FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1966726105> ?p ?o ?g. }

• W1966726105 endingPage "2029" @default.
• W1966726105 startingPage "2021" @default.
• W1966726105 abstract "Photodynamic therapy using the photosensitizer Pc 4 and red light photochemically destroys the antiapoptotic protein Bcl-2 and induces apoptosis. To characterize the requirements for photodamage, we transiently transfected epitope-tagged Bcl-2 deletion mutants into DU-145 cells. Using confocal microscopy and Western blots, wild-type Bcl-2 and mutants with deletions near the N terminus were found in mitochondria, endoplasmic reticulum, and nuclear membranes and were photodamaged. A mutant missing the C terminus, including the transmembrane domain, spread diffusely in cells and was not photodamaged. Bcl-2 missing α-helices 5/6 was also not photodamaged. Bcl-2 missing only one of those α-helices, with or without substitutions of the singlet oxygen-targeted amino acids, behaved like wild-type Bcl-2 with respect to localization and photodamage. Using green fluorescent protein (GFP)-tagged Bcl-2 or mutants in live cells, no change in either the localization or the intensity of GFP fluorescence was observed in response to Pc 4 photodynamic therapy. Western blot analysis of either GFP- or Xpress-tagged Bcl-2 revealed that the photodynamic therapy-induced disappearance of the Bcl-2 band was accompanied by the appearance of bands indicative of heavily cross-linked Bcl-2 protein. Therefore, the α5/α6 region of Bcl-2 is required for photodamage and cross-linking, and domain-dependent photodamage to Bcl-2 offers a unique mechanism for activation of apoptosis. Photodynamic therapy using the photosensitizer Pc 4 and red light photochemically destroys the antiapoptotic protein Bcl-2 and induces apoptosis. To characterize the requirements for photodamage, we transiently transfected epitope-tagged Bcl-2 deletion mutants into DU-145 cells. Using confocal microscopy and Western blots, wild-type Bcl-2 and mutants with deletions near the N terminus were found in mitochondria, endoplasmic reticulum, and nuclear membranes and were photodamaged. A mutant missing the C terminus, including the transmembrane domain, spread diffusely in cells and was not photodamaged. Bcl-2 missing α-helices 5/6 was also not photodamaged. Bcl-2 missing only one of those α-helices, with or without substitutions of the singlet oxygen-targeted amino acids, behaved like wild-type Bcl-2 with respect to localization and photodamage. Using green fluorescent protein (GFP)-tagged Bcl-2 or mutants in live cells, no change in either the localization or the intensity of GFP fluorescence was observed in response to Pc 4 photodynamic therapy. Western blot analysis of either GFP- or Xpress-tagged Bcl-2 revealed that the photodynamic therapy-induced disappearance of the Bcl-2 band was accompanied by the appearance of bands indicative of heavily cross-linked Bcl-2 protein. Therefore, the α5/α6 region of Bcl-2 is required for photodamage and cross-linking, and domain-dependent photodamage to Bcl-2 offers a unique mechanism for activation of apoptosis. photodynamic therapy phthalocyanine 4 PDT with Pc 4 Bcl-2 homology endoplasmic reticulum phosphate-buffered saline transmembrane Photodynamic therapy (PDT)1 is a novel treatment for cancer and certain noncancerous conditions that are generally characterized by overgrowth of unwanted or abnormal cells (1Dougherty T.J. Gomer C.J. Henderson B.W. Jori G. Kessel D. Korbelik M. Moan J. Peng Q. J. Natl. Cancer Inst. 1998; 90: 889-905Crossref PubMed Scopus (4719) Google Scholar, 2Oleinick N.L. Morris R.L. Belichenko I. Photochem. Photobiol. Sci. 2002; 1: 1-22Crossref PubMed Scopus (1092) Google Scholar). The procedure requires exposure of cells or tissues to a photosensitizing drug followed by irradiation with visible light of the appropriate wavelength, usually in the red or near infrared region and compatible with the absorption spectrum of the drug. Since the first modern clinical trial of PDT by Dougherty et al. (3Dougherty T.J. Cancer Res. 1978; 36: 2628-2635Google Scholar) was reported in 1978, PDT with the photosensitizer Photofrin® has been applied to many solid tumors and is approved by the United States Food and Drug Administration for the treatment of advanced esophageal, early lung, and late lung cancers. In order to enhance the efficacy of PDT and extend its applications, a variety of second generation photosensitizers, such as the silicon phthalocyanine Pc 4, are now being assessed for their efficacy in cancer therapy, and it is important to elucidate their mechanisms of action in PDT. The photosensitizers for PDT are primarily porphyrins or porphyrin-related macrocycles, such as phthalocyanines, benzoporphyrins, purpurins, and pyropheophorbides (4Berg K. Moan J. Int. J. Cancer. 