Journal of NeurochemistryLippincott—Raven Publishers, Philadelphia© 1997 International Society for Neurochemistry

Cyclosporin A, an Immunosuppressive Drug, InducesProgrammed Cell Death in Rat C6 Glioma Cells by a

Mechanism that Involves the AP- 1 Transcription Factor

Grazyna Mosieniak, *Jzabela Figiel, and Bozena Kaminska

Department of Cellular Biochemistry and * Tissue Culture Facility,Nencki Institute of Experimental Biology, Warsaw, Poland

Abstract: Cyclosporin A (CsA) is a clinically importantimmunosuppressive drug widely used to prevent graftrejection following organ orbone marrow transplantation.Although there are reports of serious neurologic alter-ations associated with the use of the drug, the precisemechanism of its action on the CNS still remains un-known. We studied the effects of CsA on the growth ofC6 glioma cells. We found that CsA inhibits the growth ofC6 glioma cells in a dose-dependent mannerand inducesmorphological changes such as shrinkage of the cellbody and loss of extensions followed by cell death. Theanalysis of DNA from CsA-treated cells revealed a ladder-like pattern of fragmented DNA. Acridine orange stainingshowed the occurrence of apoptotic changes in nuclearmorphology. Apoptotic morphological alterations wereprevented by the treatment with cycloheximide. Alto-gether, our findings suggest that the CsA-induced celldeath of C6 glioma cells bears all the features characteris-tic of programmed cell death. We also observed a sig-nificant increase in the DNA-binding activity of AP-1 dur-ing CsA-induced apoptosis. The AP-1 induction pre-ceded the appearance of apoptotic, morphologicalchanges and was accounted for by an increase in theexpression of c-Jun protein. The occurrence of increasedlevels of AP-1 complex and c-Jun protein during CsA-induced programmed cell death suggests its involvementin the induction of apoptosis. Key Words: Apoptosis—DNA fragmentation —Glioma—Cyclosporin A—Tran-scription factors—c-Jun protein.J. Neurochem. 68, 1142—1149 (1997).

Cyclosporin A (CsA) has proven to be a valuableimmunosuppressant for recipients of solid organ graftsas well as effective in the prevention of fatal graft-versus-host disease after allogeneic bone marrow trans-plantation (Powles et al., 1980; Deeg et al., 1985).The mechanism of CsA action in the immune systeminvolves the calcium/calmodulin-regulated phospha-tase—calcineurin (Liu et al., 1991; Clipstone andCrabtree, 1992; Furman et al., 1992). Calcineurin, amajor target of CsA, plays a pivotal role in transducing

membrane-associated signals to the nucleus. One ofthe putative targets of calcineurin is the cytosolic com-ponent of the nuclear factor of activated T cells. Nu-clear factor of activated T cells is one of the transcrip-tion factors required for the expression of interleukin2, the major T cell growth factor (for review, seeCrabtree and Clipstone, 1994). Interference of CsAwith the calcineurin-dependent signal transductionpathway has an inhibitory effect on T lymphocyte pro-liferation (for review, see Schreiber and Crabtree,1992; Sigal and Dumont, 1992). Moreover, it has re-cently been shown that this inhibition is followed bythe induction of apoptotic cell death in T lymphocytes(Saiagh et al., 1994).

The use of CsA for transplant patients has led to anawareness of the serious cytotoxic effects it has onthe CNS. Neurological alterations noted include motorspinal cord impairment, cerebellar-like syndrome,mental confusion, and visual disturbances (Atkinsonet al., 1984; DeGroen et al., 1987; Walker andBrochstein, 1988). In addition, several clinical reportsindicate that some patients suffer from an onset ofseizures that are generally reversible with CsA discon-tinuation (Velu et al., 1985; Vazquez de Prada et al.,1990; Ghany et al., 1991). More detailed studies ofthe neurotoxic effects of CsA revealed that the brainlesions associated with CsA appear to be focal andconcentrated in the white matter (DeGroen et al., 1987;Lind et al., 1989).

