Joan Allalunis-Turner

Intrinsic radiation sensitivity may not be the major determinant of the poor clinical outcome of glioblastoma multiforme

Abstract Purpose: Many radiobiologic mechanisms may contribute to the clinical radiation resistance of Glioblastoma Multiforme. One of them is considered to be an unusually low intrinsic radiation sensitivity. This is a collaborative study between three laboratories to evaluate the intrinsic radiation sensitivity of 85 cell lines derived from human malignant gliomas as the major cause of the poor clinical results of radiation treatment to these tumors. Methods and Materials: Fifty-one cell lines were early passage. The distribution by histologic type was: 58 glioblastoma, 17 anaplastic astrocytoma, six oligodendroglioma and four astrocytoma grade 2. The intrinsic radiation sensitivity will be expressed by the surviving fraction at 2 Gy (SF 2 ). The SF 2 has been determined for single dose irradiation for cell lines on exponential phase, under aerobic conditions, growing on plastic. The patient age, Karnofski Status, histological grade, survival, dose of irradiation for 50 patients are investigated for correlation with SF 2 of the corresponding newly established cell lines. Results: The mean SF 2 of the 85 cell lines was 0.46 (0.12 – 0.87). The mean SF 2 by histologic type was 0.50, 0.34, 0.54 and 0.38 for glioblastoma, anaplastic astrocytoma, oligodendroglioma and astrocytoma grade 2 cell lines, respectively. No correlation was found between SF 2 and the patient age or Karnofski status. The difference in SF 2 between the 58 glioblastoma and 17 anaplastic astrocytoma cell lines was significant p = 0.002. The difference in actuarial survival between glioblastoma and anaplastic astrocytoma patients was borderline of significance ( p = 0.08). The difference in SF 2 of cell lines derived from these two groups of patients was of borderline significance ( p = 0.08). The difference in radiation sensitivity for anaplastic astrocytoma and glioblastoma cell lines was clearly reflected in the difference in survival for the two groups of patients from where the cell lines were derived. However, no correlation was found between SF 2 and survival within each grade. In a multivariate analysis the age, grade and Karnofski status were found to be significant prognostic values for survival with a p values of 0.032, 0.03 and 0.038, respectively, however, the In SF 2 was not significant ( p = 0.40). The mean SF 2 of the 6 oligodendroglioma cell lines (0.54) was comparable to that of glioblastoma multiforme (0.50). The high SF 2 for oligodendroglioma does not accord with the much better clinical outcome of these tumors. Conclusions: These data on 85 malignant glioma cell lines show a very broad distribution of SFZ values for irradiation in vitro . SF 2 reflected the difference in sensitivity between AA (Grade 3) and GBM (Grade 4). This may suggest that the parameter SF 2 is useful to discriminate between the sensitivity of different grades or types of histology in vitro . However, SF 2 was not a predictor of the clinical outcome on individual basis for malignant gliomas. The in vitro studies will need to be supplemented by physiologic characterization of the tumors in vivo . Such conclusions would limit the predictive value of current radiation sensitivity assays based on in vitro dose-survival measurement for at least high grade malignant gliomas.

Lack of correlation between ATM protein expression and tumour cell radiosensitivity

PURPOSE Cells derived from individuals in which the ataxia telangiectasia (ATM) gene is mutated are hypersensitive to ionizing radiation. Whether differences in ATM protein levels exist among human malignant glioma cell lines and whether such differences are correlated with cellular radiosensitivity were determined. MATERIALS AND METHODS Polyclonal antibodies were raised to separate regions of the ATM protein. ATM protein expression in human malignant glioma cell lines, SV40 transformed normal human fibroblasts and SV40 transformed AT fibroblasts was analysed by Western blotting. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to assess the presence of ATM transcript. RESULTS While ATM protein was detected in all cell extracts, significant differences in the level of expression were observed. There was no apparent correlation between cellular radiosensitivity and differences in ATM protein levels in these human glioma cells. Extremely low levels of ATM protein were observed in M059J cells, which provide the only example of DNA-dependent protein kinase (DNA-PKcs) deficiency in a cell line of human origin. CONCLUSIONS Variations in the levels of ATM protein are insufficient to explain the differences in cellular radiosensitivity observed in a panel of human malignant glioma cell lines.

