Therapeutic Analysis of Melphalan-Resistant Human Rhabdomyosarcoma Xenograft TE-671 MR1

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(CANCER RESEARCH 51, 3906-.1909. Augusl I. 1991] Therapeutic Analysis of Melphalan-resistant Human Rhabdomyosarcoma Xenograft TE-671 MR1

Eileen R. Lilley, Gertrude B. Elion, Mark W. Dewhirst, S. Clifford Schold, Jr., M. Robert Blum, Paul M. Savina, Daniel T. Laskowitz, Darell D. Bigner, and Henry S. Friedman2 Departments of Pediatrics fE. R. L.. D. T. L., H. S. F.], Medicine Â¡G.B. E., S. C. S.], Radiology IM. H'. lÃ¬.],andPathology [S. C. S., D. D. B., H. S. F.], and the Preuss Laboratory for Brain Tumor Research Â¡D. D. B.]. Duke University Medical Center. Durham, North Carolina 27710; and Burroughs H eliconie Company Research Triangle Park. North Carolina 27709 [M. R. B.. P. M. S.J

ABSTRACT of TE-671 MR to melphalan and the modulations effective in Investigations with the melphalan-resistant human rhabdomyosarcoma overcoming this resistance. In addition to the determination of the sensitivity of TE-671 MR to a variety of agents in vivo, the xenograft TE-671 MR were carried out to identify patterns of cross- resistant tumor was compared with the sensitive line TE-671 resistance and collateral sensitivity and to define the mechanism(s) with respect to O6-alkylguanine-DNA alkyltransferase levels, mediating melphalan resistance. TE-671 MR was cross-resistant to thio- TEPA, mitomycin, vincristine, and cisplatin, and partially resistant to tumor oxygÃ©nationlevels, and ability of the tumor to transport chlorambucil and cyclophosphamide. TE-671 MR and the parent line and retain melphalan. Since combinations of melphalan and TE-671 were both resistant to l,3-bis(2-chloroethyl)-nitrosourea and VP-16 had shown synergistic activity in the TE-671 line, this expressed similar levels of O6-alkylguanine-DNA alkyltransferase. TE- combination was also examined in the resistant line. The data 671 MR retained full sensitivity to actinomycin D and demonstrated suggest that altered melphalan delivery or efflux may be a enhanced sensitivity to VP-16 compared to TE-671. Treatment of TE- contributing factor to resistance to the drug. 671 MR with melphalan plus VP-16 resulted in greater than additive growth delays. The frequency of hypoxic regions was similar in TE-671 MR and TE-671, respectively. Measurement of tumor-to-plasma levels MATERIALS AND METHODS at 180 min following i.p. administration of melphalan at 0.5 of the 10% lethal dosage showed mean tumor-to-plasma ratios of 3.81 in TE-671 Animals. Male or female athymic BALB/c mice (nu/nu genotype, 6 MR and 7.38 in TE-671, respectively. The lower drug levels in TE-671 weeks or older) were used for all studies and were maintained as MR may be contributing to the resistance to melphalan and thus indicate previously described (5). the need for further studies to define the reasons for these differences in Xenograft Transplantation and Tumor Lines. TE-671, a subline of the tumor drug level. human rhabdomyosarcoma-derived continuous cell line RD, and the melphalan-resistant line TE-671 MR, growing as s.c. xenografts, were used for all studies. The establishment and characterization of TE-671 INTRODUCTION and TE-671 MR have been described previously (4, 6-8). Xenograft The development of drug resistance with subsequent treat transplantation s.c. was performed as previously described with inocu lation volumes of 30 ^1 (9). ment failure is a major obstacle to the successful treatment of Tumor Measurements. Tumors were measured every 3-4 days with cancer. Selection of new therapies effective in bypassing or vernier calipers (Scientific Products, McGaw Park, IL) as previously overcoming clinically relevant drug resistance may be facilitated described (9). by identification of patterns of cross-resistance and collateral Drugs for Therapy Studies and Drug Toxicity. All drugs were pur sensitivity and by definition of