Matherly L H, Angeles S M, Czajkowski C A
Developmental Therapeutics Program, Michigan Cancer Foundation, Detroit 48201.
J Biol Chem. 1992 Nov 15;267(32):23253-60.
An earlier report (Matherly, L. H., Czajkowski, C. A., and Angeles, S. M. (1991) Cancer Res. 51, 3420-3426) described a K562 human erythroleukemia line (K562.4CF), characterized by an elevated uptake capacity for methotrexate (MTX) and 5-formyltetrahydrofolate, and the identification of a highly glycosylated membrane transporter (GP-MTX) by radioaffinity labeling with N-hydroxysuccinimide [3H] methotrexate. In the present study, radioaffinity-labeled GP-MTX from K562.4CF cells was isolated by Ricinus communis agglutinin I-agarose affinity chromatography, coupled with gel filtration and preparative electrophoresis. Antiserum to the purified, radio-labeled protein was raised in a rabbit and screened by immunoblot analysis of K562.4CF plasma membrane proteins or purified GP-MTX. The antiserum detected a broad GP-MTX band centered at 92 kDa on 7.5% gels. On 4-10% gels, the apparent molecular mass for GP-MTX shifted to 99 kDa. Antiserum specificity was established by quantitatively converting the immunoreactive glycoprotein in plasma membrane homogenates to its N- and O-deglycosylated forms with N- and O-glycanases, respectively. Whereas the methotrexate uptake capacity of K562.4CF cells was elevated 6.1-fold over parental cells, the GP-MTX content on immunoblots was increased approximately 3-fold. For two methotrexate-resistant K562 lines (33- and 70-fold), decreased drug uptake (28 and 18% of parental levels) closely correlated with their reduced GP-MTX contents. A GP-MTX isoform was also detected on immunoblots of membrane proteins from CCRF-CEM human lymphoblastic leukemia cells. With a transport-impaired CCRF-CEM line (13% of wild type uptake), an aberrant electrophoretic migration for GP-MTX was observed, establishing the presence of structural modifications in the transport protein. These structural differences were independent of carrier glycosylation since they were detected following the glycosidase treatments. These findings implicate important roles for distinct carrier-specific alterations in the expression of diminished drug transport in methotrexate-resistant human tumor cells.
较早的一份报告（Matherly, L. H., Czajkowski, C. A., and Angeles, S. M. (1991) Cancer Res. 51, 3420 - 3426）描述了一种K562人红白血病细胞系（K562.4CF），其特征在于对甲氨蝶呤（MTX）和5 - 甲酰四氢叶酸的摄取能力增强，并且通过用N - 羟基琥珀酰亚胺[3H]甲氨蝶呤进行放射亲和标记鉴定出一种高度糖基化的膜转运蛋白（GP - MTX）。在本研究中，通过蓖麻凝集素I - 琼脂糖亲和层析，结合凝胶过滤和制备电泳，从K562.4CF细胞中分离出放射亲和标记的GP - MTX。用纯化的放射性标记蛋白免疫家兔制备抗血清，并通过对K562.4CF质膜蛋白或纯化的GP - MTX进行免疫印迹分析来筛选。该抗血清在7.5%的凝胶上检测到一条以92 kDa为中心的宽的GP - MTX条带。在4 - 10%的凝胶上，GP - MTX的表观分子量移至99 kDa。通过分别用N - 糖苷酶和O - 糖苷酶将质膜匀浆中的免疫反应性糖蛋白定量转化为其N - 去糖基化和O - 去糖基化形式，确定了抗血清的特异性。虽然K562.4CF细胞的甲氨蝶呤摄取能力比亲代细胞提高了6.1倍，但免疫印迹上的GP - MTX含量增加了约3倍。对于两种耐甲氨蝶呤的K562细胞系（分别为33倍和70倍耐药），药物摄取减少（分别为亲代水平的28%和18%）与其GP - MTX含量降低密切相关。在CCRF - CEM人淋巴细胞白血病细胞的膜蛋白免疫印迹上也检测到一种GP - MTX同工型。对于一种转运功能受损的CCRF - CEM细胞系（摄取能力为野生型的13%），观察到GP - MTX出现异常的电泳迁移，表明转运蛋白存在结构修饰。这些结构差异与载体糖基化无关，因为它们在糖苷酶处理后仍能被检测到。这些发现表明，在耐甲氨蝶呤的人肿瘤细胞中，不同的载体特异性改变在药物转运减少的表达中起重要作用。
