Endothelial cells co-cultured with wild-type and dominant/negative p53-transfected glioblastoma cells exhibit differential sensitivity to radiation-induced apoptosis

We performed expressional profiling of isogenic glioblastoma cell lines U87-Lux8 and U87-175.4. These cell lines differ in that U87-Lux8 expresses wild-type p53 and U87-175.4 expresses a dominant-negative p53 (175(His) mutation). DNA array analysis and real-time PCR measurements demonstrated that basal expression and response to irradiation were different in these isogenic glioblastoma cell lines. These differences included genes involved in growth regulation and genes associated with cell-to-cell and cell/ECM communications. Co-cultivation of U87-175.4 and U87-Lux8 with HUVE cells demonstrated that U87-175.4 cells suppress the angiogenic phenotype of HUVEC and increase their sensitivity to radiation-induced apoptosis compared to co-culture of U87-Lux8/HUVEC. These data suggest that blockade of p53 function may alter the communication between tumor cells and endothelial cells such that endothelial cells exhibit an increase in radiosensitivity. These findings may have important implications for the treatment of glioblastoma tumors and other human cancers.     

Degree of apoptosis induced by adenovirus-mediated transduction of p53 or p73alpha depends on the p53 status of glioma cells

It has been reported that U-87MG glioma cells with wild-type p53 are resistant to p53 replacement gene therapy. As some gliomas harbor wild-type p53, it would be important to override the resistance mechanism due to wild-type p53 in glioma gene therapy. In this study, we transduced U-87MG cells or U251 glioma cells harboring mutated p53 with the p53 or p73alpha gene (a homologue of p53, that differently induces some p53-responsive genes) via adenovirus vectors (Advs) at same multiplicities of infection (MOIs) into respective cells (U-87MG: MOI 1000, U251: MOI 100), and evaluated the degree of apoptosis. The results demonstrate that the degree of apoptosis induced by Adv-mediated transduction of p53 in U-87MG cells was lower than that in U251 cells, whereas that induced by Adv-mediated transduction of p73alpha in U-87MG cells was higher than that in U251 cells. Bax expression in U-87MG and U251 cells induced by Adv-mediated transduction of p53 was almost the same as that of p73alpha. On the other hand, Adv-mediated transduction of p73alpha induced caspase-9 at higher levels than that of p53 in both cells. The results indicate that Adv-mediated transduction of p73alpha might be beneficial to overcome the resistance mechanism of glioma cells harboring wild-type p53.     

Apoptosis induced by adenovirus-mediated p53 gene transfer in human glioma correlates with site-specific phosphorylation

Therapeutic replacement of the p53 gene using an adenovirus vector (Ad-p53) may be an effective alternative to conventional therapies for the treatment of glioma. We have previously demonstrated that the introduction of Ad-p53 into glioma cells containing mutant p53 induces apoptosis, whereas glioma cells containing wild-type p53 are resistant. However, Ad-p53 will enhance the radiosensitivity of wild-type p53 glioma cells by increasing their tendency for apoptosis. The mechanism underlying these different responses to Ad-p53 has not been elucidated to date. Because phosphorylation of p53 at serines 15, 20, and 392 may play a role in regulating p53-mediated apoptotic activity, we determined the phosphorylation status of exogenous p53 in mutant and wild-type gliomas after Ad-p53 transfer. Monolayer cultures of glioma cell lines expressing mutant p53 (U251 and U373) or wild-type p53 (U87 and D54) were infected with Ad-p53 and analyzed by Western blotting. High levels of exogenous p53 were detected in both cell lines after Ad-p53 transfer. However, only apoptotic mutant p53 cells expressed high levels of phospho-Ser15-p53 and phospho-Ser20-p53. The levels of phospho-Ser15-p53 and phospho-Ser20-p53 were very low in wild-type p53 cells after Ad-p53 infection alone. When wild-type p53 glioma cells were exposed to radiation after Ad-p53 infection, phospho-Ser15-p53 and phospho-Ser20-p53 were detected at high levels, and the cells subsequently underwent apoptosis; no change in serine 392 was detected. The induction of apoptosis and the expression of phospho-Ser15 and phospho-Ser20 in these cells were also enhanced by the combination of Ad-p53 and other DNA-damaging agents such as cisplatin and bichloroethyl nitrosourea. Furthermore, the expression of phospho-Ser15-p53 and phospho-Ser20-p53 correlated with the amount of apoptosis; the apoptotic activity of p53 in glioma cells was partially inhibited by a mutation of p53 at serine 15. These results suggest that phosphorylation of p53 at serine 15 and serine 20 is critical for apoptosis induction in p53 gene therapy for gliomas.     

