The membrane attack complex of complement induces caspase activation and apoptosis.

Activation of the terminal pathway of the complement system leads to insertion of terminal complement complexes (C5b-9) into the cell membrane, which may induce cytolysis. Recent data indicate that the terminal complement pathway can also result in apoptosis in vivo. To further define the cell death pathway induced by complement, we examined induction of apoptosis by complement in vitro.

Differential regulation of phosphatidylserine externalization and DNA fragmentation by caspases in anticancer drug-induced apoptosis of rat mammary adenocarcinoma MTLn3 cells.

Caspase activation is a central event in the execution phase of apoptosis and is associated with phosphatidylserine (PS) externalization and DNA fragmentation. We investigated the role of caspase activity in anticancer drug-induced PS externalization and DNA fragmentation in MTLn3 cells. Caspase activation (DEVD-AMC cleavage) occurred in a time- and concentration-dependent manner after exposure to doxorubicin, in association with cleavage of poly(ADP) ribose polymerase and protein kinase C delta, two caspase-3 substrates.

Suppression of chemically induced apoptosis but not necrosis of renal proximal tubular epithelial (LLC-PK1) cells by focal adhesion kinase (FAK). Role of FAK in maintaining focal adhesion organization after acute renal cell injury.

Decreased phosphorylation of focal adhesion kinase (FAK) is associated with loss of focal adhesions and actin stress fibers and precedes the onset of apoptosis in renal epithelial cells caused by nephrotoxicants (Van de Water, B., Nagelkerke, J. F.

Macrophage p53 deficiency leads to enhanced atherosclerosis in APOE*3-Leiden transgenic mice.

Cell proliferation and cell death (either necrosis or apoptosis) are key processes in the progression of atherosclerosis. The tumor suppressor gene p53 is an essential gene in cell proliferation and cell death and is upregulated in human atherosclerotic plaques, both in smooth muscle cells and in macrophages. In the present study, we investigated the importance of macrophage p53 in the progression of atherosclerosis using bone marrow transplantation in APOE*3-Leiden transgenic mice, an animal model for human-like atherosclerosis.

Cleavage of the actin-capping protein alpha -adducin at Asp-Asp-Ser-Asp633-Ala by caspase-3 is preceded by its phosphorylation on serine 726 in cisplatin-induced apoptosis of renal epithelial cells.

Decreased phosphorylation of focal adhesion kinase and paxillin is associated with loss of focal adhesions and stress fibers and precedes the onset of apoptosis (van de Water, B., Nagelkerke, J. F.

Linking gene expression to mechanisms of toxicity.

Activation of gene expression is one of the earliest cellular responses to toxicity. However, our understanding of the biological and biochemical signals that activate these toxicant-responsive genes as well as the consequences of gene activation to survival of the organism remains sketchy. In this article, strategies that can be used to link changes in gene expression to biochemical mechanisms of toxicity are addressed using the hsp70 and grp78 genes as examples.

The roles of caspase-3 and bcl-2 in chemically-induced apoptosis but not necrosis of renal epithelial cells.

The kidney is a target for toxicants including cisplatin and S-(1,2-dichlorovinyl)-L-cysteine (DCVC), a metabolite of the environmental contaminant, trichloroethylene. Necrosis is well characterized in kidney cells, but pathways leading to apoptosis are less clear. Cysteine conjugates are useful toxicants because they induce either necrosis or apoptosis depending on chemical structure or antioxidant status.

Distinct endoplasmic reticulum signaling pathways regulate apoptotic and necrotic cell death following iodoacetamide treatment.

Environmental stress induces the synthesis of glucose-regulated proteins (Grps) in the endoplasmic reticulum (ER) and heat shock proteins (Hsps) in the cytoplasm. Iodoacetamide (IDAM), a prototypical alkyating agent, induces both Grp and Hsp synthesis in renal epithelial cells and causes necrosis which is prevented by prior activation of the ER stress response (pre-ER stress) [Liu, H., et al.

Hepatocyte growth factor induces tubulogenesis of primary renal proximal tubular epithelial cells.

Hepatocyte growth factor (HGF)-induced tubulogenesis has been demonstrated with renal epithelial cell lines grown in collagen gels but not with primary cultured renal proximal tubular epithelial cells (RPTEs). We show that HGF selectively induces proliferation and branching morphogenesis of primary cultured rat RPTEs. Additional growth factors including fibroblast growth factor (FGF)-1, epidermal growth factor (EGF), FGF-7, or insulin-like growth factor-1 (IGF-1) did not selectively induce tubulogenesis.

