Inhibitors of Topoisomerase II As Ph-Dependent Modulators of Etoposide-Mediated Cytotoxicity1

Vol. 5, 2899–2907, October 1999 Clinical Cancer Research 2899

Inhibitors of Topoisomerase II as pH-dependent Modulators of Etoposide-mediated Cytotoxicity1

Seppo W. Langer,2 Gu¨nther Schmidt, INTRODUCTION Morten Sørensen, Maxwell Sehested, and The catalytic cell cycle of the nuclear enzyme topo3 II is Peter Buhl Jensen the target of some of the most used antitumor agents, e.g., the Laboratory of Experimental Medical Oncology, The Finsen Center epipodophyllotoxins etoposide and teniposide (1). A problem 5074 [S. W. L., G. S., M. Sø., P. B. J.], and Department of Pathology, with these drugs is their low therapeutic index. Obviously, a The Laboratory Center 5442 [S. W. L., G. S., M. Sø., M. Se.], more successful therapy would be possible if cytotoxicity could Rigshospitalet, DK-2100 Copenhagen, Denmark be targeted specifically to tumor cells. However, tumor-specific alterations are disappointingly few. One physiological differ- ABSTRACT ence between normal cells and tumor cells is their extracellular microenvironment, where solid tumors tend to develop a pH , Chloroquine intercalates into DNA and protects cells e which is about 0.5 pH units lower than in normal tissues (2–4). against topoisomerase II (topo II) poisons such as etoposide This pH difference could thus be suitable for targeting, as by hindering the DNA cleavage reaction of this target en- suggested by Newell et al. (3). zyme. Chloroquine, in contrast to etoposide, is a weak base Etoposide-induced cell kill can be antagonized by distinct and therefore barely enters the cell when the extracellular drug types. It has been demonstrated that the intercalating drugs fluid is acidic, as is the case in most solid tumors. Such a aclarubicin, chloroquine, and the cardioprotecting agent ICRF- pH-dependent drug interaction could be useful in targeting 187 (dexrazoxane) antagonize the cytotoxicity of etoposide in the cytotoxicity of topo II poisons toward solid tumors. vitro and in vivo (5–8).These so-called catalytic inhibitors ap- Unfortunately, antagonistic chloroquine concentrations can- pear to act in a different manner on the catalytic cycle of topo II not be reached in vivo because of its unacceptable toxicity. than the classical poisons such as etoposide, teniposide, and Thus, antagonists with a higher therapeutic index are many of the anthracyclines. They inhibit the enzyme without needed. We report here on the structure-activity relation- stabilizing the cleavable DNA-enzyme complex formation, thus ship of several chloroquine and acridine analogues in a being able to antagonize the action of the topo II poisons clonogenic assay. There were major differences in the cyto- (reviewed in Ref. 9). As aclarubicin, chloroquine also antago- toxicity of the different compounds, with acridines being nizes both topo I-mediated DNA single-strand breaks and the 50-fold more toxic than the chloroquine analogues. Several cytotoxicity of camptothecin (10). compounds were, however, able to antagonize etoposide- Chloroquine is a weak base; therefore, it is ionized at low mediated cytotoxicity in a pH-dependent manner as chloro- pH and consequently is unable to transverse the plasma mem- quine. Dependency on pH was lost if the aminoalkyl side brane. The antagonistic effect on both etoposide and camptoth- arm of chloroquine was removed or lengthened by one CH 2 ecin cytotoxicity is pH dependent in clonogenic assays. As we whereas pH dependency was strong with hydroxychloro- have shown previously, chloroquine accumulation is decreased quine. In contrast, the aminoalkyl side arm was clearly 5-fold in cells when the pH is lowered from values that are dispensable in the acridines because both quinacrine and e neutral (pH ϭ 7.4) to acidic (pH ϭ 6.0). No protection against 9-aminoacridine demonstrated profound pH dependency. etoposide by chloroquine is observed at pH ϭ 6.5, whereas at The results from clonogenic assay were compared with cel- e pH ϭ 7.4, etoposide cytotoxicity is almost completely antag- lular transport measurements and topo II enzyme inhibi- e onized (7). Unfortunately, in contrast to aclarubicin (11), chlo- tion. Compounds with the most marked pH-dependent in- roquine in itself is too toxic to protect against etoposide toxicity tracellular accumulation were also the best pH-dependent in mice.4 protectors of etoposide cytotoxicity, clearly supporting the To study structure-activity relationships and develop less hypothesis that extracellular pH can be used to regulate topo toxic antagonists, we have investigated a number of quinoline II poisoning. derivatives and acridine analogues to identify critical structures for the pH-dependent antagonism of etoposide cytotoxicity with the goal of targeting acidic tumors. Received 2/24/99; revised 7/16/99; accepted 7/26/99. 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 18 U.S.C. Section 1734 solely to 3 indicate this fact. The abbreviations used are: topo, topoisomerase; pHe, extracellular 1 Supported by grants from the Danish Cancer Society and the H:S pH; kDNA, kinetochore DNA; MTD, maximal tolerated dose; Research Council. GS1, 7-chloro-4-(3-hydroxy-propylamino)quinoline; GS2, 7-chloro-4- 2 To whom requests for reprints should be addressed, at the Laboratory (5-diethylamino-1-methylpentylamino)quinoline; GS3, 7-chloro-4-(3- of Experimental Medical Oncology, The Finsen Center 5074, Rigshos- diethylamino-1-methylpropylamino)quinoline; GS4, 7-chloro-4-(NЈ-2- pitalet, 9 Blegdamsvej, DK-2100 Copenhagen, Denmark. Fax: 45- DEAE)-N-methyl)hydrazino)quinoline. 35456966; E-mail: [email protected]. 4 Unpublished observations.

