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Toxicity of chemotherapeutic drugs towards normal cells is a serious side effect of cancer treatment. Thus, finding of molecules with low toxicity for normal cells is crucial. Several natural compounds, such as flavonoid quercertin, are receiving a growing attention as "chemopreventers". Quercetin kills tumour-derived cell lines, but little is known about its effects on normal cells. Here we show that although quercetin exerts a higher apoptotic potential on leukemic cell lines than on peripheral blood mononuclear cells (PBMCs) and does not sensitize PBMCs to CD95-induced apoptosis, it is able to inhibit normal immune functions such as T cell proliferation and activation. Quercetin sensitivity is independent on cell cycle progression since it was not abrogated in serum-starved U937 cells, nor proliferating PBMCs underwent apoptosis after quercetin treatment. However, quercetin prevented PHA-induced PBMC proliferation and SEB-induced upregulation of activation markers. Our data suggest that quercetin, while incapable of inducing apoptosis in normal cells under several conditions, could interfere with effector T cell function.

Objective and Design: The present study aims at evaluating the influence of genetic polymorphisms on antiretroviral therapy (ART)-associated lipodystrophy. We included in the study 255 ICoNA. patients and we assessed the distribution of Fas -670 AG polymorphism, ApoC3 -455 CT and -482 CT polymorphisms, C161T silent substitution in the PPAR gamma gene, the Adrenergic beta3 Receptor (ARbeta3) codon 64 TC variant, and two polymorphisms in the Adrenergic beta2 Receptor (ARbeta2) codon 16 AG and codon 27 CG. Crude rates of lipoatrophy and fat accumulation and adjusted relative rates were calculated using Poisson regression., Results: In a multivariate model after adjusting for gender, HIV exposure, age, current viral load, hepatitis C virus (HCV) serology, nucleoside reverse-transcriptase inhibitor (NRTI) pair/'third drugs' currently used, months of pre-highly active antiretroviral therapy (HAART) exposures to NRTI, the following genotypes resulted protective against lipoatrophy: ApoC3 -455 CC genotype [adjusted relative risks (ARR) 0.2, 95% confidence interval (CI) 0.046-0.91 vs CT/TT, P = 0.037], ARbeta3 codon 64 TT genotype (ARR 0.39, 95% CI 0.14-1.06 vs TC/CC, P = 0.066), and Fas -670 GG genotype (ARR 0.51, 95% CI 0.26-1.01 vs AG/AA, P = 0.053). With regard to fat accumulation, in the multivariate model, the ARbeta2 codon 27 CC genotype resulted protective (ARR 0.21, 95% CI 0.08-0.51 vs CG/GG, P = 0.0006), whereas the ARbeta2 codon 16 AA genotype resulted associated with higher risk (ARR 3.72, 95% CI 1.58-8.76 vs AG/GG, P = 0.0026)., Conclusion: Our study suggests that genetic polymorphisms of genes involved in apoptosis and adipocyte metabolism are significantly related to ART associated lipodystrophy. Particularly, we evidenced a role for ApoC3 -455 in lipoatrophy and for the two variants of ARbeta2 in fat accumulation.

Different types of cells from subjects with Down syndrome (DS) have an increased susceptibility to cell death. We have studied apoptosis and mitochondrial (mt) membrane potential (DeltaPsi(m)) in peripheral blood mononuclear cells (PBMC) from DS children and age-matched healthy donors after in vitro treatment with apoptogenic molecules, along with mtDNA content. We found that PBMC from DS and healthy controls had a similar tendency to undergo apoptosis and a similar amount of mtDNA. However, in cells from DS subjects, mitochondria showed a higher loss of DeltaPsi(m), underlying the presence of an increasing susceptibility of these organelles to damaging agents.

The analysis of changes in mitochondrial membrane potential (MMP) that can occur during apoptosis provides precious information on the mechanisms and pathways of cell death. For many years, the metachromatic fluorochrome JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide) was used for this purpose. Thanks to new dyes and to the technical improvements recently adopted in several flow cytometers, it is now possible to investigate, along with MMP, a variety of other parameters. Using three sources of excitation and polychromatic flow cytometry, we have developed a protocol that can be applied to cells undergoing apoptosis. In the model of U937 cells incubated with the chemopreventive agent quercetin (3,3',4',5,7-pentahydroxyflavone), we describe the detection at the single cell level of changes in MMP (by JC-1), early apoptosis (exposition of phosphatidylserine on the plasma membrane detected by annexin-V), late apoptosis and secondary necrosis (decreased DNA content by Hoechst 33342 and permeability of the plasma membrane to propidium iodide). The procedure can be completed in less than 2 h.

