- Email: [email protected]
Nerve growth factor and forskolin prevent H2O2-induced apoptosis in PC12 cells by glutathione independent mechanism
ELSEVIER
Neuroscience Letters 212 (1996) 179-182
NEUROSCI[NC[ I[TT[RS
Nerve growth factor and forskolin prevent H202-induced apoptosis in PC 12 cells by glutathione independent mechanism Hideaki Kamata*, Chihiro Tanaka, Hitoshi Yagisawa, Hajime Hirata Department of Life Science, Faculty of Science, Himeji Institute of Technology, Akoh-gun, Hyogo 678-12, Japan
Received 8 May 1996; revised version received 12 June 1996; accepted 12 June 1996
Abstract
PCI2 cells died by apoptosis at relatively low concentrations of H202, of which cytotoxicity was effectively suppressed by nerve growth factor (NGF), forskolin, and dbt-cAMP. Treatment with NGF or forskolin for 24 h increased the level of cellular antioxidant glutathione (GSH) by 1.6-2.0-fold. However, both NGF and forskolin protected cells against H202-stress even when cellular GSH was depleted by treatment with L-buthionine-(S,R)-sulfoximine (BSO). The GSH-independent protection effects of NGF and forskolin did not require new protein or RNA synthesis. Exogenous expression of an oncogenic ras suppressed apoptosis caused by H202 indicating that Ras protein also plays a role in suppressing apoptosis caused by oxidative radical stress. Keywords: PC 12; Apoptosis; Oxidative radical stress; Ras; Glutathione; Nerve growth factor; Forskolin
Increased level of reactive oxygen intermediates (ROIs) in cells, referred to as oxidative radical stress, is cytotoxic and has been postulated to be the cause of neuronal degenerative diseases [2,5,12,15,19]. Against oxidative radical stress, cells possess several cellular defense systems including the antioxidant enzymes and antioxidants, such as glutathione (GSH), vitamin E, or fl-carotene. Nerve growth factor (NGF) rescues cells from injury by oxidative radical stress [4,10,11] and is revealed to increase both the level of cellular GSH and the activity of the antioxidant enzymes, GSH peroxidase and catalase, in neuronal cells and PC12 cells [14,17]. However, it is still unclear how oxidative radical stress induces cell death and how N G F protects cells from oxidative radical stress. Recently, it has been reported that oxidative radical stress induced apoptosis [9]. Here we investigated whether oxidative radical stress induces apoptosis in PC12 cells. PC12 cells were cultured in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% horse serum, 5% fetal bovine serum and 50/.tg/ml kanamycin. When PC12 cells were exposed to oxidative radical stress by incubation with 400/.tM H202 for 12 h, marked fragmentations of cellular DNA into sizes of multiples of 180 bp were induced as shown * Corresponding author. Tel.: +81 7915 80198; fax: +81 7915 80197; e-mail: [email protected]
in Fig. IA. However, the degree of DNA fragmentation was apparently reduced at the higher concentration of H202 (1.2 mM). At 400/~M H202, some cells had the appearance typified as apoptotic morphology with membrane fragmentation and cellular debris (data not shown). Whereas at the higher concentration of H20 2, almost all the cells showed signs of necrotic cell death with focal rupture of cell membranes and surface blebbing. It has been reported that the mitochondrial complex I inhibitors, which stimulate the production of ROIs, also cause apoptosis in PC12 cells at low concentrations and necrosis at high concentrations [7]. Thus, the dose-dependent induction of apoptosis may be a typical feature of cell death caused by oxidative radical stress. NGF and forskolin are reported to suppress the cell death caused by either serum deprivation or the withdrawal of neurotrophic factors by transcription- and translationindependent mechanism in PC12 cells [3,16]. A 3-(4,5dimethyl-2-thiazol)-2,5-diphenyl-2H tetrazolium bromide (MTr) reduction assay (Fig. 1B) and a trypan blue dye exclusion assay (Fig. 1C) revealed that NGF and forskolin promoted the survival of cells exposed to 1-1202 at concentrations of 200-800/~M which induced apoptosis. In fact, the DNA fragmentation caused by 400/.tM H202 was apparently reduced by NGF and forskolin as shown in Fig. 1A. To clarify the mechanism of these reagents to protect cells from oxidative radical stress, we estimated the level
0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940(96) 12806-8
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H. Kamata et al. / Neuroscience Letters 212 (1996) 179-182
