From the Departments of Radiation Oncology and
§ Anatomy, and the ¶ Case Western Reserve
University/Ireland Comprehensive Cancer Center, Case Western Reserve
University School of Medicine, Cleveland, Ohio 44106
Received for publication, May 28, 2002, and in revised form, October 1, 2002
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ABSTRACT |
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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 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 (1, 2). 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. (3) 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 (4, 5). 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 (6, 7). 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 (7). 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 (8, 9). One important target of
Pc 4-PDT (10) as well as PDT with certain other photosensitizers (11)
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 Structural analysis of Bcl-xL reveals its resemblance to
the bacterial pore-forming toxins, colicin and diphtheria toxin, especially in the The role of Bcl-2 in the apoptotic response caused by PDT has remained
controversial. He et al. (21) 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. (11) 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 (22). As in the study by Kim et al.
(11), 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 (10). Bcl-2 photodamage was selective, in that several other
mitochondrial proteins were not affected. Kessel and Castelli (23)
found that PDT with three different photosensitizers destroyed Bcl-2
but not Bax. In contrast, a recent report from Antieghem et
al. (24) 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.
Cell Culture--
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 (25). 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.
Plasmid Construction--
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) (26). Bcl-2 cDNA was digested with
EcoRI 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) (18, 19). The following
mutagenic primers were used: for mutant a, Bcl-2 (
Additional Bcl-2 mutants were constructed to mutate specific amino
acids. In order to generate mutant i, pUC19-Bcl-2 (
The following mutagenic primers were used: for mutant i, Bcl-2
(
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 with
DpnI (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.
Photodynamic Therapy--
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 ( Western Blot Analysis--
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.
Fluorescence Immunocytochemistry--
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.
Localization of Pc 4 in DU-145 Cells--
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.
Confocal Microscopy--
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 (27). 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 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 (27). After taking these images, the cells were
photoirradiated, and the same cells were imaged immediately and 1 h later.
Pc 4 Localizes to Mitochondria and Other Intracellular Organelles
of DU-145 Cells--
In cells, PDT causes oxidative damage to target
molecules that reside within a few nm of the sites of photoactivation
of the photosensitizer (7, 8). 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 (6) and human skin carcinoma A431 cells (7).
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 (6,
7).
Bcl-2 Mutants Missing Portions of the N-terminal Half of the
Protein Respond to Pc 4-PDT Similarly to Wild-type Bcl-2--
We
have reported that the antiapoptotic protein Bcl-2 disappears
from Western blots immediately upon Pc 4-PDT (10). 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 XpressTM
epitope 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 (29). 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 (17, 19, 28) (Fig.
3A). 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).
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 ( A Bcl-2 Mutant Missing the C-terminal 20 Amino Acids, Including the
TM Domain, Was Not Destroyed by Pc 4-PDT, Whereas Retention of Part of
the TM Domain Allows Photodamage to Occur--
A mutant (g) missing
the C-terminal region, Bcl-2 ( Deletion of Observation of GFP-Bcl-2 Fluorescence in Live Cells Revealed That
Photodamaged Bcl-2 Remains at Its Original Sites in the Nuclear
Envelope, ER, and Mitochondrial Membrane after Pc 4-PDT--
Until
this point, photodamage has been monitored by Western blot analysis,
which reveals that immediately after Pc 4-PDT, there is a nearly
complete loss of the 26-kDa protein. Since it is unlikely that this
response reflects the immediate disappearance of the protein from the
cells, we sought further clarification of the photodamage response by
monitoring the fate of GFP-tagged Bcl-2 in live cells following Pc
4-PDT. Thus, we constructed GFP-Bcl-2 and GFP-Bcl-2 mutants and
examined the localization of the GFP fusion proteins by confocal
microscopy before and after PDT. GFP-Bcl-2 localized to the nuclear
envelope, ER, and mitochondria (Fig. 6A), as previously observed by
immunohistochemical analysis of Xpress-tagged Bcl-2 in Fig.
3A. One h after Pc 4-PDT, GFP-Bcl-2 remained in the same
locations with approximately the same fluorescence intensity. It is
also apparent that the mitochondria had swollen by 1 h after Pc
4-PDT, as we had previously found in A431 cells (7), and some of the
GFP-Bcl-2 clearly outlines the swollen mitochondria (Fig.
