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Coexistence of High Levels of Apoptotic Signaling and Inhibitor of Apoptosis Proteins in Human Tumor Cells

Implication for Cancer Specific Therapy¹

Department of Surgery and Winship Cancer Institute, Emory University School of Medicine, Atlanta, Georgia 30322

	ABSTRACT

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It is well known that dysfunction of the apoptotic pathway confers apoptosis resistance and results in a low sensitivity of human cancer cells to therapeutic agents. A novel strategy to overcome the resistance is to target the apoptotic pathway directly. To identify molecular targets in the apoptotic pathway that are differentially regulated in cancer and normal cells, we have examined the levels of apoptotic effectors and inhibitors in human tumor and normal cell lines as well as in cancer and normal tissues. These include three pancreatic cancer lines (BXPC-3, MIA PaCa-2, and Panc-1), four breast cancer cell lines (MDA-MB-231, MDA-MB-435, MDA-MB-361, and MCF-7), and colon carcinoma line (SW620). Additionally, breast carcinoma tissue specimens were examined. Compared with normal human fibroblast and mammary epithelial cell lines, we detected high basal levels of caspase-3 and caspase-8 activities and active caspase-3 fragments in the tumor cell lines and cancer tissues in the absence of apoptotic stimuli. Furthermore, the tumor cells expressed high levels of survivin and XIAP, two members of the inhibitor of apoptosis (IAP) protein family. When the activity of these IAPs was blocked by expression of dominant-negative mutant survivin (survivinT34A) and XIAP-associated factor 1, respectively, apoptosis was induced in tumor but not normal cell lines. Moreover, down-regulation of both survivin and XIAP significantly enhanced tumor-cell apoptosis as compared with inhibition of either survivin or XIAP alone. These results suggest that up-regulated IAP expression counteracts the high basal caspase-3 activity observed in these tumor cells and that apoptosis in tumor cells but not normal cells can be induced by blocking IAP activity. Therefore, IAPs are important molecular targets for the development of cancer-specific therapeutic approaches.

	INTRODUCTION

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Impairment of the apoptotic signaling pathway plays an important role in the initiation and progression of human cancers (1, 2, 3, 4) . Recent studies have shown that apoptotic cell death is also critical for the response of many cancer therapeutic agents (5, 6, 7, 8) . To develop innovative strategies for cancer therapy, it is necessary to identify molecular targets in the apoptotic pathway that are differentially regulated in normal and cancer cells. Many cellular factors involved in apoptosis have been identified and their roles in the apoptotic pathway are elucidated. Apoptosis is initiated through an extrinsic pathway by the interaction of cell death receptors with their ligands or an intrinsic pathway triggered by leakage of cytochrome c from mitochondria. These result in activation of a cascade of caspase proteolysis reaction (2 , 9) . Caspases can be divided into two groups based on the length of their prodomain and substrate specificity. The initiator caspase group includes caspase-2, caspase-8, caspase-9, and caspase-10, having long NH₂-terminal prodomains, which interact with adapter molecules to form a death-inducing signaling complex. Downstream caspases such as caspase-3, caspase-6, and caspase-7 are executioner caspases that remain dormant until the initiator caspases activate them by proteolysis (9) . The activated executioner caspases cleave a number of structural and regulatory proteins, leading to apoptotic cell death (10 , 11) .

Recently, the IAP⁴ family, including XIAP, c-IAP1, c-IAP2, ML-IAP, NAIP, survivin, and Apollon, has been characterized (2 , 12, 13, 14) . These proteins contain a novel 80 amino acid motif that is defined as the BIR (12) . XIAP is the most potent caspase inhibitor in the IAP family. It has been shown that XIAP prevents the proteolytic processing of procaspase-3, procaspase-6, and procaspase-7 by binding and blocking the activity of caspase-9 through its BIR 3 domain. XIAP additionally inhibits the activity of active caspase-3 through a short linker region that precedes BIR 2 (15 , 16) . Another IAP protein, survivin, counteracts a broad range of apoptosis stimuli such as Fas stimulation, overexpression of Bax and caspases, and chemotherapy drugs (17) . At present, it is still controversial whether survivin directly binds and inhibits caspase-3. A comparison of the X-ray crystallographic structures of survivin with that of the XIAP:caspase-3 complex indicates lack of a structural basis for survivin-caspase-3 interaction (18) . XIAP is detected at a low level in normal adult tissues. Survivin is expressed during fetal development but not in most adult tissues (19) .

