title: A human lymphoid leukemia cell line with a V(D)J recombinase-mediated deletion of hprt.
abstract: 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.
authors.affiliation: Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, TN 38101, USA.
authors.forename: C L
authors.initials: CL
authors.lastname: Chen
authors.forename: M H
authors.initials: MH
authors.lastname: Woo
authors.forename: G A
authors.initials: GA
authors.lastname: Neale
authors.forename: R M
authors.initials: RM
authors.lastname: Goorha
authors.forename: J C
authors.initials: JC
authors.lastname: Fuscoe
authors.forename: F G
authors.initials: FG
authors.lastname: Behm
authors.forename: S
authors.initials: S
authors.lastname: Mathew
authors.forename: M V
authors.initials: MV
authors.lastname: Relling
mesh_terms: D000954:Antigens, Surface / Q000378:metabolism; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D001483:Base Sequence; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004273:DNA, Neoplasm / Q000235:genetics; D005047:Etoposide / Q000494:pharmacology; D005091:Exons; D017353:Gene Deletion; D016654:Genes, RAG-1; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D007231:Infant, Newborn; D015458:Leukemia, T-Cell / Q000188:drug therapy / Q000201:enzymology* / Q000235:genetics*; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D011948:Receptors, Antigen, T-Cell / Q000235:genetics; D011995:Recombination, Genetic / Q000187:drug effects; D014407:Tumor Cells, Cultured; D045567:VDJ Recombinases
chemical_list: D000954:Antigens, Surface; D000972:Antineoplastic Agents, Phytogenic; D017931:DNA Primers; D004273:DNA, Neoplasm; D011948:Receptors, Antigen, T-Cell; D005047:Etoposide; D007041:Hypoxanthine Phosphoribosyltransferase; D004254:DNA Nucleotidyltransferases; D045567:VDJ Recombinases