Biomedical subjects

Colleen Nelson

Publications and source records attributed to Colleen Nelson.

13 recordsLinked to original sources

PubMed2006

Cluster-based network model for time-course gene expression data.

We propose a model-based approach to unify clustering and network modeling using time-course gene expression data. Specifically, our approach uses a mixture model to cluster genes. Genes within the same cluster share a similar expression profile. The network is built over cluster-specific expression profiles using state-space models. We discuss the application of our model to simulated data as well as to time-course gene expression data arising from animal models on prostate cancer progression. The latter application shows that with a combined statistical/bioinformatics analyses, we are able to extract gene-to-gene relationships supported by the literature as well as new plausible relationships.

Animals↗

PubMed2006

Small interference RNA targeting heat-shock protein 27 inhibits the growth of prostatic cell lines and induces apoptosis via caspase-3 activation in vitro.

OBJECTIVES: To evaluate synthetic small interference RNA (siRNA) compounds targeting heat-shock protein 27 (Hsp27) as an alternative approach to Hsp27 'knockdown' in prostate cancer cells, as Hsp27 expression is highly up-regulated in prostate cancer cells after androgen withdrawal or chemotherapy, to become uniformly highly expressed in androgen-independent (AI) prostate cancer. MATERIALS AND METHODS: We recently showed that targeting Hsp27 by a 2'-methoxyethyl modified phosphorothioate antisense oligonucleotide, OGX-427, inhibits Hsp27 expression and enhances hormone- and chemotherapy in prostate cancer xenograft models. In the present study, a 'gene walk' screening different siRNAs was initially used in PC-3 and LNCaP cells to determine the most potent sequence to down-regulate Hsp27 mRNA and protein levels. The effects of Hsp27 silencing on in vitro growth rates were studied by tetrazolium-blue and crystal violet assays. Apoptosis was determined by single-stranded DNA nuclear and cleaved caspase-3 immunostaining, as well as flow cytometry. Spotted microarrays with 14,000 human oligonucleotides were used to examine changes in gene expression. RESULTS: Low concentrations of 1 nm siRNA decreased Hsp27 mRNA levels by 19-fold and suppressed protein expression to undetectable levels. Silencing of Hsp27 in prostate cancer cells by siRNA # 2 increased apoptotic rates 2.4-4 fold and caused 40-76% inhibition of cell growth in LNCaP and PC-3 cells. Characteristic cleavage of caspase-3 occurred after treatment with Hsp27 siRNA (1 nm). cDNA microarray analysis from LNCaP and PC-3 cell lines revealed differential gene expression profiles after Hsp27 down-regulation that could be used to identify various survival pathways involved in androgen-dependent and AI growth. CONCLUSIONS: These findings illustrate the potential utility of Hsp27-silencing therapy and highlight Hsp27 siRNA strategies as a novel and highly effective tool, with the potential for future targeted therapy in enhancing the efficacy of chemotherapy in advanced prostate cancer.

Apoptosis↗

PubMed2005

Increased Hsp27 after androgen ablation facilitates androgen-independent progression in prostate cancer via signal transducers and activators of transcription 3-mediated suppression of apoptosis.

One strategy to improve therapies in prostate cancer involves targeting cytoprotective genes activated by androgen withdrawal to delay the emergence of the androgen-independent (AI) phenotype. The objectives of this study were to define changes in Hsp27 levels after androgen ablation and to evaluate the functional relevance of these changes in AI progression. Using a tissue microarray of 232 specimens of hormone-naïve and post-hormone ablation-treated prostate cancer, we found that Hsp27 levels increase after androgen ablation to become highly expressed (>4-fold, P < or = 0.01) in AI tumors. Hsp27 overexpression rendered LNCaP cells highly resistant to androgen withdrawal both in vitro and in vivo. Tumor volume and serum prostate-specific antigen levels increased 4.3- and 10-fold faster after castration when Hsp27 was overexpressed. Treatment of LNCaP tumor cells in vitro with Hsp27 antisense oligonucleotides (ASO) or short-interfering RNA suppressed Hsp27 levels in a dose-dependent and sequence-specific manner increased the apoptotic sub-G0-G1 fraction and caspase-3 cleavage >2-fold, as well as decreased signal transducers and activators of transcription 3 (Stat3) levels and its downstream genes, c-fos and sPLA-2. The cytoprotection afforded by Hsp27 overexpression was attenuated by Stat3 knockdown using specific Stat3 ASO. Coimmunoprecipitation and immunofluorescence confirmed that Hsp27 interacts with Stat3 and that Stat3 levels correlated directly with Hsp27 levels. Hsp27 ASO treatment in athymic mice bearing LNCaP tumors significantly delayed LNCaP tumor growth after castration, decreasing mean tumor volume and serum prostate-specific antigen levels by 57% and 69%, respectively. These findings identify Hsp27 as a modulator of Stat3-regulated apoptosis after androgen ablation and as a potential therapeutic target in advanced prostate cancer.