6A). GFP-Bcl-2 (
The two mutants that did not suffer from photodamage, Bcl-2
( Photodamage to Bcl-2 Is Observed on Western Blots as the
Disappearance of the Control Band and the Appearance of High Molecular
Weight Bands--
The above data demonstrate that photodamaged Bcl-2
remains at its original sites in cells but is not found at its normal
position on Western blots. These observations suggest that there has
been a photochemical change in the protein that alters its
electrophoretic mobility. Previously, we found evidence for high
molecular weight complexes containing Bcl-2 in Western blots of Pc
4-PDT-treated Chinese hamster ovary cells (10). In order to further
characterize the photodamage, some of the previously analyzed cell
lysates (Figs. 3B, 4B, and 5B) were
reanalyzed; however, for these experiments, proteins in both the
running and stacking gels were transferred to polyvinylidene difluoride
membranes, and the entire blot was evaluated using the mouse monoclonal
anti-Xpress antibody (Fig. 6E). Essentially the same results
were obtained for cells overexpressing GFP-wild-type Bcl-2 or GFP-Bcl-2
mutant, when the entire gel was transferred and evaluated with the
anti-GFP antibody (data not shown).
As observed previously (Figs. 3B, 4B, and
5B), photodamage to wild-type Bcl-2 and to Bcl-2 ( Pc 4-PDT Does Not Directly Damage the Singlet Oxygen-sensitive
Amino Acids within the Pc 4-PDT Does Not Photodamage either Bak or Bax in Cancer
Cells--
The The present study builds on the earlier findings of Xue et
al. (10) that Pc 4-PDT causes photodestruction of Bcl-2. In both the earlier study and the present one, a maximal loss of Bcl-2 was
observed by Western blot analysis of samples taken immediately upon
photoirradiation. Furthermore, the expression of photodamage was
independent of the availability of a known caspase cleavage site.
Specifically, both Bcl-2 ( In the present study, we found that overexpressed tagged wild-type
Bcl-2 protein and overexpressed GFP-Bcl-2 protein, which localized to
the mitochondria, ER, and nuclear envelope (Figs. 3A and
6A), were photodamaged immediately and did not recover by
1 h after PDT. Since essentially all of the Bcl-2 was
photodamaged, our data indicate that Pc 4-PDT damages not only
mitochondrial membranes but also the ER and nuclear envelope. Kvam
et al. (40) have reported that PDT using
mesotetra-(3-sulfonatophenyl)porphyrin damaged the nuclear membrane and
induced DNA repair, although much subsequent work has indicated that
DNA is not an important target for cell killing by PDT (41, 42). Pc 4 binds to the same membrane systems that house Bcl-2 and therefore may
be especially well positioned to damage all cellular Bcl-2, not just
the mitochondrially bound protein.
The subcellular localization of Pc 4 is similar in DU-145 cells as in
other cell types (6, 7) (i.e. cytoplasmic membranes, including mitochondria, ER, and nuclear envelope) (Fig. 1). The reactive oxygen species produced by PDT, especially singlet oxygen, are
highly reactive and therefore cause photooxidative damage to proteins
and lipids that reside within a few nm of photosensitizer binding sites
(34, 35). A major conclusion of the present study is that the
photodamage and cross-linking of Bcl-2 are absolutely dependent upon
anchorage of the protein in membranes. We found that two membrane
anchorage domains of Bcl-2 were required for efficient photodamage. One
is the region between the BH1 and BH2 domains, corresponding to the
We hypothesize that the region between the BH1 and BH2 domains,
corresponding to the An important question is whether the photodamaged Bcl-2 retains its
antiapoptotic function. We were not able to observe morphologically typical apoptosis (i.e. chromatin condensation and
fragmentation) in DU-145 cells after PDT because of the absence of Bax
from these cells. However, the cells die after PDT without undergoing
typical apoptosis, as determined by clonogenic
assay.2 Moreover, DU-145
cells that stably overexpress Bcl-2 have a similar sensitivity to Pc
4-PDT as those expressing only endogenous Bcl-2. Although the absence
of a differential photosensitivity might be attributed to the inability
of DU-145 cells to up-regulate Bax (11), the same observation has been
made in MCF-7 cells that express adequate levels of Bax. We suggest
that Pc 4-PDT-induced photodamage eliminates the native Bcl-2 structure
and thereby its antiapoptotic activity. Since all Bcl-2 is
photodamaged, the pre-PDT level of Bcl-2 may not be relevant to
determining the cell's sensitivity to Pc 4-PDT. Support for this idea
derives from observations (43, 44) that overexpressed Bcl-2 does not protect cells against PDT-induced release of cytochrome c.
