Targeted-Simultaneous Expression of Gas1 and P53 Using a Bicistronic

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ORIGINAL ARTICLE Targeted-simultaneous expression of Gas1 and p53 using a bicistronic adenoviral vector in gliomas JA Benı´ tez, L Arregui, P Vergara and J Segovia Departamento de Fisiologı´a, Biofı´sica y Neurociencias, Centro de Investigacio´n y de Estudios Avanzados del IPN, Me´xico DF, Me´xico

The targeted expression of transgenes is one of the principal goals of gene therapy, and it is particularly relevant for the treatment of brain tumors. In this study, we examined the effect of the overexpression of human gas1 (growth arrest specific 1) and human p53 cDNAs, both under the transcriptional control of a promoter of the human glial fibrillary acidic protein (gfa2), employing adenoviral expression vectors, in glioma cells. We showed that the targeted overexpression of gas1 and p53 (AdSGas1 and AdSp53, respectively) in rat glioma cells (C6) reduced the number of viable cells and induced apoptosis. Moreover, the adenovirally targeted expression of these genes also reduced tumor growth in vivo. Unexpectedly, there was no additive effect when both gas1 and p53 were simultaneously expressed in the same cells using a bicistronic adenoviral vector. We suggest that Gas1 does not act in combination with p53 in the C6 and U373 glioma cell lines, inducing apoptosis and cell cycle arrest. Our results indicate that the targeted expression of tumor suppressor genes (gas1 and p53) regulated by the gfa2 promoter, together with adenoviral vectors may provide an interesting approach for adjuvant selective glioma gene therapy. Cancer Gene Therapy (2007) 14, 836–846; doi:10.1038/sj.cgt.7701076; published online 29 June 2007 Keywords: apoptosis; cell cycle; GFAP promoter; Gas1; p53; gliomas

