Molecular evolution of epididymal lipocalin genes localized on mouse chromosome 2

Producción CientíficaWe previously identified two murine secretory proteins, mE-RABP(Lcn5) and mEP17(Lcn8), belonging to the lipocalin family and specifically expressed in the epididymis. The genes are contiguous and localized on mouse chromosome 2. We now show that five other related lipocalin genes, Lcn9, Lcn10, Lcn11, Lcn12, and Lcn13, that evolved by in situ tandem duplication are present on the same locus. Lcn9, Lcn10, Lcn12, and Lcn13 genes, like Lcn5 and Lcn8 genes, are specifically expressed in the mouse epididymis. However, each gene has a distinct spatial expression within the epididymis and different regulation. Analysis of the human genome sequence shows the presence of genes encoding lipocalins with genomic organization, chromosomal arrangement, and orientation similar to that of the corresponding murine genes, indicating that the epididymal cluster is evolutionary conserved. A phylogenetic analysis of the new epididymal proteins reveals their spread position in the lipocalin protein family tree. This suggests the preservation of the regulatory sequences, while protein sequences have greatly diverged, reflecting functional diversity and possibly multifunctionality. In terms of the cluster ancestry, epididymal expression possibly appeared in a PGDS-like lipocalin in amniotes, and the duplications generating the cluster occurred at least in the common ancestor of rodents and primates. The presence and conservation of a cluster of five genes encoding epididymal lipocalins, differently regulated and regionalized in the epididymis, strongly suggests that these proteins may play an important role for male fertility

Human homolog of Drosophila Hairy and enhancer of split 1, Hes1, negatively regulates δ-catenin (CTNND2) expression in cooperation with E2F1 in prostate cancer

<p>Abstract</p> <p>Background</p> <p>Neuronal synaptic junction protein δ-catenin (<it>CTNND2</it>) is often overexpressed in prostatic adenocarcinomas but the mechanisms of its activation are unknown. To address this question, we studied the hypothesis that Hes1, human homolog of <it>Drosophila </it>Hairy and enhancer of split (Hes) 1, is a transcriptional repressor of δ-catenin expression and plays an important role in molecular carcinogenesis.</p> <p>Results</p> <p>We identified that, using a <it>δ-catenin </it>promoter reporter assay, Hes1, but not its inactive mutant, significantly repressed the upregulation of δ-catenin-luciferase activities induced by E2F1. Hes1 binds directly to the E-boxes on <it>δ-catenin </it>promoter and can reduce the expression of δ-catenin in prostate cancer cells. In prostate cancer CWR22-Rv1 and PC3 cell lines, which showed distinct δ-catenin overexpression, E2F1 and Hes1 expression pattern was altered. The suppression of Hes1 expression, either by γ-secretase inhibitors or by siRNA against Hes1, increased δ-catenin expression. γ-Secretase inhibition delayed S/G2-phase transition during cell cycle progression and induced cell shape changes to extend cellular processes in prostate cancer cells. In neuroendocrine prostate cancer mouse model derived allograft NE-10 tumors, δ-catenin showed an increased expression while Hes1 expression was diminished. Furthermore, <it>E2F1 </it>transcription was very high in subgroup of NE-10 tumors in which <it>Hes1 </it>still displayed residual expression, while its expression was only moderately increased in NE-10 tumors where Hes1 expression was completely suppressed.</p> <p>Conclusion</p> <p>These studies support coordinated regulation of δ-catenin expression by both the activating transcription factor E2F1 and repressive transcription factor Hes1 in prostate cancer progression.</p

Molecular cloning and hormonal regulation of a murine epididymal retinoic acid-binding protein messenger ribonucleic acid

A complementary DNA encoding the mouse epididymal secretory protein MEP 10 (mouse epididymal protein 10) was cloned and is now renamed murine epididymal retinoic acid binding protein (mE-RABP). The analysis of the predicted primary amino acid sequence showed that mE-RABP has a 75% identity with rat ESP I (epididymal secretory protein I), another epididymal retinoic acid-binding protein. The homology strongly suggests that mE-RABP is the mouse orthologue of rat ESP I. A computer analysis of the predicted three- dimensional structure confirmed that mE-RABP can accommodate retinoic acid as ligand. In the rat, ESP I messenger RNA (mRNA) is expressed in the efferent ducts and in the entire caput epididymidis. However, in the mouse, the expression of a 950-bp mE-RABP mRNA was detected only in principal cells of the mid/distal caput epididymidis, suggesting that the regulation of region- specific expression is different in rat and mouse. Northern blot analyses showed that mE-RABP gene expression is no longer detected 10 days after castration but progressively rebounds between days 15 and 60. However, mE- RABP protein could not be detected by Western blot 30 days after castration. Androgen replacement, begun 5 days after castration and continued for 4 days restored significant expression of mE-RABP mRNA. Efferent duct ligation for 10 days did not affect gene expression. Taken together, these results indicate that mE-RABP mRNA expression is regulated by androgens but not by testicular factors. The overall similarity in the primary amino acid sequence of mE-RABP with ESP I and other members of the lipocalin superfamily suggests that they are evolutionarily related

