Sosinski J, Thakar J H, Germain G S, Harwood F C, Houghton P J
Department of Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, Memphis, TN 38101.
Biochem Pharmacol. 1993 May 25;45(10):2135-42. doi: 10.1016/0006-2952(93)90027-t.
The mechanism(s) by which antitumor diarylsulfonylureas (DSU) cause cytotoxicity has been examined in GC3/c1 human colon adenocarcinoma cells and a subline selected for resistance to N-(5-indanylsulfonyl)-N'-(4-chlorophenyl)urea (ISCU). Resistance was stable in the absence of selection pressure. This mutant (designated LYC5) was 5.5-fold resistant to ISCU compared to parental GC3/c1 cells in serum containing medium when cells were exposed for 7 days. In contrast, LYC5 cells were not resistant to a 4-hr exposure to ISCU. These data indicated two possible mechanisms of action, dependent on concentration and time of exposure to ISCU. Proliferation-dependent and -independent mechanisms of cytotoxicity were identified in wild-type and resistant clones. In serum-free medium containing growth factors, the IC50 for parental cells was 0.51 microM and for LYC5 7.0 microM (13.6-fold resistance), whereas without growth factors both lines were 8- to 9-fold resistant relative to conditions of cellular proliferation. Accumulation of ISCU was similar in quiescent and proliferating cells, and was reduced only slightly in resistant LYC5 cells. Analysis of DNA by agarose gel electrophoresis showed that in GC3/c1 cells nucleosomal ladders were formed only when proliferating cells were exposed to ISCU. No nucleosomal ladders were detected in quiescent cells during exposure to toxic concentrations of drug (IC90), or after removal of ISCU and addition of serum to stimulate growth. These data indicate several mechanisms by which diarylsulfonylurea antitumor agents may cause cell death. In serum-free medium at very high concentration (IC50 approximately 370 microM) for short periods of exposure (4 hr), cytotoxicity was proliferation independent, and GC3/c1 and LYC5 cells were equally sensitive. This mechanism may relate to the uncoupling activity of ISCU. However, at pharmacological relevant concentrations, the primary mechanism was proliferation dependent and led to formation of nucleosomal DNA ladders (IC50 approximately 0.5 microM). A possible additional mechanism occurred at higher concentration (IC50 approximately 7 microM) in quiescent cells, and was not associated with DNA degradation.
已在GC3/c1人结肠腺癌细胞和一个对N-(5-茚满基磺酰基)-N'-(4-氯苯基)脲（ISCU）具有抗性的亚系中研究了抗肿瘤二芳基磺酰脲（DSU）引起细胞毒性的机制。在没有选择压力的情况下，抗性是稳定的。当细胞在含血清培养基中暴露7天时，与亲本GC3/c1细胞相比，这个突变体（命名为LYC5）对ISCU的抗性高5.5倍。相反，LYC5细胞对4小时的ISCU暴露没有抗性。这些数据表明了两种可能的作用机制，这取决于ISCU的浓度和暴露时间。在野生型和抗性克隆中鉴定出了细胞毒性的增殖依赖性和非依赖性机制。在含有生长因子的无血清培养基中，亲本细胞的IC50为0.51微摩尔，LYC5为7.0微摩尔（抗性为13.6倍），而在没有生长因子的情况下，相对于细胞增殖条件，两个细胞系的抗性均为8至9倍。ISCU在静止细胞和增殖细胞中的积累相似，并且在抗性LYC5细胞中仅略有减少。通过琼脂糖凝胶电泳分析DNA表明，在GC3/c1细胞中，仅当增殖细胞暴露于ISCU时才形成核小体梯带。在暴露于毒性浓度药物（IC90）期间，或在去除ISCU并添加血清以刺激生长后，在静止细胞中未检测到核小体梯带。这些数据表明了二芳基磺酰脲抗肿瘤剂可能导致细胞死亡的几种机制。在无血清培养基中，以非常高的浓度（IC50约为370微摩尔）进行短时间暴露（4小时）时，细胞毒性是增殖非依赖性的，GC3/c1和LYC5细胞同样敏感。这种机制可能与ISCU的解偶联活性有关。然而，在药理学相关浓度下，主要机制是增殖依赖性的，并导致核小体DNA梯带的形成（IC50约为0.5微摩尔）。在静止细胞中，在较高浓度（IC50约为7微摩尔）下可能发生另一种机制，并且与DNA降解无关。
