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Tumor Biology |
Molecular Pathology Unit, Massachusetts General Hospital, and Department of Pathology, Harvard Medical School, Boston, Massachusetts 02129 [N. M., W-h. Y., I. S.], and National Cancer Institute, Bethesda, Maryland 20892 [V. P., M. T.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recent evidence has implicated the Fas pathway in the induction of tumor cell death (4, 5, 6) . Engagement of Fas (CD95/Apo-1), a member of the tumor necrosis factor receptor superfamily (7) , by its ligand (FasL2 ) initiates the recruitment of the adaptor molecule Fas-associated death domain (8) that provides a docking surface for and facilitates the autoprocessing and activation of Fas-associated death domain-like interleukin 1ß converting enzyme or caspase-8 (6 , 9) . Activated Fas-associated death domain-like interleukin 1ß converting enzyme triggers the processing and activation of several effector caspases that cleave vital components of the cell and lead to its demise. FasL is a type II integral membrane protein expressed in activated immune cells, immune-privileged tissues (testis, eye, brain, and placenta; Ref. 10 ), and a wide variety of tumors, many of which also express Fas (11 , 12) . FasL is observed to be proteolytically cleaved from the surface of some cell types, by an enzyme(s) the biochemical characteristics of which are consistent with those of members of the MMP family (12 , 13) . A recent report has identified MMP-7 as one member of the MMP family that is capable of cleaving FasL (14) . The ability of the cleavage product, or sFasL, to induce apoptosis is significantly lower than that of its cell surface precursor (15 , 16) , suggesting that proteolytic cleavage of FasL may provide a mechanism for at least partial protection against Fas-mediated cell death.
MMPs are zinc-dependent enzymes that help regulate the turnover of ECM components. They are considered to play an important role in embryo development, morphogenesis, and tissue remodeling, as well as in tumor invasion and metastasis. Current opinion holds that MMPs promote tumor growth and metastasis by a variety of mechanisms that include ECM degradation, induction, and promotion of angiogenesis, and possibly, regulation of tumor cell growth itself (17) . All MMPs are synthesized, and most are secreted as latent proenzymes that are activated by a variety of proteases (18) . In steady-state conditions, MMP activity in tissues is nearly undetectable, partly because of low levels of constitutive expression and partly because of efficient homeostatic inhibitory mechanisms that include tissue inhibitors of metalloproteinases. Invading and metastatic tumor cells typically secrete MMPs and induce MMP production by surrounding stromal cells that may overwhelm the local tissue capacity to maintain their proteolytic activity in check. Synthetic MMPIs, including BB-94 (batimastat) and BB-2516 (marimastat), have been shown to prevent tumor cell-induced remodeling of extracellular matrix and angiogenesis and provide potentially useful reagents for the control of cancer spread (19 , 20) .
Interestingly, broad-spectrum MMPIs can block shedding of sFasL (12 , 13) , causing its accumulation on the cell surface (13) and induction of apoptosis in Fas-sensitive cells (21) . We have also observed that inhibition of sFasL shedding augments doxorubicin-induced apoptosis in Fas-sensitive tumor cells (21) , suggesting that FasL is involved in anticancer drug-induced cell death. However, the mechanism of FasL cleavage in tumor cells and its significance in cancer biology remain to be resolved. In the present work, we addressed the role of FasL and its shedding in doxorubicin-induced apoptosis in cancer cells. We show that the FasL/Fas pathway, which mediates doxorubicin-induced tumor cell death, is inhibited by exogenous or tumor cell-derived, proteolytically active MMP-7, which cleaves FasL from the tumor cell surface and reduces its efficacy in triggering Fas signals. Our results suggest that the proteolytic activity of MMP-7, which is broadly expressed in primary and especially metastatic human malignancies, may contribute to tumor resistance to cytotoxic agents and thereby constitute a potential therapeutic target, inactivation of which may enhance the efficacy of conventional cancer chemotherapy.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Western Blotting.