Although neurologic alterations associated with theuse of CsA have been frequently reported, these obser-vations are difficult to interpret owing to the small

Received July 2, 1996; revised manuscript received October 25,1996; accepted October 25, 1996.

Address correspondence and reprint requests to Dr. B. Kaminskaat Department of Cellular Biochemistry, Nencki Institute of Experi-mental Biology, Pasteura 3 St., 02-093 Warsaw, Poland.

Abbreviations used: CsA, cyclosporin A; CX, cycloheximide;EMSA, electrophoretic mobility shift assay; PBS, phosphate-buf-fered saline.

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CsA-INDUCED APOPTOSIS IN GLIOMA 1143

patient number and the modified chemotherapy regi-mens used in these studies. Several hypotheses havebeen developed to explain the toxic effects of CsA onthe nervous system cells (Brat et al., 1992), but theprecise mechanism of its action remains elusive.

To investigate the possible mechanism of the cyto-toxic effect of CsA and its contribution to CNS pathol-ogy, we investigated the effects of CsA on C6 gliomacells. Based on preliminary results that revealed bothCsA’s antiproliferative effects and ability to induce celldeath, we applied both morphological and biochemicalmethods to elucidate the underlying mechanism ofCsA-induced cell death.

MATERIALS AND METHODS

Cell culture and treatmentRat C6 glioma cells (ATCC) were cultured in Dulbecco’ s

modified Eagle’s medium (GibcoBRL, U.S.A.) supple-mented with 2 mM glutamine, 10% calf serum (Sigma,U.S.A.), and antibiotics (50 U/ml penicillin, 50 mg/mlstreptomycin). Cells were grown in 24-well or 10-cm-diam-eter culture plates (Corning, U.S.A.) in a humidified atmo-sphere of C02/air (5%/95%) and were preplated every 2—3 days. Cells were preplated 1 day before the start of theexperiment at a density of 1.2 x 10

4/cm2 to avoid contactinhibition during 3 days of observation.

CsA (Sandimmun; Sandoz; stock solution, 50 mg/ml)was added at different concentrations at 18 h after platingof cells. The effect of the compound was monitored at vari-ous times up to 72 h by phase-contrast microscopy. Cyclo-heximide (CX) was added at 1 mg/ml simultaneously withCsA or alone. In an additional set of experiments solid CsA(catalogue no. C-224; RBI), dissolved in ethanol (30 mg/ml), was applied. C6 glioma cells were treated for 24 and48 h with 75 jig/ml CsA; in parallel, cells were treated withthe corresponding amount of ethanol.

Nuclear staining of DNAFor nuclear DNA staining, thecultures were established at

adensity of 2 X l0~cells/cm2 onto 11-mm2 glass coverslipscoated with collagen and grown for 24 or 48 h without orwith treatment with 75 ,ug/ml CsA. At various time pointsculture medium was aspirated, and cells were washed withphosphate-buffered saline (PBS) and fixed with 70% ethanolfor 24 h at 4°C.Staining with acridine orange was performedaccording to the procedure of Darzynkiewicz (1990). Inbrief, cells were incubated with RNase (10 UIml) for 1 hat 37°C,washed with PBS, and incubated for 30 s with asolution containing 0.2 ml of PBS and 0.5 ml of 0.1 M HC1,followedby incubation in 2 ml of amixturecontaining 0.1 Mcitric acid, 0.02 M sodium phosphate, and 6 ~g/ml acridineorange. After 5 mm the stained cells were observed under afluorescence microscope (Nikon UFX-DX) with excitationat 450—490 nm.

