Dosimetric properties of a proton beamline dedicated to the treatment of ocular disease.

PURPOSE: A commercial proton eyeline has been developed to treat ocular disease. Radiotherapy of intraocular lesions (e.g., uveal melanoma, age-related macular degeneration) requires sharp dose gradients to avoid critical structures like the macula and optic disc. A high dose rate is needed to limit patient gazing times during delivery of large fractional dose. Dose delivery needs to be accurate and predictable, not in the least because current treatment planning algorithms have limited dose modeling capabilities. The purpose of this paper is to determine the dosimetric properties of a new proton eyeline. These properties are compared to those of existing systems and evaluated in the context of the specific clinical requirements of ocular treatments. METHODS: The eyeline is part of a high-energy, cyclotron-based proton therapy system. The energy at the entrance of the eyeline is 105 MeV. A range modulator (RM) wheel generates the spread-out Bragg peak, while a variable range shifter system adjusts the range and spreads the beam laterally. The range can be adjusted from 0.5 up to 3.4 g/cm(2); the modulation width can be varied in steps of 0.3 g/cm(2) or less. Maximum field diameter is 2.5 cm. All fields can be delivered with a dose rate of 30 Gy/min or more. The eyeline is calibrated according to the IAEA TRS-398 protocol using a cylindrical ionization chamber. Depth dose distributions and dose/MU are measured with a parallel-plate ionization chamber; lateral profiles with radiochromic film. The dose/MU is modeled as a function of range, modulation width, and instantaneous MU rate with fit parameters determined per option (RM wheel). RESULTS: The distal fall-off of the spread-out Bragg peak is 0.3 g/cm(2), larger than for most existing systems. The lateral penumbra varies between 0.9 and 1.4 mm, except for fully modulated fields that have a larger penumbra at skin. The source-to-axis distance is found to be 169 cm. The dose/MU shows a strong dependence on range (up to 4%/mm). A linear increase in dose/MU as a function of instantaneous MU rate is observed. The dose/MU model describes the measurements with an accuracy of ± 2%. Neutron dose is found to be 146 ± 102 µSv/Gy at the contralateral eye and 19 ± 13 µSv/Gy at the chest. CONCLUSIONS: Measurements show the proton eyeline meets the requirements to effectively treat ocular disease.

Functional imaging in treatment planning in radiation therapy: a review

No disponible

Treatment of rectal adenocarcinoma with endocavitary and external beam radiotherapy: results for 199 patients with localized tumors.

PURPOSE: Endocavitary radiation (RT) provides a conservative alternative to proctectomy. Although most suitable for small, mobile lesions, patients with less favorable tumors are often referred if they are poor surgical candidates. Knowing the extent to which radiation can control such tumors can be an important factor in making clinical decisions. METHODS AND MATERIALS: One hundred ninety-nine patients, who received endocavitary RT with or without external beam RT (EBRT) during 1981 through 1995, were followed for disease status for a median of 70 months, including deaths from intercurrent causes. In the early years of the study, 21 patients were treated with endocavitary RT alone, the remainder of the patients received pelvic EBRT (usually 45 Gy in 25 fractions) 5-7 weeks before endocavitary RT. RESULTS: Overall, 141 patients (71%) had local control with RT alone. Salvage surgery rendered an additional 20 patients disease free, for an ultimate local control rate of 81%. On multivariate analysis for local control (excluding surgical salvage), the most significant factors were mobility to palpation, use of EBRT, and whether pretreatment debulking of all macroscopic disease had been done (generally a piecemeal, nontransmural procedure). Of 77 cases staged by transrectal ultrasonography, the local control rate with RT alone was 100% for uT1 lesions, 85% (90% with no evidence of disease after salvage) for freely mobile uT2 lesions, and 56% (67% with no evidence of disease after salvage) for uT3 lesions and uT2 lesions that were not freely mobile. CONCLUSIONS: Patients with small mobile tumors that are either uT1 or have only a scar after debulking achieve excellent local control with endocavitary RT. About 15% of mobile uT2 tumors fail RT; therefore, careful follow-up is critical. Small uT3 tumors are appropriate for this treatment only if substantial contraindications to proctectomy are present.

