Alternative Lengthening Of Telomeres Cell Lines Research Articles

Telomere elongation involves intra-molecular DNA replication in cells utilizing alternative lengthening of telomeres

Alternative lengthening of telomeres (ALT) is a telomere length maintenance mechanism based on recombination, where telomeres use other telomeric DNA as a template for DNA synthesis. About 10% of all human tumors depend on ALT for their continued growth, and understanding its molecular details is critically important for the development of cancer treatments that target this mechanism. We have previously shown that telomeres of ALT-positive human cells can become lengthened via inter-telomeric copying, i.e. by copying the telomere of another chromosome. The possibility that such telomeres could elongate by using other sources of telomeric DNA as copy templates has not been investigated previously. In this study, we have determined whether a telomere can become lengthened by copying its own sequences, without the need for using another telomere as a copy template. To test this, we transduced an ALT cell line with a telomere-targeting construct and obtained clones with a single tagged telomere. We showed that the telomere tag can be amplified without the involvement of other telomeres, indicating that telomere elongation can also occur by intra-telomeric DNA copying. This is the first direct evidence that the ALT mechanism involves more than one method of telomere elongation.

Evidence for alternative lengthening of telomeres in liposarcomas in the absence of ALT‐associated PML bodies

Immortalized and cancer cells maintain their telomeres by activation of a telomere maintenance mechanism (TMM). In approximately 85% of cancers telomerase is activated (TA) but in some tumours, in particular sarcomas, an alternative lengthening of telomeres (ALT) pathway is used. Liposarcomas are the most common soft-tissue sarcoma in adults and they activate ALT or telomerase with equal frequency, however no TMM has been identified in approximately 50% of liposarcomas. In our study, we have shown that instability at the minisatellite MS32, usually associated with ALT activation, aids the identification of liposarcomas that have recombination-like activity at telomeres in absence of ALT associated PML-bodies (APBs). Furthermore, using single molecule telomere analysis, we have detected complex telomere mutations directly in ALT positive liposarcomas and interestingly in some liposarcomas with an unknown TMM but high MS32 instability. We have shown by sequence analysis that some of these complex telomere mutations must arise by an inter-molecular recombination-like process rather than by deletion caused by t-loop excision or by unequal telomere-sister-chromatid-exchange (T-SCE), which is known to be elevated in ALT cell lines. Preliminary evidence also suggests that inter-molecular recombination events may be processed differently in liposarcomas with APBs compared to those without. In conclusion, we have shown for the first time, that some telomerase negative liposarcomas without APBs have other features associated with ALT, indicating that the incidence of ALT in these tumours has previously been under-estimated. This has major implications for the use of cancer treatments targeted at TMMs.

Sumoylation of TRF1 Is Essential for Its Recruitment to ALT-Associated PML Bodies.

To achieve unlimited proliferative potential, most cancer cells activate telomerase to maintain telomeres. However, some cancer cells elongate telomeres through a telomerase-independent pathway termed alternative lengthening of telomeres (ALT). These ALT cells contain a novel promyelocytic leukemia (PML) body (ALT-associated PML body, APB), which comprises telomeric DNA and a number of proteins, including PML protein, the telomere binding proteins TRF1 and TRF2, replication factor A, and recombination factors Rad51, Rad52, and the Rad50/Mre11/NBS1 complex. TRF1, as the first identified telomeric binding protein, binds the duplex telomeric repeats at telomere ends. It plays an important role in telomere length control, telomeric ends shelter and cell cycle regulation. In ALT cell lines, TRF1 co-localized with PML protein at APBs, but the exact mechanism of its recruitment to APBs is not clear. Here we show that TRF1 localizes to PML bodies in about 5% of an asynchronously growing culture of U2OS cells and the percentage of cells with colocalization of TRF1 and PML bodies increases to about 40% in cells enriched in G2/M, which is consistent with the previous studies. Furthermore, our results show that TRF1 is modified by the small ubiquitin-like protein SUMO-1 in vivo and in vitro. Firstly, 293T cells were transfected with Flag-TRF1, HA-Ubc9, GFP-SUMO1 and then immunoprecipitated by using FLAG-M2 gel under denaturing conditions. Immunoblotting with GFP and Flag antibodies demonstrated that TRF1 is modified by SUMO-1 in vivo. Next, in vitro SUMO-1 conjugation assay of TRF1 was employed. The results showed that TRF1 is conjugated with SUMO-1 in the presence of purified recombinant protein SAE1/SAE2, Ubc9, SUMO1, His-TRF1 and ATP. Either SAE1/SAE2, Ubc9, SUMO-1 or ATP was omitted from the reaction abolished the sumoylation of TRF1. Previous studies have shown that sumoylation controls the recruitment of several proteins to PML bodies, so we examined whether sumoylation of TRF1 is required for its recruitment to APBs. We mutated the potential sumoylation sites of TRF1 according to the computational prediction and then transfected it into U2OS cells to examine its localization. The results showed that TRF1 mutant does not localize to PML bodies. Taken together, all these results suggest that TRF is modified by SUMO-1 and sumoylation of TRF1 is essential for its recruitment to APBs in ALT cells.