DNA fragmentation analysisTo prepare cellular DNA, the untreated cells and cells

treated with CsA (75 ~g/ml) for various times were de-tached from culture dishes in PBS using a cell scraper. Aftercentrifugation at 200 g for 5 mm, the cell pellet was washedwith PBS, resuspended in 400 ml of extraction buffer (500mM Tris-HC1, 1 mM EDTA, 10 mM NaC1, and 0.5% so-

dium dodecyl sulfate), and incubated with 100 ~.tg/mlpro-teinase K overnight at 48°C.The cell lysates were then ex-tracted with phenol, and nucleic acids were precipitated with96% ethanol. Pellets were gently resuspended overnight inTE buffer (10 M Tris-HC1 and 1 mM EDTA). Thereafter,the samples were incubated for 1 h at 37°Cwith RNase A(0.1 mg/mI). Electrophoresis conditions were as previouslydescribed (Filipkowski et al., 1994; Kaminska et al., 1994).

Preparation of protein extracts and analysis ofDNA-binding activities

At different time points after the treatment, cells werewashed with PBS, detached from culture dishes by a cellscraper in 0.5 ml of bufferA, andimmediately processed fornuclear protein extraction (Kaminska et al., 1992). Buffer Acontained 10 mM HEPES (pH 7.9), 10 mM KC1, 0.2 mMEDTA, 1 mM dithiothreitol, and protease inhibitors (0.5mM phenylmethylsulfonyl fluoride, 10 mg/ml aprotinin, 10mg/ml leupeptin, and 1 mg/ml pepstatin A; all from Sigma).After incubation for 15 mm on ice, NonidetP-40 was addedto a final concentration of 0.5% followed by centrifugationat 11,000 g for 1 mm at 4°C.The crude pellet was resus-pended in buffer B (20 mM HEPES, 0.4 M NaC1, 2 mMEDTA, 1 mM dithiothreitol, and protease inhibitors asabove) and incubatedfor 15 mm at 4°C.Aftercentrifugationfor 15 mm at 11,000 g, supernatants were frozen at —70°C.Theprotein content was estimated by themethod of Bradford(1976).

We applied the electrophoretic mobility shift assay(EMSA) to assess the DNA-binding activities of the ex-tracted proteins from the different experimental conditionsas previously described (Kaminska et al., 1995, 1996). Fivemicrograms of nuclear proteins waspreincubated for 10 mmat room temperature in binding buffer [10 mM HEPES, 25mM KC1, 0.5 mM EDTA, 0.25 ~ig/ml of bovine serumalbumin, 1 mM dithiothreitol, and 20 jig/ml of poly d (I-C)]and subsequently incubated with 0.25 ng (30,000—40,000Cerenkov cpm) of an end-labeled probe for 20 mm at roomtemperature. Commercially available double-stranded oligo-nucleotides (Stratagene, La Jolla, CA, U.S.A.) containingbinding motifs for the transcription factors AP-1 and CREBwere applied: AP-l, 5’-CTA GTG ATG AGT CAG CCGGATC-3’; CRE, 5 ‘-GAT TGG CTG ACG TCA GAGAGCT-3’. Oligonucleotides were labeled with [32P]dCTPusing the 3 ‘-end labeling kit (Promega, U.S.A.) and purifiedon spin columns (LKB Pharmacia). A series of control ex-periments with an excess of unlabeledprobe were performedto assess the specificity of binding.

Following incubation, 2 ~.tlof loading buffer containing0.3% bromophenol blue/3% glycerol was added to the sam-ples and electrophoresed at 130 V for 2 h in anondenaturing4% polyacrylamide gel. The electrophoresis was performedin a low ionic strength buffer [6.7 mM Tris-HC1 (pH 7.5),1 mM EDTA, and 3.3 mM sodium acetate]. Gels were driedand exposed to autoradiographic film (Amersham MP) withintensifying screens at —70°C overnight. To facilitate thecomparison among the different conditions, the autoradio-grams were scanned densitometrically and analyzed withL’~sageQuantsoftware (Molecular Dynamics).