Introduction reports indicating that Gas1-mediated growth arrest is p53-dependent, but that it is independent of the p53 6 Gliomas are the most common primary tumors of the transactivating function. On the other hand, Gas1- central nervous system (CNS), and glioblastoma multi- induced apoptosis is p53-independent and it has been 7 forme is the most malignant type of these tumors. Despite observed in interdigital cell death during development. maximum treatment efforts, the prognosis of patients Gas1 prevents DNA synthesis by blocking the entry of 8 with gliomas remains very poor, and the lifespan is less cells into the S phase. Recently, several reports have than 1 year after diagnosis.1 Current therapy includes shown that Gas1 is involved in programmed cell death 7,9,10 surgery, radiotherapy and chemotherapy. However, (apoptosis) in different types of cells. Furthermore, gliomas are highly refractory to conventional therapy we have shown that the targeted overexpression of gas1 due to the biological properties of the tumors, such as induces apoptosis in retrovirally tranduced C6 cells and 11 high infiltration into the brain parenchyma leading to inhibits tumor growth in vivo. Taken together, these recurrence of the tumor, and resistance to chemotherapy results suggest that the expression of the proapoptotic and radiotherapy.2 Novel strategies of therapy for gene gas1 could be a useful therapeutic tool in cancer gene gliomas have been developed including gene therapy, therapy. Furthermore, p53 is a critical component in cell virotherapy, adoptive immunotherapy, and vaccination cycle regulation and apoptosis, and is a key participant in studies. These therapeutic strategies have been translated the development of gliomas. The expression of p53 has into clinical trials.3,4 Unfortunately, to date, the results also been employed as an experimental approach to treat 12 achieved from most clinical trials have not shown gliomas and recent reports showed the relevance of substantial therapeutic efficacy. reactivating p53 to induce tumor growth regression 13,14 Growth arrest specific-1 (gas-1) is a pleiotropic GPI indicating that p53 is an important therapeutic target. (glycosil-phosphatidylinositol)-linked protein.5 There are Two major approaches have been explored in experimental gene targeting to the CNS. The first one is based on tropism-modified viral vectors that target specific cell Correspondence: Dr J Segovia, Departamento de Fisiologı´a, populations, and the second is based on the transcrip- Biofı´sica y Neurociencias, Centro de Investigacio´ n y de Estudios tional regulation of therapeutic genes using cell-type or Avanzados del IPN, Av. Instituto Polite´ cnico Nacional # 2508, 15 Me´ xico DF 07360, Me´ xico. inducible promoters. Previously, we and other groups E-mail: [email protected] have shown that a 2.2 kilobases (kb) fragment of the Received 15 March 2007; revised 8 May 2007; accepted 27 May human glial fibrillary acidic protein (GFAP) promoter 2007; published online 29 June 2007 (gfa2) is sufficient to regulate the targeted expression Gas1 and p53 expression in gliomas JA Benı´tez et al 837 of different transgenes in astrocytes, glioma cell lines from the pC53-SN3 vector, near the 30 end (168 bp) of and primary astrocyte cultures.11,16–18 Importantly, the IRES sequence.26 The human p53 was cloned into glial-specificity transcription of the gfa2 promoter was the SalI site, that was filled in with Klenow and a preserved, independently of the gene transfer method BglII restriction site of the pSgGi vector obtaining employed11,19–21 and the gfa2 promoter has been used the pSgfa2Gas1-IRES-p53 vector (pSgGi53). The cDNAs to study the role of glial cells in neurogenesis using orientation was determined by restriction mapping and transgenic mice.22 DNA sequencing. Recombinant virus was expanded in In this study, we examined the effect of different HEK293 cells and purified by ultracentrifugation on adenoviral systems to target specifically the expression a cesium chloride gradient. Viral titers were determined of both gas1 and p53 transgenes to glioma cells. We by end point cytopathic effect assay on HEK293 cells and showed that the adenovirus-mediated overexpression expressed as plaque-forming units per milliliter (pfu/ml). of gas1 (AdSGas1) in rat glioma cells (C6) and human astrocytoma cells (U373) reduced the number Cell culture and adenoviral transduction of viable cells and induced apoptosis. In addition, we C6 