Enhancement of radiation effect by heavy elements.

The enhancement of radiobiological effects by heavy elements is reviewed. As an underlying mechanism, Auger effects have been stressed which can be induced via inner-shell photoabsorption or via excitation and/or ionization by secondary electrons. Latter channel of Auger induction expands the applicability of Auger enhancing phenomena to electron and hadron therapy. After discussion on the required characteristics for radiosensitizers, possibility of nanoparticles of Au or Pt is mentioned since they could be synthesized or modified as ideal radiosensitizers.

[Thymolipoma associated with myasthenia gravis].

A 42-year-old woman was admitted to our hospital complaining of left ptosis, diplopia, and muscle weakness. A diagnosis of myasthenia gravis was made. Chest roentgenograms, computed tomography (CT), and magnetic resonance imaging (MRI) showed a large anterior mediastial mass and suggested thymolipoma. Extended thymectomy was performed via a median sternotomy. Histopathological examination revealed that the tumor consisted of mature adipose tissue and weighed 1,500 grams, in which thymic tissues with Hassall' s corpuscles but without a germinal center were contained. The histological appearance was compatible with a typical thymolipoma. This is the 24th reported case of thymolipoma associated with myasthenia gravis.

Mammalian cells loaded with platinum-containing molecules are sensitized to fast atomic ions.

PURPOSE: This work investigates whether a synergy in cell death induction exists in combining atomic ions irradiation and addition of platinum salts. Such a synergy could be of interest in view of new cancer therapy protocol based on atomic ions--hadrontherapy--with the addition of radiosensitizing agents containing high-Z atoms. The experiment consists in irradiating by fast ions cultured cells previously exposed to dichloroterpyridine Platinum (PtTC) and analyzing cell survival by a colony-forming assay. MATERIALS AND METHODS: Chinese Hamster Ovary (CHO) cells were incubated for six hours in medium containing 350 microM PtTC, and then irradiated by fast ions C(6+) and He(2+), with Linear Energy Transfer (LET) within range 2-70 keV/microm. In some experiments, dimethyl sulfoxide (DMSO) was added to investigate the role of free radicals. The intracellular localization of platinum was determined by Nano Secondary Ion Mass Spectroscopy (Nano-SIMS). RESULTS: For all LET examined, cell death rate is largely enhanced when irradiating in presence of PtTC. At fixed irradiation dose, cell death rate increases with increasing LET, while the platinum relative effect is larger at low LET. CONCLUSION: This finding suggests that hadrontherapy or protontherapy therapeutic index could be improved by combining irradiation procedure with concomitant chemotherapy protocols using platinum salts.

Irradiation of DNA loaded with platinum containing molecules by fast atomic ions C(6+) and Fe(26+).

PURPOSE: In order to study the role of the Linear Energy Transfer (LET) of fast atomic ions in platinum-DNA complexes inducing breaks, DNA Plasmids were irradiated by C(6+) and Fe(26+) ions. MATERIAL AND METHODS: DNA Plasmids (pBR322) loaded with different amounts of platinum contained in a terpyridine-platinum molecule (PtTC) were irradiated by C(6+) ions and Fe(26+) ions. The LET values ranged between 13.4 keV/microm and 550 keV/microm. In some experiments, dimethyl sulfoxide (DMSO) was added. RESULTS: In all experiments, a significant increase in DNA strand breaks was observed when platinum was present. The yield of breaks induced per Gray decreased when the LET increased. The yield of single and double strand breaks per plasmid per track increased with the LET, indicating that the number of DNA breaks per Gray was related to the number of tracks through the medium. CONCLUSIONS: These findings show that more DNA breaks are induced by atomic ions when platinum is present. This effect increases for low LET heavy atoms. As DSB induction may induce cell death, these results could open new perspectives with the association of hadrontherapy and chemotherapy. Thus the therapeutic index might be improved by loading the tumour with platinum salts.

[Usefulness of dynamic computed tomography for the diagnosis of mediastinal hemangioma].

An asymptomatic 59-year-old female was admitted with an abnormal shadow on her chest radiography. Chest computed tomography (CT) revealed a mass measuring 20 mm in the anterior mediastinum. At the arterial phase on dynamic contrast-enhanced CT (dynamic CT), the pattern of "peripheral puddles", defined as discrete well-defined peripheral enhancing globles, was found in the mass. The tumor was completely resected via a median sternotomy, and was histopathologicaly diagnosed as hemangioma. In this case, dynamic CT was very useful for the preoperative diagnosis, and then the enhancement pattern of "peripheral puddles" on dynamic CT may be a conclusive finding for the diagnosis of mediastinal hemangiomas.

