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Experimental Therapeutics |
Departments of Experimental Therapeutics [J-J. B., X-F. L., J. Y., L. W., Y. Y. and R. C. B.], Blood and Marrow Transplantation [R-Y. W., M. A.], Thoracic and Cardiac Surgery [B. F.], Biomathematics [E. N. A.], and Molecular Therapeutics [R. L.], University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Introduction of ARHI into cells that retain ARHI expression fails to inhibit clonogenic growth, consistent with the possibility that ARHI overexpression might spare normal cells and selectively inhibit growth of cancer cells (1) . Evaluation of the potential of ARHI for gene therapy requires a method for efficient transfer and high-level expression of ARHI in cancer cells. Initial attempts to package ARHI in a single adenoviral vector failed to produce high titers of virus, related in part to the viability of packaging cells. To obtain a high-titered adenovirus preparation that could achieve high levels of ARHI expression in cancer cells, we have used a binary adenoviral vector system (2) .
The present report examines the potential of ARHI for gene therapy against human ovarian and breast cancer cells grown in anchorage-dependent cultures and as heterografts in immunosuppressed mice. The mechanism by which ARHI expression inhibits cell growth has also been explored, examining possible arrest of the cell cycle and induction of apoptosis. ARHI reexpression has been shown to induce apoptosis through a caspase-independent, calpain-dependent mechanism.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Binary Adenoviral Expression System.
Initial attempts to package ARHI in 293 cells were not successful and yielded only low titers of virus, related in all probability to the toxic effects of ARHI expression on 293 packaging cells. Consequently, a binary adenoviral vector system was constructed that required infection with two different adenoviral vectors to achieve high levels of ARHI expression (9)
. Briefly, full-length ARHI cDNA was cloned into an adenoviral vector downstream of a GT (GAL4/TATA) promoter that contained five GAL4-binding sites and a TATA box (Fig. 1)
. This Ad/GT-ARHI construct exhibited very low transcriptional activity in vitro and in vivo. However, ARHI expression could be induced by coinfecting cells with Ad/GT-ARHI and with the adenoviral vector Ad/PGK-GV16 that contained a promoter from the mouse housekeeping gene PGK and a GAL4-VP16 fusion protein that can transactivate the GT promoter. Controls were provided by coinfecting cells with Ad/PGK-GV16 and with Ad/GT-LacZ, an adenovirus that contained the Escherichia coli ß-galactosidase gene under the control of a GT promoter.
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Adenovirus Coinfection and Detection of ARHI Protein.
Ovarian and breast cancer cells growing as monolayers were detached with 0.1% EDTA and 0.25% trypsin (Cellgro), washed twice in culture medium, and plated in 60-mm culture dishes with 4 ml of culture medium 24 h before infection with adenoviral vectors. Supernatant medium was aspirated, and fresh medium containing different combinations of adenoviral vectors was added to different plates. The Ad/GT-ARHI or Ad/GT-LacZ vector was mixed with the Ad/PGK-GV16 induction vector at a ratio of 2:1 unless otherwise indicated. This ratio was found to be optimal in preliminary experiments. Mixtures of the vectors are referred to as Ad/ARHI/GV16 and Ad/LacZ/GV16, respectively.
For detection of ARHI expression, cells were washed with PBS two times and lysed in radioimmunoprecipitation assay buffer [50 mM HEPES (pH 7.25), 150 mM NaCl, 50 µM ZnCl2, 50 µM NaH2PO4, 50 µM NaF, 2 mM EDTA, 1% NP40, 0.1% SDS, 0.1% sodium deoxycholate, 1 mM NaVO4, and 1 mM phenylmethylsulfonyl fluoride]. Cell lysates were centrifuged at 10,000 x g for 15 min. The protein concentration of supernatant fluid was measured by the Bradford assay (Bio-Rad). Equal amounts of supernatant protein were separated by 12% SDS-PAGE and immunoblotted with anti-ARHI murine monoclonal antibody 15E11 (1) . Horseradish peroxidase-conjugated goat antimouse IgG served as the secondary antibody for the enhanced chemiluminescence detection system (ECL; Amersham).
