FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2079273110> ?p ?o ?g. }

• W2079273110 endingPage "44128" @default.
• W2079273110 startingPage "44117" @default.
• W2079273110 abstract "Bcl-2 protein family members function either to promote or inhibit programmed cell death. Bcl-2, typically an inhibitor of apoptosis, has also been demonstrated to have pro-apoptotic activity (Cheng, E. H., Kirsch, D. G., Clem, R. J., et al. (1997) Science 278, 1966–1968). The pro-apoptotic activity has been attributed to the cleavage of Bcl-2 by caspase-3, which converts Bcl-2 to a pro-apoptotic molecule. Bcl-2 is a membrane protein that is localized in the endoplasmic reticulum (ER) membrane, the outer mitochondrial membrane, and the nuclear envelope. Here, we demonstrate that transient expression of Bcl-2 at levels comparable to those found in stably transfected cells induces apoptosis in human embryonic kidney 293 cells and in the human breast cell line MDA-MB-468 cells. Furthermore, we have targeted Bcl-2 specifically to either the ER or the outer mitochondrial membrane to test whether induction of apoptosis by Bcl-2 is dependent upon its localization within either of these membranes. Our findings indicate that Bcl-2 specifically targeted to the mitochondria induces cell death, whereas Bcl-2 that is targeted to the ER does not. The expression of Bcl-2 does result in its cleavage to a 20-kDa protein; however, mutation of the caspase-3 cleavage site (D34A) does not inhibit its ability to induce cell death. Additionally, we find that transiently expressed ER-targeted Bcl-2 inhibits cell death induced by Bax overexpression. In conclusion, the ability of Bcl-2 to promote apoptosis is associated with its localization at the mitochondria. Furthermore, the ability of ER-targeted Bcl-2 to protect against Bax-induced apoptosis suggests that the ER localization of Bcl-2 may play an important role in its protective function. Bcl-2 protein family members function either to promote or inhibit programmed cell death. Bcl-2, typically an inhibitor of apoptosis, has also been demonstrated to have pro-apoptotic activity (Cheng, E. H., Kirsch, D. G., Clem, R. J., et al. (1997) Science 278, 1966–1968). The pro-apoptotic activity has been attributed to the cleavage of Bcl-2 by caspase-3, which converts Bcl-2 to a pro-apoptotic molecule. Bcl-2 is a membrane protein that is localized in the endoplasmic reticulum (ER) membrane, the outer mitochondrial membrane, and the nuclear envelope. Here, we demonstrate that transient expression of Bcl-2 at levels comparable to those found in stably transfected cells induces apoptosis in human embryonic kidney 293 cells and in the human breast cell line MDA-MB-468 cells. Furthermore, we have targeted Bcl-2 specifically to either the ER or the outer mitochondrial membrane to test whether induction of apoptosis by Bcl-2 is dependent upon its localization within either of these membranes. Our findings indicate that Bcl-2 specifically targeted to the mitochondria induces cell death, whereas Bcl-2 that is targeted to the ER does not. The expression of Bcl-2 does result in its cleavage to a 20-kDa protein; however, mutation of the caspase-3 cleavage site (D34A) does not inhibit its ability to induce cell death. Additionally, we find that transiently expressed ER-targeted Bcl-2 inhibits cell death induced by Bax overexpression. In conclusion, the ability of Bcl-2 to promote apoptosis is associated with its localization at the mitochondria. Furthermore, the ability of ER-targeted Bcl-2 to protect against Bax-induced apoptosis suggests that the ER localization of Bcl-2 may play an important role in its protective function. endoplasmic reticulum human embryonic kidney green fluorescence protein polymerase chain reaction phosphate-buffered saline propidium iodide Bcl-2 is the founding member of a family of proteins that regulate apoptosis. Originally isolated from the t(14;18) chromosomal breakpoint in human B cell lymphomas, bcl-2 has since been established to be a proto-oncogene that prolongs cell survival by inhibiting apoptosis (1Cleary M.L. Sklar J. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7439-7443Crossref PubMed Scopus (833) Google Scholar, 2Bakhshi A. Jensen J.P. Goldman P. Wright J.J. McBride O.W. Epstein A.L. Korsmeyer S.J. Cell. 1985; 41: 899-906Abstract Full Text PDF PubMed Scopus (1010) Google Scholar, 3Tsujimoto Y. Gorham J. Cossman J. Jaffe E. Croce C.M. Science. 