Numerous in vitro studies have shown that human cell lines lacking functional ATM are extremely radiosensitive. In this issue, Moding et al. demonstrate using a murine model of sarcoma that deletion of the Atm gene has much less of a radiosensitizing effect on normal cardiac endothelia than on rapidly proliferating tumor endothelia. This work confounds our assumptions about the generality of the role of ATM in radiation sensitivity and the potential use of ATM inhibitors as radiosensitizers.
\n \n\n \n \nHypoxia (low oxygen levels) occurs in a range of biological contexts, including plants, bacterial biofilms, and solid tumors; it elicits responses from these biological systems that impact their survival. For example, conditions of low oxygen make treating tumors more difficult and have a negative impact on patient prognosis. Therefore, chemical probes that enable the study of biological hypoxia are valuable tools to increase the understanding of disease-related conditions that involve low oxygen levels, ultimately leading to improved diagnosis and treatment. While small-molecule hypoxia-sensing probes exist, the majority of these image only very severe hypoxia (<1% O2) and therefore do not give a full picture of heterogeneous biological hypoxia. Commonly used antibody-based imaging tools for hypoxia are less convenient than small molecules, as secondary detection steps involving immunostaining are required. Here, we report the synthesis, electrochemical properties, photophysical analysis, and biological validation of a range of indolequinone-based bioreductive fluorescent probes. We show that these compounds image different levels of hypoxia in 2D and 3D cell cultures. The resorufin-based probe 2 was activated in conditions of 4% O2 and lower, while the Me-Tokyo Green-based probe 4 was only activated in severe hypoxia\u25000.5% O2 and less. Simultaneous application of these compounds in spheroids revealed that compound 2 images similar levels of hypoxia to pimonidazole, while compound 4 images more extreme hypoxia in a manner analogous to EF5. Compounds 2 and 4 are therefore useful tools to study hypoxia in a cellular setting and represent convenient alternatives to antibody-based imaging approaches.
\n \n\n \n \nLocal hypoxia occurs in most solid tumors and is associated with aggressive disease and therapy resistance. Widespread changes in gene expression play a critical role in the biological response to hypoxia. However, most research has focused on hypoxia-inducible genes as opposed to those that are decreased in hypoxia. We demonstrate that chromatin accessibility is decreased in hypoxia, predominantly at gene promoters and specific pathways are impacted including DNA repair, splicing, and the R-loop interactome. One of the genes with decreased chromatin accessibility in hypoxia was DDX5, encoding the RNA helicase, DDX5, which showed reduced expression in various cancer cell lines in hypoxic conditions, tumor xenografts, and in patient samples with hypoxic tumors. Most interestingly, we found that when DDX5 is rescued in hypoxia, replication stress and R-loop levels accumulate further, demonstrating that hypoxia-mediated repression of DDX5 restricts R-loop accumulation. Together these data support the hypothesis that a critical part of the biological response to hypoxia is the repression of multiple R-loop processing factors; however, as shown for DDX5, their role is specific and distinct.
\n \n\n \n \nRadiotherapy is a critical component of many current, curative cancer treatments, yet it is accompanied by unavoidable irradiation of normal tissues. Abdominal and pelvic radiation almost always results in some dose delivered to the bowel with deleterious effects to the small and large intestines. While the likelihood of enteritis is dose dependent, there is also considerable variation between patients in both the extent of symptoms of enteritis as well as their duration. In this article, Martin and colleagues hypothesized that the radiation sensitivity of intestinal organoids could predict the sensitivity of individual patients to enteritis and have taken the first steps to develop such an assay.See related article by Martin et al., p. 1219.
\n \n\n \n \n[This corrects the article DOI: 10.3389/fonc.2019.01563.].
\n \n\n \n \nImmunopeptidomics enables the identification of peptides presented by major histocompatibility complex (MHC) molecules, offering insights into antigen presentation and immune recognition. Understanding these mechanisms in hypoxic conditions is crucial for deciphering immune responses within the tumor microenvironment. Current immunopeptidomics approaches do not capture hypoxia-induced changes in the repertoire of MHC-presented peptides. This protocol describes the isolation of MHC class I-bound peptides from in vitro hypoxia-treated cells, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. It describes optimized steps for cell lysis, immunoaffinity purification, peptide elution, and MS-compatible preparation under controlled low-oxygen conditions. The method is compatible with various quantitative mass spectrometry approaches and can be adapted to different cell types. This workflow provides a reliable and reproducible approach to studying antigen presentation under hypoxic conditions, thereby enhancing physiological relevance and facilitating deeper immunological insights. Key features \u2022 Enables isolation of MHC class I-bound peptides from cells cultured under hypoxic conditions. \u2022 Designed for low-input samples and optimized for maintaining cell viability during extended hypoxic exposure. \u2022 Compatible with label-free LC-MS/MS for detailed immunopeptidome analysis. \u2022 Adaptable to all human and murine cell lines commonly used in cancer and immunology research.
