In Vitro Prevention of the Emergence of Multidrug Resistance in a Pediatric Rhabdomyosarcoma Cell Line1

Vol. 7, 3193–3198, October 2001 Clinical Cancer Research 3193

Hilary A. Cocker, Nicki Tiffin, modulators of MDR and merit clinical studies to test this Kathy Pritchard-Jones, C. Ross Pinkerton, and hypothesis. Lloyd R. Kelland2 INTRODUCTION CRC Centre for Cancer Therapeutics, The Institute of Cancer Research [H. A. C., L. R. K.], and Section of Paediatrics, The Institute RMS is a highly malignant tumor which accounts for over of Cancer Research/Royal Marsden NHS Trust [N. T., K. P-J., half of all soft tissue sarcomas in children (1). The disease is C. R. P.], Sutton, Surrey SM2 5NG, United Kingdom commonly treated using chemotherapeutic agents, including Vinca alkaloids, anthracyclines, etoposide, and cyclophospha- mide. Initial treatment is often successful, but relapse because of ABSTRACT drug resistance remains a major obstacle to survival. In some, We have established preclinical models for the devel- but not all, studies, the expression of P-gp,3 encoded by the opment of drug resistance to vincristine (a major drug used mdr-1 gene, has been associated with adverse prognosis (2, 3). in the treatment of pediatric rhabdomyosarcoma) using cell Our recent in vitro studies have indicated that relatively low lines. The RD cell line has a mutant P53 phenotype and does expression of P-gp and MRP may contribute to resistance in not have detectable P-glycoprotein (P-gp) or multidrug re- RMS (4). sistance-related protein (MRP) despite expressing low levels Historically, studies of acquired resistance have involved of mdr-1 mRNA, which encodes P-gp and mrp1 mRNA. the development of a resistant cell line in vitro after exposure to Resistant variants of RD were derived by exposure to in- increasing drug concentrations (5, 6). When used with MDR (2) creasing concentrations of vincristine. This was repeated on efflux substrates, this method has probably led to a dispropor- six occasions, resulting in three cell lines which could toler- tionate number of resistant cell lines which express P-gp or ؋ ate 64 the IC50 concentration. Six independent agents MRP1, as this appears to be the most common mechanism of were tested for their ability to prevent the development of resistance. Evidence that this is also true in the clinic is limited, resistance in this model. Despite at least 10 attempts, resist- and clones surviving a single high dose of drug frequently show ance did not develop in the presence of the multidrug resist- other mechanisms of resistance (7). The long-term exposure ance (MDR) modulators PSC833, VX710, and XR9576. This method has also led to cell lines which overexpress very high strongly suggests that these agents may delay or even pre- levels of P-gp and MRP1, and this degree of resistance is rarely vent the development of resistance to vincristine. This was seen in clinical samples. Despite these caveats, cell lines over- also confirmed in a second rhabdomyosarcoma cell line, expressing P-gp and MRP1 have been extremely useful in Rh30. In contrast, the agents indomethacin (MRP1 modu- understanding the biology of drug resistance. lator), CGP41251 (protein kinase C inhibitor), and dexra- Expression of mdr-1 may be rapidly up-regulated in re- zoxane (putative MDR prevention agent) did not affect the sponse to chemotherapeutic agents both in vitro (8–10) and in development of resistance in the RD model. Characteriza- vivo (11). These observations may explain the lack of correlation of the resistant cell lines indicated the presence of tion between P-gp expression at the time of diagnosis (before increased mdr-1 and P-gp expression, which resulted in treatment) and eventual outcome in many studies. resistance to the agents doxorubicin, etoposide, and vincris- At least three modulators of MDR, and P-gp in particular, tine but not cisplatin. The resistance could be modulated are currently undergoing clinical trials. They include agents using PSC833 or VX710, confirming that functional P-gp is such as the nonimmunosuppressive cyclosporin A analogue, present. No apparent differences were seen between the PSC833 (Valspodar; Ref. 12), the nonmacrocyclic pipecolinate resistant cell lines derived in the absence and presence of derivative VX710 (Biricodar; Ref. 13), and the novel compound the various agents. These experiments strongly suggest XR9576 (14). These compounds are usually administered after that the development of MDR may be preventable using relapse, and their effectiveness in the clinic to date is debatable. An interesting therapeutic strategy is to try to prevent the development of MDR by treating with an MDR modulator at the onset of therapy. If resistance is intrinsic, the modulator sensi- Received 4/24/01; revised 7/20/01; accepted 7/23/01. tizes the tumor, whereas if resistance is acquired, multidrug- The costs of publication of this article were defrayed in part by the resistant cells no longer have a selective advantage. However, payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by an Institute of Cancer Research studentship. 2 To whom requests for reprints should be addressed, at CRC Center for 3 The abbreviations used are: P-gp, P-glycoprotein; RMS, rhabdomyo- Cancer Therapeutics, The Institute of Cancer Research, Cotswold Road, sarcoma; PKC, protein kinase C; MRP, multidrug resistance protein; Sutton, Surrey SM2 5NG, United Kingdom. Phone: (44) (20) 87224301; MDR, multidrug resistance; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5- Fax: (44) (20) 86421140; E-mail: [email protected]. diphenyltetrazolium bromide.

