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Biomedical subjects

Ann M Bode

Publications and source records attributed to Ann M Bode.

At least 37 records · Page 2Linked to original sources
PubMed2005

2-Arachidonoylglycerol stimulates activator protein-1-dependent transcriptional activity and enhances epidermal growth factor-induced cell transformation in JB6 P+ cells.

2-Arachidonoylglycerol (2-AG) is the most abundant endocannabinoid, and it plays a critical role in cannabinoid receptor-mediated cell signaling. Although 2-AG was shown to induce ERK activation via the cannabinoid receptor 1 (CB1), only a nonspecific CB receptor agonist and antagonist was used in those studies. Whether cannabinoid receptor 2 (CB2) is involved in 2-AG-induced ERK activation is still unclear. Moreover, whether 2-AG is involved in mediation of AP-1 activity and cell transformation is also not known. In the present study, we show that 2-AG stimulates AP-1-dependent transcriptional activity and enhances epidermal growth factor-induced cell transformation in mouse epidermal JB6 P+ Cl41 cells. Using JB6 P+ C141 cells, stably transfected with an AP-1 luciferase reporter, we found that 10 microm 2-AG induced up to a 3-fold stimulation of AP-1 transcriptional activity. The AP-1 stimulation appeared to be mediated by ERK but not JNK or p38 kinase. PD98059, a specific inhibitor of MEK1, almost completely blocked 2-AG-induced ERK phosphorylation and AP-1 activation. Using CB1/2-/- murine embryonic fibroblasts, we present the first direct evidence that both cannabinoid receptors 1 and 2 (CB1/2) are involved in 2-AG-induced ERK activation. 2-AG could not stimulate ERK phosphorylation or Fyn kinase activity in dominant negative Fyn. In addition, the Fyn inhibitor PP2 blocked 2-AG-induced Fyn kinase activity and ERK phosphorylation and activity. Small interfering RNA Fyn also suppressed 2-AG-induced ERK phosphorylation. Interestingly, 2-AG enhanced epidermal growth factor-induced AP-1 DNA binding and cell transformation. Taken together, our data provide direct evidence suggesting that 2-AG may have a novel role in cell transformation and carcinogenesis in a signaling pathway involving CB1/2 and activation of Fyn, ERKs, and AP-1.

Animals↗
PubMed2005

The p53 protein is a novel substrate of ribosomal S6 kinase 2 and a critical intermediary for ribosomal S6 kinase 2 and histone H3 interaction.

The tumor suppressor p53 protein is one of the most highly connected nodes in cellular signal transduction pathways and acts as a central regulatory switch in networks controlling cell proliferation and apoptosis. It is involved in the activation of genes that maintain control over cellular responses to DNA errors such as DNA repair, chromosomal recombination, and chromosome segregation. Here we show that ribosomal S6 kinase 2 (RSK2) activates and phosphorylates p53 (Ser15) in vitro and in vivo and colocalizes with p53 in the nucleus. Deficiency of p53 diminishes RSK2-mediated phosphorylation of histone H3 (Ser10) and adding back p53 to p53-/- embryonic fibroblasts restored phosphorylation of histone H3 at Ser10. These results show that the p53 protein is an important substrate of RSK2 and a critical intermediary in the RSK2 and histone H3 interaction. The RSK2-p53-histone H3 complex may likely contribute to chromatin remodeling and cell cycle regulation.

Animals↗
PubMed2005

Inducible covalent posttranslational modification of histone H3.