Cell Growth and Mitotic Index.
Ovarian or breast cancer cells (3–5 x 105) were seeded in 100-mm culture dishes for 20 h before virus infection. Medium was aspirated from triplicate cultures and replaced with medium supplemented with diluent, Ad/ARHI/GV16, or Ad/LacZ/GV16. At different intervals, cells were trypsinized, and viable cells that excluded trypan blue dye were counted in a hemocytometer. The average numbers of cells per milliliter were calculated and plotted as a function of time.
To measure mitotic index, cells were plated in chamber slides. After 20 h, medium was replaced and supplemented with diluent, Ad/ARHI/GV16, or Ad/LacZ/GV16. At different intervals, cells were fixed in 4% paraformaldehyde for 10 min at room temperature and stained with H&E. The number of mitoses among 1000 cells was counted and presented as the mitotic index.
Clonogenic Growth.
Ovarian or breast cancer cells (106) were incubated in 5-ml polypropylene tubes with diluent or infected with Ad/ARHI/GV16 or Ad/LacZ/GV16 for 3 h and serially diluted 5-fold. From each dilution, aliquots of 100 µl were pipetted into 6 wells of a 96-well culture plate (10)
. An additional 100 µl of media were added to each well, and cells were incubated for 21 days. Clonogenic growth of surviving cancer cells was evaluated by inverted phase microscopy, scoring the number of wells with at least one colony containing more than 30 cells. Estimates of the most probable number of residual clonogenic units were calculated by a modification of the method of Spearman and Karber (11)
. The number of clonogenic units was calculated as shown below.
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Growth of Heterografts in nu/nu Mice.
The impact of ARHI expression on tumor growth in vivo was evaluated using heterografts of the human breast cancer MDA-MB-231 in immunosuppressed nu/nu mice. All animal experiments were carried out in accordance with the Guidelines for the Care and Use of Laboratory Animals (NIH Publication 85-23) and the institutional guidelines of The University of Texas M. D. Anderson Cancer Center. Human breast cancer xenografts were established in 6–8-week-old BALB/c nu/nu mice (Charles River). MDA-MB-231 cells (5 x 106) were injected s.c. into the mammary fat pad of each mouse. Tumors were measured twice a week, and tumor volumes were calculated as (a x b x c)1/3, where a, b, and c were three diameters. When palpable tumors had grown to a diameter of 0.5 cm, each mouse received three intratumoral injections of 50 µl of PBS containing 9 x 1010 vp (1.5 x 109 pfu) of Ad/ARHI/GV16 or Ad/LacZ/GV16. Mice that received PBS or PBS containing 9 x 1010 vp (1.5 x 109 pfu) of Ad/GT-ARHI only were used as controls. Animals were sacrificed when tumors reached a diameter of 
1.5 cm.
Cell Cycle Arrest and Apoptosis.
Ovarian and breast cancer cells (3–5 x 105) were plated in 100-mm culture dishes for 20 h and incubated with diluent, Ad/ARHI/GV16, or Ad/LacZ/GV16. After infection, cells were trypsinized and collected in PBS at different intervals and fixed on ice with 1% paraformaldehyde and subsequently with 70% cold ethanol. After treatment with 10 µg/ml RNase, cells were stained with 50 µg/ml propidium iodide for cell cycle analysis using a FACScan (Becton Dickenson). Apoptotic cells were detected with a TUNEL assay, performed according to the manufacturer’s instructions (BD Pharmingen). The fraction of apoptotic cells was confirmed in some assays with Annexin V. Cells were trypsinized and stained with Annexin V as described by the kit manufacturer (Roche Biochem).
cDNA Array Analysis.