o f cellular antioxidant G S H and the activities of catalase, G S H peroxidase, and G S H reductase. N G F and forskolin did not increase the activities of these antioxidant enzymes within 24 h stimulation (data not shown), consistent with the previous report that these activities are increased after several days o f N G F stimulation, however, it was obvious that treatment with N G F or forskolin for 24 h elevated cellular G S H level by 1.6-2-fold (Fig. 2A). To elucidate the relation between the G S H level and cell viability, the cellular G S H level was reduced by various concentrations of L-buthionine-(S,R)-sulfoximine (BSO), which is a specific inhibitor of y-glutamylcysteine synthetase, the rate limiting enzyme o f G S H synthesis (Fig. 2A). Incubation with 200 ffM H20 2 had no effect on cell viability in the absence o f BSO, but, more than 50% o f cells died by the H202-treatment when cellular G S H was depleted by 1 m M B S O (Fig. 2B). However, it should be noted that both N G F and forskolin were capable of protecting cells against H202-stress, even when the cellular G S H was depleted (Fig. 2B). These results imply that GSH-independent mechanism is involved in the protective effects of N G F and forskolin against oxidative radical stress. With respect to the GSH-independent protection effects, both N G F and forskolin protected cells against H202-stress transcription- and translation-independently (Fig. 2C,D). As these survival factors also prevent serum deprivation-induced cell death [3,16], a common mechanism which does not require protein synthesis is suggested to be involved in the suppression of cell death by N G F and forskolin. Some other stimuli, such as fibroblast growth factor (FGF), dbt-cAMP, insulin, E G F and elevated K ÷, act as survival factors to suppress the cell death caused by deprivation of either serum or neurotrophic factors [3,16]. Among these factors, only NGF, forskolin and dbt-cAMP were effective in rescuing cells from actinomycin-D and cycloheximide as reported by Lindenboim et al. [13] and from the attack of H202 (Fig. 3). This result was confirmed by a trypan blue dye exclusion assay and by an analysis of cellular D N A fragmentation (data not shown). But, it is noteworthy that the other factors, which failed to suppress H202-induced apoptosis, also increased the GSH level, the increments were 1.8-fold by E G F (30 ng/ml), 1.4-fold by F G F (10 ng/ml), 1.2-fold by insulin (100/~g/ml), 2.9-fold by phorbol myristate acetate (PMA; 100 ng/ml), and 1.4fold by dexamethasone (100 nM). These results supported the GSH-independent protection mechanism of N G F and forskolin against oxidative radical stress. It is plausible that the death signal from oxidative radical stress is mediated by the pathway shared with that from the cytotoxic drugs, and converged into a final common pathway which is susceptible to NGF, forskolin, and dbt-cAMP. Several studies have revealed an essential role of cellular Ras in NGF-signaling [6,18,20]. This gives rise to the question whether Ras is involved in suppression of oxidative radical stress-induced cell death by NGF. To elucidate this, we prepared a PC12 derived cell line
PCMTras21 carrying the H - r a s gene by transfection with plasmids pMTIDrasneo which encodes the oncogenic T24
(A)
H202 [laM]
H 2 0 2 [400 p.M]
I
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9
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15
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Time [hr]
Fig. 1. (A) Soluble DNA was extracted as described [8] from PC12 cells (2 x 106/10 ml per 100-mm dish) cultured in various concentrations of H202 together with or without NGF (50 ng/ml) or forskolin (10ffM) for 12 h. Soluble DNA (20/tg) was subjected to electrophoresis, and then blotted onto a membrane Hybond-N+ (Amersham). The filter was probed with the 32p-labeled genomic DNA from PC12 cells. The soluble DNA from PC12 ceils cultured without H202 was applied to lane I. (B) PC12 cells (2 x 104/100/.d per well of 96-well plates) .were incubated with or without NGF or forskolin for 24 h, then, H202 was added to the culture. After 24 h, 0.5 mg/ml MTT was added. The generated dark crystals were dissolved by 50% isopropanol and 0.02 N HCI, and the absorbance at 595 rim, with the reference at 655 nm, was measured. (C) H202 (400/~M) was added to the culture of PC12 cells (1 x 105/4 ml per 60-mm dish) preincubated with NGF or forskolin for 24 h. Viability of the cells was estimated by dye exclusion assay using 0. 1% trypan blue solution at various times after the addition of H202.
