title: Novel use of a selectable fusion gene as an "in-out" marker for studying genetic loss in mammalian cells.
abstract: Recent demonstrations of loss of heterozygosity in a wide variety of human cancers suggest that large multilocus genetic deletions (presumably including tumor suppressor genes) constitute a major class of genetic alteration in human carcinogenesis. Here we show that a bifunctional fusion gene (Hytk), suitable for both positive and negative selection, is an effective marker for studying genetic loss in mammalian cells with minimal interference from point-mutational changes. Studies with a transgenic V79 cell line in which a single functional copy of Hytk was stably inserted into the genome in a retroviral vector showed that loss of the marker (and presumably flanking cellular genetic material) could be induced efficiently by ionizing radiation (gamma-rays and fast neutrons) but only weakly by the powerful point-mutagen benzo[a]pyrene diol epoxide. In a first application of the system, we provide evidence that radiation-induced loss can occur through an indirect mechanism after a high-frequency event. Collectively, our results suggest that the Hytk marker should be a valuable tool for studying genome position effects on the tolerance of genetic loss in cultured human cells that represent different stages in clonal evolution and tumor progression.
authors.affiliation: Human Cancer Genetics Unit, Brunel University, Uxbridge, United Kingdom.
authors.forename: D A
authors.initials: DA
authors.lastname: Trott
authors.forename: A P
authors.initials: AP
authors.lastname: Cuthbert
authors.forename: C M
authors.initials: CM
authors.lastname: Todd
authors.forename: M
authors.initials: M
authors.lastname: Themis
authors.forename: R F
authors.initials: RF
authors.lastname: Newbold
mesh_terms: D016475:3T3 Cells; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000633:toxicity; D000818:Animals; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004307:Dose-Response Relationship, Radiation; D017353:Gene Deletion; D016147:Genes, Tumor Suppressor; D005819:Genetic Markers; D014644:Genetic Variation; D006801:Humans; D051379:Mice; D009502:Neutrons; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011533:Proviruses; D012190:Retroviridae; D014162:Transfection
chemical_list: D005819:Genetic Markers; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide