Effects of subtoxic concentrations of benzoyl peroxide on cell lipid metabolism.

Benzoyl peroxide (BP), a tumor promoter, has been shown to cause free-radical-induced lipid peroxidation and membrane damage at toxic concentrations. However, its effects on lipid metabolism at concentrations that were not overtly toxic have not been investigated. The purpose of the current study was to examine the effects of BP and its final degradation product, benzoic acid (BA), on lipid metabolism. Two cell lines, hamster cheek pouch (HCP) and human monocytes (THP-1), were used to determine the effects of BP, BA, and BP combined with FeCl2 on cell lipid metabolism. Cells were exposed to BP and 14C acetate for 24 h, or cells with prelabeled lipids were harvested, and the lipids were extracted and separated with the use of thin-layer chromatography. Lipid metabolism of some neutral lipids such as triglycerides was altered for both cell types in response to BP. Also, cholesterol content was reduced in THP-1 cells and a phospholipid, phosphatidylethanolamine (PE), was reduced in HCP cells. The final degradation product of BP, BA, failed to elicit any response in lipid metabolism. Subtoxic concentrations of BP induced changes in neutral lipids such as triglycerides and cholesterol. The metabolism of major phospholipids except PE remained unchanged. The effects were related to BP and its degradation and varied with the cell type.

Transforming growth factor beta 1 dysregulation in a human oral carcinoma tumour progression model.

A human oral tumour progression model was established that consists of normal epithelial cells and three cell lines representing stages from dysplastic to metastatic cells. To investigate the impact of exogenous transforming growth factor-beta 1 on this model system, we analysed the responsiveness of those cells to transforming growth factor-beta 1 and explored the potential mechanism underlying the transforming growth factor-beta 1 activity. We found that the growth of all cell types, regardless of their stage of tumour progression, is inhibited by transforming growth factor-beta 1, although to different degrees. Transforming growth factor-beta 1 induced the expression of cyclin-dependent kinase inhibitors p15(INK4B), p21WAF1/(CIP1) and p27(KIP1). In contrast, transforming growth factor-beta 1 was found to stimulate the invasive potential of one cell type that represents the most advanced stage of tumour phenotype, suggesting that the impact of transforming growth factor-beta 1 on functional features of tumour cells other than cellular proliferation may play a significant role in the process of oral tumour progression.

The equine herpesvirus 1 immediate-early protein interacts with EAP, a nucleolar-ribosomal protein.

The equine herpesvirus 1 (EHV-1) immediate-early (IE) phosphoprotein is essential for the activation of transcription from viral early and late promoters and regulates transcription from its own promoter. The IE protein of 1487 amino acids contains a serine-rich tract (SRT) between residues 181 and 220. Deletion of the SRT decreased transactivation activity of the IE protein. Previous results from investigation of the ICP4 protein, the IE homolog of herpes simplex virus 1 (HSV-1), revealed that a domain containing a serine-rich tract interacts with EAP (Epstein-Barr virus-encoded small nuclear RNA-associated protein), a 15-kDa nucleolar-ribosomal protein (R. Leopardi, and B. Roizman, Proc. Natl. Acad. Sci. USA 93, 4572-4576, 1996). DNA binding assays revealed that (i) glutathione S-transferase (GST)-EAP disrupted the binding of HSV-1 ICP4 to its cognate DNA in a dose-dependent manner, (ii) GST-EAP interacted with the EHV-1 IE protein, but did not disrupt its binding to its cognate site in viral DNA. GST-pulldown assays indicated that the SRT of the IE protein is required for physical interaction with EAP. The IE protein and EAP colocalized in the cytoplasm of the infected equine ETCC cells at late times of the infection cycle. This latter finding may be important in EHV-1 gene regulation since late viral gene expression is greatly influenced by the EICP0 trans-activator protein whose function is antagonized by the IE protein.

Chemopreventive effects of green tea polyphenols correlate with reversible induction of p57 expression.

Green tea polyphenols are known to induce apoptosis in certain types of tumor cells. However, the mechanism(s) that enables normal cells to evade the apoptotic effect is still not understood. In this study, Western blot analysis combined with cycloheximide treatment was used to examine the effects of green tea polyphenols on the expression levels of p57, a cyclin-dependent kinase and apoptosis inhibitor, in normal human keratinocytes and in the oral carcinoma cell lines SCC25 and OSC2. The results showed that the most potent green tea polyphenol, (-)-epigallocatechin-3-gallate (EGCG), induced p57 in normal keratinocytes in a dosage- and time-dependent manner, while the levels of p57 protein in oral carcinoma cells were unaltered. The differential response in p57 induction was consistent with the apoptosis status detected by annexin V assay. The data suggest that the chemopreventive effects of green tea polyphenols may involve p57-mediated cell cycle regulation in normal epithelial cells.

Changes in cell phospholipid metabolism in vitro in the presence of HEMA and its degradation products.

