Affiliations of authors: R. Bao, D. C. Connolly, J. K. Weinstein, D. A. Pisarcik, T. C. Hamilton, (Ovarian Cancer Program), M. Murphy (Department of Pharmacology), Fox Chase Cancer Center, Philadelphia, PA; J. Green, Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute, Bethesda, MD.
Correspondence to: Thomas C. Hamilton, Ph.D., Ovarian Cancer Program, Fox Chase Cancer Center, 7701 Burholme Ave., Philadelphia, PA 19111 (tc_hamilton{at}fccc.edu).
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ABSTRACT |
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INTRODUCTION |
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Although survivin is not expressed in normal adult human tissues, it is expressed in various human cancers (4,5). Survivin expression may be activated transcriptionally (6); consequently, the survivin promoter might be a cancer-specific promoter with utility in gene therapy or oncolytic viral replication. Such a tumor-selective promoter may also be useful in tumor-prone transgenic mice by activating the expression of a marker gene at the initiation of oncogenesis. In this study, we used transfection experiments to examine whether 1092 base pairs of the 5` upstream regulatory sequence of the human survivin gene could control the expression of a reporter gene in cancer cell lines derived from tumors of the uterine cervix, breast, ovary, lung, and colon. We also evaluated the activity of the survivin promoter in a cancer cell line in vivo as a xenograft.
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MATERIALS AND METHODS |
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The following human cancer cell lines (and their tissue of origin) were used in this study: A2780, OVCAR3, OVCAR5, OVCAR8, OVCAR10, SKOV3, PEO1, and UPN251 (ovary); HT29 (colon); MCF7 (breast); H1299 (lung); and HeLa (uterus). ROSE-TAg is a tumorigenic cell line derived from Fisher 344 rat ovarian surface epithelial (ROSE) cells transformed with simian virus 40 (SV40) large T antigen (TAg) in vitro. NuTu19 and NuTu26 are spontaneously transformed cell lines derived from Fisher 344 ROSE cells (7). IG10 and IF5 are spontaneously transformed mouse ovarian surface epithelial (MOSE) cell lines (8). Mc6 is a mammary cancer cell line derived from a mammary tumor of a mammary tumor-prone C3 (1)/TAg transgenic mouse line, and Pr14 is a prostate cancer cell line derived from a prostate tumor of a prostate cancer-prone line of C3 (1)/TAg transgenic mice (9,10). Normal MOSE cells were isolated from the ovaries of C57BL/6 adult mice (8) and used for up to three passages. Normal ROSE cells were isolated from the ovaries of Fisher 344 rats (7) and used for up to five passages. Normal human ovarian surface epithelial (HOSE) cells were derived from normal human ovaries as previously described (11). NIH 3T3 is an immortalized, nontumorigenic mouse fibroblast cell line. All cell lines were maintained at 37 °C in a humidified incubator with an atmosphere of 5% CO2/95% air. HOSE, A2780, OVCAR3, OVCAR5, OVCAR8, OVCAR10, SKOV3, PEO1, UPN251, ROSE-TAg, NuTu19, NuTu26, MCF7, and normal ROSE cells were cultured in RPMI 1640 medium (GIBCO BRL, Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS). IG10, IF5, Mc6, Pr14, and MOSE cells were cultured in Dulbecco's modified Eagle medium (DMEM; GIBCO BRL) plus 5% FBS. NIH 3T3 cells were cultured in DMEM plus 10% calf serum, and H1299 and HeLa cells were cultured in DMEM plus 10% FBS. All media were supplemented with streptomycin (100 µg/mL), penicillin (100 U/mL), glutamine (0.3 mg/mL), and pork insulin (0.25 U/mL or 1x ITS [insulin, transferrin, and selenium]; GIBCO BRL, Rockville, MD).