EMSA-supershift analysis wasperformed to determine thecontribution of the c-Jun protein to the AP- 1 DNA-bindingactivity using procedures that we have previously described(Kaminska et al., 1995, 1996). Monoclonal antibody againstc-Jun protein (sc-822 X; Santa Cruz Biotechnology, Santa

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1144 G. MOSIENIAK ET AL

FIG. 1. Dose-dependent inhibition of growth of C6 glioma cellsby CsA. The inhibitory effect was estimated by measuring totalprotein content (Bradford, 1976) in untreated and CsA-treatedcultures 24 h after the drug addition. Data are mean ±SEM(bars) values from three experiments done in triplicate. C6 gli-oma cells were cultured in Dulbecco’s modified Eagle’s mediumsupplemented with 2 mMglutamine, 10% calf serum, and antibi-otics under standard conditions. Cells were preplated 1 day be-fore the start of the experiment at a density of 7 x 103/cm2.CsA (Sandimmun; Sandoz; stock solution, 50mg/mi) was addedat different concentrations.

Cruz, CA, U.S.A.) was preincubated for 1 h at 4°Cwith 5~.tgof protein extracts from untreated and treated cells forvarious times with CsA (75 ~.tg/ml).The antibody had nodetectable cross-reactivity with other Fos and Jun proteinsthat was confirmed by our western blot analysis (data notshown).

RESULTS

General description of morphological effects ofCsA on C6 glioma cells

In preliminary experiments we studied the effect ofdifferent concentrations of CsA on the growth of C6glioma cells. We found that CsA affects the growthof C6 glioma cells and produces dramatic changes incellular morphology. The inhibitory effect of CsA oncell growth was dose-dependent and manifested as astatistically significant decline in total protein contentas measured at 24 h after CsA addition (Fig. I). Insubsequent experiments, CsA, at a concentration of 75~.tg/ml, was used to characterize the time course ofmorphological and biochemical changes.

Cultures of normal cells constituted of flattenedpolygonal cells forming a network of multipolar andbranched glialprocesses (Fig. 2A). The earliestvisibleeffect of CsA was a loss of fine processes followed bythe appearance of spindle-shaped and round cells. At24 h following drug addition, the vast majority of cellshad no processes, and cells were rounded and becamedetached from the substratum (Fig. 2B). These cellswere still viable, and the integrity of the cytoplasmicand intracellular membranes remained intact becausemost of them excluded trypan blue (data not shown).At 48 h the number of cells gradually declined owingto cell death.

Characterization of cell death induced by CsA inC6 glioma cells

Because CsA-induced cell death was characterizedby morphological hallmarks typical of apoptosis rather

than necrosis, we chose to examine three criteria usu-ally adapted to identify apoptosis: dependence of celldeath on protein synthesis, appearance of pyknoticcells, and occurrence of DNA fragmentation.

We sought to determine whether CsA-induced celldeath represented passive degeneration or, alterna-tively, an active “suicide” process that might beslowed or blocked by inhibition of macromolecularsynthesis. For such a purpose, we investigated the ef-fects of CX, a reversible inhibitor of translation, onCsA-induced morphological changes of C6 gliomacells 24 h after drug addition. Figure 2D shows thatCX was partially effective in preventing morphologicalchanges induced by CsA. Most cells were alive, withclusters of phase-bright cell bodies and intact pro-cesses. CX alone did not significantly affect the mor-phology of the cells but did reduce cell growth(Fig. 2C).

The specific DNA fluorescent stain acridine orangewas used to assess changes in DNA and nuclear struc-ture following treatment with 75 ~.tg/ml CsA. Cellstreated with CsA demonstrated morphological featurestypical of apoptotic nuclei. Nuclei of control C6 cellswere large and round and exhibited diffuse, uniformstaining (Fig. 3). Cells treated with CsA for 24 and48 h contained nuclei with abnormal morphologies:“bean’ ‘-shaped nuclei with highly condensed chroma-tin or irregular clumps of dense chromatin (Fig. 3).The number of nuclei with chromatin condensationand fragmentation increased as a function of time.