Further work will be required to assess the function of photodamaged
Bcl-2. We have begun experiments to evaluate by flow cytometry the
ability of transiently expressed Bcl-2 and mutants to modify Pc
4-PDT-induced apoptosis.
The region between the BH1 and BH2 domains of Bcl-2 is very similar to
those of Bak and Bax (16). The proapoptotic protein Bak localizes to
the mitochondrial membrane but is not destroyed by Pc 4-PDT (Fig.
9A). One possible reason for the resistance of Bak to
photodamage is that it may reside in the membrane at a site far from
the sites of singlet oxygen generation. Another possibility is
suggested by the observations of Griffiths et al. (38) that
the Bak N-terminal domain regulates the conformation of the BH1 domain,
and apoptotic signals promote sequential changes in the N-terminal and
BH1 domains. In that case, conformational regulation of the
Based on analysis of the three-dimensional structure of Bax, its normal
conformation is reported to involve an interaction between the BH3
domain and the N terminus; in response to an apoptotic signal, Bax
undergoes a conformational change, which results in rotation of the BH3
domain away from the N terminus (45-48). Eskes et al. (49)
and Antonsson et al. (50) demonstrated that in apoptotic cells, Bax was not only attached to the mitochondrial membrane but also inserted into it. However, despite having an If Pc 4 were homogeneously distributed in the membranes, any
membrane-inserted protein would be susceptible to photodamage. However,
other mitochondrial membrane proteins, including the voltage-dependent anion channel and the adenine nucleotide
translocator, were not photodamaged by Pc 4-PDT (10). Thus, the
cross-linking is a relatively specific response of Bcl-2 to Pc 4-PDT,
which may result from a greater photosensitivity of the Bcl-2 complexes or greater exposure of those complexes to PDT-generated singlet oxygen.
We conclude that the -helices 5/6 was also not photodamaged.
Bcl-2 missing only one of those
-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
5/
6 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.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
-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) (12). 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 (13). 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 (14, 15).
-helical region that mediates membrane insertion of the toxins (16, 17). This region corresponds to the BH1 domain and
part of the BH2 domain of Bcl-2 and Bcl-xL, and is called
the
5/
6 region. This region is thought to
be inserted into the membrane and form ion channels (18-20). It has
been reported that mutations within the
5/
6 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 (16, 17).
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ABSTRACT
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DISCUSSION
REFERENCES
33-54)
(5'-ATCTTCTCCTCCCAGCCCGGGCAGACCCCGGCTGCCCCCGGC-3' (forward) and
5'-GCCGGGGGCAGCCGGGGTCTGCCCGGGCTGGGAGGAGAAGAT-3' (reverse)); for mutant
b, Bcl-2 (
37-63) (5'-GTGGGATGCGGGAGATGTGGGCGACCCGGTCGCCAGGACC (forward) and 5'-GGTCCTGGCGACCGGGTCGCCCACATCTCCCGCATCCCAC-3'
(reverse)); for mutant c, Bcl-2 (
10-125)
(5'-CTGGGAGAACGGGGTACGACGCGCGGGGACGCTTTGCCAC-3' (forward) and
5'-GTGGCAAAGCGTCCCCGCGCGTCGTACCCCGTTCTCCCAG-3' (reverse)); for mutant
d, Bcl-2 (
153-179) (5'-GGATTGTGGCCTTCTTTGAGTACCTGAACCGGCACCTGCAC-3' (forward) and 5'-GTGCAGGTGCCGGTTCAGGTACTCAAAGAAGGCCACAATCC-3' (reverse)); for mutant e, Bcl-2 (
153-168)
(5'-GGATTGTGGCCTTCTTTGAGCTGGTGGACAACATCGCCC-3' (forward) and
5'-GGGCGATGTTGTCCACCAGCTCAAAGAAGGCCACAATCC-3' (reverse)); for mutant f,
Bcl-2 (
168-179) (5'-GCGTCAACCGGGAGATGTCGTACCTGAACCGGCACCTGC-3' (forward) and 5'-GCAGGTGCCGGTTCAGGTACGACATCTCCCGGTTGACGC-3' (reverse)); for mutant g, Bcl-2 (
210-239)
(5'-GCCCCAGCATGCGGCCTCTGTGAAGTCAACATGCCTGCCC-3' (forward) and
5'-GGGCAGGCATGTTGACTTCACAGAGGCCGCATGCTGGGGC-3' (reverse)); for mutant
h, Bcl-2 (
218-233) (5'-ATTTCTCCTGGCTGTCTCTGGCCTATCTGAGCCACAAGTGA-3' (forward) and 5'-TCACTTGTGGCTCAGATAGGCCAGAGACAGCCAGGAGAAAT-3' (reverse)).