Western Blot Analysis
At about 70% confluence, cells were harvested with trypsin/EDTA, and PBS-washed cell pellets were stored at 70 °C until use. Whole-cell protein was extracted from the cell pellets with RIPA buffer (50 mM TrisHCl at pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% sodium dodecyl sulfate, and 1% sodium deoxycholate). Protein was also extracted from normal tissues (brain, thymus, heart, lung, liver, stomach, small intestine, bladder, kidney, ovary, oviduct, uterus, spleen, pancreas, and skeletal muscle) of female C57BL/6 mice (8 weeks, 12 months, or 16 months of age). Tissues were homogenized in a Mini Bead Beater (BioSpec Products, Inc., Bartlesville, OK) and the T-MER tissue protein extraction reagent (Pierce, Rockford, IL). For survivin detection, 30 µg of total protein extract was resolved on sodium dodecyl sulfate15% polyacrylamide gels and transferred to nitrocellulose membranes (Amersham, Piscataway, NJ). The blots were hybridized with an anti-survivin polyclonal antibody (diluted 1 : 2000; Novus Biological, Littleton, CO), followed by incubation with a peroxidase-conjugated goat anti-rabbit second antibody (diluted 1 : 5000; Amersham). The peroxidase activity was detected by the ECL method (NEN, Boston, MA).
Vector Construction
A 1092-base-pair fragment of the human survivin gene promoter (nucleotides 18212912, GenBank accession number U75285) was excised from plasmid SpI with restriction enzymes KpnI and XhoI (12). The secreted alkaline phosphatase (SEAP) expression vector under control of the survivin promoter (pSRVN-SEAP) was constructed by subcloning the KpnI-XhoI fragment into the multiple cloning site of the SEAP expression vector pSEAP-Basic (Clontech, Palo Alto, CA). To generate stable transfectants, the pSRVN-SEAP-NEO plasmid was constructed by subcloning the SRVN-SEAP sequence (a KpnI-XbaI fragment) from pSRVN-SEAP into the PC3 vector (13), a modified pcDNA3 vector (Invitrogen, San Diego, CA) without the cytomegalovirus promoter.
Transient Transfection
The pSRVN-SEAP plasmid was transiently transfected into cell lines by use of the TransIT-LT1 transfection reagent (PanVera, Madison, WI). Briefly, 3 x 105 cells were placed into each well of a six-well plate in 2 mL of complete medium. After incubation overnight, cells were 40%50% confluent, and a mixture of 2 µg of pSRVN-SEAP plasmid, 0.2 µg of pGL3 control plasmid, 6 µL of LT1 transfection reagent, and 100 µL of serum-free medium was added to each well. The pGL3 control plasmid (Promega, Madison, WI), which is a luciferase expression vector driven by the SV40 promoter, was used to assess transfection efficiency and, hence, normalize each transfection. Two other plasmids, pSEAP-Basic (a promoterless SEAP construct) and pSV40-SEAP (a SEAP expression vector with the SV40 promoter) (Clontech), were also used for each cell line as negative and positive controls, respectively. Medium (100 µL) was removed 48 hours after transfection and used to determine SEAP activity after normalization of the transfection efficiency. Briefly, the adherent cells were washed once with PBS, exposed to 1 mL of lysis buffer (Promega), and scraped from dishes with a cell scraper. After centrifugation of the cell lysates at 15 700 relative centrifugal force (rcf) for 1 minute, the supernatants were removed and stored at 70 °C until luciferase activity was assayed. Luciferase activity was determined by mixing 5 µL of supernatant with 100 µL of luciferase assay reagent (Promega) and determining the relative luminescence with a luminometer (Analytical Luminescence Laboratory, San Diego, CA). This procedure allowed us to adjust the amount of conditioned medium used to allow for differences in transfection efficiency.
