Aberrant activation of Wingless-type (Wnt)/β-catenin signaling is widespread in human cervical cancer. However, the underlying mechanisms of Wnt activation and the therapeutic potential of Wnt ...inhibition remain largely unknown. Here, we demonstrate that the Wnt inhibitory factor 1 (WIF1), a secreted Wnt antagonist, is downregulated in all human primary cervical tumors and cell lines analyzed. Our data reveal that WIF1 downregulation occurs due to promoter hypermethylation and is an early event in cervical oncogenesis. WIF1 re-expression upon 5-aza-2'-deoxycytidine treatment or WIF1 gene transfer induces significant apoptosis and G(2)/M arrest, and inhibits cervical cancer cell proliferation in vitro. Consistent with this, treatment of established mice tumor xenografts with peritumoral WIF1 gene transfer results in a significant inhibition of cancer growth and invasion. WIF1 treatment causes a significant decrease in intracellular WNT1 and TCF-4 proteins revealing novel Wnt-regulatory mechanisms. Thus, WIF1 causes a major cellular re-distribution of β-catenin and a significant inhibition of the Wnt/β-catenin pathway in tumor cells, as documented by a remarkable reversion in the expression of Wnt/β-catenin transcriptional target genes (E-cadherin, c-Myc, cyclin D1, CD44 and VEGF). Consequently, multiple critical events in tumor progression and metastasis such as cell proliferation, angiogenesis and invasion were inhibited by WIF1. In addition, WIF1 modulated the expression of specific anti-apoptotic and apoptotic proteins, thereby inducing significant apoptosis in vivo. Our findings demonstrate for the first time that WIF1 downregulation by epigenetic gene silencing is an important mechanism of Wnt activation in cervical oncogenesis. Of major clinical relevance, we show that peritumoral WIF1 gene transfer reduces not only cancer growth but also invasion in well-established tumors. Therefore, our data provide novel mechanistic insights into the role of WIF1 in cervical cancer progression, and the important preclinical validation of WIF1 as a potent drug target in cervical cancer treatment.
Abstract Objective This study aims to assess the role of polymorphisms in DNA repair genes, excision repair cross-complementation group 1 ( ERCC1 ) and methyl-methanesulfonate sensitivity 19 ( MMS19 ...), in tumor response to platinum-based chemotherapy and survival in advanced epithelial ovarian cancer (EOC). Methods Single nucleotide polymorphism (SNP) analysis was performed on the paraffin-embedded tumor tissue of women with advanced EOC, treated with platinum-based chemotherapy at the University of Oklahoma Health Sciences Center. Polymorphisms from two ERCC1 (codon-118 and C8092A) and three MMS19 (rs2211243, rs2236575 and rs872106) gene loci were evaluated by real time PCR Allelic Discrimination Assay. Results Genotyping was performed in 107 patients, 45 platinum-sensitive and 62 platinum-resistant. ERCC1 , codon-118 and C8092A genotyping was evaluable in 98 and 106 patients respectively and in all 107 patients for MMS19 polymorphisms. No differences were observed in genotype between platinum-sensitive and platinum-resistant patients. Polymorphisms in the ERCC1 , codon-118 and MMS19 genes did not correlate with overall survival (OS), although a trend toward improved progression free survival (PFS) was observed in patients expressing the minor (GG) alleles of the rs872106 MMS19 gene. Women homozygous for the ERCC1 -C8092A minor (AA) alleles had a significant increase in PFS compared to AC and CC patients and both AA and AC genotypes conferred improved survival over the major (CC) genotype. Conclusions Polymorphisms in ERCC1 , codon-118 and MMS19 genes are not associated with clinical response to platinum or survival. The ERCC1 -C8092A genotypes containing an “A” allele were associated with significant improvement in PFS and OS strengthening the value of this specific genotype in survival.
Altered signalling in B cells is a predominant feature of systemic lupus erythematosus (SLE). The genes BANK1 and BLK were recently described as associated with SLE. BANK1 codes for a B-cell-specific ...cytoplasmic protein involved in B-cell receptor signalling and BLK codes for an Src tyrosine kinase with important roles in B-cell development. To characterise the role of BANK1 and BLK in SLE, a genetic interaction analysis was performed hypothesising that genetic interactions could reveal functional pathways relevant to disease pathogenesis.
The GPAT16 method was used to analyse the gene-gene interactions of BANK1 and BLK. Confocal microscopy was used to investigate co-localisation, and immunoprecipitation was used to verify the physical interaction of BANK1 and BLK.
Epistatic interactions between BANK1 and BLK polymorphisms associated with SLE were observed in a discovery set of 279 patients and 515 controls from northern Europe. A meta-analysis with 4399 European individuals confirmed the genetic interactions between BANK1 and BLK. As BANK1 was identified as a binding partner of the Src tyrosine kinase LYN, the possibility that BANK1 and BLK could also show a protein-protein interaction was tested. The co-immunoprecipitation and co-localisation of BLK and BANK1 were demonstrated. In a Daudi cell line and primary naive B cells endogenous binding was enhanced upon B-cell receptor stimulation using anti-IgM antibodies.
This study shows a genetic interaction between BANK1 and BLK, and demonstrates that these molecules interact physically. The results have important consequences for the understanding of SLE and other autoimmune diseases and identify a potential new signalling pathway.
