Autophagic flux in glioblastoma cells.

To establish metabolic context for radiation sensitivity by measuring autophagic flux in two different glioblastoma (GBM) cell lines. Clonogenic survival curve analysis of U87 or U251 cells exposed to γ radiation, fast neutrons, a mixed energy neutron beam (METNB) or Auger electrons from a gadolinium neutron capture (GdNC) reaction suggested other factors, beyond a defective DNA damage response, contribute to cell death of U251 cells. Altered tumor metabolism (autophagy) was hypothesized as a factor in U251 cells' clonogenic response. Each of the four different radiation modalities induced an increase in the number of autophagosomes in both U87 and U251 cells. Changes in the number of autophagosomes can be explained by either induction of autophagy or alterations in autophagic flux so autophagic flux was assayed by p62 immunoblotting or in engineered GBM cells that stably express an autophagy marker protein, LC3B-eGFP-mCherry. Perturbations in later stages of autophagy in U251 cells corresponded with radiation sensitivity of U251 cells irradiated with 10 Gy γ rays. Establishment of altered autophagic flux is a useful biomarker for metabolic stress and provided metabolic context for radiation sensitization to 10 Gy γ rays. These results provide strong evidence for the usefulness of managing tumor cell metabolism as a tool for the enhancement of radiation therapy.

Necrosis is not induced by gadolinium neutron capture in glioblastoma multiforme cells.

PURPOSE: A comparative study of the effects of different radiation modalities on cell death was performed. MATERIALS AND METHODS: Radiation modalities included γ-rays, fast neutrons, a mixed energy neutron beam called the modified enhanced thermal neutron beam and the mixed beam including Auger electron irradiation by gadolinium neutron capture. U87 (human brain tumor cells) cell survival curve data were modeled to predict how cells died. Transmission electron microscopy (TEM) images were assembled into a morphology of cell death (MCD) database and used to determine the fraction of necrotic or autophagic cells. RESULTS: Linear energy transfer (LET) differences for the different radiation modalities were revealed by modeling. All radiation modalities induced autophagy but only fast neutrons induced significant levels of necrosis. No necrosis, above control levels, was found in cells irradiated with mixed beam irradiation including Auger electrons. The number of autophagosomes increased with increasing time after exposure to all radiation modalities indicating progression of autophagy but only cells irradiated with the mixed beam plus Auger electrons exhibited extreme autophagy. CONCLUSIONS: Mixed neutron beam irradiation plus Auger electron irradiation from gadolinium neutron capture is a moderately high LET modality that kills U87 cells without the induction of necrosis and with progression of autophagy to an extreme state.

Molecular and cellular effects of Auger emitters: 2008-2011.

PURPOSE: To review recent Auger emitter research with an emphasis on a review of findings on targeting and accumulation of Auger emitters in tumor cells. CONCLUSION: Significant progress can be reported for targeting methods and improvements in methods to accumulate Auger emitters in the target cells, as well as advances in dose calculations. These studies further our understanding of how Auger emitters induce cell death at a cellular and molecular level, supporting the targeted radiomedical applications of Auger emitters.

Boron neutron capture in prostate cancer cells.

A modified enhanced thermal neutron beam (METNB) assembly at Fermilab was used to irradiate borylphenylalanine (BPA) treated human prostate cancer cells, DU 145. Acceptable cellular uptake levels of BPA and no BPA cytotoxicity were observed. In the absence of BPA, the relative biological effectiveness (RBE) of the METNB was determined to be 2.3-4.8 times greater than gamma rays. An additional 1.2 or 1.4 fold relative enhancement from boron neutron capture (RE(BNC)) was observed for METNB irradiated DU 145 cells treated with 4.9 or 12mM BPA, respectively. The additional cell killing of the BPA loaded DU 145 cells by the METNB at Fermilab is evidence for a BNC enhanced cell killing.

Gadolinium neutron capture in glioblastoma multiforme cells.

PURPOSE: A proof of principle for cell killing by Gadolinium (Gd) neutron capture in Magnevist preloaded Glioblastoma multiforme (GBM) cells is provided. MATERIALS AND METHODS: U87cells were pre-loaded with 5 mg/ml Magnevist (Gd containing compound) and irradiated using an enhanced neutron beam developed at NIU Institute for Neutron Therapy at Fermilab. These experiments were possible because of an enhanced fast neutron therapy assembly designed to use the fast neutron beam at Fermilab to deliver a neutron beam containing a greater fraction of thermal neutrons and because of the development of improved calculations for dose for the enhanced neutron beam. Clonogenic response was determined. RESULTS: U87 cell survival after gamma irradiation, fast neutron irradiation and irradiation with the enhanced neutron beam in the presence or absence of Magnevist were determined. CONCLUSIONS: U87 cells were the least sensitive to gamma radiation, and increasingly sensitive to fast neutron irradiation, irradiation with the enhanced neutron beam and finally a significant enhancement in cell killing was observed for U87 cells preloaded with Magnevist. The sensitivity of U87 cells pre-loaded with Magnevist and then irradiated with the enhanced neutron beam can at least in part be attributed to the Auger electrons emitted by the neutron capture event.

Non-DNA-binding platinum anticancer agents: Cytotoxic activities of platinum-phosphato complexes towards human ovarian cancer cells.