1994; 59: 814-822Crossref PubMed Scopus (140) Google Scholar, 5Kessel D. Luo Y. J. Photochem. Photobiol. B Biol. 1998; 42: 89-95Crossref PubMed Scopus (297) Google Scholar). Because of their hydrophobic aromatic ring structures, they localize to one or more cellular membranes. We have reported that Pc 4 preferentially binds to the mitochondrial membrane, the endoplasmic reticulum, and Golgi complexes in cancer cells (6Trivedi N.S. Wang H.W. Nieminen A.L. Oleinick N.L. Izatt J.A. Photochem. Photobiol. 2000; 71: 634-639Crossref PubMed Scopus (97) Google Scholar, 7Lam M. Oleinick N.L. Nieminen A.L. J. Biol. Chem. 2001; 276: 47379-47386Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). Upon photoactivation, mitochondrial reactive oxygen species are produced, and these are critical in initiating mitochondrial inner membrane permeabilization, which leads to mitochondrial swelling, cytochrome c release to the cytosol, and apoptotic death (7Lam M. Oleinick N.L. Nieminen A.L. J. Biol. Chem. 2001; 276: 47379-47386Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). PDT produces singlet oxygen and other reactive oxygen species in the membranes and causes photooxidative damage to proteins and lipids that reside within a few nm of the photosensitizer binding sites (8Moan J. Berg K. Photochem. Photobiol. 1992; 55: 931-948Crossref PubMed Scopus (397) Google Scholar, 9He X.Y. Sikes R.A. Thomsen S. Chung L.W. Jacques S.L. Photochem. Photobiol. 1994; 59: 468-473Crossref PubMed Scopus (174) Google Scholar). One important target of Pc 4-PDT (10Xue L.Y. Chiu S.M. Oleinick N.L. Oncogene. 2001; 20: 3420-3427Crossref PubMed Scopus (204) Google Scholar) as well as PDT with certain other photosensitizers (11Kim H.R. Luo Y. Kessel D. Cancer Res. 1999; 59: 3429-3432PubMed Google Scholar) is the antiapoptotic protein Bcl-2. The Bcl-2 family consists of proteins that either promote or inhibit apoptosis. Proapoptotic members include Bax and Bak, whereas antiapoptotic members include Bcl-2 and Bcl-xL. They share 1–4 α-helical Bcl-2 homology (BH) domains, whose subtle differences in primary sequence and spatial arrangements may determine the pro- and antiapoptotic functions. The antiapoptotic proteins, like Bcl-2, possess four BH domains (BH1–BH4) (12Tsujimoto Y. Shimizu S. FEBS Lett. 2000; 466: 6-10Crossref PubMed Scopus (637) Google Scholar). The C-terminal regions of many members of the Bcl-2 family consist of a stretch of hydrophobic amino acids that serves to anchor the proteins to intracellular membranes, specifically the outer mitochondrial membrane, the endoplasmic reticulum, and the nuclear envelope (13Nguyen 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). Bcl-2 contains a transmembrane domain of 19 amino acids at the C terminus, and it has been reported that deletion of the C-terminal 22 amino acids of Bcl-2 abrogates cellular membrane attachment (14Borner C. Martinou I. Mattmann C. Irmler M. Schaerer E. Martinou J.C. Tschopp J. J. Cell Biol. 1994; 126: 1059-1068Crossref PubMed Scopus (175) Google Scholar, 15Choi W.S. Yoon S.Y. Chang I.I. Choi E.J. Rhim H. Jin B.K., Oh, T.H. Krajewski S. Reed J.C. Oh Y.J. J. Neurochem. 2000; 74: 1621-1626Crossref PubMed Scopus (25) Google Scholar). Structural analysis of Bcl-xL reveals its resemblance to the bacterial pore-forming toxins, colicin and diphtheria toxin, especially in the α-helical region that mediates membrane insertion of the toxins (16Muchmore S.W. Sattler M. Liang H. Meadows R.P. Harlan J.E. Yoon H.S. Nettesheim D. Chang B.S. Thompson C.B. Wong S.L., Ng, S.C. Fesik S.W. Nature. 