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Clinical Cancer Research Vol. 10, 1454-1462, February 2004
© 2004 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

Adenovirus-Mediated Retinoblastoma 94 Gene Transfer Induces Human Pancreatic Tumor Regression in a Mouse Xenograft Model

Josep Maria Roig1, Miguel Angel Molina1, Anna Cascante2, Joaquim Calbó1, Neus Carbó1, Uwe Wirtz3, Sunil Sreedharan3, Cristina Fillat2 and Adela Mazo1

1 Department of Biochemistry and Molecular Biology, University of Barcelona, Barcelona, Spain;2 Genes and Disease Program, Center for Genomic Regulation, Barcelona, Spain; and3 Titan Pharmaceuticals, Inc., San Francisco, California


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: Gene transfer of a truncated variant of the retinoblastoma (RB) gene encoding a Mr 94,000 protein that lacks the NH2-terminal 112 amino acid residues, termed RB94, has been shown to inhibit proliferation of several human tumor cell types. We have assessed its therapeutic effectiveness on pancreatic cancer, one of the most aggressive and therapy-resistant types of cancer. For this purpose, preclinical studies aimed to evaluate the therapeutic potential of RB94 gene transfer in pancreatic cancer were carried out.

Experimental Design: We have compared the antiproliferative effects of adenovirus-mediated gene transfer of RBwt and RB94 at the in vitro and in vivo levels in three RB-positive human pancreatic tumor cell lines: (a) NP-9; (b) NP-18; and (c) NP-31. We have also examined their effects on cell cycle and their capacity to induce apoptosis.

Results: In vitro results indicate that RB94 gene transfer has stronger antiproliferative effects compared with RBwt. RB94 transduction correlated with accumulation at the S-G2 phase of the cell cycle in the three cell lines tested and induction of apoptosis in two of them. In vivo studies show significant decreases in the growth rate of tumors treated with Ad-RB94 when compared with those treated with Ad-RBwt. Moreover, terminal deoxynucleotidyl transferase-mediated nick end labeling analyses of Ad-RB94-treated tumor sections revealed that only RB94 is able to significantly induce apoptosis.

Conclusions: RB94 gene expression has antiproliferative effects also in human pancreatic tumor cells, being more effective than wild-type RB in preventing tumor growth.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pancreatic tumors resist conventional cancer treatment modalities, such as radiotherapy and chemotherapy (1) , and cancer of the pancreas is currently the fifth leading cause of tumor-related adult death in Europe and North America (2) . Despite some minor advances in diagnosis, staging, and treatment, the prognosis remains extremely poor, with a 5-year survival rate of <1%. Therefore, new therapeutic approaches for this malignancy are urgently needed.

Adenoviruses are very efficient delivery vehicles for therapeutic gene transfer to tumors (3) , and strategies based on tumor suppressor gene transfer represent a potential modality for treating human cancers. One of such genes is retinoblastoma (RB), which codes for a key nuclear protein involved in the control of cell cycle (4 , 5) . RB protein interacts with other proteins that control cell proliferation, senescence, and death, and its activity is tightly regulated as cells progress through the cell cycle (6) . Hypophosphorylated RB proteins bind to the E2F family of transcription factors, inducing cell cycle arrest in the G1 phase. For this reason, phosphorylation of the RB protein is a key requirement for cell cycle progression and cell proliferation (5 , 7) . The RB pathway is disrupted in the majority of tumors, in which it might be an essential step in tumorigenesis (8) .

Some efforts have been undertaken for the development of antitumor strategies involving RB gene transfer (9, 10, 11) . However, the effectiveness of wild-type RB gene therapy is limited by the rapid inactivation caused by phosphorylation of the ectopically expressed RB protein (12 , 13) . Preclinical studies have also shown that the therapeutic potential of RBwt gene transfer is limited to RB-negative tumors (14) . To circumvent the problem of the functional inactivation of the RB gene product, a truncated variant, which encodes a Mr 94,000 protein that lacks the 112 NH2-terminal amino acid residues of the wild-type RB protein, was assayed. Termed RB94, this protein has been reported to remain in a hypo-phosphorylated state in transfected cells, resulting in a significantly greater half-life than that of the wild-type RB protein (15 , 16) . In addition, RB94 showed an increased antiproliferative and antitumor efficacy in RB-negative and -positive human tumor cell lines of various origins, including fibrosarcoma, osteosarcoma, bladder, breast, lung, and prostate carcinoma (15 , 16) .

