Inhibition of Glycogen Synthase Kinase-3 Increases the Cytotoxicity of Enzastaurin

Cutaneous T-cell lymphomas (CTCL) represent a spectrum of several distinct non-Hodgkin's lymphomas that are characterized by an invasion of the skin by malignant, clonal lymphocytes. Our laboratory has previously demonstrated that the protein kinase C (PKC) β inhibitor Enzastaurin increases apoptosis in malignant lymphocytes of CTCL. These results directly led to a clinical trial for Enzastaurin in CTCL in which it was well tolerated and showed modest activity. To ascertain a means of improving the efficacy of Enzastaurin, we investigated complementary signaling pathways and identified glycogen synthase kinase-3 (GSK3) as important in survival signaling in CTCL. Enzastaurin combined with GSK3 inhibitors demonstrated an enhancement of cytotoxicity. Treatment with a combination of Enzastaurin and the GSK3 inhibitor AR-A014418 resulted in upregulation of β-catenin total protein and β-catenin-mediated transcription. Inhibition of β-catenin-mediated transcription or small hairpin RNA (shRNA) knockdown of β-catenin decreased the cytotoxic effects of Enzastaurin plus AR-A014418. In addition, treatment with Enzastaurin and AR-A014418 decreased the mRNA levels and surface expression of CD44. shRNA knockdown of β-catenin also restored CD44 surface expression. Our observations provide a rationale for the combined targeting of PKC and GSK3 signaling pathways in CTCL to enhance the therapeutic outcome

A collection of bioconductor methods to visualize gene-list annotations

<p>Abstract</p> <p>Background</p> <p>Gene-list annotations are critical for researchers to explore the complex relationships between genes and functionalities. Currently, the annotations of a gene list are usually summarized by a table or a barplot. As such, potentially biologically important complexities such as one gene belonging to multiple annotation categories are difficult to extract. We have devised explicit and efficient visualization methods that provide intuitive methods for interrogating the intrinsic connections between biological categories and genes.</p> <p>Findings</p> <p>We have constructed a data model and now present two novel methods in a Bioconductor package, "GeneAnswers", to simultaneously visualize genes, concepts (a.k.a. annotation categories), and concept-gene connections (a.k.a. annotations): the "Concept-and-Gene Network" and the "Concept-and-Gene Cross Tabulation". These methods have been tested and validated with microarray-derived gene lists.</p> <p>Conclusions</p> <p>These new visualization methods can effectively present annotations using Gene Ontology, Disease Ontology, or any other user-defined gene annotations that have been pre-associated with an organism's genome by human curation, automated pipelines, or a combination of the two. The gene-annotation data model and associated methods are available in the Bioconductor package called "GeneAnswers " described in this publication.</p

Insulin resistance in H-35 rat hepatoma cells is mediated by post-receptor mechanisms

Incubation of H-35 cells with 300 ng/ml (50 nM) of insulin causes a 3-4-fold induction of tyrosine aminotransferase at 4-6 h of incubation. At 24 h the activity of transaminase returns to basal levels despite the presence of sufficient insulin to stimulate a maximal response. Furthermore, addition of 300 ng/ml of fresh insulin fails to stimulate the induction of transaminase. In contrast, the addition of 0.1 [mu]M dexamethasone to insulintreated cells stimulates the induction of tyrosine aminotransferase, indicating that the loss of responsiveness is specific to insulin action. Incubation of H-35 cells with insulin also causes a 25-30% decrease in insulin binding. This modest decrease in receptor binding is not sufficient to explain the virtually complete loss of insulin responsiveness. Hence, in H-35 hepatoma cells insulin-induced desensitization to insulin action is mediated primarily by post-receptor events.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25115/1/0000548.pd

Nuclear translocation of Atox1 potentiates activin A-induced cell migration and colony formation in colon cancer.