1996; 381: 335-341Crossref PubMed Scopus (1289) Google Scholar, 17Kroemer G. Reed J.C. Nat. Med. 2000; 6: 513-519Crossref PubMed Scopus (2784) Google Scholar). This region corresponds to the BH1 domain and part of the BH2 domain of Bcl-2 and Bcl-xL, and is called the α5/α6 region. This region is thought to be inserted into the membrane and form ion channels (18Schendel S.L. Xie Z. Montal M.O. Matsuyama S. Montal M. Reed J.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5113-5118Crossref PubMed Scopus (548) Google Scholar, 19Matsuyama S. Schendel S.L. Xie Z. Reed J.C. J. Biol. Chem. 1998; 273: 30995-31001Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 20Minn A.J. Kettlun C.S. Liang H. Kelekar A. Heiden M.G.V. Chang B.S. Fesik S.W. Fill M. Thompson C.B. EMBO J. 1999; 18: 632-643Crossref PubMed Scopus (184) Google Scholar). It has been reported that mutations within the α5/α6 region of Bcl-2 and Bcl-xL abrogate the antiapoptotic activity and block the heterodimerization with other members of the Bcl-2 family, such as Bax or Bak, which are death-promoting proteins. The region between the BH1 and BH2 domains forms part of a hydrophobic cleft and may be the site of interaction with Bax or Bak (16Muchmore S.W. Sattler M. Liang H. Meadows R.P. Harlan J.E. Yoon H.S. Nettesheim D. Chang B.S. Thompson C.B. Wong S.L., Ng, S.C. Fesik S.W. Nature. 1996; 381: 335-341Crossref PubMed Scopus (1289) Google Scholar, 17Kroemer G. Reed J.C. Nat. Med. 2000; 6: 513-519Crossref PubMed Scopus (2784) Google Scholar). The role of Bcl-2 in the apoptotic response caused by PDT has remained controversial. He et al. (21He J. Agarwal M.E. Larkin H.E. Friedman L.R. Oleinick N.L. Photochem. Photobiol. 1996; 64: 845-852Crossref PubMed Scopus (113) Google Scholar) first reported that overexpressing human Bcl-2 made Chinese hamster ovary cells more resistant to apoptosis and to loss of clonogenicity upon exposure to Pc 4-PDT. In contrast, Kim et al. (11Kim H.R. Luo Y. Kessel D. Cancer Res. 1999; 59: 3429-3432PubMed Google Scholar) found that overexpression of Bcl-2 in MCF-10A cells caused the up-regulation of Bax and enhanced cell killing and apoptosis by PDT with AlPc. Recently, Srivastava et al. reported that antisense-Bcl-2 sensitized A431 cells to Pc 4-PDT (22Srivastava M. Ahmad N. Gupta S. Mukhtar H. J. Biol. Chem. 2001; 276: 15481-15488Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). As in the study by Kim et al.(11Kim H.R. Luo Y. Kessel D. Cancer Res. 1999; 59: 3429-3432PubMed Google Scholar), the cells overexpressing Bcl-2 were more sensitive to induction of apoptosis than were the parental cells, because of up-regulation of Bax; however, no studies were done to determine whether those cells were sensitized to overall cell death. We have reported that Pc 4-PDT photodamages Bcl-2, as detected by Western blot analysis as the immediate loss of the native 26-kDa protein (10Xue L.Y. Chiu S.M. Oleinick N.L. Oncogene. 2001; 20: 3420-3427Crossref PubMed Scopus (204) Google Scholar). Bcl-2 photodamage was selective, in that several other mitochondrial proteins were not affected. Kessel and Castelli (23Kessel D. Castelli M. Photochem. Photobiol. 2001; 74: 318-322Crossref PubMed Scopus (117) Google Scholar) found that PDT with three different photosensitizers destroyed Bcl-2 but not Bax. In contrast, a recent report from Antieghem et al. (24Vantieghem A., Xu, Y. Assefa Z. Piette J. Vandenheede J.R. Merlevede W. de Witte P.A.M. Agostinis P. J. Biol. Chem. 2002; 277: 37718-37731Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar) showed that PDT with the nonporphyrin photosensitizer, hypericin, does not cause Bcl-2 destruction but produces a G2/M delay, during which Bcl-2 becomes transiently phosphorylated. Although it is now clear that Bcl-2 is one target of PDT with Pc 4 and some other photosensitizers, the mechanism of the photodestruction of Bcl-2 has not been defined. In order to elucidate the structural features that determine photosensitivity, we constructed Bcl-2 mutants by site-directed mutagenesis and examined the association between their subcellular localization and their sensitivity to photodestruction by Pc 4-PDT. Furthermore, we compared the responses of wild-type and mutant Bcl-2 to those of the proapoptotic proteins, Bak and Bax. Our photobiological analysis of the Bcl-2 family members suggests a relationship between membrane localization and photosensitivity. Human