Cholesterol, its biosynthetic precursors and the cholesterol-lowering drug compactin were able to inhibit the growth of human erythroleukemia K562 cells. Compactin, farnesyldiphosphate and cholesterol were cytotoxic by the induction of apoptosis (programmed cell death, PCD). Compactin doubled the number of apoptotic cells compared to control numbers, whereas farnesyldiphosphate and cholesterol led to a fivefold increase in PCD over the control levels. At variance with cholesterol, cholesterol esters did not affect K562 cell viability and apoptotic body formation, regardless of chain length and degree of saturation. Compactin and farnesyldiphosphate reduced the membrane cholesterol content, thus increasing membrane fluidity. Conversely, cholesterol treatment reduced the membrane fluidity by increasing cholesterol content in the lipid bilayer. Unlike farnesyldiphosphate, the other cholesterol precursors and cholesterol esters were ineffective in increasing the cholesterol content and, thereby, the fluidity of cell membranes. Compactin and cholesterol precursors, apart from farnesyldiphosphate, did not affect the amount of the farnesylated proteins Ras and lamin B in the cytosolic and the membrane fractions of K562 cell extracts, whereas farnesyldiphosphate reduced the content of both proteins in both fractions. The level of lamin B in K562 cytosol and membranes was also reduced by cholesterol treatment, which did not significantly affect the amount of Ras. These findings highlight the role of cholesterol in promoting PCD.Keywords : apoptosis ; cholesterol ; lamin B ; membrane ; Ras.Apoptosis (programmed cell death, PCD) is a genetically syl anchor, belongs to a superfamily containing more than 50 small GTPases (Peeper et al., 1997). Ras is implicated in cell controlled process, which counteracts mitosis in regulating cell number (Kerr et al., 1972). Several cell-death pathways are trig-growth control and, therefore, associated with several human cancers. Lamin B, a 67-kDa farnesylated protein, has a structural gered by membrane receptors and are executed through membrane-derived lipid messengers (Hale et al., 1996; Skowronski role in nuclear membranes (Hancock, 1993; Gibbs et al., 1994).Lamin B is cleaved by caspases (Kumar and Lavin, 1996) in the et al., 1996). Recently, we have reported profound changes in the cell membrane composition of human erythroleukemia K562 pathway of Fas-mediated PCD (Greidinger et al., 1996; Peter et al., 1996). Thus, a perturbation of cholesterol biosynthesis can cells forced into apoptosis by treatment with transforming growth factor β1, alone or in combination with cisplatin (Mac-be expected to affect the cells by altering the membrane cholesterol content (that is, membrane properties) and/or the level of carrone et al., 1996). These changes at the membrane level might be the basis for specific recognition and elimination of farnesylated proteins (that is, regulation of cell proliferation).Inhibitors of cholesterol biosynthesis, such as lovastatin and apoptotic cells by macrophage...

Background:Oxidative stress and neuronal cell death have been implicated in the pathogenesis of Alzheimer disease (AD). Considering that the glutathione transferase (GST) supergene family encodes isoenzymes that appear to be critical in protection against oxidative stress, we aimed at determining the various GSTP1, GSTM1, and GSTT1 polymorphisms and ApoE genotypes to investigate their role as susceptibility genes for late-onset AD (LOAD). Methods: We included 210 LOAD patients and 228 healthy controls matched for age, sex, and educational level in our case-control genetic association study. GSTM1 and GSTT1 genotypes were studied by conventional PCR, whereas GSTP1 and ApoE genotypes were determined by real-time PCR on the LightCycler. Results: We found a significant association between LOAD and the GSTP1*C allelic variant [odds ratio (OR) ‫؍‬ 1.9; P <0.05], but no association between the GSTM1 and GSTT1 deleted genotypes and LOAD. In addition, a preliminary result suggested that carriers of both the GSTP1*C and ApoE ⑀4 allelic variants were at increased risk of LOAD (OR ‫؍‬ 19.98; P <0.0001).

Neuroblastoma, one of the most common pediatric solid tumors, originates from the peripheral sympathetic nervous system and is responsible for approximately 15% of all childhood cancer deaths. Among the several antineoplastic drugs used in neuroblastoma chemotherapeutic protocols, topoisomerase inhibitors (i.e., etoposide) represent the most commonly used. Several resistance mechanisms limit the clinical success of topoisomerase-targeting drugs, mainly reducing the ability of neoplastic cells to start programmed cell death when exposed to antineoplastic drugs. The aim of this study was to determine, by means of proteomics, potential markers of etoposide resistance in human neuroblastoma cell lines as well as to investigate protein levels and modifications possibly involved in the onset of resistance. The etoposide resistant clone showed overexpression of the following proteins: peroxiredoxin 1, beta-galactoside soluble lectin binding protein, vimentin (three protein spots), heat shock 27 kDa protein (two protein spots) and heterogeneous nuclear ribonucleoprotein K. In addition, we also found down-regulation of dUTP pyrophosphatase. This investigation might represent a first step towards the development of novel prognostic markers of neuroblastoma chemotherapy.

