A Complex Adenovirus Vector That Delivers FASL–GFP with Combined Prostate-Specific and Tetracycline-Regulated Expression

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doi:10.1006/mthe.2001.0478, available online at http://www.idealibrary.com on IDEAL

A Complex Adenovirus Vector That Delivers FASL–GFP with Combined Prostate-Specific and Tetracycline-Regulated Expression Semyon Rubinchik,1 Danher Wang,2 Hong Yu,1 Fan Fan,1 Min Luo,1 James S. Norris,1 and Jian-yun Dong1,* 1

Department of Microbiology and Immunology, Medical University of South Carolina, Charlestown, South Carolina 29403, USA 2 GenPhar Incorporated, Mt. Pleasant, South Carolina 29464, USA *To whom correspondence and reprint requests should be addressed. Fax: (843) 792-2464. E-mail: [email protected].

Cell-type-restricted transgene expression delivered by adenovirus vectors is highly desirable for gene therapy of cancer, as it can limit cytotoxic gene expression to tumor cells. However, many tumor- and tissue-specific promoters are weaker than the constitutively active promoters and are thus less effective. To combine cell-type specificity with high-level regulated transgene expression, we have developed a complex adenoviral vector. We have placed the tetracycline transactivator gene under the control of a prostate-specific ARR2PB promoter, and a mouse Tnfsf6 (encoding FASL)–GFP fusion gene under the control of the tetracycline responsive promoter. We have incorporated both expression cassettes into a single construct. We show that FASL–GFP expression from this vector is essentially restricted to prostate cancer cells, in which it can be regulated by doxycycline. Higher levels of prostate-specific FASL–GFP expression were generated by this approach than by driving the FASL–GFP expression directly with ARR2PB. More FASL–GFP expression correlated with greater induction of apoptosis in prostate cancer LNCaP cells. Mouse studies confirmed that systemic delivery of both the prostate-specific and the prostate-specific/tet-regulated vectors was well tolerated at doses that were lethal for FASL–GFP vector with CMV promoter. This strategy should be able to improve the safety and efficacy of cancer gene therapy using other cytotoxic genes as well. Key Words: prostate-specific promoter, tetracycline expression system, combined regulation, expression amplification

INTRODUCTION Gene therapy approaches to the treatment of many types of human cancers are highly attractive because they hold the possibility of selectively targeting and killing tumor cells while avoiding high levels of systemic toxicity associated with treatments like chemotherapy and radiation. In cases in which traditional treatments have proved to be of limited benefit, as is the case for prostate cancer, development of alternatives such as gene therapy is even more important. A number of clinical trials investigating a wide variety of tumor targeting and ablation strategies is currently underway [1–3]. Adenovirus vectors (AdVs) are well suited to serve as a platform for delivery of therapeutic genes to tumor cells, as they can be grown and concentrated to very high titers, the preparations are stable and can be stored for extended periods of time, and the vectors are known to infect a wide variety of cell types, either actively dividing or quiescent

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[4,5]. Although studies have been published describing the use of AdVs that selectively replicate in tumor cells [6–8], most anti-cancer vectors are based on first-generation, replication-defective type 5 adenovirus. Because these vectors cannot replicate, one of the problems associated with their use in the treatment of large or dispersed tumors is the difficulty of infecting all or even most of the tumor cells [9–12]. For this reason, AdV-mediated tumor eradication strategies generally rely on the concept known as the “bystander effect,” which is essentially the extension of cytotoxic effects from transduced to untransduced cells [1,13]. The efficiency and range of the bystander effect depends on many factors, including the expression level of the cytotoxic gene inside the target cell [14]. Typically, AdVs are targeted by direct injection into the tumor mass, so that strong, constitutively active promoters can be used to drive the expression of the cytotoxic genes. In cases in which systemic vector delivery is required, or if there is a

