Transport of a fluorescent antifolate by methotrexate-sensitive and methotrexate-resistant human leukemic lymphoblasts.
Transport of a fluorescent antifolate by methotrexate-sensitive and methotrexate-resistant human leukemic lymphoblasts.
复制标题
甲氨蝶呤敏感和甲氨蝶呤抗性人白血病淋巴母细胞转运荧光抗叶酸剂。
DOI：
10.1016/0006-2952(86)90541-1
复制
发表时间：
1986
影响因子：
5.8
通讯作者：
Frei3rd,E
中科院分区：
文献类型：
--
作者：
Rosowsky,A;Wright,JE;Cucchi,CA;Boeheim,K;Frei3rd,E
自从最初报道[1]由甲氨蝶呤（MTX）、异硫氰酸荧光素（FITC）和1，5-戊二胺合成MTX-F以来，对二氢叶酸还原酶（DHFR*）的荧光抑制剂的兴趣已经增长了十年。结果表明，MTX-F可作为一种内示踪标记物。f~ r* D~，这导致该化合物在通过流式细胞术研究MTX抗性细胞中DHFR过量产生中的令人印象深刻的用途14-81。在我们的实验室中由FITC和MTX 19.101的lvsine类似物合成了一种相关但结构更简单的化合物PI'430（图1）。其它荧光DHFk配体通过MTX的赖氨酸和鸟氨酸类似物与丹磺酰氯反应而间接合成[H-14]。已经用PT 430作为荧光标记物进行了流式细胞术研究[9，10，15，16]，但尚未报道丹磺酰衍生物。在本文中，我们提出了一种改进的PT 430合成方法，并描述了MTX敏感的人白血病淋巴母细胞（CEM细胞）[17]和具有正常DHFR水平但MTX转运明显缺陷的耐药亚系（CEM/MTX）对其摄取的流式细胞术测量[181]。还比较了MTX对这些细胞摄取PT 430的影响。我们的结果表明，MTX转运机制缺陷的细胞可以通过与PT 430孵育后的流式细胞术分析与其他正常策勒区分开来。缩写：DHFR，氢叶酸还原酶（EC 1.5. 1.3）; MTX，甲氨蝶呤，4-氨基和脱氧-NiO-甲基蝶酰基+谷氨酸; MTX-F，荧光素-二氨基戊烷-甲氨蝶呤; PT 430，N#-（4-氨基-4-脱氧-~ 0-甲基蝶酰基）-Ne-（4 '-荧光素-硫代氨基甲酰基）-r_赖氨酸; FITC，异硫氰酸荧光素; DEAE-纤维素，N，N-二乙基氨基乙基纤维素; FBS，胎牛血清; DME，Dulbecco改良的Eagle培养基;和PBS，磷酸盐缓冲盐水。
Interest in fluorescent inhibitors of dihydrofolate reductase (DHFR*) has grown over the decade since the original report [l] of the synthesis of MTX-F from methotrexate (MTX), fluorescein isothiocyanate (FITC), and 1, 5-pentanediamine. It was shown 12.31 that MTX-F could serve as an intrace~ ular marker. f~ r* D~, and this led to impressive uses of the compound in studies of DHFR overproduction in MTX-resistant cells by flow cytometry 14-81. A compound of related but simpler structure, PI’430 (Fig: 1), was synthesized in our laboratory from FITC and the lvsine analoeue of MTX 19.101. other fluorescent DHFk ligands w&e synthesized mdepkndently by reaction of the lysine and ornithine analogues of MTX with dansyl chloride [H-14]. Flow cytometric studies have been performed with PT430 as the fluorescent marker [9, 10, 15, 16], but have not been reported for the dansyl derivatives. In this paper, we present an improved synthesis of PT430 and describe flow cytometric measurements of its uptake by MTX-sensitive human leukemic lymphoblasts (CEM cells)[17] and a resistant subline (CEM/MTX) with normal DHFR levels but a marked defect in MTX transport [181. The influence of MTX on PT430 uptake by these cells is also compared. Our results demonstrate that cells with a defect in their transport mechanism for MTX can be distinguished from otherwise normal celle by flow cytometric analysis following incubation with PT430.* Abbre~ ations: DHFR,~ hydrofolate reductase (EC 1.5. 1.3); MTX, methotrexate, 4-amino&deoxy-NiO-methylpteroyl+ glutamic acid; MTX-F, fluorescein-diaminopentane-methotrexate; PT430, N#-(4-amino-4-deoxy-~ 0-methylpteroyl)-Ne-(4’-fluore~ ceinthiocarbamyl)-r_-lysine; FITC, fluorescein isothiocyanate; DEAE-cellulose, iV, N-diethylaminoethylcellulose; FBS, fetal bovine serum; DME, Dulbecco’s modified Eagle’s medium; and PBS, phosphate-buffered saline.