Radiation-induced gamma-H2AX in mammalian cells irradiated with a synchrotron X-ray microbeam.

In order to study the radiobiological effects from low dose radiation, a cell irradiation system using synchrotron X-ray microbeam has been developed, by which cells can be recognised individually and irradiated one by one with the desired dose of monochromatic X rays. The minimum beam sizes obtained are 2 microm with the focusing optics and 5 microm square with the non-focused beam, and the beam size can be changed easily with a high-precision slit in the case of a non-focused beam. Human fibroblast cells were individually irradiated with this system, and immunostained by gamma-H2AX antibody to visualise the DNA damage. Most of the fluorescent foci were observed in a localised area in cell nuclei, the size of which was almost the same as the beam size.

[Surgical treatment for adrenal metastasis from lung cancer].

Several long-term survivors after surgical resection for a solitary adrenal metastasis from non-small cell lung cancer (NSCLC) have been reported in case reports and case series with a small number of patients. We have experienced 6 cases of patients who had adrenalectomy (ADR) for a metastasis from NSCLC. The median survival time (MST) after ADR was 24 months, and there was only 1 case of 3-year survivor. To elucidate the surgical indication and the prognostic factors of patients with a solitary adrenal metastasis from NSCLC, we analyzed 104 patients including our 6 patients who had ADR for a metastasis from NSCLC. The MST after ADR and 5-year survival were 24 months and 31%, respectively. Univariate and multivariate analysis demonstrated that lymph node metastasis at the surgery for primary lung cancer was the only significant and independent predictor of poor survival in patients after ADR. The results suggest that aggressive surgical treatment of a solitary adrenal metastasis from NSCLC may be effective when a patient have N0 disease.

Genetic alteration of the beta-catenin gene (CTNNB1) in human lung cancer and malignant mesothelioma and identification of a new 3p21.3 homozygous deletion.

The beta-catenin gene (CTNNB1) has been shown to be genetically mutated in various human malignancies. To determine whether the beta-catenin gene is responsible for oncogenesis in thoracic malignancies, we searched for the mutation in 166 lung cancers (90 primary tumors and 76 cell lines), one blastoma and 10 malignant mesotheliomas (two primary tumors and eight cell lines). Among the lung cancers, including 43 small cell lung cancers (SCLCs) and 123 non-small cell lung cancers (NSCLCs), we identified four alterations in exon 3, which is the target region of mutation for stabilizing beta-catenin. One primary adenocarcinoma had a somatic mutation from C to G, leading to an amino acid substitution from Ser to Cys at codon 37. Among the cell lines, SCLC NCI-H1092 had a mutation from A to G, leading to an Asp to Gly substitution at codon 6, NSCLC HCC15 had a mutation from C to T, leading to a Ser to Phe substitution at codon 45, and NSCLC NCI-H358 had a mutation from A to G, leading to a Thr to Ala substitution at codon 75. One blastoma also had a somatic mutation from C to G, leading to a Ser to Cys substitution at codon 37. Among the 10 malignant mesotheliomas, we identified a homozygous deletion in the NCI-H28 cell line. Cloning of the rearranged fragment from NCI-H28 indicated that all the exons except exon 1 of the beta-catenin gene are deleted and that the deletion junction is 13 kb downstream from exon 1. Furthermore, Northern blot analysis of 26 lung cancer and eight mesothelioma cell line RNAs detected ubiquitous expression of the beta-catenin messages except NCI-H28, although Western blot analysis showed that relatively less amounts of protein products were expressed in some of lung cancer cell lines. Our findings suggest that the beta-catenin gene is infrequently mutated in lung cancer and that the NCI-H28 homozygous deletion of the beta-catenin gene might indicate the possibility of a new tumor suppressor gene residing in this region at 3p21.3, where various types of human cancers show frequent allelic loss.

Fast He2+ ion irradiation of DNA loaded with platinum-containing molecules.

