Tight regulation of the therapeutic gene expression is critical in gene therapy. In this report, a doxycycline (Dox)-regulated retrovirus-mediated gene expression system was used to study the effects of suicide gene therapy on human breast cancer cell line MCF-7 and the nude mice model of implanted human breast cancer. To render the expression of suicide gene under control, we used two pseudoviruses simultaneously, RevTRE/HSVtk and RevTet-On, to infect MCF-7 cells or xenografts of nude mice. When infected by the pseudoviruses and followed by Dox and Ganciclovir (GCV) treatment, MCF-7 cells were arrested at S phase and the growth was suppressed. We then evaluated the antitumor efficiency of this system in vivo through studying the mice bearing human breast cancer xenografts. Compared with control groups, the HSVtk mRNA level increased significantly in tumor tissues, mass of the tumors shrank remarkably, and tumor necrosis features occurred after treatment with Dox and GCV. These data suggest that suicide gene therapy using the Dox-induced Tet-On-controlled HSVtk gene expression system is a feasible method to treat human breast cancer.
Suicide gene therapy involves the introduction of suicide genes into tumor cells to render them susceptible to specific antitumor drugs. The most widely used suicide gene system is the herpes simplex virus thymidine kinase/Ganciclovir (HSVtk/GCV), which accounts for about 10% of cancer gene therapy in clinical trials.1 GCV, a nontoxic prodrug, can be phosphorylated by the HSVtk protein and converted into toxic triphosphates that potently inhibit DNA polymerase, disrupt cellular DNA synthesis, and ultimately kill the cells. In addition, GCV treatment also exerts a bystander effect by which it causes cell death of not only the tk-transduced cells but also the neighboring untransduced cells.2 These properties make the HSVtk/GCV system attractive for cancer gene therapy.
The major challenge to gene-based cancer therapy is the efficient delivery and adequate expression of therapeutic genes (including suicide genes) in target tumors. Although some achievements have been made to improve transduction efficiency,3, 4 a clear mechanism on how suicide genes are regulated is not yet fully understood. Recent studies have shown that an appropriate dose of the therapeutic protein was required for successful treatment of certain disease, whereas excessive production of the protein might be toxic.5, 6 For safe treatments, recent studies have also suggested the need to maintain the protein concentrations within a therapeutic dose. This result requires the therapeutic gene expression to be regulated tightly in administration, induction, and termination.7 To achieve this set of conditions, several inducible eukaryotic gene promoters have been used to deliver genes in a regulated manner. The promoter inducers include steroid hormones, oxygen, heavy metals, and physical stimulus (e.g. radiation).8, 9, 10 However, most of these promoters are not suitable for clinical application for various reasons: First, these promoters are mammalian gene promoters, so that the exogenous regulation of the target gene through such a promoter could affect the transcription of the host endogenous genes; second, the inducers of these promoters are generally endogenous molecules (e.g. hormones, oxygen), the levels of which cannot be modulated significantly and safely. Other limitations include the potential toxicity or side effects of the inducer. Thus, a regulatory system that offers tight control of gene expression in response to inducible agents is valuable for cancer gene therapy. The tetracycline regulatory system offers such substantial regulation of target gene expression in response to doxycycline (Dox) that can be repetitively administrated.11 The Tet-On system, which is characterized by two regulatory elements, the tetracycline repressor protein (TetR) and the tetracycline operator sequence (TetO) to which TetR binds, can turn on target gene expression in the presence of Dox. Gene expression can be switched off by withdrawal of Dox. This approach works in the same way as the original tet gene expression system does and can integrate into the genome of host cells.12, 13, 14 Indeed, the low cytotoxicity of tetracycline, as well as that of its derivatives, and its high affinity for TetR have enabled the use of this antibiotic at concentrations that cause no appreciable adverse effects in transgenic animals.15, 16, 17
We previously reported that the expression of the suicide gene HSVtk was regulated by Dox in MCF-7 cells that were cotransfected with pRevTet-On and pRevTRE/HSVtk plasmids, and the antitumor effect was observed in severe combined immune deficiency (SCID) mice bearing breast cancer xenografts.18 On the basis of these findings, we asked whether retrovirus-mediated suicide gene transfer could be used to treat tumors in a more clinically relevant setting. In this study, we created a regulated suicide gene expression system with a retroviral vector and showed that tight regulation of tk gene expression could be achieved under the control of Dox. We also evaluated its antitumor effects on human breast cancer cell line MCF-7 in both in vitro and in vivo models. Our study suggests that this system offers great potential for inhibition and elimination of tumor cell growth.