and U373 cells were grown in Dulbecco’s modified found that the adenovirally mediated targeted expression Eagle F12 medium (DMEM-F12, Invitrogen, Carlsbad, of gas1 and p53 significantly inhibited tumor growth in CA) with 10% fetal calf serum (FCS, Invitrogen). nude mice. However, the effect of a bicistronic adenoviral HEK293 cells were obtained from ATCC and grown in vector (AdSGi53), which expresses both human gas1 DMEM-HG (Invitrogen) with 10% horse serum (Invitro- and human p53 regulated by the gfa2 promoter, was gen), 2 mML-glutamine (Sigma, St Louis, MO) and not different from AdSGas1 or AdSp53 monocistronic 110 mg/ml sodium pyruvate (Invitrogen) at 371Cina vectors, independently of the p53 status of the glioma 5% CO2–95% air humidified atmosphere. The conditions cell line. We suggest that Gas1 and p53 do not act for infections were as follows: cells were plated at a in combination, and that the Gas1 effect is not mediated density of 4 Â 104 cells/well into 6-well plates. Twenty- by p53 in either C6 or U373 cell lines. Therefore, there four hours later, cells were infected with different are no additive or synergic effects of Gas1 and p53 recombinant adenoviral vectors. After 4 h in the presence inducing apoptosis in glioma cells. These results of the adenovirus, the medium was changed for fresh suggest that the targeted expression of gas1 and p53 medium. Cells were harvested at different time points driven by the gfa2 promoter from adenoviral vectors may according to the experiment. provide an interesting approach for selective gene therapy against gliomas. RT-PCR and PCR assays RNA was extracted using the TriPure reagent (Roche, Mannhein, Germany). Five micrograms of total RNA were reverse-transcribed with M-MLV reverse transcrip- Materials and methods tase (Invitrogen), and 5 ml of the resulting cDNA amplified Construction of adenoviral vectors byPCR.Theprimersetsusedtoamplifygas1 were: sense The adenoviral vectors were produced according to the 50-CTGTGCCTGATGGCGCTGCTGC-30 and antisense pAdEasy System developed by Vogelstein and collabora- 50-GACTTGCTATTCTGTGCATC-30.Weamplifiedrat tors23 (www.coloncancer.org). Briefly, the gfa2-LacZ and b-actin fragment using primers reported previously,27 and gfa2-gas1 fragments were excised from the pgfa2LacZ the human b-actin fragment using the following primers: plasmid16 and pgfa2gas1 vector10 respectively, using BglII sense 50-TGGCACCACACCTTCTACA-30 and antisense and cloned into the BglII restriction site of the pShuttle 50-TCACGCACGATTTCCC-30. On the other hand, the vector to produce the AdSLacZ and the AdSGas1 gfa2-gas1 fragment was amplified from the pgfa2gas1 vector adenoviral vectors. The AdS53 vector was obtained as using primers: sense 50-GGAATTCCTGAGCTCCCACCT follows: human p53 cDNA was excised from the pC53- CCCTCT-30 and antisense 50-GGAATTCCAGTGGGAC SN3 vector24 with BamHI and cloned into the BamHI site GCGGGCTCTC-30, primers contain EcoRI linkers, for of the pgfa2MCS plasmid (unpublished results) to obtain directional cloning. The cDNA of human p53 was amplified the pgfa2p53 plasmid. Subsequent to this procedure, the from the pC53-SN3 vector24 using primers: sense 50-GCCA gfa2-p53 cassette was released with BglII and cloned into TGGAGGAGCCGCAGTCAG-30 and antisense 50-GAA the pShuttle vector. On the other hand, to insure optimal GATCTTCTGGGAGGCTGTCAGTGGGGAAC-30. expression levels of the second cistron of the AdSGi53 bicistronic vector, we inserted the gfa2-gas1 cassette, Cell viability amplified by PCR from the pgfa2gas1 plasmid, into the To determine the effect of adenovirus-mediated transgene EcoRI site of the pIRES vector (Promega, Madison, WI) expression on cell viability in C6 and U373 cells, we so that the IRES sequence started within 100 nucleo- used the 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium tides25 following the termination codon of the human bromide (MTT) and the trypan blue exclusion assays. For gas1 cDNA. Using NheI and SalI restriction endo- MTT assay, cells were seeded in 96-well plates at a density nucleases we released the gfa2Gas1IRES fragment from of 2000 cells/well. Cells were infected with AdSLacZ or the pgGas1-IRES vector and inserted it into the XbaI/SalI AdSGas1 with MOIs of 500 and 1000. Forty-eight hours restriction sites of the pShuttle vector to obtain pSgGi. later, the number of viable cells was assessed by We then placed the human p53 cDNA, amplified by PCR measuring the conversion of the tetrazolium salt MTT