Vaccinia virus-mediated expression of wild-type p53 suppresses glioma cell growth and induces apoptosis

The use of vaccinia virus vectors for cancer gene therapy may become a powerful method to achieve efficient anti-tumor effects. We used recombinant vaccinia virus expressing wild-type p53 (rVV-p53) to examine the biological effects of exogenous tumor suppressor p53 in human (U-373MG, U-87MG, LN-Z308) and rat glioma cells (9L, C6) in vitro. All glioma cell lines infected with rVV-p53 exhibited growth inhibition and underwent apoptosis as demonstrated by morphological studies using nuclear staining and flow cytometry. The key role of p53 in cell growth inhibition was confirmed as measured by colony forming efficiency. Growth inhibition and apoptosis were independent of the endogenous p53 status of the glioma cell lines.     

Phosphorylation of Thr18 and Ser20 of p53 in Ad-p53-induced apoptosis

The p53 protein plays a critical role in inducing cell cycle arrest or apoptosis. Because p53 is inactivated in human gliomas, restoring p53 function is a major focus of glioma therapy. The most clinically tested strategy for replacing p53 has been adenoviral-mediated p53 gene therapy (Ad-p53). In addition to their therapeutic implications, investigations into Ad-p53 provide model systems for understanding p53's ability to induce cell cycle arrest versus apoptosis, particularly because wild-type p53 cells are resistant to Ad-p53-induced apoptosis. Here we use Ad-p53 constructs to test the hypothesis that simultaneous phosphorylation of p53 at threonine 18 (Thr18) and serine 20 (Ser20) is causally associated with p53-mediated apoptosis. Studies using phosphorylation-specific antibodies demonstrated that p53-induced apoptosis correlates with phosphorylation of p53 at Thr18 and Ser20 but not with carboxy-terminal phosphorylation (Ser392). To prove a causal relationship between apoptosis and Thr18 and Ser20 phosphorylation of p53, the effects of an adenoviral p53 construct that was not phosphorylated (Ad-p53) was compared with a Thr18/Ser20 phosphomimetic construct (Ad-p53-18D20D) in wild-type p53 gliomas. Whereas treatment with Ad-p53 resulted only in cell cycle arrest, treatment with Ad-p53-18D20D induced dramatic apoptosis. Microarray and Western blot analyses showed that only Ad-p53-18D20D was capable of inducing expression of apoptosis-inducing proteins. Chromatin immunoprecipitation assays indicated that the protein product of Ad-p53-18D20D, but not Ad-p53, was capable of binding to apoptosis-related genes. We thus conclude that phosphorylation of Thr18 and Ser20 is sufficient for inducing p53-mediated apoptosis in glioma cells. These results have implications for p53 gene therapy and inform other strategies that aim to restore p53 function.     

Combined radiation and p53 gene therapy of malignant glioma cells

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 beta-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.     

High linear energy transfer carbon radiation effectively kills cultured glioma cells with either mutant or wild-type p53

PURPOSE: A mutation in the p53 gene is believed to play an important role in the radioresistance of many cancer cell lines. We studied cytotoxic effects of high linear energy transfer (LET) carbon beams on glioma cell lines with either mutant or wild-type p53. METHODS AND MATERIALS: Cell lines U-87 and U-138 expressing wild-type p53 and U-251 and U-373 expressing mutant p53 were used. These cells were irradiated with 290 MeV/u carbon beams generated by the Heavy Ion Medical Accelerator in the National Institute of Radiologic Science or X-rays. A standard colony-forming assay and flow cytometric detection of apoptosis were performed. Cell cycle progression and the expression of p53, p21, and bax proteins were examined. RESULTS: High LET carbon radiation was more cytotoxic than low LET X-ray treatment against glioma cells. The effects of the carbon beams were not dependent on the p53 gene status but were reduced by G(1) arrest, which was independent of p21 expression. The expression of bax remained unchanged in all four cell lines. CONCLUSION: These results indicate that high LET charged particle radiation can induce cell death in glioma cells more effectively than X-rays and that cell death other than p53-dependent apoptosis may participate in the cytotoxicity of heavy charged particles. Thus, it might prove to be an effective alternative radiotherapy for patients with gliomas harboring mutated p53 gene.     

Sensitizing glioma cells to cisplatin by abrogating the p53 response with antisense oligonucleotides

Most gene therapy strategies related to p53 concentrate on the restoration of the activity of mutant p53, as several observations indicate that tumors and cell lines having the mutant gene are resistant to chemotherapy. However, as there is also some evidence to the contrary, we studied the relationship of the p53 status to the cellular response of glioma cells that were exposed to cisplatin. At a concentration of 2.5 microg/ml (which is about half the peak pharmacological blood level reached during chemotherapy), U373MG glioma cells, which had a mutant p53 gene, were more sensitive to the drug as compared to U87MG glioma cells (with normal p53). The U373MG cells responded with apoptosis while U87MG cells responded with a G2-M arrest. In U87MG cells, blocking the p53 response by antisense oligonucleotides also sensitized the cells to 2.5 microg/ml cisplatin, and shifted the cellular response from arrest to caspase 3-mediated apoptosis. A sensitive, p53-independent, mechanism for chemotherapy-induced apoptosis suggests that, in some cases, p53 abrogation by gene therapy or small molecule-based strategies could be a viable therapeutic strategy.     