Although apoptotic cell death is often defined bymorphological criteria, a biochemical hallmark ofapoptosis is the DNA fragmentation into oligonucleo-some-length fragments. As such, we isolated and frac-tionated DNA on agarose gels from control cells andcells treated for various times with 75 ~.tg/mlCsA.This analysis revealed that DNA isolated from cellstreated with CsA for 36—48 h demonstrated the ladder-like pattern of DNA fragmentation (Fig. 4).

Induction of DNA-binding activity of AP-1transcription factor during CsA-inducedapoptosis of C6 glioma cells

As our results raised the possibility that CsA-in-duced death of C6 glioma cells belongs to the pro-grammed cell death phenomena, we used this model toassess the possible involvement of AP- 1 transcriptionfactor. Induction of AP- 1 transcription factor has beenassociated with apoptosis in CNS cells in vivo(Kamin-ska et al., 1994; Kasof et al., 1995). Nuclear extractsfrom cultures untreated or treated for different timeswith CsA were analyzed by EMSA to determine thelevel of DNA-binding activity of AP- 1. The DNA-binding activity of CREB (which is a constitutivelyexpressed transcription factor) was tested in parallelas an internal standard for the quality and quantity ofprotein extracts.

The DNA-binding activity of AP-1 was easily de-

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CsA-INDUCED APOPTOSIS IN GLIOMA 1145

FIG. 2. Morphological changes in C6 glioma cells produced by CsA and effects of CX. C6 cells were plated and cultured as describedin Materials and Methods in the absence or presence of the compounds. Cells were monitored for 24 h by phase-contrast microscopyat x16. A: Normal, untreated cells. B: In cultures treated with CsA (75 ~ig/ml)all cells have lost fine processes, and the majority ofcells have become round. C: CX (1 mg/mI) alone reduced several cells but did not alter cellular morphology. D: CX in combinationwith CsA prevented morphological changes of C6 glioma cells induced by CsA.

tectable in proliferating C6 glioma cells. CsA producedan increase of AP- 1 DNA-binding activity 6 h follow-ing drug addition, and this activity remained elevatedup to 36 h after addition of the drug (Fig. 5, lowerpanel). CsA treatment did not produce any major ef-fects on the level of CRE DNA-binding activity (Fig.5, upper panel). Competition experiments with a 20-fold excess of unlabeled probe were performed to ver-ify the specificity of the observed DNA-binding activi-ties (Fig. 5, comp. lane).

To facilitate comparison among multiple samplesfrom four sets of experiments, we performed a densi-tometry-based quantification of DNA-binding activi-ties of transcription factors. The data are based ondensitometric measurements of autoradiograms fromfour sets of experiments. In particular, experimentalmeasurements of the level of AP- 1 DNA-binding activ-ity were normalized to CRE binding activity to controlfor differences in protein loading. These measurements

were related then to control values (representing theAP- 1 level in untreated cultures, defined as 1). Resultsare expressed as mean ±SEM values (number of cul-tures = 4) and presented in Fig. 6.

Supershift experiments with c-Jun antibody wereperformed to examine the contribution of this proteinto AP- 1 DNA-binding activity during CsA-inducedapoptosis of C6 glioma cells. We demonstrated thatthis antibody has the appropriate binding characteris-tics and no detectable cross-reactivity with other mem-bers of the Fos and Jun families and can be useful todetermine the presence of c-Jun protein in the AP-Icomplex (Kaminska et al., 1996). The c-Jun antibodyalone does not produce any shifted band in the absenceof protein extract (data not shown). Five microgramsof protein nuclear extracts from untreated and treatedcells for various times with CsA (75 ~sg/ml) wereanalyzed using the supershift method. Figure 7 showsthe presenceof the c-Jun protein inAP- 1 complex from

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1146 G. MOS1ENIAK ET AL.

