SerpinB10, a Serine Protease Inhibitor, Is Implicated in UV-Induced Cellular Response.

UV-induced DNA damage response and repair are extensively studied processes, as any malfunction in these pathways contributes to the activation of tumorigenesis. Although several proteins involved in these cellular mechanisms have been described, the entire repair cascade has remained unexplored. To identify new players in UV-induced repair, we performed a microarray screen, in which we found SerpinB10 (SPB10, Bomapin) as one of the most dramatically upregulated genes following UV irradiation. Here, we demonstrated that an increased mRNA level of SPB10 is a general cellular response following UV irradiation regardless of the cell type. We showed that although SPB10 is implicated in the UV-induced cellular response, it has no indispensable function in cell survival upon UV irradiation. Nonetheless, we revealed that SPB10 might be involved in delaying the duration of DNA repair in interphase and also in S-phase cells. Additionally, we also highlighted the interaction between SPB10 and H3. Based on our results, it seems that SPB10 protein is implicated in UV-induced stress as a "quality control protein", presumably by slowing down the repair process.

SerpinB2 is involved in cellular response upon UV irradiation.

Ultraviolet light induced pyrimidine dimer is a helix distortion DNA damage type, which recruits repair complexes. However, proteins of these complexes that take part in both DNA damage recognition and repair have been well-described, the regulation of the downstream steps of nucleotide excision repair (NER) have not been clearly clarified yet. In a high-throughput screen, we identified SerpinB2 (SPB2) as one of the most dramatically upregulated gene in keratinocytes following UV irradiation. We found that both the mRNA and the protein levels of SPB2 were increased upon UV irradiation in various cell lines. Additionally, UV damage induced translocation of SPB2 from the cytoplasm to the nucleus as well as the damage induced foci formation of it. Here we show that SPB2 co-localizes with XPB involved in the NER pathway at UV-induced repair foci. Finally, we demonstrated that UV irradiation promoted the association of SPB2 with ubiquitylated proteins. In basal cell carcinoma tumour cells, we identified changes in the subcellular localization of SPB2. Based on our results, we conclude that SPB2 protein has a novel role in UV-induced NER pathway, since it regulates the removal of the repair complex from the damaged site leading to cancerous malformation.

Endoplasmic reticulum stress: major player in size-dependent inhibition of P-glycoprotein by silver nanoparticles in multidrug-resistant breast cancer cells.

BACKGROUND: Development of multidrug resistance (MDR) is a major burden of successful chemotherapy, therefore, novel approaches to defeat MDR are imperative. Although the remarkable anti-cancer propensity of silver nanoparticles (AgNP) has been demonstrated and their potential application in MDR cancer has been proposed, the nanoparticle size-dependent cellular events directing P-glycoprotein (Pgp) expression and activity in MDR cancer have never been addressed. Hence, in the present study we examined AgNP size-dependent cellular features in multidrug resistant breast cancer cells. RESULTS: In this study we report that 75 nm AgNPs inhibited significantly Pgp efflux activity in drug-resistant breast cancer cells and potentiated the apoptotic effect of doxorubicin, which features were not observed upon 5 nm AgNP treatment. Although both sized AgNPs induced significant ROS production and mitochondrial damage, 5 nm AgNPs were more potent than 75 nm AgNPs in this respect, therefore, these effects can not to be accounted for the reduced transport activity of ATP-driven pumps observed after 75 nm AgNP treatments. Instead we found that 75 nm AgNPs depleted endoplasmic reticulum (ER) calcium stores, caused notable ER stress and decreased plasma membrane positioning of Pgp. CONCLUSION: Our study suggests that AgNPs are potent inhibitors of Pgp function and are promising agents for sensitizing multidrug resistant breast cancers to anticancer drugs. This potency is determined by their size, since 75 nm AgNPs are more efficient than smaller counterparts. This is a highly relevant finding as it renders AgNPs attractive candidates in rational design of therapeutically useful agents for tumor targeting. In the present study we provide evidence that exploitation of ER stress can be a propitious target in defeating multidrug resistance in cancers.

