JBJS | Intrinsic Resistance to Chemotherapeutic Agents in Murine Osteosarcoma Cells*

The Journal of Bone & Joint Surgery, Volume 82, Issue 7

Articles | July 01, 2000

Intrinsic Resistance to Chemotherapeutic Agents in Murine Osteosarcoma Cells*

Hideyuki Takeshita, M.D.†; Katsuyuki Kusuzaki, M.D.‡; Tsukasa Ashihara, M.D.‡; Mark C. Gebhardt, M.D.§; Henry J. Mankin, M.D.§; Yasusuke Hirasawa, M.D.‡

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Investigation performed at Kyoto Prefectural University of Medicine, Kyoto, and Massachusetts General Hospital, Boston
*No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article. No funds were received in support of this study.
†Department of Orthopaedic Surgery, Otsu Municipal Hospital, 2 Chome, Motomiya, Otsu, Shiga Prefecture 520-0804, Japan. E-mail address: h2-take@kf6.so-net.ne.jp. Please address requests for reprints to H. Takeshita.
‡Departments of Orthopaedic Surgery (K. K. and Y. H.) and Pathology (T. A.), Kyoto Prefectural University of Medicine, Kawaramachi, Hirokoji, Kamikyo-ku, Kyoto 602-0841, Japan.
§Department of Orthopaedic Surgery, Gray Building 607, Massachusetts General Hospital, 55 Fruit Street, Boston, Massachusetts 02114.

The Journal of Bone & Joint Surgery. 2000; 82:963-963

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Abstract

Background: There are two general categories of drug resistance: acquired and intrinsic. The mechanisms involved in acquired drug resistance have been extensively studied, and several mechanisms have been described. However, the mechanisms responsible for intrinsic drug resistance have not been elucidated, to our knowledge. The purpose of the present study was to investigate the cytological and biochemical differences between acquired and intrinsic drug resistance in osteosarcoma cells.

Methods: We previously isolated a clonal cell line (MOS/ADR1) to study acquired resistance in osteosarcoma by exposure of parental murine osteosarcoma cells (MOS) to doxorubicin. In the present study, we cloned a new, intrinsically resistant cell line (MOS/IR1) by single-cell culture of MOS cells and we investigated the differences in cell phenotype and the mechanisms of resistance in both of these resistant clones.

Results: The MOS/ADR1 and MOS/IR1 cells were sevenfold and fivefold more resistant to doxorubicin than the parental murine osteosarcoma cells. Morphologically, the MOS/ADR1 cell line was composed of polygonal cells, whereas the MOS/IR1 cell line consisted of plump spindle cells with long cytoplasmic processes. The MOS/IR1 cells showed a much lower level of alkaline phosphatase activity than did the MOS/ADR1 and MOS cells. There were no substantial differences in the cellular DNA content or the doubling time among these three lines.

Overexpression of the P-glycoprotein involved in the function of an energy-dependent drug-efflux pump was detected in the MOS/ADR1 cells but not in the MOS/IR1 cells. After the cells were incubated with doxorubicin for one hour, the two resistant lines had less accumulation of the drug than did the parent line (p < 0.05). The addition of a P-glycoprotein antagonist, verapamil, or the depletion of cellular adenosine triphosphate resulted in a marked increase in the accumulation of doxorubicin in the MOS/ADR1 cells (p < 0.05) but not in the MOS/IR1 cells. The MOS/ADR1 cells were found to exhibit cross-resistance only to substrates for P-glycoprotein (such as doxorubicin, vincristine, and etoposide), whereas the MOS/IR1 cells were resistant to all of the drugs studied (including cisplatin and methotrexate).

The degree of drug resistance in the MOS/IR1 cells was found to be associated with the molecular weight of the drugs (p < 0.05). Permeabilization of the plasma membrane by saponin increased both the accumulation of doxorubicin (p < 0.05) and the cytotoxic activity of this drug in all lines, but the effects were most pronounced in the MOS/IR1 cells.

