Autophagy is the main driver of radioresistance of HNSCC cells in mild hypoxia.

Hypoxia poses a significant challenge to the effectiveness of radiotherapy in head and neck squamous cell carcinoma (HNSCC) patients, and it is imperative to discover novel approaches to overcome this. In this study, we investigated the underlying mechanisms contributing to x-ray radioresistance in HPV-negative HNSCC cells under mild hypoxic conditions (1% oxygen) and explored the potential for autophagy modulation as a promising therapeutic strategy. Our findings show that HNSCC cells exposed to mild hypoxic conditions exhibit increased radioresistance, which is largely mediated by the hypoxia-inducible factor (HIF) pathway. We demonstrate that siRNA knockdown of HIF-1α and HIF-1ß leads to increased radiosensitivity in HNSCC cells under hypoxia. Hypoxia-induced radioresistance was not attributed to differences in DNA double strand break repair kinetics, as these remain largely unchanged under normoxic and hypoxic conditions. Rather, we identify autophagy as a critical protective mechanism in HNSCC cells following irradiation under mild hypoxia conditions. Targeting key autophagy genes, such as BECLIN1 and BNIP3/3L, using siRNA sensitizes these cells to irradiation. Whilst autophagy's role in hypoxic radioresistance remains controversial, this study highlights the importance of autophagy modulation as a potential therapeutic approach to enhance the effectiveness of radiotherapy in HNSCC.

Synthesis and Characterization of Vanadium Nitride/Carbon Nanocomposites.

The present work focuses on the synthesis of a vanadium nitride (VN)/carbon nanocomposite material via the thermal decomposition of vanadyl phthalocyanine (VOPC). The morphology and chemical structure of the synthesized compounds were characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy dispersive spectroscopy (EDS), Fourier transformed infrared spectroscopy (FTIR), X-ray diffraction (XRD), and X-ray photoemission spectroscopy (XPS). The successful syntheses of the VOPC and non-metalated phthalocyanine (H2PC) precursors were confirmed using FTIR and XRD. The VN particles present a needle-like morphology in the VN synthesized by the sol-gel method. The morphology of the VN/C composite material exhibited small clusters of VN particles. The XRD analysis of the thermally decomposed VOPC indicated a mixture of amorphous carbon and VN nanoparticles (VN(TD)) with a cubic structure in the space group FM-3M consistent with that of VN. The XPS results confirmed the presence of V(III)-N bonds in the resultant material, indicating the formation of a VN/C nanocomposite. The VN/C nanocomposite synthesized through thermal decomposition exhibited a high carbon content and a cluster-like distribution of VN particles. The VN/C nanocomposite was used as an anode material in LIBs, which delivered a specific capacity of 307 mAh g-1 after 100 cycles and an excellent Coulombic efficiency of 99.8 at the 100th cycle.

Amino-modified upcycled biochar achieves selective chromium removal in complex aqueous matrices.

Chromium pollution of groundwater sources is a growing global issue, which correlates with various anthropogenic activities. Remediation of both the Cr(VI) and Cr(III), via adsorption technologies, has been championed in recent years due to ease of use, minimal energy requirements, and the potential to serve as a highly sustainable remediation technology. In the present study, a biochar sorbent sourced from pineapple skins, allowed for the upcycling of agricultural waste into water purification technology. The biochar material was chemically modified, through a green amination method, to produce an efficient and selective adsorbent for the removal of both Cr(VI) and Cr(III) from complex aqueous matrices. From FTIR analysis it was evident that the chemical modification introduced new C-N and N-H bonds observed in the modified biochar along with a depletion of N-O and C-H bonds found in the pristine biochar. The amino modified biochar was found to spontaneously adsorb both forms of chromium at room temperature, with binding capacities of 46.5 mg/g of Cr(VI) and 27.1 mg/g of Cr(III). Interference studies, conducted in complex matrices, showed no change in adsorption capacity for Cr(VI) in matrices containing up to 3,000× the concentration of interfering ions. Finally, Cr(III) removal was synergized to 100% adsorption at interfering ions concentrations up to 330× of the analyte, which were suppressed at higher interference concentrations. Considering such performance, the amino modified biochar achieved selective removal for both forms of chromium, showing great potential for utilization in complex chromium pollution sources.

