Telomerase-negative immortalized human cells maintain their telomeres by a mechanism known as alternative lengthening of telomeres (ALT). We report here that ALT cells contain a novel promyelocytic leukemia (PML) body (ALT-associated PML body, APB). APBs are large donut-shaped nuclear structures containing PML protein, telomeric DNA, and the telomere binding proteins human telomere repeat binding factors 1 and 2. Immunostaining showed that APBs also contain replication factor A, RAD51, and RAD52, proteins involved in DNA synthesis and recombination. During immortalization, APBs appeared at exactly the same time as activation of ALT. APBs were found in ALT tumors and cell lines but not in mortal cell strains or in telomerase-positive cell lines or tumors.
// Christine E. Napier 1 , Lily I. Huschtscha 1 , Adam Harvey 2 , Kylie Bower 1 , Jane R. Noble 1 , Eric A. Hendrickson 2 and Roger R. Reddel 1,2 1 Cancer Research Unit, Children’s Medical Research Institute, The University of Sydney, Westmead, NSW, Australia 2 Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota Medical School, Minneapolis, MN, USA Correspondence to: Roger R. Reddel , email: // Keywords : ATRX, ALT, telomere, immortalization Received : December 18, 2014 Accepted : March 20, 2015 Published : April 15, 2015 Abstract The unlimited proliferation of cancer cells requires a mechanism to prevent telomere shortening. Alternative Lengthening of Telomeres (ALT) is an homologous recombination-mediated mechanism of telomere elongation used in tumors, including osteosarcomas, soft tissue sarcoma subtypes, and glial brain tumors. Mutations in the ATRX/DAXX chromatin remodeling complex have been reported in tumors and cell lines that use the ALT mechanism, suggesting that ATRX may be an ALT repressor. We show here that knockout or knockdown of ATRX in mortal cells or immortal telomerase-positive cells is insufficient to activate ALT. Notably, however, in SV40-transformed mortal fibroblasts ATRX loss results in either a significant increase in the proportion of cell lines activating ALT (instead of telomerase) or in a significant decrease in the time prior to ALT activation. These data indicate that loss of ATRX function cooperates with one or more as-yet unidentified genetic or epigenetic alterations to activate ALT. Moreover, transient ATRX expression in ALT-positive/ATRX-negative cells represses ALT activity. These data provide the first direct, functional evidence that ATRX represses ALT.
Abstract Hereditary SDHB-mutant pheochromocytomas (PC) and paragangliomas (PG) are rare tumours with a high propensity to metastasize although their clinical behaviour is unpredictable. To characterize the genomic landscape of these tumours and identify metastasis biomarkers, we performed multi-omic analysis on 94 tumours from 79 patients using seven molecular methods. Sympathetic (chromaffin cell) and parasympathetic (non-chromaffin cell) PCPG had distinct molecular profiles reflecting their cell-of-origin and biochemical profile. TERT and ATRX-alterations were associated with metastatic PCPG and these tumours had an increased mutation load, and distinct transcriptional and telomeric features. Most PCPG had quiet genomes with some rare co-operative driver events observed, including EPAS1/HIF-2α mutations. Two mechanisms of acquired resistance to DNA alkylating chemotherapies were also detected - MGMT overexpression and mismatch repair-deficiency causing hypermutation. Our comprehensive multi-omic analysis of SDHB-mutant PCPG therefore identified features of metastatic disease and treatment response, expanding our understanding of these rare neuroendocrine tumours.
MicroRNAs (miRNAs) are a class of noncoding small RNAs that act as negative regulators of gene expression. To identify miRNAs that may regulate human cell immortalization and carcinogenesis, we performed comparative miRNA array profiling of human normal and SV40-T antigen immortalized cells. We found that miR-296 was upregulated in immortalized cells that also had activation of telomerase. By an independent experiment on genomic analysis of cancer cells we found that chromosome region (20q13.32), where miR-296 is located, was amplified in 28/36 cell lines, and most of these showed enriched miR-296 expression. Overexpression of miR-296 in human cancer cells, with and without telomerase activity, had no effect on their telomerase function. Instead, it suppressed p53 function that is frequently downregulated during human cell immortalization and carcinogenesis. By monitoring the activity of a luciferase reporter connected to p53 and p21WAF1 (p21) untranslated regions (UTRs), we demonstrate that miR-296 interacts with the p21-3′UTR, and the Hu binding site of p21-3′UTR was identified as a potential miR-296 target site. We demonstrate for the first time that miR-296 is frequently upregulated during immortalization of human cells and contributes to carcinogenesis by downregulation of p53-p21WAF1 pathway.
Some cancers use alternative lengthening of telomeres (ALT), a mechanism whereby new telomeric DNA is synthesized from a DNA template. To determine whether normal mammalian tissues have ALT activity, we generated a mouse strain containing a DNA tag in a single telomere. We found that the tagged telomere was copied by other telomeres in somatic tissues but not the germline. The tagged telomere was also copied by other telomeres when introgressed into CAST/EiJ mice, which have telomeres more similar in length to those of humans. We conclude that ALT activity occurs in normal mouse somatic tissues.
Here we describe a method for growing fibroblasts from human skin explants that increases the number of cells obtained by up to two orders of magnitude, thus increasing the amount of material available for research and diagnostic purposes and potentially for cell-based therapies. Explants can be transferred sequentially up to 80 times, if required, at which point the explants appear to be completely depleted of fibroblasts. Utilizing skin samples obtained from 16 donors, aged 18-66 years old, the first 20 transfers produced cultures with lifespan and growth characteristics that were all very similar to each other, but the cultures derived from later transfers had a decreasing replicative capacity. Final cumulative population doublings did not correlate with donor age, but correlated positively with the telomere length at early passage. We also demonstrated that explants can be transduced directly by lentiviral infection, and that cryopreserved tissue can be explanted successfully using this procedure.
Cancers that utilize the Alternative Lengthening of Telomeres (ALT) mechanism for telomere maintenance are often difficult to treat and have a poor prognosis. They are also commonly deficient for expression of ATRX protein, a repressor of ALT activity, and a component of PML nuclear bodies (PML NBs) which are required for intrinsic immunity to various viruses. Here we asked whether ATRX-deficiency creates a vulnerability in ALT cancer cells that could be exploited for therapeutic purposes. We showed in a range of cell types that a mutant herpes simplex virus type 1 (HSV-1) lacking ICP0, a protein that degrades PML NB components including ATRX, was ten- to one thousand-fold more effective in killing ATRX-deficient cells. Infection of co-cultured primary and ATRX-null cancer cells revealed that mutant HSV-1 selectively killed ATRX-null cells. Sensitivity to mutant HSV–1 infection also correlated inversely with PML protein levels, and we showed that ATRX upregulates PML expression at both the transcriptional and post-transcriptional levels. These data provide a basis for predicting, based on ATRX or PML levels, which tumors will respond to a selective oncolytic herpesvirus.
As teacher educators strive to prepare preservice teachers for careers as literacy instructors and advocates of social justice in education, critical service-learning pedagogy has been considered as an approach for teacher education programs. Tenets of academic study, reflective practice, social change, and the development of authentic relationships between universities and communities outline the structure for critical-based field experiences. What are preservice teachers learning in these spaces? How do they grow as part of critical service- learning courses? How do community organizations and members interpret experiences in the partnership, and how do they describe their roles?
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