Genomic instability in both wild-type and telomerase null MEFs
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To examine chromosome instability in the absence of telomerase, we established mouse embryonic fibroblast (MEF) lines from late generation mTR−/− and wild-type animals and examined metaphases using telomere fluorescence in situ hybridization (FISH) and spectral karyotyping (SKY). In early passages, mTR−/− G6 cell lines showed more chromosome ends with no telomere signal, more chromosome end-to-end fusions and greater radiosensitivity than wild-type lines. At later passages, however, the rate of genomic instability in the wild-type MEFs increased to a level similar or higher than seen in the mTR−/− G6 cell lines. This high degree of instability in wild-type MEF lines suggests that post-crisis MEFs should not be considered genetically defined cell lines. Surprisingly, the increased radiosensitivity seen in early passage mTR−/− G6 cultures was lost after crisis. Both post-crisis mTR−/− G6 MEFs and wild-type MEFs showed loss of p53 and γ-H2AX phosphorylation in response to irradiation, indicating a loss of DNA damage checkpoints.
KeywordsTelomere Length Mouse Embryonic Fibroblast Telomere Repeat Mouse Embryonic Fibroblast Cell H2AX Phosphorylation
We thank Drs. Michael Hemann, Forrest Spencer and members of the Greider laboratory for critical reading of the manuscript, Margaret Strong for technical support and Dr. Steve Elledge for providing the Chk2 antibody. This work was supported by NIH grant CA16519 to C.W.G.