Materials and methods
Cell line, culture and vectors
Human breast cancer cell line MCF-7, human kidney 293 cells, PA317 cells, and fibroblast cell line NIH/3T3 were from the American Type Culture Collection (ATCC; Manassas, VA). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) plus 100 U of penicillin per ml and 0.1 mg of streptomycin per ml. The cells were grown at 37°C in a humidified incubator with an atmosphere of 5% CO2. The RevTet-On system, including the plasmids of pRevTRE and pRevTet-On, was from Clontech (Palo Alto, CA).
Construction and preparation of recombinant retroviruses
The construction of recombinant retrovirus plasmid pRevTRE/HSVtk was previously described.18 In brief, the tk gene was amplified from plasmid pGEM7Z-tk by primers TK1/TK2 (TK1 IndexTerm5′-TCT ATG GAT CCA GAT CTT GGT GGC GTG AAA C-3′; TK2 IndexTerm5′-TAG CCA AGC TTT ATT GCC GTC ATA GCG C-3′, BamHI and HindIII restriction sites are shown in boldface). Then the polymerase chain reaction (PCR) product was inserted into the pRevTRE vector. The recombinant plasmid was verified by DNA sequencing.
Retroviruses were prepared using the ‘ping-pong effect’ strategy with some minor modifications.19 In brief, PA317 cells and HEK293 packaging cells were plated into six-well plate at 5 × 105 cells per well. After 12–20 h, the cells were transfected, respectively, with the plasmid pRevTRE/HSVtk and pRevTet-On by the calcium phosphate precipitation method.20 Following incubation for 8–12 h, the DNA suspension was removed, and fresh medium was added. At 48 h after transfection, medium containing ecotropic recombinant viruses was removed and used to infect PA317 amphotropic packaging cells in the presence of 8 μg of polybrene per ml. Then PA317 package cells were cloned after being screened by hygromycin B (600 μg/ml) or G418 (800 μg/ml). Cloned PA317 cells were amplified, and supernatant was collected. Three to four repeat collections were made, and the supernatant was pooled, filtered (0.45-μm-pore-size filter), and concentrated by centrifugation (80 700 g) at 4°C for 2 h. Resuspension was frozen under cold drying, and pellets were then resuspended in a small volume of fresh medium, divided into aliquots, and stored at −70°C. The titer of virus was determined in NIH/3T3 cells with different cultured times and sodium butyrate concentrations under G418 selection. The virus with the highest titer was kept at −70°C for further use.
Infection of MCF-7 cells with retroviruses
MCF-7 cells were seeded at 5 × 104 cells per well in six-well plates and virus-containing supernatants were added at 37°C for 8 h. Viral solutions were then replaced with normal medium. Then, infected cells were cloned after being screened by hygromycin B (600 μg/ml) and G 418 (800 μg/ml). Cloned cells were amplified and cultured in complement DMEM containing 10% solution of FBS. To confirm the specificity of viral infections of the target cancer cells, reverse transcription-PCR (RT-PCR) and Southern blotting assays on infected cells were performed.
