BACKGROUND: Microenvironmental conditions in normal or tumour tissues and cell lines may interfere on further biological analysis. To evaluate transcript variations carefully, it is common to use stable housekeeping genes (HKG) to normalise quantitative microarrays or real-time polymerase chain reaction results. However, recent studies argue that HKG fluctuate according to tissues and treatments. So, as an example of HKG variation under an array of conditions that are common in the cancer field, we evaluate whether hypoxia could have an impact on HKG expression. METHODS: Expression of 10 commonly used HKG was measured on four cell lines treated with four oxygen concentrations (from 1 to 20%). RESULTS: Large variations of HKG transcripts were observed in hypoxic conditions and differ along with the cell line and the oxygen concentration. To elect the most stable HKG, we compared the three statistical means based either on PCR cycle threshold coefficient of variation calculation or two specifically dedicated software. Nevertheless, the best HKG dramatically differs according to the statistical method used. Moreover, using, as a reference, absolute quantification of a target gene (here the proteinase activating receptor gene 1 (PAR1) gene), we show that the conclusions raised about PAR1 variation in hypoxia can totally diverge according to the selected HKG used for normalisation. CONCLUSION: The choice of a valid HKG will determine the relevance of the results that will be further interpreted, and so it should be seriously considered. The results of our study confirm unambiguously that HKG variations must be precisely and systematically determined before any experiment for each situation, to obtain reliable normalised results in the experimental setting that has been designed. Indeed, such assay design, functional for all in vitro systems, should be carefully evaluated before any extension to other experimental models including in vivo ones.

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Telomere maintenance is essential to preserve genomic stability and involves several telomere-specific proteins as well as DNA replication and repair proteins. The kinase ATR, which has a crucial function in maintaining genome integrity from yeast to human, has been shown to be involved in telomere maintenance in several eukaryotic organisms, including yeast, Arabidopsis and Drosophila. However, its role in telomere maintenance in mammals remains poorly explored. Here, we report by using telomere-fluorescence in situ hybridization (Telo-FISH) on metaphase chromosomes that ATR deficiency causes telomere instability both in primary human fibroblasts from Seckel syndrome patients and in HeLa cells. The telomere aberrations resulting from ATR deficiency (i.e. sister telomere fusions and chromatid-type telomere aberrations) are mainly generated during and/or after telomere replication, and involve both leading and lagging strand telomeres as shown by chromosome orientation-FISH (CO-FISH). Moreover, we show that ATR deficiency strongly sensitizes cells to the G-quadruplex ligand 360A, enhancing sister telomere fusions and chromatid-type telomere aberrations involving specifically the lagging strand telomeres. Altogether, these data reveal that ATR plays a critical role in telomere maintenance during and/or after telomere replication in human cells.