DNA is believed to be the molecular target for the cytotoxic activities of platinum (Pt) anticancer drugs. We report here a class of platinum(II)- and platinum(IV)-pyrophosphato complexes that exhibit cytotoxicity comparable with and, in some cases, better than cisplatin in ovarian cell lines (A2780, A2780/C30, and CHO), yet they do not show any evidence of covalent binding to DNA. Moreover, some of these compounds are quite effective in cisplatin- and carboplatin-resistant cell line A2780/C30. The lack of DNA binding was demonstrated by the absence of a detectable Pt signal by atomic absorption spectroscopy using isolated DNA from human ovarian cells treated with a platinum(II)-pyrophosphato complex, (trans-1,2-cyclohexanediamine)(dihydrogen pyrophosphato) platinum(II), (pyrodach-2) and from NMR experiments using a variety of nucleotides including single- and double-stranded DNA. Furthermore, pyrodach-2 exhibited reduced cellular accumulations compared with cisplatin in cisplatin- and carboplatin-resistant human ovarian cells, yet the IC(50) value for the pyrophosphato complex was much less than that of cisplatin. Moreover, unlike cisplatin, pyrodach-2 treated cells overexpressed fas and fas-related transcription factors and some proapoptotic genes such as Bak and Bax. Data presented in this report collectively indicate that pyrodach-2 follows different cytotoxic mechanisms than does cisplatin. Unlike cisplatin, pyrodach-2 does not undergo aquation during 1 week and is quite soluble and stable in aqueous solutions. Results presented in this article represent a clear paradigm shift not only in expanding the molecular targets for Pt anticancer drugs but also in strategic development for more effective anticancer drugs.

Using Hoechst 33342 to target radioactivity to the cell nucleus.

We have explored the use of Hoechst 33342 (H33342) to carry radioactivity to the cell nucleus. H33342 enters cells and targets DNA at adenine-thymine-rich regions of the minor groove. Considerable membrane blebbing and ruffling occur in CHO cells within minutes after its addition to the culture medium in micromolar quantities. Blue vesicles are apparent in the cell cytoplasm, and by 30 min the nuclei are stained dark blue. Upon its binding to DNA, a visible emission shift of the dye can be observed with fluorescence microscopy. We have radioiodinated (125I) H33342 and specifically irradiated nuclear DNA by incubating CHO cells with 125I-H33342 at 37 degrees C and accumulating 125I decays at -90 degrees C. At various times, the cells are thawed and assayed for survival (clonogenicity) and DSB (gamma-H2AX) formation. 125I-H33342 decay leads to a monoexponential decrease in cell survival with a D0 of 122 125I decays per cell and a linear increase in DNA DSB induction (equivalent to 15 gamma-H2AX foci/cell). Cell death is not modified by the radioprotective effects of H33342 because we use considerably lower concentrations than those that provide a slight protection against gamma radiation. We conclude that cell killing by 125I-H33342 and the induction of gamma-H2AX foci are highly correlated.

GammaH2AX foci induced by gamma rays and 125idU decay.

PURPOSE: GammaH2AX foci formation was investigated after gamma irradiation and after accumulating 125IdU decays to study the DNA double strand break (dsb) damage repair response in human breast cancer cells, MCF-7. MATERIALS AND METHODS: Confocal laser scanning microscopy (CLSM) was used to detect yH2AX foci formed in response to DNA dsbs induced by 0, 0.5, 1, 2 and 5 Gy gamma irradiation and 125IdU decays accumulated at -90 degrees C in human breast cancer cells, MCF-7. 125IdU treated cells were labeled with 4 different concentrations of 125IdU and then accumulated decays for 6, 19 or 35 days. gammaH2AX foci formation time for all experiments was 1 hour at 37 degrees C. Visual confirmation of gammaH2AX foci was achieved by digital imaging (histogram analysis or profile analysis) and by standardizing the scored number of foci. The average numbers of gammaH2AX foci formed per cell after gamma irradiation or accumulated (125)IdU decays were determined by counting red voxels or counting gammaH2AX foci in propidium iodide (PI) counterstained nuclei by eye in optically sectioned cells. RESULTS: Control, unirradiated MCF-7 cells had an average of 1.7 gammaH2AX foci per cell and an average of 32 yH2AX foci were scored for cells irradiated with 1 Gy gamma rays. The data for doses up to approximately 1 Gy was a good linear fit (r2 =0.97) indicating that the assay is sensitive to low doses of gamma rays. The average number of gammaH2AX foci scored in control cells that were frozen and thawed but not irradiated (=2.3) was not statistically significantly different from controls that were not frozen and thawed. The average number of yH2AX foci was linearly related (r2 = 0.98) to low numbers (< 200 decays/cell) of 125IdU decays indicating that the assay is also sensitive to low numbers of accumulated 125IdU decays. At 125I decays greater than 200 decays/cell, the average number of yH2AX foci plateaued. Regression analysis of the data for 0-140 125IdU decays per cell was used to calculate the rate of yH2AX foci formation (=0.26 foci per 125I decay). CONCLUSIONS: The gammaH2AX foci formation assay is sensitive to low doses of gamma rays and accumulated 125I decays. When 125IdU decays were accumulated at -90 degrees C (to overcome confounding DNA damage repair processes that occur during simultaneous 125IdU incorporation and decay accumulation at 37 degrees C), 0.26 gammaH2AX foci were formed per 125IdU decay. Methods used to incorporate 125I decay may modulate the number of gammaH2AX foci scored in cells.