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Ionizing radiation is a mainstay of high-grade glioma therapy. The current standard radiotherapeutic schedule involves a total 60 Gy split in 2.0 Gy fractions delivered on weekdays for six weeks. Thereafter, almost invariably the tumor relapses and progresses. In vitro studies have demonstrated that the therapeutic effectiveness of ionizing radiation towards high-grade glioma cells is greatly increased by splitting the total dose in fractions ten times smaller [0.1-0.5 Gy instead of standard 2.0 Gy-ultra-hyper-fractionated radiotherapy (ultra-hyper-FRT)]. Recently, it became possible to consistently translate this therapeutic effect to the animal setting, by using glioma-initiating cell-driven faithful animal modeling. A re-analysis of the literature reporting radiotherapeutic clinical trials also suggests that the lower the average fraction size, the higher is the achievable overall survival of patients. However, average fraction sizes ≤ 0.5 Gy have never been thoroughly investigated in the clinics. We propose to study in the clinical setting the therapeutic effect of an ultra-hyper-FRT schedule promptly extending the conventional radiation component of the current guidelines ("Stupp") therapeutic protocol.

High-grade gliomas (HGGs; WHO grades III and IV) are invariably lethal brain tumors. Low-dose hyper-radiosensitivity (HRS) of HGG is a well-established phenomenon in vitro. However, possibly linked to the unavailability of accurate animal models of the diseases, this therapeutic effect could not be consistently translated to the animal setting, thus impairing its subsequent clinical development. The purpose of this study was to develop radiotherapeutic (RT) schedules permitting to significantly improve the overall survival of faithful animal models of HGG that have been recently made available. We used primary glioma initiating cell (GIC)-driven orthotopic animal models that accurately recapitulate the heterogeneity and growth patterns of the patients' tumors, to investigate the therapeutic effects of low radiation doses toward HGG. With the same total dose, RT fractions ≤0.5 Gy twice per week [ultra-hyper-fractionation (ultra-hyper-FRT)] started at early stages of tumor progression (a condition that in the clinical setting often occurs at the end of the guidelines treatment) improved the effectiveness of RT and the animal survival in comparison to standard fractions. For the same cumulative dose, the use of fractions ≤0.5 Gy may permit to escape one or more tumor resistance mechanisms thus increasing the effectiveness of RT and the overall animal survival. These findings suggest investigating in the clinical setting the therapeutic effect of an ultra-hyper-FRT schedule promptly extending the conventional RT component of the current guideline ("Stupp") therapeutic protocol.

High-grade gliomas (World Health Organization grades III and IV) are the most frequent and fatal brain tumors, with median overall survivals of 24–72 and 14–16 months, respectively. We reviewed the progress in the diagnosis and prognosis of high-grade gliomas published in the second half of 2021. A literature search was performed in PubMed using the general terms “radio* and gliom*” and a time limit from 1 July 2021 to 31 December 2021. Important advances were provided in both imaging and non-imaging diagnoses of these hard-to-treat cancers. Our prognostic capacity also increased during the second half of 2021. This review article demonstrates slow, but steady improvements, both scientifically and technically, which express an increased chance that patients with high-grade gliomas may be correctly diagnosed without invasive procedures. The prognosis of those patients strictly depends on the final results of that complex diagnostic process, with widely varying survival rates.

Albeit the effort to develop targeted therapies for patients with high-grade gliomas (WHO grades III and IV) is evidenced by hundreds of current clinical trials, radiation remains one of the few effective therapeutic options for them. This review article analyzes the updates on the topic “radiotherapy of high-grade gliomas” during the period 1 January 2021–30 June 2021. The high number of articles retrieved in PubMed using the search terms (“gliom* and radio*”) and manually selected for relevance indicates the feverish research currently ongoing on the subject. During the last semester, significant advances were provided in both the preclinical and clinical settings concerning the diagnosis and prognosis of high-grade gliomas, their radioresistance, and the inevitable side effects of their treatment with radiation. The novel information concerning tumor radiosensitization was of special interest in terms of therapeutic perspective and was discussed in detail.

Ataxia Telangiectasia and Rad3 related protein (ATR) is a central mediator of the response to DNA damage that may cause the quiescent resistance of cancer initiating cells to genotoxic radiotherapy. NVP-BEZ235 is a dual PI3K/mTOR inhibitor that also effectively targets ATR with IC50 = 21 × 10− 9 M in cells. AZD6738 does not target significantly PI3K/mTOR-related kinases but specifically inhibits ATR with IC50 = 74 × 10− 9 M in cells. Both drugs have been proposed as radiosensitizers of different tumors including glioblastoma (GB), the most malignant brain tumor. In order to study the radiosensitizing properties of ATR inhibitors NVP-BEZ235 and AZD6738 towards GB, we have preliminarily investigated their capacity to penetrate the brain after systemic administration. Tumor-free CD-1 mice were inoculated i.p. with 25 mg/Kg body weight of NVP-BEZ235 or AZD6738. 1, 2, 6 and 8 h later, blood was collected by retro-orbital bleeding after which the mice were euthanized and the brains explanted. Blood and brain samples were then extracted and NVP-BEZ235 and AZD6738 concentrations determined by High Performance Liquid Chromatography/Mass Spectrometry. We found for NVP-BEZ235 and especially for AZD6738, elevated bioavailability and effective brain penetration after intraperitoneal administration. Albeit low drug and radiation dosages were used, a trend to toxicity of NVP-BEZ235 followed by ionizing radiation (IR) towards mice bearing primary glioma initiating cells (GIC)-driven orthotopic tumors was yet observed, as compared to AZD6738 + IR and vehicle+IR. Survival was never improved with median values of 99, 86 and 101 days for vehicle+IR, NVP-BEZ235 + IR and AZD6738 + IR-treated mice, respectively. Although the present results indicate favorable pharmacokinetics properties of ATR inhibitors NVP-BEZ235 and AZD6738, they do not lend support to their use as radiosensitizers of GB.