Conclusions: Taken together, this data suggests that reduced drug accumulation in the MOS/IR1 cells may be due to the effect of decreased permeability of the plasma membrane on the transport of drugs from the extracellular environment into the cytosol of the cell and that this may be the mechanism responsible for intrinsic resistance to multiple drugs in the MOS/IR1 cell line.

Clinical Relevance: Current drug treatment for human osteosarcoma may include multiple chemotherapeutic agents, such as doxorubicin, cisplatin, and methotrexate. These drugs exhibit different cytotoxic actions and, thus, the mechanisms of resistance to individual drugs vary. Clinical resistance to multidrug chemotherapy may be observed in tumors that recur after repetitive chemotherapy and in previously untreated tumors. In the former group, a tumor cell may express multidrug resistance by combining several different mechanisms due to its exposure to various drugs. In the latter group, however, this is not likely. Decreased intracellular drug accumulation due to reduced permeability of the plasma membrane, found in the MOS/IR1 cells, is one possible mechanism and may explain the intrinsic resistance to multidrug chemotherapy for the treatment of osteosarcoma. Further study regarding the resistance mechanism in the MOS/IR1 cells may help to overcome the intrinsic drug resistance in osteosarcoma.

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Introduction

Introduction | Materials and Methods | Results | Discussion | References

The prognosis of patients with osteosarcoma is closely associated with the response of the tumor cells to chemotherapy. Although the use of multiple chemotherapeutic agents for the treatment of osteosarcoma has substantially improved the outcome for these patients, more than 30 percent of the tumors still appear to be resistant to current chemotherapy regimens^16,22.

Tumor cells may become resistant to drugs, after an initial response, during repetitive chemotherapy (acquired resistance), or they may be intrinsically resistant without previous exposure to drugs (intrinsic resistance). The mechanisms involved in acquired drug resistance have been extensively studied^6,8,12,18 because cells with acquired drug resistance can be produced experimentally by the successive exposure of parent cells to a particular drug in vitro⁴. Several mechanisms of acquired drug resistance have been demonstrated, including reduced intracellular accumulation of drugs caused by membrane drug-efflux pumps such as P-glycoprotein¹² and multidrug-resistance-associated protein⁸, changes in the expression of enzymes involved in the glutathione detoxification pathway⁶, and altered function of the nuclear enzyme topoisomerases that are the targets for intercalating or nonintercalating drugs¹⁸. In contrast, few studies have examined the mechanisms responsible for intrinsic drug resistance¹⁰. The reason that some tumor cells are inherently resistant without previous drug treatment is not known.

We previously isolated multidrug-resistant clones from the Massachusetts General Hospital (MGH) murine osteosarcoma cells by means of repetitive short-term exposure of the parent cells to doxorubicin followed by clonal analysis^26,28. The surviving clones overexpressed the membrane P-glycoprotein, an adenosine triphosphate-dependent drug-efflux pump, which actively transports and extrudes structurally unrelated drugs from cells, rendering the cells multidrug resistant. The P-glycoprotein-mediated multidrug resistance is the best characterized mechanism in acquired drug resistance, and it is believed to be at least partially responsible for clinically encountered drug resistance^1,7. In the present study, we cloned a new, intrinsically drug-resistant cell line by single-cell culture of parental MGH osteosarcoma cells without exposure to any drugs. This cell line does not have an energy-dependent drug-efflux pump involving P-glycoprotein but still manifests a multidrug-resistant phenotype and a decrease in intracellular drug accumulation. To the best of our knowledge, such intrinsic resistance has not been reported. The purpose of our study was to investigate the cytological and biochemical differences between acquired and intrinsic drug resistance in MGH murine osteosarcoma cells.

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Table I: In Vitro Characteristics of the Cell Lines

*IC50 = drug concentration yielding 50 percent growth inhibition. Doxorubicin values were evaluated with use of the tetrazolium colorimetric assay.Immunofluorescence staining of P-glycoprotein (Pgp) with the C219 antibody. A plus sign indicates that more than 50 percent of the cells were stained positively with C219, and a minus sign indicates that none of the cells were stained positively with C219.ALP = cellular alkaline phosphatase activity.