The cytotoxicity of γ-secretase inhibitor I to breast cancer cells is mediated by proteasome inhibition, not by γ-secretase inhibition

IntroductionNotch is a family of transmembrane protein receptors whose activation requires proteolytic cleavage by γ-secretase. Since aberrant Notch signaling can induce mammary carcinomas in transgenic mice and high expression levels of Notch receptors and ligands correlates with overall poor clinical outcomes, inhibiting γ-secretase with small molecules may be a promising approach for breast cancer treatment. Consistent with this hypothesis, two recent papers reported that γ-secretase inhibitor I (GSI I), Z-LLNle-CHO, is toxic to breast cancer cells both in vitro and in vivo. In this study, we compared the activity and cytotoxicity of Z-LLNle-CHO to that of two highly specific GSIs, DAPT and L-685,458 and three structurally unrelated proteasome inhibitors, MG132, lactacystin, and bortezomib in order to study the mechanism underlying the cytotoxicity of Z-LLNle-CHO in breast cancer cells.MethodsThree estrogen receptor (ER) positive cell lines, MCF-7, BT474, and T47D, and three ER negative cell lines, SKBR3, MDA-MB-231, and MDA-MB-468, were used in this study. Both SKBR3 and BT474 cells also overexpress HER2/neu. Cytotoxicity was measured by using an MTS cell viability/proliferation assay. Inhibition of γ-secretase activity was measured by both immunoblotting and immunofluorescent microscopy in order to detect active Notch1 intracellular domain. Proteasome inhibition was determined by using a cell-based proteasome activity assay kit, by immunoblotting to detect accumulation of polyubiquitylated protein, and by immunofluorescent microscopy to detect redistribution of cellular ubiquitin.ResultsWe found that blocking γ-secretase activity by DAPT and L-685,458 had no effect on the survival and proliferation of a panel of six breast cancer cell lines while Z-LLNle-CHO could cause cell death even at concentrations that inhibited γ-secretase activity less efficiently. Furthermore, we observed that Z-LLNle-CHO could inhibit proteasome activity and the relative cellular sensitivity of these six breast cancer cell lines to Z-LLNle-CHO was the same as observed for three proteasome inhibitors. Finally, we found that the cell killing effect of Z-LLNle-CHO could be reversed by a chemical that restored the proteasome activity.ConclusionsWe conclude that the cytotoxicity of Z-LLNle-CHO in breast cancer cells is mediated by proteasome inhibition, not by γ-secretase inhibition.

Hypoxic regulation of cytoglobin and neuroglobin expression in human normal and tumor tissues

BackgroundCytoglobin (Cygb) and neuroglobin (Ngb) are recently identified globin molecules that are expressed in vertebrate tissues. Upregulation of Cygb and Ngb under hypoxic and/or ischemic conditions in vitro and in vivo increases cell survival, suggesting possible protective roles through prevention of oxidative damage. We have previously shown that Ngb is expressed in human glioblastoma multiforme (GBM) cell lines, and that expression of its transcript and protein can be significantly increased after exposure to physiologically relevant levels of hypoxia. In this study, we extended this work to determine whether Cygb is also expressed in GBM cells, and whether its expression is enhanced under hypoxic conditions. We also compared Cygb and Ngb expression in human primary tumor specimens, including brain tumors, as well as in human normal tissues. Immunoreactivity of carbonic anhydrase IX (CA IX), a hypoxia-inducible metalloenzyme that catalyzes the hydration of CO2 to bicarbonate, was used as an endogenous marker of hypoxia.ResultsCygb transcript and protein were expressed in human GBM cells, and this expression was significantly increased in most cells following 48 h incubation under hypoxia. We also showed that Cygb and Ngb are expressed in both normal tissues and human primary cancers, including GBM. Among normal tissues, Cygb and Ngb expression was restricted to distinct cell types and was especially prominent in ductal cells. Additionally, certain normal organs (e.g. stomach fundus, small bowel) showed distinct regional co-localization of Ngb, Cygb and CA IX. In most tumors, Ngb immunoreactivity was significantly greater than that of Cygb. In keeping with previous in vitro results, tumor regions that were positively stained for CA IX were also positive for Ngb and Cygb, suggesting that hypoxic upregulation of Ngb and Cygb also occurs in vivo.ConclusionsOur finding of hypoxic up-regulation of Cygb/Ngb in GBM cell lines and human tumor tissues suggests that these globin molecules may be part of the repertoire of defense mechanisms that allow cancer cells to survive in hypoxic microenvironments.

