Combined radiation and p53 gene therapy of malignant glioma cells

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More than half of malignant gliomas reportedly have alterations in the p53 tumor suppressor gene. ... human glioma cells containing normal or mutated p53.
Combined radiation and p53 gene therapy of malignant glioma cells Behnam Badie,1 Chern Sing Goh,1 Jessica Klaver,1 Hans Herweijer,2 and David A. Boothman3 1

Neuro-oncology Laboratory, Department of Neurological Surgery, 2The Waisman Center, and 3Department of Human Oncology, University of Wisconsin School of Medicine, Madison, Wisconsin 53792. More than half of malignant gliomas reportedly have alterations in the p53 tumor suppressor gene. Because p53 plays a key role in the cellular response to DNA-damaging agents, we investigated the role of p53 gene therapy before ionizing radiation in cultured human glioma cells containing normal or mutated p53. Three established human glioma cell lines expressing the wild-type (U87 MG, p53wt) or mutant (A172 and U373 MG, p53mut) p53 gene were transduced by recombinant adenoviral vectors bearing human p53 (Adp53) and Escherichia coli b-galactosidase genes (AdLacZ, control virus) before radiation (0 –20 Gy). Changes in p53, p21, and Bax expression were studied by Western immunoblotting, whereas cell cycle alterations and apoptosis were investigated by flow cytometry and nuclear staining. Survival was assessed by clonogenic assays. Within 48 hours of Adp53 exposure, all three cell lines demonstrated p53 expression at a viral multiplicity of infection of 100. p21, which is a p53-inducible downstream effector gene, was overexpressed, and cells were arrested in the G1 phase. Bax expression, which is thought to play a role in p53-induced apoptosis, did not change with either radiation or Adp53. Apoptosis and survival after p53 gene therapy varied. U87 MG (p53wt) cells showed minimal apoptosis after Adp53, irradiation, or combined treatments. U373 MG (p53mut) cells underwent massive apoptosis and died within 48 hours of Adp53 treatment, independent of irradiation. Surprisingly, A172 (p53mut) cells demonstrated minimal apoptosis after Adp53 exposure; however, unlike U373 MG cells, apoptosis increased with radiation dose. Survival of all three cell lines was reduced dramatically after .10 Gy. Although Adp53 transduction significantly reduced the survival of U373 MG cells and inhibited A172 growth, it had no effect on the U87 MG cell line. Transduction with AdLacZ did not affect apoptosis or cell cycle progression and only minimally affected survival in all cell lines. We conclude that responses to p53 gene therapy are variable among gliomas and most likely depend upon both cellular p53 status and as yet ill-defined downstream pathways involving activation of cell cycle regulatory and apoptotic genes.

Key words: Adenoviral vectors; brain neoplasm; gene therapy; glioma; p53; radiation.

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utations of the p53 tumor suppressor gene have been reported in more than half of all human cancers, including brain, lung, breast, prostate, thyroid, bladder, colon, liver, leukemia, head and neck, and ovarian cancers.1,2 p53 may play a central role in cell cycle regulation, DNA repair, and programmed cell death after DNA damage. Alteration of its function can also lead to genomic instability, which is thought to be an early event in glioma oncogenesis.3 Normal p53 function is important in the cellular response to genotoxic agents causing DNA damage. The p53 protein acts as a transcription factor, inducing growth arrest by modulating specific downstream target genes, including p21, which arrests cells in late G1 by inhibiting cyclin-dependent kinase activity,4,5 or by up-regulating GADD45, which Received November 5, 1997; accepted May 4, 1998. Address correspondence and reprint requests to Dr. Behnam Badie, H4/330 Clinical Science Center, Department of Neurological Surgery, University of Wisconsin, 600 Highland Avenue, Madison, WI 53792-3232. E-mail address: [email protected] © 1999 Stockton Press 0929-1903/99/$12.00/10

