ABSTRACT
From yeast to mammals, autophagy is an important mechanism for sustaining cellular homeostasis through facilitating the degradation and recycling of aged and cytotoxic components. During ...autophagy, cargo is captured in double‐membraned vesicles, the autophagosomes, and degraded through lysosomal fusion. In yeast, autophagy initiation, cargo recognition, cargo engulfment, and vesicle closure is Atg8 dependent. In higher eukaryotes, Atg8 has evolved into the LC3/GABARAP protein family, consisting of 7 family proteins LC3A(2 splice variants), LC3B, LC3C, GABARAP, GABARAPL1, and GABARAPL2. LC3B, the most studied family protein, is associatedwith autophagosome development and maturation and is used tomonitor autophagic activity. Giventhe high homology, the other LC3/GABARAP family proteins are often presumed to fulfill similar functions. Nevertheless, substantial evidence shows that the LC3/GABARAP family proteins are unique in function and important in autophagy‐independent mechanisms. In this review, we discuss the current knowledge and functions of the LC3/GABARAP family proteins. We focus on processing of the individual family proteins and their role in autophagy initiation, cargo recognition, vesicle closure, and trafficking, a complex and tightly regulated process that requires selective presentation and recruitment of these family proteins. In addition, functions unrelated to autophagy of the LC3/GABARAP protein family members are discussed.—Schaaf, M. B. E., Keulers, T. G, Vooijs, M. A., Rouschop, K. M. A. LC3/GABARAP family proteins: autophagy‐(un)related functions. FASEB J. 30, 3961–3978 (2016). www.fasebj.org
Multiple Myeloma (MM) is an incurable plasma cell malignancy residing within the bone marrow (BM). We aim to develop allogeneic Natural Killer (NK) cell immunotherapy for MM. As the BM contains ...hypoxic regions and the tumor environment can be immunosuppressive, we hypothesized that hypoxia inhibits NK cell anti-MM responses.
NK cells were isolated from healthy donors by negative selection and NK cell function and phenotype were examined at oxygen levels representative of hypoxic BM using flowcytometry. Additionally, NK cells were activated with IL-2 to enhance NK cell cytotoxicity under hypoxia.
Hypoxia reduced NK cell killing of MM cell lines in an oxygen dependent manner. Under hypoxia, NK cells maintained their ability to degranulate in response to target cells, though, the percentage of degranulating NK cells was slightly reduced. Adaptation of NK- or MM cells to hypoxia was not required, hence, the oxygen level during the killing process was critical. Hypoxia did not alter surface expression of NK cell ligands (HLA-ABC, -E, MICA/B and ULBP1-2) and receptors (KIR, NKG2A/C, DNAM-1, NCRs and 2B4). It did, however, decrease expression of the activating NKG2D receptor and of intracellular perforin and granzyme B. Pre-activation of NK cells by IL-2 abrogated the detrimental effects of hypoxia and increased NKG2D expression. This emphasized that activated NK cells can mediate anti-MM effects, even under hypoxic conditions.
Hypoxia abolishes the killing potential of NK cells against multiple myeloma, which can be restored by IL-2 activation. Our study shows that for the design of NK cell-based immunotherapy it is necessary to study biological interactions between NK- and tumor cells also under hypoxic conditions.
The epidermal growth factor receptor (EGFR) is amplified or mutated in various human epithelial tumors. Its expression and activation leads to cell proliferation, differentiation, and survival. ...Consistently, EGFR amplification or expression of EGFR variant 3 (EGFRvIII) is associated with resistance to conventional cancer therapy through activation of pro-survival signaling and DNA-repair mechanisms. EGFR targeting has successfully been exploited as strategy to increase treatment efficacy. Nevertheless, these targeting strategies have only been proven effective in a limited percentage of human tumors.
Recent knowledge indicates that EGFR deregulated tumors display differences in autophagy and dependence on autophagy for growth and survival and the use of autophagy to increase resistance to EGFR-targeting drugs. In this review the dependency on autophagy and its role in mediating resistance to EGFR-targeting agents will be discussed. Considering the current knowledge, autophagy inhibition could provide a novel strategy to enhance therapy efficacy in treatment of EGFR deregulated tumors.
Mounting evidence suggests that one of the ways that cells adapt to hypoxia is through alternative splicing. The aim of this study was firstly to examine the effect of hypoxia on the alternative ...splicing of cancer associated genes using the prostate cancer cell line PC3 as a model. Secondly, the effect of hypoxia on the expression of several regulators of splicing was examined.