mechanisms mediating drug chased from commercial sources except chlorambucil and melphalan, resistance (1). Identification of cross-resistance and collateral which were provided by Burroughs Wellcome, Research Triangle Park, sensitivity allows the use of agents to which the tumor is still NC, and bleomycin, which was provided by Bristol-Myers, Wallingford, sensitive, while avoiding those drugs to which the tumor has CT. The lethal toxicity of individual drugs was assessed by probit developed cross-resistance. Definition of mechanisms mediat analysis (10). A minimum of 4 dose levels with 10 animals/dose was used to calculate the LDm1 of each drug. The LD,o for the individual ing drug resistance may allow for modulations effective in drugs was found to be as follows: actinomycin D, 0.5 mg/m2; BCNU, overcoming resistance. Despite the activity of bifunctional al- 100 mg/m2; bleomycin, 330 mg/m2; chlorambucil, 130 mg/m2; cispla kylating agents against a broad spectrum of human neoplasms, tin, 32.5 mg/m2; cyclophosphamide, 1391 mg/m2; ifosfamide, 2015 resistance to these agents frequently develops, with subsequent mg/m2; melphalan, 71.3 mg/m2; mitomycin, 15.7 mg/m2; thio-TEPA, relapse and death. The majority of laboratory models of al- 61.8 mg/m2; vincristine, 9.8 mg/m2; and VP-16, 170 mg/m2. The dose kylator resistance have resulted from in vitro exposure of cell administered in each single-agent experiment was 100% of the calcu lines to these agents (2). However, drug resistance may develop lated LD,O. In combination studies with VP-16 and melphalan, the through mechanisms that are only operational in vivo (3). The doses used were 0.50 LD10of VP-16 with varying fractions (0.10, 0.25, establishment of a human rhabdomyosarcoma xenograft, TE- 0.375, 0.50, and 0.75) of the LD,o of melphalan. Drugs were given as 671 MR, with melphalan resistance generated in vivo, has a single dose on Day 1 except for daily doses of actinomycin D, on provided a model for the therapeutic analysis of melphalan Days 1-5; VP-16 on Days 1, 4, and 7; and bleomycin on Days 1, 4, resistance in a human neoplasm (4). and 7. When used in combination with VP-16, melphalan was given on Day 1 and VP-16 was given on Days 1, 4, and 7. Drugs were adminis The current studies were conducted to define the response of tered by i.p. injection in a volume of 90 ml/m2. All drugs were dissolved TE-671 MR to a broad spectrum of chemotherapeutic agents in 0.9% saline except melphalan, which was dissolved in 17% dimethyl and to attempt to identify both the mechanisms of resistance sulfoxide; BCNU, which was dissolved in 0.12% ethanol; and chlor Received 2/19/91; accepted 5/23/91. ambucil, which was dissolved in 17% dimethyl sulfoxide. The costs of publication of this article were defrayed in part by the payment Tumor Therapy. Groups of 8-10 randomly assigned mice bearing of page charges. This article must therefore be hereby marked advertisement in TE-671 or TE-671 MR tumors were treated by i.p. injection with accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 'This work was supported by NIH Grants CAI 1898, CA44640, NS00958. chemotherapeutic compounds according to the doses and schedules CA40355, NS20023: ACS Research Grant CH-403: and Bristol-Myers Research described above when the median tumor volume exceeded 200 mm3. Grant 100-R18. 2To whom requests for reprints should be addressed, at P. O. Box 2916, 'The abbreviations used are: LD,u, dose lethal to 10?; of treated animals; Department of Pediatrics, Duke University Medical Center. Durham, NC 27710. BCNU, 1.3-bis(2-chloroethyl)-nitrosourea. 3906