BackgroundColorectal cancer remains a deadly cancer due to metastatic disease. To understand the molecular mechanisms of metastasis in colon cancer, we investigated whether the copper chaperone antioxidant-1 (Atox1) protein plays a role in this process. Recent findings indicate that Atox1 protein has transcription factor activities and plays a vital role in cell proliferation in cancer cells. However, the role of Atox1 in metastasis has not been examined.MethodsAtox1 expression was determined by immunofluorescence in a tissue microarray generated from a spectrum of CRC patients. Subcellular fractionation of colon cancer cell lines SW480 and SW620 cells was used to examine the cellular location of Atox1 in the face of activin A, a cytokine that stimulates colon cancer metastasis. Atox1 expression was genetically manipulated and cellular migration measured through trans-well assay and proliferation measured by colony formation assays.ResultsHere we demonstrate that in patients with metastatic colon cancer, there is a significant increase in the expression of nuclear Atox1. Interestingly, the metastatic CRC cell line SW620 has increased nuclear localization of Atox1 compared to its related non-metastatic cell line SW480. Further, inhibition of endogenous Atox1 by siRNA in SW620 decreased colony formation and reactive oxygen species generation via decreased expression of Atox1 targets cyclin D1 and NADPH oxidase subunit p47 phox, respectively. Additionally, overexpression of nuclear-targeted but not copper binding domain-mutated Atox1 in SW480 cells increased colony formation and cell migration that was further augmented by activin A stimulation, a known enhancer of colon cancer metastasis.ConclusionsOur findings suggest that nuclear Atox1 might be a new therapeutic target as well as a new biomarker for metastatic colorectal cancer

Integrative Gene Expression Profiling Reveals G6PD-Mediated Resistance to RNA-Directed Nucleoside Analogues in B-Cell Neoplasms

<div><p>The nucleoside analogues 8-amino-adenosine and 8-chloro-adenosine have been investigated in the context of B-lineage lymphoid malignancies by our laboratories due to the selective cytotoxicity they exhibit toward multiple myeloma (MM), chronic lymphocytic leukemia (CLL), and mantle cell lymphoma (MCL) cell lines and primary cells. Encouraging pharmacokinetic and pharmacodynamic properties of 8-chloro-adenosine being documented in an ongoing Phase I trial in CLL provide additional impetus for the study of these promising drugs. In order to foster a deeper understanding of the commonalities between their mechanisms of action and gain insight into specific patient cohorts positioned to achieve maximal benefit from treatment, we devised a novel two-tiered chemoinformatic screen to identify molecular determinants of responsiveness to these compounds. This screen entailed: 1) the elucidation of gene expression patterns highly associated with the anti-tumor activity of 8-chloro-adenosine in the NCI-60 cell line panel, 2) characterization of altered transcript abundances between paired MM and MCL cell lines exhibiting differential susceptibility to 8-amino-adenosine, and 3) integration of the resulting datasets. This approach generated a signature of seven unique genes including <em>G6PD</em> which encodes the rate-determining enzyme of the pentose phosphate pathway (PPP), glucose-6-phosphate dehydrogenase. Bioinformatic analysis of primary cell gene expression data demonstrated that G6PD is frequently overexpressed in MM and CLL, highlighting the potential clinical implications of this finding. Utilizing the paired sensitive and resistant MM and MCL cell lines as a model system, we go on to demonstrate through loss-of-function and gain-of-function studies that elevated G6PD expression is necessary to maintain resistance to 8-amino- and 8-chloro-adenosine but insufficient to induce <em>de novo</em> resistance in sensitive cells. Taken together, these results indicate that G6PD activity antagonizes the cytotoxicity of 8-substituted adenosine analogues and suggests that administration of these agents to patients with B-cell malignancies exhibiting normal levels of <em>G6PD</em> expression may be particularly efficacious.</p> </div

Increased G6PD expression is observed in MM and CLL patient samples.

<p>(A) G6PD expression was interrogated in clinical myeloma samples through querying the dataset ‘Zhan Myeloma 3’ for G6PD probe set ‘202275_at’ in the Oncomine™ database. MM samples exhibit a 4.745 fold increase in G6PD expression relative to normal plasma cells (NPC). MGUS: Monoclonal Gammopathy of Undetermined Significance. According to the authors Zhan et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041455#pone.0041455-Zhan1" target="_blank">[31]</a>, the diagnosis of MGUS included the following criteria: levels of monoclonal protein ≤30 g/L, plasma cell bone marrow infiltration <10%, and no incidence of related organ or tissue impairment. (B) G6PD expression was interrogated in clinical CLL samples through querying the dataset ‘Haslinger Leukemia’ for G6PD probe set ‘38043_at’ in the Oncomine™ database. CLL samples exhibit a 2.319 fold increase in G6PD expression relative to normal B lymphocytes (NBL). Data in parts (A) and (B) are log₂ transformed and median-centered. Boxes indicate 95 and 5 percentiles and median expression values. Whiskers represent minimum and maximum expression values. * <i>P</i><.05 ** <i>P</i><.01 *** <i>P</i><.005.</p