W Stremmel

Drug-induced apoptosis in hepatoma cells is mediated by the CD95 (APO-1/Fas) receptor/ligand system and involves activation of wild-type p53.

Chemotherapeutic drugs are cytotoxic by induction of apoptosis in drug-sensitive cells. We investigated the mechanism of bleomycin-induced cytotoxicity in hepatoma cells. At concentrations present in the sera of patients during therapy, bleomycin induced transient accumulation of nuclear wild-type (wt) p53 and upregulated expression of cell surface CD95 (APO-1/Fas) receptor in hepatoma cells carrying wt p53 (HepG2). Bleomycin did not increase CD95 in hepatoma cells with mutated p53 (Huh7) or in hepatoma cells which were p53-/- (Hep3B). In addition, sensitivity towards CD95-mediated apoptosis was also increased in wt p53 positive HepG2 cells. Microinjection of wt p53 cDNA into HepG2 cells had the same effect. In contrast, bleomycin did not enhance susceptibility towards CD95-mediated apoptosis in Huh7 and in Hep3B cells. Furthermore, bleomycin treatment of HepG2 cells increased CD95 ligand (CD95L) mRNA expression. Most notably, bleomycin-induced apoptosis in HepG2 cells was almost completely inhibited by antibodies which interfere with CD95 receptor/ligand interaction. These data suggest that apoptosis induced by bleomycin is mediated, at least in part, by p53-dependent stimulation of the CD95 receptor/ligand system. The same applies to other anti-cancer drugs such as cisplatin and methotrexate. These data may have major consequences for drug treatment of cancer and the explanation of drug sensitivity and resistance.

Copper in disorders with neurological symptoms: Alzheimer’s, Menkes, and Wilson diseases

Copper is an essential element for the activity of a number of physiologically important enzymes. Enzyme-related malfunctions may contribute to severe neurological symptoms and neurological diseases: copper is a component of cytochrome c oxidase, which catalyzes the reduction of oxygen to water, the essential step in cellular respiration. Copper is a cofactor of Cu/Zn-superoxide-dismutase which plays a key role in the cellular response to oxidative stress by scavenging reactive oxygen species. Furthermore, copper is a constituent of dopamine-beta-hydroxylase, a critical enzyme in the catecholamine biosynthetic pathway. A detailed exploration of the biological importance and functional properties of proteins associated with neurological symptoms will have an important impact on understanding disease mechanisms and may accelerate development and testing of new therapeutic approaches. Copper binding proteins play important roles in the establishment and maintenance of metal-ion homeostasis, in deficiency disorders with neurological symptoms (Menkes disease, Wilson disease) and in neurodegenerative diseases (Alzheimers disease). The Menkes and Wilson proteins have been characterized as copper transporters and the amyloid precursor protein (APP) of Alzheimers disease has been proposed to work as a Cu(II) and/or Zn(II) transporter. Experimental, clinical and epidemiological observations in neurodegenerative disorders like Alzheimers disease and in the genetically inherited copper-dependent disorders Menkes and Wilson disease are summarized. This could provide a rationale for a link between severely dysregulated metal-ion homeostasis and the selective neuronal pathology.

Involvement of the CD95 (APO-1/Fas) Receptor and Ligand System in Helicobacter pylori -induced Gastric Epithelial Apoptosis

Helicobacter pylori infection is associated with chronic gastritis, peptic ulceration, and gastric carcinoma. The potential role of CD95-mediated apoptosis was investigated in a panel of gastric biopsies obtained from patients with H. pylori-associated chronic gastritis (n = 29) and with noninfected normal mucosa (n = 10). Immunohistochemistry revealed increased CD95 receptor expression in epithelial and lamina propria cells in chronic gastritis. By in situ hybridization, CD95 ligand mRNA was absent or low in normal mucosa but expressed at high levels in lamina propria lymphocytes and, unexpectedly, in epithelial cells in chronic gastritis. Apoptotic cells were rare in normal mucosa but were observed regularly in chronic gastritis in close proximity to CD95 ligand mRNA expression throughout the epithelial and lamina propria cells. In a functional analysis gastric epithelial cell lines were incubated with supernatants of H. pylori. Treatment with the cytotoxic isolate H. pylori 60190 but not with the noncytotoxic isolate Tx30a upregulated CD95 in up to 50% of gastric epithelial cells and induced apoptosis in these cells. H. pylori-induced apoptosis was partially prevented by blocking CD95, demonstrating the functional role of the CD95 system. These findings suggest that H. pylori-associated chronic gastritis involves apoptosis of gastric epithelial cells by activation of the CD95 receptor and ligand system.

