The Absence of Oligonucleosomal DNA Fragmentation during Apoptosis of IMR-5 Neuroblastoma Cells
2001; Elsevier BV; Volume: 276; Issue: 25 Linguagem: Inglês
10.1074/jbc.m100072200
ISSN1083-351X
AutoresVı́ctor J. Yuste, José R. Bayascas, Núria Llecha, I. Prieto Sánchez, Jacint Boix, Joan X. Comella,
Tópico(s)Microtubule and mitosis dynamics
ResumoCaspase-activated DNase is responsible for the oligonucleosomal DNA degradation during apoptosis. DNA degradation is thought to be important for multicellular organisms to prevent oncogenic transformation or as a mechanism of viral defense. It has been reported that certain cells, including some neuroblastoma cell lines such as IMR-5, enter apoptosis without digesting DNA in such a way. We have analyzed the causes for the absence of DNA laddering in staurosporine-treated IMR-5 cells, and we have found that most of the molecular mechanisms controlling apoptosis are well preserved in this cell line. These include degradation of substrates for caspases, blockade of cell death by antiapoptotic genes such as Bcl-2 or Bcl-XL, or normal levels and adequate activation of caspase-3. Moreover, these cells display normal levels of caspase-activated DNase and its inhibitory protein, inhibitor of caspase-activated DNase, and their cDNA sequences are identical to those reported previously. Nevertheless, IMR-5 cells lose caspase-activated DNase during apoptosis and recover their ability to degrade DNA when human recombinant caspase-activated DNase is overexpressed. Our results lead to the conclusion that caspase-activated DNase is processed during apoptosis of IMR-5 cells, making these cells a good model to study the relevance of this endonuclease in physiological or pathological conditions. Caspase-activated DNase is responsible for the oligonucleosomal DNA degradation during apoptosis. DNA degradation is thought to be important for multicellular organisms to prevent oncogenic transformation or as a mechanism of viral defense. It has been reported that certain cells, including some neuroblastoma cell lines such as IMR-5, enter apoptosis without digesting DNA in such a way. We have analyzed the causes for the absence of DNA laddering in staurosporine-treated IMR-5 cells, and we have found that most of the molecular mechanisms controlling apoptosis are well preserved in this cell line. These include degradation of substrates for caspases, blockade of cell death by antiapoptotic genes such as Bcl-2 or Bcl-XL, or normal levels and adequate activation of caspase-3. Moreover, these cells display normal levels of caspase-activated DNase and its inhibitory protein, inhibitor of caspase-activated DNase, and their cDNA sequences are identical to those reported previously. Nevertheless, IMR-5 cells lose caspase-activated DNase during apoptosis and recover their ability to degrade DNA when human recombinant caspase-activated DNase is overexpressed. Our results lead to the conclusion that caspase-activated DNase is processed during apoptosis of IMR-5 cells, making these cells a good model to study the relevance of this endonuclease in physiological or pathological conditions. caspase-activated DNase acetyl-Asp[OMe]-Glu[OMe]-Val-Asp[OMe]-7-amino-4-trifluoromethyl coumarin extracellular-regulated kinase human CAD inhibitor of the caspase-activated DNase human ICAD 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide nuclei isolation buffer phosphate-buffered saline staurosporine benzyloxycarbonyl-Val-Ala- Asp[OMe]-fluoromethylketone phenylmethylsulfonyl fluoride 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid 1,4-piperazinediethanesulfonic acid Programmed cell death or apoptosis is characterized by a set of morphological and biochemical events that are critical for embryonic development and tissue homeostasis in metazoans (reviewed in Refs. 1Steller H. Science. 