Cytometric methods to analyze ionizing-radiation effects.

Four cytometric assays for the assessment of radiation-induced DNA damage in individual cells are presented. Two of these, the alkaline and neutral comet assays, are useful for the detection of DNA damage due to very low radiation doses and promise to be useful for the quantitation of genomic damage after clinically or environmentally relevant exposures. The other two, the halo and halo-comet assays, reveal aspects of chromatin structure in the presence of DNA damage that reflect differences in intrinsic cellular radiosensitivity. Further development of these assays used alone, or in combination, should eventually lead to the definition of readily measurable cytometric parameters that will be useful as predictive markers for cellular responses to DNA damaging agents.

Neoplastic transformation in C3H 10T(1/2) cells after exposure to 835.62 MHz FDMA and 847.74 MHz CDMA radiations.

The effect of radiofrequency (RF) radiation in the cellular phone communication range (835.62 MHz frequency division multiple access, FDMA; 847.74 MHz code division multiple access, CDMA) on neoplastic transformation frequency was measured using the in vitro C3H 10T(1/2) cell transformation assay system. To determine if 835.62 MHz FDMA or 847.74 MHz CDMA radiations have any genotoxic effects that induce neoplastic transformation, C3H 10T(1/2) cells were exposed at 37 degrees C to either of the above radiations [each at a specific absorption rate (SAR) of 0.6 W/kg] or sham-exposed at the same time for 7 days. After the culture medium was changed, the cultures were transferred to incubators and refed with fresh growth medium every 7 days. After 42 days, the cells were fixed and stained with Giemsa, and transformed foci were scored. To determine if exposure to 835.62 MHz FDMA or 847.74 MHz CDMA radiation has any epigenetic effects that can promote neoplastic transformation, cells were first exposed to 4.5 Gy of X rays to induce the transformation process and then exposed to the above radiations (SAR = 0.6 W/kg) in temperature-controlled irradiators with weekly refeeding for 42 days. After both the 7-day RF exposure and the 42-day RF exposure after X irradiation, no statistically significant differences in the transformation frequencies were observed between incubator controls, the sham-exposed (maintained in irradiators without power to the antenna), and the 835.62 MHz FDMA or 847.74 MHz CDMA-exposed groups.

The human Hsp70B gene at the HSPA7 locus of chromosome 1 is transcribed but non-functional.

The human heat-inducible Hsp70B and Hsp70B' genes were co-localized to 1q23.1 by in situ hybridization. However, though transcripts from Hsp70B could be detected in heat-shocked cells, DNA sequence analyses of both the gene and cDNA copies of the mRNA indicate the gene is non-functional. Moreover, mouse homologues of Hsp70B/B' were not detected by Southern blot analysis, suggesting Hsp70B/B' arose from either Hsp70-1or Hsp70-2 after the divergence of mice and humans.

Cell phones and cancer: what is the evidence for a connection?

There have been allegations in the media and in the courts that cell phones and other types of hand-held transceivers are a cause of cancer. There have also been numerous public objections to the siting of TV, radio and cell phone transmission facilities because of a fear of cancer induction. A recent publication in Radiation Research by Repacholi et al. (147, 631-640, 1997) which suggests that exposure to radiofrequency (RF) radiation may increase lymphoma incidence in mice has contributed to this controversy. The goal of this review is to provide biomedical researchers a brief overview of the existing RF radiation-cancer studies. This article begins with a brief review of the physics and technology of cell phones. It then reviews the existing epidemiological studies of RF radiation, identifying gaps in our knowledge. Finally, the review discusses the cytogenetics literature on RF radiation and the whole-animal RF-radiation carcinogenesis studies. The epidemiological evidence for an association between RF radiation and cancer is found to be weak and inconsistent, the laboratory studies generally do not suggest that cell phone RF radiation has genotoxic or epigenetic activity, and a cell phone RF radiation-cancer connection is found to be physically implausible. Overall, the existing evidence for a causal relationship between RF radiation from cell phones and cancer is found to be weak to nonexistent.

