Cell fate regulated by nuclear factor-κB- and activator protein-1-dependent signalling in human melanocytes exposed to ultraviolet A and ultraviolet B.

BACKGROUND: Ultraviolet (UV) radiation constitutes an important risk factor for malignant melanoma, but the wavelength responsible for the initiation of this disease is not fully elucidated. Solar UV induces multiple signalling pathways that are critical for initiation of apoptotic cell death as a cellular defence against malignant transformation. OBJECTIVES: To evaluate the involvement of the transcription factors nuclear factor (NF)-κB and activator protein (AP)-1 in the signalling pathways induced by UVA or UVB irradiation in human melanocytes. METHODS: Primary cultures of normal human melanocytes were irradiated with UVA or UVB, and the concomitant DNA damage and redox alterations were monitored. The resulting activation of the NF-κB and AP-1 signalling pathways and subsequent apoptosis were studied. RESULTS: UVB irradiation causes DNA damage detected as formation of cyclobutane pyrimidine dimers, while UVA induces increased levels of 8-hydroxydeoxyguanosine and lipid peroxidation. UVA and UVB initiate phosphorylation of c-Jun N-terminal protein kinase and extracellular signal-regulated kinase, and the apoptosis signalling pathways converge into a common mechanism. Downregulation of c-Jun suppresses AP-1-mediated signalling and prevents apoptosis upstream of lysosomal and mitochondrial membrane permeabilization, whereas inhibition of NF-κB by SN50 increases apoptosis. CONCLUSIONS: We conclude that AP-1 induces proapoptotic signalling, whereas NF-κB is a key antiapoptotic/prosurvival factor in both UVA- and UVB-induced cellular damage in human melanocytes, which might in turn impact melanoma development and progression.

The histone deacetylase inhibitor trichostatin A reduces lysosomal pH and enhances cisplatin-induced apoptosis.

High activity of histone deacetylases (HDACs) has been documented in several types of cancer and may be associated with survival advantage. In a head and neck squamous cell carcinoma cell line, cisplatin-induced apoptosis was augmented by pretreatment with the HDAC inhibitor trichostatin A. Apoptosis was accompanied by lysosomal membrane permeabilization (LMP), as shown by immunoblotting of the lysosomal marker protease cathepsin B in extracted cytosol and by immunofluorescence. Moreover, LAMP-2 (lysosomal associated membrane protein-2) was translocated from lysosomal membranes and found in a digitonin extractable fraction together with cytosolic proteins and pretreatment with trichostatin A potentiated the release. Overall, protein level of LAMP-2 was decreased during cell death and, interestingly, inhibition of cysteine cathepsins, by the pan-cysteine cathepsin inhibitor zFA-FMK, prevented loss of LAMP-2. The importance of LAMP-2 for lysosomal membrane stability, was confirmed by showing that LAMP-2 knockout MEFs (mouse embryonic fibroblasts) were more sensitive to cisplatin as compared to the corresponding wildtype cells. Trichostatin A reduced lysosomal pH from 4.46 to 4.25 and cell death was prevented when lysosomal pH was increased by NH(4)Cl, or when inhibiting the activity of lysosomal proteases. We conclude that trichostatin A enhances cisplatin induced cell death by decreasing lysosomal pH, which augments cathepsin activity resulting in reduced LAMP-2 level, and might promote LMP.

Glutathione in the blood and cerebrospinal fluid: a study in healthy male volunteers.

Glutathione (GSH) is an important regulator of intracellular redox homeostasis. In the brain, glutathione is considered a major antioxidant, which is also found at high concentrations in the extracellular environment. Altered GSH balance in plasma, blood and cerebrospinal fluid (CSF) has been observed in several disorders suggesting that an impaired antioxidant function is part of the pathophysiology. The aim of the present study was to investigate a possible relationship between glutathione in plasma and CSF. Blood samples were collected from 26 healthy male volunteers at 8a.m., noon, 4p.m. and 8 p.m. At 8a.m. the following morning, blood was drawn and three 6-ml fractions of CSF were collected by lumbar puncture. In CSF, a disrupted gradient was found showing the highest glutathione concentration in the second compared to the first and third fraction (P<0.002). Moreover, correlation and regression analyses between glutathione in plasma and CSF revealed an association between the third fraction CSF and plasma glutathione 8 p.m. the day before lumbar puncture. Thus, if carefully standardised due to the disrupted gradient in CSF, it might be possible to estimate glutathione levels in CSF by analysing plasma in healthy males.