1985; 229: 1390-1393Crossref PubMed Scopus (836) Google Scholar, 4Vaux D.L. Cory S. Adams J.M. Nature. 1988; 335: 440-442Crossref PubMed Scopus (2730) Google Scholar). The protection from apoptosis conferred by Bcl-2 upon withdrawal of cytokine was found to be a consequence of its dysregulated overexpression in B cell lymphomas. The promotion of cell survival by Bcl-2 has been recapitulated in many cell line model systems in which Bcl-2 overexpression has been enforced by stable transfection. Although Bcl-2 has been found to inhibit cell death induced by a wide variety of apoptotic signals in many cell types, the mechanism of its protective action still remains unclear. Bcl-2 family members are characterized by containing at least one of four Bcl-2 homology domains (BH1-BH4). Some of these proteins, such as Bax and Bak, function to promote apoptosis, whereas others like Bcl-2 and Bcl-XL inhibit apoptosis (5Oltvai Z.N. Milliman C.L. Korsmeyer S.J. Cell. 1993; 74: 609-619Abstract Full Text PDF PubMed Scopus (5865) Google Scholar, 6Farrow S.N. White J.H. Martinou I. Raven T. Pun K.T. Grinham C.J. Martinou J.C. Brown R. Nature. 1995; 374: 731-733Crossref PubMed Scopus (470) Google Scholar, 7Boise L.H. Gonzalez-Garcia M. Postema C.E. Ding L. Lindsten T. Turka L.A. Mao X. Nunez G. Thompson C.B. Cell. 1993; 74: 597-608Abstract Full Text PDF PubMed Scopus (2929) Google Scholar). The subcellular localization of these proteins provides important clues as to how these proteins function. Certain pro-apoptotic proteins, such as Bax, and anti-apoptotic proteins, such as Bcl-2, bear a C-terminal transmembrane-targeting domain that allows them to be inserted into the cytosolic face of intracellular membranes (8Nguyen M. Millar D.G. Yong V.W. Korsmeyer S.J. Shore G.C. J. Biol. Chem. 1993; 268: 25265-25268Abstract Full Text PDF PubMed Google Scholar, 9Nechushtan A. Smith C.L. Hsu Y.T. Youle R.J. EMBO J. 1999; 18: 2330-2341Crossref PubMed Scopus (627) Google Scholar). Bcl-2 is found in several intracellular membranes including the endoplasmic reticulum (ER),1 the outer mitochondrial membrane, and the nuclear envelope (10Akao Y. Otsuki Y. Kataoka S. Ito Y. Tsujimoto Y. Cancer Res. 1994; 54: 2468-2471PubMed Google Scholar, 11Krajewski S. Tanaka S. Takayama S. Schibler M. Fenton W. Reed J. Cancer Res. 1993; 53: 4701-4714PubMed Google Scholar). In contrast, Bax is not located in the ER or perinuclear membrane but remains in the cytoplasm until an apoptotic signal is generated and induces its translocation to mitochondria (12Wolter K. Hsu Y. Smith C. Nechushtan A. Xi X. Youle R. J. Cell Biol. 1997; 139: 1281-1292Crossref PubMed Scopus (1577) Google Scholar, 13Gross A. Jockel J. Wei M. Korsmeyer S. EMBO J. 1998; 17: 3878-3885Crossref PubMed Scopus (966) Google Scholar). The oligomerization of Bax in the mitochondrial membrane has been shown to induce cytochrome c release and the subsequent steps in the execution phase of apoptosis (13Gross A. Jockel J. Wei M. Korsmeyer S. EMBO J. 1998; 17: 3878-3885Crossref PubMed Scopus (966) Google Scholar, 14Antonsson B. Montessuit S. Lauper S. Eskes R. Martinou J.C. Biochem. J. 2000; 345: 271-278Crossref PubMed Scopus (564) Google Scholar). The pro-apoptotic activity of other proteins, such as Bid and Bak, have been associated with their regulated targeting to the mitochondrial membrane (15Li H. Zhu H. Xu C.J. Yuan J. Cell. 1998; 94: 491-501Abstract Full Text Full Text PDF PubMed Scopus (3790) Google Scholar, 16Luo X. Budihardjo I. Zou H. Slaughter C. Wang X. Cell. 1998; 94: 481-490Abstract Full Text Full Text PDF PubMed Scopus (3080) Google Scholar). Thus, mitochondria appear to be a target for mediators of apoptosis and occupy a central role in the apoptotic pathway (17Green D. Reed J. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar). The structures of both pro-apoptotic and anti-apoptotic proteins, Bax and Bcl-XL, respectively, have been determined and found to be strikingly similar (18Muchmore S. Sattler M. Liang H. Meadows R. Harlan J. Yoon H. Nettsheim D. Chang B. Thompson C. Nature. 