Abstract
Activation of telomerase or alternative lengthening of telomeres (ALT) is necessary for tumours to escape from dysfunctional telomere-mediated senescence. Anti-telomerase drugs might be effective in suppressing tumour growth in approximately 85–90% of telomerase-positive cancer cells. However, there are still chances for these cells to bypass drug treatment after switching to the ALT mechanism to maintain their telomere integrity. But the mechanism underlying this switch is unknown. In this study, we used telomerase-positive cancer cells (HTC75) to discover the mechanism of the telomerase-ALT switch by inducing telomere-specific DNA damage, alpha-thalassemia X-linked syndrome protein (ATRX) knockdown and deletion of death associated protein (DAXX). Surprisingly, two important ALT hallmarks in the ALT-like HTC75 cells were observed after treatments: ALT-associated promyelocytic leukaemia bodies (APBs) and extrachromosomal circular DNA of telomeric repeats. Moreover, knocking out hTERT by utilizing the CRISPR/Cas9 technique led to telomere elongation in a telomerase-independent manner in ALT-like HTC75 cells. In summary, this is the first report to show that inducing telomeric DNA damage, disrupting the ATRX/DAXX complex and inhibiting telomerase activity in telomerase-positive cancer cells lead to the ALT switch.
Highlights
alternative lengthening of telomeres (ALT)-positive cells typically contain abnormally heterogeneous telomeres, ALT-associated promyelocytic leukaemia bodies (APBs) and extrachromosomal TTAGGG repeats (ECTRs)[18,19]
The expression level of ATRX and DAXX proteins in three ΔT +shA +DAXX knock out (D-KO) HTC75 cell clones were nearly undetectable by western blotting (WB) (Fig. 1D)
To better understand the mechanism that leads to the telomerase-ALT switch in telomerase-positive cancer cells, we constructed different cell lines with specific telomeric DNA damage, ATRX knockdown, DAXX deletion or to generate hTERT knock out (TERT KO)
Summary
ALT-positive cells typically contain abnormally heterogeneous telomeres, ALT-associated promyelocytic leukaemia bodies (APBs) and extrachromosomal TTAGGG repeats (ECTRs)[18,19]. Somatic mutations of the histone variant H3.3, alpha-thalassemia X-linked syndrome protein (ATRX) and death associated protein (DAXX) have been found in ALT cancers, including pancreatic neuroendocrine (panNET) cancers and glioblastoma multiforme (GBM) cancers[30,31]. They are chromatin remodeling factors at telomeres, which are responsible for ALT activity[32,33]. To determine the mechanism by which telomerase-positive cancer cells switch to ALT and to elucidate the mechanism of ALT induction, we induced telomere-specific DNA damage, disrupted the function of the ATRX/ DAXX complex and inhibited telomerase activity in telomerase positive cancer cells, which successfully transformed a telomerase-positive cell line into a ALT-positive cell line
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