OBJECTIVES: Diacylglycerol-kinase (DAG-kinase) is an enzyme that phosphorylates diacylglycerol (DAG) to phosphatidic acid (PA), which serves as a precursor to phosphoglycerides involved in cell signaling or as cell membrane structural components. DAG-kinase can be inhibited by diacylethylene glycols (DAEG). We hypothesize that 2-hydroxyethyl methacrylate (HEMA) may alter phosphorylation of DAG to PA following intracellular formation of DAEG. METHODS: Cultured rabbit kidney (RK13) epithelial cells were treated with HEMA, EG, or known inhibitors of DAG-kinase for 24 h, then exposed to [32P]O4- in the presence of a synthetic diacylglycerol for 2 h. Other cultures were radiolabeled with [3H]-oleic acid for 24 h, then exposed to HEMA for an additional 24 h. The cells were harvested and the lipids extracted. Radioactive lipids were separated by thin layer chromatography, located by autoradiography, and quantitated as cpm/ug protein. Cell cultures treated with HEMA were homogenized and the DAG-kinase activity was assayed and expressed as cpm/ug protein. Data were analyzed by one-way ANOVA and Newman-Keuls Multiple Comparison Test. RESULTS: Cultures exposed to HEMA or known DAG-kinase inhibitors exhibited reduced incorporation of radioactivity in the PA fraction compared to control cultures. Direct assays of DAG-kinase activity from cells exposed to HEMA demonstrated decreased enzyme activity. Evaluation of cell phospholipid synthesis showed altered formation of phosphatidylethanolamine and phosphatidylcholine. SIGNIFICANCE: Results suggest that HEMA impairs formation of PA, possibly by acylation of EG released by hydrolysis of the HEMA and resultant production of the inhibitor DAEG. The decreased availability of PA may alter PA-dependent cell structural lipid pathways and lipid-dependent signaling pathways, altering cell growth.

The EICP22 protein of equine herpesvirus 1 physically interacts with the immediate-early protein and with itself to form dimers and higher-order complexes.

The EICP22 protein (EICP22P) of Equine herpesvirus 1 (EHV-1) is an early protein that functions synergistically with other EHV-1 regulatory proteins to transactivate the expression of early and late viral genes. We have previously identified EICP22P as an accessory regulatory protein that has the ability to enhance the transactivating properties and the sequence-specific DNA-binding activity of the EHV-1 immediate-early protein (IEP). In the present study, we identify EICP22P as a self-associating protein able to form dimers and higher-order complexes during infection. Studies with the yeast two-hybrid system also indicate that physical interactions occur between EICP22P and IEP and that EICP22P self-aggregates. Results from in vitro and in vivo coimmunoprecipitation experiments and glutathione S-transferase (GST) pull-down studies confirmed a direct protein-protein interaction between EICP22P and IEP as well as self-interactions of EICP22P. Analyses of infected cells by laser-scanning confocal microscopy with antibodies specific for IEP and EICP22P revealed that these viral regulatory proteins colocalize in the nucleus at early times postinfection and form aggregates of dense nuclear structures within the nucleoplasm. Mutational analyses with a battery of EICP22P deletion mutants in both yeast two-hybrid and GST pull-down experiments implicated amino acids between positions 124 and 143 as the critical domain mediating the EICP22P self-interactions. Additional in vitro protein-binding assays with a library of GST-EICP22P deletion mutants identified amino acids mapping within region 2 (amino acids [aa] 65 to 196) and region 3 (aa 197 to 268) of EICP22P as residues that mediate its interaction with IEP.

Alterations in cell lipid metabolism by glycol methacrylate (HEMA).

Components of dental resins such as dimethylaminoethyl methacrylate (DMAEMA) can alter cell lipid composition, presumably by esterase-mediated hydrolysis. The resulting dimethylethanolamine is incorporated into cell phospholipids, while the methacrylic acid may alter several metabolic pathways. We hypothesize that HEMA is cleaved in a similar manner and the released ethylene glycol is incorporated into cell lipids, yielding phosphatidylethylene glycol (PtEG), and the methacrylic acid alters other lipid pathways in a manner similar to that of methacrylic acid released from hydrolysis of DMAEMA. Cultures of hamster buccal pouch (HCP) and rabbit kidney (RK13) epithelial cells were exposed to subtoxic concentrations of HEMA in the presence of [14C]-acetate or [3H]-oleic acid. Other cultures were prelabeled with [14C]-acetate followed by exposure to various concentrations of HEMA. Cell lipids were extracted by the method of Bligh and Dyer and separated by thin layer chromatography on silica gel K-6 plates or SG-81 silica gel loaded chromatography paper. The fate of the ethylene glycol was traced using [14C]-ethylene glycol. Radioactive lipids were located using autoradiography and known standard lipids and quantitated by liquid scintillation spectrometry. In the presence of HEMA several classes of lipids were altered. Among the neutral lipids, the most notable changes involved sterol precursors, triglycerides, fatty acids, and cholesterol esters, while phosphatidylcholine was affected among the phospholipids. The results differed quantitatively between the two cell types. Results also suggest that EG, including that released by hydrolysis of HEMA, is incorporated into cell phospholipids, producing PtEG. The changes in neutral lipid labeling may occur by alteration of lipid synthetic pathways utilizing acetyl Co-A as well as inhibition of enzymes involved in synthesis of cholesterol from sterol precursors and hydrolysis of cholesterol esters. Synthesis of PtEG may take place via phospholipase D-mediated headgroup exchange. Alterations in the cellular lipids may affect cell membrane properties and associated cell functions.

