Mechanisms of Resistance to Etoposide and Teniposide in Acquired Resistant Human Colon and Lung Carcinoma Cell Lines'

[CANCER RESEARCH 51, 5275-5284, October 1, 1991] Mechanisms of Resistance to Etoposide and Teniposide in Acquired Resistant Human Colon and Lung Carcinoma Cell Lines'

Byron H. Long,2 Lotte Wang, Aurelio Lorico,3 Richard C. C. Wang, Michael G. Brattain, and Anna Maria Casazza Departments of Experimental Therapeutics [B. H. L., A. L., R. C. C. H'., A. M. C.], Cellular and Molecular Biology Â¡L.U'.J, Oncology Drug Discovery, Pharmaceutical Research Institute, Bristol-Meyers Squibb Company, Wallingford, Connecticut 06492, and Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030 [M. G. B.]

ABSTRACT mediate relates to the cytotoxicity of certain antitumor drugs, including VP-16 and VM-26. Evidence for this is provided by Stable acquired resistance to etoposide (VP-16) or teniposide (VM- the correlation between topoisomerase II intermediate stabili 26) in 11(1116 human colon carcinoma cells and A549 human lung adenocarcinoma cells, was previously obtained by weekly 1-h exposures zation in vitro, DNA breakage in cells, and cytotoxicity of to cither drug (B. H. Long, Nati. Cancer Inst. Monogr., 4: 123-127, different analogues in various cell lines (2,13,20-22). However, 1987). The purpose of this study was to identify possible mechanisms of cytotoxicity does not appear to directly result from the inhibi resistance present in these cells by using human nutrÃ¬andtopoisomerase tion of the catalytic activity of topoisomerase II but rather II DNA probes, antibodies to these gene products, and P4 phage un- results from a yet unknown subsequent event related to the knotting assay for topoisomerase II activities. HCT116(VP)35 cells were level of stabilized DNA-topoisomerase II intermediate (21-23). 9-, 7-, and 6-fold resistant to VP-16, VM-26, and Adriamycin, respec Cellular resistance to the cytotoxic effects of topoisomerase tively, and showed no cross-resistance to colchicine and actinomycin D. II active drugs correlates with lower enzyme levels in resistant These cells had no differences in mdrl gene, mdrl mRNA, or P- cells (21-26). This observation is compatible with the hypoth glycoprotein levels but displayed decreased levels of topoisomerase II esis that cytotoxicity is not related to the inhibition of catalytic mRNA and enzyme activity without any alteration of drug sensitivity displayed by the enzyme. HCT116(VM)34 cells were 5-, 7-, and 21-fold activity but is more closely related to the presence of stabilized resistant to VP-16, VM-26, and Adriamycin; were cross-resistant to DNA-enzyme intermediate. Cells containing lower levels of colchicine (7-fold) and actinomycin D (18-fold); and possessed a 9-fold topoisomerase II would have less stabilized intermediate capa increase in mdrl mRNA and increased P-glycoprotcin without evidence ble of contributing to cytotoxicity. Not surprisingly, mutated of mdrl gene amplification. No alterations in topoisomerase II gene or enzymes that are less sensitive to drug or ATP have been found mRNA levels, enzyme activity, or drug sensitivity were observed. in drug resistant cells, as well (27-30). The presence of elevated A549(VP)28 and A549(VM)28 cells were 8-fold resistant to VP-16 and levels of P-glycoprotein, either by gene amplification or by over VM-26 and 3-fold resistant to Adriamycin. Both lines were not cross- expression of the mdrl gene that codes for P-glycoprotein has resistant to colchicine or actinomycin D but were hypersensitive to ci.\- been related to drug resistance by causing decreased intracel- platinum. No alterations in mdrl gene, mdrl mRNA, or P-glycoprotein lular accumulation of cytotoxic drugs, including those that levels, but lower topoisomerase II mRNA levels and decreased enzyme inhibit topoisomerase II (see Refs. 31 and 32). activities, were observed. Of the four acquired resistant cell lines, resist ance is likely related to elevated imlrl expression in one line and to We describe here the characterization of four human cell lines displaying very stable levels of acquired resistance to VP- decreased topoisomerase II expression in the other three lines. 16 and VM-26 for known mechanisms of resistance to these drugs. Relatively low levels of acquired resistance were devel INTRODUCTION oped by intermittent drug exposures over a 6-month period in The active anticancer drugs VP-164 and VM-26 have been order to obtain resistant lines with mechanisms of resistance closely reflecting those that may arise in the clinic. Further identified as potent inhibitors of eukaryote topoisomerase II (1-5). This inhibition results in the stabilization of the transient more, cell lines with low levels of resistance should be more likely to have only one mechanism of resistance, thus allowing covalent intermediate formed between the enzyme and its DNA substrate (6-9). The formation of this covalent DNA-protein such lines to serve as model cell lines for a given mechanism of drug resistance. Such appears to be the case in that three of the complex is readily observable in intact cells by using standard four sublines studied displayed decreased levels of topoisomer alkaline DNA elution techniques (10, 11). In fact, alkaline DNA ase II without any indication of altered mdrl gene expression, elution methodology played a major role in identifying topo isomerase II as the target for VP-16 and VM-26 (1-3, 5, 12, and the fourth line had elevated levels of mdrl mRNA and P-glycoprotein without changes in topoisomerase II levels 13) and DNA intercalating drugs (14-19). It is generally rec or activities. ognized that inhibition of topoisomerase II resulting in stabili zation of a normally transient, covalent, DNA-enzyme inter- MATERIALS AND METHODS Received1/31/90:accepted7/17/91. The costs of publication of this article were defrayed in part by the payment Cell Culture and Radioactive Labeling. Human lung adenocarcinoma of page charges. This article must therefore be hereby marked advertisement in cell line A549 and human colon carcinoma cell line HCT116 were accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' Portions of the research presented in this publication were supported by a grown and maintained in McCoy's Tissue Culture Medium 5A (Gibco, grant from the USPHS, National Cancer Institute. Grant CA-40449. Grand Island. NY) containing 10% heat-inactivated fetal bovine serum, 2To whom requests for reprints should be addressed. â€¢¿'Presentaddress: C.R.O.. Via Pedemontana Occ.. 22081 Aviano (PN). Italy. penicillin, and streptomycin and supplemented with pyruvate, essential 4The abbreviations used are: VP-16, 4'-demethylepipodophyllotoxin-4-(4.6- and nonessential amino acids, and vitamins. KB human carcinoma cell O-ethylidene-d-t)-glucopyranoside): VM-26, 4'-demethylepipodophyllotoxin-4- lines KB-3-1 (parental). KB-ChR-8-5, and KB-ChR-8-5-l 1 (kindly pro (4.6-O-thenylidene-fi-n-glucopyranoside); PFM. porfiromycin; ADM. adriamy- vided by M. Gottesman. NIH, Bethesda, MD) were, likewise, main cin; actD, actinomycin D; col, colchicine; MMC, mitomycin C; csPt. c/'i-diami- tained in McCoy's Medium 5A, supplemented as described above. Drug nodichloroplatinum II; P-glycoprotcin, a M, 170,000 glycoprotein associated with the multidrug resistance phenotype; mdrl. the gene encoding the P-glycoprotein resistant cell lines A549(VP)28, A549(VM)28, HCT116(VP)35. and phenotype; cDNA, complementary DNA; NEN, New England Nuclear, Boston. HCT116-(VM)34 were derived from parental cell lines by 1-h exposures MA. to the respective drugs once each week, followed by recovery in drug- 5275

