Photodynamic therapy (PDT) is a promising cancer treatment involving the administration of a tumor-localizing photosensitizer followed by the photochemical generation of cytotoxic singlet oxygen. PDT elicits strong transcriptional activation of a variety of genes including stress response genes belonging to the glucose-regulated protein (grp) family. Oxidative stress and hypoxia can activate GRP-78, and both of these physiological insults occur in treated tissue during and/or after PDT. In the current study, we evaluated the grp promoter as a PDT-inducible molecular switch for controlled expression of the herpes simplex virus-thymidine kinase (HSV-tk) suicide gene in mouse mammary adenocarcinoma (TSA) cells and tumors stably transduced with the G1NaGrpTk retroviral expression vector. We also examined whether PDT-inducible expression of HSV-tk, together with systemic administration of ganciclovir, could enhance the tumoricidal responsiveness of PDT. Inducible expression of HSV-tk was observed after PDT in stably transduced TSA cells grown in culture and in TSA tumors growing in BALB/c mice. We also observed enhanced tumoricidal activity in mice with TSA tumors containing the G1NaGrpTk expression vector treated with PDT plus ganciclovir when compared with either treatment alone. Our results confirm that the grp promoter was able to effectively function as a molecular switch for the inducible expression of the HSV-tk gene after exposure to PDT.

PDT3 is a localized cancer treatment involving the systemic administration of a tumor-localizing photosensitizer followed by focal light activation (1, 2). This procedure results in the photochemical generation of cytotoxic singlet oxygen within target tissue. The photosensitizer, Photofrin, is approved by the Food and Drug Administration for PDT treatment of endobronchial and esophageal carcinomas (1). PDT clinical trials using PH as well as a variety of second-generation photosensitizers are showing promise in treating malignancies of the brain, peritoneal cavity, skin, bladder, and head and neck, as well as for nononcological disorders such as age-related macular degeneration and psoriasis (1, 2). Initial treatment responses after PDT are routinely positive; however, recurrences can occur, and therefore methods to improve PDT responsiveness are needed.

Positive clinical results have encouraged an expanded mechanistic analysis of cellular and tissue responses associated with PDT as well as the identification of in vitro and in vivo targets of PDT-mediated cytotoxicity (3). Mitochondria, lysosomes, and the plasma membrane are all identified as subcellular PDT targets (2). PDT is an efficient and rapid inducer of apoptosis and necrosis (4). PDT is also a strong activator of genes encoding for transcription factors, cytokines, and stress-induced proteins (5, 6, 7, 8). Members of both the heat shock protein and GRP families are overexpressed after PDT. These proteins function as chaperones of nascent proteins and are also involved in protecting cells by binding to denatured proteins and assisting in proper refolding (7, 8).

Numerous preclinical studies indicate that PDT induces both direct tumor cell kill and vascular damage (2, 3, 9). Vascular damage within tumor tissue is accompanied by tumor tissue hypoxia (10, 11). Likewise, photochemical depletion of oxygen occurs during light illumination at high dose rates (10, 12). Therefore, PDT results in both oxidative stress and hypoxia within target tissue.

The current study builds upon our observation that PDT is an effective inducer of GRP expression and directly examines the effectiveness of the grp78 promoter to drive inducible gene expression after PDT (8). Several inducible gene expression strategies are being examined for potential therapeutic applications. These procedures allow for temporal and spatial regulation of therapeutic gene activation, which minimizes potential systemic toxicity of cytotoxic gene products. Promising preclinical gene therapy studies have combined radiotherapy together with the radiation-inducible egr-1 promoter to induce therapeutic gene expression (13). We and others have shown that hyperthermia and/or PDT can induce selective and temporal expression of heterologous genes under the control of the heat shock protein promoter (14, 15, 16). Recent experiments have also shown strong inducible expression of transgenes under the control of the grp78 promoter or multiple copies of the hypoxic response element in hypoxic tumor tissue (17, 18). We have attempted to take advantage of the fact that both PDT-mediated oxidative stress and PDT-mediated tumor tissue hypoxia can transcriptionally activate GRP-78 (19). We hypothesized that combining PDT with inducible gene therapy using the grp78 promoter to drive selective expression of HSV-tk would improve tumor treatment responses.

Drugs and Reagents.

