ΔNp63 transcriptionally regulates ATM to control p53 Serine-15 phosphorylation

Background: Delta Np63 alpha is an epithelial progenitor cell marker that maintains epidermal stem cell self-renewal capacity. Previous studies revealed that UV-damage induced p53 phosphorylation is confined to Delta Np63 alpha-positive cells in the basal layer of human epithelium. Results: We now report that phosphorylation of the p53 tumour suppressor is positively regulated by Delta Np63 alpha in immortalised human keratinocytes. Delta Np63 alpha depletion by RNAi reduces steady-state ATM mRNA and protein levels, and attenuates p53 Serine-15 phosphorylation. Conversely, ectopic expression of Delta Np63 alpha in p63-null tumour cells stimulates ATM transcription and p53 Serine-15 phosphorylation. We show that ATM is a direct Delta Np63 alpha transcriptional target and that the Delta Np63 alpha response element localizes to the ATM promoter CCAAT sequence. Structure-function analysis revealed that the Delta Np63-specific TA2 transactivation domain mediates ATM transcription in coordination with the DNA binding and SAM domains. Conclusions: Germline p63 point mutations are associated with a range of ectodermal developmental disorders, and targeted p63 deletion in the skin causes premature ageing. The Delta Np63 alpha-ATM-p53 damage-response pathway may therefore function in epithelial development, carcinogenesis and the ageing processes

Host Factors Required for Modulation of Phagosome Biogenesis and Proliferation of Francisella tularensis within the Cytosol

Francisella tularensis is a highly infectious facultative intracellular bacterium that can be transmitted between mammals by arthropod vectors. Similar to many other intracellular bacteria that replicate within the cytosol, such as Listeria, Shigella, Burkholderia, and Rickettsia, the virulence of F. tularensis depends on its ability to modulate biogenesis of its phagosome and to escape into the host cell cytosol where it proliferates. Recent studies have identified the F. tularensis genes required for modulation of phagosome biogenesis and escape into the host cell cytosol within human and arthropod-derived cells. However, the arthropod and mammalian host factors required for intracellular proliferation of F. tularensis are not known. We have utilized a forward genetic approach employing genome-wide RNAi screen in Drosophila melanogaster-derived cells. Screening a library of ∼21,300 RNAi, we have identified at least 186 host factors required for intracellular bacterial proliferation. We silenced twelve mammalian homologues by RNAi in HEK293T cells and identified three conserved factors, the PI4 kinase PI4KCA, the ubiquitin hydrolase USP22, and the ubiquitin ligase CDC27, which are also required for replication in human cells. The PI4KCA and USP22 mammalian factors are not required for modulation of phagosome biogenesis or phagosomal escape but are required for proliferation within the cytosol. In contrast, the CDC27 ubiquitin ligase is required for evading lysosomal fusion and for phagosomal escape into the cytosol. Although F. tularensis interacts with the autophagy pathway during late stages of proliferation in mouse macrophages, this does not occur in human cells. Our data suggest that F. tularensis utilizes host ubiquitin turnover in distinct mechanisms during the phagosomal and cytosolic phases and phosphoinositide metabolism is essential for cytosolic proliferation of F. tularensis. Our data will facilitate deciphering molecular ecology, patho-adaptation of F. tularensis to the arthropod vector and its role in bacterial ecology and patho-evolution to infect mammals

Evasion of IFN-γ Signaling by Francisella novicida Is Dependent upon Francisella Outer Membrane Protein C