Animals↗

PubMed2005

Timing is everything: preclinical evidence supporting simultaneous rather than sequential chemohormonal therapy for prostate cancer.

PURPOSE: Androgen ablation is the mainstay of systemic therapy for prostate cancer, with cytotoxic therapies reserved for hormone-refractory disease. It is not clear, however, that this is the most appropriate sequence of interventions for this disease. This study addresses the ideal timing of systemic treatments in the Shionogi and LNCaP xenograft models. We explored the hypothesis that stress-induced gene expression changes after chemotherapy can induce a hormone-independent phenotype. EXPERIMENTAL DESIGN: Three groups of mice bearing either Shionogi or LNCaP xenografts were treated with (a) initial castration and delayed paclitaxel, (b) initial paclitaxel and delayed castration, or (c) simultaneous castration plus paclitaxel. End points were time to tumor progression and time to sacrifice. Microarray and reverse transcription-PCR analyses were carried out to assess changes in gene expression induced by paclitaxel. RESULTS: Mice receiving simultaneous therapy showed a significant improvement in median time to progression (TTP: Shionogi, 65 versus 38 days, P = 0.004; LNCaP, 105 versus 70 days, P = 0.032) and time to sacrifice (Shionogi, 83 versus 66 days, P < 0.014) versus best sequential therapy. A marked lack of response to castration was observed after initial paclitaxel therapy. Gene expression and reverse transcription-PCR studies confirmed that several genes known to play a role in androgen independence were up-regulated in response to paclitaxel exposure. CONCLUSIONS: In laboratory models of prostate cancer, simultaneous androgen deprivation plus paclitaxel is more effective than sequential treatments. These findings provide preclinical proof-of-principle for ongoing clinical trials addressing the role and timing of systemic therapies in prostate cancer.

Androgens↗

PubMed2005

Akt phosphorylates the Y-box binding protein 1 at Ser102 located in the cold shock domain and affects the anchorage-independent growth of breast cancer cells.

Akt/PKB is a serine/threonine kinase that promotes tumor cell growth by phosphorylating transcription factors and cell cycle proteins. There is particular interest in finding tumor-specific substrates for Akt to understand how this protein functions in cancer and to provide new avenues for therapeutic targeting. Our laboratory sought to identify novel Akt substrates that are expressed in breast cancer. In this study, we determined that activated Akt is positively correlated with the protein expression of the transcription/translation factor Y-box binding protein-1 (YB-1) in primary breast cancer by screening tumor tissue microarrays. We therefore questioned whether Akt and YB-1 might be functionally linked. Herein, we illustrate that activated Akt binds to and phosphorylates the YB-1 cold shock domain at Ser102. We then addressed the functional significance of disrupting Ser102 by mutating it to Ala102. Following the stable expression of Flag:YB-1 and Flag:YB-1 (Ala102) in MCF-7 cells, we observed that disruption of the Akt phosphorylation site on YB-1 suppressed tumor cell growth in soft agar and in monolayer. This correlated with an inhibition of nuclear translocation by the YB-1(Ala102) mutant. In conclusion, YB-1 is a new Akt substrate and disruption of this specific site inhibits tumor cell growth.

Breast Neoplasms↗

PubMed2005

Mechanisms of the development of androgen independence in prostate cancer.

The effectiveness of androgen ablation in the management of advanced prostate cancer is of limited duration, with the median length of response being only 18-24 months. The transition of the prostate cancer cell to an androgen independent phenotype is a complex process that involves selection and outgrowth of pre-existing clones of androgen-independent cells (clonal selection) as well as adaptive up-regulation of genes that help the cancer cells survive and grow after androgen ablation (adaptation). These two mechanisms share an important pre-requisite characteristic: prostate cancers are heterogeneous tumours comprised of various subpopulations of cells that respond differently to androgen withdrawal therapy. This tumour heterogeneity may reflect either a multifocal origin, adaptation to environmental stimuli, and/or genetic instability of the initial cancer. This review will reexamine the different mechanisms that enable prostate cancer cells to proliferate in an androgen depleted environment.

Androgens↗

PubMed2005

Osteopontin expression correlates with melanoma invasion.

