Apoptosis has classically been viewed as a process not involving mitochondria, whereas the implication of mitochondrial dysfunction in necrosis has been recognized for several decades. Recently, it has become clear that apoptosis implies a disruption of mitochondrial membrane intregrity that is decisive for the cell death process. Cytofluorometric methods assessing the mitochondrial membrane function and structure can be employed to demonstrate that, at least in most models of apoptosis, mitochondrial changes precede caspase and nuclease activation. Moreover, pharmacological and genetic experiments suggest that the loss of mitochondrial membrane integrity is a critical event of the apoptotic process, beyond or at the point of no return of programmed cell death. Inhibitors of the mitochondrial megachannel (= permeability transition pore) can prevent both the mitochondrial and the post-mitochondrial manifestations of apoptosis.

Apoptosis↗

PubMed1998

Study on the Sternberg paradigm in cirrhotic patients without overt hepatic encephalopathy.

Memory dysfunction is reported in cirrhotics. The aim of this paper was to increase insight into memory function of cirrhotic patients without overt hepatic encephalopathy. Eighty-six consecutive cirrhotics without overt hepatic encephalopathy (aged 54+/-10 yr., mean+/-s.d.) and 28 controls (52+/-10 yr.) with comparable education level were enrolled. Seventeen patients were class A, 55 class B, 14 class C according to Child-Pugh classification; 29 had alcoholic cirrhosis. The presence of subclinical signs of central nervous system dysfunction were assessed by Number Connection Test (NCT) and quantified EEG analysis. Memory scanning was evaluated by reaction times (RTs) in the Sternberg paradigm. MANOVA analysis showed that RTs were higher (F1,99=11, p<0.01) and time outs (TOs) more frequent (F1,110=10, p<0.01) in cirrhotics than in controls, whereas button press errors (BPEs) did not differ significantly (F1,110=2, p=n.s.). In cirrhotics, an interaction Child-Pugh class x memory set size was found (F2,146=4, p<0.05), showing exceedingly delayed RTs with greater memory set size in class C patients. Patients with altered NCT had significantly prolonged RTs (F1,71=4, p<0.05) and more TOs (F1,82=11, p<0.01) than patients with normal NCT. Cirrhotics with altered EEG had significantly prolonged RTs (F2,70=6, p<0.01). RTs were found to be correlated to alpha relative power (r=-0.4, p<0.01) and theta relative power (r=0.4, p<0.01). In conclusion, cirrhotics without over encephalopathy, but with NCT or EEG alterations, perform a computerized digit recognition task more slowly and with higher TOs than cirrhotic patients with normal NCT or EEG. In severe liver insufficiency (class C cirrhotics) also an impairment of memory scanning was detected. Sternberg test performance correlates with NCT and quantitative EEG parameters.

Adult↗

PubMed1998

Radiotherapy with or without androgen deprivation in the treatment of localized adenocarcinoma of the prostate.

This study analyzes the results of disease relapse and survival in two series of patients treated between 1974 and 1991 with definitive irradiation, with or without early androgen deprivation, for carcinoma of the prostate localized to the pelvis. All 264 patients were irradiated to the prostate and pelvic lymph nodes with a dose of 50 to 54 Gy in 25 to 27 fractions, followed by a 16- to 20-Gy boost in 8 to 10 fractions to the prostate and periprostatic region. Ninety percent of patients received a total dose to the prostate (pelvis + boost) of 70 Gy. Ninety-nine of the 264 patients underwent early androgen deprivation. The endocrine manipulation program was initiated 0 to 9 months before the beginning of the radiotherapy course and was continued for 2 or more years or until disease progression. All patients who relapsed after radiotherapy alone received late hormonal manipulation. After a median follow-up of 100 months, no difference in the incidence of local and distant failure rate and cancer-specific mortality was detected between the two treatment groups. The local and distant failure rates were, respectively, 19% and 40% in patients who had undergone radiotherapy and early androgen deprivation and 20% and 36% in patients who received radiotherapy alone. Cancer mortality was similar, with 35% and 30% of deaths in the former and latter group, respectively. Death for intercurrent disease, however, was significantly more frequent (p = 0.03) in patients treated with radiotherapy and hormones (19%) than in those who received radiotherapy alone (8%). Actuarial analysis of both metastasis-free and disease-free survival detected no difference between the two treatment groups, with 10-year rates of 53.3% and 42.5%, respectively, in the radiation-alone group and 45.5% and 47%, respectively, in the radiation-plus-androgen deprivation group. A statistically significant difference (p = 0.03) in overall survival in favor of patients treated with radiotherapy alone was noted, with a 10-year rate of 47%, compared with 26% observed in the radiotherapy-plus-androgen deprivation group. In conclusion, results of our study confirm numerous reports based on retrospective analyses that failed to show any benefit of hormonal management adjuvant to a definitive irradiation. The disappointing finding was the significantly better overall survival in patients who underwent radiotherapy alone.