PURPOSE: The association of radiotherapy and chemotherapy is an attractive approach to improve the therapeutic index of the treatment of tumors. A lot of work has been devoted to investigate the effects of X-ray, gamma-ray and neutron irradiation of DNA or living cells loaded with different chemical compounds containing heavy atoms like platinum. No such studies exist presently when fast atomic ions are chosen as ionizing particles. In the present work, we investigate quantitatively the increase of DNA breaks in complexes of plasmid-DNA loaded with platinum atoms under irradiation by fast atomic He2+ ions. MATERIALS AND METHODS: DNA Plasmids (pBR322) are incubated in solutions containing different concentrations of terpyridine platinum (PtTC). In some preparations, dimethyl sulfoxide (DMSO), a free radical scavenger, has been added in order to investigate the role of the free radicals. The complexes of DNA plasmids loaded with high-Z atoms are irradiated under atmospheric conditions by He2+ ions at an energy of 143 MeV/amu and a linear energy transfert (LET) of 2.24 keV/microm. Analysis of DNA damage--single and double strand breaks--is made by electrophoresis on agarose gels. RESULTS: The results show a significant increase in DNA strand breaks when platinum is present, indicating a radiosensitization by the high Z atoms. The increase in DNA damages is attributed to inner-shell ionization of a platinum atom by secondary electrons emitted along the He2+ tracks followed by an Auger deexcitation, leading, thus, to a local amplification of the radiative effects close to the DNA. The contributions of scavengeable--solvant mediated--indirect effects and non-scavengeable effects (direct ionization) are quantitatively evaluated. CONCLUSION: Enhancement of DNA breaks in plasmids loaded with heavy atoms like platinum and irradiated by atomic ions are observed. This finding suggests an enhancement of cell death rate will occur under irradiation by atomic ions when the cells contain high-Z atoms located close to DNA due to the increase of the DNA breaks.

[Pulmonary pleomorphic carcinoma; report of a case].

We report a case of a 64-year-old man with pleomorphic carcinoma of the lung and thymic cyst. He was admitted to our hospital because of an abnormal shadow observed on chest X-ray. Computed tomography (CT) showed a mass lesion located in the right upper lobe and a non-invasive anterior mediastinal tumor adjacent to the left brachiocepharic vein. On enhanced CT, the lung mass showed central low-attenuation areas with a substantial enhancement in the periphery. Preoperative transbronchial blushing cytology of the mass revealed adenocarcinoma. With a diagnosis of primary lung cancer (cT3N0M0) and mediastinal tumor, an operation was performed through a median sternotomy. The mediastinal tumor was excised and a right upper lobectomy and were also accomplished, because the lung tumor did not show adhesion or pleural invasion. Histopathologic examination of the resected specimen revealed that the lung tumor composed of a mixture of spindle and giant cell features and contained a component of adenocarcinoma and squamous cell carcinoma. This finding yielded a pathological diagnosis of pleomorphic carcinoma (pT2N0M0). The mediastinal tumor was diagnosed as thymic cyst. The postoperative course was uneventful, and he is currently well 6 months after surgery.

[Carney triad; report of a case].

We report a case of Carney triad which is very rare disease composed of gastrointestinal stromal tumor (GIST), pulmonary chondroma and paraganglioma. A 15-year-old girl was reffered for treatment with multiple tumors in the left lung. At the age of 13, she underwent total gastrestomy for GIST. At that time multiple pulmonary tumors have already developed and made a diagnosis of chondroma. Progressive enlargement of their size and persistent bloody sputum made her received operation. Finally she underwent left pneumonectomy. In general all 3 tumors have manifested for a long time. Gastric tumors and paragangliomas are often lethal. This shows the necessity of intensive and long-term follow-up.

Real-time observation of irradiated HeLa-cell modified by fluorescent ubiquitination-based cell-cycle indicator using synchrotron X-ray microbeam.

Fluorescent ubiquitination-based cell-cycle indicator (FUCCI) human cancer (HeLa) cells (red indicates G1; green, S/G2) were exposed to a synchrotron X-ray microbeam. Cells in either G1 or S/G2 were irradiated selectively according to their colour in the same microscopic field. Time-lapse micrographs of the irradiated cells were acquired for 24 h after irradiation. For fluorescent immunostaining, phosphorylated histone proteins (γ-H2AX) indicated the induction of DNA double-strand breaks. The cell cycle was arrested by irradiation at S/G2. In contrast, cells irradiated at G1 progressed to S/G2. The foci were induced in cells irradiated at both G1 and S/G2, suggesting that the G1-S (or S) checkpoint pathway does not function in HeLa cells due to the fact that the cells are functionally p53 deficient, even though X-ray microbeam irradiation significantly induces double-strand breaks. These results demonstrate that single FUCCI cell exposure and live cell imaging are powerful methods for studying the effects of radiation on the cell cycle.

Enhancement of X-ray-induced breaks in DNA bound to molecules containing platinum: a possible application to hadrontherapy.