A human cell line that maintains telomeres in the absence of telomerase and of key markers of ALT

In human somatic cells proliferation results in telomere shortening due to the end replication problem and the absence of adequate levels of telomerase activity. The progressive loss of telomeric DNA has been associated with replicative senescence. Maintenance of telomere structure and function is, therefore, an essential requisite for cells that proliferate indefinitely. Human cells that have acquired the immortal phenotype mostly rely on telomerase to compensate for telomere shortening with cell division. However, a certain percentage of immortalized cell lines and human tumors maintain their telomeres by Alternative Lengthening of Telomeres (ALT), a mechanism not fully understood but apparently based on homologous recombination. Here, we report the isolation of an immortal human cell line that is derived from an ALT cell line but maintains telomeres in the absence of key features of ALT and of telomerase. The properties of these cells suggest that the identification of ALT cells may not be reliably based on known ALT markers. This finding is of relevance for discriminating between the mortal and immortal phenotype among telomerase-negative cells in vitro and in vivo, particularly in regard to the development of pharmacological approaches for cancer treatment based on telomerase inhibition.

Reversible Conversion of Immortal Human Cells from Telomerase-Positive to Telomerase-Negative Cells

Immortal cell lines and tumors maintain their telomeres via the telomerase pathway or via a telomerase-independent pathway, referred to as alternative lengthening of telomeres (ALT). Here, we show the reversible conversion of the human papillomavirus type 16 E6-induced immortal human fibroblasts E6 Cl 6 from telomerase-positive (Tel(+)) to telomerase-negative (Tel(-)) cells. Tel(+) cells converted spontaneously to Tel(-) cells that reverted to Tel(+) cells following treatment with trichostatin A (TSA) and/or 5-aza-2'-deoxycytidine (5-AZC), which induced the reversion from complete to partial methylation of the CpG islands of the human telomerase reverse transcriptase (hTERT) promoter in Tel(-) E6 Cl 6 cells. Tel(-) E6 Cl 6 cells lacked the phenotypes characteristic of ALT cell lines such as very long and heterogenous telomeres and ALT-associated promyelocytic leukemia nuclear bodies (APB) but grew for >240 population doublings (PD) after they became telomerase negative. The ratios of histone H3 (H3) lysine (K) 9 methylation to each of H3-K4 methylation, H3-K9 acetylation, and H3-K14 acetylation of the chromatin containing the hTERT promoter in Tel(-) E6 Cl 6 cells and ALT cell lines were greater than those in Tel(+) cells and decreased following treatment with TSA and/or 5-AZC, inversely corresponding to telomerase activity. Our findings suggest the possibility that human tumors may be able to reversibly interconvert their telomere maintenance phenotypes by chromatin structure-mediated regulation of hTERT expression.