The physiological state of a eukaryotic cell is determined by endogenous and exogenous signals, and often the endpoint of the pathways that transmit these signals is DNA. DNA is organized into chromatin, a nucleoprotein complex, which not only facilitates the packaging of DNA within the nucleus but also serves as an important factor in the regulation of gene function. The nucleosome is the basic unit of chromatin and generally consists of approximately two turns of DNA wrapped around an octamer of core histone proteins. Each histone also contains an accessible N-terminal tail that extends outside the chromatin complex and is subject to posttranslational modifications that are crucial in the regulation of gene expression. Two distinct categories of histone posttranslational modification have been observed: (i) inducible or stimulation-dependent and (ii) mitosis-dependent. Stimulation by mitogens or stress leads to rapid transient posttranslational modifications of histones, in particular histone H3, which are mechanistically and temporarily distinct from modifications associated with mitosis. This Review focuses mainly on the inducible phosphorylation of histone H3 brought about by different stimuli, such as epidermal growth factor, 12-O-tetradecanoylphorbol-13-acetate, arsenite, or ultraviolet radiation. We examine the most recent, and at times controversial, research data concerning the identity of the histone H3 kinases responsible for this phosphorylation. In addition, the interdependence of phosphorylation and acetylation will be discussed in light of data showing patterns of inducible modification at specific genes.

Acetylation↗
PubMed2005

The intermediate filament protein vimentin is a new target for epigallocatechin gallate.

Epigallocatechin gallate (EGCG) is the major active polyphenol in green tea. Protein interaction with EGCG is a critical step in the effects of EGCG on the regulation of various key proteins involved in signal transduction. We have identified a novel molecular target of EGCG using affinity chromatography, two-dimensional electrophoresis, and mass spectrometry for protein identification. Spots of interest were identified as the intermediate filament, vimentin. The identification was confirmed by Western blot analysis using an anti-vimentin antibody. Experiments using a pull-down assay with [3H]EGCG demonstrate binding of EGCG to vimentin with a Kd of 3.3 nm. EGCG inhibited phosphorylation of vimentin at serines 50 and 55 and phosphorylation of vimentin by cyclin-dependent kinase 2 and cAMP-dependent protein kinase. EGCG specifically inhibits cell proliferation by binding to vimentin. Because vimentin is important for maintaining cellular functions and is essential in maintaining the structure and mechanical integration of the cellular space, the inhibitory effect of EGCG on vimentin may further explain its anti-tumor-promoting effect.

Amino Acid Sequence↗
PubMed2005

Phosphorylation of Ser28 in histone H3 mediated by mixed lineage kinase-like mitogen-activated protein triple kinase alpha.

The mitogen-activated protein kinase cascades elicit modification of chromatin proteins such as histone H3 by phosphorylation concomitant with gene activation. Here, we demonstrate for the first time that the mixed lineage kinase-like mitogen-activated protein triple kinase (MLTK)-alpha phosphorylates histone H3 at Ser28. MLTK-alpha but neither a kinase-negative mutant of MLTK-alpha nor MLTK-beta interacted with and phosphorylated histone H3 in vivo and in vitro. When overexpressed in 293T or JB6 Cl41 cells, MLTK-alpha phosphorylated histone H3 at Ser28 but not at Ser10. The interaction between MLTK-alpha and histone H3 was enhanced by stimulation with ultraviolet B light (UVB) or epidermal growth factor (EGF), which resulted in the accumulation of MLTK-alpha in the nucleus. UVB- or EGF-induced phosphorylation of histone H3 at Ser28 was not affected by PD 98059, a MEK inhibitor, or SB 202190, a p38 kinase inhibitor, in MLTK-alpha-overexpressing JB6 Cl41 cells. Significantly, UVB- or EGF-induced phosphorylation of histone H3 at Ser28 was blocked by small interfering RNA of MLTK-alpha. The inhibition of histone H3 phosphorylation at Ser28 in the MLTK-alpha knock-down JB6 Cl41 cells was not due to a defect in mitogen- and stress-activated protein kinase 1 or 90-kDa ribosomal S6 kinase (p90RSK) activity. In summary, these results illustrate that MLTK-alpha plays a key role in the UVB- and EGF-induced phosphorylation of histone H3 at Ser28, suggesting that MLTK-alpha might be a new histone H3 kinase at the level of mitogen-activated protein kinase kinase kinases.

Amino Acid Sequence↗
PubMed2004

The signal transduction networks required for phosphorylation of STAT1 at Ser727 in mouse epidermal JB6 cells in the UVB response and inhibitory mechanisms of tea polyphenols.

