Telomere dysfunction is typically studied under conditions in which a component of the six-subunit shelterin complex that protects chromosome ends is disrupted. The nature of spontaneous telomere ...dysfunction is less well understood. Here we report that immortalized human cell lines lacking wild-type p53 function spontaneously show many telomeres with a DNA damage response (DDR), commonly affecting only one sister chromatid and not associated with increased chromosome end-joining. DDR(+) telomeres represent an intermediate configuration between the fully capped and uncapped (fusogenic) states. In telomerase activity-positive (TA(+)) cells, DDR is associated with low TA and short telomeres. In cells using the alternative lengthening of telomeres mechanism (ALT(+)), DDR is partly independent of telomere length, mostly affects leading strand-replicated telomeres, and can be partly suppressed by TRF2 overexpression. In ALT(+) (but not TA(+)) cells, DDR(+) telomeres preferentially associate with large foci of extrachromosomal telomeric DNA and recombination proteins. DDR(+) telomeres therefore arise through different mechanisms in TA(+) and ALT(+) cells and have different consequences.
Protein colocalisation is often studied using pixel intensity-based coefficients such as Pearson, Manders, Li or Costes. However, these methods cannot be used to study object-based colocalisations in ...biological systems. Therefore, a novel method is required to automatically identify regions of fluorescent signal in two channels, identify the co-located parts of these regions, and calculate the statistical significance of the colocalisation. We have developed MatCol to address these needs. MatCol can be used to visualise protein and/or DNA colocalisations and fine tune user-defined parameters for the colocalisation analysis, including the application of median or Wiener filtering to improve the signal to noise ratio. Command-line execution allows batch processing of multiple images. Users can also calculate the statistical significance of the observed object colocalisations compared to overlap by random chance using Student's t-test. We validated MatCol in a biological setting. The colocalisations of telomeric DNA and TRF2 protein or TRF2 and PML proteins in >350 nuclei derived from three different cell lines revealed a highly significant correlation between manual and MatCol identification of colocalisations (linear regression R
= 0.81, P < 0.0001). MatCol has the ability to replace manual colocalisation counting, and the potential to be applied to a wide range of biological areas.
Acquisition of replicative immortality is currently regarded as essential for malignant transformation. This is achieved by activating a telomere lengthening mechanism (TLM), either telomerase or ...alternative lengthening of telomeres, to counter normal telomere attrition. However, a substantial proportion of some cancer types, including glioblastomas, liposarcomas, retinoblastomas, and osteosarcomas, are reportedly TLM-negative. As serial samples of human tumors cannot usually be obtained to monitor telomere length changes, it has previously been impossible to determine whether tumors are truly TLM-deficient, there is a previously unrecognized TLM, or the assay results are false-negative. Here, we show that a subset of high-risk neuroblastomas (with ∼50% 5-year mortality) lacked significant TLM activity. Cancer cells derived from these highly aggressive tumors initially had long telomeres and proliferated for >200 population doublings with ever-shorter telomeres. This indicates that prevention of telomere shortening is not always required for oncogenesis, which has implications for inhibiting TLMs for cancer therapy.
Display omitted
•A subset of neuroblastomas lack a telomere length maintenance mechanism•These cancers with “ever-shorter telomeres (EST)” are highly aggressive•Telomere length measurement fails to distinguish EST from ALT cancers•Activation of telomerase or ALT can rescue the EST phenotype
Dagg et al. find that a subset of highly malignant neuroblastomas (survival ∼50% despite intensive treatment) lack an effective telomere length maintenance mechanism. Their cells undergo continuous telomere shortening throughout >200 population doublings, challenging the concept that activation of a mechanism to prevent telomere shortening is essential for oncogenesis.