Mutation in mitochondrial complex I ND6 subunit is associated with defective response to hypoxia in human glioma cells

BackgroundHypoxia-tolerant human glioma cells reduce oxygen consumption rate in response to oxygen deficit, a defense mechanism that contributes to survival under moderately hypoxic conditions. In contrast, hypoxia-sensitive cells lack this ability. As it has been previously shown that hypoxia-tolerant (M006x, M006xLo, M059K) and -sensitive (M010b) glioma cells express differences in mitochondrial function, we investigated whether mitochondrial DNA-encoded mutations are associated with differences in the initial response to oxygen deficit.ResultsThe mitochondrial genome was sequenced and 23 mtDNA alterations were identified, one of which was an unreported mutation (T-C transition in base pair 14634) in the hypoxia-sensitive cell line, M010b, that resulted in a single amino acid change in the gene encoding the ND6 subunit of NADH:ubiquinone oxidoreductase (Complex I). The T14634C mutation did not abrogate ND6 protein expression, however, M010b cells were more resistant to rotenone, an agent used to screen for Complex I mutations, and adriamycin, an agent activated by redox cycling. The specific function of mtDNA-encoded, membrane-embedded Complex I ND subunits is not known at present. Current models suggest that the transmembrane arm of Complex I may serve as a conformationally driven proton channel. As cellular respiration is regulated, in part, by proton flux, we used homology-based modeling and computational molecular biology to predict the 3D structure of the wild type and mutated ND6 proteins. These models predict that the T14634C mutation alters the structure and orientation of the trans-membrane helices of the ND6 protein.ConclusionComplex I ND subunits are mutational hot spots in tumor mtDNA. Genetic changes that alter Complex I structure and function may alter a cells ability to respond to oxygen deficit and consolidate hypoxia rescue mechanisms, and may contribute to resistance to chemotherapeutic agents that require redox cycling for activation.

Notch signaling as a therapeutic target for breast cancer treatment

Aberrant Notch signaling can induce mammary gland carcinoma in transgenic mice, and high expressions of Notch receptors and ligands have been linked to poor clinical outcomes in human patients with breast cancer. This suggests that inhibition of Notch signaling may be beneficial for breast cancer treatment. In this review, we critically evaluate the evidence that supports or challenges the hypothesis that inhibition of Notch signaling would be advantageous in breast cancer management. We find that there are many remaining uncertainties that must be addressed experimentally if we are to exploit inhibition of Notch signaling as a treatment approach in breast cancer. Nonetheless, Notch inhibition, in combination with other therapies, is a promising avenue for future management of breast cancer. Furthermore, since aberrant Notch4 activity can induce mammary gland carcinoma in the absence of RBPjκ, a better understanding of the components of RBPjκ-independent oncogenic Notch signaling pathways and their contribution to Notch-induced tumorigenesis would facilitate the deployment of Notch inhibition strategies for effective treatment of breast cancer.

