Impairment of methotrexate (MTX)-polyglutamate formation of MTX-resistant K562 cell lines.
Impairment of methotrexate (MTX)-polyglutamate formation of MTX-resistant K562 cell lines.
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DOI：
10.1111/j.1349-7006.1988.tb01549.x
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发表时间：
1988-11
期刊：
影响因子：
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通讯作者：
Koizumi S
中科院分区：
文献类型：
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作者：
Koizumi S
我们检测了5个耐甲氨蝶呤(MTX)的K562细胞亚系对MTX的耐药机制。根据克隆形成试验，这些抗甲氨蝶呤克隆的IC50为10到40μMmTx，表明与亲本细胞系相比，抗性是2,000到5,000倍。这些耐MTX的K562细胞株的倍增时间(27-60小时)长于亲本K562细胞株(24小时)。抗性细胞1小时的MTX积累量是亲本细胞的70-80%。为研究甲氨蝶呤多聚谷氨酰胺(MTX-PG)的形成，用~3H-甲氨蝶呤(1或10μM)在胸腺嘧啶核苷和脱氧肌苷存在下与耐药细胞孵育24小时以防止细胞毒性。用高压液相色谱技术分析MTX(-Glu1)和多聚谷氨酸代谢产物(MTX-Glu2、-Glu3、-Glu4和-Glu5)。经10μ甲氨蝶呤作用24小时后，细胞内甲氨蝶呤总浓度达39~89nmoL/g蛋白，仅为亲代K562细胞甲氨蝶呤浓度的20~40%。高效液相色谱分析显示，在5株MTX耐药的K562细胞中，MTX-PGs(MTX-Glu3、-Glu4和-Glu5)的大分子形式不到2%，而亲本细胞中MTX-Glu3-5占细胞内总MTX的46%。这些数据表明，MTX-PG形成的损害和转运的改变，可能是人类白血病细胞对这种药物产生高水平耐药性的一种特殊机制。
We examined the mechanism of methotrexate (MTX) resistance in five K562 cell subclones resistant to MTX. Based on a clonogenic assay, the IC50s of these MTX‐resistant clones were 10 to 40μMMTX, indicating 2,000 to 5,000‐fold resistance as compared to that of the parent cell line. The doubling times of these MTX‐resistant K562 cell lines are longer (27–60 hr) than that of the parent K562 cell line (24 hr). One‐hour MTX accumulation in the resistant cells was 70–80% of that in parent cells. To investigate the formation of MTX‐polyglutamates (MTX‐PGs), resistant cells were incubated with 3H‐MTX (1 or 1OμM) for 24 hr in the presence of thymidine and deoxyinosine to prevent cytotoxicity. MTX (‐Glu1) and the polyglutamate metabolites (MTX‐Glu2, ‐Glu3, ‐Glu4 and ‐Glu5) were analyzed by a high‐pressure liquid chromatography (HPLC) technique. After a 24‐hr incubation with 10μM MTX, the total concentration of intracellular MTX reached 39 to 89 nmol/g protein, only 20 to 40% of the MTX level of the parent K562 cells. The HPLC analysis revealed that less than 2% of intracellular MTX was in the form of high‐molecular MTX‐PGs (MTX‐Glu3, ‐Glu4 and ‐Glu5) in the five MTX‐resistant K562 cell lines, while in the parent cells MTX‐Glu3–5 comprised 46% of the total intracellular MTX. These data indicate the possibility that impairment of MTX‐PG formation, with transport alteration, may be a special mechanism for the high level of resistance to this agent in human leukemic cells.