J Neurooncol (2007) 82:239–248 DOI 10.1007/s11060-006-9279-x
LAB INVESTIGATION – HUMAN/ANIMAL TISSUE
Exogenous wt-p53 enhances the antitumor effect of HSV-TK/ GCV on C6 glioma cells Qiang Huang Æ Peiyu Pu Æ Zhibo Xia Æ Yongping You
Received: 24 July 2006 / Accepted: 2 October 2006 / Published online: 11 November 2006 Springer Science+Business Media B.V. 2006
Abstract Objective To study on the antitumor effect of combining wt-p53 gene with suicide gene therapy (HSVtk+GCV) for malignant gliomas. Methods AdCMV-p53 was transfected into C6 glioma cells at MOI of (Multiplicity of infection) 0(G100), 10(TPG1), 100(TPG2), then AdCMV-tk was transducted to C6 glioma cells of G100, TPG1 and TPG2, respectively, at MOI of 100. The C6 glioma cells tranfected with both AdCMV-p53 and AdCMV-tk were exposed to various concentration of GCV. The cell survival rate was measured by MTT assay in vitro. Rat glioma model was established by injecting 5 · 105 C6 glioma cells into right caudate nucleus of SD rats. AdCMV-p53 and AdCMV-tk were injected into glioma on day 5 and 6, respectively. On day 7, ganciclovir (GCV) was administrated intraperitoneally at 15 mg/ kg/day for 14 days. The survival time of all rats was observed. The growth of intracerebral tumors was monitored dynamically by enhanced MRI. Cell apoptosis was evaluated by TUNEL method. Expression of HSV-tk gene was identified by in situ hybridization and expression of exogenous p53 gene was detected with Western blotting. Results In vitro, wt-p53 significantly enhanced antitumor effect of HSV-tk/GCV. The concentration of Q. Huang (&) P. Pu Z. Xia Y. You Department of Neurosurgery, Tianjin Medical University General Hospital, 154 Anshan Road, Tianjin 300052, People’s Republic of China e-mail:
[email protected] Q. Huang P. Pu Z. Xia Y. You Laboratory of Neuro-Oncology, Tianjin Neurological Institute, Tianjin 300052, People’s Republic of China
GCV for ID50 of TPG2 cells (0.001 lg/ml GCV) was 10 times lower than that for the cells of tk-GCV group (MOI = 100), while the concentration of GCV for ID100 of TPG2 (0.01 lg/ml GCV) and TPG1(0.1 lg/ml GCV) was 100 and 10 times lower than that for the cells of tk-GCV group (MOI = 100), respectively. Apoptosis of C6 glioma cells also could be induced by transfection with wt-p53 gene slightly. For in vivo study, the survival time of tumor-bearing rats treated with HSV-TK/GCV or wt-p53 combined with HSV-TK/GCV was significantly prolonged and the intracerebral tumors were regressed and disappeared earlier in the combined gene therapy group than those in the HSV-TK/GCV therapy group as shown in enhanced MRI. However, only half dose of GCV for the rats treated with both wt-p53 and HSV-TK/GCV was needed to obtain the same efficacy as those rats treated with HSV-TK/GCV alone. These results indicate that the transfection of wt-p53 potentiates the effect of HSV-TK/GCV therapy. Conclusions The combination of HSV-tk/GCV system with wt-p53 gene transduction is optimal for clinical therapeutic trials of suicide gene therapy for malignant gliomas. Keywords Glioma wt-p53 HSV-tk/GCV Suicide gene therapy Combined gene therapy
Introduction Malignant glioma, the most common type of intracranial tumor, accounts for 30–50% of primary brain tumor in adults, with high morbidity, mortality and extremely grim prognosis. Recent advances in the
123
240
neurosurgical technique, radiation therapy and chemotherapy have failed to improve substantially the poor prognosis of patients with this malignant tumor. The life expectancy of patients with glioblastoma is less than 1 year from the time of diagnosis, and the 5-year survival rate is less than 5%. With the development of molecular biology and molecular genetics, the molecular events of glioma have been partially elucidated. Meanwhile, the technique of gene transfer and DNA recombination etc has been improved. All these progress provides the theoretic foundation and technical support for the development of gene therapy for malignant gliomas. In recent years, gene therapy of tumor is the hotspot for studying the treatment of tumors and suicide gene therapy using HSV-TK/GCV system is one of the most favorite strategies for treatment of malignant gliomas. The thymidine kinase enzyme (TK) produced by the Herpes simplex virus (HSV) is harmless to human body because of lacking it’s substrate. However, in cells transfected with HSV-TK gene and followed by receiving ganciclovir (GCV), the TK is able to catalyze the non-toxic prodrugs GCV to produce a cytotoxic nucleotide (GCV triphosphate, GCVTP). The nucleotide is incorporated into the DNA of dividing cells during replication where it halts DNA synthesis and results in cell death mainly by apoptosis. This is of particular significance in the brain where the normal neurons surrounding the tumor are non-proliferative and therefore not susceptible to toxic metabolites. The therapeutic effect is further strengthened by a bystander effect [1, 2]. Suicide gene therapy (HSV-tk/GCV) has revealed the encouraging therapeutic efficacy on glioma in vitro and in vivo studies. However, several phase I/II clinic trials did not show satisfactory results that HSV-tk/ GCV can prolong the survival time slightly [3–14], so it is imperative to optimize the protocols of HSV-tk/ GCV aiming at improving its curative effect. P53 may play a central role in cell cycle regulation, DNA repair, genomic stability and inducing programmed cell death after DNA damage. In a normal cell, irradiation or other DNA-damaging agents can induce a p53-dependent cell cycle arrest, which presumably allows enough time to repair the damaged cellular genome. Alternatively, if DNA damage is beyond repair, p53-dependent apoptosis may occur [15]. We presume that wt-p53 is able to enhance the antitumor effect of HSVTK/GCV, because a unrepaired DNA damage caused by triphosphate GCV might induce p53-dependent apoptosis. So in the present study, therapeutic effect of recombinant adenovirus-mediated HSV-tk gene
123
J Neurooncol (2007) 82:239–248
combining with p53 gene for gliomas followed by treatment with GCV has been studied.