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tissues or to eliminate infiltrating immune cells [25,26]. Nevertheless, a large number of established cancer cell lines and primary tumor isolates are sensitive to FASL-induced apoptosis. Currently, we are investigating the feasibility of using adenovirus-mediated FASL gene therapy for the treatment of several types of cancers, including prostate cancer. Expression of FASL in vector-transduced cells generates a significant bystander effect. Because both ligand and receptor are membrane proteins, TNFRSF6-induced apoptosis is mediated through cell–cell contact, which means that the cell expressing FASL can induce apoptosis in several cells that are in close contact with it and express TNFRSF6. In addition, FASLexpressing cells may exfoliate membrane microvesicles containing FASL that can reach and eliminate more distant cells [27–29]. However, expression FIG. 1. Structures of adenovirus vectors. We assembled vector genomes in vitro using the pL-Ad and pR-Ad shuttle vectors. The resulting vectors are E1-deleted and have a deletion in the E3 region (E3 of FASL in some cells can result in accupromoter is retained). They also lack all of the E4 ORFs, except orf6, which is expressed from the E4 mulation of a soluble version of this promoter. protein in the extracellular medium. This is usually the result of metalloproteinase cleavage of the full-length FASL just outside the cell membrane [30,31]. The soluble carconcern that significant amounts of vector may “leak” boxy-terminal half of FASL does not induce apoptosis from the injection site and cause damage to non-tumor efficiently, and in fact inhibits cytotoxicity of the memcells, additional mechanisms to limit cytotoxic effects to brane-bound FASL [30,31]. tumor cells are used. One such approach, known as tranBecause TNFRSF6 and FASL interact at the cell surface scriptional targeting, uses cell-type- or tumor-specific proin a stoichiometric manner (that is, there is no enzymoters to selectively express the transgenes in target cells matic turnover), we hypothesized that unlike the case of [15–17]. Although most tumor-specific promoters are not prodrug-converting enzymes, increasing the expression as strong as constitutive promoters such as of cytotoxic agents such as FASL in target cells can lead cytomegalovirus (CMV), some reports suggest that the to a larger bystander effect and thus more effective vecability of vectors using these promoters to drive prodrugtors. In a previous study, we described an adenovirus converting enzymes (for example, HSV1-TK) to induce vector that delivers a fusion protein combining mouse cytotoxicity is not diminished [18]. FASL with green fluorescent protein (GFP). The expresFASL (also known as CD95L; encoded by the gene sion of this FASL–GFP protein was controlled by a tetraTNFSF6) is one of the cytotoxic agents being investigated cycline-regulated expression system [32]. Under induced for the treatment of various tumors [19,20]. This type II conditions, this system generated very high levels of membrane protein is a member of the tumor necrosis facFASL–GFP expression and induced apoptosis in many tor family [21] and, on binding to its receptor, TNFRSF6 different prostate cancer cell lines [33], including those (also known as FAS, APO-1, and CD95), it induces apopthat have previously been described as expressing tosis in receptor-expressing cells through a complex cashuman FASL and being resistant to TNFRSF6-mediated pase activation pathway [22]. Expression of FASL is priapoptosis [34,35]. More recently, we have characterized marily limited to activated T-lymphocytes and natural the activity of a modified probasin promoter, ARR2PB, killer (NK) cells, where it acts as one of the two major in an adenovirus vector and established that the activcytotoxic mechanisms for eliminating target cells [23] as ity of this promoter in prostate-derived LNCaP cells was well as a feedback mechanism to prevent the overprolifsignificantly higher than that of the natural prostate eration of the immune cells by inducing their apoptosis specific antigen (PSA) promoter but not as high as those at the end of the immune response [24]. However, certain of the CMV and activated tetracycline response element tumor cells are also known to express FASL, which they (TRE) promoters [36]. can potentially use to help them to invade surrounding

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FIG. 2. The rAd/FASL–GFPPS and rAd/FASL–GFPPS/TR vectors mediate prostate-specific expression of FASL– GFP. We seeded several different cell lines in 12-well plates and transduced them with indicated AdVs at a MOI of 100 in the presence of 10 nM DHT. We then examined cells with fluorescent microscopy 48 hours post-transduction.

Here, we make an AdV construct with ARR2PB driving the expression of FASL–GFP protein and show that, while transgene expression and associated cytotoxicity are completely restricted to LNCaP cells, the extent of that activity is lower than that achieved using the CMV promoter. We subsequently test the concept of a complex AdV that could deliver FASL–GFP expression driven by an advanced regulation system that combined the high prostate-cell specificity of ARR2PB with the regulated, high-level gene expression of a tet-responsive promoter. We characterize the activity of this vector, which demonstrates high-level, FASL–GFP expression and cytotoxicity in LNCaP cells that can be regulated by varying the concentrations of doxycycline. Both vectors with prostate cancer cell targeted expression had substantially lower systemic toxicity compared with the CMV promoter vector in a mouse model.

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RESULTS ARR2PB Restricts FASL–GFP Expression to LNCaP Cells The androgen-regulated synthetic ARR2PB promoter has been characterized as highly prostate-specific and has a high rate of transgene expression in LNCaP cells in transient DNA transfection assays [37]. We cloned ARR2PB to drive the expression of FASL–GFP fusion protein and inserted this expression cassette near the right inverted terminal repeat (r-ITR) of our Ad5-derived AdV (Fig. 1) to take advantage of the reduced E1A enhancer interference at that site [36]. The prostate-specific (PS) rAd/FASL–GFPPS vector propagated efficiently in 293CrmA cells, with very low levels of GFP fluorescence observed (data not shown). When we used it to transduce several cultured cell lines, we detected FASL–GFP expression in prostate cancer LNCaP cells only, confirming the specificity of the ARR2PB promoter (Fig. 2). However,

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FIG. 3. Pro-apoptotic activity of rAd/FASL–GFPPS is restricted to LNCaP cells but is less effective than that of rAd/FASL– GFPCMV. We seeded LNCaP, SF763, and A549 cells in 96-well plates and transduced them with the indicated vectors at MOIs of 30, 90, and 270 the next day. Culture media contained 10 nM DHT. At 48 h post-transduction, we determined cell viability using an MTS assay. Average cell viability is shown as a percentage of living cells ± SEM (n = 4). Viability of mock-transduced cells was taken as 100%.

qualitative observations of GFP fluorescence indicated that the rAd/FASL–GFPPS vector generated less FASL–GFP expression in LNCaP cells than rAd/FASL–GFPCMV (Fig. 1), a vector with a strong constitutive CMV promoter driving FASL–GFP expression (Fig. 2). To characterize the pro-apoptotic activity of rAd/FASL–GFPPS, we transduced three cell lines with that vector, rAd/FASL–GFPCMV, or control rAd/GFP vector (Fig. 1) at several multiplicities of infection (MOI) and quantitatively determined the effect on cell viability using colorimetric soluble tetrazolium compound (MTS) assay (Fig. 3). The control vector had a very small negative effect on the viability of all three cell lines, and rAd/FASL–GFPPS transduction had a similar minor effect on viability of either glioma SF763 cells (82.6% versus 75.2% at MOI 270, P value > 0.19) or lung carcinoma A549 (75.7% versus 61.6%, P value < 0.01) cells (Fig. 3). Viability of LNCaP cells was reduced by 21% to 57% (depending on MOI) following rAd/FASL–GFPPS transduction compared with rAd/GFP. However, rAd/FASL–GFPCMV was more effective than the prostate-specific vector in LNCaP cells (Fig. 3), reducing cell viability to 9.5% compared with the latter’s 36.6% (P value < 0.005). These findings indicate that