Potential of adenoviral p53 gene therapy and irradiation for the treatment of malignant gliomas

We investigated the combined effects of p53 gene transfer and irradiation and its still unclear interaction mechanism in human gliomas. Four human glioma cell lines expressing mutant type p53 (U373 and A172) and wild-type p53 (D54MG and EFC-2) were transfected by adenoviral vectors bearing p53 gene at 50 multiplicity of infection. Two days after transfection, cells were irradiated (3, 6, and 9 Gy). The cytotoxicity was evaluated by clonogenic assay. The quantitative analysis of apoptosis and cell cycle analysis were performed using flow cytometry. Irradiation combined with adenoviral p53 transfection significantly increased cytotoxicity, which was additive in cell lines with wild-type p53 and more than additive in cell lines with mutant p53. The combination of two modalities increased the apoptotic population by 14% in A172 cells and 20% in D54 MG cells, which were the sum of apoptosis from each modality. Adenoviral p53 transfection increased the G1 phase fraction and concomitant decrease of radioresistant S phase fraction in A172 and D54MG cells. Our study demonstrated that p53 gene transfer combined with irradiation increased absolute cytotoxicity in human glioma cells used in this experiment. The interaction mechanism for increased cytotoxicity involved, in part, increased apoptosis and change of cell cycle profile.     

冬凌草甲素上调p53转录活性促进胶质瘤细胞凋亡的作用与分子机制探讨

目的:探讨冬凌草甲素抑制胶质瘤细胞U-87 MG、A-172活性的分子机制。方法:CCK8法检测不同浓度的冬凌草甲素对胶质瘤细胞U-87 MG、A-172活力的影响。选择20 μmol/L的冬凌草甲素分别处理胶质瘤细胞U-87 MG、A-172 0、6、12 h后,Western blotting法检测p53及其下游p21、Bax蛋白的表达情况,同时检测Cleaved PARP、Cleaved Caspase3的表达情况,实时荧光定量PCR法检测p53 mRNA水平的改变。应用p53转录活性抑制剂Pifithrin-α（PFT-α)预处理胶质瘤细胞U-87 MG 24 h后,Western blotting法检测冬凌草甲素对p53及其下游蛋白、Cleaved PARP、Cleaved Caspase3表达情况的影响。结果:冬凌草甲素对U-87 MG、A-172细胞的活性具有明显的抑制作用,冬凌草甲素作用24 h对胶质瘤细胞U-87 MG、A-172的IC50均介于20~30 μmol/L。冬凌草甲素可以上调p53及其下游p21、Bax蛋白并具有时间依赖性,而不改变p53的mRNA水平。此外,冬凌草甲素可以诱导胶质瘤细胞U-87 MG、A-172凋亡。p53转录活性抑制剂可以废除冬凌草甲素对p53及其下游p21、Bax蛋白的上调作用,同时也减弱了冬凌草甲素对胶质瘤细胞U-87 MG的促凋亡作用。结论:冬凌草甲素可能通过上调p53蛋白表达、增强其转录活性,促进胶质瘤细胞凋亡,而对胶质瘤细胞活性产生抑制效应。     

Effects of adenoviral wild-type p53 gene transfer in p53-mutated lymphoma cells

The present study assessed the role of adenoviral vector-mediated wild-type p53 gene transfer in B lymphoma cells. Deficiency of p53-mediated cell death is common in human cancer contributing to both tumorigenesis and chemoresistance. Lymphoma cells are being considered as suitable targets for gene therapy protocols. Recently, we reported an adenoviral protocol leading to highly efficient gene transfer to B lymphoma cells. All lymphoma cell lines (n=5) tested here showed mutations in the p53 gene locus. The aim of this work was to transduce lymphoma cells with the wild-type p53 gene. Using this protocol, 88% of Raji, 75% of Daudi, and 45% of OCI-Ly8-LAM53 cells were transfected with the reporter gene green fluorescent protein at a multiplicity of infection of 200. The expression of green fluorescent protein in CA46 and BL41 cells was 27% and 42%, respectively. At this multiplicity of infection, growth characteristics of lymphoma cell lines were not changed significantly. In contrast, cells transduced with wild-type p53 gene showed an inhibition of proliferation as well as an increase in apoptosis. Cell loss by apoptosis after p53 gene transfer was up to 40% as compared to transduction with an irrelevant vector. In addition, we determined the effects of DNA damage produced by the DNA topoisomerase II inhibitor etoposide on wild-type p53 transfected lymphoma cells. In Ad-p53-transfected Raji cells, treatment with the drug resulted in a marked increase of cell loss in comparison to Ad-beta-Gal-transfected cells (45% vs. 77%). Interestingly, performing cytotoxicity studies, we could show an increased sensitivity of Raji and Daudi cells against immunological effector cells. In conclusion, transduction of wild-type p53 into lymphoma cells expressing mutated p53 was efficient and led to inhibition of proliferation and increase in apoptotic rate in some cell lines dependent on p53 mutation. This protocol should have an impact on the use of lymphoma cells in cancer gene therapy protocols.     