Fig. 1 Chemical structure of the chloroquine and acridine analogues. Arrows and figures refer to the “Discussion” section.

MATERIALS AND METHODS the results were checked by fast atom proton bombardment mass Cell Lines spectroscopy and elementary analysis: Human small cell lung cancer OC-NYH (GLC-2; Ref. 12) GS1. A mixture of 3-hydroxypropylamine (0.75 g; 10 and NCI-H69 (13) were grown in RPMI 1640 supplemented mmol), 4,7-dichloroquinoline (1.98 g; 10 mmol) and phenol with 10% FCS plus penicillin and streptomycin. (0.94 g; 10 mmol) was heated with stirring at 140°C for 5 h. The reaction mixture was cooled to 100°C, poured into water, and Drugs/Chemicals stirred to induce crystallization. It was then washed with water The chemical structures of the quinolines and acridines are and filtered, and the solid was recrystallized several times from shown in Fig. 1. All purchased and synthesized drugs were of at 96% ethanol to give 1.79 g (76%) and finally dissolved in water. least 97% purity. Amodiaquine [7-chloro-4-(3-diethyl-amino- GS2. A mixture of 4-diethylamino-2-aminohexane (0.4 methyl-4-hydroxy-anilino)quinoline]; primaquine [8(4-amino- g; 2.3 mmol), 4,7-dichloroquinoline (0.46 g; 2.3 mmol), and phenol (0.22 g; 2.3 mmol) was heated at 160°C for 6 h. The 1-methylbutylamino)-6-methoxyquinoline, 2H3PO4]; quinine [␣-(hydroxy-(6-methoxy-quinolinyl-4)methyl)-3-vinylquinu- reaction mixture was cooled and poured into 30% aqueous clidine]; quinacrine [mepacrine; 3-chloro-9-(4-diethyl- sodium hydroxide and stirred for 1 h. The aqueous solution was amino-1-methylbutylamino)-7-methoxyacridine, 2HCl]; and extracted with ether, and the ether extracts were washed with chloroquine [7-chloro-4(4-diethylamino-1-methylbutylamin- water and dried over sodium sulfate. Evaporation of the solvent gave an oil that was distilled at 160–180°C and 0.06 mm Hg. o)quinoline, 2H3PO4] and -dichloroquinoline were purchased from Sigma Chemical Co. (St. Louis, MO). 9-Aminoacridine, The oil solidified on standing, and the product was recrystallized from hexane:benzene 1:1 to give 0.44 g (57%) and then dis- HCl, H20 was purchased from Aldrich (Steinheim, Germany). Hydroxychloroquine [7-chloro-4(4-(ethyl-(2-hydroxyethyl)amino- solved in water.

