Susana Bueno

Effects of dutasteride on the expression of genes related to androgen metabolism and related pathway in human prostate cancer cell lines

SummaryAndrogens play an important role in controlling the growth of the normal prostate gland and in the pathogenesis of benign prostate hyperplasia, and prostate cancer. Although testosterone is the main androgen secreted from the testes, dihydrotestosterone (DHT), a more potent androgen converted from testosterone by 5α-reductase isozymes, type I and II, is the major androgen in the prostate cells. The aim of this study is to investigate the cellular and molecular effects of dutasteride, a potent inhibitor of 5α-reductase type I and type II, in androgen-responsive (LNCaP) and androgen-unresponsive (DU145) human prostate cancer(PCa) cell lines. The expression pattern of 190 genes, selected on the basis of their proved or potential role in prostate cancerogenesis related to androgen signalling, were analysed using a low density home-made oligoarray (AndroChip 2). Our results show that dutasteride reduces cell viability and cell proliferation in both cell lines tested. AndroChip 2 gene signature identified in LNCaP a total of 11 genes differentially expressed (FC ≥ ±1.5). Eight of these genes, were overexpressed and three were underexpressed. Overexpressed genes included genes encoding for proteins involved in biosynthesis and metabolism of androgen (HSD17B1;HSD17B3;CYP11B2), androgen receptor and androgen receptor co-regulators (AR;CCND1), and signal transduction(ERBB2; V-CAM; SOS1) whereas, underexpressed genes (KLK3; KLK2; DHCR24) were androgen-regulated genes (ARGs). No differentially expressed genes were scored in DU145. Microarray data were confirmed by quantitative real-time PCR assay (QRT-PCR). These data offer a selective genomic signature for dutasteride treatment in prostate epithelial cells and provide important insights in prostate cancer pathophysiology.

Valproic acid induces neuroendocrine differentiation and UGT2B7 up-regulation in human prostate carcinoma cell line

Prostate cancer originates as an androgen-dependent hyperproliferation of the epithelial cells of the gland and it evolves in an androgen-independent, highly aggressive cancer for which no successful therapy is available to date. Neuroendocrine (NE) differentiation plays an important role in the progression of prostate cancer to an androgen-independent state with profound impact on prostate cancer (CaP) therapies. Actually, new approaches on treating advanced prostate cancer are focused on modulators of epigenetic transcriptional regulation. A new class of antitumoral agents is emerging: histone deacetylase (HDAC) inhibitors are interesting for their ability to arrest cell growth, to induce cell differentiation, and in some cases, to induce apoptosis of cancer cells. We studied the effect of valproic acid (VPA), an inhibitor of HDAC, in the human prostate androgen-dependent cancer cell line LNCaP. We observed that VPA promotes neuroendocrine-like differentiation associated with an increase in the expression of neuron-specific enolase, a decrease in prostate-specific antigen, and a down-regulation of androgen receptor protein, suggesting a modulation in the responsiveness to androgen therapy. Furthermore, selective gene expression profiling using a low-density microarray showed that VPA was able to modulate the expression of different androgen metabolism genes. We observed a down-regulation of androgen receptor coregulator (ARA24) and prostate-specific antigen, and an up-regulation of some of the UDP-glucuronosyltransferases (UGT2B11 and UGT2B7) implicated in catabolism of dihydrotestosterone (DHT) was detected. Even though UGT2B7 has only about one-tenth to one-hundredth the activity of UGT2B15 and 2B17 toward active androgens and we did not found any modulation in gene expression of these enzymes, it can be hypothesized that VPA might enhance DHT catabolism in this in vitro model and induces NE differentiation. Our data seem to raise concern about CaP treatment with VPA.

The open state of human topoisomerase I as probed by molecular dynamics simulation

The open state of human topoisomerase I has been probed by molecular dynamics simulation, starting from the coordinates of the closed structure of the protein complexed with DNA, after elimination of the 22-bp DNA duplex oligonucleotide. A repulsion force between the two lips of the protein has been introduced for a short time to induce destabilization of the local minimum, after which an unperturbed simulation has been carried out for 10 ns. The simulation shows that the protein undergoes a large conformational change due to rearrangements in the orientation of the protein domains, which however move as a coherent unit, fully maintaining their secondary and tertiary structures. Despite movements between the domains as large as 80–90 Å, the catalytic pentad remains preassembled, the largest deviation of the active site backbone atoms from the starting crystallographic structure being only 1.7 Å. Electrostatic calculation of the open protein structure shows that the protein displays a vast positive region with the active site residues located nearly at its center, in a conformation perfectly suited to interact with the negatively charged supercoiled DNA substrate.

