DNA double-strand breaks (DSBs) induced by photon irradiation are the most deleterious damage for cancer cells and their efficient repair may contribute to radioresistance, particularly in hypoxic conditions. Carbon ions (C-ions) act independently of the oxygen concentration and trigger complex- and clustered-DSBs difficult to repair. Understanding the interrelation between hypoxia, radiation-type, and DNA-repair is therefore essential for overcoming radioresistance. The DSBs signaling and the contribution of the canonical non-homologous end-joining (NHEJ-c) and homologous-recombination (HR) repair pathways were assessed by immunostaining in two cancer-stem-cell (CSCs) and non-CSCs HNSCC cell lines. Detection and signaling of DSBs were lower in response to C-ions than photons. Hypoxia increased the decay-rate of the detected DSBs (γH2AX) in CSCs after photons and the initiation of DSB repair signaling (P-ATM) in CSCs and non-CSCs after both radiations, but not the choice of DSB repair pathway (53BP1). Additionally, hypoxia increased the NHEJ-c (DNA-PK) and the HR pathway (RAD51) activation only after photons. Furthermore, the involvement of the HR seemed to be higher in CSCs after photons and in non-CSCs after C-ions. Taken together, our results show that C-ions may overcome the radioresistance of HNSCC associated with DNA repair, particularly in CSCs, and independently of a hypoxic microenvironment.

Depuis un certain nombre d’années, l’université connaît de nombreuses mutations qui font évoluer l’enseignement et favorisent le développement d’un objet nouveau : la pédagogie universitaire. Les pratiques d’enseignement se modifient sous l’influence des changements sociaux et sociétaux : massification, arrivée des nouvelles technologies, changement du rapport au savoir, aspirations nouvelles et mobilité accrue des étudiants, exigence de pédagogies diversifiées, etc. De nouvelles formes d’enseignement apparaissent (apprentissage par problèmes, classe inversée, formations à distance, hybrides ou co-modales…), mais ce sont aussi les modalités d’évaluation et les espaces d’apprentissage qui changent. Et si, il y a quelques années encore, la formation des enseignants universitaires à la pédagogie était inexistante, des centres d’appui à la pédagogie se créent maintenant dans le monde entier, quelle que soit la typologie des établissements, tandis qu’émerge la question de la valorisation de l’engagement pédagogique des enseignants.

Background: Hadrontherapy is an alternative to radiotherapy in the treatment of Head-and-Neck cancers (HNSCC), because of accurate ballistic and high biological efficiency, even in hypoxic tumor areas. These cancers are of poor prognosis because of a high risk of recurrence related to the presence of cancer stem cells (CSCs) located in hypoxic niches. Aim of this work was to determine the molecular specificities of the response to carbon-ion irradiation versus photons in HNSCC cancer cell lines and their CSCs’ subpopulation, under hypoxic and normoxic conditions. Methods : SQ20B, FaDu cells, and their CSCs were irradiated with photons or carbon-ion (290MeV/n,NIRS) in normoxia or hypoxia (1%O2). Cell survival curves, expression of HIF-1a reactive oxygen species (ROS) and Migration/Invasion processes were quantified. Results/Conclusions: For CSCs and non-CSCs, an oxygen-enhancement-ratio (OER) upper than 1.2 was measured in response to photons, associated with stabilization of HIF-1α. This stabilization, depending on the ROS production, appears earlier in CSCs. Inhibition of HIF-1α expression results in decreased survival in HNSCC-CSCs after both type of radiation under hypoxia associated with a significant increase in residual DNA-DSBs. Furthermore, a relationship is demonstrated between HIF-1α expression and the DSBs’ detection and repair by the Homologous-Recombination. Finally, the dense and homogeneous ROS production induced by photons, essential for HIF-1α stabilization, leads to the activation of the 3 major epithelio-mesenchymal transition (EMT) signaling pathways (STAT3,MEK/p38/JNK,Akt/mTOR). At the opposite, the ROS concentrated into the carbon ion tracks are insufficient to activate HIF-1α and the upstream EMT pathways. All these results, supported by Monte-Carlo simulations, converge towards the central role of spatial ROS distribution at the nanometric scale to explain the specificities of the molecular response to carbon ions. Their therapeutic advantage may result both from unrepaired complex-DNA lesions and the non-activation of ROS-dependent-signaling pathways involved in tumor cell defense. Supported by Labex PRIMES-ANR-11-LABX-0063;ANR-11-IDEX-0007;ITMO-Cancer-AVIESAN