Telomerase-Independent Telomere Length Maintenance in the Absence of Alternative Lengthening of Telomeres–Associated Promyelocytic Leukemia Bodies

Immortal tumor cells and cell lines employ a telomere maintenance mechanism that allows them to escape the normal limits on proliferative potential. In the absence of telomerase, telomere length may be maintained by an alternative lengthening of telomeres (ALT) mechanism. All human ALT cell lines described thus far have nuclear domains of unknown function, termed ALT-associated promyelocytic leukemia bodies (APB), containing promyelocytic leukemia protein, telomeric DNA and telomere binding proteins. Here we describe telomerase-negative human cells with telomeres that contain a substantial proportion of nontelomeric DNA sequences (like telomerase-null Saccharomyces cerevisiae survivor type I cells) and that are maintained in the absence of APBs. In other respects, they resemble typical ALT cell lines: the telomeres are highly heterogeneous in length (ranging from very short to very long) and undergo rapid changes in length. In addition, these cells are capable of copying a targeted DNA tag from one telomere into other telomeres. These data show that APBs are not always essential for ALT-mediated telomere maintenance.

A Novel Telomere Structure in a Human Alternative Lengthening of Telomeres Cell Line

Cancer cells require mechanisms to maintain telomeres. Most use telomerase, but 5% to 20% of tumors use alternative lengthening of telomeres (ALT), a telomerase-independent mechanism that seems to depend on recombination. ALT is characterized by amplification of telomere TTAGGG repeats to lengths beyond 50 kb, by elevated rates of telomere recombination, and by nuclear structures called ALT-associated promyelocytic leukemia bodies. In Saccharomyces cerevisiae, survivors of telomerase inactivation also use recombination to maintain telomeres. There are two types of survivors, which differ in telomere structure. The first possesses telomere repeats and the Y' subtelomeric element amplified together as a tandem array at chromosome termini (type I), and the other possesses amplification of telomeric repeats alone (type II), similar to previously described human ALT cells. Here, we describe the first human ALT cell line having "tandem array" telomeres with a structure similar to that of type I yeast survivors. The chromosome termini consist of a repeat unit containing approximately 2.5 kb of SV40 DNA and a variable amount of TTAGGG sequence repeated in tandem an average of 10 to 20 times. Similar to previously described ALT cells, they show evidence of telomere recombination, but unlike standard ALT cells, they lack ALT-associated promyelocytic leukemia bodies and their telomeres are transcribed. These findings have implications for the pathogenesis and diagnosis of cancer.

Telomeric DNA in ALT Cells Is Characterized by Free Telomeric Circles and Heterogeneous t-Loops

A prerequisite for cellular immortalization in human cells is the elongation of telomeres through the upregulation of telomerase or by the alternative lengthening of telomeres (ALT) pathway. In this study, telomere structure in multiple ALT cell lines was examined by electron microscopy. Nuclei were isolated from GM847, GM847-Tert, and WI-38 VA13 ALT cells, psoralen photo-cross-linked in situ, and the telomere restriction fragments were purified by gel filtration chromatography. Examination of telomere-enriched fractions revealed frequent extrachromosomal circles, ranging from 0.7 to 56.8 kb. t-loops were also observed, with the loop portion ranging from 0.5 to 70.2 kb. The total length of the loop plus tail of the t-loops corresponded to the telomere restriction fragment length from the ALT cell lines as determined by pulsed-field gel electrophoresis. The presence of extrachromosomal circles containing telomeric DNA was confirmed by two-dimensional pulsed-field gel electrophoresis. These results show that extrachromosomal telomeric DNA circles are present in ALT nuclei and suggest a roll-and-spread mechanism of telomere elongation similar to that seen in previous observations of multiple yeast species. Results presented here also indicate that expression of telomerase in GM847 cells does not affect t-loop or extrachromosomal circle formation.

P53 differentially inhibits cell growth depending on the mechanism of telomere maintenance.