1995; 267: 1445-1449Crossref PubMed Scopus (2431) Google Scholarand 2Jacobson M.D. Weil M. Raff M.C. Cell. 1997; 88: 347-354Abstract Full Text Full Text PDF PubMed Scopus (2409) Google Scholar). Apoptosis has also been involved as an important phenomenon in many different human diseases (reviewed in Ref. 3Fadeel B. Orrenius S. Zhivotovsky B. Biochem. Biophys. Res. Commun. 1999; 266: 699-717Crossref PubMed Scopus (213) Google Scholar). Shrinkage and fragmentation of the nucleus as well as the cell body and extensive degradation of chromosomal DNA are some of the most characteristic features of apoptosis (4Wyllie A.H. Kerr J.F. Currie A.R. Int. Rev. Cytol. 1980; 68: 251-306Crossref PubMed Scopus (6725) Google Scholar). DNA fragmentation is a two-step process in which the DNA is first cleaved into 50–300-kilobase pair fragments (high molecular weight DNA degradation), that are subsequently degraded into smaller fragments of oligonucleosomal size (DNA ladder) (reviewed in Ref. 5Walker P.R. Sikorska M. Biochem. Cell Biol. 1997; 75: 287-299Crossref PubMed Scopus (144) Google Scholar). It is considered that only the high molecular weight DNA degradation is essential for cell death, because there are several cell types that never produce DNA ladders with treatments that induce the typical nuclear and cytoplasmatic changes of apoptosis. These cell types include the breast carcinoma-derived cell line MCF-7 (6Janicke R.U. Sprengart M.L. Wati M.R. Porter A.G. J. Biol. Chem. 1998; 273: 9357-9360Abstract Full Text Full Text PDF PubMed Scopus (1720) Google Scholar), the human androgen-independent prostatic cancer cell DU-145 (7Oberhammer F. Wilson J.W. Dive C. Morris I.D. Hickman J.A. Wakeling A.E. Walker P.R. Sikorska M. EMBO J. 1993; 12: 3679-3684Crossref PubMed Scopus (1161) Google Scholar), the human neuronal-like cell NT2 (8Walker P.R. Leblanc J. Carson C. Ribecco M. Sikorska M. Ann. N. Y. Acad. Sci. 1999; 887: 48-59Crossref PubMed Scopus (39) Google Scholar), and some human neuroblastomas such as IMR-5 and IMR-32 (9Boix J. Llecha N. Yuste V.J. Comella J.X. Neuropharmacology. 1997; 36: 811-821Crossref PubMed Scopus (77) Google Scholar). Recently, an endonuclease that is activated specifically by caspase-3 during apoptosis has been described in human and mouse. The human gene for this protein has been named caspase-activated nuclease (10Halenbeck R. MacDonald H. Roulston A. Chen T.T. Conroy L. Williams L.T. Curr. Biol. 1998; 8: 537-540Abstract Full Text Full Text PDF PubMed Google Scholar) or DFF40 (DNA fragmentationfactor, 40 kDa subunit) (11Liu X. Li P. Widlak P. Zou H. Luo X. Garrard W.T. Wang X. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8461-8466Crossref PubMed Scopus (502) Google Scholar), whereas the mouse homologue has been named caspase-activated DNase (CAD)1 (12Enari M. Sakahira H. Yokoyama H. Okawa K. Iwamatsu A. Nagata S. Nature. 1998; 391: 43-50Crossref PubMed Scopus (2807) Google Scholar). CAD is maintained inactive in the cytoplasm of normal cells because of its association with a protein that acts as a chaperone and inhibitor. This protein has been named the inhibitor of CAD (ICAD) in the mouse (13Sakahira H. Enari M. Nagata S. Nature. 1998; 391: 96-99Crossref PubMed Scopus (1424) Google Scholar) or DFF45 (DNA fragmentationfactor, 45 kDa subunit) in humans (14Liu X. Zou H. Slaughter C. Wang X. Cell. 1997; 89: 175-184Abstract Full Text Full Text PDF PubMed Scopus (1650) Google Scholar). Here we will refer to it as ICAD. It has been demonstrated that the enzymatic activity of CAD is induced when caspase-3 cleaves ICAD at two specific aspartic residues (Asp117 and Asp224), thus releasing CAD (12Enari M. Sakahira H. Yokoyama H. Okawa K. Iwamatsu A. Nagata S. Nature. 