Differential cytotoxic activity of a novel palladium-based compound on prostate cell lines, primary prostate epithelial cells and prostate stem cells

The outcome for patients with advanced metastatic and recurrent prostate cancer is still poor. Therefore, new chemotherapeutics are required, especially for killing cancer stem cells that are thought to be responsible for disease recurrence. In this study, we screened the effect of a novel palladium-based anticancer agent (Pd complex) against six different prostate cancer cell lines, and primary cultures from seven Gleason 6/7 prostate cancer, three Gleason 8/9 prostate cancer and four benign prostate hyperplasia patient samples, as well as cancer stem cells selected from primary cultures. MTT and ATP viability assays were used to assess cell growth and flow cytometry to assess cell cycle status. In addition, immunofluorescence was used to detect γH2AX nuclear foci, indicative of DNA damage, and Western blotting to assess the induction of apoptosis and autophagy. The Pd complex showed a powerful growth-inhibitory effect against both cell lines and primary cultures. More importantly, it successfully reduced the viability of cancer stem cells as first reported in this study. The Pd complex induced DNA damage and differentially induced evidence of cell death, as well as autophagy. In conclusion, this novel agent may be promising for use against the bulk of the tumour cell population as well as the prostate cancer stem cells, which are thought to be responsible for the resistance of metastatic prostate cancer to chemotherapy. This study also indicates that the combined use of the Pd complex with an autophagy modulator may be a more promising approach to treat prostate cancer. In addition, the differential effects observed between cell lines and primary cells emphasise the importance of the model used to test novel drugs including its genetic background, and indeed the necessity of using cells cultured from patient samples

Regulation of the stem cell marker CD133 is independent of promoter hypermethylation in human epithelial differentiation and cancer

BackgroundEpigenetic control is essential for maintenance of tissue hierarchy and correct differentiation. In cancer, this hierarchical structure is altered and epigenetic control deregulated, but the relationship between these two phenomena is still unclear. CD133 is a marker for adult stem cells in various tissues and tumour types. Stem cell specificity is maintained by tight regulation of CD133 expression at both transcriptional and post-translational levels. In this study we investigated the role of epigenetic regulation of CD133 in epithelial differentiation and cancer.MethodsDNA methylation analysis of the CD133 promoter was done by pyrosequencing and methylation specific PCR; qRT-PCR was used to measure CD133 expression and chromatin structure was determined by ChIP. Cells were treated with DNA demethylating agents and HDAC inhibitors. All the experiments were carried out in both cell lines and primary samples.ResultsWe found that CD133 expression is repressed by DNA methylation in the majority of prostate epithelial cell lines examined, where the promoter is heavily CpG hypermethylated, whereas in primary prostate cancer and benign prostatic hyperplasia, low levels of DNA methylation, accompanied by low levels of mRNA, were found. Moreover, differential methylation of CD133 was absent from both benign or malignant CD133+/α2β1integrinhi prostate (stem) cells, when compared to CD133-/α2β1integrinhi (transit amplifying) cells or CD133-/α2β1integrinlow (basal committed) cells, selected from primary epithelial cultures. Condensed chromatin was associated with CD133 downregulation in all of the cell lines, and treatment with HDAC inhibitors resulted in CD133 re-expression in both cell lines and primary samples.ConclusionsCD133 is tightly regulated by DNA methylation only in cell lines, where promoter methylation and gene expression inversely correlate. This highlights the crucial choice of cell model systems when studying epigenetic control in cancer biology and stem cell biology. Significantly, in both benign and malignant prostate primary tissues, regulation of CD133 is independent of DNA methylation, but is under the dynamic control of chromatin condensation. This indicates that CD133 expression is not altered in prostate cancer and it is consistent with an important role for CD133 in the maintenance of the hierarchical cell differentiation patterns in cancer

Differential cytotoxic activity of a novel palladium-based compound on prostate cell lines, primary prostate epithelial cells and prostate stem cells