Increasing evidence demonstrated that IAPs, especially XIAP and survivin, are up-regulated in many human tumor types (4 , 13 , 20 , 21) . A recent study showed up-regulation of survivin in 77% of pancreatic duct cell adenocarcinoma, and survivin was expressed in early stages of neoplastic transition in pancreatic cancer cells. On the other hand, survivin was not detected in normal pancreatic tissues (4) . Approximately 71% of breast cancers were positive for survivin expression while surrounding normal tissues are negative. An increase in the level of survivin contributed to a higher apoptotic threshold and survival of the breast cancer cells (21) . Survivin was also detected in 64% of human colorectal cancers. Five-year survival rate in the stage II patients with positive survivin were significantly lower than that of the survivin-negative patients (20) . Furthermore, a high level of XIAP was also detected in human tumors and has been shown to confer the resistance to chemotherapy drugs (22, 23, 24) .

A logical strategy to overcome resistance to apoptosis is to directly target the apoptotic pathway. Gene therapy vectors have been used to deliver apoptosis inducer genes such as Fas ligand, Bax, and caspases; induction of apoptosis was observed in various human tumor cell lines after transduction of these genes (25, 26, 27, 28) . Another promising approach is to decrease the apoptotic threshold in tumor cells by counteracting IAP function. A XAF1, which is present in many normal tissues but is low or absent in tumors, abolishes the function of XIAP through direct interaction of the proteins (29 , 30) . In addition, expression of antisense or mutant survivin genes inhibited tumor proliferation and induced apoptosis in vitro, as well as in animal tumor models (31) . One of the mutants contains an amino acid substitution from threonine (T) to alanine (A) at 34 position (survivinT34A). Expression of this construct blocked phosphorylation of survivin on Thr³⁴, resulting in dissociation of survivin-caspase-9 complex and induction of caspase-9-dependent apoptosis in tumor cells (32) . Interestingly, expression of survivin T34A selectively induced apoptotic cell death in tumor cells but not in normal cells (31) .

Although induction of apoptotic cell death by expression of either XAF1 or dominant-negative survivin has been demonstrated in human tumor cells, mechanisms for selective induction in tumor cells are largely unknown. In this study, we compared the levels and activities of apoptotic effectors and inhibitors in human cancer cell lines and tissues with normal cell lines and tissues. We demonstrated that human tumor cell lines have constitutively activated caspase activities in the absence of apoptotic stimuli and yet are not undergoing apoptosis. These cell lines also have high levels of survivin and XIAP. Therefore, we hypothesized that inhibition of IAPs could induce apoptotic cell death selectively in tumor cells that have an activated apoptotic signaling. Results of our study additionally demonstrated that expression of IAP-counteracting proteins such as survivinT34A and XAF1 could indeed lead to specific apoptosis in the tumor cell lines.

	MATERIALS AND METHODS

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Human Tumor and Normal Cell Lines
Pancreatic cancer cell lines BXPC-3, MIA PaCa-2, and Panc-1, colon cancer cell line SW620, and normal immortalized human mammary epithelial cell line MCF-10A were obtained from the ATCC (Manassas, VA). Breast cancer cell lines MDA-MB-231, MDA-MB-361, and MCF-7 were from ATCC, and MDA-MB-435 was kindly provided by Dr. Zhen Fan (M. D. Anderson Cancer Center, Houston, TX). Primary normal human dermal fibroblast cell line HDF was purchased from Emory University Skin Disease Center (Atlanta, GA). BXPC-3 and SW620 cell lines were cultured in RPMI medium, and MIA PaCa-2 cells were cultured in DMEM (Mediatech, Herndon, VA). Human breast cancer cell lines were maintained in DMEM:Ham’s F-12 medium (50:50; Mediatech). All above media were supplemented with 10% FBS (Hyclone, Logan, UT), 2 mM L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin (Mediatech). HDF was maintained in DMEM with 20% FBS. MCF-10A cells were cultured in DMEM:Ham’s F-12 medium supplemented with 20 ng/ml epidermal growth factor, 500 ng/ml hydrocortisone, 100 ng/ml cholera toxin, 10 µg/ml insulin, 2 mM L-glutamine, and 5% FBS.

