Ionizing radiation inhibits chemotherapy-induced apoptosis in cultured glioma cells: implications for combined modality therapy.

Surgical resection followed by radiation therapy is the mainstay of treatment for glioblastoma multiforme (GBM), the most aggressive of the malignant gliomas. The poor clinical response of GBM and the intrinsic radiation resistance of this tumor type have prompted clinical investigations seeking to define the role of chemotherapy in the treatment of GBM. In this study, we examined the cytotoxic response of GBM-derived cell lines to treatment with both radiation and chemotherapy. We observed that the sensitivity of glioma cells to cisplatin- and FAS-induced apoptosis was diminished by prior treatment with ionizing radiation. Radiation conferred resistance to cisplatin and FAS cytotoxicity in a dose- and time-dependent manner. Radiation diminished the cisplatin-induced cytotoxicity of malignant glioma cells but failed to alter the cisplatin susceptibility of normal primary human astrocytes. Given the role of p53 in the response of cells to irradiation, we evaluated whether p53 function affects the observed radiation-induced resistance to cisplatin. By examining isogenic cell lines differing only in p53 function, we demonstrated that radiation conferred resistance to cisplatin independently of p53. Current clinical strategies in the treatment of astrocytic tumors, which include combined modality therapy, have been empirically derived from limited clinical experience. Further understanding of the molecular determinants of apoptosis associated with combined modality therapy may guide the design of more efficacious multimodality protocols.

Fas (APO-1/CD95) signaling pathway is intact in radioresistant human glioma cells.

Radiation-induced apoptosis can be mediated through pathways initiated by either DNA damage or ceramide-induced Fas signaling. Glioblastoma multiforme is a primary brain tumor that is highly resistant to irradiation, and U-87 MG, SF126, and T98G are glioblastoma-derived cell lines that mimic this characteristic. We found that these radioresistant glioma cells are susceptible to Fas-mediated cell death induced by treatment with either anti-Fas antibody or exogenous ceramide. Fas-mediated cell death in these cell lines is p53-independent. These data demonstrate that apoptosis can be induced by ceramide and mediated through the Fas pathway in glioma cells, although high-dose ionizing radiation fails to trigger this pathway.

Cell cycle synchrony unmasks the influence of p53 function on radiosensitivity of human glioblastoma cells.

Although ionizing radiation causes DNA damage that can play a role in tumorigenesis, such irradiation is also an important modality of cancer therapy. We studied the radiation response of the U-87 MG human glioblastoma cell line and transfected derivatives in which p53 function had been inactivated. Although little effect of p53 on the radiation sensitivity of asynchronously growing cultures could be detected, inactivation of p53 resulted in a large increase in clonogenic survival when cells synchronized by mitotic selection were irradiated in early G1. The radiation dose sufficient to reduce cellular clonogenicity by 1 log in cells expressing functional p53 was 3.26 +/- 0.12 Gy, whereas a much higher dose (7.41 +/- 0.44 Gy) was required to achieve the same killing effect in cells in which p53 was inactivated. Apoptosis was excluded as a probable mechanism contributing to the radiosensitivity of these cells. Fluorescence-activated cell sorter analysis, continuous labeling with tritiated thymidine, and time-lapse videomicroscopy documented the first example of a prolonged p53-dependent G1 arrest induced by ionizing radiation during the first postirradiation cell cycle of tumor cells, suggesting a role for G1 arrest in determining the sensitivity of these cells to irradiation.

p27Kip1 is required for PTEN-induced G1 growth arrest.

The tumor suppressor PTEN is one of the most commonly inactivated genes in human cancer. Glioblastoma multiforme cells harboring mutant PTEN have abnormally high levels of 3' phosphoinositides and elevated protein kinase B activity. Expression of wild-type PTEN in glioma cells, containing endogenous mutant PTEN, reduces 3' phosphoinositides levels, inhibits PKB activity, and induces G1 cell cycle arrest. We investigated the mechanism of the PTEN-induced growth arrest in glioma cell lines. Expression of PTEN is associated with increased expression of p27Kip1, decreased expression of cyclins A and D3, inhibition of cdk2 activity, and dephosphorylation of pRb. Inactivation of p53, by the human papilloma virus E6 oncoprotein, does not prevent PTEN-induced G1 arrest, implying that p53 is not required for G1 arrest. In contrast, p27Kip1 antisense oligonucleotides abrogated the growth arrest induced by PTEN. Furthermore, blocking p27Kip1 expression prevented the PTEN-induced reduction of cyclin-dependent kinase 2 activity, indicating that p27Kip1 functions upstream of cyclin-dependent kinase 2 in the PTEN regulatory cascade. These results implicate p27Kip1 as a critical mediator of PTEN-induced G1 arrest.