181
H. Kamata et al. / Neuroscience Letters 212 (1996) 179-182
gene under influence of the metallothionein promoter. As shown in Fig. 4, an MT'I" reduction assay showed that PCMTras21 was slightly resistant to H202stress, actinomycin-D, and cycloheximide. Activation of the MTID promoter by ZnCI2 resulted in PCMTras21 more resistant to both H202-stress and these cytotoxic drugs. Similar results were also obtained in the experiments using a trypan blue dye exclusion assay (data not shown). Furthermore, the expression of oncogenic ras in H-ras
(A)
80 60 40 20
o~ 100
30 D:
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• : NGF [] : Forskolin
e-
20
.=_o 80 -~ 6o ~- 40
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20
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-r" (/)
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10 100 BSO [~tM]
1
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(B) 120
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~80 O
00
~ 60
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i
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1000
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tO
9_ 5" E "u
: NGF : Forskolin
0
z
0.8 ~ 0.6 eo
:= 0.4
-~m
=
Fig. 3. Comparison of the effects of several growth factors and reagents on the viability of PC12 cells under H202-stress or in the presence of cytotoxic drugs. PC 12 cells (2 x 104/100/tl per well of 96-well plates) were incubated with NGF (50 ng/ml), forskolin (10/~M), dbt-cAMP (1 mM), bFGF (10 ng/ml), EGF (30 ng/ml), insulin (100 mg/ml), KCI (50mM), PMA (100ng/ml), IL-1 (20ng/ml), tumor necrosis factor (TNF) (20 ng/ml), or dexamethasone (DEX) (100 nM) for 24 h. Cell viability was assessed by MTT reduction following incubation with H202 (800ktM) for 24 h, or with actinomycin-D (1 ~M), or cycloheximide (10/~M) for 60 h. The data are represented as means _+SD for triplicate cultures.
"O
0.2 0 (D)
400 600 H202 [~tM]
800
1000
1.0 0
g
200
PC12 cells has been reported to suppress cell death in serum-free medium [16]. Thus, NGF protect cells from oxidative radical stress independently of GSH, and Ras may be involved in the suppression of apoptosis by NGF.
.
BSO+Cycloheximide ~
8
0.4 0.6 f 0.2 0.0
0
200
400 H202
600 [laM]
800
1000
= : NGF -- : Forskolin
Fig. 2. Dose-dependent effect of BSO on cellular GSH levels and on cell viability under H202-stress. (A) PC12 cells (1 × 106/4 ml per 60mm dish) were treated with various concentrations of BSO together with NGF or forskolin for 24 h, then the GSH levels were determined by a method originally described by Anderson [l]. (B) PCI2 cells (2 x 104/100/zl per well of 96-well plates) were treated with various concentrations of BSO in the presence or absence of NGF or forskolin lor 24 h. Then H20 2 (200/~M) was added to the medium and further incubated for 24 h. (C) Actinomycin-D (0.1 /~g/ml) or cycloheximide (1/~g/ml) together with BSO (100/zM) were added to PCI2 cell culture (2 × 104 cells/100/~1 per well of 96-well plates) 2 h before the addition of NGF (50 ng/ml) or forskolin (10/~M). Twelve hours after, various concentrations of H202 were added and incubated further for 12 h. The data are represented as means + SD in triplicate experiments.
182
1-1. Kamata et aL / Neuroscience Letters 212 (1996) 179-182
(A) 120 %.100
~
80
0
~ 6o ~ 40 20 0 0
200
400
600 800 H202 [~M]
1000
1200
(B) 120 100 =
80
0
6o 40
20 0t 0
i
0.1
t
h
i
1 10 100 1000 10000 Actinomycin-D [ng/ml]
(C) 120 100~
"E 80 .o
s= so 40
~
20 1
10 100 I000 10000100000 Cycloheximide [ng/ml] x : PCMT --o-- : PCMTras21 : PCMTras21/ZnCI2
Fig. 4. PCMTras21 cells were PCl2-derived cells harboring the H-ras gene and PCMT cells were control PCl2-derived cells transfected with the control vector plasmid pMTIDneo. ZnCI 2 (200/zM) was added to the cell cultures (2 x 104/100/zl per well of 96-well plates) and the cells were incubated for 24 h. Various concentrations of H202 (A), actinomycin-D (B), and cycloheximide (C) were added to the cultures. The data are represented as means _+SD for triplicate cultures.
W e t h a n k Dr. K . M a r u y a m a for p r o v i d i n g the p l a s m i d p M T I D - r a s , a n d t h e R I K E N Cell B a n k for p r o v i d i n g P C 12 cells. [1] Anderson, M.E., Determination of glutathione and glutathione disulfide in biological samples, Methods Enzymoi., 113 (1985) 548-555. [2] Deng, H.-X., Hentati, A., Tainer, J.A., lqbal, Z., Cayabyab, A., Hung, W.-Y., Getzoff, E.D., Hu, P., Herzfeldt, B., Roos, R.P., Warner, C., Deng, G., Soriano, E., Smyth, C., Parge, H.E., Ahmed, A., Roses, A.D., Hallewell, R.A., Perieak-Vance, M.A. and Siddique, T., Amyotrophic lateral sclerosis and structural defects in Cu/Zn superoxide dismutase, Science, 261 (1993) 1047-1051. [3] Edwards, S.N., Buckmaster, A.E. and Tolkovsky, A.M., The death program in cultured sympathetic neurons can be suppressed at the posttranslational level by nerve growth factor, cyclic AMP, and depolarization, J. Neurochem., 57 (1991) 2140-2143.