SKOv3 ovarian cancer cells (9 x 106) were plated in 150-mm culture dishes and infected with Ad/ARHI/GV16 or Ad/LacZ/GV16 at 3,000 vp (50 pfu)/cell or 6,000 vp (100 pfu)/cell. After 3 days, total RNA was extracted using a Totally RNA Kit (Ambion). After DNase digestion, cDNA was generated from total RNA, labeled with Cy3 or Cy5 dye, and hybridized to a cDNA expression array that spotted 2304 known and uncharacterized genes in duplicate. The signal:noise ratio measurement provided by the quantification software (ArrayVision; Imaging Research, Inc., St. Catherine’s, Ontario, Canada) is defined as: spot density minus background density, divided by the SD of the background density. A "smooth t-statistic" was applied to correctly determine gene expression profiles on each array. A T-score was calculated as follows: T-score = (log(A) - log(B))/
, where is the SD across the entire array, log(A) is the log intensity of Cy3 dye (which labeled the cDNA sample from Ad/LacZ/GV16-infected cells), and log(B) is the log intensity of Cy5 dye (which labeled the cDNA sample from Ad/ARHI/GV16-infected cells). All procedures were carried out in the M. D. Anderson Cancer Center Genomics Core Facility.
Calpain Expression and Lack of Caspase Activation.
Ovarian or breast cancer cells (3–5 x 105) were plated in 100-mm culture dishes and incubated with diluent or infected with Ad/ARHI/GV16 or Ad/LacZ/GV16. DOV13 cells and MDA-MB-231 cells were incubated with 4000 vp (67 pfu)/cell, and SKOv3 cells were incubated with 6000 vp (100 pfu)/cell. At different intervals, cells were lysed, and proteins were analyzed by Western blot for caspase and calpain. Antibodies reactive with caspase 3, 6, 7, 8, 9, or 12 (Cell Signaling) and calpain (Chemicon) were incubated with immunoblotted protein. Goat antimouse or antirabbit IgG were used to develop the reactions. Enhanced chemiluminescence (Amersham) was used to detect proteins recognized by the immunological reagents. For analysis of PARP binding to cleaved DNA, cells were stained with a rabbit polyclonal anti-PARP antibody (Roche Biochem), and antibody binding was detected with fluoresceinated sheep antirabbit IgG. After washing, cells were examined using a fluorescence microscope.
Inhibition of Calpain Activity.
DOV13 and MDA-MB-231 cells (3–5 x 105) were plated in 100-mm culture dishes and incubated overnight to allow the formation of an adherent monolayer. Cancer cells were preincubated with calpain inhibitor I (5 µM), PD150606 (5 µM), or PD151746 (5 µM). Negative controls were provided by incubating cells for 2 h with diluent alone, with the inactive calpain inhibitor analogue PD145305 (5 µM), or the pan-caspase inhibitor Z-VAD-fmk (5 µM). After 2 h, medium containing the inhibitors or diluent was removed, and cells were incubated with fresh medium alone, Ad/ARHI/GV16, or Ad/LacZ/GV16 at 4000 vp (67 pfu)/cell. After 1 h, inhibitor or DMSO was added back to each dish. At 72 h postinfection, apoptosis was determined by flow cytometry using Annexin V.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Coinfection of SKOv3 ovarian cancer cells with Ad/LacZ/GV16 failed to induce detectable ARHI on Western blot analysis (Fig. 2)
. Infection with Ad/ARHI/GV16 produced a time- and dose-dependent expression of ARHI (Fig. 2)
. Neither Ad/GT-ARHI nor Ad/PGK-GV16 alone produced detectable ARHI expression (data not shown). With Ad/ARHI/GV16, ARHI protein was strongly expressed at 24 h (Fig. 2A)
and could be detected as early as 12 h after infection. ARHI expression could be readily detected for 5 days (Fig. 2A)
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. Increasing numbers of particles produced more intense ARHI expression with a maximum at 8000 particles (133 pfu)/cell (Fig. 2B
; additional data not shown). Using the same dual vector system, similar levels of ARHI could be reexpressed in MDA-MB-231 breast cancer cells and DOV13 ovarian cancer cells. The relative efficiency of adenovirus-mediated gene transfer was also assessed as the percentage of 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside-stained cells after infection with adenovirus Ad/LacZ/GV16 containing bacterial LacZ gene. Differences were detected between the cell lines tested. More than 90% of MDA-MB-231 breast cancer cells or DOV13 ovarian cancer cells were LacZ positive with a MOI of 4000 vp (67 pfu)/cell. A higher MOI of 6000 vp (100 pfu)/cell was needed to obtain the same level of transduction in SKOv3 ovarian cancer cells, correlating with the number of vp required for optimal ARHI expression. Consequently, different MOIs were used in additional studies of growth inhibition and apoptosis with each cell line.