Melanoma is one of the most aggressive cancers affecting humans. Although early melanomas are curable with surgical excision, metastatic melanomas are associated with high mortality. The mechanism of melanoma development, progression, and metastasis is largely unknown. In order to uncover genes unique to melanoma cells, we used high-density DNA microarrays to examine the gene expression profiles of metastatic melanoma nodules using benign nevi as controls. Over 190 genes were significantly overexpressed in metastatic melanomas compared with normal nevi by at least 2-fold. One of the most abundantly expressed genes in metastatic melanoma nodules is osteopontin (OPN). Immunohistochemistry staining on tissue microarrays and individual skin biopsies representing different stages of melanoma progression revealed that OPN expression is first acquired at the step of melanoma tissue invasion. In addition, blocking of OPN expression by RNA interference reduced melanoma cell numbers in vitro. Our observations suggest that OPN may be acquired early in melanoma development and progression, and may enhance tumor cell growth in invasive melanoma.

Adult↗

PubMed2004

Androgen responsiveness of Renilla luciferase reporter vectors is promoter, transgene, and cell line dependent.

Renilla based reporters are frequently used as transfection controls for luciferase transcriptional reporter assays. However, recent evidence suggests that a commonly used reporter (HSV-thymidine kinase driven Renilla) is responsive to androgen receptor (AR) and glucocorticoid receptors in the presence of the cognate ligands, dihydrotestosterone (DHT) and dexamethasone (DEX), respectively [1]. We further validate this important technical difficulty by illustrating that in LNCaP prostate cancer cells, spurious Renilla luciferase activity is a function of (a) the promoter driving Renilla expression, (b) the presence of co-transfected transgenes, and (c) the androgen responsiveness of the cell line used. Using inhibitors of transcription and translation we showed that transcript interference or translational modulation is not a major means by which androgens affect Renilla luciferase activity. As luciferase reporter assays are a frequent means of studying transcriptional co-regulation in the highly androgen dependent LNCaP cell line, our data serves as a cautionary note that alternative normalization techniques should be employed to avoid misinterpretation of data.

Androgens↗

PubMed2004

Development and application of a salmonid EST database and cDNA microarray: data mining and interspecific hybridization characteristics.

We report 80,388 ESTs from 23 Atlantic salmon (Salmo salar) cDNA libraries (61,819 ESTs), 6 rainbow trout (Oncorhynchus mykiss) cDNA libraries (14,544 ESTs), 2 chinook salmon (Oncorhynchus tshawytscha) cDNA libraries (1317 ESTs), 2 sockeye salmon (Oncorhynchus nerka) cDNA libraries (1243 ESTs), and 2 lake whitefish (Coregonus clupeaformis) cDNA libraries (1465 ESTs). The majority of these are 3' sequences, allowing discrimination between paralogs arising from a recent genome duplication in the salmonid lineage. Sequence assembly reveals 28,710 different S. salar, 8981 O. mykiss, 1085 O. tshawytscha, 520 O. nerka, and 1176 C. clupeaformis putative transcripts. We annotate the submitted portion of our EST database by molecular function. Higher- and lower-molecular-weight fractions of libraries are shown to contain distinct gene sets, and higher rates of gene discovery are associated with higher-molecular weight libraries. Pyloric caecum library group annotations indicate this organ may function in redox control and as a barrier against systemic uptake of xenobiotics. A microarray is described, containing 7356 salmonid elements representing 3557 different cDNAs. Analyses of cross-species hybridizations to this cDNA microarray indicate that this resource may be used for studies involving all salmonids.

Animals↗

PubMed2003

An androgen-dependent upstream enhancer is essential for high levels of probasin gene expression.

Previously we reported that a small rat probasin (PB) promoter (-426 to +28 bp, -426PB) would target androgen- regulated prostate-specific expression in transgenic mice. Later we demonstrated that a large (L) fragment (-10806 to +28 bp, LPB) of the PB promoter would target high levels of gene expression to the prostate in transgenic mice. These results suggested that optimal transcription of the PB gene depended on the presence of enhancer regions upstream of the proximal promoter. To identify these enhancers, the LPB fragment was sequenced and the enhancer activities of restriction fragments were characterized in cell lines. Two nonconventional androgen receptor binding sites (ARBSs), ARBS-3 and ARBS-4, in an upstream androgen-dependent enhancer of the PB gene were identified. One site functions as a weak steroid response element in both LNCaP and MCF-7 cells; another site acts as a strong steroid response element, which preferentially responds to androgen and is preferentially activated in LNCaP cells. These two new ARBSs interact in a cooperative manner with the previously described androgen response region (ARR) (defined by -244 to -96 bp) that contains ARBS-1; ARBS-2; and two lower-affinity ARR binding sites, G-1 and G-2 sites. We conclude that the context in which the ARR binding sites are present is pivotal in determining their effect on transcriptional regulation. Thus, the -705/+28 PB promoter contains a second ARR, PB enhancer element (-705/-426 PB), in addition to the first described ARR. The PB promoter creates a model that contains six AR binding sites that function in a cooperative manner for maximum androgen-regulated prostate-specific gene transcription.