PC3 cells were grown in 1% oxygen in a hypoxic chamber for 48 h, RNA extracted and sent for high throughput PCR analysis at the RNomics platform at the University of Sherbrooke, Canada. Genes whose exon inclusion rate PSI (ψ) changed significantly were identified, and their altered exon inclusion rates verified by RT-PCR in three cell lines. The expression of splice factors and splice factor kinases in response to hypoxia was examined by qPCR and western blotting. The splice factor kinase CLK1 was inhibited with the benzothiazole TG003.
In PC3 cells the exon inclusion rate PSI (ψ) was seen to change by > 25% in 12 cancer-associated genes; MBP, APAF1, PUF60, SYNE2, CDC42BPA, FGFR10P, BTN2A2, UTRN, RAP1GDS1, PTPN13, TTC23 and CASP9 (caspase 9). The expression of the splice factors SRSF1, SRSF2, SRSF3, SAM68, HuR, hnRNPA1, and of the splice factor kinases SRPK1 and CLK1 increased significantly in hypoxia. We also observed that the splice factor kinase CLK3, but not CLK2 and CLK4, was also induced in hypoxic DU145 prostate, HT29 colon and MCF7 breast cancer cell lines. Lastly, we show that the inhibition of CLK1 in PC3 cells with the benzothiazole TG003 increased expression of the anti-apoptotic isoform caspase 9b.
Significant changes in alternative splicing of cancer associated genes occur in prostate cancer cells in hypoxic conditions. The expression of several splice factors and splice factor kinases increases during hypoxia, in particular the Cdc-like splice factor kinases CLK1 and CLK3. We suggest that in hypoxia the elevated expression of these regulators of splicing helps cells adapt through alternative splicing of key cancer-associated genes. We suggest that the CLK splice factor kinases could be targeted in cancers in which hypoxia contributes to resistance to therapy.
Through the use of modified nucleotides, synthetic nucleic acids have found several fields of application within biotechnology and in the pharmaceutical industry. We have previously introduced ...nucleotides with an additional functional nucleobase linked to C2' of arabinonucleotides (
). These double-headed nucleotides fit neatly into DNA·DNA duplexes, where they can replace the corresponding natural dinucleotides and thus condense the molecular information. Here, we introduce a 2'-deoxy version of the
design with inversion of the C2' stereochemistry (
) with the aim of obtaining improved RNA recognition. Specifically,
analogues with cytosine or isocytosine attached to C2' of 2'-deoxyuridine (
and
) were synthesized and evaluated in duplexes. Whereas the
design did not outperform the
design in terms of mimicking dinucleotides in nucleic acid duplexes, it was able to engage in reverse Watson-Crick pairing using its 2'-base. This was evident from the ability of the
cytosine to form stable mis-matching base pairs with opposite cytosines identified as hemiprotonated C·C
pairs. Furthermore, specific base-pairing with guanine was only observed for the isocytosine-bearing
monomer. Very stable duplexes were obtained with
monomers in each strand indicating that fully modified double-headed nucleic acid sequences could be based on the
design.
Planet-forming disks are not isolated systems. Their interaction with the surrounding medium affects their mass budget and chemical content. In the context of the ALMA-DOT program, we obtained ...high-resolution maps of assorted lines from six disks that are still partly embedded in their natal envelope. In this work, we examine the SO and SO2 emission that is detected from four sources: DG Tau, HL Tau, IRAS 04302+2247, and T Tau. The comparison with CO, HCO+, and CS maps reveals that the SO and SO2 emission originates at the intersection between extended streamers and the planet-forming disk. Two targets, DG Tau and HL Tau, offer clear cases of inflowing material inducing an accretion shock on the disk material. The measured rotational temperatures and radial velocities are consistent with this view. In contrast to younger Class 0 sources, these shocks are confined to the specific disk region impacted by the streamer. In HL Tau, the known accreting streamer induces a shock in the disk outskirts, and the released SO and SO2 molecules spiral toward the star in a few hundred years. These results suggest that shocks induced by late accreting material may be common in the disks of young star-forming regions with possible consequences for the chemical composition and mass content of the disk. They also highlight the importance of SO and SO2 line observations in probing accretion shocks from a larger sample.