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Human T-cell Lymphotrophic Virus 1 (HTLV-1) is the etiologic agent of Adult-T cell Leukemia/Lymphoma (ATL). Therapeutic options for ATL patients are very limited and in aggressive forms of the disease survival rate is only 10% to 30% with conventional chemotherapies and bone marrow transplantation. Although some clinical trials gave encouraging results regarding the efficacy of new treatments, most of them are lifelong, aggressive and failed to achieve a significant impact on long-term survival. Consequently, new treatments for ATL patients are needed to limit relapses and side effects. Specific HTLV-1 cellular immune response is dramatically impaired in ATL patients, which could favor the initiation and the progression of the disease. Hence, stimulating immune responses against HTLV-1 can be an appropriate therapeutic option to treat ATL. THERAVECTYS has developed an anti-HTLV-1 vaccine, based on its lentiviral vector technology inducing a broad, intense and long-lasting cellular immune response after intra-muscular injection. THERAVECTYS was the first company to have launched a clinical trial based on lentiviral vectors technology with the THV01 vaccine for the treatment of HIV (NCT02054286). Results obtained demonstrated both safety and immunogenicity of THV01 in human, with polyfunctional and multi-specific CD4 and CD8 T-cells responses. The anti-HTLV-1 lentiviral vector, THV02 vaccine, encodes for a unique polypeptide derived from Tax, HBZ, p12I and p30II proteins, involved in HTLV-1 pathogenicity and known to be recognized by the immune system of HTLV-1 infected patients. Our preclinical results have demonstrated that THV02 can induce a cellular immune response in C57Bl/6j and BalbC mice and in Sprague Dawley rats, as demonstrated by IFN-γ Elispot. Safety of the THV02 vaccine has been demonstrated during carcinogenicity and regulatory GLP preclinical toxicity studies. Biodistribution and shedding studies demonstrated the very limited diffusion of THV02 after injection, its fast clearance and a non-dissemination in body fluids. As no relevant ATL immunocompetent animal model is available to assess the anti-tumor effect of THV02, THERAVECYTS is developing an ex-vivo efficacy model using blood samples of ATL patients. Briefly, monocyte-derived dendritic cells (MDDC) from blood of ATL patients are purified by isolation of CD14 positive cells from PBMC and differentiation in the presence of IL4 and GM-CSF. MDDC are then transduced with lentiviral vectors encoding for the anti-HTLV-1 antigen and maturation is induced upon TNFa and PGE2 exposure before the co-culture with autologous CD8+ T-cells for stimulation of the cellular immune response. Then, stimulated CD8+ are co-cultured with autologous CD4+ CD25+ ATL cells and the cytotoxic activity is monitored by flow cytometry. Preliminary results demonstrated that MDDC from a chronic ATL patient can be efficiently transduced and matured as attested by the CD40, CD86, HLA-DR, -A, -B and C markers on their surface. In addition, we have observed a specific stimulation of the CD8+, ie an increase of IFNg, TNFa, IL2 and perforin in the media of the co-culture of CD8+ with MDDC expressing anti-HTLV-1 antigen. These data are very encouraging and demonstrate for the first time the feasibility to develop an ex vivo model to assess vaccine efficacy using ATL blood sample. The development of this model is ongoing using several ATL donors representing the different subtypes of the disease and will be presented at the meeting. Regarding the indication and the safety profile of THV02, THERAVECTYS plans to begin a clinical trial in Q4 2015. This assay will be an open-label, dose escalation phase I/II study to assess the safety and the immunogenicity (cellular immune response) of the THV02 vaccination as a treatment of ATL patients. All ATL subtypes will be considered since THV02 vaccine can be combined with conventional ATL treatments. In addition, as the THV02 antigen contains peptides derived from Tax but also HBZ, p12I and p30II viral proteins, all ATL patients can be treated whatever the status of Tax expression. As secondary objectives, both humoral immune response and clinical effect will be assessed. HTLV-1 RNA expression and clonality of HTLV-1 infected cells will be studied as exploratory objectives. Finally, up to 16 patients will be enrolled in France, UK, French Guiana, Martinique and Guadeloupe before doing a phase of extension cohort in US. Disclosures No relevant conflicts of interest to declare.

Interstitial telomeric sequences (ITSs) in hamster cells are hot spots for spontaneous and induced chromosome aberrations (CAs). Most data on ITS instability to date have been obtained in DNA repair-proficient cells. The classical non-homologous end joining repair pathway (C-NHEJ), which is the principal double strand break (DSB) repair mechanism in mammalian cells, is thought to restore the morphologically correct chromosome structure. The production of CAs thus involves DNA-PKcs-independent repair pathways. In our current study, we investigated the participation of DNA-PKcs from the C-NHEJ pathway in the repair of spontaneous or radiation-induced DSBs in ITSs using wild-type and DNA-PKcs mutant Chinese hamster ovary cells. Our data demonstrate that DNA-PKcs stabilizes spontaneous DSBs within ITSs from the chromosome 9 long arm, leading to the formation of terminal deletions. In addition, we show that DNA-PKcs-dependent C-NHEJ is employed following radiation-induced DSBs in other ITSs and restores morphologically correct chromosomes, whereas DNA-PKcs independent mechanisms co-exist in DNA-PKcs proficient cells leading to an excess of CAs within ITSs.