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The Journal of Bone and Joint Surgery 82:963 (2000)
© 2000 The Journal of Bone and Joint Surgery, Inc.

Intrinsic Resistance to Chemotherapeutic Agents in Murine Osteosarcoma Cells*

Hideyuki Takeshita, M.D.{dagger}, Katsuyuki Kusuzaki, M.D.{ddagger}, Tsukasa Ashihara, M.D.{ddagger}, Mark C. Gebhardt, M.D.§, Henry J. Mankin, M.D.§ and Yasusuke Hirasawa, M.D.{ddagger}

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.
{dagger}Department of Orthopaedic Surgery, Otsu Municipal Hospital, 2 Chome, Motomiya, Otsu, Shiga Prefecture 520-0804, Japan. E-mail address: h2-take{at}kf6.so-net.ne.jp Please address requests for reprints to H. Takeshita.
{ddagger}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.

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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