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although rAd/FASL–GFPPS can direct FASL–GFP expression specifically to prostate-derived LNCaP cells, it generates less expression in those cells than the CMV promoter, and subsequently is less effective in inducing cell death through apoptosis. A Direct Correlation between FASL–GFP Expression Levels and Apoptosis in LNCaP Cells We next confirmed and characterized the relationship between the level of FASL–GFP expression in LNCaP cells and the loss of cell viability. Previous studies have reported that LNCaP cells co-expressed TNFRSF6 and FASL on the cell surface [34] and were resistant to anti-TNFRSF6 antibodies that were capable of inducing apoptosis in other cancer cells [35]. These cells, therefore, have a substantial level of resistance to TNFRSF6mediated apoptosis. However, we have previously shown that LNCaP cells could be forced into apoptosis by transduction with an AdV that generated very high expression levels of FASL–GFP using a tet-regulated expression system [33]. To regulate FASL–GFP expression levels in our study, we constructed an AdV with constitutively expressed reverse tet transactivator (rtTA) in the E1 region and TRE promoter regulated FASL–GFP near the right ITR (Fig. 1). In this type of tet-regulated system, known as “Tet-ON,” rtTA must bind to the tetracycline analog doxycycline before it can bind to the TRE and activate transcription [38]. To characterize the correlation between the expression levels of FASL–GFP and the induction of apoptosis in LNCaP cells, we infected these cells with rAd/FASL–GFPrtTA at a MOI of 100, and cultured them at different doxycycline concentrations. Parallel cell samples were quantitatively analyzed for the extent of FASL–GFP expression (as a function of GFP fluorescence) and cell viability (Fig. 4). We found that LNCaP cells infected at the same MOI showed substantial increases in GFP fluorescence with increasing doxycycline concentrations (Fig. 4A), and that there was a strong correlation between increasing levels of FASL–GFP expression and increasing levels of apoptosis (Fig. 4B). In the absence of doxycycline (background TRE promoter activity) only 27% of LNCaP cells were nonviable, whereas at 1 mg/ml doxycycline, more than 70% of cells became apoptotic (Fig. 4B). Similar correlations between FASL–GFP expression and apoptosis were observed in MCF-7 and SF763 cells (data not shown), which have also been reported to be somewhat resistant to FasL activity. Therefore, increasing the levels of FASL–GFP expression in “TNFRSF6-resistant” cells such as LNCaP can significantly improve induction of apoptosis in those cells, necessitating the development of a strategy that could increase the activity of ARR2PB.

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AdV with Combined Prostate-Specific and Tet-Regulated FASL–GFP Expression Generates More FASL–GFP and High Apoptosis Levels in LNCaP Cells To verify relative activity of several different promoters in LNCaP cells, we transfected these cells with plasmid constructs carrying a SEAP reporter gene under the control of the ARR2PB promoter, TRE promoter, or human CMVi/e promoter. We used A549 cells as a non-prostate cell control. All SEAP vectors were cotransfected with pL-Ad-C.rtTA plasmid vector, which contains the reverse tet transactivator under the control of the CMV promoter. The ARR2PB promoter generated approximately 32% and 0.08% of CMV activity in LNCaP and A549 cells, respectively, in the presence of DHT (Fig. 5A). In the absence of androgen activation, this activity was 1.4% and 0.08% of CMV activity. The TRE promoter had 1.7% and 2.3% of CMV activity in LNCaP and A549 cells, respectively, in the absence of doxycycline (Fig. 5A). However, when the rtTA was activated by the addition of doxycycline to culture medium, TRE activity reached 138% and 270% of CMV activity in LNCaP and A549 cells, respectively (Fig. 5A). These observations confirmed that although ARR2PB is a relatively strong promoter, its activity in LNCaP cells is lower than that of the activated TRE promoter. Therefore, we decided to combine the prostate specificity of ARR2PB with the high expression levels and small drug molecule regulatability of the tetracycline system. The resulting vector, rAd/FASL–GFPPS/TR, can deliver FASL–GFP with a prostate-specific and tetracycline-regulated (PS/TR) expression pattern (Fig. 1). To construct this AdV, we placed the tetracycline-repressed transactivator (tTA) gene under the control of the ARR2PB promoter in the E1 region of the vector, whereas Tnfrsf6–GFP and the TRE promoter that drives it were cloned near the right ITR. In LNCaP cells (or in any cell with active androgen receptor) in the presence of androgen such as DHT, tTA will be expressed and will be able to bind to TRE and activate high levels of expression of FASL–GFP. In non-prostate cells, no tTA will be made, and only background levels of FASL–GFP expression will occur. Similarly, FASL–GFP expression is reducible to background levels in prostate-derived cells by the addition of tetracycline or doxycycline, which binds to tTA and prevents its interaction with the TRE. When compared with rAd/FASL–GFPPS, rAd/FASL–GFPPS/TR generated up to sevenfold more FASL–GFP expression in LNCaP cells transduced at a MOI of 100, as determined by quantitative analysis of GFP fluorescence (Fig. 5B). Analysis of cell viability has determined that in LNCaP cells transduced at a MOI of 50, rAd/FASL–GFPPS/TR killed 56% of the cells, whereas rAd/FASL–GFPPS killed 32% (P value < 0.02, Fig. 6). In fact rAd/FASL–GFPPS/TR performed slightly better than the rAd/FASL–GFPCMV (viability of 44% and 48%, respectively, P value < 0.007) at a MOI of 50 (Fig. 6). Transduction of LNCaP cells with a control rAd/GFP vector had little effect on cell viability.