Cancer Gene Therapy Gas1 and p53 expression in gliomas JA Benı´tez et al 838 into formazan. Cells were incubated with 25 mg of MTT in agreement with the Guide for the Care and Use in 50 ml of medium for 4 h at 371C, the medium was aspi- of Laboratory Animals of National Institute of Health rated and the formazan crystals were dissolved in 150 ml (NIH) and the internal (Cinvestav) guidelines. C6 of DMSO. Absorbance was read at 595 nm using an cells 1 Â 106 were subcutaneously inoculated into the automated microplate reader (Bio-Rad). We also deter- dorsal flank of 6–8 week-old nu/nu mice. When the mined the number of viable cells 24, 48 and 72 h after tumors reached 4–5 mm (large axis), animals were divided infection using the trypan blue exclusion assay. Assays into five groups and 3 days later were injected with 50 ml were performed in quadruplicate. of PBS (control) or 1 Â 108 pfu/50 ml of adenovirus AdSLacZ, AdSGas1, AdS53 and AdSGi53 intratumorally Cytotoxicity assay every other day for a total of three injections. Tumor size Lactate dehydrogenase (LDH) activity was determined was measured every second day for 21 days after the first using the CytoTox 96 non-radioactive cytotoxicity assay viral injection. Tumor volume was calculated according 2 kit (Promega). to the formula V ¼ (Dmax)(Dmin) /(0.5), where Dmax and Dmin represent the large and small axes, respectively. Apoptosis assays Forty-eight hours after transduction, we measured the Immunohistochemical analysis percentage of apoptotic cells by flowcytometry (FACS- Mice were anesthetized with an overdose of pentobarbital Calibur, Becton-Dickinson, Franklin Lakes, NJ) using (460 mg/kg) and killed 24 days after the first viral the Annexin-V-FLUOS staining kit (Roche). We also treatment. Mice were perfused with 0.9% saline solution identified apoptotic cells by fluorescence microscopy. followed by 4% p-formaldehyde in PBS (pH 7.4) and 4 Briefly, cells were plated at densities of 2 Â 10 cells/well tumors were dissected out. For immunohistochemistry, 2 on 100 mm coverslips in 24 wells plates and 48 h after 14-mm tissue sections were processed for dual immuno- transduction non fixed cells were processed using the fluorescence. An anti-human p53 monoclonal antibody Annexin-V-FLUOS staining kit (Roche) or using an (1:50, Santa Cruz Biotechnology) was revealed with a in situ cell death detection kit based on the TdT-mediated Texas Red streptavidin-conjugated secondary antibody dUTP nick end labeling (assay) (TUNEL) method (1:300 Vector Laboratories), and a polyclonal anti-Gas1 according to the manufacturer’s protocol (Roche). Cells antibody, produced by ProSci Incorporated using the were counterstained with 4,6-diamidino-2-phenylindole human specific peptide sequence DDGVPHPPRPGSGA (DAPI) to reveal nuclei. (1:100), was used. The presence of Gas1 was revealed with a fluorescein streptavidin-conjugated secondary antibody BrdU incorporation assay (1:300 Vector Laboratories). Tissue sections were counter- Cells were plated as described previously for the apoptosis stained with DAPI. assay, and incubated with 50 mM of BrdU for 6 h at 371C. Forty-eight hours after transduction, cells were analyzed for BrdU incorporation. We used a BrdU staining kit (Zymed) to reveal positive cells, according to the Results manufacturer’s instructions. A total of 200–300 cells by Construction and expression of monocistronic and coverslips were examined to determine the percentage of bicistronic adenoviral vectors containing gas1 and p53 cells that incorporated BrdU. cDNAs, regulated by the gfa2 promoter We obtained replication-defective recombinant adeno- Western blot analysis virus using the AdEasy System.29 Figure 1a shows a Total protein was isolated using the TriPure reagent schematic representation of the adenoviral vectors used (Roche) according to the manufacturer’s instructions. in this work. The AdSGas1 and AdS53 monocistronic Fifty micrograms of total protein were separated on 8% adenoviral vectors express gas1 and p53 cDNAs driven by SDS-PAGE gels and transferred into nitrocellulose the gfa2 promoter, respectively. The AdSGi53 bicistronic membranes (Bio-Rad). Membranes were blocked for 1 h adenovirus expresses both gas1 and p53 transgenes, regu- at room temperature in 5% nonfat milk/TBST (0.05% lated by the gfa2 promoter (Figure 1a). The bicistronic Tween-20, TBS) and incubated with the primary anti- adenoviral vector contains an internal ribosome entry site bodies overnight at 41C. Blots were then incubated with (IRES) of the encephalomyocarditis virus (ECMV), which a peroxidase-coupled secondary antibody followed by allows translation of both cDNAs from a single mRNA.30 enhanced chemiluminescence detection (Perkin Elmer, We also constructed AdSLacZ adenovirus that expresses Boston, MA), according to the manufacturer’s instruc- b-galactosidase driven by the gfa2 promoter, as a control tions. The primary antibodies were anti-human p53 1:500 vector (Figure 1a). (Santa Cruz Biotechnology, Santa Cruz, CA), which To determine transgene expression, we transduced C6 recognizes both mutant and wild-type forms, anti-b and U373 cells, rat and human glioma-derived lines galactosidase 1:500 (Promega) and anti-b actin 1:600.28 respectively, with different adenoviral vectors. Figure 1b shows the expression of the LacZ protein in C6 and U373- Subcutaneous tumors cells transduced with the AdSLacZ vector. Human gas1 Experiments were performed according to current Mexi- mRNA was identified in both AdGas1 and AdSGi53 can legislation NOM-062-ZOO-1999 (SAGARPA), and glioma-transduced cells using RT-PCR assays (Figure 1c).