This article has two objectives : to explain the engineering and pedagogical design of an online continuous training system for academics, beginners or not, to university pedagogy, and to evaluate its declared uses from a professional development perspective. Created by the Ministry of Higher Education in France, in the context of the decree of May 2017 about obligation of continuous training for new academics, it is based on the creation of the MOOC Training to teach in higher education, and on local activities organized by the pedagogy support services of several French higher education institutions. A quantitative and qualitative survey on uses of the MOOC, conducted during its first session 2017/2018 on a sample of participants, identified self-regulated learning projects with an extrinsic motivation, as part of a professionalization process but facing several barriers (situational, institutional and epistemic).

Background: Head and neck squamous cell carcinoma (HNSCC) are resistant to standard treatments, partly due to cancer stem cells (CSCs) localised in hypoxic niches. Compared to X-rays, carbon ion irradiation relies on better ballistic properties, higher relative biological effectiveness and the absence of oxygen effect. Hypoxia-inducible factor-1 α (HIF-1 α ) is involved in the resistance to photons, whereas its role in response to carbon ions remains unclear. Methods: Two HNSCC cell lines and their CSC sub-population were studied in response to photons or carbon ion irradiation, in normoxia or hypoxia, after inhibition or not of HIF-1 α . Results: Under hypoxia, compared to non-CSCs, HIF-1 α is expressed earlier in CSCs. A combined effect photons/hypoxia, less observed with carbon ions, results in a synergic and earlier HIF-1 α expression in both subpopulations. The diffuse ROS production by photons is concomitant with HIF-1 α expression and essential to its activation. There is no oxygen effect in response to carbon ions and the ROS localised in the track might be insufficient to stabilise HIF-1 α . Finally, in hypoxia, cells were sensitised to both types of radiations after HIF-1 α inhibition. Conclusions: Hypoxia-inducible factor-1 α plays a main role in the response of CSCs and non-CSCs to carbon ion and photon irradiations, which makes the HIF-1 α targeting an attractive therapeutic challenge.

Apoptosis has been reported in oocytes and human preimplantation embryos both in vitro and in vivo. BCL-2 family proteins are likely to play a pivotal role in controlling oocyte and early embryo degeneration. However, no BCL-2-related survival factors have been identified that would specifically function during oocyte maturation, after fertilization and during early embryogenesis. Here, we performed a comprehensive tissue expression pattern analysis of the BCL-2 family at the mRNA level. While expression of various members was detected in human oocytes and during early primate embryogenesis, our data indicate that BCL2L10 is the predominant maternally loaded Bcl-2 family transcript, revealing an evolutionary conserved expression profile at the egg-to-zygote transition. We provide evidence that BCL2L10 is associated with the microtubule binding protein translationally controlled tumor protein and mitochondria, with a stage-specific redistribution along the pericortical regulatory ooplasm. In dying oocytes, BCL2L10 colocalized with proapoptotic BAX and neutralization of BCL2L10 accelerated oocyte death. We propose BCL2L10 as a novel and prime candidate related to oocyte maturation, fertility, and embryo developmental competence.

Research demonstrated that the function of mitochondria extends well beyond that of being cell powerhouses and revealed that these organelles fulfil a dual role in both cellular life and death. In most vertebrates, execution of the mitochondrial pathway of apoptosis requires permeabilization of the mitochondrial outer membrane, an event which allows for the release of a variety of intramembrane space proteins, leading to the activation of caspases and ultimately cell demise. Bcl-2 family proteins, which include pro- and antiapoptotic members, positively or negatively regulate mitochondrial outer membrane permeabilization, i.e. a barrier to apoptosis induction. Over-expression of Bcl-2 and Bcl-x(L) is associated with tumor progression and may be responsible for drug resistance, making pro-survival Bcl-2 family members important targets for the development of anticancer agents. Pharmacological apoptosis modulation by manipulation of pro-apoptotic Bcl-2 family proteins, with the goal to treat disorders associated with uncontrolled cell death or to kill unwanted cells, is likely to represent an additional research focus in the coming years. The purpose of this review is to describe, with examples taken from recent patents, novel strategies for targeting the Bcl-2 family of apoptotic regulators through peptide-based approaches and selective delivery of functional nucleic acids.