Materials and methods Recombinant adenoviral vectors The HSVtk gene was isolated from the plasmid pBlueScriptII-TK (a gift from Tang MD, Alabama State University) with restriction enzymes HindIII and XbaI and ligated into pACCMVpLpA to give rise to the plasmid pACCMV-tk. The pACCMV-p53 was constructed from pACCMVpLpA and pC53-SN3 by the same method. Recombinant adenovirus was generated in 293 human kidney cells by Lipofectaminemediated homologous recombination between pACCMV-tk or pACCMV-p53 and pJM17. Recombinant viruses were identified by Southern blot, amplified and titrated. Ad.CMVbgal recombinant adenovirus harboring the Escherichia coli LacZ gene encoding b-galactosidase (bgal) was also constructed by inserting the LacZ gene, digested the pSV-bgal with restriction enzymes KpnI and XbaI, into pACCMVpLpA. Viral titer was determined by plaque-assay on 293 cells and expressed as plaque formation units (p.f.u.)/ml. Cell cultures C6 rat glioma cells and 293 human kidney cells were grown in DMEM supplemented with 10% fetal calf serum, 100 lg/ml streptomycin and 100 U/ml penicillin. The cells were maintained at 37C in a humidified atmosphere of 5% CO2. In vitro studies C6 rat glioma cells were plated on the coverglass. Triplicate coverglasses were infected with Ad.CMVbgal at multiplicity of infections (MOI) of 0, 10, 100. Twenty-four hours later, cells were washed with PBS, fixed in 0.4% formaldehyde and 0.2% glutaraldehyde, and incubated with X-gal solution (5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl2, 1 mg/ml 5-bromo-4-chloro-3-indolyl b-D-galactopyranoside in PBS) for 12 h. C6 rat glioma cells were trypsinized and harvested, seeded at a density of 4 · 103 cells/well in 96-well culture dishes. Twelve hours later, triplicate wells were infected with Ad.CMVbgal at moi of 0, 10, 100 as empty vector control group. Triplicate wells were infected with Ad.CMVtk or Ad.CMVp53 at moi of 0,
J Neurooncol (2007) 82:239–248
241
10, 100 as control group, and triplicate wells were infected with Ad.CMVp53 at moi of 0(G100), 10(TPG1), 100(TPG2) first, then with Ad.CMVtk at moi of 100 as treatment groups. Twelve hours later, cells were incubated at 37C in the presence of various concentrations of ganciclovir (from 10–3 to 103 lg/ml). Fresh medium containing various concentrations of GCV were added every other day. The percentage of surviving cells was measured using a method based on the metabolism, by living cells, of the mitochondrial substrate 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) into formazan, which is detected by measurement of the absorbency at 570 nm at every 24 h from 24 to 144 h.
1 · 108 p.f.u. of Ad.CMVp53 in 50 ll buffer were directly injected into the growing tumor at 5 points.
In vivo studies
HSV-TK + p53 + half dose of GCV treatment group (TPGH)
Male SD rats (250–300 g) were anesthetized by intraperitoneal injection of chloral hydrate and were placed in a stereotactic apparatus. Using a Hamilton syringe, 5 · 105 C6 glioma cells in 20 ll serum free DMEM were injected through a burr hole into the right caudate nucleus (3 mm lateral and 1 mm anterior to the bregma, 5 mm deep from the dura) over 10 min. Then the syringe was retracted over 5 min. The burr hole was filled with bone wax. The SD rats were divided into six groups, each group consists of 10 rats. Blank control group (C) Only 5 · 105 C6 glioma cells were injected. Empty vector control group (B) On day 5 after the implantation of glioma cells, approximate 1 · 108 p.f.u. of Ad.CMVbgal in 50 ll buffer containing 10 mM Tris–Cl pH 7.5, 1 mM MgCl2 and 135 mM NaCl were directly injected into the growing tumor. The injection was performed at 5 points along the needle tract (6, 5.5, 5, 4.5, 4 mm deep from the dura), over a period of 10 min. Afterwards, the needle was left in place for 5 min and was then retracted over 5 min. The burr hole was also blocked with bone wax. Two days later ganciclovir (GCV) was administrated intraperitoneally at 30 mg/kg/day for consecutive 14 days. p53 group Rats were injected with wild-type C6 glioma cells in right caudate nucleus. On day 5, approximate
HSV-TK + full dose of GCV treatment group (TGF) The same procedures were used as B group except replacement of Ad.CMVbgal with Ad.CMVtk. HSV-TK + half dose of GCV treatment group (TGH) The same procedures were performed as TGF group. Instead of using full dose of GCV (30 mg/kg/day), half dose of GCV (15 mg/kg/day) was administrated intraperitoneally.