Background: Until now, the simultaneous analysis of several parameters during apoptosis, including DNA content and mitochondrial membrane potential (DeltaPsi), has not been possible because of the spectral characteristics of the commonly used dyes. Using polychromatic flow cytometry based upon multiple laser and UV lamp excitation, we have characterized cells with different DeltaPsi during apoptosis., Methods: U937 cells were treated with the flavonoid quercetin (Qu) and stained with JC-1 to detect DeltaPsi, propidium iodide (PI) for cell viability, Hoechst 33342 for DNA content, Annexin V conjugated with Alexa Fluor-647 for detection of phosphatidilserine (PS) exposure, marker of early apoptosis, or Mitotracker Deep Red for the determination of mitochondrial mass., Results: Treatment with Qu provoked the onset of three cell populations with different DeltaPsi: (1) healthy cells, with normal DeltaPsi, DNA content and physical parameters, high mitochondrial mass, PI- and Annexin V-negative; (2) cells with intermediate DeltaPsi and normal DNA content, but with physical parameters typical of apoptotic cells and low mitochondrial mass; most of them were PI+ and Annexin V+; (3) cells with collapsed DeltaPsi that had low mitochondrial mass and were Annexin-V+, PI+; half of them showed diminished DNA content. Similar results, i.e. the presence of cells with intermediate DeltaPsi, were observed in other models of apoptosis., Conclusions: During Qu-induced apoptosis, loss of DeltaPsi, PS exposure, and decrease of mitochondrial mass are early events that precede permeability to PI and loss of DNA. Populations of cells with different DeltaPsi, as revealed by flow cytometry after JC-1 staining, differed also for other parameters associated to apoptosis. Thus, the simultaneous analysis of several parameters by polychromatic flow cytometry permits a better identification of many stages of cell death, and, more in general, allows to evaluate the eventual heterogenic sensibility of the population under study to a given compound.

The onset of resistance to drug-induced apoptosis of tumour cells is a major problem in cancer therapy. We studied a drug-selected clone of promyelocytic HL-60 cells, called HCW-2, which display a complex resistance to a wide variety of apoptosis-inducing agents and we found that these cells show a dramatic increase in the expression of heat shock proteins (Hsps) 70 and 27, while the parental cell line does not. It is known that stress proteins such as Hsps can confer resistance to a variety of damaging agents other than heat shock, such as TNF-alpha, monocyte-induced cytotoxicity, and also play a role in resistance to chemotherapy. This elevated expression of Hsps is paralleled by an increased activity of mitochondrial metabolism and pentose phosphate pathway, this latter leading to high levels of glucose-6-phosphate dehydrogenase and, consequently, of glutathione. Thus, the apoptotic-deficient phenotype is likely because of the presence of high levels of stress response proteins and GSH, which may confer resistance to apoptotic agents, including chemotherapy drugs. Moreover, the fact that in HCW-2 cells Hsp70 are mainly localised in mitochondria may account for the increased performances of mitochondrial metabolism. These observations could have some implications for the therapy of cancer, and for the design of combined strategies that act on antioxidant defences of the neoplastic cell.

Cardiolipin (CL) is essential for the functionality of several mitochondrial proteins. Its distribution between the inner and outer leaflet of the mitochondrial internal membrane is crucial for ATP synthesis. We have investigated alterations in CL distribution during the early phases of apoptosis. Using two classical models (staurosporine-treated HL-60 cells and tumor necrosis factor alpha-treated U937 cells), we found that in apoptotic cells CL moves to the outer leaflet of mitochondrial inner membrane in a time-dependent manner. This occurs before the appearance of apoptosis markers such as plasma-membrane exposure of phosphatidylserine, changes in mitochondrial membrane potential, DNA fragmentation, but after the production of reactive oxygen species. The exposure of a phospholipid on the outer surface during apoptosis thus occurs not only at the plasma membrane level but also in mitochondria, reinforcing the hypothesis of mitoptosis as a crucial regulating system for programmed cell death, also occurring in cancer cells after treatment with antineoplastic agents.