Preclinical studies on RB94 gene transfer were also performed with non-small lung and bladder carcinoma human tumor xenografts but with little evaluation of the mechanism of action (15 , 16) . Recently, adenovirus-mediated RB94 (Ad-RB94) gene transfer has been examined in human head and neck squamous cell carcinoma, bladder cancer cell lines, and tumor xenografts (17 , 18) . RB94 gene expression resulted in cell cycle arrest in the G2 phase, a late decrease in telomerase activity, the induction of apoptosis in 33% of treated tumors, and tumor regression in nude mice bearing head and neck cancer xenografts (17) . Zhang et al. (18) have shown that Ad-RB94-induced bladder cancer cell death is associated with a caspase-dependent pathway and early marked telomere erosion and chromosomal crisis. Nevertheless, no alterations in telomerase activity were found at the time points in which RB94 produced significant cytotoxicity (17 , 18) . Additional evidences are needed to better understand the exact mechanisms of RB94-induced cytotoxicity, being also of great interest to ascertain the therapeutic potential of RB94 on other cancer types.

In the present study, we have evaluated the antiproliferative effects of adenovirus-mediated RB94 (Ad-RB94) or wild-type RB (Ad-RBwt) gene transfer in RB-positive human pancreatic cell lines and mouse xenografts. We have examined changes in cell cycle distribution and apoptotic levels by Annexin-V binding, poly (ADP-ribose) polymerase cleavage, and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) analyses. Our results indicate that treatment with Ad-RB94 but not Ad-RBwt had significant effects on the induction of apoptosis and/or S-G2 cell cycle arrest in all three human pancreatic cancer cell lines tested. In addition, treatment with Ad-RB94 significantly reduced the growth of human pancreatic xenografts in nude mice. These results, consistent with those found by others on different tumor types, demonstrate that gene expression of the NH2-terminal truncated RB protein (RB94) could have a significant therapeutic potential for the treatment of human pancreatic cancer.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines.
NP-9, NP-18, and NP-31 are RB-positive cell lines derived from human pancreatic adenocarcinomas, which had been perpetuated as xenografts in nude mice and further characterized for different oncogene and tumor suppressor profiles (19) . It has been established that both NP-9 and NP-31 are p16null, whereas NP-18 is p16wt (19 , 20) . NP-9 cells were grown in DMEM:F12 medium (1:1), and NP-18 and NP-31 in RPMI 1640, all supplemented with 10% fetal bovine serum and antibiotics, in a humidified atmosphere containing 5% CO2 at 37°C.

Replication-Defective Adenoviruses and Infection Conditions.
Recombinant Ad-lacZ was generated in our laboratory by cotransfection of the pCCMVpLpA plasmid containing the lacZ cDNA with pJM17 into subconfluent cultures of 293 cells as described (21) . Ad5CMV-RBwt construct (Ad-RBwt; Ref. 9 ) was a generous gift from Dr. J. Fueyo (M. D. Anderson Cancer Center, Houston, TX), and Ad5tTACMV-RB94 (Ad-RB94) was provided by Titan Pharmaceuticals, Inc., (South San Francisco, CA; Ref. 15 ). All viruses were cloned, amplified, and titered by plaque assay on the 293 cell line.

NP cells were plated 24 h before viral infection in medium with 10% fetal bovine serum. The viral stock was diluted to reach the desired multiplicity of infection (MOI), added to cultures in serum-free medium, and incubated for 2 h at 37°C. Mock-infected cells were incubated with serum-free medium. Infection was stopped by adding medium supplemented with 10% heat shock-inactivated (30 min at 56°C) fetal bovine serum.

Cell Growth and Dose-Response Analysis.
Cells were seeded at a density of 15,000/well (for NP-18) or 20,000/well (for NP-9 and NP-31) in 24-well tissue culture plates. After 24 h, quadruplicate wells were infected with Ad-RBwt or Ad-RB94.