Tumor cells are characterized by deregulated proliferation and resistance to proapoptotic stimuli. The Bcl-2 family of antiapoptotic proteins is overexpressed in a large number of chemoresistant tumors. Downregulation or inhibition of antiapoptotic proteins might result in the sensitization of cancer cells to chemotherapeutic agents. In the present study, we took advantage of the peptide aptamer strategy to target Nr-13, a Bcl-2 antiapoptotic protein involved in neoplastic transformation by the Rous sarcoma virus. We isolated peptide aptamers that behave as Nr-13 regulators, in vitro and in mammalian cells in culture. Some of these aptamers have potential proapoptotic activities. These data suggest that peptide aptamers targeting the Bcl-2 family of apoptosis inhibitors may be useful for the development of anticancer molecules.

XPG is the human endonuclease that cuts 3' to DNA lesions during nucleotide excision repair. Missense mutations in XPG can lead to xeroderma pigmentosum (XP), whereas truncated or unstable XPG proteins cause Cockayne syndrome (CS), normally yielding life spans of <7 years. One XP-G individual who had advanced XP/CS symptoms at 28 years has been identified. The genetic, biochemical, and cellular defects in this remarkable case provide insight into the onset of XP and CS, and they reveal a previously unrecognized property of XPG. Both of this individual's XPG alleles produce a severely truncated protein, but an infrequent alternative splice generates an XPG protein lacking seven internal amino acids, which can account for his very slight cellular UV resistance. Deletion of XPG amino acids 225 to 231 does not abolish structure-specific endonuclease activity. Instead, this region is essential for interaction with TFIIH and for the stable recruitment of XPG to sites of local UV damage after the prior recruitment of TFIIH. These results define a new functional domain of XPG, and they demonstrate that recruitment of DNA repair proteins to sites of damage does not necessarily lead to productive repair reactions. This observation has potential implications that extend beyond nucleotide excision repair.

Of the eight human genes implicated in xeroderma pigmentosum, defects in XPG produce some of the most clinically diverse symptoms. These range from mild freckling to severe skeletal and neurologic abnormalities characteristic of Cockayne syndrome. Mildly affected xeroderma pigmentosum group G patients have diminished XPG endonuclease activity in nucleotide excision repair, whereas severely affected xeroderma pigmentosum group G/Cockayne syndrome patients produce truncated XPG proteins that are unable to function in either nucleotide excision repair or the transcription-coupled repair of oxidative lesions. The first two xeroderma pigmentosum group G patients, XP2BI and XP3BR, were reported before the relationship between xeroderma pigmentosum group G and Cockayne syndrome was appreciated. Here we provide evidence that both patients produce truncated proteins from one XPG allele. From the second allele, XP2BI generates full-length XPG of 1186 amino acids containing a single L858P substitution that has reduced stability and greatly impaired endonuclease activity. In XP3BR, a single base deletion and alternative splicing at a rare noncanonical AT-AC intron produces a 1185 amino acid protein containing 44 internal non-XPG residues. This protein is stably expressed but it also has greatly impaired endonuclease activity. These four XPG products can thus account for the severe ultraviolet sensitivity of XP2BI and XP3BR fibroblasts. These cells, unlike those from xeroderma pigmentosum group G/Cockayne syndrome patients, are capable of limited transcription-coupled repair of oxidative lesions. Our results suggest that the L858P protein in XP2BI and the almost full-length XPG protein in XP3BR are responsible for this activity and for the absence of severe early onset Cockayne syndrome symptoms in these patients.

Nr-13 is an anti-apoptotic member of the Bcl-2 family previously shown to interact with Bax. The biological significance of this interaction was explored both in yeast and vertebrate cells and revealed that Nr-13 is able to counteract the pro-apoptotic activity of Bax. The Bax-interacting domain has been identified and corresponds to alpha-helices 5 and 6 in Nr-13. Site-directed mutagenesis has revealed that the N-terminal region of Nr-13 is essential for activity and corresponds to a genuine Bcl-2 homology domain (BH4). The modelling of Nr-13, based on its similarity with other Bcl-2 family proteins and energy minimization, suggests the possibility of electrostatic interactions between the two N-terminal-conserved domains BH4 and BH3. Disruption of these interactions severely affects Nr-13 anti-apoptotic activity. Together our results suggest that electrostatic interactions between BH4 and BH3 domains play a role in the control of activity of Nr-13 and a subset of Bcl-2 family members.