Francisella tularensis is a Gram-negative facultative intracellular bacterium and the causative agent of the lethal disease tularemia. An outer membrane protein (FTT0918) of F. tularensis subsp. tularensis has been identified as a virulence factor. We generated a F. novicida (F. tularensis subsp. novicida) FTN_0444 (homolog of FTT0918) fopC mutant to study the virulence-associated mechanism(s) of FTT0918.The ΔfopC strain phenotype was characterized using immunological and biochemical assays. Attenuated virulence via the pulmonary route in wildtype C57BL/6 and BALB/c mice, as well as in knockout (KO) mice, including MHC I, MHC II, and µmT (B cell deficient), but not in IFN-γ or IFN-γR KO mice was observed. Primary bone marrow derived macrophages (BMDM) prepared from C57BL/6 mice treated with rIFN-γ exhibited greater inhibition of intracellular ΔfopC than wildtype U112 strain replication; whereas, IFN-γR KO macrophages showed no IFN-γ-dependent inhibition of ΔfopC replication. Moreover, phosphorylation of STAT1 was downregulated by the wildtype strain, but not the fopC mutant, in rIFN-γ treated macrophages. Addition of NG-monomethyl-L-arginine, an NOS inhibitor, led to an increase of ΔfopC replication to that seen in the BMDM unstimulated with rIFN-γ. Enzymatic screening of ΔfopC revealed aberrant acid phosphatase activity and localization. Furthermore, a greater abundance of different proteins in the culture supernatants of ΔfopC than that in the wildtype U112 strain was observed.F. novicida FopC protein facilitates evasion of IFN-γ-mediated immune defense(s) by down-regulation of STAT1 phosphorylation and nitric oxide production, thereby promoting virulence. Additionally, the FopC protein also may play a role in maintaining outer membrane stability (integrity) facilitating the activity and localization of acid phosphatases and other F. novicida cell components

Anterior gradient protein 3 is associated with less aggressive tumors and better outcome of breast cancer patients

Joanna Obacz,1 Veronika Brychtova,1 Jan Podhorec,1 Pavel Fabian,2 Petr Dobes,1 Borivoj Vojtesek,1 Roman Hrstka1 1Regional Centre for Applied Molecular Oncology (RECAMO), 2Department of Pathology, Masaryk Memorial Cancer Institute, Brno, Czech Republic Abstract: Anterior gradient protein (AGR) 3 is a highly related homologue of pro-oncogenic AGR2 and belongs to the family of protein disulfide isomerases. Although AGR3 was found in breast, ovary, prostate, and liver cancer, it remains of yet poorly defined function in tumo-rigenesis. This study aimed to determine AGR3 expression in a cohort of 129 primary breast carcinomas and evaluate the clinical and prognostic significance of AGR3 in these tumors. The immunohistochemical analysis revealed the presence of AGR3 staining to varying degrees in 80% of analyzed specimens. The percentage of AGR3-positive cells significantly correlated with estrogen receptor, progesterone receptor (both P<0.0001) as well as low histological grade (P=0.003), and inversely correlated with the level of Ki-67 expression (P<0.0001). In the whole cohort, AGR3 expression was associated with longer progression-free survival (PFS), whereas AGR3-positive subgroup of low-histological grade tumors showed both significantly longer PFS and overall survival. In conclusion, AGR3 is associated with the level of differentiation, slowly proliferating tumors, and more favorable prognosis of breast cancer patients. Keywords: AGR3, patient survival, protein disulfide isomerase, ER-positive breast cancer, immuno­histochemistr

Significant overexpression of Hsp110 gene during colorectal cancer progression

Colorectal cancer (CRC) is one of the most frequent malignant diseases in the world. Metastatic spread of the cancer to the lymph nodes is a crucial factor for progression and therapeutic management of the disease. We analysed gene expression profiles of CRC patiens by low-density cancer-focused oligonucleotide microarrays to identify new predictive markers of the extent of the disease and for better understanding of CRC progression. Relative expression levels of 440 genes known to be involved in cancer biology were obtained by low-density oligonucleotide microarrays from 20 tumor samples. Statistical analysis of gene expression data identified 3 genes (HSP110, HYOU1 and TCTP) significantly up-regulated in primary tumors of patients who developed lymph node metastasis. We have shown, for the first time, that up-regulation HSP110 and HYOU1 expression is associated with lymph node involvement in CRC. We validated the differences in HSP110 expression in an independent group of 30 patients of all clinical stages by real-time PCR. We identified significant up-regulation of HSP110 expression in colorectal tumors compared to adjacent non-tumoral tissue (p<0.0003). We observed significant differences of HSP110 gene expression between metastatic and localized disease (p=0.031) and negative trend of HSP110 gene expression and overall survival of CRC patients. We suggest that HSP110 gene is a promising molecular predictor in CRC