To determine growth curves, cells were transfected with adenovirus at 50 MOI or mock infected, and quadruplicate wells from each treatment were counted on days 2, 3, and 4 after infection. To determine dose-response curves, cells were transfected at different MOIs and counted 4 days after infection. Cell numbers were assessed in a Multisizer auto-analyzer (Coulter Corp., Hialeah, FL). Adenoviral MOIs that produced ID50 were estimated from the dose-response curves by standard nonlinear regression using an adapted Hill equation.

Cell Cycle Analysis.
Cells were seeded in triplicate (125,000 cells/dish for NP-18 and 250,000 cells/dish for NP-9 and NP-31 lines) in 6-cm dishes. Cultured cells were harvested at different times after viral infection and stained in Tris-buffered saline containing propidium iodide (50 µg/ml), RNase A (10 µg/ml), and Igepal CA-630 (0.1%) for 1 h at 4°C. All measurements of cell cycle distribution were performed on an EPICS-XL flow cytometer (Coulter). Data from >=10,000 cells were collected and analyzed by Multicycle software (Phoenix Flow Systems, San Diego, CA).

Western Blot Assay.
Cells were seeded (7.5 x 105 for NP-18 and 1.5 x 106 for NP-9 and NP-31) in 100-mm dishes. At each time point, treated cells were washed twice with PBS, harvested in 0.5 ml of cold lysis buffer [10 mM Tris (pH 7.5), 400 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% Igepal CA-630, 5 mM NaF, 1 mM sodium orthovanadate, 1 mM DTT, and one protease inhibitor cocktail tablet (Roche, Mannheim, Germany) per 10 ml of buffer], and incubated on ice for 15 min. After removal of cell debris by centrifugation at 14,000 rpm, the protein concentration in cell lysates was determined by the Bradford assay. Total extracts of control cells were obtained at day 0. Ad-RBwt- and Ad-RB94-transduced cells were collected at different times after infection. Samples containing equal amounts of protein were mixed with loading buffer with 5% 2-mercaptoethanol, heated for 5 min at 100°C, and loaded onto an 8% SDS-PAGE gel. Electrophoretic transfer onto nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany) was followed by immunoblotting with anti-poly (ADP-ribose) polymerase (PharMingen, 7D3–6) or anti-RB (IF-8; Santa Cruz Biotechnology, Santa Cruz, CA) monoclonal antibodies and detection with an HRP-conjugated antimouse IgG (DAKO Corp, Carpinteria, CA). The chemiluminescent signal was developed using the enhanced chemiluminescence detection system (Amersham, Arlington Heights, IL).

Annexin-V Assays.
Cells were seeded in triplicate (125,000 cells/dish for NP-18 and 250,000 cells/dish for NP-9 and NP-31 lines) in 6-cm dishes. Apoptosis was assessed by Annexin-V-FITC binding (Genzyme Corp., Cambridge, MA). Floating and attached cells were collected at different times after infection and washed in PBS, and 1 x 106 cells were resuspended in 1 ml of binding buffer containing 0.5 µg/ml Annexin-V-FITC and 5 µg/ml propidium iodide. Cells were incubated in the dark for 1 h at room temperature. Triplicate dishes for each treated sample were analyzed with an EPICS-XL flow cytometer (Coulter). Propidium iodide-positive cells were considered the necrotic population, and only propidium iodide-negative and Annexin-V-FITC-positive cells were considered as the apoptotic population.

Tumor Xenograft Studies.
NP-9 or NP-18 cells were trypsinized and resuspended in serum-free medium. Male BALB/c nude mice were injected s.c. into each posterior flank region with 1.5 x 107 tumor cells in 0.1 ml of medium. Tumors were allowed to grow, and gene transfer treatments were initiated when they reached a mean volume of 40 mm3. The tumors were divided into three groups (n = 8/group): (a) one group was used as a control and received intratumoral injections of Ad-lacZ; (b) the second group was treated with intratumoral injections of Ad-RBwt; and (c) the third group was injected intratumorally with Ad-RB94. In all cases, the respective recombinant adenoviruses were administered by four injections of 5 µl (5 x 109 plaque-forming units/ml) in different regions of the tumor mass, using a Hamilton syringe at days 0, 7, and 14 after the establishment of the groups. NP-18 and NP-9 tumors were measured every other day over the next 25 or 50 days, respectively. Volumes were calculated according to the formula: V (mm3) = larger diameter (mm) x smaller diameter2 (mm2) x {pi}/6. Animal care and use were in accordance with recommendations from the Proper Care and Use of Laboratory Animals Committee. Data significance was evaluated using the Mann-Whitney nonparametric test.