In search of human homologues of the anti-apoptotic protein Nr-13, we have characterized a human EST clone that potentially encodes a protein, which is the closest homologue of Nr-13 among the Bcl-2 family members, to date known, in humans. Phylogenetic analyses suggest Human nrh, Mouse diva/boo and Quail nr-13 to be orthologous genes. The nrh gene has the same overall organization as nr-13 and diva/boo with one single intron interrupting the ORF at the level of the Bcl-2-homology domain BH2. RT–PCR-based analysis of nrh expression indicated that this gene is preferentially expressed in the lungs, the liver and the kidneys. Interestingly, two in frame ATG codons can lead potentially to the synthesis of two products, one of them lacking 10 aminoacids at the N-terminal end. Sequence alignment with Nr-13 and Diva/Boo in addition to secondary structure prediction of the nrh transcript suggested that the shortest protein will be preferentially synthetized. Immunohistochemical analyses have revealed that Nrh is associated with mitochondria and the nuclear envelope. Moreover, Nrh preferentially associates with the apoptosis accelerator Bcl-Xs and behaves as an inhibitor of apoptosis both in yeast and vertebrate cells.

The human XPG endonuclease cuts on the 3' side of a DNA lesion during nucleotide excision repair. Mutations in XPG can lead to the disorders xeroderma pigmentosum (XP) and Cockayne syndrome. XPG shares sequence similarities in two regions with a family of structure-specific nucleases and exonucleases. To begin defining its catalytic mechanism, we changed highly conserved residues and determined the effects on the endonuclease activity of isolated XPG, its function in open complex formation and dual incision reconstituted with purified proteins, and its ability to restore cellular resistance to UV light. The substitution A792V present in two XP complementation group G (XP-G) individuals reduced but did not abolish endonuclease activity, explaining their mild clinical phenotype. Isolated XPG proteins with Asp-77 or Glu-791 substitutions did not cleave DNA. In the reconstituted repair system, alanine substitutions at these positions permitted open complex formation but were inactive for 3' cleavage, whereas D77E and E791D proteins retained considerable activity. The function of each mutant protein in the reconstituted system was mirrored by its ability to restore UV resistance to XP-G cell lines. Hydrodynamic measurements indicated that XPG exists as a monomer in high salt conditions, but immunoprecipitation of intact and truncated XPG proteins showed that XPG polypeptides can interact with each other, suggesting dimerization as an element of XPG function. The mutation results define critical residues in the catalytic center of XPG and strongly suggest that key features of the strand cleavage mechanism and active site structure are shared by members of the nuclease family.

Xeroderma pigmentosum (XP) patients have defects in nucleotide excision repair (NER), the versatile repair pathway that removes UV-induced damage and other bulky DNA adducts, patients with Cockayne syndrome (CS), another rare sun-sensitive disorder, are specifically defective in the preferential removal of damage from the transcribed strand of active genes, a process known as transcription-coupled repair, These two disorders are usually clinically and genetically distinct, but complementation analyses have assigned a few CS patients to the rare XP groups B, D, or G, The XPG gene encodes a structure-specific endonuclease that nicks damaged DNA 3' to the lesion during NER, Here we show that three XPG/CS patients had mutations that would produce severely truncated XPG proteins, In contrast, two sibling XPG patients without CS are able to make full-length XPG, but with a missense mutation that inactivates its function in NER, These results suggest that XPG/CS mutations abolish interactions required for a second important XPG function and that it is the loss of this second function that leads to the CS clinical phenotype.

Loss of heterozygosity (LOH) was evaluated in 174 breast and ovarian tumors derived from 94 families with at least 3 first-degree relatives affected with either of these cancers. By linkage analysis 26 families were identified as having a high I posterior probability of being due to BRCA1 (the breast/ovarian cancer susceptibility locus on 17q12-21) with lod scores varying from 0.51 to 9.49. Tumor genotypes were determined at at least 2 constitutionally heterozygous markers flanking BRCA1 in a total of 58 tumors from these families. These tumors were derived from 52 patients, the BRCA1 mutation carrier status of which was evidenced by DNA sequencing in 20, and inferred by reconstructing haplotypes in the remainder. LOH was detected in 50 (86%) tumors, and invariably involved the wild-type allele. Where informative, this allele was of paternal origin in 33 cases and of maternal origin in 10 cases. These results strongly suggest that BRCA1 is a tumor suppressor gene and that LOH is greatly favored to fully inactivate it. (C) 1995 Wiley-Liss, Inc.