Telomere stabilization is critical for tumorigenesis. A number of tumors and cell lines use a recombination-based mechanism, alternative lengthening of telomeres (ALT), to maintain telomere repeat arrays. Current data suggest that the mutation of p53 facilitates the activation of this pathway. In addition to its functions in response to DNA damage, p53 also acts to suppress recombination, independent of transactivation activity, raising the possibility that p53 might regulate the ALT mechanism via its role as a regulator of recombination. To test the role of p53 in ALT we utilized inducible alleles of human p53. We show that expression of transactivation-incompetent p53 inhibits DNA synthesis in ALT cell lines but does not affect telomerase-positive cell lines. The expression of temperature-sensitive p53 in clonal cell lines results in ALT-specific, transactivation-independent growth inhibition, due in part to the perturbation of S phase. Utilizing chromatin immunoprecipitation assays, we demonstrate that p53 is associated with the telomeric complex in ALT cells. Furthermore, the inhibition of DNA synthesis in ALT cells by p53 requires intact specific DNA binding and suppression of recombination functions. We propose that p53 causes transactivation-independent growth inhibition of ALT cells by perturbing telomeric recombination.

Effects of reconstitution of telomerase activity on telomere maintenance by the alternative lengthening of telomeres (ALT) pathway.

Telomere length maintenance is essential for cellular immortalization, and thus tumorigenesis. Most human tumors and immortal cell lines maintain their telomeric DNA via the activity of a specialized reverse transcriptase, telomerase. Stabilization of telomeric repeat tracts may also be achieved through a telomerase-independent mechanism, referred to as alternative lengthening of telomeres (ALT). ALT cells are telomerase negative and are characterized by extremely long and heterogeneously sized telomeres and novel multiprotein structures called ALT-associated PML nuclear bodies which are unique to ALT cells. To determine if reconstitution of telomerase activity suppressed ALT and restored wild-type telomere lengths, we introduced the catalytic subunit of telomerase into two ALT cell lines. Initially, two clonal lines exhibited enrichment of shorter telomeres while maintaining a population of ultra-long telomeres similar to that observed in the parental line, suggesting that telomerase is stabilizing the shorter telomeres in the population. Telomere length in the third clonal line was not detectably different from that in the parental cell line. One clonal line with a phenotype of shorter telomeres maintained this pattern over time in culture while the second gradually reverted to the parental ALT telomere length pattern, concurrent with reduction of telomerase activity. All clones continued to maintain ALT-associated PML nuclear bodies regardless of whether telomerase was present. The data suggest that introduction of telomerase activity alone is not sufficient to completely repress ALT, that telomerase acts preferentially on the shortest telomeres in the culture and that the ALT and telomerase pathways may be present concurrently in mammalian cells.

Telomerase Can Inhibit the Recombination-based Pathway of Telomere Maintenance in Human Cells

Telomere length can be maintained by telomerase or by a recombination-based pathway. Because individual telomeres in cells using the recombination-based pathway of telomere maintenance appear to periodically become extremely short, cells using this pathway to maintain telomeres may be faced with a continuous state of crisis. We expressed telomerase in a human cell line that uses the recombination-based pathway of telomere maintenance to test whether telomerase would prevent telomeres from becoming critically short and examine the effects that this might have on the recombination-based pathway of telomere maintenance. In these cells, telomerase maintains the length of the shortest telomeres. In some cases, the long heterogeneous telomeres are completely lost, and the cells now permanently contain short telomeres after only 40 population doublings. This corresponds to a telomere reduction rate of 500 base pairs/population doubling, a rate that is much faster than expected for normal telomere shortening but is consistent with the rapid telomere deletion events observed in cells using the recombination-based pathway of telomere maintenance (Murnane, J. P., Sabatier, L., Marder, B. A., and Morgan, W. F. (1994) EMBO J. 13, 4953-4962). We also observed reductions in the fraction of cells containing alternative lengthening of telomere-associated promyelocytic leukemia bodies and extrachromosomal telomere repeats; however, no alterations in the rate of sister chromatid exchange were observed. Our results demonstrate that human cells using the recombination-based pathway of telomere maintenance retain factors required for telomerase to maintain telomeres and that once the telomerase-based pathway of telomere length regulation is engaged, recombination-based elongation of telomeres can be functionally inhibited.

Coexistence of alternative lengthening of telomeres and telomerase in hTERT-transfected GM847 cells.