1-methylbutylamino)quinoline, H2S04] was obtained from Erco- GS3. A mixture of 4-diethylamino-2-aminobutane (0.5 g; pharm (Kvistgaard, Denmark). [14C]Chloroquine was obtained 3.5 mmol), 4,7-dichloroquinoline (0.63 g; 3.5 mmol), and phe- from Amersham (Buckinghamshire, United Kingdom). Aclarubi- nol (0.33 g; 3.5 mmol) was heated at 160°C for 6 h. The reaction cin was purchased from Lundbeck (Copenhagen, Denmark), and mixture was cooled and poured into 30% aqueous sodium ICRF-187 was from Chiron (Amsterdam, the Netherlands). hydroxide and stirred for 1 h. The aqueous solution was ex- The following compounds where synthesized de novo, and tracted with ether, and the ether extracts were washed with water

and dried over sodium sulfate. Evaporation of the solvent gave Table 1 MTDs of the drugs used in the clonogenic assay

an oil that was distilled at 150–180°C and 0.07 mm Hg. The oil Drug MTD (␮M) solidified on standing, and the product was recrystallized from Quinine 650 hexane:benzene 1:1 to give 0.66 g (62%) and dissolved in Hydroxychloroquine 550 DMSO. Primaquine 550 GS4. A mixture of N-(2-DEAE)-N-methylhydrazine Chloroquine 500 (1.00 g; 6.6 mmol), 4,7-dichloroquinoline (1.19 g; 6.0 mmol), GS3 500 and phenol (2.8 g; 30 mmol) was heated with stirring at 150°C GS4 500 GS1 300 for 7 h. The reaction mixture was cooled, poured into 10% 4,7-Dichloroquinoline 50 aqueous KOH, and extracted with dichloromethane. The organic GS2 25 extract was washed with water and dried over sodium sulfate. Quinacrine 20 Evaporation of the solvent gave an oil that was distilled at 9-Aminoacridine 15 Amodiaquine 10 140–150°C and 0.07 mm Hg. The oil solidified on standing, and the product was recrystallized from hexane to give 0.59 g (32%) and finally dissolved in DMSO.

8), 120 KCl, 10 mM MgCl , 1.0 mM ATP, 0.5 mM DTT, and 30 Clonogenic Assay 2 mg of BSA/ml]. After addition of stop buffer/loading dye mix Drug cytotoxicity was assessed by colony formation in soft (5% Sarkosyl, 0.0025% bromphenol blue, and 25% glycerol), agar with a feeder layer containing sheep RBCs as described samples were loaded on 1% agarose/0.5% ethidium bromide previously (14). Single-cell suspensions in RPMI 1640 supple- gels and run in TBE buffer containing 0.5 mg/ml ethidium mented with 10% FCS (complete medium) were exposed for 1 h bromide at 100 V for ϳ50 min and photographed under UV to the drugs at 37°C at the desired pH and concentrations and light. In addition, loading wells were cut out and scintillation washed twice with complete medium at 37°C at pH 7.4. The pH 4 counted to obtain numerical values. was adjusted with 5 M HCl. Approximately 2 ϫ 10 cells were plated to obtain 3000–4000 colonies in the control dishes. The colonies were counted after 3 weeks incubation, and survival RESULTS was calculated as compared with control cells. All experiments The structure-related strategy used in testing the different were done in triplicate. compounds is outlined in Fig. 1. Dose-finding Studies. Initially, the MTD of the modulator drugs were determined in a series of clonogenic assays (not Drug Accumulation shown). MTD was defined as the highest achievable dose in the Two million cells in single-cell suspension in 2 ml of PBS pH range of 6.3–7.4, resulting in less than LD20. These concen- containing dialyzed 5% FCS were incubated for 30 min with trations (Table 1) were the ones subsequently used in the clo- DNase I, type II (Sigma D4527). Hereafter, we essentially nogenic assays, both alone and in combination with 20 ␮M followed the procedures described by Skovsgaard (15). Twenty etoposide. ␮ l of the drug were added to produce the following final Amodiaquine, GS2, and the neutral compound 4,7-dichlo- concentrations: 350 ␮g/ml primaquine, 20 ␮g/ml quinacrine, 10 roquinoline, were far more potent (MTD, 10–50 ␮M) than the ␮g/ml amodiaquine, and 5 ␮g/ml 9-aminoacridine. Cells were rest of the 4-aminoquinolines (MTD, 300–550 ␮M). The acri- ϫ then pelleted at 3000 g for 2 min at 4°C and washed twice dines 9-aminoacridine and quinacrine also were potent cytotoxic with 7 ml of ice-cold Ringer’s solution at 4°C to remove the compounds, with MTDs of 15 and 20 ␮M, respectively. extracellular drug. The drug then was extracted from the cells The pH-dependent Cytotoxicity and Antagonism of with3mlof0.3M HCl in 50% ethanol solution. Etoposide. We then performed a series of clonogenic assays The total fluorescence of the supernatant solution after to determine the pH-dependent cytotoxicity of etoposide alone ϫ centrifugation at 5000 g for 10 min was determined in and in combination with the various drugs, as depicted in Fig. 2. duplicate by spectrofluorophotometry (9-aminoacridine excita- As shown previously, etoposide was more toxic at neutral than tion and emission wavelengths 373 nm and 456 nm; quinacrine at acidic extracellular values. It appears that the cytotoxicity of 438 nm and 456 nm, respectively). Spectrophotometric meas- etoposide is somewhat variable from experiment to experiment, urements of extracts of primaquine and amodiaquine were done most likely attributable to the fact that the experiments were in triplicate at wavelengths of 335 nm and 344 nm, respectively. 14 conducted over a period of several months. For the experiments Accumulation of [ C]chloroquine was assessed by liquid to be suitable for direct comparison, the required etoposide scintillation counting as described previously (7). cytotoxicity had to exceed 90% at all pH levels. The ability of the drugs to antagonize 20 ␮M etoposide is illustrated in Fig. 3, Decatenation Assay showing the fold protection, which was calculated as the relative topo II catalytic activity was measured by kDNA decat- survival of colonies treated with the combination of modulator enation. 3H-labeled kDNA was isolated from Crithidia fascicu- drug and etoposide to the survival when treated with etoposide

