Inhibitors of histone deacetylase arrest cell cycle and induce apoptosis in cervical carcinoma cells circumventing human papillomavirus oncogene expression.

Histone deacetylase (HDAC) inhibitors sodium butyrate and trichostatin A arrest human papillomavirus (HPV)-positive carcinoma cells in G1 to S transition of the cell cycle, which is paralleled by an up-regulation of the cyclin-dependent kinase inhibitors (CKIs) p21CIP1 and p27KIP1 as well as the complete loss of cdk2 activity. Although HPV expression was hitherto thought to be required to maintain a proliferative phenotype of these cells, cdk2 suppression is achieved even in the presence of ongoing viral transcription. While CKIs normally cannot exert their cdk2-inhibitory function in the presence of the viral oncoprotein E7, co-immunoprecipitation experiments revealed that E7 binding is prevented. Increase of p27KIP1 correlates with down-regulation of p45SKP2, a component of the ubiquitin-protein ligase SCF(SKP2) controlling the half-life of regulatory proteins during the cell cycle. HDAC inhibition also triggered an E7-dependent degradation of pRb, while the levels of E2F remained unaffected. The presence of free intracellular E2F and the concomitant up-regulation of CKIs during G1 arrest results in a 'conflicting growth situation', which finally renders the cells to undergo apoptosis. These data provide novel molecular insights into how the transforming potential of HPV can be bypassed and open new therapeutical perspectives for the treatment of cervical cancer.

Differential transcriptional regulation of the monocyte-chemoattractant protein-1 (MCP-1) gene in tumorigenic and non-tumorigenic HPV 18 positive cells: the role of the chromatin structure and AP-1 composition.

The expression of the monocyte-chemoattractant-protein-1 (MCP-1) is closely linked with a non-tumorigenic phenotype in somatic cell hybrids made between the human papillomavirus type 18 (HPV 18) positive cervical carcinoma cell line HeLa and normal human fibroblasts. In contrast, MCP-1 transcription is absent in tumorigenic segregants derived from the same hybrids or in parental HeLa cells. Selectivity of MCP-1 transcription, which is regulated at the level of initiation of transcription, is mainly based on differences in the location and extension of DNAse I-hypersensitive regions (DHSR) at both ends of the gene. While TNF-alpha only moderately increases the sensitivity of pre-existing 5'-DHSRs, a 3'-end DHSR became strongly induced exclusively in non-malignant hybrids. DNA sequencing showed that the 3'-DHSR coincides with an additional AP-1 site located approximately 600 bp downstream of the polyadenylation site. Analyses of AP-1 composition revealed that MCP-1 is only expressed in those cells where jun-family members were mainly heterodimerized with the fos-related protein fra-1. In contrast, in tumorigenic cells the 1: 1 ratio between jun and fra-1 is disturbed and the MCP-1 gene is no longer expressed. Hence, alterations in the heterodimerization pattern of AP-1 and its selective accessibility to opened chromatin may represent a novel regulatory pathway in the regulation of chemokines in malignant and non-malignant HPV-positive cells.

Genetic complementation to non-tumorigenicity in cervical-carcinoma cells correlates with alterations in AP-1 composition.

The transcription factor AP-1 represents a central key element in the expression of human pathogenic papillomaviruses (HPV). We here propose a novel role for AP-1 as an essential component of an intracellular surveillance mechanism negatively controlling the proliferation of HPV-positive cells under in vivo conditions. The dissection of AP-1 composition in cervical-carcinoma cells revealed an inverse relationship between the Fos-related antigen Fra-1 and the tumorigenic phenotype. Cervical-carcinoma cell lines were either negative or expressed only low amounts of Fra-1 (jointly with c-Fos) within their AP-1 complexes. Somatic-cell hybridization technique was used to fuse different HPV-positive malignant cell lines. This resulted either in tumorigenic hybrids or in cells in which the malignant phenotype of the parental fusion partners was completely suppressed. The monitoring of AP-1 composition in electrophoretic mobility super-shift assays showed that the amount of Fra-1 was substantially increased within the AP-1 complex of non-malignant cells. In contrast, Fra-1 was even diminished in malignant hybrids, while c-Fos remained expressed. This correlation suggests that the concentration of Fra-1 within the AP-1 transcription complex might be an important marker for predicting the in vivo growth properties of HPV-positive cells.