Culturing of diagnostic muscle biopsies as spheroid-like structures: a pilot study of morphology and viability.

OBJECTIVE: The aim of this study was to establish three-dimensional cultures originating from muscle biopsies and evaluate the viability and morphology. METHOD: Muscle biopsies from patients with suspected neuromuscular disorders were obtained and established as primary muscle tissue cultures. Tissue pieces, 1-2 mm of diameters, were placed in culture medium and subjected to sporadic stirring to prevent attachment and outgrowth as monolayer cells. Morphology and ability to attach to the surface were investigated by light microscopy. Viability was evaluated by (99m)Tc-tetrofosmin uptake. After 1 month, histology was evaluated by light microscopy and immunocytochemistry. The findings of a healthy muscle and a dystrophic muscle were compared. RESULTS: Initially, the tissue pieces were unshaped but formed spheroid-like structures during the culture period. For dystrophic muscle, attachment capacity to the surface was initially potent and decreased during the culture period, whereas control muscle showed weak attachment from the start that increased during the culture period. The uptake of (99m)Tc-tetrofosmin increased in control muscle, while it decreased in dystrophic muscle, during the culture period. The histological investigation demonstrated larger destruction of myofiber, weaker satellite cell activation and reduced myofiber regeneration in the dystrophic muscle as compared to the control muscle. CONCLUSION: The cellular components of the muscle tissue can survive and proliferate as spheroid-like primary cultures. The cellular composition resembles the in vivo condition, which allows studies of degeneration of the original fibers, and activation and proliferation of the satellite cells. The culture system may provide better understanding of the degeneration and regeneration processes in different muscle disorders and allow investigations of pharmacological interventions.

Effects of inhibitors of the arachidonic acid cascade on primary muscle culture from a Duchenne muscular dystrophy patient.

The aim of this study was to elucidate the mechanisms of action for potential targets of therapeutic intervention related to the arachidonic acid cascade in muscular dystrophy. Primary cultures from a Duchenne patient were used to study the expression of dystrophin-1, utrophin, desmin, neonatal myosin heavy chain (MHCn) and Bcl-2 during inhibition of phospholipase A2 (PLA2), cyclooxygenase (COX) and lipoxygenase (LOX). Hypo-osmotic treatment was applied in order to trigger Ca2+ influx and PLA2 activity. Inhibition of PLA2 and LOX with prednisolone and nordihydroguaiaretic acid (NDGA) caused a semi-quantitative increase of utrophin and Bcl-2-, and a dose-dependent, quantitative increase of desmin expression, an effect that was augmented by hypo-osmotic treatment. Our results indicate that LOX inhibitors, similarly to corticosteroids, can be beneficial in the treatment of muscular dystrophies.

Ultraviolet (UV)A- and UVB-induced redox alterations and activation of nuclear factor-kappaB in human melanocytes-protective effects of alpha-tocopherol.

BACKGROUND: Despite compelling evidence that ultraviolet (UV) irradiation causes melanoma the knowledge concerning reaction pathways and signalling transduction in melanocytes is still limited. OBJECTIVES: To evaluate the protective capacity of alpha-tocopherol and beta-carotene during UVA and UVB irradiation of human melanocytes in vitro. METHODS: Primary cultures of normal human melanocytes were irradiated by different wavelengths within the UV spectrum (UVA 6 J cm(-2), UVB 60 mJ cm(-2)). Redox alterations and apoptosis were studied and the protective potential of alpha-tocopherol and beta-carotene was evaluated. RESULTS: UVA and UVB irradiation decreased the intracellular concentration of reduced glutathione and activated the transcription factor nuclear factor (NF)-kappaB, detected as the increased level of the p65 subunit and translocation to the nucleus. This coincided with a rise in the level of gamma-glutamyl-cysteine-synthetase, the rate-limiting enzyme of the glutathione synthesis. UVA and UVB caused apoptotic cell death as detected by nuclear fragmentation and caspase activation 24 h postirradiation. Pretreatment with alpha-tocopherol prevented UVA- and UVB-induced glutathione loss, NF-kappaB translocation and diminished apoptosis, but beta-carotene did not show a similar protective capacity. Further, exposure to alpha-tocopherol by itself reduced cell proliferation rate. CONCLUSIONS: UVA and UVB irradiation affected the intracellular redox state and increased the frequency of apoptosis in human melanocytes in vitro. alpha-Tocopherol might be a useful substance in protecting melanocytes from UV-induced damage.