1996; 381: 335-341Crossref PubMed Scopus (1286) Google Scholar, 19Suzuki M. Youle R.J. Tjandra N. Cell. 2000; 103: 645-654Abstract Full Text Full Text PDF PubMed Scopus (906) Google Scholar). Although pro-apoptotic and anti-apoptotic proteins share structural similarities and are both found in mitochondrial membranes, it is not understood how they can function in opposing ways to regulate apoptosis. Recent evidence has indicated, however, that Bcl-2 under certain conditions can function as a pro-apoptotic molecule. Bcl-2 and Bcl-XL can be cleaved by caspase-3 and thus be converted to a pro-apoptotic protein similar to Bax (20Cheng E.H. Kirsch D.G. Clem R.J. Ravi R. Kastan M.B. Bedi A. Ueno K. Hardwick J.M. Science. 1997; 278: 1966-1968Crossref PubMed Scopus (1005) Google Scholar, 21Clem R.J. Cheng E.H. Karp C.L. Kirsch D.G. Ueno K. Takahashi A. Kastan M.B. Griffin D.E. Earnshaw W.C. Veliuona M.A. Hardwick J.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 554-559Crossref PubMed Scopus (471) Google Scholar). Conversely, Bax has also been shown to inhibit neuronal cell death when infected with Sinbis virus (22Lewis J. Oyler G.A. Ueno K. Fannjiang Y.R. Chau B.N. Vornov J. Korsmeyer S.J. Zou S. Hardwick J.M. Nat. Med. 1999; 5: 832-835Crossref PubMed Scopus (97) Google Scholar). These observations suggest that members of the Bcl-2 family have reversible roles in the regulation of apoptosis and have the potential to function in a pro-apoptotic or anti-apoptotic capacity. Here, we demonstrate that acute Bcl-2 expression by transient transfection induces cell death in the human breast carcinoma cell line MDA-MB-468 and human embryonic kidney (HEK) 293 cells. By specifically targeting Bcl-2 either to the ER or the mitochondrial membrane, we find that this induction of apoptosis by Bcl-2 is associated with its localization at the mitochondrial membrane. Transient overexpression of either wild-type or mitochondrially targeted Bcl-2 is sufficient to induce cell death and is not inhibited by the mutation of the caspase-3 cleavage site (D34A). Furthermore, ER-targeted Bcl-2 was found to inhibit cell death induced by Bax overexpression, whereas wild-type and mitochondrially targeted Bcl-2 did not. Thus, ER-targeted Bcl-2 does confer protection against apoptosis by Bax overexpression, suggesting that the ER localization of Bcl-2 may play a significant role in its inhibition of cell death pathways mediated by Bax. HEK 293 cells were cultured in Dulbecco's modified Eagle's medium buffered with 25 mm HEPES that was supplemented with 10% fetal bovine serum and 2 mml-glutamine. African green monkey kidney cells (COS-7) were cultured in Dulbecco's modified Eagle's medium, and human breast epithelial cells MDA-MB-468 were cultured in improved minimum Eagle's medium; both were supplemented with 10% fetal bovine serum, 2 mml-glutamine, and 1% penicillin/streptomycin sulfate. Cell lines were maintained at 37 °C with 7% CO2. MDA-MB-468 cells stably expressing Bcl-2 were generated by transfecting pSFFV-Bcl-2 expression vector (provided by Roger Miesfeld, University of Arizona) with FuGENE6 (Roche Molecular Biochemicals). Transfected cells were subjected to selection in 0.8 mg/ml G418 (Life Technologies, Inc.). All GFP expression vectors described in this work were constructed with the pEmd-C1 expression vector. This expression vector uses the Emerald GFP variant in which GFP was enhanced for higher quantum efficiency (Packard Instrument Co., Meriden, CT). The ER-targeted GFP-Bcl-2 expression vector (pGFP-Bcl-2Cb5) was constructed by the following cloning strategy. The coding region of Bcl-2 was PCR-amplified from the plasmid pB4, which contains the full-length human bcl-2 cDNA (ATCC, Manasass, VA). All PCR reactions were performed using Vent polymerase (New England Biolabs, Beverly, MA). The forward primer used in the PCR amplification contains a BglII restriction site, which ligates in frame with GFP. The resulting PCR fragment was digested with BglII and BbsI, which deletes the C-terminal 658–720 base pairs (corresponding to amino acids 210–239) and generates a BglII overhang at the 5′ end. The nucleotides 297–401 of human cytochrome b5 that encode amino acids 100–134 (ITTIDSSSSWWTNWVIPAISAVAVALMYRLYMAED) were PCR-amplified from an expressed sequence tag obtained from ATCC containing a partial cDNA of human cytochrome b5. This ER-targeting signal sequence (underlined) is identical to that of rat cytochrome b5, with the exception of the hydrophobic membrane anchor domain that has been demonstrated previously not to have a role in determining membrane specificity (23Mitoma J. Ito A. EMBO J. 1992; 11: 4197-4203Crossref PubMed Scopus (103) Google Scholar). The oligonucleotides used to amplify the ER-targeting signal generated a BbsI restriction site at the 5′ end and an EcoRI site at the 3′ end. These PCR fragments were then ligated in frame in the expression vector pEmd-C1, which was digested with BglII and EcoRI. The FLAG epitope-tagged ER-targeted Bcl-2 was constructed similarly using the expression vector pCMV-Tag2B (Stratagene, La Jolla, CA), with the exception that Bcl-2 BgII PCR fragment was ligated into a BamHI site in pCMV-Tag2B. The wild-type GFP-Bcl-2 expression vector was created by PCR amplification of the human Bcl-2-coding region from pB4 with oligonucleotides that generated a BglII at the 5′ end and an EcoRI site at the 3′ end. The PCR fragment was ligated in frame with GFP in the BglII/EcoRI-digested pEmd-C1 and BamHI/EcoRI-digested pCMV-Tag2B expression vectors. To generate the mitochondrially targeted GFP-Bcl-2 expression vector (pGFP-Bcl-2MAOB), Bcl-2 was PCR-amplified, and restriction was digested as described for pEmd-Bcl-2Cb5. Nucleotides 1473–1562 that encode amino acids 492–520 (LLRLIGLTTIFSATALGFLAHKRGLLVRV) of monoamine oxidase B was PCR-amplified from an expressed sequence tag obtained from Genome Systems, Inc., which contains a partial cDNA of human monoamine oxidase B. Oligonucleotides used to amplify this fragment generated BbsI and EcoRI restriction sites that were ligated in frame with Bcl-2 into pEmd-C1 and pCMV-Tag2B. Bcl-2, cytochrome b5, and monoamine oxidase B-targeting sequences were also fused in frame with GFP alone. The C-terminal domain of Bcl-2 used in this expression vector included amino acids 217–239. These constructs were made by PCR amplifying the targeting sequences using oligonucleotides containing a BglII restriction site, which enables in-frame ligation with GFP. To generate the GFP-Bax, the full coding region of Bax was amplified by PCR from pSFFV-Bax, a plasmid containing Bax cDNA, with oligonucleotides, which created a 5′ BglII site and a 3′EcoRI site. The BglII site of the Bax PCR fragment enabled an in-frame ligation with GFP into the pEmd-C1 expression vector digested with BglII and EcoRI. The GFP-Bcl-XL expression vector was generated by PCR amplification of Bcl-XL from pBS-Bcl-XL, which contains the Bcl-XL cDNA, to generate a PCR fragment with 5′ BglII and 3′ EcoRI restriction sites. This fragment was subsequently subcloned into pEmd-C1 as described above. All expression vectors were confirmed by sequence analysis. The D34A mutation was generated in each GFP and FLAG Bcl-2 expression vector using the QuickChangeTM site-directed mutagenesis kit (Stratagene). The mutagenic oligonucleotide 5′-CGAGTGGGATGCGGGAG CTGTGGGCGCCGCG-3′ containing a single A–C base change was used to replace aspartate 34 to alanine. The mutation on each plasmid was verified by sequence analysis. For live cell fluorescence imaging of COS-7 cells transiently transfected with GFP-Bcl-2 expression vectors, cells were plated on 35-mm coverslip dishes (MatTek Corp., Ashland, MA) and were used for transient transfection experiments the following day. Cells were transfected with 2 μg of plasmid DNA using FuGENE6 transfection reagent (Roche Molecular Biochemicals). 16–24 h after transfection, cells were incubated with 100 nm MitoTracker Red (Molecular Probes, Inc., Eugene, OR) for 15 min at 37 °C. Cells were then washed twice with media. For immunofluorescence microscopy of MDA-MB-468 cells stably expressing Bcl-2, cells were grown on 35-mm coverslip dishes and fixed in 95% methanol. Cells were then blocked with 5% goat serum and 0.1% Triton X-100 in phosphate buffered saline (PBS) for 30 min. Bcl-2 was detected by incubating the cells with a dilution of 1:500 anti-human Bcl-2 monoclonal antibody (6C8) (Pharmingen) for 1 h. Cells were washed three times and then incubated with 1:500 dilution of anti-hamster IgG (Pharmingen) for 1 h. Cells were washed three times with PBS and then incubated for 30 min with 1:500 dilution of anti-mouse Alexa488 (Molecular Probes, Inc.). HEK 293 cells were plated on 35-mm coverslip dishes, and 2 days later, they were transiently transfected with GFP-Bcl-2 constructs. 