On day 5 after injection of glioma cells, 40 ll of AdCMV-p53 were directly injected into the growing tumors and the next day, 40 ll of AdCMV-tk were injected into the growing tumors. On day 7, ganciclovir (GCV) was administrated intraperitoneally at 15 mg/ kg/day for 14 days. The general behavior and the survival of the rats in each group; the findings on MR imaging; the HSV-tk and p53 gene expression and apoptosis in the developing gliomas were observed. The volume of tumors in each group of rats was measured at regular intervals by using high-resolution MR imaging. This procedure has the advantage of allowing a comparison of tumor size at different periods in the same animal. Magnetic resonance imaging was performed in the rats by using a 1.5-T system. The tumor volume was estimated using the formula: Volume of tumor = (L · W2)/2 (L: the length of tumor and W: the width of tumor in enhanced MRI coronal scanning) [33]. The histopathological changes were examined using paraffin-embedded sections stained with hematoxylin and eosin. Cell apoptosis were detected on frozen sections by using the methods described earlier. In situ hybridization C6 glioma cells were plated on the coverglass and incubated at 37C in a humidified atmosphere of 5% CO2, then cells were infected with Ad.CMV-tk at multiplicity of infections (moi) of 100. Twenty-four hours later, cells were fixed with 4% paraformaldehyde–PBS (20 min) and washed in PBS. The slides were pre-hybridized in a solution containing 50% formamide, 1· Denhardt’s solution, 10% dextran
123
242
sulfate, 0.3 M NaCl, 10 mM Tris–HCl (pH 7.5), 5 mM EDTA, and 100 lg/ml salmon sperm cDNA at 42C for 4 h. Hybridization was performed in the same solution containing digoxigenin-labeled DNA probe (tk cDNA 1.8 kb) at 42C for 24 h. After hybridization, the slides were blocked with 1% (w/v) blocking reagent in 100 mM Tris–HCl and 150 mM NaCl (pH 7.5) for 30 min and incubated with mouse antidigoxigenin antibody (Boehringer Mannheim) for 1 h at room temperature. After rinsing, all slides were stained with NBT/BCIP. Western blotting analysis After C6 glioma cells were infected with Ad.CMV-p53 at multiplicity of infections (moi) of 0, 10, 100 for 24, 48, 72 h, cells were washed twice with PBS and harvested in lysis buffer (50 mM Tris–HCl pH 7.5, 250 mM NaCl, 0.1% NP-40, 5 mM EDTA, 10 lg/ml aprotinin, and 1 mM PMSF). Bicinchoninic acid protein assay kit (Sigma) was used to determine protein concentration, equal amounts of protein (approximately 50 lg) were subjected to SDS-PAGE on 10% acrylamide gel, and the separated proteins were transferred to PVDF membrane. Membranes were incubated with p53 antibody (1:500 dilution) followed by sheep anti-mouse IgG conjugated with horseradish peroxidase. P53 protein was detected using a Super Signal protein detection kit (Pierce, USA). After washing with buffer, the PVDF membrane was rehybridized with the primary antibody for b-actin (1:500 dilution) followed by the same procedures as described above. The relative quantification was determined as the density of p53 divided by the density of b-actin. Southern blot analysis Two hundred and ninety-three cells were seeded in 100 ml cell culture flasks. When 293 cells grew to approximately 85% confluence, 200 ll of AdCMV-TK or AdCMV-p53 were added. After incubation for 36 h, genomic DNA of adenovirus was extracted with the lysis solution containing 0.6% SDS, 10 mM EDTA, 100 lg/ml proteinase K. The products were separated by electrophoresis in 0.7% agarose gel and transferred to a nylon membrane. Hybridization was performed at 42C for 12 hin a solution containing 5· SSC , 5· Denhardt, 0.02% SDS, 1· blocking reagent, 50% formamide. The membrane was blocked with 1% (w/v) blocking reagent in 100 mM Tris–HCl and 150 mM NaCl (pH 7.5) for 30 min and incubated with mouse antidigoxigenin antibody (Boehringer Mannheim) for
123
J Neurooncol (2007) 82:239–248
1 h at room temperature. After rinsing, the membrane was stained with NBT/BCIP. The TUNEL method Cells or frozen tissue slides were fixed with 4% paraformaldehyde–PBS (20 min), washed in PBS and incubated with TdT and biotinylated dUTP (Boehringer Mannheim) at 37C for 45 min. Then they were rinsed, incubated with 50 ll converter-POD at 37C for 45 min and stained with DAB at 37C for 30 min. Finally, they were counterstained with hematoxylin. After the reaction, the nuclei of apoptotic cells were stained brown and that of normal cells were stained blue. Statistical analysis All data were analyzed with SPSS 10.0 statistics software. The result are presented as mean ± SE of the mean. Average results were evaluated by one-way analysis of variance (ANOVA) and Bonferroni t test. Survival data of rat bearing-tumor were analyzed with Kaplan–Meier survival plot with log rank regression.
