Topoisomerase I-Related Parameters and Camptothecin Activity in the Colon Carcinoma Cell Lines from the National Cancer Institute Anticancer Screen1

[CANCER RESEARCH 55, 2116-2121. May 15. 1995] Topoisomerase I-related Parameters and Camptothecin Activity in the Colon Carcinoma Cell Lines from the National Cancer Institute Anticancer Screen1

Francois Goldwasser, Insoo Bae, Monica Valenti, Keila Torres, and Yves Pommier2

DNA Topology Section, Laboratory of Molecular Pharmacology DTP. Division of Cancer Treatment. National Cancer Institute. NIH, BethesJa. Maryland 20892-4255

ABSTRACT as cleavable complex. CPT specifically and reversibly stabilizes cleavable complexes by inhibiting their religation (11, 16-18). The Camptothecin (CPT) derivatives are a new family of anticancer agents mechanism of CPT cytotoxicity is thought to be the consequence of a which are selective inhibitors of DNA topoisomerase I (topi) and have collision between moving replication forks and CPT-stabilized cleav entered clinical trials with promising results. The cellular determinants able complexes (19-21). for CPT activity were studied in the seven cell lines of the National Cancer Institute anticancer screen. These cell lines exhibit natural differences A better understanding of the cellular determinants for CPT activity in sensitivity to CPT and can be divided into three groups, according is needed for optimal clinical use and to address the controversy to their increasing resistance: colo205, SW620, HCT116drug sensitivity assays for the clinic. One way to mRNA, measured by Northern blotting analysis, and topi protein levels, answer these questions is to compare topi-related parameters in cell measured by Western blotting, varied by 2-fold or less among the cell lines lines which naturally exhibit differences in CPT sensitivity. Using the and were correlated neither with the CPT cytotoxicity nor the levels of seven colon cell lines of the NCI anticancer cell screen as a tool cleavable complexes measured by alkaline elution in the various cell lines. (miniclinic), we studied the topi-related parameters: topi mRNA and An overall log-linear correlation was observed between CPT-induced topi-cleavable complexes and growth inhibition, indicating the impor protein levels, cell doubling-time, and cell cycle distribution that are tance of cleavable complex formation rather than topi levels for cell already used in ongoing clinical studies and measurement of cleavable killing in this panel of cell lines. Also, some cell lines displayed marked complex formation. We find that topi levels are not predictive of drug growth inhibition differences with minimal differences in cleavable com cytotoxicity but rather an overall log-linear relationship between plexes and S-phase fraction, suggesting that parameters downstream from CPT-induced cleavable complexes and cytotoxicity. These observa the cleavable complexes are also critical for CPT cytotoxicity. tions suggest that measurement of topi-induced DNA damage rather than topi levels should be monitored during clinical use of CPT and INTRODUCTION derivatives. A higher benefit of extended CPT exposure rather than dose escalation is also suggested by the present results. During the last decade, cancer chemotherapy lacked original mol ecules to improve the prognosis of solid tumors and metastatic can cers. Recently, two new families of cytotoxic agents have been MATERIALS AND METHODS discovered, taxol and CPT3 derivatives. CPT derivatives have a Drugs and Chemicals. CPT and ['HJCPT (labeled at position 9; specific unique mechanism of action, and their original spectrum of activity, activity, 17 Ci/mmol) were a kind gift from Drs. Monroe E. Wall and Mansukh both in xenografts (1) and