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FIG. 4. Correlation between FASL–GFP expression and cell viability in LNCaP cells. We seeded cells in 12-well plates. The next day, we infected cells at a MOI of 100 with rAd/FASL–GFPrtTA. We added doxycycline to wells at the concentrations indicated. At 48 h post-transduction, we collected all cells from each well, washed then with 1 PBS, and resuspended them in 200 l of 1 PBS. (A) We transferred half of each sample to a well of a black 96-well plate and determined GFP fluorescence. Average fluorescence activity is shown in relative fluorescence units ± SEM (n = 3). Fluorescence of mock-transduced cells was taken as background and subtracted from other samples. (B) We transferred the other half of each sample to a well of a clear 96-well plate which contained 20 l of MTS reagent per well. After 1 hour incubation at 37C, we determined cell viability. Average cell viability is shown as a percentage of living cells ± SEM (n = 3). Viability of mocktransduced cells was taken to be 100%.

The Basal Activity of the TRE Promoter Is Higher Than That of the ARR2PB Promoter Although rAd/FASL–GFPPS/TR generated very high levels of FASL–GFP expression in only LNCaP cells, low levels of expression were detectable in other (non-prostate) cell lines (Fig. 2). Quantitative cell viability assays in A549 and SF763 cells confirmed that transduction with rAd/FASL–GFPPS/TR vector resulted in greater reduction of cell viability than transduction with rAd/FASL–GFPPS at the same MOI (Fig. 6). However, pro-apoptotic activity of rAd/FASL–GFPPS/TR in non-prostate cells was significantly less than that induced by rAd/FASL–GFPCMV (Fig. 6), so that a large overall gain in activity in prostate versus

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non-prostate cells was realized. To determine whether the low level of FASL–GFP expression observed in nonprostate cells transduced with rAd/FASL–GFPPS/TR was due to the basal activity of the TRE promoter or if some leaky expression of the tTA protein has occurred, we infected LNCaP, SF763, and MCF-7 cells with rAd/FASL–GFPrtTA and rAd/FASL–GFPPS/TR vectors at a MOI of 200 in the presence or absence of doxycycline (Fig. 7). Under conditions that suppressed the binding of the tet transactivator to the TRE promoter (uninduced), both vectors reduced cell viability to a similar extent in all three cell lines (Fig. 7). When induced, the levels of apoptosis generated by rAd/FASL–GFPrtTA reached 85–90% in all cell lines, whereas rAd/FASL–GFPPS/TR generated similar levels of cell death only in LNCaP cells (Fig. 7), demonstrating a preferential induction of FASL–GFP in prostate-derived cells. Levels of cell death in rAd/FASL–GFPPS/TR-transduced MCF-7 and SF763 cells remained essentially the same under both induced and uninduced conditions, indicating that tTA was not expressed in these nonprostate cells and that the observed FASL–GFP expression was the result of basal transcriptional activity of the TRE promoter.

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FIG. 5. Comparison between ARR2PB and TRE promoter activities. (A) We seeded A549 and LNCaP cells in 12-well plates and transfected them the next day with plasmid vectors containing SEAP under the control of different promoters. Where two plasmids were co-transfected, a 1:1 molar ratio was used. The average SEAP activity is shown as relative luminescence units ± SEM (n = 3). The column on the right indicates the activity of each promoter in the two cell lines as it relates to the activity of the hCMVie promoter in each cell line: 1, cells transfected with pL-Ad.C.rtTA alone to determine background activity; 2, pR-Ad.C.SP (hCMVie) and pL-Ad.C.rtTA + 10 nM DHT; 3, pR-Ad.P.SP (ARR2PB) and pL-Ad.C.rtTA; 4, pRAd.P.SP (ARR2PB) and pL-Ad.C.rtTA + 10 nM DHT; 5, pR-Ad.T.SP (TRE) and pL-Ad.C.rtTA + 2 g/ml doxycycline; 6, pR-Ad.T.SP (TRE) and pL-Ad.C.rtTA. (B) We seeded LNCaP cells in a 24-well plate at 5  104 cells per well and infected them the next day with rAd/FASL–GFPPS or rAd/FASL–GFPPS/TR at a MOI of 100. DHT was added to the final concentration of 10 nM. After 48 h, we collected all cells from each well, washed them with 1 PBS, resuspended them in 100 l of 1 PBS, and transferred them to a well of a black 96-well plate. Shown is average GFP fluorescence activity in relative fluorescence units ± SEM (n = 3). 1, Cells transduced with rAd/FASL–GFPPS; 2, cells transduced with rAd/FASL–GFPPS/TR; 3, mock-transduced LNCaP cells.