1998; 391: 43-50Crossref PubMed Scopus (2807) Google Scholar, 15McIlroy D. Sakahira H. Talanian R.V. Nagata S. Oncogene. 1999; 18: 4401-4408Crossref PubMed Scopus (111) Google Scholar). Activated CAD degrades the DNA at the internucleosomal regions, and analysis of this DNA in agarose gels shows a characteristic ladder pattern (14Liu X. Zou H. Slaughter C. Wang X. Cell. 1997; 89: 175-184Abstract Full Text Full Text PDF PubMed Scopus (1650) Google Scholar). Although, caspase-3 seem necessary for the laddering degradation of DNA, it is considered that apoptotic cell death can proceed without caspase-3 in some cell types. For example, cells derived from caspase-3 −/− mice or cells defective in this caspase (MCF-7) die without displaying oligonucleosomal DNA degradation when treated with pro-apoptotic stimuli (6Janicke R.U. Sprengart M.L. Wati M.R. Porter A.G. J. Biol. Chem. 1998; 273: 9357-9360Abstract Full Text Full Text PDF PubMed Scopus (1720) Google Scholar, 16Woo M. Hakem R. Soengas M.S. Duncan G.S. Shahinian A. Kagi D. Hakem A. McCurrach M. Khoo W. Kaufman S.A. Senaldi G. Howard T. Lowe S.W. Mak T.W. Genes Dev. 1998; 12: 806-819Crossref PubMed Scopus (765) Google Scholar). Moreover, several types of cells derived from the ICAD-deficient mice lack internucleosomal DNA degradation, although these mice do not show major defects in development (17Zhang J. Liu X. Scherer D.C. van Kaer L. Wang X. Xu M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12480-12485Crossref PubMed Scopus (159) Google Scholar). Very little information exists about the cell death process without DNA fragmentation, but it is assumed that the oligonucleosomal DNA degradation confers some advantages to the organism. Thus, for example, it has been proposed that DNA degradation may minimize the risk of transferring oncogenes from a doomed cell to an adjacent healthy cell or to a phagocyte (18de la Taille A. Chen M.W. Burchardt M. Chopin D.K. Buttyan R. Cancer Res. 1999; 59: 5461-5463PubMed Google Scholar). On the other hand, it has also been proposed that the failure to digest chromatin could be the basis for the pathogenesis of some autoimmune diseases in which antibodies against the heterochromatin are generated (19Herrmann M. Voll R.E. Zoller O.M. Hagenhofer M. Ponner B.B. Kalden J.R. Arthritis Rheum. 1998; 41: 1241-1250Crossref PubMed Scopus (714) Google Scholar). We have reported previously that human neuroblastoma IMR-5 does not show oligonucleosomal DNA fragmentation nor condensation of nuclear chromatin into the rounded masses typical of apoptosis when cells were treated with staurosporine (STP) (9Boix J. Llecha N. Yuste V.J. Comella J.X. Neuropharmacology. 1997; 36: 811-821Crossref PubMed Scopus (77) Google Scholar). In the present report we have analyzed several regulatory mechanisms that control apoptosis in the IMR-5 cell line. We have not found major defects in caspase-3 protein levels or its protease activity, and caspase inhibitors or antiapoptotic genes such as Bcl-2 or Bcl-XL also block cell death. Moreover, ICAD or CAD protein levels are comparable with other cell lines that display oligonucleosomal degradation after induction of apoptosis, and the primary sequences of these two proteins are the same as that reported previously (GenBankTM accession numbers NM 004402 for CAD and NM 004401 for ICAD) (10Halenbeck R. MacDonald H. Roulston A. Chen T.T. Conroy L. Williams L.T. Curr. Biol. 1998; 8: 537-540Abstract Full Text Full Text PDF PubMed Google Scholar, 11Liu X. Li P. Widlak P. Zou H. Luo X. Garrard W.T. Wang X. