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(Journal of Leukocyte Biology. 2003;73:399-406.)
© 2003 by Society for Leukocyte Biology

Possible contribution of apoptosis-inducing factor (AIF) and reactive oxygen species (ROS) to UVB-induced caspase-independent cell death in the T cell line Jurkat

Hideaki Murahashi*,{dagger}, Hiroshi Azuma*, Naoufal Zamzami{ddagger}, Ko-ji Furuya§, Kenji Ikebuchi*, Miki Yamaguchi*, Yoshiko Yamada*, Norihiro Sato*, Mitsuhiro Fujihara*, Guido Kroemer{ddagger} and Hisami Ikeda*

* Hokkaido Red Cross Blood Center, Sapporo, Japan;
{dagger} Research and Development Laboratory, Nipro Corporation, Kusatsu, Japan;
§ Hokkaido Institute of Public Health, Sapporo, Japan; and
{ddagger} CNRS-UMR 1599, Institut Gustave Roussy, Villejuif, France

Correspondence: Hiroshi Azuma, M.D., Hokkaido Red Cross Blood Center, Yamanote 2-2 Nishi-ku, Sapporo 063-0002, Japan. E-mail: azuma{at}hokkaido.bc.jrc.or.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We analyzed the mechanism of UVB-induced cell death using the Jurkat T cell line. Apoptosis was assessed by measuring phosphatidylserine (PS) externalization, caspase activity, the decrease in mitochondrial membrane potential ({Delta}{Psi}m), nucleosomal DNA fragmentation, and morphological changes such as chromatin condensation. The mitochondrio-nuclear translocation of apoptosis-inducing factor (AIF) was evaluated by confocal laser microscopy. The cell death pattern of UVB-irradiated cells was similar to the Fas-induced cell death pattern. However, zVAD-fmk inhibited the nucleosomal fragmentation of DNA but not the externalization of PS, decrease in {Delta}{Psi}m, or mitochondrio-nuclear translocation of AIF. N-acetyl L-cysteine significantly inhibited the translocation of AIF induced by UVB. These results suggested that caspase-dependent and -independent pathways were involved in UVB-induced cell death in Jurkat cells, and the mitochondrio-nuclear translocation of AIF was associated with the latter pathway. In addition, reactive oxygen species generated by UVB might be involved in inducing the mitochondrio-nuclear translocation of AIF.

Key Words: reactive oxygen species • mitochondria • caspase inhibitor • N-acetyl L-cysteine


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ultraviolet (UV) irradiation induces various cellular responses including the activation of protein kinases, production of transcription factors, and induction of proinflammatory cytokines [1 2 3 ]. These biological responses are closely related to reactive oxygen species (ROS) generated by UV [4 5 6 7 8 ]. In addition, some investigators have suggested a close relation between ROS and cellular apoptotic events [9 , 10 ].

Near UV (UVB and UVA) not UVC is applied to the treatment of graft versus host disease and intractable allergic or asthmatic dermatitis. In addition, UVB but not UVC was reported to inactivate bone marrow T lymphocytes while sparing hematopoietic precursor cells [11 ]. We have confirmed this result in our experimental system [12 ]. Thus, we paid attention to the biological effect of UVB on lymphocytes. There have been several studies examining the UVB-induced apoptosis of lymphocytes [12 13 14 15 16 17 ]. Recent studies have shown that UVB is capable of inducing an aggregation of death receptors such as Fas in the absence of its ligand, resulting in the activation of executioner caspases [18 , 19 ]. Thus, caspase activation appeared to be essential for the UVB-induced apoptotic cell death.

However, it has been reported that the death of dendritic cells induced by UVB irradiation cannot be inhibited by the caspase inhibitor [20 ]. This suggests the contribution of a caspase-independent pathway to UVB-induced cell death.

Recently, a new factor involved in the caspase-independent cell death process was identified and was named apoptosis-inducing factor (AIF) [21 ]. This factor is translocated from mitochondria to the nucleus in response to various stimuli, resulting in the induction of phosphatidylserine (PS) externalization, chromatin condensation, and a decrease in mitochondrial membrane potential ({Delta}{Psi}m) without nuclear body formation or nucleosomal DNA fragmentation in the absence of caspase activity [21 22 23 ].