in the first clinical trials, have born new C. Wani (Research Triangle Institute, Research Triangle Park, NC). Stock hopes for therapeutic improvements (2, 3). Phase I/II clinical trials of solutions of CPT were prepared in DMSO at 10 mM. [mei/iv/-'H]thymidine, CPT derivatives are in progress, and promising activity has been 2-['4C)lhymidine (specific activity, 20 and 0.05 Ci/mmol, respectively), and [a-32P]dATP (specific activity, 6000 Ci/mmol) were purchased from NEN observed in various malignancies and especially in colorectal carci nomas (4-8). Irinotecan (CPT-11) already appears as the most active Research Products (Boston, MA). anticancer agent in colon cancer and is efficient in 5-fluorouracil- Cell Culture and Radiolabeling. All cell lines, colo2Q5, SW620, HCT116, HT29, HCC2998, HCT15 and KM12, were provided by Dr. Dominic refractory colon cancer patients (9). Scudiero (NCI, Frederick, MD) and were grown in monolayer cultures in CPT was isolated by Monroe E. Wall, Mansukh C. Wani, and RPMI 1640 supplemented with 5% heat-inactivated PCS and 2 mM glutamine. co-workers 30 years ago as the active alkaloid of extracts from the No antibiotic was added to the medium. The cells were trypsinized and passed Chinese tree Camptotheca acuminata (10). Almost 10 years later, 2-3 times a week. CPT was found to inhibit selectively a new target, eukaryotic DNA Growth Inhibition Assays. Approximately 10s cells were seeded in T25 topi (11). Topi is ubiquitous and is required for Drosophila devel flasks, and cell counts were checked from control flasks every 24 h. When the opment (12). Topi relaxes DNA supercoiling by making transient cells were in exponential growth phase and reached 4 x 10s cells/flask (usually single-strand breaks (13, 14, 15). These breaks are coupled with the after 48 h), treatments were performed with various CPT concentrations for 1 transient formation of a covalent DNA-enzyme intermediate termed or 24 h at 37Â°C.Drug treatments were ended by rinsing cells twice in preheated PBS; then they were diluted in 5 ml of preheated medium. Every 24 h, for each concentration of CPT, one separate T25 flask was taken, and the cells were Received 11/18/94; accepted 3/16/95. trypsinized and counted for 4 days using a Coulter Counter (Coulter Electron 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 in accordance with ics Inc., Hialeah, FL). Growth inhibition was calculated as: N3-Co/C3-Co, 18 U.S.C. Section 1734 solely to indicate this fact. where N3 is the number of cells in the CPT-treated sample at day 3, C3 the 1Supported in part by a fellowship from the French Ligue Nationale contre le Cancer number of control cells at day 3, and C0 the cell number at day 0, before drug and from the National Cancer Institute/European Organisation for Research and Treat treatment. When the amount of cells was less than the initial number seeded, ment of Cancer (to F. G.) and by a fellowship from Associazione Italiana per la Ricerca sul Cancro, Milano, Italy (to M. V.). the growth inhibition appeared as more than 100%, reflecting the cytotoxic 2 To whom requests for reprints should be addressed at. Laboratory of Molecular effect. Pharmacology, National Cancer Institute. Bldg. 37, Room 5C25, NIH, Bethesda, MD Cell Cycle Determination. Cells were harvested, washed in ice-cold PBS 20892-4255. -1The abbreviations used are: CPT, Camptothecin; topi, topoisomerase I; NCI, National (pH 7.4), fixed in 70% ethanol, washed twice in ice-cold PBS, and treated with RNase (30 min at 37Â°C;500 units/ml; Sigma Chemical Co., St. Louis, MO); Cancer Institute; DPC, DNA protein cross-links; Gig,,, concentration of CPT that inhibits 90% growth. cellular DNA was stained with 500 /il of 50 fig/ml propidium iodide. Cells 2116

