Fig. 4.
Nuclear localization of the EBNA1 probes and their in vivo antitumor activities. (A–C) Two-photon fluorescence imaging of ZRL5P2, ZRL5P4, and ZRL5P6 in living EBV-positive (A) NPC43 cells and (B) C666-1 cells and EBV-negative (C) HONE-1 cells. ZRL5Pn, signal emitted from the respective EBNA1 probe. DRAQ5 is a fluorescent dye used to label the cell nuclei of the living cells as indicated. (D) In vitro emission spectra (from confocal microscopy) of ZRL5P2, ZRL5P4, and ZRL5P6 in the nucleus of EBV-positive NPC43 and C666-1 and EBV-negative HONE-1 cells. Emission intensity was much greater for ZRL5P4 and ZRL5P6 in EBV-positive cells. (E) In vivo antitumor activity of ZRL5P2, ZRL5P4, and ZRL5P4. Mice transplanted with C666-1–derived tumors were treated twice weekly with 4 µg per injection of the probes for 18 d. Throughout the treatment period, tumor volumes were measured. At the experimental endpoint, tumors were excised. Data are expressed as the means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 vs. control (0.1% DMSO). (Scale bars, 10 mm.) (F) Representative photographs of tumors. (G) Representative H&E staining images of tumor sections derived from the above in vivo animal study. Cell necrosis (acellular areas indicated by asterisk) was observed in the tumor nodules treated with ZRL5P4 and ZRL5P6. T, adjacent area with tumor cells. Magnification, 400×. (Scale bars, 20 µm.) (H) Response of plasma EBV DNA levels in mice transplanted with C666-1 cells after the treatment of ZRL5P4. The circulating EBV DNA level of each mouse (DMSO #1, #2; ZRL5P4 #1, #2) is shown.