Signal transducers and activators of transcription (STATs) play a critical role in signal transduction pathways. STATs are a family of cytoplasmic proteins with roles as signal messengers and transcription factors that participate in normal cellular responses to cytokines and growth factors. Phosphorylation of STAT1 at Ser727 is essential for its activation and occurs in response to stress signals, inflammation or infection. We observed that UVB induced phosphorylation of STAT1 (Ser727) in mouse epidermal JB6 Cl41 cells. This stimulation was inhibited by PD98059 and UO126, wortmannin, LY294002, SB202190 and SP600125 and dominant negative mutants of ERK2 (DNM-ERK2), p38 (DNM-p38) and JNK1 (DNM-JNK1). The response was absent in Jnk1(-/-) or Jnk2(-/-) knockout cells, but was unaffected by a dominant negative mutant of the phosphatidylinositol-3 kinase (PI-3K) p85 subunit (DNM-Deltap85). STAT1 (Ser727) phosphorylation was also blocked in a Rsk2(-) cell line. In Pdk1(-/-) cells STAT1 was not activated by UVB stimulation compared with strong activation in Pdk1(+/+) cells. Our data indicate that phosphorylation of STAT1 (Ser727) occurs through PI-3K, ERKs, p38 kinase, JNKs, PDK1 and p90RSK2 in the cellular response to UVB. We also show an inhibitory effect of theaflavins and EGCG on UVB-induced STAT1 (Ser727), ERKs, JNKs, PDK1 and p90RSK2 phosphorylation.

Animals↗
PubMed2004

Regulation of ultraviolet B-induced phosphorylation of histone H3 at serine 10 by Fyn kinase.

Ultraviolet B (UVB) induces phosphorylation of histone H3 at serine 10, and mitogen-activated protein kinases are involved in this signal transduction pathway. Here we provide evidence that Fyn kinase, a member of the Src kinase family, is involved in the UVB-induced phosphorylation of histone H3 at serine 10. UVB distinctly increased Fyn kinase activity and phosphorylation. Fyn kinase inhibitors 4-amino-5-(4-chlorophenyl)-7(t-butyl)pyrazol(3,4-d)pyramide and leflunomide, an Src kinase inhibitor, suppressed both UVB-induced phosphorylation of histone H3 at serine 10 and Fyn kinase activity and phosphorylation. UVB-induced phosphorylation of histone H3 at serine 10 was blocked by either a dominant-negative mutant of Fyn (DNM-Fyn) kinase or small interfering RNA of Fyn kinase. UVB-induced phosphorylation and activities of ERKs and protein kinase B/Akt were markedly inhibited by DNM-Fyn kinase. However, DNM-Fyn kinase did not inhibit UVB-induced phosphorylation of p38 MAPK or c-Jun N-terminal kinases. Active Fyn kinase phosphorylated histone H3 at serine 10 in vitro, and the phosphorylated Fyn kinase could translocate into the nucleus of HaCaT cells. These results indicate that Fyn kinase plays a key role in the UVB-induced phosphorylation of histone H3 at serine 10.

Animals↗
PubMed2004

Involvement of ERKs and mitogen- and stress-activated protein kinase in UVC-induced phosphorylation of ATF2 in JB6 cells.

Activating transcription factor 2 (ATF2) has been shown to regulate gene expression in the cellular response to environmental stresses such as ultraviolet (UV) irradiation. However, the signal transduction mechanism of ATF2 activation by UV is not as yet completely understood. In the present study, we provide evidence showing that UVC-stimulated phosphorylation of ATF2 (Thr71) was to varying degrees prevented by a dominant negative mutant of p38beta kinase, c-Jun N-terminal kinase 1 (JNK1) or extracellular signal-regulated kinase 2 (ERK2). The phosphorylation was also suppressed by PD98059, an MEK inhibitor, or H89, a potent inhibitor of mitogen- and stress-activated protein kinase 1 (MSK1), and a C- or N-terminal 'kinase-dead' mutant of MSK1 (MSK1-Cd or MSK1-Nd). Furthermore, co- immunoprecipitation experiments revealed a potential intracellular signaling complex consisting of ATF2 and ERKs and/or MSK1. In vitro kinase assays revealed that ERK1, ERK2 and MSK1, like p38 kinase and JNK2, directly phosphorylate ATF2 at Thr71, but addition of RSK2 or Akt1 had almost no effect. Active kinase immunoprecipitated by an MSK1, ERKs or p38 antibody from an extract of JB6 cells irradiated by UVC can directly phosphorylate ATF2 at Thr71, suggesting UVC induces a direct phosphorylation of ATF2 by ERKs or MSK1. Overall, our results reveal that MSK1 and ERKs, like p38 kinase and JNKs, are required for ATF2 phosphorylation (Thr71) in the UVC response.

