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Biomedical subjects

Richard B Gaynor

Publications and source records attributed to Richard B Gaynor.

At least 19 recordsLinked to original sources
PubMed2006

IKKalpha regulates the mitotic phase of the cell cycle by modulating Aurora A phosphorylation.

The IKK complex includes two catalytic components, IKKalpha and IKKbeta, in addition to the scaffold protein IKKgamma/NEMO. Even though IKKalpha and IKKbeta share significant sequence homology, they have distinct biological roles with IKKbeta regulates the classical pathway of NF-kappaB activation and IKKalpha regulates the alternative pathways. In addition, it has been shown that the IKKs regulate the proliferation of both normal and tumor cells; however, the mechanisms by which the IKKs regulate the cell cycle remain to be further defined. Here, we demonstrate that IKKalpha, but not IKKbeta, has role in regulating the M phase of the cell cycle. IKKalpha siRNA knock -down resulted in increased numbers of cells in the G(2)/M phase of the cell cycle as compared to control and IKKbeta siRNA transfected HeLa cells. This effect was associated with upregulation of cyclin B1 and Plk1 protein levels and increased histone H3 phosphorylation, consistent with a potential role of IKKalpha in the regulation of M phase regulatory factors. IKKalpha was found to be associated with Aurora A in the centrosome and regulate Aurora A phosphorylation at threonine residue 288, a site which is important in modulating its kinase activity. Taken together, these data provide the evidence that IKKalpha regulates the M phase of the cell cycle by modulating Aurora A phosphorylation and activation leading to the regulation of the M phase of the cell cycle.

Aurora Kinases↗
PubMed2006

IKK alpha regulates estrogen-induced cell cycle progression by modulating E2F1 expression.

The IkappaB kinase (IKK) complex consists of the catalytic subunits IKKalpha and IKKbeta and a regulatory subunit, IKKgamma/NEMO. Even though IKKalpha and IKKbeta share significant sequence similarity, they have distinct biological roles. It has been demonstrated that IKKs are involved in regulating the proliferation of both normal and tumor cells, although the mechanisms by which they function in this process remain to be better defined. In this study, we demonstrate that IKKalpha, but not IKKbeta, is important for estrogen-induced cell cycle progression by regulating the transcription of the E2F1 gene as well as other E2F1-responsive genes, including thymidine kinase 1, proliferating cell nuclear antigen, cyclin E, and cdc25A. The role of IKKalpha in regulating E2F1 was not the result of reduced levels of cyclin D1, as overexpression of this gene could not overcome the effects of IKKalpha knock-down. Furthermore, estrogen treatment increased the association of endogenous IKKalpha and E2F1, and this interaction occurred on promoters bound by E2F1. IKKalpha also potentiated the ability of p300/CBP-associated factor to acetylate E2F1. Taken together, these data suggest a novel mechanism by which IKKalpha can influence estrogen-mediated cell cycle progression through its regulation of E2F1.

Animals↗
PubMed2005

Selective identification of secreted and transmembrane breast cancer markers using Escherichia coli ampicillin secretion trap.

Secreted and cell surface proteins play important roles in cancer and are potential drug targets and tumor markers. Here, we describe a large-scale analysis of the genes encoding secreted and cell surface proteins in breast cancer. To identify these genes, we developed a novel signal sequence trap method called Escherichia coli ampicillin secretion trap (CAST). For CAST, we constructed a plasmid in which the signal sequence of beta-lactamase was deleted such that it does not confer ampicillin resistance. Eukaryotic cDNA libraries cloned into pCAST produced tens of thousands of ampicillin-resistant clones, 80% of which contained cDNA fragments encoding secreted and membrane spanning proteins. We identified 2,708 unique sequences from cDNA libraries made from surgical breast cancer specimens. We analyzed the expression of 1,287 of the 2,708 genes and found that 166 were overexpressed in breast cancers relative to normal breast tissues. Eighty-five percent of these genes had not been previously identified as markers of breast cancer. Twenty-three of the 166 genes (14%) were relatively tissue restricted, suggesting use as cancer-specific targets. We also identified several new markers of ovarian cancer. Our results indicate that CAST is a robust, rapid, and low cost method to identify cell surface and secreted proteins and is applicable to a variety of relevant biological questions.

Ampicillin Resistance↗
PubMed2005

Formation of an IKKalpha-dependent transcription complex is required for estrogen receptor-mediated gene activation.