Cell line	IC50 of Doxorubicin* (Î§ per mL)	Pgp	DNA Index	Doubling Time (hrs.)	ALP (Î per min. per mg protein)
MOS	0.022	-	1.41	20.2	1.27
MOS/ADR1	0.160	+	1.37	18.3	2.64
MOS/IR1	0.110	-	1.37	19.6	0.11

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Table I: In Vitro Characteristics of the Cell Lines

Cell line	IC50 of Doxorubicin* (Î§ per mL)	Pgp	DNA Index	Doubling Time (hrs.)	ALP (Î per min. per mg protein)
MOS	0.022	-	1.41	20.2	1.27
MOS/ADR1	0.160	+	1.37	18.3	2.64
MOS/IR1	0.110	-	1.37	19.6	0.11

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Fig. 1-A:: Figs. 1-A, 1-B, and 1-C: Phase-contrast photomicrographs of the cultured cells.
Fig. 1-A: The parent murine osteosarcoma cell line (MOS). This cell line was composed of a mixed-cell population, including small round cells, large polygonal cells, and a few plump spindle cells.

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Fig. 1-B: The osteosarcoma cell line with acquired resistance to doxorubicin (MOS/ADR1) that was produced by exposure of the MOS cells to doxorubicin followed by cloning of a single cell. This cell line displayed a relatively homogeneous population of polygonal cells.

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Fig. 1-C: The osteosarcoma cell line with intrinsic resistance to doxorubicin (MOS/IR1) that was isolated from the MOS cells without drug treatment. This cell line consisted of plump spindle cells with long cytoplasmic processes.

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Fig. 2-A:: Figs. 2-A and 2-B: Bar graphs showing the intracellular accumulation of doxorubicin (DOX).
Fig. 2-A: Accumulation after the cells were incubated for one hour with [¹⁴C]-labeled doxorubicin. CPM = counts per minute. * = p < 0.05 for the difference compared with the MOS cells.

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Fig. 2-B:: Effects of adding the P-glycoprotein antagonist verapamil and the effects of depletion of cellular adenosine triphosphate (ATP) on the accumulation of doxorubicin, as described in the Materials and Methods section of this paper. ** = p < 0.05 for the difference compared with the control cells.

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Fig. 3:: Graph showing the retention of doxorubicin (DOX). The cells were preloaded for one hour with [¹⁴C]-labeled doxorubicin with depletion of cellular adenosine triphosphate, washed with phosphate-buffered saline solution, and incubated in drug-free medium. Active extracellular transport of doxorubicin was detected in the MOS/ADR1 cells but not in the MOS/IR1 cells.

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Fig. 4:: Bar graph showing the difference in the spectrum of cross-resistance between the MOS/IR1 and the MOS/ADR1 cells. The results are expressed as the ratios of the drug concentrations yielding 50 percent growth inhibition (IC50) of the drug-resistant cell lines (MOS/ADR1 and MOS/IR1) to those of the parent cells (MOS), as determined with the tetrazolium colorimetric assay. The molecular weights of the drugs are given in parentheses. There was a significant association between the degree of drug resistance in the MOS/IR1 cells and the molecular weight of the drugs (p < 0.05). ACD = actinomycin D, VCR = vincristine, ETP = etoposide, DOX = doxorubicin, MTX = methotrexate, CDDP = cisplatin, IFO = ifosfamide, and 5FU = 5-fluorouracil.

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Fig. 5:: Graph showing the cytotoxicity of saponin in the cell lines. The addition of saponin at a concentration of more than twenty micrograms per milliliter caused substantial inhibition of growth in all three cell lines.

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Fig. 6:: Bar graph showing the effect of saponin on accumulation of doxorubicin (DOX). The addition of saponin at a concentration of fifteen micrograms per milliliter resulted in a marked increase in the intracellular accumulation of doxorubicin in all three cell lines. * = p < 0.05 for the difference compared with the control cells.