The Ataxia Telangiectasia Mutated (ATM)-mediated DNA damage response (DDR) is a major mechanism of resistance of glioblastoma (GB) - initiating cells (GICs) to radiotherapy. The closely related Ataxia Telangiectasia and Rad3-related protein (ATR) is also involved in tumor resistance to radio- and chemotherapy. It has been shown that pharmacological inhibition of ATM protein may overcome the DDR-mediated resistance in GICs and significantly radiosensitize GIC-driven GB. Albeit not essential for life as shown by the decade-long lifespan of AT patients, the ATM protein may be involved in a number of important functions other than the response to DNA damage. We discuss our current knowledge about the toxicity of pharmacologic inhibition of ATM and ATR proteins.

If cancer is hard to be treated, brain cancer is even more, caused by the inability of many effective drugs given systemically to cross the blood brain and blood tumor barriers and reach adequate concentrations at the tumor sites. Effective delivery of drugs to brain cancer tissues is thus a necessary, albeit not sufficient, condition to effectively target the disease. In order to analyze the current status of research on drug delivery to high grade gliomas (HGG-WHO grades III and IV), the most frequent and aggressive brain cancers, a literature search was conducted in PubMed using the terms: "drug delivery and brain tumor" over the publication year 2015. Currently explored drug delivery techniques for HGG include the convection and permeabilization-enhanced deliveries, drug-releasing depots and Ommaya reservoirs. The efficacy/safety ratio widely varies among these techniques and the success of current efforts to increase this ratio widely varies as well.

Genotoxic anticancer drugs explicate their effects damaging DNA, thus triggering a coordinated signal-transduction network called DNA Damage Response (DDR). Ataxia Telangiectasia Mutated (ATM) protein plays a central role in this response: activated by DNA damage, ATM phosphorylates itself and downstream effectors that arrest cell cycle allowing for DNA repair or, should DNA damage be too severe and not retrievable, inducing apoptosis. ATM is a worth-investigating target for tumor radio- and chemosensitization. During last years, pharmaceutical industries and research laboratories have developed a series of small molecules, capable to inhibit ATM with increasing specificity. Several preclinical studies have demonstrated that these inhibitors alone or in association with other treatments may improve therapeutic outcomes. In this review we discuss ATM inhibitors so far developed, focussing on recent acquisitions on their potential antineoplastic usefulness.

It has been reported that the ATM kinase inhibitor KU60019 preferentially radiosensitizes orthotopic high grade gliomas (HGG) driven by established U87 and U1242 cell lines bearing specific TP53 mutations. We wished to determine whether those results could be extended to tumors driven by primary glioma initiating cells (GIC) that closely mimic clinical tumors. Orthotopic HGG were developed in immunodeficient non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice by intracranial injection of primary GIC isolated from the adult glioblastoma COMI (acronym of patient’s name) and the pediatric anaplastic astrocytoma 239/12. Similar to the clinical tumors of origin, the orthotopic tumors COMI and 239/12 displayed different growth properties with a voluminous expansive lesion that exerted considerable mass effect on the adjacent structures and an infiltrating, gliomatosis-like growth pattern with limited compressive attitude, respectively. Significant elongations of median animal survival bearing the adult COMI tumor was observed after one KU60019 convection enhanced delivery followed by total 7.5 Gy of ionizing radiation delivered in fifteen 0.5 Gy fractions, as compared to animals treated with vehicle + ionizing radiation (105 vs 89 days; ratio: 0.847; 95% CI of ratio 0.4969 to 1.198; P:0.0417). Similarly, a trend to increased median survival was observed with the radiosensitized pediatric tumor 239/12 (186 vs 167 days; ratio: 0.8978; 95% CI of ratio: 0.5352 to 1.260; P: 0.0891). Our results indicate that radiosensitization by KU60019 is effective towards different orthotopic gliomas that faithfully mimic the clinical tumors and that multiple GIC-based animal models may be essential to develop novel therapeutic protocols for HGG transferable to the clinics.

It has been reported that the ATM kinase inhibitor KU60019 preferentially radiosensitizes orthotopic high grade gliomas (HGG) driven by established U87 and U1242 cell lines bearing specific TP53 mutations. We wished to determine whether those results could be extended to tumors driven by primary glioma initiating cells (GIC) that closely mimic clinical tumors. Orthotopic HGG were developed in immunodeficient non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice by intracranial injection of primary GIC isolated from the adult glioblastoma COMI (acronym of patient's name) and the pediatric anaplastic astrocytoma 239/12. Similar to the clinical tumors of origin, the orthotopic tumors COMI and 239/12 displayed different growth properties with a voluminous expansive lesion that exerted considerable mass effect on the adjacent structures and an infiltrating, gliomatosis-like growth pattern with limited compressive attitude, respectively. Significant elongations of median animal survival bearing the adult COMI tumor was observed after one KU60019 convection enhanced delivery followed by total 7.5 Gy of ionizing radiation delivered in fifteen 0.5 Gy fractions, as compared to animals treated with vehicle + ionizing radiation (105 vs 89 days; ratio: 0.847; 95% CI of ratio 0.4969 to 1.198; P:0.0417) [ARRIVE 16]. Similarly, a trend to increased median survival was observed with the radiosensitized pediatric tumor 239/12 (186 vs 167 days; ratio: 0.8978; 95% CI of ratio: 0.5352 to 1.260; P: 0.0891) [ARRIVE 16]. Our results indicate that radiosensitization by KU60019 is effective towards different orthotopic gliomas that faithfully mimic the clinical tumors and that multiple GIC-based animal models may be essential to develop novel therapeutic protocols for HGG transferable to the clinics.