there are several reasons why this strategy may fail. The tumor land) and indomethacin (Sigma Chemical Co.) were dissolved in may initially possess multiple mechanisms of resistance, or absolute ethanol to give stock solutions of 1 and 50 mM, respec- alternative mechanisms of resistance may arise as frequently tively. Dexrazoxane (Chiron, Harefield, United Kingdom) was Ϫ and as quickly as MDR. Despite this, there may be a subpopu- dissolved in water to give a stock solution of 20 mg ml 1. lation of tumors for which this strategy is beneficial and results XR9576 (Xenova, Slough, United Kingdom) and CGP41251 in longer disease-free survival or even a cure. There is limited (Ciba Geigy, Basle, Switzerland) were dissolved in DMSO to clinical evidence that this is the case (15). give stock solutions of 5 and 1 mM, respectively. All these drugs This study has developed an in vitro model for the devel- were stored at Ϫ20°C, with the exception of vincristine, which opment of resistance to vincristine in pediatric RMS. Vincristine was stored at 4°C. Immediately before use, the drugs were was selected because it is widely used in treatment and is one of diluted with RPMI 1640 prepared as for cell culture, with the the most effective agents. This model has been used to test the exception of vincristine, which was diluted in RPMI 1640 ability of six agents to prevent resistance, including the modulators without any supplements. PSC833, VX710, and XR9576. In addition, the MRP1 modula- MTT (Sigma Chemical Co.) was dissolved in PBS to Ϫ1 tor indomethacin (16) was included, and because of the postu- produce a stock solution of 5 mg ml , which was stored at 4°C. 6 lated role of PKC in MDR (17), the inhibitor CGP41251 was Development of Resistant Cell Lines. Cells (1 ϫ 10 ) 2 also included (18). Finally, a recent study suggested that dexra- were seeded in a 75-cm tissue culture flask. After 24 h, vin- zoxane, a compound used to minimize the cardiotoxicity of cristine was added at the IC50 concentration (10 nM, as deter- doxorubicin, may also prevent the development of MDR in vitro mined using the MTT assay; see below). The growth medium (19), and this compound has also been tested. and vincristine were replaced weekly if necessary. When the These studies have been used to investigate whether these cells grew to confluence, they were harvested by trypsinization, 6 agents prevent or delay development of MDR and whether 1 ϫ 10 cells were reseeded, and the vincristine dose was alternative mechanisms of resistance arise. Previous studies doubled. In this manner, cell lines were developed, which were ϫ have shown that resistance can arise in the presence of modu- tolerant of 64 the IC50 concentration. These cell lines were lators (20, 21), so this study investigates how frequently this grown in drug-free medium for 1 week before experiments. occurs and whether alternative mechanisms of resistance de- Various agents were tested for prevention of development velop as quickly. of resistance. The growth of cell lines in the presence of these agents was monitored for 4 weeks to ensure no effects on growth when used alone. The concentrations used were 2 ␮M MATERIALS AND METHODS PSC833, 2 ␮M VX710, 10 ␮M indomethacin, 2 ␮M XR9576, 10 Cell Lines. This study uses the cell lines RD and Rh30, nM CGP41251, and 200 nM dexrazoxane. These agents were which have been described previously (4). The cell line RD was diluted in tissue culture medium and added to the cells with the obtained from the American Type Culture Collection (Manas- vincristine. sas, VA), and Rh30 was a gift from Dr. P. J. Houghton (St. Real-time PCR. Relative expression of the mdr-1 and Jude’s Children’s Research Hospital). The human ovarian car- mrp1 genes was measured using TaqMan Real-time PCR (PE cinoma cell lines CH1 and its multidrug-resistant variant Biosystems, Foster City, CA), using the Applied Biosystems CH1DoxR were used as negative and positive controls for Prism 7700 sequence detection system. Total RNA (1 ␮g) was mdr-1 and P-gp expression (22). The human non-small cell lung reverse transcribed in a 20-␮l volume using Superscript reverse carcinoma cell line CORL23 and its multidrug-resistant variant transcriptase (Life Technologies, Inc.) and random hexanucle- CORL23/R were used as negative and positive controls for otides (Amersham Pharmacia Biotech United Kingdom, Ltd., mrp1 expression (23). Bucks, United Kingdom), according to the manufacturer’s in- All cell lines were maintained in RPMI 1640 (Sigma structions. Real-time PCR for mdr-1 was performed in a final Chemical Co., Poole, Dorset, United Kingdom) supplemented volume of 25 ␮l containing 1 ϫ TaqMan Universal PCR Master with 10% fetal bovine serum (Life Technologies, Inc., Scotland, Mix (PE Biosystems), 900 nM forward primer (5Ј-AAAAGT- United Kingdom) and 2 mML-glutamine (Sigma Chemical Co.). GAAAAAGATAAGAAGGAAAAGAAA-3Ј), 900 nM reverse Cells were grown as attached monolayers and were incubated at primer (5Ј-CACCATATACAACTTGTCAAGCCAA-3Ј), and Ј 37°C in a humidified atmosphere with 5% CO2. All cell lines 150 nM FAM/TAMRA dual-labeled oligonucleotide probe (5 - were routinely screened for Mycoplasma by PCR assay (Strat- FAM-TTGAATAGCGAAACATTGAAAATACACTGACA- agene, Cambridge, United Kingdom). GTTG-TAMRA-3Ј). Real-time PCR for mrp1 was performed in Drugs and Chemicals. Vincristine (David Bull Labora- a final volume of 25 ␮l containing 1 ϫ TaqMan Universal PCR tories, Dublin, Ireland) and Etoposide (Bristol Myers Squibb Master Mix, 300 nM forward primer (5Ј-AGATGACACCTCT- Pharmaceuticals, Hounslow, United Kingdom) stock solutions CAACAAAACCA-3Ј), 300 nM reverse primer (5Ј-AGGTCTGC- were obtained as pharmacy preparations at concentrations of CCAGCAGACG-3Ј), and 50 nM FAM/TAMRA dual-labeled 1.083 and 34 mM, respectively. Doxorubicin (Sigma Chemical oligonucleotide probe (5Ј-FAM-AACTGCCTTGGGATTTTT- Co.) was dissolved in water to give a stock solution of 1 mM. GCTGTGGA-TAMRA-3Ј). Both primer and probe sets ampli- Cisplatin (Johnson Matthey Technology Center, Reading, fied across an exon/exon boundary to prevent amplification of United Kingdom) was dissolved in 0.9% saline to give a stock genomic DNA. cDNA (0.1 ␮l) was used in each reaction. To solution of 1 mM. VX710 (Vertex Pharmaceuticals, Inc., Cam- measure the total amount of sample RNA present, levels of 18S bridge, MA) was dissolved in saline to give a 50 mM stock rRNA were quantitated in each reaction using TaqMan rRNA solution. PSC833 (Novartis Pharmaceuticals, Basel, Switzer- Control Reagents with a VIC/TAMRA oligonucleotide probe

