Raghbir S. Athwal, Arbansjit K Sandhu, Deepthi Reddy, Neena Deoghare and G. Pal Kaur
Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA 19140
We have concluded the production of mouse/human monochromosomal hybrid cell lines. The current panel is comprised of chromosomes 1-3, 5-17, 19-21, X and Y. In addition to the monochromosomal hybrid cell lines, we have also assembled a panel of normal human diploid cell lines each carrying gpt integrated into a different chromosome. These panels are currently used to map genes involved in cellular senescence which may be lost during the evolution of human tumors.
Human cells in culture have a limited life span, as do the cells of other species. After a number of generations normal cells display morphological changes, cessation of proliferation and senescence. In comparison many tumor cells have overcome senescence and can grow continuously in culture and in vivo. Thus cellular immortalization may represent a critical step in tumor progression. We have identified genes on human chromosomes 3, 6, 9, 13 and 17 which restore senescence when introduced into ovarian tumor cells. One of these genes which restores senescence in human as well in rat ovarian and breast tumor cells has been mapped to 6ql6-21.
Single gpt tagged normal human chromosomes, present in mouse/human monochromosomal hybrid cells, were introduced into human and rat ovarian tumor cells via microcell fusion. Chromosome transfer clones were isolated by growth in the medium containing mycophenolic acid (25ug/ml) and xanthine (70ug/ml, MX medium) to select for gpt. Introduction of chromosome 3, 6, 9, 13 or 17 led to the senescence of both human and rat tumor cells while transfer of chromosomes 10 or 14 had no effect on morphology or growth potential of these cells. The reappearance of tumor type cells concordant with the loss of donor human chromosome further confirmed the presence of a cellular senescence genes on these chromosomes. Immortal revertant clones also appeared among senescent cells maintained in MX medium to retain introduced chromosome. These revertant clones seem to arise due to deletions in donor chromosomes. Detailed analysis of the chromosome 6 revertant clones using microsatellite markers to assess minimum chromosomal deletion revealed that a senescence gene may be located in the region 6ql6-21. Microcell transfer of a chromosomal region 6ql3-21 (gpt tagged) into human and rat ovarian tumor cells induced cell senescence while introduction of another chromosome 6 lacking this region had no effect on cell growth. These results indicate that a gene which impart senescence to ovarian tumor cells is present in the chromosomal region 6ql3-21.