[4] Enokido, Y. and Hatanaka, H., High oxygen atmosphere for neuronal cell culture with nerve growth factor. II. Survival and growth of clonal rat pheochromocytoma PC12h cells, Brain Res., 536 (1990) 23-29. [5] Fahn, S. and Cohen, G., The oxidant stress hypothesis in Parkinson's disease: evidence supporting it, Ann. Neurol., 32 (1992) 804-812. [6] Hagag, N., Halegoua, S. and Viola, M., Inhibition of growth factor-induced differentiation of PC12 cells by microinjection of antibody to ras p21, Nature, 319 (1986) 680-682. [7] Hartley, A., Stone, J.M., Heron, C., Cooper, J.M. and Schapira, A.H.V., Complex I inhibitors induce dose-dependent apoptosis in PCI2 cells: relevance to Parkinson's disease, J. Neurochem., 63 (1994) 1987-1990. [8] Hockenbery, D., Nunez, G., Milliman, C., Schreiber, R.D. and Korsmeyer, S.J., Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death, Nature, 348 (1990) 334336. [9] Hockenbery, D.M., Oltvai, Z.N., Yin, X.-M., Milliman, C.L. and Korsmeyer, S.J., Bcl-2 functions in an antioxidant pathway to prevent apoptosis, Cell, 75 (1993) 241-251. [10] Jackson, G.R., Apffel, L., Werrbach-Perez, K. and Perez-Polo, J.R., Role of nerve growth factor in oxidant-antioxidant balance and neuronal injury. 1. Stimulation of hydrogen peroxide resistance, J. Neurosci. Res., 25 (1990) 360-368. [11] Jackson, G.R., Werrbach-Perez, K. and Perez-Polo, J.R., Role of nerve growth factor in oxidant-antioxidant balance and neuronal injury. 1I. A conditioning lesion paradigm, J. Neurosci. Res., 25 (1990) 369-374. [12] Jenner, P., Schapira, A.H.V. and Marsden, C.D., New insights into the cause of Parkinson's disease, Neurology, 42 (1992) 2241-2250. [13] Lindenboim, L., Haviv, R. and Stein, R., Inhibition of druginduced apoptosis by survival factors in PCI2 cells, J. Neurochem., 64 (1995) 1054-1063. [14] Pan, Z. and Perez-Polo, R., Role of nerve growth factor in oxidant homeostasis: glutathione metabolism, J. Neurochem., 61 (1993) 1713-1721. [15] Rosen, D.R., Siddique, T., Patterson, D., Figlewicz, D.A., Sapp, P., Hentati, A., Donaldson, D., Goto, J., O'Regan, J.P., Deng, H.-X., Rahmani, Z., Krizus, A., McKenna-Yasek, D., Cayabyab, A., Gaston, S.M., Berger, R., Tanzi, R.E., Halperin, J.J., Herzfeldt, B., Bergh, R.V.d., Hung, W.-Y., Bird, T., Deng, G., Mnlder, D.W., Smyth, C., Laing, N.G., Soriano, E., Pericak-Vance, M.A., Haines, J., Rouleau, G,A., Gusella, J.S., Horvitz, H.R. and Brown, Jr., R.H., Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis, Nature, 362 (1993) 59-62. [16] Rukenstein, A., Rydel, R.E. and Greene, L.A., Multiple agents rescue PCI2 cells from serum-free cell death by translation- and transcription-independent mechanisms, J. Neurosci., 11 (1991) 2552-2563. [17] Sampath, D., Jackson, G.R., Werrbach-Perez, K. and Perez-Polo, J.R., Effects of nerve growth factor on glutathione peroxidase and catalase in PC 12 cells, J. Neurochem., 62 (1994) 2476-2479. [18] Thomas, S.M., DeMarco, M., D'Arcangelo, G., Halegoua, S. and Brugge, J.S., Ras is essential for nerve growth factor- and phorbol ester-induced tyrosine phosphorylation of MAP kinases, Cell, 68 (1992) 1031-1040. [19] Wiedau-Pazos, M., Goto, J.J., Rabizadeh, S., Gralla, E.B., Roe, J.A., Lee, M.K., Valentine, J.S. and Bredesen, D.E., Altered reactivity of superoxide dismutase in familial amyotrophic lateral sclerosis, Science, 271 (1996) 515-518. [20] Wood, K.W., Sarnecki, C., Roberts, T.M. and Blenis, J,, ras Mediates nerve growth factor receptor modulation of three signaltransducing protein kinases: MAP kinase, Raf-l, and RSK, Cell, 68 (1992) 1041-1050.