Based on these reports, we focused on AIF and tried to elucidate its role in UV-induced cell death in Jurkat cells. Here, we present data suggesting an association of AIF in a caspase-independent pathway in UVB-induced Jurkat cell death and the possible involvement of ROS in the mitochondrio-nuclear translocation of AIF induced by UVB irradiation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture and UVB irradiation
The human T cell line, Jurkat, was cultured in RPMI 1640 (Gibco-BRL, Grand Island, NY) containing 10% fetal calf serum (FCS) and antibiotics (100 IU/ml penicillin and 100 mg/ml streptomycin) at 37ºC in a humidified atmosphere containing 5% CO2. UV irradiation was performed under conditions described elsewhere [12 ]. Briefly, UV irradiation was performed using four UVB lamps (FL15E UV-320, UVP Inc., San Gabriel, CA), which emit most of their energy within the UV-B range (300–320 nm) with an emission peak at 315 nm. UVB intensity was measured using a UVX digital radiometer (UVP Inc.). Before exposure to UV irradiation, cells were washed twice with plain RPMI-1640 medium and in the same medium. They were seeded in 60 mm tissue-culture dishes or 48-well plates at a concentration of 1 x 106 cells/ml and irradiated through serum-free medium at a depth of 1 mm. Subsequently, an equal volume of medium containing 20% FCS was added, and the incubation was continued. Throughout this study, a dose of 300 J/m2 UVB was used, based on findings of pilot experiments where this dose induced cell death in a substantial percentage of cells. Control cells were subjected to an identical procedure without exposure to UV. Similarly, cell death was induced by anti-Fas monoclonal antibody (mAb) CH-11 (Immunotech, Marseille, France) at a concentration of 20 ng/ml or diamide (Sigma Chemical Co., St. Louis, MO) at a concentration of 500 µM.

Assay for caspase-3, -8, and -9 (-like) protease activities
Caspase-3, -8, and -9 (-like) protease activities were measured using a CPP32/caspase-3, FLICE/caspase-8, and caspase-9/Mch6 colorimetric protease assay kit (MBL, Nagoya, Japan), respectively, according to the manufacturer’s protocol. Jurkat cells were treated with CH-11 or irradiated with UVB and incubated at 37ºC for various periods. Cells were collected at given times and washed twice with phosphate-buffered saline (PBS(–)), after which they were pelleted and lysed. Lysates were centrifuged at 10,000 g for 1 min, and supernatants were collected. The supernatants containing 50–100 µg protein were incubated at 37ºC for 2 h with 200 µM DEVD-pNA (for caspase-3), IETD-pNA (for caspase-8), or LEHD-pNA (for caspase-9) substrate, respectively, in 96-well plates (Corning Inc., Corning, NY). The substrate was hydrolyzed, and a colored product was formed in proportion to the activity of caspase or other related caspases in the sample. Substrate hydrolysis was determined by measuring sample absorbance at 405 nm using a spectrophotometer (TiterTek Multiskan® MCC/340, Flow Laboratories Inc., McClean, VA).

Evaluation of apoptosis by flow cytometry
To detect apoptotic cells, fluorescein isothiocyanate-conjugated Annexin V (AV; Boehringer Mannheim, Germany) and 2 µg/ml propidium iodide (PI; Sigma Chemical Co.) were used. Cells were incubated for 6 or 24 h after UVB irradiation or treatment with CH-11 antibody. The cells were then stained with AV and PI solution. Two-color flow cytometry was performed to distinguish between early apoptotic cells (positive for AV binding and negative for PI staining, AV+/PI-) and late apoptotic or necrotic cells (AV+/PI+) using a Cytoron (Ortho Diagnostic Systems K.K., Raritan, NJ).

Treatment of cells with inhibitors
Jurkat cells were suspended at a density of 1 x 106 cells/ml in plain RPMI-1640 zVAD-fmk (MBL) and N-acetyl L-cysteine (NAC; Sigma Chemical Co.) for at least 30 min or 1 h, respectively, before stimulation. Experiments were then performed in the continuous presence of these reagents.