Complexes made of DNA and chloroterpyridine platinum (PtTC) bound to plasmid DNA were placed in aqueous solution and irradiated with monochromatic X rays tuned to the resonant photoabsorption energy of the L(III) shell of the platinum atom. The number of single- and double-strand breaks (SSBs and DSBs) induced by irradiation on a supercoiled DNA plasmid was measured by the production of the circular-nicked and linear forms. To distinguish the contribution of the direct effects of ionization from the indirect effects due to a free radical attack, experiments were also performed in the presence of a hydroxyl free radical scavenger, dimethyl sulfoxide (DMSO). An enhancement of the number of SSBs and DSBs was observed when the plasmids contained the platinum intercalating molecules. A quantitative analysis was made to evaluate the respective contributions of the direct effects (Auger effect) and the indirect effects (free radical attack) to the number of DNA strand breaks. Even when off-resonant X rays were used, the strand break efficiency remained higher than expected based upon the absorption cross section, suggesting that the platinum bound to DNA might be increasing the yield of strand breaks. A mechanism is suggested that involves photoelectrons generated from the ionization of water which efficiently ionize platinum atoms. If this mechanism is correct, then heavy atoms, with a large cross section for ionization by electrons that are bound to the DNA, should behave as a radiosensitizer. This observation may provide insight into understanding the effects of new radiotherapy protocols, related chemotherapeutic agents such as cisplatin, and conventional radiotherapy for the treatment of tumors. A possible way to deliver the dose selectively in a well-defined volume, which uses the properties of the linear energy transfer of atomic ions interacting with matter, is suggested.

Inactivation action spectra of Bacillus subtilis spores with monochromatic soft X rays (0.1-0.6 nm) of synchrotron radiation.

Five types of Bacillus subtilis spores differing in DNA repair and recombinational capacities were exposed in vacuum to monochromatic soft X rays from synchrotron radiation. The inactivation rate constants were obtained from exposure-survival curves upon irradiations at 12 wavelengths in the range of 0.1000 nm (12.40 keV) to 0.6000 nm (2.066 keV). Spores of two repair-deficient strains, UVS (uvrA ssp) and UVP (uvrA ssp polA), exhibited almost equal sensitivities to those of wild-type UVR+, while those of two recombination-deficient strains, RCE (recE) and RCF (recF), exhibited higher sensitivities in the whole wavelength range. This suggested that the repair of DNA damage produced by soft X rays was dependent on the recombinational capabilities. Inactivation action spectra based on photon fluence showed that the effectiveness of the radiation increased as the wavelengths became longer. Abrupt changes in the effectiveness occurred around the wavelengths corresponding to the absorption edges of K-shell electrons of phosphorus and calcium. In both cases, the sensitivity was the highest at the wavelengths of the resonance absorption peak, the next highest at those of the higher energy, and the lowest at the lower energy. Mass energy absorption coefficients of spores were obtained from the transmission of a flake made of spores. They were used to derive inactivation action spectra based on absorbed doses. In these spectra, basal levels of the sensitivity seemed constant, and enhancements of the sensitivity were observed consistent with the absorption by calcium and phosphorus. Thus calcium and phosphorus atoms were the predominant targets for the absorption events leading to the inactivation of spores in the wavelength range examined.

Strand break induction by photoabsorption in DNA-bound molecules.

Dried samples of a DNA-chloroterpyridine platinum complex were irradiated with monochromatic X rays tuned to the photoabsorption resonance of the L(III) inner shell of the platinum atom. The number of single- and double-strand breaks (SSBs and DSBs) triggered by the Auger effect in supercoiled DNA plasmids was measured by the production of circular nicked and linear forms. To probe the specific contribution of the L(III) inner-shell excitation of the platinum atom, photon wavelengths were tuned on the resonance energy (on peak) and below (off peak). The quantum yields of the resonance radiation were typically found to be 11 for the SSBs and 1 for the DSBs. The DSB-to-SSB ratio increased by 20% when switching from off-resonance to on-resonance irradiation.

Oxidation yield of the ferrous ion in a Fricke solution irradiated with monochromatic synchrotron soft X-rays in the 1.8-10 keV region.

The oxidation yield of ferrous ion in a Fricke solution was measured in the soft X-ray region from 1.8 to 10 keV. The standard Fricke solution was irradiated with monochromatic X-rays from synchrotron radiation. The yield decreased with decreasing X-ray energy, as several theoretical calculations have predicted. No significant changes were observed in the K-shell photo-absorption of the iron and sulphur contained in the standard Fricke solution. The high-LET nature of soft X-rays is discussed.

Biological effects of Auger processes of bromine on yeast cells induced by monochromatic synchrotron X-rays.