Activating Transcription Factor 2↗
PubMed2004

A novel role for mixed-lineage kinase-like mitogen-activated protein triple kinase alpha in neoplastic cell transformation and tumor development.

Previously, no member of the mixed-lineage kinase (MLK) protein family was known to function as an oncogene. Here, we demonstrate that MLK-like mitogen-activated protein triple kinase (MLTK)-alpha, a member of the MLK family, induced neoplastic cell transformation and tumorigenesis in athymic nude mice. Introduction of small interference RNA (siRNA)-MLTK-alpha into MLTK-alpha-overexpressing cells dramatically suppressed cell transformation. Nuclear accumulation of the pHisG-MLTK-alpha fusion protein was observed after epidermal growth factor or 12-O-tetradecanoylphorbol-13-acetate treatment. Phosphorylation of downstream mitogen-activated protein kinase-targeted transcription factors including c-Myc, Elk-1, c-Jun, and activating transcription factor (ATF) 2 was also differentially enhanced in MLTK-alpha-overexpressing cells exposed to epidermal growth factor or 12-O-tetradecanoylphorbol-13-acetate stimulation compared with cells expressing mock vector or siRNA-MLTK-alpha. Very importantly, MLTK-alpha-overexpressing cells formed fibrosarcomas when injected s.c. into athymic nude mice, whereas almost no tumor formation was observed in mice that received injections of mock or siRNA-MLTK-alpha stably transfected cells. These results are the first to indicate that MLTK-alpha plays a key role in neoplastic cell transformation and cancer development.

Animals↗
PubMed2004

Caffeine inhibits cell proliferation by G0/G1 phase arrest in JB6 cells.

Caffeine is a major biologically active constituent in coffee and tea. Because caffeine has been reported to inhibit carcinogenesis in UVB-exposed mice, the cancer-preventing effect of caffeine has attracted considerable attention. In the present study, the effect of caffeine in quiescent (G0 phase) cells was investigated. Pretreatment with caffeine suppressed cell proliferation in a dose-dependent manner 36 h after addition of fetal bovine serum as a cell growth stimulator. Analysis by flow cytometry showed that caffeine suppressed cell cycle progression at the G0/G1 phase, i.e., 18 h after addition of fetal bovine serum, the percentages of cells in G0/G1 phase in 1 mM caffeine-treated cells and in caffeine-untreated cells were 61.7 and 29.0, respectively. The percentage of cells in G0/G1 phase at 0 h was 75.5. Caffeine inhibited phosphorylation of retinoblastoma protein at Ser780 and Ser807/Ser811, the sites where retinoblastoma protein has been reported to be phosphorylated by cyclin-dependent kinase 4 (cdk4). Furthermore, caffeine inhibited the activation of the cyclin D1-cdk4 complex in a dose-dependent manner. However this compound did not directly inhibit the activity of this complex. In addition, caffeine did not affect p16INK4 or p27Kip1 protein levels, but inhibited the phosphorylation of protein kinase B (Akt) and glycogen synthase kinase 3beta. Our results showed that caffeine suppressed the progression of quiescent cells into the cell cycle. The inhibitory mechanism may be due to the inhibition of cell growth signal-induced activation of cdk4, which may be involved in the inhibition of carcinogenesis in vivo.

Animals↗
PubMed2004

Marine alkaloid polycarpine and its synthetic derivative dimethylpolycarpine induce apoptosis in JB6 cells through p53- and caspase 3-dependent pathways.