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8461-8466Crossref PubMed Scopus (502) Google Scholar, 14Liu X. Zou H. Slaughter C. Wang X. Cell. 1997; 89: 175-184Abstract Full Text Full Text PDF PubMed Scopus (1650) Google Scholar, 20Mukae N. Enari M. Sakahira H. Fukuda Y. Inazawa J. Toh H. Nagata S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9123-9128Crossref PubMed Scopus (172) Google Scholar, 21Liu X. Zou H. Widlak P. Garrard W. Wang X. J. Biol. Chem. 1999; 274: 13836-13840Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). However, IMR-5 cells rapidly lose CAD during STP-induced apoptosis. We also observed that IMR-5 cells recover their ability to degrade DNA into oligonucleosomal fragments when engineered to overexpress mouse or human CAD, pointing out the importance of the relative levels of this nuclease. In conclusion, we demonstrate that processing of CAD could be a new regulatory step in the control of apoptosis and specifically during oligonucleosomal DNA degradation. Moreover, because the transgenic mouse with the deletion of the CAD gene is not currently available, IMR-5 cells could serve as an alternative model to test the relevance of oligonucleosomal degradation of DNA in normal cells or in pathological process such as viral infection. Human SH-SY5Y and IMR-5 cell lines employed in the present study were kindly provided by Dr. Dionisio Martı́n-Zanca (Salamanca, Spain). The cells were routinely grown in 75-cm2 culture flasks (Sarstedt, Newton, NC) containing 10 ml of Dulbecco's modified Eagle's medium supplemented with 2 mml-glutamine, antibiotics, and heat-inactivated fetal bovine serum (Life Technologies, Inc.). IMR-5 cells were grown in the presence of 10% (v/v) fetal bovine serum, whereas SH-SY5Y cells were grown in 15% (v/v) fetal bovine serum. Medium was routinely changed every 3 days. Cells were maintained at 37 °C in a saturating humidity atmosphere containing 95% air and 5% CO2. For the different experiments, cells were grown at the adequate cell densities in culture dishes or multiwell plates (Corning, NY) using the same culture conditions as described above. MTT is a water-soluble tetrazolium salt that is reduced by metabolically viable cells to a colored, water-insoluble formazan salt. The procedure employed for this assay was the same as that described by Boix et al. (9Boix J. Llecha N. Yuste V.J. Comella J.X. Neuropharmacology. 1997; 36: 811-821Crossref PubMed Scopus (77) Google Scholar). For trypan blue staining, cells were seeded in 24-multiwell plates at 5 × 104 cells/well. After 24 h of seeding, cells were treated with STP at the adequate doses and times. Then cells were gently dissociated with a blue tip in their own culture medium, and a sample (100 μl) was taken and mixed with 20 μl of trypan blue solution (0.4%) (Sigma). Ten μl of the resulting cell suspension were counted with an hemocytometer. The results were expressed as percentages of trypan blue-stained cells over the total number of cells. Nuclear morphology was assessed by staining cells with the Hoechst 33258, which is also known as bisbenzimide ([2′-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5′-bi-1H-benzimidazole trihydrochloride]) as established in our laboratory (9Boix J. Llecha N. Yuste V.J. Comella J.X. Neuropharmacology. 1997; 36: 811-821Crossref PubMed Scopus (77) Google Scholar). The normal or apoptotic cell nuclei were visualized with an Olympus microscope equipped with epifluorescence optics under UV illumination. Control or STP-treated cells growing in 35-mm culture dishes (1 × 106 cells) were gently pelleted by centrifugation and washed twice with phosphate-buffered saline (PBS) (150 mm NaCl, 2.7 mm KCl, 8 mm Na2HPO4, 1.5 mmKH2PO4, pH 7.2). Fixation was performed with 100 mm phosphate buffer, pH 7.4, containing 2.5% glutaraldehyde for 30 min at 4 °C. The pellets were rinsed twice with cold PBS, postfixed in buffered