Oligonucleotide overloading results in type I interferonopathies such as the Aicardi-Goutiéres Syndrome, a progressive encephalopathy determined by an immune response against endogenous DNA/RNA molecules. No therapy targeting pathogenic mechanisms is available for affected patients. Accordingly, we set up an in vitro/in vivo experimental model aimed at reproducing the pathogenic mechanisms of type I interferonopathies, in order to develop an effective pharmacological modulation and toxicological alterations caused by intracranial delivery of encapsulated CpG. The in vitro model used Aicardi-Goutiéres Syndrome immortalized lymphocytes activated by interferon I and co-cultured with human astrocytes; lymphocyte neurotoxicity was attenuated by the calcineurin-inhibitor Tacrolimus and by the anti-interferon monoclonal antibody Sifalimumab. The in vivo model was set up in mice by subcutaneous injection of encapsulated CpG oligonucleotides; the immune-stimulating activity was demonstrated by cytometric analysis in the spleen. To mime pathogenesis of type I interferonopathies in the central nervous system, CpG oligonucleotides were administered intracranially in mice. In the brain, CpG overload induced a rapid activation of macrophage-like microglial cells and focal accumulation mononuclear cells. The subcutaneous administration of Tacrolimus and, more potently, Sifalimumab attenuated CpG-induced brain alterations. These findings shed light on molecular mechanisms triggered by oligonucleotides to induce brain damage. Monoclonal antibodies inhibiting interferon seem a promising therapeutic strategy to protect brain in type I interferonopathies.

Glioblastoma (GB – WHO grade IV) is the most frequent and lethal primary brain tumour with median overall survival of 7–15 months after diagnosis. As in other cancer research areas, an overwhelming amount of pre-clinical research acquisitions in the GB field have not been translated to patients' benefit, potentially due to inappropriate treatment schedules and/or trial designs in the clinical setting. The recent failure of promising anti-VEGF bevacizumab to improve GB patients' overall survival recapitulates this sense of frustration. The following measures are proposed: 1. to change Phase II design. Bevacizumab and other drugs may have failed in Phase III just because of an inappropriate clinical treatment schedule adopted. Multiple-step Phase II clinical trials allowing more thorough definition of treatment protocols to be extensively studied in Phase III should be designed. 2. to monitor standards of care by documenting survival rates of GB patients in European Cancer Units, in order to homogenize GB treatment quality to the highest possible level all over EU28. 3. to introduce the therapeutic impact factor (TIF) and therapeutic (t) index bibliometric parameters, in order to orientate pre-clinical research toward more therapy-focussed activities.

Whilst knowledge of basic biology, diagnosis and prognosis of glioblastoma (GB - WHO grade IV) are steadily improving, advancements of therapy are discouragingly slow, with the only significant novelty during last ten years represented by introduction of temozolomide in chemotherapy. In order to analyze the current status of clinical research on GB, a literature search was conducted in PubMed using the terms: "glioma AND trial" over a 500 day period elapsing from Jan 1, 2013 to May 15, 2014 and results of Phase I, II and III trials were reviewed. Results in the pediatric setting were included as well. It was concluded that, as in other cancer research areas, an overwhelming amount of pre-clinical research acquisitions in the GB field are not presently translated to improved patients' survival. In order to explore novel therapeutic avenues for this deadly tumour, two innovative medicinal chemistry approaches are proposed and discussed: a) Specific glioma initiating cell-radiosensitization by ATM inhibitors [1] and b) Specific glioma initiating cell-chemotherapeutic targeting by MYC inhibitors [2].

// Richard E. Kast 1 , Nicolas Skuli 2 , Georg Karpel-Massler 3 , Guido Frosina 4 , Timothy Ryken 5 and Marc-Eric Halatsch 3 1 IIAIGC Study Center, Burlington, VT, USA 2 INSERM, Centre de Recherches en Cancerologie de Toulouse, CRCT, Inserm/Universite Toulouse III, Paul Sabatier, Hubert Curien, Toulouse, France 3 Department of Neurosurgery, Ulm University Hospital, Albert-Einstein-Allee, Ulm, Germany 4 Mutagenesis & Cancer Prevention Unit, IRCCS Azienda Ospedaliera Universitaria San Martino, IST Istituto Nazionale per la Ricerca sul Cancro, Largo Rosanna Benzi, Genoa, Italy 5 Department of Neurosurgery, University of Kansas, Lawrence, KS, USA Correspondence to: Richard E. Kast, email: // Keywords : AMPK, EIS regimen, EMT, glioma, glioblastoma Received : March 02, 2017 Accepted : May 12, 2017 Published : June 01, 2017 Abstract This paper outlines a treatment protocol to run alongside of standard current treatment of glioblastoma- resection, temozolomide and radiation. The epithelial to mesenchymal transition (EMT) inhibiting sextet, EIS Regimen, uses the ancillary attributes of six older medicines to impede EMT during glioblastoma. EMT is an actively motile, therapy-resisting, low proliferation, transient state that is an integral feature of cancers’ lethality generally and of glioblastoma specifically. It is believed to be during the EMT state that glioblastoma’s centrifugal migration occurs. EMT is also a feature of untreated glioblastoma but is enhanced by chemotherapy, by radiation and by surgical trauma. EIS Regimen uses the antifungal drug itraconazole to block Hedgehog signaling, the antidiabetes drug metformin to block AMP kinase (AMPK), the analgesic drug naproxen to block Rac1, the anti-fibrosis drug pirfenidone to block transforming growth factor-beta (TGF-beta), the psychiatric drug quetiapine to block receptor activator NFkB ligand (RANKL) and the antibiotic rifampin to block Wnt- all by their previously established ancillary attributes. All these systems have been identified as triggers of EMT and worthy targets to inhibit. The EIS Regimen drugs have a good safety profile when used individually. They are not expected to have any new side effects when combined. Further studies of the EIS Regimen are needed.