Transcriptional activation of TRADD mediates p53-independent radiation-induced apoptosis of glioma cells.

Survival of patients with Glioblastoma Multiforme (GM), a highly malignant brain tumor, remains poor despite concerted efforts to improve therapy. The median survival of patients with GM has remained approximately 1 year regardless of the therapeutic approach. Since radiation therapy is the most effective adjuvant therapy for GM and nearly half of GM tumors harbor p53 mutations, we sought to identify genes that mediate p53-independent apoptosis of GM cells in response to ionizing radiation. Using broad-scale gene expression analysis we found that following radiation treatment, TRADD expression was induced in a uniquely radiosensitive GM cell line but not in radioresistant GM cell lines. TRADD over-expression killed GM cells and activated NF-kappa B. We found that blocking the TRADD-mediated pathway using a dominant-negative mutant of FADD (FADD-DN) enhanced radiation resistance of GM cells, as reflected in both susceptibility to apoptosis and clonogenic survival following irradiation. Conversely, stable expression of exogenous TRADD enhanced radiation-induced apoptosis of GM cell lines, reflecting the biological significance of TRADD regulation in p53-independent apoptosis. These findings generate interest in utilizing TRADD in gene therapy for GM tumors, particularly in light of its dual function of directly inducing rapid apoptosis and sensitizing GM cells to standard anti-neoplastic therapy.

Inactivation of p53 is associated with decreased levels of radiation-induced apoptosis in medulloblastoma cell lines.

Radiation is the primary therapeutic modality for children with medulloblastoma, a pediatric brain tumour. We examined the response of four medulloblastoma cell lines to ionising radiation. Our evaluation utilising flow cytometry, morphological analysis and terminal deoxynucleotidyl transferase assays demonstrated that medulloblastoma cells undergo radiation-induced apoptosis. p53 mediates radiation-induced apoptosis in many cell types, and p53 mutations have been associated with increased resistance to ionising radiation. p53 mutations are rare in medulloblastoma. We found that wildtype p53 is required for high levels of apoptosis in medulloblastoma, and cell lines in which p53 had been inactivated by mutation had very low levels of apoptosis. Inactivation of endogenous wildtype p53 in medulloblastoma cells by introduction of a dominant negative mutant of p53 decreased the level of radiation-induced apoptosis. Our results suggest that the sensitivity of medulloblastoma to irradiation involves p53-mediated apoptosis and that p53 gene status may be a predictor of response to radiation therapy.

Functional and clinical outcomes of limb-sparing therapy for pediatric extremity sarcomas.

PURPOSE: To determine the clinical and functional outcomes of children undergoing limb-sparing therapy for extremity sarcomas. METHODS AND MATERIALS: We retrospectively reviewed 30 patients, age < or = 21 years, who were treated between l979 and l998 with external beam radiotherapy as a component of limb-sparing therapy for primary sarcomas of the extremity at UCSF. Included were patients for whom complete follow-up and functional outcome assessments were available. We assessed the patterns of failure, overall survival, disease-free survival, local control, and limb function. RESULTS: At a median follow-up of 3 years, 12 of the 30 patients recurred: 3 locally, 8 distantly, and 1 with synchronous local and distant disease as site of first progression. Eighteen patients were alive with no evidence of disease. The median overall survival was 10 years, with a median disease-free survival of 8 years. Functional outcome assessment revealed 15 patients retained excellent, 12 good, 1 fair, and 2 poor limb function. CONCLUSION: In pediatric patients receiving limb-sparing therapy, 90% maintained excellent or good limb function without compromising survival, demonstrating the validity of limb preservation in children with extremity sarcomas.

p53 function influences the effect of fractionated radiotherapy on glioblastoma tumors.

PURPOSE: Glioblastoma multiforme brain tumors (GM) are treated with a spectrum of fractionation regimens based on the clinical and anatomical characteristics of the tumor but rarely based on the molecular characteristics of the individual neoplasm. This study tests the hypothesis that the response of cell lines derived from GM to fractionated radiotherapy depends on the function of wild-type p53 (wt p53), a tumor suppressor gene frequently mutated in GM tumors. METHODS & MATERIALS: Isogenic derivatives of glioblastoma cells differing only in p53 function were prepared using a retroviral vector expressing a dominant negative mutant of p53 (mt p53). Radiation survival in vitro was quantitated using linear quadratic and repair-saturation mathematical models. Apoptosis was assayed by a terminal deoxynucleotide transferase-labeling technique and chromatin morphology. RESULTS: We have previously reported the generation of isogenic GM cell lines differing only in p53 function. U87-175.4, lacking wt p53 function, had a significantly lower alpha/beta value than U87-LUX.8, expressing functional wt p53, leading us to hypothesize that fractionated irradiation would preferentially spare GM cells harboring mt p53 compared with those expressing functional, wt p53. Survival curves following either 2.0 Gy or 3.5 Gy/fraction demonstrated that lack of functional wt p53 was associated with resistance to fractionated irradiation. Radiation-induced apoptosis could not account for the observed differences in clonogenic survival. Rather, our data suggested that a deficit in the G1-checkpoint contributed to increased resistance to fractionated irradiation of cells expressing mutant p53. CONCLUSIONS: The effect of fractionated radiotherapy in GM may depend on the function of the tumor suppressor gene p53. A potential clinical consequence of these findings is that hyperfractionation regimens may provide a therapeutic advantage specifically for tumors expressing wt p53 whereas a radiotherapy course of fewer, larger fractions may be appropriate for the treatment of tumors carrying p53 mutations. Further studies are needed to confirm our proposal that the p53 status of GM tumors can be used to guide our choice of fractionation schemes.