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Fig. 7:: Bar graph showing the effect of saponin on resistance to doxorubicin (DOX). The addition of saponin at a concentration of fifteen micrograms per milliliter enhanced the cytotoxicity of doxorubicin in all three cell lines, but the effect was most pronounced in the MOS/IR1 cells. IC50 = drug concentration yielding 50 percent growth inhibition.

Materials and Methods

Introduction | Materials and Methods | Results | Discussion | References

Establishment of the cell lines: The parental cell line, MGH murine osteosarcoma cells (MOS), was derived from a radiation-induced murine osteosarcoma². The P-glycoprotein-positive drug-resistant cell line (MOS/ADR1) was developed by six pulse exposures of the MOS cells to doxorubicin, as described previously²⁶. The P-glycoprotein-negative drug-resistant cell line designated MOS/IR1 was cloned directly from the MOS cells by a limiting dilution method of clonal analysis with use of ninety-six-well plates. The plates were inspected microscopically on a daily basis to identify the wells containing a single cell. These clones were shown to be P-glycoprotein-negative by immunofluorescence staining with the monoclonal antibody C219 to P-glycoprotein (described below). In a preliminary survey to select the drug-resistant clones, sensitivity to doxorubicin of the cloned cell lines (eight cell lines) was compared with results in the parental MOS cells with use of the tetrazolium colorimetric assay described below. Only one clone demonstrated resistance to the drug, and it was selected for additional studies. All of the cell lines were stored in liquid nitrogen, and the experiments were performed within a month after the cells were thawed. The cell lines were maintained at 37 degrees Celsius in a humidified incubator containing 5 percent carbon dioxide in Dulbecco's modified Eagle medium supplemented with fifteen-millimolar HEPES buffer, 10 percent fetal calf serum, and an antibiotic solution of penicillin (100 units per milliliter) and streptomycin (fifty micrograms per milliliter). All of the experiments were performed during the exponential growth phase.

Staining for P-glycoprotein: Cells grown on coverslips were fixed with acetone for thirty minutes at room temperature and were stained with the indirect immunofluorescence method. The primary monoclonal antibody C219 (Centcor Diagnostics, Malvern, Pennsylvania) was applied for twenty hours at 4 degrees Celsius at a concentration of five micrograms per milliliter. After the cells were washed with phosphate-buffered saline solution, they were incubated with fluorescein isothiocyanate-conjugated F(ab)Â¢2 goat anti-mouse IgG at a concentration of thirty-five micrograms per milliliter (Caltag Laboratories, San Francisco, California) for one hour at room temperature and then washed again with phosphate-buffered saline solution. The coverslips were mounted on glass slides, and P-glycoprotein immunofluorescence was examined with a fluorescence microscope.

Cellular DNA content, doubling time, and alkaline phosphatase activity: Cellular DNA content²⁰, doubling time²⁰, and cellular alkaline phosphatase activity^23,26 were determined as previously described.

Effect of verapamil and energy depletion on the intracellular accumulation of [¹⁴C]-labeled doxorubicin: Cells grown in twenty-four-well plates were incubated for one hour in medium A, B, or C. Medium A was a growth medium containing [¹⁴C]-labeled doxorubicin at a concentration of ten micrograms per milliliter, medium B was medium A with verapamil at a concentration of ten micrograms per milliliter, and medium C was medium A without glucose but containing sodium azide (ten-millimolar) and 2-deoxy-d-glucose (one milligram per milliliter). The cells were then washed with ice-cold phosphate-buffered saline solution and trypsinized. Each well was washed twice with cold phosphate-buffered saline solution, and the cells as well as both washes were placed in a scintillation vial. Scintillation cocktail was added and mixed, and the radioactivity of the samples was determined with a liquid scintillation counter. Cells treated with nonradioactive doxorubicin were also trypsinized, and the number of cells was counted with a hemocytometer. The results were expressed as counts per minute per 10⁴ cells.