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1995 American Association for Cancer Research. 1)1 IIRMINANT OF SENSITIVITY TO CAMPTOTHECIN were stored at 4Â°Cprior to analysis. Cell cycle determinations were performed 24 hours 1 hour using a Becton Dickinson fluorescence-activated cell analyzer, and data were interpreted using the SFIT model program provided by the manufacturer. Results represent the mean of triplicate determinations in which a minimum of 15,000 cells were assayed for each determination. Immunoblot Analysis of DNA Topi. Immunoblots were performed as described previously (22, 23). The whole cell lysates from each cell line were subjected to gradient 4-12% SDS-PAGE, and the proteins were transferred to a nitrocellulose filter (Immobilon-P; Millipore Corporation, Bedford, MA). The filters were incubated overnight at 4Â°Cin PBS containing 5% (w/v) milk and mouse monoclonal topi antibodies (C21), kindly provided by Dr. Yung- chi Cheng (Yale University, New Haven, CT; Ref. 24). Membranes were then 0.02 0.04 O.OE 0.08 0.1 incubated for l h in PBS with sheep peroxidase-conjugated secondary anti- mouse IgM antibody (Amersham, IL). Development was performed according Camptothecin (uM) to the manufacturer specifications using Amersham films. Fig. 1.Growth inhibition after 1and 24 h exposure to CPT of the seven colon cell lines Northern Blot Analysis of DNA Topi. Total RNA was extracted from of the NCI Anticancer Drug Screen. Symbols are Cola 205 (Â»),SW620(â€¢),HCT1I6(A), exponentionally growing cells according to standard procedures (25). mRNA Â«729(â€¢),HCC2998(O), HCT15 (A), and KM12 (O). was isolated using oligo d(T) column (oligotex-dT; Qiagen, Chatsworth, CA). Two fig mRNA were subjected to electrophoresis in a 1% agarose gel con taining 0.66 M formaldehyde and then transferred to a nylon membrane was eluted with tetrapropylammonium hydroxide-EDTA (pH 12.2) without (Duralose-UV; Stratagene, La Jolla. CA). The filter was hybridized with each SDS. Fractions were collected at 3-h intervals for 15 h. DPC frequencies were 12P-labeled cDNA probe in 50% formamide at 43Â°Cfor 24 h. Human topi calculated according to the bound-to-one terminus model and expressed in cDNA (TIB) used as a probe was kindly provided by Dr. William C. Earnshaw rad-equivalents (28, 29) (Johns Hopkins University School of Medicine, Baltimore, MD; Ref. 26). Human G3PDH cDNA probe was purchased from Clontec (Palo Alto, CA) and used as internal standard. The filter was then washed twice in 2X SSC-0.1% RESULTS SDS and then twice in 0.5X SSC-0.1% SDS at 65Â°C[IX SSC = 0.15 M NaCl-0.015 M sodium citrate (pH 7.0)]. Membranes were scanned using Differential Sensitivity of the Colon Cell Lines of the NCI Anticancer Screen Cell Screen and Time-dependence of Campto- Phosphorlmager (Molecular Dynamics, Sunnyvale, CA), and quantification was performed using IMAGE QUANT Ver.3.22. The expression level of topi thecin-induced Growth Inhibition. Fig. 1 shows growth inhibition was calculated as the ratio between pixels of the topi band and those of the assays after 1 and 24 h of exposure to CPT. The GI,,, was 14- to corresponding G3PDH band. 40-fold higher after 1-h exposure compared to 24-h exposure. The CPT Uptake. This technique was similar to that used previously by Pom ranking of the cell lines was the same in both assays, and after a 1-h mier et al. (27). Briefly, IO7 cells were incubated in 10 ml medium for l h in exposure to CPT, the KM 12 cells were 17 times more resistant than presence of 1 JAMmixture of unlabeled and labeled CPT. At the end of the the colo205 cells (Table 1). Three cell lines, colo205, HCT116 and treatment, cells were scraped, centrifuged, and resuspended in 1 ml of CPT- SW620, were very sensitive, with GIW (24 h) less than 0.03 /AMand containing medium. Nine hundred /*! of the cell suspension was layered above GÃ¬.Â«,(1h) less than 0.6 /AMCPT. Two cell lines, HT29 and HCC2998, 0.5 ml of silicone oil (Versitube F50; General Electric Co., Waterford, NY) in a microcentrifuge tube and centrifuged for 5 min at 12,000 X g. The bottom were intermediate, with GIyo (24 h) between 0.045 to 0.065 /XMand of the tube was cutoff and placed in a liquid counting scintillation vial. The cell Gly,, (1 h) between 0.7 and 0.9 /UMCPT. Two cell lines were more pellet was solubili/al by the addition of 2.5 ml of 0.4 M NaOH and incubated resistant, HCT15 and KM12, with GI<)(1(24h) higher than 0.1 /IM CPT overnight at 37Â°Cbefore counting. and GIW (1 h) around 3 /XMCPT. Determination of Drug-stabilized Cleavable Complexes Frequency. Topi mRNA and Protein Expression and Cell Cycle Kinetics Nondeproteinizing, DNA-denaturing alkaline elution was used to determine Do Not Predict Cell Sensitivity to CPT. As shown in Table 1, the the amount of DPC as described previously (28, 29). Briefly, colon cells were range of S-phase fraction was 38-52% within these cells. High labeled with 0.02 fiCi/ml of [14C]thymidine for 1 to 2 doubling times at 37Â°C. S-phase fraction (more than 45%) was observed both in sensitive cells All cells were then chased in nonradioactive medium for at least 4 h before (SW620) or in the most resistant cells (KM 12). Low S-phase fraction drug treatment. After a 1-h CPT treatment, the cells were irradiated on ice with could be associated with high sensitivity (colo205) or resistant phe- 30 Gy prior to elution. Cells were gently layered onto polyvinylchloride- acrylic copolymer filters (Metrice! DM-800; Gelman Sciences, Ann Arbor, notype (HCT15). Doubling times were not correlated with CPT sen MI) and lysed using a solution containing 0.2% sodium sarkosyl-2 M NaCl- sitivity either (Table 1). 0.04 M EDTA (pH 10.0) in the absence of proteinase K. CPT concentrations Topi detection by Western blot analysis showed the expected M, were maintained constant until cell lysis to avoid topl-cleavable complex 100,000 size in all cell lines with minor proteolysis products (Fig. 2). reversal that is known to take place even at 4Â°Cafter drug dilution (30). DNA Little variation was seen among the seven cell lines. Topi protein