168-179) was
amplified, and to generate mutant j, pUC19-Bcl-2 (
153-168) was
amplified as a template for the QuikChangeTM site-directed
mutagenesis kit.
168-179, F153L,M157I,C158V)
(5'-CTTCTTTGAGCCGGTGGGGTCATCGTTGTGGAGAGCGTC-3' (forward) and
5'-GACGCTCTCCACAACGATGACCCCACCGAGCTCAAAGAAG-3' (reverse)); for mutant
j, Bcl-2 (
153-168, W176L,M177L)
(5'-GTGGACAACATCGCCCTGTTACTGACTGAGTACCTGAACC-3') (forward) and
5'-GGTTCAGGTACTCAGTCAGTAACAGGGCGATGTTGTCCAC-3' (reverse)).
max 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.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
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Fig. 1.
Pc 4 localizes to cytoplasmic membranes.
DU-145 cells were loaded with 200 nM Pc 4 for 16 h and
100 nM MitoTracker Green. The image of Pc 4 displayed a punctate pattern, but it did not completely co-localize
with MitoTracker Green. Scale bar, 5 µm.
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Fig. 2.
Design of Bcl-2 mutants. A,
the positions of the deleted regions within the human Bcl-2 protein are
depicted. B, the mutants (a-h) are indicated
with reference to the amino acids deleted from each.
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Fig. 3.
The 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
( 33-54), Bcl-2 (
37-63), or Bcl-2 (
10-125). 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 (t0),
and 1 h after (t1h) Pc 4-PDT. The control
received Pc 4 but was not irradiated (
PDT).
33-54), Bcl-2 (
37-63) and
Bcl-2 (
10-125)) were immediately photodamaged by Pc 4-PDT (Fig.
3B). 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. 3B). 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 (30-32), 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.
210-239), including the entire TM
domain, was found throughout the cell in a diffuse nonmitochondrial
pattern, as previously reported for a similar mutant (33) (Fig.
4A). 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. 4B). However,
another C-terminal mutant (mutant h, Bcl-2 (
218-233)), 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 membranes. These results indicate that
for Pc 4-PDT to cause photodamage, membrane anchorage of the protein
may be essential, which requires at least part of the C-terminal TM
domain.
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Fig. 4.
The subcellular localization and
photosensitivity of mutant Bcl-2 missing portions of the C-terminal
region of the protein. DU-145 cells were transiently transfected
with the pcDNA4/HisMax expression vector containing Bcl-2
( 210-239) or Bcl-2 (
218-233). A, 24 h after
transfection, subcellular localization of the 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 (t0), and 1 h
(t1h) after Pc 4-PDT. The control received Pc 4 but
was not irradiated (
PDT).
-Helices 5 and 6 in the Region between the BH1 and
BH2 Domains Eliminates the Ability of Pc 4-PDT to Photodamage
Bcl-2--
The region between the BH1 and BH2 domains, which contains
two core hydrophobic
-helices (
5 and
6), is required for membrane insertion and channel
formation (17-19). Images of mutant d (Bcl-2 (
153-179)), which
lacks both of the
-helices, showed only punctate fluorescence in the
cytoplasm that completely overlapped with the fluorescence of
MitoTracker Green, indicating that Bcl-2 (
153-179) localized to the
mitochondrial membrane but not to the ER or nuclear envelope (Fig.
5A). Although Bcl-2
(
153-179) retains the C-terminal TM domain and binds to
mitochondria, it was not photodamaged by Pc 4-PDT (Fig. 5B).