Groups of matched animals served as controls and received the drug D, 1 of 20; ifosfamide, 3 of 20; chlorambucil, 3 of 30; VP-16 vehicle on Day 1. (1.0 LD,o), 2 of 17; melphalan (1.0 LD10), 3 of 28; melphalan Assessment of Response. Response of xenografts was assessed by plus VP-16 (0.75 LD,o Â±0.5 LD,0), 2 of 9. Mean nadir weight growth delay [(T â€”C)being the difference in days between the median loss was less than 15% in all groups except cyclophosphamide time for the tumors of treated (T) and control (C) animals to reach a (19.7%), vincristine (15.7%), actinomycin D (19.1%), bleomy- volume of 5 times greater than the volume at the time of treatment], cin (24.9%), and melphalan plus VP-16 (0.75 LD,0 Â±0.50 and by treated versus control tumor regressions. Statistical analyses were performed as previously described (11). LD,o)(19.6%). Melphalan Levels. Athymic nude mice bearing s.c. TE-671 MR Single-Agent Therapy. The response of TE-671 MR and TE- xenografts were given i.p. injections of melphalan at 0.5 of the LD,0 671 to chemotherapy is summarized in Table 1. Results of when the median tumor volumes exceeded 200 mm3. Groups of 3 mice previously published experiments showing the response of the each were anesthetized with halothane; blood was obtained by cardiac parent line, TE-671, to chlorambucil, cyclophosphamide, ifos puncture; and the xenografts were resected at 30, 60, 120, 180, and famide, thio-TEPA, and vincristine are shown for comparative 240 min following injection of the drug. Plasma obtained from the blood samples was frozen (â€”70*C)for subsequent measurement of purposes (4, 16). In addition to resistance to melphalan, TE- melphalan levels. The harvested xenografts were homogenized in a 671 MR demonstrated a similar degree of resistance to the Brinkman Polytron in 10% perchloric acid (5:1, v/w), and the homog- alkylating agents chlorambucil, cyclophosphamide, and ifos enates were frozen (-70Â°C) for subsequent melphalan assays. Mel famide. Three other alkylating agents active in the parent line, phalan measurements were performed by high pressure liquid chro- thio-TEPA, cisplatin, and mitomycin, were ineffective in TE- matography as previously described (12). Mean tumor-to-plasma mel 671 MR. BCNU, which was inactive against TE-671, was phalan ratios were calculated from the ratios obtained in individual similarly inactive against TE-671 MR. TE-671 MR was cross- mice. O'-Alkylguanine-DNA Alkyltransferase. Two TE-671 xenografts and resistant to bleomycin, retained sensitivity to actinomycin D, two TE-671 MR xenografts were resected and snap-frozen in liquid and demonstrated collateral sensitivity to VP-16. nitrogen for subsequent determination of O6-alkylguanine-DNA alkyl- Combination Therapy. The response of TE-671 MR to VP- transferase levels. Enzyme levels were determined as previously de 16 (0.50 LDio) alone, to varying fractions of the LDio of scribed (13). melphalan alone, or to VP-16 (0.50 LD,0) plus melphalan at Tumor OxygÃ©nationStudies. Oxygen tension was measured in TE- various fractions of the LDio is summarized in Table 2. The 671 and TE-671 MR tumors by using a polarographic technique (14). combination of melphalan and 0.50 LD,0 VP-16 was more than Briefly, the measurement relies on ionization of molecular oxygen at a additive, with the greatest combined effect found at the 0.25- polarizing voltage of 0.7 V. The amount of current which flows at the 0.50 LD10 dose range of melphalan. These results are similar polarizing voltage is directly proportional to the oxygen concentration to the effects seen with the same drug combinations against near the sensing electrode. Clark style microelectrodes (Diamond Gen TE-671, where a synergistic interaction was shown previously eral Corp., Ann Arbor, MI) (15), with tip diameters ranging from 50 to 150 Mm,were used for this study. All measurements were made in a (17). radiofrequency-shielded enclosure which was securely grounded. This Melphalan Levels. Melphalan levels in the tumors and was done to prevent stray electromagnetic radiation from interfering plasma of mice bearing TE-671 MR were compared with those with the measurements. Electrodes were calibrated in saline and bubbled previously found in mice bearing tumors of the parent line