Table 1 Topi-related parameters in the colon cell lines of the NCI Anticancer Drug Screen

GI90, h(/IM)"GI9024 â€ž(^M)Â°Doubling (h)S-phasetime fractionTopi protein''Topi HiRNA*CPT uptake'CPT7r(1 Â±110.21105HCT1160.40.02180.411.30.853Â±280.7573SW6200.50.016210.510.80.815Â±21537HT290.70.047200.521136Â±1.41384HCC29980.90.065330.450.60.722Â±4.23209HCT152.6>fl.l210.380.850.724Â±18345KM123.4Xl.l200.461.45132Â±162350 (LIM)CC0.5 (rad-eq)''colo2050.20.005230.410.91.139 " Ninety % growth inhibitory concentrations of CPT after 1- or 24-h exposure. Topi mRNA and protein expressions relative to the HT29 cells. ' Measured after l JIMCPT treatment for 1 h and expressed in pmol/10 cells. Mean Â±SDof at least two independent experiments. i/CPT7S(),CPT concentration which induces a 750 rad-equivalent DPC. CC05, DPC (cleavable complex) frequency (in rad-eq) after treatment with 0.5 JIMCPT for I h.

2117

(HCT116 and SW620) of topi mRNA. In contrast, the most resistant cell line, KM12, had the same level of topi mRNA than the HT29 cells and only 15% less than the most sensitive colo205 cell line. Topi mRNA and Protein Expression and CPT Uptake Are Not Willi Correlated with Topi Cleavable Complexes. Topi activity was assayed by measuring the amount of drug-sensitive (chromatin-asso- 220 ciated), enzyme associated with cleavable complexes (or DPC) in cells by alkaline elution. Fig. 3 shows the dose-response curves of 100 CPT-stabilized cleavable complexes in the seven cell lines over a wide range of CPT concentration. Generally, the more sensitive cells 66 had high frequencies of DPC, while the more resistant cells had low 46 frequencies. The range in DPC frequency within the seven cell lines was best detected at 10 JAMCPT and reached 2.5-fold (788 to 1900 rad-eq). The colo205 cells had a particular behavior, with a highest frequency of DPC between 0.1 and l /AMand a plateau above 0.5 JAMCPT. While topi mRNA and protein expressions varied only within a factor of 2 in the seven cell lines, the amount of cleavable complexes could vary by 5.3-fold at 0.5 JAMCPT (Fig. 3). Thus, the relationship between topi mRNA/protein expression and the amount of cleavable complexes was very different from one cell line to another. The topi mRNA level was well correlated with cleavable complexes in the HCC2998 and HCT15 cells (low expression, low amount of cleavable complexes). On the other hand, SW620 had low topi mRNA and protein levels with high amounts of cleavable complexes. For three cell lines, the relationship depended on the concentration of camptothecin used; for colo205, topi mRNA and cleavable complexes at low concentrations of CPT were relatively high, while cleavable com plexes tended to plateau above 0.5 JAMCPT. HT29 and KM 12 had similar topi mRNA expression and comparable cleavable complexes up to l JAMCPT, while at high concentrations of CPT, HT29 produced 1.6-fold more cleavable complexes than KM 12. Topi protein levels were consistent with topi mRNA levels (Table 1; Fig. 2). However, the KM12 cell line which exhibited a low level of cleavable complexes (Fig. 3) and the lowest sensitivity to CPT (Table 1; Fig. 1) had the highest topi protein level (Fig. 2). Taken together, these results demonstrate no relationship between topi mRNA or protein expression and topi-induced cleavable complexes Fig. 2. Topi protein and mRNA expression in the seven colon cell lines of the NCI in cells. Anticancer Drug Screen. Top, immunoblot probed with mouse monoclonal topi antibody. Cells (5 X IO5) were lysed, directly loaded, and electrophoresed in a gradient 4-12% SDS CPT uptake was determined using [3H]CPT. Table 1 shows the polyacrylamide gel. Numbers lo the right correspond to the migration positions of results from at least two independent experiments. CPT uptake varied molecular mass markers. Middle, quantitation of topi protein levels as determined by only within a factor of 2 in the colon cell lines. The sensitive HCT116 densitometry. Values shown are from wide beam densitometric scanning of individual lanes. Values from a typical experiment are expressed relative to HT29. Botlmn, quanti- talion of topi mRNA from Northern blot analysis. Values from a typical experiment are expressed relative to HT29. 2000 expression did not correlate with sensitivity since the HCT116-sensitive cells and the resistant KM 12 had the same protein expression (Table 1). Therefore, topi expression does not predict cell sensitivity in this panel of cell lines. Even when taking into account both topi expression and S-phase fraction, cell sensitivity could not be predicted â€žE loco- .a u since KM 12 had both the highest topi expression and highest S-phase g o. fraction, while SW620 had the same S-phase fraction but a lower topi expression. 500 H Topi mRNA levels were measured by Northern and dot-blot anal ysis and normalized to the HT29 cell line topi mRNA level (Fig. 2; Table 1). The predicted 4.1-kilobase size was observed for topi 0.01 10 mRNA in all cell lines (data not shown). In agreement with the topi protein levels, topi mRNA levels exhibited little (approximately 30%) Camptothecin (uM) variation among the cell lines, in contrast with the large range of Fig. 3. CPT-stabilized cleavable complexes in the seven colon cell lines of the NCI sensitivity (Fig. 1; Table 1). The relative topi mRNA levels were not Anticancer Drug Screen. Cleavable complexes were measured as DPC by alkaline elution after 60-min CPT treatments. Individual points represent the mean of two to four correlated with CPT sensitivity; when compared to the HT29 cells, the independent experiments with a 10% variability. Symbols are Colo 205 (*), HCTII6 most sensitive cells could have higher levels (colo205) or lower levels (A), SW620 (â€¢),HT29 (â€¢).HCC2998 (D). HCTI5 (A), and KM12 (O). 2118