FIG. 4. CsA induces ladder-likeDNA fragmentation in C6 gliomacells. Gel electrophoresis was per-formed on DNA extracted fromcontrol C6 glioma cells and cellstreated with CsA (75 ~.tg/ml)at dif-ferent times after the drug addi-tion. DNA was resolved in 2%agarose gel in the presence ofethidium bromide and visualizedunder UV light. Ml and M2 repre-sent DNA molecular weight mark-ers: a 1-kb DNA ladder and 123-bp ladder, respectively.

ence of a more slowly migrating “supershifted” bandreflects the presence of a particular member of the AP-1 complex (Lukasiuk and Kaczmarek, 1994; Kaminskaet al., 1996).

DISCUSSION

CsA induces apoptotic cell death ofrat C6 glioma cells

We report here that CsA, a powerful immunosup-pressant, induces an apoptotic cell death of C6 gliomacells. It has repeatedly been stressed that there is nosingle marker of apoptosis and that only the combina-tion of several ones can be used to define the process

FIG. 3. Alterations in nuclear morphology induced by C5A inC6 glioma cells. Fluorescence microscopy of cells stained withacridine orange revealed cell nuclei deformation and chromatincondensation 24 and 48 h after CsA treatment. Arrows incx~atetypical apoptotiC cell nuclei.

untreated proliferating C6 cells. Further illustrated isthe significant increase of c-Jun content throughoutCsA-induced apoptosis with a peak at 6 h followingdrug addition. It has been demonstrated that the pres-

FIG. 5. Levels of CRE and AP-1 DNA-binding activities duringCsA-induced apoptosis in C6 glioma cells. The levels of DNA-binding activities of transcription factors were measured byEMSAat various times following CsA treatment. Five microgramsof nuclear protein extracts from untreated and CsA-treated cul-tures were processed in parallel. The experiment was repeatedfour times on independent cultures. The lane marked Comp.represents a competition assay with a 20-fold excess of unla-beled, specific oligonucleotides.

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CsA-INDUCED APOPTOSIS IN GLIOMA 1147

FIG. 6. Densitometry-based quantification ofAP-1 DNA-bindingactivity. The data are based on densitometric measurements ofautoradiograms from four sets of experiments. In a particularexperiment measurements of the AP-1 DNA-binding activitywere normalized to CREbinding activity for differences in proteinloading and related to control values (representing the AP-l levelin untreated cultures, defined as 1). Data are mean ±SEM (bars)values (number of Cultures = 4).

(Kerr et al., 1972; Wyllie et a!., 1980). CsA-inducedcell death shows all the features characteristic ofapoptosis: (a) Cells growing in monolayer roundedup with loss of junctions with neighboring cells anddetached from the substratum; (b) their nuclei under-went condensation and fragmentation of chromatin;(c) death was accompanied by the appearance of aDNA ladder on gel electrophoresis; and (d) appear-ance of apoptotic morphological changes was pre-vented by the addition of CX, an inhibitor of proteinsynthesis.

Apoptosis, an active way of cell death, is often pre-sented as being in contrast to necrosis, with the latteroccurring rapidly after an insult, not requiring proteinsynthesis, and not involving chromatin fragmentation.The long time course required for the cell death tooccur and the early reversibility by an inhibitor ofprotein synthesis suggest that CsA-induced cell deathis an active process involving specific alterations inprotein and gene expression. In fact, we have foundan induction of the DNA-binding activity of the AP-1 transcription factor to occur 6 h after CsA treatment.This induction preceded the appearance of apoptoticchanges in cellular and nuclear morphology.

CsA use for immunosuppression is linked to CNSpathology as evidenced by a syndrome of confusion,agitation, psychosis, seizures, and paralysis. In thisstudy Sandimmun, a clinical formulation of CsA, wasused in most experiments. Our results raise a possibil-ity that the side effects of the drug are due to promotionof apoptotic cell death of brain glia and possibly othercell types. An induction of the programmed cell deathin C6 glioma cells that requires an induction of newgenes and proteins suggests a specific mechanism forCsA action. Our preliminary studies showed a specificdecrease of the nuclear factor of activated T cellsDNA-binding activity in C6 cells following CsA treat-ment (G.M. and B.K., unpublished data). Indeed, it

has been recently demonstrated that CsA (20 bIM) in-duces apoptosis in murine cortical cell cultures (Mc-Donald et al., 1995). It is interesting that oligodendro-cytes exhibited heightened vulnerability (greater thanthat of cortical neurons) to CsA-induced apoptosis.

