Telomere shortening necessitates that tumor cells activate a telomere maintenance mechanism (TMM) to support immortalization. Although most tumor cells activate expression of the enzyme telomerase, some cells elongate telomeres in a telomerase-independent manner, termed alternative lengthening of telomeres (ALT). Previous studies have evaluated the presence of telomerase or ALT mechanisms or both in a variety of tumor types. Our studies also show that TMMs are not mutually exclusive in some tumors. In contrast, our IHC analyses of human sarcomas identified a subset of tumors with some cells containing ALT-associated PML bodies, a hallmark of ALT, and separate cells expressing telomerase in the same tumor. By using a second set of human osteosarcomas, we merged IHC and biochemical analyses to characterize more fully the tumor TMM. The IHC data reveal the presence of both telomerase- and ALT-positive tumor cells in samples that demonstrate characteristics of both telomerase and ALT in biochemical assays. These assays, which measure telomere length and telomerase activity of tumor extracts, are conventionally used to classify tumor TMM. Our results suggest that TMM is not a single or perhaps static characteristic of some tumors and that TMM heterogeneity should be considered in tumor stratification. Furthermore, clinical interest in telomere-based therapies may necessitate accurate characterization of tumor TMM before treatment to maximize therapeutic efficacy.

Telomere maintenance can occur in the presence of telomerase or in its absence, termed alternative lengthening of telomeres (ALT). ALT adds telomere repeats using recombination-based processes and DNA repair proteins that function in homologous recombination. Our previous work reported that the RecQ-like BLM helicase is required for ALT and that it unwinds telomeric substrates in vitro. WRN is also a RecQ-like helicase that shares many biochemical functions with BLM. WRN interacts with BLM, unwinds telomeric substrates, and co-localizes to ALT-associated PML bodies (APBs), suggesting that it may also be required for ALT processes. Using long-term siRNA knockdown of WRN in three ALT cell lines, we show that some, but not all, cell lines require WRN for telomere maintenance. VA-13 cells require WRN to prevent telomere loss and for the formation of APBs; Saos-2 cells do not. A third ALT cell line, U-2 OS, requires WRN for APB formation, however WRN loss results in p53-mediated apoptosis. In the absence of WRN and p53, U-2 OS cells undergo telomere loss for an intermediate number of population doublings (50–70), at which point they maintain telomere length even with the continued loss of WRN. WRN and the tumor suppressor BRCA1 co-localize to APBs in VA-13 and U-2 OS, but not in Saos-2 cells. WRN loss in U-2 OS is associated with a loss of BRCA1 from APBs. While the loss of WRN significantly increases telomere sister chromatid exchanges (T-SCE) in these three ALT cell lines, loss of both BRCA1 and WRN does not significantly alter T-SCE. This work demonstrates that ALT cell lines use different telomerase-independent maintenance mechanisms that variably require the WRN helicase and that some cells can switch from one mechanism to another that permits telomere elongation in the absence of WRN. Our data suggest that BRCA1 localization may define these mechanisms.

Telomeres protect chromosome termini to maintain genomic stability and regulate cellular lifespan. Maintenance of telomere length is required for neoplastic cells after the acquisition of mutations that deregulate cell cycle control and increase cellular proliferation, and can occur through expression of the enzyme telomerase or in a telomerase-independent manner termed alternative lengthening of telomeres (ALT). The precise mechanisms that govern the activation of ALT or telomerase in tumor cells are unknown, although cellular origin may favor one or the other mechanisms. ALT pathways are incompletely understood to date; however, recent publications have increasingly broadened our understanding of how ALT is activated, how it proceeds, and how it influences tumor growth. Specific mutational events influence ALT activation, as mutations in genes that suppress recombination and/or alterations in the regulation of telomerase expression are associated with ALT. Once engaged, ALT uses DNA repair proteins to maintain telomeres in the absence of telomerase; experiments that manipulate the expression of specific proteins in cells using ALT are illuminating some of its mechanisms. Furthermore, ALT may influence tumor growth, as experimental and clinical data suggest that telomerase expression may favor tumor progression. This review summarizes recent findings in mammalian cells and models, as well as clinical data, that identify the genetic mutations permissive to ALT, the DNA repair proteins involved in ALT mechanisms and the importance of telomere maintenance mechanisms for tumor progression. A comprehensive understanding of the mechanisms that permit tumor cell immortalization will be important for identifying novel therapeutic targets in cancer.

Telomeres, the terminal-most structures of chromosomes, are critical to maintain the integrity of chromosomal DNA. As tumor cells bypass normal cell cycle controls and expand their proliferative capacity, accelerated telomere erosion necessitates the activation of a telomere maintenance mechanism. This mechanism can be telomerase-dependent or -independent. Telomerase-independent maintenance is termed alternative lengthening of telomeres, or ALT, and may represent an amalgamation of several recombination-associated pathways. ALT likely uses other telomeres or extrachromosomal telomeric DNA as a template for recombination-based addition of telomere repeats to a shortened chromosome. While most human tumors use telomerase to maintain telomere length, a subset uses ALT. Osteosarcomas are more commonly in this category, as almost 60% demonstrate ALT characteristics. Previous studies to analyze mechanisms of telomere maintenance have shown that while some tumors are ALT or telomerase-dependent, a small number demonstrates mixed Results: both a heterogeneous telomere length consistent with ALT and telomerase activity, incompatible with ALT, or vice versa (reviewed in Henson and Reddel, 2010). These results suggest the idea of a mixed population of cells within some tumors that uses both telomerase-dependent and -independent telomere maintenance mechanisms; however, this has not yet been confirmed. Our studies have used a human osteosarcoma tissue microarray to demonstrate both ALT and telomerase-dependent cells within the same tumor. Tumor sections were stained for PML, TRF2 and telomerase, followed by analysis with the Nuance Multispectral Imaging System. Some tumors (4/6) displayed robust staining and colocalization of PML and TRF2, indicative of ALT-associated PML bodies (APBs), a marker of ALT cells. Some of these tumors (3/4) also contained cells staining strongly for telomerase that were exclusive of ALT-specific colocalization. These two types of cells within this subset of tumors were distinct from one another. To confirm these initial findings, five frozen human osteosarcoma samples were evaluated to correlate in situ staining results with classical methods of gross telomere classification, characterization of telomere length via telomere restriction fragment (TRF) Southern blot and identification of telomerase activity via the telomere repeat amplification protocol (TRAP). Two tumors displayed ALT characteristics only, exhibiting heterogeneous telomere lengths with TRF Southern blots and negative findings with TRAP; one tumor displayed positive TRAP results and more homogeneous telomere lengths associated with telomerase activity. Of the remaining two tumors, one was characterized by telomerase-associated homogenous telomere length and one was characterized by telomerase-independent heterogeneous telomere length by TRF Southern blots; both tumor lysates displayed variable and weak telomerase activity with TRAP. These data suggest that cells within these tumors use more than one mechanism of telomere maintenance. Tumor sections from these five osteosarcomas are now being analyzed using our in situ immunohistochemistry methods. Given recent interest in telomerase-targeted cancer therapies, the coexistence of both telomere maintenance mechanisms within a single tumor would have significant implications for understanding the mechanisms of telomere maintenance and for developing precise therapies to target ALT mechanisms of telomere elongation. Henson, J. D. and Reddel, R. R. Assaying and investigating Alternative Lengthening of Telomeres activity in human cells and cancers. FEBS Lett., 584: 3800–3811, 2010. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B57.