We have previously shown that pharmacological inhibition of ataxia telangiectasia mutated (ATM) protein sensitizes glioblastoma-initiating cells (GICs) to ionizing radiation (IR). Herein, we report the experimental conditions to overcome GIC radioresistance in vitro using the specific ATM inhibitor KU-60019, two major determinants of the tumor response to this drug and the absence of toxicity of this treatment in vitro and in vivo. Repeated treatments with KU-60019 followed by IR substantially delayed GIC proliferation in vitro and even eradicated radioresistant cells, whereas GIC treated with vehicle plus radiation recovered early and expanded. The tumor response to the drug occurred under a cutoff level of expression of TP53 and over a cutoff level of expression of phosphatidylinositol 3-kinase (PI3K). No increased clastogenicity or point mutagenicity was induced by KU-60019 plus radiation when compared to vehicle plus radiation. No significant histological changes to the brain or other organs were observed after prolonged infusion into the brain of KU-60019 at millimolar concentrations. Taken together, these findings suggest that GIC-driven tumors with low expression of TP53 and high expression of PI3K might be effectively and safely radiosensitized by KU-60019.

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Previous studies have shown that tumor-driving glioma stem cells (GSC) may promote radio-resistance by constitutive activation of the DNA damage response started by the ataxia telangiectasia mutated (ATM) protein. We have investigated whether GSC may be specifically sensitized to ionizing radiation by inhibiting the DNA damage response. Two grade IV glioma cell lines (BORRU and DR177) were characterized for a number of immunocytochemical, karyotypic, proliferative and differentiative parameters. In particular, the expression of a panel of nine stem cell markers was quantified by reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry. Overall, BORRU and DR177 displayed pronounced and poor stem phenotypes, respectively. In order to improve the therapeutic efficacy of radiation on GSC, the cells were preincubated with a nontoxic concentration of the ATM inhibitors KU-55933 and KU-60019 and then irradiated. BORRU cells were sensitized to radiation and radio-mimetic chemicals by ATM inhibitors whereas DR177 were protected under the same conditions. No sensitization was observed after cell differentiation or to drugs unable to induce double-strand breaks (DSB), indicating that ATM inhibitors specifically sensitize glioma cells possessing stem phenotype to DSB-inducing agents. In conclusion, pharmacological inhibition of ATM may specifically sensitize GSC to DSB-inducing agents while sparing nonstem cells.

Oxidative damage plays a pathogenic role in various chronic degenerative diseases. Oxidative damage targeting trabecular meshwork (TM) cells as a consequence of mitochondrial damage is a pathogenic mechanism for glaucoma, the most common cause of irreversible blindness worldwide. Consequences of oxidative damage are attenuated by endocellular activities involved in scavenging reactive oxidative species and DNA repair. Selected bacterial genes are highly efficient at protecting cells from oxidative DNA damage. This situation occurs for Escherichia coli formamidopyrimidine DNA glycosylase (FPG), a major DNA glycosylase that repairs oxidatively damaged DNA. Accordingly, this study was aimed at transfecting human TM cells (HTMC) with Fpg in order to increase their resistance to oxidative damage. This study demonstrates that it is feasible to increase resistance of HTMC to endogenous oxidative damage by gene transfection. These findings bear relevance for primary and secondary prevention of degenerative glaucomas and other degenerative diseases where oxidative damage plays a pathogenic role.

Repair of the oxidized purine 8-oxo-7,8-dihydro-2'-deoxyguanosine is inefficient in cells belonging to both complementation groups A and B of Cockayne syndrome (CS), a developmental and neurological disorder characterized by defective transcription-coupled repair. We show here that both CS-A and CS-B cells are also defective in the repair of 5-hydroxy-2'-deoxycytidine (5-OHdC), an oxidized pyrimidine with cytotoxic and mutagenic properties. The defect in the repair of oxidatively damaged DNA in CS cells thus extends to oxidized pyrimidines, indicating a general flaw in the repair of oxidized lesions in this syndrome. The defect could not be reproduced in in vitro repair experiments on oligonucleotide substrates, suggesting a role for both CS-A and CS-B proteins in chromatin remodeling during 5-OHdC repair. Expression of Escherichia coli formamidopyrimidine DNA glycosylase (FPG) or endonuclease III complemented the 5-OHdC repair deficiency. Hence, the expression of a single enzyme, FPG from E. coli, stably corrects the delayed removal of both oxidized purines and oxidized pyrimidines in CS cells.

