Lack of TRF2 in ALT cells causes PML-dependent p53 activation and loss of telomeric DNA

Alternative lengthening of telomere (ALT) tumors maintain telomeres by a telomerase-independent mechanism and are characterized by a nuclear structure called the ALT-associated PML body (APB). TRF2 is a component of a telomeric DNA/protein complex called shelterin. However, TRF2 function in ALT cells remains elusive. In telomerase-positive tumor cells, TRF2 inactivation results in telomere de-protection, activation of ATM, and consequent induction of p53-dependent apoptosis. We show that in ALT cells this sequence of events is different. First, TRF2 inactivation/silencing does not induce cell death in p53-proficient ALT cells, but rather triggers cellular senescence. Second, ATM is constitutively activated in ALT cells and colocalizes with TRF2 into APBs. However, it is only following TRF2 silencing that the ATM target p53 is activated. In this context, PML is indispensable for p53-dependent p21 induction. Finally, we find a substantial loss of telomeric DNA upon stable TRF2 knockdown in ALT cells. Overall, we provide insight into the functional consequences of shelterin alterations in ALT cells

Inhalation therapy in the next decade : Determinants of adherence to treatment in asthma and COPD

Peer reviewedPublisher PD

Development of the International Severe Asthma Registry (ISAR) : A Modified Delphi Study

This study is cofunded by Optimum Patient Care Global and AstraZeneca.Peer reviewedPostprin

Deconstructing the ALT-associated promyelocytic leukemia biodies

Cells in which Alternative lengthening of telomeres (ALT) is activated have been reported to contain a novel PML nuclear body (PML-NB) known as ALT-associated PML body (APB). These large, donut-shaped nuclear structures are unique to telomerase-negative cells and contain, among others, PML, telomeric DNA, the telomere binding proteins TRF1 and TRF2, MRE11 and NBS1. APBs can be correlated with ALT in that they appear when the telomere length pattern becomes characteristic of ALT positive cells and are not found in telomerase positive cell lines. The exact role of these bodies in telomere maintenance and/or protection is as yet unclear as there have been few studies carried out to determine the functional involvement of the specific components. Our study aims at the identification of the essential components of this structure and, ultimately, at studying the effect of APB alterations on telomere length and genomic stability. We began by determining the percentage of APB-positive cells within our chosen cell lines, and found that there were a higher proportion of APB positive cells than reported in the literature. In order to determine the role of PML in formation of APB, we employed RNA interference oligonucleotides directed against nuclear PML. Interestingly, decreased speckled distribution of MRE11 and TRF1 was observed in PML-depleted cells. In contrast, TRF2 was not affected, and its down-regulation has no clear effect on MRE11 or TRF1 localization. TRF2 silencing results in telomere erosion, thus suggesting that in ALT cells TRF2 regulates telomere length. In addition, TRF2 depletion causes inhibition of colony formation, increased cell size, p21 upregulation and induction of genomic instability. Although in ALT cells active ATM is found at APBs, it is only following TRF2 silencing that the ATM target p53 becomes phosphorylated and activated. This study sheds new light on the role of the APB components PML and TRF2 in ALT cells

Initiating or changing to a fixed-dose combination of fluticasone propionate/formoterol over fluticasone propionate/salmeterol : a real-life effectiveness and cost impact evaluation

With institutional support from NAPP Pharmaceutical Group Ltd. All named authors meet the International Committee of Medical Journal Editors (ICMJE) criteria for authorship for this manuscript, take responsibility for the integrity of the work and have given final approval to the version to be published. The authors would like to thank Rosalind Bonomally for medical writing support.Peer reviewedPostprin

Conservative site-specific and single-copy transgenesis in human LINE-1 elements

Genome engineering of human cells plays an important role in biotechnology and molecular medicine. In particular, insertions of functional multi-transgene cassettes into suitable endogenous sequences will lead to novel applications. Although several tools have been exploited in this context, safety issues such as cytotoxicity, insertional mutagenesis and off-target cleavage together with limitations in cargo size/expression often compromise utility. Phage λ integrase (Int) is a transgenesis tool that mediates conservative site-specific integration of 48 kb DNA into a safe harbor site of the bacterial genome. Here, we show that an Int variant precisely recombines large episomes into a sequence, termed attH4X, found in 1000 human Long INterspersed Elements-1 (LINE-1). We demonstrate single-copy transgenesis through attH4X-targeting in various cell lines including hESCs, with the flexibility of selecting clones according to transgene performance and downstream applications. This is exemplified with pluripotency reporter cassettes and constitutively expressed payloads that remain functional in LINE1-targeted hESCs and differentiated progenies. Furthermore, LINE-1 targeting does not induce DNA damage-response or chromosomal aberrations, and neither global nor localized endogenous gene expression is substantially affected. Hence, this simple transgene addition tool should become particularly useful for applications that require engineering of the human genome with multi-transgenes.MOE (Min. of Education, S’pore)Published versio

Fractional exhaled nitric oxide as a predictor of response to inhaled corticosteroids in patients with non-specific respiratory symptoms and insignificant bronchodilator reversibility : a randomised controlled trial

Funding: Sponsored by OPRI with partial funding by Circassia and study drugs provided by TEVA.Peer reviewedPostprin