The outcome for patients with advanced metastatic and recurrent prostate cancer is still poor. Therefore, new chemotherapeutics are required, especially for killing cancer stem cells that are thought to be responsible for disease recurrence. In this study, we screened the effect of a novel palladium-based anticancer agent (Pd complex) against six different prostate cancer cell lines, and primary cultures from seven Gleason 6/7 prostate cancer, three Gleason 8/9 prostate cancer and four benign prostate hyperplasia patient samples, as well as cancer stem cells selected from primary cultures. MTT and ATP viability assays were used to assess cell growth and flow cytometry to assess cell cycle status. In addition, immunofluorescence was used to detect gamma H2AX nuclear foci, indicative of DNA damage, and Western blotting to assess the induction of apoptosis and autophagy. The Pd complex showed a powerful growth-inhibitory effect against both cell lines and primary cultures. More importantly, it successfully reduced the viability of cancer stem cells as first reported in this study. The Pd complex induced DNA damage and differentially induced evidence of cell death, as well as autophagy. In conclusion, this novel agent may be promising for use against the bulk of the tumour cell population as well as the prostate cancer stem cells, which are thought to be responsible for the resistance of metastatic prostate cancer to chemotherapy. This study also indicates that the combined use of the Pd complex with an autophagy modulator may be a more promising approach to treat prostate cancer. In addition, the differential effects observed between cell lines and primary cells emphasise the importance of the model used to test novel drugs including its genetic background, and indeed the necessity of using cells cultured from patient samples.YÖKYorkshire Cancer Research Core GrantUK Research & Innovation (UKRI) Medical Research Council UK (MRC) European Commission (G0900871)UK Research & Innovation (UKRI) Medical Research Council UK (MRC) (G0900871

Distinct Steps of Neural Induction Revealed by Asterix, Obelix and TrkC, Genes Induced by Different Signals from the Organizer

The amniote organizer (Hensen's node) can induce a complete nervous system when grafted into a peripheral region of a host embryo. Although BMP inhibition has been implicated in neural induction, non-neural cells cannot respond to BMP antagonists unless previously exposed to a node graft for at least 5 hours before BMP inhibitors. To define signals and responses during the first 5 hours of node signals, a differential screen was conducted. Here we describe three early response genes: two of them, Asterix and Obelix, encode previously undescribed proteins of unknown function but Obelix appears to be a nuclear RNA-binding protein. The third is TrkC, a neurotrophin receptor. All three genes are induced by a node graft within 4–5 hours but they differ in the extent to which they are inducible by FGF: FGF is both necessary and sufficient to induce Asterix, sufficient but not necessary to induce Obelix and neither sufficient nor necessary for induction of TrkC. These genes are also not induced by retinoic acid, Noggin, Chordin, Dkk1, Cerberus, HGF/SF, Somatostatin or ionomycin-mediated Calcium entry. Comparison of the expression and regulation of these genes with other early neural markers reveals three distinct “epochs”, or temporal waves, of gene expression accompanying neural induction by a grafted organizer, which are mirrored by specific stages of normal neural plate development. The results are consistent with neural induction being a cascade of responses elicited by different signals, culminating in the formation of a patterned nervous system

Multiple novel prostate cancer susceptibility signals identified by fine-mapping of known risk loci among Europeans

Genome-wide association studies (GWAS) have identified numerous common prostate cancer (PrCa) susceptibility loci. We have fine-mapped 64 GWAS regions known at the conclusion of the iCOGS study using large-scale genotyping and imputation in 25 723 PrCa cases and 26 274 controls of European ancestry. We detected evidence for multiple independent signals at 16 regions, 12 of which contained additional newly identified significant associations. A single signal comprising a spectrum of correlated variation was observed at 39 regions; 35 of which are now described by a novel more significantly associated lead SNP, while the originally reported variant remained as the lead SNP only in 4 regions. We also confirmed two association signals in Europeans that had been previously reported only in East-Asian GWAS. Based on statistical evidence and linkage disequilibrium (LD) structure, we have curated and narrowed down the list of the most likely candidate causal variants for each region. Functional annotation using data from ENCODE filtered for PrCa cell lines and eQTL analysis demonstrated significant enrichment for overlap with bio-features within this set. By incorporating the novel risk variants identified here alongside the refined data for existing association signals, we estimate that these loci now explain ∼38.9% of the familial relative risk of PrCa, an 8.9% improvement over the previously reported GWAS tag SNPs. This suggests that a significant fraction of the heritability of PrCa may have been hidden during the discovery phase of GWAS, in particular due to the presence of multiple independent signals within the same regio