PURPOSE: Polycarpine from ascidian Polycarpa aurata was previously found to be active against different human tumor cells. In this study, we investigated the antitumor mechanisms of polycarpine and its synthetic derivative, desmethoxyethoxy-polycarpine (dimethylpolycarpine), through the induction of apoptosis. This new knowledge regarding the proapoptotic action of polycarpine and dimethylpolycarpine should lead to a better understanding of their effects and development of a new class of anticancer drugs. METHODS: Apoptosis was clearly observed by flow cytometry and Western blotting using an antibody against cleaved caspase-3 as an apoptotic marker. RESULTS: Polycarpines differentially activated p38 kinase, JNKs, and ERKs in JB6 Cl 41 cells. The polycarpines-induced apoptosis was decreased in cells expressing a dominant-negative mutant of JNK. Both compounds stimulated p53-dependent transcriptional activity and phosphorylation. Induction of p53-phosphorylation at serine 15 was suppressed in JNKI and JNK2 knockout cells. Furthermore, polycarpines were unable to induce apoptosis in p53-deficient MEFs in contrast to a strong induction of apoptosis in wild type MEFs, suggesting that p53 is involved in apoptosis induced by polycarpines. The p53 phosphorylation in turn was mediated by activated JNKs. CONCLUSIONS: These results indicate that all three MAPK signaling pathways are involved in the response of JB6 cells to treatment with polycarpines. Evidence also supports a proapoptotic role of the JNKs signaling pathway in vivo and clearly indicates that JNKs are required for phosphorylation of c-Jun, activation of p53, and subsequent apoptosis induced by polycarpines.

Alkaloids↗
PubMed2004

Post-translational modification of p53 in tumorigenesis.

Interest in the tumour suppressor p53 has generated much information regarding the complexity of its function and regulation in carcinogenesis. However, gaps still exist in our knowledge regarding the role of p53 post-translational modifications in carcinogenesis and cancer prevention. A thorough understanding of p53 will be extremely useful in the development of new strategies for treating and preventing cancer, including restoration of p53 function and selective killing of tumours with mutant TP53.

Acetylation↗
PubMed2003

Induction of apoptosis by caffeine is mediated by the p53, Bax, and caspase 3 pathways.

Caffeine is a key component of many popular drinks, especially tea and coffee. Previous reports have shown that caffeine may contribute to the chemopreventive effect of tea in animals. Here, we report that treatment with low concentrations of caffeine induced apoptosis in JB6 Cl41 cells. JB6 Cl41 cells were starved in 0.1% fetal bovine serum/MEM for 72 h and then treated with 50-450 microM caffeine for 24 h. Cells showed the typical DNA laddering pattern and other characteristics of apoptosis. The IC(50) of caffeine on JB6 Cl41 cells was 2.7 mM. Induction of apoptosis by caffeine appeared to be p53-dependent because cells lacking p53 (p53(-/-)) showed no signs of apoptosis after treatment with caffeine. Immunoprecipitation assays and Western blot analysis showed that caffeine induced phosphorylation of p53 at Ser(15) in JB6 Cl41 cells. The same low concentration of caffeine that was effective for inducing phosphorylation of p53 was also shown to increase p53 activation. Expression of Bax, another p53 target, distinctly increased in a time- and dose-dependent manner. Cleaved caspase 3 was also increased in a time- and dose-dependent manner. These data show that a low concentration of caffeine can induce p53-dependent apoptosis in JB6 cells through the Bax and caspase 3 pathways.

Animals↗
PubMed2003

p38 Mitogen-activated protein kinase regulation of JB6 Cl41 cell transformation promoted by epidermal growth factor.