Cancer Gene Therapy Gas1 and p53 expression in gliomas JA Benı´tez et al 839

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Fold 1.0 1.2 2.3 4.5 Figure 1 Construction of adenoviral vectors and transgene expression in C6 and U373 transduced cells. (a) Schematic representation of the expression cassettes in adenovirus vectors: AdSgfa2LacZ (AdSLacZ), AdSgfa2Gas1 (AdSGas1), AdSgfa2p53 (AdS53), and AdSgfa2Gas1- IRES-p53 (AdSGi53). In each vector the gfa2 promoter drives the expression of the transgenes. (b) Western blot analysis of b-galactosidase expression in C6 and U373 cells 48 h after infection. (c) Reverse transcription (RT)-PCR assays to determine the expression of human gas1 in transduced cells. The RT-PCR amplified a fragment of 604 bp in transduced cells. (d) Western blot analysis of the expression of human p53 in C6 and U373 cells 48 h after transduction. Treatments are indicated in each panel. U/t, untreated cells, M, molecular weight, (À) negative control for RT-PCR, no DNA added.

Western-blot analysis was used to confirm the expres- 1000 in our assays to obtain a therapeutic effect. sion of human p53 protein in AdS53 and AdSGi53 C6 Moreover, other groups have used MOI as high as and U373-transduced cells (Figure 1d). The human p53 2000–3000 to obtain therapeutic effects using adeno- protein was expressed exclusively in C6 cells that were virus and tissue-specific promoters.31,32 To correlate the transduced with AdS53 and AdSGi53 adenovirus. We effect of gas1 expression on cell viability we measured also identified that the level of human p53 was higher the release of LDH from glioma-transduced cells, as an in AdS53 (2.3 fold) and AdSGi53 (4.5 fold) U373- indicator of cell death. Figure 2c shows a significant transduced cells compared with control cells. These results increase of LDH release from AdSGas1-transduced C6 demonstrate the capacity of adenovirus to express and U373 cells at 1000 MOI, compared with AdSLacZ. transgenes driven by the gfa2 promoter. These results indicate that the reduction of cell viability was associated with an increase of cell death as Effects of the adenovirally mediated gene transfer of determined by the LDH assay in glioma cell lines treated gas1 and p53 to glioma cell lines in cell viability, with adenovirus. To evaluate the effect of gas1 and p53 in proliferation and cell-cycle progression C6 cells transduced with adenoviral vectors, we deter- We first compared the effect of gas1 expression on cell mined the viability of cells at different time points after viability in AdSGas1-transduced cells with different transduction. We found that Gas1 and p53 decreased multiplicity of infections (MOIs), using the MTT assay. the number of viable C6 cells as a function of time. Forty Treatment with AdSGas1 employing 500 MOI and eight hours after transduction AdSGas1 and AdS53 1000 MOI significantly reduced the viability of C6 cells reduced the number of viable cells by 52.574.5 and 48 h after transduction compared with the control vector 43.175.0%, respectively (Figure 2d), compared with AdSLacZ (Figure 2a). However, in U373-transduced control. Also, AdSGi53 inhibits proliferation of C6 cells cells we only observed decreases of cell viability with by as much 35.075.5%, 48 h after infection (Figure 2d). 1000 MOI using the AdSGas1 vector (Figure 2b). We However, we only found a significant cell reduction determined that the efficiency of transduction of C6 and in AdSGas1 (26.278.6%) and AdSGi53 (21.2475.7%) U373 cells was approximately 50 and 100% respectively, U373-transduced cells compared with control vector but the levels of GFAP expression were lower in U373 AdSLacZ (Figure 2e). We determined this effect for compared with C6 cells (as determined by immuno- upto 72 h after transduction for each adenoviral vector cytochemistry, data not shown), therefore we used MOI tested.

Cancer Gene Therapy Gas1 and p53 expression in gliomas JA Benı´tez et al 840 abc1.50

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02448 72 96 02448 72 96 Hours in culture Hours in culture Figure 2 Gas1 and p53 decrease the number of viable cells and induce cell death 48 and 72 h after infection in C6 and U373 cells. (a and b) Cytotoxic effect of AdSLacZ and AdSGas1 in astrocytoma cell lines using different MOIs. C6 (a) and U373 (b) cells were infected with MOIs of 500 or 1000. Cell viability was assessed 48 h after transduction, using the 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. (c) Increased lactate dehydrogenase (LDH) activity released from C6 and U373 cells transduced with AdSGas1. C6 and U373 cells were infected with AdSGas1 at a MOI of 1000. LDH activity was measured 48 h after infection. C6 (d) and U373 (e) cells were transduced with a MOI of 1000. Cell viability was determined 24, 48 and 72 h after transduction by trypan blue exclusion. Arrows indicate time of infection. Data are means7s.e.m. determined from three experiments. Analysis of variance (ANOVA), followed by Tukey. *Po0.05, **Po0.01 ***Po0.001, with respect to both untreated (À) and AdSLacZ.

We next tested whether the decrease in cell proliferation tronic vector had the same effect on the proliferation rate results from a reduction in the proliferation rate or to an in C6 and U373 cells as the monocistronic vectors. increase in apoptosis. To evaluate the rate of proliferation, we determined the number of cells that incorporated Adenovirus-mediated Gas1 and p53 expression BrdU, as a direct measure of cells undergoing DNA in C6 cells induce apoptosis synthesis at a given time. Figure 3a shows a representative Several groups have shown that Gas1 is involved in cell BrdU immunocytochemistry of treated C6 cells, in which death and apoptosis.7,33 To determine whether the we can observe a decrease in the number of BrdU-positive decrease of cell proliferation and number of viable cells cells (cells with dark nuclei) that overexpress gas1 and that express gas1 and p53 is related to apoptosis, we p53. When we determined the percentage of cells that analyzed the translocation of phosphatidylserine to the incorporated BrdU, we found that AdSGas1, AdS53 and external side of the cell membrane using Annexin V AdSGi53 groups incorporated 40.574.4, 47.674.5 and staining as an indicator of apoptosis by FACS analysis in 45.478.5%, respectively less BrdU than the AdSLacZ- C6 cells. Using Annexin V-conjugated FITC and propi- treated group (Figure 3b), but there was no significant dium iodide (PI) staining, we found that the percentage of difference between them. We also determined that early- and late-apoptotic cells increased when cells were AdSGas1, AdS53 and AdSGi53 inhibited the prolifera- transduced with AdSGas1, AdS53 and AdSGi53 (13.5, tion rate of U373 cell by 29.5712.9, 16.777.1 and 24.97 18.4 and 15.2% respectively), compared with and trans- 5.5% respectively, compared with control group (Ad- duced with control vector (1.4% in AdSLacZ-transduced SLacZ) (Figure 3c). These data indicate that both gas1 cells) (Figure 4a). Using fluorescence microscopy, no and p53 expression inhibits the G1–S transition of cell positive staining to Annexin V was observed in AdSLacZ cycle in C6 cells, but in U373 cells only gas1 was capable C6-transduced cells (data not shown), but we found of significantly inhibit cell-cycle progression, and that the positive staining in cells overexpressing gas1 and p53,or targeted coexpression of gas1 and p53 using the bicis- both (Figure 4b). In addition, we used TUNEL staining