The biological effects of inner-shell ionization in bromine atoms incorporated into DNA in the form of bromodeoxyuridine monophosphate (BrdUMP), induced by monochromatized synchrotron X-rays, were studied using a deoxythymidine monophosphate (dTMP)-permeable mutant of yeast, Saccharomyces cerevisiae. The BrdUMP-incorporated yeast cells were irradiated with monochromatic X-rays of 13.51 or 13.45 keV, between which the bromine K-absorption edge (13.47 keV) is located. The cells were 1.07 times more sensitive to irradiation by 13.51 keV X-rays than at 13.45 keV, while dTMP-incorporated cells did not show any difference in sensitivity. In the presence of a radioprotector during irradiation, BrdUMP-incorporated cells showed a larger enhancement (1.20). These enhancements observed in the bromine-incorporated cells cannot be explained only by an increase of the absorbed dose due to a substitution of CH3 group of thymine by bromine. It may be concluded that a major part of the enhancement was caused by inner-shell photoionization, followed by an Auger cascade of the bromine in the DNA. The quantum yield of lethality caused by the photoabsorption of bromine K-shell is not affected by the presence of cysteamine, suggesting the biological enhancement by the Auger processes may not be influenced by chemical protection.

Mutagenic and transforming effects of soft-X-rays with resonance energy of phosphorus K-absorption edge.

Syrian golden hamster embryo (SHE) cells were exposed to synchrotron-produced monochromatic X-rays at 5.747 (2.159 keV), 5.763 (2.153 keV) and 5.779 A (2.147 keV). Although X-rays of all wavelengths induced mutations and chromatid aberrations in a dose-dependent manner, when cells were irradiated with 2.153 keV X-rays, which correspond to the resonance energy of the phosphorus K-absorption edge, the frequencies of mutation and chromatid aberration at equal dose levels were higher than for X-rays of the other wavelengths. At equal survival levels, however, there was no difference in the frequencies of mutations and chromatid aberrations in cells irradiated with soft X-rays. On the other hand, the frequency of morphological transformation in cells irradiated with 2.147 keV X-rays was higher than those irradiated with 2.153 keV and 2.159 keV X-rays. The relative biological effectiveness compared to cobalt-60 gamma-rays in morphological transformation was 2.8 for 2.147 keV, 1.1 for 2.159 keV and 1.0 for 2.153 keV at a 37% survival level.

Single- and double-strand breaks in pBR322 plasmid DNA by monochromatic X-rays on and off the K-absorption peak of phosphorus.

Using a synchrotron irradiation system pBR322 plasmid DNA was irradiated under vacuum by monochromatic X-rays having five specific photon energies (2.147, 2.153, 2.159, 2.168 and 2.199 keV) both on and off the K-absorption peak (2.153 keV) of phosphorus. The single- and double-strand breaks (ssb and dsb) were measured as conversions of the closed circular form of DNA (form I) to open circular (form II) and linear (form III) forms respectively. Exposures to induce one strand break per molecule were lowest at the peak (2.153 keV), and highest at 2.147 keV; the ratios were 2.7 for ssb and 3.0 for dsb. The exposures for dsb were 21-26 times higher than those for ssb. When the exposures were converted to absorbed doses in grays the absorbed doses per ssb were almost independent of photon energy. This result indicates that a certain absorbed dose was necessary to induce a ssb, regardless of whether photons were absorbed by the K-shell of phosphorus or by other shells, or by other atoms. However, the absorbed dose per dsb at 2.147 keV was 1.17 times higher than that averaged over four X-ray energies above 2-153 keV, indicating that the K-shell absorption, and the subsequent Auger event, efficiently induce dsb. The results are also discussed concerning the number of photo-absorptions of the constituent atoms per DNA strand break.

Identity of dimeric dihydrodiol dehydrogenase as NADP(+)-dependent D-xylose dehydrogenase in pig liver.

Dimeric dihydrodiol dehydrogenases (DDs, EC 1.3.1.20), which oxidize trans-dihydrodiols of aromatic hydrocarbons to the corresponding catechols, have been molecularly cloned from human intestine, monkey kidney, pig liver, dog liver, and rabbit lens. A comparison of the sequences with the DNA sequences in databases suggested that dimeric DDs constitute a novel protein family with 20 gene products. In addition, it was found that dimeric DD oxidizes several pentoses and hexoses, and the specificity resembles that of NADP(+)-dependent D-xylose dehydrogenase (EC 1.1.1.179) of pig liver. The inhibition of D-xylose dehydrogenase activity in the extracts of monkey kidney, dog liver and pig liver, its co-purification with dimeric DD activity from pig liver, and kinetic analysis of the D-xylose reduction by pig dimeric DD indicated that the two enzymes are the same protein.