Linda S. Yasui

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 IC50 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.

Relative biological effectiveness of accumulated 125IdU and 125I-estrogen decays in estrogen receptor-expressing MCF-7 human breast cancer cells

Abstract Yasui, L. S., Hughes, A. and DeSombre, E. R. Relative Biological Effectiveness of Accumulated 125IdU and 125I-Estrogen Decays in Estrogen Receptor-Expressing MCF-7 Human Breast Cancer Cells. The therapeutic potential for delivering a cytotoxic dose of radiation (using the decay of Auger-electron emitters) to the cell nucleus of cancer cells that express estrogen receptors (ERs) by radiolabeled estrogen was investigated in the ER-expressing human breast cancer cell line, MCF-7. The radiolabeled estrogen/ER complex irradiates the cell nucleus by binding specific DNA sequences called estrogen response elements (EREs). Cell clonogenicity and induction of DNA double-strand breaks (DSBs) by γ radiation or accumulation of 125I-iododeoxyuridine (125IdU) or E-17α[125I]iodovinyl-11βmethoxyestradiol (125IVME2) decays were determined. MCF-7 cells were efficiently killed by accumulation of 125IdU (D0 = 30 decays per cell) and 125IVME2 decays (D0 = 28 decays per cell). DNA DSBs were induced by the accumulation of 125IdU (approximately 3750 decays per cell required to reduce the mean value of the elution profile to 50%) or 125IVME2 decays (approximately 465 decays per cell required to reduce the mean value to 50%). For survival of MCF-7 cells after γ irradiation, the D0 was 1 Gy, and approximately 65 Gy was required to reduce the mean value to 50% for induction of DSBs. The RBE values for cell killing and induction of DSBs by 125IVME2 relative to γ radiation were 4.8 and 18.8, respectively. The RBE values for cell killing and induction of DSBs by 125IdU relative to γ radiation were 4.5 and 2.3, respectively. Cell killing in a manner similar to that induced by high-LET radiation and the high RBE for induction of DSBs by 125IVME2 in the ER-expressing MCF-7 cells provide a biological rationale for the use of Auger electron-emitting radionuclides covalently bound to estrogen to deliver a cytotoxic dose of radiation to ER-positive cancers.

Nuclear Scaffold Organization in the X-ray Sensitive Chinese Hamster Mutant Cell Line, xrs-5

Nuclear organization was probed in the radiation-sensitive Chinese hamster ovary CHO) cell line, xrs-5, and compared with parental CHO K1 cells using the resinless section technique and DNase I digestions. The resinless section data showed no gross morphological differences in core filaments from the nuclear scaffolds of unirradiated CHO K1 and xrs-5 cells. However, the nuclear scaffolds of irradiated xrs-5 cells (1 Gy) had significantly increased ground substance. Irradiated and unirradiated CHO K1 cell nuclear scaffolds were morphologically identical. These data suggest that both CHO K1 and xrs-5 cell nuclear scaffolds had internal nuclear scaffolding networks that could provide DNA attachment sites. The rate of DNase I digestion of unirradiated CHO K1 and xrs-5 was not significantly different, but the extent of digestion was greater in unirradiated CHO K1 cells that in xrs-5 cells suggesting that less xrs-5 cell chromatin at DNA attachment points is accessible to the enzyme DNase I. The extents of digestion in irradiated (1 Gy) CHO K1 and xrs-5 cell nuclei also differed but the relationship was reversed. The irradiated xrs-5 cell samples were digested to a greater extent compared with CHO K1 cells. These chromatin digestion data suggest that the matrix attachment regions in xrs-5 cells are different from CHO K1 cells. The different DNA attachment organization in the xrs-5 cells may play a role in modulating radiation sensitivity.

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

Abstract 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.

γH2AX foci induced by γ rays and 125IdU decay

Purpose: γH2AX foci formation was investigated after γ 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 γH2AX foci formed in response to DNA dsbs induced by 0, 0.5, 1, 2 and 5 Gy γ irradiation and 125IdU decays accumulated at −90°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. γH2AX foci formation time for all experiments was 1 hour at 37°C. Visual confirmation of γH2AX foci was achieved by digital imaging (histogram analysis or profile analysis) and by standardizing the scored number of foci. The average numbers of γH2AX foci formed per cell after γ irradiation or accumulated 125IdU decays were determined by counting red voxels or counting γH2AX 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 γH2AX foci per cell and an average of 32 γH2AX foci were scored for cells irradiated with 1 Gy γ 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 γ rays. The average number of γH2AX 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 γH2AX 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 γH2AX foci plateaued. Regression analysis of the data for 0–140 125IdU decays per cell was used to calculate the rate of γH2AX foci formation (=0.26 foci per 125I decay). Conclusions: The γH2AX foci formation assay is sensitive to low doses of γ rays and accumulated 125I decays. When 125IdU decays were accumulated at −90°C (to overcome confounding DNA damage repair processes that occur during simultaneous 125IdU incorporation and decay accumulation at 37°C), 0.26 γH2AX foci were formed per 125IdU decay. Methods used to incorporate 125I decay may modulate the number of γH2AX foci scored in cells.