Cells were scraped, centrifuged briefly, and lysed for 30 min on ice in
a lysis buffer [50 mM Tris-HCl (pH 8), containing 120
mM NaCl and 1% NP40], supplemented with the Complete-TM
mixture of proteinase inhibitors (Boehringer Mannheim). The samples
were cleared by microcentrifugation (14,000 rpm for 30 min at 4°C)
and assessed for protein concentration. Thirty µg of protein/sample
were electrophoresed in a 12% SDS-PAGE and electroblotted onto
nitrocellulose membranes. The membranes were exposed overnight at 4°C
to the primary antibodies, which were, respectively, the rabbit
polyclonal anti-FasL antibody Q20 (Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA), the mouse monoclonal anti-MMP7 antibody Ab-1 (Oncogene
Research, Cambridge, MA), and the goat polyclonal anti-actin antibody
C-11 (Santa Cruz Biotechnology). All primary antibodies were used at
1:1000 dilution. After washing in PBS, the respective secondary
peroxidase-labeled antibody was applied at 1:10,000 dilution for 1 h at room temperature. The proteins were visualized with the enhanced
chemiluminescence technique (Amersham Pharmacia Biotech, Piscataway,
NJ).
Construction of the Fas-IgG Fusion Plasmids and Transient
Expression in Tumor Cells.
A soluble Fas-IgG chimeric molecule (FasRg) was developed as described
previously (22)
. Briefly, the sequence encoding the
extracellular domain of Fas was amplified by PCR using human Fas cDNA
as template and appropriate synthetic oligonucleotide primers, designed
to include an XhoI site (forward primer) and a
BamHI site (reverse primer) to facilitate incorporation into
the immunoglobulin expression vector (22)
. The primers
used were: forward, cac gac ctc gag atg ctg ggc atc tgg acc ctc cta;
and reverse, cac ggg atc ctc ttt gca ctt ggt gtt gct ggt gag. Amplified
cDNA was subjected to XhoI/BamHI digestion and
ligated to Xho/BamHI-cut Rg expression vector. Construction
and expression of CD8-IgG was described previously (22)
.
SK-N-MC and SW-480 cells were transfected with pCDM-Fas-Ig or pCDM-CD8-IgG using Superfect reagent (Qiagen, Valencia, CA) and treated with doxorubicin (0.1 or 0.5 µg/ml, respectively) for an additional 48 or 72 h, respectively. Cell death was assessed using the MTT assay as described previously (21) .
Treatment with Doxorubicin and Purified Active MMPs.
SK-N-MC and SW-480 cells were incubated in serum-free DMEM with
doxorubicin (0.1 or 0.5 µg/ml, respectively) for 48 or 72 h,
respectively, in the presence or absence of human purified active
MMP-2, MMP-7, or MMP-9 (Oncogene Research; 10 nM). Cell
death was estimated using the MTT assay, as described previously
(21)
.
In Vitro FasL Cleavage Assay.
Human active MMP-2, MMP-7, and MMP-9 were tested for the ability to
digest human recombinant FasL in a 1x assay buffer [50 mM
Tricine buffer (pH 7.5), containing 0.2 M NaCl, 10
mM CaCl2, 5 µM
ZnSO4, 0.05% Brij-35 (polyoxyethylene lauryl
ether), and 0.02% NaN3], in the presence or
absence of the metalloproteinase inhibitor BB-3103 (British Biotech
Pharmaceuticals Ltd., Oxford, United Kingdom; 100 µM).
All reactions were terminated with Laemmli buffer and subjected to
SDS-PAGE. FasL was detected by immunoblotting as described.
Detection of Cell Surface FasL Expression.
SK-N-MC cells were incubated with or without MMP-7 (10 nM)
for 3 h in serum-free DMEM. Subsequently, cell surface FasL levels
were detected as described previously (21)
. Briefly, cell
surface proteins were biotinylated by incubating in 0.5 mg/ml
Sulfo-NHS-LC-Biotin (Pierce, Rockford, IL) in PBS for 30 min at room
temperature, as described previously (13)
.
