ALT-associated Promyelocytic Leukemia Nuclear Bodies Research Articles

ZBTB40 is a telomere-associated protein and protects telomeres in human ALT cells

Alternative lengthening of telomeres (ALTs) mechanism is activated in some somatic, germ cells, and human cancer cells. However, the key regulators and mechanisms of the ALT pathway remain elusive. Here we demonstrated that ZBTB40 is a novel telomere-associated protein and binds to telomeric dsDNA through its N-terminal BTB (BR-C, ttk and bab) or POZ (Pox virus and Zinc finger) domain in ALT cells. Notably, the knockout or knockdown of ZBTB40 resulted in the telomere dysfunction-induced foci and telomere lengthening in the ALT cells. The results also show that ZBTB40 is associated with ALT-associated promyelocytic leukemia nuclear bodies, and the loss of ZBTB40 induces the accumulation of the ALT-associated promyelocytic leukemia nuclear bodies in U2OS cells. Taken together, our results implicate that ZBTB40 is a key player of telomere protection and telomere lengthening regulation in human ALT cells.

Telomerase Is a Prognostic Marker of Poor Outcome and a Therapeutic Target in Neuroblastoma.

Telomere maintenance is a hallmark of high-risk neuroblastoma; however, the contribution of telomerase and alternative lengthening of telomeres (ALT) to clinical phenotypes has remained unclear. We aimed to determine the clinical relevance of telomerase activation versus ALT as biomarkers in pretreatment neuroblastoma and to assess the potential value of telomerase as a therapeutic target. The genomic status of TERT and MYCN was assessed in 457 pretreatment neuroblastomas by fluorescence in situ hybridization. ALT was examined in 273 of 457 tumors by detection of ALT-associated promyelocytic leukemia nuclear bodies, and TERT expression was determined by microarrays in 223 of these. Cytotoxic effects of telomerase-interacting compounds were analyzed in neuroblastoma cell lines in vitro and in vivo. We detected TERT rearrangements in 46 of 457 cases (10.1%), MYCN amplification in 93 of 457 cases (20.4%), and elevated TERT expression in tumors lacking TERT or MYCN alterations in 10 of 223 cases (4.5%). ALT activation was found in 49 of 273 cases (17.9%). All these alterations occurred almost mutually exclusively and were associated with unfavorable prognostic variables and adverse outcome. The presence of activated telomerase (ie, TERT rearrangements, MYCN amplification, or high TERT expression without these alterations) was associated with poorest overall survival and was an independent prognostic marker in multivariable analyses. We also found that the telomerase-interacting compound 6-thio-2'-deoxyguanosine effectively inhibited viability and proliferation of neuroblastoma cells bearing activated telomerase. Similarly, tumor growth was strongly impaired upon 6-thio-2'-deoxyguanosine treatment in telomerase-positive neuroblastoma xenografts in mice. Our data suggest telomerase activation and ALT define distinct neuroblastoma subgroups with adverse outcome and that telomerase may represent a promising therapeutic target in many high-risk neuroblastomas.

Telomere Maintenance-Associated PML Is a Potential Specific Therapeutic Target of Human Colorectal Cancer

Telomere length maintenance is essential for cell proliferation, which is particularly prominent in cancer. We validate that the primary colorectal tumors exhibit heterogeneous telomere lengths but mostly (90%) short telomeres relative to normal tissues. Intriguingly, relatively short telomeres are associated with tumor malignancy as indicated by poorly differentiated state, and these tumors contain more cancer stem-like cells (CSLCs) identified by several commonly used markers CD44, EPHB2 or LGR5. Moreover, promyelocytic leukemia (PML) and ALT-associated PML nuclear bodies (APBs) are frequently found in tumors with short telomeres and high proliferation. In contrast, distant normal tissues rarely or only minimally express PML. Inhibition of PML and APBs by an ATR inhibitor decreases proliferation of CSLCs and organoids, suggesting a potential therapeutic target to progressive colorectal tumors. Together, telomere maintenance underling tumor progression is connected with CSLCs.