DNA damage in rat brain cells after in vivo exposure to 2450 MHz electromagnetic radiation and various methods of euthanasia.

The present study was done to confirm the reported observation that low-intensity acute exposure to 2450 MHz radiation causes DNA single-strand breaks (Lai and Singh, Bioelectromagnetics 16, 207-210, 1995). Male Sprague-Dawley rats weighing approximately 250 g were irradiated with 2450 MHz continuous-wave (CW) microwaves for 2 h at a specific absorption rate of 1.2 W/kg in a cylindrical waveguide system (Guy et al., Radio Sci. 14, 63-74, 1979). There was no associated rise in the core body temperature of the rats. After the irradiation or sham treatments, rats were euthanized by either CO2 asphyxia or decapitation by guillotine (eight pairs of animals per euthanasia group). After euthanasia the brains were removed and immediately immersed in cold Ames medium and the cells of the cerebral cortex and the hippocampus were dissociated separately and subjected to the alkaline comet assay. Irrespective of whether the rats were euthanized by CO2 asphyxia or decapitated by guillotine, no significant differences were observed between either the comet length or the normalized comet moment of cells from either the cerebral cortex or the hippocampus of sham-treated rats and those from the irradiated rats. However, the data for the rats asphyxiated with CO2 showed more intrinsic DNA damage and more experiment-to-experiment variation than did the data for rats euthanized by guillotine. Therefore, the guillotine method of euthanasia is the most appropriate in studies relating to DNA damage. Furthermore, we did not confirm the observation that DNA damage is produced in cells of the rat cerebral cortex or the hippocampus after a 2-h exposure to 2450 MHz CW microwaves or at 4 h after the exposure.

Detection of DNA damage by the alkaline comet assay after exposure to low-dose gamma radiation.

The alkaline comet assay as described by Olive et al. (Exp. Cell Res. 198, 259-267, 1992) was used to detect DNA damage in cells exposed to low doses (0-5 cGy) of gamma radiation. Experiments were performed using lymphocytes isolated from whole blood of rats. The comet parameters, normalized comet moment and comet length, described by Kent et al. (Int. J. Radiat. Biol. 67, 655-660, 1995), were used as measurements of DNA damage. It was observed that the alkaline comet assay can detect DNA damage at doses as low as 0.6 cGy. The results of the experiments using low-dose gamma radiation are comparable with published results obtained using the alkaline comet assay according to the method of Singh et al. (Int. J. Radiat. Biol. 66, 23-28, 1994). Based on this observation and analysis of results published previously, we conclude that the version of the alkaline comet assay described by Olive et al. is as sensitive as other modifications of the comet assay reported in literature for the detection of DNA damage in cells exposed to low doses of ionizing radiation.

Nuclear matrix as a target for hyperthermic killing of cancer cells.

The nuclear matrix organizes nuclear DNA into operational domains in which DNA is undergoing replication, transcription or is inactive. The proteins of the nuclear matrix are among the most thermal labile proteins in the cell, undergoing denaturation at temperatures as low as 43-45 degrees C, i.e. relevant temperatures for the clinical treatment of cancer. Heat shock-induced protein denaturation results in the aggregation of proteins to the nuclear matrix. Protein aggregation with the nuclear matrix is associated with the disruption of many nuclear matrix-dependent functions (e.g. DNA replication, DNA transcription, hnRNA processing, DNA repair, etc.) and cell death. Heat shock proteins are believed to bind denatured proteins and either prevents aggregation or render aggregates more readily dissociable. While many studies suggest a role for Hsp70 in heat resistance, we have recently found that nuclear localization/delocalization of Hsp70 and its rate of synthesis, but not its amount, correlate with a tumor cell's ability to proliferate at 41.1 degrees C. These results imply that not only is the nuclear matrix a target for the lethal effects of heat, but it also is a target for the protective, chaperoning and/or enhanced recovery effects of heat shock proteins.