Endothelin-1 and smooth muscle cells: induction of jun amino-terminal kinase through an oxygen radical-sensitive mechanism.

Endothelin-1 (ET-1) has been proposed to contribute to atherogenesis and plaque rupture in coronary heart disease through activation of mitogen-activated protein kinases (MAPKs) in smooth muscle cells (SMCs). Reactive oxygen species (ROS) have been shown to be important signal transduction molecules in SMCs. Thus, the present study aimed to assess the role of ROS in ET-1-mediated activation of c-Jun amino-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) 1/2. Rat SMCs were exposed to ET-1 over time at concentrations from 10(-6) to 10(-10) mol/L, and MAPK activity was quantified. Activation of JNK and ERK was observed with a maximum stimulation at 10(-7) mol/L ET-1. JNK and ERK were activated by ET-1 binding to a single receptor (ET-1A) but differed in their downstream mechanisms: only JNK activation was sensitive to the radical scavenger N-acetylcysteine and diphenylene iodonium, an inhibitor of NADPH oxidase, indicating a role for ROS. The downstream MAPK effector and proinflammatory transcription factor, the activator protein-1 complex, was maximally activated 2 hours after the addition of ET-1. It was mainly composed of the JNK substrate c-Jun, and activation was also dependent on ROS formation. We suggest that plaque activation by ET-1 can be mediated through ROS. It can be hypothesized that the clinical benefit of antioxidants in the treatment of atherogenesis may partially depend on neutralization of ET-1-mediated ROS production.

Differential activation of mitogen-activated protein kinases in smooth muscle cells by angiotensin II: involvement of p22phox and reactive oxygen species.

The atherogenic effect of the renin-angiotensin system can be explained, in part, by the influence of its effector, angiotensin II (Ang II), on vascular smooth muscle cell (VSMC) growth. There is evidence that reactive oxygen species (ROS) play a role in the atherogenesis and activation of mitogen-activating protein (MAP) kinases, which are involved in proliferation and differentiation. The study was performed to further characterize the role of ROS in Ang II-mediated MAP kinase activation and the regulation of the transcription factor activator protein-1 (AP-1). Rat VSMCs were stimulated with Ang II. The activities of MAP kinases were assessed by Western blot analysis or by immunocomplex kinase assay. AP-1 binding was determined by using an electrophoretic mobility shift assay. Rat VSMCs were treated with Ang II-activated MAP kinases, extracellular signal-regulated kinase (ERK), c-Jun amino terminal kinase (JNK), p38 MAP kinase (p38 MAPK), and their downstream effector, AP-1. Interestingly, only the activation of ERK1/2, but not JNK or p38 MAPK, was tyrosine kinase, protein kinase C, and MEK1/2 dependent. Ang II also induced the rapid formation of ROS, which could be inhibited by a specific antibody as well as by antisense against the p22phox subunit of the NAD(P)H oxidase. JNK and p38 MAPK, but not ERK, activation was inhibited by an inhibitor of NAD(P)H oxidase. Antisense against p22phox also solely inhibited p38 MAPK but did not affect ERK. The results indicate that in VSMCs, Ang II activates MAP kinases and AP-1 through different pathways; the results further suggest that ROS, generated by p22phox, mediate Ang II-induced JNK and p38 MAPK activation, which may contribute to the pathogenesis of atherosclerosis.

Conversion of HPV 18 positive non-tumorigenic HeLa-fibroblast hybrids to invasive growth involves loss of TNF-alpha mediated repression of viral transcription and modification of the AP-1 transcription complex.