Withaferin-A kills cancer cells with and without telomerase: chemical, computational and experimental evidences

Maintenance of telomere length is the most consistent attribute of cancer cells. Tightly connected to their capacity to overcome replicative mortality, it is achieved either by activation of telomerase or an Alternative mechanism of Lengthening of Telomeres (ALT). Disruption of either of these mechanisms has been shown to induce DNA damage signalling leading to senescence or apoptosis. Telomerase inhibitors are considered as potential anticancer drugs but are ineffective for ALT cancers (~15% of all cancers). Withaferin-A (Wi-A), a major constituent of the medicinal plant, Withania somnifera (Ashwagandha), has been shown to exert anti-tumour activity. However, its effect on either telomerase or ALT mechanisms has not been investigated. Here, by using isogenic cancer cells with/without telomerase, we found that Wi-A caused stronger cytotoxicity to ALT cells. It was associated with inhibition of ALT-associated promyelocytic leukemia nuclear bodies, an established marker of ALT. Comparative analyses of telomerase positive and ALT cells revealed that Wi-A caused stronger telomere dysfunction and upregulation of DNA damage response in ALT cells. Molecular computational and experimental analyses revealed that Wi-A led to Myc-Mad mediated transcriptional suppression of NBS-1, an MRN complex protein that is an essential component of the ALT mechanism. The results suggest that Wi-A could be a new candidate drug for ALT cancers.

Alternative lengthening of telomeres in neuroblastoma cell lines is associated with a lack of MYCN genomic amplification and with p53 pathway aberrations.

Alternative lengthening of telomeres (ALT) is a telomerase-independent telomere length maintenance mechanism that enables the unlimited proliferation of a subset of cancer cells. Some neuroblastoma (NB) tumors appear to maintain telomere length by activating ALT. Of 40 NB cell lines, we identified four potential ALT cell lines (CHLA-90, SK-N-FI, LA-N-6, and COG-N-291) that were telomerase-negative and had long telomeres (a feature of ALT cells). All four cell lines lacked MYCN amplification and were p53 non-functional upon irradiation. Two of these cell lines (CHLA-90 and SK-N-FI) were positive for C-circles (telomeric DNA circles) and ALT-associated promyelocytic leukemia nuclear bodies, both of which are phenotypic characteristics of ALT. Mutation of ATRX (associated with ALT in tumors) was only found in CHLA-90. Thus, the ALT phenotype in NB may not be limited to tumors with ATRX mutations but is associated with a lack of MYCN amplification and alterations in the p53 pathway.

Detection of ALT Associated Promyelocytic Leukemia Nuclear Bodies (APBs) by Immunofluorescence-FISH (IF-FISH)

The activation of functions that counteract the physiological shortening of telomeres in rapidly proliferating cell is prerequisite for the progression of cancer cells to full malignancy (Collado et al., 2007). In most human cancers, the length of telomere is maintained through up-regulation of telomerase whereas a telomerase-independent pathway, termed Alternative Lengthening of Telomeres (ALT) is active in about 10-15% of cancers (Johnson and Broccoli, 2007; Heaphy et al., 2011). One characteristic feature of ALT is the formation of ALT-associated Promyelocytic Leukemia nuclear bodies (APBs) (Lang et al., 2010; Yeager et al., 1999). APBs contain Promyelocytic Leukemia nuclear bodies (PML-NB) components such as PML, SP100 and SUMO, telomeric DNA and telomere associated proteins including the shelterin components TRF1, TRF2, POT1, TIN2, TPP1 and Rap1 (Yeager et al., 1999). In addition, APBs contain proteins involved in DNA repair. In particular, the presence of components of the homologous recombination machinery suggests that APBs may promote telomere elongation by facilitating the homologous recombination of telomeric templates (Nabetani et al., 2004; Stavropoulos et al., 2002). This is also supported by the requirement of the homologous recombination-associated MRN complex for APB formation (Wu et al., 2000). Furthermore, APBs are suggested to be active sites of ATM and ATR dependent DNA repair (Nabetani et al., 2004). Finally, the number of APBs increases in G2 phase of the cell cycle when recombination is mainly active (Grobelny et al., 2000). We have shown that infection of normal and malignant B lymphocytes with the human oncogenic herpesvirus Epstein-Barr virus (EBV) is associated with the induction of APBs and with numerous signs of chromosomal and genomic instability (Kamranvar et al., 2007; Kamranvar and Masucci, 2011; Kamranvar et al., 2013). Here we describe a method for detection of APBs in human B-lymphocytes. The method can be applied with minor modifications to different cell types including adherent, suspension and primary cells.