Measurement of DNA damage after exposure to 2450 MHz electromagnetic radiation.

Recent reports suggest that exposure to 2450 MHz electromagnetic radiation causes DNA single-strand breaks (SSBs) and double-strand breaks (DSBs) in cells of rat brain irradiated in vivo (Lai and Singh, Bioelectromagnetics 16, 207-210, 1995; Int. J. Radiat. Biol. 69, 513-521, 1996). Therefore, we endeavored to determine if exposure of cultured mammalian cells in vitro to 2450 MHz radiation causes DNA damage. The alkaline comet assay (single-cell gel electrophoresis), which is reportedly the most sensitive method to assay DNA damage in individual cells, was used to measure DNA damage after in vitro 2450 MHz irradiation. Exponentially growing U87MG and C3H 10T1/2 cells were exposed to 2450 MHz continuous-wave (CW) radiation in specially designed radial transmission lines (RTLs) that provided relatively uniform microwave exposure. Specific absorption rates (SARs) were calculated to be 0.7 and 1.9 W/kg. Temperatures in the RTLs were measured in real time and were maintained at 37 +/- 0.3 degrees C. Every experiment included sham exposure(s) in an RTL. Cells were irradiated for 2 h, 2 h followed by a 4-h incubation at 37 degrees C in an incubator, 4 h and 24 h. After these treatments samples were subjected to the alkaline comet assay as described by Olive et al. (Exp. Cell Res. 198, 259-267, 1992). Images of comets were digitized and analyzed using a PC-based image analysis system, and the "normalized comet moment" and "comet length" were determined. No significant differences were observed between the test group and the controls after exposure to 2450 MHz CW irradiation. Thus 2450 MHz irradiation does not appear to cause DNA damage in cultured mammalian cells under these exposure conditions as measured by this assay.

Measurement of DNA damage after exposure to electromagnetic radiation in the cellular phone communication frequency band (835.62 and 847.74 MHz).

Mouse C3H 10T1/2 fibroblasts and human glioblastoma U87MG cells were exposed to cellular phone communication frequency radiations to investigate whether such exposure produces DNA damage in in vitro cultures. Two types of frequency modulations were studied: frequency-modulated continuous-wave (FMCW), with a carrier frequency of 835.62 MHz, and code-division multiple-access (CDMA) centered on 847.74 MHz. Exponentially growing (U87MG and C3H 10T1/2 cells) and plateau-phase (C3H 10T1/2 cells) cultures were exposed to either FMCW or CDMA radiation for varying periods up to 24 h in specially designed radial transmission lines (RTLs) that provided relatively uniform exposure with a specific absorption rate (SAR) of 0.6 W/kg. Temperatures in the RTLs were monitored continuously and maintained at 37 +/- 0.3 degrees C. Sham exposure of cultures in an RTL (negative control) and 137Cs gamma-irradiated samples (positive control) were included with every experiment. The alkaline comet assay as described by Olive et al. (Exp. Cell Res. 198, 259-269, 1992) was used to measure DNA damage. No significant differences were observed between the test group exposed to FMCW or CDMA radiation and the sham-treated negative controls. Our results indicate that exposure of cultured mammalian cells to cellular phone communication frequencies under these conditions at an SAR of 0.6 W/kg does not cause DNA damage as measured by the alkaline comet assay.

A comparison of the modes and kinetics of heat-induced cell killing in HeLa and L5178Y cells.