Efflux of doxorubicin: The cells were exposed for one hour to [¹⁴C]-labeled doxorubicin in medium C, washed with phosphate-buffered saline solution, and incubated for another thirty minutes in medium A without doxorubicin. At subsequent intervals, [¹⁴C]-labeled doxorubicin retained by the cells was measured with a liquid scintillation counter.

Multidrug-resistant phenotype: The multidrug-resistant phenotype of the cell lines was determined with the tetrazolium colorimetric assay described by Hansen et al.¹⁴. The assay is dependent on the reduction of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma Chemical, St. Louis, Missouri) by the mitochondrial dehydrogenase of viable cells to a blue formazan product, the concentration of which can be measured with spectrophotometry. After incubation of the cells with anticancer drugs, the cellular activity was determined with this assay. The cells were seeded in ninety-six-well plates at a density of 2 Â¥ 10³ cells in 100 microliters of medium per well. After thirty-six hours, the culture medium was removed and was replaced with medium containing varying concentrations of anticancer drugs, and the cultures were incubated for an additional seventy-two hours. Twenty-five microliters of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide at a concentration of five milligrams per milliliter in phosphate-buffered saline solution was then added to each well. After two hours of incubation at 37 degrees Celsius, 100 microliters of the extraction buffer (N,N-dimethyl formamide and sodium dodecyl sulfate [Sigma Chemical] at pH 4.7) was added. After twenty-four hours of incubation at 37 degrees Celsius, the optical density at 570 nanometers was measured with use of a microplate reader, with the extraction buffer as the blank. Growth inhibition by anticancer drugs was calculated by the formula: cytostasis (percent) = (1 - A/B) 100, where A is the absorbance of treated cells and B is the absorbance of control cells. The drug concentrations yielding 50 percent growth inhibition were then determined from the dose-response curve by plotting the cytostasis against the drug concentration.

Effect of plasma-membrane permeabilization by saponin on the accumulation and efficacy of doxorubicin: First, growth inhibition by saponin alone was examined in order to determine the nontoxic doses of the drug. The cells were incubated in various concentrations of saponin for one hour, washed with phosphate-buffered saline solution, and incubated with saponin-free medium for an additional seventy-two hours. Cytotoxic concentrations of saponin were then determined with the tetrazolium colorimetric assay. The effect of saponin was assessed by comparing the concentration of doxorubicin yielding 50 percent growth inhibition in the absence and presence of the nontoxic dose of saponin. Changes in the intracellular accumulation of doxorubicin caused by saponin were also evaluated with cytofluorometry, as described previously²⁶.

Statistical analysis: The data was analyzed with use of software programs from StatSoft (Tulsa, Oklahoma) on a personal computer (T2000Sxe; Toshiba, Tokyo, Japan). The relationship between parameters was assessed by linear regression analysis. The Student t test was performed to analyze differences in the data. P values of less than 0.05 were considered significant.

Results

Introduction | Materials and Methods | Results | Discussion | References

Morphological appearance of the cell lines: Of the several clones isolated from the parent cell line (MOS), only one clone, designated MOS/IR1, was intrinsically resistant to doxorubicin. This clone exhibited several interesting features that were not observed in the MOS/ADR1 cells with acquired drug resistance. There was a remarkable difference in cell morphology between the cell lines. The parent cells were morphologically heterogeneous (Fig. 1-A); they consisted mainly of large polygonal and small round cells but also had a very small portion of plump spindle cells. In contrast, the MOS/ADR1 clone displayed a relatively homogeneous cell population of polygonal cells (Fig. 1-B. The MOS/IR1 clone was composed of plump spindle cells with long cytoplasmic processes (Fig. 1-C).