Adenocarcinoma↗

PubMed1997

The apoptosis-necrosis paradox. Apoptogenic proteases activated after mitochondrial permeability transition determine the mode of cell death.

Mitochondrial alterations including permeability transition (PT) constitute critical events of the apoptotic cascade and are under the control of Bcl-2 related gene products. Here we show that induction of PT is sufficient to activate CPP32-like proteases with DEVDase activity and the associated cleavage of the nuclear DEVDase substrate poly(ADP-ribose) polymerase (PARP). Thus, direct intervention on mitochondria using a ligand of the mitochondrial benzodiazepin receptor or a protonophore causes DEVDase activation. In addition, the DEVDase activation triggered by conventional apoptosis inducers (glucocorticoids or topoisomerase inhibitors) is prevented by inhibitors of PT. The protease inhibitor N-benzyloxycabonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD.fmk) completely prevents the activation of DEVDase and PARP cleavage, as well as the manifestation of nuclear apoptosis (chromatin condensation, DNA fragmentation, hypoploidy). In addition, Z-VAD.fmk delays the manifestation of apoptosis-associated changes in cellular redox potentials (hypergeneration of superoxide anion, oxidation of compounds of the inner mitochondrial membrane, depletion of non-oxidized glutathione), as well as the exposure of phosphatidylserine residues in the outer plasma membrane leaflet. Although Z-VAD.fmk retards cytolysis, it is incapable of preventing disruption of the plasma membrane during protracted cell culture (12-24 h), even in conditions in which it completely blocks nuclear apoptosis (chromatin condensation and DNA fragmentation). Electron microscopic analysis confirms that cells treated with PT inducers alone undergo apoptosis, whereas cells kept in identical conditions in the presence of Z-VAD.fmk die from necrosis. These observations are compatible with the hypothesis that PT would be a rate limiting step in both the apoptotic and the necrotic modes of cell death. In contrast, it would be the availability of apoptogenic proteases that would determine the choice between the two death modalities.

Amino Acid Chloromethyl Ketones↗

PubMed1997

Glutathione depletion is an early and calcium elevation is a late event of thymocyte apoptosis.

According to current understanding, several metabolic alterations form part of the common phase of the apoptosis process. Such alterations include a disruption of the mitochondrial transmembrane potential (delta psi(m)), a depletion of nonoxidized glutathione (GSH) levels, an increase in the production of reactive oxygen species (ROS), and an elevation in cytosolic free Ca2+ levels. Using a cytofluorometric approach, we have determined each of these parameters at the single cell level in thymocytes or T cell hybridoma cells undergoing apoptosis. Regardless of the apoptosis induction protocol (glucocorticoids, DNA damage, Fas cross-linking, or CD3epsilon cross-linking), cells manifest a near-to-simultaneous delta psi(m) dissipation and GSH depletion early during the apoptotic process. None of the protocols for apoptosis inhibition (antioxidants, delta psi(m) stabilization, Bcl-2 hyperexpression, or inhibition of IL-1-converting enzyme) allowed for the dissociation of delta psi(m) disruption and GSH depletion, indicating that both parameters are closely associated with each other. At a later stage of the apoptotic process, cells manifest a near-simultaneous increase in ROS production and intracellular Ca2+ levels. Whereas the thapsigargin- or ionophore-induced elevation of calcium levels has no immediate consequence on delta psi(m') cellular redox potentials, or ROS production, pro-oxidants and menadione, an inducer of mitochondrial superoxide anion generation, cause a rapid (15 min) Ca2+ elevation. Together, these data suggest a two-step model of the common phase of apoptosis. After an initial delta psi(m) dissipation linked to GSH depletion (step 1), cells hyperproduce ROS with an associated disruption of Ca2+ homeostasis (step 2).

Animals↗

PubMed1997

Bcl-2 and Bcl-XL antagonize the mitochondrial dysfunction preceding nuclear apoptosis induced by chemotherapeutic agents.

A number of apoptosis-inducing agents used in cancer therapy (etoposide, doxorubicin, 1-beta-D-arabinofuranosylcytosine), as well as the proapoptotic second messenger ceramide, induce a disruption of the mitochondrial transmembrane potential (delta psi m) that precedes nuclear DNA fragmentation. This effect has been observed in tumor cell lines of T-lymphoid, B-lymphoid, and myelomonocytic origin in vitro. Circulating tumor cells from patients receiving chemotherapy in vivo also demonstrate a delta psi m disruption after in vitro culture that precedes nuclear apoptosis. Transfection-enforced hyperexpression of the proto-oncogenes bcl-2 and bcl-XL protects against chemotherapy-induced apoptosis, at both the level of the mitochondrial dysfunction preceding nuclear apoptosis and the level of late nuclear apoptotic events. Bcl-2-mediated inhibition of ceramide-induced delta psi m disruption is observed in normal as well as anucleate cells, indicating that bcl-2 acts on an extranuclear pathway of apoptosis. In contrast to Bcl-2 and Bcl-XL, hyperexpression of the protease inhibitor cytokine response modifier A fails to protect tumor cells against chemotherapy-induced delta psi m disruption and apoptosis, although cytokine response modifier A does prevent the delta psi m collapse and posterior nuclear apoptosis triggered by cross-linking of Fas/Apo-1/CD95. In conclusion, delta psi m disruption seems to be an obligatory step of early (pre-nuclear) apoptosis, and delta psi m is stabilized by two members of the bcl-2 gene family conferring resistance to chemotherapy.