The mode and kinetics of cell killing in HeLa and L5178Y cells were investigated to elucidate possible relationships between the mechanisms and modes of heat-induced cell death. L5178Y cells were heat-shocked for either 24 min at 43 degrees C or 30 min at 45 degrees C, while HeLa cells were given only the latter treatment. The degree of heat-induced excess nuclear protein correlated with cell death in HeLa cells but not in L5178Y cells. This difference suggests that the mechanism of cell death differs between these cell lines. With L5178Y cells the main mode of death after treatment at 43 degrees C was apoptosis with little progression of cells through the cell cycle. However, after treatment at 45 degrees C these cells died by necrosis without progressing through the cell cycle. HeLa cells were found to die by a mechanism other than apoptosis after significant progression of cells through the cell cycle and perturbation of the normal distribution of cells in the phases of the cell cycle (specifically, the fraction of cells in S and G2 phase increased 50% and 30%, respectively, prior to the occurrence of measurable cell death). These results are consistent with the hypothesis that the response to injury which has the potential to be lethal varies between different cell types, and results in the induction of different pathways leading to cell death.

DNA supercoiling changes and nuclear matrix-associated proteins: possible role in oncogene-mediated radioresistance.

PURPOSE: Transfection with either H-ras or H-ras and c-myc has been shown to confer radioresistance in rat embryonal cells (REC). REC primary, transfected with either c-myc, H-ras or cotransfected with c-myc and H-ras (in ascending order of radioresistance and tumorigenicity), were used as an in vitro model system to determine if nuclear matrix-mediated higher order DNA organization contributes to oncogene-mediated radioresistance. METHODS AND MATERIALS: DNA damage induction and repair were measured by the alkaline and neutral filter elution assays. Analysis of the ability of DNA loop domains to undergo supercoiling changes in the presence of radiation-induced damage was determined by the fluorescent halo assay (FHA). Because DNA loops are organized by the nuclear matrix (NM), a study of NM-associated proteins by high resolution two-dimensional gel electrophoresis was performed. RESULTS: Induction and repair rates of DNA single- and double-strand breaks were similar for the relatively radiosensitive c-myc transfected and the radioresistant c-myc + H-ras transfected cells. However, the degree of inhibition of DNA supercoil rewinding in the presence of radiation-induced damage was less in the radioresistant cells and was inversely correlated with survival. A progressive loss of NM-associated proteins was observed, which correlated with increasing radioresistance and tumorigenicity in these cell lines. In addition, some protein changes were consistent with the possibility that these changes could be involved in DNA anchoring. CONCLUSIONS: Increased radioresistance associated with increasing tumorigencity in these oncogene-transfected cell lines could be due to changes in NM-mediated DNA organization, possibly via differences in NM protein composition that occur following oncogenic transfection.

DNA supercoiling changes and nucleoid protein composition in a group of L5178Y cells of varying radiosensitivity.

Cell of the radioresistant L5178Y-R, -S35, -SR and M10(neo 5)-1 and radiosensitive L5178Y-S, M10 and LX830 cell lines were used to investigate the relationship between radiosensitivity and DNA supercoiling ability mediated by the nuclear matrix within chromatin loops containing DNA damage. The ability of DNA loops to undergo changes in supercoiling in the presence of radiation-induced damage revealed that in all cases the degree of inhibition of supercoil rewinding was greater in the radiosensitive cells. Since the amount of DNA damage induced per unit dose is known to be equal in all these cell lines, the same number of DNA lesions produced a greater loss of topological constraint in the radiosensitive cells. The differential loss of DNA supercoiling ability could be due to differences in DNA-nuclear matrix anchor points. High-resolution two-dimensional gel electrophoresis of nucleoid proteins showed numerous reproducible differences in nuclear matrix protein between the cell lines studied. A total of nine proteins were associated with nucleoids from L5178Y-R cells and absent from L5178Y-S nucleoids. None of them, however, correlated absolutely with radioresistance. Thus, unlike previous studies in CHO cells, no candidates for the conveyance of cellular radiosensitivity that were single proteins were detected. However, these results are consistent with the hypothesis that stability of DNA loop domains in the presence of DNA damage is a determinant of the outcome of radiation-induced DNA damage.

