Withanolide E sensitizes renal carcinoma cells to TRAIL-induced apoptosis by increasing cFLIP degradation.

Withanolide E, a steroidal lactone from Physalis peruviana, was found to be highly active for sensitizing renal carcinoma cells and a number of other human cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. Withanolide E, the most potent and least toxic of five TRAIL-sensitizing withanolides identified, enhanced death receptor-mediated apoptotic signaling by a rapid decline in the levels of cFLIP proteins. Other mechanisms by which TRAIL sensitizers have been reported to work: generation of reactive oxygen species (ROS), changes in pro-and antiapoptotic protein expression, death receptor upregulation, activation of intrinsic (mitochondrial) apoptotic pathways, ER stress, and proteasomal inhibition proved to be irrelevant to withanolide E activity. Loss of cFLIP proteins was not due to changes in expression, but rather destabilization and/or aggregation, suggesting impairment of chaperone proteins leading to degradation. Indeed, withanolide E treatment altered the stability of a number of HSP90 client proteins, but with greater apparent specificity than the well-known HSP90 inhibitor geldanamycin. As cFLIP has been reported to be an HSP90 client, this provides a potentially novel mechanism for sensitizing cells to TRAIL. Sensitization of human renal carcinoma cells to TRAIL-induced apoptosis by withanolide E and its lack of toxicity were confirmed in animal studies. Owing to its novel activity, withanolide E is a promising reagent for the analysis of mechanisms of TRAIL resistance, for understanding HSP90 function, and for further therapeutic development. In marked contrast to bortezomib, among the best currently available TRAIL sensitizers, withanolide E's more specific mechanism of action suggests minimal toxic side effects.

Isolation and characterization of a glycopeptide from human senescent erythrocytes.

A glycopeptide (called "senescence-factor glycopeptide", SF-G) has been isolated from a tryptic digest of human erythrocytes by specific adsorption and elution from immobilized peanut lectin. SF-G was detectable in old but not in young erythrocytes isolated from the same unit of blood. It is present in small quantities, less than 1% of the D-galactose oxidase-borotritide-labeled D-galactosyl residues of erythrocytes. SF-G is free of sialic acid but is quite distinct from a similar glycopeptide isolated from completely desialylated erythrocytes. SF-G binds to spleen monocytes, and this property is abolished upon treatment of SF-G with beta-galactosidase. Some, but not all, of the oligosaccharide chains of the SF-G are of the O-glycosyl type, being released by an endo-N-acetyl-alpha-D-galactosaminidase.

Differential acute and chronic response of protein kinase C in cultured neonatal rat heart myocytes to alpha 1-adrenergic and phorbol ester stimulation.

Both alpha 1-adrenergic agonists (e.g. norepinephrine, NE*) and tumor-promoting phorbol esters (e.g. phorbol myristate acetate, PMA) are known to activate protein kinase C (PKC) (Abdel-Latif, 1986, Niedel and Blackshear, 1986). However, alpha 1 agonists and PMA produce very different effects on cardiac function (see Simpson, 1985; Benfey, 1987; Meidell et al., 1986; Leatherman et al., 1987; Yuan et al., 1987; for examples). PKC activation in heart cells has been studied only for PMA treated perfused heart (Yuan et al., 1987). Therefore, acute activation and chronic regulation of PKC by NE and PMA were compared in cultured neonatal rat heart myocytes. NE acutely and transiently activated PKC, as measured by translocation of PKC activity to the cell particulate fraction (Niedel and Blackshear, 1986). Particulate PKC activity peaked at 23% of total after NE for 30 s, as compared with 8% for control (P less than 0.001). By contrast, acute PKC activation by PMA was more pronounced and persistent, with particulate PKC activity 62% of total at 5 min (P less than 0.001). Calcium/lipid-independent kinase activity increased acutely with PMA, but not with NE. Chronic treatment with NE (24 to 48 h) increased total per cell PKC activity and 3H-phorbol dibutyrate (PDB) binding sites, an index of the number of PKC molecules (Niedel and Blackshear, 1986), by 30 to 60% over control (all P less than 0.05 to 0.01). In contrast with NE, chronic treatment with PMA down-regulated PKC, reducing total per cell PKC activity and 3H-PDB binding sites to 3% and 12% of control, respectively (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular weight dependence of hyaluronic acid produced during oncogenic transformation.

Cultured chicken embryo fibroblasts synthesize two distinct molecular size classes of hyaluronic acid. The high molecular weight material (form I, 2.98 x 10(6) is the predominant species synthesized by transformed cells, whereas form II (1.42 x 10(5)) is the major product of non-transformed cells. A shift to synthesis of predominantly form I hyaluronic acid is an early transformation event in cells infected with LA24 Rous sarcoma virus and maintained at the permissive temperature for transformation (35 degrees C). Form I hyaluronic acid exhibits greater binding to preparations of cellular fibronectin and to both normal and transformed cells than does form II. Both forms bind more to transformed cells than to normal, uninfected cells. Hyaluronic acid (predominantly form I) isolated from transforming cells stimulates proliferation in growth-retarded, non-transformed cells.