Evaluation of {Delta}{Psi}m
To detect any decrease in {Delta}{Psi}m, Jurkat cells were preincubated for 30 min in the absence or presence of zVAD-fmk. Subsequently, they were mixed with CH-11 (20 ng/ml) or irradiated by UVB (300 J/m2) and further incubated. Cells were collected at given times and exposed to the potent fluorescence dye, 3,3'-dihexyloxacarbocyanin iodide (DiOC6, Lambda, Graz, Austria) at 40 nM for 15 min at 37ºC. After a wash with PBS (–) once, the cells were analyzed on a flow cytometer by measuring the fluorescence at 525 nm with an excitation wavelength of 488 nm.

Detection of nucleosomal fragmentation of DNA
Jurkat cells were seeded at a density of 1.8 x 105 cells/plate in 24-well plates and incubated for 7 or 18 h after UVB irradiation in the absence or presence of zVAD-fmk. Subsequently, cells were collected and pelleted. After lysis, the fragmented DNA was selectively extracted using ApopLadder ExTM (TaKaRa, Kyoto, Japan) according to the manufacturer’s protocol. Following electrophoresis of the fragmented DNA on 1.5% agarose gels and staining of the gels with ethidium bromide, bands were visualized under UV light.

Analysis of morphological changes of the nucleus
Jurkat cells were incubated in the absence or presence of 2 µM zVAD-fmk or 15 mM NAC for 1 h. Subsequently, they were mixed with CH-11 (20 ng/ml) or irradiated with UVB (300 J/m2) and further incubated for 24 h. Following centrifugation onto microscope slides using a Cytospin 2 centrifuge (Shandon Inc., Pittsburgh, PA), the cells were stained with May-Grunwald Giemsa solution (Merck, Darmstadt, Germany) and analyzed by light microscopy (Olympus BX50, Tokyo, Japan).

Confocal immunofluorescence microscopy of AIF localization
Jurkat cells were incubated in the absence or presence of 20 µM zVAD-fmk or 15 mM NAC for 1 h. Subsequently, they were irradiated with UVB (300 J/m2) and further incubated for 4 h. Cells were collected and washed twice with PBS, seeded at a density of 2 x 104 cells/well in 14-well slide glasses, and air dried. They were fixed with 4% paraformaldehyde and 0.19% picric acid in PBS for 30 min at room temperature, permeabilized with 0.1% sodium dodecyl sulfate in PBS for 10 min at room temperature, and then incubated in 3% bovine serum albumin (BSA) in PBS for 30 min at room temperature. Subsequently, cells were incubated with rabbit anti-AIF antiserum [21 , 22 ], diluted 1:200 in PBS containing 0.1% BSA for 10 h at 4ºC in a humidified chamber. After three washes in PBS containing 0.02% Tween 20, cells were incubated with 1:200-diluted Alexa Fluor® 568 goat anti-rabbit immunoglobulin G (IgG; Molecular Probes, Leiden, The Netherlands) and 1:200-diluted Alexa Fluor® 568 goat anti-mouse IgG (Molecular Probes) for 1 h at room temperature, with protection against light. After three washes in PBS containing 0.02% Tween 20, cells were incubated with 25 nM Sytox Green (Molecular Probes) for 15 min at room temperature. After three washes in PBS containing 0.02% Tween 20, slide glasses were mounted onto microscope slides and analyzed using an inverted laser scanning microscope (Fluoview, Olympus).

Statistical analysis
Statistical analyses were performed using the Mann-Whitney U-test (StatView, Version J4.02), with a P value of less than 0.05 taken to indicate that the value of the test sample was significantly different from that of the relevant control.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
UVB irradiation induces apoptosis associated with activation of caspases in Jurkat cells
To confirm that UVB induces the apoptosis of Jurkat cells, cells were irradiated at a dose of 300 J/m2. Cell death was then detected by monitoring PS externalization by AV binding and destruction of the cellular membrane by PI staining. Early apoptotic cells (AV+/PI-) were detected 6 h after UVB irradiation. The late apoptotic cells (AV+/PI+) became detectable 24 h after UVB irradiation (Fig. 1 ). These changes were similar to those of CH-11-induced cell death in Jurkat cells (data not shown). We next examined the time courses of changes in caspase-3, -8, and -9 (-like) activities after UVB irradiation and compared the results with those of CH-11 treatment. After UVB irradiation, an elevation of caspase-3, -8, and -9 (-like) activities was detected, reaching a peak at 9–12 h. These patterns of caspase activation were similar to those of CH-11 treatment (Fig. 2 ).