KDM5B Is Essential for the Hyperactivation of PI3K/AKT Signaling in Prostate Tumorigenesis

KDM5B (lysine[K]-specific demethylase 5B) is frequently upregulated in various human cancers including prostate cancer. KDM5B controls H3K4me3/2 levels and regulates gene transcription and cell differentiation, yet the contributions of KDM5B to prostate cancer tumorigenesis remain unknown. In this study, we investigated the functional role of KDM5B in epigenetic dysregulation and prostate cancer progression in cultured cells and in mouse models of prostate epithelium–specific mutant Pten/Kdm5b. Kdm5b deficiency resulted in a significant delay in the onset of prostate cancer in Pten-null mice, whereas Kdm5b loss alone caused no morphologic abnormalities in mouse prostates. At 6 months of age, the prostate weight of Pten/Kdm5b mice was reduced by up to 70% compared with that of Pten mice. Pathologic analysis revealed Pten/Kdm5b mice displayed mild morphologic changes with hyperplasia in prostates, whereas age-matched Pten littermates developed high-grade prostatic intraepithelial neoplasia and prostate cancer. Mechanistically, KDM5B governed PI3K/AKT signaling in prostate cancer in vitro and in vivo. KDM5B directly bound the PIK3CA promoter, and KDM5B knockout resulted in a significant reduction of P110α and PIP3 levels and subsequent decrease in proliferation of human prostate cancer cells. Conversely, KDM5B overexpression resulted in increased PI3K/AKT signaling. Loss of Kdm5b abrogated the hyperactivation of AKT signaling by decreasing P110α/P85 levels in Pten/Kdm5b mice. Taken together, our findings reveal that KDM5B acts as a key regulator of PI3K/AKT signaling; they also support the concept that targeting KDM5B is a novel and effective therapeutic strategy against prostate cancer

Measurement and comparison of individual external doses of high-school students living in Japan, France, Poland and Belarus -- the "D-shuttle" project --

Twelve high schools in Japan (of which six are in Fukushima Prefecture), four in France, eight in Poland and two in Belarus cooperated in the measurement and comparison of individual external doses in 2014. In total 216 high-school students and teachers participated in the study. Each participant wore an electronic personal dosimeter "D-shuttle" for two weeks, and kept a journal of his/her whereabouts and activities. The distributions of annual external doses estimated for each region overlap with each other, demonstrating that the personal external individual doses in locations where residence is currently allowed in Fukushima Prefecture and in Belarus are well within the range of estimated annual doses due to the background radiation level of other regions/countries

ROCK Inhibitor Y-27632 Suppresses Dissociation-Induced Apoptosis of Murine Prostate Stem/Progenitor Cells and Increases Their Cloning Efficiency

Activation of the RhoA/ROCK signaling pathway has been shown to contribute to dissociation-induced apoptosis of embryonic and neural stem cells. We previously demonstrated that approximately 1 out of 40 Lin−Sca-1+CD49fhigh (LSC) prostate basal epithelial cells possess the capacities of stem cells for self-renewal and multi-lineage differentiation. We show here that treating LSC cells with the ROCK kinase inhibitor Y-27632 increases their cloning efficiency by 8 fold in an in vitro prostate colony assay. Y-27632 treatment allows prostate colony cells to replate efficiently, which does not occur otherwise. Y-27632 also increases the cloning efficiency of prostate stem cells in a prostate sphere assay and a dissociated prostate cell regeneration assay. The increased cloning efficiency is due to the suppression of the dissociation-induced, RhoA/ROCK activation-mediated apoptosis of prostate stem cells. Dissociation of prostate epithelial cells from extracellular matrix increases PTEN activity and attenuates AKT activity. Y-27632 treatment alone is sufficient to suppress cell dissociation-induced activation of PTEN activity. However, this does not contribute to the increased cloning efficiency, because Y-27632 treatment increases the sphere-forming unit of wild type and Pten null prostate cells to a similar extent. Finally, knocking down expression of both ROCK kinases slightly increases the replating efficiency of prostate colony cells, corroborating that they play a major role in the Y-27632 mediated increase in cloning efficiency. Our study implies that the numbers of prostate cells with stem/progenitor activity may be underestimated based on currently employed assays, supports that dissociation-induced apoptosis is a common feature of embryonic and somatic stem cells with an epithelial phenotype, and highlights the significance of environmental cues for the maintenance of stem cells