ARHI Reexpression Inhibits Growth of Ovarian and Breast Cancer Cells.
The dual adenovirus system was used to determine the impact of ARHI reexpression on the growth of ovarian and breast cancer cell lines. Infection with Ad/ARHI/GV16 (4000 vp, 67 pfu) markedly inhibited anchorage-dependent growth of DOV13 ovarian cancer cells (Fig. 3A)
, MDA-MB-231 breast cancer cells (Fig. 3B)
, and SKOv3 ovarian cancer cells (Fig. 3C)
between 3 and 5 days after infection. The number of uninfected DOV13 cells increased 347% over 5 days, whereas the number of DOV13 cells infected with Ad/ARHI/GV16 increased 30% (Fig. 3A)
. Coinfection with Ad/LacZ/GV16 produced slight but significant inhibition, seen most clearly on days 4 and 5. Consequently, adenoviral infection or overexpression of the bacterial galactosidase protein may also partially inhibit growth of ovarian and breast cancer cell lines in culture.
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. At an optimal dose of 4000 vp (67 pfu)/cell, reexpression of ARHI inhibited clonogenic growth by about 2 log units (99%), whereas incubation with Ad/LacZ/GV16 inhibited clonogenic growth by <0.3 log units (50%). Reexpression of ARHI exerted quantitatively different effects in other cancer cell lines. Coinfection with Ad/ARHI/GV16 inhibited clonogenic growth of SKOv3 cells by 1.5 log units (data not shown).
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. Less marked inhibition of tumor growth was observed with the Ad/GT-ARHI vector alone or with Ad/LacZ/GV16. Significantly greater inhibition was observed after treatment with Ad/ARHI/GV16 than after treatment with Ad/GT-ARHI alone or with Ad/LacZ/GV16.
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. No G0-G1 arrest was produced by infection with Ad/LacZ/GV16 at any time interval studied. An increase in G2-M cells was observed after coinfection with either Ad/ARHI/GV16 or Ad/LacZ/GV16 when compared with mock-infected controls, consistent with an effect of adenovirus infection. When a mitotic index was calculated, ARHI expression reduced mitotic cells by 44–72% between 1 and 5 days after infection (Table 1)
. During the first 2 days, infection with Ad/Lacz/GV16 also decreased the mitotic index, but this nonspecific effect of viral infection or overexpression of foreign protein was not observed on days 3–5. Taken together, these data indicate that reexpression of ARHI induces G1 arrest in MDA-MB-231 cells for at least 64 h and decreases the mitotic index.
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. At 72 h after infection, about 30% of ARHI-infected MDA-MB-231 cells had undergone apoptosis, and this percentage increased to more than 45% by day 5 after infection. An excess of apoptotic cells was not observed after mock infection or after infection with Ad/LacZ/GV16. ARHI reexpression induced apoptosis in other cancer cell lines, albeit at a different level. In SKOv3, a maximum of 11% apoptotic cells was observed after Ad/ARHI/GV16 infection, with baseline rates of apoptosis of around 2% for Ad/LacZ/GV16 and mock-infected cells (Fig. 7A)
. Consequently, growth inhibition may relate to both cell cycle arrest and apoptosis, depending on the interval after infection and on the cell line.
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, active fragments of caspase 3 (Fig. 9A)
, 6 (data not shown), 7 (Fig. 9B)
, 8 (Fig. 9C)
, 9 (data not shown), and 12 (Fig. 9D)
were not found on Western analysis studied at multiple intervals after infection.