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1995 American Association for Cancer Research. DETERMINANT OF SENSITIVITY TO CAMPTOTHECIN cells had a 2-fold higher CPT uptake. This was associated with high DISCUSSION cleavable complexes and might participate to their high sensitivity. On In this study, using the colon cell lines from the NCI anticancer the other hand, SW620, another sensitive cell line, had the lowest CPT screen, we have found a log-linear relationship between CPT-induced uptake. The other cell lines did not differ significantly one from the DNA cleavable complexes and growth inhibition. Topi mRNA and other. Therefore, no simple relationship could be drawn either be protein expression and S-phase fraction were not predictive for sen tween CPT uptake and cleavable complexes or between CPT uptake sitivity to CPT. Moreover, we have confirmed the time-dependency of and CPT sensitivity. CPT-induced toxicity and that optimum toxicity can be achieved with Log-linear Relationship between Frequency of CPT-induced low CPT concentration for a long treatment period. Cleavable Complexes and Cytotoxicity. Fig. 4 represents the The time-dependence of the camptothecin-induced growth inhibi amount of cleavable complexes as a function of growth inhibition tion appears in the experiments comparing the toxicity of 1 versus 24 over a wide range of CPT concentrations. Taken all the cell lines h exposure to CPT. Twenty-four h exposure required about 14 to 40 together, in spite of their various sensitivity to CPT, the best repre times lower concentrations of CPT to achieve similar cytotoxicity. sentation was log-linear. When the concentration of drug increased, These data are in agreement with the model of cytotoxicity of CPT the amount of stabilized cleavable complexes was enhanced and so proposed earlier (19-21) that only the cleavable complexes that are did the growth inhibition. More than 100% growth inhibition was reached by moving replication forks lead to DNA damage. This model found at the highest drug concentrations for the most sensitive cell provides arguments to administer CPT derivatives by continuous lines, as the number of cells counted 72 h after drug treatment was less infusion rather than bolus administration (2, 3, 31). However, l h than the number of seeded cells. exposure to CPT inhibited the growth of a larger cell fraction than The log-linearity of this relationship indicates that identical in expected from the fraction of cells in S-phase. We had already noticed creases in cleavable complexes have different consequences on tox- this apparent discrepancy in studies with CPT and its more potent icity, depending on the range of cleavable complexes. The same derivative, 10,11-methylenedioxycamptothecin in HT29 cells and increase in DNA damage is associated with a higher increase in suggested that S-phase fraction, as defined by flow cytometry, prob toxicity in the lower range than in the higher range of cleavable ably underestimates the fraction of cells synthesizing DNA (32). S-phase fraction also was not correlated with CPT cytoxicity in cell complexes. Moreover, low amounts of cleavable complexes could be enough to induce strong growth inhibition. lines with similar topi levels. This is in agreement with studies on However, close examination of the data shows that additional xenografts models in which CPT derivatives have shown high effi ciency despite the low S-phase fraction usually observed in these factors are probably involved. For instance, approximately 100 rad- models (3). Hence, CPT may also be toxic outside of S-phase, through equivalent cleavable complexes (corresponding to approximately one cleavable complex/106 nucleotides; Refs. 28 and 29) induced 40% collisions between topi-cleavable complexes and DNA repair pro cesses which physiologically appear at any phase of the cell cycle. growth inhibition in SW620 and less than 25% inhibition in HT29 and This possibility would be consistent with the topi down-regulation KM 12 cells. However, these three cell lines had similar S-phase observed after ionizing radiation-induced DNA damage and its cor fractions. This suggests that their differences in sensitivity are also relation with better survival (33). related to differential response to DNA damage. Differences in p53 Topi expression has been found to be decreased in CPT-resistant status are probably not critical in this panel of cell lines since HCT116 cell lines selected after exposure to the drug (24, 34, 35). However, is the only wild-type p53 line, while the other cell lines have mutant selection might introduce a bias, since to select a highly resistant cell p53 (data not shown). line, one topi alÃeleis expected to be silent or lost while the other is mutated (51); therefore, for experimental reasons, topi expression is expected to be decreased. Here, we used a panel of cell lines which exhibit natural differences in sensitivity to CPT. This situation should 140-.120 be clinically more relevant. According to the replication fork collision COL0205Aâ€¢ ,A SW620 A model (21), it is expected that the greater the S-phase fraction and the -Ci HCT116 _ higher the topi expression, the more likely CPT will be toxic. How â€¢HT29|D â€¢AHCC2998 A* ever, we found that topi mRNA and protein expressions are not 100^C HCT15 â€¢â€¢*> predictive for cell sensitivity; low topi expression could be associated O KM12 l^"*â€¢ '.JC with high sensitivity to CPT (colo205 cells), and high expression 80-g5 could be observed with resistance (KM12 cells). Our findings in the Aâ€¢ AOn4 colon cell lines is in opposition with previous observations, which

:1 60 suggested a good predictive value of topi expression for cellular or :20 gD tumor sensitivity (36, 37). Differences between cell type is not sur prising, taking into account the importance of individual downstream signaling and repair pathways (21). Both topi expression and S-phase fraction are probably too much upstream from the lethal events to -â€¢Â» AO have any predictive value in many cases. The ability of the cells to repair the CPT-induced DNA damage and their sensitivity to undergo apoptosis are also critical parameters for CPT sensitivity. 100 1000 3000 Topi mRNA and protein expression were not found to predict the Cleavable Complexes (rad-eq) amount of CPT-induced cleavable complexes, the latter being the best Fig. 4. Relationship between drug-stabilized cleavable complexes and growth inhibi predictors of CPT cytotoxicity. Differential CPT uptake might ex tion. A composite figure was obtained using the data from Figs. I and 3 and various CPT plain, in part, higher detection of cleavable complexes in HCTI 16 concentrations for the seven colon ceil lines of the NCI Anticancer Drug Screen. Symbols are COLO205( Â«),SW620(â€¢),HCTI16(A), HT29 (â€¢).HCC2998(D), HCT15 (A), and cells. However, no correlation between CPT uptake and the amount of KM12 (O). CPT-stabilized cleavable complexes was found since SW620 and 2119