These results indicate that the two core hydrophobic regions between
the BH1 and BH2 domains are required for Bcl-2 photodamage, and the
C-terminal TM domain may not be a phototarget site. Interestingly,
Bcl-2 proteins (mutants e and f) missing only one of the
-helices
(Bcl-2 (
153-168) and Bcl-2 (
168-179)) behaved like wild-type
Bcl-2 (Fig. 5A). Figs. 2 and 7 show that the 16 amino acids
(positions 153-168) deleted in mutant e correspond to part of the
5 region, and the 12 amino acids (positions 168-179) deleted in mutant f correspond to part of the
6 region.
Both of these proteins localized similarly to wild-type Bcl-2 and could be destroyed by Pc 4-PDT (Fig. 5, A and B). These
results indicate that both
5- and
6-helices may be required for attachment of the protein
to the ER and nuclear membrane but that either the
5 or
the
6 domain is sufficient for photodamage through
attachment to or insertion into the mitochondrial membrane.
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Fig. 5.
The subcellular localization and
photosensitivity of mutant Bcl-2 missing portions of the
5/
6
region between the BH1 and BH2 domains. DU-145 cells were
transiently transfected with the pcDNA4/HisMax expression vector
containing Bcl-2 (
153-179), Bcl-2 (
153-168), or Bcl-2
(
168-179). A, 24 h after the transfection,
subcellular localization of Bcl-2 mutant protein was determined by
imaging the fluorescence of Texas Red, and localization of mitochondria
was indicated by 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 PDT (200 nM Pc 4 plus 200 mJ/cm2 photoirradiation)
24 h after the transfection. The Bcl-2 level was examined using a
mouse monoclonal anti-Xpress tag antibody by Western blot analysis
before (
PDT), immediately after (t0),
and 1 h after (t1h) Pc 4-PDT. The control
received Pc 4 but was not irradiated (
PDT).
33-54), which was photodamaged by Pc
4-PDT (Fig. 3B), also did not disappear from its normal binding sites after PDT (Fig. 6B); this mutant revealed the
same localization pattern as wild-type GFP-Bcl-2. We observed neither aggregation of organelles nor large complexes of GFP-Bcl-2, despite greater irregularity in the shape of the nuclear membrane and swelling
of the mitochondria after PDT (Fig. 6, A and
B).
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Fig. 6.
The subcellular localization and
photosensitivity of GFP-Bcl-2 and GFP-Bcl-2 mutants before and 1 h
after PDT. DU-145 cells were transiently transfected with an
expression vector encoding Bcl-2, Bcl-2 ( 33-54), Bcl-2
(
153-179), or Bcl-2 (
210-239) as a fusion protein with GFP at
the N terminus. A-D, 8 h after the transfection, 200 nM Pc 4 was added to the cultures, which were further
incubated for 16 h and incubated with 100 nM
MitoTracker Red for 45 min. Then cultures were irradiated with 200 mJ/cm2 red light. In living DU-145 cells, localizations of
Bcl-2 and Bcl-2 mutants were determined by observing GFP fluorescence,
and localizations of mitochondria were determined by observing
fluorescence of MitoTracker Red. GFP and MitoTracker Red images were
overlaid to show colocalization in yellow. In cells
transfected with GFP-Bcl-2 (A), GFP-Bcl-2 (
33-54)
(B), GFP-Bcl-2 (
153-179) (C), or GFP-Bcl-2
(
210-239) (D), images were collected before and 1 h
after PDT. Scale bar, 5 µm. E, whole
cell lysates used in previous experiments (Figs. 3B,
4B, and 5B) were loaded to 10% SDS-PAGE gels.
Proteins in both the running and stacking gels were transferred to
polyvinylidene difluoride membranes. The Bcl-2 level was examined using
a mouse monoclonal anti-Xpress antibody by Western blot analysis before
(
PDT), immediately after (t0), and 1 h after
(t1h) PDT. The membrane was reprobed with anti-actin
to control for loading. Control cells received Pc 4 but were not
irradiated (
PDT).
153-179) and Bcl-2 (
210-239) (Figs. 4B and
5B), were also examined as GFP-tagged proteins by confocal
microscopy in live cells (Fig. 6, C and D).
Similar to the photodamageable Bcl-2 species (Fig. 6, A and
B), the localization and intensity of the nonphotodamageable mutants were identical before and after PDT (Fig. 6, C and
D), and the localizations were similar to those observed in
Figs. 4A and 5A. For the four GFP-Bcl-2 species
examined in Fig. 6, images of GFP intensity and localization obtained
immediately after Pc 4-PDT were not different from those at 1 h
after PDT, except for less mitochondrial swelling at the early time
(data not shown). These results indicate that Pc 4-PDT-damaged Bcl-2 remains at its previous sites in the cell.