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1991 American Association for Cancer Research. VP-16 AND VM-26 RESISTANT HUMAN CELL LINES free medium. Each cycle was repeated for 29, 34, or 35 weeks, as kyo, Japan) and reactive against an external epitope of P-glycoprotein previously described (22). The generation times for HCT116. in living cells, was used for these studies (35,36). Exponentially growing HCT1 16(VP)35, HCT1 16(VM)34, A549, A549(VP)28, and cells released from flasks with trypsin-EDTA were resuspended at 5 x A549(VM)28 cells were 24, 34, 23, 28, 37, and 30 h, respectively. Cells IO6cells/ml in Dulbecco's phosphate-buffered saline (Gibco) containing intended for alkaline elution experiments were labeled separately for 3 5% fetal bovine serum. A 1:250 dilution (final concentration of 100 days with 0.01 /iCi/ml |'4C]thymidine in 60-cm: plastic culture dishes ng/^l) of MRK16 and 20 ^1 (2 Mg)of antibody were incubated with 0.1 to be used for experimental purposes or with 0.1 /jCi/ml [3H]thymidine ml of cells (5 x IO5 cells) on ice for 30 min. After washing twice in in 75-cm: plastic culture flasks to be used for reference purposes (33). phosphate-buffered saline containing serum, the cells were incubated Cytotoxicity Assay. The cytotoxic effects of VP-16, VM-26, and six on ice for 30 min with 100 n\ of a 1:40 dilution of fluorescein isothio- other anticancer drugs or cytotoxic agents on the parental and acquired cyanate-conjugated goat anti-mouse IgG (F(ab'): fragment) (TAGO resistant cells were determined by assessing inhibition of cell prolifer Inc., Burlingame, CA), then washed and resuspended in 0.5 to 1.0 ml ation in 25-cnr flasks 5 to 7 days following drug treatment. Cells were of McCoy's medium containing 10% fetal bovine serum. Indirect im- incubated with drug for l h followed by incubation in drug-free medium munofluorescence analysis was carried out on a Facstar flow cytometer until the flasks containing untreated cells were 50 to 80% confluent, as (Becton Dickinson Immunocytometry Systems. Mountainview, CA) previously described (21). Following the cell proliferation period, the after counting 10,000 cells. surviving cells were released from flasks with trypsin-EDTA, fixed with Assay for Topoisomerase II Activities. To assay topoisomerase II formaldehyde, and counted by using a Model Zf Coulter Counter activities in the different cell lines, 2 x IO7 log phase cells in 75-cnr (Hialcah, FL). Average counts obtained from duplicate flasks of cells tissue culture flasks were washed twice with ice-cold phosphate-buffered exposed to a given drug concentration were expressed as a percentage saline, then incubated with reticulocyte swelling buffer composed of 5 of the average number of untreated control cells from four flasks for niM KH:PO4 (pH 7.5), 2 mM MgCI:, 0.5 mM dithiothreitol, 0.1 mM each experiment. These percentages were plotted as probit values versus ethyleneglycol bis(ÃŸ-aminoethylether)-A',A;,A7'>A''-tetraacetic acid, 0.1 log of drug concentration to obtain the concentration of drug necessary mM EDTA, 10 mM 2-mercaptoethanol, and 1 mM phenylmethylsulfo- to inhibit cell proliferation by 50%. nyl fluoride on ice for 10 min before scraping off the surface of the Alkaline Million Assays for DNA Breakage in Intact Cells. DNA flasks. The cells were Dounce homogenized with 10 strokes of a tight break production was quantified by standard alkaline elution techniques pestle at 4Â°Cand were subsequently washed 3 times with nuclear wash (10, 11). Logarithmically growing cells, containing [MC]DNA were buffer composed of 5 mM KH2PO4 (pH 7.5), 1 mM ethyleneglycol bis(/3- exposed to various concentrations of VP-16 and VM-26 for 1 h. Cells aminoethyl ether)-A',A',A",A''-tetraacetic acid, 1 mM EDTA, 10 mM 2- were harvested and layered onto polycarbonate filters with reference mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, and 10% glyc- cells containing ['HjDNA and exposed to 300 rads of 7-radiation as an erol on ice. Disruption of the cell membranes was checked by phase- internal elution standard. Cells were lysed with 2% sodium dodecyl contrast microscopy. After washing twice by centrifugation and resus- sulfate, 25 mM EDTA (pH 9.6) containing proteinase K, and the DNA pension in nuclear wash buffer, the nuclei were resuspended in nuclear remaining on the filters was slowly eluted by pumping at 40 Â¿il/min, wash buffer containing 0.5 M NaCl (final concentration). The salt using a buffer composed of tetrapropylammonium hydroxide and extraction was performed for 30 min at 4Â°C.Extracted nuclei and EDTA, which was adjusted to pH 12.1 to reveal both single and double insoluble material were removed by centrifugation at 600 x g for 20 strand DNA breaks. Quantification of DNA breaks was achieved by min, then at 12,000 x g for 10 min at 4T. Protein quantification was relating slopes of DNA elution cunes obtained following drug treat assessed by the method of Bradford (37). ment to those obtained following exposure to different doses of y- The strand-passing activities of topoisomerase II in serially diluted radiation and calculation of true double/single strand break ratio was aliquots of 0.35 M NaCl extracts were monitored by using the P4 DNA achieved, as previously described (3, 13, 21, 34). unknotting assay. Reactions were initiated upon addition of 5 fil of VP-16 and Porfiromycin Influx and Efflux. Cells plated at a density of 5 x IO6cells in each 75-cm: flask on the previous day were incubated each diluted sample to 20 M' of reaction mixture containing 50 mM at 37'C with 5 Â¿IM('HJVP-16or ['Hjporfiromycin (Moravek Biochem- Tris-HCl (pH 7.5), 100 mM NaCl, 10 mM MgCl:, 0.5 mM EDTA, 30 Mg/ml bovine serum albumin, 0.5 mM dithiothreitol. 1 mM ATP, and icals. Brea, CA) for 0 to 30 min. The cells were immediately rinsed 4 2 Â¿ig/mlP4 knotted DNA. After incubation at 37Â°Cfor 30 min, the times with ice-cold phosphate-buffered saline at the end of the incuba reactions were stopped by addition of 5 n\ of stopping buffer consisting tion period, then lysed with 0.1 MNaOH. For efflux studies, cells were of 50% glycerol, 5% sodium dodecyl sulfate, 30 mM EDTA, and 0.25% incubated with radiolabeled drug for 30 min; washed once with warm, drug-free medium; and incubated at 37Â°Cfor 0 to 30 min, then rinsed bromphenol blue. The DNA products were then separated by electrophoresis in 0.9% agarose gels in buffer consisting of 80 mM Tris-HCl and processed as above. Intracellular contents of drug expressed in pmol/lO6 cells were calculated from the specific radioactivities of each (pH 8.0), 40 mM boric acid, and 2 mM EDTA. Photographic negatives drug. Cell volumes were determined by suspending 1 x IO7 cells in from ethidium bromide-stained gels were scanned with an LKB Ultra- warm medium containing 5 pCi of 'H2O and incubating at 37Â°Cfor 30 scan XL densitometer. The linear P4 band provided an arbitrary min, after which 2 ^Ci of [14C)mannitol were added, the cells were separation point between the knotted and the unknotted DNA topoiso- mers. The topoisomerase II activities were calculated as the ratio sedimented by centrifugation, and the pellets were dissolved in 0.1 M NaOH. Radioactivities for AHand '4C were determined by scintillation between the unknotted and the knotted forms after control background subtraction, and 1 unit of enzyme activity was defined as the concen counting. Cell volumes were calculated from the formula: tration capable of unknotting 50% of the P4 knotted DNA in 30 min cell volume/1011 cells under the conditions described above. Slot-Blot Analysis of mdrl and Topoisomerase II nRNA Levels. Both 'H dpm in pellet l4Cdpm in pellet RNA and DNA were extracted from 5 x IO7 cells by using the Total 'H dpm/ml in suspension Total I4Cdpm/ml in suspension guanidinium isothiocyanate method (38). After ultracentrifugation, Total cell number x 10"" RNA pellets were resuspended in 0.3 M sodium acetate, pH 6.0, and precipitated with 70% ethanol. DNA samples in CsCI solution were Intracellular drug concentrations were calculated by dividing the intra- ethanol precipitated then treated with proteinase K. After phenol/ cellular contents by the cell volume (22). chloroform extractions, DNA was recovered by ethanol precipitation. Flow Cytometric Analysis for P-glycoprotein. Flow cytometry was Polyadenylate-containing mRNA was isolated from total RNA ex used to quantify expression of P-glycoprotein on the surface of cells tracted from A549 cells by using a QuickPrep mRNA purification kit reacted first with a mouse monoclonal antibody against P-glycoprotein, (Pharmacia) and was stored as an ethanol precipitate at -70Â°C. then stained with a fluorescent second antibody. Mouse monoclonal The 1.2-kilobase clone pCA12-2 cDNA for human multidrug resist antibody MRK16. generously provided by Dr. T. Tsuruo (Japanese ant gene mdrl was kindly provided by Dr. T. Tsuruo. The cDNA was Foundation for Cancer Research, Kami-Ikebukuro, Toshima-Ku, To labeled by random priming with [Â«-'-PjdATP (3000 Ci/mmol; NEN). 5276