Table 1 Attempts to derive vincristine-resistant derivatives of the cell line RD in the absence and presence of various agents. Duration of attempt represents the time between seeding the first flask and discarding the last flask No. of Average duration of No. of resistant Agent present attempts attempts (days) cell lines obtained None 6 52 3 PSC833 25 34 0 VX710 18 45 0 XR9576 11 17 0 CGP41251 5 47 1 Indomethacin 4 31 1 Dexrazoxane 2 43 1 Fig. 1 Time course of development of resistance in the RD cell line the point at which a ,ء .(repeated in three independent experiments (1–3 subline was derived. VCR tolerated, the concentration of vincristine in the growth medium. (PE Biosystems). Relative expression of mdr-1 and mrp1 was calculated from standard curves generated from cell lines CH1DoxR and CORL23/R, respectively. Immunoblotting. Immunoblotting was carried out as described previously (4). The antibody C219 (Centocor Diagnos- tics, Malvern, PA) was used to detect P-gp, the antibody MRPm6 (Monosan, Uden, the Netherlands) was used to detect MRP1, and B-1-5-2 (Sigma Chemical Co.) was used to detect ␣-tubulin to ensure equal loading of wells in all experiments. Growth Inhibition Assay. The MTT assay (24), using 4 days continuous exposure, was used to measure growth inhibition, as described previously (4). The effects of all of the modulators PSC833 and VX710 on growth inhibition were also tested by this method. The modulators were added immediately before addition of the cytotoxic agent at a concentration which kills Ͻ10% of cells (2 ␮M). Statistical Analysis. Where appropriate, statistical sig- nificance was tested using a two-tailed Student’s t test.