OsO4, dehydrated in graded acetone, and embedded in Durcupan ACM resin (Fluka, Buchs, Switzerland). Ultrathin sections were obtained, mounted in copper grids, and counterstained with uranyl acetate and lead citrate. The specimens were observed with a Zeiss EM910 electron microscope. Cells grown in 35-mm culture dishes (1 × 106 cells) were collected in their culture medium, pelleted at 400 × g for 5 min, and rinsed twice with PBS. The pellet was homogenized in 600 μl of lysis buffer (0.5 m EDTA, 1% lauryl sarcosine, 10 mmTris-HCl, pH 9.5) by pipetting through a blue cone. Homogenates were clarified by centrifuging at 13,000 × g for 15 min, and supernatants were collected to a new tube in which they were extracted with phenol:chloroform:isoamyl alcohol (25:24:1) (Iberlabo, Madrid, Spain). DNA was precipitated with two volumes of cold ethanol and 0.5 volumes of 7.5 m ammonium acetate. Precipitated DNA was washed once in 70% ethanol and resuspended in 1 mmEDTA, 10 mm Tris-HCl, pH 8.0 containing DNase-free RNase at a final concentration of 20 μg/ml. DNA was analyzed in 1.5% agarose gel in 1 mm EDTA, 40 mm Tris acetate, pH 7.6. Approximately 2 × 106 cells/condition were detached from the 60-mm culture dish, pelleted at 400 × g for 5 min, and washed twice with PBS. Then cells were lysed with 100 μl of RIPA buffer (10 mm Tris-HCl, pH 7.4, 1 mm EDTA, 150 mm NaCl, 1% Nonidet P-40, 1% deoxycholate, 0.1% SDS, 1 mm PMSF) for nuclear protein extracts or with Nonidet P-40 buffer (10 mm Tris-HCl, pH 7.4, 5 mm EDTA, 50 mm NaCl, 5 mm dithiothreitol, 1 mmPMSF, 0.05% Nonidet P-40) for cytosolic protein extracts. The pellets were clarified by centrifuging at 13,000 × g for 10 min at 4 °C. The protein in the supernatants was quantitated using the Bio-Rad DC protein assay, and 25–50 μg of protein were loaded in SDS-polyacrylamide gels. The proteins were electrophoresed and electrotransferred to polyvinylidene difluoride Immobilon filters (Millipore, Bedford, MA) with a semidry apparatus following the instructions of the supplier (Hoefer, Amersham Pharmacia Biotech). Then filters were reacted with the appropriate specific primary antibodies and incubated with the adequate secondary antibodies conjugated with peroxidase (Sigma). Finally, immunoblots were developed with the SuperSignal West Dura Extended Duration Substrate (Pierce) for the detection of caspase-3 fragments and CAD and with the ECL system (Amersham Pharmacia Biotech) for the rest. We developed a new method to quantify DEVD-directed caspase activity. The cells were grown onto 96-well multiplates at 4 × 104 cells/well. After 24 h, the cells were treated with STP and then lysed by adding 1 volume of Ac-DEVD-afc lysis buffer 2-fold concentrated containing the fluorogenic substrate Ac-DEVD-afc (Enzyme Systems Products, Livermore, CA). Ac-DEVD-afc 2 × lysis buffer buffer is 40 mmHepes/NaOH, pH 7.2, 300 mm NaCl, 20 mmdithiothreitol, 10 mm EDTA, 0.2% CHAPS, 2% Nonidet P-40, 20% sucrose plus 50 μm of Ac-DEVD-afc. The plates were incubated for 12 h at 37 °C and then read in a Bio-Tek FL 600 fluorimeter (Izasa, Spain) at 360 nm (40 nm bandwidth) of excitation and 530 nm (25 nm bandwidth) of emission. For quantitative DEVD-like activity in cell lysates, cells were seeded onto 35-mm culture plates and, after treatment, were detached, washed twice with PBS, and lysed with Ac-DEVD-afc lysis buffer without the Ac-DEVD-afc. The supernatant was clarified by centrifuging at 13,000 × g in a microcentrifuge at 4 °C, and the protein was quantitated using the Bio-Rad protein assay Bradford-based method. Twenty-five μg of protein were added to 100 μl of the same lysis buffer containing 20 μm of Ac-DEVD-afc. The samples were incubated at 37 °C for 6 h and read with the fluorimeter. Nuclei