Stable Transfection
The pSRVN-SEAP-NEO plasmid was linearized with restriction enzyme PvuI and purified by phenolchloroform extraction and ethanol precipitation. Before electroporation, subconfluent A2780 cells were trypsinized, washed twice with PBS, and resuspended at 10 x 106 cells in 0.7 mL of PBS. The cell suspension was transferred into a Gene Pulser cuvette (Bio-Rad Laboratories, Hercules, CA), and 5 µg of linearized pSRVN-SEAP-NEO or control vector PC3 was added. After 10 minutes on ice, the cells were subjected to electroporation by using the Gene Pulser II System (Bio-Rad Laboratories) at 250 V/cm and 975 µF and then plated in three 10-mm Petri dishes with complete medium. One day later, medium was changed to complete growth medium supplemented with G418 at 500 µg/mL. After 2 weeks, the G418-resistant clones were isolated with cloning cylinders. SEAP activity in the conditioned medium from individual clones was determined when the cells were nearly confluent.
Animal Study
Female CB17/ICR SCID (severe combined immunodeficient) mice, approximately 8 weeks of age and weighing approximately 20 g, were used to establish orthotopic ovarian tumors. All these mice were bred in the Laboratory Animal Facility at Fox Chase Cancer Center, were maintained in specific pathogen-free conditions, and received commercial food and water ad libitum. Institution guidelines were followed in handling the animals. To establish the orthotopic tumors, cultured A2780 transfectants (two SRVN-SEAP-NEO clones, A2780SSN1 and A2780SSN2, and one vector control clone, A2780PC3) were harvested with 0.05% trypsin-EDTA (GIBCO BRL), washed in PBS, and resuspended in RPMI-1640 complete medium at 40 x 106 cells per milliliter. Before intrabursal implantation of tumor cells, eight SCID mice were anesthetized with a 15 : 3 : 5 : 152 mixture of ketamine-HCl (100 mg/mL), acepromazine malleate (10 mg/mL), xylazine hydrochloride (20 mg/mL) (Fort Dodge Animal Health, Fort Dodge, IA), and 0.9% normal saline, injected intraperitoneally at 10 µL/g of body weight. The skin was disinfected with Wescodyne and 70% ethanol. A small incision was made on one side of the back to locate the ovary. The oviduct was held with small forceps, and a 26-gauge needle connected to a syringe was inserted into the oviduct and was passed through the infundibulum until the needle tip reached the space between the bursa and the ovary. Approximately 20 µL of the cell suspension (about 0.8 x 106 cells) was injected into the intrabursal space. The needle was slowly removed, the ovary was replaced in the abdominal cavity, and the body wall was closed with sutures. One ovary of each animal was injected.
Plasma for SEAP analysis was obtained by orbital puncture with heparinized glass tubes (Fisher Scientific, Pittsburgh, PA) on days 0, 1, 3, 6, and 9 after cell implantation. About 20 µL of plasma was obtained after the blood was centrifuged at 4500 rcf for 7 minutes (14). Animals were sacrificed 14 days after implantation; ovaries were removed, embedded in paraffin, and sectioned for histopathologic analysis.
SEAP Assay
SEAP activity in culture medium or plasma was determined by a chemiluminescence or fluorescence method using Great Escape SEAP kits from Clontech (15). In brief, 5-µL samples were mixed with 45 µL of dilution buffer and incubated in a oven at 70 °C for 45 minutes. Sixty microliters of assay buffer containing L-homoarginine was then added. After a 5-minute incubation at room temperature, the samples were exposed to 60 µL of chemiluminescent substrate CSPD (disodium 3-[4-methoxyspiro{1.2-dioxetane-3,2`-(5`-chloro)tricyclo(3.3.1.1)-decan}4-yl]phenyl phosphate) (1.25 mM) or 3 µL of fluorescent substrate 4-methylumbelliferyl phosphate (Clontech). Chemiluminescence was measured with a luminometer (Analytical Luminescence System) after a 10-minute incubation at room temperature.
After a 60-minute incubation in the dark, fluorescence was measured with a CytoFluor II fluorometer (Bio-Rad Laboratories) with excitation and emission wavelengths of 360 nm and 449 nm, respectively. SEAP activity was determined from a standard curve.