A 21-mcr oligonucleotide probe for the human topoisomerase II gene observed for csPt that accompanied resistance to VP-16 and with the sequence 5'-TCG TGG ACT AGC AGA ATC CTT-3' begin VM-26. A similar observation was made for the inherently VP- ning at nucleotide 2389 of the cDNA sequence described by Tsai- Pflugfelder et al. (39) was synthesized and end labeled with [7-<2P]- 16 resistant human small cell lung carcinoma cell line SW900, which is hypersensitive to MMC and csPt due to less efficient dATP (6000 Ci/mmol; NEN), using terminal deoxynucleotidyl trans- ferase (BRL, Bethesda, MD). A 1.8-kilobase cDNA fragment of the 3'- repair of the monoadducts before DNA cross-links were formed (23). The other three acquired resistant cell lines are cross- terminal one-third of human topoisomerase II mRNA (ZII-1.8) cloned into the EcoRl site of Bluescript SK(+) plasmid, generously supplied resistant only to the other demethylepipodophyllotoxin and to by Dr. Leroy Liu, was isolated from the purified plasmid by EcoRl ADM. digestion and purification in 1% SeaKem GTG (FMC Bioproducts. Alkaline Elution Analysis of Topoisomerase II Inhibition. It is Rockland, ME) agarosc gel. This probe was radiolabeled by random well documented that VP-16 and VM-26 are potent inhibitors priming by using the Klenow fragment of DNA polymerase Â«in the of eukaryote topoisomerase II (1-5), resulting in the stabiliza- presence of [