Genetic, epigenetic and transcriptional comparison of esophagus tumor­associated and adjacent normal myofibroblasts.

Myofibroblasts (MFs) are present in healthy tissues and are also key components of the tumor microenvironment. In the present study a comparative analysis of MFs obtained from various gastrointestinal tumor tissues and from tumor­adjacent normal tissues of cancer patients was performed, with the aim to evaluate differences in MF morphology, gene expression profile and function. The goal was to correlate the observed morphological and functional variations with the underlying genetic and epigenetic backgrounds. The mutation frequency of MFs was assessed by next generation sequencing. The transcript levels of cancer­specific genes were determined by TaqMan array and quantitative polymerase chain reaction. Epigenetic modifications were analyzed by immunocytochemistry and western blotting. The migratory capacity of MFs was assessed by scratch assay, whereas matrix metalloproteinase expression and activity were obtained by quantitative polymerase chain reaction and zymography. The results of the present study demonstrate that MFs were present in an increased number and with altered morphology in tumor samples compared with the healthy tissue. Although the detected number of mutations in tumor­associated and normal tissue­derived MFs did not differ markedly, shifts in the level of specific acetylated and methylated histone proteins, namely decreased levels of trimethylated H3K9 and acetylated H4K16 were demonstrated in tumor­associated MFs. Transcript levels of several tumor­specific genes involved in metastasis, regulation of cellular growth, apoptosis, as well as in hypoxia­angiogenesis were altered in tumor­derived MF cultures. Increased mRNA levels were obtained and activity of matrix metalloproteases in tumor­derived MFs and these cells also exhibited a higher migratory capacity compared with the normal MFs. In summary, the results of the present study indicate that tumor­associated MFs display an altered phenotype compared with healthy tissue derived counterparts. The results imply that epigenetic rather than genetic alterations are associated with the development of the distinct expressional and functional features, which define this MF phenotype in the tumor microenvironment.

Biosynthesized silver and gold nanoparticles are potent antimycotics against opportunistic pathogenic yeasts and dermatophytes.

BACKGROUND: Epidemiologic observations indicate that the number of systemic fungal infections has increased significantly during the past decades, however in human mycosis, mainly cutaneous infections predominate, generating major public health concerns and providing much of the impetus for current attempts to develop novel and efficient agents against cutaneous mycosis causing species. Innovative, environmentally benign and economic nanotechnology-based approaches have recently emerged utilizing principally biological sources to produce nano-sized structures with unique antimicrobial properties. In line with this, our aim was to generate silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs) by biological synthesis and to study the effect of the obtained nanoparticles on cutaneous mycosis causing fungi and on human keratinocytes. METHODS: Cell-free extract of the red yeast Phaffia rhodozyma proved to be suitable for nanoparticle preparation and the generated AgNPs and AuNPs were characterized by transmission electron microscopy, dynamic light scattering and X-ray powder diffraction. RESULTS: Antifungal studies demonstrated that the biosynthesized silver particles were able to inhibit the growth of several opportunistic Candida or Cryptococcus species and were highly potent against filamentous Microsporum and Trichophyton dermatophytes. Among the tested species only Cryptococcus neoformans was susceptible to both AgNPs and AuNPs. Neither AgNPs nor AuNPs exerted toxicity on human keratinocytes. CONCLUSION: Our results emphasize the therapeutic potential of such biosynthesized nanoparticles, since their biocompatibility to skin cells and their outstanding antifungal performance can be exploited for topical treatment and prophylaxis of superficial cutaneous mycosis.

Biological activity of green-synthesized silver nanoparticles depends on the applied natural extracts: a comprehensive study.