RESULTS The first aim of this study was to develop an in vitro model for the development of resistance to vincristine in RMS. Previ- ous publications have suggested that expression of mdr-1 may be rapidly expressed after exposure to chemotherapeutic agents (8–10). Despite this, we did not observe rapid (Յ24 h) up- regulation of mdr-1 or mrp1 after treatment with vincristine in three RMS cell lines: RD, SCMC, and Rh30 (data not shown). The cell line RD was used to produce a model for the development of resistance to vincristine after long-term expo- Fig. 2 A, time course of development of resistance to VCR in the RD sure. This cell line has a mutation in the P53 gene, which leads cell line in the absence (Ⅺ) or presence of 2 ␮M PSC833 (F), 2 ␮M Œ ␮ ࡗ to a loss of function (4). It expresses low levels of the mdr-1 and VX710 ( ), or 2 M XR9576 ( ). The attempt with the longest duration is shown. B, time course of development of resistance to VCR mrp1 genes, but the P-gp and MRP1 proteins are not detectable in the RD cell line in the absence (Ⅺ) or presence of 10 nM CGP41251 by immunoblotting. Exposure to vincristine was carried out (Œ), 10 ␮M indomethacin (F), or 200 nM dexrazoxane (ࡗ). according to a set protocol. Three independent experiments were established simulta- neously, and this resulted in three resistant cell lines from a total of six attempts (Table 1). Resistance developed rapidly on all assay. In the presence of the MDR modulators PSC833, VX710, three occasions (Fig. 1), and the resulting cell lines are desig- and XR9576, resistance did not develop despite at least 10 nated RD(1), RD(2), and RD(3). Resistance appeared to stabi- attempts (Table 1 and Fig. 2A). In contrast, the use of the agents lize over a period of Ն3 months; the lines were not maintained indomethacin (MRP1 modulator), CGP41251 (PKC inhibitor), under selective pressure. and dexrazoxane (putative MDR prevention agent) did not pre- This model was then used to test various agents for their vent the development of resistance to vincristine. This occurred ability to prevent the development of resistance. All agents were after comparatively few attempts (Table 1) and developed as used at concentrations which are nontoxic in a 28-day growth rapidly as in the absence of any agents (Fig. 2B).

doxorubicin, etoposide, and vincristine was significantly re- duced in all of the resistant cell lines (Fig. 4, B–D, respectively). This strongly suggests that the increased expression of P-gp is responsible for the functional resistance. To confirm this, the P-gp modulators PSC833 and VX710 were tested in the resistant cell lines. The sensitivity of all of the resistant cell lines to vincristine could be modulated between 85-fold and 1191-fold using these agents, confirming that P-gp is the major mechanism of resistance (Fig. 4E). A second RMS cell line, Rh30, was used to test the hypothesis that P-gp modulators may prevent the development of resistance. The first attempt to develop a vincristine-resistant variant of Rh30 led to a resistant cell line (Fig. 5). In contrast, vincristine-resistant cell lines did not develop in the presence of PSC83 or VX710 despite six and four attempts, respectively.

DISCUSSION The mechanisms by which RMS cells become resistant to chemotherapeutic agents are relatively unclear. Recent studies have indicated that rapid up-regulation (Յ24 h) of mdr-1 after doxorubicin treatment may contribute to resistance both in vitro (10) and in vivo (11). This hypothesis was tested using three RMS cell lines with varying vincristine treatment dose and duration, but up-regulation of mdr-1 or mrp1 was not found. The cell line RD was then used to test whether exposure to vincristine for at least three weeks would lead to drug resistance. The