Neuroscience Letters 212 (1996) 179-182
NEUROSCI[NC[ I[TT[RS
Nerve growth factor and forskolin prevent H202-induced apoptosis in PC 12 cells by glutathione independent mechanism Hideaki Kamata*, Chihiro Tanaka, Hitoshi Yagisawa, Hajime Hirata Department of Life Science, Faculty of Science, Himeji Institute of Technology, Akoh-gun, Hyogo 678-12, Japan
Received 8 May 1996; revised version received 12 June 1996; accepted 12 June 1996
Abstract
PCI2 cells died by apoptosis at relatively low concentrations of H202, of which cytotoxicity was effectively suppressed by nerve growth factor (NGF), forskolin, and dbt-cAMP. Treatment with NGF or forskolin for 24 h increased the level of cellular antioxidant glutathione (GSH) by 1.6-2.0-fold. However, both NGF and forskolin protected cells against H202-stress even when cellular GSH was depleted by treatment with L-buthionine-(S,R)-sulfoximine (BSO). The GSH-independent protection effects of NGF and forskolin did not require new protein or RNA synthesis. Exogenous expression of an oncogenic ras suppressed apoptosis caused by H202 indicating that Ras protein also plays a role in suppressing apoptosis caused by oxidative radical stress. Keywords: PC 12; Apoptosis; Oxidative radical stress; Ras; Glutathione; Nerve growth factor; Forskolin
Increased level of reactive oxygen intermediates (ROIs) in cells, referred to as oxidative radical stress, is cytotoxic and has been postulated to be the cause of neuronal degenerative diseases [2,5,12,15,19]. Against oxidative radical stress, cells possess several cellular defense systems including the antioxidant enzymes and antioxidants, such as glutathione (GSH), vitamin E, or fl-carotene. Nerve growth factor (NGF) rescues cells from injury by oxidative radical stress [4,10,11] and is revealed to increase both the level of cellular GSH and the activity of the antioxidant enzymes, GSH peroxidase and catalase, in neuronal cells and PC12 cells [14,17]. However, it is still unclear how oxidative radical stress induces cell death and how N G F protects cells from oxidative radical stress. Recently, it has been reported that oxidative radical stress induced apoptosis [9]. Here we investigated whether oxidative radical stress induces apoptosis in PC12 cells. PC12 cells were cultured in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% horse serum, 5% fetal bovine serum and 50/.tg/ml kanamycin. When PC12 cells were exposed to oxidative radical stress by incubation with 400/.tM H202 for 12 h, marked fragmentations of cellular DNA into sizes of multiples of 180 bp were induced as shown * Corresponding author. Tel.: +81 7915 80198; fax: +81 7915 80197; e-mail: [email protected]
in Fig. IA. However, the degree of DNA fragmentation was apparently reduced at the higher concentration of H202 (1.2 mM). At 400/~M H202, some cells had the appearance typified as apoptotic morphology with membrane fragmentation and cellular debris (data not shown). Whereas at the higher concentration of H20 2, almost all the cells showed signs of necrotic cell death with focal rupture of cell membranes and surface blebbing. It has been reported that the mitochondrial complex I inhibitors, which stimulate the production of ROIs, also cause apoptosis in PC12 cells at low concentrations and necrosis at high concentrations [7]. Thus, the dose-dependent induction of apoptosis may be a typical feature of cell death caused by oxidative radical stress. NGF and forskolin are reported to suppress the cell death caused by either serum deprivation or the withdrawal of neurotrophic factors by transcription- and translationindependent mechanism in PC12 cells [3,16]. A 3-(4,5dimethyl-2-thiazol)-2,5-diphenyl-2H tetrazolium bromide (MTr) reduction assay (Fig. 1B) and a trypan blue dye exclusion assay (Fig. 1C) revealed that NGF and forskolin promoted the survival of cells exposed to 1-1202 at concentrations of 200-800/~M which induced apoptosis. In fact, the DNA fragmentation caused by 400/.tM H202 was apparently reduced by NGF and forskolin as shown in Fig. 1A. To clarify the mechanism of these reagents to protect cells from oxidative radical stress, we estimated the level
0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940(96) 12806-8
180
H. Kamata et al. / Neuroscience Letters 212 (1996) 179-182