p53-dependent G1 arrest and p53-independent apoptosis influence the radiobiologic response of glioblastoma.

PURPOSE: Loss of the p53 tumor suppressor gene has been associated with tumor progression, disease relapse, poor response to antineoplastic therapy, and poor prognosis in many malignancies. We have investigated the contribution of p53-mediated radiation-induced apoptosis and G1 arrest to the well described radiation resistance of glioblastoma multiforme (GM) cells. METHODS AND MATERIALS: Radiation survival in vitro was quantitated using linear quadratic and repair-saturation mathematical models. Isogenic derivatives of glioblastoma cells differing only in their p53 status were generated using a retroviral vector expressing a dominant negative mutant of p53. Radiation-induced apoptosis was assayed by Fluorescence-activated cell sorter (FACS) analysis, terminal deoxynucleotide transferase labeling technique, and chromatin morphology. Cells were synchronized in early G1 phase and mitotic and labeling indices were measured. RESULTS: Radiation-induced apoptosis of GM cells was independent of functional wild-type p53 (wt p53). Decreased susceptibility to radiation-induced apoptosis was associated with lower alpha values characterizing the shoulder of the clonogenic radiation survival curve. Using isogenic GM cells differing only in their p53 activity, we found that a p53-mediated function, radiation-induced G1 arrest, could also influence the value of alpha and clonogenic radiation resistance. Inactivation of wt p53 function by a dominant negative mutant of p53 resulted in a significantly diminished alpha value with no alteration in cellular susceptibility to radiation-induced apoptosis. The clonal derivative U87-LUX.8 expressing a functional wt p53 had an alpha (Gy-1) value of 0.609, whereas the isogenic clonal derivative U87-175.4 lacking wt p53 function had an alpha (Gy-1) value of 0.175. CONCLUSION: We conclude that two distinct cellular responses to radiation, p53-independent apoptosis and p53-dependent G1-arrest, influence radiobiological parameters that characterize the radiation response of glioblastoma cells. Further understanding of the molecular basis of GM radiation resistance will lead to improvement in existing therapeutic modalities and to the development of novel treatment approaches.

Intraoperative radiation therapy for high-risk pediatric neuroblastoma.

PURPOSE: To evaluate the efficacy of intraoperative radiation therapy (IORT) in the treatment of high-risk pediatric neuroblastoma. METHODS AND MATERIALS: Between 1986 and 1998, 23 children received IORT for pediatric neuroblastoma. Electron beam energies ranged from 4 MeV to 16 MeV and median dose was 10 Gy (7-16 Gy). RESULTS: Twenty-one of 23 patients were classified as high-risk. A gross total resection (GTR) was achieved in 18 patients, of whom 6 experienced disease recurrence, 2 of which included a locoregional relapse as a component of failure. Fourteen of 18 patients receiving IORT after a GTR are disease-free survivors. A second subset of 5 patients had a subtotal resection (STR), with gross residual disease remaining after surgery. All 5 patients recurred locally, and all died of their disease. IORT was extremely well-tolerated in our cohort. Surgical resection and IORT resulted in the narrowing of the abdominal aorta and an atrophic kidney in 1 patient. CONCLUSIONS: For high-risk neuroblastoma patients, IORT as the only radiotherapy to the primary, produced excellent local control after a GTR. However, IORT as the sole radiotherapy to the primary was inadequate for patients with extensive adenopathy or an STR. In this setting, we are exploring the use of IORT as a boost in conjunction with external beam radiation therapy.

Cytogenetic damage and the radiation-induced G1-phase checkpoint.