Dephosphorylation of focal adhesion kinase (FAK) and loss of focal contacts precede caspase-mediated cleavage of FAK during apoptosis in renal epithelial cells.

The relationship between focal adhesion protein (FAK) activity and loss of cell-matrix contact during apoptosis is not entirely clear nor has the role of FAK in chemically induced apoptosis been studied. We investigated the status of FAK phosphorylation and cleavage in renal epithelial cells during apoptosis caused by the nephrotoxicant dichlorovinylcysteine (DCVC). DCVC treatment caused a loss of cell-matrix contact which was preceded by a dissociation of FAK from the focal adhesions and tyrosine dephosphorylation of FAK.

Cisplatin effects on F-actin and matrix proteins precede renal tubular cell detachment and apoptosis in vitro.

In primary cultures of porcine proximal tubular kidney cells and LLC-PK1 cells cisplatin (5 - 50 microM) caused apoptosis and cell detachment; in both systems cell detachment occurred, preceded by a loss of cytoskeletal F-actin stress fibers within 4 - 6 h, and a reduction of mRNA encoding for fibronectin, collagen a2 type (IV) and laminin B2 within 17 - 41 h. Prevention of F-actin damage by phalloidin prevented nuclear fragmentation, suggesting a relation between F-actin damage and apoptosis. Overexpression of Bcl-2 also prevented apoptosis, but did not prevent damage to the F-actin skeleton or the reduction of mRNA expression of the matrix proteins.

Endoplasmic reticulum stress proteins block oxidant-induced Ca2+ increases and cell death.

Oxidants are important human toxicants. Increased intracellular free Ca2+ may be critical for oxidant toxicity, but this mechanism remains controversial. Furthermore, oxidants damage the endoplasmic reticulum (ER) and release ER Ca2+, but the role of the ER in oxidant toxicity and Ca2+ regulation during toxicity is also unclear.

Transformation-sensitive changes in expression, localization, and phosphorylation of adducins in renal proximal tubule epithelial cells.

Adducins are cytoskeletal proteins that facilitate interactions between spectrin and actin to form the subcortical membrane skeleton. We recently determined that alpha- and gamma-adducins are among a group of PKC substrates that we have designated "STICKS" (substrates that interact with C-kinase). To study the role of adducins and their regulation by protein kinase C (PKC) in carcinogenesis, we compared the content, localization, and phosphorylation of alpha- and gamma-adducins in primary renal proximal tubule epithelial (RPTE) cells and oncogene-altered derivative lines.

Endoplasmic reticulum chaperones GRP78 and calreticulin prevent oxidative stress, Ca2+ disturbances, and cell death in renal epithelial cells.

Activation of stress response genes can impart cellular tolerance to environmental stress. Iodoacetamide (IDAM) is an alkylating toxicant that up-regulates expression of hsp70 (Liu, H., Lightfoot, D.

Cisplatin-induced nephrotoxicity in porcine proximal tubular cells: mitochondrial dysfunction by inhibition of complexes I to IV of the respiratory chain.

Cisplatin-induced nephrotoxicity was studied in porcine proximal tubular cells, focusing on the relationship between mitochondrial damage, reactive oxygen species (ROS) and cell death. Cisplatin specifically affected mitochondrial functions: complexes I to IV of the respiratory chain were inhibited 15 to 55% after 20 min of incubation with 50 to 500 microM, respectively. As a result, intracellular ATP was decreased to 70%.

Comparative cytotoxicity of 14 novel selenocysteine se-conjugates in rat renal proximal tubular cells.

Recently, Se-substituted selenocysteine conjugates were proposed as potential prodrugs to target biologically active selenol compounds to tissues containing high activities of cysteine conjugate beta-lyases, such as the kidneys. However, several selenium compounds are known to be relatively toxic compounds. In the present study, the cytotoxicity of 14 selenocysteine Se-conjugates was determined in freshly isolated rat renal proximal tubular cells (RPTC).

F-actin disorganization in apoptotic cell death of cultured rat renal proximal tubular cells.

The mechanism of nephrotoxin-induced apoptosis was studied in rat renal proximal tubular cells (PTC) exposed to the nephrotoxin S-(1,2-dichlorovinyl)-L-cysteine (DCVC). After a 6-h incubation, DCVC caused a condensation of heterochromatin and a fragmentation of the nucleus in 84 and 16% of the cells, respectively, which is indicative of apoptosis. This was confirmed biochemically by agarose gel electrophoresis demonstrating the formation of DNA fragments with multiples of 200 bp.