o f cellular antioxidant G S H and the activities of catalase, G S H peroxidase, and G S H reductase. N G F and forskolin did not increase the activities of these antioxidant enzymes within 24 h stimulation (data not shown), consistent with the previous report that these activities are increased after several days o f N G F stimulation, however, it was obvious that treatment with N G F or forskolin for 24 h elevated cellular G S H level by 1.6-2-fold (Fig. 2A). To elucidate the relation between the G S H level and cell viability, the cellular G S H level was reduced by various concentrations of L-buthionine-(S,R)-sulfoximine (BSO), which is a specific inhibitor of y-glutamylcysteine synthetase, the rate limiting enzyme o f G S H synthesis (Fig. 2A). Incubation with 200 ffM H20 2 had no effect on cell viability in the absence o f BSO, but, more than 50% o f cells died by the H202-treatment when cellular G S H was depleted by 1 m M B S O (Fig. 2B). However, it should be noted that both N G F and forskolin were capable of protecting cells against H202-stress, even when the cellular G S H was depleted (Fig. 2B). These results imply that GSH-independent mechanism is involved in the protective effects of N G F and forskolin against oxidative radical stress. With respect to the GSH-independent protection effects, both N G F and forskolin protected cells against H202-stress transcription- and translation-independently (Fig. 2C,D). As these survival factors also prevent serum deprivation-induced cell death [3,16], a common mechanism which does not require protein synthesis is suggested to be involved in the suppression of cell death by N G F and forskolin. Some other stimuli, such as fibroblast growth factor (FGF), dbt-cAMP, insulin, E G F and elevated K ÷, act as survival factors to suppress the cell death caused by deprivation of either serum or neurotrophic factors [3,16]. Among these factors, only NGF, forskolin and dbt-cAMP were effective in rescuing cells from actinomycin-D and cycloheximide as reported by Lindenboim et al. [13] and from the attack of H202 (Fig. 3). This result was confirmed by a trypan blue dye exclusion assay and by an analysis of cellular D N A fragmentation (data not shown). But, it is noteworthy that the other factors, which failed to suppress H202-induced apoptosis, also increased the GSH level, the increments were 1.8-fold by E G F (30 ng/ml), 1.4-fold by F G F (10 ng/ml), 1.2-fold by insulin (100/~g/ml), 2.9-fold by phorbol myristate acetate (PMA; 100 ng/ml), and 1.4fold by dexamethasone (100 nM). These results supported the GSH-independent protection mechanism of N G F and forskolin against oxidative radical stress. It is plausible that the death signal from oxidative radical stress is mediated by the pathway shared with that from the cytotoxic drugs, and converged into a final common pathway which is susceptible to NGF, forskolin, and dbt-cAMP. Several studies have revealed an essential role of cellular Ras in NGF-signaling [6,18,20]. This gives rise to the question whether Ras is involved in suppression of oxidative radical stress-induced cell death by NGF. To elucidate this, we prepared a PC12 derived cell line
PCMTras21 carrying the H - r a s gene by transfection with plasmids pMTIDrasneo which encodes the oncogenic T24
(A)
H202 [laM]
H 2 0 2 [400 p.M]
I
I o
I
~]
ogo.o
°
i ~
CXl ,~t
~
Z
co
uO°
--2.0 --1.4 --1.1 --0.9 --0.6
--0.3 (kbp)
))
(B) 120
100
~
£
80
•-g 60 40 2O
0 0
200
400
600 H202 [~tM]
800
1000
1200
120
(c)
:-
lO~
¢
: NGF :
•~
'
80
~ 60 N
g ~
40 20 i
t
3
6
I
9
12
15
18
21
24
Time [hr]
Fig. 1. (A) Soluble DNA was extracted as described [8] from PC12 cells (2 x 106/10 ml per 100-mm dish) cultured in various concentrations of H202 together with or without NGF (50 ng/ml) or forskolin (10ffM) for 12 h. Soluble DNA (20/tg) was subjected to electrophoresis, and then blotted onto a membrane Hybond-N+ (Amersham). The filter was probed with the 32p-labeled genomic DNA from PC12 cells. The soluble DNA from PC12 ceils cultured without H202 was applied to lane I. (B) PC12 cells (2 x 104/100/.d per well of 96-well plates) .were incubated with or without NGF or forskolin for 24 h, then, H202 was added to the culture. After 24 h, 0.5 mg/ml MTT was added. The generated dark crystals were dissolved by 50% isopropanol and 0.02 N HCI, and the absorbance at 595 rim, with the reference at 655 nm, was measured. (C) H202 (400/~M) was added to the culture of PC12 cells (1 x 105/4 ml per 60-mm dish) preincubated with NGF or forskolin for 24 h. Viability of the cells was estimated by dye exclusion assay using 0. 1% trypan blue solution at various times after the addition of H202.