In vitro characteristics of the cell lines: The tetrazolium colorimetric assay demonstrated that the MOS/ADR1 and MOS/IR1 cells were sevenfold and fivefold more resistant to doxorubicin than the parent cells were (Table I). The expression of P-glycoprotein was detected in most of the MOS/ADR1 cells, as shown previously²⁶, but not in any of the parental MOS cells or the MOS/IR1 cells. Cytofluorometric studies demonstrated that these three cell lines displayed similar DNA content and had an aneuploid DNA index ranging from 1.37 to 1.41. They also had similar doubling times (range, 18.3 to 20.2 hours). Cellular alkaline phosphatase activity compared with that in the parent cell line was higher in the acquired-resistance clone MOS/ADR1 but much lower in the intrinsically resistant cell line MOS/IR1 (Table I).

Accumulation of doxorubicin: After cells had been incubated in medium containing [¹⁴C]-labeled doxorubicin for one hour, intracellular accumulation of doxorubicin in the MOS/IR1 and MOS/ADR1 cells was 67 and 51 percent of that in the parent cells, respectively (p < 0.05) (Fig. 2-A). Addition of verapamil significantly increased the accumulation of doxorubicin in the MOS/ADR1 cells (p < 0.05) but not in the MOS/IR1 or MOS cells (Fig. 2-B). Depletion of cellular adenosine triphosphate resulted in an increase in the accumulation of doxorubicin in all cell lines (p < 0.05). However, the effects were most pronounced in the MOS/ADR1 cells.

Efflux of doxorubicin: Preloading of the cells with [¹⁴C]-labeled doxorubicin and depletion of adenosine triphosphate, followed by incubation in drug-free medium, resulted in a marked decrease in intracellular doxorubicin in the MOS/ADR1 cells (Fig. 3). There was no substantial difference in the efflux of doxorubicin between the MOS and the MOS/IR1 cells. This data indicated that a decrease in intracellular doxorubicin in the MOS/IR1 cells (Fig. 2-A) did not result from an increase in efflux of the drug.

Spectrum of cross-resistance: The spectrum of cross-resistance of the cell lines was evaluated with the tetrazolium colorimetric assay. The ratios of the concentrations of the drugs yielding 50 percent inhibition of cell growth for the resistant cell lines to those for the parent cell line were calculated (Fig. 4). Both the MOS/IR1 and the MOS/ADR1 cells displayed the multidrug-resistant phenotype. However, there was a difference in the spectrum of cross-resistance between the two cell lines. The acquired-resistance cell line MOS/ADR1 was resistant only to substrates for P-glycoprotein such as doxorubicin, vincristine, and etoposide. In contrast, the MOS/IR1 cells were resistant to all of the drugs studied, including cisplatin and methotrexate. In addition, there was a significant correlation between the degree of resistance and the molecular weight of the drugs in the MOS/IR1 cells (p < 0.05).

Reversal of doxorubicin resistance by membrane permeabilization: The data presented above suggested that drug resistance in the MOS/IR1 cells might result from a decrease in the transport of drugs across the cell membrane from the extracellular space to the intracellular space. To test this hypothesis, we studied the effect of saponin, a membrane-permeabilization agent, on the accumulation and efficacy of doxorubicin. As a first step, we examined the cytotoxicity of saponin alone with the tetrazolium colorimetric assay. Because saponin at concentrations of more than twenty micrograms per milliliter is highly toxic to cells, we used a concentration of fifteen micrograms per milliliter in the present study (Fig. 5). This concentration significantly increased the intracellular accumulation of doxorubicin (p < 0.05) (Fig. 6) and, thus, enhanced the efficacy of doxorubicin in all of the cell lines (Fig. 7). These effects of saponin, however, were most pronounced in the MOS/IR1 cells.

Discussion

Introduction | Materials and Methods | Results | Discussion | References

The present study demonstrated differences in the mechanisms of drug resistance and cell phenotype between the two murine osteosarcoma cell lines, MOS/IR1 and MOS/ADR1, derived from the same parent cell line, MOS. The MOS/ADR1 clone was produced after exposure of the MOS cells to doxorubicin, whereas the MOS/IR1 clone was isolated directly from the MOS cells without exposure to any drug. The MOS/ADR1 clone showed acquired drug resistance, whereas the MOS/IR1 clone showed intrinsic drug resistance.