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  		Figure 1. Induction of apoptotic cell death by UVB irradiation. Jurkat cells were exposed to UVB at a dose of 300 J/m2. After incubation for 6 or 24 h, cells were stained with AV and PI solution. The early apoptotic cells (AV+/PtdIns- cells) were detected at 6 h. After 24 h, early apoptotic and late apoptotic or necrotic cells were observed.

 


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  		Figure 2. Evaluation of caspase-3, -8, and -9 (-like) activities. Caspase-3, -8, and -9 (-like) activities of Jurkat cells were measured at the times indicated after treatment with CH-11 (20 ng/ml; A) or UVB irradiation (300 J/m2; B). The pattern of elevation of caspase (-like) activity induced by UVB irradiation was quite similar to that induced by CH-11 treatment. Data shown are representative of four independent experiments.

 
Inhibitory effect of zVAD-fmk on PS externalization and DNA fragmentation
To clarify whether a caspase-independent pathway is involved in UVB-induced cell death, we assessed the inhibitory effects of zVAD-fmk on UVB-induced cell death in comparison with those on CH-11-induced cell death. CH-11-induced PS externalization was significantly (P<0.01) inhibited by 2 µM zVAD-fmk (Fig. 3A ). In contrast, UVB-induced PS externalization was not inhibited (P=0.347) by 2 µM zVAD-fmk (Fig. 3B) . Even at 20 µM, zVAD-fmk could not inhibit the UVB-induced PS externalization (Fig. 3D) . The same results were obtained with UVB irradiation at various doses (100–400 J/m2; Fig. 3C ). These findings indicated that UVB-induced PS externalization can be executed through a caspase-independent pathway. In addition, cells with PS externalization were observed 7 h after UVB irradiation in the absence or presence of 2 µM zVAD-fmk (Fig. 4A ). However, nucleosomal DNA fragmentation was detected only in the cells exposed to UVB in the absence of zVAD-fmk (Fig. 4B) . We confirmed that no DNA fragmentation was detected in zVAD-fmk-treated cells analyzed 18 h after UVB exposure (Fig. 4C) . These results suggested that even 2 µM zVAD-fmk was enough to block the caspase activity, and PS externalization can occur without caspase activity after UVB irradiation.



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  		Figure 3. Effects of zVAD-fmk on UVB-induced cell death. Jurkat cells were preincubated for 30 min in the absence (open bars) or presence (hatched bars) of 2 µM (A–C) or 20 µM (D) zVAD-fmk. Subsequently, they were mixed with CH-11 (20 ng/ml; A), irradiated at a specific UVB dose (300 J/m2; B and D), and irradiated at the indicated doses of UVB (C). After incubation for 24 h, cells positive for AV binding were detected by flow cytometry. zVAD-fmk did not inhibit UVB-induced apoptosis. The bars indicate mean percentages ± SD (n=5 for A and B; n=3 for D). (C) The data are representative of two independent experiments.

 


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  		Figure 4. Inhibition of DNA fragmentation by zVAD-fmk. (A) Jurkat cells were preincubated for 30 min in the absence or presence of 2 µM zVAD-fmk, subjected to UVB irradiation (300 J/m2), and further incubated at 37ºC. After 7 h of incubation, the cells were stained with AV and PI solution. After 7 (B) and 18 (C) h incubation, cells were harvested, and fragmented DNA was selectively extracted and visualized as described in Materials and Methods. Lanes 1 and 5, Molecular marker (ØX174-HaeIII digest); lanes 2 and 6, UVB-/zVAD-; lanes 3 and 7, UVB+/zVAD-; lanes 4 and 8, UVB+/zVAD+. The appearance of early apoptotic cells after UVB irradiation was confirmed in the absence and presence of zVAD-fmk. However, DNA ladder formation was seen only in the cells without zVAD-fmk (lane 3). Data shown are representative of two independent experiments.