The p97-Ataxin 3 complex regulates homeostasis of the DNA damage response E3 ubiquitin ligase RNF8.

The E3 ubiquitin ligase RNF8 (RING finger protein 8) is a pivotal enzyme for DNA repair. However, RNF8 hyper-accumulation is tumour-promoting and positively correlates with genome instability, cancer cell invasion, metastasis and poor patient prognosis. Very little is known about the mechanisms regulating RNF8 homeostasis to preserve genome stability. Here, we identify the cellular machinery, composed of the p97/VCP ubiquitin-dependent unfoldase/segregase and the Ataxin 3 (ATX3) deubiquitinase, which together form a physical and functional complex with RNF8 to regulate its proteasome-dependent homeostasis under physiological conditions. Under genotoxic stress, when RNF8 is rapidly recruited to sites of DNA lesions, the p97-ATX3 machinery stimulates the extraction of RNF8 from chromatin to balance DNA repair pathway choice and promote cell survival after ionising radiation (IR). Inactivation of the p97-ATX3 complex affects the non-homologous end joining DNA repair pathway and hypersensitises human cancer cells to IR. We propose that the p97-ATX3 complex is the essential machinery for regulation of RNF8 homeostasis under both physiological and genotoxic conditions and that targeting ATX3 may be a promising strategy to radio-sensitise BRCA-deficient cancers.

The Cellular Response to Complex DNA Damage Induced by Ionising Radiation.

Radiotherapy (ionising radiation; IR) is utilised in the treatment of ~50% of all human cancers, and where the therapeutic effect is largely achieved through DNA damage induction. In particular, complex DNA damage (CDD) containing two or more lesions within one to two helical turns of the DNA is a signature of IR and contributes significantly to the cell killing effects due to the difficult nature of its repair by the cellular DNA repair machinery. The levels and complexity of CDD increase with increasing ionisation density (linear energy transfer, LET) of the IR, such that photon (X-ray) radiotherapy is deemed low-LET whereas some particle ions (such as carbon ions) are high-LET radiotherapy. Despite this knowledge, there are challenges in the detection and quantitative measurement of IR-induced CDD in cells and tissues. Furthermore, there are biological uncertainties with the specific DNA repair proteins and pathways, including components of DNA single and double strand break mechanisms, that are engaged in CDD repair, which very much depends on the radiation type and associated LET. However, there are promising signs that advancements are being made in these areas and which will enhance our understanding of the cellular response to CDD induced by IR. There is also evidence that targeting CDD repair, particularly through inhibitors against selected DNA repair enzymes, can exacerbate the impact of higher LET, which could be explored further in a translational context.

Targeting Metal Impurities for the Detection and Quantification of Carbon Black Particles in Water via spICP-MS.

Carbon black (CB) is a nanomaterial with numerous industrial applications and high potential for integration into nano-enabled water treatment devices. However, few analytical techniques are capable of measuring CB in water at environmentally relevant concentrations. Therefore, we intended to establish a quantification method for CB with lower detection limits through utilization of trace metal impurities as analytical tracers. Various metal impurities were investigated in six commercial CB materials, and the Monarch 1000 CB was chosen as a model for further testing. The La impurity was chosen as a tracer for spICP-MS analysis based on measured concentration, low detection limits, and lack of polyatomic interferences. CB stability in water and adhesion to the spICP-MS introduction system presented a challenge that was mitigated by the addition of a nonionic surfactant to the matrix. Following optimization, the limit of detection (64 µg/L) and quantification (122 µg/L) for Monarch 1000 CB demonstrated the applicability of this approach to samples expected to contain trace amounts of CB. When compared against gravimetric analysis and UV-visible absorption spectroscopy, spICP-MS quantification exhibited similar sensitivity but with the ability to detect concentrations an order of magnitude lower. Method detection and sensitivity was unaffected when dissolved La was spiked into CB samples at environmentally relevant concentrations. Additionally, a more complex synthetic matrix representative of drinking water caused no appreciable impact to CB quantification. In comparison to existing quantification techniques, this method has achieved competitive sensitivity, a wide working range for quantification, and high selectivity for tracing possible release of CB materials with known metal contents.

HECTD1 promotes base excision repair in nucleosomes through chromatin remodelling.