Radiation sensitivity correlates with changes in DNA supercoiling and nucleoid protein content in cells of three Chinese hamster cell lines.

We have investigated the composition of nuclear matrix proteins and DNA supercoiling characteristics of cell lines expressing altered radiation sensitivity. Chinese hamster ovary cell lines 4364 (wild-type), XR-1 (DSB repair-deficient, radiosensitive) and XR-122 (a radioresistant variant of XR-1 bearing human chromosome 5) were used as a model to study the relationship between intrinsic radiation sensitivity and the level of DNA supercoiling ability within chromatin loops and the composition of nuclear matrix proteins. Analysis of the ability of DNA loop domains to undergo changes in DNA supercoiling in the presence of DNA damage revealed that the degree of inhibition of loop rewinding was greater in the radiation-sensitive cells (XR-1) compared to the radiation-resistant cells (4364 and XR-122). Furthermore, the loop-rewinding characteristics correlated inversely with the clonogenic survival of these cells after exposure to ionizing radiation. Since DNA loops are anchored to the nuclear matrix by protein-DNA anchor points, a study of the nuclear matrix proteins by high-resolution 2D-PAGE was conducted for these cells to determine whether differential inhibition of loop rewinding could be due to differences in the DNA loop-protein anchor points in these cells. The XR-1 cells showed an overall absence of 13 proteins compared to the 4364 cells. Of these 13, 5 were restored in XR-122 cells. These results are consistent with the hypothesis that stability of the DNA loop domains in the presence of DNA damage contributes to the expression of potentially lethal damage by ionizing radiation.

Variation in heat sensitivity through the cell cycle of M10 and Burkitt P3HR-1 cells.

The cell-cycle age response to 44 or 45 degrees C hyperthermic treatment was studied in M10, a mutant of mouse L5178Y cells, and human Burkitt P3HR-1 cells synchronized by centrifugal elutriation. Survival response to 44 degrees C hyperthermia or radiation showed that Burkitt cells were relatively hyperthermic resistant and average in radiosensitivity, while M10 cells were relatively hyperthermic sensitive and substantially radiosensitive. A typical age-response to hyperthermia through the cell-cycle was observed in M10 cells, although no significant variation in the response to hyperthermia was demonstrated in Burkitt P3HR-1 cells. Combined effect of procaine with 44 degrees C 20-min treatment for Burkitt P3HR-1 cells showed a substantial enhancement of cytotoxicity in S phase cells. The probable implication of membrane composition, membrane changes due to heat and its modifying agents are discussed. Furthermore, when hyperthermia was combined with radiation in Burkitt P3HR-1 cells, cytotoxic enhancement was observed in G1/S boundary phase, not in S phase.

Cell-cycle dependence of heat-induced interphase death in mouse L5178Y cells.

Cell lysis and eosin staining were observed in L5178Y cells within the first 3 h of post-hyperthermia incubation at 37 degrees C, after which both leveled to a plateau. Lysis and eosin staining were proportional to the severity of heat in asynchronous cells, whereas it was maximum in the most heat-sensitive M phase, intermediate in S, and least in heat-resistant G1 for the same heat treatment. Further, leakage of labeled [3H]thymidine and a decrease in radioactivity retained within heated cells coincided with an increase in eosin staining, indicating that the dye uptake was due to membrane damage. It was presumed that the eosin-stained fraction represented dead cells. The percentage eosin-stained cells reached a plateau, and this level was used to determine survival; when the results were compared with those obtained by the colony formation method, they were identical. By comparing the two survival assay methods we concluded that cell death after hyperthermia in L5178Y cells is mainly by interphase death in all phases of the cell cycle. The reasons for this conclusion are that a reduction in survival could be detected within one generation of L5178Y cells by the eosin staining method, and the survival values obtained by this method were identical to those obtained by the colony formation method.