The IkappaB kinases IKKalpha and IKKbeta regulate distinct cytoplasmic and nuclear events that are critical for cytokine-mediated activation of the NF-kappaB pathway. Because the IKKs have previously been demonstrated to associate with the nuclear hormone receptor coactivator AIB1/SRC-3, the question of whether either IKKalpha or IKKbeta may be involved in increasing the expression of hormone-responsive genes was addressed. We demonstrated that IKKalpha, in conjunction with ERalpha and AIB1/SRC-3, is important in activating the transcription of estrogen-responsive genes, including cyclin D1 and c-myc, to result in the enhanced proliferation of breast cancer cells. Estrogen treatment facilitated the association of IKKalpha, ERalpha, and AIB1/SRC-3 to estrogen-responsive promoters and increased IKKalpha phosphorylation of ERalpha, AIB1/SRC-3, and histone H3. These results suggest that IKKalpha plays a major role in regulating the biological effects of estrogen via its promoter association and modification of components of the transcription complex.

Apoptosis↗
PubMed2005

Exclusive ubiquitination and sumoylation on overlapping lysine residues mediate NF-kappaB activation by the human T-cell leukemia virus tax oncoprotein.

The transcription factor NF-kappaB is critical for the induction of cancer, including adult T-cell leukemia, which is linked to infection by human T-cell leukemia virus type 1 and the expression of its regulatory protein Tax. Although activation of the NF-kappaB pathway by Tax involves its interaction with the regulatory subunit of the IkappaB kinase (IKK) complex, NEMO/IKKgamma, the mechanism by which Tax activates specific cellular genes in the nucleus remains unknown. Here, we demonstrate that the attachment of SUMO-1 to Tax regulates its localization in nuclear bodies and the recruitment of both the RelA subunit of NF-kappaB and free IKKgamma in these nuclear structures. However, this sumoylation step is not sufficient for the activation of the NF-kappaB pathway by Tax. This activity requires the prior ubiquitination and colocalization of ubiquitinated Tax with IKK complexes in the cytoplasm and the subsequent migration of the RelA subunit of NF-kappaB to the nucleus. Thus, the ubiquitination and sumoylation of Tax function in concert to result in the migration of RelA to the nucleus and its accumulation with IKKgamma in nuclear bodies for activation of gene expression. These modifications may result in targets for the treatment of adult T-cell leukemia.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Enhanced chemosensitivity to irinotecan by RNA interference-mediated down-regulation of the nuclear factor-kappaB p65 subunit.

In preclinical tumor models, inhibition of nuclear factor-kappaB (NF-kappaB) has been associated with increased sensitivity to chemotherapeutic agents such as irinotecan (CPT-11). This is based on the fact that a variety of chemotherapy agents such as CPT-11 activate NF-kappaB to result in the expression of genes such as c-IAP1 and c-IAP2 that might be responsible for the inhibition of chemotherapy-induced apoptosis. In this study, RNA interference [small interfering RNA (siRNA)] was used to down-regulate the NF-kappaB p65 subunit in the HCT116 colon cancer cell line, and its role, in the presence and absence of CPT-11, was assessed on cell growth and apoptosis. Reduction of endogenous p65 by siRNA treatment significantly impaired CPT-11-mediated NF-kappaB activation, enhanced apoptosis, and reduced colony formation in soft agar. Furthermore, the in vivo administration of p65 siRNA reduced HCT116 tumor formation in xenograft models in the presence but not the absence of CPT-11 administration. These data indicate that the administration of siRNA directed against the p65 subunit of NF-kappaB can effectively enhance in vitro and in vivo sensitivity to chemotherapeutic agents.

Agar↗
PubMed2004

IkappaB kinases: key regulators of the NF-kappaB pathway.

The nuclear factor (NF)-kappaB pathway is important for the expression of a wide variety of genes that are involved in the control of the host immune and inflammatory response, and in the regulation of cellular proliferation and survival. The constitutive activation of this pathway is associated with inflammatory and autoimmune diseases, such as asthma, rheumatoid arthritis and inflammatory bowel disease, in addition to atherosclerosis, Alzheimer's disease, cancer and diabetes. One of the key steps in activating the NF-kappaB pathway is the stimulation of the IkappaB (inhibitor of kappaB) kinases. Recent data indicate that these kinases activate the NF-kappaB pathway through distinct steps that are operative in both the cytoplasm and the nucleus. A better understanding of the mechanisms that activate this pathway provides the potential for defining new therapeutic targets that might prevent the aberrant activation of NF-kappaB in a variety of human diseases.

