DNA damage foci in karyogamic syncytia elicited by HIV-1 Env.

<p>A. ATM and histone H2AX phosphorylation. Syncytia were generated by coculture of HeLa CD4 and HeLa Env cells (36 h), followed by immunofluorescence double staining for the detection of ATMS1981P and γ-H2AX. Representative pre-karyogamic (pre-KG), karyogamic (KG), and apoptotic cells are shown. The inserts depict the staining with the chromatin-specific dye Hoechst 33342. B. Biochemical evidence for ATM and H2AX phosphorylation. Extemporaneous 1∶1 mixtures of HeLa Env and HeLa CD4 single cells (SC) or syncytia (48h, which are mixtures of pre-KG and KG cells) were subjected to immunoblot detection of total ATM, ATMS1981, total H2AX and γ-H2AX. C. Quantitation of ATM and H2AX phosphorylation in distinct categories of syncytia, as determined in A. D. Time course of ATM, H2AX and caspase activation in syncytia, as determined by immunofluorescence stainings with anti-ATMS1981P, anti-γ-H2AX (as in A) and an antibody only recognizing active caspase-3. In addition, karyogamy and apoptotic chromatin condensation was assessed (X±SD, n = 3). E. Role of cell fusion in ATM activation. Single cell (SC) or HeLa Env/HeLa CD4 cocultures were performed in standard conditions (as in A), in the absence or presence of the fusion inhibitor AMD3100. Alternatively, HeLa CD4 and HeLa Env cells were subjected to polyethylene glycol (PEG)-enforced cell fusion, either separatedly or in mixed cultures, maintained in the absence or presence of AMD3100. Twenty four hours and 36 hours later, karyogamy, nuclear apoptosis, ATM phosphorylation was assessed by immunostaining. F,G. Inhibition of ATM activation by cell cycle arrest. Hela CD4 and HeLa Env cells were exposed twice overnight to thymidine (1 mM), which leads to an accumulation (synchronization) of cells in the early S-phase. Then, synchronized (“T-blocked”) or non-synchronized cells were cocultured in the absence or in the presence of thymidine (to leave or maintain the cell cycle blockade). Note that continuous blockade prevents BrdU incorporation (F), karyogamy as well as the phosphorylation of ATM (G). Results are means±SD of three independent experiments, each performed in triplicate.