Bi-modal strategy of gastrulation in reptiles

[Background]: Amniote gastrulation is often described with respect to human, mouse and chick development by the presence of the primitive streak, a posterior-to-anterior midline morphological cell ingression feature that has come to define Amniote gastrulation. How this midline, ingression-based strategy of gastrulation evolved from the ancestral blastopore, a circumferential involution event in Anamniotes, is unknown. However, within the Amniote clade there exists a more diverse range of gastrulation strategies than just the primitive streak. Investigating gastrulation in a wider range of Amniotes provides a way to understand evolutionary transition from blastopore to the primitive streak. [Results]: We analysed early to late gastrulation stages of Chamaeleo calyptratus, showing their unique morphology through confocal imaging of F-actin and laminin-stained embryos to visualise cell morphology and assess basal lamina integrity. We analysed the expression pattern of core mesodermal markers Brachyury and Fgf8 and complimented this analysis with that of the turtle, Trachemys scripta. [Conclusions]: Our analysis suggests that reptile gastrulation is bi-modal; primary internalization occurs anteriorly by means of an incomplete blastopore-like opening, while posteriorly the cells undergo ingression in the Brachyury-expressing blastoporal plate. This strategy stands mid-way between Anamniotes and Avians/Mammals, suggesting that blastoporal plate is a precursor of the avian primitive streak.funded by: The Biotechnology and Biological Sciences Research Council. Grant Number: BB/J00989X/1; The Spanish Ministry of Science and Innovation. Grant Number: BFU2010-19656; A Royal Society International Exchanges Award. Grant Number: IE121500; The Stowers Institute for Medical Research.Peer Reviewe

Neural induction by the node and placode induction by head mesoderm share an initial state resembling neural plate border and ES cells

Significance It is generally believed that the outcome of many inductive interactions occurring during development is largely dependent on the responding tissue, the source of the signals playing a relatively minor part. Here, we compare induction of the neural plate by the node, and of placodes by the head mesoderm, and show that both inducing tissues elicit a similar initial response but that they later diverge. We characterize the initial common state by a variety of methods and show its similarity to ES cells, suggesting that these inductions may begin with a common “reprogramming” step. This initial state also shares many features in common with the border of the neural plate, suggesting that this region retains features of a “ground state.”</jats:p

Regulation of <i>Obelix</i> by various secreted factors.

<p><b>A–P</b>. The ability of various peptide factors to induce <i>Obelix</i> expression was tested by local application of beads soaked in the protein or pellets of COS-1 cells transfected with a construct encoding the factor into the area opaca of a host embryo (A). Examples of FGF4 beads (B), Chordin (C) and Noggin (D) cells, FGF8/control beads (E), Dickkopf (F), Cerberus (G) cells and HGF/SF beads (H) are shown. I–P show sections through the grafted region of the embryos in B–H at the levels indicated. <b>Q–U</b>. Co-transplantation of a quail Hensen's node with beads soaked in the FGF inhibitor SU5402 has little or no effect: Obelix is still induced (Q–U). Q shows a grafted embryo fixed after 6 hours, and R is an example of an embryo grown overnight after the graft. S–U are sections through these embryos at the levels indicated in Q and R. Quail cells are stained with QCPN (brown). Note that some probes attach non-specifically to some types of beads and to COS cell pellets (eg. panels K–H).</p

Molecular characterization of Obelix.

<p><b>A.</b> Sequence alignment of Obelix protein (AY103477) with ESTs for EIF1A-related proteins from several species. Residues are displayed in different colours based on different aminoacid families and degree of homology is represented by conservation of these sites. Conserved OB-like domain is shown as a block in the alignment. Species are abbreviated as follows: ag, <i>Anopheles</i> mosquito (BM594550); bt, cow (BF043073); ce, <i>C. elegans</i> (AV203381); ci, <i>Ciona</i> (AV841463); dm, <i>Drosophila melanogaster</i> (BE977318); dr, zebrafish (BM859434); hs, human (BG149615); mm, mouse (BI103120); ss, pig (BG610103); rn, rat (BF420639); xl, <i>Xenopus laevis</i> (BG730245); xt, <i>Xenopus tropicalis</i> (AL637659). <b>B.</b> Phylogenetic tree with bootstrap values comparing the full-length sequences of Obelix in a variety of species, showing that eIF1A and Obelix segregate into two distinct sub-classes of OB-containing proteins. The LG model was used to construct the tree and bootstrap values were calculated from 1000 replicates.</p