Loss of heterozygosity at loci on chromosome 4, a common genetic event during the spontaneous immortalization of mouse embryonic fibroblasts.
Spontaneously immortalized fibroblast cell lines derived from embryonic tissues of C3D2F1, mice were analyzed for loss of heterozygosity (LOH) at multiple chromosomal loci to identify candidate suppressor loci for immortalization. Among 47 simple sequence repeat (SSR) loci selected for screening, those on chromosome 4 exhibited an exceptionally high LDH incidence of up to 89%. Only four other chromosomes (8, 11, 12, and 18) showed LOH, with the highest incidence being 33%. To further localize candidate suppressor genes on mouse chromosome 4, detailed deletion mapping was performed with 18 cell lines and 14 SSR markers. The greatest LOH incidence (94%) was observed at the D4Mit14 locus located on distal chromosome 4, indicating that a major suppressor gene resides in this region. On the other hand, at the D4Mit77 locus, 30 cM proximal to the D4Mit14 locus, we found the SSR to be homozygously lost in 39% of the cell lines. Because the D4Mit77 is tightly linked to the tumor suppressor gene p16, for which homozygous deletion has been reported in various human tumor cell lines, we also examined our fibroblast cell lines for gross aberrations of the p16 gene by using the Southern blot method. The p16 gene was found to be homozygously deleted in 56% of the cell lines. Although this result implies that the p16 gene plays a role as a suppressor gene for immortalization, the combined incidence of LOH and homozygous deletion at the D4Mit77 locus was 72%, which is significantly lower than the observed incidence at the D4Mit14 locus. Consequently, we concluded that immortalization of mouse embryonic fibroblasts may involve more than one suppressor gene on chromosome 4.
Obata
M
M
Department of Pathology, Asahikawa Medical College, Japan.
Lee
G H
GH
Kanda
H
H
Kitagawa
T
T
Ogawa
K
K
D000818:Animals; D015139:Blotting, Southern; D002352:Carrier Proteins / Q000235:genetics; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D002869:Chromosome Aberrations; D002875:Chromosomes; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004622:Embryo, Mammalian; D005260:Female; D005347:Fibroblasts / Q000166:cytology* / Q000648:ultrastructure*; D017353:Gene Deletion; D016147:Genes, Tumor Suppressor; D016678:Genome; D006579:Heterozygote; D006720:Homozygote; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains
D002352:Carrier Proteins; D019941:Cyclin-Dependent Kinase Inhibitor p16