The oligonucleotide probe was evaluated for specificity for human Tablc 1 Comparison of median cytotoxic concentrations of common anticancer topoisomerase II by comparing its ability with that of the cDNA drugs in parental and acquired resistant cell lines fragment to detect the 6.2-kilobase human topoisomerase II mRNA Cells were exposed to different concentrations of drug for l h in tissue culture after electrophoresis in a 1.2% agarose gel containing 1.28% formal flasks before allowing growth to continue for 5 to 9 days in the absence of drug. Median cytotoxic drug concentrations were derived from at least 3 separate dehyde, as described by Henderson et al. (40). Following electropho experiments as described in "Materials and Methods." Each value has a standard resis, the RNA was transferred to Duralose-UV nylon-reinforced nitro deviation of less than 20%. cellulose membranes (Stratagene, La Jolla, CA) by capillary blotting VP-16 VM-26 ADM ActD Colchicine MMC csPt without prior alkaline treatment. RNA was bound to the membranes Cell line (fiM) (fiM) (JJM) (/Â¿g/ml) UJg/ml) (MM) by UV cross-linking by using a Stratalink apparatus (Stratagene). The HCT116HCTI16(VP)35HCTI16(VM)34A549A549(VP)28A549(VM)28108653865670.85.65.30.54.13.40.42.58.60.72.32.30.030.010.550.060.090.030.20.31.30.30.40.31.30.91.21.30.22.610662629 blot to be evaluated by using the oligo probe was incubated in 6 ml of prehybridization solution at 37Â°C,then in 6 ml of hybridization solution containing radiolabeled probe at a final concentration of 5 x 10'' cpm/ ml for 22 h at 37Â°C,asdescribed by Henderson et al. (40). After several washes in 1x SSPE (0.15 Msodium chloride, 0.01 Msodium phosphate, and 0.002 M EDTA) containing 0.5% sodium dodecyl sulfate and 0.1% nonfat dry milk, the blot was washed first at 37Â°Cthen at 42Â°Cfor 30 min in 0.1 x SSPE and 0.5% sodium dodecyl sulfate. Autoradiography was conducted by placing the nitrocellulose sheet between two DuPont Cronex Lightning Plus intensifying screens obtained from Sigma (St. Louis, MO) and Xomat-AR film sheets (Kodak) and exposed at -70Â°C overnight. Slot blots of DNA and RNA in quantities ranging from 5 to 0.5 ^g per slot on nitrocellulose filter membranes were prepared by using a Manifold II Slot Blotter (Schleicher & Schnell. Keene. NH). which were processed according to the manufacturer's protocol. Hybridization conditions were essentially the same for both RNA and DNA blots, depending on the probe used in each case. Filters were prehybridized, hybridized, and washed as described previously (41). This oligo probe identified two EcoRl restriction fragments in digested cellular DNA, following electrophoresis in agarose gels and transfer to nitrocellulose filters. The filters were hybridized overnight at 37Â°Cand washed at 40Â°Cfor 20 min in 0.1 x SSC (75 mM sodium chloride and 7.5 mM 10 20 30 40 sodium citrate). The DNA blots were normalized with a chicken muscle 50 glyceraldehyde-3-phosphate dehydrogenase cDNA probe obtained from Drug Concentration (u.M) Dr. K. Mulder (Bristol-Baylor Laboratory, Baylor College of Medicine, Houston, TX). End-labeled oligodeoxythymidylate20 (Pharmacia, Pis- cataway, NJ) was used to normalize RNA blots (42). Quantification of slot hybridization results was accomplished by densitometric analyses of the autoradiographs by using a Bio-Rad (Rockville, NY) Model 620 video densitometer.