BiotecVisions 2012, February

BiotecVisions 2012, February

A three‐dimensional colocalization RNA interference screening platform to elucidate the alternative lengthening of telomeres pathway

A high-content colocalization RNA interference screen based on automatic three-color confocal fluorescence microscopy was developed to analyze the alternative lengthening of telomeres (ALT) pathway. Via this pathway telomerase-negative cancer cells can maintain their telomeres and with it their unlimited proliferative potential. A hallmark of ALT cells is the colocalization of promyelocytic leukemia (PML) nuclear bodies with telomeres to form ALT-associated PML nuclear bodies (APBs). In our screen, the presence of APBs was used as a marker to identify proteins required for the ALT mechanism. A cell-based assay and an automatic confocal image acquisition procedure were established. Using automatic image analysis based on 3D parametric intensity models to identify APBs, we conducted an unbiased and quantitative analysis of nine different candidate genes. A comparison with the literature and manual analysis of the gene knockdown demonstrates the reliability of our approach. It extends the available repertoire of high-content screening to studies of cellular colocalizations and allows the identification of candidate genes for the ALT mechanism that represent possible targets for cancer therapy.

Induction of alternative lengthening of telomeres-associated PML bodies by p53/p21 requires HP1 proteins

Alternative lengthening of telomeres (ALT) is a recombination-mediated process that maintains telomeres in telomerase-negative cancer cells. In asynchronously dividing ALT-positive cell populations, a small fraction of the cells have ALT-associated promyelocytic leukemia nuclear bodies (APBs), which contain (TTAGGG)n DNA and telomere-binding proteins. We found that restoring p53 function in ALT cells caused p21 up-regulation, growth arrest/senescence, and a large increase in cells containing APBs. Knockdown of p21 significantly reduced p53-mediated induction of APBs. Moreover, we found that heterochromatin protein 1 (HP1) is present in APBs, and knockdown of HP1α and/or HP1γ prevented p53-mediated APB induction, which suggests that HP1-mediated chromatin compaction is required for APB formation. Therefore, although the presence of APBs in a cell line or tumor is an excellent qualitative marker for ALT, the association of APBs with growth arrest/senescence and with “closed” telomeric chromatin, which is likely to repress recombination, suggests there is no simple correlation between ALT activity level and the number of APBs or APB-positive cells.

Multiple Mechanisms of Telomere Maintenance Exist and Differentially Affect Clinical Outcome in Diffuse Malignant Peritoneal Mesothelioma

This study aims to investigate the prevalence of the two known telomere maintenance mechanisms, telomerase activity (TA) and alternative lengthening of telomeres (ALT), and to assess their prognostic relevance in diffuse malignant peritoneal mesothelioma (DMPM). In 44 DMPM specimens obtained from 38 patients, TA was determined using the telomeric repeat amplification protocol and ALT was detected by assaying ALT-associated promyelocytic leukemia nuclear bodies. The prognostic significance of telomere maintenance mechanisms was analyzed by Cox regression in the overall series and in a subset of 29 patients who underwent a uniform treatment regimen consisting of cytoreductive surgery and hyperthermic i.p. chemotherapy. Telomere maintenance mechanisms were detectable in 86.4% of DMPM: ALT or TA alone was found in 18.2% or 63.6% of lesions, respectively, whereas two cases (4.6%) were ALT+/TA+. TA and ALT proved to be inversely associated (P = 0.002). In the overall series, TA was prognostic for 4-year relapse (TA+ versus TA-, hazard ratio, 3.30; 95% confidence interval, 1.23-8.86; P = 0.018) and cancer-related death (TA+ versus TA-, hazard ratio, 3.56; 95% confidence interval, 1.03-12.51; P = 0.045), whereas ALT failed to significantly affect clinical outcome. These results held true also in the subset of patients submitted to uniform treatment with cytoreductive surgery and hyperthermic i.p. chemotherapy. Our results indicate that both known telomere maintenance mechanisms, TA and ALT, are present in DMPM and differentially affect patient prognosis.