Cathepsin D mediates cytochrome c release and caspase activation in human fibroblast apoptosis induced by staurosporine.

There is increasing evidence that proteases other than caspases, for example, the lysosomal cathepsins B, D and L, are involved in apoptotic cell death. In the present study, we present data that suggest a role for cathepsin D in staurosporine-induced apoptosis in human foreskin fibroblasts. Cathepsin D and cytochrome c were detected partially released to the cytosol after exposure to 0.1 muM staurosporine for 1 h. After 4 h, activation of caspase-9 and -3 was initiated and later caspase-8 activation and a decrease in full-length Bid were detected. Pretreatment of cells with the cathepsin D inhibitor, pepstatin A, prevented cytochrome c release and caspase activation, and delayed cell death. These results imply that cytosolic cathepsin D is a key mediator in staurosporine-induced apoptosis. Analysis of the relative sequence of apoptotic events indicates that, in this cell type, cathepsin D acts upstream of cytochrome c release and caspase activation.

Chlamydia trachomatis-induced apoptosis occurs in uninfected McCoy cells late in the developmental cycle and is regulated by the intracellular redox state.

Infections with the obligate intracellular bacterium Chlamydia trachomatis are characterized by avoidance of fusion between chlamydia-containing endosomes and lysosomes, bacterial persistence and development of post-infectious sequelae. In this report we show that C. trachomatis induces apoptosis in McCoy and HeLa cells. Apoptosis was monitored by three different techniques; enzyme-linked immunoassay (EIA) of fragmented nucleosomes, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) and flow cytometry of propidium iodide-stained cells. Apoptosis occurred in uninfected cells, was induced late in the chlamydial developmental cycle, beyond 24 h post-infection and was dependent on bacterial protein synthesis. Apoptosis was not significantly increased in infected, inclusion-containing cells. Treatment of cells with the antioxidants ascorbic acid (10 microM) and alpha-tocopherol (10 microM) reduced the degree of apoptosis. These results suggest that host cells infected with C. trachomatis generate proapoptotic stimuli that induce apoptosis in uninfected, neighbouring cells and that the redox state of the cell is a regulator in chlamydia-induced apoptosis.

Inhibition of cathepsin D prevents free-radical-induced apoptosis in rat cardiomyocytes.

Apoptosis was inhibited in rat cardiomyocytes pretreated with the aspartic protease inhibitor pepstatin A and subsequently exposed to naphthazarin (5,8-dihydroxy-1,4-naphthoquinone). Cathepsin D was released from lysosomes to the cytosol upon exposure to naphthazarin, and the enzyme activity decreased simultaneously. Later, cathepsin D reappeared in granules of increased size, and enzyme activity was restored. Activation of caspase-3-like proteases was detected, and the number of cells showing apoptotic morphology increased with time. Pepstatin A pretreatment did not prevent release of cathepsin D from lysosomes but did significantly inhibit subsequent naphthazarin-induced caspase activation and apoptotic morphology. This suggests that cathepsin D exerts its apoptosis-stimulating effect upstream of caspase-3-like activation.

Anthraquinone cytotoxicity and apoptosis in primary cultures of rat hepatocytes.