In transient expression assays for transcription, a vector carrying the experimental reporter is usually co-transfected with a second vector containing a distinct reporter gene as a control. The ...second reporter is linked to a constitutive promoter driving a low-level transcription that is unresponsive to the experimental trans-acting transcription factors used. The use of dual reporters enables the normalization of the experimental gene transcription with respect to the control reporter transcription. This method is expected to minimize the inherent variability in transfection data caused by changes in cell density and viability, cell lysis, and the recovery of samples at various stages of the experiment. Here, we report that one of the most widely used internal control reporters, the
luciferase plasmid (pRL-TK), is unresponsive to human tumor suppressor protein p53, a potent transcriptional regulator; however, the reporter transcription is enhanced by another well-known transcriptional regulator, the adenoviral 12S E1A oncoprotein, thereby disqualifying pRL-TK as a universal internal control reporter for transcription assays. Our results reveal the necessity of stringent experiments to test the responsiveness of internal control plasmids to transcriptional regulators present in the assay to avoid the misinterpretation of transcriptional analysis data.
We have previously identified massively expressed 24- and 20.5-kDa male-specific proteins in submandibular salivary glands (SMG) of adult hamsters. Here we report the cloning of the cDNA encoding the ...24-kDa protein which we have now found to be a heterogenously N-glycosylated form of the 20.5-kDa protein. The deduced amino acid sequence indicated that the protein is a member of the lipocalin family, the two most related lipocalins being rat odorant-binding protein of nasal mucosa and aphrodisin, a pheromonal protein present in vaginal discharge and saliva of female hamsters. Northern blot analysis showed that cognate mRNA is expressed in hamster SMG and lacrimal gland (LG) displaying marked sex-hormonal repression. The sex-hormonal repression patterns showed similarities and dissimilarities between SMG and LG but they were, respectively, similar to the sex-hormonal repression pattern noted for the SMG 24/20.5-kDa male-specific proteins and for an abundant female-specific 20-kDa LG secretory protein. These SMG and LG proteins were found to be immunologically similar and secretion of the SMG proteins in saliva and their excretion in urine was detected. The male-specific and abundant expression of the SMG proteins were seen at and after sexual maturity but was not dependent on androgens. Surprisingly, a temporary male-like expression of these SMG proteins was seen in lactating females which was obliterated by oestrogen administration. Our results show that despite differences in their repression by sex hormones, the gene for SMG 24/20.5-kDa proteins is similar or identical to that of LG 20-kDa protein and their marked repression by both androgens and oestrogens might be at the transcriptional level. Moreover, they might be excellent models with which to study sex hormone repression of gene expression at the molecular level. The results of homology search and the male- and lactation-specific pressure of the SMG proteins in adult saliva and urine suggests a possibility of their involvement in olfaction-mediated chemical communication between hamsters.
In transient expression assays for transcription, a vector carrying the experimental reporter is usually co-transfected with a second vector containing a distinct reporter gene as a control. The ...second reporter is linked to a constitutive promoter driving a low-level transcription that is unresponsive to the experimental trans-acting transcription factors used. The use of dual reporters enables the normalization of the experimental gene transcription with respect to the control reporter transcription. This method is expected to minimize the inherent variability in transfection data caused by changes in cell density and viability, cell lysis, and the recovery of samples at various stages of the experiment. Here, we report that one of the most widely used internal control reporters, the Renilla luciferase plasmid (pRL-TK), is unresponsive to human tumor suppressor protein p53, a potent transcriptional regulator; however, the reporter transcription is enhanced by another well-known transcriptional regulator, the adenoviral 125 EIA oncoprotein, thereby disqualifying pRL-TK as a universal internal control reporter for transcription assays. Our results reveal the necessity of stringent experiments to test the responsiveness of internal control plasmids to transcriptional regulators present in the assay to avoid the misinterpretation of transcriptional analysis data.
One of the major features of cancer is Otto Warburg's observation that many tumors have increased extracellular acidification compared to healthy tissues. Since Warburg's observation, the importance ...of extracellular acidification in cancer is now considered a hallmark of cancer. Human MAP3K4 functions upstream of the p38 and JNK mitogen activated protein kinases (MAPKs). Additionally, MAP3K4 is required for cell migration and extracellular acidification of breast cancer cells in response to HER2/HER3 signaling. Here, we demonstrate that GIT1 interacts with MAP3K4 by immunoprecipitation, while cellular lactate production and the capacity of MCF-7 cells for anchorage independent growth in soft agar were dependent on GIT1. Additionally, we show that activation of HER2/HER3 signaling leads to reduced expression of lactate receptor (GPR81) mRNA and that both, GIT1 and MAP3K4, are necessary for constitutive expression of GPR81 mRNA. Our study suggests that targeting downstream proteins in the HER2/HER3-induced extracellular lactate signaling pathway may be a way to inhibit the Warburg Effect to disrupt tumor growth.
Applications that range over various mists are frequently observed to be powerless against security dangers. Such profoundly heterogeneous situations need a fine-grained access control system like ...Attribute-based Access Control for upholding security. One of the most encouraging applications in distributed computing is online information sharing. Step by step instructions to share client information securely and productively has turned out to be one of the most testing issues in distributed computing. Be that as it may, this postures new provokes identified with making secure and solid information stockpiling over questionable specialist co-ops. The principle objective of distributed computing is the means by which to verify, secure the information and procedure. In this study, we find the issue of guaranteeing the respectability of data in cloud computing. Specifically, we consider techniques for decreasing the weight of producing a consistent measure of metadata at the customer side. Ascribe division chooses whether to momentarily disavow client approval as indicated by property subset. Furthermore, we propose another model called Policy-based Attribute Based Access Control (Pa-ABAC) utilizing half breed property based encryption. Our goal is to officially determine the conduct of various components of the proposed model in a cloud domain. This is imperative to build up a substantial security arrangement for a cloud situation free from determination mistakes and irregularities.
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