TE- with gas mixtures certified for 0, 5, 10, and 21% oxygen prior to and 671. The tumor-to-plasma melphalan ratio for the TE-671 MR after each experiment. The calibration temperature was 37Â°C.Calibra line was not significantly different from that obtained in the tions were also performed in vivo. Measurements over exposed, resting TE-671 line at 30, 60, and 120 min post-melphalan injection, muscle in a film of saline were assumed to be equivalent to room air but was significantly lower than that of TE-671 at 180 and 240 (21% oxygen). At the end of each experiment, the animals were eu thanized with a barbiturate overdose. Measurements taken in deep min postinjection (3.18 Â±0.967 versus 7.38 Â±1.72 at 180 min; thigh muscle 10-30 min after euthanasia were assumed to be at 0% Ã›2. undetectable versus 4.67 Â±3.40 at 240 min) (Table 3). The calibrations performed in vivo did not always fit the in vitro curves, O6-Alkylguanine-DNA Alkyltransferase Levels. The mean which was likely due to electrolyte differences between the saline and O6-alkylguanine-DNA alkyltransferase in the TE-671 MR was the animal. Thus, all data were fit to the in vivo curves, and the in vitro calibrations were used to verify that the electrode was functioning properly before and after each experiment. The electrode sensitivities averaged 4.9 Â±3.3 (SD) pA/mm Hg. Table 1 Chemotherapeutic response of s.c. TE-671 MR and TE-671 xenografts Animals bearing 5- to 7-mm s.c. tumors in the lateral flank were T-C (days) anesthetized by using pentobarbitol sodium (40 mg/kg i.p. injection). Agent"MelphalanChlorambucilCyclophosphamideIfosfamideThio-TEPAMitomycinVincristineCisplatinBleomycinBCNUActinomycinMR*'C5.6, The animals were placed on a constant temperature pad (37Â°C)and 10.85.59.5, covered to prevent hypothermia. An incision was made over a lateral 10.6a17.3, surface of the tumor. The oxygen microelectrodes were inserted through 11.48.9, \SA'i11.3, the skin and fascia incision and into the tumors, using the tip for blunt 10.01.5(NS)f0.7 IS.S1*7.1, \2-ff115.5, dissection. Before a measurement was taken, the tip was retracted (NS)3.4, 19.214.3, slightly to prevent pressure effects on the microvessels. Two to 4 radial 6.9"1.6(NS)6.30.2 ll.i"16.711.21.4(NS)9.5, profiles were made in each tumor at 1-mm intervals. After each profile was taken, the tip of the electrode was rinsed with 0. l N HC1 followed by saline to prevent buildup of protein on the tip. All resultant data (NS)9.4, DVP-16TE-671 10.57.3. 10.74.0. from these experiments were converted to mm Hg O2. 9.9TE-671*34.910.2. 5.3 " All agents were administered i.p. at their respective LD,0. * Tâ€”C (days): the difference in days between the median time for the tumors RESULTS of treated (T) and control (C) animals (n = 8 to 10) to reach a volume 5 times greater than the volume at the time of treatment. Duplicate values represent Drug Toxicity. Twenty-two deaths in 326 tumor-bearing results of 2 separate experiments. treated animals resulted from drug toxicity at the doses given ' P values for all growth delays (7"- C) in comparison to untreated controls in "Materials and Methods" with the following distribution: were <0.05 unless otherwise noted. NS, not significant; P > 0.05. " Previously published (T - C) values for TE-671 and TE-671 MR shown here cyclophosphamide, 4 of 20; vincristine, 4 of 10; actinomycin for comparative purposes (4. 9). 3907

