The study finds that telomeres can set damage thresholds for cancer cells over which they cannot continue dividing

Telomeres represent the region that forms the end of chromosomes and is protected by telomere binding proteins. These proteins protect telomeres from being recognized as double-strand breaks of DNA. During somatic cell division, the end of the telomere cannot proliferate by the late filament of the replication fork, causing the telomere to shorten after each cell cycle.

Stady: The alternative lengthening of the telomere machinery threatens the integrity of the telomere if it is not constrained properly. Image Credit: nobeastsofierce / Shutterstock

If cells continue to multiply, telomere depletion can lead to cellular senescence. This phenomenon can be a hindrance to the formation of tumors. However, to achieve replicative immortality, telomere shortening is overcome by cancer cells. This is mostly accomplished by activating telomerase or using a telomerase-independent but homologous recombination-based pathway associated with the extension or maintenance of telomeres. This mechanism is known as alternative telomere lengthening (ALT).

Alternate lengthening of telomere carcinomas (ALT)

ALT cancers achieve immortality by re-lengthening telomeres at the G2 and M phases of the cell cycle via the breakage-induced replication (BIR) pathway. Previous studies have elucidated the toxic nature of the ALT mechanism. High-copy, RNA-rich ALT telomeres: DNA hybrids (telR loops).

A decrease in FANCM, a highly conserved protein within the anemia tumor suppressor (FA) pathway, and depletion of the RNAseH1 endonuclease lead to enhanced telR loops, telomere instability, and ALT activity.

newly PNAS The study hypothesized that ALT may compromise telomere integrity if not restricted appropriately. It has been shown that inhibition of telomere repeat-containing RNA (TERRA) transcription can attenuate ALT activity. For this purpose, the scientists published transcriptional activator-like effectors targeting TERRA promoters comprising CpG-rich and 29-bp repeats (T-TALEs) and incorporating them into the transcriptional repression domain.

Main results

A terminally fused T-TALE was developed with nuclear localization signaling (NLS), RNA polymerase II transcriptional activator VP64, and human influenza hemagglutinin epitope (HA). The scientists cloned a gene downstream of the inducible doxycycline (Dox), which was used to develop two independent ALT-derived cloned cell lines, U2OS called vp6 and vp30.

Western blotting with the help of anti-HA antibody was used to check induced gene expression. Cells were treated with dox for 24 h, and TERRA qRT-PCR was performed. TERRA transcripts from 29-bp subtelomeres were 3–10-fold higher than that of untreated cells. However, TERRA of 29-bp free subtelomeres remained unchanged.

Northern blot hybridization with probes was able to identify TERRA from the 10q sub-telomere or (UUAGGG) n sequence consisting of all TERRA molecules, which showed an increase in TERRA species upon dox treatment. This result indicates that this system can effectively improve TERRA transcription in U2OS cells.

The accumulation of the replication stress marker (RPA32) phosphorylated at serine 33 (pSer33), and the DNA damage marker histone H2AX phosphorylated at serine 139 (γH2AX) were monitored at telomeres. ALT was monitored by measuring ALT-bound PML antibodies, and events of de novo synthesis of telomeric DNA in G2 phase were analyzed by EdU incorporation.

Dox treatments have not been found to be effective on TERRA, telomere stability, and ALT levels. DNA fluorescence on site Hybridization (FISH) experiments revealed that inhibition of TERRA transcription enhanced the number of chromosome ends devoid of telomeric DNA signals that could be detected at the metaphase stage of the cell cycle. The results documented in this study are consistent with a previous study that revealed that inhibiting TERRA transcription can improve ALT and promote progressive telomere shortening.

It was observed that accumulation of telomere-free ends (TFEs) in vp6 and vp30 cells, treated with dox for nine days, had no significant effect in nls1 cells. This result indicated that the mechanism of telomere loss, other than repetitive shortening, could be activated upon elevated telomere transcription.

The current study revealed that Dox treatment and a Mus81 structure-specific endonuclease bound to ALT telomeres enhanced the frequency of Mus81 telomeric foci in vp6 and vp30 cells. Mus81 depletion caused an increase of TFEs in nls1 and vp30 cells, which were not subjected to dox treatment.

The decrease in Mus81 prevented TFE accumulation in vp30 cells treated with dox without avoiding the induction of TERRA transcription or the accumulation of pSer33 and H2AX at telomeres. This finding suggested that Mus81 could play an important role in telomere loss events associated with enhanced TERRA transcription.


Based on the empirical results of the current study and reports from other similar studies, the authors proposed a model for ALT-based studies. This study provided evidence and confirmed that the mechanism of ALT is directly related to molecular events that can compromise telomere stability. Thus, ALT must be appropriately controlled to allow telomere elongation and unlimited cell division without excessive telomere loss. TERRA transcripts have been identified as a versatile target for the treatment of ALT cancer.