We compared three different anthraquinones, rhein (4,5-dihydroxy-anthraquinone-2-carboxylic acid), danthron (1,8-dihydroxy-anthraquinone) and chrysophanol (1,8-dihydroxy-3-methylanthraquinone), with respect to their toxicity and ability to induce apoptosis in primary cultures of rat hepatocytes. Rhein was the most effective in producing free radicals, and was the only one of the tested anthraquinones that could induce apoptosis. Addition of 50 microM rhein to hepatocyte cultures led to depletion of intracellular reduced glutathione (GSH) and ATP and accumulation of lipid peroxidation products. The substances N,N'-diphenyl-p-phenylenediamine (DPPD), dithiothreitol (DTT), nifedipine and desferal all protected the hepatocytes, i.e. prevented viability loss and ATP depletion, and decreased the GSH depletion. Cultures exposed to rhein for 15 min and subsequently rinsed and incubated for 16 h under normal culture conditions (complete medium) exhibited apoptosis, as shown by DNA fragmentation, nuclear condensation and positive TUNEL reaction. Pretreatment with the antioxidant DPPD and the iron-chelator desferal gave complete protection against apoptosis. No signs of oxidative cell damage were detected when the cultures were exposed to danthron or chrysophanol. All three anthraquinones did, however, cause an immediate increase in the intracellular Ca2+ concentration. We conclude that rhein, which contains one carboxyl group, is a suitable substrate for one-electron-reducing enzymes and an effective redox cycler, which leads to the production of oxygen-derived free radicals that eventually induce apoptotic cell death.

Lysosomal release of cathepsin D precedes relocation of cytochrome c and loss of mitochondrial transmembrane potential during apoptosis induced by oxidative stress.

Apoptosis was induced in human foreskin fibroblasts by the redox-cycling quinone naphthazarin (5,8-dihydroxy-1,4-naphthoquinone). Most of the cells displayed ultrastructure typical of apoptosis after 8 h of exposure to naphthazarin. Apoptosis was inhibited in fibroblasts pretreated with the cathepsin D inhibitor pepstatin A. Immunofluorescence analysis of the intracellular distribution of cathepsin D revealed a distinct granular pattern in control cells, whereas cells treated with naphthazarin for 30 min exhibited more diffuse staining that corresponded to release of the enzyme from lysosomes to the cytosol. After 2 h, release of cytochrome c from mitochondria to the cytosol was indicated by immunofluorescence. The membrane-potential-sensitive probe JC-1 and flow cytometry did not detect a permanent decrease in mitochondrial transmembrane potential (delta psi(m)) until after 5 h of naphthazarin treatment. Our findings show that, during naphthazarin-induced apoptosis, lysosomal destabilization (measured as release of cathepsin D) precedes release of cytochrome c, loss of delta psi(m), and morphologic alterations. Moreover, apoptosis could be inhibited by pretreatment with pepstatin A.

Protection by alpha-tocopherol but not ascorbic acid from hydrogen peroxide induced cell death in normal human breast epithelial cells in culture.

Alpha-tocopherol and ascorbic acid have been suggested to play a role in breast cancer prevention due to their antioxidative capacity. Increased exposure to endogenous and exogenous sex steroids is a known risk factor for breast cancer. We have studied the effects of alpha-tocopherol and ascorbic acid on hydrogen peroxide induced cell death in sex hormone treated normal breast epithelial cells in culture. We found that alpha-tocopherol but not ascorbic acid alone protected the cells. The effect of alpha-tocopherol increased when ascorbic acid was added to the cultures. The hydrogen peroxide degradation rate decreased in cultures treated with alpha-tocopherol alone and in combination with ascorbic acid compared to cells grown in medium or with ascorbic acid only. Oestradiol and progesterone treatment did not influence the results. Possible beneficial effects of combining various antioxidants, endogenous as well as exogenous, on human breast tissue need to be investigated further both in vivo and in vitro.

Impact of oestradiol and progesterone on antioxidant activity in normal human breast epithelial cells in culture.

The risk of developing breast cancer increases after long term use of oestrogen and progestagen, and carcinogenesis in the breast is partly due to oxidative damage to DNA bases. Therefore, we studied the effects of 17 beta-oestradiol and progesterone on the antioxidative status and the vulnerability to oxidative stress exhibited by normal human breast epithelial cells in culture. After exposure to hydrogen peroxide, cells grown with oestradiol alone or with both oestradiol and progesterone showed significantly decreased viability compared to cells grown in medium without added hormones. There was, however, no difference in hydrogen peroxide degradation rate between controls and hormone treated cultures. When desferrioxamine was added, the viability increased and the hydrogen peroxide degradation rate decreased. The levels of several antioxidants were altered in cells grown in the presence of oestradiol and progesterone: the concentrations of glutathione reductase and catalase decreased significantly while the levels of glutathione peroxidase and reduced glutathione did not change. The alterations in enzyme activity and cell vulnerability were more pronounced in cultures treated with a combination of oestradiol and progesterone. We conclude that the redox balance in the cultured normal human breast epithelial cells was altered by treatment with oestradiol and progesterone, and that this change led to the increased death of cells subsequently exposed to hydrogen peroxide. This effect may have implications for sex hormone dependent diseases of the breast.