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inhibits DNA synthesis and may activate nucleotide excision repair.6 Alternatively, p53-mediated regulation of apoptosis is not well understood, and some evidence implies that transactivation of the bax gene (a member of the Bcl-2 family that, in contrast with Bcl-2, has the ability to induce apoptosis) plays a role.7 Loss of these p53-dependent regulatory functions may, therefore, lead to tumor radioresistance and account for the association between p53 mutations and the poorer prognosis observed in some tumors. Despite recent advances in p53 biology, the relationship between p53 gene expression and cellular radiosensitivity remains unclear. Reports of increased, decreased, and no apparent differences in radiosensitivities have all been associated with p53 function.8 –10 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 serve a protective role and prevent the transfer of new mutations to daughter cells. In a neoplastic cell bearing the wild-type

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(wt) p53 gene, however, such a response could have both beneficial and harmful therapeutic consequences. After DNA damage (chemotherapy or irradiation), p53-dependent G1 arrest could confer therapeutic resistance by providing tumor cells with enough time to undergo DNA repair before cell division. Conversely, p53-dependent apoptosis could confer therapeutic sensitivity to the tumor cells. In a neoplastic cell bearing a nonfunctional p53 gene, the opposite may be true: cells may become sensitive by not arresting in G1 for repair or may become resistant by averting the apoptosis induced by irradiation or chemotherapy. An overexpression of p53 by gene therapy techniques may, therefore, have both beneficial and harmful therapeutic consequences and may depend upon the cell type and endogenous nature of p53 and other cell cycle and apoptotic regulatory genes.11 Although the antineoplastic role of p53 gene therapy has been documented in various tumor models, its effect on the radiosensitization of gliomas has not been studied thoroughly. Adenoviral vectors encoding the human p53 gene (Adp53) have been used to significantly inhibit p53mut gliomas cells in vitro and in vivo.12–15 When tested on p53wt gliomas, however, Adp53 induced only a slight growth inhibition.15,16 This lack of response in p53wt tumors may prove to be a significant limitation in a clinical application of p53 gene therapy. Because p53 is thought to be a mediator of radiation-induced apoptosis, we hypothesized that its overexpression may lead to the sensitization of both p53mut- and also p53wt-bearing gliomas. In this study, Adp53 was used to transfer the wt human p53 gene into human glioma cell lines with differing p53 functions before radiation. Although p53 overexpression led to apoptosis and/or radiosensitization of p53mut cell lines (U373 MG and A172), it had little effect in p53wt-expressing cells (U87 MG).

In vitro gene delivery In vitro transduction was performed as described previously.17 Briefly, nonconfluent monolayers in six-well plates were incubated in 1.5 mL of sera-free culture medium with or without the vectors (i.e., AdLacZ or Adp53) at a multiplicity of infection of 100 for 60 minutes at 37°C. After this incubation, 1.5 mL of medium containing 10% fetal calf serum was added to each plate. Some cells were then irradiated 4 hours later at different intensities using a 137Cs irradiator (Sheppard, Glendale, Calif) at a dose rate of 5.94 Gy/min. Other mockirradiated cells were treated in a similar manner without ionizing radiation. Cells were then collected by trypsinization after 48 hours and stained for flow cytometry as described below.

Flow cytometry At 48 hours after irradiation, adherent cells were harvested by trypsinization and resuspended with the floating cells in 10 mM tris(hydroxymethyl)aminomethane (Tris) (pH 7.0) and 150 mM NaCl (Tris saline). After several washings, cells were centrifuged and resuspended in 1 mL of fixative solution (100 mL of Tris saline and 900 mL of absolute ethanol) and stored at 220°C. Before flow cytometry, cells were washed and incubated for 15 minutes in phosphate citric acid buffer (0.192 M Na2HPO4 and 4 mM citric acid) and then resuspended in propidium iodine (PI) solution (50 mg/mL PI, 1 mg/mL ribonuclease A, 10 mM ethylenediaminetetraacetic acid, and 0.2% Nonidet P-40 in phosphate-buffered saline (PBS)). Cells were incubated in the dark for 1 hour at 4°C and then analyzed with a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, Calif) equipped with a 15-mW, 488-nm air-cooled argon ion laser. At least 10,000 events were acquired using Lysis II software (Becton Dickinson). Orange fluorescence (PI, FL2) was collected using a 585 6 42-nm BP filter, and DNA content was measured using FL2 area versus FL2 width for doublet discrimination. Data were analyzed using Modfit software (Verity Software House, Topsham, Me). Each experiment was performed in triplicate and repeated at least twice.