 
Inhibitory effect of zVAD-fmk on {Delta}{Psi}m
A decrease in {Delta}{Psi}m was reported to be a key event in the caspase-independent pathway of cell death [21 , 24 25 26 ]. Thus, we investigated the changes in {Delta}{Psi}m on UVB irradiation. UVB and CH-11 stimulation induced decreases in {Delta}{Psi}m in a time-dependent manner. However, the kinetics of the appearance of {Delta}{Psi}mlow cells appeared to differ between the CH-11 stimulation and UVB stimulation. zVAD-fmk did not inhibit the appearance of {Delta}{Psi}mlow cells at all at any time point. However, at 24 h, the percentage of {Delta}{Psi}mlow cells induced by CH-11 treatment was significantly inhibited by zVAD-fmk (Fig. 5A ). Even in the presence of 20 µM zVAD-fmk, the percentage of cells was not inhibited at all (Fig. 5B) . These results suggested that the UVB-induced decrease in {Delta}{Psi}m also occurred via a caspase-independent pathway.



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  		Figure 5. Effects of zVAD-fmk on the decrease in {Delta}{Psi}m induced by UVB. Jurkat cells were preincubated for 30 min in the absence or presence of 2 µM zVAD-fmk (A) or the indicated concentration of zVAD-fmk (B), subsequently mixed with CH-11 (20 ng/ml) or irradiated with UVB (300 J/m2) and incubated further. Cells were collected at the indicated times (A) or at 24 h (B) and exposed to DiOC6. The fluorescence of DiOC6 was monitored by flow cytometry. zVAD-fmk did not inhibit the decrease in {Delta}{Psi}m induced by UVB irradiation. Data shown are representative of two independent experiments.

 
Inhibitory effects of zVAD-fmk on the morphological changes of the nucleus
We next investigated whether UVB-induced morphological changes in the nucleus could be blocked by 2 µM zVAD-fmk. Jurkat cells were exposed to UVB at a dose of 300 J/m2 in the absence or presence of zVAD-fmk. Twenty-four hours after the exposure, chromatin condensation associated with nuclear body formation was observed in the absence and presence of zVAD-fmk (Fig. 6B and 6C ). These observations suggested that the apoptotic nuclear change after UVB irradiation occurred in a caspase-independent manner.



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  		Figure 6. Effects of zVAD-fmk on UVB-induced morphological changes of the nucleus. Jurkat cells were preincubated for 1 h with or without zVAD-fmk (2 µM) or NAC (15mM). Subsequently, they were irradiated with UVB (300 J/m2) and further incubated for 24 h and sujected to microscopic examination. Chromatin condensation was clearly observed in the absence (B) and presence (C) of zVAD-fmk compared with control (A). Chromatin condensation was less severe in the presence of NAC (D).

 
Involvement of AIF in the caspase-independent pathway of apoptosis induced by UVB
We focused on the localization of AIF before and after UVB irradiation. As reported previously [22 ], AIF was basically localized in the mitochondria (Fig. 7A ). However, regardless of the presence of 20 µM zVAD-fmk, translocation of AIF to the nucleus was observed 4 h after UVB irradiation (Fig. 7B and 7C) . The same results were obtained in the presence of 2 µM zVAD-fmk (data not shown). In contrast, AIF remained in mitochondria 4 h after UVB irradiation in the presence of NAC (Fig. 7D) .



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  		Figure 7. Translocation of AIF from mitochondria to the nucleus after UVB irradiation. Jurkat cells were preincubated for 1 h with medium only, zVAD-fmk (20 µM) or NAC (15 mM). Subsequently, they were irradiated with UVB (300 J/m2) and further incubated for 4 h. Then, AIF was stained with antibodies or fluorescent dye as described in Materials and Methods. (A) Control; (B) UVB irradiation only; (C) UVB irradiation + zVAD-fmk; (D) UVB irradiation + NAC. The nucleus (green) was distinguishable from AIF (red), which was present in the perinulear area (cytoplasm; A). The translocation of AIF from mitochondria to the nucleus after UVB irradiation was confirmed with or without zVAD-fmk (B and C). In contrast, the AIF was still present in the perinuclear area in a substantial number of cells in the presence of NAC (D). Data shown are representative of four independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Cell death induced by treatment with CH-11 and irradiation with UVB appeared to be quite similar with respect to the kinetics of PS externalization and the kinetics of caspase activation. In addition, morphological changes to the nucleus were similar (data not shown). This suggested that as in a keratinocyte cell line [18 ], death receptor aggregation and subsequent death receptor-induced signal complex (DISC) formation played an important role in UVB-induced cell death in Jurkat cells.

