Rufloxacin induced photosensitization in bio-models of increasing complexity.

Rufloxacin belongs to the class of fluoroquinolones that act mainly as specific inhibitors of bacterial Topoisomerase II. These drugs are widely known to be involved in various diseases ranging from cutaneous reactions to aging. The type II photosensitizing activity of Rufloxacin has been already demonstrated on calf thymus DNA and free nucleosides. The aim of this study is to examine in control untreated and UVA irradiated human fibroblasts the modifications on DNA status induced by Rufloxacin added in the culture medium. This allows to investigate the photosensitizing activity of Rufloxacin in a more complex cell model. Fibroblasts, either in the presence or in the absence of Rufloxacin, were exposed to UVA irradiation for different times. An experimental protocol was followed in order to evaluate the amount of single-strand breaks (SSB) and double-strand breaks (DSB) DNA fragmentation by comet assay, and plasmid photocleavage. The presence of oxidized bases was also evaluated using the 8-OH-dGuo test. The comet assay test was also employed to assess cellular repair capacity. The intracellular drug concentration was verified by HPLC-MS. The results confirming the role of Rufloxacin as photosensitizer were: (i) a time-dependent increase in DNA fragmentation when fibroblasts were irradiated in the presence of Rufloxacin; (ii) the efficiency of the cellular repair machinery to be exhaustive after 2 h (whereas no correlation between irradiation time and DNA damage repair was observed with a higher level of DNA fragmentation after shorter irradiation times); (iii) the increased number of cells exhibiting high DNA fragmentation, seen as comets with long tails, was not accompanied by a similar large extent of oxidised DNA base formation, as measured by 8-OH-dGuo analysis; (iv) the double helix SSB, formed in plasmid photosensitization, agreed with the comet assay results, pointing out a good correlation among the cell system and the simpler models used.     

RFX proteins, a novel family of DNA binding proteins conserved in the eukaryotic kingdom.

Until recently, the RFX family of DNA binding proteins consisted exclusively of four mammalian members (RFX1-RFX4) characterized by a novel highly conserved DNA binding domain. Strong conservation of this DNA binding domain precluded a precise definition of the motif required for DNA binding. In addition, the biological systems in which these RFX proteins are implicated remained obscure. The recent identification of four new RFX genes has now shed light on the evolutionary conservation of the RFX family, contributed greatly to a detailed characterization of the RFX DNA binding motif, and provided clear evidence for the function of some of the RFX proteins. RFX proteins have been conserved throughout evolution in a wide variety of species, including Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans, mouse and man. The characteristic RFX DNA binding motif has been recruited into otherwise very divergent regulatory factors functioning in a diverse spectrum of unrelated systems, including regulation of the mitotic cell cycle in fission yeast, the control of the immune response in mammals, and infection by human hepatitis B virus.     

Assembly of the RFX complex on the MHCII promoter: role of RFXAP and RFXB in relieving autoinhibition of RFX5

The RFX complex is key component of a multi-protein complex that regulates the expression of the Major Histocompatibility Class II (MHCII) genes, whose products are essential for the initiation and development of the adaptive immune response. The RFX complex is comprised of three proteins--RFX5, RFXAP, and RFXB--all of which are required for expression of MHCII genes. We have used electrophoretic mobility shift assays to characterize the DNA binding of RFX5 and the complexes it forms with RFXB and RFXAP, to the proximal regulatory region of the MHCII promoter. DNA binding of RFX5 is inhibited by domains flanking its DNA binding domain, and both RFXAP and RFXB are required to overcome the inhibition of both domains. We provide evidence that a single RFX complex binds to the proximal regulatory region of the MHCII promoter and identify regions of the DNA that are important for high affinity binding of the RFX complex. Together, our results provide the most detailed view to date of the assembly of the RFX complex on the MHCII promoter and how its DNA binding is regulated.     

Characterization of the RFX complex and the RFX5(L66A) mutant: implications for the regulation of MHC class II gene expression

Major histocompatability complex class II (MHCII) molecules are an essential component of the mammalian adaptive immune response. The expression of MHCII genes is regulated by a cell-specific multiprotein complex, termed the MHCII enhanceosome. The heterotrimeric RFX complex is the key DNA-binding component of the MHCII enhanceosome. The RFX complex is comprised of three proteins, RFXB, RFXAP, and RFX5, all of which are required for DNA binding and activation of MHCII gene expression. Static light scattering and chemical cross-linking of the three RFX proteins show that RFXB and RFXAP are monomers and that RFX5 dimerizes through two separate domains. One of these domains, the oligomerization domain, promotes formation of a dimer of dimers of RFX5. In addition, we show that the RFX complex forms a 2:1:1 complex of RFX5.RFXAP.RFXB, which can associate with a further dimer of RFX5 to form a 4:1:1 complex through the oligomerization domain of RFX5. On the basis of these studies, we propose DNA-binding models for the interaction between the RFX complex and the MHCII promoter including a DNA looping model. We also provide direct evidence that the RFX5(L66A) point mutation prevents dimerization of the RFX complexes and propose a model for how this results in a loss of MHCII gene expression.     

Spectroscopic and biological studies of a novel synthetic chlorin derivative with prospects for use in PDT

Photosensitizers with desirable combinations of chemical, photophysical and biological properties are essential for improving the efficacy of photodynamic therapy (PDT) against various cancers. Chlorins seem to be promising candidates for photodynamic therapy (PDT) owing to their photophysical properties. This paper reports spectroscopic and biological properties of a novel synthetic chlorin derivative. Cytotoxicity, phototoxicity as well as subcellular localization of the novel derivative was studied using Lewis lung carcinoma cultured cells (LLC). In the examined concentration range no significant cytotoxic effects were found but high phototoxicity was observed. Confocal laser scanning microscopy demonstrated that the compound, upon entering cells, was localized in the perinuclear cytoplasm of LLC cells. Using fluorescent microscopy we investigated the impact of PDT based on the novel compound upon cytoskeleton and DNA structure of LLC cells. Our results indicate that liposomes are effective in transferring the chlorin photosensitizer into the studied cells, leading to their high photosensitization, whereas the non-carrier delivery mode (i.e., DMSO) is rather useless for such purposes.     

Mitochondria and plasma membrane as targets of UVA-induced toxicity of neuroleptic drugs fluphenazine, perphenazine and thioridazine

In order to gain insights into the mechanism of phototoxicity of the neuroleptic drugs fluphenazine, perphenazine and thioridazine in cultured cells, studies were performed with murine 3T3 fibroblasts, aimed at identifying some cellular targets responsible for photoinduced cell death and possible cytotoxic reactive species involved in the photosensitization process. 3T3 fibroblasts incubated with 5 microM drugs and irradiated with UVA light (up to 8 J/cm2) underwent cell death, the extent of which depended on light dose. Of the three drugs, fluphenazine exhibited the highest phototoxicity and 100% cell death was achieved with a light dose of 5 J/cm2. Superoxide dismutase and alpha-tocopherol exerted a dose-dependent protective effect against drug phototoxicity, whereas N-acetylcysteine failed to do so. These findings indicate that superoxide anion and other free radical intermediates, generated in lipophilic cellular environments, play a role in photoinduced toxicity. Phototreatment of drug-loaded cells induces release of the cytosolic enzyme lactate dehydrogenase and causes loss of activity of mitochondrial NADH dehydrogenase, indicating that plasma membrane and mitochondria are among the targets of the phototoxicity of these drugs.