The importance of DNA repair as a resistance mechanism in gliomas, the most aggressive form of brain tumor, is a clinically relevant topic. Recent studies show that not all cells are equally malignant in gliomas. Certain subpopulations are particularly prone to drive tumor progression and resist chemo- and radiotherapy. Those cells have been variably named cancer stem cells or cancer-initiating cells or tumor-propagating cells, owing to their possible (but still uncertain) origin from normal stem cells. Although DNA repair reduces the efficacy of chemotherapeutics and ionizing radiation toward bulk gliomas, its contribution to resistance of the rare glioma stem cell subpopulations is less clear. Mechanisms other than DNA repair (in particular low proliferation and activation of the DNA damage checkpoint response) are likely main players of resistance in glioma stem cells and their targeting might yield significant therapeutic gains. (Mol Cancer Res 2009;7(7):989–99)

Stem cells build and maintain organisms. Accordingly, they are particularly well-protected from damage to DNA and other cellular components. This feature becomes a serious drawback when stem cells transform and develop cancer, because they resist to radiation and chemotherapy. Various mechanisms ensure protection of stem cells. In normal stem cells enhanced DNA repair is often one of them. Whether the same holds for cancer stem cells still is an open question.

Cockayne syndrome (complementation groups A and B) is a rare autosomal recessive DNA repair disorder characterized by photosensitive skin and severely impaired physical and intellectual development. The Cockayne syndrome A and B proteins intervene in the repair of DNA modifications that block the RNA polymerase in transcribed DNA sequences (transcription-coupled repair). Recent results suggest that they also have a more general role in the repair of oxidative DNA base modifications. Although the phenotypical consequences of defective repair of oxidatively damaged DNA in Cockayne syndrome are not determined, accumulation of oxidized lesions might contribute to delay the physical and intellectual development of these patients. To conceive new therapeutic strategies for this syndrome, we are investigating whether the oxidatively damaged DNA repair defect in Cockayne syndrome might be complemented by heterologous repair proteins, such as the Escherichia coli formamidopyrimidine-DNA glycosylase and endonuclease III. The complementation studies may shed light on the important lesions for the Cockayne syndrome phenotype and offer new tools for future therapies aimed at counteracting the consequences of oxidatively damaged DNA accumulation.

Repair of the oxidized purine 8-oxo-7,8-dihydroguanine (8-oxoGua) is inefficient in cells belonging to the B complementation group of Cockayne syndrome (CS-B), a developmental and neurological disorder characterized by defective transcription-coupled repair. We show here that cells belonging to the A complementation group (CS-A) are also defective in repair of 8-oxoGua and we demonstrate that expression of the Escherichia coli formamidopyrimidine DNA glycosylase (FPG) completely corrects the repair deficiency in both CS-A and CS-B cells. Phenotypically, CS-A cells are normally sensitive to toxicity and micronuclei induced by the oxidizing agent potassium bromate. CS-B cells display sensitivity to elevated concentrations of potassium bromate but this is not compensated by FPG expression, suggesting toxicity of lesions that are not FPG substrates. The data indicate that 8-oxoGua is not a major toxic and clastogenic lesion in CS cells.

Gene therapy, the treatment of disorders or pathophysiologic states on the basis of the transfer of genetic information, has been thoroughly investigated for the treatment of lung illnesses, e.g. cystic fibrosis, alpha1-antitrypsin deficiency-related emphysema and cancer. Transfer of genetic information may be further used to elevate the level of protection of normal lung tissues in at risk individuals, with preventing purposes. This concept can be described by the term "gene prophylaxis". Lying at the gas-exchange interface, lung epithelia may be at risk of oxidation-induced mutagenesis. Further, inflammation processes possibly consequent on smoking liberate reactive oxygen species (ROS) that multiply the carcinogenic effects of tobacco. Some studies report in lung cancer patients an high frequency of variations of the 8-oxoguanine DNA glycosylase (hOGG1) gene that encodes a sluggish glycosylase for oxidized purines. Unlike dietary interventions with antioxidant drugs that only allow temporary oxy-radical scavenging, reinforcing the DNA repair capacity in lung epithelia may afford long-term, steady protection from ROS-generated mutagenesis and carcinogenesis. In this regard, the Escherichia coli formamidopyrimidine DNA glycosylase (FPG) is a possible tool. FPG is 80-fold faster than hOGG1 in repairing oxidized purines and has broader substrate specificity. Cell culture studies have shown that FPG can be expressed in mammalian cells where it accelerates DNA repair and abates mutagenicity of a wide range of DNA damaging agents. Spontaneous mutagenesis drops too. Prophylaxis of oxidative DNA damage and mutation could be achieved in lung epithelia and other tissues of at-risk individuals by FPG expression. Currently available vehicles for this peculiar type of gene therapy are briefly surveyed.

High grade gliomas (HGGs) are fatal brain tumors due to their infiltration capacity and the presence of resistant cell populations. Further, the brain is naturally protected from many exogenous molecules by the brain blood barrier (BBB), which limits or cancels passage of cytotoxic drugs to the tumor sites. In order to cope with the latter problem, nanoparticle (NP)-based carriers are intensively investigated, due to multiple possibilities to drive them across the BBB to the tumor sites and drop cytotoxic molecules there. The current status of research on NP for drug delivery to HGGs has been analyzed. The results indicate gold, lipids and proteins as three main materials featuring NP formulations for HGG treatment. Albeit specific drug targeting to HGG cells may have not been so far significantly improved, NP may help drugs crossing the BBB and enter the brain thus potentially fixing at least one part of the problem. From the Clinical Editor High grade gliomas (HGG) are very aggressive tumours and current therapy remains unsatisfactory. The advance in nanomedicine has allowed the development of novel treatment modalities. In this review article, the authors outlined the current status in using nanoparticle (NP)-based carriers for drug delivery to HGG. This should help readers to understand and develop ideas for further drug carrier designs.