Results Identification of recombinant adenoviral vector Recombinant adenoviral vectors containing HSV-tk and p53 gene obtained by homologous recombination were approximate 10.6 kb. Southern blot analysis confirmed that AdCMV-tk and AdCMV-p53 only had HSV-tk and p53 gene, respectively. The viral titer of AdCMV-tk and AdCMV-p53 was 2.6 · 109 and 1.4 · 109 p.f.u./ml, respectively. The antitumor effect of wt-p53 for C6 cells in vitro Western blotting analysis demonstrated that p53 expression of C6 cells was lower when moi = 0, meanwhile, when MOI = 10 and 100, the expression of p53 protein increased with times and was up to the high level at 72 h after transfection. MTT assay showed that the suppression of exogenous wt-p53 on growth of C6 glioma cells was limited, the highest suppressive rate was approximate 40% at 48–72 h at MOI = 100. C6 cells re-proliferated significantly after 96 h. The apoptosis of C6 cells detected by TUNEL method also exhibited that exogenous wt-p53 induced increase of apoptotic cells slightly (Fig. 1).
J Neurooncol (2007) 82:239–248
243
Fig. 1 (A) Western blotting analysis showed that p53 protein expression in C6 cells was lowered when moi = 0, meanwhile, in case of MOI = 10, 100, the expression of p53 protein increased with times and was up to the high level at 72 h after transfection (1: Wildtype C6; 2, 3: C6 transducted with wt-p53 at 24 h; 4, 5: transducted with wt-p53 at 48 h; 6, 7: transducted with wt-p53 at 72 h; 2, 4, 6: MOI = 10 AdCMV-p53; 3, 5, 7: MOI = 100 AdCMV-p53). (B) MTT results showed that the repression of exogenous wt-p53 to C6 glioma cells was limited, the highest repressive rate was approximate 40% at 48–72 h at MOI = 100. C6 cells re-proliferated significantly after 96 h. (C) The TUNEL assay approved that exogenous wt-p53 could increase the apoptosis of C6 cells slightly
Exogenous wt-p53 enhanced the antitumor effect of HSV-TK/GCV on C6 glioma cells in vitro To evaluate the antitumor effect of HSV-TK/GCV combined with wt-p53 on C6 glioma, we set up two combined therapeutic groups, one was TPG1 that C6 cells was infected with AdCMV-p53 at MOI of 10 and AdCMV-tk at MOI of 100, the other was TPG2 that C6 cells was infected with AdCMV-p53 at MOI of 100 and AdCMV-tk at MOI of 100. Because C6 glioma cell growth could be inhibited by either wt-p53 or HSV-TK/ GCV alone, we supposed two results of the combined therapy to be occurred. One was that the combined antitumor effect of wt-p53 and HSV-TK/GCV was simply the additive effect of single wt-p53 and HSVTK/GCV therapy, the other one was that wt-p53 combined with HSV-TK/GCV therapy could bring the synergistic antitumor effect which would be superior to the additive effect. In order to differentiate the two possible results, the C6 cells infected with AdCMV-p53 at the same time and MOI was used as the control group. (the cells survival rate = A value of the experiment group/A value of the control group · 100%. TPG1and TPG2 used the A value of C6 cells infected by AdCMV-p53 at MOI of 10, MOI of 100, respectively, at the same time as control).
We demonstrated that wt-p53 combined with HSVTK/GCV gene therapy resulted in the super-additive antitumor effect. X-gal staining showed that 100% C6 cells were transducted with AdCMV-bgal at MOI of 100. In situ hybridization also showed that 100% C6 cells expressed HSV-tk gene after transducted with AdCMV-TK at MOI = 100. The cell survival rate examined by MTT assay revealed that the concentration of GCV for ID50 of TPG2 cells (0.001 lg/ml GCV) was 10 times lower than that for the cells of tkGCV group (MOI = 100) at 144 h, while the concentration of GCV for ID100 of TPG2 (0.01 lg/ml GCV) and TPG1 (0.1 lg/ml GCV) was 100 and 10 times lower, respectively, than that for the cells of tk-GCV group (MOI = 100) at 144 h. The significant antitumor effect was demonstrated at 48 h in TPG1 and TPG2 after adding GCV (P<0.05). As shown by the TUNEL assay, almost 100% C6 cells occurred apoptosis after treatment with GCV of 0.01 lg/ml at 96 h in TPG2 group that is comparable to the apoptosis induced by GCV of 1 lg/ml at 120 h in G100 group (Fig. 2). Taken together, the results demonstrated that exogenous wt-p53 strengthened the sensitivity of C6 glioma cells to HSV-TK/GCV and increase the apoptosis of C6 glioma cells after treatment with HSV-TK/GCV in vitro.