The relationship between cell transformation and p38 MAP kinase, a major mitogen-activated protein (MAP) kinase pathway converting signals of various extracellular stimuli into expression of specific target genes through activation of transcription factors, still remains unclear. The aim of the present study was to investigate the role of the p38 MAP kinase pathway in epidermal growth factor (EGF)-induced cell transformation in JB6 cells. Our data show that a dominant negative mutant of p38 MAP (DN-p38) kinase inhibits EGF-promoted JB6 Cl41 cell transformation and that SB202190, an inhibitor of p38 MAP kinase, also inhibits JB6 Cl41 cell transformation in a dose-dependent manner. Moreover, our results show that DN-p38 MAP kinase inhibits the phosphorylation of EGF-stimulated activating transcription factor-2 (ATF-2) and signal transducer and activator of transcription 1 (STAT1). Additionally, DN-p38 MAP kinase inhibits EGF-induced phosphorylation of c-Myc (Thr58/Ser62). Gel shift assays indicate that DN-p38 MAP kinase inhibits EGF-induced activator protein-1 (AP-1) DNA binding in a dose-dependent manner. These results show that p38 MAP kinase plays a key role in the regulation of EGF-induced cell transformation in JB6 cells through regulation of phosphorylation of p38 MAP kinase and activation of its target genes in phosphorylation, c-Myc cell transformation-related genes, and AP-1 binding ability.

Activating Transcription Factor 2↗
PubMed2003

Ataxia telangiectasia mutated proteins, MAPKs, and RSK2 are involved in the phosphorylation of STAT3.

Phosphorylation at Ser(727) is known to be required for complete activation of STAT3 by diverse stimuli including UV irradiation, but the kinase(s) responsible for phosphorylating STAT3 (Ser(727)) is still not well discerned. In the present study, we observed that activation of ATM is required for a UVA-stimulated increase in Ser(727) phosphorylation of STAT3 as well as in activation and phosphorylation of p90 ribosomal protein S6 kinases (RSKs). Moreover, UVA-stimulated activation of upstream kinases, such as c-Jun N-terminal kinases (JNKs) and ERKs, involved in mediating phosphorylation of RSKs and STAT3 was defective or delayed in ATM-deficient cells. Furthermore, we provide evidence that RSK2-deficient cells were defective for UV-induced Ser(727) phosphorylation of STAT3, and the defect was restored after ectopic expression of transfected full-length RSK2. In vitro experiments showed that active RSK2 and JNK1 induce the phosphorylation of STAT3 precipitates from immunoprecipitation but not from glutathione S-transferase (GST) pull-down. Interestingly, the GST fusion STAT3 proteins mixed together with STAT3 immunoprecipitates can be phosphorylated by JNK. However, the in vitro phosphorylation of STAT3 was reduced by the GST-STAT3 beta protein, a dominant negative form of STAT3. Taken together, our results demonstrate that the STAT3 phosphorylation at Ser(727) is triggered by active RSK2 or JNK1 in the presence of a downstream kinase or a cofactor, and thereby the intracellular phosphorylation process is stimulated through a signaling pathway involving ATM, MAPKs, RSK2, and an as yet unidentified kinase or cofactor. Additionally, RSK2-mediated phosphorylation of STAT3 (Ser(727)) was further determined to be required for basal and UVA-stimulated STAT3 transcriptional activities.

Acute-Phase Proteins↗
PubMed2003

Mitogen-activated protein kinase activation in UV-induced signal transduction.

Experimental evidence supported by epidemiological findings suggests that solar ultraviolet (UV) irradiation is the most important environmental carcinogen leading to the development of skin cancers. Because the ozone layer blocks UVC (wavelength, 180 to 280 nm) exposure, UVA (UVA I, 340 to 400 nm; UVA II, 320 to 340 nm) and UVB (280 to 320 nm) are probably the chief carcinogenic components of sunlight with relevance for human skin cancer. Substantial contributions to the elucidation of the specific signal transduction pathways involved in UV-induced skin carcinogenesis have been made over the past few years, and most evidence suggests that the cellular signaling response is UV wavelength-dependent. The mitogen-activated protein kinase (MAPK) signaling cascades are targets for UV and are important in the regulation of the multitude of UV-induced cellular responses. Experimental studies have used a range of UVA, UVB, UVC, and various combinations in multiple doses, and the observed effects on activation and phosphorylation of MAPKs are varied. This review focuses on the mechanistic data supporting a role for MAPKs in UV-induced skin carcinogenesis. Progress in understanding the mechanisms of UV-induced signal transduction could lead to the use of these protein kinases as specific targets for the prevention and control of skin cancer.

Animals↗