Diddens H, Niethammer D, Jackson R C
Cancer Res. 1983 Nov;43(11):5286-92.
Methotrexate (MTX)-resistant sublines of malignant human cells were selected in vitro by stepwise increase in drug concentration in the medium. By this procedure a subline of Burkitt's lymphoma cells (RAJI) was made 290-fold resistant (RAJI/MTX-R), T-cell leukemia cells (CCRF-CEM) were obtained 210-fold resistant (CEM/MTX-R), and 3 MTX-resistant human osteosarcoma lines were selected: TE-85/MTX-R (19-fold resistant; relative to wild-type); MG-63/MTX-R (8-fold resistant); and SAOS-2/MTX-R (200-fold resistant). We also studied a B-cell lymphoblastoid line, WI-L2/m4, that was 13,000-fold resistant. Assay of cellular dihydrofolate reductase (DHFR) showed the following pattern of activity in resistant cell lines, relative to parental cell activity: RAJI/MTX-R, 550-fold increased; CEM/MTX-R, unchanged; TE-85/MTX-R, 4-fold increased; MG-63/MTX-R, 6-fold increased; SAOS-2/MTX-R, unchanged; and WI-L2/m4, 110-fold increased. Measurement of MTX membrane transport showed decreased uptake in CEM/MTX-R and SAOS-2/MTX-R, relative to parental cell lines. The other DHFR-overproducing cells all gave normal initial MTX uptake rates but increased total uptake. The DHFR-overproducing lines all had significant cross-resistance to both metoprine and trimetrexate; the two lines with defective MTX transport were not cross-resistant, and the CEM/MTX-R cells showed collateral sensitivity to these agents. Only minor cross-resistance to homofolic acid was found in all MTX-resistant lines. The highly MTX-resistant RAJI/MTX-R and WI-L2/m4 cells showed minor cross-resistance to the dual inhibitor of thymidylate synthetase and DHFR, CB3717 (5- and 15-fold, respectively). These studies demonstrated that, depending upon the mechanism of resistance, MTX-resistant human tumor cells may be effectively killed by antifolates with different routes of uptake into cells, or with a different enzyme target. Thus, there are at least three functionally distinct classes of folate antagonist with antitumor activity.
通过逐步提高培养基中药物浓度，在体外筛选出对甲氨蝶呤（MTX）耐药的人恶性细胞亚系。通过此方法，伯基特淋巴瘤细胞亚系（RAJI）产生了290倍耐药性（RAJI/MTX-R），T细胞白血病细胞（CCRF-CEM）产生了210倍耐药性（CEM/MTX-R），并筛选出3种耐MTX的人骨肉瘤细胞系：TE-85/MTX-R（19倍耐药；相对于野生型）；MG-63/MTX-R（8倍耐药）；以及SAOS-2/MTX-R（200倍耐药）。我们还研究了一种B细胞淋巴母细胞系WI-L2/m4，其耐药性达13000倍。对细胞二氢叶酸还原酶（DHFR）的检测显示，相对于亲代细胞活性，耐药细胞系中的酶活性呈现以下模式：RAJI/MTX-R，增加550倍；CEM/MTX-R，无变化；TE-85/MTX-R，增加4倍；MG-63/MTX-R，增加6倍；SAOS-2/MTX-R，无变化；WI-L2/m4，增加110倍。对MTX膜转运的检测显示，相对于亲代细胞系，CEM/MTX-R和SAOS-2/MTX-R中MTX的摄取减少。其他DHFR过度表达的细胞初始MTX摄取率均正常，但总摄取量增加。DHFR过度表达的细胞系对甲氨蝶呤和三甲曲沙均有显著的交叉耐药性；MTX转运缺陷的两个细胞系无交叉耐药性，且CEM/MTX-R细胞对这些药物表现出间接敏感性。在所有耐MTX的细胞系中，仅发现对同型叶酸有轻微交叉耐药性。高度耐MTX的RAJI/MTX-R和WI-L2/m4细胞对胸苷酸合成酶和DHFR的双重抑制剂CB3717表现出轻微交叉耐药性（分别为5倍和15倍）。这些研究表明，根据耐药机制的不同，耐MTX的人肿瘤细胞可能会被通过不同细胞摄取途径或针对不同酶靶点的抗叶酸药物有效杀死。因此，至少有三类功能不同的具有抗肿瘤活性的叶酸拮抗剂。