181
H. Kamata et al. / Neuroscience Letters 212 (1996) 179-182
gene under influence of the metallothionein promoter. As shown in Fig. 4, an MT'I" reduction assay showed that PCMTras21 was slightly resistant to H202stress, actinomycin-D, and cycloheximide. Activation of the MTID promoter by ZnCI2 resulted in PCMTras21 more resistant to both H202-stress and these cytotoxic drugs. Similar results were also obtained in the experiments using a trypan blue dye exclusion assay (data not shown). Furthermore, the expression of oncogenic ras in H-ras
(A)
80 60 40 20
o~ 100
30 D:
m
• : NGF [] : Forskolin
e-
20
.=_o 80 -~ 6o ~- 40
(3b
E
20
10
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0
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10 100 BSO [~tM]
1
1000
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(B) 120
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~80 O
00
~ 60
4o ~
:
20 i
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i
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1000
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0
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-~m
=
Fig. 3. Comparison of the effects of several growth factors and reagents on the viability of PC12 cells under H202-stress or in the presence of cytotoxic drugs. PC 12 cells (2 x 104/100/tl per well of 96-well plates) were incubated with NGF (50 ng/ml), forskolin (10/~M), dbt-cAMP (1 mM), bFGF (10 ng/ml), EGF (30 ng/ml), insulin (100 mg/ml), KCI (50mM), PMA (100ng/ml), IL-1 (20ng/ml), tumor necrosis factor (TNF) (20 ng/ml), or dexamethasone (DEX) (100 nM) for 24 h. Cell viability was assessed by MTT reduction following incubation with H202 (800ktM) for 24 h, or with actinomycin-D (1 ~M), or cycloheximide (10/~M) for 60 h. The data are represented as means _+SD for triplicate cultures.
"O
0.2 0 (D)
400 600 H202 [~tM]
800
1000
1.0 0
g
200
PC12 cells has been reported to suppress cell death in serum-free medium [16]. Thus, NGF protect cells from oxidative radical stress independently of GSH, and Ras may be involved in the suppression of apoptosis by NGF.
.
BSO+Cycloheximide ~
8
0.4 0.6 f 0.2 0.0
0
200
400 H202
600 [laM]
800
1000
= : NGF -- : Forskolin
Fig. 2. Dose-dependent effect of BSO on cellular GSH levels and on cell viability under H202-stress. (A) PC12 cells (1 × 106/4 ml per 60mm dish) were treated with various concentrations of BSO together with NGF or forskolin for 24 h, then the GSH levels were determined by a method originally described by Anderson [l]. (B) PCI2 cells (2 x 104/100/zl per well of 96-well plates) were treated with various concentrations of BSO in the presence or absence of NGF or forskolin lor 24 h. Then H20 2 (200/~M) was added to the medium and further incubated for 24 h. (C) Actinomycin-D (0.1 /~g/ml) or cycloheximide (1/~g/ml) together with BSO (100/zM) were added to PCI2 cell culture (2 × 104 cells/100/~1 per well of 96-well plates) 2 h before the addition of NGF (50 ng/ml) or forskolin (10/~M). Twelve hours after, various concentrations of H202 were added and incubated further for 12 h. The data are represented as means + SD in triplicate experiments.
182
1-1. Kamata et aL / Neuroscience Letters 212 (1996) 179-182
(A) 120 %.100
~
80
0
~ 6o ~ 40 20 0 0
200
400
600 800 H202 [~M]
1000
1200
(B) 120 100 =
80
0
6o 40
20 0t 0
i
0.1
t
h
i
1 10 100 1000 10000 Actinomycin-D [ng/ml]
(C) 120 100~
"E 80 .o
s= so 40
~
20 1
10 100 I000 10000100000 Cycloheximide [ng/ml] x : PCMT --o-- : PCMTras21 : PCMTras21/ZnCI2
Fig. 4. PCMTras21 cells were PCl2-derived cells harboring the H-ras gene and PCMT cells were control PCl2-derived cells transfected with the control vector plasmid pMTIDneo. ZnCI 2 (200/zM) was added to the cell cultures (2 x 104/100/zl per well of 96-well plates) and the cells were incubated for 24 h. Various concentrations of H202 (A), actinomycin-D (B), and cycloheximide (C) were added to the cultures. The data are represented as means _+SD for triplicate cultures.
W e t h a n k Dr. K . M a r u y a m a for p r o v i d i n g the p l a s m i d p M T I D - r a s , a n d t h e R I K E N Cell B a n k for p r o v i d i n g P C 12 cells. [1] Anderson, M.E., Determination of glutathione and glutathione disulfide in biological samples, Methods Enzymoi., 113 (1985) 548-555. [2] Deng, H.-X., Hentati, A., Tainer, J.A., lqbal, Z., Cayabyab, A., Hung, W.-Y., Getzoff, E.D., Hu, P., Herzfeldt, B., Roos, R.P., Warner, C., Deng, G., Soriano, E., Smyth, C., Parge, H.E., Ahmed, A., Roses, A.D., Hallewell, R.A., Perieak-Vance, M.A. and Siddique, T., Amyotrophic lateral sclerosis and structural defects in Cu/Zn superoxide dismutase, Science, 261 (1993) 1047-1051. [3] Edwards, S.N., Buckmaster, A.E. and Tolkovsky, A.M., The death program in cultured sympathetic neurons can be suppressed at the posttranslational level by nerve growth factor, cyclic AMP, and depolarization, J. Neurochem., 57 (1991) 2140-2143.
