Grem J L, Voeller D M, Geoffroy F, Horak E, Johnston P G, Allegra C J
National Cancer Institute-Navy Medical Oncology Branch, National Naval Medical Center, Bethesda, Maryland 20889-5105.
Br J Cancer. 1994 Dec;70(6):1075-84. doi: 10.1038/bjc.1994.451.
We examined the cytotoxicity and biochemical effects of the lipophilic antifol trimetrexate (TMQ) in two human colon carcinoma cell lines, SNU-C4 and NCI-H630, with different inherent sensitivity to TMQ. While a 24 h exposure to 0.1 microM TMQ inhibited cell growth by 50-60% in both cell lines, it did not reduce clonogenic survival. A 24 h exposure to 1 and 10 microM TMQ produced 42% and 50% lethality in C4 cells, but did not affect H630 cells. Dihydrofolate reductase (DHFR) and thymidylate synthase were quantitatively and qualitatively similar in both lines. During drug exposure, DHFR catalytic activity was inhibited by > or = 85% in both cell lines; in addition, the reduction in apparent free DHFR binding capacity (< or = 20% of control), depletion of dTTP, ATP and GTP pools and inhibition of [6-3H]deoxyuridine incorporation into DNA were similar in C4 and H630 cells. TMQ produced a more striking alteration of the pH step alkaline elution profile of newly synthesised DNA in C4 cells compared with 630 cells, however, indicating greater interference with DNA chain elongation or more extensive DNA damage. When TMQ was removed after a 24 h exposure to 0.1 microM, recovery of DHFR catalytic activity and apparent free DHFR binding sites was evident over the next 24-48 h in both cell lines. With 1 and 10 microM, however, persistent inhibition of DHFR was evident in C4 cells, whereas DHFR recovered in H630 cells. These data suggest that, although DHFR inhibition during TMQ exposure produced growth inhibition, DHFR catalytic activity 48 h after drug removal was a more accurate predictor of lethality in these two cell lines. Several factors appeared to influence the duration of DHFR inhibition after drug removal, including initial TMQ concentration, declining cytosolic TMQ levels after drug removal, the ability to acutely increase total DHFR content and the extent of TMQ-mediated DNA damage. The greater sensitivity of C4 cells to TMQ-associated lethality may be attributed to the greater extent of TMQ-mediated DNA damage and more prolonged duration of DHFR inhibition after drug exposure.
我们研究了亲脂性抗叶酸药三甲曲沙（TMQ）对两种对TMQ固有敏感性不同的人结肠癌细胞系SNU - C4和NCI - H630的细胞毒性及生化效应。虽然在两种细胞系中，24小时暴露于0.1微摩尔/升的TMQ均使细胞生长抑制50 - 60%，但并未降低克隆形成存活率。24小时暴露于1微摩尔/升和10微摩尔/升的TMQ分别使C4细胞产生42%和50%的致死率，但对H630细胞无影响。两种细胞系中的二氢叶酸还原酶（DHFR）和胸苷酸合成酶在数量和质量上相似。在药物暴露期间，两种细胞系中DHFR的催化活性均被抑制≥85%；此外，C4细胞和H630细胞中表观游离DHFR结合能力的降低（≤对照的20%）、dTTP、ATP和GTP池的消耗以及[6 - ³H]脱氧尿苷掺入DNA的抑制情况相似。然而，与630细胞相比，TMQ使C4细胞中新合成DNA的pH步移碱性洗脱图谱发生了更显著的改变，这表明对DNA链延伸的干扰更大或DNA损伤更广泛。当在24小时暴露于0.1微摩尔/升后去除TMQ时，在接下来的24 - 48小时内，两种细胞系中DHFR催化活性和表观游离DHFR结合位点均明显恢复。然而，对于1微摩尔/升和10微摩尔/升的情况，C4细胞中DHFR持续受到抑制，而H630细胞中的DHFR得以恢复。这些数据表明，虽然TMQ暴露期间对DHFR的抑制导致了生长抑制，但去除药物48小时后DHFR的催化活性是这两种细胞系中致死率更准确的预测指标。几个因素似乎影响了去除药物后DHFR抑制的持续时间，包括初始TMQ浓度、去除药物后胞质中TMQ水平的下降、急性增加总DHFR含量的能力以及TMQ介导的DNA损伤程度。C4细胞对TMQ相关致死性的更高敏感性可能归因于TMQ介导的DNA损伤程度更大以及药物暴露后DHFR抑制持续时间更长。