Cancer Gene Therapy Gas1 and p53 expression in gliomas JA Benı´tez et al 841

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(-) AdSLacZ AdSGas1 AdS53 AdSGi53 (-) AdSLacZ AdSGas1 AdS53 AdSGi53 Figure 3 Gas1 and p53 retard the proliferation rate of C6 and U373 cells. (a) Representative coverslips of C6 cells immunostained for BrdU 48 h after infection. C6 cells were plated on coverslips and transduced with the indicated adenovirus vector. Scale bar, 50 mm. (b and c) Analysis of the proliferation index. Incorporation of BrdU in AdSGas1, AdS53, AdSGi53 and AdSLacZ treated cells, 48 h after infection in both C6 (b) and U373 (c) cells. (n ¼ 200–300 cells in each coverslip). Data are means7s.e.m. of three experiments. Analysis of variance (ANOVA), followed by Tukey. *Po0.05, **Po0.01, ***Po0.001 compared to both (À) untreated and AdSLacZ. to identify cells undergoing DNA fragmentation, as we admininistered the adenoviral vectors intratumorally another marker of an apoptotic process. In Figure 4c every other day for a total of three injections. We we observe that AdSGas1, AdS53 and AdSGi53 trans- monitored the size of the tumors for 24 days after the duced cells were positive for TUNEL staining. Therefore, first injection. At the end of the experiment, mice were these assays indicate that the reduction in the number killed and tumors dissected out. The treatments with of viable cells 48 and 72 h after viral transduction with AdSGas1 or AdS53 resulted in 64.9717.4 and 59.57 gas1 and p53 transgenes (Figure 2) is related to both cell 26.1% suppression of tumor growth, which was signifi- cycle inhibition and programmed cell death. cantly different compared with the untreated and LacZ groups, 21 days after the first injection (Figure 5). A GFAP promoter drives gas1 and p53 expression Unexpectedly, the group treated with AdSGi53, which in vivo and suppresses xenograft tumor growth using expresses both gas1 and p53 from the same gfa2 adenoviral vectors promoter, showed a smaller inhibition of tumor growth To evaluate the effect of the targeted expression of gas1 (46.3715.9% compared with the untreated group), that and p53 in an in vivo glioma model using xenograft is different from both the independent administration tumors, we subcutaneously established C6 tumors in nude of Gas1 and p53. The tumor suppression effect using mice, and treated them with the different adenoviral bicistronic adenovirus correlates with the effect on cell vectors. We injected C6 cells into the flanks of nude mice, proliferation (Figure 2d). We next confirmed gas1 and and when the tumors reached 4–5 mm of longer axis, p53 transgene expressions in tumors by immunohisto-

Cancer Gene Therapy Gas1 and p53 expression in gliomas JA Benı´tez et al 842 FL1-Height (3) vs FL2-Height (4) FL1-Height (3) vs FL2-Height (4) FL1-Height (3) vs FL2-Height (4) 104 104 104

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Figure 4 Apoptotic characteristics of C6 cells transduced with AdSGas1, AdS53 and AdSGi53. (a) The Annexin-V-FLUOS staining kit was used to determine the expression of apoptotic characteristics on C6 cells, 48 h after infection with the vectors indicated in the panel. Cells were incubated with Annexin V and propidium iodide (PI) and stained cells were analyzed by flow cytometry. The percentage of early- and late- apoptotic cells are indicated on each dot plot. (b) A representative example of C6 cells stained with the Annexin V and counterstained with 4,6- diamidino-2-phenylindole (DAPI), 48 h after infection. Scale bar, 20 mm. (c) Representative slides of DNA fragmentation in treated C6 cells stained using the TdT-mediated dUTP nick end labeling (TUNEL) assay, cells were counterstained with DAPI. Cells that overexpress gas1 and p53 are positive for the TUNEL assay. Scale bar 50 mm. Treatments are indicated on the left side of each panel.

chemical staining. Intratumoral injection of AdSGas1 and and in an in vivo glioma model with transgenes driven by AdSGi53 resulted in specific human Gas1 expression in gfa2, a glial-specific promoter, and that the results of both treated tumors (Figure 6a). In addition, we observed growth arrest and apoptosis induced by the overexpres- human p53-specific expression in tumors treated with sion of gas1 and p53. AdS53 and AdSGi53 (Figure 6b). Furthermore, we showed colocalization of Gas1 and p53 expression in AdSGi53-treated tumors (Figure 6c). Taken together, our Discussion results showthat tumor growthsuppression results of both cell cycle arrest and apoptosis induced by the We report that the effect of gas1 (AdSGas1) on cell overexpression of gas1 and p53 in glioma cells. viability and tumor growth inhibition was similar to the In conclusion, our results demonstrate that AdSGas1 effect of p53 driven by the gfa2 promoter (AdS53). Taken and AdS53 have tumor suppressor activity both in vitro together, these results confirm the role of gas1 as a tumor