Apoptosis Determination by TUNEL Assay.
TUNEL analysis was performed using an in situ death detection kit (Roche Molecular Biochemicals) according to the manufacturer’s instructions. Briefly, frozen tissue sections (5 µm) were fixed in 4% paraformaldehyde for 20 min at room temperature, incubated with blocking solution (3% H2O2 in methanol), and then permeabilized for 2 min on ice with 0.1% Triton X-100 in 0.1% sodium citrate. The TUNEL reaction mixture was prepared using a ratio 9:1 buffer-to-enzyme, and sections were incubated in a humidified chamber for 1 h at 37°C. After rinsing three times for 5 min with PBS, sections were mounted using diaminophenylindole in the mounting medium, and apoptotic nuclei were visualized under a fluorescent microscope. A negative control was established using the labeling solution without terminal transferase on sections as described above.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Antiproliferative Effects of RB94 or RBwt Gene Expression on Human Pancreatic Tumor Cells.
Recombinant adenoviruses were used to compare the effects of RB94 and RBwt gene transfer in human pancreatic tumor cells NP-9, NP-18, and NP-31. In pilot studies with cell cultures infected with Ad-RBwt or Ad-RB94 at a MOI of 50, we observed that Ad-RB94 was significantly more potent in inhibiting tumor cell growth than Ad-RBwt in all three cell lines tested (Fig. 1A)Citation . The magnitude of growth inhibition was cell type dependent, NP-18 being the most sensitive of all. Inhibition of cell proliferation correlated with striking morphological changes in the Ad-RB94-transduced cultures (Fig. 1B)Citation . In the case of NP-9 cells, after Ad-RB94 infection, cells appeared enlarged with prominent nuclei, a morphological change commonly observed in cells arrested at G2 phase. In contrast, a significant proportion of NP-18 cells and, to a lesser extent, NP-31 cells reflected features typical of apoptosis; individual cells shrunk and separated from neighboring cells, then appeared refringent, and finally detached from the monolayer by day 2 or 3 after infection with Ad-RB94. When cultures were infected with Ad-RBwt, no morphological changes were apparent in NP-18 and NP-31 cells, and only NP-9 cells transiently showed a morphology similar to that observed in the Ad-RB94-infected cells.



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  		Fig. 1. Comparison of Ad-RBwt and Ad-RB94 gene transfer effects on the proliferation and morphology of human pancreatic tumor cell lines. A, growth curves for NP-9, NP-18, and NP-31 cells transduced at a MOI of 50 and counted at 1, 2, 3, and 4 days after transduction. ——, mock infected; —, Ad-RB94 treated; - - -, Ad-RBwt treated. Data are expressed as cell number ± SEM. B, morphology of pancreatic tumor cells after being mock infected or infected with Ad-RB94 or Ad-RBwt at a MOI of 50. Pictures were taken 2 (NP-18) or 4 days (NP-9 and NP-31) after infection.

 
Induction of apoptosis by Ad-RB94 was confirmed by Annexin-V labeling and poly (ADP-ribose) polymerase cleavage. Annexin-V-FITC binding assay revealed that RB94 gene transfer significantly increased the percentage of apoptotic cells in NP-18 and NP-31 but not in NP-9 cultures (Fig. 2A)Citation . In contrast, no significant increase in apoptosis was observed in any of the cell lines transfected with Ad-RBwt. Cell extracts were also prepared at different times postinfection and analyzed by Western blot for evidence of poly (ADP-ribose) polymerase cleavage, a target of caspase-3 during apoptosis. As seen in Fig. 2BCitation , an increase in the Mr 85,000 proteolytic product was observed in NP-18 and NP-31 cultures infected with Ad-RB94, confirming that the induction of apoptosis observed correlates with the expression of the RB94 transgene in these cell lines.