Hif1α down-regulation is associated with transposition of great arteries in mice treated with a retinoic acid antagonist

BackgroundCongenital heart defect (CHD) account for 25% of all human congenital abnormalities. However, very few CHD-causing genes have been identified so far. A promising approach for the identification of essential cardiac regulators whose mutations may be linked to human CHD, is the molecular and genetic analysis of heart development. With the use of a triple retinoic acid competitive antagonist (BMS189453) we previously developed a mouse model of congenital heart defects (81%), thymic abnormalities (98%) and neural tube defects (20%). D-TGA (D-transposition of great arteries) was the most prevalent cardiac defect observed (61%). Recently we were able to partially rescue this abnormal phenotype (CHD were reduced to 64.8%, p = 0.05), by oral administration of folic acid (FA). Now we have performed a microarray analysis in our mouse models to discover genes/transcripts potentially implicated in the pathogenesis of this CHD.ResultsWe analysed mouse embryos (8.5 dpc) treated with BMS189453 alone and with BMS189453 plus folic acid (FA) by microarray and qRT-PCR. By selecting a fold change (FC) ≥ ± 1.5, we detected 447 genes that were differentially expressed in BMS-treated embryos vs. untreated control embryos, while 239 genes were differentially expressed in BMS-treated embryos whose mothers had also received FA supplementation vs. BMS-treated embryos. On the basis of microarray and qRT-PCR results, we further analysed the Hif1α gene. In fact Hif1α is down-regulated in BMS-treated embryos vs. untreated controls (FCmicro = -1.79; FCqRT-PCR = -1.76; p = 0.005) and its expression level is increased in BMS+FA-treated embryos compared to BMS-treated embryos (FCmicro = +1.17; FCqRT-PCR = +1.28: p = 0.005). Immunofluorescence experiments confirmed the under-expression of Hif1α protein in BMS-treated embryos compared to untreated and BMS+FA-treated embryos and, moreover, we demonstrated that at 8.5 dpc, Hif1α is mainly expressed in the embryo heart region.ConclusionsWe propose that Hif1α down-regulation in response to blocking retinoic acid binding may contribute to the development of cardiac defects in mouse newborns. In line with our hypothesis, when Hif1α expression level is restored (by supplementation of folic acid), a decrement of CHD is found. To the best of our knowledge, this is the first report that links retinoic acid metabolism to Hif1α regulation and the development of D-TGA.

Gene expression profile study in CFTR mutated bronchial cell lines

Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis conductance transmembrane regulator (CFTR). Symptoms are pancreatic insufficiency, chronic obstructive lung disease, liver disease, chronic sinusitis and infertility in male patients. The phenotypic variability may be explained only in part by the more than 1200 CFTR mutations, which are grouped into six different classes, according to their effect on the protein ranging from a severe (no synthesis or blocked processing) to mild mutation (altered conductance or reduced synthesis). However, it is now accepted that other genes (CF modifiers) influence the phenotypic spectrum of the disease. In order to identify CF modifier genes, we built a low-density home-made oligoarray containing 144 genes selected according to biochemical criteria and evaluated their expression in two CF bronchial epithelial cell lines (CuFi1 F508del/F508del; CuFi3 F508del/R553X). If we consider both cell lines, 38 genes (26.3%) show an altered expression pattern with a threshold >±1.5. Of these 38 genes, 12 are altered in CuFi1, and 26 in CuFi3. Some of these genes share the same expression pattern in both cell lines, while others have a different behaviour. These results were validated by a QRT-PCR assay (R2CuFi1=0.81 and R2CuFi3=0.91). These data could suggest that the presence of a class I allele (R553X) determines a more profound alteration of gene expression pattern than the presence of a class II allele (F508del). The identification of the genes altered by a specific CF mutation could lead to the development of a pharmacological approach specific for different CFTR genotypes.

A specific transcriptional response of yeast cells to camptothecin dependent on the Swi4 and Mbp1 factors

Topoisomerase I (Top1) is the specific target of the anticancer drug camptothecin (CPT) that interferes with enzyme activity promoting Top1-mediated DNA breaks and inhibition of DNA and RNA synthesis. To define the specific transcriptional response to CPT, we have determined the CPT-altered transcription profiles in yeast by using a relatively low concentration of the drug. CPT could alter global expression profiles only if a catalytically active Top1p was expressed in the cell, demonstrating that drug interference with Top1 was the sole trigger of the response. A total of 95 genes showed a statistically-significant alterations. Gene Ontology term analyses suggested that the cell response was mainly to the inhibition of nucleic acid synthesis and cell cycle progression. Promoter sequence analyses of the 22 up-regulated genes and expression studies in gene-deleted strains showed that the transcription factors, Swi4p and Mbp1p, mediate at least partially the transcriptional response to CPT. The MBP1 gene deletion abrogates a transient cell growth delay caused by CPT whereas the SWI4 gene deletion increases yeast resistance to CPT. Thus, the findings show that yeast cells have a highly selective and sensitive transcriptional response to CPT depending on SWI4 and MBP1 genes suggesting a complex regulation of cell cycle progression by the two factors in the presence of CPT.