RESULTS Cross-Resistance Properties of Acquired Resistant Human Carcinoma Cell Lines. Table 1 reveals that the four acquired resistant cell lines previously described (22) are 5- to 9-fold less sensitive to VP-16 than the parental lines. This resistance is stable and has not diminished over months of continuous culture. Data reported in Table 1 show that an extensive multidrug resistance profile was observed for only one line, HCT116(VM)34, which was resistant to all of the drugs shown in Table 1 except csPt and M MC. This cell line is 20-fold Drug Concentration (u,M) resistant to ADM, while displaying only a 9-fold resistance to Fig. 1. Total DNA breaks in cells exposed to different concentrations of VP- VM-26, the pressuring agent. One interesting observation re 16 (A) or VM-26 (B). Cells were incubated in cell culture dishes with different concentrations of drug for l h and then processed for alkaline clution at pH 12.1 garding this cross-resistance profile was the increased sensitivity to reveal total cellular DNA breaks, as described in "Materials and Methods." 5277

5-1 to cellular exportation by the transport protein P-glycoprotein (for reviews see Refs. 31, 32, 43, 44) is unknown. Experiments performed on the HCTl 16 parental cell line and its two sublines are illustrated in Fig. 2. When cells were incubated with 5 Â¿IM VP-16 (Fig. 2A) or PFM (Fig. 2Ã„),the drugs rapidly accumu lated in parental cells, reaching plateaus in 10 to 20 min. When the medium-containing drug was exchanged for drug-free me dium, the intracellular concentrations of both VP-16 and PFM rapidly decreased to nearly undetectable levels for VP-16 and to 10% for PFM within 30 min. However, little drug entered the cells upon addition of drug to the medium or exited from the cells at 4Â°Cwhen the cells were loaded with drug at 37Â°C. Similar results were seen by Yallowich and Ross (45, 46). It can be seen from Fig. 3 that both HCT116(VM)34 and HCT116(VP)35 had significantly decreased intracellular accumulations of VP-16, which was especially true of HCTl 16(VM)34 cells, relative to their parental line. However, only the HCT116(VM)34 line displayed a significantly lower accumulation of PFM, a finding consistent with the multidrug resistance properties of this line and the lack of multidrug resistance properties for the HCTl 16(VP)35 cell line (Table 1). Both A549 acquired resistant lines displayed no abnormal accumulations of PFM but A549(VP)29 accumulated signifi cantly less VP-16 than the parental line (Fig. 3).

