L'expression de la désaminase de cytosine de l'ADN Apobec3B est induite par les adénovirus

Somatic mutations, which are responsible for the emergence and development of cancers, have multiple origins, such as the exposition to carcinogenic agents or the DNA polymerase infidelity during replication. After the spontaneous mutations associated with age, the second most prevalent mutational process has been attributed to the deaminase activity of APOBEC3 proteins (Apolipoprotein B Editing Catalytic subunits 3, A3). This family of enzymes, which contains 7 members (A3A, B, C, DE, F, G and H) in humans and involved in innate immune defenses against viruses, converts cytosine bases to uracil in single stranded DNA contexts. Genomic mutations associated with APOBEC3 are observed in human papillomavirus (HPV) positive head and neck squamous cell carcinomas, whereas they are lacking in HPV negative oropharyngeal cancer. This study established the first link between viral infection and APOBEC3-associated mutations. Although these mutations are also common in non-small cell lung cancer, no link has yet been established with a possible viral infection. The aim of this project was to test whether adenoviral strains (HAdV) frequently involved in lower respiratory tract infections (HAdV-C2, HAdV-C5 and HAdV-B3) induce the expression of APOBEC3 enzymes in pulmonary epithelial cells. We founded that A3B transcripts are the only ones that are upregulated upon adenoviral infection in A549 cancerous pulmonary epithelial cells. We also detected the expression of A3B at the protein level upon infection by the B3 and C2 strains. Very interestingly, the induced A3B proteins showed a slight different molecular weight. We hypothesize that this difference might be caused by different post-translational modifications. Although they must be confirmed by other experiments, these results are very encouraging and may be the starting point for the first link between viral infection and APOBEC3-associated mutation in non-small cell lung cancer. Liste des abréviations Abréviation Nom complet % Pourcent °C Degré Celsius μ Micro A APOBEC A549 Adenocarcinomic human alveolar basal epithelial cells AAV Virus adéno-associé ADN/DNA Acide désoxyribonucléique ADNc ADN complémentaire AdV Adénovirus AID Désaminase à activation induite ApoB Apolipoprotéine B APOBEC Apolipoprotein B mRNA Editing enzyme ARNm Acide ribonucléique messager BAK BCL-2 antagonist/killer BAX BCL-2 associated X-protein bp Paire de bases BSA Bovine serum albumin CAR Coxsackie/Adenovirus receptor CBNPC Cancer bronchique à non petites cellules CBPC Cancer bronchique à petites cellules CBX Chromobox CCD charge coupled devices CTSD Cathepsin D Da Dalton DMEM Dulbecco’s Modified Eagle’s Medium DTT Dithiothréitol E. coli Escherichia coli EDTA Acide éthylène diamine tétraacétique eIF Facteur d’initiation de la traduction ER Estrogen receptor FOS FBJ Murine Osteosarcoma Viral Oncogene Homolog FX Facteur de coagulation X G Accélération de la pesanteur GREB Gene Regulated By Estrogen In Breast Cancer h Heure HAdV Adénovirus humain HBEC3-KT Human bronchial epithelial cells HBV Virus de l’hépatite B HED HEPES-EDTA-DTT HEK 293T Human embryonic kidney 293T HEPES Acide 4-(2-hydroxyéthyl)-1-pipérazine éthane sulfonique His Histidine HPRT Hypoxanthine phosphoribosyltransferase HPV Human papillomavirus HTLV-1 Human T-lymphotropic virus 1 IFN Interféron Ig Immunoglobuline IL Interleukine K kDa kb Kilobase LTR Long terminal repeats M Milli M Molarité shARN Short hairpin RNA MLP Promoteur tardif majeur MOI Multiplicity of infection N Nano NEB New England Biolabs NFκB Nuclear factor kappa B NHEJ Jonction des régions non-homologues nm Nanomètre NPC Nuclear Pore Complex PBS Phosphate buffered saline PCR Réaction en chaîne par polymérisation Pen-Strep Pénicilline-streptavidine PKR Protéine kinase R Pmol Picomole pRB Protéine du rétinoblastome Pten Homologue de phosphatase et tensine RNAse Ribonucléase ROS Dérivé réactif de l’oxygène SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Ta Température d’appariement TBE Tris, Borate, EDTA TBST Tris-Buffered Saline Tween 20 TFF1 Trefoil factor 1 Tm Température de fusion TNF Facteur de nécrose tumorale Tris Trishydroxyméthylaminométhane UDG Uracil DNA glycosylase V Volt VA-RNA Virus associated-RNA VIH Virus à immunodéficience humaine α Alpha β Béta κ Kappa