We have recently reported that glioblastoma (GB)-initiating cells (GIC) with low expression and/or mutation of TP53 and high expression of PI3K ("responder" genetic profile) can be effectively and safely radiosensitized by the ATM inhibitor KU60019. We report here on drug's diffusion and elimination from the animal body and brain, its effects on orthotopic GB and efficacy toward pediatric GIC. Healthy mice were infused by convection enhanced delivery (CED) with KU60019 and the drug kinetics followed by high performance liquid chromatography-mass spectrometry. Already at the end of CED, KU60019 had diffused from the injection site to the ipsilateral and, to a lower extent, controlateral hemisphere. After 24 hr, no drug could be detected all over the brain or in other organs, indicating rapid draining and excretion. After intraperitoneal injection, traces only of KU60019 could be detected in the brain, indicating inability to cross the brain-blood barrier. Consistent with the induction of cell cycle progression previously observed in vitro, KU60019 stimulated proliferation of orthotopic GB cells with the highest effect observed 96 hr after drug delivery. Adult GIC with high expression of TP53 and low expression of PI3K could be radiosensitized by KU60019, although less promptly than GIC bearing the "responder" profile. Consistent with the kinetics of proliferation induction, the highest radiosensitizing effect was observed 96 hr after delivery of KU60019 to GIC. Pediatric GIC could be similarly radiosensitized after exposure to KU60019. The results indicate that ATM inhibition may allow to radiosensitize a wide range of adult and pediatric high-grade gliomas.

Lying at the gas-exchange interface, lung epithelia may be at risk of oxidation-induced mutagenesis. Further, inflammation processes possibly consequent on smoking liberate reactive oxygen species that multiply the carcinogenic effects of tobacco. DNA repair mechanisms play a major role in counteracting the deleterious effects of oxidative DNA damage. Some studies find positive associations between lung cancer and variations in the human 8-oxoguanine DNA glycosylase (hOGG1) gene that encodes a major DNA glycosylase for oxidized lesions with sluggish kinetics properties. The bacterial homologue formamidopyrimidine-DNA glycosylase (FPG) is 80-fold faster than hOGG1 in repairing mutagenic oxidative lesions. Cell-culture studies have shown that FPG can be expressed in mammalian cells, where it accelerates DNA repair and abates mutagenicity of a wide range of DNA-damaging agents. Prophylaxis of oxidative DNA damage and mutation could be achieved in lung epithelia and other tissues of at-risk individuals by expression of the FPG protein. Currently available vehicles for this peculiar type of gene therapy are briefly surveyed.

The S3 ribosomal protein of Drosophila melanogaster possesses various DNA repair activities, including the capacity to incise at apurinic/apyrimidinic (AP) sites and 8-oxo-7,8-dihydroguanine (8-oxoG) residues. We have recently hypothesized that this multifunctional protein may improve the efficiency of DNA base excision repair (BER) in mammalian cells. We have investigated the effect of pure GST-tagged Drosophila S3 on BER of different endogenous lesions performed by human and mouse cell extracts. Drosophila S3 significantly accelerated the BER of 8-oxoG (initiated by the bifunctional glycosylase OGG1). The stimulating effect was linked to the capacity of S3 to remove the 8-oxoG lesion and cleave the resulting AP site, rather than acceleration of downstream steps of the BER pathway (e.g., removal of 3′ blocking fragments). No stimulating effect was observed on the BER of uracil, natural AP sites, and β-lyase-cleaved AP sites. Heterologous expression of Drosophila S3 may be used to enhance 8-oxoG repair in human cells. Environ. Mol. Mutagen. 42:50–58, 2003. © 2003 Wiley-Liss, Inc.

Glioblastoma multiforme (WHO grade IV) is a highly lethal brain tumor. Its malignancy is in part due to cell populations refractory to radiotherapy and chemotherapy, which in some patients display stem properties (glioma stem cells-GSC). We and others have recently shown that a major mechanism of resistance of GSC to therapies resides in their slow proliferation that in turn is linked to constitutive activation of the DNA damage response. FancD2, a central player of the Fanconi anemia pathway, is induced when replication forks stall at DNA damage sites.We have analyzed the kinetics of FancD2 induction in two glioma cell lines with pronounced (Borru) and poor (DR177) stem phenotypes, by fluorescence analysis of nuclear foci.FancD2 activation was significantly delayed in Borru, consistent with the slow replication fork progression in these cells. On the contrary, no significant difference between Borru and DR177 was observed for pH2AX nuclear foci formation that hallmarks a number of DNA structure variations including double-strand breaks.GSC display reduced FancD2 activation following radiation damage, most probably due to their elongated cell cycle.

DNA damage of endogenous origin may significantly contribute to human cancer. A major pathway involved in DNA repair of endogenous damage is DNA base excision repair (BER). BER is rather efficient in human cells but a certain amount of endogenous damage inevitably escapes mending and likely contributes to human carcinogenesis. Apart from some glycosylases that are particularly sluggish (e.g. 8-oxoG DNA glycosylase), recent work suggests that the general rate-limiting steps of BER may be trimming of 2-deoxyribose 5-phosphate in case the process is started by a monofunctional glycosylase or trimming of a 3'-blocking fragment, in case BER is started by a bifunctional glycosylase or in the case of single-strand breaks produced by free radical attack. Overexpression of the 5'-deoxyribophosphodiesterase (dRPase) domain of DNA polymerase beta, on the one hand, and of yeast APN1 protein, containing an efficient 3' repair activity, on the other, may lead to improved BER in mammals. The recently characterized S3 protein of Drosophila, containing both dRPase and 3'-trimming activities, could also be considered for overexpression studies. The possible protecting role of enhanced BER could be investigated in cultured rodent embryonic fibroblasts undergoing spontaneous transformation, a most interesting system that merits rediscovery.