Telomerase is a ribonucleoprotein complex that maintains the length and integrity of telomeres, and thereby enables cellular proliferation. Understanding the regulation of telomerase in hematopoietic ...cells is relevant to the pathogenesis of leukemia, in which telomerase is constitutively activated, as well as bone marrow failure syndromes that feature telomerase insufficiency. Past studies showing high levels of telomerase in human erythroblasts and a prevalence of anemia in disorders of telomerase insufficiency provide the rationale for investigating telomerase regulation in erythroid cells. Here it is shown for the first time that the telomerase RNA-binding protein dyskerin (encoded by
) is dramatically upregulated as human hematopoietic stem and progenitor cells commit to the erythroid lineage, driving an increase in telomerase activity in the presence of limiting amounts of
mRNA. It is also shown that upregulation of
was necessary for expansion of glycophorin A
erythroblasts and sufficient to extend telomeres in erythroleukemia cells. Chromatin immunoprecipitation and reporter assays implicated GATA1-mediated transcriptional regulation of
in the modulation of telomerase in erythroid lineage cells. Together these results describe a novel mechanism of telomerase regulation in erythroid cells which contrasts with mechanisms centered on transcriptional regulation of
that are known to operate in other cell types. This is the first study to reveal a biological context in which telomerase is upregulated by
and to implicate
in telomerase regulation. The results from this study are relevant to hematopoietic disorders involving
mutations,
deregulation and/or telomerase insufficiency.
Alternative Lengthening of Telomeres (ALT) is a non-telomerase mechanism of telomere lengthening that occurs in about 10% of cancers overall and is particularly common in astrocytic brain tumors and ...specific types of sarcomas. Somatic cell hybridization analyses have previously shown that normal telomerase-negative fibroblasts and telomerase-positive immortalized cell lines contain repressors of ALT activity, indicating that activation of ALT results from loss of one or more unidentified repressors. More recently, ATRX or DAXX was shown to be mutated both in tumors with telomere lengths suggestive of ALT activity and in ALT cell lines. Here, an ALT cell line was separately fused to each of four telomerase-positive cell lines, and four or five independent hybrid lines from each fusion were examined for expression of ATRX and DAXX and for telomere lengthening mechanism. The hybrid lines expressed either telomerase or ALT, with the other mechanism being repressed. DAXX was expressed normally in all parental cell lines and in all of the hybrids. ATRX was expressed normally in each of the four telomerase-positive parental cell lines and in every telomerase-positive hybrid line, and was abnormal in the ALT parental cells and in all but one of the ALT hybrids. This correlation between ALT activity and loss of ATRX expression is consistent with ATRX being a repressor of ALT.
There is limited knowledge about cancer patients' experiences of uncertainty while waiting for genome sequencing results, and whether prolonged uncertainty contributes to psychological factors in ...this context. To investigate uncertainty in patients with a cancer of likely hereditary origin while waiting for genome sequencing results, we collected questionnaire and interview data at baseline, and at three and 12 months follow up (prior to receiving results). Participants (
= 353) had negative attitudes towards uncertainty (
= 4.03, SD 0.68) at baseline, and low levels of uncertainty at three (
= 8.23, SD 7.37) and 12 months (
= 7.95, SD 7.64). Uncertainty about genome sequencing did not change significantly over time
(210) = 0.660,
= 0.510. Greater perceived susceptibility for cancer
(348) = 0.14,
<
.01, fear of cancer recurrence
(348) = 0.19,
<
.01, perceived importance of genome sequencing
(350) = 0.24,
<
.01, intention to change behavior if a gene variant indicating risk is found
(349) = 0.29,
<
.01, perceived ability to cope with results
(349) = 0.36,
<
.01, and satisfaction with decision to have genome sequencing
(350) = 0.52,
<
.01 were significantly correlated with negative attitudes towards uncertainty at baseline. Multiple primary cancer diagnoses B = -2.364 -4.238, -0.491,
= 0.014, lower perceived ability to cope with results B = -0.1.881 -3.403, -0.359,
= 0.016 at baseline, greater anxiety about genome sequencing (avoidance) B = 0.347 0.148, 0.546,
= 0.0012 at 3 months, and greater perceived uncertainty about genome sequencing B = 0.494 0.267, 0.721
= 0.000 at 3 months significantly predicted greater perceived uncertainty about genome sequencing at 12 months. Greater perceived uncertainty about genome sequencing at 3 months significantly predicted greater anxiety (avoidance) about genome sequencing at 12 months B = 0.291 0.072, 0.509,
= 0.009. Semi-structured interviews revealed that while participants were motivated to pursue genome sequencing as a strategy to reduce their illness and risk uncertainty, genome sequencing generated additional practical, scientific and personal uncertainties. Some uncertainties were consistently discussed over the 12 months, while others emerged over time. Similarly, some uncertainty coping strategies were consistent over time, while others emerged while patients waited for their genome sequencing results. This study demonstrates the complexity of uncertainty generated by genome sequencing for cancer patients and provides further support for the inter-relationship between uncertainty and anxiety. Helping patients manage their uncertainty may ameliorate psychological morbidity.