Although a decrease in intracellular drug accumulation is associated with multidrug resistance in both the MOS/IR1 and the MOS/ADR1 cells, the mechanisms are quite different. The assays used to examine the cellular uptake (Fig. 2-A and Fig. 2-B) and retention (Fig. 3) of doxorubicin indicated that reduced accumulation of the drug in the MOS/IR1 cells is due to the decreased entry of the drug into the cell and not to the increased efflux of the drug mediated by energy (adenosine triphosphate)-dependent pump mechanisms involving P-glycoprotein or multidrug-resistance-associated protein. It is believed that drugs enter the cell through passive diffusion, facilitated diffusion, and active transport³. Of these, passive diffusion is the most important transport mechanism and may depend on several factors, including the molecular weight of the drugs, membrane lipid composition, and membrane fluidity²⁴. The present study demonstrated that the MOS/IR1 cells were resistant to all of the drugs tested and that the resistance level is significantly related to the molecular weight of these drugs (p < 0.05). Moreover, permeabilization of the plasma membrane by saponin markedly increased intracellular accumulation of doxorubicin and enhanced the cytotoxic activity of this drug. Saponin is known to act mainly by solubilizing cholesterol, leaving much of the membrane structure intact¹⁵. Therefore, this data suggests that the intrinsic multidrug resistance in the MOS/IR1 cells is probably due to decreased plasma-membrane permeability, resulting in the restriction of passive diffusion of the drugs.

Current drug treatment for osteosarcoma may include multiple chemotherapeutic agents, such as doxorubicin, cisplatin, and methotrexate. These drugs possess different mechanisms of cellular toxicity and, thus, the mechanisms of resistance to the individual drugs vary. For example, resistance to doxorubicin is known to be caused by overexpression of P-glycoprotein¹² or multidrug-resistance-associated protein²⁹ or by a decreased level of topoisomerase II⁹, resistance to cisplatin is known to be caused by enhanced activity of DNA repair system²¹ or by an increased level of metallothionein¹⁷ or glutathione¹¹, and resistance to methotrexate is known to be caused by increased activity of the targeting enzyme dihydrofolate reductase¹³. In osteosarcomas that recur following repetitive chemotherapy with these drugs, a tumor cell may express multidrug resistance by combining several different mechanisms because of previous exposure to the drugs. However, in previously untreated osteosarcomas, this is not likely. Thus, we hypothesize that intrinsic resistance of osteosarcoma cells to multidrug chemotherapy may be caused by more common underlying mechanisms. One of these mechanisms may be decreased plasma-membrane permeability, as shown in the present study.

We recently showed that an in vitro analysis of the intracellular accumulation of doxorubicin in human osteosarcoma cells can predict the response of the tumor cells to preoperative multidrug chemotherapy¹⁹. In that study, tumors with decreased intracellular accumulation of doxorubicin had a poor response to multidrug treatment with doxorubicin and cisplatin. It is therefore likely that the reduced accumulation of doxorubicin in tumor cells predicts resistance not only to doxorubicin but also to cisplatin. These results suggest that human osteosarcoma cells may express intrinsic multidrug resistance by the mechanism observed in MOS/IR1 cells.

It may be difficult to overcome such drug resistance. Although the plasma membrane-selective detergents such as saponin and digitonin are known to be effective in increasing membrane permeability, they are highly cytotoxic and their use may be strictly limited to laboratory investigations. One possible method for clinical use may be electroporation, in which direct-current electric pulses result in nonspecific plasma-membrane permeabilization⁵. The use of electric pulses with cytotoxic drugs (electrochemotherapy) has been proven to increase drug accumulation in tumor cells and, thus, to potentiate the antitumor effectiveness of the drugs. This method has been tested in clinical trials on certain cancers, including malignant melanoma and squamous-cell carcinoma²⁵. Although the effect of electrochemotherapy may be influenced by the location and size of the tumor, it may be valuable to apply this method to the treatment of drug-resistant osteosarcoma.

