Tew K D, O'Brien M, Laing N M, Shen H
Department of Pharmacology, Fox Chase Cancer Center, Philadelphia, PA 19111, USA.
Chem Biol Interact. 1998 Apr 24;111-112:199-211. doi: 10.1016/s0009-2797(97)00162-2.
Maintenance of cellular homeostasis is a critical survival trait when cells are exposed to electrophilic chemicals. Because conjugation and elimination of these toxins is dependent upon sequential and coordinated metabolic pathways, acquired resistance through a gradual adaptive response would rarely be expected to be the consequence of changes in one gene product. Human HT29 colon cancer cells chronically exposed to EA have acquired resistance to the drug. Commensurate with resistance, EA is more effectively conjugated to GSH and effluxed from the resistant cells. Using directed and random (differential display) approaches, a number of detoxification and/or protective gene products have been shown to be expressed at elevated levels. These include gamma-GCS (approximately 3-fold), GST-pi (approximately 3-fold), MRP (approximately 3-fold), NQO1 (approximately 3-fold), DDH (20-fold), and SSP 3521, a transcriptional regulator (approximately 3-fold). Multiple mechanisms contribute to these increases, including enhanced transcriptional rate and prolonged mRNA and protein half lives. Further indications for the involvement of transcriptional regulators is found in HL60 adriamycin-resistant cells which overexpress MRP, GST-pi and gamma-GCS and also have 15-20-fold more DNA-dependent protein kinase. It is possible that this enzyme serves as an early stress response gene which may activate downstream transcription factors. Intriguingly, the catalytic subunit of DNA-dependent protein kinase has a high avidity for [35S]azidophenacyl-GSH. High levels of GSH conjugates indicate cell stress and it would seem reasonable to speculate that DNA-dependent protein kinase may serve as a receiver and transmitter of signals which contribute to drug resistance and maintain cell viability.
当细胞暴露于亲电化学物质时，维持细胞内稳态是一项关键的生存特性。由于这些毒素的结合和消除依赖于连续且协调的代谢途径，因此通过逐渐适应性反应获得的耐药性很少会被认为是一种基因产物变化的结果。长期暴露于依他硝唑（EA）的人HT29结肠癌细胞已获得对该药物的耐药性。与耐药性相一致的是，EA更有效地与谷胱甘肽（GSH）结合并从耐药细胞中流出。使用定向和随机（差异显示）方法，已证明许多解毒和/或保护基因产物的表达水平升高。这些包括γ-谷氨酰半胱氨酸合成酶（γ-GCS，约3倍）、谷胱甘肽S-转移酶π（GST-π，约3倍）多药耐药相关蛋白（MRP，约3倍）、醌氧化还原酶1（NQO1，约3倍）、双脱氢酶（DDH，20倍）以及一种转录调节因子SSP 3521（约3倍）。多种机制导致了这些增加，包括转录速率增强以及mRNA和蛋白质半衰期延长。在耐阿霉素的HL60细胞中发现了转录调节因子参与其中的进一步迹象，这些细胞过表达MRP、GST-π和γ-GCS，并且DNA依赖性蛋白激酶也多15 - 20倍。这种酶有可能作为一种早期应激反应基因，可能激活下游转录因子。有趣的是，DNA依赖性蛋白激酶的催化亚基对[35S]叠氮苯甲酰-GSH具有高亲和力。高水平的GSH结合物表明细胞应激，推测DNA依赖性蛋白激酶可能作为信号的接受者和传递者，这有助于耐药性并维持细胞活力似乎是合理的。