first dose given to the cells was the IC50 concentration (10 nM), ϫ Fig. 3 Real-time PCR (A) for expression of mdr-1 gene (open bars) and this was doubled until 64 IC50 was reached. In this and mrp1 gene (solid bars) and immunoblotting (B) for P-gp and MRP1 experiment, resistance to vincristine developed rapidly on three in resistant sublines of RD derived in the absence or presence of 10 nM independent occasions. CGP41251, 10 ␮M indomethacin, or 200 nM dexrazoxane. mdr-1 ex- The use of modulating agents in the clinic is usually limited pression is relative to CH1DoxR, and mrp1 expression is relative to to patients who have relapsed after prior chemotherapy. In these CORL23/R. Values are the mean Ϯ SD of three independent experi- -significantly different from parent line. Positive controls for patients the tumor may already have acquired diverse mecha ,ء .ments expression were included in each experiment. nisms of resistance after exposure to cytotoxic drugs. A strategy to overcome this problem is to use modulators at the onset of treatment to prevent the survival of a multidrug-resistant clone. However, it is unclear how frequently, and how quickly, alter- These experiments led to the development of six resistant native mechanisms of resistance would arise. Previous studies cell lines which could tolerate 640 nM vincristine in the growth have reported that it is possible to develop resistant cell lines in medium. Three cell lines were derived in the absence of any the presence of modulators (20, 21) but do not describe whether agents, and three were derived in the presence of indomethacin, alternative mechanisms of resistance were readily expressed. CGP41251, or dexrazoxane. These cell lines were characterized To test this in vitro, RD was exposed to vincristine as to determine the mechanisms of resistance. All of the resistant before but in the presence of either PSC833, VX710, or cell lines expressed significantly higher levels of mdr-1 than the XR9576. Despite at least 10 attempts, in each case no resistant parent cell line (Fig. 3A). Three resistant cell lines expressed cell lines developed in the presence of these agents. This is significantly lower mrp1 than the parent line, but the fold probably because the main mechanism of vincristine resistance change was small (ϳ2-fold) and probably reflects the selection in these lines is P-gp overexpression. However, it is interesting of a clonal population. The increased expression of mdr-1 leads that alternative mechanisms of resistance did not develop, and if to overexpression of P-gp in all six resistant lines, but no this were true in the clinic, it would represent an increase in the changes in MRP1 expression were observed (Fig. 3B). The total time to relapse and possibly cure rate. We have also confirmed PKC activity, which may be linked to MDR, remained un- these observations in a second RMS cell line, Rh30. changed (data not shown). Three agents, which are not conventional P-gp modulators, Growth inhibition assays were performed to measure the were also tested. These were CGP41251 (PKC inhibitor), indo- sensitivity of the resistant cell lines to a variety of chemother- methacin (MRP1 modulator), and dexrazoxane, which is nor- apeutic agents. Changes in sensitivity to cisplatin, which is not mally used to prevent the cardiotoxicity of doxorubicin but a P-gp substrate, were comparatively small and showed no which may also prevent resistance (19). Dexrazoxane was used consistent patterns (Fig. 4A), suggesting they are attributable to at the same concentration as in previous studies but was added clonal variation. In contrast, sensitivity to the MDR substrates weekly rather than every 3 days, according to the standard

Fig. 4 Sensitivity of RD and its vincristine-resistant variants derived in the absence or presence of 10 nM CGP41251, 10 ␮M indomethacin, or 200 nM dexrazoxane to cisplatin (A), doxorubicin (B), etoposide (C), and vincristine (D). IC50 values given are the mean ϮSD from three independent experiments. significantly different from ,ء parental cell line. E, modulation of sensitivity to vincristine in RD and its vincristine-resistant variants derived in the absence or presence of 10 nM CGP41251, 10 ␮M indomethacin, or 200 nM dexrazoxane using 2 ␮M PSC833 (open bars)or2␮M VX710 (solid bars). Values given are the mean Ϯ SD of three independent experiments. Sensitization ratio ϭ (S.R.) IC50 in the absence of modulator:IC50 in the presence of modulator.

protocol. None of these agents prevented the development of cisplatin, which is not a substrate for P-gp. In all cases, the resistance to vincristine, and resistance developed as quickly as sensitivity to vincristine could be modulated using PSC833 and in the absence of any agents. This indicates that none of the VX710, confirming the presence of functional P-gp. There were pathways affected by these agents (e.g., PKC and MRP1) are no apparent differences between cell lines developed in the involved in the development of resistance in this cell line, presence and absence of various agents, confirming that these although dexrazoxane may be effective if added more fre- agents do not affect the development of resistance. quently. It also indicates that the ability to prevent resistance is In conclusion, these studies demonstrate that in vitro re- specific to modulators of P-gp and not a general finding. sistance to vincristine does not develop rapidly in RMS cell The resistant cell lines resulting from these experiments lines but emerges after a long continuous exposure. The devel- were characterized, and all were found to express mdr-1 and opment of resistance can be prevented using modulating agents, P-gp at higher levels than the parental cell line. This led to such as PSC833, VX710, and XR9576, but not agents which resistance to vincristine, etoposide, and doxorubicin but not affect other pathways, such as indomethacin and CGP41251.

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Hilary A. Cocker, Nicki Tiffin, Kathy Pritchard-Jones, et al.

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