Knockdown of Cytoglobin Expression Sensitizes Human Glioma Cells to Radiation and Oxidative Stress

Cytoglobin is a recently identified vertebrate globin whose functions include scavenging reactive oxygen and nitrosative species. In tumor cells, CYGB may function as a tumor suppressor gene. Here we show that knockdown of cytoglobin expression can sensitize human glioma cells to oxidative stress induced by chemical inhibitors of the electron transport chain and as well can increase cellular radiosensitivity. When treated with antimycin A, an inhibitor of the mitochondrial electron transport chain, cytoglobin-deficient cells showed significantly higher H2O2 levels, whereas H2O2 levels were significantly reduced in cytoglobin-overexpressing cells. In addition, cytoglobin knockdown significantly decreased the doubling time of glioma cell lines, consistent with a putative tumor suppressor function. These finding suggest that modulating cytoglobin levels may be a promising treatment strategy for sensitizing human glioma cells to oxidative stress that is induced by ionizing radiation, certain chemotherapies and ischemia-reperfusion.

Quantitative analysis reveals asynchronous and more than DSB-associated histone H2AX phosphorylation after exposure to ionizing radiation.

Abstract Han, J., Hendzel, M. J. and Allalunis-Turner, J. Quantitative Analysis Reveals Asynchronous and more than DSB-Associated Histone H2AX Phosphorylation after Exposure to Ionizing Radiation. Radiat. Res. 165, 283–292 (2006). Rapid phosphorylation of histone H2AX after exposure of cells to ionizing radiation occurs at DSB sites and extends to a region including as much as 30 Mbp of chromatin to form visible microscopic structures called γ-H2AX foci. Although the kinetics of total cellular histone H2AX phosphorylation after irradiation has been characterized, we still know little about the phosphorylation kinetics of individual γ-H2AX foci. In addition, there are hundreds of smaller γ-H2AX foci that are not associated with DNA double-strand breaks. We refer to these sites as DSB-unrelated γ-H2AX foci. By using indirect immunofluorescence microscopy, deconvolution and three-dimensional image analysis, we established an objective method to quantitatively analyze each γ-H2AX focus as well as to discriminate DSB-related γ-H2AX foci from DSB-unrelated γ-H2AX foci. Using this method, we found that histone H2AX phosphorylation at different DSB sites was asynchronous after exposure to ionizing radiation. This may reflect the heterogeneous characteristic of free DNA ends that are generated under these conditions. In addition, we found that increased histone H2AX phosphorylation also occurred outside of DSB sites after exposure to ionizing radiation. The function of this DSB-unassociated phosphorylation is not known.

Expression and hypoxic up‐regulation of neuroglobin in human glioblastoma cells

Neuroglobin is a recently identified globin molecule that is expressed predominantly in the vertebrate brain. Neuroglobin expression increases in oxygen‐deprived neurons, suggesting it protects neurons from ischemic cell death. We report that neuroglobin transcript and protein are expressed in human glioblastoma cells, and that this expression increases in hypoxia in vitro. We also show that neuroglobin is up‐regulated in hypoxic microregions of glioblastoma tumor xenografts. Our finding of hypoxic up‐regulation of neuroglobin in human glioblast oma cells may provide insight into how tumor cells adapt to and survive in hypoxic microenvironments.

Intact G2-phase checkpoint in cells of a human cell line lacking DNA-dependent protein kinase activity.

Cells respond to radiation-induced DNA damage in a cell cycle phase-specific manner as shown by (1) variation in radiosensitivity across the cell cycle and (2) checkpoints in G1 and G2 phase at which arrest of progression of cells through the phases of the cell cycle occurs. We studied these processes in cells of human glioma cell lines which lack (M059J(PK-)) or express (M059K(PK+)) DNA-dependent protein kinase (DNA-PK) activity. Cell populations enriched with cells of a specific cell cycle phase were y-irradiated and analyzed for cell survival. Although both cell lines were relatively sensitive in G1 phase and resistant in S phase, the differential sensitivity was greater in M059J(PK-) cells. In the studies on checkpoints, unsynchronized cells were irradiated and examined for evidence of cell cycle arrest. Neither cell line showed a postirradiation G1-phase arrest, presumably because of mutant p53 status. For M059J(PK-) cells, all doses tested (2.5-10 Gy) resulted in a significant increase in the proportion of G2/M-phase cells; however, for M059K(PK+) cells, a significant increase in G2/M phase was observed only after 10 Gy. These results suggest that the ability to activate the G2-phase checkpoint remains intact in cells which lack DNA-PK activity.