Table 2 Chemotherapeutic responses ofs.c. TE-6 71 MR xenografts to varying doses ofmelphalan plus t'P-16 (0.50 LD,,,) understanding of patterns of cross-resistance, collateral sensi Treatment" tivity, and mechanisms of resistance is, therefore, essential for (fraction LD,0) the optimal use of current drugs, as well as for the development of modulations effective in overcoming resistance. Melphalan0.1000.100.2500.250.37500.3750.5000.500.7500.75VP-1600.500.5000.500.5000.500.5000.500.5000.500.50T-C"0.3 (NSC'3.74.11.9 Exposure to a single chemotherapeutic agent may result in (NS)0/8 cross-resistance to other drugs with different structures and (NS)0/9 (NS)0/9 mechanisms of action. This phenomenon has generally been seen with the use of natural products and their derivatives and (NS)1.6 (NS)0/9 is often associated with the presence of P-glycoprotein, which (NS)8.6'4.24.312.5'4.32.811.3'10.04.315.0'Regressions'0/9(NS)1/9 (NS)0/10 functions as an efflux pump (18). The development of resistance to an alkylating agent has typically not been shown to lead to (NS)0/10 (NS)6/10i0/10 cross-resistance to other alkylating agents (2, 19, 20). The melphalan-resistant xenograft TE-671 MR retained sensitivity to actinomycin D, exhibited collateral sensitivity to VP-16, but (NS)0/10(NS)(,/\Of4/10 demonstrated complete or partial cross-resistance to a series of alkylating agents. The marked cross-resistance to mitomycin C was not mediated by changes in tumor oxygÃ©nationsince TE- (NS)0/10 (NS)5/7 671 MR was not more oxygenated than the parent line. Indeed, the tumor oxygÃ©nationmeasurements tended to be lower in 0 Experiment regimen: VP-16. Days 1. 4. 7; melphalan. Day 1 (i.p.). TE-671 MR than in TE-671, as shown by lower pO; profiles * T - C (days): the difference in days between the median time for the tumors of treated (7Â°)andcontrol (O animals to reach a volume 5 times greater than the and lower average median values (Fig. 1). The precise mecha volume at the time of treatment. nism of this cross-resistance to mitomycin C remains unclear. ' Number of animals with regressing tumors/number of surviving treated Although Dean et al. (21) have demonstrated that enhanced animals. Regressing tumors have at least 2 consecutively decreasing volume measurements. nitrosourea cytotoxicity in a melphalan-resistant Walker 256 d P values for all experiments were <0.01 unless otherwise designated. NS. not carcinoma is mediated by a decrease in O6-alkylguanine-DNA significant; P> 0.01. 'Growth delay (7"- C) for melphalan plus VP-16 significantly longer than alkyltransferase levels, this was not true in TE-671 MR, which melphalan alone: P< 0.01. demonstrated BCNU resistance and 06-alkylguanine-DNA al Degressions for melphalan plus VP-16 significantly greater than melphalan kyltransferase levels virtually identical to the parent line. alone: P< 0.01. Bleomycin is an antineoplastic antibiotic which requires the presence of a reducing agent, such as glutathione, for cytotoxic action (22, 23). Since a glutathione-rich human ovarian carci Table 3 Melphalan tumor-to-plasma ratios MR0.954 noma cell line was previously shown to be bleomycin sensitive Time (min)3060 (24), TE-671 MR, which also exhibits increased glutathione Â±0.262Â° Â±0.320 2.11 Â±0.885 1.82 Â±0.624 levels relative to the parent line (4), was treated with bleomycin 120 4.59 Â±1.30 4.11Â±0.0213.18 to determine if this xenograft demonstrated collateral sensitiv 180 7.38 Â±1.72 Â±0.967 b ity to bleomycin. Bleomycin was not, however, effective against 240TE-6710.997 4.67 Â±3.40TE-671 TE-671 MR. Patterns of cross-resistance and collateral sensi " Mean Â±SD. * Plasma and tumor melphalan levels were below the limit of detection in this tivity are, therefore, poorly predictable and may possibly depend assay (0.2 ^M). on the specific tumor histiotype. TE-671 MR demonstrated collateral sensitivity to VP-16 with growth delays approximately twice that observed in the 1282 fmol/mg protein, compared with 1324 fmol/mg protein parent line. VP-16 is an epipodophyllotoxin with cytotoxic in the parent line, TE-671 (P > 0.05). properties due to inhibition of topoisomerase II activity. High Tumor OxygÃ©nationStudies. In both xenograft lines, pO2 values were lower near the tumor center than at the edge (Fig. 1). TE-671 MR tended to have lower values near the center than TE-671, but the differences between average or median values were not significant (P > 0.05). Hypoxie regions with less than 10 mm Hg were observed in 4 of 5 TE-671 MR tumors and 2 of 6 TE-671 tumors. The overall frequencies of hypoxic regions were 17 of 92 (18%) and 13 of 73 (17%) for TE-671 MR and TE-671, respectively. The mean and SD of median pO: values for TE-671 MR tumors was 32.9 Â±21.0 mm Hg. I 40H The mean and SD of pO2 values for TE-671 was 49.2 Â±46 mm Hg. The large standard deviations are indicative of large varia TE-671 MR tions in oxygÃ©nationbetween tumors. TE-671