Oxidative stress causes relocation of the lysosomal enzyme cathepsin D with ensuing apoptosis in neonatal rat cardiomyocytes.

Exposing neonatal rat heart myocytes to the redox cycling quinone naphthazarin (5,8-dihydroxy-1,4-naphthoquinone) for 15 to 45 minutes led to a time-dependent release of cathepsin D from many secondary lysosomes to the cytosol, as analyzed by morphometry. Cathepsin D was detected electron microscopically using a pre-embedding immunostaining technique that utilizes antibodies conjugated to ultra-small (0.8-nm) gold particles and subsequent silver enhancement. The exposure to naphthazarin also caused a decrease in both the pH and the ATP level of the cells within the same time frame. Lipid peroxidation was, however, not detected. Pretreatment of the cultures with alpha-tocopherol succinate prevented cathepsin D relocation, as shown by immunofluorescence. After exposure to naphthazarin, cells were washed, and normal culture conditions were re-established for 18 hours. Many cells then showed apoptotic morphology (ie, cellular shrinkage and chromatin condensation) as analyzed by Giemsa staining. Also, 41% of the cells stained positive with the TUNEL technique, and DNA fragmentation was detected by separation of intact and fragmented DNA. Apoptosis was significantly decreased in cultures pretreated with alpha-tocopherol succinate.

A pre-embedding technique for immunocytochemical visualization of cathepsin D in cultured cells subjected to oxidative stress.

We describe a pre-embedding immunocytochemical method for visualization of the lysosomal enzyme cathepsin D in cultured cells. The protein was demonstrated at both light and electron microscopic levels by neutral-pH silver enhancement of ultrasmall (0.8-nm) gold particles conjugated to the antibodies. The best morphological preservation and the highest labeling density were achieved by initial fixation for 20 min at 4C in 4% paraformaldehyde (PFA) and 0. 05% glutaraldehyde (GA) in 0.15 M sodium cacodylate buffer, followed by permeabilization in sodium borohydride. Three cell types were used: human foreskin fibroblasts, histocytic lymphoma (J-774) cells, and primary rat heart myocytes. In all three, cathepsin D was demonstrated in lysosome-like structures. The rat heart myocytes were also exposed to the redox cycling substance naphthazarin (5, 8-dihydroxy-1,4-naphthoquinone) to induce oxidative stress. This was done for such a short period of time that the cells initially did not show any signs of morphological damage and retained normal plasma membrane stability, although an early and clear redistribution of cathepsin D from membrane-bound structures to the cytosol was apparent. This redistribution was followed by cell degeneration and, eventually, by cell death.

A short exposure to a high-glucose milieu stabilizes the acidic vacuolar apparatus of insulinoma cells in culture to ensuing oxidative stress.

It was recently suggested that extracellular hydrogen peroxide, after diffusing into and throughout adjacent cells--which may be the case if they have only a weak capacity to degrade hydrogen peroxide--labilizes their lysosomal compartment due to its content of low-molecular-weight iron in redox-active form. The iron would be present as a consequence of normal autophagocytotic degradation of various iron-containing metalloproteins. Beta- and insulinoma cells are especially vulnerable to oxidative stress, since they possess only low capacity to degrade hydrogen peroxide, and, perhaps, since they normally have a certain degree of autophagocytotic degradation of secretory granules with some iron content--crinophagy. The toxicity to beta cells of oxidative stress, such as an exposure to alloxan, that results in extracellular formation of hydrogen peroxide, is considerably reduced if animals are initially given an intravenous bolus dose of glucose, temporarily bringing up the blood level to about 20 mM. In this study it was demonstrated that already as short an exposure as 30 min to 20 mM D-glucose reduces the sensitivity of HIT and NIT insulinoma cells in culture to a subsequent exposure to hydrogen peroxide. In parallel, exposure to such a high-glucose medium also reduces their desferrioxamine-available amount of iron and, moreover, stabilizes their lysosomal membranes against oxidative stress--thus preventing diffusion to the cytosol of damaging lysosomal contents following iron-catalyzed, Fenton-type, intralysosomal reactions. We suggest that both general autophagocytotic turnover and, in particular, crinophagy of secretory granules are decreased by an increased glucose concentration of the surrounding milieu, with attendant reduced amounts of intralysosomal low-molecular-weight iron and, thus, diminished sensitivity to oxidative stress.