Increased cancer risk associated with germ-line p53 mutation was linked to a deficit in the ability to maintain genomic stability. Accordingly, normal fibroblasts from cancer-prone individuals accumulate genomic aberrations with concomitant loss of wild-type p53 allele during in vitro culture. We tested whether such changes also occur in EBV-immortalized lymphoblastoid cells. Both normal and p53 germ-line mutant lymphoblastoid cells maintained functional p53 and genomic stability during long term in vitro culture. These unexpected differences between fibroblastic and lymphoblastic cells suggest that phenotypic expression of p53 deficiency is cell type specific. This could contribute to selective tissular localization of tumours observed in patients with Li-Fraumeni syndrome despite the presence of a mutant p53 allele in all cells.

The breast-ovary cancer-family syndrome is a dominant predisposition to cancer of the breast and ovaries which has been mapped to chromosome region 17q12-q21. The majority, but not all, of breast-ovary cancer families show linkage to this susceptibility locus, designated BRCA1. We report here the results of a linkage analysis of 145 families with both breast and ovarian cancer. These families contain either a total of three or more cases of early-onset (before age 60 years) breast cancer or ovarian cancer. All families contained at least one case of ovarian cancer. Overall, an estimated 76% of the 145 families are linked to the BRCA1 locus. None of the 13 families with cases of male breast cancer appear to be linked, but it is estimated that 92% (95% confidence interval 76%-100%) of families with no male breast cancer and with two or more ovarian cancers are linked to BRCA1. These data suggest that the breast-ovarian cancer-family syndrome is genetically heterogeneous. However, the large majority of families with early-onset breast cancer and with two or more cases of ovarian cancer are likely to be due to BRCA1 mutations.

Loss of heterozygosity (LOH) was evaluated in 174 breast and ovarian tumors derived from 94 families with at least 3 first‐degree relatives affected with either of these cancers. By linkage analysis 26 families were identified as having a high posterior probability of being due to BRCAI (the breadovarian cancer susceptibility locus on 17q 12–21) with lod scores varying from 0.5 1 to 9.49. Tumor genotypes were determined at at least 2 constitutionally heterozygous markers flanking BRCA I in a total of 58 tumors from these families. These tumors were derived from 52 patients, the BRCAI mutation carrier status of which was evidenced by DNA sequencing in 20, and inferred by reconstructing haplotypes in the remainder. LOH was detected in 50 (86%) tumors. and invariably involved the wild‐type allele. Where informative, this allele was of paternal origin in 33 cases and of maternal origin in I0 cases. These results strongly suggest that BRCA I is a tumor suppressor gene and that LOH is greatly favored to fully inactivate it. © 1995 Wiley‐Liss, Inc.

Codon 257 of the p53 gene is an extremely rare target for somatic mutations (accounting for only two of 1600 published mutations). We report here two constitutional mutations both affecting the second nucleotide of codon 257. A thymine to adenine transversion resulting in an amino acid change from leucine to glutamine was found in one proband who developed multiple independent malignant tumors (osteosarcoma, phyllodes tumor, soft-tissue sarcoma). Her mother died of early-onset breast cancer. In the other case, a deletion resulting in a frameshift in the C-terminal coding region of p53 was found in a woman who was diagnosed with breast cancer at age 34. This woman belongs to a family with features of Li-Fraumeni syndrome. In both cases, the p53 mutations identified in the proband was found in other members of the family. Codon 257, even if rarely mutated in somatic cells, may thus be an important target for germ-line mutations.

Previous studies have shown p53 germ-line mutations in some familial cancer aggregations with or without the Li-Fraumeni syndrome (LFS). Such mutations were also reported in children and young adults with second malignant neoplasms (SMN). This led us to screen for p53 germ-line mutations in a group of seven patients affected with SMN, but characterized by an older age of onset than in the previous reports. No mutation was found in exons 4 to 8 and their boundaries using the single-strand conformation polymorphism technique. Our results give strong evidence for genetic heterogeneity of SMN, probably related to the age of cancer onset.