Base excision repair (BER) is the major cellular DNA repair pathway that recognises and excises damaged DNA bases to help maintain genome stability. Whilst the major enzymes and mechanisms co-ordinating BER are well known, the process of BER in chromatin where DNA is compacted with histones, remains unclear. Using reconstituted mononucleosomes containing a site-specific synthetic abasic site (tetrahydrofuran, THF), we demonstrate that the DNA damage is less efficiently incised by recombinant AP endonuclease 1 (APE1) when the DNA backbone is facing the histone core (THF-in) compared to that orientated away (THF-out). However, when utilizing HeLa whole cell extracts, the difference in incision of THF-in versus THF-out is less pronounced suggesting the presence of chromatin remodelling factors that stimulate THF accessibility to APE1. We subsequently purified an activity from HeLa cell extracts and identify this as the E3 ubiquitin ligase, HECTD1. We demonstrate that a recombinant truncated form of HECTD1 can stimulate incision of THF-in by APE1 in vitro by histone ubiquitylation, and that siRNA-mediated depletion of HECTD1 leads to deficiencies in DNA damage repair and decreased cell survival following x-ray irradiation, particularly in normal fibroblasts. Thus, we have now identified HECTD1 as an important factor in promoting BER in chromatin.

TRIM26 Maintains Cell Survival in Response to Oxidative Stress through Regulating DNA Glycosylase Stability.

Oxidative DNA base lesions in DNA are repaired through the base excision repair (BER) pathway, which consequently plays a vital role in the maintenance of genome integrity and in suppressing mutagenesis. 8-oxoguanine DNA glycosylase (OGG1), endonuclease III-like protein 1 (NTH1), and the endonuclease VIII-like proteins 1-3 (NEIL1-3) are the key enzymes that initiate repair through the excision of the oxidized base. We have previously identified that the E3 ubiquitin ligase tripartite motif 26 (TRIM26) controls the cellular response to oxidative stress through regulating both NEIL1 and NTH1, although its potential, broader role in BER is unclear. We now show that TRIM26 is a central player in determining the response to different forms of oxidative stress. Using siRNA-mediated knockdowns, we demonstrate that the resistance of cells to X-ray radiation and hydrogen peroxide generated as a consequence of trim26 depletion can be reversed through suppression of selective DNA glycosylases. In particular, a knockdown of neil1 or ogg1 can enhance sensitivity and DNA repair rates in response to X-rays, whereas a knockdown of neil1 or neil3 can produce the same effect in response to hydrogen peroxide. Our study, therefore, highlights the importance of TRIM26 in balancing cellular DNA glycosylase levels required for an efficient BER response.

microRNA-184 is induced by store-operated calcium entry and regulates early keratinocyte differentiation.

Extracellular calcium (Ca2+ ) and store-operated Ca2+ entry (SOCE) govern homoeostasis in the mammalian epidermis. Multiple microRNAs (miRNA) also regulate epidermal differentiation, and raised external Ca2+ modulates the expression of several such miRNAs in keratinocytes. However, little is known about the regulation of miR-184 in keratinocytes or the roles of miR-184 in keratinocyte differentiation. Here we report that exogenous Ca2+ stimulates miR-184 expression in primary epidermal keratinocytes and that this occurs in a SOCE-dependent manner. Levels of miR-184 were raised by about 30-fold after exposure to 1.5 mM Ca2+ for 5 days. In contrast, neither phorbol ester nor 1,25-dihydroxyvitamin D3 had any effect on miR-184 levels. Pharmacologic and genetic inhibitors of SOCE abrogated Ca2+ -dependent miR-184 induction by 70% or more. Ectopic miR-184 inhibited keratinocyte proliferation and led to a fourfold increase in the expression of involucrin, a marker of early keratinocyte differentiation. Exogenous miR-184 also triggered a threefold rise in levels of cyclin E and doubled the levels of γH2AX, a marker of DNA double-strand breaks. The p21 cyclin-dependent kinase inhibitor, which supports keratinocyte growth arrest, was also induced by miR-184. Together our findings point to an SOCE:miR-184 pathway that targets a cyclin E/DNA damage regulatory node to facilitate keratinocyte differentiation.