Sulfo-NHS-LC-Biotin does not cross the cell membrane because of its
negative charge, assuring that intracellular proteins are not
biotinylated. Then, the cells were washed three times in cold PBS,
scraped, centrifuged briefly, and lysed for 30 min on ice in a lysis
buffer [50 mM Tris-HCl (pH 8), containing 120
mM NaCl and 1% Igepal], supplemented with the Complete-TM
mixture of proteinase inhibitors (Boehringer Mannheim). The samples
were cleared by centrifugation (14000 rpm for 30 min at 4°C) and
assessed for protein concentration. Biotinylated proteins, representing
the cell surface proteins, were immunoprecipitated with
Streptavidin-agarose for 2 h at 4°C and separated by SDS-PAGE,
and FasL levels were detected as described previously.
Cloning and Expression of Human MMP-7.
The full-length human MMP-7 sequence was amplified by PCR using an HT29
colon carcinoma expression library cloned into the pCDM8 vector and
appropriate synthetic oligonucleotide primers corresponding to
sequences encoding the NH2- and COOH-terminal
extremities of the precursor protein and cloned into the mammalian
expression vector pcDNA3.1-TOPO-V5-His (Invitrogen, Carlsbad, CA). The
correct orientation and sequence were verified by restriction analysis
and dideoxy sequencing, respectively. A construct expressing
constitutively active, V5-tagged MMP-7 (pcDNA3.1-caMMP7) was created by
removing nucleotides 64–282, corresponding to the inhibitory prodomain
(amino residues 22–94), using the Excite site-directed mutagenesis kit
(Stratagene, La Jolla, CA). The deletion was verified by sequencing.
For verification of enzymatic activity, NIH3T3 cells were transfected
with pcDNA3.1-caMMP7 or empty vector with Lipofectamine-Plus (Life
Technologies, Inc., Gaithersburg, MD). Forty-eight h later, the cells
were harvested, and zymographic analysis performed as described
previously (23)
, revealed strong MMP activity,
corresponding to MMP-7 in cells transfected with pcDNA3.1-caMMP7 but
not the empty vector. For use as a negative control, pcDNA3.1-caMMP7
was mutated at the active center (Glu 215 to Ala; pcDNA3.1-MMP7mut215)
using the QuikChange site-directed mutagenesis kit (Stratagene).
SK-N-MC cells were transfected with pcDNA3.1-caMMP7, pcDNA3.1-MMP7mut215, or empty vector using Superfect. Forty-eight h later, the cells were treated with or without doxorubicin (0.1 µg/ml) for additional 48 h. Cell death was evaluated with MMT as described previously.
Immunoprecipitation of MMP-7 and FasL.
SK-N-MC cells were transfected with pcDNA3.1-MMP7mut215-V5 or empty
vector using Superfect. Forty-eight h later, the cells were lysed and
incubated with 1 µg/ml anti-V5 monoclonal antibody overnight. After
precipitation with protein G-Sepharose and SDS-PAGE, immunoblotting
analysis for FasL was performed.
Transfection with Antisense MMP7.
The full-length human MMP-7 sequence, amplified by PCR as described
previously, was cloned in the antisense orientation into the mammalian
expression vector pcDNA3.1-TOPO-V5-His (Invitrogen) and transiently
transfected into the SK-N-MC cells using Superfect. Forty-eight h after
the initiation of the transfection, the cells were treated with or
without doxorubicin (0.1 µg/ml) for an additional 48 h. Cell
death was evaluated with MMT as described previously. The inhibition of
MMP7 expression was verified by Western blotting.
To delineate the role of FasL in the protective effect that MMP7 was found to have on doxorubicin-induced apoptosis, the same transient transfection experiment was performed in SK-N-MC cells that had been stably transfected previously with the pcDNA3.1 vector carrying the human FasL sequence in the antisense orientation and, thus, lacked FasL. As a control, SK-N-MC cells stably transfected with the pcDNA3.1 vector carrying the chloramphenicol acetyltransferase gene were used.
Immunohistochemistry.
Ten formalin-fixed, paraffin-embedded specimens removed from lung
metastases of Ewing’s sarcoma patients were obtained from the files of
the Laboratory of Pathology at the National Cancer Institute and
stained for MMP-7 expression with the monoclonal antibody Ab-1
(dilution, 1:100). The avidin-peroxidase method was used, as described
previously (12)
.
Statistical Analysis.