were obtained as described previously (22Lazebnik Y.A. Cole S. Cooke C.A. Nelson W.G. Earnshaw W.C. J. Cell Biol. 1993; 123: 7-22Crossref PubMed Scopus (426) Google Scholar, 23Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4463) Google Scholar) with minor modifications. Cells were grown in 75-cm2 culture flasks, and 1 × 108 cells were harvested by centrifugation at 200 ×g for 5 min. After three washes with PBS and one with nuclei isolation buffer (NIB; 10 mm Pipes, pH 7.4, 10 mm KCl, 2 mm MgCl2, 1 mm dithiothreitol, 10 μm cytochalasin B, 1 mm PMSF), cells were resuspended in 10 volumes of NIB and transferred to a Dounce-type homogenizer. Cells were swelled for 30 min at 4 °C and gently lysed with 25 strokes of the pestle. Then nuclei were layered over a 30% (w/v) sucrose cushion in NIB and centrifuged at 800 × g for 10 min. Supernatant was discarded, and the pellet was washed three times in NIB. Finally, nuclei were resuspended in nuclear storage buffer (10 mmPipes, pH 7.4, 80 mm KCl, 20 mm NaCl, 250 mm sucrose, 5 mm EGTA, 1 mmdithiothreitol 0.5 mm spermidine, 0.2 mmspermine, 1 mm PMSF, 50% (v/v) glycerol) at 1 × 108 nuclei/ml and kept at −80 °C until used. Cytosolic extracts were obtained from cells treated with 100 nm of STP for 24 h or from control untreated cells. Cells were collected and centrifuged at 200 ×g for 5 min, washed three times with PBS, resuspended in a buffer containing 10 mm Hepes/KOH, pH 7.2, 2 mmMgCl2, 5 mm EGTA, 50 mm NaCl, 5 mm dithiothreitol, 1 mm PMSF, 20% glycerol, 1% Nonidet P-40, and swelled at 4 °C for 15 min. The lysates were centrifuged in a microcentrifuge at 4 °C, and the supernatants were quantitated with Bio-Rad protein assay Bradford-based method. To reconstitute the system, cytosolic extracts and purified nuclei from the different conditions were adequately combined. Thus, 150 μg of protein from cytosolic extracts and 5 × 105 of nuclei were incubated at 37 °C for 2 h in a buffer containing an ATP-regenerating system (2 mm ATP, 10 mmphosphocreatine, 50 μg/ml creatine kinase) and 1 mg/ml of bovine serum albumin. Reactions were stopped by adding 5 mm of EDTA, and the DNA was extracted with phenol:chloroform:isoamyl alcohol (25:24:1) and ethanol precipitation. DNA laddering was analyzed in a 1.8% agarose gel. One microgram of RNA from either IMR-5 or SH-SY5Y was treated with 2 units of DNase RNase-free (Amersham Pharmacia Biotech) and was reverse transcribed using 10 pmol of the specific downstream primer (ICAD-R or CAD-R; see below) for 1 h at 42 °C. Approximately 10 ng of cDNA was amplified by polymerase chain reaction in a PerkinElmer thermal cycler 2400 with 200 nm each primer. The polymerase chain reaction conditions were 94 °C for 20 s, 65 °C for 20 s, and 72 °C for 1 min for 35 cycles in 50 mm Tris-HCl, pH 9.0, 2.5 mm MgCl2, 15 mm(NH4)2SO4, 0.1% Triton X-100, and 1 unit of DyNAzime-EXT DNA polymerase (Finnzymes). Primers used were CAD-F (TGCAATGCTCCAGAAGCCCAAGAGC) and CAD-R (TCACTGGCGTTTCCGCACAGGCTG), which amplify a band of 1120 base pairs corresponding to the whole open reading frame of CAD (GenBankTM accession number NM 004402); and ICAD-F (CGCTCCGGCCTCCCGCGACTTCTCG) and ICAD-R (GGCGTGAGCCACTGCGCCTGGCCAA), which cover a 1353-base pair region containing the ICAD open reading frame (GenBankTM accession number NM 004401). The polymerase chain reaction products were automatically sequenced in both direction in an ABI PRISM 310 automatic sequence analyzer (PerkinElmer). MTbcl-2TKNeo (24Vaux D.L. Cory S. Adams J.M. Nature. 1988; 335: 440-442Crossref PubMed Scopus (2729) Google Scholar) and pSFFVNeobcl-XL (25Benito A. Silva M. Grillot D. Nuñez G. Fernandez-Luna J.L. Blood. 