Cytotoxicity of 125 I-oestrogen decay in non-oestrogen receptor-expressing human breast cancer cells, MDA-231 and oestrogen receptor-expressing MCF-7 cells

Purpose : To compare the cytotoxicity of 125 I-oestrogen (E-17 α [ 125 I]iodovinyl-11 β methoxyoestradiol or 125 IVME2) decay accumulation in human breast adenocarcinoma cells that do not express oestrogen receptor (ER) (MDA-231 cells) with human breast adenocarcinoma cells that do express ER (MCF-7 cells). Materials and methods : MDA-231 cells were labelled with 125 IVME2 or [ 125 I]iododeoxyuridine (125 IdU), frozen for decay accumulation, thawed and then plated for colony formation. γ-irradiation survival was also determined. A whole-cell 3 H-oestrogen-binding assay and a specific-binding assay were used to detect ER. Results : No MDA-231 cell killing by accumulated 125 IVME2 decays (up to 440 dpc) was observed but ER-positive MCF-7 cells were killed by 125 IVME2 (D o =28 dpc). MDA-231 cells were not significantly more radioresistant to γ-rays (D o =1.7Gy for MDA-231 cells; 1 Gy for MCF-7 cells) or to 125 IdU decays (D o = 44dpc for MDA-231 cells; 30 dpc for MCF-7 cells). No ER were detected in MDA-231 cells. Conclusions : ER-negative cells, MDA-231, are not killed by 125 IVME2 decay accumulation. It is speculated that without ER (required to translocate the 125 IVME2 to its nuclear target), formation of the 125 IVME2-ER-DNA oestrogen-response element (ERE) complex and subsequent specific irradiation of the DNA at the ERE cannot occur. These results support the hypothesis that the nuclear genome is a critical target for radiation-induced cell death.

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 γ 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 γ 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.

Dna Damage Induction by 125I-Estrogen

DNA damage induced by the radioactive decay of 125I-estrogen (125I-VME2) in an estrogen receptor expressing CHO cell line, CHO-ER, was measured. 125I-VME2 targeted 125I atoms proximal to DNA estrogen response elements (EREs). 125I decays were accumulated at -135 degrees C, and thereafter assayed by alkaline and neutral filter elution techniques to measure DNA single strand break (ssb) and double strand break (dsb) induction respectively. Increasing DNA damage (both ssbs and dsbs) was detected after exposure of cells to increasing concentrations of 125I-VME2. DNA ssb and dsb dose-response curves for 125I-VME2 were multiphasic. The rates of DNA damage induction by the decay of 125I-VME2 was determined by comparing slopes of all data or by comparing initial slopes. DNA ssb induction per 125I-VME2 decay was approximately 2 times greater compared with DNA dsb induction. 125I-VME2 decay induced approximately 4-8 times more DNA dsbs than 125IUdR decay.

Cytotoxicity of 125I decay in the DNA double strand break repair deficient mutant cell line, xrs-5

The survival of parental Chinese hamster ovary (CHO) K1 cells and the DNA double strand break (DSB) repair deficient mutant, xrs-5 was determined after accumulation of 125I decays. Both CHO and xrs-5 cells were extremely sensitive to accumulated 125I decays. The D0 values for CHO and xrs-5 cells were 40 and approximately 7 decays per cell, respectively. The difference in cell survival between CHO and xrs-5 cells was not due to differences in overall 125IUdR incorporation, differences in labelling index (LI) or differences in plating efficiency (PE). Relative biological effectiveness (RBE) values calculated relative to 137Cs gamma radiation survival values (D0 and D10) were higher in xrs-5 cells compared with CHO cells. Although both CHO and xrs-5 cells have high RBE values that correspond to a high sensitivity of CHO and xrs-5 cells to 125I decay. The higher RBE observed for xrs-5 cells in combination with the known repair defect in xrs-5 cells support the idea that unrepaired DNA double strand breaks are lethal to the cell.