Western blotting MATERIALS AND METHODS

Cell culture and adenoviral vectors The human glioma cell lines U87 MG, A172, and U373 MG were obtained from the American Type Culture Collection (Manassas, Va) and grown in Dulbecco’s modified Eagle medium supplemented with 10% fetal calf serum (Life Technologies, Gaithersburg, Md), penicillin G (100 U/mL), and streptomycin (100 mg/mL) at 37°C. These cell lines were selected because of their known p53 gene status and efficient transduction with adenoviruses. U87 MG expresses wt p53, whereas A172 and U373 MG express mutant forms.15 Adenoviral vector propagation and isolation have been described previously.12 The adenoviral vector AdHCMVsp1LacZ (AdLacZ) contains an expression cassette encoding the Escherichia coli b-galactosidase gene, driven by the immediate early cytomegalovirus promoter upstream from a polyadenylation signal. This vector served as a control in our experiments and was used to confirm the transduction efficiencies of the glioma cell lines as reported by other investigators.15 Adp53 has a similar construction, except that it contains a 1.4-kilobase pair sequence encoding the human wt p53 protein instead of the b-galactosidase gene.

Both adherent and floating cells were collected as described above, washed in PBS, and lysed in RIPA buffer (37.5 mM NaCl, 12.5 mM Tris base, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 10 mL/mL of phenylmethylsulfonyl fluoride, aprotinin, leupeptin, and pepstatin) at 4°C for 30 minutes. Lysates were centrifuged at 10,000 3 g for 15 minutes, sample protein concentrations were defined spectrophotometrically using a bovine serum albumin protein assay (Pierce, Rockford, Ill), and a 50-mg aliquot of total protein was separated in 10% (for p53 and p21) or 15% (for Bax) SDS-polyacrylamide gel electrophoresis under reducing conditions after a 3-minute boil in SDS sample buffer. Gels were then electroblotted onto Immobilon-P transfer membranes (Millipore, Bedford, Mass). After blocking for nonspecific binding with 5% milk and 0.05% Tween-20 in PBS, membranes were incubated with either mouse monoclonal anti-human p53 antibodies (Santa Cruz Biotechnology, Santa Cruz, Calif), anti-human p21WAF1 antibodies (Oncogene Science, Uniondale, NY), or polyclonal anti-human Bax antibodies (Santa Cruz Biotechnology) as recommended by the supplier. Horseradish peroxidase-conjugated goat anti-mouse IgG antibody was then used to visualize the protein signal using SuperSignal chemiluminescent substrate (Pierce).

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Hoechst staining for apoptotic cells Cells in monolayers were washed in PBS and fixed (150 mL methanol and 50 mL glacial acetic acid) for $2 hours at 4°C before incubating them with freshly made Hoechst 33258 stain (10 mg/mL in PBS, Sigma, St. Louis, Mo). The stain was removed after a 15-minute incubation, and cells were washed with PBS and visualized with a fluorescent microscope under N-propyl gallate (0.1 M solution in 80% glycerol).

Clonogenic survival assays After treatments with various vectors and/or radiation (as indicated), cells were harvested by trypsinization, replated at a density of 3000 cells/100-mm Petri dish in triplicate, and grown under the above culture conditions. After 2 weeks, plates were washed with PBS, fixed, and stained with Coomassie blue. The number of colonies containing .50 normal-appearing cells was counted.