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with a T-score of -8.452 after infection with 50 pfu and -12.568 after infection with 100 pfu. ARHI-induced up-regulation of calpain was confirmed by Western blot analysis (Fig. 9E)
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both inhibited ARHI-induced apoptosis, whereas the inactive analogue PD145305 did not (Fig. 10C)
. Caspase inhibitor Z-VAD-fmk failed to inhibit ARHI-induced apoptosis (Fig. 10D)
. Taken together, these data suggest that reexpression of ARHI induces caspase-independent, calpain-dependent apoptosis. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Inhibition of tumor growth could be related to at least two mechanisms. Reexpression of ARHI protein caused cell cycle arrest and apoptosis in MDA-MB-231 breast cancer cells. Similar results were observed in ovarian cancer cell lines. Apoptosis often involves activation of caspases (12) . A careful examination of caspase expression and cleavage failed to reveal activation of caspase 3, 6, 7, 8, 9, or 12 after ARHI reexpression. By contrast, calpain was overexpressed and cleaved in cancer cells after expression of ARHI. Several calpain inhibitors prevented ARHI-induced apoptosis, but the pan-caspase inhibitor Z-VAD-fmk did not.
Calpains are a family of calcium-activated cysteinyl proteases that include several tissue-specific isoforms (n-calpain) and two ubiquitous isozymes (µ-calpain and m-calpain; Ref. 12 ). Calpains are distributed widely throughout the cytosol of many cell types. Activation of calpain is an early event in the cellular response to a variety of stressful and potentially pathogenic conditions. Prolonged activation of calpains can lead to cell death (13) .
Calpains are regulated by Ca2+ and target cellular proteins that are instrumental for maintaining the integrity of the cytoskeleton, making them attractive candidates for mediating certain events during apoptosis. Calpain I may be activated in the cytosol or when bound to the cell membrane, whereas calpain II activation occurs primarily at membrane sites. Our results showed ARHI stimulated only calpain I. Of interest are recent reports that inhibitors of JNK activation block calpain and caspase-induced apoptosis in neurons (14) . Reexpression of ARHI has been shown to induce JNK activation,5 and this may be an important mechanism for activating calpains.
Calpains have been implicated in apoptosis based on two types of observations: (a) the activation of calpains during cell death; and (b) the inhibition of apoptotic execution by various calpain inhibitors. In this study, we provide evidence that calpain is involved in ARHI-induced apoptosis, demonstrating that ARHI-infected ovarian and breast cancer cells exhibit increased amounts of calpain, calpain is cleaved, and inhibition of calpain activity reduces apoptosis. Currently available calpain inhibitors include modified peptides that compete for the active site of the protease (15 , 16) and inhibitors that do not bind to the active site, whose selectivity comes from their unique interaction with the calcium-binding domains of calpain, a feature not found in other protease inhibitors. In our experiments, both classes of calpain inhibitor resulted in significant reduction of apoptosis induced by ARHI infection. Known calpain substrates include cytoskeletal associated proteins, kinases and phosphatases, membrane receptors, transporters, and oncogenes.
Recently, Gil-Parrado et al. (17) reported that calpains may cleave BCL-2 and permit the translocation of Bax to the mitochondria. Bid may also be cleaved and presumably translocated to the mitochondria, amplifying the apoptotic signaling pathway. In the case of ARHI-induced apoptosis, these mechanisms may not be relevant because caspase activation was not observed.
Taken together, our data indicate that reexpression of ARHI in cells that have lost expression of this gene can produce a modest G0-G1 cell cycle arrest and more marked induction of apoptosis. Inhibition of tumor cell growth appears to be related predominantly to caspase-independent, calpain-dependent apoptotic events.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by NIH Grants CA64602 (to R. C. B.) and CA80957 (to Y. Y.), Cancer Center Support Grant CA16672 (to M. A.), and a grant from the Susan G. Komen Breast Cancer Foundation (to Y. Y.) 
2 To whom requests for reprints should be addressed, at Office of Translational Research, Box 355, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 792-7743; Fax: (713) 745-2107; E-mail: rbast{at}mdanderson.org
3 The abbreviations used are: JNK, c-Jun-NH2-terminal kinase; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; PARP, poly(ADP-ribose) polymerase; vp, virus particle(s); pfu, plaque-forming unit(s); MOI, multiplicity of infection.
4 Y. Yu, J. J. Bao and R. C. Bast, unpublished data.
Received 6/11/02. Accepted 10/10/02.
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REFERENCES |
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