TIME (MIN) Fig. 2. Intraccllular accumulation and efflux of radiolabeled VP-16 (.â€¢()and porfiromycin (K). C'clls in cell culture flasks were incubated with 5 ^M |'H]VP- 16 or porfiromycin for different times up to 30 min at 37Â°Ctoassess intracellular accumulation, or for 30 min then for different lengths of time in drug-free medium at 37"C to assess efflux of drug from cells. Points, means of 3 separate experi ments. â€¢¿ Â».HCT116; O O. HCTl 16(VM)34: â€¢¿ â€¢¿HCTII6(VP)35 at 37-C. â€¢¿ â€¢¿.HCTl16: O O, HCTl I6(VM)34; â€¢¿ â€¢¿.HCTl16(VP)35 with either influx or efflux conducted at 0 instead of .V7Â°C. tion of the intermediate formed between the enzyme and its DNA substrate (6-9). This stabilized intermediate is observable in intact cells through the use of alkaline elution techniques (1- 3, 5, 13, 21, 34). Using alkaline elution to compare the forma tion of stabilized enzyme-DNA intermediates in acquired resistant cells with those formed in parental HCTl 16 cells, we find that HCT116(VM)34 cells require 40-fold and HCTl 16(VP)35 require 100-fold more VP-16 to produce com parable numbers of breaks (Fig. \A). Similar observations can be made for VM-26, as well (Fig. \B). In addition, nuclei from HCTlia HOT (VP) HCT (VM) HCT116(VP)35 cells produced substantially lower levels of Celi Line DNA breaks when alkaline elution studies were conducted on isolated nuclei incubated with different concentrations of VP-16 VP-16 or VM-26.5 PFM Intracellular Accumulation and Efflux of VP-16 and Porfiro Fig. 3. Intracellular accumulation of radiolabeled VP-16 and porfiromycin. mycin. Decreased drug accumulation may account for the much The levels of intracellular accumulation of [JH]VP-16 and I'Hlporfiromycin in lower levels of topoisomerase II inhibition observed in acquired the different cell lines after a 30-min incubation at 37*C Â»eredetermined as resistant HCTl 16 cells. Therefore, influx and efflux of radio shown in Fig. 2. Values shown for intracellular accumulation concentrations active VP-16 and PFM in the acquired resistant cells were represent mean values obtained from at least 3 separate experiments for each cell line. Significance of differences were determined by using protected Fisher least studied. The MMC analogue PFM, which is readily prepared significant difference one way analysis of the variants. P values for uptake by the in a radioactive form, was used in these studies as an alternative acquired resistant cell line relative to the corresponding parental cell line were calculated and are shown below for each cell line, with * denoting significant anticancer drug with a mechanism of action unrelated to topo differences. HCT(VP) is HCT116(VP)35 (P = 0.002Â«for VP-16 and 0.37 for isomerase II inhibition. The susceptibility of MMC and PFM PFM): HCT(VM) is HCTl 16(VM)34 (P = 0.0003Â«for VP-16 and 0.0042Â«for PFM): A549(VP) is A549(VP)29 (P = 0.014Â«for VP-16 and 0.035 for PFM); ' B. Long, unpublished results. and A549IVM) is A549(VM)29 (P = 0.17 for VP-16 and 0.18 for PFM). 5278

300 300 HCT116 A549

V ""I TT7Ti 10' 10" 10Â° 10' 10' 103 10* FL1 FL1

300 300 HCT116(VP)35 A549(VP)29

Fig. 4. Cellular content of P-glycoprotein. The relative cellular contents of P-glycoprotein were determined by indirect immunolabeling by using MRK16 monoclonal antibody against P- glycoprotein and fluorescein isothiocyanate con jugated anti-mouse IgG (F(ab'); fragment) and cell sorting in a flow cytometer, as described in "Materials and Methods."

TTT7] ÃœB

10Â° 10' 10! IO3 10 102 FL1 FL1

300

Flow Cytometric Analysis for P-glycoprotein, The expression P4 phage DNA into the circular, covalently closed DNA form of P-glycoprotein on the surface of cells was evaluated by using (47). Fig. 5 shows results obtained with nuclear extracts from flow cytometry of indirect immunofluorescent stained cells. In HCT116 parental and acquired resistant cell lines. Undiluted these studies, mouse monoclonal antibody MRK16, which rec extract caused complete conversion of the knotted P4 phage ognizes an external epitope of human P-glycoprotein in living DNA used as substrate to the covalently closed, circular, un- cells, was reacted first with the cells, then stained with a knotted DNA product. Progressive serial dilutions of each fluorescein-conjugated F(ab')2 fragment of goat anti-mouse an extract resulted in the dilution of enzyme activities until less tibody. Both parental lines HCT116 and A549 contain little, if than 50% of the substrate was converted into the unknotted any, P-glycoprotein on the cell surface (Fig. 4). Of the three DNA product. It is clear from Fig. 5 that the level of enzyme acquired resistant cell lines evaluated in this manner, only present in salt extracts of HCT116(VM)34 nuclei was essen HCT116(VM)34 clearly displayed increased amounts of P- tially the same as that obtained from HCT116 nuclei, whereas glycoprotein on the cell surface, whereas both HCT116(VP)35 substantially less enzyme activity was extracted from the nuclei and A549(VP)29 had essentially the same levels of P-glycopro obtained from HCT116(VP)35 cells (Fig. 5). Densitometric tein as their parental lines (Fig. 4). scans of the negatives of photographs taken of the gel shown Topoisomerase II Activities. The activities of topoisomerase in Fig. 5 and of a gel obtained from an experiment comparing II in 0.35 M NaCl extracts of nuclei isolated from parental and the enzyme activities in A549 parental and acquired resistant acquired resistant cell lines were compared by using the topo cell lines (not shown) provides a means of quantifying and isomerase II specific unknotting reaction. In this assay, only comparing enzyme activities in the different cell lines (Table topoisomerase II activity is capable of converting highly knotted 2). As suggested from Fig. 5, salt extracts of nuclei obtained 5279

< HCT116 HCT116Ã•VMI34 HCT116IVPÃŒ35 < UABCDEFGH I JKLMN 0 P Q

Fig. 5. Topoisomerasc II activities in nu clear extracts. Topoisomerasc* II activities in Unknotted DNA- washed nuclei were extracted with 0.5 M NaCI. Serial dilutions of extracts were incubated with 0.5 jig of knotted P4 DNA for 30 min at 37'C. after which the knotted and unknotted forms were separated by agarose gel electrophoresis and then stained with cthidium bromide to visualize the DNA. as described in "Materials Knotted DNA and Methods." A, F, and /., 700 ng; B, G. and A/. 350 ng; (. H, and N. 175 ng; D, I, and O. 88 ng: /:, J, and P. 44 ng; and A'and Q, 22 ng of nuclear protein extracted from the cell lines shown in the figure.