lata (American Type Culture Collection, Manassas, VA) as alone at neutral (pH 7.3–7.5) as well as acidic pHe (pH 6.3–6.5). described by Sahai and Kaplan (16). Briefly, purified topo IIa The minor change of the chloroquine alkyl side chain resulting was incubated with 0.2 mg of kDNA for 15 min at 37°C in a in hydroxychloroquine (step 2 in Fig. 1) as well as the change of final volume of 20 ml in a buffer containing [50 mM Tris-Cl (pH the ring structure (step 8, quinacrine) did not have any signifi-

Fig. 2 Results of the clonogenic assays. Bars, SE of triplicate platings.

Fig. 2 Continued.

Fig. 3 The fold protection of the different compounds in acidic (the left of each pair of

columns) and neutral (right columns) pHe. The fold protection expresses the degree of protection conferred by the combination of the modulator compound ϩ20 ␮M etoposide to the cytotoxicity of 20 ␮M etoposide alone.

Fig. 4 Relative intracellular drug accumulation as a function of pHe. Ordinates, pHe; abscissae, relative intracellular drug concentration. Bars, SE of two or three measurements.

cant impact on the marked ability to antagonize the cytotoxicity pronounced effect on antagonism. Although a weak antagonist, of etoposide at neutral pH in vitro. Removal of the side chain the cytotoxicity of amodiaquine in combination with etoposide from the acridine ring system (step 9, 9-aminoacridine) did not was pH dependent as opposed to primaquine (step 10), which

influence the antagonism or pH dependency. At neutral pHe the was in accordance with the measurements of intracellular drug fold protection for these three compounds were in the range of accumulation (Fig. 4). The most pronounced structure-activity

80–430, declining to 1–4 at acidic pHe. relationship was seen in step 4; the elongation of the alkyl side The insertion of a ring structure instead of an alkyl side chain by only one CH2 in GS2 resulted in complete loss of chain at the four positioned substituent in the quinoline ring etoposide antagonism and far greater toxicity as compared with (steps 7 and 11; amodiaquine and quinine, respectively) had a the rest of the 4-aminoquinolines, apart from 4,7-dichloroquino-

Fig. 5 Effect of reducing test drug concentration from MTD to one-fifth of the MTD on pH dependency and/or etoposide antagonism. a, chloroquine; b, quinacrine; and c, summary table.

line. This neutral compound likewise had only weak antagonis- shows the results from the assay using chloroquine as the test tic properties with relatively small pH variation (step 1). drug. The pH dependency and the antagonism of etoposide Shortening of the alkyl side chain, on the other hand, had cytotoxicity are clearly reduced at one-fifth of the MTD. Dose- no effect at all (GS4, step 6) or a slightly diminishing influence dependent reduction of etoposide antagonism and pH depen- on the antagonism (GS1 and GS3, steps 5 and 3, respectively). dency were features of all of the antagonists apart from quina- The potency of these drugs was comparable. crine (Fig. 5b) and 9-aminoacridine. These two acridines To rule out the possibility that our findings were cell line exhibited totally unchanged antagonism as well as pH depen- specific, we also conducted a small series of clonogenic assays dency at the low concentrations. with the more commonly used NCI-H69. These showed that Drug Accumulation. Next, measurements of intracellu- both chloroquine and 9-aminoacridine, but not primaquine or lar drug accumulation as a function of extracellular pH were GS2, protected against etoposide toxicity at neutral pH (not performed with chloroquine, primaquine, amodiaquine, quina- shown). crine, and 9-aminoacridine. The intracellular concentrations at Antagonism and pH-dependent Cytotoxicity at Low neutral pH were highest in all experiments; the results are Dose. We repeated the clonogenic assays at pH 7.4 and 6.7 in presented as the relative values compared with neutral values two doses, i.e., MTD and one-fifth of the MTD. In this way, we (Fig. 4). The accumulation of primaquine was almost independ- were able determine whether the etoposide antagonism and/or ent of extracellular acidity, whereas lowering of the pHe had an pH-dependent cytotoxicity was dose dependent as well. Fig. 5a obvious inhibitory effect on cellular uptake of the other drugs