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Background and aim: Inflammation has been extensively implicated in the pathogenesis of Alzheimer's disease (AD). Although there is evidence of a key role for cytokines in neuroinflammation processes, so far the proinflammatory cytokine interleukin (IL)-18 has not been associated with AD. The aim of this study was to investigate the impact of two polymorphisms of the human IL-18 gene promoter at positions 2607 (C/A) and 2137 (G/C) on both susceptibility to and progression of AD. Results: The results revealed that the genotype distribution of the 2607 (C/A) polymorphism was different between patients with AD and control subjects (x 2 = 7.99, df = 2, p = 0.0184). In particular, carriers of the CC genotype were at increased risk of developing AD (OR 2.33; 95% CI 1.29 to 4.22; p = 0.0052). The observed genotypes were in Hardy-Weinberg equilibrium, as for the 2607 polymorphism, whereas the 2137 polymorphism appeared in Hardy-Weinberg disequilibrium only in the patient group (p = 0.0061). Finally, in a 2 year follow-up study, the 2137 CC genotype was strongly and specifically associated with a faster cognitive decline (F = 4.024; df = 4,192; p = 0.0037 for time by IL-18 2137 G/C group interaction) with no interaction effect with the apolipoprotein E e4/non-e4 allele presence. Conclusion: As IL-18 cytokine promoter gene polymorphisms have been previously described to have functional consequences on IL-18 expression, it is possible that individuals with a prevalent IL-18 gene variant have a dysregulated immune response, suggesting that IL-18 mediated immune mechanisms may play a crucial role in AD.

Glutathione S-transferases (GSTs, EC 2.5.1.18) belong to a large family of functionally different enzymes that catalyze the S-conjugation of glutathione with a wide variety of electrophilic compounds including carcinogens and anticancer drugs. Drug resistance may result from reduction in apoptosis of neoplastic cells when exposed to antineoplastic drugs. The c-Jun N-terminal Kinase (JNK) belongs to the family of stress kinases and has been shown to be required for the maximal induction of apoptosis by DNA-damaging agents. Recently, an inhibition of JNK activity by GST P1-1, which was reversed by polymerization induced by oxidative stress, has been reported in 3T3-4A mouse fibroblast cell lines. The finding that GST P1-1 might inhibit JNK activity and that it is frequently highly expressed in tumor tissues suggests its possible implication in "apoptosis resistance" during antineoplastic therapy. We investigated the modulation of GST P1-1 during apoptosis in a neoplastic T-cell line (Jurkat) induced by hydrogen peroxide and etoposide. Apoptosis was paralleled by the appearance of a dimeric form of GST P1-1 on western blotting, associated with an increase in the Km(GSH) and a reduction in GST P1-1 specific activity toward 1-chloro-2,4-dinitrobenzene, which reached statistical significance only in H(2)O(2)-treated cells. Our data seem to suggest that H(2)O(2) and etoposide may partly act through a process of partial inactivation of the GST P1-1, possibly involving the "G" site in the process of dimerization, and thus favoring programmed cell death.

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Background and Aims: Increased generation of reactive oxygen species and mitochondrial dysfunction may underlie the pathophysiology of Friedreich's ataxia, the most common inherited ataxia, due to GAA expansion in a gene coding for a mitochondrial protein (frataxin), implicated in the regulation of iron metabolism. Because iron overload would cause oxidative stress in Friedreich's ataxia, we investigated the enzyme antioxidant system in the blood of 14 patients by determining superoxide dismutase, glutathione peroxidase, and glutathione trasferase catalytic activities. We also studied the glutathione S-transferase genotype polymorphism in order to evaluate its possible influence on enzyme activity. Methods: Blood samples were obtained from 14 unrelated patients with Friedreich's ataxia and 21 age matched healthy subjects. Antioxidant enzyme determinations were spectrophotometrically assayed using specific substrates; the glutathione S-transferase genotype polymorphism was analysed by endonuclease restriction mapping of exon 5 and 6 amplification products.Results: There was a significant elevation of the superoxide dismutase/glutathione peroxidase activity ratio (0.037 (0.01) v 0.025 (0.008) of controls) and an 83% rise of glutathione transferase specific activity (0.22 (0.1) v 0.12 (0.03) nmol/min/mg protein) in blood of patients with Friedreich's ataxia than in the controls. The genotype polymorphism of glutathione S-transferase enzyme did not show any relevant differences when compared to that of healthy subjects. Conclusions: Data show an impairment in vivo of antioxidant enzymes in patients with Friedreich's ataxia and provide evidence of an increased sensitivity to oxidative stress, supporting a consistent role of free radical cytotoxicity in the pathophysiology of the disease.