The photosensitizer Photofrin porfimer sodium was a gift from QLT PhotoTherapeutics, Inc. (Vancouver, British Columbia, Canada) and Axcan Scandipharm, Inc. (Birmingham, AL). The drug was dissolved in 5% dextrose in water to make a 2.5-mg/ml stock solution. Ganciclovir was obtained from Roche Laboratories, Inc. (Nutley, NJ). A 50-mg/ml stock solution of GCV was diluted with normal saline to obtain a 10-mg/ml working solution. The calcium ionophore A-23187 was obtained from Calbiochem (La Jolla, CA), and a stock solution was prepared in ethanol at a concentration of 1 mg/ml.

Cell Culture and in Vivo Tumor Models.

The TSA mouse mammary adenocarcinoma cell line was used in all in vitro and in vivo experiments (20). Cells were grown as a monolayer in DMEM supplemented with 4.5 mg/ml glucose, 10% FCS, 2 mm glutamine, and 1% penicillin-streptomycin-neomycin. TSA cells transduced with the G1NaGrpTk vector were maintained in the same culture conditions along with 600 mg/ml of G418. Mammary carcinomas were generated by injection of 106 TSA cells or TSA cells infected with the G1NaGrpTk-inducible expression vector in the hind right flank of 8–12-week-old female BALB/c mice.

Treatment Protocols.

In vitro photosensitization experiments involved seeding 2 × 106 cells into 100-mm plastic Petri dishes and incubating overnight in complete growth medium to allow for cell attachment. PDT treatments included incubating cells in the dark at 37°C for 16 h with PH (25 μg/ml) in medium containing 5% FCS. Cells were then incubated for an additional 30 min in growth media containing 10% FCS and rinsed in medium without serum. The cells were then exposed to graded doses of red light (570–650 nm) generated by a parallel series of red Milar-filtered 30 W fluorescent bulbs and delivered at a dose rate of 0.35 mW/cm2(14). Cell protein was collected at various time intervals after treatment.

In vivo PDT tumor treatments were performed as reported previously on tumors measuring 6 mm in diameter (21). Mice were randomly placed in groups receiving either no treatment, PDT alone, PDT plus GCV, or GCV alone. PDT included an i.v. injection of PH (5 mg/kg), followed 24 h later with tumor-localized laser irradiation using an argon-pumped dye laser emitting red light at 630 nm. A nonthermal light dose rate of 75 mW/cm2 and a total light dose of 200 J/cm2 were used for all in vivo PDT treatments. GCV was administered as a daily 100 mg/kg i.p. injection starting 1 h before PDT light exposure. After treatment, tumors were measured three times/week. Cures were defined as being disease free for at least 40 days after PDT. Previous studies in our laboratory have shown that mouse tumor responses observed 40 days after PDT treatment remain constant when analyzed at 90 days.

Western Blot Analysis.

HSV-tk expression was monitored by Western immunoblot analysis as described previously (14). Control and treated cells were scraped off Petri dishes and transferred to 15-ml tubes. Cell pellets were lysed in SDS loading buffer (4% SDS, 10% glycerol, 4% 2-mercaptoethanol, 0.125 m Tris base, and 0.02% bromphenol blue, pH 6.8). Tumors from treated mice were collected at various times after treatment and homogenized with a Polytron in 1× commercial lysis buffer (Promega Corp., Madison WI). Protein concentrations were determined using Bio-Rad protein analysis solution (Bio-Rad, Hercules, CA). Protein samples (30 μg) were size separated on 10% discontinuous polyacrylamide gels and transferred overnight to nitrocellulose membranes. Filters were blocked for 1 h with 5% nonfat milk and then incubated for 2 h with either rabbit polyclonal anti-TK antibody (provided by W. Summers, Yale University, New Haven, CT) or mouse monoclonal antiactin antibody (clone C-4; ICN, Aurora, OH). Filters were then incubated with antirabbit or antimouse IgG (Sigma Chemical Co., St. Louis, MO), and the resulting complexes were visualized by enhanced chemiluminescence autoradiography (Amersham Life Science, Chicago, IL).

Statistical Analysis.

The PDT treatment response data were evaluated using the log-rank test.