AdVs with PS or PS/TR Regulation of FASL–GFP Expression Engender Substantially Reduced Systemic Toxicity The presence of low, nonspecific background transcriptional activity inherent in the TRE promoter raised a question of whether the regulated transcriptional amplification of tissue-specific promoters generated vectors that were truly safer than those with strong, ubiquitously active promoters. To address this issue, we injected groups of three male BALB/c mice through the tail vein with 2  108 and 109 IP of each of the rAd/FASL–GFPPS, rAd/FASL–GFPPS/TR, and rAd/FASL–GFPCMV vectors. All three mice injected with 109 IP of rAd/FASL–GFPCMV vector died less than 24 hours postinjection, whereas all other mice remained viable and had normal feeding and grooming activity. All remaining mice were sacrificed at 48 hours postinjection and frozen sections of their livers were examined for morphology with hematoxylin and eosin staining or for the extent of apoptosis with FITC-labeling TUNEL assay (Fig. 8). Liver sections of animals injected with either buffer only (Figs. 8A and 8B) or 109 IP of rAd/FASL–GFPPS (Figs. 8C and 8D) showed essentially normal histology and a very low background of apoptotic nuclei. In contrast, liver sections of animals injected with 109 IP of rAd/FASL–GFPCMV vector indicated a massive amount of tissue disruption and apoptosis (Figs. 8G and 8H). Finally, sections of livers from animals injected with 109 IP of rAd/FASL–GFPPS/TR vector have an overall normal appearance (Fig. 8E) with only a slightly higher than background level of apoptotic nuclei (Fig. 8F), suggesting that, despite background activity of the TRE promoter, the rAd/FASL–GFPPS/TR vector achieves a major improvement in safety when compared with constitutive promoter systems such as CMV.

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FIG. 6. Comparison of the pro-apoptotic activity of prostate-specific FASL–GFP expressing AdVs. We seeded cells in 96-well plates and infected them with indicated vectors at MOIs of 10 and 50 the next day. Culture medium contained 10 nM DHT. We determined cell viability using an MTS assay.

Discussion There is a great deal of interest in using gene therapy approaches to treat different types of cancer that may be resistant to more conventional treatments [1,2]. Adenovirus-based vectors have been used extensively as delivery vehicles to carry various cytotoxic genes to the target tumor cells [4,5]. To make such therapy safer for nontumor tissues, transcriptional targeting—in which tumorspecific promoters are used to direct the expression of the lethal genes—has been used [15–17]. Although most tissue-specific promoters are not as strong as the constitutively active promoters, reports on the use of targeted expression of prodrug converting enzymes such as TK seem to suggest that promoter strength has a minor if any role in determining vector efficacy [18]. We are interested in AdV-mediated delivery of FASL activity to force cancer cells to undergo apoptosis. FASL is a membrane protein that upon binding to its receptor, TNFRSF6 (a membrane protein on the surface of a target cell), activates a signal cascade that results in apoptosis of the target cell. As the TNFRSF6–FASL interaction is stoichiometric, we felt it was likely that vector efficacy could be improved by increasing the rate of FASL expression in transduced cells. In a previous study, we have placed a FASL–GFP fusion protein under the control of a tet-regulated expression system in a single complex adenovirus vector and were able to generate very high transgene expression levels in several prostate cancer cell lines in vitro [33]. We have found that apoptosis could be successfully induced in most of the cell lines tested, including those that were previously reported to be resistant to TNFRSF6-mediated apoptosis [39–41]. Although complete resistance to FASL signal can arise if a mutation is severe enough to generate a block in the TNFRSF6 pathway [42,43], it is possible that in many

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FIG. 7. Activity of rAd/FASL–GFPPS/TR and rAd/FASL–GFPrtTA vectors in the presence and absence of tTA- or rtTA-mediated TRE activation. We seeded MCF7, SF763, and LNCaP cells in 96-well plates and infected them with rAd/FASL–GFPrtTA or rAd/FASL–GFPPS/TR at a MOI of 200. After 48 h, we determined cell viability by MTS assay. Average cell viability is shown as a percentage of living cells ± SEM (n = 4). Viability of mock-transduced cells was taken to be 100%. 1, rAd/FASL–GFPPS/TR plus 2 g/ml doxycycline (uninduced TRE); 2, rAd/FASL–GFPPS/TR without doxycycline (induced TRE); 3, rAd/FASL–GFPrtTA without doxycycline (uninduced TRE); 4, rAd/FASL–GFPrtTA plus 2 g/ml doxycycline (induced TRE).

tumor cells resistance to TNFRSF6-mediated apoptosis is partial and may result from a reduced level of TNFRSF6 expression on their surface. Alternatively, tumor cells may express soluble forms of TNFRSF6 or FASL, which can antagonize the activity of the membrane-bound molecules to some extent [30,31,44]. We hypothesized that such partial resistance may be overcome by a combination of several features of our tet-regulated FASL–GFP expressing vector, among them a very high level of FASL–GFP expression. To develop new vectors specifically designed to use FASL–GFP expression to treat prostate cancer with improved safety, we had considered transcriptional targeting. Prostate-specific promoters are extremely selective for cells of prostate origin that express androgen receptor, and are therefore an excellent choice for limiting vectordelivered toxic gene expression to prostate cancer cells [45,46]. However, their activity is low compared with that of the CMV promoter [46]. Stronger, modified prostatespecific promoters have been developed, among them the ARR2PB modified probasin promoter [37]. To test the activity of these promoters in vitro, an LNCaP prostatecancer-derived cell line is typically used. LNCaP cells contain an active androgen receptor and express most prostate cancer markers, such as PSA and PMSA [47]. However, this cell line is resistant to FAS-mediated apoptosis, and is known to secrete soluble FASL [34]. We have constructed an AdV with the ARR2PB promoter driving FASL–GFP expression and compared its activity with that of the AdV with CMV promoter in several cell lines. As expected, ARR2PB completely limited FASL–GFP expression to LNCaP cells (Fig. 2) and was