The role of a purinergic P2z receptor in calcium-dependent cell killing of isolated rat hepatocytes by extracellular adenosine triphosphate.

Extracellular adenosine triphosphate (ATP o) (0.4 mmol/L), a P2-purinergic receptor agonist, induces cytolysis in several cell types including isolated rat hepatocytes. In this study, we investigated the P2-receptor involved in ATP o-induced, Ca2+-dependent cytotoxicity in hepatocytes.

Alkylation-induced oxidative cell injury of renal proximal tubular cells: involvement of glutathione redox-cycle inhibition.

The nephrotoxicant S-(1,2-dichlorovinyl)-L-cysteine (DCVC) is an alkylating agent that causes oxidative stress and subsequently death of renal proximal tubular cells (PTC). In this paper the role of inhibition of the glutathione redox cycle (GSH-reductase (GRd) and -peroxidase (GPx) in the development of DCVC-induced oxidative cell injury is described. DCVC inhibited both GRd and GPx activity in PTC.

Inhibition of succinate:ubiquinone reductase and decrease of ubiquinol in nephrotoxic cysteine S-conjugate-induced oxidative cell injury.

The role of complex II in the cellular protection against oxidative stress was investigated in freshly isolated rat renal proximal tubular cells (PTC) with the use of the nephrotoxin S-(1,2-dichlorovinyl)-L-cysteine (DCVC). DCVC caused oxidative stress in PTC as determined by flow cytometry with dihydrorhodamine-123; this fluorescent probe is readily oxidized by primary hydroperoxides such as those formed during lipid peroxidation. The oxidative stress could be prevented by inhibition of the beta-lyase-mediated formation and covalent binding to cellular macromolecules of reactive DCVC metabolites, with amino oxyacetic acid (AOA), or by the antioxidant N,N'-diphenyl-p-phenylenediamine.

In vivo and in vitro detachment of proximal tubular cells and F-actin damage: consequences for renal function.

We investigated the relationship between F-actin damage and cell detachment using nephrotoxic L-cysteine S-conjugates. In vivo S-(1,2-dichlorovinyl)-L-cysteine (DCVC) induced loss of F-actin in the S3 segment of the proximal tubule in the outer stripe of the outer medulla, which was associated with loss of the brush border and loss of cells from the basement membrane. In vitro DCVC caused the detachment of primary cultured rat renal proximal tubular cells (PTC), which was clearly associated with F-actin damage.

Role of mitochondrial Ca2+ in the oxidative stress-induced dissipation of the mitochondrial membrane potential. Studies in isolated proximal tubular cells using the nephrotoxin 1,2-dichlorovinyl-L-cysteine.

The relationship between mitochondrial Ca2+, oxidative stress, and a dissipation of the mitochondrial membrane potential (delta psi) was investigated in proximal tubular kidney cells. Freshly isolated proximal tubular cells from rat kidney were exposed to the nephrotoxin 1,2-dichlorovinyl-L-cysteine (DCVC). DCVC stimulated the formation of hydroperoxides as determined by flow cytometry using the hydroperoxide-sensitive compound dichlorofluorescein.

Activation of rat Kupffer cells to tumoricidal cells by the immunomodulator muramyl tripeptide-phosphatidylethanolamine incorporated into the novel drug carrier lactosylated low density lipoprotein.

Lactosylated low density lipoprotein (lac-LDL) is a potential carrier for the site-specific delivery of lipophilic drugs to liver macrophages (Kupffer cells). In the present study we evaluated the application of lac-LDL as a carrier to target the immunomodulator muramyl tripeptide-phosphatidylethanolamine (MTP-PE) to rat Kupffer cells, to specifically activate these cells to tumor-killing cells. The drug carrier 125I-labeled lac-LDL interacted with a galactose-specific recognition system on isolated rat Kupffer cells.

Mitochondrial K+ as modulator of Ca(2+)-dependent cytotoxicity in hepatocytes. Novel application of the K(+)-sensitive dye PBFI (K(+)-binding benzofuran isophthalate) to assess free mitochondrial K+ concentrations.

In isolated rat hepatocytes a sustained high increase in intracellular free Ca2+ ([Ca2+]i), induced by extracellular ATP, is associated with mitochondrial dysfunction and cell death. The Ca(2+)-induced effects are Pi-dependent and less severe when the intracellular K+ content is low. In this study, the involvement of mitochondrial K+ processing in Ca(2+)-induced loss of mitochondrial membrane potential (MMP) and viability was investigated.