33-54)
was revealed as a reduction in intensity of the control band, whereas
there was no PDT-induced change in intensity for the nonphotodamageable
species, Bcl-2 (
153-179) and Bcl-2 (
210-239). In addition, it
is apparent that photodamage was accompanied by the appearance of high
molecular mass (>250-kDa) bands in the regions corresponding to
the top of the running gel and the stacking gel, and these were
observed in samples taken both immediately after and 1 h after PDT
(Fig. 6E). These results indicate that Pc 4-PDT forms large
cross-linked complexes of Bcl-2 or Bcl-2 (
33-54), but not of Bcl-2
(
153-179) or Bcl-2 (
210-239). The large complexes include Bcl-2
and possibly neighboring proteins and are sufficiently large that in
many cases, they are unable to enter the gel. Therefore, photodamaged
Bcl-2 seems to have disappeared only insofar as it has been converted from its native size into large complexes, but it does not disappear from the cells. The absence of cross-link formation with Bcl-2 (
153-179) and Bcl-2 (
210-239) (Fig. 6E) suggests
that the membrane anchorage domains are required for formation of the
membrane-associated protein complexes that are susceptible to
photocross-linking. In the case of Bcl-2 (
210-239), the absence of
membrane anchorage (Fig. 6D) prevents photodamage via
photoirradiated membrane-localized Pc 4. However, in the case of Bcl-2
(
153-179), which binds to the mitochondrial membrane (Fig.
6C), the absence of photodamage reveals an important role
for the
5/
6 domain. Either this domain is
the direct target of photodynamic damage, or it is needed to form the
photosensitive structure.
5 and
6
-Helices--
PDT generates singlet oxygen and other reactive
oxygen species at photosensitizer binding sites (34, 35). In order to determine whether or not the
5 and
6
-helices are damaged directly by Pc 4-PDT-produced singlet oxygen,
we modified Bcl-2 mutants e and f to construct mutants i and j by
site-directed mutagenesis. Fig. 7 shows
the amino acid sequence of the relevant parts of the
5/
6 region of Bcl-2, in which the
hydrophobic amino acids are coded red, and the
1O2-reactive amino acids (Cys, Phe, His, Met,
Trp, and Tyr) (36) are shown in single-letter code and
underlined in blue. The amino acids Phe-153,
Met-157, Cys-158, Trp-176, and Met-177 are both singlet oxygen-targeted
and either hydrophobic or polar but uncharged. Therefore, the following
amino acid substitutions were made in Bcl-2: within the
5-helix, Phe-153
Leu, Met-157
Ile, and Cys-158
Val, and within the
6-helix, Trp-176
Leu and
Met-177
Leu. In each case, the singlet oxygen-targeted amino acid
was changed to a hydrophobic one that is poorly reactive with singlet oxygen (Fig. 7). Using mutants i and j, we then tested the hypothesis that Pc 4 binds to the
5/
6 region or to
proteins near that region, and Pc 4-PDT directly damages the region,
especially the singlet oxygen-sensitive amino acids within it, causing
Bcl-2 to cross-link to itself or nearby proteins. Bcl-2 mutants i and j
localized similarly to wild-type Bcl-2, as judged by immunocytochemical analysis with the anti-Xpress antibody (data not shown). However, it
was found that Bcl-2 mutants i and j were both susceptible to
photodamage and formed high molecular weight cross-linked protein complexes upon PDT (Fig. 8). These
results demonstrate that direct singlet oxygen damage to susceptible
amino acids within the
5/
6 region is not
essential for Bcl-2 photocross-linking, and this region may not be the
direct target of Pc 4-PDT.
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Fig. 7.
Amino acid sequence of the
5/
6
region of wild-type Bcl-2 deleted in mutant d and schema of the deleted
and/or substituted amino acids in mutants e, f, i, and j. The 27 amino acids (positions 153-179) of Bcl-2 corresponding to
-helices
5 and 6 are indicated. Hydrophobic amino acids are in red,
and blue underlining indicates singlet
oxygen-targeted amino acids among the 27 amino acids (positions
153-179) between the BH1 and BH2 domains. For each mutant, a
red line indicates the deleted region, and numbers indicate
amino acid positions in the full sequence of wild-type Bcl-2. In mutant
i, within
helix 5, Phe-153
Leu, Met-157
Ile, Cys-158
Val, and the amino acids 168-179 are deleted. In mutant j, within
-helix 6, Trp-176
Leu, Met-177
Leu, and the amino acids
153-168 are deleted.