[4] Enokido, Y. and Hatanaka, H., High oxygen atmosphere for neuronal cell culture with nerve growth factor. II. Survival and growth of clonal rat pheochromocytoma PC12h cells, Brain Res., 536 (1990) 23-29. [5] Fahn, S. and Cohen, G., The oxidant stress hypothesis in Parkinson's disease: evidence supporting it, Ann. Neurol., 32 (1992) 804-812. [6] Hagag, N., Halegoua, S. and Viola, M., Inhibition of growth factor-induced differentiation of PC12 cells by microinjection of antibody to ras p21, Nature, 319 (1986) 680-682. [7] Hartley, A., Stone, J.M., Heron, C., Cooper, J.M. and Schapira, A.H.V., Complex I inhibitors induce dose-dependent apoptosis in PCI2 cells: relevance to Parkinson's disease, J. Neurochem., 63 (1994) 1987-1990. [8] Hockenbery, D., Nunez, G., Milliman, C., Schreiber, R.D. and Korsmeyer, S.J., Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death, Nature, 348 (1990) 334336. [9] Hockenbery, D.M., Oltvai, Z.N., Yin, X.-M., Milliman, C.L. and Korsmeyer, S.J., Bcl-2 functions in an antioxidant pathway to prevent apoptosis, Cell, 75 (1993) 241-251. [10] Jackson, G.R., Apffel, L., Werrbach-Perez, K. and Perez-Polo, J.R., Role of nerve growth factor in oxidant-antioxidant balance and neuronal injury. 1. Stimulation of hydrogen peroxide resistance, J. Neurosci. Res., 25 (1990) 360-368. [11] Jackson, G.R., Werrbach-Perez, K. and Perez-Polo, J.R., Role of nerve growth factor in oxidant-antioxidant balance and neuronal injury. 1I. A conditioning lesion paradigm, J. Neurosci. Res., 25 (1990) 369-374. [12] Jenner, P., Schapira, A.H.V. and Marsden, C.D., New insights into the cause of Parkinson's disease, Neurology, 42 (1992) 2241-2250. [13] Lindenboim, L., Haviv, R. and Stein, R., Inhibition of druginduced apoptosis by survival factors in PCI2 cells, J. Neurochem., 64 (1995) 1054-1063. [14] Pan, Z. and Perez-Polo, R., Role of nerve growth factor in oxidant homeostasis: glutathione metabolism, J. Neurochem., 61 (1993) 1713-1721. [15] Rosen, D.R., Siddique, T., Patterson, D., Figlewicz, D.A., Sapp, P., Hentati, A., Donaldson, D., Goto, J., O'Regan, J.P., Deng, H.-X., Rahmani, Z., Krizus, A., McKenna-Yasek, D., Cayabyab, A., Gaston, S.M., Berger, R., Tanzi, R.E., Halperin, J.J., Herzfeldt, B., Bergh, R.V.d., Hung, W.-Y., Bird, T., Deng, G., Mnlder, D.W., Smyth, C., Laing, N.G., Soriano, E., Pericak-Vance, M.A., Haines, J., Rouleau, G,A., Gusella, J.S., Horvitz, H.R. and Brown, Jr., R.H., Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis, Nature, 362 (1993) 59-62. [16] Rukenstein, A., Rydel, R.E. and Greene, L.A., Multiple agents rescue PCI2 cells from serum-free cell death by translation- and transcription-independent mechanisms, J. Neurosci., 11 (1991) 2552-2563. [17] Sampath, D., Jackson, G.R., Werrbach-Perez, K. and Perez-Polo, J.R., Effects of nerve growth factor on glutathione peroxidase and catalase in PC 12 cells, J. Neurochem., 62 (1994) 2476-2479. [18] Thomas, S.M., DeMarco, M., D'Arcangelo, G., Halegoua, S. and Brugge, J.S., Ras is essential for nerve growth factor- and phorbol ester-induced tyrosine phosphorylation of MAP kinases, Cell, 68 (1992) 1031-1040. [19] Wiedau-Pazos, M., Goto, J.J., Rabizadeh, S., Gralla, E.B., Roe, J.A., Lee, M.K., Valentine, J.S. and Bredesen, D.E., Altered reactivity of superoxide dismutase in familial amyotrophic lateral sclerosis, Science, 271 (1996) 515-518. [20] Wood, K.W., Sarnecki, C., Roberts, T.M. and Blenis, J,, ras Mediates nerve growth factor receptor modulation of three signaltransducing protein kinases: MAP kinase, Raf-l, and RSK, Cell, 68 (1992) 1041-1050.