RESULTS

p53, p21, and Bax expression Consistent with other reports,15 all three cell lines demonstrated $80% transduction with AdLacZ at a multiplicity of infection of 100 (data not shown). Effective viral transduction was also confirmed by the elevation of p53 in all cell lines within 48 hours of Adp53 exposure (Fig 1, lanes 1 and 6). Neither radiation nor AdLacZ had any effect on endogenous or exogenous p53 expression (Fig 1, lanes 2– 4 and 7–9). The biological activity of exogenous p53 was tested by examining cellular p21 content, which is a known p53inducible cell cycle regulator.4 p53 overexpression led to elevations in p21 protein in all three cell lines, suggesting intact p53 transcriptional activity (Fig 1). Although X-radiation had no effect on p21 levels in transduced cells, it slightly increased endogenous p21 expression by A172 and U87 MG cells, suggesting the presence of p53-independent mechanisms in regulating this protein.18 Because Bax has been suggested to play a role in p53-mediated apoptosis,7 we examined its expression in all three cell lines and found little change in its steadystate protein levels after radiation or Adp53 treatments.

Apoptosis Nuclear fragmentation suggestive of apoptosis was most evident in Adp53-exposed U373 MG cells (Fig 2A) and in A172 cells receiving combined treatment (data not shown). Flow cytometry was used to quantitate apoptosis by measuring the sub-G1 cell population. Regardless of irradiation dose, p53 gene therapy induced profound apoptosis in U373 MG cells (Fig 3), so that all cells died within 48 hours of transduction. Alternatively, Adp53 induced only low levels of apoptosis in A172 cells when administered alone; however, when given before radiation, Adp53 significantly increased cell death (Fig 3). In contrast, U87 MG cells showed mild apoptosis with Adp53, radiation, or combined treatment. In every cell line, AdLacZ had mild cytopathic effects and nuclear fragmentation.

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Figure 1. Western immunoblots of U373 MG, A172, and U87 MG cell lines demonstrating expression of p53, p21, and Bax. Mockinfected (lanes 1– 4), AdLacZ-infected (lane 5), and Adp53-infected (lanes 6 –10) cells were irradiated at various intensities and were checked for each protein after 48 hours.

Cell cycle The effect of ionizing radiation and p53 gene therapy on cell cycle distribution was examined by flow cytometry (Fig 4). Within 48 hours of irradiation (20 Gy), a 3- to 6-fold increase in G2/M cells was noted in U87 MG and U373 MG cells (Fig 5, A and B, respectively). This G2/M checkpoint most likely represented a p53-independent mechanism, because U373 MG cells express a mutant p53 protein and U87 MG cells did not demonstrate elevations in p53 protein after X-radiation. Alternatively, A172 cells demonstrated mild G1 arrest after irradiation (Fig 5C). This G1 arrest may have been mediated by elevations in p21 protein, which functions as a G1 checkpoint regulator5 and was elevated in this cell line after irradiation (Fig 1). Because A172 cells have a mutation in the p53 gene, this observation supports the presence of other p53-independent G1 arrest mechanisms, possibly working through p21.18 The effect of p53 gene therapy on cell cycle regulation was evident in all three cell lines. A 30% increase in G1 arrest was noted in A172 cells after Adp53 treatment, and this increase did not change with the addition of

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Figure 3. Flow cytometry was used to quantitate the apoptotic response of each treatment by measuring the sub-G1 cell population. U373 MG cells underwent significant apoptosis after Adp53 infection. Although radiation had no effect on these cells, it potentiated the p53-induced apoptosis in A172 cells. U87 MG cells showed no nuclear fragmentation even when both agents were combined. Error bars represent SE.

mediated p21 expression, which plays a role in cell cycle regulation after radiation.