The radiobiology of HPV-positive and HPV-negative head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, with reported incidences of ~800 000 cases each year. One of the critical determinants in patient response to radiotherapy, particularly for oropharyngeal cancers, is human papillomavirus (HPV) status where HPV-positive patients display improved survival rates and outcomes particularly because of increased responsiveness to radiotherapy. The increased radiosensitivity of HPV-positive HNSCC has been largely linked with defects in the signalling and repair of DNA double-strand breaks. Therefore, strategies to further radiosensitise HPV-positive HNSCC, but also radioresistant HPV-negative HNSCC, have focussed on targeting key DNA repair proteins including PARP, DNA-Pk, ATM and ATR. However, inhibitors against CHK1 and WEE1 involved in cell-cycle checkpoint activation have also been investigated as targets for radiosensitisation in HNSCC. These studies, largely conducted using established HNSCC cell lines in vitro, have demonstrated variability in the response dependent on the specific inhibitors and cell models utilised. However, promising results are evident targeting specifically PARP, DNA-Pk, ATR and CHK1 in synergising with radiation in HNSCC cell killing. Nevertheless, these preclinical studies require further expansion and investigation for translational opportunities for the effective treatment of HNSCC in combination with radiotherapy.

ATM-dependent downregulation of USP7/HAUSP by PPM1G activates p53 response to DNA damage.

The deubiquitylation enzyme USP7/HAUSP plays a major role in regulating genome stability and cancer prevention by controlling the key proteins involved in the DNA damage response. Despite this important role in controlling other proteins, USP7 itself has not been recognized as a target for regulation. Here, we report that USP7 regulation plays a central role in DNA damage signal transmission. We find that stabilization of Mdm2, and correspondingly p53 downregulation in unstressed cells, is accomplished by a specific isoform of USP7 (USP7S), which is phosphorylated at serine 18 by the protein kinase CK2. Phosphorylation stabilizes USP7S and thus contributes to Mdm2 stabilization and downregulation of p53. After ionizing radiation, dephosphorylation of USP7S by the ATM-dependent protein phosphatase PPM1G leads to USP7S downregulation, followed by Mdm2 downregulation and accumulation of p53. Our findings provide a quantitative transmission mechanism of the DNA damage signal to coordinate a p53-dependent DNA damage response.

Base Excision Repair in Chromatin and the Requirement for Chromatin Remodelling.

Base excision repair (BER) is a co-ordinated DNA repair pathway that recognises and repairs chemically modified bases and DNA single strand breaks. It is essential for the maintenance of genome integrity and thus in the prevention of the development of human diseases, including premature ageing, neurodegenerative diseases and cancer. Within the cell, DNA is usually packaged with histone proteins to form chromatin which imposes major constraints on the capacity of cells to perform BER. Therefore chromatin remodelling, stimulated through histone post-translational modifications (PTMs) or ATP-dependent chromatin remodelling complexes (ACRs), are required to stimulate access to the DNA damage and therefore enhance the BER process. Despite this, the molecular mechanisms through which this is co-ordinated and the specific enzymes that promote chromatin remodelling required for BER remain elusive. In this review, we summarise the multitude of in vitro studies utilising mononucleosome substrates containing site-specific DNA base damage that demonstrate the requirement for chromatin remodelling to facilitate BER, particularly in occluded regions. We also highlight preliminary evidence to date for the identity of ACRs, their mechanisms and the role of histone PTMs in modulating the cellular capacity for BER.

FLASH Radiotherapy: Current Knowledge and Future Insights Using Proton-Beam Therapy.

FLASH radiotherapy is the delivery of ultra-high dose rate radiation several orders of magnitude higher than what is currently used in conventional clinical radiotherapy, and has the potential to revolutionize the future of cancer treatment. FLASH radiotherapy induces a phenomenon known as the FLASH effect, whereby the ultra-high dose rate radiation reduces the normal tissue toxicities commonly associated with conventional radiotherapy, while still maintaining local tumor control. The underlying mechanism(s) responsible for the FLASH effect are yet to be fully elucidated, but a prominent role for oxygen tension and reactive oxygen species production is the most current valid hypothesis. The FLASH effect has been confirmed in many studies in recent years, both in vitro and in vivo, with even the first patient with T-cell cutaneous lymphoma being treated using FLASH radiotherapy. However, most of the studies into FLASH radiotherapy have used electron beams that have low tissue penetration, which presents a limitation for translation into clinical practice. A promising alternate FLASH delivery method is via proton beam therapy, as the dose can be deposited deeper within the tissue. However, studies into FLASH protons are currently sparse. This review will summarize FLASH radiotherapy research conducted to date and the current theories explaining the FLASH effect, with an emphasis on the future potential for FLASH proton beam therapy.