It has been shown previously that some immortalized human cells maintain their telomeres in the absence of significant levels of telomerase activity by a mechanism referred to as alternative lengthening of telomeres (ALT). Cells utilizing ALT have telomeres of very heterogeneous length, ranging from very short to very long. Here we report the effect of telomerase expression in the ALT cell line GM847. Expression of exogenous hTERT in GM847 (GM847/hTERT) cells resulted in lengthening of the shortest telomeres; this is the first evidence that expression of hTERT in ALT cells can induce telomerase that is active at the telomere. However, rapid fluctuation in telomere length still occurred in the GM847/hTERT cells after more than 100 population doublings. Very long telomeres and ALT-associated promyelocytic leukemia (PML) bodies continued to be generated, indicating that telomerase activity induced by exogenous hTERT did not abolish the ALT mechanism. In contrast, when the GM847 cell line was fused with two different telomerase-positive tumor cell lines, the ALT phenotype was repressed in each case. These hybrid cells were telomerase positive, and the telomeres decreased in length, very rapidly at first and then at the rate seen in telomerase-negative normal cells. Additionally, ALT-associated PML bodies disappeared. After the telomeres had shortened sufficiently, they were maintained at a stable length by telomerase. Together these data indicate that the telomerase-positive cells contain a factor that represses the ALT mechanism but that this factor is unlikely to be telomerase. Further, the transfection data indicate that ALT and telomerase can coexist in the same cells.

ALT-associated PML bodies are present in viable cells and are enriched in cells in the G(2)/M phase of the cell cycle.

Telomere maintenance is essential for the unlimited proliferative potential of human cells, and hence immortalization. However, a number of tumors, tumor-derived cell lines and in vitro immortalized cell lines have been described that do not express detectable telomerase activity. These lines utilize a mechanism, termed Alternative Lengthening of Telomeres (ALT), to provide telomere maintenance. A subset of the cells in each ALT cell line contain a novel form of the promyelocytic leukemia nuclear body (PML NB) in which telomeric DNA and the telomere binding proteins TRF1 and TRF2 co-localize with the PML protein, termed ALT-associated PML bodies (AA-PBs). In contrast, in non-ALT, telomerase-positive cell lines these telomeric proteins and the PML NB occupy distinct and separate subnuclear domains. PML NBs have been implicated in terminal differentiation, growth suppression and apoptosis. The role, if any, of AA-PBs in telomere maintenance or culture viability in telomerase negative cell lines is unclear, but it has been suggested that cells containing these structures are no longer viable and are marked for eventual death. We utilized a series of human ovarian surface epithelium (HOSE) cell lines that use ALT for telomere maintenance to determine if AA-PBs are indeed markers of cells in these cultures that are no longer cycling. We show that AA-PB positive cells incorporate BrdU and thus are able to carry out DNA replication. In addition, AA-PBs are present in mitotic cells and the frequency of cells containing these structures is increased when cultures are enriched for cells in the G(2)/M phase of the cell cycle suggesting that the formation of AA-PBs is coordinately regulated with the cell cycle. Finally, we demonstrate that the majority of the AA-PB positive cells in the culture are not destined for immediate apoptosis. Taken together the data argue against AA-PBs marking cells destined for death and, instead, raise the possibility that these structures may be actively involved in telomere maintenance via the ALT pathway.

Telomere maintenance by recombination in human cells.

Telomeres of eukaryotic chromosomes contain many tandem repeats of a G-rich sequence (for example, TTAGGG in vertebrates). In most normal human cells, telomeres shorten with each cell division, and it is proposed that this limits the number of times these cells can replicate. Telomeres may be maintained in germline cells, and in many immortalized cells and cancers, by the telomerase holoenzyme (first discovered in the ciliate Tetrahymena), which uses an RNA subunit as template for synthesis of telomeric DNA by the reverse transcriptase catalytic subunit. Some immortalized human cell lines and some tumours maintain their telomeres in the absence of any detectable telomerase activity by a mechanism referred to as alternative lengthening of telomeres (ALT). Here we show that DNA sequences are copied from telomere to telomere in an immortalized human ALT cell line, indicating that ALT occurs by means of homologous recombination and copy switching.