Survival

Figure 2. Nuclear morphology of Adp53-treated U373 MG cells (A) demonstrated significant fragmentation (arrows) that was not seen in nonirradiated control cells (B) or irradiated (10 Gy) cells (C). Hoechst staining was performed at 48 hours after Adp53 or X-radiation.

radiation (Figs 4 and 5C). In U87 MG and U373 MG cells, Adp53 did not increase G1 arrest by itself. However, when administered before radiation, Adp53 did increase the G1 cells by 2- to 4-fold (Fig 4 and Fig 5, A and B). This observation is most likely due to Adp53-

Clonogenic assays were used to examine the long-term significance of the observed apoptosis and cell cycle changes before and after the treatments described above. Adp53 transduction reduced the survival of U373 MG and A172 cells by 90% and 60%, respectively, but did not effect the U87 MG cells (Fig 6). Moreover, radiation did not significantly potentiate the growth inhibition of Adp53, even in A172 cells, where it increased Adp53-mediated apoptosis (Fig 3). DISCUSSION Malignant brain tumors are ideal candidates for gene therapy because they only recur locally and rarely me-

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Figure 4. Representative DNA histograms of glioma cells at 48 hours after treatments with irradiation (20 Gy) and/or Adp53. Numbers represent the percentage of cells in the G0/G1 and G2/M phases.

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Figure 5. Effect of Adp53 and radiation on cell cycle distribution. Radiation-induced G2/M arrest was seen in both U87 MG cells (A) and U373 MG cells (B) but not in A172 cells (C). Error bars represent SE.

tastasize to other organs. At the present time, cancer gene therapy can be accomplished through either: (a) cytoreductive techniques, in which genes are delivered to interrupt cancer cell growth either directly via suicide genes or indirectly through immunomodulator genes, or (b) corrective gene therapy, in which defective or abnormal genes in neoplastic cells are inactivated or supplemented with genes that can reverse the loss of growthcontrol mechanisms. Tumor suppressor genes and antisense oncogenes are among the candidates for corrective gene therapy. Inactivation of the p53 tumor suppressor gene may play a role in the tumorigenicity of most cancers. The p53 gene encodes a nuclear protein that binds to and modulates the expression of other genes that are important for DNA repair,6 cell division,19 and cell death by apoptosis.20 Reconstitution of the p53 gene into p53mut tumor cells may, therefore, lead to a re-establishment of these regulatory functions and may prove to be an effective antineoplastic strategy. Exogenous p53 gene expression has been found to induce apoptosis or sensitize p53-negative cells to DNAdamaging agents in several tumor models, including lung,21 cervical,22 prostate,23 and head and neck tumors.24,25 In gliomas expressing p53mut, Adp53 has been shown to induce apoptosis in vitro and partially inhibit tumor growth in vivo.12,13,15,16 When human glioma cells were transduced with Adp53 in vitro, most p53mutexpressing cells showed significant apoptosis and growth suppression. Similarly, Gjerset et al14 used Adp53 to induce apoptosis and sensitize T98G glioma cells (p53mut) to cisplatin in vitro. None of these studies, however, showed any apoptosis in p53wt-expressing cells. Moreover, some p53mut cells, such as A172, were more resistant to such therapy.15 In this study, we tested the possibility that p53 overexpression may increase the sensitivity of these Adp53-resistant cells to X-radiation. Three glioma cell lines with variable p53 activities and responses to Adp53 were selected to investigate the role of Adp53 radiosensitization. In U373 MG cells (p53mut), Adp53 induced apoptosis and cell death within 48 hours of viral exposure. These observations were consistent with those reported previously.15 This p53-mediated apoptosis was probably mediated through pathways independent of Bax, because no changes in the expression of this protein were detected. Although radiation alone did not induce apoptosis within this time frame, it did result in G2/M arrest, confirming the lack of p53 function as a G1/S checkpoint in this p53mut cell line. Moreover, X-radiation significantly suppressed U373 MG growth in vitro. Whether this growth arrest was due to delayed radiation-mediated apoptosis that was not detected under these experimental conditions or due to the observed G2/M arrest cannot be concluded from our studies. Because most radiation-sensitive cells such as thymocytes and medulloblastomas undergo apoptosis within a few hours of radiation exposure,20,26 it is more likely that the observed G2/M arrest was responsible for this growth inhibition.