1996; 87: 3837-3843Crossref PubMed Google Scholar) plasmids have been described previously. DNA inserts were subcloned into the pcDNA3 mammalian expression vector. (Invitrogen BV, Leek, The Netherlands). We also cloned ICAD and CAD full-length open reading frame sequence into the pcDNA3 vector. The constructs were named pcDNA3/bcl-2, pcDNA3/bcl-XL, pcDNA3/hCAD, and pcDNA3/hICADL. IMR-5 cells were transfected with different constructs using LipofectAMINE Plus reagent (Life Technologies, Inc.). Stably transfected cells were selected with 500 μg/ml G-418 (geneticin) (Life Technologies, Inc.) and were used as a pool. STP is an unspecific protein kinase inhibitor that has been widely used as a classic and potent inductor of apoptosis in many different cell types (26Bertrand R. Solary E. O'Connor P. Kohn K.W. Pommier Y. Exp. Cell Res. 1994; 211: 314-321Crossref PubMed Scopus (471) Google Scholar, 27Koh J.Y. Wie M.B. Gwag B.J. Sensi S.L. Canzoniero L.M. Demaro J. Csernansky C. Choi D.W. Exp. Neurol. 1995; 135: 153-159Crossref PubMed Scopus (234) Google Scholar, 28Prehn J.H. Jordan J. Ghadge G.D. Preis E. Galindo M.F. Roos R.P. Krieglstein J. Miller R.J. J. Neurochem. 1997; 68: 1679-1685Crossref PubMed Scopus (116) Google Scholar, 29Deshmukh M. Johnson Jr., E.M. Cell Death. Differ. 2000; 7: 250-261Crossref PubMed Scopus (57) Google Scholar). In a previous report, we characterized STP-induced cell death in several human neuroblastoma cell lines (9Boix J. Llecha N. Yuste V.J. Comella J.X. Neuropharmacology. 1997; 36: 811-821Crossref PubMed Scopus (77) Google Scholar) and found two of them, IMR-5 and IMR-32, that underwent atypical apoptotic cell death. Here we further characterize this cell death to determine precisely which can be the alteration responsible of this phenotype. SH-SY5Y neuroblastoma-derived cell line was used as a control. STP induces cell death with a similar dose-dependent profile in both cell lines as measured by MTT assay or trypan blue staining (Fig.1, A and B, respectively). Furthermore, for a given dose of STP (125 nm), the time course of cell death was comparable in both SH-SY5Y and IMR-5 cells, and about half the cells initially plated died after 12–18 h (Fig. 1 C). However, nuclear morphology and DNA laddering were two distinguishable features between these two cell lines upon STP treatment (9Boix J. Llecha N. Yuste V.J. Comella J.X. Neuropharmacology. 1997; 36: 811-821Crossref PubMed Scopus (77) Google Scholar). A ladder pattern of DNA fragmentation was clearly visible in STP-treated SH-SY5Y cells, whereas it never appeared in IMR-5 cells at any of the concentrations or times tested (Fig.1 D). When the nuclear chromatin was stained with Hoechst 33258, SH-SY5Y nuclei exhibited the typical apoptotic morphology, whereas IMR-5 chromatin was found to be condensed in the marginal zones of the nucleus (Fig. 2 A). The ultrastructural analysis of IMR-5 cells treated with STP revealed important nuclear modifications; chromatin was condensed and formed marginal and irregular masses of heterochromatin in a coarse pattern (Fig. 2 B) that has been previously defined as stage I nuclear condensation (30Susin S.A. Daugas E. Ravagnan L. Samejima K. Zamzami N. Loeffler M. Costantini P. Ferri K.F. Irinopoulou T. Prevost M.C. Brothers G. Mak T.W. Penninger J. Earnshaw W.C. Kroemer G. J. Exp. Med. 2000; 192: 571-580Crossref PubMed Scopus (662) Google Scholar). However, the characteristic high nuclear chromatin condensation typical of apoptosis (defined previously as stage II (30Susin S.A. Daugas E. Ravagnan L. Samejima K. Zamzami N. Loeffler M. Costantini P. Ferri K.F. Irinopoulou T. Prevost M.C. Brothers G. Mak T.W. Penninger J. Earnshaw W.C. Kroemer G. J. Exp. Med. 2000; 192: 571-580Crossref PubMed Scopus (662) Google Scholar)) was never found in STP-treated IMR-5 cells, although it was