Requirement for p38 and p44/p42 mitogen-activated protein kinases in RAGE-mediated nuclear factor-kappaB transcriptional activation and cytokine secretion.

Advanced glycation end product (AGE) activation of the signal-transducing receptor for AGE (RAGE) has been linked to a proinflammatory phenotypic change within cells. However, the precise intracellular signaling pathways involved have not been elucidated. We demonstrate here that human serum albumin modified with N(epsilon)-(carboxymethyl)lysine (CML), a major AGE adduct that progressively accumulates with aging, diabetes, and renal failure, induced nuclear factor (NF)-kappaB-driven reporter gene expression in human monocytic THP-1 cells. The NF-kappaB response was blocked with a synthetic peptide corresponding to the putative ligand-binding domain of RAGE, with anti-RAGE antiserum, and by coexpression of truncated receptors lacking the intracellular domain. Signal transduction from RAGE to NF-kappaB involved the generation of reactive oxygen species, since reporter gene expression was blocked with the antioxidant N-acetyl-L-cysteine. CML-modified albumin produced rapid transient activation of tyrosine phosphorylation, extracellular signal-regulated kinase 1 and 2, and p38 mitogen-activated protein kinase (MAPK), but not c-Jun NH(2)-terminal kinase. RAGE-mediated NF-kappaB activation was suppressed by the selective p38 MAPK inhibitor SB203580 and by coexpression of a kinase-dead p38 dominant-negative mutant. Activation of NF-kappaB by CML-modified albumin increased secretion of proinflammatory cytokines (tumor necrosis factor-alpha, interleukin-1beta, and monocyte chemoattractant protein-1) severalfold, and inhibition of p38 MAPK blocked these increases. These results indicate that p38 MAPK activation mediates RAGE-induced NF-kappaB-dependent secretion of proinflammatory cytokines and suggest that accelerated inflammation may be a consequence of cellular activation induced by this receptor.

Heterogeneous apoptotic responses of prostate cancer cell lines identify an association between sensitivity to staurosporine-induced apoptosis, expression of Bcl-2 family members, and caspase activation.

BACKGROUND: The goal of this work was to identify mechanisms for the inability of metastatic prostate cancer cells to engage the apoptotic pathway following hormonal or cytotoxic therapy. METHODS: Genotypically diverse cell lines isolated from patients with metastatic disease were used. RESULTS: The LNCaP and TsuPr(1) lines exhibited quintessential apoptotic features in response to the pleiotropic apoptotic inducer staurosporine (STS): rapid cytochrome c translocation to the cytosol, proteolytic processing and catalytic activation of caspase-3 and -7, proteolytic inactivation of the death substrates DNA fragmentation factor (DFF) and poly-ADP-ribose polymerase (PARP), and TUNEL-positive polyfragmented nuclei. In contrast, DU-145 and PC-3 cells exhibited few, if any, of these features, while appearing necrotic by confocal microscopy. The presence of caspase-3 and -7 without proteolytic processing suggested that the apoptotic blockade was upstream of executioner caspases in these resistant cell lines. To identify the locus of this block, Western blot analysis of cytochrome c subcellular localization and of pro- and antiapoptotic Bcl-2 family members was performed, and suggested that heterogeneous expression of these proteins might be the underlying mechanism for apoptotic resistance to STS in these cell lines. Thus, the absence of the proapoptotic Bax in DU-145 cells indicated a mechanism for apoptotic resistance of these cells. Similarly, decreased Bax expression during STS treatment, coupled with overexpression of the antiapoptotic Bcl-x(L) and inability to translocate cytochrome c to the cytosol, provided a mechanism for the insensitivity of PC-3 cells. CONCLUSIONS: These observations suggest that activation of the apoptotic machinery in metastatic prostate cancer cell lines may be determined by expression levels of Bcl-2 family members, by the ability of cytochrome c to translocate to the cytosol, and by the ability of the caspase pathway to react in response to activation of the mitochondrial phase.

Signaling pathway activated during apoptosis of the prostate cancer cell line LNCaP: overexpression of caspase-7 as a new gene therapy strategy for prostate cancer.