prostate cancer DU-145 cells were grown in Dulbecco's modified Eagle's medium containing 5% fetal bovine serum. Human breast cancer MCF-7 cells transfected with human procaspase-3 cDNA (MCF-7c3 cells) were cultured in RPMI 1640 medium containing 10% fetal bovine serum (25Xue L.Y. Chiu S.M. Oleinick N.L. Exp. Cell Res. 2001; 263: 145-155Crossref PubMed Scopus (121) Google Scholar). Human breast epithelial MDA-MB-468 cells were cultured in Dulbecco's modified Eagle's medium. All cultures were maintained in a humidified atmosphere at 37 °C with 5% CO2. An expression vector housing full-length human Bcl-2 cDNA inserted at the EcoRI site, pUC19-Bcl-2, was kindly provided by Dr. C. W. Distelhorst (Case Western Reserve University) (26Wang N.S. Unkilla M.T. Reineks E.Z. Distelhorst C.W. J. Biol. Chem. 2001; 276: 44117-44128Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Bcl-2 cDNA was digested withEcoRI and cloned into the mammalian expression vector pcDNA4/HisMax (Invitrogen), which encodes an N-terminal peptide containing a polyhistidine metal-binding tag and the Xpress epitope. It was also cloned into the N-terminal green fluorescent protein (GFP) mammalian expression vector pEGFP-C3 (Clontech, Palo Alto, CA). To generate the Bcl-2 mutants, mutations were amplified from pUC19-Bcl-2 using the QuikChangeTM site-directed mutagenesis kit (Stratagene, La Jolla, CA) (18Schendel S.L. Xie Z. Montal M.O. Matsuyama S. Montal M. Reed J.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5113-5118Crossref PubMed Scopus (548) Google Scholar, 19Matsuyama S. Schendel S.L. Xie Z. Reed J.C. J. Biol. Chem. 1998; 273: 30995-31001Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The following mutagenic primers were used: for mutant a, Bcl-2 (Δ33–54) (5′-ATCTTCTCCTCCCAGCCCGGGCAGACCCCGGCTGCCCCCGGC-3′ (forward) and 5′-GCCGGGGGCAGCCGGGGTCTGCCCGGGCTGGGAGGAGAAGAT-3′ (reverse)); for mutant b, Bcl-2 (Δ37–63) (5′-GTGGGATGCGGGAGATGTGGGCGACCCGGTCGCCAGGACC (forward) and 5′-GGTCCTGGCGACCGGGTCGCCCACATCTCCCGCATCCCAC-3′ (reverse)); for mutant c, Bcl-2 (Δ10–125) (5′-CTGGGAGAACGGGGTACGACGCGCGGGGACGCTTTGCCAC-3′ (forward) and 5′-GTGGCAAAGCGTCCCCGCGCGTCGTACCCCGTTCTCCCAG-3′ (reverse)); for mutant d, Bcl-2 (Δ153–179) (5′-GGATTGTGGCCTTCTTTGAGTACCTGAACCGGCACCTGCAC-3′ (forward) and 5′-GTGCAGGTGCCGGTTCAGGTACTCAAAGAAGGCCACAATCC-3′ (reverse)); for mutant e, Bcl-2 (Δ153–168) (5′-GGATTGTGGCCTTCTTTGAGCTGGTGGACAACATCGCCC-3′ (forward) and 5′-GGGCGATGTTGTCCACCAGCTCAAAGAAGGCCACAATCC-3′ (reverse)); for mutant f, Bcl-2 (Δ168–179) (5′-GCGTCAACCGGGAGATGTCGTACCTGAACCGGCACCTGC-3′ (forward) and 5′-GCAGGTGCCGGTTCAGGTACGACATCTCCCGGTTGACGC-3′ (reverse)); for mutant g, Bcl-2 (Δ210–239) (5′-GCCCCAGCATGCGGCCTCTGTGAAGTCAACATGCCTGCCC-3′ (forward) and 5′-GGGCAGGCATGTTGACTTCACAGAGGCCGCATGCTGGGGC-3′ (reverse)); for mutant h, Bcl-2 (Δ218–233) (5′-ATTTCTCCTGGCTGTCTCTGGCCTATCTGAGCCACAAGTGA-3′ (forward) and 5′-TCACTTGTGGCTCAGATAGGCCAGAGACAGCCAGGAGAAAT-3′ (reverse)). Additional Bcl-2 mutants were constructed to mutate specific amino acids. In order to generate mutant i, pUC19-Bcl-2 (Δ168–179) was amplified, and to generate mutant j, pUC19-Bcl-2 (Δ153–168) was amplified as a template for the QuikChangeTM site-directed mutagenesis kit. The following mutagenic primers were used: for mutant i, Bcl-2 (Δ168–179, F153L,M157I,C158V) (5′-CTTCTTTGAGCCGGTGGGGTCATCGTTGTGGAGAGCGTC-3′ (forward) and 5′-GACGCTCTCCACAACGATGACCCCACCGAGCTCAAAGAAG-3′ (reverse)); for mutant j, Bcl-2 (Δ153–168, W176L,M177L) (5′-GTGGACAACATCGCCCTGTTACTGACTGAGTACCTGAACC-3′) (forward) and 5′-GGTTCAGGTACTCAGTCAGTAACAGGGCGATGTTGTCCAC-3′ (reverse)). The reactions were carried out by 24 cycles of 30 s at 95 °C, 1 min at 55 °C, 12 min at 68 °C using Pfu polymerase (Stratagene, La Jolla, CA). The amplified products were digested withDpnI (Stratagene, La Jolla, CA), and the products were transfected into XL1-Blue supercompetent cells (Stratagene, La Jolla, CA). The amplified regions were confirmed by DNA sequence analysis and digested with EcoRI and then cloned to the mammalian expression vectors, pCDNA4/HisMax and pEGFP-C3. The phthalocyanine photosensitizer Pc 4, HOSiPcOSi(CH3)2(CH2)3N(CH3)2, was provided by Dr. M. E. Kenney (Department of Chemistry, Case Western Reserve University). Pc 4 was dissolved in dimethyl formamide