AP-1 represents a transcription factor, which plays a pivotal role in initiating and maintaining the expression of human papillomavirus (HPV) oncoproteins E6 and E7 during HPV-linked carcinogenesis of the uterine cervix. AP-1 stands as a synonym for different proteins such as c-Jun, JunB, JunD, c-Fos, FosB as well as the Fos-related antigens Fra-1 and Fra-2, which can either homo- or heterodimerize to build up a functional transcription complex. AP-1 is mainly considered as a positive regulator, which binds to cognate DNA sequences within the viral upstream regulatory region. By using non-tumorigenic HeLa-fibroblast hybrids ('444'), their tumorigenic segregants ('CGL3') as well as HPV 18 positive HeLa cells as a experimental model system, evidence is provided that AP-1 composition differs considerably between these cell lines. In nuclear extracts obtained from non-tumorigenic cells, Jun-family members (in the order c-Jun>JunD>JunB) were mainly heterodimerized with Fra-1, a protein, known to be involved in the abrogation of AP-1 activity under certain experimental conditions. In contrast, Fra-1 concentration is low in extracts from tumorigenic cells. Conversely, c-Fos, the canonical dimerization partner of Jun proteins is expressed in substantial quantity in HeLa- and 'CGL3' cells, but it is completely absent in AP-1 complexes from non-tumorigenic '444' cells. Ectopical expression of c-fos under a heterologous promoter in '444'-cells induces tumorigenicity and a change of the Jun/Fra-1 ratio towards a constellation initially detected in 'CGL3'-and HeLa cells. Furthermore, conversion to tumorigenicity is accompanied with a resistance against TNF-alpha, a cytokine, capable to selectively suppress HPV 18 transcription in formerly non-malignant cells. These data propose a novel role for AP-1 as an essential component of an inter- and intracellular surveillance mechanism negatively controlling HPV transcription in non-tumorigenic cells.

Import of firefly luciferase into mammalian peroxisomes in vivo requires nucleoside triphosphates.

The insect enzyme firefly luciferase (FL), which is known to be imported into mammalian peroxisomes as well, was introduced into the cytoplasm of chinese hamster ovary cells and human skin fibroblasts by microinjection. This model system was used to study the nucleoside triphosphate dependence of peroxisomal protein import by immunofluorescence staining of FL following depletion of cellular ATP. In energized cells a punctate staining pattern of the enzyme is observed between 30 min and 1 week after microinjection suggesting an organellar localization of FL. Evidence for its peroxisomal localization was gained by comparison of the FL staining pattern with that of catalase, a peroxisomal marker. Differential permeabilization of cells with digitonin prior to immunofluorescence staining demonstrated the intraperoxisomal localization of microinjected FL and excluded the possibility that FL is merely adhering at the cytosolic face of peroxisomes without being imported. Depletion of cellular ATP by the metabolic inhibitors 2-deoxyglucose and NaN3 completely prevented import of FL into peroxisomes whereas upon reenergizing the cells FL import was restored. The import steps that may be responsible for the observed energy dependence are discussed.

Import of firefly luciferase into peroxisomes of permeabilized Chinese hamster ovary cells: a model system to study peroxisomal protein import in vitro.

A model system for the study of in vitro peroxisomal protein import is described. Chinese hamster ovary (CHO) cells were permeabilized by the bacterial toxin streptolysin O (SLO) and their peroxisomes thus became accessible for a model import protein. Firefly luciferase (FL), which was previously shown to be imported into mammalian peroxisomes in vivo, was used as the reporter protein. When SLO-permeabilized CHO cells were incubated with FL in the presence of an ATP-regenerating system, import of FL could be documented by immunofluorescence staining of the cells with monospecific anti-FL antiserum. FL import increased with time and was dependent on temperature and the presence of hydrolysable ATP, which could not be replaced by GTP. The model system functioned reproducibly and should be of use for investigating fundamental questions on the mechanism of peroxisomal protein import.

Peroxisomal fatty acid oxidation and inhibitors of the mitochondrial carnitine palmitoyltransferase I in isolated rat hepatocytes.