from HCTl 16(VP)35 cells had almost one-half of the topoi- parental line KB-3-1 were evaluated for topoisomerase II and somerase II activity found in extracts from HCTl 16 nuclei. mdrl mRNA levels. The KB-8-5 cell line was reported to Topoisomerase II activities in similar salt extracts of nuclei display enhanced expression without gene amplification and from HCTl 16 and HCTl 16(VP)35 cells normalized for activity the KB-8-5-11 line display enhanced expression of the mdrl were equally sensitive to different concentrations of VP-16 up gene with gene amplification (48). As previously reported, to 200 //M,6indicating that resistance is not due to altered drug KB-8-5-11 cells contained very high levels, KB-8-5 cells con sensitivity. tained intermediate levels, and the parental cell line contained Although salt extracts from A549 nuclei had 3.5 times more barely detectable levels of mdrl mRNA (48). Although activity than HCTl 16 parental cells, the salt extracts obtained HCTl 16(VM)34 cells clearly overexpressed mdrl mRNA, no from nuclei of both A549 acquired resistant cell lines contained amplification of the mdrl gene was detected from DNA slot- only one-fourth as much enzyme activity (Table 2). It is para blot analysis (results not shown). In fact, only KB-8-5-11 cells doxical that HCTl 16 cells have low topoisomerase II activity of all of the cell lines tested contained amplification of the mdrl relative to A549 cells and not be resistant to VP-16 and VM- gene, in agreement with Shen et al. (48). 26. This difference is presently under investigation. Quantification of the differences seen in Fig. 6 by scanning Slot-Blot Analysis of mdrl and Topoisomerase II mRNA these autoradiograms with a densitometer revealed a direct Levels. The observation that 0.35 M NaCI extracts of nuclei correlation between elevated mdrl mRNA levels and P-gly- from acquired resistant cells contain substantially less enzyme coprotein on the cell surface, decreased intracellular drug activity than similar extracts from parental cells could be ac accumulation, and the presence of a multidrug resistance phe- counted for by differences in extractability or differences in notype (Table 3). These properties were observed only of catalytic activities between parental and their respective ac HCT116(VM)34 cells. Likewise, a direct correlation was ob quired resistant cells. Slot-blot analysis of topoisomerase II and served between decreased topoisomerase II mRNA levels and mdrl mRNA levels in parental and acquired resistant cells were enzyme activity in salt extracts of nuclei and cross-resistance conducted to address this question. Although the mdrl cDNA only to topoisomerase II inhibitors (Table 3). The other three probe used in this study has been well characterized (48), the acquired resistant cell lines possessed these properties, in that oligodeoxyribonucleotide probe for human topoisomerase II they were cross-resistant to VP-16, VM-26, and Adriamycin was heretofore undescribed. The specificity of this probe was but were sensitive to actinomycin D and colchicine and dis verified by Northern transfer analysis of mRNA extracted from played varying degrees of collateral hypersensitivities to cis- A549 cells and purified by passage through an oligodeoxythy- platinum. midine column. A single mRNA specie was detected that mi grated in an agarose gel to the same extent (Fig. 6/1) as a band Table 2 Topoisomerase II activities obtained in 0.35 M MaCI extracts of parental identified by a cDNA probe for human topoisomerase II (Fig. and acquired resistant cell lines 6Ã„). Activity is expressed as enzyme units per ng of protein in each extract. A unit Fig. 7 shows that topoisomerase II mRNA levels in of activity is defined as that activity capable of converting 0.5 Â¿igofknotted P4 DNA to the unknotted form in 30 min at 37Â°Cunder optimal conditions. HCT116(VP)35, A549(VM)29, and A549(VP)29 cells were lower than in the respective parental cells. When the same Activity mRNAs on the filter were evaluated with a DNA probe recog Cell line (units/jig) nizing mdrl gene expression, only HCT116(VM)34 revealed HCTl 16 14 HCT116(VP)35 8 increased levels of mdrl mRNA (Fig. 7). For comparison HCT116(VM)34 15 purposes, RNA extracted from human KB cells displaying A549 50 different degrees of acquired resistance to colchicine and the A549(VP)28 II ' R. Woessner, unpublished results. A549(VM)28 12 5280