Animals↗
PubMed2003

Role of the TAB2-related protein TAB3 in IL-1 and TNF signaling.

The cytokines IL-1 and TNF induce expression of a series of genes that regulate inflammation through activation of NF-kappaB signal transduction pathways. TAK1, a MAPKKK, is critical for both IL-1- and TNF-induced activation of the NF-kappaB pathway. TAB2, a TAK1-binding protein, is involved in IL-1-induced NF-kappaB activation by physically linking TAK1 to TRAF6. However, IL-1-induced activation of NF-kappaB is not impaired in TAB2-deficient embryonic fibroblasts. Here we report the identification and characterization of a novel protein designated TAB3, a TAB2-like molecule that associates with TAK1 and can activate NF-kappaB similar to TAB2. Endogenous TAB3 interacts with TRAF6 and TRAF2 in an IL-1- and a TNF-dependent manner, respectively. Further more, IL-1 signaling leads to the ubiquitination of TAB2 and TAB3 through TRAF6. Cotransfection of siRNAs directed against both TAB2 and TAB3 inhibit both IL-1- and TNF-induced activation of TAK1 and NF-kappaB. These results suggest that TAB2 and TAB3 function redundantly as mediators of TAK1 activation in IL-1 and TNF signal transduction.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Nuclear role of I kappa B Kinase-gamma/NF-kappa B essential modulator (IKK gamma/NEMO) in NF-kappa B-dependent gene expression.

The I kappa B kinase (IKK) complex, which is composed of the two kinases IKK alpha and IKK beta and the regulatory subunit IKK gamma/nuclear factor-kappa B (NF-kappa B) essential modulator (NEMO), is important in the cytokine-induced activation of the NF-kappa B pathway. In addition to modulation of IKK activity, the NF-kappa B pathway is also regulated by other processes, including the nucleocytoplasmic shuttling of various components of this pathway and the post-translational modification of factors bound to NF-kappa B-dependent promoters. In this study, we explored the role of the nucleocytoplasmic shuttling of components of the IKK complex in the regulation of the NF-kappa B pathway. IKK gamma/NEMO was demonstrated to shuttle between the cytoplasm and the nucleus and to interact with the nuclear coactivator cAMP-responsive element-binding protein-binding protein (CBP). Using both in vitro and in vivo analysis, we demonstrated that IKK gamma/NEMO competed with p65 and IKK alpha for binding to the N terminus of CBP, inhibiting CBP-dependent transcriptional activation. These results indicate that, in addition to the key role of IKK gamma/NEMO in regulating cytokine-induced IKK activity, its ability to shuttle between the cytoplasm and the nucleus and to bind to CBP can lead to transcriptional repression of the NF-kappa B pathway.

Blotting, Western↗
PubMed2003

Protein phosphatase 2Cbeta association with the IkappaB kinase complex is involved in regulating NF-kappaB activity.

The NF-kappaB pathway is important in the control of the immune and inflammatory response. One of the critical events in the activation of this pathway is the stimulation of the IkappaB kinases (IKKs) by cytokines such as tumor necrosis factor-alpha and interleukin-1. Although the mechanisms that modulate IKK activation have been studied in detail, much less is known about the processes that down-regulate its activity following cytokine treatment. In this study, we utilized biochemical fractionation and mass spectrometry to demonstrate that protein phosphatase 2Cbeta (PP2Cbeta) can associate with the IKK complex. PP2Cbeta association with the IKK complex led to the dephosphorylation of IKKbeta and decreased its kinase activity. The binding of PP2Cbeta to IKKbeta was decreased at early times post-tumor necrosis factor-alpha treatment and was restored at later times following treatment with this cytokine. Experiments utilizing siRNA directed against PP2Cbeta demonstrated an in vivo role for this phosphatase in decreasing IKK activity at late times following cytokine treatment. These studies are consistent with the ability of PP2Cbeta to down-regulate cytokine-induced NF-kappaB activation by altering IKK activity.

Amino Acid Sequence↗
PubMed2003

Increased toxicity and lack of efficacy of Rofecoxib in combination with chemotherapy for treatment of metastatic colorectal cancer: A phase II study.