\n \n\n \n \nPURPOSE: Human papillomavirus negative (HPV-ve) head and neck squamous cell carcinoma (HNSCC) has a poor prognosis compared with HPV+ve HNSCCs. Expression of p16 in HPV+ve HNSCC is thought to mediate radiosensitivity via inhibition of cyclin-dependent kinase (CDK) 4/6. We used a clinically approved CDK4/CDK6 inhibitor, palbociclib, and assessed its effect on radiosensitivity in HNSCC. METHODS AND MATERIALS: The effect of palbociclib on radiosensitivity was determined in HPV-ve and HPV+ve HNSCC cell lines using colony survival assays, immunofluorescent staining of repair proteins, homologous recombination assays, cell cycle, and metaphase spread analyses. RESULTS: Only HPV-ve HNSCC cells were radiosensitized by palbociclib, which also occurred at hypoxic levels associated with radioresistance. Palbociclib led to decreased induction of BRCA1 and RAD51 after irradiation. Homologous recombination was diminished and repair of radiation-induced DNA damage was delayed in the presence of palbociclib, leading to increased chromosomal damage. Failure to repair radiation-induced damage led to cell death as a result of mitotic catastrophe. CONCLUSIONS: Here, we highlight a therapeutic strategy to improve the radiosensitivity of HPV-ve HNSCC, a patient group that has an unmet and urgent need for improved radiation therapy efficacy.
\n \n\n \n \nAberrant activation of the hypoxia-inducible transcription factor HIF-1 and dysfunction of the tumor suppressor p53 have been reported to induce malignant phenotypes and therapy resistance of cancers. However, their mechanistic and functional relationship remains largely unknown. Here, we reveal a mechanism by which p53 deficiency triggers the activation of HIF-1-dependent hypoxia signaling and identify zinc finger and BTB domain-containing protein 2 (ZBTB2) as an important mediator. ZBTB2 forms homodimers via its N-terminus region and increases the transactivation activity of HIF-1 only when functional p53 is absent. The ZBTB2 homodimer facilitates invasion, distant metastasis, and growth of p53-deficient, but not p53-proficient, cancers. The intratumoral expression levels of ZBTB2 are associated with poor prognosis in lung cancer patients. ZBTB2 N-terminus-mimetic polypeptides competitively inhibit ZBTB2 homodimerization and significantly suppress the ZBTB2-HIF-1 axis, leading to antitumor effects. Our data reveal an important link between aberrant activation of hypoxia signaling and loss of a tumor suppressor and provide a rationale for targeting a key mediator, ZBTB2, to suppress cancer aggressiveness.
\n \n\n \n \nRegions of hypoxia occur in most tumors and are a predictor of poor patient prognosis. Hypoxia-activated prodrugs (HAPs) provide an ideal strategy to target the aggressive, hypoxic, fraction of a tumor, while protecting the normal tissue from toxicity. A key challenge associated with the development of novel HAPs, however, is the ability to visualize the delivery of the prodrug to hypoxic regions and determine where it has been activated. Here, we report a modified version of the commonly used nitroimidazole bioreductive group that incorporates the fluoroethyl epitope of the antibody-based hypoxia imaging agent, EF5. Attachment of this group to the red fluorescent dye, dicyanomethylene (DCM), enabled us to correlate the release of the DCM dye with imaging of the reduced bioreductive group using the EF5 antibody. This study confirmed that the antibody was imaging reduction and fragmentation of the pro-fluorophore. We next employed the modified bioreductive group to synthesize a new prodrug of the KDAC inhibitor Panobinostat, EF5-Pano. Release of EF5-Pano in hypoxic multiple myeloma cells was imaged using the EF5 antibody, and the presence of an imaging signal correlated with apoptosis and a reduction in cell viability. Therefore, EF5-Pano is an imageable HAP with a proven cytotoxic effect in multiple myeloma, which could be utilized in future in vivo experiments.
\n \n\n \n \nHypoxic stress, like DNA damage, induces p53 protein accumulation and p53-dependent apoptosis in oncogenically transformed cells. Unlike DNA damage, hypoxia does not induce p53-dependent cell cycle arrest, suggesting that p53 activity is differentially regulated by these two stresses. Here we report that hypoxia induces p53 protein accumulation, but in contrast to DNA damage, hypoxia fails to induce endogenous downstream p53 effector mRNAs and proteins. Hypoxia does not inhibit the induction of p53 target genes by ionizing radiation, indicating that p53-dependent transactivation requires a DNA damage-inducible signal that is lacking under hypoxic treatment alone. At the molecular level, DNA damage induces the interaction of p53 with the transcriptional activator p300 as well as with the transcriptional corepressor mSin3A. In contrast, hypoxia primarily induces an interaction of p53 with mSin3A, but not with p300. Pretreatment of cells with an inhibitor of histone deacetylases that relieves transcriptional repression resulted in a significant reduction of p53-dependent transrepression and hypoxia-induced apoptosis. These results led us to propose a model in which different cellular pools of p53 can modulate transcriptional activity through interactions with transcriptional coactivators or corepressors. Genotoxic stress induces both kinds of interactions, whereas stresses that lack a DNA damage component as exemplified by hypoxia primarily induce interaction with corepressors. However, inhibition of either type of interaction can result in diminished apoptotic activity.
