Trf1 Is Not Required for Proliferation or Functional Telomere Maintenance in Chicken DT40 Cells

Abstract

The telomere end-protection complex prevents the ends of linear eukaryotic chromosomes from degradation or inappropriate DNA repair. The homodimeric double-stranded DNA-binding protein, Trf1, is a component of this complex and is essential for mouse embryonic development. To define the requirement for Trf1 in somatic cells, we deleted Trf1 in chicken DT40 cells by gene targeting. Trf1-deficient cells proliferated as rapidly as control cells and showed telomeric localization of Trf2, Rap1, and Pot1. Telomeric G-strand overhang lengths were increased in late-passage Trf1-deficient cells, although telomere lengths were unaffected by Trf1 deficiency, as determined by denaturing Southern and quantitative FISH analysis. Although we observed some clonal variation in terminal telomere fragment lengths, this did not correlate with cellular Trf1 levels. Trf1 was not required for telomere seeding, indicating that de novo telomere formation can proceed without Trf1. The Pin2 isoform and a novel exon 4, 5-deleted isoform localized to telomeres in Trf1-deficient cells. Trf1-deficient cells were sensitive to DNA damage induced by ionizing radiation. Our data demonstrate that chicken DT40 B cells do not require Trf1 for functional telomere structure and suggest that Trf1 may have additional, nontelomeric roles involved in maintaining genome stability.

Doxorubicin induces senescence or apoptosis in rat neonatal cardiomyocytes by regulating the expression levels of the telomere binding factors 1 and 2

“…Using the siRNA technique, we have confirmed that high levels of TRF2 downregulation induce apoptosis and low levels of TRF2 downregu- lation induce senescence, and we also found that the selective knockdown of TRF1 induces neither senescence nor apoptosis. These results are in agreement with a number of studies finding that a dominant negative allele of human TRF1 does not affect the growth and viability of a variety of primary and transformed human cells (12,25,26,55).…”

Investigating the Reproducibility of In Vitro Cell Biology Assays Using Mathematical Models

“…Although there are many properties of cells that are affected by the passaging process [21,31,36,45,88,95,99,129], we choose to focus on how passaging affects the cell proliferation rate. In our model, when a cell proliferates, the daughter cells directly inherit the same proliferation rate as the mother cell.…”

“…During the passaging process, cells are lifted, split into smaller proportions, and re-grown in new cell culture flasks [8,9]. It is reported that cell characteristics change at high passage numbers, and seemingly contradictory effects have been observed in experiments of passaging cell lines [21,31,45,73,88,95,99]. For example, many studies report that cell proliferation decreases with the increasing passage number [31,45,95,99], whereas Lin et al find that cell proliferation increases at high passage numbers [73].…”

“…Considering that the passaging process involves a combination of chemical (e.g. the usage of trypsin) and mechanical disturbances known to disrupt normal cell behavior, it is reasonable to incorporate some kind of damage mechanism into the passage simulations [8,21,31,45,88,95,99]. However, the exact cause and the form of the passage-induced damage have not been established.…”

“…There are many ways in which passaging can affect cells. For example, primary cells, which are directly isolated from living tissues [41], undergo morphological changes and cumulative damage as the passage number increases [21,31,45,62,68,88,95,99]. As a result, the cell morphology, migration rate and proliferation rate can become increasingly varied, which is thought to increase the heterogeneity in cell lines [31,45,68,95,99].…”