Impact of IUdR on Rat 9L glioma cell survival for 25-35 keV photon-activated auger electron therapy.

The goal of the current study was to measure the energy dependence of survival of rat 9L glioma cells labeled with iododeoxyuridine (IUdR) that underwent photon-activated Auger electron therapy using 25-35 keV monochromatic X rays, i.e., above and below the K-edge energy of iodine. Rat 9L glioma cells were selected because of their radioresistance, ability to be implanted for future in vivo studies and analogy to radioresistant human gliomas. Survival curves were measured for a 4 MV X-ray beam and synchrotron produced monochromatic 35, 30 and 25 keV X-ray beams. IUdR was incorporated into the DNA at levels of 0, 9 and 18% thymidine replacement for 4 MV and 35 keV and 0 and 18% thymidine replacement for 30 and 25 keV. For 10 combinations of beam energy and thymidine replacement, 62 data sets (3-13 per combination) provided 776 data points (47-148 per combination). Survival versus dose data taken for the same combination, but on different days, were merged by including the zero-dose points in the nonlinear, chi-squared data fitting using the linear-quadratic model and letting the best estimate to the zero-dose plating efficiency for each of the different days be a fitting parameter. When comparing two survival curves, the ratio of doses resulting in 10% survival gave sensitization enhancement ratios (SER10) from which contributions due to linear energy transfer (LET) (SER10,LET), IUdR radiosensitization (SER10,RS), the Auger effect (SER10,AE) and the total of all effects (SER10,T) were determined. At 4 MV and 35, 30 and 25 keV, SER10,LET values were 1.00, 1.08 ± 0.03, 1.22 ± 0.02 and 1.37 ± 0.02, respectively. At 4 MV SER10,RS values for 9 and 18% IUdR were 1.28 ± 0.02 and 1.40 ± 0.02, respectively. Assuming LET effects were independent of percentage IUdR and radiosensitization effects were independent of energy, SER10,AE values for 18% IUdR at 35, 30 and 25 keV were 1.35 ± 0.05, 1.06 ± 0.03 and 0.98 ± 0.03, respectively. The value for 9% IUdR at 35 keV was 1.01 ± 0.04. First, we found the radioresistant rat 9L glioma cell line exhibited an SER10 due to the Auger effect of 1.35 at (35 keV, 18% IUdR) and an SER10 due to the radiosensitizing effect of 1.40 at (4 MV, 18% IUdR), both significantly less than values for previously reported cell lines. These low individual values emphasize the benefit of their combined value (SER10 of approximately 1.9) for achieving clinical benefit. Second, as expected, we observed that energies below the K-edge of iodine (25 and 30 keV), for which there are L, M and higher shell photoelectric events creating Auger electrons, show no promise for Auger electron therapy. Third, to proceed with future in vivo studies, additional data from 35-65 keV are needed to determine the optimal X-ray energy for IUdR Auger electron therapy. Only then can there be an answer to the question, how well the energy dependence of in vitro survival data supports the potential for photon-activated Auger electron therapy with IUdR in cancer radiotherapy.

Dependence of cell survival on iododeoxyuridine concentration in 35-keV photon-activated Auger electron radiotherapy.

PURPOSE: To measure and compare Chinese hamster ovary cell survival curves using monochromatic 35-keV photons and 4-MV x-rays as a function of concentration of the radiosensitizer iododeoxyuridine (IUdR). METHODS AND MATERIALS: IUdR was incorporated into Chinese hamster ovary cell DNA at 16.6 ± 1.9%, 12.0 ± 1.4%, and 9.2 ± 1.3% thymidine replacement. Cells were irradiated from 1 to 8 Gy with 35-keV synchrotron-generated photons and conventional radiotherapy 4-MV x-rays. The effects of the radiation were measured via clonogenic survival assays. Surviving fraction was plotted vs. dose and fit to a linear quadratic model. Sensitization enhancement ratios (SER(10)) were calculated as the ratio of doses required to achieve 10% surviving fraction for cells without and with DNA-incorporated IUdR. RESULTS: At 4 MV, SER(10) values were 2.6 ± 0.1, 2.2 ± 0.1, and 1.5 ± 0.1 for 16.6%, 12.0%, and 9.2% thymidine replacement, respectively. At 35 keV, SER(10) values were 4.1 ± 0.2, 3.0 ± 0.1, and 2.0 ± 0.1, respectively, which yielded SER(10) ratios (35 keV:4 MV) of 1.6 ± 0.1, 1.4 ± 0.1, and 1.3 ± 0.1, respectively. CONCLUSIONS: SER(10) increases monotonically with percent thymidine replacement by IUdR for both modalities. As compared to 4-MV x-rays, 35-keV photons produce enhanced SER(10) values whose ratios are linear with percent thymidine replacement and assumed to be due to Auger electrons contributing to enhanced dose to DNA. Although this Auger effectiveness factor is less than the radiosensitization factor of IUdR, both could be important for the clinical efficacy of IUdR radiotherapy.