Cancer Gene Therapy Gas1 and p53 expression in gliomas JA Benı´tez et al 843 suppressor gene. There is yet no complete description of dorsal somite. More recently, we35 and subsequently the molecular mechanism by which Gas1 induces cell others36 reported the structural similarities between Gas1 arrest and apoptosis. Lee and coworkers34 reported that and the GDNF family of coreceptors (GFR-a) and shown Gas1 antagonizes SHH (Sonic hedgehog) function in the that Ret-mediated pathways may be altered by Gas1.36 These data suggest that Gas1 may interfere in the GDNF- mediated signaling, and there is evidence indicating that Ret-mediated signaling pathways may be altered by Gas1.36 These newevidences may explain the reduction of cell proliferation and tumor growth suppression induced by Gas1 in our model. Previous results have shown that GDNF and Ret are highly expressed in C6 cells,37,38 and that GDNF exerts a powerful proliferative effect on them.39 Therefore, we propose that the effects of GDNF on the proliferation of C6 cells is antagonized by the overexpression of gas1 and results in cell death (in preparation). We cannot exclude at this point that the inhibition of GDNF signaling by Gas1 also results in autophagic cell death. Some reports indicate that the deprivation of GDNF from cells in culture induces autophagic cell death.40–42 Using vital staining with acridine orange for the detection of autophagy,43,44 we found that C6 cells that overexpress gas1 showacidic vesicle organelles (AVO) in the cytoplasm, a characteristic of autophagic process (data no shown). These results suggest that Gas1 Figure 5 In vivo anti-tumoral action of Gas1 and p53. Growth blocks GDNF-signaling pathways, and may induce curves of C6 cells tumors subcutaneously implanted in nude mice, autophagy in C6 cells. This is the first time that a link and treated with the indicated adenovirus. Mice that were injected between Gas1 and autophagic cell death is proposed. with AdSGas1, AdS53 and ASGi53 showed a slower rate of tumor growth compared with the control groups, LacZ-treated and More experiments are necessary to elucidate the relation- untreated. Arrows show the different application of the adenovirus ship between Gas1 and GDNF in both apoptosis and (n ¼ 8–9 mice per group). Two-way analysis of variance (ANOVA), autophagic cell death. followed by Bonferroni. *Po0.05, **Po0.01, ***Po0.001 compared On the other hand, our results showthat the coexpres- with both (À) untreated and AdSLacZ. sion of gas1 and p53 in the same cells using a bicistronic

DAPI Anti-Gas1 DAPI Anti-hp53 AdSGas1 AdSGi53 AdS53 AdSGi53

Anti-Gas1 Anti-hp53 DAPI Overlap AdSGi53

Figure 6 Analysis of gas1 and p53 expression in tumor samples by immunostaining. (a) Representative slides of tumors stained with an antibody against human Gas1. Only tumors that overexpressed gas1 were positive for Gas1 immunostaining. Scale bar, 50 mm. (b) Representative slides of tumors stained with an antibody against human p53. The human p53 transgene was expressed in tumors treated with AdS53 and AdSGi53 adenoviral vectors. Scale bar 50 mm. (c) Tumors infected with AdSGi53 bicistronic adenovirus co-expressed gas1 and p53 in the same cells. Arrows indicate co-localization of Gas1 and p53 in tumor slides. Scale bar 30 mm. Treatments are indicated on the left side of each panel.