Quantitative comparisons were examined with the ANOVA method, followed
by Duncan’s test.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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50% of the cells undergo
apoptosis, as determined by MTT and terminal
deoxynucleotidyltransferase-mediated nick end labeling assays. We have
shown previously that expression of both Fas and FasL is increased in
SK-N-MC cells treated with cytotoxic agents (24)
. To
provide evidence that FasL up-regulation and its subsequent interaction
with Fas are required for anticancer drug-induced apoptosis, we
transiently transfected the SK-N-MC cell line with an expression vector
containing a cDNA encoding the extracellular domain of human Fas
ligated to the constant region of the Fc fragment of human IgG1
(Fas-IgG). Expression of this construct leads to production of a
soluble Fas-IgG chimeric molecule (FasRg; Ref. 22
) that
may act as a decoy inhibitor of Fas activation. A CD8-IgG construct
(22)
was used as a control. Transient expression of FasRg,
but not CD8Rg, reduced doxorubicin-induced SK-N-MC cell death,
supporting the notion that Fas/FasL plays a role in
doxorubicin-associated cytotoxicity (Fig. 1A)
.
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and to induce
SW-480 cell death, albeit more slowly (after 72–96 h of incubation).
Similar to SK-N-MC cells, doxorubicin-induced apoptosis was inhibited
by transient expression of FasRg but not CD8Rg (Fig. 1C)
,
suggesting that Fas engagement by FasL is not a cell type-restricted
event in cytotoxic drug-mediated tumor cell death. This notion is
supported by reports of a similar role for a Fas/FasL interaction in
chemotherapy-induced cell death in leukemic (25)
, hepatoma
(26)
, and neuroblastoma cells (27)
and brain
tumors (28)
.
Proteolytically Active MMP-7 Protects Ewing’s Sarcoma and Colon
Carcinoma Cells from Doxorubicin-induced Apoptosis and Cleaves FasL.
Our observations that FasL is cleaved from the cell surface by an
MMP-like enzyme and that FasL shedding protects tumor cells from
undergoing apoptosis in culture, whereas inhibition of FasL shedding by
a synthetic MMPI sensitizes tumor cells to doxorubicin (12
, 21)
, raised the possibility that MMP activity may augment cancer
cell resistance to cytotoxic drugs. We therefore tested the effect of
MMPs that are commonly expressed by tumor cells and that have been
implicated in tumor progression, including MMP-2, MMP-7, and MMP-9, on
the survival of doxorubicin-treated SK-N-MC and SW-480 cells.
Exogenously added purified active human MMP-7, but not MMP-2 or MMP-9,
inhibited doxorubicin-induced death of SK-N-MC (Fig. 2A)
and SW-480 (Fig. 2B)
cells.
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19,000), and cleavage was completely inhibited by the
hydroxamic-acid based MMP inhibitor BB-3103 (Fig. 3A)
. Because we have shown previously that MMP inhibitors
increase the levels of FasL on the surface of SK-N-MC cells
(21)
, we assessed the effect of proteolytically active
MMP7 on cell surface FasL expression. As expected, surface FasL levels
were reduced in SK-N-MC cells incubated with active MMP7 (Fig. 3B)
.
|
.
To demonstrate physical interaction between MMP-7 and FasL in
vivo, we performed coimmunoprecipitation experiments.
Enzymatically inactive, V5-tagged MMP-7 was transiently expressed in
SK-N-MC cells, and 48 h later, the cells were harvested and lysed,
and MMP-7 was immunoprecipitated using an anti-V5 monoclonal antibody.
Immunoprecipitates were subjected to SDS-PAGE, transferred onto
blotting filters, and tested for the presence of FasL by Western blot
analysis using anti-FasL antibody (Fig. 3D)
. FasL was
observed to coimmunoprecipitate with MMP-7, providing evidence that the
two molecules interact in tumor cells.
The Protective Effect of MMP-7 on Doxorubicin-induced Apoptosis
Is FasL dependent.
