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Published on: December 14, 2019
[Primary study on establishment of namalwa 12/MTX resistant cell strain and its mechanism]
Jing Chen1, Long-jun Gu, Da-ming Ying
1Shanghai Xinhua Hospital, Shanghai Children's Medical Center, Shanghai Second Medical University, Shanghai 200092, P. R. China. zzhangd@online.sh.cn
Background & Objective:
Methotrexate has been used to treat malignant tumor for 50 years. Different mechanisms of methotrexate resistance has been known in various kinds of leukemias. Because of individualized chemotherapy according to different drug metabolism in different leukemia patients, the treatment became more reasonable and scientific and the treatment effect has been gradually improved. However, there was few report about its drug resistance mechanism in lymphoma. This study was designed to establish Burkitt's lymphoma methotrexate resistant cell strain--Namlwa 12/MTX and study its resistant mechanism.
Methods:
Namlwa 12/MTX resistant cell strain was established by repeated impulsed exposure to methotrexate. The difference of the methotrexate effect on Namlwa 12/MTX and Namlwa cell strains were evaluated with MTT method. The level of dihydrofolate reductase (DHFR) mRNA expression was assayed with RT-PCR. The function of the reduced folate carrier (RFC) were tested with 3H-MTX labeling and counted with beta-liquid scintillation counter. The capacity to form the polyglutamate methotrexate (MTXPG) in these cell strains was assayed with 3H-MTX labeling and high pressure liquid chromatography (HPLC) separation.
Results:
More than 20 times methotrexate resistance was found in Namalwa 12/MTX cell strain in comparison with the Namalwa cell strain. This resistance was not associated with the dysfunction of the RFC, but was closely associated with the amount of MTXPG formed. The amount of total MTXPG (MTXPG1-6) formed in these 2 cell strains was (1583 +/- 26) pmol/10(9) tumor cells and (4453 +/- 236) pmol/10(9) tumor cells, respectively (P < 0.05), while the amount of long chain MTXPG (MTXPG4-6) formed only accounted for 5.5% and 25.4% of the MTXPG1-6, respectively (P < 0.05). The level of DHFR mRNA expression was gradually increased with the formation of the drug resistance in Namalwa 12/MTX.
Conclusion:
Synthesizing difficulty of MTXPG and over-expression of DHFR mRNA level result in methotrexate resistance in Namalwa 12/MTX cell strain.
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