24 h after transfection, cells were incubated with 0.5 μg/ml Hoechst 33342 for 30 min at 37 °C. Cells were subsequently washed with media and observed under a fluorescence microscope. Fluorescence images were taken on a Zeiss Axiovert S100 epifluorescence microscope (Zeiss, Thornwood, NY). GFP, Bcl-2 immunofluorescence, and accompanying MitoTracker Red images were obtained using a 63Xoil/1.4N.A. objective. Hoechst 33342 DNA-staining images were obtained using a 40Xoil/1.3N.A. objective. All filter sets were obtained from Omega Optical (Brattleboro, VT). GFP fluorescence was detected with an XF23 filter cube (excitation = 485 nm, emission = 535 nm). MitoTracker Red fluorescence was detected with XF67 dichroic/emission filter and 575DF25 excitation filter (excitation = 575 nm, emission = 630 nm). Hoechst 33342 fluorescence was detected with the XF67 dichroic/emission filter and 340 excitation filter (excitation = 340 nm, emission = 470 nm). Bcl-2 immunofluorescence images in MDA-MB-468 cells were taken on a COHU 4910 monochrome CCD camera. All other images were taken on a Hamamatsu ORCA C4742–95-cooled CCD camera operating with Simple PCI software (Compix, Inc., Cranberry Township, PA). Image processing was performed using Adobe Photoshop 5.5. HEK 293 cells were plated onto 60-mm culture dishes and were used for transient transfection the following day. Cells were transfected as described previously for fluorescence microscopy. 24 h after transfection, cells were harvested for analysis by flow cytometry. Typically, transfection efficiencies ranged from 30–50%. Cells were washed twice with PBS and subsequently fixed and permeabilized in 95% methanol. Cells were washed twice with PBS after fixation and resuspended in PBS, 20 μg/ml RnaseA, and 1 mm EDTA for 15 min at 37 °C. Cells were then stained with 50 μg/ml propidium iodide (PI) and 0.05% Nonidet P-40. Cells transfected with GFP-Bcl-2 or FLAG-Bcl-2 expression vectors were analyzed for GFP or AlexaFluor488 and PI fluorescence on a Coulter XL flow cytometer with a 15-milliwatt air-cooled argon laser (excitation = 488 nm). GFP fluorescence was measured using a 525-nm band pass filter, and PI fluorescence was measured using a 620-nm band pass filter. For comparison of Bcl-2 expression levels in transient and stably transfected MDA-MB-468 cells, cell were grown in 100-mm Petri dishes. The parental cells were transiently transfected with 5.6 μg of pGFP-Bcl-2 using FuGENE6 transfection reagent. After 24 h, both transiently transfected and stably transfected cells were harvested and fixed in 95% methanol. Cells were blocked with 5% goat serum and 0.1% Triton X-100 in PBS. Cells were bound with 1:500 dilution of anti-human Bcl-2 antibody (6C8) followed by 1:500 dilution of mouse anti-hamster IgG. After washing three times with PBS, cells were bound with 1:500 dilution of anti-mouse Alexa488 and then stained with 5 μg/ml propidium iodide. In the Bax/Bcl-2 co-transfection experiments, HEK 293 cells were co-transfected with 0.5 μg of pGFP-Bax and 1.5 μg of pFLAG-Bcl-2 expression vectors. After 24 h, cells were fixed and permeabilized in 95% methanol. Cells that were positive for GFP fluorescence were gated for DNA content analysis. Data analysis of results obtained by fluorescence-activated cell sorter was performed using WinMDI 2.7 software program (written by Joseph Trotter, Scripps Research Institute, La Jolla, CA). Histograms of untransfected and transfected cells were plotted by gating cells that were positive or negative for GFP and/or AlexaFluor488 fluorescence, respectively. HEK 293 cells were transfected as described previously with either GFP-Bcl-2 or FLAG-Bcl-2 expression vectors and harvested after 24 h. For GFP-Bax/FLAG-Bcl-2 co-transfection experiments, cells were transfected with 0.5 μg of pGFP-Bax and 1.5 μg of pFL-Bcl-2 plasmid DNA. Cells were lysed in radioimmune precipitation buffer, and 50 μg of protein of each lysate was separated by SDS-polyacrylamide gel electrophoresis (14% acrylamide). The protein was then electrophoretically blotted to a polyvinylidene difluoride membrane (Millipore) and incubated with a 1:1000 dilution of anti-human Bcl-2 monoclonal antibody (6C8) followed by 1:2000 dilution of an anti-hamster horseradish peroxidase conjugate (Pharmingen). Bax was detected by an anti-Bax polyclonal antibody (N-20) (Santa Cruz Biotechnologies) diluted 1:1000 followed by an anti-rabbit horseradish peroxidase conjugate (Amersham Pharmacia Biotech). Detection of Bcl-2 or Bax on the membrane was performed using ECL (Amersham Pharmacia Biotech). To study the localization of Bcl-2 in cells that stably overexpress Bcl-2, we transfected a human breast epithelial cell line MDA-MB-468 with a Bcl-2 expression vector and selected for stable expression by growing the cells in G418. These cells do not endogenously express detectable levels of Bcl-2 and previously have been shown to be protected against apoptosis when Bcl-2 is stably transfected (24Wolf C.M. Eastman A. Biochem. Biophys. Res. Commun. 