Background: Apoptosis plays a major role in depleting CD4(+) lymphocytes during infection with HIV-1. Few data exist on its role during HIV infection of children. Sensitivity of peripheral blood lymphocytes (PBLs) to apoptotic stimuli and the importance of the patient's age remain unclear., Objectives: We sought to analyze the following: (1) markers of cell death-activation (CD95, CD45 isoforms, and CD28) in PBLs from vertically HIV-infected children of different ages before highly active antiretroviral therapy; (2) changes in other PBL populations; (3) PBL sensitivity to cell death and mitochondrial damages; and (4) role of age during progression of infection., Methods: Cell culture techniques and flow cytometry were used to analyze surface antigens, PBL susceptibility to apoptosis, or PBL susceptibility to change of mitochondrial membrane potential., Results: Donor age had a strong negative correlation with numbers of CD4(+) and CD8(+) T cells. Virgin T lymphocyte (CD45RA(+), CD95(-)) levels and those of CD95(+) cells showed no correlation with the children's clinical status but did show a correlation with patient age. CD28(-) T lymphocytes were markedly augmented in HIV-infected children but were unrelated to stage of infection or age. A relevant decrease in B lymphocytes and an increase in natural killer cells were also found. Finally, PBLs from HIV-positive children had a marked tendency to undergo apoptosis and mitochondrial damage., Conclusion: Changes in PBL phenotype, increased expression of CD95, and high sensitivity to apoptosis suggest that a precocious aging of the immune system occurs in HIV-infected children.

Human immunodeficiency virus-type 1 (HIV-1) infection is characterized by increased immune cell apoptosis. Apoptosis can be triggered by signals that arise from within the cell, or by signals that are elicited by binding of extracellular "death ligands" to their "death receptors," most of which belong to the tumor necrosis factor (TNF)-receptor family, such as CD95 (Fas/Apo-1). In immune cells the oligomerization of CD95, induced by its ligand CD95L, and the recruitment of different intracytoplasmic molecules that in turn activate FLICE/caspase 8 are crucial. To study the role of CD95/CD95L interactions during HIV-1 infection, we developed an original method based upon quantitative-competitive (QC) RT-PCR that allowed us to quantify the amounts of mRNA coding for the total (tCD95) and membrane (mCD95) forms of CD95. We first studied the expression of different forms of CD95 mRNA in a classical model of chronic HIV infection using two infected cell lines of different origin--lymphocytic (ACH-2) or monocytic (U1). We have shown that infected cells of monocytic origin preferentially produce the "protective" (soluble) form of CD95, and no detectable CD95L mRNA, while lymphoid cells produce more mRNA for the membrane form of CD95 (which triggers apoptosis) along with low but detectable amounts of CD95L mRNA. One can hypothesize that a complex balance exists between pro-apoptotic events, perhaps triggered by the host to limit viral production, and anti-apoptotic events likely triggered by the virus to increase its production and survival. In cells of monocytic origin, which act as a reservoir for the virus, the anti-apoptotic molecules are favored; in cells of lymphocytic origin, molecules with an apoptotic meaning are prevalent.

The Fas antigen (CD95) is a cell surface receptor that mediates cell apoptosis signalling. Recent investigations have shown that Fas-regulated apoptosis was linked to neurodegenerative lesions in the brain of patients with Alzheimer’s disease (AD). Here data regarding the association of two polymorphisms of the Fas promoter region with AD patient’s cognitive deterioration are reported. The polymorphism at position –1377 was associated with the risk of developing AD and with a differential rate of cognitive decline during a 2-year follow-up. The polymorphism at position –670 was not associated with the risk of AD and with the cognitive decline during the follow-up. Our data suggest that different genetic background in the Fas gene may influence the risk and clinical progression of the disease by affecting neurodegenerative processes leading to neuronal loss. Copyright © 2006 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Apoptosis plays a crucial role in immunosenescence, as also evidenced by the increased expression of Fas in lymphocytes from aged people. However, little is known about the genetic regulation of Fas and its ligand, FasL. We have studied their polymorphisms in 50 centenarians and 86 young donors living in Northern Italy. The first Fas polymorphism, at position -670, has in Caucasian a heterozigosity of 51%; the second, at -1377 position, has the wild type allele (G) with a very high frequency (83%) respect to the mutant allele. Genotype and allele distribution for both polymorphisms were similar in controls and centenarians. Similar results were found as far as two FasL polymorphisms (IVS2nt-124 and IVS3nt169) are concerned. On the whole, our data suggest that Fas and FasL polymorphisms, as well as their haplotypes, are unlikely to be associated with successful human longevity. [ABSTRACT FROM AUTHOR]