\n \n\n \n \nHypoxia is a growth inhibitory stress associated with multiple disease states. We find that hypoxic stress actively regulates transcription not only by activation of specific genes but also by selective repression. We reconstituted this bimodal response to hypoxia in vitro and determined a mechanism for hypoxia-mediated repression of transcription. Hypoxic cell extracts are competent for transcript elongation, but cannot assemble a functional preinitiation complex (PIC) at a subset of promoters. PIC assembly and RNA polymerase II C-terminal domain (CTD) phosphorylation were blocked by hypoxic induction and core promoter binding of negative cofactor 2 protein (NC2 alpha/beta, Dr1/DrAP1). Immunodepletion of NC2 beta/Dr1 protein complexes rescued hypoxic-repressed transcription without alteration of normoxic transcription. Physiological regulation of NC2 activity may represent an active means of conserving energy in response to hypoxic stress.
\n \n\n \n \nCaspase-mediated proteolysis of cytoskeletal proteins during apoptosis appears to be commonplace. Enlarging on previous studies we have shown here that gamma catenin, like beta catenin, was degraded during cisplatin-induced apoptosis, initially giving a major product of 75 kDa. This truncated protein could be co-immunoprecipitated with alpha catenin. Addition of caspase inhibitors to cells in the presence of cisplatin appreciably reduced the proteolysis of gamma catenin as well as the level of apoptosis. Only limited degradation of alpha catenin was observed even at very late times when over 90% of cells in the culture were apoptotic. Immunohistochemical staining showed that during apoptosis there was a relocation of alpha, beta, and gamma catenin from the periphery of the cell to the cytoplasm, at the same time as other morphological changes commonly associated with apoptosis occurred. Interestingly, the changes in localisation of the catenins preceded proteolysis by several hours. In the presence of cisplatin and caspase inhibitor no change in distribution of catenins was observed, suggesting that re-localisation requires caspase activity but not necessarily directed against beta and gamma catenins.
\n \n\n \n \nThe link between hypoxic conditions and radiation sensitivity is well-established, however the dynamic nature of hypoxia is often overlooked. The contribution of acute/transient hypoxia versus chronic conditions to radiosensitivity has been investigated by Wadsworth et al. using two hypoxia markers and pentoxifylline to increase blood flow to regions of transient hypoxia.
\n \n\n \n \nThe 45,000 Mr protein ASP (Apoptosis Specific Protein) was observed at high levels in both human and rat cells undergoing apoptosis but not in viable or necrotic cells. The protein was identified by means of a cross reaction with a c-jun antibody. The gene has been cloned by means of screening a human foetal liver expression library with the c-jun antibody. The gene identified is 2.9 Kb and encodes a protein of approximately 33 KD. The difference between this and the expected 45 KD molecule has been attributed to post-translational modification. Northern blot analysis shows products of 2.9 Kb and 1.7 Kb to be present in viable human tissue suggesting the presence of splice variants. RT-PCR has shown the message to be present in all the cell types tested (human and rat) including viable cells. The protein encoded by the gene has been isolated after cloning into the pQE-31 His tag expression vector and expression in E. coli. Polyclonal antibodies are being raised.
\n \n\n \n \nThe alternative lengthening of telomeres (ALT) pathway is a telomerase-independent mechanism for immortalization in cancer cells and is commonly activated in low-grade and high-grade glioma, as well as osteosarcoma. The ALT pathway can be activated under various conditions and has often been shown to include mutational loss of ATRX. However, this is insufficient in isolation and so other cellular event must also be implicated. It has been shown that excessive accumulation of DNA:RNA hybrid structures (R-loops) and/or formation of DNA-protein crosslinks (DPCs) can be other important driving factors. The underlying cellular events leading to R-loop and DPC formation in ALT cancer cells to date remain unclear. Here, we demonstrate that excessive cellular reactive oxygen species (ROS) is an important causative factor in the evolution of ALT-telomere maintenance in ATRX-deficient glioma. We identified three sources of elevated ROS in ALT-positive gliomas: co-mutation of SETD2, downregulation of DRG2, and hypoxic tumour microenvironment. We demonstrate that elevated ROS leads to accumulation of R-loops and, crucially, resolution of R-loops by the enzyme RNase H1 prevents ALT pathway activity in cells exposed to elevated ROS. Further, we found a possible causal link between the formation of R-loops and the accumulation of DPCs, in particular, formation of TOP1 complexes covalently linked to DNA (Top1cc). We also demonstrate that elevation of ROS can trigger over-activity of the ALT pathway in osteosarcoma and glioma cell lines, resulting in excessive DNA damage and cell death. This work presents important mechanistic insights into the endogenous origin of excessive R-loops and DPCs in ALT-positive cancers, as well as highlighting potential novel therapeutic approaches in these difficult-to-treat cancer types.
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