A growth factor for cardiac myocytes is produced by cardiac nonmyocytes.

Cardiac nonmyocytes, primarily fibroblasts, surround cardiac myocytes in vivo. We examined whether nonmyocytes could modulate myocyte growth by production of one or more growth factors. Cardiac myocyte hypertrophic growth was stimulated in cultures with increasing numbers of cardiac nonmyocytes. This effect of nonmyocytes on myocyte size was reproduced by serum-free medium conditioned by the cardiac nonmyocytes. The majority of the nonmyocyte-derived myocyte growth-promoting activity bound to heparin-Sepharose and was eluted with 0.75 M NaCl. Several known polypeptide growth factors found recently in cardiac tissue, namely acidic fibroblast growth factor (aFGF), basic FGF (bFGF), platelet-derived growth factor (PDGF), tumor necrosis factor alpha (TNF alpha), and transforming growth factor beta 1 (TGF beta 1), also caused hypertrophy of cardiac myocytes in a dose-dependent manner. However, the nonmyocyte-derived growth factor (tentatively named NMDGF) could be distinguished from these other growth factors by different heparin-Sepharose binding profiles (TNF alpha, aFGF, bFGF, and TGF beta 1) by neutralizing growth factor-specific antisera (PDGF, TNF alpha, aFGF, bFGF, and TGF beta 1), by the failure of NMDGF to stimulate phosphatidylinositol hydrolysis (PDGF and TGF beta 1), and, finally, by the apparent molecular weight of NMDGF (45-50 kDa). This nonmyocyte-derived heparin-binding growth factor may represent a novel paracrine growth mechanism in myocardium.

Chronic ethanol exposure increases levels of protein kinase C delta and epsilon and protein kinase C-mediated phosphorylation in cultured neural cells.

Exposure to ethanol for several days increases the number and function of dihydropyridine-sensitive Ca2+ channels in excitable tissues. In the neural cell line PC12, this process is blocked by inhibitors of protein kinase C (PKC), suggesting that PKC mediates ethanol-induced increases in Ca2+ channels. We report that treatment with 25-200 mM ethanol for 2-8 days increased PKC activity in PC12 cells and NG108-15 neuroblastoma-glioma cells. Detailed studies in PC12 cells showed that ethanol also increased phorbol ester binding and immunoreactivity to PKC delta and PKC epsilon. These changes were associated with increased PKC-mediated phosphorylation. Ethanol did not activate the enzyme directly, nor did ethanol increase levels of diacylglycerol. Ethanol-induced increases in PKC levels may promote up-regulation of Ca2+ channels, and may also regulate the expression and function of other proteins involved in cellular adaptation to ethanol.

A protein kinase C isozyme is translocated to cytoskeletal elements on activation.

Protein kinase C (PKC)1 isozymes comprise a family of related cytosolic kinases that translocate to the cell particulate fraction on stimulation. The activated enzyme is thought to be on the plasma membrane. However, phosphorylation of protein substrates occurs throughout the cell and is inconsistent with plasma membrane localization. Using an isozyme-specific monoclonal antibody we found that, on activation, this PKC isozyme translocates to myofibrils in cardiac myocytes and to microfilaments in fibroblasts. Translocation of this activated PKC isozyme to cytoskeletal elements may explain some of the effects of PKC on cell contractility and morphology. In addition, differences in the translocation site of individual isozymes--and, therefore, phosphorylation of different substrates localized at these sites--may explain the diverse biological effects of PKC.

Transcription of early developmental isogenes in cardiac myocyte hypertrophy.

We have developed a cell culture system to study molecular mechanisms important in myocardial hypertrophy. alpha 1-Adrenergic receptor stimulation produces hypertrophy of neonatal rat cardiac myocytes. Myocyte hyperplasia is not induced by alpha 1 stimulation, although alpha 1-adrenergic receptor-mediated DNA synthesis and cell division have been observed in other types of cells. The myocyte hypertrophic response does not require contractile activity. Activation of the alpha 1 receptor also produces highly specific alterations in gene expression, as measured at the mRNA and protein levels. In particular, there is selective up-regulation of two contractile protein isogenes that are expressed in vivo during early development and in pressure-load hypertrophy, skeletal alpha-actin and beta-myosin heavy chain. Studies with an in vitro transcription assay indicate that stimulation of the alpha 1-adrenergic receptor leads to a distinctive temporal sequence of transcriptional activation. Transcription of the skeletal alpha-actin isogene is induced preferentially to that of cardiac alpha-actin. Thus, early developmental isogene induction in alpha 1-stimulated hypertrophy reflects a fundamental change in the transcriptional program of the cardiac myocyte nucleus. The goal now is to define an intracellular pathway connecting the alpha 1-adrenergic receptor in the plasma membrane to activation of RNA polymerase II on the skeletal alpha-actin gene in the cardiac myocyte nucleus. There is evidence that protein kinase C may be one component of this pathway. A model for alpha 1-mediated transcription is presented.