Ataxia telangiectasia (A-T) cells are sensitive to a broad range of free-radical-producing and alkylating agents. Damage caused by such agents is in part repaired by base excision [base excision repair (BER)]. Two BER pathways have been demonstrated in mammalian cells: a single-nucleotide-insertion pathway and a long-patch pathway involving resynthesis of 2–10 nucleotides. Although early studies failed to detect DNA-repair defects in A-T cells exposed to ionizing radiation and radiomimetic agents, more recent experiments performed in non-dividing A-T cells and the demonstrated interaction of the A-T-mutated protein (ATM) with the BRCA1 gene product suggest that a DNA-repair defect may underlie, at least in part, the radiation sensitivity in A-T cells. We have analysed BER of a single abasic site or a single uracil in two A-T families, using an in vitro BER system. In both families, the mutation involved was homozygous and completely inactivated the ATM protein. No difference was observed between affected individuals and heterozygous or homozygous wild-type relatives in their capacity to perform DNA repair by either one-nucleotide insertion or the long-patch pathway. Hence, the putative DNA-repair defect in A-T cells, if any, does not involve BER.

A significant contribution to human mutagenesis and carcinogenesis may come from DNA damage of endogenous, rather than exogenous, origin. Efficient repair mechanisms have evolved to cope with this. The main repair pathway involved in repair of endogenous damage is DNA base excision repair. In addition, an important contribution is given by O6-alkylguanine DNA alkyltranferase, that repairs specifically the miscoding base O6-alkylguanine. In recent years, several attempts have been carried out to enhance the efficiency of repair of endogenous damage by overexpressing in mammalian cells single enzymatic activities. In some cases (e.g. O6-alkylguanine DNA alkyltransferase or yeast AP endonuclease) this approach has been successful in improving cellular protection from endogenous and exogenous mutagens, while overexpression of other enzymatic activities (e.g. alkyl N-purine glycosylase or DNA polymerase β) were detrimental and even produced a genome instability phenotype. The reasons for these different outcomes are analyzed and alternative enzymatic activities whose overexpression may improve the efficiency of repair of endogenous damage in human cells are proposed.

Defective DNA repair has been suggested as a possible predisposing factor for breast cancer. We have investigated the repair of the frequent endogenous lesions abasic sites in sporadic early onset breast cancer patients and matched control individuals. No significant difference was observed between the abasic site repair capacities of peripheral blood lymphocytes from cases and controls. Repair of abasic sites was also studied in tumor and surrounding normal tissues of the patients. The 2 tissues showed marked differences in histology and protein composition with a fibro-collagenous component varying from sample to sample but invariably higher in normal tissues as compared with the adjacent tumor. These differences involved the need to calculate the repair activities of tissues on the basis of cellular DNA content for comparison purposes. After doing so, tumor and normal tissues exhibited similar abasic site repair capacities, whereas lymphocytes showed a repair capacity significantly lower than tissues. We conclude that early onset sporadic breast cancer patients show no evident defect in repair of abasic sites at the level of both lymphocytes and tumor.

Although the prognosis for malignant gliomas is normally dismal, it's not infrequent in neurooncologist's experience to find cases with unusually prolonged survival. In order to understand what factors influence survival of high grade glioma patients, a cohort of 196 high (III-IV) grade glioma patients was investigated for possible association between (1) survival and age at diagnosis; (2) survival and micronuclei in tumor tissue; (3) survival and gender; (4) micronuclei in tumor tissue and age at diagnosis.Patients diagnosed at an older age (64 years) had a significantly higher hazard as compared to younger patients (or=64 years), indicating that older patients survived shorter. On the contrary, no association was found between survival and micronuclei or gender.Survival analysis was performed by the Cox' proportional hazards regression model.Age at diagnosis, together with other established prognostic factors such as histologic characteristics, extent of surgery and Karnofsky Performance Score may to a certain extent predict survival of high grade glioma patients.

Mammalian DNA ligase III exists as two distinct isoforms denoted alpha and beta. Both forms possess a motif that is homologous to the putative zinc finger present in poly(ADP-ribose) polymerase. Here, the role of this motif in the binding and ligation of nicked DNA and RNA substrates in vitro has been examined in both isoforms. Disruption of the putative zinc finger did not affect DNA ligase III activity on nicked DNA duplex, nor did it abolish DNA ligase III-alpha activity during DNA base excision repair in a cell-free assay. In contrast, disruption of this motif reduced 3-fold the activity of both DNA ligase III isoforms on nicked RNA present in RNA/DNA homopolymers. Furthermore, whereas disruption of the motif did not prevent binding of DNA ligase III to nicked DNA duplex, binding to nicked RNA homopolymers was reduced approximately 10-fold. These results suggest that the putative zinc finger does not stimulate DNA ligase III activity on simple nicked DNA substrates, but indicate that this motif can target the binding and activity of DNA ligase III to nicked RNA homopolymer. The implications of these results to the cellular role of the putative zinc finger are discussed.

