c h e m o t h e r a p y u s i n a t i o n t h e r a p i e s , a t l e a s t w i t h c i s p l aTt ai nx oa ln ad nTd a sx uo cl c. u m b e d t o p r o g r e s s i v e d i s e a only a selected population of any clinical in cell lines, only with the careful study response will further understanding of ther be possible. A patient’s failure to respond due to the inherent chemoresistance of the consequently exhibits less DNA fragmenta rest. Further study of the UL-2 cell line u n d e r s t a n d t h e s i g n i fi c a n c e o f t h e i r i n a b i a p o p t oi ns i vs i t r o . Another area of interest between the cell lines is that of FIG. 11. Western immunoblot results for the p53 tumor suppressor doubling times. OVCAR-3, SKOV-3, and UL-1 all have doubling times of approximately 24 hr, whereas UL-2 has gene protein in the individual ovarian cancer cell lines studied.

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a longer doubling time of 48 hr. The biological nature of cell turnover may have a role in cell cycle arrest and apoptosis. Most of the UL-2 cells encountered G2/M arrest rather than G1 arrest, and may account for the relative inability of this cell line to undergo apoptosis. Even with manipulation of drug exposure times and concentrations, most UL2 cell death after chemotherapy was necrosis not apoptosis (data not shown), again possibly relating to the lack of an efficient mechanism for apoptosis. Our studies included the relationship between various doses and the amount of apoptosis observed following treatment. Doses at 1 1 LD50 were extensively studied and the same trends at lesser amounts were noted. Dosages as high as 10 1 LD50 were studied; however, the amount of apoptosis and other parameters did not offer any additional information. At least in the cell lines studied, increasing the concentrations of cisplatin and Taxol did not result in a change of apoptotic ability. In sequencing experiments, doses from LD10 to LD50 were effective in showing the interaction of the two agents as shown under Results; however, higher doses did not demonstrate significant differences. Two recent articles in the literature have documented apoptosis in ovarian cancer cell lines [11, 12]. Investigation by Havrilesky et al. [11] demonstrated the presence of apoptosis in some cell lines in response to cisplatin or Taxol. Additional investigations by Ormerod et al. [12] recently described the role of apoptosis in relation to cisplatin sensitive and resistant ovarian cancer cell lines. In each of these studies, SKOV-3 was used as the representative resistant cell line to cisplatin and Taxol. Our work verifies the induction of apoptosis by cisplatin and Taxol in ovarian cancer cell lines. In contrast to other reports, we have analyzed only the low-molecularweight DNA to demonstrate fragmentation and quantitation which provides a more direct measure of chemotherapyinduced apoptosis in these ovarian cancer cell lines. p53 has been implicated as a tumor suppressor gene which directs the cell toward apoptosis [20–22]. Variable degrees of apoptosis which have been identified may correlate with mutational events of the p53 gene with a reported range of 40–80% being present in ovarian carcinomas [23–27]. p53 product accumulates through a posttranslational stabilization mechanism and arrests the cell cycle at G1 to allow for repair of damaged DNA [25]. OVCAR-3 cells have been shown to contain a transition in codon 248 of exon 7 with associated allelic loss; while SKOV-3 has a probable genetic rearrangement leading to the absence of detectable p53 [28]. Our findings indicate that both UL-1 and UL-2 have genetic mutations of the p53 gene by direct sequencing of exons 4– 8 (manuscript in preparation). These findings demonstrate that p53 mutations alone do not appear to direct the cell toward apoptosis. This continues to support the concept that apoptosis is a complex set of events and that while p53 may play an important role, it is not the only gene responsible for programmed cell death. Although it is currently unclear

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what determines the threshold for cells to undergo apoptosis upon cell damage, different cell types appear to be genetically wired for a specific ‘‘set point’’ of apoptotic induction via the regulation of other genes such myc, bcl-2, mdm-2, WAF-1, and bax and possibly others [29, 30]. Although progress has been made in the treatment of ovarian cancer, appearance of resistant cell populations resulting in failure of therapies is common. The data presented here provide a rationale for understanding the variable responses of these ovarian cancer cell lines to cisplatin or Taxol. The ability to achieve a significant therapeutic response may ultimately be the consequence of cellular and genetic alterations that accompany these malignant cells, such as those seen in the p53 gene and others. Further work will be undertaken to attempt to clarify the precise processes involved in apoptosis in these ovarian cancer cell lines, so that future strategies in therapy may encompass the concept of chemosensitivity as indexed by the amount of apoptosis needed to ultimately eradicate the tumor. ACKNOWLEDGMENT The authors gratefully acknowledge the expert technical assistance of Chris Worth in the flow cytometric analysis of these ovarian cancer cell lines.

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