Nutrient deprivation of cultured rat hepatocytes increases the desferrioxamine-available iron pool and augments the sensitivity to hydrogen peroxide.

Primary cultures of rat hepatocytes were subjected to amino acid and serum deprivation for 4 h. This treatment augmented the sensitivity to ensuing hydrogen peroxide exposure for 30 min. The by nutrient deprivation-increased autophagocytosis was confirmed by transmission electron microscopy and uptake of the lysosomotropic weak base acridine orange within the intracellular acidic vacuolar apparatus. The desferrioxamine-available pool of iron increased 2.5-fold during deprivation, compared with control cells. Furthermore, amino acid deprivation increased the cellular protein turnover, measured by radioactive labeling with -3H-Leu. Exposure to 40 microM ascorbic acid specifically decreased the turnover of ferritin, as estimated by enzyme-linked immunosorbent assay, and prevented an increase of the desferrioxamine-available iron pool, resulting in protection against hydrogen peroxide-induced cell killing. Thus, hepatocytes with nutrient deprivation-enhanced autophagocytosis contain a larger pool of catalytically active iron than control cells. This iron pool is mainly derived from the turnover (autophagocytosis) of cytosolic ferritin and is probably situated in the lysosomes. Furthermore, nutrient-deprived cells show augmented sensitivity to hydrogen peroxide-induced oxidative stress, since the enhanced availability of iron in low molecular weight form results in an increased potential of intralysosomal Fenton chemistry, that may cause lysosomal rupture with release of potent hydrolytic enzymes.

Variability of glutathione levels in normal breast tissue and subcutaneous fat during the menstrual cycle: an in vivo study with microdialysis technique.

A small increase in the risk of breast cancer has been reported after long term use of combined estrogen-progestagen treatment. Free oxygen radicals and antioxidants such as glutathione are involved in the regulation of proliferation and apoptosis and thereby in carcinogenesis. To study whether the glutathione levels are sex hormone dependent, we used the microdialysis technique to measure the in vivo concentrations of glutathione in breast tissue and sc fat during the menstrual cycle. Six healthy women (23-32 yr old) were investigated early in the follicular phase and the midluteal phase. Two 60-min fractions each were collected by microdialysis of periumbilical fat and breast tissue, respectively. The samples were stored at -70 C and analyzed by high performance liquid chromatography. Glutathione concentrations increased in the midluteal phase compared to those in the follicular phase in both adipose tissue and breast tissue (P < 0.05). The variability of glutathione levels during the menstrual cycle, with higher levels late in the menstrual cycle, indicates that the antioxidant system could be sex hormone dependent. This may be of importance in breast cancer development.

Microfluorometry using fluorescein diacetate reflects the integrity of the plasma membrane in UVA-irradiated cultured skin fibroblasts.

The damaging effect of long-wave ultraviolet radiation (UVA) on the plasma membranes of cultured human skin fibroblasts was evaluated by cytofluorometry performed after vital staining with fluorescein diacetate. The damage was associated with lipid peroxidation, as shown by accumulation of malondialdehyde and 4-hydroxyalkenals; such accumulation was induced by a UVA dose of only 8 J/cm2. Pretreatment with the effective membrane peroxidation inhibitor alpha-tocopherol (added in the form of alpha-tocopherol succinate) or the singlet oxygen quencher beta-carotene protected the cells from membrane damage. Further, depletion of intracellular glutathione by exposure of the cells to buthionine sulfoximine, an inhibitor of gamma-glutamylcysteine synthetase aggravated the membrane-damaging effects. The results confirm the photodynamic effects of UVA, which presupposes the excitation of endogenous photosensitizer(s) and the production of reactive oxygen species. The present results indicate that this method of detection of alterations in plasma membrane stability may be highly suitable for studying various photobiological phenomena and for use as a model for testing substances that could protect skin from UVA damage. The trypan blue exclusion test proved to be too insensitive to detect these changes.