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  		Fig. 2. Evaluation of apoptosis in Ad-RBwt- and Ad-RB94-treated cells by Annexin-V-FITC labeling and poly(ADP-ribose) polymerase cleavage. Cells were treated with virus at a MOI of 50. In A, apoptosis was assessed at indicated time points after infection by Annexin-V FITC and propidium iodide staining followed by flow cytometry. The population represented corresponds to the early apoptotic cells (propidium iodide negative, Annexin-V positive). White bars, mock-infected cells; gray bars, Ad-RBwt; black bars, Ad-RB94. B, cleavage of poly(ADP-ribose) polymerase in Ad-RB94- and Ad-RBwt-infected NP-18 and NP-31 cell lines. Protein extracts were prepared at different time points after transduction, and 40 µg of total protein were electrophoresed. Control (Cnt) represents mock-infected cell extracts at time 0.

 
Comparison of the Effects of RB94 or RBwt Gene Expression on Cell Cycle and Apoptosis.
To better compare the effects of Ad-RB94 and Ad-RBwt transfection, dose-response experiments were performed. Values of MOI corresponding to ID50 were determined, and, as expected, significant differences in sensitivity to Ad-RB94 and Ad-RBwt were observed in all three pancreatic cancer cell lines (Fig. 3)Citation . This was particularly striking for the NP-18 cell line, because the ID50 for Ad-RB94 was ~100-fold lower than the ID50 for Ad-RBwt. Ad-RB94 showed ID50 values that were 2- and 10-fold lower than Ad-RBwt in NP-9 and NP-31 cells, respectively.



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  		Fig. 3. Dose-response curves of NP-9, NP-18, and NP-31 cells transduced with Ad-RBwt or Ad-RB94. Cells were seeded in quadruplicate in 24-well plates. After 24 h, cells were infected with Ad-RB94 or Ad-RBwt at different MOIs, and viable cells were counted 96 h after transduction. Cell number in mock-infected cultures was considered as 100%. {blacktriangleup} —{blacktriangleup}, Ad-RB94; {triangleup} – – {triangleup}, Ad-RBwt. Values of ID50 are expressed as means ± SEM; statistical significance of the results: P < 0.05 for NP-9; P < 0.01 for NP-31; P < 0.001 for NP-18.

 
From these results on, each cell line was infected with both viruses at the MOI corresponding to their own ID50 for Ad-RB94. At different times postinfection, we analyzed the expression of RB94 and RBwt by Western blot using a monoclonal antibody that recognizes the COOH-terminal region of both proteins. No changes in the endogenous levels of Mr 110,000 RB of NP-18 and NP-31 transduced with Ad-RBwt were observed (data not shown), and only in NP-9, the levels of the Mr 110,000 RB protein increased at day 1 postinfection, after which it returned to, or even fell below, endogenous levels (Fig. 4A)Citation . Expression of the Mr 94,000 RB94 in NP-9, NP-18, and NP-31 cells reached a peak at day 1 or 2 postinfection with Ad-RB94, and the protein could be still detected 4 days afterwards (Fig. 4B)Citation .



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  		Fig. 4. Western blot analyses of RB94 and RBwt in human pancreatic cancer cells transduced with Ad-RBwt or Ad-RB94. Cell cultures were mock infected or infected with Ad-RBwt or Ad-RB94 at a dose corresponding with the ID50 of the Ad-RB94 in each cell line. Protein extracts were prepared at different time points after transduction, and 40 µg of total protein were electrophoresed. IF-8 monoclonal antihuman retinoblastoma was used as primary antibody (Santa Cruz Biotechnology). A, RBwt levels in NP-9 cells infected with Ad-RBwt; B, RB94 levels in NP-9, NP-18, and NP-31 cells transduced with Ad-RB94.

 
Flow cytometry profiles of NP-9 cells treated with Ad-RBwt demonstrated a significant but transient increase in the G2 population 2 and 3 days after infection, returning to a normal distribution by day 4. This transient accumulation in the G2 phase was not accompanied by an increase in subG1 population. In the case of NP-18 and NP-31 cell lines, no significant changes in their cell cycle profiles were observed after Ad-RBwt infection (Fig. 5A)Citation .