Human Normal and Breast Cancer Tissues
Frozen human breast cancer and normal tissues were obtained from the Emory Healthcare Tissue Procurement Facility according to an approved Institutional Review Board protocol. The identification number and pathological diagnosis of each sample were provided by the Tissue Procurement Facility. Paired breast cancer and normal tissues were obtained from the patients during operation to resect the breast cancer. The normal tissues were selected from the normal breast areas by the pathologists at Emory University. Some of the breast cancer tissue samples were obtained from the breast cancer patients during surgical resection of the cancer without obtaining paired normal samples. Normal breast tissues from normal subjects undergoing breast reduction surgery were also collected for this study.

Detection of Caspase Activity in Human Tumor and Normal Cell Lines or in Breast Cancer and Normal Tissues
Caspase-3, caspase-8, and caspase-9 activities were examined either in cell lysates or in viable cells by fluorometric assays.

Determination of Caspase Activity in Solution Using Cell Lysates.
Human tumor and normal cell lines were cultured in specific media as described above. To examine the basal caspase-3, caspase-8, and caspase-9 activities, the cell pellets from each cell line at 80% confluence were collected and lysed with the lysis buffer containing 10 mM Tris-HCl (pH 7.5), 10 mM NaH₂PO4/NaHPO₄ (pH 7.5), 130 mM NaCl, 1% Triton X-100, and 10 mM Na₂P₂O₇ 10 H₂O. The tissue lysates were prepared from frozen normal and cancer tissues by homogenizing in the lysis buffer. Caspase activity in the cell lysates was examined using substrates specific for caspase-3-like (Ac-DEVD-AFC), which detects activities of caspase-3, caspase-7, and caspase-10, caspase-8 (Ac-LETD-AFC), or caspase-9 activity (Ac-LEHD-AFC) according to a standard protocol (Bio-Rad Laboratories, Hercules, CA). The results were measured by a fluorescence microplate reader (Bioteck FL600 Fluorometer, Winooski, VT). RFUs were standardized to RFU/µg or RFU/10 µg protein. For each experiment, control groups with specific caspase inhibitors, including caspase-3 inhibitor (Z-DEVD-CHO; BD PharMingen, San Diego, CA), caspase-8 inhibitor (Z-LETD-CHO; BD PharMingen), and caspase-9 inhibitor (Z-LEHD-CHO; Alexis Biochemicals, San Diego, CA), were done to ensure specificity of the assay.

Examination of Intracellular Caspase Activity in Viable Human Tumor Cell Lines.
Intracellular caspase-3 activity in viable tumor or normal cell lines were examined by a Caspase-3 Intracellular Activity Kit II using PhiPhiLux-G₂D₂ as a substrate (OncoImmunin, Inc., Gaithersburg, MD). Tumor cell lines were cultured on chamber slides to 80% confluence and then reacted with the caspase-3-activity detection kit for 60 min according to the methods provided by the manufacture. After washing with PBS and counterstaining with 10 µg/ml Hoechst 33342 (Molecular Probes, Inc., Eugen, OR), the slides were observed under an inverted fluorescence microscope (Nikon Eclipse E800; Nikon Instrument, Inc., Melville, NY). The fluorescence intensity in the cells represents the level of active caspase-3. Fluorescence images were taken using Optronics Magnafire digital imaging system (Meyer Instrument, Houston, TX).

	Construction of Plasmids for Expression of Caspase Genes and Examination of the Effect of Expression of Caspase Genes on Human Tumor and Normal Cell Lines

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The pcDNA3 plasmids containing a procaspase-3 gene or an autocatalytic Rev-caspase-3 gene, which was engineered by switching the position of the small subunit to the front of the large subunit, were kindly provided by Dr. Emad S. Alnemri at Thomas Jefferson University in Philadelphia, Pennsylvania (33) . Procaspase-9 gene was obtained from Dr. Xiaodong Wang at University of Texas Southwestern Medical Center (Dallas, TX). Procaspase-8 gene was obtained from Dr. Xiaojing Li at Emory University (Atlanta, GA). The procaspase-3, procaspase-8, procaspase-9, and Rev-caspase-3 genes were cloned into an Adtrack-CMV vector, which also expressed a GFP marker gene (Ref. 34 ; provided by Dr. Bert Vogelstein at Johns Hopkins University, Baltimore, MD). The positive clones were selected and amplified for transfection study. To determine the effect of expression of caspase genes on tumor and normal cell lines, the plasmids were transfected into cultured tumor or normal cell lines in 24-well tissue culture plates using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Forty-eight h after transfection, the cells were stained with Hoechst 33342 and examined under an inverted fluorescence microscope. The percentage of apoptotic cells within transfected cell populations was determined by counting the number of GFP-positive cells with apoptotic nuclear morphology within all GFP-positive cells.