DISCUSSION

Chemotherapy has played a dramatic role in increasing dis ease-free survival in a variety of human neoplasms, particularly pO2 (mm Hg) in the treatment of childhood malignancies (2). However, drug Fig. 1. Cumulative frequency distribution of pO2 values for TE-671 MR and resistance often develops in tumors which were initially chem- TE-671 s.c. tumors (5 to 7 mm). There were no significant differences between the two lines. Five to six animals per group were studied with 10 to 20 measure osensitive and results in eventual treatment failure (1). An ments per mouse. Points, averages; bars, SEM. 3908

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1991 American Association for Cancer Research. ANALYSIS OF MELPHALAN-RESISTANT XENOGRAFT levels of topoisomerase II are associated with increased sensi glioma-derived cell line. J. Neuropathol. Exp. Neurol., 40: 410-427, 1981. tivity to the effects of VP-16 (25). Furthermore, a nitrogen McAllister. R. M., Isaacs, H., Rongey, R.. Peer, M., Au, W., Soulcup, S. W., and Gardner, M. B. Establishment of a human medulloblastoma cell line. mustard-resistant Burkitt's lymphoma cell line, Raji-HN2, was Int. J. Cancer, 20: 206-212, 1977. found to be collaterally sensitive to VP-16 by virtue of elevated Friedman, H. S., Bigner, S. H., McComb, R. D., Schold, S. C., Jr., Pasternak, expression and transcription of the topoisomerase gene (26, J. F., Groothuis, D. R., and Bigner, D. D. A model for human medulloblas toma: growth, morphology, and chromosomal analysis in vitro and in athymic 27). Previous studies with the parent line TE-671 have dem mice. J. Neuropathol. Exp. Neurol., 42:485-503, 1983. onstrated the synergistic interaction between VP-16 and mel- Stratton, M. R., Darling, J., Pilkington, G. J., Lantos, P. L., Reeves, B. P., phalan (17), and greater than additive growth delays were seen and Cooper, C. S. Characterization of the human cell line TE-671. Carci- with this combination against TE-671 MR. nogenesis (Lond.), 10: 899-905, 1989. Friedman, H. S., Schold, S. C., Jr., and Bigner, D. D. Chemotherapy of Multiple mechanisms of alkylator resistance are documented subcutaneous and intracranial human medulloblastoma xenografts in and include increased glutathione content, increased or altered athymic nude mice. Cancer Res., 46: 224-228, 1986. activity of glutathione-S-transferase, increased repair of drug- 10. Skipper, H. E., and Schmidt, L. H. A manual on quantitative drug evaluation in experimental tumor systems. Cancer Chemother. Rep., 17: 1-143, 1962. induced DNA damage, and decreased uptake of drug (2). Re 11.Friedman, H. S., Schold, S. C., Jr., Varia, M., and Bigner, D. D. Chemo ported mechanisms of resistance to melphalan have included therapy and radiation therapy of human medulloblastoma in athymic nude increased levels of glutathione or glutathione-S'-transferase, in mice. Cancer Res., 43: 3088-3093, 1983. creased repair of DNA cross-links, and decreased melphalan 12. Friedman, H. S., Skapek, S. X.. Colvin, O. M., Elion, G. B., Blum, M. R., Savina, P. M., Hilton, J., Schold, S. C., Jr., Kurtzberg, J., and Bigner, D. D. transport (28-31). Previous studies have shown that melphalan Melphalan transport, glutathione levels, and glutathione-5-transferase activ resistance in TE-671 MR is not associated with an altered ity of human medulloblastoma. Cancer Res., 48: 5397-5402, 1988. 