Preclinical and clinical models have demonstrated that cyclooxygenase-2 (COX-2) is overexpressed in primary and metastatic colorectal tumors. In preclinical models, there appears to be additive or synergistic effects when combining 5-Fluorouracil (5-FU) with nonsteroidal anti-inflammatory agents (NSAIDs) for the treatment of colorectal neoplasms. This data raised the question as to whether adding a COX-2 inhibitor to 5-FU-based regimens would increase the response rates with an acceptable toxicity profile in patients with metastatic colon cancer. In the current study, patients with metastatic colorectal cancer, who were either untreated or previously treated (more than 1 year ago) with adjuvant 5-FU and Leucovorin (LV) received 5-FU and LV (Mayo regimen) in addition to Rofecoxib. Tumor samples from all patients exhibited evidence of moderate COX-2 over-expression. 4 patients entered on the study developed upper gastrointestinal bleeding (grade III). Other toxicities included grade II stomatitis (3 patients), grade II thrombocytopenia (1 patient), grade II diarrhea (2 patients) and grade I nausea (1 patient). There were no partial or complete responses in the first 10 patients entered on the study so the study was terminated (probability of success < 0.3 with type 1 error of 0.05 and power of 0.8). Thus, Rofecoxib did not appear to increase antitumor activity and resulted in increased gastrointestinal toxicity when combined with 5-FU/LV. Future studies will need to consider the added gastrointestinal toxicity of Rofecoxib when combined with chemotherapy for the treatment of patients with colorectal cancer.

Aged↗
PubMed2003

Heat shock protein 27 association with the I kappa B kinase complex regulates tumor necrosis factor alpha-induced NF-kappa B activation.

Heat shock protein 27 (Hsp27) is a ubiquitously expressed member of the heat shock protein family that has been implicated in various biological functions including the response to heat shock, oxidative stress, and cytokine treatment. Previous studies have demonstrated that heat shock proteins are involved in regulating signal transduction pathways including the NF-kappa B pathway. In this study, we demonstrated that Hsp27 associates with the I kappa B kinase (IKK) complex and that this interaction was stimulated by tumor necrosis factor alpha treatment. Phosphorylation of Hsp27 by the kinase mitogen-activated protein kinase-activated protein kinase 2, a downstream substrate of the mitogen-activated protein kinase p38, enhanced the association of Hsp27 with IKK beta to result in decreased IKK activity. Consistent with these observations, treatment of cells with a p38 inhibitor reduced the association of Hsp27 with IKK beta and thus resulted in increased IKK activity. These studies indicate that Hsp27 plays a negative role in down-regulating IKK signaling by reducing its activity following tumor necrosis factor alpha stimulation.

Base Sequence↗
PubMed2003

Functional correlates of mutations in beta-catenin exon 3 phosphorylation sites.

beta-Catenin-mediated signaling can be constitutively activated by truncation or mutation of serine and threonine residues in exon 3. Mutations in this region are observed in many human tumors. Examination of the locations of these mutations reveals interesting patterns; specifically, Ser45 and Thr41 appear more frequently in malignant tumors, and Ser37 and Ser33 are more common in benign entities. To test whether these patterns represent functional differences in beta-catenin signaling mechanisms, we generated mutations of each of these residues. Stable transformation of Madin-Darby canine kidney cells showed a transformed phenotype with each of the four mutations, as assessed by growth in soft agar and collagen. Functional assays including proliferation assays, cell shedding assays, and wounding assays demonstrated two groups. Ser45 and Thr41 represent a more transformed phenotype, whereas Ser37 and Ser33 behaved similarly to the vector in these assays. Assessment of downstream genes demonstrated increased activation of the beta-catenin target gene cyclin D1 by Ser45. Finally, we examined the kinase activity of I kappa B kinase-alpha and found that this kinase, unlike glycogen synthase kinase-3 beta, appears to preferentially phosphorylate Ser45 and Thr41, independent of priming by casein kinase-1. We conclude that these sites may represent an alternative (non-wnt) signaling pathway, which may be inappropriately activated in tumors with mutations of these residues.

Animals↗
PubMed2003

Histone H3 phosphorylation by IKK-alpha is critical for cytokine-induced gene expression.