Enhanced caspase-8 recruitment to and activation at the DISC is critical for sensitisation of human hepatocellular carcinoma cells to TRAIL-induced apoptosis by chemotherapeutic drugs

AbstractTumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) exhibits potent antitumour activity upon systemic administration in mice without showing the deleterious side effects observed with other apoptosis-inducing members of the TNF family such as TNF and CD95L. TRAIL may, thus, have great potential in the treatment of human cancer. However, about 60% of tumour cell lines are not sensitive to TRAIL. To evaluate the mechanisms of tumour resistance to TRAIL, we investigated hepatocellular carcinoma (HCC) cell lines that exhibit differential sensitivity to TRAIL. Pretreatment with chemotherapeutic drugs, for example, 5-fluorouracil (5-FU), rendered the TRAIL-resistant HCC cell lines sensitive to TRAIL-induced apoptosis. Analysis of the TRAIL death-inducing signalling complex (DISC) revealed upregulation of TRAIL-R2. Caspase-8 recruitment to and its activation at the DISC were substantially increased after 5-FU sensitisation, while FADD recruitment remained essentially unchanged. 5-FU pretreatment downregulated cellular FLICE-inhibitory protein (cFLIP) and specific cFLIP downregulation by small interfering RNA was sufficient to sensitise TRAIL-resistant HCC cell lines for TRAIL-induced apoptosis. Thus, a potential mechanism for TRAIL sensitisation by 5-FU is the increased effectiveness of caspase-8 recruitment to and activation at the DISC facilitated by the downregulation of cFLIP and the consequent shift in the ratio of caspase-8 to cFLIP at the DISC.

Reactive Oxygen Intermediates Are Involved in the Induction of CD95 Ligand mRNA Expression by Cytostatic Drugs in Hepatoma Cells

Oxidative stress has been associated with the induction of programmed cell death. The CD95 ligand/receptor system is a specific mediator of apoptosis. We have used the model of drug-induced apoptosis to assess whether the CD95 ligand mRNA is induced by reactive oxygen intermediates. Treatment of HepG2 hepatoma cells with bleomycin induced the production of reactive oxygen intermediates and, as an additional parameter of oxidative stress, resulted in glutathione (GSH) depletion. In parallel, CD95 ligand mRNA expression was induced. In a similar fashion CD95 ligand mRNA expression increased after treatment with H2O2. Additional treatment with the antioxidant and GSH precursor N-acetylcysteine resulted in partial restoration of intracellular GSH levels and in reduced induction of CD95 ligand mRNA. Induction of CD95 ligand mRNA by bleomycin was further reduced by combined treatment withN-acetylcysteine and deferoxamine. These data suggest a direct role of oxygen radicals in the induction of the CD95 ligand.

Efficacy of ursodeoxycholic acid treatment and endoscopic dilation of major duct stenoses in primary sclerosing cholangitis: An 8-year prospective study

BACKGROUND/AIMS Primary sclerosing cholangitis is characterized by progressive fibrotic inflammation and obliteration of intra- and extrahepatic bile ducts. Ursodeoxycholic acid treatment leads to improvement of biochemical parameters of cholestasis and in part also of liver histology. During treatment, obstruction of major ducts may lead to deterioration of liver function, which may be prevented by endoscopic dilation of the stenoses. Controlled trials for evaluation of the beneficial effects of ursodeoxycholic acid treatment and of endoscopic measures in patients with major duct stenoses have become clinically difficult. Estimation of survival probabilities without treatment allows comparison of actuarial survival with the estimated survival probabilities. METHODS/RESULTS We studied survival in 65 patients with PSC treated with ursodeoxycholic acid (750 mg/day) and by endoscopic measures whenever necessary. Patients with decompensated cirrhosis in whom transplantation was foreseen were excluded. The study was started in May 1987 and the mean follow-up period was 45.0+/-3.5 (mean+/-SEM) months. Liver histology was performed in each of the patients before entry into the study and revealed that 21% were in stage 1, 37% in stage 2, 21% in stage 3 and 20% in stage 4. Of 65 patients, 12 had major duct stenosis at entry and another 11 developed major duct stenosis during ursodeoxycholic acid treatment, which was successfully treated by repeated endoscopic balloon dilations. The actuarial Kaplan-Meier survival probabilities without liver transplantation after treatment with ursodeoxycholic acid and dilation of major duct stenoses were significantly improved compared to the predicted survival rates with p=0.001. CONCLUSIONS Ursodeoxycholic acid does not prevent major bile duct occlusion. When ursodeoxycholic acid treatment and endoscopic opening of duct stenoses are combined, survival may be significantly improved.