123
244
J Neurooncol (2007) 82:239–248
Fig. 2 (A) The survival curves at 48 h. The survival rate of C6 cells in TPG1 (tranducted by AdCMV-TK at moi of 10 + AdCMV-p53 at moi of 100) and TPG2 (transducted by AdCMV-TK at moi of 100 + AdCMV-p53 at moi of 100) was reduced significantly compared to G100 (transducted by AdCMV-TK at moi of 100) when exposed to GCV at 101, 102, 103 lg/ml (P<0.05) at 48 h. (B) The survival curves at 144 h. The survival rate of C6 cells in TPG1 and TPG2 was reduced
significantly compared to G100 when exposed to GCV at 10–3, 10–2, 10–1 lg/ml (P < 0.05) and total C6 cells in three groups nearly were killed when exposed to GCV at 100, 101, 102, 103 lg/ ml. (C) The TUNEL assay showed that almost 100% C6 cells occurred apoptosis after exposed to GCV of 0.01 lg/ml at 96 h in TPG2 group, compared to GCV of 1 lg/ml at 120 h in G100 group
Exogenous wt-p53 enhanced the antitumor effect of HSV-TK/GCV on C6 glioma cells in vivo
TGH group and two control groups (P > 0.05). 5/7 rats of TGF group survived over 120 days, another two rats died of peritonitis at day 64 and 87, respectively, without tumor in brain. All seven rats of TPGH group survived over 120 days. Rats of TGF group and TPGH group had a longer survival time compared to two control groups, p53 group and TGH group (P < 0.05). But there was no significant difference between TPGH and TGF group (P > 0.05) (Fig. 3A). MRI showed that gliomas of TPGH group disappeared within 4 weeks while those of TGF group disappeared within 8 weeks (Fig. 4). C6 gliomas grew enough large to be detected by Enhanced MRI at 1 week following 5 · 105 C6 glioma cells were injected into rat brain. The volume of C6 glioma was calculated by enhanced MRI at 2 weeks after injection of C6 glioma cells into the brain. There were no significant differences in tumor volumes among p53 group, TGH group and two control groups (P > 0.05) or between TPGH group and TGF group.
We demonstrated that the C6 glioma cells transfected with AdCMV-p53 were expressed wt-p53 proteins in vivo by western blotting, meanwhile, the C6 glioma cells was successfully transfected with adCMV-bgal detected by X-gal staining and with adCMV-TK by in situ hybridizaiton in vivo. The results also showed that the expression of tk/bgal was highest near the needle tract and the farther from the needle tract, the lower the expression of tk/bgal was. Our results showed that wt-p53 could enhance the antitumor effect of HSV-TK/GCV in vitro, the concentration of GCV for ID100 of TPG2 was 0.01 lg/ml. So TPGH group was set up in vivo, and TGH was used as control group. The median survival time (MST) of two control groups was 16 days. MST of p53 group was 18 days. The MST of TGH group (n = 9) was 16 days. There was no significant difference among p53 group,
123
J Neurooncol (2007) 82:239–248
Fig. 3 (A) Kaplan–Meier survival plots of rats bearing brain tumor treated with wt-p53 combining with tk-GCV. (B) The volume of brain tumor 2 weeks after implantation and treatment. The volume of tumor has no significant difference among p53 group, TGH group and two control groups (P > 0.05),
245
meanwhile, between TPGH group and TGF group. However, the tumor volume of TPGH group and TGF group was significantly smaller than that of p53 group, TGH group and two control groups (P < 0.05)
Fig. 4 Enhanced MRI coronal scanning of tumor. (A) Blank control group (C) at 2 weeks. (B) Empty vector control group (B) at 2 weeks. (C) p53 group at 2 weeks. (D) HSV-TK + half dose (15 mg/ kg/day) of GCV treatment group (TGH) at 2 weeks. (E) HSV-TK + full dose (30 mg/ kg/day) of GCV treatment group (TGF) at 2 weeks. (F) TGF group at 4 weeks. (G) TGF group at 8 weeks. (H) HSV-TK + p53+half dose (15 mg/kg/day) of GCV treatment group (TPGH) at 2 weeks. (I) TPGH group at 4 weeks. Enhanced MRI showed that intracranial tumor of TPGH group disappeared at 4 weeks, a very small tumor in TGF group at 4 week and disappeared at 8 weeks
However, the tumor volume of TPGH group and TGF group was significantly smaller than that of p53 group, TGH group and two control groups (P < 0.05) (Fig. 3B). The number of apoptotic cells in TPGH group and
TGF group were much more than that of other four groups examined by the TUNEL method. These data suggest that even the half dose of GCV was used for the wt-p53 combined with HSV-TK/GCV suicide gene
123
246
therapy, the therapeutic effect would be the same as that of the full dose of GCV used for single HSV-TK/ GCV gene therapy. So wt-p53 significantly enhanced the antitumor effect of HSV-tk/GCV therapeutic system in vivo.