Goals of the present study were to document whether PDT could induce selective expression of an HSV-tk transgene that was under the control of the grp promoter and to determine whether such gene expression could enhance PDT effectiveness. A variety of gene therapy strategies offer potential clinical benefits in the treatment of solid tumors, including the combination of conventional tumor treatments with gene therapy (13, 14, 15, 16, 17, 18). Improvements in PDT responsiveness may also be obtained by combining this physical treatment with spatially controlled therapeutic gene therapy. The suicide gene HSV-tk, in combination with the prodrug GCV, has been studied extensively in preclinical and clinical gene therapy studies (22). Replicating DNA is the primary target for GCV in tumors expressing HSV-tk. Nucleoside analogues such as GCV are phosphorylated by HSV-tk. This leads to DNA incorporation of the phosphorylated GCV during S-phase and subsequent cytotoxicity by inhibiting DNA chain elongation. Cell death is most often apoptotic and independent of p53 status (23). A bystander effect for HSV-tk plus GCV results in cytotoxicity to HSV-tk-negative cells as well as to HSV-tk-positive cells (23). This phenomenon helps to compensate for the low transfer rate of genes into target tumor tissue. Numerous other cytotoxic gene products or prodrugs are also being examined in preclinical and clinical gene therapy trials. However, the nonselective toxicity of the therapeutic gene products being examined often necessitates that one minimize the level of systemic gene expression (15). Constitutive expression of some therapeutic genes can induce uncontrolled toxicity to normal tissue as well as to targeted tissue. Likewise, gene therapy expression vectors can leak out of tissue after direct injection. Therefore, interest in methods to minimize systemic toxicity using spatially inducible expression systems has increased.

We have performed proof of principle experiments using tumor cells stably transduced with the HSV-tk suicide gene controlled by the grp promoter. GRPs are stress-inducible proteins localized to the endoplasmic reticulum (24). These calcium-binding proteins serve as chaperones and assist in the folding and assembly of nascent proteins. The grp78 promoter has been shown to be highly inducible under glucose starvation conditions and within the environment of large solid tumors (17). The grp promoter has several unique characteristics, which suggested that it might also be an effective inducible promoter to study with PDT. GRPs are transcriptionally activated by both oxidative stress and hypoxia (8, 19). PDT produces both photochemically generated singlet oxygen and tumor tissue hypoxia secondary to vascular damage (1). The 695-bp grp-78 promoter fragment inserted into the G1NaGrpTk retroviral construct used in this study contains three copies of the endoplasmic reticulum stress response elements, a TATA element, as well as binding sites for CCAAT, Sp1, and cyclic AMP-responsive element binding protein transcription factors (17, 24). Synthetic endoplasmic reticulum stress response element consisting of two units of a 19-bp sequence motif (CCAAT)N9(CCACG) are responsive to glucose starvation (25). The removal of the noninducible elements is currently being examined in the context of further enhancing the selective inducibility of the grp promoter.

Our first set of experiments examined the in vitro effectiveness of PDT to induce expression of HSV-tk under the transcriptional control of the grp promoter (23). Fig. 1 shows Western analysis results demonstrating that PDT efficiently induced HSV-tk expression in TSA cells stably transduced with the G1NA grp-Tk retroviral vector. The PDT doses (0–630 J/m2) used in these experiments induced from 0 to 80% cytotoxicity, as measured by a clonogenic assay (data not shown). Fig. 1,A shows HSV-tk expression results for transduced cells collected 12 h after exposure to increasing PDT doses. The calcium ionophore, A-23187, functions as a strong transcriptional activator of grp-78 and served as a positive control for in vitro inducibility of HSV-tk under the control of the grp promoter. In vitro PDT doses corresponding to 20–50% survival (i.e., 315–420 J/m2) were associated with considerable HSV-tk expression. Higher PDT doses resulted in reduced HSV-tk expression, probably because of the severity of the PDT cytotoxic response. Minimal constitutive HSV-tk expression was observed in untreated transduced cells. Likewise, HSV-tk expression was not initiated by PH incubation alone. Kinetic analysis of HSV-tk expression is shown in Fig. 1 B for transduced cells exposed to a PDT light dose of 315 J/m2. This PDT dose resulted in 20–30% lethality. HSV-tk expression was observed from 8 to 36 h after treatment.