Loss of the Urothelial Differentiation Marker FOXA1 Is Associated with High Grade, Late Stage Bladder Cancer and Increased Tumor Proliferation

Approximately 50% of patients with muscle-invasive bladder cancer (MIBC) develop metastatic disease, which is almost invariably lethal. However, our understanding of pathways that drive aggressive behavior of MIBC is incomplete. Members of the FOXA subfamily of transcription factors are implicated in normal urogenital development and urologic malignancies. FOXA proteins are implicated in normal urothelial differentiation, but their role in bladder cancer is unknown. We examined FOXA expression in commonly used in vitro models of bladder cancer and in human bladder cancer specimens, and used a novel in vivo tissue recombination system to determine the functional significance of FOXA1 expression in bladder cancer. Logistic regression analysis showed decreased FOXA1 expression is associated with increasing tumor stage (p<0.001), and loss of FOXA1 is associated with high histologic grade (p<0.001). Also, we found that bladder urothelium that has undergone keratinizing squamous metaplasia, a precursor to the development of squamous cell carcinoma (SCC) exhibited loss of FOXA1 expression. Furthermore, 81% of cases of SCC of the bladder were negative for FOXA1 staining compared to only 40% of urothelial cell carcinomas. In addition, we showed that a subpopulation of FOXA1 negative urothelial tumor cells are highly proliferative. Knockdown of FOXA1 in RT4 bladder cancer cells resulted in increased expression of UPK1B, UPK2, UPK3A, and UPK3B, decreased E-cadherin expression and significantly increased cell proliferation, while overexpression of FOXA1 in T24 cells increased E-cadherin expression and significantly decreased cell growth and invasion. In vivo recombination of bladder cancer cells engineered to exhibit reduced FOXA1 expression with embryonic rat bladder mesenchyme and subsequent renal capsule engraftment resulted in enhanced tumor proliferation. These findings provide the first evidence linking loss of FOXA1 expression with histological subtypes of MIBC and urothelial cell proliferation, and suggest an important role for FOXA1 in the malignant phenotype of MIBC

Tissue-Specific Transcriptional Targeting of a Replication-Competent Retroviral Vector

The inability of replication-defective viral vectors to efficiently transduce tumor cells in vivo has prevented the successful application of such vectors in gene therapy of cancer. To address the need for more efficient gene delivery systems, we have developed replication-competent retroviral (RCR) vectors based on murine leukemia virus (MLV). We have previously shown that such vectors are capable of transducing solid tumors in vivo with very high efficiency. While the natural requirement of MLV infection for cell division imparts a certain degree of specificity for tumor cells, additional means for confining RCR vector replication to tumor cells are desirable. Here, we investigated the parameters critical for successful tissue-specific transcriptional control of RCR vector replication by replacing various lengths of the MLV enhancer/promoter with sequences derived either from the highly prostate-specific probasin (PB) promoter or from a more potent synthetic variant of the PB promoter. We assessed the transcriptional specificity of the resulting hybrid long terminal repeats (LTRs) and the cell type specificity and efficiency of replication of vectors containing these LTRs. Incorporation of PB promoter sequences effectively restricted transcription from the LTR to prostate-derived cells and imparted prostate-specific RCR vector replication but required the stronger synthetic promoter and retention of native MLV sequences in the vicinity of the TATA box for optimal replicative efficiency and specificity. Our results have thus identified promoter strength and positioning within the LTR as important determinants for achieving both high transduction efficiency and strict cell type specificity in transcriptionally targeted RCR vectors

Candidate metastasis suppressor genes uncovered by array comparative genomic hybridization in a mouse allograft model of prostate cancer

Background: The purpose of this study was to identify candidate metastasis suppressor genes from a mouse allograft model of prostate cancer (NE-10). This allograft model originally developed metastases by twelve weeks after implantation in male athymic nude mice, but lost the ability to metastasize after a number of in vivo passages. We performed high resolution array comparative genomic hybridization on the metastasizing and non-metastasizing allografts to identify chromosome imbalances that differed between the two groups of tumors. Results: This analysis uncovered a deletion on chromosome 2 that differed between the metastasizing and non-metastasizing tumors. Bioinformatics filters were employed to mine this region of the genome for candidate metastasis suppressor genes. Of the 146 known genes that reside within the region of interest on mouse chromosome 2, four candidate metastasis suppressor genes (Slc27a2, Mall, Snrpb, and Rassf2) were identified. Quantitative expression analysis confirmed decreased expression of these genes in the metastasizing compared to non-metastasizing tumors. Conclusion: This study presents combined genomics and bioinformatics approaches for identifying potential metastasis suppressor genes. The genes identified here are candidates for further studies to determine their functional role in inhibiting metastases in the NE-10 allograft model and human prostate cancer.Medicine, Faculty ofUrologic Sciences, Department ofOther UBCNon UBCReviewedFacult