Chen C L, Woo M H, Neale G A, Goorha R M, Fuscoe J C, Behm F G, Mathew S, Relling M V
Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, TN 38101, USA.
Mutat Res. 1998 Jul 17;403(1-2):113-25. doi: 10.1016/s0027-5107(98)00062-1.
Large deletions of exons 2 and 3 of the hprt gene are the most common type of hprt mutation in lymphocytes of newborn infants, and their frequency increases in cultured human T-lymphoid cells as a result of exposure to etoposide. Sequenced PCR products for these deletions are consistent with a V(D)J recombinase-mediated mechanism underlying their genesis. Herein, we describe the isolation and characterization of an etoposide-induced mutant CEM cell line that is clonal for a V(D)J recombinase-mediated exon 2 + 3 deletion. Human CCRF-CEM cells were exposed to 5 muM etoposide for 4 h, selected in 6-thioguanine, and an exon 2 + 3 deletion mutant was isolated through serial limiting dilution, using a PCR-based assay for detection of the exon 2 + 3 deletion. Untreated CEM cells and cells treated with 6-thioguanine alone were similarly subcultured. The exon 2 + 3 deletion-containing line was termed SJCEM808 and had a slightly longer doubling time than the control lines, tended to clump in suspension, and was characterized by cell membrane blebbing. Compared to the parent line, SJCEM808 had similar cytogenetic abnormalities, lower CD2, CD1, and CD10 expression, and negligible RAG-1 expression. However, RAG-1 expression was down-regulated in some untreated parental subclones following similar subculturing. The sequence of the exon 2 + 3 deletion mutation exhibited nucleotide insertions, and the breakpoints were adjacent to heptamer signal recognition sequences in intact hprt, consistent with a V(D)J recombinase-mediated mechanism underlying its genesis. There were no MLL gene or interlocus T-cell receptor (TCR) rearrangements. These results indicate that non-homologous recombination following etoposide treatment is neither necessarily accompanied by other large DNA rearrangements nor simply a pre-lethal event, and this cell line may serve as a useful tool for studying illegitimate V(D)J recombinase-mediated deletions.
次黄嘌呤磷酸核糖转移酶（hprt）基因外显子2和3的大片段缺失是新生儿淋巴细胞中最常见的hprt基因突变类型，由于暴露于依托泊苷，其在培养的人T淋巴细胞中的频率会增加。这些缺失的测序PCR产物与V（D）J重组酶介导的其发生机制一致。在此，我们描述了一种依托泊苷诱导的突变CEM细胞系的分离和特性，该细胞系对于V（D）J重组酶介导的外显子2 + 3缺失是克隆性的。将人CCRF - CEM细胞暴露于5μM依托泊苷4小时，在6 - 硫鸟嘌呤中进行筛选，并通过连续有限稀释分离出外显子2 + 3缺失突变体，使用基于PCR的检测方法来检测外显子2 + 3缺失。未处理的CEM细胞和仅用6 - 硫鸟嘌呤处理的细胞进行类似的传代培养。含有外显子2 + 3缺失的细胞系称为SJCEM808，其倍增时间比对照细胞系略长，倾向于在悬浮液中聚集，并具有细胞膜泡状化的特征。与亲代细胞系相比，SJCEM808具有相似的细胞遗传学异常、较低的CD2、CD1和CD10表达以及可忽略不计的RAG - 1表达。然而，在类似传代培养后，一些未处理的亲代亚克隆中RAG - 1表达下调。外显子2 + 3缺失突变的序列显示出核苷酸插入，并且断点与完整hprt中的七聚体信号识别序列相邻，这与V（D）J重组酶介导的其发生机制一致。不存在MLL基因或基因间T细胞受体（TCR）重排。这些结果表明，依托泊苷处理后的非同源重组既不一定伴随着其他大的DNA重排，也不只是一个濒死事件，并且该细胞系可作为研究非法V（D）J重组酶介导的缺失的有用工具。