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Fig. 8.
The photosensitivity of mutants i and j, in
which the singlet oxygen-targeted amino acids were replaced. Eight
h after transfection of pcDNA4/HisMax-Bcl-2 mutant i or j, 200 nM Pc 4 was added to the cultures, which were further
incubated for 16 h. Then cultures were irradiated with 200 mJ/cm2 red light and collected before ( PDT),
immediately after (t0) and 1 h after
(t1h) PDT. The control received Pc 4 but was not
irradiated (
PDT). The samples were loaded to 10% SDS-PAGE
gel, and the contents of the complete gel, including the stacking gel,
were transferred to a polyvinylidene difluoride membrane. The Bcl-2
level was examined using a mouse monoclonal anti-Xpress antibody, and
loading was checked by reprobing with anti-actin.
5/
6 region has an amino
acid sequence with high homology to the corresponding region of other
Bcl-2 family members, such as Bcl-xL, Bax, and Bak (16).
Bak, a proapoptotic protein that localizes to the mitochondrial
membrane (37, 38), was not photodamaged in DU-145 cells by PDT (Fig.
9A). Pc 4-PDT-induced photodamage to Bak was also not found in MCF-7c3 and MDA-MB-468 cells,
demonstrating that the resistance of Bak to photodestruction is a
general phenomenon (Fig. 9A). The proapoptotic protein Bax is normally found in the cytosol, although Bax has a hydrophobic C-terminal domain similar to those of Bcl-2 and Bak (16, 37, 39). Since
DU-145 cells are Bax-null (27, 29), the fate of Bax was explored in
MCF-7c3 cells, with the result that no photodamage was found (Fig.
9B). After an apoptotic stimulus, Bax can translocate to the
mitochondrial membrane from the cytoplasm, and we hypothesized that it
might be possible to photodamage Bax once it had bound to the
mitochondria. Therefore, 4 h before PDT, we initiated apoptosis in
MCF-7c3 cells with 100 nM staurosporine. Although Bax
translocated to the mitochondria by 4 h after the addition of
staurosporine to the medium (data not shown), it was not cross-linked
by Pc 4-PDT (Fig. 9C). These data suggest that the
hydrophobic
5/
6 region of Bax may not be
inserted to the membrane, and/or in the case of Bax the region may
reside in the membrane far from the sites where singlet oxygen is
generated by Pc 4-PDT.
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Fig. 9.
Resistance of Bak and Bax to photodamage by
Pc 4-PDT. A, DU-145 cells, MCF-7c3 cells, and
MBA-MB-468 cells were exposed to 200 nM Pc 4. After 16 h, the cells were irradiated with red light (200 mJ/cm2).
Immediately after or 1 h after PDT, cells were collected, and Bak
levels were examined by Western blot analysis. B, MCF-7c3
cells were treated with either 200 or 400 nM Pc 4. After
16 h, the cells were irradiated with red light (200 mJ/cm2). Immediately after PDT, cells were harvested, and
Bax levels were analyzed by Western blot. C, MCF-7c3 cells
were exposed to 200 or 400 nM Pc 4. After 12 h, 100 nM staurosporine was added to the cultures, and 4 h
later, the cells were irradiated with red light (200 mJ/cm2). Immediately after PDT, the cells were collected,
and Bax levels were analyzed by Western blot. Controls included cells
treated only with PDT or only with staurosporine
(STS).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
33-54) and Bcl-2 (
10-125) are missing
Asp-34, a target of caspase-3 (30-32), but were photodamaged as
efficiently as wild-type Bcl-2, and Bcl-2 (
33-54) was photodamaged as efficiently as Bcl-2 (
37-63), a mutant of similar size and placement in the molecule that retains Asp-34 (Fig. 3, A and
B). These results confirm the findings of Xue et
al. (10) that Bcl-2 photodamage was manifested immediately upon
photoirradiation, in MCF-7 cells lacking procaspase-3, in cells treated
with a variety of protease inhibitors, and in cells that were
photoirradiated in the cold (10). Thus, Bcl-2 photodamage is a direct
result of photodynamic action and does not require protease or other enzymatic activity.