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  		Fig. 5. Cell cycle profiles and Annexin-V binding in NP-9, NP-18, and NP-31 cell cultures transduced with Ad-RBwt and Ad-RB94. Cell cultures were mock infected or infected with Ad-RBwt or Ad-RB94 at a dose corresponding with the ID50 of the Ad-RB94 in each cell line and analyzed at different time points after transduction. A, cell cycle distribution assessed by flow cytometry analysis of propidium iodide-stained cells. White bars, the percentage of total cells in G1 block (G0-G1 phases); black bars, the percentage of total cells in S-G2 block (that covers the S, G2, and M phases). In B, apoptosis was assessed at indicated time points after infection by Annexin-V FITC and propidium iodide staining followed by flow cytometry. 1, numbers correspond to percentages of early apoptotic cells (propidium iodide negative, Annexin-V positive).

 
In clear contrast, infection with Ad-RB94 induced sustained changes in the flow cytometry profiles of all three pancreatic tumor cell lines. These alterations in cell cycle distribution were marked and cell type specific. RB94 gene expression resulted in an accumulation of NP-9, NP-18, and NP-31 cells at the S-G2 phase of the cell cycle. In addition, an increase in the subG1 population was observed in NP-18 and NP-31 cells, indicating that RB94 was able to induce apoptosis even at a MOI at which RBwt did not induce apoptosis. Annexin-V analysis revealed that the percentage of apoptosis for NP-31 cells increased to 10% at day 2 postinfection and remained at this level. For NP-18 cells, the percentage reached 24% 3 days after infection. No apoptotic population was observed in NP-9 cells infected with Ad-RB94, or in all three pancreatic tumor cell lines when infected with Ad-RBwt, at any of the times tested (Fig. 5B)Citation .

In Vivo Analysis of RB94 or RBwt Gene Transfer in Human Pancreatic Cancer Xenografts.
We examined the ability of Ad-RB94 or Ad-RBwtgene transfer to inhibit the growth of solid tumors in an in vivo model. NP-9 or NP-18 cancer cells, representative of different responses to RB94 treatments in vitro, were injected s.c. into nude mice, and tumors were allowed to establish until they reached a mean volume of 40 mm3. Then, tumors were treated with intratumoral injections of Ad-lacZ, Ad-RBwt, or Ad-RB94. As expected, Ad-lacZ-treated animals showed the maximal tumor growth rate. No significant reduction in NP-9 and NP-18 tumor growth was observed in animals treated with Ad-RBwt. In contrast, there was a marked inhibition in the growth of NP-18 and NP-9 tumors (P < 0.01 and P < 0.02 at the end of the treatment, respectively) in the Ad-RB94-treated mice. Statistically significant differences (P < 0.05) started at day 7 in NP-18-derived tumors and at day 39 in NP-9 xenografts (Fig. 6A)Citation . TUNEL analysis of tumor xenografts was performed to determine whether treatments induce apoptosis in vivo (Fig. 6B)Citation . Mice were sacrificed on day 1 after the third virus injection, and TUNEL-stained tumor sections revealed a marked increase in the percentage of cells that underwent apoptosis in Ad-RB94-treated NP-18 tumors. A small increase in apoptosis was also observed in NP-18 xenografts treated with Ad-RBwt. In contrast, no apoptotic cells were detected in any of the treated NP-9 tumors (data not shown).



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  		Fig. 6. Evaluation of Ad-lacZ-, Ad-RBwt-, or Ad-RB94-mediated gene transfer in human pancreatic cancer tumor xenografts. In A, cells were injected s.c. in the dorsal area of male BALB/c nude mice. Treatments started when the xenografts had reached a mean volume of 40 mm3 (day 0). Adenoviruses were administered intratumorally at days 0, 7, and 14. Ad-lacZ (——), Ad-RBwt (- - -), or Ad-RB94 (—) treatments are represented for NP-9 (a) or NP-18 (b) tumors (n = 8). Data are expressed as a percentage of initial tumor growth (means ± SEM). *, statistical significance (P < 0.05) when compared with the control group by the Mann-Whitney test. B, apoptosis detection by terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) staining of NP-18 human pancreatic tumor xenograft sections. Terminal deoxynucleotidyltransferase-terminal staining of ornithine carbamyl transferase-embedded sections from NP-18 s.c. tumors harvested 16 days after the first treatment with Ad-lacZ (a and b), Ad-RBwt (c and d), or Ad-RB94 (e and f). TUNEL-positive cells were detected with an FITC-conjugated antibody (b, d, and f), and the tissue samples were counterstained with 4',6-diamidino-2-phenylindole (DAPI; a, c, and e). Magnification: x100.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A great deal of effort is currently being devoted to the development of new treatment modalities for human cancer, and these include gene transfer approaches. A variety of viral or nonviral vectors, tissue-specific gene promoters, and tumor suppressor genes has been tested in preclinical models with different degrees of success. One candidate gene for cancer gene therapy is the RB tumor suppressor gene. Several studies have led to the recognition of its importance in cell cycle control and cell proliferation, and the RB pathway has been found altered in the majority of human tumors (8) . However, the wild-type Mr 110,000 RB protein has shown limited efficacy as a tumor growth suppressor because of its rapid phosphorylation and subsequent inactivation in RB-positive and some RB-negative cells (12 , 13) .