	Adenoviral Vector Production

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AdXAF1 vector was provided by Dr. Robert Korneluk (Solange Gauthier-Karsh Molecular Genetics Laboratory, Children’s Hospital of Eastern Ontario, Ottawa, Ontario, Canada). AdsurvivinT34A vector was produced in our laboratory using the AdEasy system, obtained from Dr. Bert Vogelstein at Johns Hopkins University, according to a standard protocol (34) . SurvivinT34A gene was generated by PCR-directed mutagenesis from pcDNA3-survivin plasmid containing a wild-type survivin gene (Dr. Dario Altieri, Yale University, New Heaven, CT; Ref. 32 ). The survivin T34A gene was cloned into the Adtrack-CMV shuttle plasmid and then transferred to AdEasy 1 plasmid through homogeneous recombination as described previously (34) . Adenoviral vector expressing the survivinT34A gene (AdsurvivinT34A) was produced by transfecting the AdEasy plasmid containing the survivinT34A gene into a human embryonic kidney cell line 293 (ATCC). The viral vectors were additionally amplified in 293 cell line, and high titer viral vectors were purified by centrifugation and CsCI banding.

	Examination of Effects of Expression of SurvivinT34A and XAF1 Genes on Human Tumor and Normal Cell Lines

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To determine the percentage of apoptotic cell death induced by expression of survivinT34A and/or XAF1 genes, 3 x 10³/well of human tumor or normal cells were plated in 96-well tissue culture plates. The cells were transduced with AdsurvivinT34A or AdXAF1 vector at a MOI of 100 pfu. Some groups received both AdsurvivinT34A and AdXAF1 vectors at MOI of 100 pfu. Seven days after the viral vector transduction, the percentage of viable cells was determined by staining cells with crystal violet followed by extraction of the dye for measuring absorbance at 590 nm with a microplate reader (µQuart; Biotek Instrument, Inc.). The percentages of viable cells in experimental groups were calculated using the absorbance value from the AdGFP control group as 100%.

	Western Blot Analysis of Protein Levels

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Human tumor and normal cells were lysed in the lysis buffer containing 50 mM HEPES, 50 mM NaCl, 5 mM EDTA, 10 mM Na₂P₂O₇ 10 H₂O, 50 mM NaF, 1 mM NaVO₄, 1% Triton X-100, and protease inhibitor mixture tablets (Complete mini; Roche Molecular Biochemical, Indianapolis, IN). Tissue lysates of frozen normal and breast cancer tissues were obtained by adding the lysis buffer to the tissue powder produced through hammering liquid nitrogen treated tissues slices. Protein concentrations of the resulting lysates were determined using Bio-Rad protein assay kit. Fifty µg of protein were resolved on 12–15% polyacrylamide SDS gels and then transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories). The membranes were blocked with 5% of nonfat milk in Tris-buffered saline for 1 h and then incubated from 1 h to overnight with primary antibodies, including goat antihuman survivin (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anticleaved caspase-3 fragments (Cell Signaling Technology, Inc., Beverly, MA), rabbit anti-XIAP or XAF (Dr. Robert Korneluk), and mouse monoclonal antibodies to human PARP (BD PharMingen), ß-tubulin, or ß-actin (Sigma Chemical Co., St. Louis, MO). After washing the membranes with Tris-buffered saline three times, the membranes were incubated with horseradish peroxidase conjugated with antigoat, rabbit, or mouse secondary antibody for 1 h. The levels of specific proteins in each lysate were detected by enhanced chemiluminescence using ECL plus (Amersham International, Buckingham, United Kingdom) followed by autoradiography.