to 0.5 mm. Cells were loaded with Pc 4 by the addition of an aliquot of the stock solution to the culture medium 16 h before irradiation. For all experiments, the light source was an EFOS light-emitting diode array (EFOS; Mississauga, Canada) delivering red light (λmax 670–675 nm; bandwidth at half-maximum 24 nm; fluence rate at the level of the cell monolayer, 6–7 milliwatts/cm2). The dose of PDT used in most of these experiments (200 nm Pc 4 plus 200 mJ/cm2) was demonstrated to produce 98 ± 4% killing of MCF-7c3 cells and 92 ± 2% killing of DU-145 cells, as determined by clonogenic assay. Cells were harvested by centrifugation and washed twice with ice-cold phosphate-buffered saline (PBS). The cell pellets were incubated in a lysis buffer (50 mm Tris-HCl, pH 7.5, 120 mm NaCl, 1% Triton X-100, 0.2% SDS, 0.5% deoxycholate, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride, 100 mm NaF) on ice for 30 min and then sonicated. The protein content of the whole cell lysates was measured using the BCA protein assay reagent (Pierce). An aliquot (20 μg) of the whole cell lysate was separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were incubated with one of the following antibodies: mouse monoclonal anti-Xpress antibody (Invitrogen), rabbit polyclonal anti-human Bax antibody, rabbit polyclonal anti-human Bak antibody, mouse monoclonal anti-GFP antibody, and mouse monoclonal anti-actin antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at appropriate concentrations for 1 h. After rinsing with PBS containing 0.1% (v/v) Triton X-100, the membranes were incubated with anti-mouse or anti-rabbit immunoglobulin G conjugated to horseradish peroxidase for 1 h at room temperature. The membranes were washed and developed with Western blotting enhanced chemiluminescence detection reagents (Amersham Biosciences). Independent experiments were repeated at least three times. DU-145 cells were grown on glass coverslips and then were transiently transfected with an expression vector (pcDNA/HisMax) encoding wild-type Bcl-2 or Bcl-2 mutants. Eight h after the transfection, Pc 4 was added to the cultures, which were further incubated for 16 h and then incubated with 100 nm MitoTracker Green (Molecular Probes, Inc., Eugene, OR) for 45 min at 37 °C. The coverslips were washed in PBS and fixed in 1% formaldehyde for 30 min. After rinsing twice with PBS, the fixed cells were incubated in IFA buffer (PBS containing 1% bovine serum albumin, 0.1% Tween 20) for 10 min and then IFA containing mouse anti-XpressTM antibody (1:300 dilution; Invitrogen, CA) for 1 h at room temperature. After rinsing with IFA buffer to remove excess unbound antibody, the coverslips were incubated for at least 1 h at room temperature in IFA containing the second antibody, which was anti-mouse IgG conjugated to Texas Red (1:300 dilution; Vector Laboratories, Burlingame, CA). In all experiments, there was no detectable immunofluorescence signal above background in the absence of anti-XpressTM antibody. To investigate the subcellular sites of Pc 4 localization, DU-145 cells were plated on 35-mm coverslip dishes (MatTek Corp., Ashland, MA) and exposed to 200 nm Pc 4 at 37 °C for 16 h. To assess specific localization to mitochondria, cells were also incubated with 100 nm MitoTracker Green for 45 min at 37 °C. All fluorescence images were acquired using a Zeiss LSM 510 inverted laser-scanning confocal fluorescence microscope in the Case Western Reserve University Ireland Comprehensive Cancer Center confocal microscopy facility. A ×63 numerical aperture 1.4 oil immersion planapochromat objective was used. Confocal images of Pc 4 fluorescence were collected using 633-nm excitation light from a HeNe laser and a 650-nm long pass filter. Images of MitoTracker Green fluorescence were collected using 488-nm excitation light from an argon laser and a 500–550-nm band pass barrier filter. To investigate the localization of wild-type Bcl-2 and mutants, images of Texas Red were collected using 543-nm excitation light from a HeNe laser and a 560-nm long pass filter. For live cell fluorescence imaging of DU-145 cells transiently transfected with GFP-wild-type Bcl-2 or with GFP-Bcl-2 mutants, cells were plated on 35-mm glass bottom dishes (MatTek Cop., Ashland, MA) and transiently transfected (27Usuda J. Chiu S.M. Azizuddin K. Xue L.Y. Lam M. Nieminen A.