In vitro GCV sensitivity
Infected MCF-7 cells were plated on 96-well plates at a density of 104 cells/well. The cells were treated with 1 μg of GCV (Sigma, St Louis, MO) per ml 48 h after inducing with different Dox (Sigma) concentrations ranging from 0 to 1200 ng/ml. Then the cells were incubated with 0.5% 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma) for 4 h; cell viability was determined by the MTT assay. In brief, the medium was removed and 100 μl of 0.04 M dimethyl sulfoxide solution was added, followed by incubation at 37°C for another 4 h. The absorbance of the reaction solution was measured at 570 nm. To calculate the percentage of living cells, we treated cells with GCV for 48 h, and viable cells were counted using the trypan blue dye exclusion method. The cell number in experimental wells was divided by the cell number in control wells. All experiments were performed in quadruplicate.
Cell cycle analysis
The cell cycle distribution was analyzed by fluorescence-activated cell sorter (FACS) analysis. About 106 cells were harvested by trypsinization, washed with phosphate-buffered saline (PBS) and then centrifuged at 2000 r.p.m. for 10 min. Cell pellets were resuspended in 80% ethanol and stored at 4°C for 24 h. Cells were washed twice with PBS and resuspended in PBS containing 100 μg of RNase A per ml. They were then incubated at 37°C for 30 min and stained with propidium iodide (PI). The DNA content, which was indicated by the extent of incorporation of PI, was measured using the FACS scan, and analyzed with Cell Fit software (version 2.0). Cell cycle analyses were performed in triplicate.
RNA extraction and RT-PCR analyses
Expression of the HSVtk gene in the infected MCF-7 cells and implanted tumors of nude mice was analyzed with RT-PCR. RNAs were extracted from the tumor tissues or cultured cells using TRIzol reagents (Invitrogen, La Jolla, CA). HSVtk gene expression was detected by RT-PCR with nested primers TK3/TK4 (a DNA fragment of 420 bp). For internal control, β-actin was amplified along with HSVtk. The primers for β-actin were as follows: IndexTerm5′-TGA CGG TCA GGT CAT CAC TAT CGG CAA TGA-3′ (sense) and IndexTerm5′-TTG ATC TTC ATG GTG (AGC) TAG GAG CGA GGG CA-3′ (antisense) (a DNA fragment of 260 bp). Before reverse transcription, residual genomic DNA was removed by RQ DNase (Promega, Madison, WI). cDNA was synthesized from total RNA (1.5 μg) with AMV reverse transcriptase (Promega) in 20 μl of reaction mixture by standard protocol utilizing oligo(dT)15 as primer. PCR was performed with 5 μl of RT products in 50 μl of reaction system using Taq DNA polymerase. The conditions for amplification were 94°C for 30 s, 56°C for 45 s, and 72°C for 40 s for 30 cycles. PCR products were run on 1.5% agarose gel containing 0.5 μg of ethidium bromide per ml.
Establishment of human breast cancer models in nude mice and GCV treatment
BALB/c nude mice were from the Lab Animal Center of the Academy of Medical Sciences of China (Shanghai, China). The mice were housed in vinyl cages equipped with air filter lids, which were kept in laminar airflow hoods and maintained under pathogen-limiting conditions. All mice were cared for according to institutional guidelines, and all food, water, caging, and bedding were sterilized before use. All animal experiments were approved by the National Animal Care and Use Committee of China.