We studied the molecular mechanisms of apoptosis in the prostate cancer cell line LNCaP and whether overexpression of caspase activity could force this cell line to undergo apoptosis. The inhibitor of phosphomevalonate decarboxylase, sodium phenylacetate, and the protein kinase inhibitor staurosporine induced (a) release of cytochrome c from the mitochondria to the cytosol; (b) reduction in mitochondrial transmembrane potential; (c) proteolytic processing of caspase-3 and -7 but not -2; (d) cleavage of the DEVD substrate and the death substrates poly(ADP-ribose) polymerase and DNA fragmentation factor; and (e) apoptosis. The panspecific inhibitor of caspase activation N-benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (z-VAD-FMK) prevented all of these events except release of mitochondrial cytochrome c into the cytosol. None of these apoptotic signaling events were elicited by staurosporine or sodium phenylacetate treatment of LNCaP-Bcl-2 cells that overexpress the oncoprotein Bcl-2. Because caspase-7 is activated in every model of apoptosis that we have characterized thus far, we wished to learn whether overexpression of this protease could directly cause apoptosis of LNCaP cells. By using a replication-defective adenovirus, overexpression of caspase-7 protein in both LNCaP and LNCaP-Bcl-2 cells was accompanied by induction of cleavage of the DEVD substrate and TUNEL. These studies have demonstrated that caspase-7 and -3 are critical mediators of apoptosis in LNCaP cells. Caspase-7 was proteolytically activated in every model of apoptosis that we have developed, and the overexpression of it induced apoptosis of LNCaP and LNCaP-Bcl-2 cells. Thus, adenoviral-mediated transfer of caspase-7 may offer a new effective approach for the treatment of prostate cancer.

Caspase-7 is activated during lovastatin-induced apoptosis of the prostate cancer cell line LNCaP.

The goals of this work were to establish a reproducible and effective model of apoptosis in a cell line derived from advanced prostate cancer and to study the role of the caspase family of proteases in mediating apoptosis in this system. The study involved the use of the prostate cancer cell line LNCaP. Apoptosis was induced using the hydroxymethyl glutaryl CoA reductase inhibitor, lovastatin, and was evaluated by agarose gel electrophoresis of genomic DNA, morphological criteria, and terminal deoxynucleotidyl transferase-mediated nick end labeling. Caspases were studied by catalytic activity, mRNA induction, and protein processing. Lovastatin (30 microM) was an effective inducer of apoptosis, causing changes that were evident after 48 h and essentially complete after 96-120 h of treatment. These effects were prevented by the simultaneous addition of mevalonate (300 microM) to the culture medium. Lovastatin induced a proteolytic activity that was able to cleave the enzyme poly(ADP-ribose) polymerase and the substrate Z-DEVD-AFC, which is modeled after the P1-P4 amino acids of the poly(ADP-ribose) polymerase cleavage site. Caspase-7, but not caspase-3, underwent proteolytic activation during lovastatin-induced apoptosis, an effect prevented by mevalonate. Caspase-7 was the only detected interleukin 1beta converting enzyme family protease with DEVD cleavage activity that exhibited lovastatin-induced mRNA up-regulation. Again, mevalonate blocked this effect. Lovastatin-induced apoptosis also was prevented when the caspase inhibitors Z-DEVD-CH2F or Z-VAD-CH2F (100 microM) where added to the medium. These studies have identified lovastatin as a powerful inducer of apoptosis in the cell line LNCaP. Caspase activation was a necessary event for LNCaP cells to undergo apoptosis during treatment with lovastatin. Of the caspases tested, only caspase-7 underwent proteolytic activation after stimulation with lovastatin. Identification of caspase-7 as a potential mediator of lovastatin-induced apoptosis broadens our knowledge of the molecular events associated with programmed cell death in a cell line derived from prostatic epithelium.

Physiological responsiveness during hypnosis.

4 physiological measures--electromyogram, respiration rate, heart rate, and skin conductance--were recorded for 11 high and 11 low hypnotizable Ss. It was hypothesized (a) that physiological responsiveness during hypnosis would vary depending on the nature of the task instructions, and (b) that high hypnotizable Ss would show more physiological responsiveness than low hypnotizable Ss. The first hypothesis was substantiated across all 4 measures. Only heart rate levels supported the second hypothesis. The results are discussed as they relate to the 2 hypotheses and to future research.