-L. Oleinick N.L. Photochem. Photobiol. 2002; 76: 217-223Crossref PubMed Scopus (63) Google Scholar). Eight h after the transfection, Pc 4 was added to the cultures, which were further incubated for 16 h and then incubated with 100 nmMitoTracker Red (Molecular Probes) for 45 min at 37 °C. Images of GFP fluorescence and MitoTracker Red fluorescence were collected using the same filter settings as for MitoTracker Green plus Texas Red, respectively (27Usuda J. Chiu S.M. Azizuddin K. Xue L.Y. Lam M. Nieminen A.-L. Oleinick N.L. Photochem. Photobiol. 2002; 76: 217-223Crossref PubMed Scopus (63) Google Scholar). After taking these images, the cells were photoirradiated, and the same cells were imaged immediately and 1 h later. In cells, PDT causes oxidative damage to target molecules that reside within a few nm of the sites of photoactivation of the photosensitizer (7Lam M. Oleinick N.L. Nieminen A.L. J. Biol. Chem. 2001; 276: 47379-47386Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, 8Moan J. Berg K. Photochem. Photobiol. 1992; 55: 931-948Crossref PubMed Scopus (397) Google Scholar). We have previously shown that Pc 4 binds to mitochondria but also to various other organellar membranes, including the nuclear membrane and ER/Golgi membranes of murine lymphoma L5178Y-R cells (6Trivedi N.S. Wang H.W. Nieminen A.L. Oleinick N.L. Izatt J.A. Photochem. Photobiol. 2000; 71: 634-639Crossref PubMed Scopus (97) Google Scholar) and human skin carcinoma A431 cells (7Lam M. Oleinick N.L. Nieminen A.L. J. Biol. Chem. 2001; 276: 47379-47386Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). Therefore, we first examined the localization of Pc 4 in DU-145 cells using confocal microscopy (Fig. 1). To assess whether Pc 4 binds to the mitochondria, DU-145 cells were co-loaded with MitoTracker Green, a mitochondrion-specific dye. The images of Pc 4 displayed a punctate pattern, but Pc 4 fluorescence only partially co-localized with MitoTracker Green fluorescence. These results suggest that in DU-145 cells Pc 4 localizes not only to mitochondria but also to the endoplasmic reticulum (ER), Golgi complexes, nuclear membrane, and possibly other intracellular organelles but to neither the plasma membrane nor the nucleus. The data are consistent with our earlier findings in other cell lines (6Trivedi N.S. Wang H.W. Nieminen A.L. Oleinick N.L. Izatt J.A. Photochem. Photobiol. 2000; 71: 634-639Crossref PubMed Scopus (97) Google Scholar,7Lam M. Oleinick N.L. Nieminen A.L. J. Biol. Chem. 2001; 276: 47379-47386Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). We have reported that the antiapoptotic protein Bcl-2 disappears from Western blots immediately upon Pc 4-PDT (10Xue L.Y. Chiu S.M. Oleinick N.L. Oncogene. 2001; 20: 3420-3427Crossref PubMed Scopus (204) Google Scholar). To elucidate the mechanism of the photodamage and reveal the target site, we constructed Bcl-2 mutants in which regions of the protein are deleted (Fig. 2, A and B). We used pcDNA4/HisMax plasmid, which encodes the XpressTMepitope and a polyhistidine metal-binding tag at the N-terminal region of the multiple cloning site. We transiently transfected pcDNA4/HisMax-full-length human Bcl-2 (239 amino acids) and Bcl-2 mutants into DU-145 cells, which have very low endogenous levels of Bcl-2 (29Rokhlin O.W. Bishop G.A. Hostager B.S. Waldschmidt T.J. Sidorenko S.P. Pavloff N. Kiefer M.C. Umansky S.R. Glover R.A. Cohen M.B. Cancer Res. 1997; 57: 1758-1768PubMed Google Scholar). The subcellular localization of Bcl-2 in DU-145 cells was determined by immunocytochemical analysis with the anti-Xpress antibody and confocal microscopy. Bcl-2 co-localized with mitochondria and also localized to the nuclear envelope and other cellular organelles, as previously reported (17Kroemer G. Reed J.C. Nat. Med. 2000; 6: 513-519Crossref PubMed Scopus (2784) Google Scholar, 19Matsuyama S. Schendel S.L. Xie Z. Reed J.C. J. Biol. Chem. 1998; 273: 30995-31001Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 28Oleinick N.L. Antunez A.R. Clay M.E. Rihter B.D. Kenney M.E. Photochem. Photobiol. 