Discussion HSV-tk/GCV gene therapy for malignant gliomas has been demonstrated highly effective in a variety of animal models. HSV-tk/GCV approach significantly prolonged the survival time of the glioma-bearing animals, and about 20–100% of the tumor eradication rate was achieved due to the difference of vector systems, animal models, and dose of GCV [7–13]. Despite promising results in experimental studies, clinical trials of suicide gene therapy with HSV-tk/GCV system in brain tumor patients have generally been disappointing. A much smaller, Phase I study of adeno.tk injection into malignant gliomas demonstrated that the approach was safe but not very effective [4]. A small, uncontrolled trial of HSV-tk gene therapy in malignant gliomas demonstrated better efficiency of replicationdeficient adenovirus vector as compared with a retrovirus vector [5]. Another randomized, controlled trial of AdvHSV-tk gene therapy in human malignant gliomas reported that it could improve survival [6], However, a large, controlled, multicentric Phase III study of adjuvant suicide gene therapy with HSV-TK/ GCV system in 248 patients with glioblastoma multiforme had been conducted and did not show any benefit of the injection of HSV-tk3 retrovirus vectorproducing cells [3], and it was found in some re-operated and autopsied cases the transduction efficiency of HSV-tk gene was very low, that might also be one of the important factors for the failure of this novel approach. Many studies tried to improve the therapeutic efficacy of HSV-TK/GCV by combining with some other gene therapy approaches, such as IL2, IL4, TNFa, CD/ 5-FU [16–20]. In the present study, we tried to enhance sensitivity of the tk+ cells to GCV by introducing wt-p53. wt-p53 can induce the cells which contains un-repaired DNA damages to apoptosis, meanwhile, incorporation of GCVTP into the DNA of dividing cells results in an irreversible DNA damages to wt-p53. So theoretically, wt-p53 has ability to enhance the efficacy of HSV-TK/GCV therapy. It was reported that TK/GCV-induced apoptosis requires p53, which then triggered the translocation of CD95 to the cell surface resulting in death receptor-mediated activation of caspases [21]. However, Xie et al. reported that
123
J Neurooncol (2007) 82:239–248
co-expression of p53 may not necessarily improve the efficacy of adenovirus-mediated CD/5FC and HSVTK/GCV suicide gene therapies to human Hep3B and SK-OV-3 cells in vitro and in vivo [22]. Fischer et al. reported that TK/GCV- and CD/5-FC-induced apoptosis required neither p53 nor death receptors, but converged at a mitochondrial pathway triggered by different mechanisms of modulation of Bcl-2 proteins. They showed that exposure to GCV and 5-FC resulted in a weak p53 response in wild-type p53-expressing U87 cells although apoptosis was efficiently induced. But they did not evaluate the effect of exogenous wtp53 to TK/GCV-induced apoptosis [23]. So the molecular mechanism of TK/GCV-induced apoptosis is controversial and not yet fully understood. We constructed AdCMV-tk and AdCMV-p53 recombinant adenoviral vectors and examined the efficacy of tk combining with wt-p53 to C6 glioma cells in vitro. In the present experiment, exogenous wt-p53 can inhibit the proliferation of C6 cells without p53 mutation transiently and increase their apoptosis slightly in vitro, and wt-p53 cannot prolong the survival time of C6 glioma-bearing rats. The similar results that unlike gliomas containing mutant p53, glioma cell lines harboring wild-type p53 alleles do not undergo apoptosis after infection with Ad-p53 were reported by several studies. Thus these wild-type p53 cells are resistant to the apoptosis-inducing effects of Ad-p53 [24–26]. But Li et al. reported that wt-p53 significantly inhibited the proliferation of U87 cells expressing wtp53 and induced apoptosis [27]. Although the different responses of gliomas with mutant p53 and wild-type p53 to Ad-p53 alone have been known for some time, the mechanism underlying these differences has not been elucidated to date. We observed that the significant enhanced antitumor effect started from 48 to 144 h when C6 cells were treated with wt-p53 combining with HSV-tk/GCV in vitro. Exogenous wt-p53 significantly enhanced the sensitivity of tk+ C6 cells to GCV (10 times and 100 times) in vitro. Apoptosis was induced in almost 100% C6 cells transducted with wtp53 and HSV-tk gene after exposure to GCV of 0.01 lg/ml at 96 h, that is comparable to tk+ C6 cells exposure to GCV of 1 lg/ml at 120 h without wt-p53 transduction. A bystander effect, that is HSV-tk non-expressing cells near HSV-tk expressing cells also can be killed, is mainly responsible to a dramatic tumor ablation or regression of the HSV-tk/GCV system found in animal experiment. Two major mechanisms have been proposed to explain the bystander effect. One involves the transfer of phosphorylated GCV through a gap junction. The other involves uptake of phosphorylated
J Neurooncol (2007) 82:239–248
GCV through apoptotic vesicles between HSV-tk expressing cells and non-expressing cells [28–31]. We suppose that the better efficacy of wt-p53 and HSV-tk combined gene therapy would be achieved in vivo when gene transduction used by two vectors which contained wt-p53 and HSV-tk gene, respectively, compared that used by a single adenovirus or other eukaryotic expression vectors containing wt-p53 and HSV-tk gene simultaneously. As the p53 and HSV-tk genes are transducted into cells with two separate vectors, it is possible that some cells are transducted by tk or by wt-p53 gene only in vivo. We demonstrate that exogenous wt-p53 remarkably enhance the sensitivity of C6 cells to lower dose of GCVTP in vitro. Some tk– C6 cells which got lower dose of GCVTP through gap junction or uptake of apoptotic vesicles might not be killed when single TK gene therapy was used. However, these tk– C6 cells might got wt-p53 gene when gene transduction used by two vectors which contained wt-p53 and HSV-tk gene separately, so they might be killed. Thus, the bystander effect of HSV-TK/GCV could be enhanced when HSV-TK/GCV combines with wt-p53 therapy. This is the reason why we constructed AdCMV-tk and AdCMV-p53 recombinant adenoviral vector separately. van Dillen et al. reported that HSV-tk/GCV-induced bystander effect is influenced by p53 mutations. They found that compared to cell lines without p53 mutation, GCV sensitivity was significantly decreased in cell lines with p53 mutation. Additionally, a significant bystander effect (relative increase in cell kill) was found in cell lines without p53 mutation [32]. Our experiment in vivo also demonstrated that the effect of the half dose of GCV (15 mg/ kg/day) used in wt-p53 and HSV-tk/GCV combination therapy was the same as that of the full dose of GCV (30 mg/kg/day) used in single HSV-tk/GCV therapy, which can eradicate gliomas of all treated tumorbearing rats. In conclusion, exogenous wt-p53 is able to enhance the sensitivity of C6 glioma cells to HSV-TK/GCV therapy and increase the apoptosis of C6 glioma cells after treatment with HSV-TK/GCV in vitro and in vivo. The combination of HSV-tk/GCV system with wt-p53 gene transduction may be a promising approach for treatment of malignant gliomas.