The importance of MMP7 in conferring protection against anticancer
drug-induced apoptosis was further underscored by observing the effect
of specific inhibition of MMP7 expression on the survival of SK-N-MC
cells after exposure to doxorubicin. The coding sequence of human MMP-7
was cloned in antisense orientation into the mammalian expression
vector pcDNA3.1 and was transiently expressed in SK-N-MC cells. The
resulting transfectants displayed reduced expression of MMP-7 protein
and an increased sensitivity to doxorubicin compared with cells
transfected with the expression vector alone (Fig. 4A)
. To further determine whether MMP-7-dependent reduction in
doxorubicin-induced tumor cell death implicates FasL, antisense MMP-7
cDNA was transiently expressed in SK-N-MC cells that lack FasL
expression attributable to the stable expression of a plasmid carrying
the human FasL sequence in antisense orientation. These cells were
found to be resistant to doxorubicin and were not sensitized by
transient transfection of antisenseMMP7 (Fig. 4B)
,
supporting the notion that the protective effect of MMP7 is mediated by
FasL processing.
|
. Of 10 tumor specimens of the Ewing’s sarcoma family that
were evaluated immunohistochemically, MMP-7 immunoreactivity was found
in 9 (Fig. 5B)
.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Shedding of FasL from the cell surface has been observed in activated immune cells (13) and tumor cells (12) and may provide a mechanism for partial protection of Fas-sensitive cells from apoptosis (21) . Biochemical evidence has suggested that FasL shedding is catalyzed by matrix metalloproteinase type enzymatic activity (12 , 13 , 21) , and a recent report has shown that MMP-7 cleaves FasL in vitro and colocalizes with FasL on prostate epithelial cells (14) . In our study, MMP-7 expressed in tumor cells was found to coimmunoprecipitate with FasL and to reduce FasL protein expression on the tumor cell surface. The observed reduction in cell surface FasL expression was most likely attributable to proteolytic cleavage, because MMP-7 cleaved FasL in vitro, consistent with findings by others (14) . Because soluble FasL displays reduced proapoptotic potency compared with its cell surface precursor (12 , 13 , 15 , 16 , 21) , MMP-7-mediated FasL cleavage could provide tumor cells with a mechanism to evade apoptosis induced by the concomitant expression (constitutive or cytotoxic drug-induced) of functional Fas and FasL, thereby facilitating tumor survival and progression. This notion is supported by the observations that exogenous, proteolytically active MMP-7, but not MMP-2 or MMP-9, inhibited doxorubicin-induced tumor cell death and that this inhibitory effect could be reproduced by overexpression of active MMP-7 but not of an enzymatically inactive mutant.
Recent work by others (14) suggests that MMP-7-mediated FasL cleavage enhances prostate involution, and that the apoptotic index of involuting prostate epithelium in MMP-7-deficient mice is reduced. Taken at face value, these observations suggest that MMP-7 expression correlates with increased apoptosis, which would appear to contradict our present findings. However, in vivo behavior of tissues in MMP-7 null mice may be subject to the action of potential compensatory mechanisms for the loss of MMP-7. Moreover, normal epithelial cells and tumor cells may display different sensitivity to signals induced by soluble FasL. It is therefore possible that by cleaving FasL, MMP-7 may help promote involution of normal tissues while contributing to the survival of neoplastic cells. MMP-7 is widely expressed in human cancer, including colorectal (30) , gastric (30) , esophageal (31) , and endometrial carcinomas as well as in Ewing’s sarcoma cell lines and metastatic tumor specimens, as we showed in this study. In support of our present findings, MMP-7 expression levels have been shown to correlate with the clinical aggressiveness of various tumor types (31) , and overexpression of MMP-7 in colon carcinoma cells increased their tumorigenicity in vivo without altering their invasiveness in vitro (32) . These observations support the notion that tumor progression associated with MMP-7 activity may not be attributable to proteolytic degradation of a structural component of the ECM but rather may relate to the regulation of tumor cell survival itself. Consistent with this possibility, we found that down-regulation of MMP-7 protein levels, by transfection of a plasmid carrying the human MMP-7 sequence in antisense orientation sensitized tumor cells to doxorubicin, supporting our previous finding that inhibition of FasL shedding by a broad-spectrum synthetic MMPI sensitizes tumor cells to doxorubicin (21) . Taken together, these observations suggest that specific inhibition of MMP7 may provide a potentially effective approach toward increasing the efficacy of chemotherapy in at least a subset of carcinomas.
