Bender R A, Makula D M
Br J Cancer. 1978 Mar;37(3):403-10. doi: 10.1038/bjc.1978.60.
L1210 leukaemia cells were preloaded with [(3)H methotrexate] (MTX) to saturate high-affinity intracellular sites, and were then incubated with [(3)H]MTX to determine the steady-state intracellular MTX concentrations at extracellular concentrations ranging from 10 μM to zero. In addition, incubations to generate incomplete saturation of high-affinity intracellular MTX-binding sites were also carried out. Following determination of the total intracellular MTX, cells were pulsed with deoxyuridine (UdR) and its incorporation into DNA examined to assess the role of exchangeable and bound intracellular MTX on DNA synthesis. Further, cell pellets were disrupted and dihydrofolate reductase (DHFR) activity determined under each experimental condition. Extracellular MTX concentrations in excess of 1 μM depressed UdR incorporation to <2% of control, but incorporation rapidly recovered to 62% of control at the point of MTX-DHFR equivalence, and exceeded control values when all high-affinity sites were not saturated. DHFR activity was undetectable at extracellular MTX concentrations >0.50 μM, and never exceeded 6.09% of control at the "equivalence point" where all high-affinity sites were saturated. When less than 10% of potential inhibitor sites were occupied, enzyme activity increased rapidly, but never reached control. However, 5% of the DHFR activity was sufficient to permit UdR incorporation to continue at 50% of control levels, and UdR incorporation returned to control levels at 20% of the DHFR activity. The relationship between cellular MTX content and DNA synthesis or DHFR activity is sigmoid, suggesting a reversible interaction between enzyme and inhibitor. This lends support to the notion that "free" intracellular MTX is necessary for a maximal antitumour effect, and may explain its role in "high-dose" MTX therapy in man.
将L1210白血病细胞预先用[³H甲氨蝶呤]（MTX）加载以饱和高亲和力细胞内位点，然后用[³H]MTX孵育，以确定细胞外浓度从10μM到零范围内的稳态细胞内MTX浓度。此外，还进行了孵育以产生高亲和力细胞内MTX结合位点的不完全饱和。在测定细胞内总MTX后，用脱氧尿苷（UdR）脉冲细胞并检测其掺入DNA的情况，以评估可交换和结合的细胞内MTX对DNA合成的作用。此外，将细胞沉淀破碎并在每个实验条件下测定二氢叶酸还原酶（DHFR）活性。细胞外MTX浓度超过1μM时，UdR掺入量降至对照的<2%，但在MTX - DHFR等效点时掺入量迅速恢复到对照的62%，当所有高亲和力位点未饱和时超过对照值。细胞外MTX浓度>0.50μM时检测不到DHFR活性，在所有高亲和力位点饱和的“等效点”，其活性从未超过对照的6.09%。当潜在抑制剂位点被占据不到10%时，酶活性迅速增加，但从未达到对照水平。然而，5%的DHFR活性足以使UdR掺入以对照水平的50%继续进行，并且当DHFR活性为20%时，UdR掺入恢复到对照水平。细胞内MTX含量与DNA合成或DHFR活性之间的关系呈S形，表明酶与抑制剂之间存在可逆相互作用。这支持了“游离”细胞内MTX对于最大抗肿瘤作用是必需的这一观点，并可能解释其在人类“高剂量”MTX治疗中的作用。