Female BALB/c nude mice (4 weeks old) were given a single priming dose of cyclophosphamide (100 mg/kg of body weight, subcutaneously (s.c.)) 3 days prior to inoculation of cells to deplete the natural killer cells of mice. The MCF-7 cells were trypsinized, washed with DMEM three times to remove the enzyme, and harvested by centrifugation. The cell pellets were resuspended in serum-free DMEM medium. After a short period of ether inhalation anesthesia, 7 × 106 MCF-7 cells in 0.2 ml of suspension were injected s.c. to the left-flank regions of nude mice to generate s.c. tumors. Tumor diameters were monitored every 3 days with vernier caliper measurements. When s.c. tumors reached 7–8 mm in diameter, intratumoral injections of retroviruses containing HSVtk and Tet-On or DMEM (mock group) were performed four times (twice a week). Then, 32 mice were randomly divided into four groups and each group had eight mice: GCV+Dox, GCV+viruses, GCV+Dox+viruses, and DMEM only. All mice were induced with Dox (20 mg/kg) for 1 week prior to intraperitoneal administration of GCV (100 mg/kg) twice daily for 14 days. The volumes of tumor were calculated using the formula: V=L × W2 π/2, where L is the length and W is the width of the tumor (all in millimeters). Mean value of tumor volume was plotted in each group.
Subcutaneous tumors were excised from nude mice with different treatments. Tumors were rinsed twice in normal saline (NS) and then fixed in 4% polyparaformaldehyde for 12 h and embedded in paraffin. Sections were stained with hematoxylin and eosin for light microscopic observation.
Results were expressed as means±s.d. The statistical difference between the groups was determined by one-way ANOVA test. Differences among groups were considered statistically different at P<0.05.
Generation of pseudoviruses and titer determinations
The pseudoviruses, RevTRE, RevTRE/HSVtk, and RevTet-On, were produced from package cells PA317 using the ‘ping-pong effect’ strategy. To obtain the highest titers of retroviral particles, we optimized the conditions of viral preparation by collecting culture supernatants in different cultured times and sodium butyrate concentrations. We found that the highest titer of retrovirus stocks, about 1.2 × 106 CFU per ml, can be obtained in PA317 culture medium by using the ‘ping-pong effect’ technique at 30 h and 10 mM sodium butyrate concentration.
Sensitivity of MCF-7 cells infected by Dox-regulated pseudoviruses to GCV
The MCF-7 cells infected by recombinant pseudoviruses RevTRE/HSVtk and RevTet-On exhibited high sensitivity to the cytotoxicity effect of GCV at 48 h with Dox induction. Cell proliferation was significantly inhibited by 1 μg of GCV per ml at Dox induction concentrations from 800 to 1200 ng/ml examined with the trypan blue dye exclusion method (Figure 1). All infected MCF-7 cells exhibited sensitivity to GCV in a Dox dosage-dependent manner. Drug effect (cytotoxicity) was enhanced with increasing Dox concentrations. The most suitable concentration of Dox for inducing tk gene expression in MCF-7 cells was 1000 ng/ml in these experiments. We also examined the proliferative abilities of HSVtk-expressing cells by using the MTT assay. Similar growth-suppressive effects were observed after GCV treatment induced by different concentrations of Dox (data not shown). The levels of HSVtk mRNA in MCF-7 cells at different Dox concentrations were determined by relatively quantitative RT-PCR. The transcription level of this gene appeared to be directly correlated with the level of Dox exposure (Figure 2). In pseudovirus-infected MCF-7 cells, the expression of the tk gene exhibited a sigmoidal concentration–effect relationship with Dox induction. To further define at which stage the cells were mostly arrested by Dox induction, we characterized the cell cycle populations by flow cytometry (FCM). With treatment of GCV at 2 μg/ml for 48 h plus 1 μg/ml Dox, 38.2% of the virus-infected MCF-7 cells were at S phase. There was a significant increase of S phase cells, compared with 23.1% of MCF-7 cells and 25.3% of virus-infected MCF-7 cells without Dox/GCV treatment at S phase (P<0.001) (Figure 3). These results indicated that Dox stimulation greatly facilitated the cells to be arrested at S phase. In addition, both HSVtk and Tet-On genes were integrated into the MCF-7 genome as analyzed with Southern blotting (data not shown). Thus, the pseudoviruses were capable of rendering human breast cancer cells sensitive to GCV in a dose-dependent manner with Dox induction.
