PCNA damage caused by antineoplastic drugs

Abstract

Structurally diverse chemotherapeutic and chemopreventive drugs, including camptothecin, doxorubicin, sanguinarine, and others, were found to cause covalent crosslinking of proliferating cell nuclear antigen (PCNA) trimers in mammalian cells exposed to fluorescent light. This PCNA damage was caused by both nuclear and cytoplasmically localizing drugs. For some drugs, the PCNA crosslinking was evident even with very brief exposures to laboratory room lighting. In the absence of drugs, there was no detectable covalent crosslinking of PCNA trimers. Other proteins were photocrosslinked to PCNA at much lower levels, including crosslinking of additional PCNA to the PCNA trimer. The proteins photo-crosslinked to PCNA did not vary with cell type or drug. PCNA was not crosslinked to itself or to other proteins by superoxide, hydrogen peroxide or hydroxyl radicals, but hydrogen peroxide caused mono-ubiquitination of PCNA. Quenching of PCNA photo-crosslinking by histidine, and enhancement by deuterium oxide, suggest a role for singlet oxygen in the crosslinking. SV40 large T antigen hexamers were also efficiently covalently photo-crosslinked by drugs and light. Photodynamic crosslinking of nuclear proteins by cytoplasmically localizing drugs, together with other evidence, argues that these drugs may reach the nucleoplasm in amounts sufficient to photodamage important chromosomal enzymes. The covalent crosslinking of PCNA trimers provides an extremely sensitive biomarker for photodynamic damage. The damage to PCNA and large T antigen raises the possibility that DNA damage signaling and repair mechanisms may be compromised when cells treated with antineoplastic drugs are exposed to visible light.

Oxidative Stress in Mammalian Cells Impinges on the Cysteines Redox State of Human XRCC3 Protein and on Its Cellular Localization

“…Because photosensitization by UVA is known to generate mainly 1 O 2 [4], we examined its implication in hXRCC3 oxidation by irradiating CHO cells in the presence of increasing concentrations of NaN 3 (Figure 5A) or L-Histidine (Figure 5B), two quenchers of 1 O 2 [40], [41], or of N-acetyl-L-cysteine (NAC) (Figure 5C) that scavenges free radicals [42]. Following quantifications, we found that hXRCC3 oxidation by UVA radiation was significantly prevented by increasing the concentration of NaN 3 or L-Histidine but not of NAC (Figure 5D).…”

Nicotine Mediates Hypochlorous Acid-Induced Nuclear Protein Damage in Mammalian Cells

“…Treatment of CV-1 mammalian cells with Nic-Cl (1mM) induced a high molecular weight PCNA antibody-reactive band migrating at 93 kDa. This is the well-established molecular weight of the covalently crosslinked PCNA trimer [41]. The formation of the PCNA trimer was robust as detected by Western blotting (Fig 2 a) and was found to be dose-dependent (Fig 3 a).…”

“…Covalent crosslinking of PCNA subunit, thus, interferes with PCNA ring opening and, consequently, with its function. Crosslinking of PCNA subunits is thus considered as a critical damage to PCNA [27, 41]. Because PCNA is a nuclear protein, PCNA crosslinking has been used as a sensitive marker for intracellular protein damage induced by different cytotoxic compounds [27, 41].…”

“…Crosslinking of PCNA subunits is thus considered as a critical damage to PCNA [27, 41]. Because PCNA is a nuclear protein, PCNA crosslinking has been used as a sensitive marker for intracellular protein damage induced by different cytotoxic compounds [27, 41]. While covalent crosslinking of PCNA produces mainly trimers with a molecular weight of 93 kDa, other forms such as PCNA dimers and double trimers have also been reported [27, 41, 42].…”

“…Because PCNA is a nuclear protein, PCNA crosslinking has been used as a sensitive marker for intracellular protein damage induced by different cytotoxic compounds [27, 41]. While covalent crosslinking of PCNA produces mainly trimers with a molecular weight of 93 kDa, other forms such as PCNA dimers and double trimers have also been reported [27, 41, 42]. In the current work, PCNA was used to study the ability of nicotine to mediate HOCl-induced intracellular protein damage in intact cultured mammalian lung and kidney cells.…”