	RESULTS

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Many Tumor Cell Lines Had High Basal Levels of Caspase Activity but Were Still Able to Survive.
At first, we examined the basal level activity of caspase-3, caspase-8, and caspase-9 in cell lysates of different tumor cell lines, as well as in normal cell lines using substrates specific for each caspase. Our results demonstrated that a considerable basal level of caspase activity could be detected in the tumor cell lines. Using caspase activity of normal primary human fibroblast (HDF) and normal mammary epithelial cell line (MCF-10A) as references, we found that most human tumor cell lines examined showed a higher basal level of caspase-3 activity relative to normal cell lines (Table 1) . Up-regulation of caspase-8 activity was also detected in five of seven tumor cell lines. Colon cancer SW620 and pancreatic cancer MIA PaCa-2 cell lines showed very high levels of both caspase-8 and caspase-3 activities. On the other hand, the basal level of caspase-9 activity was only slightly higher in some tumor cell lines than that in the normal cell lines. To determine whether our observation in vitro reflected the situation in living cells, we examined intracellular caspase activity in viable cells using an Intracellular Caspase-3 Activity Kit II. In combination with Hoechst 33342 staining of cell nuclei, we found that intracellular caspase-3 activity was detected in many human tumor cell lines but not in HDF or MCF-10A cell line (Fig. 1) . Although some tumor cells had a very high level of intracellular caspase-3 activity, most of the tumor cells displayed from low to intermediate levels of caspase-3 activity (Fig. 1) . We consistently observed a subpopulation of cultured tumor cells that had a high level of caspase-3 activity but were not apoptotic cells (Fig. 1 , arrows). We additionally demonstrated the presence of cleaved active caspase-3 fragments in cultured human tumor cells by Western blot analysis, using a rabbit polyclonal antibody specific for cleaved caspase-3 fragments (17–19 kDa). We detected from low to intermediate levels of cleaved caspase-3 fragments in BXPC-3, MIA PaCa-2, and SW620 cell lines (Fig. 2) . However, active caspase-3 fragments were not detected in HDF cells. We also found that although active caspase-3 fragments were present in the tumor cell lines, we did not detect cleaved PARP fragments (85 kDa) in BXPC-3 and MIA PaCa-2 cells, and only a very low level of cleaved PARP fragments was detected in SW620 cells (Fig. 2) .

View larger version (94K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Detection of intracellular caspase-3 activity in viable cells by a fluorometric assay using Caspase-3 Intracellular Activity Kit II. The cells were cultured on chamber slides for 24 h and then the substrate for caspase 3 was added into the culture medium for 60 min. After staining with Hoechst 33342 (blue), the cells were then observed under an inverted fluorescence microscope. Arrows indicate tumor cells with a high level of intracellular caspase 3 activity (red, cytoplasmic staining) but are not apoptotic (blue, nuclei).

View larger version (51K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Examination of the levels of active caspase-3 and cleaved PARP fragments in tumor cell lines by Western blot analysis. The tumor cell lines cultured at 70% confluence were collected for Western blot analysis. The level of cleaved caspase-3 fragments (17 and 19 kDa) and PARP (85 kDa) in the cell lysates was examined using antibodies specific for active caspase-3 or PARP. Normal cell line HDF was used as a control. A positive control sample was obtained from treating the herpes simplex virus thymidine kinase gene-modified BXPC-3 cells with 5 µg/ml ganciclovir (GCV) for 2 days, which induced apoptotic cell death in the cells (49) . Although detectable basal level of active caspase-3 was seen in BXPC-3 cells, a marked increase in the level of cleaved caspase-3 and PARP p85 fragments was detected in BXPC-3 cells undergoing GCV-induced apoptosis.

View larger version (28K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Examination of the levels of survivin and XIAP expression in human tumor and normal cell lines by Western blot analysis. A high level of survivin (16.5 kDa) was detected in all breast, pancreatic, and colon cancer cell lines examined. Expression of survivin was very low in normal cell lines such as MCF-10A and HDF. Expression of XIAP (53 kDa) was found in both normal and tumor cell lines. However, the level of XIAP was up-regulated in all tumor cell lines.

View larger version (48K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Detection of the levels of survivin and active caspase-3 in breast normal and cancer tissues by Western blot analysis and a fluorometric assay. A, levels of survivin and active caspase-3 fragments in paired normal and cancer tissue samples from the breast cancer patients. B, levels of survivin and active caspase-3 fragments in breast cancer tissues and normal breast tissues obtained from breast reduction surgery. C, comparison of average caspase-3 activity detected by a fluorometric assay in cell lysates obtained from normal and breast cancer tissues. A and B, 50 µg of total protein from tissue lysates were examined for the levels of survivin and active caspase-3 fragments by Western blot analysis. N, normal tissue; C, cancer tissue; LN, draining lymph node. The same lysate samples were also examined for the caspase-3 activity using a fluorometric assay. The RFU/µg of protein for each sample is also shown in A and B. C, average caspase-3 activity from 15 normal and 18 cancer tissue samples were compared. The caspase-3 activity was >2-fold higher in the tumor tissues than that of the normal tissues. There was a significant difference in the caspase-3 activity between breast cancer and normal tissues (Student’s t test, P < 0.005).