Androgen-Binding Protein↗

PubMed2003

The p38 mitogen-activated protein kinase regulates interleukin-1beta-induced IL-8 expression via an effect on the IL-8 promoter in intestinal epithelial cells.

Several lines of evidence implicate the p38 mitogen-activated protein kinase (p38 MAPK) in the proinflammatory response to bacterial agents and cytokines. Equally, the transcription factor, nuclear factor (NF)-kappaB, is recognized to be a critical determinant of the inflammatory response in intestinal epithelial cells (IECs). However, the precise inter-relationship between the activation of p38 MAPK and activation of the transcription factor NF-kappaB in the intestinal epithelial cell (IEC) system, remains unknown. Here we show that interleukin (IL)-1beta activates all three MAPKs in Caco-2 cells. The production of IL-8 and monocyte chemotactic protein 1 (MCP-1) was attenuated by 50% when these cells were preincubated with the p38 MAPK inhibitor, SB 203580. Further investigation of the NF-kappaB signalling system revealed that the inhibitory effect was independent of the phosphorylation and degradation of IkappaBalpha, the binding partner of NF-kappaB. This effect was also independent of the DNA binding of the p65 Rel A subunit, as well as transactivation, determined by an NF-kappaB luciferase construct, using both SB 203580 and dominant-negative p38 MAPK. Evaluation of IL-8 and MCP-1 RNA messages by reverse transcription-polymerase chain reaction (RT-PCR) revealed that the inhibitory effect of SB 203580 was associated with a reduction in this parameter. Using an IL-8-luciferase promoter construct, an effect of p38 upon its activation by both pharmacological and dominant-negative p38 construct co-transfection was demonstrated. It is concluded that p38 MAPK influences the expression of chemokines in intestinal epithelial cells, through an effect upon the activation of the chemokine promoter, and does not directly involve the activation of the transcription factor NF-kappaB.

Cell Line↗

PubMed2003

Antisense targets to enhance hormone and cytotoxic therapies in advanced prostate cancer.

The main obstacle to improved survival of advanced prostate cancer is our failure to prevent or treat its progression to its lethal and untreatable stage of androgen independence. New therapeutic agents designed to prevent androgen-independent progression are required. Accelerated identification and characterization of cancer-relevant molecular targets has sparked considerable interest in the development of new generations of anti-cancer agents that specifically inhibit a progression-relevant target. Antisense oligonucleotides, short synthetic stretches of chemically modified DNA capable of specifically hybridizing to the mRNA of a chosen cancer-relevant target gene. promise to show enhanced specificity for malignant cells with a more favorable side-effect profile due to well-defined and tailored modes of action. Although not all of the challenges have been met to date, emerging clinical evidence supports the premise that antisense oligonucleotides stand a realistic chance of emerging as major partners of rationally designed anti-cancer regimens. The status of antisense targeting of several genes, including bcl-2, bcl-xL, clusterin, androgen receptor and IGFBPs, relevant to prostate and other cancers, are reviewed.

Androgen Antagonists↗

PubMed2002

Acrylamide capture of DNA-bound complexes: electrophoretic purification of transcription factors.

We have developed a rapid nonradioactive electrophoretic technique to analyze proteins within DNA-binding complexes, acrylamide capture of DNA-binding complexes (ACDC), using Acrydite-linked DNA-binding targets. The method is highly sensitive and easily adaptable to virtually any protein-DNA interaction. The utility of this technique is illustrated using recombinant and full-length androgen receptors and associated co-regulatory proteins present within nuclear extracts. In brief proteins were incubated with DNA-binding targets in which one oligonucleotide was synthesized with an Acrydite moiety at the 5' end to allow for covalent linkage to acrylamide. Alternatively, gene promoter regions were amplified with an Acrydite-modified PCR primer to analyze protein-DNA complexes. The DNA-binding reaction was polymerized into an acrylamide matrix within the well of a precast gel. Proteins complexed to the Acrydite DNA are trapped and purified by the electrophoretic migration of unbound proteins. Proteins captured in the Acrydite-DNA can be eluted and identified by Western analysis or 2-D gel electrophoresis. The advantages of this technique are that it is rapid, adaptable, sensitive, unlimited by the size of the DNA or protein complex, and can be used to detect tertiary interactions with co-regulatory factors and unidentified proteins. These features make the ACDC technique a powerful tool for transcription factor research.

Acrylamide↗