Due to obvious disadvantages of the classical chemical methods, green synthesis of metallic nanoparticles has attracted tremendous attention in recent years. Numerous environmentally benign synthesis methods have been developed yielding nanoparticles via low-cost, eco-friendly, and simple approaches. In this study, our aim was to determine the suitability of coffee and green tea extracts in green synthesis of silver nanoparticles as well as to compare the performance of the obtained materials in different biological systems. We successfully produced silver nanoparticles (C-AgNP and GT-AgNP) using coffee and green tea extracts; moreover, based on our comprehensive screening, we delineated major differences in the biological activity of C-AgNPs and GT-AgNPs. Our results indicate that although GT-AgNPs exhibited excellent antimicrobial activity against all the examined microbial pathogens, these particles were also highly toxic to mammalian cells, which limits their potential applications. On the contrary, C-AgNPs manifested substantial inhibitory action on the tested microbes but were nontoxic to human and mouse cells, indicating an outstanding capacity to discriminate between potential pathogens and mammalian cells. These results clearly show that the various green materials used for stabilization and for reduction of metal ions have a defining role in determining and fine-tuning the biological activity of the obtained nanoparticles.

Ecdysone induced gene expression is associated with acetylation of histone H3 lysine 23 in Drosophila melanogaster.

Posttranslational modification of histones regulates transcription but the exact role that acetylation of specific lysine residues plays in biological processes in vivo is still not clearly understood. To assess the contribution of different histone modifications to transcriptional activation in vivo, we determined the acetylation patterns on the ecdysone induced Eip74EF and Eip75B genes in Drosophila melanogaster larvae by chromatin immunoprecipitation. We found that acetylation of histone H3 lysine 23 is localized to promoters and correlates with endogenous ecdysone induced gene activation. In contrast, acetylation of lysines 8, 12 and 16 of histone H4 and lysine 9 of histone H3 showed minor differences in their distribution on the regulatory and transcribed regions tested, and had limited or no correlation with ecdysone induced transcriptional activity. We found that dCBP, which is encoded by the nejire gene, acetylates H3 lysine 23 in vivo, and silencing of nejire leads to reduced expression of the Eip74EF and Eip75B genes. Our results suggest that acetylation of specific lysine residues of histones contribute specifically to the dynamic regulation of transcription. Furthermore, along with previous studies identify CBP dependent H3 lysine 23 acetylation as an evolutionarily conserved chromatin modification involved in steroid induced gene activation.

The dissociable RPB4 subunit of RNA Pol II has vital functions in Drosophila.

RNA polymerase II (Pol II) is composed of a ten subunit core and a two subunit dissociable subcomplex comprising the fourth and seventh largest subunits, RPB4 and RPB7. The evolutionary highly conserved RPB4/7 heterodimer is positioned in the Pol II such that it can make contact with various factors involved in RNA biogenesis and is believed to play roles both during the process of transcription and post-transcription. A detailed analysis of RPB4/7 function in a multicellular eukaryote, however, is lacking partly because of the lack of a suitable genetic system. Here, we describe generation and initial analysis of Drosophila Rpb4 mutants. In the fly, RPB4 is a product of a bicistronic gene together with the ATAC histone acetyltransferase complex constituent ADA2a. DmAda2a and DmRpb4 are expressed during fly development at different levels. The structure of mature mRNA forms suggests that the production of DmADA2a and DmRPB4-specific mRNAs is ensured by alternative splicing. Genetic analysis indicates that both DmRPB4 and DmADA2a play essential roles, because their absence results in lethality in early and late larval stages, respectively. Upon stress of high temperature or nutritional starvation, the levels of RPB4 and ADA2a messages change differently. RPB4 colocalizes with Pol II to several sites on polytene chromosomes, however, at selected locus, the abundances of Pol II and RPB4 vary greatly. Our data suggest no tight functional link between DmADA2a and DmRPB4, and reveal differences in the abundances of Pol II core subunits and RPB4 localized at specific regions on polytene chromosomes, supporting the suggested role of RPB4 outside of transcription-engaged Pol II complexes.