References 1. Moolten FL (1994) Drug sensitivity (bsuicideQ) genes for selective cancer chemotherapy. Cancer Gene Ther 1:279–287 2. Hakumaki JM, Poptani H, Sandmair AM, Yla-Herttuala S, Kauppinen RA (1999) 1H MRS detects polyunsaturated fatty acid accumulation during gene therapy of glioma:
247
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
implications for the in vivo detection of apoptosis. Nat Med 5:1323–1327 Rainov NGA (2000) Phase III clinical evaluation of herpes simplex virus type 1 thymidine kinase and ganciclovir gene therapy as an adjuvant to surgical resection and radiation in adults with previously untreated glioblastoma multiforme. Hum Gene Ther 11:2389–2401 Trask TW, Trask RP, Aguilar-Cordova E, Shine HD, Wyde PR, Goodman JC, Hamilton WJ, Rojas-Martinez A, Chen SH, Woo SL, Grossman RG (2000) Phase I study of adenoviral delivery of the HSV-tk gene and ganciclovir administration in patients with current malignant brain tumors. Mol Ther 1:195–203 Sandmair AM, Loimas S, Puranen P, Immonen A, Kossila M, Puranen M, Hurskainen H, Tyynela K, Turunen M, Vanninen R, Lehtolainen P, Paljarvi L, Johansson R, Vapalahti M, Yla-Herttuala S (2000) Thymidine kinase gene therapy for human malignant glioma, using replication-deficient retroviruses or adenoviruses. Hum Gene Ther 11:2197–2205 Immonen A, Vapalahti M, Tyynel K, Hurskainen H, Sandmair A, Vanninen R, Langford G, Murray N, Yla-Herttuala S (2004) AdvHSV-tk gene therapy with intravenous ganciclovir improves survival in human malignant glioma: a randomised, controlled study. Mol Ther 10:967–972 Namba H, Iwadate Y, Tagawa M, Kimura M, Shimizu H, Sato Y, Sueyoshi K, Sakiyama S (1996) Evaluation of the bystander effect in experiment brain tumors bearing HSVTK gene by serial MRI. Hum Gene Ther 7:1847–1852 Cool V, Pirotte B, Gerard C, Dargent JL, Baudson N, Levivier M, Goldman S, Hildebrand J, Brotchi J, Velu T (1996) Curative potenial of HSVtk gene transfer in rats with 9L gliosarcoma. Hum Gene Ther 7:627–635 Di Meco F, Benedetti S, Colombo MP, Finocchiaro G (1997) Perspectives for the gene therapy of malignant gliomas by suicide gene transfer. J Neurosurgical Sci 41:227–234 Rosolen A, Frascella E, di Francesco C, Todesco A, Petrone M, Mehtali M, Zacchello F, Zanesco L, Scarpa M (1998) In vitro and in vivo antitumor effect of retrovirus-mediated herpes simplex thymidine kinase gene-transfer in human medulloblastoma. Gene Ther 5:113–120 Vincent AJ, Vogels R, Someren GV, Esandi MC, Noteboom JL, Avezaat CJ, Vecht C, Bekkum DW, Valerio D, Bout A, Hoogerbrugge PM (1996) Herpes simplex virus thymidine kinase gene therapy for rat malignant brain tumors. Hum Gene Ther 7:197–205 Maron A, Gustin T, Le Roux A, Mottet I, Dedieu JF, Brion JP, Demeure R, Perricaudet M, Octave JN (1996) Gene therapy of rat C6 glioma using adenovirus-mediated transfer of the HSV-tk gene: long-term follow-up by magnetic resonance imaging. Gene Ther 3:315–322 Perez-Cruet MJ, Trask TW, Chen SH, Goodman JC, Woo SL, Grossman RG, Shine HD (1994) Adenovirus-mediated gene therapy of experimental gliomas. J Neurosci Res 39:506–511 Smitt PS, Driesse M, Wolbers J, Kros M, Avezaat C (2003) Treatment of relapsed malignant glioma with an adenoviral vector containing the herpes simplex thymidine kinase gene followed by ganciclovir. Mol Ther 7:851–858 Symonds H, Krall L, Remington L, Saenz-Robles M, Lowe S, Jacks T, Dyke TV (1994) p53-dependent apoptosis suppresses tumor growth and progression in vivo. Cell 78:703– 711 Niranjan A, Moriuchi S, Lunsford LD, Kondziolka D, Flickinger JC, Fellows W, Rajendiran S, Tamura M, Cohen JB, Glorioso JC (2000) Effective treatment of experimental glioblastoma by HSV verctor-mediated TNFa and HSV-tk
123
248
17.
18.
19.
20.
