Viral Oncogene–Induced DNA Damage Response Is Activated in Kaposi Sarcoma Tumorigenesis
Figure 5
Antiproliferative Checkpoints Are Activated in KSHV-Infected ECs
(A) hT-HDMECs, hT-HDMEC–p53CTer, hT-HDMEC–LT, and hT-HDMEC–E6/E7 were infected with rKSHV.219 virus and grown for 9 d. Proliferation of these cells in relation to noninfected cells was determined by the MTT assay at 10, 12, 14, and 16 d after infection.
(B) Top panels: KSHV-ECs grown for 7 d and stained with Hoechst. GFP is expressed from the recombinant virus. Arrows indicate binucleated cells. Scale bar = 50 μm. Lower panels: Noninfected (No virus) and KSHV-ECs were analysed for centrosomes by anti–γ-tubulin antibodies (red) and DNA by Hoechst staining (blue) at day 7. Scale bar = 5 μm.
(C) KSHV-ECs were treated with wortmannin (200 nM) or caffeine (4 mM) for 24 h, and analysed for the centrosome numbers by γ-tubulin antibodies. Untreated as well and noninfected (No virus) ECs were used as controls. Percentage of cells with aberrant centrosome numbers is shown as an average of two independent experiments and analysis of at least 200 cells per sample.
(D) KSHV-ECs grown for 2 wk (early) or approximately 10 wk (late), and their passage-matched, noninfected ECs were labeled with anti-53BP1 antibodies to address activation of the DNA damage response. The bar graph shows the percentage of cells with intranuclear 53BP1-positive foci. About 200 cells were counted per sample. The insert depicts noninfected (ECs) and late KSHV-ECs showing intranuclear 53BP1 foci in the latter one (bottom right panel). Scale bar = 20 μm.