Cell lipid alterations resulting from prolonged exposure to dimethylaminoethylmethacrylate.

Dimethylaminoethylmethacrylate (DMAEMA), a commonly-used component of visible-light polymerized dental resins, has the potential to elute and interact with tissue cells to cause cytotoxicity or sublethal metabolic changes. Short-term exposure of cultured oral epithelial cells to sublethal DMAEMA concentrations has been shown previously to affect cell neutral lipid and phospholipid metabolism, resulting in accumulation of significant quantities of dimethylphosphatidylethanolamine (DMPE). In non-treated cells, DMPE is a transient intermediate in phospholipid metabolism and is not detectable by standard methods. In the current study, the effects of prolonged exposure of cells to DMAEMA, and the mechanisms for formation of DMPE in the presence of DMAEMA were examined. Exposure of a keratinizing hamster buccal cheek pouch cell line (HCP cells) to 0.8 mM DMAEMA for 2, 3, 7, and 14 days resulted in reduced incorporation of [14C]acetate into several classes of phospholipids. DMPE was detectable at all time points in DMAEMA-exposed cultures and comprised between 12.48% and 18.33% of the total radiolabeled phospholipids. The results of short-term exchange experiments indicated that headgroup exchange was not the major reaction responsible for formation of DMPE in DMAEMA-treated cells; rather the formation appeared to occur through typical phospholipid metabolic pathways. The cells appeared able to re-establish and maintain homeostasis in the presence of this altered cell lipid composition.

Structural and antigenic identification of the ORF12 protein (alpha TIF) of equine herpesvirus 1.

The equine herpesvirus 1 (EHV-1) homolog of the herpes simplex virus type 1 (HSV-1) tegument phosphoprotein, alpha TIF (Vmw65; VP16), was identified previously as the product of open reading frame 12 (ORF12) and shown to transactivate immediate early (IE) gene promoters. However, a specific virion protein corresponding to the ORF12 product has not been identified definitively. In the present study the ORF12 protein, designated ETIF, was identified as a 60-kDa virion component on the basis of protein fingerprint analyses in which the limited proteolysis profiles of the major 60-kDa in vitro transcription/ translation product of an ORF12 expression vector (pT7-12) were compared to those of purified virion proteins of similar size. ETIF was localized to the viral tegument in Western blot assays of EHV-1 virions and subvirion fractions using polyclonal antiserum and monoclonal antibodies generated against a glutathione-S-transferase-ETIF fusion protein. Northern and Western blot analyses of EHV-1-infected cell lysates prepared under various metabolic blocks indicated that ORF12 is expressed as a late gene, and cross reaction of polyclonal anti-GST-ETIF with a 63.5-kDa HSV-1 protein species suggested that ETIF and HSV-1 alpha TIF are antigenically related. Last, DNA band shift assays used to assess ETIF-specific complex formation indicated that ETIF participates in an infected cell protein complex with the EHV-1 IE promoter TAATGARAT motif.

Effects of an aminomethacrylate on epithelial cell lipid metabolism.

Methacrylates can affect cell functions by surfactant-like effects or by altering cell lipid composition. Dimethylaminoethyl methacrylate (DMAEMA), an activator widely used in visible-light polymerized dental resins has been shown to elute readily into aqueous environments. The current study examined the metabolism of this material by oral epithelial cells (HCP) and its subsequent effects on cell lipids. Cells were plated in culture medium, then exposed to DMAEMA in the presence of 14C-acetate, a precursor which labeled the cell lipids. Other cultures were prelabeled with radioisotope, then exposed to DMAEMA. After incubation, the cell lipids were extracted and separated by TLC. Radioactive lipids were located and quantitated. Exposure of the cells to DMAEMA resulted in decreased synthesis of cholesterol with a concomitant increase in sterol precursors. Cholesterol esters and triacylglycerides also increased. Among the polar lipids, phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE) decreased in response to DMAEMA. However, dimethylphosphatidyl ethanolamine (DMPE), a precursor of PC not detectable in control cultures, accumulated to a significant extent in cells exposed to DMAEMA. Furthermore, changes in PC and DMPE levels persisted in the cells for at least 48 h after removal of the DMAEMA. The results indicate that DMAEMA produces alterations in the relative amounts of several cellular neutral and polar lipids. Such alterations, especially of the normal phospholipid composition, along with an alteration in cellular cholesterol, could result in altered membrane-associated cell functions.