KM12 cells had same cleavable complexes from 0.01 to 5 /XMCPT, Kantarjian, H. M., Beran, M., Ellis, A., O'Brien, S., Cazenave, L, Koller, C., RÃ•OS, while KM 12 had higher CPT uptake. Since the discrepancy between M. B., Plunkett, W., Keating, M. J., and Estey, E. H. Phase I study of topotecan, a new topoisomerase I inhibitor, in patients with refractory or relapsed acute leukemia. topi expression and topi-cleavable complexes could not be explained Blood, 81: 1146-1151, 1993. by differential CPT transport, this observation underlies the impor Masuda, N., Fukuoka, M., Kusunoki, Y.. Matsui, K., Takifuji, N., Kudoh, S., Negoro, tance of posttranslational modifications for topi regulation. Dephos- S., Nishiola, M., Nakagawa, K., and Takada, M. CPT11, a new derivative of camptothecin, for previously untreated non-small-cell lung cancer. J. Clin. Oncol., 10: phorylation abolishes catalytic activity and CPT sensitivity (38, 39, 1225-1229, 1992. 40, 41), while phosphorylation by protein kinase C increases the Lynch, T. J. J., Kalish, L., Strauss, G., Elias, A., Skarin, A., Shulman, L. N., Posner, M., and Frei, E. Phase II study of topotecan in metastatic non-small-cell lung cancer. formation of topi-cleavable complexes (41). Poly(ADP-ribosyl)ation J. Clin. Oncol., 12: 347-352, 1994. inhibits topi catalytic activity (42-45), and 3-aminobenzamide, a Abigerges, D., Chabot, G. G., Armand, J. P., Herait, P., Gouyette, A., and GandÃa,D. poly(ADP ribose) inhibitor, reduces the amount of CPT-induced Phase 1 and pharmacological studies of the camptothecin analog irinotecan adminis tered every 3 weeks in cancer patients. J. Clin. Oncol., 13: 210-221, 1995. cleavable complexes (46). The complexity of topi regulation is also 10. Wall, M. E., Wani, M. C., Cooke, C. E., Palmer, K. H., McPhail, A. T., and Slim, exemplified by the importance of chromatin structure that limits and G. A. The isolation and structure of camptothecin, a novel alkaloidal leukemia and tumor inhibitor from Camptotheca acuminata. J. Am. Chem. Soc., 88: 3888-3890, regulates the association of topi with its DNA target. Cleavable 1966. complexes may be prevented to form and/or change their chromatin Hsiang, Y. H., Hertzberg, R., Hecht, S., and Liu, L. F. Camptothecin induces distribution depending upon nucleosome structure (47), concentra protein-linked DNA breaks via mammalian DNA topoisomerase I. J. Biol. Chem., 260: 14873-14878, 1985. tions of polyamines (48), and DNA methylation (49). Therefore, it is 12. Lee, M. P., Brown, S. D., and Hsieh, T-S. DNA topoisomerase I is essential in important to bear in mind ,for clinical trials of CPT and its derivatives, Drosophila melanogaster. Proc. Nati. Acad. Sci. USA, 90: 6656-6660, 1993. 13. Champoux, J. Mechanistic aspects of type-I topoisomerases. In: J. C. Wang and N. R. that topi expression, although convenient for measurement, may not Cozarelli (eds.), DNA Topology and Its Biological Effects, pp. 217-242. Cold Spring be well correlated with drug efficacy. Harbor, NY: Cold Spring Harbor Laboratory, 1990. In contrast to topi expression, the amount of functional topl- 14. Wang, J. C. Recent studies of DNA topoisomerases. Biochim. Biophys. Acta, 909: 1-9, 1987. cleavable complexes, measured in cells by alkaline elution, was a 15. Wang, J. C. DNA topoisomerases: why so many? J. Biol. Chem., 266: 6659-6662, relatively good parameter to predict sensitivity to CPT; the higher was 1991. the amount of cleavable complexes stabilized by the drug, the greater 16. Pommier. Y., and Tanizawa, A. Mammalian DNA topoisomerase I and its inhibitors. In: !. Hickman and T. Tritton (eds.), Cancer Chemotherapy, pp. 214-250. Oxford: was the cell sensitivity. This parameter might be interesting to con Blackwell Scientific Publications, Ltd., 1993. sider to monitor drug sensitivity of individual patients. However, 17. Chen, A. Y., and Liu, L. F. DNA Topoisomerases: essential enzymes and lethal alkaline elution is a long method that works best when cellular DNA targets. Annu. Rev. Pharmacol. Toxicol., 94: 194-218, 1994. 18. Porter, S. E., and Champoux, J. J. The basis for camptothecin enhancement of DNA is labeled (28) and, therefore, cannot be transferred to the clinic. breakage by eukaryotic topoisomerase I. Nucleic Acids. Res., 17: 8521-8532, 1989. Others methods to measure drug-induced DNA damages are war 19. Holm, C., Covey, J. M., Kerrigan, D., and Pommier, Y. Differential requirement of DNA replication for the cytotoxicity of DNA topoisomerase I and II inhibitors in ranted. Several authors have suggested to use PCR techniques and Chinese hamster DC3F cells. Cancer Res., 49: 6365-6368, 1989. have shown that, in very precise experimental conditions, the reduc 20. Hsiang, Y-H., Lihou, M. G., and Liu, L. F. Arrest of DNA replication by drug- tion of DNA amplification is proportional to the amount of DNA stabilized topoisomerase I-DNA cleavable complexes as a mechanism of cell killing by camptothecin. Cancer Res., 49: 5077-5082, 1989. damage (50). 