Implanted human breast cancers were sensitive to GCV treatment in vivo
To test the in vivo antitumor effect of the regulatory HSVtk/Tet-On/GCV system, we established the human breast cancer models in nude mice. During the first 14 days after GCV administration, tumor volume in all groups increased almost at the same rate. However, in the GCV+Dox+viruses group, the volume of tumors shrank remarkably after 30 days of GCV treatment and the tumor growth rate was decreased after treatment with GCV compared with the control groups (Figure 4). In the control groups, tumors grew continuously up to 2 cm in diameter on day 40, when the mice were killed. To evaluate the efficiency of gene transfer in vivo, we used RT-PCR to detect the expression of transferred genes. We found that HSVtk mRNA was remarkably more highly expressed in tumors of the GCV+Dox+viruses group than those in other three groups (Figure 5). HSVtk expression in both DMEM and GCV+Dox groups was undetectable, and the low-level expression of HSVtk was maintained in the GCV+viruses group without induction of Dox. After induction of Dox, the HSVtk level was enhanced by approximately four-fold in tumors subjected to treatment with GCV+viruses. As expected, without HSVtk expression, the growth of parental MCF-7 cells was not affected by GCV and tumors continued to grow until the mice were killed. The group injected with pseudoviruses followed by GCV and Dox treatment resulted in rapid regression of all tumors by day 30 (Figure 4). The mass of tumors in the treatment group decreased remarkably compared with control groups, respectively (P<0.05), among which 89% of tumor xenografts were suppressed and 17% were completely regressed. Taken together, these data showed that the tk gene was expressed in a regulated manner in the nude mice model of implanted human breast cancer that was infected with pseudoviruses and that human breast cancer cells (MCF-7) expressing the HSVtk gene can be eradicated by administration of GCV.
Histological analysis of human breast cancer xenografts
The BALB/c nude mice with human breast cancer xenografts were given sodium pentobarbital (50 mg/kg, intraperitoneally (i.p.)) anesthesia to excise subcutaneous tumors. Tumors were fixed in 4% polyparaformaldehyde and embedded in paraffin, and the sections were stained with hematoxylin and eosin for histological examination. There were few infiltrating cells in the tumors of control groups. Conversely, marked infiltration of leukocytes was observed throughout the tumors injected with Dox followed by GCV treatment, and breast cancer cells were severely damaged (Figure 6). Furthermore, many acidophilic glasslike MCF-7 cells with condensed nuclei were observed in the tumors, suggesting that the HSVtk/GCV system also induced apoptotic death of MCF-7 cells.
Applications of chemotherapy agents or other antitumor drugs suggested that some kinds of cancer could be cured if higher doses of active reagents were delivered safely. However, high doses of chemotherapy agents and some antitumor drugs are associated with significant toxicity to normal organs, host morbidity, and even treatment-related mortality.6, 21, 22 Suicide gene therapy is one of the safety choices among the treatment options for malignant diseases. HSVtk/GCV system is one of the most commonly used suicide gene therapy systems and is suitable for the treatment of breast cancer.23 Although considerable efforts were made in the improvement of suicide gene expression,24, 25 few approaches had been undertaken to evaluate the suicide gene expression under delicate control. While a dose–response relationship exists in chemotherapy, it may be possible that this relationship reaches a plateau at a higher dose due to limitations of normal organ toxicity. The Tet-On system, which uses an E. coli gene regulatory system,11 appears to fulfill the criteria for clinical application with several advantages:6, 7, 11, 26 (1) gene expression is easily regulated by administration of Dox; (2) Dox is minimally toxic and has been widely used as an antibiotic; and (3) Dox acts specifically on the target gene and does not activate other cellular genes. Dox can activate the tet-response element and significantly enhance the target gene expression in cells in a dose-dependent manner. Thus, specific delivery of therapeutic reagents to cancer cells under delicate control could potentially avoid host toxicity and improve therapeutic outcome for patients.