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FIG. 8. Characterization of liver toxicity mediated by adenovirus vectors expressing FASL–GFP following systemic delivery in a mouse model. We injected male BALB/c mice through the tail vein with 200 l of 1 HBS containing no vector (A, B), 109 IP of rAd/FASL–GFPPS (C, D), 109 IP of rAd/FASL–GFPPSTR (E, F), or 109 IP of rAd/FASL–GFPCMV (G, H). (A, C, E and G) Hematoxylin and eosin staining; (B, D, F and H) FITC staining of apoptotic nuclei. We injected all mice with 1 l/g body weight of 10 mM DHT solution. We killed mice and harvested their livers 48 h after vector injection, except in the case of rAd/FASL–GFPCMV-injected mice, where this occurred at 24 h due to the death of the animals. We stained frozen sections of the livers with hematoxylin and eosin or conducted an in situ TUNEL assay on the section. Shown are photographs of sections as examined under the microscope.

essentially nontoxic to all other cell lines. At the same time, this vector generated less FASL–GFP expression in LNCaP cells than the CMV-driven construct (Fig. 2) and, more importantly, less apoptotic activity at the same MOI (Fig. 3). Using an AdV with a tet-regulated FASL–GFP expression system, we subsequently confirmed that in LNCaP cells (as well as in several other cell lines with partial resistance to TNFRSF6-mediated apoptosis) levels of FASL–GFP expression directly correlated with those of apoptosis (Fig. 4). Analysis of transient transfection promoter studies has indicated that in LNCaP cells DHT-activated ARR2PB could reach slightly more that 30% of CMV activity, whereas induced TRE promoter was more than fourfold more active than ARR2PB (Fig. 5A). Based on these observations, we decided to combine the prostate cell

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specificity of ARR2PB with the high expression levels of the tet-regulated expression system in a single AdV. We have constructed a vector with ARR2PB directing the expression of tTA protein while the FASL–GFP was placed under the control of the TRE promoter (Fig. 1). The TRE/FASL–GFP expression cassette was cloned near the right ITR of the Ad5 genome, to minimize transcription interference from the E1A enhancer. The new vector with the prostate-specific tet-regulated (PS/TR) expression system generated up to sevenfold more FASL–GFP expression in LNCaP cells then the ARR2PB vector (Figs. 2 and 5B) and was substantially more effective in inducing apoptosis of those cells, even surpassing the pro-apoptotic activity of the vector with CMV-driven FASL–GFP expression (Fig. 6). However, the PS/TR system was not as specific for

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prostate-derived cells as the ARR2PB, with a low background of FASL–GFP expression being observed in most of the non-prostate cells examined (Fig. 2). This background expression, in turn, resulted in decreased viability of the transduced non-prostate cells, with the levels of cell death somewhere between those generated by CMV and ARR2PB vectors (Fig. 6). We confirmed that this nonspecific activity was the result of the basal activity of the TRE promoter (Fig. 7). Background transcriptional activity of the tet-regulated system is an intrinsic property of the TRE promoter when the transducing DNA is in a non-integrated state [48] and therefore it is a feature of most currently available adenovirus vectors that use tet regulation. Clearly, these vectors would benefit from the development of mechanisms that would significantly reduce or eliminate basal TRE activity in the absence of induction. We are currently working on incorporating such mechanisms into the next generation of our vectors. In their role as anticancer agents, rAd/FASL–GFPTET and rAd/FASL–GFPrtTA vectors were envisioned to combine very high levels of transgene expression with increased safety, as transgene expression could be reduced to the basal TRE level if necessary. In practice, this would require a realization that something went wrong after the vector was injected into the patient, and by then significant damage could already occur. The rAd/FASL–GFPPS/TR vector was designed to solve this problem by generating very high levels of FASL–GFP expression in prostate-derived tumor cells but only background TRE activity in any non-prostate cells infected. To demonstrate that a substantial improvement in reducing vector-mediated systemic toxicity was achieved, we injected tail veins of male BALB/c mice with our FASL–GFP expressing vectors. All three mice injected with 109 IP of rAd/FASL–GFPCMV died in less than 24 hours, whereas mice injected with similar doses of either rAd/FASL–GFPPS or rAd/FASL–GFPPS/TR under conditions in which these vectors would be fully active in prostate cells (that is, with DHT and in the absence of doxycycline) survived with no observable negative effects. Histological examination of liver sections and apoptosis-detecting in situ TUNEL assay (Fig. 8) revealed that rAd/FASL–GFPCMV generated a massive amount of tissue damage with clear evidence of apoptosis. A similar dose of rAd/FASL–GFPPS resulted in no detectable change in morphology and no increase in the level of apoptosis, whereas rAd/FASL–GFPPS/TR seemed to produce no morphological changes and only a minor increase in apoptosis level. These results indicate that mouse liver cells are able to tolerate low amounts of FASL expression without triggering apoptotic cascade, but that apoptosis initiates once a certain level of FASL activity is reached. They also imply that the background activity of the TRE promoter is sufficiently low (at this level of vector load) to avoid this threshold in most infected liver cells in vivo. In principle, the approach we have used in this study— significantly increasing the activity of a cell-type-specific