1993; 57: 242-247Crossref PubMed Scopus (247) Google Scholar) (Fig. 3 A). All of the mutants with deletions in the N-terminal half of Bcl-2 (mutants a, b, and c) localized in DU-145 cells similarly to wild-type Bcl-2. In control DU-145 cells transfected with empty vector, there was no detectable immunofluorescence signal above background (data not shown).Figure 3The subcellular localization and photosensitivity of mutant Bcl-2 having deletions within the N-terminal half of the protein. DU-145 cells were transiently transfected with the pcDNA4/HisMax expression vector containing Bcl-2, Bcl-2 (Δ33–54), Bcl-2 (Δ37–63), or Bcl-2 (Δ10–125). A, 24 h after transfection, subcellular localization of wild-type Bcl-2 and Bcl-2 mutant proteins was determined by imaging the fluorescence of Texas Red, and localization of mitochondria was determined by imaging the fluorescence of MitoTracker Green. Texas Red and MitoTracker Green images were overlaid to show the extent of co-localization in yellow. Scale bar, 5 μm. B, other groups of cells were exposed to Pc 4-PDT (200 nm plus 200 mJ/cm2 photoirradiation) 24 h after transfection. The Bcl-2 level was examined using a mouse monoclonal anti-Xpress tag antibody by Western blot analysis before (−PDT), immediately after (t 0), and 1 h after (t 1h) Pc 4-PDT. The control received Pc 4 but was not irradiated (−PDT).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Twenty-four h after the transfection, we performed PDT. The dose of PDT in all experiments (200 nm Pc 4 plus 200 mJ/cm2) was demonstrated to produce 92 ± 2% killing of untransfected DU-145 cells, as determined by clonogenic assay. The extent of photodamage was assessed by Western blot analysis. Similarly to the overexpressed wild-type Bcl-2, mutants with deletions in the N-terminal half of Bcl-2 (Bcl-2 (Δ33–54), Bcl-2 (Δ37–63) and Bcl-2 (Δ10–125)) were immediately photodamaged by Pc 4-PDT (Fig. 3 B). One h after PDT, neither the wild-type Bcl-2 protein nor any of the mutant proteins had been restored to their previous levels (Fig. 3 B). These results indicate that there is no essential target site of Pc 4-PDT in the N-terminal half of Bcl-2, and furthermore, Asp-34, a known caspase cleavage site (30Kirsch D.G. Doseff A. Chau B.N. Lim D.S. SouzaPinto N.C. Hansford R. Kastan M.B. Lazebnik Y.A. Hardwick J.M. J. Biol. Chem. 1999; 274: 21155-21161Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar, 31Cheng 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, 32Grandgirard D. Studer E. Monney L. Belser T. Fellay I. Borner C. Michel M.R. EMBO J. 1998; 17: 1268-1278Crossref PubMed Scopus (173) Google Scholar), is not required for photodamage to Bcl-2 by Pc 4-PDT. Moreover, since essentially all of the Bcl-2 in the cell is damaged by the chosen dose of Pc 4-PDT, the results show that all of the sites of Bcl-2 localization are accessible for photodamage and that Pc 4 must reside within a few nm of Bcl-2 binding sites in the mitochondrial, ER, and nuclear membranes. A mutant (g) missing the C-terminal region, Bcl-2 (Δ210–239), including the entire TM domain, was found throughout the cell in a diffuse nonmitochondrial pattern, as previously reported for a similar mutant (33Conus S. Kaufmann T. Fellay I. Otter I. Rosse T. Borner C. EMBO J. 2000; 19: 1534-1544Crossref PubMed Scopus (39) Google Scholar) (Fig. 4 A). Although some of the image of this mutant appeared punctate, there was no co-localization with MitoTracker Green. Mutant g displayed apparently complete resistance to photodamage by Pc 4-PDT, since there was no decrease in the amount of the protein either immediately after or 1 h after treatment of the cells with Pc 4-PDT (Fig. 4 B). However, another C-terminal mutant (mutant h, Bcl-2 (Δ218–233)), was found in mitochondrial and nuclear membranes and was destroyed immediately after PDT (Fig. 4, A and B). Mutant h retains 4 amino acids (positions 234–237) within the TM domain, which is evidently sufficient for attachment to the membran" @default.
• W1966726105 created "2016-06-24" @default.
• W1966726105 creator A5023315520 @default.
• W1966726105 creator A5037099107 @default.
• W1966726105 creator A5043087658 @default.
• W1966726105 creator A5055853672 @default.
• W1966726105 creator A5079216912 @default.
• W1966726105 creator A5085124229 @default.
• W1966726105 date "2003-01-01" @default.
• W1966726105 modified "2023-10-17" @default.