The second set of experiments examined the in vivo efficiency of PDT to function as a molecular switch for the inducible expression of HSV-tk. Stably transduced TSA cells were injected into the hind flank of BALB/c mice and produced reproducible solid tumors amenable to PDT. PDT treatments were started when tumors reached 6 mm in diameter. Fig. 2,A shows HSV-tk expression 12 h after PDT doses ranging from 50 to 350 J/cm2. PDT produced significant HSV-tk expression over a large range of light doses. The exceptionally strong in vivo inducible expression of HSV-tk may be related to the fact that PDT induces both oxidative stress and tumor tissue hypoxia (3, 9, 10, 11). Both of these stress conditions can activate GRP-78 (8, 19). Fig. 2 B shows the kinetics of PDT-inducible HSV-tk expression in treated tumor tissue. Expression is observed within 12 h of treatment and continues to be observed 36 h after treatment. PDT elicits rapid hemorrhagic necrosis in tumor tissue, and therefore, analysis of HSV-tk expression at time intervals longer than 36 h was not possible because of the lack of removable tissue (2, 3). Nevertheless, HSV-tk expression in any remaining viable tumor cells would likely occur at time periods longer than 36 h because of the prolonged hypoxia induced by PDT (1, 3). These experiments demonstrate a strong and prolonged expression of a therapeutic transgene under the control of the grp promoter.

We next examined the therapeutic efficacy of the PDT-inducible gene therapy in BALB/c mice transplanted with s.c. tumors derived from stably transduced TSA cells. Tumors were again treated when they reached 6 mm in diameter. Fig. 3 shows tumor response data for mice treated with PDT alone, GCV alone, or a combination of PDT plus GCV. The PDT dose (5 mg/kg, 200 J/cm2) was chosen because: (a) it elicited a positive inducible HSV-tk expression in tumor tissue as shown in Fig. 2; and (b) it produces a 50% cure rate in this tumor model when used alone. We have shown previously that this tumor response level allows for effective evaluation of combination therapies involving PDT and antiangiogenic therapy (26). A standard multiday administration regimen of GCV was used (17, 20). GCV by itself did not result in any cures in the transduced TSA tumors, probably because of the minimal level of hypoxia in these 6-mm lesions. PDT alone resulted in a 50% tumor cure rate, whereas the combination of PDT and GCV resulted in 100% cures. The combination of PDT plus PDT-inducible gene therapy showed a significantly enhanced tumor response compared with PDT alone (P = 0.0066). Treatment of nontransduced parental tumors with GCV did not affect PDT sensitivity (data not shown). These results indicate that HSV-tk gene expression under the control of the grp promoter was effective at enhancing the tumoricidal response of PDT when the prodrug GCV was added to the treatment regimen.

PDT-responsive, promoter-mediated gene activation has been demonstrated previously using the heat shock protein promoter (14). The results of the current study extend these observations and demonstrate the effectiveness of PDT to activate an HSV-tk gene controlled by the grp promoter. This study also illustrates the feasibility of using PDT-controlled, HSV-tk suicide gene therapy in the treatment of solid tumors. Clinically relevant PDT doses and treatment parameters were used in this study, suggesting that PDT-inducible gene therapy could be expected to function within current clinical treatment protocols. Studies are in progress to evaluate the in vivo responsiveness of PDT-mediated gene therapy after direct injection of adenoviral expression vectors.

Fig. 1.

Inducible expression of HSV-tk is observed in TSA G1NaGrpTk cells after exposure to PDT. Cell lysates were assayed for tk and actin levels by Western immunoblot analysis. A, transduced cells were exposed to increasing PDT doses (0–6.3 × 10−2 J/cm2) and collected 12 h after treatment. Transduced cells exposed to the calcium ionophore A23187 for either 16 or 20 h served as a positive control for grp promoter activation and TK expression. Nontransduced parental TSA cells did not exhibit TK expression. B, transduced cells were exposed to a 3.15 × 10−2 J/cm2 PDT dose and collected at increasing time intervals (0–72 h) after treatment. Expression of actin was used to monitor protein loading.

Fig. 1.

Inducible expression of HSV-tk is observed in TSA G1NaGrpTk cells after exposure to PDT. Cell lysates were assayed for tk and actin levels by Western immunoblot analysis. A, transduced cells were exposed to increasing PDT doses (0–6.3 × 10−2 J/cm2) and collected 12 h after treatment. Transduced cells exposed to the calcium ionophore A23187 for either 16 or 20 h served as a positive control for grp promoter activation and TK expression. Nontransduced parental TSA cells did not exhibit TK expression. B, transduced cells were exposed to a 3.15 × 10−2 J/cm2 PDT dose and collected at increasing time intervals (0–72 h) after treatment. Expression of actin was used to monitor protein loading.