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promoter by coupling it to the tet-regulated expression system—can be used with a variety of promoters and cytotoxic genes in those cases in which higher transgene expression would render oncolytic vectors more effective. If the issue of basal TRE expression can be successfully resolved, additional applications in other areas of gene therapy are also possible, as the system combines tissue/cell-type targeting with a relatively easy and safe method of regulating transgene expression from very low to very high levels. We have confirmed here that the increased promoter activity generates higher levels of FASL–GFP expression, which correlate with reduced cell viability in cell lines partially resistant to TNFRSF6-mediated apoptosis. The vector which combines prostate-specific promoter activity with the tet-induced expression system delivered FASL–GFP expression and induced cell death in prostate cancer cells at levels significantly higher than those provided by the vector using ARR2PB directly, while at the same time demonstrating a comparable level of safety in a systemic toxicity mouse model. Meanwhile, the rAd/FASL–GFPPS vector has demonstrated prostate cell specificity, but proved to be less effective in killing LNCaP cells when delivering FASL–GFP expression. Its high level of specificity would be suitable to delivery of a gene with very high cytotoxic activity, for example the Escherichia coli purine nucleoside phosphorylase [49]. These vectors provide a choice of safer platforms for delivery of gene therapeutics towards the treatment of prostate cancer. The strategy of amplifying and regulating the activity of tissuespecific promoters may also be applicable to gene therapy of other types of cancer.

MATERIALS

AND

METHODS

Cell lines. The androgen-responsive prostate cancer cell line LNCaP was obtained from Urocor (Oklahoma City, OK). Human A549 lung adenocarcinoma and embryonic kidney-derived HEK293 cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD). A variant of HEK293 cell line stably transformed with Cowpox virus cytokine response modifier protein, 293CrmA, has been described [32]. Human breast cancer cell line MCF-7 was obtained from the ATCC (HTB-22). The SF763 cell line was derived from a primary brain astrosarcoma isolate at the UCSF Medical Center. All cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Mediatech, Inc., Herndon, VA) supplemented with 10% cosmic calf serum (CCS) from HyClone (Logan, UT) and 1% penicillin/streptomycin (Mediatech, Inc.). Plasmid vectors. The pSEAP2-Basic vector was purchased from Clontech (Palo Alto, CA). The pUHD10-3 (containing the TRE promoter), pUHD151 (containing tTA gene) and pUHD17-1 (containing rtTA gene) vectors were contributed by Hermann Bujard (Center for Molecular Biology, University of Heidelberg, Heidelberg, Germany). The ARR2PB (0.45-kb) promoter was developed in the laboratory of Robert J. Matusik (Department of Cell Biology, Vanderbilt University Medical Center, Nashville, TN), who contributed the pARR2PB.PolI.TRZ-SK vector. ARR2PB is based on the minimal probasin promoter with a duplicated probasin Androgen Response Region (ARR) upstream of it [50]. Construction of pL-Ad-CMV, pL-AdC.GFsL, pL-Ad-C.GFP.B, pL-Ad-C.tTA, pR-Ad-mcs, and pR-Ad-T.GFsL vectors has been described [32]. We excised the TRE promoter from pUHD103, the CMV promoter from pL-Ad-CMV, and the ARR2PB promoter from

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doi:10.1006/mthe.2001.0478, available online at http://www.idealibrary.com on IDEAL