1999; 254: 821-827Crossref PubMed Scopus (18) Google Scholar). The subcellular localization of Bcl-2 in these cells was determined by immunofluorescence microscopy (Fig. 1). To visualize mitochondria, cells were loaded with MitoTracker Red, a fluorescent dye that binds specifically to mitochondrial membranes. The pattern of Bcl-2 was found to co-localize with mitochondria as well as with a more extensive reticular network resembling the ER (Fig. 1,A–C). The localization of ER membranes was determined by immunofluorescent detection of ER resident proteins using an anti-KDEL antibody (Fig. 1, D–F). Bcl-2 immunolocalization studies were also performed on control cells transfected with empty vector; however, there was no detectable immunofluorescence signal above background, indicating that there is little, if any, endogenous Bcl-2 in the parental cell line. The pattern of Bcl-2 localization that we observe in the stably transfected MDA-MB-468 cells is thus consistent with previous reports that Bcl-2 is localized to both mitochondrial and ER membranes (10Akao Y. Otsuki Y. Kataoka S. Ito Y. Tsujimoto Y. Cancer Res. 1994; 54: 2468-2471PubMed Google Scholar, 11Krajewski S. Tanaka S. Takayama S. Schibler M. Fenton W. Reed J. Cancer Res. 1993; 53: 4701-4714PubMed Google Scholar). It was observed that transfection of Bcl-2 in MDA-MB-468 cells initially resulted in a large number of dead cells relative to those that were transfected with a control vector. This was quantified by transfecting these cells with a GFP-Bcl-2 fusion protein and analyzing GFP fluorescent cells by flow cytometry (Fig.2). After 24 h, cells were harvested, fixed, and stained with PI, a fluorescent dye that binds to DNA. A hallmark of apoptotic cell death is the enzymatic cleavage of chromosomal DNA by endonucleases activated by the caspase cascade. This degradation of chromosomal DNA results in a decrease in total DNA content to a level that is lower than the DNA content of a cell in G1 phase (25Sherwood S.W. Schimke R.T. Methods Cell Biol. 1995; 46: 77-97Crossref PubMed Scopus (88) Google Scholar). Consequently, the DNA content of apoptotic cells appears in a DNA content profile as a peak below the DNA content of cells in G1 phase. By gating GFP-positive and GFP-negative cells, we determined that 22.9% of cells expressing GFP-Bcl-2 were apoptotic compared with 7.7% apoptotic cells in the population that did not express GFP-Bcl-2 (Fig. 2 A). Because MDA-MB-468 cells demonstrate low transfection efficiency, we chose to test whether this effect is observed in other cell types. Thus, we repeated this transient transfection assay in HEK 293 cells and compared the effect of expression of several Bcl-2 family members including Bax, a known pro-apoptotic protein, and another anti-apoptotic protein Bcl-XL. Bax, Bcl-2, and Bcl-XL were expressed as GFP fusion proteins, and their pattern of expression was observed by fluorescence microscopy in MCF-7 cells, which are well suited for imaging (Fig. 2 B). GFP-Bax was found to be diffusely localized throughout the cytoplasm, and in some cells, it exhibited a punctate pattern, which probably reflects its translocation to the mitochondrial membrane as described previously (12Wolter K. Hsu Y. Smith C. Nechushtan A. Xi X. Youle R. J. Cell Biol. 1997; 139: 1281-1292Crossref PubMed Scopus (1577) Google Scholar). GFP-Bcl-2 was found to be localized both in mitochondria, which surround the nucleus, as well as the ER, which resembles an extensive network throughout the cell. Unlike Bcl-2, GFP-Bcl-XL was found to be diffused throughout the cell but was also found in the mitochondrial membranes that surround the nucleus. These expression vectors were subsequently transiently transfected in HEK 293 cells. After 24 h, the cells were harvested and analyzed by flow cytometry. The results indicate that the expression of Bax and Bcl-2 induces apoptosis, whereas Bcl-XL expression did not induce apoptosis significantly above levels observed for GFP transfection alone (Fig. 2 C). These