Adenovirus-mediated transfer of p33ING1 with p53 drastically augments apoptosis in gliomas.

The p53 tumor suppressor gene is an important target for the gene therapy of cancers, and clinical trials targeting this gene have been conducted. Some cancers, however, are refractory to p53 gene therapy. Therefore, it has been combined with other therapies, including chemotherapy and radiotherapy, to enhance the cytopathic effect of p53 induction. The p33ING1 gene cooperates with p53 to block cell proliferation. In this study, we investigated whether adenovirus (Adv)-mediated coinduction of p33ING1 and p53 enhances apoptosis in glioma cells (U251 and U-373 MG), which showed no genetic alterations but low expression levels of p33ING1. Although the single infection of Adv for p33ING1 (Adv-p33) at a multiplicity of infection (MOI) of 100, or Adv for p53 controlled by myelin basic protein (MBP) promoter (Adv-MBP-p53), a glioma-specific promoter, at a MOI of 50, did not induce apoptosis in U251 and U-373 MG glioma cells; coinfection of Adv-p33 and Adv-MBP-p53 at the same MOIs induced drastically enhanced apoptosis in both cell lines. Apoptosis was not induced in NGF-treated PC-12 cells infected with a high MOI (300) of Adv-p33 nor in those coinfected with Adv-p33 (100) and Adv-MBP-p53 (50). Coinfection of Adv-p33 and Adv-MBP-p53 demonstrated morphological mitochondrial damage during the initial stage of apoptosis, which likely led to apoptotic cell death. Our results indicate that this coinfection approach can be used as a modality for the gene therapy of gliomas, sparing damage to normal tissues.     

Adenovirus-mediated overexpression of p14(ARF) induces p53 and Bax-independent apoptosis

The human INK4a gene locus encodes two structurally unrelated tumor suppressor proteins, p16(INK4a) and p14(ARF), which are frequently inactivated in human cancer. Whereas p16(INK4a) acts through engagement of the Rb-cdk4/6-cyclin D pathway, both the pro-apoptotic and cell cycle-regulatory functions of p14(ARF) were shown to be primarily dependent on the presence of functional p53. Recent reports have also implicated p14(ARF) in p53-independent mechanisms of cell cycle regulation and apoptosis induction, respectively. To further explore the pro-apoptotic function of p14(ARF) in relation to functional cellular p53, we constructed a replication-deficient adenoviral vector for overexpression of p14(ARF) (Ad-p14(ARF)). As expected, Ad-p14(ARF) efficiently induced apoptosis in p53/Rb wild-type U-2OS osteosarcoma cells at low multiplicities of infection. Interestingly, Ad-p14(ARF) also induced apoptosis in both p53-deleted SAOS-2 osteosarcoma cells and HCT116 colon cancer cells with a bi-allelic knock-out of p53 (HCT116-p53(-/-)). Similarly, adenovirus-mediated overexpression of p14(ARF) induced apoptosis in p53/Bax-mutated DU145 prostate cancer cells as well as in HCT116 cells devoid of functional Bax (HCT116-Bax(-/-)). Restoration of Bax expression by retroviral gene transfer in DU145 cells did not further enhance p14(ARF)-triggered cell death. Infection with Ad-p14(ARF) induced activation of mitochondrial permeability shift transition, caspase activation and apoptotic DNA fragmentation irrespective of the presence or absence of either Bax or functional cellular p53. Nevertheless, overexpression of the anti-apoptotic Bcl-2 homolog Bcl-x(L) markedly inhibited p14(ARF)-induced apoptosis. This may indicate that p14(ARF) triggers a so far unknown activator of mitochondrial apoptosis which can be inhibited by Bcl-2 but which acts either independently or downstream of Bax. Taken together, this report demonstrates the participation of signaling pathways apart from the p53/Mdm-2 rheostat and Bax in p14(ARF)-mediated apoptosis.