NOVEL USE OF A SELECTABLE FUSION GENE AS AN IN-OUT MARKER FOR STUDYING GENETIC LOSS IN MAMMALIAN-CELLS

Recent demonstrations of loss of heterozygosity in a wide variety of h uman cancers suggest that large multilocus genetic deletions (presumab ly including tumor suppressor genes) constitute a major class of genet ic alteration in human carcinogenesis. Here we show that a bifunctiona l fusion gene (Hytk), suitable for both positive and negative selectio n, 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 f ast neutrons) but only weakly by the powerful point-mutagen benzo[a]py rene diol epoxide. in a first application of the system, we provide ev idence that radiation-induced loss can occur through an indirect mecha nism after a high-frequency event. Collectively, our results suggest t hat the Hytk marker should be a valuable tool for studying genome posi tion effects on the tolerance of genetic loss in cultured human cells that represent different stages in clonal evolution and tumor progress ion. (C) 1995 Wiley-Liss, Inc.