USP47 is a deubiquitylating enzyme that regulates base excision repair by controlling steady-state levels of DNA polymerase ß.

DNA base excision repair (BER) is an essential cellular process required for genome stability, and misregulation of BER is linked to premature aging, increased rate of mutagenesis, and cancer. We have now identified the cytoplasmic ubiquitin-specific protease USP47 as the major enzyme involved in deubiquitylation of the key BER DNA polymerase (Pol ß) and demonstrate that USP47 is required for stability of newly synthesized cytoplasmic Pol ß that is used as a source for nuclear Pol ß involved in DNA repair. We further show that knockdown of USP47 causes an increased level of ubiquitylated Pol ß, decreased levels of Pol ß, and a subsequent deficiency in BER, leading to accumulation of DNA strand breaks and decreased cell viability in response to DNA damage. Taken together, these data demonstrate an important role for USP47 in regulating DNA repair and maintaining genome integrity.

Ubiquitylation-dependent regulation of NEIL1 by Mule and TRIM26 is required for the cellular DNA damage response.

Endonuclease VIII-like protein 1 (NEIL1) is a DNA glycosylase involved in initiating the base excision repair pathway, the major cellular mechanism for repairing DNA base damage. Here, we have purified the major E3 ubiquitin ligases from human cells responsible for regulation of NEIL1 by ubiquitylation. Interestingly, we have identified two enzymes that catalyse NEIL1 polyubiquitylation, Mcl-1 ubiquitin ligase E3 (Mule) and tripartite motif 26 (TRIM26). We demonstrate that these enzymes are capable of polyubiquitylating NEIL1 in vitro, and that both catalyse ubiquitylation of NEIL1 within the same C-terminal lysine residues. An siRNA-mediated knockdown of Mule or TRIM26 leads to stabilisation of NEIL1, demonstrating that these enzymes are important in regulating cellular NEIL1 steady state protein levels. Similarly, a mutant NEIL1 protein lacking residues for ubiquitylation is more stable than the wild type protein in vivo We also demonstrate that cellular NEIL1 protein is induced in response to ionising radiation (IR), although this occurs specifically in a Mule-dependent manner. Finally we show that stabilisation of NEIL1, particularly following TRIM26 siRNA, contributes to cellular resistance to IR. This highlights the importance of Mule and TRIM26 in maintaining steady state levels of NEIL1, but also those required for the cellular DNA damage response.

CHIP-mediated degradation and DNA damage-dependent stabilization regulate base excision repair proteins.

Base excision repair (BER) is the major pathway for processing of simple lesions in DNA, including single-strand breaks, base damage, and base loss. The scaffold protein XRCC1, DNA polymerase beta, and DNA ligase IIIalpha play pivotal roles in BER. Although all these enzymes are essential for development, their cellular levels must be tightly regulated because increased amounts of BER enzymes lead to elevated mutagenesis and genetic instability and are frequently found in cancer cells. Here we report that BER enzyme levels are linked to and controlled by the level of DNA lesions. We demonstrate that stability of BER enzymes increases after formation of a repair complex on damaged DNA and that proteins not involved in a repair complex are ubiquitylated by the E3 ubiquitin ligase CHIP and subsequently rapidly degraded. These data identify a molecular mechanism controlling cellular levels of BER enzymes and correspondingly the efficiency and capacity of BER.

ARF induction in response to DNA strand breaks is regulated by PARP1.

