A study of photodynamic damage to the DNA replication system .

摘要

Photodynamic therapy (PDT) is a promising clinical modality for killing unwanted cells, especially cancer cells by the combined use of light, photosensitizers and molecular oxygen. In the process of PDT, energy flows from light-activated triplet state photosensitizers to the triplet ground state molecular oxygen by the type II pathway, converting it to singlet oxygen, which can damage cancer cells by directly reacting with nearby biomolecules, or indirectly by destroying tumor vasculature and invoking systemic immune responses. To improve the efficiency of PDT in clinical practice, we studied photodynamic damage to important nuclear proteins involved in DNA replication/repair, and identified the [Ru(tpy)(pydppn)]2+complex as a very promising photosensitizer.;PCNA, p53, SV40 large T-antigen, topoisomerase I and lamin B were identified as cellular protein targets for photodynamic damage caused by proflavine and visible light. Following photodynamic damage, p53 was detected in a distinct covalent crosslinking profile, with p53 tetramers and dimers being the predominant forms; SV40 large T-antigen was mainly crosslinked into a hexamer while lamin B was crosslinked to dimers, trimers and tetramers.;[Ru(tpy)n(pydppn)2-n]2+ (n = 0, 1) complexes have been reported to be capable of photosensitizing the generation of singlet oxygen with near 100% efficiency in vitro. In our study, [Ru(tpy)(pydppn)]2+ caused efficient covalent oligomerization of cellular PCNA and p53 in light. In the cell lysates, both [Ru(tpy)(pydppn)] 2+ and [Ru(pydppn)2]2+ were able to produce efficient PCNA and p53 photodynamic crosslinking. Cellular PCNA photocrosslinking caused by [Ru(tpy)(pydppn)]2+ increased linearly with [Ru(tpy)(pydppn)] 2+ concentration, time of uptake, or visible light exposure. The inclusion of azide, a singlet oxygen quencher, led to significant suppression of p53 photocrosslinking, suggesting that singlet oxygen is the reactive agent causing p53 photocrosslinking. [Ru(tpy)(pydppn)]2+ caused efficient photodynamic protein-DNA crosslinking in cells, which increased with increasing levels of photodynamic damage. Photodynamic damage by [Ru(tpy)(pydppn)] 2+ resulted in inhibition of DNA replication in a biphasic manner in mammalian cells. Another ruthenium containing complex, [Ru(bpy)2(CH 3CN)2]Cl2 inhibited SV40 viral DNA replication to a great extent in a light-dependent manner, but no protein damage of the types found for photodynamic ruthenium complexes (protein-protein crosslinking, protein-DNA crosslinking) was detected by Western blotting.;We also studied the contribution of photodynamic properties of certain topoisomerase poisons in improving their cytotoxicity. It was shown that the topoisomerase II poison ellipticine reduced cell survival to a significantly greater extent when irradiated with visible light than in the dark conditions. However, the topoisomerase-DNA-ellipticine cleavable complex appears not to be the relevant cytotoxic target.