The human AP endonuclease (APE) activity counteracts the mutagenic and cytotoxic effects of the frequent genomic lesions abasic (AP) sites. In order to investigate the interindividual variability of APE levels, a simple and quantitative assay was developed. Crude lymphocyte extracts were incubated with a depurinated or a control supercoiled plasmid substrate, and the accumulation of nicked circular plasmid forms was monitored by agarose gel electrophoresis. The detected incision activity was AP sites-dependent and EDTA-sensitive. The unit of enzymatic activity was defined as that amount able to incise 1 ng of plasmid DNA carrying 1 AP site/plasmid at 30 degrees C in 10 min. The assay was used to measure the APE activity in 10 healthy individuals ages 25-48 years. Values ranged from 0.38 to 0.94 units/ng protein, with a mean value of 0.62. The use of the assay for screening of people with DNA base excision repair (BER) defects is proposed.

To study novel treatments for high grade gliomas (WHO grade III and IV) we need animal models of those disorders. Orthotopic tumors in mouse or rats seem at present the most reliable in vivo glioma model in order to develop specific therapies. The orthotopic tumor characteristics should yet closely mimic the human glioma features (in particular infiltrating growth and neovascularization). In this regard, glioma cell lines with stem properties (glioma stem cells - GSC) may fulfill at best those needs. Orthotopic gliomas developed from some widely used, established non-stem cell lines showing poor infiltrating capacity are not suitable. Therapeutic and diagnostic procedures recently developed using orthotopic rodent glioma tumors along their potentialities and pitfalls are analyzed.

We have previously shown that DNA repair of oxidized bases (either purines or pyrimidines) is inefficient in cells from patients with Cockayne syndrome (cs), a rare developmental and neurological genetic disorder. Here, we show for the first time that resolution of ionizing radiation (IR)-induced pH2AX foci, an indicator of DNA double-strand breaks, is significantly delayed in IR-treated cells belonging to the B complementation group of cs (csb). Using alkaline single-cell gel electrophoresis, which predominantly detects single-strand breaks, we further demonstrate elevated DNA breakage in csb cells early after irradiation. Both the delayed resolution of pH2AX foci and the early DNA breakage of csb cells were partially complemented by expression of wild-type CSB protein. Hence, the csb mutation impairs resolution of pH2AX foci and causes DNA fragility, broadening the spectrum of lesions whose processing is altered in this syndrome. © 2011 IUBMB IUBMB Life, 63(4): 272–276, 2011

Patients with glioblastoma multiforme (GBM - WHO grade IV) seldom recover. This is due to the infiltrative nature of these tumours and the presence of cellular populations with ability to escape therapies and drive tumour recurrence and progression. In some cases, these resistant cells exhibit stem properties [glioma stem cells (GSC)]. This article aims at discussing relevant issues on GSC resistance to current therapies and outlines possible and promising avenues in regard to novel therapeutic strategies, such as pharmacological, immunological and viral interventions.

DNA repair is a double-edged sword in stem cells. It protects normal stem cells in both embryonic and adult tissues from genetic damage, thus allowing perpetuation of intact genomes into new tissues. Fast and efficient DNA repair mechanisms have evolved in normal stem and progenitor cells. Upon differentiation, a certain degree of somatic mutations becomes more acceptable and, consequently, DNA repair dims. DNA repair turns into a problem when stem cells transform and become cancerous. Transformed stem cells drive growth of a number of tumours (e.g., high grade gliomas) and being particularly resistant to chemo- and radiotherapeutic agents often cause relapses. The contribution of DNA repair to resistance of these tumour-driving cells is the subject of intense research, in order to find novel agents that may sensitize them to chemotherapy and radiotherapy.

A recently developed in vitro excision-repair system was used to investigate the effect of the topoisomerase poisons VM 26, fostriecin and camptothecin on DNA repair replication carried out by Chinese hamster ovary cell extracts. VM 26 and camptothecin partially inhibit topoisomerases II and I respectively, which are present in the repair-competent extracts, but have only slight effects on the repair efficiency. On the contrary, the antitumor drug fostriecin markedly affects repair replication but, in contrast to a previous report, does not seem to have, under the experimental conditions used, any inhibitory effect on topoisomerase II. This lack of correlation between the ability to inhibit DNA topoisomerases and the effect on DNA repair replication suggests that topoisomerases should not play a primary role in mammalian excision repair. The use of cleavable-complex stabilizing poisons to investigate the role of eukaryotic topoisomerases in DNA excision repair is discussed.

It has been reported that cancer stem cells may contribute to glioma radioresistance through preferential activation of the DNA damage checkpoint response and an increase in DNA repair capacity. We have examined DNA repair in five stem and nonstem glioma cell lines. The population doubling time was significantly increased in stem compared with nonstem cells, and enhanced activation of Chk1 and Chk2 kinases was observed in untreated CD133+ compared with CD133− cells. Neither DNA base excision or single-strand break repair nor resolution of pH2AX nuclear foci were increased in CD133+ compared with CD133− cells. We conclude that glioma stem cells display elongated cell cycle and enhanced basal activation of checkpoint proteins that might contribute to their radioresistance, whereas enhanced DNA repair is not a common feature of these cells. (Mol Cancer Res 2009;7(3):383–92)

An in vitro system allowing human cell extracts to carry out excision repair in ultraviolet light-irradiated, closed circular DNA was used to study the influence of transcription on repair synthesis. RNA synthesis from plasmid DNA containing bacterial transcription units was obtained by addition of Escherichia coli RNA polymerase and ribonucleoside triphosphates to the repair-incubation mixture. No increase in UV-stimulated repair replication was observed under transcriptional conditions; in fact, an inhibition of repair replication occurred, possibly due to impairment of DNA polymerization. This is unlike the in vivo situation where preferential DNA repair of transcribed genes has been found after UV-irradiation. Possible factors important for preferential repair of expressed genes in mammalian cells are discussed.