Telomere Maintenance Mechanisms in Liposarcomas: Association with Histologic Subtypes and Disease Progression

Human cancer cells maintain telomeres by telomerase activity (TA) or by alternative lengthening of telomeres (ALT). We proposed to define the prevalence of the two telomere maintenance mechanisms (TMM), to assess their association with histology, and to compare their prognostic relevance in a series of 93 patients with liposarcoma. ALT was detected by assaying ALT-associated promyelocytic leukemia nuclear bodies and TA was assayed using the telomeric repeat amplification protocol. ALT or TA was found in 25.9% or 26.6% of 139 tested liposarcoma lesions, respectively. Three lesions were ALT+/TA+ whereas approximately 50% of lesions did not show any known TMM. TMM phenotype was consistent during disease progression. ALT was prevalent in dedifferentiated and in grade 3 liposarcomas whereas TA prevailed in most round-cell myxoid and in grade 2 liposarcomas. ALT and TA incidence was similar in primary and recurrent lesions whereas metastases were more frequently TA+ than ALT+ (59% versus 18%; P = 0.04). TMM presence negatively affected patient prognosis (P = 0.001): increased mortality was associated with positivity for TA (P = 0.038) or ALT (P < 0.0001) compared with TMM absence. ALT proved to be a stronger prognostic discriminant of increased mortality than TA even when adjusted for tumor location, grade, and histology (hazard ratio for cause-specific death, 3.58 versus 1.15). Our results indicate that ALT can support fully malignant liposarcomas and is associated with unfavorable disease outcome.

Association of MutantTP53with Alternative Lengthening of Telomeres and Favorable Prognosis in Glioma

The molecular basis for alternative lengthening of telomeres (ALT), a prognostic marker for glioma patients, remains unknown. We examined TP53 status in relation to telomere maintenance mechanism (TMM) in 108 patients with glioblastoma multiforme and two patients with anaplastic astrocytoma from New Zealand and United Kingdom. Tumor samples were analyzed with respect to telomerase activity, telomere length, and ALT-associated promyelocytic leukemia nuclear bodies to determine their TMM. TP53 mutation was analyzed by direct sequencing of coding exons 2 to 11. We found an association between TP53 mutation and ALT mechanism and between wild-type TP53 and telomerase and absence of a known TMM (P < 0.0001). We suggest that TP53 deficiency plays a permissive role in the activation of ALT.

Multiple Mechanisms of Telomere Maintenance Exist in Liposarcomas

Telomeres are specialized nucleoprotein complexes that protect and confer stability upon chromosome ends. Loss of telomere function as a consequence of proliferation-associated sequence attrition results in genome instability, which may facilitate carcinogenesis by generating growth-promoting mutations. However, unlimited cellular proliferation requires the maintenance of telomeric DNA; thus, the majority of tumor cells maintain their telomeres either through the activity of telomerase or via a mechanism known as alternative lengthening of telomeres (ALT). Recent data suggest that constitutive telomere maintenance may not be required in all tumor types. Here we assess the role and requirement of telomere maintenance in liposarcoma. Tumor samples were analyzed with respect to telomerase activity, telomere length, and the presence of ALT-specific subcellular structures, ALT-associated promyelocytic leukemia nuclear bodies. This multi-assay assessment improved the accuracy of categorization. Our data reveal a significant incidence (24%) of ALT-positive liposarcomas, whereas telomerase is used at a similar frequency (27%). A large number of tumors (49%) do not show characteristics of telomerase or ALT. In addition, telomere length was always shorter in recurrent disease, regardless of the telomere maintenance mechanism. These results suggest that approximately one half of liposarcomas either employ a novel constitutively active telomere maintenance mechanism or lack such a mechanism. Analysis of recurrent tumors suggests that liposarcomas can develop despite limiting or undetectable activity of a constitutively active telomere maintenance mechanism.

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.