Our observations do not exclude the possibility that other MMPs may play a similar role to that of MMP-7 uncovered here in other cell types in which cytotoxic drugs exert part or most of their effect through the Fas pathway. It is also possible that MMP-7 may cooperate with other MMPs in the catalysis of FasL shedding. Nevertheless, the regulation by a specific MMP of FasL cleavage and cytotoxic drug-induced apoptosis may not only shed new light onto the complex mechanisms by which MMP activity controls tumor invasion and metastasis but provide a potential target for the development of new therapeutic approaches that may significantly augment the efficacy of conventional chemotherapy.
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at Department of Adult Oncology, Dana-Farber Cancer
Institute, Mayer Building, Room M557, 44 Binney Street, Boston, MA
02115. Phone: (617) 632-2681; Fax: (617) 632-2140; E-mail: mitsiades{at}netscape.net 
2 The abbreviations used are: FasL, Fas ligand;
sFasL, soluble FasL; MMP, matrix metalloproteinase; MMPI, MMP
inhibitor; ECM, extracellular matrix; Rg, receptor globulin; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
Received 8/28/00. Accepted 11/27/00.
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 L. E. Wroblewski, P.-J. M. Noble, A. Pagliocca, D. M. Pritchard, C. A. Hart, F. Campbell, A. R. Dodson, G. J. Dockray, and A. Varro Stimulation of MMP-7 (matrilysin) by Helicobacter pylori in human gastric epithelial cells: role in epithelial cell migration J. Cell Sci., July 15, 2003; 116(14): 3017 - 3026. [Abstract] [Full Text] [PDF]
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 N. Mitsiades, C. S. Mitsiades, V. Poulaki, D. Chauhan, G. Fanourakis, X. Gu, C. Bailey, M. Joseph, T. A. Libermann, S. P. Treon, et al. Molecular sequelae of proteasome inhibition in human multiple myeloma cells PNAS, October 29, 2002; 99(22): 14374 - 14379. [Abstract] [Full Text] [PDF]
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T. Vargo-Gogola, B. Fingleton, H. C. Crawford, and L. M. Matrisian Matrilysin (Matrix Metalloproteinase-7) Selects for Apoptosis-resistant Mammary Cells in Vivo Cancer Res., October 1, 2002; 62(19): 5559 - 5563. [Abstract] [Full Text] [PDF]
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G. Hashimoto, I. Inoki, Y. Fujii, T. Aoki, E. Ikeda, and Y. Okada Matrix Metalloproteinases Cleave Connective Tissue Growth Factor and Reactivate Angiogenic Activity of Vascular Endothelial Growth Factor 165 J. Biol. Chem., September 20, 2002; 277(39): 36288 - 36295. [Abstract] [Full Text] [PDF]
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F. Zuo, N. Kaminski, E. Eugui, J. Allard, Z. Yakhini, A. Ben-Dor, L. Lollini, D. Morris, Y. Kim, B. DeLustro, et al. Gene expression analysis reveals matrilysin as a key regulator of pulmonary fibrosis in mice and humans PNAS, April 30, 2002; 99(9): 6292 - 6297. [Abstract] [Full Text] [PDF]
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 D. Sheppard Uses of Expression Microarrays in Studies of Pulmonary Fibrosis, Asthma, Acute Lung Injury, and Emphysema : Roger S. Mitchell Lecture Chest, March 1, 2002; 121(2007): 21S - 25S. [Abstract] [Full Text] [PDF]
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C. J. Morrison, G. S. Butler, H. F. Bigg, C. R. Roberts, P. D. Soloway, and C. M. Overall Cellular Activation of MMP-2 (Gelatinase A) by MT2-MMP Occurs via a TIMP-2-independent Pathway J. Biol. Chem., December 7, 2001; 276(50): 47402 - 47410. [Abstract] [Full Text] [PDF]
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) as compared with the empty vector (
;
P < 0.001). In contrast, the latter
cells, which lacked FasL expression, were constitutively
resistant to doxorubicin and were not sensitized by transfection of
pcDNA3.1-antisense MMP-7. Mean values from triplicate experiments are
shown; bars, SD.