21.
22.
23.
24.
J Neurooncol (2007) 82:239–248 gene transfer in combination with radiosurgery and ganciclovir administration. Mol Ther 2:114–120 Uckert W, Kammertons T, Haack K, Qin Z, Gebert J, Schendel DJ, Blankenstein T (1998) Double suicide gene (cytosine deaminase and herpes simplex virus thymidine kinase) but not single gene transfer allows reliable elimination of tumor cells in vivo. Hum Gene Ther 9:855–865 Terao S, Shirakawa T, Goda K, Kamidono S, Fujisawa M, Gotoh A (2005) Recombinant interleukin-2 enhanced the antitumor effect of ADV/RSV-HSV-tk/ACV therapy in a murine bladder cancer model. Anticancer Res 25:2757– 2760 Colombo F, Barzon L, Franchin E, Pacenti M, Pinna V, Danieli D, Zanusso M, Palu G (2005) Combined HSV-TK/ IL-2 gene therapy in patients with recurrent glioblastoma multiforme: biological and clinical results. Cancer Gene Ther 12:835–848 Benedetti S, Dimeco F, Pollo B, Cirenei N, Colombo BM, Bruzzone MG, Cattaneo E, Vescovi A, Didonato S, Colombo MP, Finocchiaro G (1997) Limited efficacy of the HSVtk/GCV system for gene therapy of malignant gliomas and perspectives for the combined transduction of the IL-4 gene. Hum Gene Ther 8:1345–1353 Beltinger C, Fulda S, Kammertoens T, Meyer E, Uckert W, Debatin KM (1999) Herpes simplex virus thymidine kinase/ ganciclovir-induced apoptosis involves ligand-independent death receptor aggregation and activation of caspases. Proc Natl Acad Sci USA 96:8699–8704 Xie YJ, Gilbert JD, Kim JH, Freytag SO (1999) Efficacy of adenovirus-mediated CD/5-FC and HSV-1 thymidine kinase/ ganciclovir suicide gene therapies concomitant with p53 gene therapy. Clinical Cancer Res 5:4224–4232 Fischer U, Steffens S, Frank S, Rainov NG, Schulze-Osthoff K, Kramm CM (2005) Mechanisms of thymidine kinase/ ganciclovir and cytosine deaminase/5-fluorocytosine suicide gene therapy-induced cell death in glioma cells. Oncogene 24:1231–1243 Kock H, Harris MP, Anderson SC, Machemer T, Hancock W, Sutjipto S, Wills KN, Gregory RJ, Shepard HM, Westphal M, Maneval DC (1996) Adenovirus-mediated p53 gene transfer suppress growth of human glioblastoma cells in vitro and in vivo. Int J Cancer 67:808–815
123
25. Gomez-Manzano C, Fueyo J, Kyritsis AP, Steck PA, Roth JA, McDonnell TJ, Steck KD, Levin VA, Yung WK (1996) Adenovirus-mediated transfer of the p53 gene produces rapid and generalized death of human glioma cells via apoptosis. Cancer Res 56:694–699 26. Harris MP, Sutjipto S, Wills KN, Hancock W, Cornell D, Johnson DE, Gregory RJ, Shepard HM, Maneval DC (1996) Adenovirus-mediated p53 gene transfer inhibits growth of human tumor cells expressing mutant p53 protein. Cancer Gene Ther 3:121–130 27. Li H, Lochmuller H, Yong VW, Karpati G, Nalbantoglu J (1997) Adenovirus-mediated wild-type p53 gene transfer and overexpression induces apoptosis of human glioma cells independent of endogenous p53 status. J Neuropathol Exp Neurol 56:872–878 28. Bi WL, Parysek LM, Warnick R, Stambrook PJ (1993) In vitro evidence that metabolic cooperation is responsible for the bystander effect observed with HSV-TK retroviral gene therapy. Hum Gene Ther 4:725–731 29. Touraine RL, Ishii-Morita H, Ramsey WJ, Blaese RM (1998) The bystander effect in the HSVtk/ganciclovir system and its relation to gap junctional communication. Gene Ther 5:1705–1711 30. Touraine RL, Vahanian N, Ramsey WJ, Blaese RM (1998) Enhancement of the herpes simplex virus thymidine kinase/ ganciclovir bystander effect and its antitumor efficacy in vivo by pharmacologic manipulation of gap junctions. Hum Gene Ther 9:2385–2391 31. Freeman SM, Abboud CN, Whartenby KA, Packman CH, Koeplin DS, Moolten FL, Abraham GN (1993) The bystander effect: tumor regression when a fraction of tumor mass is genetically modified. Cancer Res 53:5274–5283 32. van Dillen IJ, Mulder NH, Sluiter WJ, Meijer C, de Jong S, Loncarek J, Mesnil M, de Vries EF, Vaalburg W, Hospers GA (2005) Influence of p53 status on the HSV-Tk/GCVinduced bystander effect in a panel of human ovarian carcinoma cell lines. Oncol Res 15:151–159 33. Mishima K, Asai A, Sugiyama A, Miyagi Y, Kitanaka C, Kagaya S, Kirino T, Kuchino Y (1996) Increased expression of schwannoma-derived growth factor (SDGF) mRNA in rat tumor cells: involvement of SDGF in the growth promotion of rat gliomas. Int J Cancer 66:352–357