Previous studies have suggested that the 5'-truncated variant of the RB gene encoding the NH2-terminal-truncated Mr 94,000 RB94 protein is a good candidate as a therapeutic gene for cancer (15 , 18) . The RB94 protein has a longer cellular half-life than the wild-type RB protein and tends to remain longer in the active hypophosphorylated forms. It has also been shown to inhibit growth not only of RB-negative but also of RB-positive tumor cells (15 , 16) . Tumor cells with endogenous RBwt are characterized by high rates of RB phosphorylation attributable, among other reasons, either to high G1-cyclin-dependent kinase activities or the lack of the cyclin kinase inhibitor, p16. Indeed, p16 is the most frequently tumor suppressor function lost in pancreatic carcinomas (>90%), whereas the incidence of RB mutations is very low (6%; Ref. 22 ) Therefore, it is of great interest to assess the therapeutical potential of the RB94 protein on RB-positive pancreatic cancer models.

The preclinical studies presented here demonstrate that Ad-RB94 gene transfer significantly suppressed growth of human pancreatic carcinoma cells, both in vitro and in nude mice xenografts. In addition, treatment with Ad-RB94 is markedly more effective in tumor cell growth suppression than treatment with the virus carrying the wild-type RB gene (Ad-RBwt). In the three cell lines tested, NP-9, NP-18, and NP-31, Ad-RB94 treatment had a striking effect on cell cycle distribution, resulting in an increase in cells arrested at the S-G2 phase. These alterations in cell cycle profiles induced by RB94 gene transfer fit well with the differences in proliferation inhibition (see Figs. 1Citation and 5Citation ). In NP-18 and NP-31 but not NP-9 cells, there was also an induction of apoptotic cell death. The lack of apoptosis in NP-9 cells may be related to an intrinsic antiapoptotic feature that makes this cell line extremely resistant to cell death. Indeed, NP-9 cells have been shown extensively resistant to apoptosis induction by the reintroduction of other tumor suppressor genes, such as p53 and p16 (20 , 21) .

Interestingly, antitumor effects were observed in both NP-9 and NP-18 xenografts treated with Ad-RB94, but apoptosis was only induced in NP-18 tumors. This is in good agreement with the in vitro results. Curiously, a slight TUNEL-positive signal was detected in RBwt-treated NP18 tumors. Although we do not have an explanation for this differential behavior in vitro and in vivo, it is conceivable that RBwt overexpression in cancer cells that are embedded in a tumoral mass may modulate the complex signaling network that controls cell fate in a different manner than it does in vitro. On the other hand, we have shown previously that the genetic background of the NP-9 tumor cells makes them resistant to programmed cell death in response to transfer of other tumor suppressor genes (20 , 21) . Indeed, we propose that NP-9 tumors undergo a cell cycle arrest in response to RB94 reintroduction as it could be expected from the in vitro results.

