Chromosomal Instability Induced by Overexpression of Telomere Repeats Binding Factor 1.

Abstract

Telomere dysfunction has been associated with chromosomal stability in process of oncogenesis. Telomere, a special nucleoprotein complex at the termini of linear eukaryotic chromosomes, functions to prevent chromosomes from degradation by endogenous nucleases, fusion and recombination of chromosome ends, triggering DNA damage response and checkpoint-induced cell cycle arrest or apoptosis. Telomere homoeostasis and length are maintained by a set of telomere binding proteins. Among these, telomere repeats binding factor 1(TRF1) serves as a negative regulator of telomere length since TRF1 overexpression would elicit the shortening of telomere length in telomerase-positive cells. To date, the expression level of TRF1 in human cancers remains controversial and its role and mechanism in tumorigenesis are poorly understood. To answer these questions, we introduced EGFP-tagged TRF1 plasmid into HeLa cells to establish a stable cell line with TRF1 overexpression. Positive clones were selected by G418(1mg/ml) and GFP fusion protein was confirmed by immunoblotting analysis with anti-GFP antibodies. Interestingly, these cells demonstrated a higher percentage of mitotic abnormality including chromosome misalignment, anaphase bridging, lagging chromosome and multipolar spindles than controls after over 10 passages(20.0% vs 1.5%, p<0.01). To further clarify if this phenomenon resulted from chromosomal fusion, metaphase chromosome spreads were prepared. As expected, the TRF1 stable cells showed an elevated frequency of dicentric chromosomes compared with untransfected cells, which implicated chromosomal end-to-end fusion in these cells. Our results suggested high expression of TRF1 would induce chromosome missegregation at mitosis, a feature of chromosomal instability which is proposed to be a critical step in carcinogenesis. These findings bring fourth a new hint for our further understanding of carcinogenesis led by telomere dysfunction.