1234 5678 DISCUSSION Two primary and well-documented mechanisms have been ORIGIN - described by which cancer cells resist the cytotoxic effects of the anticancer drug VP-16. The initial work from the labora tories of Biedler and Ling revealed that cells resistant to high concentrations of one cytotoxic agent were generally highly resistant to many structurally different natural product cytotox- ins due to the presence of a specific membrane protein (49, 50). This extensively studied drug resistance mechanism is charac terized by decreased intracellular accumulation of drug facili tated by overexpression of the human mdrl gene, causing overproduction of P-glycoprotein. This cell membrane protein acts as an export pump for a wide variety of unrelated foreign natural products (for reviews see Refs. 31, 32, 43, 44). By maintaining lower intracellular levels of drug, less drug would be available to the target, which has been shown to be topoisom- 3.5 Kb - erase II (1-5). A second mechanism has come to light recently, which relates directly to the target enzyme, namely that either low enzyme levels (24-26) or altered sensitivity of the enzyme 1.7 Kb - for the drug (27-29) confer resistance to that drug. This mech anism also confers a form of multidrug resistance, in that resistance to one topoisomerase II inhibitor through decreased or altered topoisomerase activity generally translates into re sistance to most other topoisomerase II inhibitors (51). Of the four acquired resistant cell lines described here, three are resistant to VP-16 and VM-26 by a decreased topoisomerase II expression mechanism without any involvement of an mdrl- related mechanism of resistance and one line is resistant to VP- 16 and VM-26 in part, if not exclusively, through an mdrJ,

topoll mdrl

Fig. 6. Northern blot analysis of mRNA isolated from A549 cells examined HCT116 with a radiolabcled oligonucleotide probe (A) or a cDNA probe (B). I.ane 1, 10 ng of total cellular RNA; Lane 2, 2 >ig; Lane 3, 4 fig; and Lane 4. 8 n% of polyadenylated RNA. RNA aliquots for A and B were subjected to electrophoresis HCT116(VP)35 in the same agarose gel. After transfer of RNA to nitrocellulose membranes by HCT116(VM)34 â€”¿ _ capillary blotting, the membrane was cut in half and probed separately, as described in "Materials and Methods." Kb, kilobase.

MOSER â€”¿ â€”¿ Table 3 Relative levels of genes, gene expression, protein levels, and enzyme activities in parental and acquired resistant cell lines VACOS â€”¿ Values represent ratios of resistant cell contents or activities over those of the respective parental cell. ND, no data; >, elevated levels of protein; <, decreased levels of protein, and =, ratios deviating less than I0"Ãfrom l.O. mdrl topoll CelllineHCT116HCT116(VM)34HCT116(VP)35A549A549(VM)29A549(VP)29DNARNA9= RNA> DNA A549 A549(VP)29 = 1.40.70.40.5Protein=

== =Protein KB3-1 KB8-5 Therefore, three of the four acquired resistant cell lines we KB8-5-11 â€”¿ ^ have studied are resistant to VP-16 and VM-26 in part, if not exclusively, by a decreased topoisomerase II expression mech Fig. 7. Composite presentation of slot-hot analysis of topoisomerase II and anism without any involvement of an wi/r7-related mechanism muri gene expression. Total RNA was extracted from cells and 0.5 to 5.0 Mgof RNA were placed in each slot over a nitrocellulose filter membrane. After blotting of resistance. Conversely, the HCT116(VM)34 line is resistant and drying, slots on the filter were analyzed for topoisomerase II. mdrl. and total to VP-16 and VM-26 in part, if not exclusively through an polyadenylatc-containing mRNA levels using <;P-labeled oligonucleotide. cDNA, and oligodeoxythymidine probes, respectively. X-ray films were exposed to the mdrl mechanism reflecting decreased intracellular drug accu washed and dried filter to obtain autoradiograms. See "Materials and Methods" mulation, rather than a topoisomerase II related mechanism. for further details. 5281

rather than a topoisomerase 11related mechanism. It should be We are presently utilizing these resistant cell lines as solid noted that efforts were taken to develop low levels of resistance tumor model systems for the evaluation of VP-16 analogues in so as to limit the number of mechanisms of resistance function vitro and in vivo, both as subrenal capsule and as s.c. implants ing in a given cell line. Furthermore, the resistance described (65). for these acquired resistant sublines is stable and has not diminished substantially over 1 year in culture. Therefore, it is possible to make some generalizations about cross-resistance ACKNOWLEDGMENTS properties displayed by the two different mechanisms. The excellent technical assistance of Steve T. Musial. Patrick O'Mal- First, the Wr/-resistant cell subline HCT116(VM)34, which ley, and Laura A. Newhouse is gratefully acknowledged. is 5- to 6-fold resistant to VP-16 and VM-26, is cross-resistant to ADM, actD, and colchicine but not to MMC or csPt (Table REFERENCES 1). It is well recognized that elevated P-glycoprotein does not confer resistance to csPt, but the lack of resistance to MMC 1. Long. B. H.. and Minocha, A. Inhibition of topoisomerase II by VPI6-213 cannot be explained in light of the observation of substantially (etoposide), VM26 (tcniposide). and structural congeners as an explanation for in rivo DNA breakage and cytotoxicity. Proc. Am. Assoc. Cancer Res., decreased accumulation of PFM by these cells (Figs. 2 and 3). 2*321, 1983. The higher resistance to ADM, actD, and colchicine (21-, 18-, 2. Minocha. A., and Long, B. H. Inhibition of the DNA catenation activity of type II topoisomerase by VPI6-123 and VM26. Biochem. Biophys. Res. and 7-fold, respectively) relative to resistance to VP-16 and Commun.. 122: 165-170, 1984. 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Byron H. Long, Lotte Wang, Aurelio Lorico, et al.

Cancer Res 1991;51:5275-5283.

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