In the context of immunosuppressive drug treatmentand side effects of CsA, it is critical to know whatpart of Sandimmun, a clinical formulation of CsA thatcontains emulsifier, is responsible for the toxic effect.Although the emulsifier for CsA in clinical intravenousformulations, Cremophor, has recently come into ques-tion as a possible source of neurologic side effectsin immunosuppressant therapy (Brat et al., 1992), aserious toxicity of CsA provided orally in milk ordrinking water has been described (Atkinson et a!.,1984; Murren et al., 1991). We can rather excludethe possibility of cytotoxic effects of emulsifier in ourexperiments, because we found that pure CsA (75 ,ug/ml) exerts the same effect on glioma cells.

Involvement of AP-1 transcription factor in CsA-induced apoptosis of C6 glioma cells

An induction of AP-I transcription factor has beenassociated with apoptosis of the CNS cells in vivo(Kaminska et al., 1994; Kasof et al., 1995). Theinvolvement of c-Jun protein in apoptotic cell deathhas been suggested by both direct and indirect lines ofevidence. The increased level of c-Junprotein has beentemporarily associated with neuronal death (Dragunowet al., 1993; Dragunow and Preston, 1995; Andersonet a!., 1996; Soriano eta!., 1996). Moredirect evidenceimplicating AP- 1 and its c-Jun component in apoptoticevents comes from Collotta et al. (1992), who demon-strated an inhibition of apoptosis of lymphoid cells byantisense oligonucleotides directed against c-fos andc-jun mRNA. More recently, Estus et a!. (1994) have

FIG. 7. The contribution of c-Jun protein to AP-l DNA-bindingactivity during CsA-induced apoptosis of C6 glioma cells. Thepresence of c-Jun in the AP-i Complex was established in 5 ~igof protein nuclear extracts from cells untreated and treated for24 h with CsA (75 ~tg/ml)using the supershift method. The arrowindicates the position ofa retarded band. Commercially available(Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.) monoclonalantibody against c-Jun was used. Antibody (1 mg/i ml) wasaffinity-purified by the manufacturer and has no detectablecross-reactivity with other members ofthe Jun and Fos families.

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1148 G. MOSIENIAK ET AL.

reported that antibodies against c-Jun protein protectedsympathetic neurons from apoptosis induced by nervegrowth factor deprivation. Furthermore, Ham et a!.(1995) have shown that a dominant, negative mutantof c-jun (which abolishes the activity of AP- 1 com-plex) has a similar protective effect.

We have demonstrated a specific increase of DNA-binding activity of AP- 1 in C6 glioma cells duringCsA-induced apoptosis. The AP- 1 DNA-binding activ-ity peaks at 6 h following the drug addition and remainselevated for up to 36 h thereafter. It is interesting thatthe CsA-induced AP- 1 content increase precedes theappearance of apoptotic alterations in cellular and nu-clear morphology. The c-Jun protein is present in theAP-l complex from untreated, proliferating C6 cells.CsA treatment results in a significant increase in thec-Jun contribution to the DNA-binding activity of AP-1. This demonstrates that the observed increase of theDNA-binding activity of AP- 1 is at least partially ac-counted for by the increase of c-Jun expression. Ourresults suggest that up-regulation of AP- 1 expressionand in particular its c-Jun component plays a pivotalrole in initiating downstream events responsible forapoptotic cell death.

Acknowledgment: These studieswere supported by grant4.PO5A.037.09 from the KBN (State Committee of ScientificResearch, Poland). We would like to thank Dr. L. Kaczma-rek for critical reading of the manuscript.

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