Silicon Phthalocyanine Pc 4 and Red Light Causes Apoptosis in HIV-infected Cells

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Abstract
The silicon phthalocyanine HOSiPcOSi(CH3)2(CH2)3 N(CH3)2 (Pc 4), is being studied as a photosensitizer for virus inactivation in red blood cell concentrates (RBCC). The RBCC spiked with cell-free human immunodeficiency virus (HIV) or with HIV actively replicating in the T-lymphocytic cell line CEM can be successfully inactivated (> or = 6 log10) when exposed to 2 microM Pc 4 and 90 J/cm2 red light (600-800 nm). Inactivation of > or = 6 log10 inducible HIV in the latently infected promonocytic cell line U1 occurred at 22.5 J/cm2 (H. Margolis-Nunno et al., Transfusion 36, 743-750, 1996). In order to understand the reason for the increased susceptibility of U1 to photosensitized inactivation we looked for induction of apoptosis by photodynamic treatment (PDT). Agarose gel electrophoresis was used to observe the appearance of a characteristic 180-200 base pair DNA ladder, which can indicate apoptosis. Using this assay it is shown that Pc 4 treatment induced apoptosis in U1 cells in a light dose-dependent manner, starting 30 min after light exposure. Using the ApopTag Plus kit (which attaches a fluorescent label to the 3'-OH ends of the degraded DNA) and flow cytometry, the percentage of cells undergoing apoptosis was quantitated. At 10.5 J/cm2, 3 h after light exposure, about 92.5% of the cells were apoptotic. Under these conditions 99% of the cells eventually die. The CEM cells similarly treated underwent apoptosis at slower kinetics and required higher light doses. Other cell lines latently infected with HIV (ACH-2 and OM 10.1) were as sensitive as U1 to HIV inactivation by Pc 4-PDT (H. Margolis-Nunno et al., Transfusion 36, 743-750, 1996) and underwent apoptosis at a similar kinetic. These results suggest that the enhanced inactivation of HIV in latently infected cells compared to CEM cells by Pc 4-PDT may be due, at least in part, to apoptosis in the former.