pARR2PB.PolI.TRZ-SK. We then cloned these promoters into the MCS (multiple-cloning site) of pSEAP2-Basic, generating pT.SP, pC.SP, and p2Pb.SP, respectively. We deleted the CMV promoter/enhancer from pL-Ad-CMV to generate pL-Ad-mcs. We cloned the rtTA gene from pUHD17-1 into pL-Admcs to produce pL-Ad-C.rtTA. We removed the entire SEAP expression unit (containing transcription blocker (TB), promoter, SEAP gene, and SV40pA) from the vectors p2Pb.SP, pT.SP, and pC.SP using NotI and SalI, and cloned each promoter element separately into pL-Ad-mcs and pR-Ad-mcs (cut with NotI and XhoI) to generate L-Ad and R-Ad series of constructs, respectively. We replaced SEAP in pL-Ad.2Pb.SP with the tTA gene from pUHD15-1 to generate pL-Ad-2Pb.tTA. Similarly, we replaced SEAP in pR-Ad-2Pb.SP with FASL–GFP to generate pR-Ad-2Pb.GFsL. Adenovirus vectors. We assembled adenovirus vectors using the following combinations of pL-Ad and pR-Ad shuttle vectors: pL-Ad-C.GFsL and pRAd-mcs to construct rAd/FASL-GFPCMV, pL-Ad-C.rtTA and pR-Ad-T.GFsL to construct rAd/FASL-GFPrtTA, pL-Ad-mcs and pR-Ad-2Pb.GFsL to construct rAd/FASL-GFPPS, and pL-Ad-2Pb.tTA and pR-Ad-T.GFsL to construct rAd/FASL-GFPPS/TR. Construction of rAd/GFP has been described [32]. We assembled the genomes of recombinant Ad vectors in vitro and transfected them into 293CrmA cells as described [32,36]. We propagated the cultures until virus-induced cytopathic effects (CPE) were observed. We amplified primary vector stocks according to established techniques, banding twice using CsCl gradient ultracentrifugation and desalting twice using PD-10 columns (Amersham Pharmacia Biotech, San Francisco, CA) against 1 HEPES saline buffer (HBS; 21 mM HEPES, 140 mM NaCl, 5mM KCl, 0.75 mM Na2HPO4.H20 and 0.1% (w/v) dextrose) according to the manufacturer’s instructions. We titrated the vectors on 293 cells based on GFP fluorescence and scored the titers as infectious particles (IP) per ml. Vector structures are diagrammed in Fig. 1. Transient transfections and viral vector infections. We seeded cells in 12well plates (Greiner, Frickenhausen, Germany) at 1  105 cells per well. The next day, we transfected cells with plasmid constructs using SuperFect reagent (Qiagen, Valencia, CA) according to the manufacturer’s instructions. At this time, we also added additional assay components such as dihydrotestosterone (DHT, Sigma, St. Louis, MO) or doxycycline (Sigma). We transfected control wells with pL-Ad-C.Gf.B construct and monitored transfection efficiency by visualizing GFP fluorescence in cells after 24 hours using Axiovert-25 fluorescent microscope (Zeiss Optical Systems, Inc., Thornwood, NY) and FITC excitation/emission filter set (Chroma Technology Corp., Brattleboro, VT). Alternatively, if cells were to be infected with rAd vectors, we treated two wells from each plate with a trypsin-containing solution to detach the cells and subsequently counted those cells using a counting chamber slide (VWR Scientific Products, Atlanta, GA). We then infected the remaining wells with the appropriate rAd vector at a desired MOI. We monitored the efficiency of infection after 24 h as described above for transient transfections. When required, we took photographs (100 magnification) of infected cells using an attached Pixera Professional digital camera (Pixera Corp., Los Gatos, CA). Quantitative GFP fluorescence assay. To determine relative expression levels of the FASL–GFP protein, we seeded 5  104 cells per well into 24well plates (Greiner). The next day, we detached cells from two wells with trypsin and counted them to determine average cell number per well. We infected the remaining wells with viral vectors at desired the MOI. We added doxycycline to appropriate wells to the required final concentrations. After 48 h, we removed cells from each well, transferred them to 1.5-ml Eppendorf tubes, washed them once with 1 PBS, and resuspended the cells in 100 l of 1 PBS. We next transferred each cell sample to a well of a 96-well black plate (BMG Labtechnologies, Durham, NC). We then determined relative GFP fluorescence using Fluostar dual fluorescence/absorbance plate reader (BMG Labtechnologies) with 485 nm excitation and 520 nm emission filter set. Secreted alkaline phosphatase assay. We seeded cells in 12-well plates and transfected them as described above. After 24 h, we removed the culture medium, washed the cells with 1 PBS, and added 1 ml of fresh medium to each well. If necessary for the particular experiment, components such as DHT and/or doxycycline were added. After another 24 h, we collected and froze 200 l samples of the culture medium. We determined SEAP activity using the Great EscAPe chemiluminescence detection kit (Clontech)

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according to the manufacturer’s instructions and using AutoLumat LB953 luminometer (EG&G, Gaithersburg, MD). SEAP activity was reported as relative luminescence units. Cell viability assay. To determine cytotoxic activity of rAd vectors expressing FASL–GFP protein, we seeded 5  103 cells/well into 96-well plates (Greiner) using medium supplemented with 10 nM DHT and 2 g/ml doxycycline where necessary. The next day, we detached cells from four wells with trypsin and counted them to determine the average cell number per well. We infected the remaining wells with viral vectors at the desired MOI. After 48 h, we removed culture medium from wells and replaced it with 100 l serum-free medium and 20 l of MTS reagent. We incubated plates at 37C for 1 h, and measured absorbance at 492 nm for each well (related to number of viable cells) using Fluostar dual fluorescence/absorbance plate reader. The ratio of absorbance in wells transduced with adenovirus vectors to that of uninfected cells was used to determine percentage of viable cells remaining in vector-treated wells. In vivo systemic toxicity assay. We purchased male BALB/c mice from Charles River Laboratory (Charleston, SC). These mice were 8 weeks old and weighed 25.6 ± 2.3 g. We injected groups of three mice through the tail vein with either 2  108 or 1  109 IP of the desired viral vector in 200 l of 1 HBS. As a control, we injected 200 l of 1 HBS into the tail veins of one group of mice. Where indicated, we injected doxycycline (1 mg/ml in H2O) intraperitoneally at 4 g/g body weight immediately following vector injection (t = 0 h) and again the next day (t = 24 h). We sacrificed mice by cervical dislocation at t = 48 h, and removed their livers, except in the cases where mortality occurred before that time point. We covered the livers with Tissue-Tek O.C.T. Compound (Sakura Finetek USA, Inc., Torrance, CA) and snap-froze them on dry ice. We prepared frozen sections (6 m) using a Cryostat microtome, adhered them to glass slides and stained them with hematoxylin and eosin according to established procedures. Alternatively, to measure apoptosis induction, we labeled nuclear sections with FITC using the In Situ Death Detection kit, POD (Roche Diagnostics Corp., Indianapolis, IN), according to the manufacturer’s instructions. RECEIVED FOR PUBLICATION AUGUST 2; ACCEPTED SEPTEMBER 13, 2001.

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