MECHANISM OF DRUG RESISTANCE OF METHOTREXATE IN OSTEOSARCOMA CELLS

Research Project

Project/Area Number	06671442
Research Category	Grant-in-Aid for General Scientific Research (C)
Allocation Type	Single-year Grants
Research Field	Orthopaedic surgery
Research Institution	NIIGATA UNIVERSITY SCHOOL OF MEDICINE
Principal Investigator	TAKAHASHI Hideaki NIIGATA UNIVERSITY SCHOOL OF MEDICINE,DEPARTMENT OF ORTHOPAEDIC SURGERY,PROFESSOR, 医学部, 教授 (50018397)
Co-Investigator(Kenkyū-buntansha)	HOTTA Tetsuo NIIGATA UNIVERSITY SCHOOL OF MEDICINE,DEPARTMENT OF ORTHOPAEDIC SYRGERY,INSTRUCT, 医学部, 助手 (00272815)
Project Period (FY)	1994 – 1995
Project Status	Completed (Fiscal Year 1995)
Budget Amount *help	¥2,100,000 (Direct Cost: ¥2,100,000) Fiscal Year 1995: ¥700,000 (Direct Cost: ¥700,000) Fiscal Year 1994: ¥1,400,000 (Direct Cost: ¥1,400,000)
Keywords	OSTEOSARCOMA / CELL CULTURE / DRUG RESISTANCE / METHOTREXATE / DIHYDROFOLATE REDUCTASE / ENZYME INDUCTION
Research Abstract	An basic research of drug resistance of methotrexate (MTX) in osteosarcoma cells was performed on the hypothesis of induction of dihydofolate reductase, which was inhibitted by MTX.MTX-resistant human osteosarcoma cell line designated NOS-1 and MTX-sensitive mouse osteosarcoma cell line designated Dunn were used for this study. At first, the sensitivity against MTX was studied in several concentration of MTX.Dunn osteosarcoma cells were completely killed by the treatment of 5 mug/ml of MTX.On the other hand, NOS-1 cells could not be killed at the same dose, but showed slight growth inhibition. G_2 block was observed in the growth kinetic analysis by flow cytometry. The expression of p-glycoprotein, multidrug reisistant protein, was also observed in MOS-1 cells. The enzyme activity of cell extracts was measured by a spectrophotometory using MTX assay kit, (YATRON). MOS-1 cells showed from 1.5 times to 2 times of enzyme activity compared with Dunn cells. But this assay system was quite unstable and the reproducibility was very low. So now we are continuting to measure the activity over and over. Simultaneously, we are checking reagents of assay system. Anyway, two possibility of active drug clearance and induction of enzyme were suggested for the mechanisum of drug resistance of MTX in osteosarcoma cells. We are going to measure the concentlation of MTX in both MOS-1 and Dunn osteosarcoma cells after treatment of MTX.It should clarify the possibility of active clearance of MTX.