• W1966726105 title "Domain-dependent Photodamage to Bcl-2" @default.
• W1966726105 cites W1550247880 @default.
• W1966726105 cites W1916661321 @default.
• W1966726105 cites W1965217295 @default.
• W1966726105 cites W1965634795 @default.
• W1966726105 cites W1990397750 @default.
• W1966726105 cites W1990427071 @default.
• W1966726105 cites W1993977807 @default.
• W1966726105 cites W1994611236 @default.
• W1966726105 cites W1998887606 @default.
• W1966726105 cites W2007030237 @default.
• W1966726105 cites W2014586657 @default.
• W1966726105 cites W2018559975 @default.
• W1966726105 cites W2028305534 @default.
• W1966726105 cites W2031315018 @default.
• W1966726105 cites W2032221839 @default.
• W1966726105 cites W2044505588 @default.
• W1966726105 cites W2049295460 @default.
• W1966726105 cites W2050681211 @default.
• W1966726105 cites W2054709426 @default.
• W1966726105 cites W2067622439 @default.
• W1966726105 cites W2079273110 @default.
• W1966726105 cites W2086365629 @default.
• W1966726105 cites W2086405807 @default.
• W1966726105 cites W2088805436 @default.
• W1966726105 cites W2090288682 @default.
• W1966726105 cites W2092759765 @default.
• W1966726105 cites W2093382456 @default.
• W1966726105 cites W2102684933 @default.
• W1966726105 cites W2106697922 @default.
• W1966726105 cites W2114001234 @default.
• W1966726105 cites W2115472797 @default.
• W1966726105 cites W2126798834 @default.
• W1966726105 cites W2130361718 @default.
• W1966726105 cites W2136269288 @default.
• W1966726105 cites W2140967934 @default.
• W1966726105 cites W2141086199 @default.
• W1966726105 cites W2169404281 @default.
• W1966726105 cites W2171365668 @default.
• W1966726105 cites W2326387362 @default.
• W1966726105 cites W23937241 @default.
• W1966726105 cites W4211024586 @default.
• W1966726105 cites W4232376605 @default.
• W1966726105 cites W4246832840 @default.
• W1966726105 cites W4385202450 @default.
• W1966726105 cites W80462140 @default.
• W1966726105 doi "https://doi.org/10.1074/jbc.m205219200" @default.
• W1966726105 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12379660" @default.
• W1966726105 hasPublicationYear "2003" @default.
• W1966726105 type Work @default.
• W1966726105 sameAs 1966726105 @default.
• W1966726105 citedByCount "110" @default.
• W1966726105 countsByYear W19667261052012 @default.
• W1966726105 countsByYear W19667261052013 @default.
• W1966726105 countsByYear W19667261052014 @default.
• W1966726105 countsByYear W19667261052015 @default.
• W1966726105 countsByYear W19667261052016 @default.
• W1966726105 countsByYear W19667261052017 @default.
• W1966726105 countsByYear W19667261052018 @default.
• W1966726105 countsByYear W19667261052019 @default.
• W1966726105 countsByYear W19667261052020 @default.
• W1966726105 countsByYear W19667261052021 @default.
• W1966726105 countsByYear W19667261052023 @default.
• W1966726105 crossrefType "journal-article" @default.
• W1966726105 hasAuthorship W1966726105A5023315520 @default.
• W1966726105 hasAuthorship W1966726105A5037099107 @default.
• W1966726105 hasAuthorship W1966726105A5043087658 @default.
• W1966726105 hasAuthorship W1966726105A5055853672 @default.
• W1966726105 hasAuthorship W1966726105A5079216912 @default.
• W1966726105 hasAuthorship W1966726105A5085124229 @default.
• W1966726105 hasBestOaLocation W19667261051 @default.
• W1966726105 hasConcept C12554922 @default.
• W1966726105 hasConcept C134306372 @default.
• W1966726105 hasConcept C185592680 @default.
• W1966726105 hasConcept C33923547 @default.
• W1966726105 hasConcept C36503486 @default.
• W1966726105 hasConcept C70721500 @default.
• W1966726105 hasConcept C86803240 @default.
• W1966726105 hasConcept C95444343 @default.
• W1966726105 hasConceptScore W1966726105C12554922 @default.
• W1966726105 hasConceptScore W1966726105C134306372 @default.
• W1966726105 hasConceptScore W1966726105C185592680 @default.
• W1966726105 hasConceptScore W1966726105C33923547 @default.
• W1966726105 hasConceptScore W1966726105C36503486 @default.
• W1966726105 hasConceptScore W1966726105C70721500 @default.
• W1966726105 hasConceptScore W1966726105C86803240 @default.
• W1966726105 hasConceptScore W1966726105C95444343 @default.
• W1966726105 hasIssue "3" @default.

• next