To determine whether exogenous SEAP could be separated from endogenous placental alkaline phosphatase of pregnant animals, plasma from two C57BL/6 pregnant mice at embryonic day 12, one normal control mouse, and one CB17/ICR SCID mouse carrying an A2780SEAP13 cell implant (14), was isolated. Five microliters of plasma was mixed with 45 µL of dilution buffer, and the mixture was heated to 70 °C for 0, 20, 40, or 60 minutes. Alkaline phosphatase activity was determined as described above.
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RESULTS |
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To determine how frequently the increased expression of survivin is detected in cancer cell lines compared with normal tissues and cells, protein extracts were prepared from cells and tissues derived from different species and subjected to western blot analysis. As shown in Fig. 1, A, survivin protein was detected as an intense band at 16.5 kDa in all the human ovarian cancer cell lines, including A2780, OVCAR3, OVCAR5, OVCAR8, OVCAR10, SKOV3, PEO1, and UPN251, but not in the normal HOSE cells. An intense 16.5-kDa survivin band was also detected in transformed rat ovarian surface epithelial cell lines (ROSE-TAg, NuTu19, and NuTu26), but only a faint band was detected in early-passage normal ROSE cells (Fig. 1, B
). Consistent with our findings in normal and transformed ROSE cells, an intense survivin band was detected in the transformed MOSE cell lines (IG10 and IF5), but only a faint band was detected in the normal MOSE cells (Fig. 1, C
). An intense survivin band was also detected in tumor cell lines from transgenic mice prone to develop mammary tumors (Mc6) (9) or prostate tumors (Pr14) (10). A faint survivin band was detected in nontumorigenic mouse fibroblast cell line NIH 3T3 (Fig. 1, C
).
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Levels of survivin protein in normal tissues from 8-week-old female C57BL/6 mice were determined by western blot analysis. No survivin protein was detected in brain, heart, lung, liver, stomach, intestine, bladder, kidney, ovary, oviduct, uterus, pancreas, or skeletal muscle. Survivin was detected in the thymus and, to a lesser extent, in the spleen (Fig. 2, A). Because age-associated thymic atrophy could result in a decrease in survivin protein in the thymus, we evaluated survivin expression in 12-month-old and 16-month-old C57BL/6 mice and detected a marked reduction of survivin protein in mature, as opposed to young adult, mice (Fig. 2, B
).
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We constructed the pSRVN-SEAP plasmid to determine whether the survivin promoter functioned in cancer cells. Promoter activity was determined from the SEAP activity in conditioned medium from transiently transfected cells. In A2780 cells transfected with the promoterless pSEAP-Basic plasmid, SEAP expression was almost baseline (Table 1). In several other cancer cell lines, SEAP expression was also almost baseline, but in others, the promoterless plasmid had some activity. In all cancer cell lines transfected with a plasmid containing the survivin promoter (i.e., pSRVN-SEAP), SEAP expression was fivefold to about 400-fold higher than that observed with the promoterless pSEAP-Basic plasmid (Table 1
; Fig. 3, A
). However, early-passage normal ROSE and MOSE cells similarly transfected showed less SEAP expression when transfected with pSRVN-SEAP than with pSEAP-Basic (Table 1
). To determine the relative promoter activity of the survivin promoter compared with the relatively strong SV40 viral promoter, we transfected the various cell lines with the SV40 promoter-driven SEAP expression plasmid pSV40-SEAP and measured SEAP expression. The survivin promoter was more active in the cancer cell lines, and the SV40 promoter was more active in the nontransformed cell lines (i.e., NIH 3T3, ROSE, and MOSE cells) (Table 1
; Fig. 3
).
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To determine whether the survivin promoter could induce enough SEAP activity to monitor tumor growth in vivo, we created stable A2780 transfectants harboring stably integrated SRVN-SEAP-NEO. Two clones (A2780SSN1 and A2780SSN2) were selected because of their relatively high SEAP production (Fig. 4, A). These two SRVN-SEAP-NEO clones and one vector control clone (A2780PC3) were used to generate orthotopic ovarian tumors by injection into the intrabursal space of mouse ovaries to mimic early ovarian cancer. After tumor cell implantation, plasma was collected at designated intervals to measure SEAP activity. SEAP activity was detected as early as 24 hours in animals implanted with 0.8 x 106 cells from either of the two SRVN-SEAP-NEO clones and increased with time and tumor growth. In contrast, SEAP activity was not detected in the animal injected with the vector control clone (Fig. 4, B and C
). Paraffin sections prepared from ovaries removed on day 14 had small tumors in the intrabursal cavity in all mice injected with a pSRVN-SEAP-NEO clone (A2780SSN) or the vector control clone (A2780PC3) (Fig. 4, D
). Contralateral ovaries were normal.