results are also consistent with previous findings in which infection of glioblastoma cells with an adenoviral Bcl-2 expression vector induced apoptosis, whereas infection with a Bcl-XL expression vector did not (26Shinoura N. Yoshida Y. Nishimura M. Muramatsu Y. Asai A. Kirino T. Hamada H. Cancer Res. 1999; 59: 4119-4128PubMed Google Scholar). It should also be noted that we obtained similar results in transient transfections of MCF-7 cells, although cell death was found to occur more slowly possibly because of the deficiency of caspase-3 in MCF-7 cells. To test whether Bcl-2 can block apoptosis in MDA-MB-468 cells, cells stably overexpressing Bcl-2 and cells containing a control vector were treated with 1 μm staurosporine or 100 nm thapsigargin (Fig. 3). After 48 h, control cells treated with either staurosporine or thapsigargin were found to exhibit typical morphological features of apoptosis, such as lifting from the surface, condensed chromatin, and fragmentation into apoptotic bodies. However, apoptosis was significantly inhibited in Bcl-2 expressing cells treated with either staurosporine or thapsigargin. Thus, as expected, Bcl-2 expressed stably results in the protection of MDA-MB-468 cells from programmed cell death. The discrepancy between the cytotoxic effect of transient Bcl-2 expression and its protective effect in stably expressing cells may be attributed to a difference in expression levels. This view was also shared by others who have previously reported the toxic effect of transient Bcl-2 expression (26Shinoura N. Yoshida Y. Nishimura M. Muramatsu Y. Asai A. Kirino T. Hamada H. Cancer Res. 1999; 59: 4119-4128PubMed Google Scholar, 27Uhlmann E.J. Subramanian T. Vater C.A. Lutz R. Chinnadurai G. J. Biol. Chem. 1998; 273: 17926-17932Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Thus, we examined whether the toxicity of transient transfection of Bcl-2 required expression at much higher levels than that observed in stably transfected cells. To compare Bcl-2 expression levels between transiently and stably transfected MDA-MB-468 cells, Bcl-2 protein was detected on a single cell basis by using an anti-human Bcl-2 monoclonal antibody followed by a secondary antibody-Alexa488 conjugate and measuring fluorescence intensity by flow cytometry (Fig.4 A). It has been previously demonstrated that antibody detection of Bcl-2 protein levels by flow cytometry correlates well with results obtained by Western blot (28Dragowska W.H. Lopes de Menezes D.E. Sartor J. Mayer L.D. Cytometry. 2000; 40: 346-352Crossref PubMed Scopus (20) Google Scholar). Analysis of Bcl-2 expression level by this method revealed that 80% of transiently transfected cells expressed Bcl-2 within the same range of Bcl-2 levels detected in stably overexpressing cells (Fig.4 B). Expression levels attained in transiently transfected cells appear to vary over a wider range compared with stably transfected cells, however, only 20% of transiently transfected cells attain Bcl-2 expression levels higher than what is observed in stably transfected cells. When transient Bcl-2-expressing cells that express the same levels as stably expressing cells were analyzed for cell death (Fig. 4 C), we found that ∼20% of these cells were apoptotic, indicating that most of the apoptotic cells expressed the same levels of Bcl-2 as stably expressing cells. The cell death-inducing effect of many pro-apoptotic Bcl-2 family proteins has been found to be a result of their association with the mitochondrial membrane. Bax, a cytosolic protein, translocates to the mitochondrial membrane when the cell receives an apoptotic signal (12Wolter K. Hsu Y. Smith C. Nechushtan A. Xi X. Youle" @default.
• W2079273110 created "2016-06-24" @default.
• W2079273110 creator A5030489938 @default.
• W2079273110 creator A5033663842 @default.
• W2079273110 creator A5067803417 @default.
• W2079273110 creator A5080092755 @default.
• W2079273110 date "2001-11-01" @default.
• W2079273110 modified "2023-10-18" @default.
• W2079273110 title "Transient Expression of Wild-type or Mitochondrially Targeted Bcl-2 Induces Apoptosis, whereas Transient Expression of Endoplasmic Reticulum-targeted Bcl-2 Is Protective against Bax-induced Cell Death" @default.
• W2079273110 cites W1481679077 @default.
• W2079273110 cites W1530373462 @default.
• W2079273110 cites W161538641 @default.
• W2079273110 cites W1731365463 @default.