It is proposed that genomic integrity is preserved after DNA damage in a variety of ways. X irradiation induces a p53-dependent G1-phase cell cycle checkpoint which putatively allows time for repair of DNA damage. The p53 protein is also involved in the initiation of apoptosis after radiation-induced DNA damage, presumably leading to the elimination of lethally damaged cells from the irradiated population. To test the hypothesis that repair occurs in the additional time provided by the activation of the G1-phase checkpoint, we investigated whether the presence of a G1-phase arrest modified the frequency and type of chromosomal rearrangements at the first mitosis after irradiation. Isogenic cell lines derived from the same human glioma cell line, but differing in p53 status, were used. Purified G1-phase cells, isolated by centrifugal elutriation and X-irradiated, were studied. The wild-type p53 cell line demonstrated a dose-dependent arrest during G1 phase, as determined by flow cytometry. These cells remained in G1-phase as long as 48 h after irradiation. Cells expressing a dominant-negative p53 mutation accumulated to a much lesser extent in G1 phase after irradiation. Cells lacking the G1-phase checkpoint showed increased survival at all radiation doses. There were no significant differences in the type or frequency of total chromosomal aberrations in mitotic cells from either cell line after 1,2,4 or 6 Gy X rays, as measured by conventional cytogenetic analysis. There was an increase, however, in the number of reciprocal translocations in mitotic cells with mutant p53 (lacking a G1-phase checkpoint), as measured by fluorescence in situ hybridization with a chromosome 4-specific DNA library, but only after 6 Gy. The results suggest that the presence of a well-defined p53-dependent G1-phase arrest does not reduce chromosomal aberrations caused by low doses of ionizing radiation markedly, but may reduce the overall degree of survival by triggering other G1-phase events.

P53-independent apoptosis: a mechanism of radiation-induced cell death of glioblastoma cells.

PURPOSE: Radiation therapy, though routinely used in the treatment of patients with glioblastoma multiforme, is of limited efficacy in extending patients' lives. In this study we investigated the mechanism by which ionizing radiation causes death of glioblastoma cells in the hope of ultimately altering the intrinsic radioresistance of glioblastoma tumors. METHODS: Radiation survival in vitro was quantitated using linear quadratic and repair-saturation mathematical models. Radiation-induced apoptosis was assayed by fluorescence-activated cell sorter analysis, terminal deoxynucleotide transferase labeling technique, and chromatin morphology. Cellular distribution within the cell cycle was quantitated by dual labeling with propidium iodide and bromodeoxyuridine. RESULTS: We examined whether in vitro clonogenic radioresistance of glioblastoma would reflect their susceptibility to radiation-induced apoptosis and their ability to undergo a G1 arrest--two cellular functions associated with wild-type p53 expression. We demonstrated that apoptosis contributed to the cytocidal effect of ionizing radiation on glioblastoma cells. The apoptosis observed in glioblastoma cell lines occurred in the absence of wild-type p53 expression. We identified a glioblastoma cell line expressing wild-type p53 and found that it did not exhibit radiation-induced apoptosis but rather underwent a prolonged G1 arrest not observed in any glioblastoma cell line lacking wild-type p53 expression. CONCLUSION: Apoptosis is an important component of the lethal effect of ionizing radiation on glioblastoma cells and does not require wild-type p53 expression. Glioblastoma expressing wild-type p53 exhibited no apoptosis, even after high radiation doses, but rather underwent a prolonged G1 arrest. The observation of p53-independent apoptosis and p53-dependent Gi arrest in glioblastoma cells have important radiobiologic and clinical implications.

Inhibition of apoptosis by the retinoblastoma gene product.

Tissue homeostasis and the prevention of neoplasia require regulatory co-ordination between cellular proliferation and apoptosis. Several cellular proteins, including c-myc and E2F, as well as viral proteins such as E1A, have dual functions as positive regulators of apoptosis and proliferation. The product of the retinoblastoma tumor suppressor gene, pRb, binds these proteins and is known to function in growth suppression. To examine whether pRb may function as a negative regulator of both proliferation and apoptosis, we analyzed apoptosis induced in transfected derivatives of the human osteosarcoma cell line SAOS-2. Ionizing radiation induced apoptosis in a time- and dose-dependent manner in SAOS-2 cells, which lack pRb expression. In both a transient and stable transfection assay, SAOS-2 derivatives expressing wild-type (wt) pRb exhibited increased viability and decreased apoptosis following treatment at a variety of radiation doses. Expression in SAOS-2 of a mutant pRb that fails to complex with several known binding partners of pRb, including E1A and E2F, did not protect SAOS-2 cells from apoptosis. Radiation exposure induced a G2 arrest in SAOS-2 and in derivatives expressing pRb. Inhibition of DNA synthesis and cell cycle progression by aphidicolin treatment failed to protect SAOS-2 cells or pRb-expressing isolates from undergoing apoptosis. Our data document a novel function for pRb in suppressing apoptosis and suggest that several proteins shown to induce apoptosis, including E1A, E2F and c-myc, may do so by interfering with the protective function of pRb.