Lily Hu

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.

Cytotoxicity and cell-cycle effects of paclitaxel when used as a single agent and in combination with ionizing radiation☆

PURPOSE This study aimed to determine the extent of paclitaxel-induced cytotoxicity and cell-cycle perturbations when used alone and in combination with radiation in human glioma cells. METHODS AND MATERIALS The effect of paclitaxel alone on three human glioma cells lines--SF-126, U-87 MG, and U-251 MG--was assessed after 24, 48, 72, or 96 h treatment. For experiments in combination with radiation, cells were exposed to either a long (48-h) or short (8-h) duration of paclitaxel treatment prior to irradiation. Cell survival was determined by clonogenic assay. Cell cycle perturbations were assessed by using flow cytometry to measure the proportion of cells in G1, S, and G2/M phases. RESULTS When cells were treated with paclitaxel alone for > or = 24 h, cytotoxicity increased up to a threshold dose, after which it plateaued. When treatment duration was < or = 24 h, cytotoxicity was appreciably greater in U-251 MG cells than in SF-126 and U-87 MG cells. After 24 h of paclitaxel treatment, cells in plateau phase growth had increased survival compared to cells in log phase growth. In contrast, after 8 h paclitaxel treatment, mitotic cells had reduced survival compared to cells from an asynchronous population. Cell-cycle perturbations were consistent with the presence of a mitotic block after paclitaxel treatment, although changes in other cell-cycle phase fractions varied among cell lines. For experiments in combination with radiation, cytotoxicity was increased when cells were irradiated after 48 h of paclitaxel treatment but not after 8 h of treatment. CONCLUSION The duration of paclitaxel treatment and the location of cells in the cell cycle modify the degree of radiation cytotoxicity. The mechanisms of paclitaxel cytotoxicity are likely to be multifactorial because varying effects are seen in different cell lines. Furthermore, it is clear that simply increasing the number of cells in G2/M is insufficient in itself to increase the response of cells to radiation.

Functionality of hypoxia-induced BAX expression in a human glioblastoma xenograft model.

The effectiveness of radiation therapy for human brain tumors is limited by the presence of radiation-resistant hypoxic cells. In order to improve patient outcomes, therapeutic methods that increase hypoxic cell killing must be developed. To investigate the possibility of using the hypoxic tumor microenvironment itself as a target for gene therapy, we stably transfected U-251 MG human glioblastoma cells with constructs containing the suicide gene Bax under the regulation of a nine-copy concatemer of hypoxia responsive elements (HREs). Previously, we demonstrated that the expression of BAX protein under anoxic conditions in transfected U-251 MG clones leads to increased cell killing in vitro. Our recent studies revealed that HIF-1α induction under anoxic conditions occurs prior to the increase in BAX expression, thereby implicating HIF-1 induction as the basis of BAX upregulation. To test the effect of BAX-mediated cell killing in vivo, we implanted five stably transfected clones subcutaneously into the flanks of athymic mice. Compared to nontransfected controls, tumor growth in four of five clones was significantly retarded. Histopathological analysis demonstrated decreased hypoxic fractions and increased amounts of apoptosis in clone-derived tumors. These results suggest that the tumor microenvironment is sufficiently hypoxic to trigger HRE-mediated cell killing via the BAX apoptotic pathway.

Development of a hypoxia-inducible cytosine deaminase expression vector for gene-directed prodrug cancer therapy

One important feature of human solid tumors is the presence of a hypoxic microenvironment. Under hypoxia, genes that contain a hypoxia-response element (HRE) can be activated by the binding of hypoxia-inducible factor-1. To reach the goal of selectively killing tumor cells in a hypoxic microenvironment using a gene therapy approach, we developed a cytosine deaminase (CD) gene construct (pH9YCD2) that contains an HRE gene enhancer. CD is an enzyme that catalyzes the conversion of noncytotoxic 5-fluorocytosine (5-FC) to the cytotoxic and radiosensitizing drug 5-fluorouracil (5-FU). Yeast CD was cloned into an SV40 promoter-based mammalian expression vector, and an HRE enhancer was inserted in front of the promoter. Human glioblastoma U-87 MG cells were transfected with pH9YCD2. Western blots revealed that CD was strongly expressed under hypoxic conditions (0.3–1% O2), whereas only minor CD expression was seen under normoxic conditions. To confirm that the expressed CD enzyme retains catalytic activity, we performed a 5-FC/5-FU-conversion assay in which 5-FC was incubated with the lysates of pH9YCD2-transfected cells. The percentage of conversion from 5-FC to 5-FU was 63% under hypoxia versus 13% under normoxia. In vitro, cell viability and colony-forming efficiency assays demonstrated that the gene construct was able to significantly kill glioblastoma cells in a hypoxia-dependent manner. In addition, 5-FC treatment of hypoxic pH9YCD2-transfected cells produced a marked bystander effect, which could be a distinct advantage for gene therapy. If this construct exhibits antitumor efficacy in vivo, it may have promise as an antitumor agent in humans.