View larger version (32K): [in this window] [in a new window] [Download PPT slide]   Fig. 5. Induction of apoptotic cell death by overexpression of procaspase-8, procaspase-9, and procaspase-3 genes or an autocatalytic Rev-caspase-3 gene. Human tumor and normal cell lines were cultured in 24-well plates for 24 h and then transfected with 1 µg of AdtrackCMV plasmids containing procaspase-8, procaspase-9, procaspase-3, or Rev-caspase-3 gene. At 48 h after transfection, the cells were stained with Hoechst 33342, and the percentage of apoptotic cells in transfected cell population, which were cells expressing GFP marker gene (green) and showing apoptotic nuclei as detected by Hoechst 33342 staining (blue), was examined in each transfected group. The numbers in the figure represent mean values from the examination of three to five fields under a x40 lens with total cell numbers from 80 to 300 depending on transfection efficiency for tumor cell lines.

View larger version (54K): [in this window] [in a new window] [Download PPT slide]   Fig. 6. Effects of expression of survivinT34A and/or XAF1 genes on human tumor and normal cell lines. A and B, induction of apoptotic cell death in human pancreatic, colon, and breast tumor cell lines by overexpression of survivinT34A and/or XAF1 genes. The tumor and normal cell lines were cultured in 96-well plates for 24 h and then transduced with the adenoviral vector at a MOI of 100 pfu. Over 80% of the cells were transduced with the adenoviral vectors as determined from AdGFP or AdsurvivinT34A vector-transduced groups. The percentage of viable cells at 7 days after transduction was determined by staining the cells with crystal violet followed by extraction of the dye for measuring absorbance at 590 nm. The absorbance value of the cells transduced with control vector AdGFP serves as a relative cell number of 100%. Each value in the bar figure represents a mean value of three to four repeat samples. Similar results were found in three repeat experiments. Student’s t test was used to determine the significant difference between the percentage of viable cells in the groups treated with AdsurvivinT34A alone or ADXAF1 alone with the groups received both AdsurvivinT34A and AdXAF1. A significant difference in the percentage of viable cells was detected in the groups transduced with both AdsurvivinT34A and AdXAF1 as compared with the groups transduced with AdsurvivinT34A or AdXAF1 vector alone (Student’s t test: MIA PaCa-2, P < 0.05; SW620, P < 0.005; MDA-MB-231, P < 0.01). C, detection of expression of survivinT34A or XAF1 in normal cell lines after transduction with either AdsurvivinT34A or AdXAF1 vectors by Western blot analysis. MDA-MB-231 and MCF-10A cells were transduced with AdsurvivinT34A, AdXAF1, AdGFP, or AdLacZ vectors at MOI of 100 pfu for 2 days. The level of survivin or XAF1 expression was examined by Western blot analysis using antibodies specific for XAF1 and survivin. Because MCF-10A cells expressed a very low level of survivin, an increase in the level of survivin represents the amount of survivinT34A expressed from the AdsurvivinT34A vector.

View larger version (32K): [in this window] [in a new window] [Download PPT slide]   Fig. 7. Examination of the levels of caspase-3 and caspase-9 activity in human tumor and normal cell lines transduced with adenoviral vectors for 3 days. A, caspase-3 activity detected in cell lysates of AdsurvivinT34A or XAF1-transduced tumor and normal cell lines. B, detection of caspase-9 activity in cell lysates of AdsurvivinT34A or XAF1-transduced tumor and normal cell lines. The cell lysates from each treatment group were examined for caspase-3 or caspase-9 activity with caspase-specific substrates. The numbers in the figure represent mean values of three repeat groups (RFU/10 µg of protein). Similar results were obtained from repeat experiments.

	DISCUSSION

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To address this question, we have examined the regulatory factors in the apoptotic pathways in human tumor and normal cells. We found that in comparison with normal cell lines and tissues, many tumor cell lines and tissues had activated apoptotic signaling such as high levels of caspase-8 and caspase-3 activities in the absence of apoptotic stimuli. Experimental results from our study additionally revealed that these human tumor cell lines and tissues also had high levels of survivin and XIAP. On the basis of our observations, we hypothesized that the genetic and molecular changes associated with malignant transformation lead to activation of the apoptotic cascade, yet the cancer cells are able to avoid death by up-regulating IAPs, which inhibits activity of the caspases and blocks apoptotic cascades. We additionally hypothesized that we could restore the final apoptotic events specifically in tumor cells by inhibiting IAP function through expression of IAP-counteracting proteins such as survivinT34A and XAF1.