Results in this report suggest that the HSVtk/Tet-On/GCV system is a feasible method in the treatment of breast cancer and other solid tumors. Our data show that there is an apparent dose-dependent relationship between HSVtk gene expression and Dox concentration in infected MCF-7 cells when the concentration of GCV is at 1 μg/ml. These findings indicate that HSVtk expression may selectively enhance the sensitivity of breast cancer cells to GCV in vitro, in agreement with other results showing that the Tet-On system has low basal transcriptional activity and high inducibility.27
It is well recognized that the tumor growth rate is the balance between cell proliferation and cell death. Breast cancer gene therapy has generally been considered to be tumorostatic, thus implying that the predominant mechanism is involved in the induction of cell death or apoptosis.28, 29 Recently, however, there have been several studies showing that breast cancer is to some extent a cell cycle disease.30, 31 The cell cycle coordination takes place mainly at G1/S and G2/M phase transitions by a series of checkpoints. It has been shown that the activities of many regulatory factors of checkpoints are lost or arrested during the process of tumorigenesis32 and that some of the antitumor agents can restore the altered regulatory checkpoints.33 Many antitumor agents can arrest and regulate the cell cycle.34, 35 The results in our report demonstrate that the application of the HSVtk/Tet-On/GCV system can prolong S phase of MCF-7 cells. Our findings are consistent with the previous reports, showing that S-phase arrest was noticeable and a significant increase in cell granularity was observed in cells treated with either GCV or 5-FC.36 Thus, our system may provide an alternative strategy to the treatment of human breast cancer.
A fundamental problem in cancer gene therapy is the efficiency of gene delivery. A human breast cancer model in SCID mice in our pervious trials was established by inoculating MCF-7 cells that were cotransduced with pRevTet-On and pRevTRE/HSVtk plasmids.18 In that report, we showed that the amount of expressed HSVtK gene could be regulated by Dox in a dose-dependent manner in transfected MCF-7 cells and in SCID mice, and it was shown that MCF-7 cells expressing the HSVtk gene can be eradicated by administration of GCV and induction with Dox in vitro and in vivo. However, this method of gene delivery is not practical in the clinic. Intratumoral injection is a routine method for local gene delivery that may improve interstitial transport of viral vectors in tumor tissues and reduce systemic toxicity.37 Therefore, we constructed and produced the recombinant pseudoviruses to transfer HSVtk and Tet-On genes. To examine whether these infectious pseudoviruses carrying HSVtk and Tet-On genes could control tumor growth rate in vivo, we established a human breast cancer model in nude mice by implanting MCF-7 cells. Nude mice bearing tumors were injected with the retroviruses RevTRE/HSVtk and RevTet-On and treated with GCV after consecutive induction with Dox. The antitumor treatment occurred with an acceptable toxicity level, and the treated mice achieved long-term survival. The tumor mass of the treatment group decreased remarkably compared with that of control groups; the suppression rate of xenograft tumor growth reached up to 89%. The HSVtk/Tet-On/GCV system may also be used for efficient external control of transgenic expression. These data indicate that our suicide gene system is a powerful tool to inhibit cell growth and induce apoptosis in breast cancer gene therapy. Although there was a leakage expression without induction of Dox when the HSVtk and Tet-On genes were transferred, the HSVtk expression level was too low to affect the growth of tumor cells in vivo. We could not get a complete eradication for all tumors in these experiments, but we did see a partial response. Our results show that direct intratumoral injection is a simple and practical gene delivery method in solid tumor gene therapy and that the HSVtk/Tet-On/GCV system can serve as a potential strategy for breast cancer treatment. The expression level of the HSVtk gene is positively associated with tumor cell killing. Enhanced HSVtk gene expression leads to increased generation of phosphorylated GCV and results in heightened sensitivity to GCV, which may be the major mechanism whereby this system works.38
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This work was supported by the grant from CMB (China Medical Board of New York Inc., Grant # 99-698).
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