The ARF tumour suppressor protein, the gene of which is frequently mutated in many human cancers, plays an important role in the cellular stress response by orchestrating up-regulation of p53 protein and consequently promoting cell-cycle delay. Although p53 protein function has been clearly linked to the cellular DNA damage response, the role of ARF protein in this process is unclear. Here, we report that arf gene transcription is induced by DNA strand breaks (SBs) and that ARF protein accumulates in response to persistent DNA damage. We discovered that poly(ADP-ribose) synthesis catalysed by PARP1 at the sites of unrepaired SBs activates ARF transcription through a protein signalling cascade, including the NAD(+)-dependent deacetylase SIRT1 and the transcription factor E2F1. Our data suggest that poly(ADP-ribose) synthesis at the sites of SBs initiates DNA damage signal transduction by reducing the cellular concentration of NAD(+), thus down-regulating SIRT1 activity and consequently activating E2F1-dependent ARF transcription. Our findings suggest a vital role for ARF in DNA damage signalling, and furthermore explain the critical requirement for ARF inactivation in cancer cells, which are frequently deficient in DNA repair and accumulate DNA damage.

Regulation of base excision repair proteins by ubiquitylation.

Human cellular DNA is under constant attack from both endogenous and exogenous mutagens, and consequently the base excision repair (BER) pathway plays a vital role in repairing damaged DNA bases, sites of base loss (apurinic/apyrimidinic sites) and DNA single strand breaks of varying complexity. BER thus maintains genome stability, and prevents the development of human diseases, such as premature aging, neurodegenerative diseases and cancer. Indeed, there is accumulating evidence that misregulation of BER protein levels is observed in cells and tissues from patients with these diseases, and that post-translational modifications, particularly ubiquitylation, perform a key role in controlling BER protein stability. This review will summarise the presently available data on ubiquitylation of some of the key BER proteins, and the functional consequences of this modification.

Regulation of oxidative DNA damage repair by DNA polymerase λ and MutYH by cross-talk of phosphorylation and ubiquitination.

It is of pivotal importance for genome stability that repair DNA polymerases (Pols), such as Pols λ and ß, which all exhibit considerably reduced fidelity when replicating undamaged DNA, are tightly regulated, because their misregulation could lead to mutagenesis. Recently, we found that the correct repair of the abundant and highly miscoding oxidative DNA lesion 7,8-dihydro-8-oxo-2'-deoxyguanine (8-oxo-G) is performed by an accurate repair pathway that is coordinated by the MutY glycosylase homologue (MutYH) and Pol λ in vitro and in vivo. Pol λ is phosphorylated by Cdk2/cyclinA in late S and G2 phases of the cell cycle, promoting Pol λ stability by preventing it from being targeted for proteasomal degradation by ubiquitination. However, it has remained a mystery how the levels of Pol λ are controlled, how phosphorylation promotes its stability, and how the engagement of Pol λ in active repair complexes is coordinated. Here, we show that the E3 ligase Mule mediates the degradation of Pol λ and that the control of Pol λ levels by Mule has functional consequences for the ability of mammalian cells to deal with 8-oxo-G lesions. Furthermore, we demonstrate that phosphorylation of Pol λ by Cdk2/cyclinA counteracts its Mule-mediated degradation by promoting recruitment of Pol λ to chromatin into active 8-oxo-G repair complexes through an increase in Pol λ's affinity to chromatin-bound MutYH. Finally, MutYH appears to promote the stability of Pol λ by binding it to chromatin. In contrast, Pol λ not engaged in active repair on chromatin is subject for proteasomal degradation.

Phosphorylation of PNKP by ATM prevents its proteasomal degradation and enhances resistance to oxidative stress.

We examined the mechanism regulating the cellular levels of PNKP, the major kinase/phosphatase involved in the repair of oxidative DNA damage, and find that it is controlled by ATM phosphorylation and ubiquitylation-dependent proteasomal degradation. We discovered that ATM-dependent phosphorylation of PNKP at serines 114 and 126 in response to oxidative DNA damage inhibits ubiquitylation-dependent proteasomal degradation of PNKP, and consequently increases PNKP stability that is required for DNA repair. We have also purified a novel Cul4A-DDB1 ubiquitin ligase complex responsible for PNKP ubiquitylation and identify serine-threonine kinase receptor associated protein (STRAP) as the adaptor protein that provides specificity of the complex to PNKP. Strap(-/-) mouse embryonic fibroblasts subsequently contain elevated cellular levels of PNKP, and show elevated resistance to oxidative DNA damage. These data demonstrate an important role for ATM and the Cul4A-DDB1-STRAP ubiquitin ligase in the regulation of the cellular levels of PNKP, and consequently in the repair of oxidative DNA damage.