Cytokine-induced activation of the IkappaB kinases (IKK) IKK-alpha and IKK-beta is a key step involved in the activation of the NF-kappaB pathway. Gene-disruption studies of the murine IKK genes have shown that IKK-beta, but not IKK-alpha, is critical for cytokine-induced IkappaB degradation. Nevertheless, mouse embryo fibroblasts deficient in IKK-alpha are defective in the induction of NF-kappaB-dependent transcription. These observations raised the question of whether IKK-alpha might regulate a previously undescribed step to activate the NF-kappaB pathway that is independent of its previously described cytoplasmic role in the phosphorylation of IkappaBalpha. Here we show that IKK-alpha functions in the nucleus to activate the expression of NF-kappaB-responsive genes after stimulation with cytokines. IKK-alpha interacts with CREB-binding protein and in conjunction with Rel A is recruited to NF-kappaB-responsive promoters and mediates the cytokine-induced phosphorylation and subsequent acetylation of specific residues in histone H3. These results define a new nuclear role of IKK-alpha in modifying histone function that is critical for the activation of NF-kappaB-directed gene expression.

Animals↗
PubMed2003

Identification of NF-kappa B-regulated genes induced by TNFalpha utilizing expression profiling and RNA interference.

Tumor necrosis factor alpha (TNF alpha) is a proinflammatory cytokine with important roles in regulating inflammatory responses as well as cell cycle proliferation and apoptosis. Although TNFalpha stimulates apoptosis, it also activates the transcription factor NF-kappa B, and studies have shown that inhibition of NF-kappa B potentiates the cytotoxicity of TNFalpha. Since several chemotherapy agents act like TNFalpha to both promote apoptosis and activate NF-kappa B, understanding the role of NF-kappa B in suppressing apoptosis may have significant clinical applications. To understand the effects of stimulation with TNFalpha and the role of NF-kappa B in regulating this response, a 23k human cDNA microarray was used to screen TNFalpha-inducible genes in HeLa cells. Real-time PCR verified expression changes in 16 of these genes and revealed three distinct temporal patterns of expression after TNFalpha stimulation. Using RNA interference to disrupt expression of the p65 subunit of NF-kappa B, all but two of the genes were shown to depend on this transcription factor for their expression, which correlated well with the existence of NF-kappa B binding sites in most of their promoters. Inflammatory, proapoptotic, and antiapoptotic genes were all shown to be regulated by NF-kappa B, demonstrating the wide variety of targets activated by NF-kappa B signaling and the necessity of differentiating among these genes for therapeutic purposes.

Computer Systems↗
PubMed2003

TAK1 is critical for IkappaB kinase-mediated activation of the NF-kappaB pathway.

Cytokine treatment stimulates the IkappaB kinases, IKKalpha and IKKbeta, which phosphorylate the IkappaB proteins, leading to their degradation and activation of NF-kappaB regulated genes. A clear definition of the specific roles of IKKalpha and IKKbeta in activating the NF-kappaB pathway and the upstream kinases that regulate IKK activity remain to be elucidated. Here, we utilized small interfering RNAs (siRNAs) directed against IKKalpha, IKKbeta and the upstream regulatory kinase TAK1 in order to better define their roles in cytokine-induced activation of the NF-kappaB pathway. In contrast to previous results with mouse embryo fibroblasts lacking either IKKalpha or IKKbeta, which indicated that only IKKbeta is involved in cytokine-induced NF-kappaB activation, we found that both IKKalpha and IKKbeta were important in activating the NF-kappaB pathway. Furthermore, we found that the MAP3K TAK1, which has been implicated in IL-1-induced activation of the NF-kappaB pathway, was also critical for TNFalpha-induced activation of the NF-kappaB pathway. TNFalpha activation of the NF-kappaB pathway is associated with the inducible binding of TAK1 to TRAF2 and both IKKalpha and IKKbeta. This analysis further defines the distinct in vivo roles of IKKalpha, IKKbeta and TAK1 in cytokine-induced activation of the NF-kappaB pathway.

Enzyme Activation↗
PubMed2003

Methylation of SPT5 regulates its interaction with RNA polymerase II and transcriptional elongation properties.

SPT5 and its binding partner SPT4 function in both positively and negatively regulating transcriptional elongation. The demonstration that SPT5 and RNA polymerase II are targets for phosphorylation by CDK9/cyclin T1 indicates that posttranslational modifications of these factors are important in regulating the elongation process. In this study, we utilized a biochemical approach to demonstrate that SPT5 was specifically associated with the protein arginine methyltransferases PRMT1 and PRMT5 and that SPT5 methylation regulated its interaction with RNA polymerase II. Specific arginine residues in SPT5 that are methylated by these enzymes were identified and demonstrated to be important in regulating its promoter association and subsequent effects on transcriptional elongation. These results suggest that methylation of SPT5 is an important posttranslational modification that is involved in regulating its transcriptional elongation properties in response to viral and cellular factors.

Arginine↗