The MOS/IR1 cells had several other interesting features. The cells were composed of plump spindle cells with characteristic, elongated cytoplasmic processes, whereas the MOS/ADR1 cells consisted of polygonal cells. In the MOS/IR1 cells, actin filaments were diffusely spread throughout the cytoplasm. In contrast, the MOS/ADR1 cells had a number of well organized actin stress fibers²⁷. Furthermore, the MOS/IR1 cells exhibited very low levels of alkaline phosphatase activity compared with the MOS/ADR1 cells (Table I). This data suggests that the MOS/IR1 line may consist of a less differentiated cell population compared with the parent and the MOS/ADR1 cells. However, there were no substantial differences in cell kinetics: the parent and the two drug-resistant lines had similar aneuploid DNA indices and similar doubling times in vitro. It would be interesting to investigate the differences in histological characteristics and malignant phenotypes among the cell lines.

In conclusion, we have demonstrated that intrinsic resistance and acquired resistance to multiple drugs in MGH murine osteosarcoma cells may be caused by different cellular mechanisms. Our data suggests that reduced drug accumulation in MOS/IR1 cells is probably due to decreased plasma-membrane permeability and, hence, is a mechanism responsible for intrinsic resistance to multiple drugs. Further study of the resistance mechanism in MOS/IR1 cells may help to overcome intrinsic resistance in human osteosarcoma.

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Introduction | Materials and Methods | Results | Discussion | References

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Table I: In Vitro Characteristics of the Cell Lines

Cell line	IC50 of Doxorubicin* (Î§ per mL)	Pgp	DNA Index	Doubling Time (hrs.)	ALP (Î per min. per mg protein)
MOS	0.022	-	1.41	20.2	1.27
MOS/ADR1	0.160	+	1.37	18.3	2.64
MOS/IR1	0.110	-	1.37	19.6	0.11

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Table I: In Vitro Characteristics of the Cell Lines

Cell line	IC50 of Doxorubicin* (Î§ per mL)	Pgp	DNA Index	Doubling Time (hrs.)	ALP (Î per min. per mg protein)
MOS	0.022	-	1.41	20.2	1.27
MOS/ADR1	0.160	+	1.37	18.3	2.64
MOS/IR1	0.110	-	1.37	19.6	0.11

Bosch, I., and Croop, J.: P-glycoprotein multidrug resistance and cancer. Biochim. Biophys. Acta,1288: 37-F54, 1996.128837 1996

Charles, S. M., and Kenneth, H. C.: Glutathione S-transferase and drug resistance. Cancer Cells,2: 15-22, 1990.215 1990 [PubMed]

Gottesman, M. M., and Pastan, I.: Biochemistry of multidrug resistance mediated by the multidrug transporter. Ann. Rev. Biochem.,62: 385-427, 1993.62385 1993 [PubMed]

Jacob, M. C.; Favre, M.; and Bensa, J. C.: Membrane cell permeabilization with saponin and multiparametric analysis by flow cytometry. Cytometry,12: 550-558, 1991.12550 1991 [PubMed]

Lai, G. M.; Ozols, R. F.; Smith, J. F.; Yong, R. C.; and Hamilton, T. C.: Enhanced DNA repair and resistance to cisplatin in human ovarian cancer. Biochem. Pharmacol.,,37: 4597-4600, 1988.374597 1988

Lowly, O. H.: Micromethods for the assay of enzymes. II. Specific procedures. Alkaline phosphatase. Meth. Enzymol.,4: 371-372, 1957.4371 1957

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Hideyuki Takeshita, Katsuyuki Kusuzaki, Tsukasa Ashihara, Mark C. Gebhardt, Henry J. Mankin, Yasusuke Hirasawa; Intrinsic Resistance to Chemotherapeutic Agents in Murine Osteosarcoma Cells*. The Journal of Bone & Joint Surgery. 2000 Jul;82(7):963-963.

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