Weidner D A, Sommadossi J P
Department of Pharmacology, Center for AIDS Research, Birmingham, Alabama.
Mol Pharmacol. 1990 Dec;38(6):797-804.
We previously demonstrated that 3'-azido-3'-deoxythymidine (AZT) inhibits proliferation of human bone marrow progenitor cells in vitro and that incorporation of AZT into nuclear DNA may be one mechanism responsible for AZT-induced bone marrow toxicity [Antimicrob. Agents Chemother. 31:452-454 (1987); Mol. Pharmacol. 36:9-14 (1989)]. The present study explores possible genetic mechanisms involved in AZT-induced anemia by evaluating the effects of AZT on globin gene expression at both the transcriptional and the translational levels in butyric acid-induced K-562 human erythroleukemia cells. AZT, at concentrations ranging from 10 to 250 microM, was added to cells 25 hr after initiation of induction of hemoglobin (Hb) synthesis with 1.4 mM butyric acid. Hb synthesis, as measured by benzidine staining, was inhibited by AZT in a dose- and time-dependent manner in these cells. AZT inhibition of cell growth was not the major contributing factor in the net inhibition of Hb synthesis in K-562 cells. As assessed by Northern blot analysis, AZT inhibition of Hb synthesis was associated with a decrease in globin mRNA steady state levels without inhibition of total RNA synthesis or actin mRNA steady state levels. In particular, a decrease of globin mRNA levels of 23% by 25 microM AZT was observed, reaching a maximum inhibition of 59% in the presence of 250 microM AZT. In vitro translation experiments demonstrated that essentially all nonglobin translatable mRNAs were not inhibited by AZT concentrations as high as 250 microM, whereas globin mRNAs coding for epsilon, zeta, A gamma, G gamma, and alpha chains were substantially inhibited to similar levels by AZT, in a dose-dependent manner. Transcriptional run-on studies with isolated nuclei from AZT-treated K-562 cells demonstrated a 20 and 50% inhibition of in vitro synthesized globin transcripts from cells exposed to 25 and 100 microM AZT, respectively. 2',3'-Dideoxycytidine also inhibited K-562 cell growth in the same concentration range as AZT but, of importance, had no effects on Hb production. These data suggest that inhibition of globin gene expression may play a role in the cytotoxicity of AZT to the erythroid cell.
我们先前已证明，3'-叠氮-3'-脱氧胸苷（AZT）在体外可抑制人骨髓祖细胞的增殖，且AZT掺入核DNA可能是其所致骨髓毒性的一种机制[《抗菌剂与化疗》31:452 - 454(1987);《分子药理学》36:9 - 14(1989)]。本研究通过评估AZT对丁酸诱导的K - 562人红白血病细胞中珠蛋白基因在转录和翻译水平上的影响，探讨AZT诱导贫血可能涉及的遗传机制。在用1.4 mM丁酸诱导血红蛋白（Hb）合成25小时后，将浓度范围为10至250 microM的AZT添加到细胞中。通过联苯胺染色测定，在这些细胞中，AZT以剂量和时间依赖性方式抑制Hb合成。在K - 562细胞中，AZT对细胞生长的抑制并非Hb合成净抑制的主要促成因素。通过Northern印迹分析评估，AZT对Hb合成的抑制与珠蛋白mRNA稳态水平的降低相关，而总RNA合成或肌动蛋白mRNA稳态水平未受抑制。特别是，观察到25 microM AZT使珠蛋白mRNA水平降低23%，在250 microM AZT存在时最大抑制率达到59%。体外翻译实验表明，高达250 microM的AZT浓度基本上不会抑制所有非珠蛋白可翻译mRNA，而编码ε、ζ、Aγ、Gγ和α链的珠蛋白mRNA会被AZT以剂量依赖性方式显著抑制至相似水平。对经AZT处理的K - 562细胞分离的细胞核进行转录延伸研究表明，暴露于25和100 microM AZT的细胞中，体外合成的珠蛋白转录本分别受到20%和50%的抑制。2',3'-双脱氧胞苷在与AZT相同的浓度范围内也抑制K - 562细胞生长，但重要的是，对Hb产生没有影响。这些数据表明，珠蛋白基因表达的抑制可能在AZT对红系细胞的细胞毒性中起作用。