Fig. 6 The relative inhibition of topo II activity at different doses in a decatenation assay. Left columns, dose ϭ MTD; middle columns, dose ϭ 2 ϫ MTD; right columns, dose ϭ 5 ϫ MTD. Bars, range in two independent assays. Data not available for GS4, quinine, and where indicated by ૺ.

tested. In particular, the effect was pronounced on 9-aminoacri- mediated decatenation of kDNA. In contrast, 9-aminoacridine dine and quinacrine, with a 75 and 50% reduction at pH 6.9 and and quinacrine as well as the chloroquine analogues exhibit 6.7, respectively. dose-dependent inhibition. Thus, the lack of antagonism of GS2 Effect of the Test Drugs on topo II-mediated kDNA in the clonogenic assays could be explained by low drug uptake. Decatenation. Finally, the relative inhibition of the test drugs Amodiaquine, which displays weak antagonistic properties in

on topo II-mediated decatenation of the kDNA network at the the clonogenic assays at neutral pHe, presumably exerts its three dose levels is shown in Fig. 6. Chloroquine, OH-chloro- effect by another mechanism other than interference with the quine, GS3, 9-aminoacridine, and quinacrine exhibited dose- DNA-topo II complex. dependent inhibition of the topo II enzyme activity, whereas Compared with the majority of the 4-aminoquinolines, the amodiaquine, and GS2 did not. Primaquine and 4,7-dichloro- acridine-based compounds (quinacrine and 9-aminoacridine) are quinoline were only tested at two dose levels because of a lack cytotoxic at very low concentrations. Interestingly, the pro- of solubility but appeared not to affect the decatenation at these nounced etoposide antagonism exhibited by both drugs does not concentrations. GS1 was only a weak inhibitor. seem to relate to an alkyl side chain as seen with the tested 4-aminoquinolines. The results from the clonogenic assays can DISCUSSION be related to the measurements of intracellular drug accumula- We have shown previously that the antimalarial chloro- tion and thereby confirm the role of the cell membrane in the quine antagonizes etoposide-mediated cytotoxicity in a dose- regulation of the pH-dependent antagonism of etoposide antag- dependent manner (7). Furthermore, the antagonism is almost onism. For example, the pH-dependent drug transport profile is

complete at pHe 7.4, whereas the protection is abolished at pHe most marked for 9-aminoacridine. At low pH, the measured 6.5. It would therefore be expected that other quinoline com- intracellular concentration is indeed minimal. However, the pounds could have the same effect. clonogenic assays measuring dose-relationship showed com- Despite minor changes in the alkyl side chain of the chlo- pletely unchanged pH dependency and etoposide antagonism for roquine molecule, i.e., shortening and hydroxylation as de- 9-aminoacridine and quinacrine, even at doses as low as one- scribed, the 4-aminoquinolines retain their ability to protect cells fifth of the MTD, in contrast to the dose-dependent antagonism against etoposide-mediated cytotoxicity in a pH-dependent of chloroquine. Therefore, the combined effects of the accumu- manner in vitro. However, major changes, i.e., insertion of a lation and drug potency profile conveniently link the results of ring structure in the side chain (amodiaquine) or lengthening or the transport measurements and the clonogenic assays. even removal of the alkyl side chain of chloroquine (GS2 and The clinical usefulness of cytotoxic drugs is limited by 4,7-dichloroquinoline, respectively), markedly reduced etopo- development of drug resistance and by unwanted side effects. side antagonism. The cytotoxicity of these drugs is enhanced as Both problems are at least somewhat (and inversely) related to well. The decatenation assay measures the direct effect of the dose levels. Accordingly, it would be desirable to be able to test drugs on the catalytic cycle of topo II. Even at high con- target cytotoxicity drugs directly to the malignant tissue, thus centrations, amodiaquine and GS2 do not inhibit the topo II- reducing systemic side effects, and furthermore attempt to in-