13. Schold. S. C., Jr., Brent, T. P., Von Hofe, E., Friedman, H. S., Mitra, S., karyotype, increased expression of mdr\, or increased glutathi Bigner, D. D., Swenberg, J. A., and Kleihues, P. O'-Alkylguanine-DNA one-S'-transferase (4). A 2-fold increase in glutathione content alkyltransferase and sensitivity to procarbazine in human brain-tumor xen in TE-671 MR was observed, but this alteration cannot yet be ografts. J. Neurosurg., 70: 573-577, 1989. directly implicated as responsible for the observed resistance, 14. Willard. H. H., Merritt, L. L., and Dean, J. A. Instrumental Methods of Analysis. New York: Van Nostrand Co., 1969. since buthionine sulfoximine-mediated glutathione depletion 15. Baumgartl, H.. and Lubbers, D. W. Platinum needle electrodes for polaro- enhances melphalan activity in both TE-671 and TE-671 MR graphic measurement of local O2 pressure in cellular range of living tissue: xenografts (4). Our current studies show lower concentrations its construction and properties. In: E. Gnalger and H. Forstner (eds.), Polarographic Oxygen Sensors: Aquatic and Physiologic Applications, p. of melphalan into resistant tumors compared with the parent 37-65. Heidelberg: Springer-Verlag, 1983. line and suggest that decreased melphalan delivery (as a con 16. Friedman, H. S.. Colvin, O. M., Skapek, S. X., Ludeman, S. M., Elion, G. sequence of altered tumor blood flow or intracellular drug B., Schold, S. C, Jr., Jacobsen, P. F., Muhlbaier, L. H., and Bigner, D. D. Experimental chemotherapy of human medulloblastoma cell lines and trans- transport) or enhanced melphalan efflux may be contributing plantable xenografts with bifunctional alkylating agents. Cancer Res.. 48: to melphalan resistance in TE-671 MR. 4189-4195, 1988. The extent to which TE-671 MR illustrates clinically relevant 17. Lilley, E. R., Rosenberg, M. C, Elion, G. B., Colvin, O. M., Bigner, D. D., and Friedman, H. S. Synergistic interactions between cyclophosphamide or mechanisms of melphalan resistance and cross-resistance is melphalan and VP-16 in a human rhabdomyosarcoma xenograft. Cancer presently undefined. The general heterogeneity of tumors and Res., 50: 284-287, 1989. the wide spectrum of mechanisms mediating alkylating agent 18. Moscow, J. A., and Cowan, K. H. Multidrug resistance. J. Nati. Cancer Inst., 80: 14-19, 1987. resistance in various tumor histiotypes suggests that identifi 19. Bergsagel. D. E., Cowan. D. H., and Hasselback, R. Plasma cell myeloma: cation of the mechanisms most responsible for clinical drug response of melphalan-resistant patients to high-dose intermittent cyclo resistance may be difficult. Nevertheless, studies of the charac phosphamide. Can. Med. Assoc. J., 107: 851-855, 1972. teristics and mechanisms of melphalan resistance (specifically 20. Schabel, F. M., Griswold, D. P., Jr., CorbeÂ«,T. H., and Laster, W. R., Jr. Increasing therapeutic response rates to anticancer drugs by applying the the factors responsible for the decreased melphalan uptake in basic principles of pharmacology. Pharmacol. Ther., 20: 283-305, 1983. TE-671 MR) in this and other tumor models provide a greater 21. Dean. S. W., Gibson, N. W., and Tew, K. D. Selection of nitrogen mustard resistance in a rat tumor cell line results in loss of guanine O'-alkyltransferase understanding of alkylator resistance generally and may lead to activity. Mol. Pharmacol.. 30: 77-80, 1986. useful clinical applications in some tumors. 22. Antholine, W. E., Petering, D. H., Saryan, L. H., and Brown, C. E. Interac tions among iron(ll) bleomycin, Lewis bases, and DNA. Proc. Nati. Acad. Sci. USA, 78: 7517-7520, 1981. ACKNOWLEDGMENTS 23. Caspar), W. J., Lanzo, D. A., and Niziak, C. Intermediates in the ferrous oxidase cycle of bleomycin. Biochemistry, 20: 3868-3875, 1981. We wish to acknowledge Ann Tamariz for editorial assistance on 24. 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Eileen R. Lilley, Gertrude B. Elion, Mark W. Dewhirst, et al.

Cancer Res 1991;51:3906-3909.

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