Involvement of ATM and NBS1 in syncytial apoptosis observed in HIV-1 infected human lymphoblasts.

<p>PHA/IL-2 lymphoblasts obtained from healthy donors were infected with HIV-1 WT and HIV1 IN^D64V at a MOI of 10 for 48 h and then stained for the indicated markers. A. DNA damage foci containing ATMS1981P in syncytia were identified by immunofluorescence after infection of PHA/IL-2 lymphoblasts with HIV-1 WT and HIV1 IN^D64V. B. Comparative quantification of DNA damage foci induced by infection with HIV-1 WT and HIV1 IN^D64V at a MOI of 10 for 48 h. The phosphorylation status of ATM, H2AX and apoptosis were quantified (X±SD, n = 3 experiments). C. Effect of the ATM inhibitor KU-55933 on the apoptosis of HIV-1 infected primary T lymphoblasts. PHA/IL-2 lymphoblasts from healthy donors (n = 3) were infected with the indicated HIV-1 strain (MOI 5, 48 h), followed by Hoechst 33342 staining and quantification of syncytia exhibiting apoptotic chromatin condensation. D. Reduced syncytial apoptosis of Herpes virus saimiri (HVS)-transformed T cells from A-T patients. CD4⁺ T lymphoblasts from an A-T patient and a healthy donor (Co.) were infected for 24 hours with HIV-1 WT or HIV1 IN^D64V at a MOI of 10, followed by determination of the frequency of nuclear apoptosis among syncytia by Hoechst 33342 staining. E,F. Comparison of T lymphoblasts from healthy and A-T patients. PHA/IL-2 lymphoblasts from controls or A-T patients (n = 3) were infected by HIV-1 WT, and the frequency of syncytia with Tunel⁺ nuclei was assessed. Representative syncytia are shown in E and quantitative data (X±SD, n = 3) are reported in F. G. Decreased HIV-1-induced apoptosis of immortalized T cells obtained from a patent with Nijmegen breakage syndrome (NBS). Cells from an NBS patient and a healthy donor (Co.) were infected for 48h hours with HIV-1 WT at a MOI of 10, and the frequency of syncytia bearing Tunel-positive nuclei was evaluated. H. Comparison of primary T lymphoblasts from healthy and NBS donors. PHA/IL-2 lymphoblasts from controls (n = 2) or NBS patients (n = 2) were infected. The frequency of syncytial apoptosis was determined by TUNEL assay. Asterisks indicate significant (p<0.01) effects of KU-55933, NBS or A-T-mutations as compared to the respective untreated or unmutated control cells.