Adult↗

PubMed1997

Peripheral benzodiazepine receptors in isolated human pancreatic islets.

Peripheral benzodiazepine receptors have been shown in some endocrine tissues, namely the testis, the adrenal gland, and the pituitary gland. In this work we evaluated whether peripheral benzodiazepine receptors can be found in the purified human pancreatic islets and whether they may have a role in insulin release. Binding of the isoquinoline compound [3H]1-(2-chlorophenyl-N-methyl-1-methyl-propyl)-3- isoquinolinecarboxamide (13H]PK-11195) a specific ligand of peripheral benzodiazepine receptors, to cellular membranes was saturable and Scatchard's analysis of the saturation curve demonstrated the presence of a single population of binding sites, with an affinity constant value of 9.20 +/- 0.80 nM and a maximum number of binding sites value of 8913 +/- 750 fmol/mg of proteins. PK-11195 and 7-chloro-1,3-dihydro-1-methyl-5-(p-chlorophenyl)-2H-1,4- benzodiazepine-2-on (Ro 5-4864) significantly potentiated insulin secretion from freshly isolated human islets at 3.3 mM glucose. These results show the presence of peripheral benzodiazepine receptors in purified human pancreatic islets and suggest their role in the mechanisms of insulin release.

Benzodiazepinones↗

PubMed1997

Redox regulation of apoptosis: impact of thiol oxidation status on mitochondrial function.

The probability that a cell will undergo apoptosis is in part dictated by the cellular redox potential, which is mainly determined by the reduction and oxidation of thiol residues on glutathione and proteins. We and others have recently shown that mitochondria play a critical role in the apoptotic cascade. Here, we address the question as to whether thiol modification regulates apoptosis and in which cellular compartment apoptosis-regulatory thiols are localized. To resolve this problem, we employed the divalent thiol-reactive agent diamide, which causes thiol cross-linking and thus mimics disulfide bridge formation, and a panel of monovalent thiol-reactive compounds (which impede disulfide bridge formation due to thiol oxidation), one of which is specifically targeted to the mitochondrial matrix. Our data indicate that thymocyte apoptosis induced by diamide mimics natural apoptosis in the sense that mitochondrial transmembrane potential (delta psi(m)) disruption precedes nuclear chromatin degradation; that monovalent thiol-reactive compounds inhibit apoptosis induced by diamide, glucocorticoids, irradiation, and topoisomerase inhibition; that the critical thiols determining cell fate after exposure to diamide, glucocorticoids, or DNA damage are likely to be located in the mitochondrial matrix; and that thiol oxidation and reduction are critical for apoptosis induction by some stimuli (glucocorticoids, DNA damage), but not by Fas/CD95 cross-linking. Taken together, these findings suggest that, at least in some pathways of apoptosis, mitochondrial thiols constitute a critical sensor of the cellular redox potential.

Animals↗

PubMed1997

Effects of glibenclamide and metformin (alone or in combination) on insulin release from isolated human pancreatic islets.

Isolated human pancreatic islets were prepared by collagenase digestion and density gradient purification, and the effects of glibenclamide (0.5 and 5.0 mumol/l) and metformin (20 and 200 mumol/l), alone or in combination, on insulin release were evaluated at varying glucose concentrations. At 3.3 mmol/l glucose level, the addition of 5.0 mumol/l glibenclamide or 5.0 mumol/l glibenclamide plus 200 mumol/l metformin caused a significant increase of insulin release, compared with glucose alone. At 16.7 mmol/l glucose concentration, a significant increase of insulin secretion, compared with glucose alone, was produced by the addition of either 5.0 mumol/l glibenclamide, 200 mumol/l metformin, or both 5.0 mumol/l glibenclamide and 200 mumol/l metformin. The effect of the combination of the two drugs was significantly higher than that with either drug used alone. Thus, glibenclamide was shown to have an insulinotropic effect on human islets at both low and high glucose concentrations, and metformin at high glucose concentrations. A possible synergistic effect of glibenclamide and metformin at high glucose concentrations is also suggested.

Cell Separation↗

PubMed1997

Maintenance of in vitro and in vivo viability of bovine islets during prolonged (3 months) culture.

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Animals↗