Hypoxia-Induced BAX Overexpression and Radiation Killing of Hypoxic Glioblastoma Cells

Abstract Chen, J. K., Hu, L. J., Wang, J., Lamborn, K. R., Kong, E. L. and Deen, D. F. Hypoxia-Induced BAX Overexpression and Radiation Killing of Hypoxic Glioblastoma Cells. Radiat. Res. 163, 644–653 (2005). One major challenge in treating glioblastoma multiforme (GBM) has been the presence of radiation-resistant hypoxic cells. The pro-apoptosis protein BAX has been reported to be a possible radiation sensitizer of cancer cells; however, to our knowledge, no studies have reported on the effects of BAX on radiation sensitivity under hypoxic conditions. Therefore, in this study, we specifically overexpressed murine Bax in hypoxic cells in an attempt to enhance radiation-induced cell killing. We have previously stably transfected U-251 MG and U-87 MG human GBM cells with constructs containing murine Bax under the regulation of nine copies of hypoxia-responsive elements (HREs). During hypoxia, the transcriptional complex hypoxia-inducible factor 1 (HIF1) forms and binds to HRE; this binding facilitates the transcription of downstream genes. In the experiments reported here, two protocols were used. In the first protocol, parent and clone cells were exposed to graded doses of X rays under hypoxic conditions, left hypoxic for 0, 4, 16 or 24 h, and then assayed for clonogenic cell survival. In the second protocol, cells were incubated under hypoxic conditions for 20 h, irradiated with graded doses under hypoxia, then left in hypoxic conditions for 4 h before being assayed for clonogenic cell survival. Western blots showed that we had successfully increased Bax expression in both U-251 MG and U-87 MG Bax clone cells after 16 h of hypoxic incubation, yet dose–response curves showed no difference in radiation-induced cell killing between control non-Bax-expressing pNeo clone cells and the U-251 MG Bax clone cells using either protocol. In U-87 MG cells, the first protocol showed no difference in radiation response between control pNeo clone cells and the Bax clone, similar to the results obtained in U-251 cells. However, the second protocol revealed that Bax overexpression did render these cells more sensitive to radiation under hypoxic conditions. Therefore, we conclude that whether Bax is a radiation enhancer under hypoxia not only is cell line-dependent but also depends on when the Bax overexpression occurs.

Radiopotentiation of human brain tumor cells by sodium phenylacetate.

Phenylacetate (PA) inhibits the growth of tumor cells in vitro and in vivo and shows promise as a relatively nontoxic agent for cancer treatment. A recent report shows that prolonged exposure of cells to low concentrations of PA can enhance the radiation response of brain tumor cells in vitro, opening up the possibility of using this drug to improve the radiation therapy of brain tumor patients. We investigated the cytotoxicity produced by sodium phenylacetate (NaPA) alone and in combination with X-rays in SF-767 human glioblastoma cells and in two medulloblastoma cell lines, Masden and Daoy. Exposure of all three cell lines to relatively low concentrations of NaPA for up to 5 days did not enhance the subsequent cell killing produced by X-irradiation. However, enhanced cell killing was achieved by exposing either oxic or hypoxic cells to relatively high drug concentrations ( > 50-70 mM) for 1 h immediately before X-irradiation. Because central nervous system toxicity can occur in humans at serum concentrations of approximately 6 mM PA, translation of these results into clinical trials will likely require local drug-delivery strategies to achieve drug concentrations that can enhance the radiation response. The safety of such an approach with this drug has not been demonstrated.

Chromosome transfer experiments link regions on chromosome 7 to radiation resistance in human glioblastoma multiforme

Glioblastoma multiforme (GM) is the most lethal form of brain tumor, with a median survival of approximately 1 year. Treatment options are limited. Radiation therapy is a common form of treatment, but many tumors are resistant. In earlier studies, we found that gain of chromosome 7 is associated with radiation resistance in human primary GM. In this study, we extend that result to a model system in which we transferred chromosome 7 to recipient cells and confirmed radiation resistance as a function of chromosome 7 gain. We identified three candidate regions on chromosome 7 that conferred radiation resistance in our model system.

Radiopotentiation of human brain tumor cells by the spermine analog N1,N14-bis(ethyl)homospermine.

PURPOSE To determine whether the cytotoxicity produced by radiation can be increased by the spermine analog N1,N14-bis(ethyl)homospermine (BE-4-4-4). METHODS AND MATERIALS Two human tumor cell lines, SF-126 and U-251 MG, were either treated with 0.1 or 0.4 microM BE-4-4-4 for 3 or 4 days, or with 0.2 microM BE-4-4-4 for 4 days. At the end of BE-4-4-4 treatment, cells were irradiated and assayed immediately. Polyamine levels, cell survival, and cell number were determined. RESULTS In SF-126 cells, treatment with 0.2 microM BE-4-4-4 for 4 days killed about 50% of the cells and also increased the cytotoxicity of radiation. The dose enhancement ratio was approximately 1.3:1.5, which is similar to that reported for alpha-difluoromethylornithine. Polyamine levels were partially depleted, and growth was inhibited to about 60% of control levels. Pretreatment of cells with either 0.1 or 0.4 microM BE-4-4-4 for 3 or 4 days produced less of an increase in radiation-induced cytotoxicity, even though these exposures killed 30-40% or 60-90% of the cells, respectively. Similar treatment with 0.1-0.4 microM BE-4-4-4 in U-251 MG cells had minimal effects on cytotoxicity and growth inhibition, while treatment with 1.0 microM and 2.0 microM BE-4-4-4 for 4 days produced more than a 50% depletion in polyamine levels and partial inhibition in growth, but failed to demonstrate radiopotentiation. CONCLUSION The cytotoxic polyamine analog BE-4-4-4 can increase the cytotoxicity caused by radiation in at least one cell line. The amount of potentiation depends on the concentration of the analog, with the most occurring at the intermediate concentration. Because we did not observe potentiation in both cell lines, and because of the dose dependence seen in SF-126 cells, the clinical efficacy produced by combined BE-4-4-4 and radiation protocols may be limited.