Cancer Gene Therapy Gas1 and p53 expression in gliomas JA Benı´tez et al 844 adenoviral vector has a similar effect both on cell getting us closer to the fulfillment of the expectations proliferation and tumor growth as the individual expres- generated by this newbranch of molecular medicine. sion of each one of these transgenes in the C6 glioma Gene therapy approaches for the treatment of cancer model. We suggest that Gas1 and p53 act independently include the combination of the expression of a therapeutic and that the Gas1 effect is not mediated by p53, at least transgene together with the administration of pharmaco- not in this model, in contrast with previous reports, in logical agents, or the expression of more than one which different cell lines were used.6,45 These results are therapeutic transgene. There are several examples of in agreement with previous reports which demonstrated successful coexpression of transgenes to increase their that the ectopic expression of wild-type (wt) p53 in glioma individual therapeutic efficacy, using both viral and cell lines with wild-type p53, such as C6 cells,46 or mutant non-viral systems.60–63 The success of these approaches p53, like U373 cells47 leads to cell death by apoptosis48 or will depend on the additive or synergistic effects of the growth arrest,46 depending on the final expression levels transgenes used, the specific tumor, the efficiency of of p53,49 as well as by the expression of other genes (that the gene delivery system, and case by case analysis will be is, p21 and PUMA) involved in the p53 pathways.50 necessary to determine the efficacy of the combinations. In this study, we show that Gas1 does not require p53 In general, a gene therapy-corrective approach for to induce apoptosis and cycle arrest in glioma cells. gliomas involves the restoration of apoptotic pathways Among the possible interpretations of the present data and/or the correction of cell cycle control. Thus, the that deserve to be further explored there are: that a kinetic targeted expression of gas1 using the gfa2 promoter to effect may participate, so that Gas1 and p53-induced drive its expression is an interesting approach to treat intracellular signaling have different kinetics, and the gliomas, because Gas1 blocks the proliferative action of faster system cancels out the effect of the other. Another GDNF, which is implicated in glioma cell migration and possibility is related to interference of signal transduction proliferation.39,64 Also, it is reported that Gas1 anta- pathways. As we have discussed, there is evidence gonizes the Shh pathway,34,65 which is involved in brain indicating that Gas1 alters the GDNF intracellular tumor formation and tumor growth.66,67 Moreover, Gas1 signaling cascades, and that Gas1 inhibits the GDNF- inhibits cell proliferation and induces cell death (apopto- induced activation of Akt35,36; (manuscript in prepara- sis and/or autophagy) even in the absence of active p53. tion). One of the effects of Akt activation is to release Additionally, we also propose the targeted overexpression NF-kB from IkB, thus allowing it to perform its trans- of p53 using the gfa2 promoter as an alternative treatment criptional activities.51 Although NF-kB activity is tradi- for gliomas, because p53 is the most common tumor tionally associated with cell survival pathways, it has suppressor gene inactivated in gliomas and it has a critical been shown that it is required for efficient p53-induced role in the progression of these tumors. Recent reports apoptosis in some cellular systems.52,53 Thus, Gas1 could, showed the relevance of p53 in tumor growth and by its inhibition of GDNF signaling, decrease the activity indicated that reactivation of p53 induces tumor growth of NF-kB and thus impede the apoptotic process induced regression and cell death.13,14 Therefore, according to by p53. Finally, an extremely interesting hypothesis is that these and previous results we propose that the targeted Ret acts as dependence receptor,54 since it has been shown expression of gas1 and p53 driven by the gfa2 promoter that Ret induces apoptosis in the absence of GDNF.55 In using adenovirus as the gene transfer system is an the present situation, the absence of GDNF binding or interesting adjuvant strategy for the selective gene therapy activity would be due to Gas1 expression, and Ret will set of gliomas. the cell into an apoptotic process independent of p53. Targeted gene expression aims at achieving the expression of therapeutic transgenes in specific and restricted Acknowledgements cell populations, thus sparing all other cells of the We thank Dr B Vogelstein (Johns Hopkins University) unwanted effects of the gene product. This concept is for providing us with the reagents (pAdEasy system) to particularly relevant for the treatment of cancer, because construct the adenoviral vectors. The p53 plasmid (pC53- the principal objective of the therapy is to eliminate the SN3) was kindly provided by Dr A Gutie´ rrez (Centro tumor cells, and the optimal therapeutic outcome is to Nacional de Rehabilitacio´ n), and the b-actin antibody destroy cancer cells without harming healthy cells.15 Some was a gift from Dr M Herna´ ndez (Cinvestav). We thank advantages of cell-specific promoters are that the trans- RSa´ nchez for laboratory assistance. This work was gene can be expressed, not only in the therapeutically partially supported by a CONACyT grant 42721-M (JS). appropriate cells, but also in a regulated manner and when necessary, for prolonged periods, compared with the use of viral promoters. However, occasionally one References promoter may be active in different cell types, and its transcription can be downregulated by epigenetic proces- 56 1 Lopez-Gonzalez MA, Sotelo J. Brain tumors in Mexico: sing. Transcriptional regulation of transgene expression characteristics and prognosis of glioblastoma. Surg Neurol has been extensively used to express therapeutic trans- 10,11,18,57–59 2000; 53: 157–162. genes in gliomas using the GFAP promoter. 2 Maher EA, Furnari FB, Bachoo RM, Rowitch DH, Louis Reaching the goals of targeted gene expression will greatly DM, Cavenee WK et al. Malignant glioma: genetics and increase the specificity and safety of gene therapy, thus biology of a grave matter. Genes Dev 2001; 15: 1311–1333.

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