Wang Jianyong, Chen Tao, Honma Masamitsu, Chen Ling, Moore Martha M
Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.
Environ Mol Mutagen. 2007 Apr-May;48(3-4):248-57. doi: 10.1002/em.20263.
3'-Azido-3'-deoxythymidine (AZT), a nucleoside analogue used for the treatment of acquired immunodeficiency syndrome (AIDS), induced a significant dose-related increase in the thymidine kinase (Tk) mutant frequency (MF) in L5178Y/Tk(+/-) 3.7.2C mouse lymphoma cells. Treatment with 1 mg/ml (3,742 muM) AZT for 24 hr resulted in a MF of 407 x 10(-6) compared to a control MF of 84 x 10(-6). The MFs of the large and small colony mutants resulting from AZT exposure were 142 x 10(-6) and 265 x 10(-6), respectively. One hundred and fifty mutants from the 1 mg/ml (3,742 muM) AZT-treated culture and sixty-nine mutants from independent untreated cultures were isolated and analyzed. LOH analysis using a heteromorphic microsatellite locus located in the Tk gene was performed to determine the presence or absence of the Tk(+) allele. Eight other microsatellite markers spanning the entire mouse chromosome 11 also were examined for heterozygosity to determine the extent of LOH. In addition, Tk gene dosage analysis was conducted using Real-Time PCR in those mutants showing LOH at the Tk locus. The presence of only one Tk allele based on Real-Time PCR indicated that the mutant resulted from deletion while the presence of two alleles was consistent with a recombination event. More mutants from the AZT-treated culture showed Tk LOH than did independent mutants from the untreated cultures (91% vs. 64%) and the induced mutants also showed distinct chromosome 11 LOH patterns. The mutation spectrum of mutants from AZT-treated cells was also significantly different from that of spontaneous mutants. More deletions and fewer intragenic mutations were observed in the mutants from the AZT-treated culture than independent mutants from the untreated control. Our data indicate that AZT primarily induced LOH mutations in L5178Y mouse lymphoma cells and a large number of LOH mutations resulted from deletions.
3'-叠氮-3'-脱氧胸苷（AZT）是一种用于治疗获得性免疫缺陷综合征（艾滋病）的核苷类似物，它在L5178Y/Tk(+/-) 3.7.2C小鼠淋巴瘤细胞中可引起胸苷激酶（Tk）突变频率（MF）显著的剂量相关性增加。用1 mg/ml（3742 μM）的AZT处理24小时后，突变频率为407×10⁻⁶，而对照突变频率为84×10⁻⁶。AZT处理后产生的大菌落突变体和小菌落突变体的突变频率分别为142×10⁻⁶和265×10⁻⁶。从1 mg/ml（3742 μM）AZT处理的培养物中分离出150个突变体，并从独立的未处理培养物中分离出69个突变体进行分析。使用位于Tk基因中的多态性微卫星位点进行杂合性缺失（LOH）分析，以确定Tk(+)等位基因的存在与否。还检查了跨越整个小鼠11号染色体的其他8个微卫星标记的杂合性，以确定LOH的范围。此外，对那些在Tk基因座显示LOH的突变体，使用实时定量PCR进行Tk基因剂量分析。基于实时定量PCR仅存在一个Tk等位基因表明该突变体是由缺失导致的，而存在两个等位基因则与重组事件一致。与未处理培养物中的独立突变体相比，AZT处理培养物中的更多突变体显示出Tk基因杂合性缺失（91%对64%），并且诱导产生的突变体也显示出明显不同的11号染色体LOH模式。AZT处理细胞产生的突变体的突变谱也与自发突变体的显著不同。与未处理对照的独立突变体相比，在AZT处理培养物的突变体中观察到更多的缺失和更少的基因内突变。我们的数据表明，AZT主要在L5178Y小鼠淋巴瘤细胞中诱导LOH突变，并且大量的LOH突变是由缺失引起的。