clearly apparent in SH-SY5Y (Fig. 2 B).Figure 2Absence of high nuclear chromatin condensation in IMR-5 cells treated with STP. SH-SY5Y cells (left column) and IMR-5 cells (right column) were treated with 100 nm STP for 24 h (all panels in A and bottom panels inB) or left untreated (top panels inB). Then cells were stained with Hoechst 33258 (A) or included in Durcupan and processed for electron microscopy (B). Note that treatment of IMR-5 cells with STP induces an incomplete condensation of the nuclear chromatin. Thelower panels in A are high magnifications of the cells framed in upper panels. The bars indicate 20 μm in the top panels in A and 2 μm forall panels in B.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To rule out the possibility that the lack of oligonucleosomal DNA fragmentation could be a specific feature of STP treatment, we tested a set of apoptotic inducers at different doses and times. These included DNA-damaging agents (camptothecin (100 μm), etoposide (100 μm), N-nitroso-N-methylurea (5 mm), and cisplatin (50 μm)), protein kinase inhibitors (H-7 (100 μm) and roscovitine (50 μm)), cytoskeleton-disrupting agents (vinblastine (100 μm), nocodazole (50 μm), and colchicine (10 μg/ml)), and macromolecular synthesis inhibitors (cycloheximide (2 μg/ml) and actinomicin D (5 μg/ml)). (The highest doses tested are indicated in parentheses.) None of these compounds was able to induce ladder formation in IMR-5 cells at the doses and times that efficiently promoted laddering in SH-SY5Y (data not shown). Moreover, all of these drugs were capable of inducing cell death in both SH-SY5Y and IMR-5 cells as measured by trypan blue and MTT assays (data not shown). Thus, the inability of STP to induce an apoptotic phenotype in IMR-5 cells seemed to be due to an intrinsic defect of these cells rather than to an incapacity of STP to lead the nuclear apoptotic changes. Antiapoptotic members of the Bcl-2 family have been shown to protect several cell models from STP-induced apoptosis (31Boix J. Fibla J. Yuste V.J. Piulats J.M. Llecha N. Comella J.X. Exp. Cell Res. 1998; 238: 422-429Crossref PubMed Scopus (26) Google Scholar, 32Gross A. McDonnell J.M. Korsmeyer S.J. Genes Dev. 1999; 13: 1899-1911Crossref PubMed Scopus (3254) Google Scholar). To gain further information on the cell death induced by STP in our experimental system, we analyzed the ability of Bcl-XL or Bcl-2 to prevent STP-induced IMR-5 cell death. For this purpose, we obtained stable pools of transfected cells with any of these two genes, and STP-induced cell death was measured using the MTT assay (Fig. 3 A). Both Bcl-2 and Bcl-XL overexpression afforded a significant protection against cell death at all the concentrations of STP tested. At the highest doses, Bcl-2 was slightly more efficient than Bcl-XL. However, neither of the two proteins was able to prevent cell death when the STP concentrations were higher than 5 μm. Western blot analysis of the transfected cells showed increased levels of the corresponding protein (Fig. 3 B). It has been demonstrated that caspase-3-deficient mice or MCF-7 cells enter apoptosis with an atypical morphology (similar to the one described above for IMR-5 cells) and are unable to degrade DNA into oligonucleosomal fragments (6Janicke R.U. Sprengart M.L. Wati M.R. Porter A.G. J. Biol. Chem. 1998; 273: 9357-9360Abstract Full Text Full Text PDF PubMed Scopus (1720) Google Scholar, 16Woo M. Hakem R. Soengas M.S. Duncan G.S. Shahinian A. Kagi D. Hakem A. McCurrach M. Khoo W. Kaufman S.A. Senaldi G. Howard T. Lowe S.W. Mak T.W. Genes Dev. 1998; 12: 806-819Crossref PubMed Scopus (765) Google Scholar). Therefore, an alteration in the caspase-3 function seemed a good e
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