Transient Activation of the ALT Pathway in Human Primary Fibroblasts Exposed to High-LET Radiation

Abstract

It is well established that high-LET radiations efficiently induce chromosome aberrations. However, data on the effect of protons on telomere maintenance, as involved in genomic stability, are scarce and contradictory. Here we demonstrate that high-LET protons induce telomere lengthening in human primary fibroblasts and that this elongation does not involve the telomerase enzyme, supporting the hypothesis that high-LET radiations are able to activate a telomerase-independent mechanism. In tumor cells that lack telomerase, one or more non-telomerase mechanisms for telomere maintenance are present, which are termed alternative lengthening of telomeres (ALT). Since ALT cells are characterized by recombinational events at telomeres, known as telomeric-sister chromatid exchanges (T-SCE), and colocalization of telomeres and premyelocytic leukemia protein (PML), we analyzed both T-SCE and PML. Our results show that high-LET protons induce a 2.5-fold increase of T-SCE and a colocalization of PML protein and telomeric DNA. Furthermore, our data show that the ALT pathway can be activated in human primary cells after induction of severe DNA damage. Thus, since telomeres are known to be involved in chromosome maintenance, the present work may contribute in the elucidation of the mechanism by which ionizing radiation induces genomic instability.

Telomere loss, not average telomere length, confers radiosensitivity to TK6-irradiated cells

“…Since it has been shown that radiations induce upregulation of telomerase [36,39] in haematopoietic cells, we investigated whether such an effect of stabilization of longer telomeres could have occurred in our experimental conditions, through an increase in telomerase activity. However, in agreement with our previous results on telomerase-negative HFFF2 fibroblasts [31], we found that such an enzyme was not upregulated after exposure to doses up to 10 Gy of X-rays. This led us to speculate about the possibility that cells with longer telomeres already present in the general population behave as more radioresistant cells and hence are selected by irradiation and therefore expand in the growing population.…”

“…q-FISH staining was performed as previously described [31]. Briefly, chromosome spreads were hybridized with fluorescent PNA telomeric probes and chromosome 2 centromeric PNA probe (DAKOCytomatation), used as references for telomere lengths quantification.…”

“…The software calculates telomere lengths as the ratio between the fluorescence of each telomere signal and the fluorescence of the centromere of chromosome 2, used as the internal reference in each metaphase analyzed. Data were expressed as percentage (T/C%) [31,32].…”

Telomere Maintenance Mechanisms in Soft Tissue Sarcomas

“…The spatio-temporal dynamics of telomeric DNA association with PML bodies have been recently described, and the authors of this study conclude that telomere recombination takes place in these structures (Draskovic et al, 2009). Furthermore, new PML bodies form at telomeric DNA regardless of which TMM is active (Brouwer et al, 2009) and APBs form transiently in human diploid fibroblasts following high LET radiation (Berardinelli et al, 2010), suggesting that the association of telomeric DNA with PML bodies may be a component of a DNA damage response. Indeed, telomeres are transiently recognized as DNA damage during normal DNA replication (Verdun & Karlseder, 2006).…”

DDM1 guards against telomere truncation in Arabidopsis

“…There is mounting evidence that telomeres are responsive to environmental stress through recombinational mechanisms. For example, both telomere shortening and increased telomere sister chromatid exchange are triggered by ionizing radiation and oxidative stress in human cells (Berardinelli et al 2010; Coluzzi et al 2017). In addition, telomeres in plants deficient in the TEN1 component of the CST telomere end-binding complex undergo acute shortening upon heat shock; telomere truncation coincides with an increased level of ECTCs (Lee et al 2016).…”