Fatty acid oxidation was studied in the presence of inhibitors of carnitine palmitoyltransferase I (CPT I), in normal and in peroxisome-proliferated rat hepatocytes. The oxidation decreased in mitochondria, as expected, but in peroxisomes it increased. These two effects were seen, in variable proportions, with (+)-decanoylcarnitine, 2-tetradecylglycidic acid (TDGA) and etomoxir. The decrease in mitochondrial oxidation (ketogenesis) affected saturated fatty acids with 12 or more carbon atoms, whereas the increase in peroxisomal oxidation (H2O2 production) affected saturated fatty acids with 8 or more carbon atoms. The peroxisomal increase was sensitive to chlorpromazine, a peroxisomal inhibitor. To study possible mechanisms, palmitoyl-, octanoyl- and acetyl-carnitine acyltransferase activities were measured, in homogenates and in subcellular fractions from control and TDGA-treated cells. The palmitoylcarnitine acyltransferase was inhibited, as expected, but the octanoyltransferase activity also decreased. The CoA derivative of TDGA was synthesized and tentatively identified as being responsible for inhibition of the octanoylcarnitine acyltransferase. These results show that inhibitors of the mitochondrial CPT I may also inhibit the peroxisomal octanoyl transferase; they also support the hypothesis that the octanoyltransferase has the capacity to control or regulate peroxisomal fatty acid oxidation.

Detection of an ATPase activity in rat liver peroxisomes.

An ATPase co-sedimenting with rat liver peroxisomes has been detected after subcellular fractionation. The activity is Mg2+ dependent, with pH optimum of 7.5 and is inhibited by NEM and DCCD but not by oligomycin. Partial inhibition of the mitochondrial ATPase allows to detect the peroxisomal activity in the gradients. Protease inactivation and solubilization data suggests that the activity resides in a protein of the peroxisomal membrane, exposed to the cytosol.

Resistance of Pseudomonas aeruginosa to beta-lactam antibiotics.

A series of experiments were performed with P. aeruginosa to demonstrate which of the biochemical mechanisms are responsible for the resistance to the beta-lactam antibiotics. The constitutive beta-lactamase was isolated and characterized for the strain used as type OXA whose pI was 7.1, with a molar mass of 49 kg/mol. The strain was also submitted to a series of treatments with Tris and Tris-EDTA to disrupt the outer membrane. The treated cells demonstrated a ten-fold reduction in the MIC with cloxacillin, six-fold with penicillin, and five-fold with oxacillin. At least two different biochemical mechanisms were responsible for the resistance to the beta-lactams studied which could be important in the prevalence of P. aeruginosa in nosocomial infections.

Protein phosphorylation in peroxisomes.

The possible presence of phosphorylated proteins in peroxisomes was studied in hepatocytes from nafenopin-treated and normal rats. A 63 kDa phosphorylated protein was consistently and exclusively found in the membrane of peroxisomes from hepatocytes incubated in the presence of 32P-phosphate. The peroxisomes were isolated in metrizamide isopycnic gradients of postnuclear supernatants and were subfractionated by alkaline extraction to separate the membrane and the matrix proteins. Polyacrylamide gel electrophoresis, autoradiography and densitometry were employed to characterize the proteins. The 63 kDa membrane protein copurifies with peroxisomes in metrizamide gradients and apparently can be phosphorylated, in purified peroxisomes, with ATP and catalytic subunit of cAMP-dependent protein kinase.

Large cation-selective pores from rat liver peroxisomal membranes incorporated to planar lipid bilayers.

Fusion of a highly purified fraction of rat liver peroxisomal membranes to planar lipid bilayers incorporates large, cation-selective voltage-dependent pores. The PK/PCl ratio of these pores, estimated in KCl gradients, is close to 4. The pores display several conductance states and spend most of the time open at voltages near 0 mV, closing at more positive and negative voltages. At voltages near 0 mV the most frequent open state has a conductance of 2.4 nS in 0.3 M KCl. At voltages more positive and more negative than 10 mV the most frequent open state displays a conductance of 1.2 nS in 0.3 M KCl. With these results pore diameters of 3 and 1.5 nm, respectively, can be estimated. We suggest that these pores might account for the unusually high permeability of peroxisomes to low molecular weight solutes. Fusion also incorporates a perfectly anion-selective, two-open states channel with conductances of 50 and 100 pS in 0.1 M KCl.