Proper chromatin function and maintenance of genomic stability depends on spatiotemporal coordination between the transcription and replication machinery. Loss of this coordination can lead to DNA ...damage from increased transcription-replication collision events. We report that deregulated transcription following BRD4 loss in cancer cells leads to the accumulation of RNA:DNA hybrids (R-loops) and collisions with the replication machinery causing replication stress and DNA damage. Whole genome BRD4 and γH2AX ChIP-Seq with R-loop IP qPCR reveals that BRD4 inhibition leads to accumulation of R-loops and DNA damage at a subset of known BDR4, JMJD6, and CHD4 co-regulated genes. Interference with BRD4 function causes transcriptional downregulation of the DNA damage response protein TopBP1, resulting in failure to activate the ATR-Chk1 pathway despite increased replication stress, leading to apoptotic cell death in S-phase and mitotic catastrophe. These findings demonstrate that inhibition of BRD4 induces transcription-replication conflicts, DNA damage, and cell death in oncogenic cells.
Several clinically used drugs are mitotoxic causing mitochondrial DNA (mtDNA) variations, and thereby influence cancer treatment response. We hypothesized that radiation responsiveness will be ...enhanced in cellular models with decreased mtDNA content, attributed to altered reactive oxygen species (ROS) production and antioxidant capacity. For this purpose BEAS-2B, A549, and 143B cell lines were depleted from their mtDNA (ρ0). Overall survival after irradiation was increased (p<0.001) for BEAS-2B ρ0 cells, while decreased for both tumor ρ0 lines (p<0.05). In agreement, increased residual DNA damage was observed after mtDNA depletion for A549 and 143B cells. Intrinsic radiosensitivity (surviving fraction at 2Gy) was not influenced. We investigated whether ROS levels, oxidative stress and/or antioxidant responses were responsible for altered radiation responses. Baseline ROS formation was similar between BEAS-2B parental and ρ0 cells, while reduced in A549 and 143B ρ0 cells, compared to their parental counterparts. After irradiation, ROS levels significantly increased for all parental cell lines, while levels for ρ0 cells remained unchanged. In order to investigate the presence of oxidative stress upon irradiation reduced glutathione: oxidized glutathione (GSH:GSSG) ratios were determined. Irradiation reduced GSH:GSSG ratios for BEAS-2B parental and 143B ρ0, while for A549 this ratio remained equal. Additionally, changes in antioxidant responses were observed. Our results indicate that mtDNA depletion results in varying radiation responses potentially involving variations in cellular ROS and antioxidant defence mechanisms. We therefore suggest when mitotoxic drugs are combined with radiation, in particular at high dose per fraction, the effect of these drugs on mtDNA copy number should be explored.
Different measures for quantifying the percentage of people with a disability in surveys result in diverging estimates of prevalence and disability-related inequalities. Thus understanding the ...implications of using different disability measures is of vital policy importance. This study is the first to investigate the within-survey variation in disability prevalence based on two internationally recognized measures: the Washington Group Short Set (WGSS) and the Global Activity Limitation Indicator (GALI). It is also the first to examine the disability-related inequality in voter turnout, based on official validated voter records.
We use data on 11,308 25-54-year-old respondents from the 2016 wave of the Survey of Health, Impairment and Living Conditions in Denmark (SHILD) to estimate the disability prevalence based on the WGSS and the GALI. Moreover, we investigate health characteristics of individuals with a disability according to the two measures and inequalities in two central social policy success parameters: voter turnout and employment.
The WGSS estimates higher disability prevalence (10.6%) than the GALI (5.5%). Only 2.5% of the sample are in both groups, implying that largely, different individuals are defined as having a disability depending on which measure is used. The health profiles of the two groups also differ, as people with a GALI-defined disability are significantly more likely to report a severe mental illness or a major physical health problem. The GALI estimates indicate larger inequalities between people with and without a disability than the WGSS for the probability of being employed, whereas there are no significant differences for voter turnout.
The choice of disability measure strongly influences within-survey estimates of disability prevalence, the health profile of the defined groups, and inequalities in outcomes. The WGSS underrepresents the number of people suffering from severe mental illness. Estimated inequalities in employment are larger for the GALI than for the WGSS.
•The estimated prevalence of disability varies by choice of disability measure.•Different people are defined as having a disability subject to which measure is used.•The choice of measure strongly influences the health profile of the defined groups.•Inequalities between people with and without disability vary across measures.
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