The role of RB in controlling the G1 cell cycle checkpoint is well established. Phosphorylation in late G1, executed by members of the cyclin-dependent family of kinases, lowers RB’s affinity for transcription factors, such as E2F-1, leading to activation of genes whose products are required to enter in the S phase (23) . It is therefore not surprising that, on reintroduction of RBwt gene, RB-defective cells arrest in G1 (7) . However, there is growing evidence that RB also has regulatory effects beyond the G1-S boundary, mediating additional cell division control at the S and G2 phases (24, 25, 26, 27) . It has also been described that a partially phosphorylated RB mutant allows cells to enter S phase but not to complete it, leading to the hypothesis that control of S phase entry and completion are separable events that could be mediated by distinct downstream pathways (28 , 29) . The accumulation of cells in S-G2 phase observed in all cell lines after RB94 transduction is in good agreement with the existence of these additional actions of RB. In fact, RB94 has been described as a poor substrate for cyclin-dependent kinases, despite lacking only 1 (Thr 5) of the 16 phosphorylation sites reported for the RBwt protein (15) . Therefore, RB94 might behave in a similar way to the phosphorylation mutants described by Chew et al. (29) , allowing passage of cells through the G1 checkpoint but preventing exit from the S phase. Likewise, recent studies using a NH2-terminal truncated RB variant (deleted residues 1–378) show that this region may be dispensable for the proliferation suppression function (30) . However, the NH2-terminal region is necessary for the interaction with a kinase active in G2-M named RbK (31) and proteins, such as MCM7, involved in the initiation of chromosomal DNA replication (32) . The lack of the NH2 terminus might then be related to the inability of RB94-transduced cells to complete the S phase, supporting the idea of a role for RB in S-G2 phase in addition to its known activities in G1.

RB exerts antiapoptotic actions by controlling E2F activity, whose deregulation may promote apoptosis by p53-dependent and -independent mechanisms (33, 34, 35) . Given that RB94 does not arrest cells in G1, it is conceivable that E2F-1 activity is not down-regulated. Thus, apoptosis induction might arise from the generation of a conflict between the continued cell cycle progression and RB activities involved in E2F-independent pathways, as it has been proposed previously (36) . When we introduced the RB94 gene into pancreatic tumor cell lines, apoptosis was induced in two of the three cell lines examined. Furthermore, our in vivo results clearly demonstrate that RB94 is also able to induce apoptosis in a human pancreatic tumor model. Apoptosis induction has also been observed after RB94 gene transfer in head and neck cancer (17) . The authors argue that the inability of the tumor cell lines transfected with Ad-RB94 to exit chromosomal replication, along with inhibition observed previously of telomerase activity by RB94 gene expression, could potentially trigger the induction of apoptosis in susceptible tumor cells (17) . However, in RB94-treated bladder cancer cells (18) , apoptosis was not related with telomerase activity inhibition at the time points in which RB94 produced significant cytotoxicity. The evidences for the involvement of caspases and the time points at which RB94 has induced the cytotoxic effects are in good agreement to those shown in the present study with pancreatic cancer cells. Therefore, early mechanisms that do not involve telomerase activity modulation might be responsible for the RB94-induced apoptosis observed in these cancer cells. Although no evidence of such a mechanism has been demonstrated in our models, it is conceivable that similar processes are taking place in pancreatic cancer cell lines.

In conclusion, we present evidence that adenovirus-mediated RB94 gene transfer significantly inhibits human pancreatic cancer cell proliferation and tumor growth in nude mice. As expected, Ad-RBwt is much less effective than Ad-RB94. The effects are elicited by arresting cells at the S-G2 cell cycle phases and, depending on the genetic background of the cancer cell, triggering tumor cell apoptosis. Although further research is required to better elucidate the molecular pathways underlying these RB94 effects, from the results presented here, it is worthy noting that RB94 is emerging as a promising clinical application for pancreatic cancer.


   ACKNOWLEDGMENTS
 
We thank Jaume Comas, who is in charge of the flow cytometry section at the Scientific-technic Services from the University of Barcelona, and Robin Rycroft for editorial support.


   FOOTNOTES
 
Grant support: Grants from the Spanish Ministry of Science and Technology, FEDER (SAF-2002-04122), Instituto de Salud Carlos III (G03/156), and Titan Pharmaceuticals, Inc. Ad-RBwt was a generous gift from Dr. J. Fueyo (M. D. Anderson Cancer Center).

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.

Note: A. Cascante and J. Calbó are both recipients of predoctoral fellowships from the Spanish Ministry of Education and Culture.

Requests for reprints: Adela Mazo, Department of Biochemistry and Molecular Biology, C/Martí i Franqués, 1, E-08028-Barcelona, Spain. Phone: 34-93-402.12.10; Fax: 34-93-402.12.19; E-mail: adela{at}bq.ub.es

Received 7/ 2/03; revised 9/17/03; accepted 10/ 2/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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