A New Concept of Cellular Uptake and Intracellular Trafficking of Long-Chain Fatty Acids

Fatty acids are the main structural and energy sources of the human body. Within the organism, they are presented to cells as fatty acid: albumin complexes. Dissociation from albumin represents the first step of the cellular uptake process, involving membrane proteins with high affinity for fatty acids, e.g., fatty acid translocase (FAT/CD 36) or the membrane fatty acid-binding protein (FABPpm). According to the thus created transmembrane concentration gradient, uncharged fatty acids can flip-flop from the outer leaflet across the phospholipid bilayer. At the cytosolic surface of the plasma membrane, fatty acids can associate with the cytosolic FABP (FABPc) or with caveolin-1. Caveolins are constituents of caveolae, which are proposed to serve as lipid delivery vehicles for subcellular organelles. It is not known whether protein (FABPc)- and lipid (caveolae)-mediated intracellular trafficking of fatty acids operates in conjunction, or in parallel. Channeling fatty acids to the different metabolic pathways requires activation to acyl-CoA. For this process, the family of fatty acid transport proteins (FATP 1-5/6) might be relevant because they have been shown to possess acyl-CoA synthetase activity. Their variable N-terminal signaling sequences suggest that they might be targeted to specific organelles by anchoring in the phospholipid bilayer of the different subcellular membranes. At the highly conserved cytosolic AMP-binding site of FATP, fatty acids are activated to acyl-CoA for subsequent metabolic disposition by specific organelles. Overall, fatty acid uptake represents a continuous flow involving the following: dissociation from albumin by membrane proteins with high affinity for fatty acids; passive flip-flop across the phospholipid bilayer; binding to FABPc and caveolin-1 at the cytosolic plasma membrane; and intracellular trafficking via FABPc and/or caveolae to sites of metabolic disposition. The uptake process is terminated after activation to acyl-CoA by the members of the FATP family targeted intracellularly to different organelles.

TAp73/ΔNp73 influences apoptotic response, chemosensitivity and prognosis in hepatocellular carcinoma

We investigated the mechanisms by which TAp73β and dominant-negative p73 (ΔNp73) regulate apoptosis. TAp73β transactivated the CD95 gene via the p53-binding site in the first intron. In addition, TAp73β induced expression of proapoptotic Bcl-2 family members and led to apoptosis via the mitochondrial pathway. Endogenous TAp73 was upregulated in response to DNA damage by chemotherapeutic drugs. On the contrary, ΔNp73 conferred resistance to chemotherapy. Inhibition of CD95 gene transactivation was one mechanism by which ΔNp73 functionally inactivated the tumor suppressor action of p53 and TAp73β. Concomitantly, ΔNp73 inhibited apoptosis emanating from mitochondria. Thus, ΔNp73 expression in tumors selects against both the death receptor and the mitochondrial apoptosis activity of TAp73β. The importance of these data is evidenced by our finding that upregulation of ΔNp73 in hepatocellular carcinoma patients correlates with reduced survival. Our data indicate that ΔNp73 is an important gene in hepatocarcinogenesis and a relevant prognostic factor.

Mice with targeted disruption of the fatty acid transport protein 4 (Fatp 4, Slc27a4) gene show features of lethal restrictive dermopathy

The fatty acid transport protein family is a group of evolutionarily conserved proteins that are involved in the cellular uptake and metabolism of long and very long chain fatty acids. However, little is known about their respective physiological roles. To analyze the functional significance of fatty acid transport protein 4 (Fatp4, Slc27a4), we generated mice with a targeted disruption of the Fatp4 gene. Fatp4-null mice displayed features of a neonatally lethal restrictive dermopathy. Their skin was characterized by hyperproliferative hyperkeratosis with a disturbed epidermal barrier, a flat dermal–epidermal junction, a reduced number of pilo-sebaceous structures, and a compact dermis. The rigid skin consistency resulted in an altered body shape with facial dysmorphia, generalized joint flexion contractures, and impaired movement including suckling and breathing deficiencies. Lipid analysis demonstrated a disturbed fatty acid composition of epidermal ceramides, in particular a decrease in the C26:0 and C26:0-OH fatty acid substitutes. These findings reveal a previously unknown, essential function of Fatp4 in the formation of the epidermal barrier.

Cellular uptake of fatty acids driven by the ER-localized acyl-CoA synthetase FATP4.

Long-chain fatty acids are important metabolites for the generation of energy and the biosynthesis of lipids. The molecular mechanism of their cellular uptake has remained controversial. The fatty acid transport protein (FATP) family has been named according to its proposed function in mediating this process at the plasma membrane. Here, we show that FATP4 is in fact localized to the endoplasmic reticulum and not the plasma membrane as reported previously. Quantitative analysis confirms the positive correlation between expression of FATP4 and uptake of fatty acids. However, this is dependent on the enzymatic activity of FATP4, catalyzing the esterification of fatty acids with CoA. Monitoring fatty acid uptake at the single-cell level demonstrates that the ER localization of FATP4 is sufficient to drive transport of fatty acids. Expression of a mitochondrial acyl-CoA synthetase also enhances fatty acid uptake, suggesting a general relevance for this mechanism. Our results imply that cellular uptake of fatty acids can be regulated by intracellular acyl-CoA synthetases. We propose that the enzyme FATP4 drives fatty acid uptake indirectly by esterification. It is not a transporter protein involved in fatty acid translocation at the plasma membrane.