• W2079273110 cites W1913661233 @default.
• W2079273110 cites W1915733116 @default.
• W2079273110 cites W1965217295 @default.
• W2079273110 cites W1967354499 @default.
• W2079273110 cites W1975356481 @default.
• W2079273110 cites W1988355439 @default.
• W2079273110 cites W1988800817 @default.
• W2079273110 cites W1991913775 @default.
• W2079273110 cites W1999197294 @default.
• W2079273110 cites W2003301112 @default.
• W2079273110 cites W2009183764 @default.
• W2079273110 cites W2012173926 @default.
• W2079273110 cites W2012974735 @default.
• W2079273110 cites W2013065884 @default.
• W2079273110 cites W2016575124 @default.
• W2079273110 cites W2028526641 @default.
• W2079273110 cites W2032049833 @default.
• W2079273110 cites W2051258629 @default.
• W2079273110 cites W2052853635 @default.
• W2079273110 cites W2062853648 @default.
• W2079273110 cites W2063176993 @default.
• W2079273110 cites W2067805812 @default.
• W2079273110 cites W2073028327 @default.
• W2079273110 cites W2077987011 @default.
• W2079273110 cites W2092759765 @default.
• W2079273110 cites W2098160574 @default.
• W2079273110 cites W2113999599 @default.
• W2079273110 cites W2114001234 @default.
• W2079273110 cites W2130361718 @default.
• W2079273110 cites W2132411546 @default.
• W2079273110 cites W2134872883 @default.
• W2079273110 cites W2134979684 @default.
• W2079273110 cites W2140967934 @default.
• W2079273110 cites W2169404281 @default.
• W2079273110 cites W2188651990 @default.
• W2079273110 cites W2315241706 @default.
• W2079273110 cites W2318252390 @default.
• W2079273110 cites W53848852 @default.
• W2079273110 cites W80462140 @default.
• W2079273110 doi "https://doi.org/10.1074/jbc.m101958200" @default.
• W2079273110 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11546793" @default.
• W2079273110 hasPublicationYear "2001" @default.
• W2079273110 type Work @default.
• W2079273110 sameAs 2079273110 @default.
• W2079273110 citedByCount "166" @default.
• W2079273110 countsByYear W20792731102012 @default.
• W2079273110 countsByYear W20792731102013 @default.
• W2079273110 countsByYear W20792731102014 @default.
• W2079273110 countsByYear W20792731102015 @default.
• W2079273110 countsByYear W20792731102016 @default.
• W2079273110 countsByYear W20792731102017 @default.
• W2079273110 countsByYear W20792731102018 @default.
• W2079273110 countsByYear W20792731102019 @default.
• W2079273110 countsByYear W20792731102020 @default.
• W2079273110 countsByYear W20792731102021 @default.
• W2079273110 countsByYear W20792731102022 @default.
• W2079273110 countsByYear W20792731102023 @default.
• W2079273110 crossrefType "journal-article" @default.
• W2079273110 hasAuthorship W2079273110A5030489938 @default.
• W2079273110 hasAuthorship W2079273110A5033663842 @default.
• W2079273110 hasAuthorship W2079273110A5067803417 @default.
• W2079273110 hasAuthorship W2079273110A5080092755 @default.
• W2079273110 hasBestOaLocation W20792731101 @default.
• W2079273110 hasConcept C104317684 @default.
• W2079273110 hasConcept C111919701 @default.
• W2079273110 hasConcept C1491633281 @default.
• W2079273110 hasConcept C158617107 @default.
• W2079273110 hasConcept C185592680 @default.
• W2079273110 hasConcept C190283241 @default.
• W2079273110 hasConcept C2780799671 @default.
• W2079273110 hasConcept C2909544034 @default.
• W2079273110 hasConcept C31573885 @default.
• W2079273110 hasConcept C41008148 @default.
• W2079273110 hasConcept C55493867 @default.
• W2079273110 hasConcept C6856738 @default.
• W2079273110 hasConcept C86803240 @default.
• W2079273110 hasConcept C95444343 @default.
• W2079273110 hasConceptScore W2079273110C104317684 @default.
• W2079273110 hasConceptScore W2079273110C111919701 @default.
• W2079273110 hasConceptScore W2079273110C1491633281 @default.
• W2079273110 hasConceptScore W2079273110C158617107 @default.
• W2079273110 hasConceptScore W2079273110C185592680 @default.
• W2079273110 hasConceptScore W2079273110C190283241 @default.
• W2079273110 hasConceptScore W2079273110C2780799671 @default.
• W2079273110 hasConceptScore W2079273110C2909544034 @default.

• next