21. Pommier, Y., Leteurtre, F., Fesen, M., Fujimori, A., Bertrand, R., Solary, E., Kohl- In conclusion, we are using the cell lines of the NCI anticancer hagen, G., and Kohn, K. W. Cellular determinants of sensitivity and resistance to screen as a tool (miniclinic) to study the cellular determinants for DNA topoisomerase inhibitors. Cancer Invest., 12: 530-542, 1994. 22. Sambrook, J.. Fritsch, E. F., and Maniatis, T. Detection and analysis of proteins expressed sensitivity to CPT and its derivatives. In vivo cleavable complexes from cloned genes. In: N. Ford (ed.), Molecular Cloning. A Laboratory Manual. Vol. 3, were the best topi phenotypic parameter to predict cell sensitivity to pp. 62-63. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1989. CPT in this panel of colon cell lines. The log-linear relationship 23. Tanizawa, A., and Pommier, Y. Topoisomerase I alteration in a camptothecin- resistant cell line derived from Chinese hamster DC3F cells in culture. Cancer Res., between CPT-induced DNA damage and survival and the differences 52: 1848-1854, 1992. in CPT toxicity observed after 1 and 24 h exposure suggest that 24. Chang, J-Y., Dethlefsen, L. A., Barley, L. R., Zhou, B. S., and Cheng, Y-C. Characterization of camptothecin-resistant Chinese hamster lung cells. Biochem. increases in drug exposure are probably more cytotoxic and more Pharmacol., 43: 2443-2452, 1992. clinically relevant than dose escalation. 25. Chomczynski, P., and Sacchi, N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 762: 156-159, 1987. D'Arpa, P., Machlin, P. S., Ratrie, H., Ill, Rothfield, N. F., Cleveland, D. W., and ACKNOWLEDGMENTS 26. Earnshaw, W. C. cDNA cloning of human DNA topoisomerase I: catalytic activity of a We thank Dr. D. Scudiere (NCI, Frederick, MD), who provided the cell 67.7-kDa carboxyl-terminal fragment. Proc. Nati. Acad. Sci. USA, 85: 2543-2547,1988. lines, and Dr. Y-C. Cheng (Yale University, New Haven, CT) for the 27. Pommier, Y., Zwelling, L. A., Mattern, M. R., Erickson, L. C., Kerrigan, D., Schwartz, R., and Kohn, K. W. Effects of dimethyl sulfoxide and thiourea upon generous gift of topi monoclonal antibodies. A special thanks to Dr. K. W. intercalator-induced DNA single-strand breaks in mouse leukemia (L1210) cells. Kohn (Laboratory of Molecular Pharmacology, NCI) for discussions and Cancer Res., 43: 5718-5724, 1983. support during the course of this work. 28. Kohn, K. W., Ewig, R. A. G., Erickson, L. C., and Zwelling, L. A. Measurement of strand breaks and crosslinks by alkaline elution. In: E. C. Friedberg and P. C. Hanawalt (eds.), DNA Repair: A Laboratory Manual of Research Procedures, pp. REFERENCES 379-401. New York, NY: Marcel Dekker, Inc., 1981. 29. Bertrand, R., and Pommier, Y. Assessment of DNA damage in mammalian cells by 1. Giovanella, B. C, Stehlin, J. S.. Wall, M. E., Wani, M. C, Nicholas, A. W., Liu, L. F., DNA filter elution methodology. In: G. P. Studzinski (eds.), Cell Growth and Silber, R., and Potmesil, M. DNA topoisomerase I-targeted chemotherapy of human Apoptosis, Vol. 6, pp. 97-117. Newark, NJ: Oxford University Press, 1995. colon cancer in xenografts. Science (Washington DC). 246.- 1046-1048, 1989. 30. Covey, J. M., Jaxel, C., Kohn, K. W., and Pommier, Y. Protein-linked DNA strand 2. Potmesil, M. Camptothecins: from the bench research to the hospital wards. Cancer breaks induced in mammalian cells by camptothecin, an inhibitor of topoisomerase I. Res., 54: 1431-1439, 1994. Cancer Res., 49: 5016-5022, 1989. 3. Slichenmyer, W. J., Rowinsky, E. K., Donehower, R. C., and Kaufmann, S. H. The 31. HÃ¶chster, H., Liebes, L., Speyer, J., Sorich, J., Taubes, B., Oratz, R., Wernz, J., current status of camptothecin analogs as anticancer agents. J. Nati. Cancer. Inst., 85: Chachoua, A., Raphael, B., Vinci, R. Z., and Blum, R. H. Phase I trial of low-dose 271-287, 1993. continuous topotecan infusion in patients with cancer: an active and well tolerated 4. Rowinsky, E. K., Grochow, L. B., Hendricks, C. B.. Ettinger, D. S., Forastiere, A. A.. regimen. J. Clin. Oncol., 12: 553-559, 1994. O'Connor, P. M., Nieves-Neira, W., Kerrigan, D., Bertrand, R., Goldman, J., Kohn, Hurwitz. L. A., McGuire, W. P., Sartorius, S. E., Lebejko, B. G., Kaufman, S. H., and 32. Donehower, R. C. Phase I and pharmacologie study of topotecan. J. Clin. Oncol., 10: K. W., and Pommier, Y. S-phase population analysis does not correlate with the 647-656, 1992. cytotoxicity of camptothecin and 10,11-methylenedioxycamptothecin in human colon 5. Blaney, S. M., Balis, F. M., Cole, D. E., Craig, J. M., Reid, J. M., Ames, M. M., carcinoma HT-29 cells. Cancer Commun., 3: 233-240, 1991. Drailo, M., Reaman, G., Hammond, D., and Poplack, D. G. Pediatrie phase 1trial and 33. Boothman. D. A., Fukunaga, N.. and Wang, M. Down-regulation of topo pharmacokinetic study of topotecan administered as a 24-hour continuous infusion. isomerase I in mammalian cells following ionizing radiation. Cancer Res., 54: Cancer Res., 53: 1031-1036, 1993. 4618-4626, 1994.