crease their doses to overcome drug resistance. As opposed to fon-m-anisidide in human small cell lung cancer cell lines and on chloroquine and hydroxychloroquine, the pH-dependent etopo- topoisomerase II-mediated DNA cleavage. Cancer Res, 50: 3311–3316, side antagonism of the acridines, i.e., quinacrine and 9-amino- 1990. acridine, was present at low concentrations. This might be 7. Jensen, P. B., Sørensen, B. S., Sehested, M., Grue, P., Demant, E. J. F., and Hansen, H. H. Targeting the cytotoxicity of topoisomerase beneficial in a toxicological sense, so that the potential of using II-directed epipodophyllotoxins to tumor cells in acidic environments. these drugs clinically as an instrument for targeted chemother- Cancer Res., 54: 2959–2964, 1994. apy could be studied in preclinical animal tests. Hence, these in 8. Sehested, M., Jensen, P. B., Sørensen, B. S., Holm, B., Friche, E., vitro results do encourage further in vivo studies of these two and Demant, E. J. F. Antagonistic effect of the cardioprotector (ϩ)-1,2,- drugs in particular. In contrast, the de novo synthesized drugs, bis(3,5-dioxopiperazinyl-1-yl)propane (ICRF-187) on DNA breaks and i.e., GS1, GS2, GS3, and GS4 do not seem to be improved cytotoxicity induced by the topoisomerase II directed drugs daunorubi- agents. cin and etoposide (VP-16). Biochem. Pharmacol., 46: 389–393, 1993. Other catalytic inhibitors of topo II can protect against 9. Andoh, T., and Ishida, R. Catalytic inhibitors of DNA topoisomerase II. Biochim. Biophys. Acta, 1400: 155–171, 1998. etoposide cytotoxicity in mice. Thus, aclarubicin is an effective 10. Sorensen, M., Sehested, M., and Jensen, P. B. pH-dependent regu- protector (11) that intercalates in the same manner as chloro- lation of camptothecin-induced cytotoxicity and cleavable complex for- quine, i.e., it interferes with the initial binding of topo to DNA mation by the antimalarial agent chloroquine. Biochem. Pharmacol., 54: (17, 18). However, this antagonism is not pH dependent. Fur- 373–380, 1997. thermore, the bisdioxopiperazine ICRF-187 (dexrazoxane) is a 11. Holm, B., Jensen, P. B., Sehested, M., and Hansen, H. H. In vivo very potent catalytic inhibitor in vivo. It exerts its effect much inhibition of etoposide-mediated apoptosis, toxicity and antitumor effect later in the catalytic cycle of topo II, i.e., by locking the topo II by the topoisomerase II-uncoupling anthracycline aclarubicin. Cancer after religation (17, 19–22). However, ICRF-187 is not a weak Chemother. Pharmacol., 34: 503–508, 1994. base, and its interaction with etoposide and other topo II poisons 12. de-Leij, L., Postmus, P. E., Buys, C. H., Elema, J. D., Ramaekers, F., Poppema, S., Brouwer, M., van der Veen, V., Mesander, G., and The, is not dependent on pH. We believe that the possibility of using T. H. Characterization of three new variant type cell lines derived from the naturally occurring differences in pHe between malignant small cell carcinoma of the lung. Cancer Res., 45: 6024–6033, 1985. and nonmalignant cells deserves attention. By demonstrating 13. Carney, D. N., Gazdar, A. F., Bepler, G., Guccion, J. G., Marangos, that several quinolines and acridines can antagonize the topo II P. J., Moody, T. W., Zweig, M. H., and Minna, J. D. Establishment and poison etoposide in a pH-dependent manner in vitro, the track identification of small cell lung cancer cell lines having classic and has been laid for further preclinical animal testing of this hy- variant features. Cancer Res., 45: 2913–2923, 1985. pothesis. 14. Roed, H., Christensen, I. J., Vindeløv, L. L., Spang-Thomsen, M., and Hansen, H. H. Interexperiment variation and dependence on culture conditions in assaying the chemosensitivity of human small cell lung ACKNOWLEDGMENTS cancer cell lines. Biochem. 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Seppo W. Langer, Günther Schmidt, Morten Sørensen, et al.

Clin Cancer Res 1999;5:2899-2907.

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