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To determine whether endogenous alkaline phosphatase activity could be separated from transgenic SEAP activity, we used heat to inactivate the endogenous activity. Plasma from normal control mice, pregnant mice, and a mouse carrying A2780SEAP13 cells (14) was treated with heat for 0, 20, 40, or 60 minutes, and alkaline phosphatase activity was determined. As shown in Fig. 5, alkaline phosphatase activity in plasma of normal and pregnant mice decreased quickly after the heat treatment at 70 °C and was still low 40 minutes later. However, plasma alkaline phosphatase activity in the mouse xenografted with A2780SEAP13 cells had not decreased after 60 minutes of heat treatment. Therefore, exogenous SEAP activity can be monitored during tumor development and effectively separated from endogenous alkaline phosphatase activity by heat treatment.
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DISCUSSION |
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A SAGE (serial analysis of gene expression) analysis found that survivin transcripts were the fourth most frequently overexpressed transcript in common human cancers (e.g., melanoma and cancers of the colon, brain, breast, and lung) relative to levels in normal cells (5), suggesting that survivin was a potential target for cancer therapy. If increased survivin activity is controlled transcriptionally, then the survivin promoter might control transgene expression in a cancer-specific manner. Transcriptional regulation of survivin expression in cancer cells has indeed been reported (6). Using approximately 1 kilobase of the 5` upstream regulatory region of the survivin gene to drive SEAP expression in ovarian, mammary, colon, lung, and uterine cervical cancer cell lines, we have shown that the survivin promoter can control gene expression regardless of tumor type, mechanism of oncogenesis, and species, and we have confirmed that survivin expression appears to be, at least in part, transcriptionally activated.
In contrast to adult tissues, where survivin expression is largely limited to activation during oncogenesis, in the human fetus, survivin is abundantly expressed in apoptosis-regulated tissues. Similarly, survivin was nearly ubiquitously expressed in embryonic mouse tissues at an early gestational stage (embryonic day 11.5) but was later expressed more selectively (16). Increased survivin expression and survivin promoter activity in cancer cell lines indicate that transcriptional factors needed for survivin transcription reappear or are reactivated during oncogenesis. The approximately 1-kilobase fragment of the survivin promoter used in this study overlaps with the 5` portion of the gene studied by Li and Altieri (6) and contains the CHR (cell cycle gene homology region) and abundant SP1 and CDE (6) as well as E2F (12) transcription factor binding sites that they believe are responsible for controlling the transcription of survivin.
As indicated above, our interest in the survivin promoter first arose because of a desire to drive transgene expression in a cancer-specific manner for cancer gene therapy, to improve gene delivery progress, to specifically regulate expression of transgenes to limit the toxicity of therapeutic genes such as herpes simplex virus thymidine kinase, and to create a tumor-selective replicative oncolytic virus. We believe that the survivin promoter's specificity and expression in many early-stage cancers make it an excellent candidate for these purposes (17,19). Finally, we believe that this cancer-specific reporter gene system could have major implications for monitoring tumor initiation and progression in tumor-prone transgenic animals.
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NOTES |
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We thank Dr. Randy Hardy for helpful discussion with regard to the development and involution of the mouse thymus. We also thank Dr. Paul Terranova for providing the transformed IG10, IF5, and MOSE cell lines and helpful discussion with regard to isolation and culture of primary MOSE cells.
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REFERENCES |
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Manuscript received September 28, 2001; revised January 25, 2002; accepted February 4, 2002.
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