Cellular apoptosis or survival results from a balance between apoptotic and survival factors. One of the logical therapeutic approaches for induction of apoptosis in tumor cells is to activate caspases. Several studies have shown that overexpression of initiator caspase-8 and caspase-9 or executioner caspase-3 and caspase-7 genes induced apoptotic cell death in human tumor cell lines (28 , 38) . Expression of a modified, constitutively active caspase-3 gene (Rev-caspase 3) induced apoptosis in the absence of upstream stimuli in 293 and MCF-7 cells (33) .

In this study, we examined apoptosis induction by expression of procaspase-8, procaspase-9, and procaspase-3 and Rev-caspase-3 genes in human tumor and normal cell lines. We found that although expression of procaspase-8, procaspase-9, and Rev-caspase-3 genes induced apoptotic cell death in both normal and tumor cell lines, cells with low levels of survivin and XIAP were more susceptible to apoptosis induction after expression of caspase genes. For example, we found that normal cell lines were more sensitive to apoptosis induction than that in tumor cells after overexpression of Rev-caspase-3 gene. On the other hand, tumor cells with high levels of survivin and XIAP such as SW620 and MIA PaCa-2 displayed a low sensitivity to apoptosis induction by overexpression of caspase genes. Thus, our results suggest that activation or expression of caspases alone is not a powerful strategy for induction of apoptosis in cancer cells and does not have specificity for tumor cells.

Recently, studies have revealed that tumor cells have acquired various resistant mechanisms to apoptosis. The upstream defects found in tumor cells include loss or mutation of cell death receptors and decrease in the level of Bax protein (39 , 40) . Deletion or silencing of the caspase-8 gene was discovered in childhood neuroblastoma (41) . Although different upstream deficiencies were found in selective types of tumor cell lines and tissues, it seemed that up-regulation of IAPs was a common feature for the majority tumor types. Therefore, targeting IAPs should be a feasible approach for developing novel therapeutic strategies for many cancer types. At present, the role and mechanism of increases in IAP proteins during the process of tumorigenesis are still unknown. In particular, it is not known how survivin is up-regulated in tumors because this protein is not expressed in most normal adult tissues.

Our results that human tumor cells have both activated apoptotic signaling and elevated antiapoptotic factors are in agreement with those of several recent studies that detected a high level of caspase expression or active caspase-3 in various human tumor tissues. Studies from several laboratories showed that caspase-3 expression was up-regulated in breast cancer cell lines, breast ductal carcinoma in situ, and invasive breast carcinomas by Western blot analysis and immunohistochemical staining (42, 43, 44) . Moreover, high levels of proapoptotic factors such as Bax and active caspase-3 and caspase-6 were detected in >90% of human breast cancer tissues (45) . A strong positive correlation was also found between the levels of XIAP and cleaved caspase-3 in breast cancer tissues (45) . It has also been shown that caspase-3 was overexpressed in pancreatic cancer tissues as compared with that of normal pancreas tissues (4) .

Importantly, the results of our study demonstrated, for the first time, the presence of activated apoptotic signaling such as activated caspase-3 in human tumor cells that are not apoptotic. At present, the mechanism activating the apoptotic pathway remains unclear. Because apoptotic cell death is a common pathway to eliminate the abnormal cells, it is possible that during the process of tumorigenesis, abnormalities in the tumor cells cause activation of the apoptotic pathway and induce cell death in the majority of transformed cells. In this view, only a small fraction of the transformed cells that have up-regulated their antiapoptotic mechanisms such as IAPs are able to survive and develop into a tumor mass.

Our finding that both apoptotic and antiapoptotic factors are up-regulated in human tumor cells but not in normal cells can potentially lead to the development of novel tumor-specific therapies. Because tumor cells already have activated upstream apoptotic signaling and a relatively high level of active caspases, it is possible that potentiating caspase activity by down-regulation or counteracting IAP function could induce apoptosis specifically in tumor cells. A recent study showed that expression of the survivinT34A gene by adenoviral vector induced apoptotic cell death specifically in human tumor cell lines but not in several normal cell lines (31) . In our experimental system, we observed similar results after expression of counteracting IAP proteins such as survivinT34A and XAF1 in human tumor and normal cell lines.