A Tool for Sheep Product Quality: Custom Microarrays from Public Databases

Milk and dairy products are an essential food and an economic resource in many countries. Milk component synthesis and secretion by the mammary gland involve expression of a large number of genes whose nutritional regulation remains poorly defined. The purpose of this study was to gain an understanding of the genomic influence on milk quality and synthesis by comparing two sheep breeds with different milking attitude (Sarda and Gentile di Puglia) using sheep-specific microarray technology. From sheep ESTs deposited at NCBI, we have generated the first annotated microarray developed for sheep with a coverage of most of the genome.

Microarray gene expression profiling of neural tissues in bovine spastic paresis

BackgroundBovine Spastic Paresis (BSP) is a neuromuscular disorder which affects both male and female cattle. BSP is characterized by spastic contraction and overextension of the gastrocnemious muscle of one or both limbs and is associated with a scarce increase in body weight. This disease seems to be caused by an autosomal and recessive gene, with incomplete penetration, although no genes clearly involved with its onset have been so far identified. We employed cDNA microarrays to identify metabolic pathways affected by BSP in Romagnola cattle breed. Investigation of those pathways at the genome level can help to understand this disease.ResultsMicroarray analysis of control and affected individuals resulted in 268 differentially expressed genes. These genes were subjected to KEGG pathway functional clustering analysis, revealing that they are predominantly involved in Cell Communication, Signalling Molecules and Interaction and Signal Transduction, Diseases and Nervous System classes. Significantly enriched KEGG pathway’s classes for the differentially expressed genes were calculated; interestingly, all those significantly under-expressed in the affected samples are included in Neurodegenerative Diseases. To identify genome locations possibly harbouring gene(s) involved in the disease, the chromosome distribution of the differentially expressed genes was also investigated.ConclusionsThe cDNA microarray we used in this study contains a brain library and, even if carrying an incomplete transcriptome representation, it has proven to be a valuable tool allowing us to add useful and new information to a poorly studied disease. By using this tool, we examined nearly 15000 transcripts and analysed gene pathways affected by the disease. Particularly, our data suggest also a defective glycinergic synaptic transmission in the development of the disease and an alteration of calcium signalling proteins. We provide data to acquire knowledge of a genetic disease for which literature still presents poor results and that could be further and specifically analysed in the next future. Moreover this study, performed in livestock, may also harbour molecular information useful for understanding human diseases.

Transcriptomic analysis of two sheep breeds during lactation, using a new custom microarray platform

Abstract We aim at understanding the genomic influence on milk quality and synthesis by comparing two sheep breeds using sheep-specific microarray technology. From sheep ESTs deposited at NCBI we generated a chip carrying about 22,000 non-redundant features in quadruplicate, achieving very good technical outcomes. Oligos were in situ generated on chip using the Combimatrix equipment. We analysed the mammary transcriptome in individuals of two sheep breeds at two lactation stages, to identify genes controlling milk production and metabolic pathways in which these genes are involved. With |FC|>1.4, and p-value.0.05, 142 and 14 genes resulted differentially expressed in stages 01 and 02, respectively.

Abstract 637: Antisense transcripts and R-loops caused by DNA topoisomerase I inhibition by camptothecin at human active CpG island promoters.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC DNA Topoisomerase I (Top1) is specifically inhibited by camptothecin (CPT), a natural product with effective anticancer activity. CPT can stabilize a DNA-Top1 DNA cleavage complex that can lead to irreversible DNA breakage at replication forks, and transcription-dependent genome instability. The bulk of cellular Top1 activity is required at transcribing regions to modulate DNA supercoils generated by elongating RNA polymerases. However, the molecular effects of CPT inhibition of Top1 at transcriptional levels are not fully established. By next generation sequencing analyses of bisulfite-treated RNAs extracted from human HCT116 cancer cells, we here show that CPT promoted high levels of antisense tags specifically at active promoters containing CpG islands (CGI). We found several not annotated transcripts close to Refseq genes, the majority of which were promoter-associated antisense transcripts increased by CPT. We identified 256 and 84 of such transcripts in HCT116 cells and Top1-silenced HCT116-siRNATop1 cells, respectively. We validated CPT-stimulated antisense transcripts by PCR in both cell lines, and found that several of them could form R-loops at corresponding genomic regions by affinity purification with a recombinant inactive mutant RNaseH1. Drug effects were independent from replication, and required both Top1 and ongoing transcription. In addition, CPT induced an immediate burst and a subsequent rapid reduction of Top1-DNA cleavage complexes at active, but not inactive, promoters indicating a partial, time-dependent removal of Top1 from chromatin. Our findings demonstrate that Top1 activity prevents accumulation of antisense R-loops and RNAs at human active CGI promoters by relaxing negative DNA supercoils. The transcriptional CPT effects can contribute to drug therapeutic activity in cancer and other diseases. We will discuss our findings in relation to transcription regulation and genome instability caused by interference with Top1 activity. Citation Format: Jessica Marinello, Giovanni Chillemi, Susana Bueno, Stefano G. Manzo, Giovanni Capranico. Antisense transcripts and R-loops caused by DNA topoisomerase I inhibition by camptothecin at human active CpG island promoters. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 637. doi:10.1158/1538-7445.AM2013-637