Oxidative phosphorylation analysis: assessing the integrated functional activity of human skeletal muscle mitochondria--case studies.

Oxidative phosphorylation analysis, performed on freshly-isolated mitochondria, assesses the integrated function of the electron transport chain (ETC) coupled to ATP synthesis, membrane transport, dehydrogenase activities, and the structural integrity of the mitochondria. In this review, a case study approach is employed to highlight detection of defects in the adenine nucleotide translocator, the pyruvate dehydrogenase complex, fumarase, coenzyme Q function, fatty acid metabolism, and mitochondrial membrane integrity. Our approach uses the substrates glutamate, pyruvate, 2-ketoglutarate (coupled with malonate), malate, and fatty acid substrates (palmitoylcarnitine, octanoylcarnitine, palmitoyl-CoA (with carnitine), octanoyl-CoA (with carnitine), octanoate and acetylcarnitine) in addition to succinate, durohydroquinone and TMPD/ascorbate to uncover metabolic defects that would not be apparent from ETC assays performed on detergent-solubilized mitochondria.

The peripheral benzodiazepine receptor in photodynamic therapy with the phthalocyanine photosensitizer Pc 4.

The peripheral benzodiazepine receptor (PBR) is an 18 kDa protein of the outer mitochondrial membrane that interacts with the voltage-dependent anion channel and may participate in formation of the permeability transition pore. The physiological role of PBR is reflected in the high-affinity binding of endogenous ligands that are metabolites of both cholesterol and heme. Certain porphyrin precursors of heme can be photosensitizers for photodynamic therapy (PDT), which depends on visible light activation of porphyrin-related macrocycles. Because the apparent binding affinity of a series of porphyrin analogs for PBR paralleled their ability to photoinactivate cells, PBR has been proposed as the molecular target for porphyrin-derived photocytotoxicity. The phthalocyanine (Pc) photosensitizer Pc 4 accumulates in mitochondria and structurally resembles porphyrins. Therefore, we tested the relevance of PBR binding on Pc 4-PDT. Binding affinity was measured by competition with 3H-PK11195, a high-affinity ligand of PBR, for binding to rat kidney mitochondria (RKM) or intact Chinese hamster ovary (CHO) cells. To assess the binding of the Pc directly, we synthesized 14C-labeled Pc 4 and found that whereas Pc 4 was a competitive inhibitor of 3H-PK11195 binding to the PBR, PK11195 did not inhibit the binding of 14C-Pc 4 to RKM. Further, 14C-Pc 4 binding to RKM showed no evidence of saturation up to 10 microM. Finally, when Pc 4-loaded CHO cells were exposed to activating red light, apoptosis was induced; Pc 4-PDT was less effective in causing apoptosis in a companion cell line overexpressing the antiapoptotic protein Bcl-2. For both cell lines, PK11195 inhibited PDT-induced apoptosis; however, the inhibition was transient and did not extend to overall cell death, as determined by clonogenic assay. The results demonstrate (1) the presence of low-affinity binding sites for Pc 4 on PBR; (2) the presence of multiple binding sites for Pc 4 in RKM and CHO cells other than those that influence PK11195 binding; and (3) the ability of high supersaturating levels of PK11195 to transiently inhibit apoptosis initiated by Pc 4-PDT, with less influence on overall cell killing. We conclude that the binding of Pc 4 to PBR is less relevant to the photocytotoxicity of Pc 4-PDT than are other mitochondrial events, such as photodamage to Bcl-2 and that the observed inhibition of Pc 4-PDT-induced apoptosis by PK11195 likely occurs through a mechanism independent of PBR.