However, a broad caspase inhibitor, zVAD-fmk, could not inhibit UVB-induced apoptotic changes while it inhibited CH-11-induced apoptotic changes (Figs. 3 4 5) . This strongly suggested that a caspase-independent pathway executes the events involved in UVB-induced cell death in Jurkat cells.

In this sense, it should be stressed that the mitochondrio-nuclear translocation of AIF was clearly observed even in the presence of zVAD-fmk 4 h after UVB irradiation. Considering the reported features of AIF, this can explain why zVAD-fmk failed to inhibit the cell death events induced by UVB. Thus, we concluded that the mitochondrio-nuclear translocation of AIF is associated with the UVB-induced apoptosis of Jurkat cells.

It is interesting that similar observations were reported in UVC-induced apoptosis in human carcinoma cells by Sasai et al. [27 ]. When HeLa cells or MCF-7 cells were irradiated by UVC, the release of AIF from mitochondria occurred within 4 h, as in Jurkat cells irradiated by UVC. However, it occurred without elevation of caspase-3 activity, and the nuclei of these carcinoma cells showed abnormal nuclear condensation without fragmented nuclei. Although Sasai et al. [27 ] used UVC instead of UVB, we consider that their observations can support our conclusion.

It has been reported that zVAD-fmk failed to inhibit the UVB-induced release of cytochrome c from mitochondria in the T cell line CEM [28 ]. According to this report, it seemed that UVB had the potential to perturb the mitochondrial membrane, resulting in the opening of mitochondrial permeability transition (PT) pores. As in cytochrome c, AIF resides in an intermembrane space of mitochondria [21 ], it is conceivable that like cytochrome c, AIF can be released from mitochondria through the mitochondrial PT pore opened after UVB irradiation in Jurkat cells.

It was of interest that NAC could almost completely block the mitochondrio-nuclear translocation of AIF observed 4 h after UVB irradiation. This result was quite reproducible. NAC is a scavenger of ROS, and we previously confirmed that 15 mM, the concentration used in our experiment, was sufficient to completely scavenge ROS generated by exposing Jurkat cells to a thioloxidant, diamide (500 µM) [29 ] (data not shown). Considering this with the fact that UVB is known to generate ROS, it was most likely that ROS generated by UVB functions as one of the triggers for the mitochondrio-nuclear translocation of AIF. The finding that chromatin condensation observed in the presence of NAC (Fig. 6D) appeared to be less severe than that observed in the presence or absence of zVAD-fmk may support this.

Our findings that UVB-induced cell death in Jurkat cells was not inhibited by zVAD-fmk is inconsistent with the report that UVB-induced cell death in keratinocytes was completely inhibited by zVAD-fmk [18 ]. The following explanation can be made. Keratinocytes may be type I cells, in which DISC is produced in sufficient amounts and plays a major role in apoptotic cell death, and the mitochondrial pathway might be an amplifying event only [30 ]. In contrast, Jurkat cells are reported to be type II cells [30 ]. The mitochondrial pathway plays a critical role in the apoptotic cell death of this type of cell. Thus, perturbation of the mitochondrial membrane must be essential for the Jurkat cell to undergo apoptosis. As described above, we thought that UVB might perturb the mitochondrial membrane in Jurkat cells even in the presence of a broad caspase inhibitor zVAD-fmk (in other words, without the action of a truncated bid that is cleaved by apical caspase-8), leading to the release of AIF from mitochondria within 4 h after UVB stimulation. Our observation that the increased percentage of {Delta}{Psi}mlow cells occurred much earlier and faster in UVB-irradiated cells than in CH-11-stimulated cells might at the very least reflect a direct or a caspase-independent perturbation of the mitochondrial membrane by UVB irradiation.

Taking all these results into consideration, we conclude that UVB-induced cell death could occur through a caspase-independent pathway in Jurkat cells, the mitochondrio-nuclear translocation of AIF is associated with this pathway, and the ROS generated by UVB may be involved in triggering its translocation.


   ACKNOWLEDGEMENTS
 
This study was supported in part by European Commission Grant QLG1-1999-00739 (to G. K.).

Received July 2, 2002; revised November 14, 2002; accepted November 26, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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