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Fig. 2.

Inducible expression of HSV-tk is observed in TSA G1NaGrpTk tumors after in vivo PDT treatment. Tumor lysates were assayed for TK and actin levels by Western immunoblot analysis. Localized PDT was performed on tumors measuring 6 mm in diameter. A, tumors were exposed to increasing doses of PDT (5 mg/kg PH; 0–350 J/cm2) and collected 12 h after treatment. B, for TK kinetic analysis, tumors were exposed to a 100 J/cm2. PDT dose and tumor lysates were collected at increasing time intervals (0–36 h) after treatment. Expression of actin was used to monitor protein loading.

Fig. 2.

Inducible expression of HSV-tk is observed in TSA G1NaGrpTk tumors after in vivo PDT treatment. Tumor lysates were assayed for TK and actin levels by Western immunoblot analysis. Localized PDT was performed on tumors measuring 6 mm in diameter. A, tumors were exposed to increasing doses of PDT (5 mg/kg PH; 0–350 J/cm2) and collected 12 h after treatment. B, for TK kinetic analysis, tumors were exposed to a 100 J/cm2. PDT dose and tumor lysates were collected at increasing time intervals (0–36 h) after treatment. Expression of actin was used to monitor protein loading.

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Fig. 3.

Inducible gene therapy using the grp promoter to drive expression of the HSV-tk suicide gene enhances the tumoricidal action of PDT. Treatment of TSA G1NaGrpTk tumors with a combination of PDT and GCV resulted in increased tumor cures compared with PDT treatment alone. BALB/c mice with 6-mm diameter TSA G1NaGrpTk tumors were exposed to either PDT + GCV (10 daily injections of GCV (100 mg/kg per dose) commencing 1 h before a single PDT treatment (5 mg/kg PH, 200 J/cm2; n = 13); PDT alone (the identical PDT treatment without GCV; n = 10), or GCV alone (10 daily injections of GCV; n = 8). Mice were monitored for tumor recurrences three times per week for 40 days. ∗, statistically significant difference in percentage of cures between PDT alone versus PDT + GCV (P = 0.0066).

Fig. 3.

Inducible gene therapy using the grp promoter to drive expression of the HSV-tk suicide gene enhances the tumoricidal action of PDT. Treatment of TSA G1NaGrpTk tumors with a combination of PDT and GCV resulted in increased tumor cures compared with PDT treatment alone. BALB/c mice with 6-mm diameter TSA G1NaGrpTk tumors were exposed to either PDT + GCV (10 daily injections of GCV (100 mg/kg per dose) commencing 1 h before a single PDT treatment (5 mg/kg PH, 200 J/cm2; n = 13); PDT alone (the identical PDT treatment without GCV; n = 10), or GCV alone (10 daily injections of GCV; n = 8). Mice were monitored for tumor recurrences three times per week for 40 days. ∗, statistically significant difference in percentage of cures between PDT alone versus PDT + GCV (P = 0.0066).

Close modal

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 investigation was performed in conjunction with the Clayton Foundation for Research and was supported in part by USPHS Grants RO-1 CA-31230, RO-1 CA-27607, and PO-1 CA59318 from the NIH, Office of Naval Research Grant N000014-91-J-4047 from the Department of Defense, United States Army Medical Research Grant BC981102 from the Department of Defense, the Neil Bogart Memorial Fund of the T. J. Martell Foundation for Leukemia, Cancer and AIDS Research, Susan Komen Breast Cancer Grant BCTR 2000359, and the Las Madrinas Endowment for Experimental Therapeutics in Ophthalmology.

3

The abbreviations used are: PDT, photodynamic therapy; GCV, ganciclovir; grp, glucose regulated protein; HSV, herpes simplex virus; tk, thymidine kinase; PH, Photofrin porfimer sodium.

We thank QLT PhotoTherapeutics, Inc., Vancouver, British Columbia, Canada and Axcan Scandipharm, Inc., Birmingham, AL for the generous gift of Photofrin; William C. Summers, Yale University, for providing HSV-tk antibody; Earl Leonard for assistance with statistical analysis; and Angela Ferrario for helpful discussions.

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