At present, the mechanisms of induction of apoptotic cell death by expression of survivinT34A or XAF1 have yet to be further determined. It seemed that a marked increase in caspase-3 and caspase-9 activity played a role in the apoptotic cell death of AdXAF1-transduced tumor cells because expression of XAF1 increased activity of both caspase-3 and caspase-9 in the tumor cell lines. Our result that expression of survivinT34A induced activation of caspase-9 in the tumor cell lines is also consistent with a recent study showing that association of survivin with a cellular protein, hepatitis B X-interacting protein, is required for binding survivin to procaspase-9. However, expression of survivinT34A abolishes the association between hepatitis B X-interacting protein and survivin and therefore activates caspase-9 (46) . Although caspase-9 activity was increased by expression of survivinT34A, the caspase-3 activity was not significantly increased in the tumor cell lines. It is possible that the presence of a high level of XIAP in the tumor cells prevents additional activation of caspase-3. However, expression of survivinT34A inhibits the survivin function, which allows the proceeding of the apoptosis cascade in the tumor cells with a high basal level of caspase activity. Consistent with this assumption, recent studies showed that survivin level in tumor cells was regulated by phosphorylation of survivin Thr³⁴. Expression of survivinT34A or treatment with a cyclin-dependent kinase inhibitor, flavopiridol, suppressed the phosphorylation of endogenous survivin at the Thr³⁴ position and resulted in loss of survivin expression and induction of apoptosis in the tumor cells (47 , 48) .

Furthermore, because survivin is not detected in most normal adult tissues, down-regulation of survivin function by expression of the survivinT34A gene should have a minimum effect on normal cells. Accordingly, expression of the survivin T34A gene did not induce apoptotic cell death in immortalized normal human mammary epithelial cell line MCF-10A and normal fibroblast cell line HDF, although we have detected a weak expression of survivin in those cells. XAF1 counteracts the function of XIAP through competing for binding to caspases (30) . It has been shown that XAF1 is expressed in normal cells and tissues but is down-regulated in tumor cell lines and tissue (29) . Thus, expression of XAF1 may induce apoptosis in tumor cells with a low level or lack of XAF1 without affecting the normal cells.

In summary, we have demonstrated that IAPs are potentially novel molecular targets for the development of tumor-specific therapies. Significantly, we have detected the coexistence of both activated apoptotic signaling and up-regulated antiapoptotic factors in many human tumor cell lines and tissues but not in normal cells. Down-regulation of IAP function using agents such as small molecules, small peptides, RNA interference, and gene therapy vectors may change the balance of the apoptotic pathway and induce cell death specifically in tumor cells.

	ACKNOWLEDGMENTS

 
We thank Dr. Styblo Toncred and Beth Sumpter for providing breast cancer and normal tissues. We also thank Drs. Harry Findley, Margaret K. Offermann, Haian Fu, and Ruoxiang Wang for helpful discussions and critical reviewing the manuscript.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This research is supported by funds from the Avon Foundation and NIH, National Cancer Institute Grant R29 CA 80017.

² To whom requests for reprints should be addressed, at Department of Surgery and Winship Cancer Institute, Emory University School of Medicine, Room C 4088, 1365 C Clifton Road, Northeast, Atlanta, GA 30322. Phone: (404) 778-4269; Fax: (404) 778-5048; E-mail: Lyang02{at}emory.edu

³ Present address: Huntsman Cancer Institute, Room 5-D, University of Utah, 2000 Circle of Hope, Salt Lake City, UT 84112. Phone: (801) 581-7084; Fax: (801) 585-7477; E-mail: Hui.Yan{at}hci.utah.edu

⁴ The abbreviations used are: IAP, inhibitor of apoptosis; BIR, baculoviral inhibitor of apoptosis repeat; FBS, fetal bovine serum; RFU, relative fluorescence unit; GFP, green fluorescence protein; XAF1, XIAP-associated factor 1; ATCC, American Type Culture Collection; MOI, multiplicity of infection, pfu, plaque-forming unit, PARP, poly(ADP-ribose) polymerase.

Received 1/23/03. Revised 7/25/03. Accepted 8/ 1/03.

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Top ABSTRACT INTRODUCTION MATERIALS AND METHODS Construction of Plasmids for... Adenoviral Vector Production Examination of Effects of... Western Blot Analysis of... RESULTS DISCUSSION REFERENCES

 

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