2120

34. Tamura, H-O., Kohchi, C., Yamada, R., Ikeda, T., Koiwa, O., Patterson, E., Keene, 43. Ferro, A. M., and Olivera, B. M. Poly(ADP-ribosylation) of DNA topoisomerase I J. D., Okada, K., Kjeldsen, E., Nishikawa, K., and Andoh, T. Molecular cloning of a from calf thymus. J. Biol. Chem., 259: 547-554. 1984. cDNA of a camptothecin-resistanl human DNA lopoisomerase I and identification of 44. Kasid, U. N., Halligan, B., Liu, L. F., Dritschilo, A., and Smulson, M. Poly(ADP- mutation sites. Nucleic Acids Res., 19: 69-75, 1991. ribose)-mediated post-translational modification of chromatin-associated human 35. Tanizawa, A., Bertrand, R., Kohlhagen, G., Tabuchi, A., Jenkins, J., and Pommier, Y. topoisomerase I. Inhibitory effects on catalytic activity. J. Biol. Chem., 264: Cloning of Chinese hamster DNA topoisomerasc I cDNA and identification of a 18687-18692, 1989. single point mutation responsible for camptothecin resistance. J. Biol. Chem., 268: 45. Krupila. G., and Cerutti. P. ADP-ribosylalion of ADPR-lransferase and topoisomer 25463-25468, 1993. ase I in intact mouse epidermal cell JB6. Biochemistry, 28: 2034-2040, 1989. 36. Madden. K. R.. and Champoux. J. J. Over-expression of human lopoisomerase I in 46. Maltern, M. R., Mong, S. M., Barlus. H. F., Mirabelli, C. K., Crooke, S. T., and baby hamster kidney cells: hypersensitivity to clonal isolates to camptothecin. Cancer Res., 52.- 525-532, 1992. Johnson, R. K. Relationship between the intracellular effects of camptothecin and the inhibition of DNA topoisomerasc I in cultured L1210 cells. Cancer Res., 47: 37. Husain. I., Mohler, J. L., Seigier, H. F.. and Besterman, J. M. Elevation of topoi- 1793-1798, 1987. somerase I messenger RNA, protein, and catalytic activity in human tumors: dem onstration of tumor-type specificity and implications for cancer chemotherapy. Can 47. Jaxel, C., Capranico. G., Wassermann, K., Kerrigan. D.. Kohn, K. W.. and Pommier. cer Res., 54: 539-546, 1994. Y. DNA sequence at sites of topoisomerase I cleavage induced by camptothecin in SV40 DNA. In: M. Potmesil and K. W. Kohn (eds.), DNA Topoisomerases in Cancer, 38. Durban, E., Mills, J. S., Roll, D., and Busch, H. Phosphorylation of purified Novikoff hepatoma topoisomerase I. Biochem. Biophys. Res. Commun., ///: 897-905, 1983. pp. 182-195. New York: Oxford University Press, 1991. 39. Tse-Dinh, Y. C., Wong, T. W., and Goldberg, A. R. Virus- and cell-encoded tyrosine 48. Srivenugopal, K. S.. and Morris, D. R. Differential modulation by spermidine of protein kinases inactivate DNA topoisomerases in vitro. Nature (Lond.), 312: reactions catalyzed by type 1 prokaryotic and eukaryolic topoisomerases. Biochem 785-786, 1984. istry, 24: 4766-4771, 1985. 40. Kaiserman, H. B., Ingebritsen, T. S., and Benbow, R. M. Regulation of Xenopus 49. Leteurtre, F., Kohlhagen, G., Fesen, M. R., Tanizawa, A., Kohn, K. W., and Pommier, lat'vis DNA topoisomerase I activity by phosphorylation in vitro. Biochemistry, 27: Y. Effects of DNA methylation on topoisomerase I and II cleavage activities. J. Biol. 3216-3222, 1988. Chem., 269: 7893-7900, 1994. 41. Pommier, Y., Kerrigan, D., Hartmann, K. Dâ€žand Glazer, R. I. Phosphorylation of 50. Jennerwein, M. M.. and Eastman. A. A polymerase chain reaction-based method lo mammalian DNA topoisomerase I and activation by protein C. J. Biol. Chem., M: detect cisplatin adducts in specific genes. Nucleic Acids Res., 19: 6209-6214, 1991. 9418-9422, 1990. 51. Fujimori, A., Harker, W. G., Kohlhagen, G., Hoki, Y., and Pommier, Y. Mutation at 42. Ferro, A. M., Higgins, N. P., and Olivera, B. M. Poly (ADP-ribosylation) of a DNA the catalytic site of topoisomerase I in CEM/C2, a human leukemia cell line resistant topoisomerase I. J. Biol. Chem., 258: 6000-6003, 1983. to camplothecin. Cancer Res., 55: 1339-1346, 1995.

2121

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1995 American Association for Cancer Research. Topoisomerase I-related Parameters and Camptothecin Activity in the Colon Carcinoma Cell Lines from the National Cancer Institute Anticancer Screen

François Goldwasser, Insoo Bae, Monica Valenti, et al.

Cancer Res 1995;55:2116-2121.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/55/10/2116

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/55/10/2116. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.