Montine T J, Borch R F
Department of Pharmacology, University of Rochester School of Medicine and Dentistry, New York 14642.
Cancer Res. 1988 Nov 1;48(21):6017-24.
Confluent LLC-PK1 cells express many characteristics of renal proximal tubule epithelia. We report here the use of this cell line to investigate the possible mechanisms of cis-diamminedichloroplatinum(II) (DDP)-induced nephrotoxicity and diethyldithiocarbamate (DDTC) amelioration. Cells were exposed to platinum-based drug for 1 h and then incubated in drug-free medium until assayed. There was a 10-h delay between DDP exposure and onset of toxicity which then continued to develop. Viability at 72 h was 97 +/- 2 (SD), 68 +/- 3, 33 +/- 3, and 10 +/- 2% of control after treatment with 100, 200, 300, and 400 microM DDP, respectively. trans-Diamminedichloroplatinum(II) was 5-fold less toxic than DDP and diammine(1,1-cyclobutanedicarboxylato)platinum(II) (CBDCA) was nontoxic at concentrations up to 2.0 mM. Incubation of cells with DDTC (3 mM) for 1 h immediately following DDP exposure resulted in chelation of 43% of total intracellular platinum by DDTC and significantly increased 72 h viability; 97 +/- 2, 88 +/- 3, and 42 +/- 2% of control for 200, 300, and 400 microM DDP, respectively. DDP treatment of L1210 cells yielded equivalent total intracellular platinum levels, but Pt(DDTC)2 concentrations were one-tenth those in LLC-PK1 cells after subsequent treatment with DDTC (3 mM). Immediate reduction of protein and RNA, but not DNA, synthesis by DDP was concentration dependent over the same range as viability. DDP-induced increases in unscheduled DNA synthesis also did not correlate with cytotoxicity. The inhibition of protein synthesis was unchanged by pretreatment with the RNA synthesis inhibitor actinomycin D.DDTC (3 mM) exposure produced an immediate and persistent DDP dose modification of 1.6 in protein but not RNA synthesis. CBDCA (0.5 to 1.0 mM) had no effect on protein, RNA, or DNA synthesis and only slight stimulatory effects on unscheduled DNA synthesis. DDTC alone (3-3000 microM) caused significant reduction in DNA synthesis and unscheduled DNA synthesis. Neither sodium thiosulfate nor 2-mercaptoethane-sulfonate had any effect on DDP-induced cytotoxicity or inhibition of protein, RNA, or DNA synthesis when incubated immediately after DDP, even though these drugs achieved intracellular concentrations at which DDTC was protective. These data indicate that quiescent LLC-PK1 cells are a good in vitro model for the study of DDP-induced nephrotoxicity and its modulation by thiol rescue agents and that DDP inhibition and DDTC rescue of posttranscriptional processes correlate best with viability in LLC-PK1 cells.
汇合的 LLC-PK1 细胞表达肾近端小管上皮细胞的许多特征。我们在此报告使用该细胞系研究顺二氯二氨铂（II）（DDP）诱导的肾毒性及二乙基二硫代氨基甲酸盐（DDTC）改善作用的可能机制。细胞暴露于铂类药物 1 小时，然后在无药物培养基中孵育直至检测。DDP 暴露与毒性发作之间有 10 小时延迟，之后毒性继续发展。用 100、200、300 和 400 μM DDP 处理后，72 小时的细胞活力分别为对照的 97±2（标准差）、68±3、33±3 和 10±2%。反式二氯二氨铂（II）的毒性比 DDP 低 5 倍，二氨（1,1 - 环丁烷二羧酸根）铂（II）（CBDCA）在浓度高达 2.0 mM 时无毒。DDP 暴露后立即用 DDTC（3 mM）孵育细胞 1 小时，导致 DDTC 螯合了细胞内总铂的 43%，并显著提高了 72 小时的细胞活力；200、300 和 400 μM DDP 处理后的细胞活力分别为对照的 97±2、88±3 和 42±2%。用 DDP 处理 L1210 细胞后，细胞内总铂水平相当，但后续用 DDTC（3 mM）处理后，LLC-PK1 细胞中 Pt(DDTC)2 的浓度是 L1210 细胞中的十分之一。在与细胞活力相同的范围内，DDP 对蛋白质和 RNA 合成的即时抑制（但对 DNA 合成无抑制）呈浓度依赖性。DDP 诱导的非计划 DNA 合成增加也与细胞毒性无关。用 RNA 合成抑制剂放线菌素 D 预处理对蛋白质合成的抑制无影响。DDTC（3 mM）暴露对蛋白质合成产生即时且持续的 DDP 剂量修正因子 1.6，但对 RNA 合成无影响。CBDCA（0.5 至 1.0 mM）对蛋白质、RNA 或 DNA 合成无影响，对非计划 DNA 合成仅有轻微刺激作用。单独的 DDTC（3 - 3000 μM）导致 DNA 合成和非计划 DNA 合成显著减少。DDP 处理后立即孵育时，硫代硫酸钠和 2 - 巯基乙烷磺酸盐对 DDP 诱导的细胞毒性或蛋白质、RNA 或 DNA 合成抑制均无任何影响，尽管这些药物达到了 DDTC 具有保护作用的细胞内浓度。这些数据表明，静止的 LLC-PK1 细胞是研究 DDP 诱导的肾毒性及其由硫醇救援剂调节的良好体外模型，并且 DDP 对转录后过程的抑制和 DDTC 的救援与 LLC-PK1 细胞的活力最相关。