5- and
6-helices, and the other is the
C-terminal TM domain, especially four amino acids (positions 234-237)
within the TM domain. The C-terminal TM domain is required for Bcl-2 to
be photodamaged and to become cross-linked. However, mutant h, which is
missing 15 amino acids from the TM domain, was photodamaged (Fig.
4B). These results suggest that the missing 15 amino acids
(positions 219-233) are not the target sites of Pc 4-PDT, and the four
retained amino acids (positions 234-237) are sufficient for Bcl-2
photodamage. If those four amino acids were the binding sites of Pc 4 and the targeted region for Pc 4-PDT, mutant d, Bcl-2 (
153-179),
which has the full C-terminal TM domain and localizes to mitochondria,
would have been photodamageable and capable of forming cross-links upon
PDT. Moreover, other Bcl-2 family members that have a similar
C-terminal TM domain to that of Bcl-2 should also have been
photodamaged. Therefore, we conclude that the four amino acids within
the C-terminal TM are sufficient to attach Bcl-2 to the membrane in a
photodamageable form, but that region is not the target site of Pc
4-PDT.
5- and
6-helices, is
the more important region for forming the photodamageable structure.
Furthermore, Pc 4 may bind to the membrane close to the region where
Bcl-2 is located. Muchmore et al. (16) have reported that
Bcl-2 forms pores in the cytoplasmic membranes where it localizes, and
the region between the BH1 and BH2 domains of Bcl-2 is inserted into the membranes and regulates cell death. Mutations within that region,
which are similar to Bcl-2 (
153-179), abrogate the antiapoptotic activity (18-20). Some reports have shown that mutants missing the
C-terminal TM domain but retaining the region of the
5-
and
6-helices have antiapoptotic activity (14, 15). We
suggest that the
5/
6 region of Bcl-2 is
very important to its antiapoptotic activity, but its role in
determining the subcellular localization of Bcl-2 is still not well
defined. Conus et al. (33) reported that Bcl-2 missing the
region between the BH1 and BH2 domains (i.e. similar to
Bcl-2 (
153-179)) localized to mitochondria. They suggested that the
mutant Bcl-2 perhaps had a higher affinity for mitochondria than for
other membranes, like ER and nuclear envelope. Wang et al.
(26) showed that ER-targeted Bcl-2 prevented cell death induced by Bax
overexpression, whereas mitochondrion-targeted Bcl-2 was necessary for
Bax-induced cell death. In our study, Bcl-2 missing the
5/
6 region attached to the mitochondria
but not to the ER or the nuclear membrane (Fig. 5B).
5/
6 region of Bak by the N-terminal
domain may prevent insertion into the membrane.
5/
6 region similar to that of Bcl-2 and
apoptosis-induced translocation to the mitochondria in response to
staurosporine, Bax was not photodamaged by Pc 4-PDT (Fig.
7C) (10) or by PDT with AlPc (11).
5/
6 region of Bcl-2
is not the direct target of Pc 4-PDT but contributes to photodamage by
forming essential interactions with other domains of Bcl-2. From our
photobiological analysis of Bcl-2 family members, it appears that
cross-link formation of Bcl-2 might play an important role in the
induction of apoptosis by PDT, either by blocking its antiapoptotic
function or by forming a proapoptotic form of Bcl-2.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants R01 CA83917 (to N. L. O.), P01 CA48735 (to N. L. O.), R01 NS39469 (to A.-L. N.), and P30 CA43703.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Radiation
Oncology, Case Western Reserve University School of Medicine (BRB-324),
10900 Euclid Ave., Cleveland, OH 44106-4942. Tel.: 216-368-1117; Fax:
216-368-1142; E-mail: nlo@po.cwru.edu.
Published, JBC Papers in Press, October 11, 2002, DOI 10.1074/jbc.M205219200
2 L. Y. Xue, J. Usuda, S. Chiu, and N. L. Oleinick, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PDT, photodynamic therapy; Pc 4, phthalocyanine 4; Pc 4-PDT, PDT with Pc 4; BH, Bcl-2 homology; ER, endoplasmic reticulum; PBS, phosphate-buffered saline; TM, transmembrane.
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