Tumor mutation profiling (MP) is often conducted on tissue from biopsies conducted for clinical purposes (diagnostic tissue). We aimed to explore the views of patients with cancer on who should own ...tumor biopsy tissue, pay for its storage, and decide on its future use; and determine their attitudes to and predictors of undergoing additional biopsies if required for research purposes. In this mixed methods, cross-sectional study, patients with advanced solid cancers enrolled in the Molecular Screening and Therapeutics Program (n = 397) completed a questionnaire prior to undergoing MP (n = 356/397). A subset (n = 23) also completed a qualitative interview. Fifty percent of participants believed they and/or relatives should own and control access to diagnostic tissue. Most (65.5%) believed the government should pay for tissue preparation. Qualitative themes included (1) custodianship of diagnostic tissue, (2) changing value of tissue across time and between cultures, (3) equity regarding payment, and (4) cost-benefit considerations in deciding on additional biopsies. Policy and regulation should consider patient perspectives. Extension of publicly funded health care to include tissue retrieval for clinical trials should be considered.
•WGS-based tools can be used to accurately measure telomere content.•Motif_counter, TelSeq, Computel, and qMotif perform comparably.•Telomere length analysis by WGS can be applied to large-scale ...sequencing projects.
Telomeres are regions of repetitive DNA at the ends of human chromosomes that function to maintain the integrity of the genome. Telomere attrition is associated with cellular ageing, whilst telomere maintenance is a prerequisite for malignant transformation. Whole genome sequencing (WGS) captures sequence information from the entire genome, including the telomeres, and is increasingly being applied in research and in the clinic. Several bioinformatics tools have been designed to determine telomere content and length from WGS data, and include Motif_counter, TelSeq, Computel, qMotif, and Telomerecat. These tools utilise different approaches to identify, quantify and normalise telomeric reads; however, it is not known how they compare to one another. Here we describe the details and utility of each tool, and directly compare WGS telomere length output with laboratory-based telomere length measurements. In addition, we evaluate the accessibility, practicality, speed, and additional features of each tool. Each tool was tested using a range of telomere read extraction criteria, to determine the optimal parameters for the specific WGS read length. The aim of this article is to improve the accessibility of WGS telomere length measurement tools, which have the potential to be applied to WGS cohorts for clinical as well as research benefit.
Purpose
The introduction of comprehensive tumour genomic profiling (CGP) into clinical oncology allows the identification of molecular therapeutic targets. However, the potential complexity of ...genomic results and their implications may cause confusion and distress for patients undergoing CGP. We investigated the experience of advanced cancer patients receiving CGP results in a research setting.
Methods
Semi-structured interviews with 37 advanced cancer patients were conducted within two weeks of patients receiving CGP results. Interviewees were purposively sampled based on CGP result, cancer type, age and gender to ensure diversity. Themes were derived from interview transcripts using a framework analysis approach.
Results
We identified six themes: (1) hoping against the odds; (2) managing expectations; (3) understanding is cursory; (4) communication of results is cursory; (5) genomics and incurable cancer; and (6) decisions about treatment.
Conclusion
Despite enthusiasm regarding CGP about the hope it provides for new treatments, participants experienced challenges in understanding results, and acceptance of identified treatments was not automatic. Support is needed for patients undergoing CGP to understand the implications of testing and cope with non-actionable results.
PDF