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The administration of radiation after p53 treatment in U373 MG cells did not potentiate apoptosis. This observation indicates either that p53 cannot act as a radiosensitizer in this cell line or perhaps that the apoptotic pathways were already saturated due to p53 expression. Adp53 treatment did, however, lead to G1 arrest in nonapoptotic cells after X-radiation. This change in cell cycle profile was most likely due to p53-mediated p21 expression, which is known to play a role in the G1/S checkpoint. It is not clear why most transduced U373 MG cells underwent apoptosis while some were arrested in G1. It is possible that these responses are dependent upon the temporal relationship of X-radiation and p53 expression to the cell cycle. In contrast to U373 MG cells, A172 cells, which also express p53mut, demonstrated little apoptosis after Adp53 treatment. This Adp53 resistance has been reported previously by Gomez-Manzano et al15, who noted that A172 cells die 8 days after Adp53 exposure, compared with 3 days seen in the more sensitive U373 MG cells. Because we did not detect any apoptosis within 48 hours of exposure to Adp53, we believe that this growth suppression was, in part, due to p21-mediated G1 arrest. Following radiation (10 Gy), A172 cells demonstrated a 30% increase in G1 cells and a 50% increase in G2/M cells. These cell cycle changes may have been a result of radiation-mediated p21 expression through a p53-independent mechanism that has been reported previously in another cell line.27 Interestingly, p53 expression before X-radiation significantly increased the apoptotic response to radiation in this cell line. This increase in apoptosis, however, was not related to changes in Bax expression, and other mechanisms such as activation of CPP32b (caspase 3) may have played a role.28 Moreover, this radiosensitization did not translate into further growth inhibition as detected by clonogenic assays, suggesting that activation of cell cycle checkpoints may be as important as apoptosis in determining the radiation sensitivity of gliomas.29 As reported by others,15,16 U87 MG cells showed the least response to Adp53 treatment, probably due to lack of apoptosis and weak G1 arrest. After X-radiation, U87 MG cells, like U373 MG cells, demonstrated a significant increase in G2/M arrest. This finding seemed to be in contrast to the G1 arrest reported by Yount et al30 in U87 MG cells expressing p53wt but not in those with an inactivated p53 gene. The discrepancy in our findings could be explained by the fact that we did not detect any p53 up-regulation by these cells after radiation, confirming that expression of this gene is important for G1 arrest. In fact, when both p53 and p21 were up-regulated by Adp53 transduction, this G1 checkpoint function was re-established in these cells. Whether our U87 MG cells have additional defects in radiation-mediated p53 expression (such as a mutation in the ATM gene) needs to be investigated. Otherwise, our results are in agreement with Yount et al, who also failed to observe any radiation-induced apoptosis or any radiosensitization of U87 MG cells when p53 was reactivated. This finding sug-

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Figure 6. Clonogenic assay revealing growth inhibition by Adp53 in U373 MG and A172 cells but not in U87 MG cells. The inability of Adp53 to radiosensitize these cell lines under these conditions is also demonstrated. Error bars represent SE.

gests that Adp53 therapy may not be appropriate for p53wt tumors under these conditions. We conclude that the response to combined Adp53 and radiation therapies may be variable among gliomas. This therapy may not be an effective treatment for p53wt-expressing tumors, and its efficacy on p53mut tumors may be limited not only by transduction efficiency and levels of p53 expression in vivo but also by the activation of other cell cycle regulatory and apoptotic machinery.

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ACKNOWLEDGMENTS We thank Pat Giuliani and Michael Liebman for editorial support. This work was supported in part by National Cancer Institute Grant P30 CA14520 and the University of Wisconsin Comprehensive Cancer Center (to B.B.).

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