- Email: [email protected]
Exposure to low dose (1-10 cGy) ionizing radiation: assessment of effects in humans and relevance to cancer
Proceedings of the 44th Annual ASTRO Meeting
Results: 1) Nuclear PP1 is activated in response to IR through dephosphorylation of the Thr-320 C-terminal residue. In Jurkat cells and ATM cells transfected with full-length ATM, IR resulted in dephosphorylation of the Thr-320 site and activation of PP1c. However, in AT cells IR failed to induce dephosphorylation of this site or activation of PP1c. 2) PP1c is physically associated with HDAC1 as measured by microcystin affinity chromatography and co-immunoprecipitation. IR induces disassociation of HDAC1 and PP1c in an ATM -dependent manner. IR also induced action of HDAC1. 3) PP1c is physically associated with Rb as measured by co-immunoprecipitation. IR induces disassociation of Rb and PP1c in a time-dependent and reversible manner. Conclusions: PP1 provides a fundamental link between the upstream DNA damage-sensing protein ATM and downstream checkpoint effector proteins Rb and HDAC1.
87
Exposure to Low Dose (1-10 cGy) Ionizing Radiation: Assessment of Effects in Humans and Relevance to Cancer
Z. Goldberg1, C.W. Schwietert1, R.L. Stern1, M. Arnold2, C.L. Hartmann Siantar2, R. Cary3, M. Descalle2, B.E. Lehnert3 1 Dept. of Radiation Oncology, University of California Davis, Sacramento, CA, 2Laurence Livermore National Laboratory, Livermore, CA, 3Los Alamos National Laboratory, Los Alamos, NM Purpose/Objective: Significant biological effects can occur in animals, animal cells, immortalized human cell lines and primary human cells after exposure to doses of ionizing radiation (IR) in the ⬍1-10 cGy range. However, it is unknown how these observations mimic or even relate to actual human responses when exposed to radiation in vivo. Yet this information is crucial in determining safe exposure levels for health and regulatory purposes. Thus, human translational data must be obtained with which to correlate in vitro experimental findings and evaluate their applicability to “real life”. Materials/Methods: Using human skin samples, irradiated in vivo during therapeutic radiation, gene expression changes are assessed by microarray hybridization technology. Preliminary studies, for system validation, have been completed on tissue samples obtained from aesthetic surgery. Core biopsies of 3 mm diameter are obtained and subject to mock irradiation (IR), 1 cGy, 10 cGy or 100 cGy. RNA is extracted at multiple time points, out to 24 hours post IR, allowing for a temporal profile of gene expression changes to be determined and the stability of message in unirradiated samples to be verified. The RNA is hybridized to a 25,000 cDNA microarray. The physics components of the study use film and ion chamber measurements to validate the PEREGRINE Monte Carlo simulations that determine the biopsy site for the actual patient sample collection. Results: Both depth dose and profile curves have been measured experimentally and simulated using the PEREGRINE 3D Monte Carlo code. PEREGRINE simulations can model dose delivered in areas beyond the field edge, underneath blocks, collimator jaws, or both within 5-15% of measured values. Depth doses show an initial rapid rise in dose, which peaks at less than 2 cm depth, followed by a sharp decrease in dose to a 30-40% of dmax at a depth of 5 cm. Beyond 5 cm, the depth dose curves increase minimally with depth. The biologic components of the model have been successfully tested. RNA can be extracted from small tissue samples and amplified with fidelity. Tissues from different areas of the body have been sampled and compared within a given individual to examine homogeneity of the skin response. Interarray variability has been examined and quantified within our system. Preliminary gene expression microarray hybridization data have suggested that as many as 116 genes have altered expression of at least a 2-fold extent following exposure to as little as 1 cGy. Conclusions: PEREGRINE is capable of determining within 5-15% the actual site of 1-10 cGy dose delivery to skin in areas outside of the treatment beam. Towards minimizing uncertainties in the final analysis, exit-surface collection of the samples is planned. Coupled with real time TLD measurements, we anticipate overall dosimetric uncertainties of 5%. We have validated the feasibility of testing the biologic components of the model, using an ex vivo irradiation model, as well as by showing gene changes in skin at 1 cGy. Skin samples exposed to IR in vivo can now be obtained from patients undergoing therapeutic IR to begin to define the cellular response to low dose IR in a complex biologic system.
88
Glioblastoma Cell Line Killing by Fractionated Radiation Does Not Follow the Assumption of Equal Effect Per Fraction
M. Sarvi, L. Sappelsa, E.R. Blazek Department of Radiation Oncology, Rush University, Chicago, IL Purpose/Objective: The survival of cells exposed to fractionated irradiation is termed effective survival. It is generally assumed that the effective surviving fraction (ESF) can be calculated using the linear-quadratic (LQ) model for the survival from one dose. This model assumes that n fractions of dose d are separated sufficiently for complete repair of sublethal damage, and that successive fractions have equal effect: ESFLQ(n ⫻ d) ⫽ [exp(-␣d–d2)]n (1) The aim of this study was to measure ESF curves for two glioblastoma multiforme (GBM) cell lines, U87MG and T98G, and to compare these curves to those expected from Eq. 1 using values of ␣ and determined from our single-fraction survival curves. Materials/Methods: T98G and U87MG obtained from the ATCC were harvested using trypsin-EDTA, and single-cell suspensions were counted by hemacytometer. Suspensions were serially diluted into 96-well culture plates at different densities for each dose and fractionation schedule, and irradiations were performed with a 137Cs irradiator at 0.8 Gy/min at room temperature. For single-dose experiments, duplicate plates received 2-18 Gy at 2-Gy increments. Fractionated irradiations were performed daily (including weekends) as follows: Duplicate plates received either 1-30 fractions of 2 Gy/d, 1-15 fractions of 4 Gy/d, or 1-10 fractions of 6 Gy/d. Four weeks after the last radiation fraction, cells were stained with crystal violet. In the dilution clonogenic assay, the well control fraction WCF is the fraction of all wells without any colonies of at least 50 cells.
53
Results: 1) Nuclear PP1 is activated in response to IR through dephosphorylation of the Thr-320 C-terminal residue. In Jurkat cells and ATM cells transfected with full-length ATM, IR resulted in dephosphorylation of the Thr-320 site and activation of PP1c. However, in AT cells IR failed to induce dephosphorylation of this site or activation of PP1c. 2) PP1c is physically associated with HDAC1 as measured by microcystin affinity chromatography and co-immunoprecipitation. IR induces disassociation of HDAC1 and PP1c in an ATM -dependent manner. IR also induced action of HDAC1. 3) PP1c is physically associated with Rb as measured by co-immunoprecipitation. IR induces disassociation of Rb and PP1c in a time-dependent and reversible manner. Conclusions: PP1 provides a fundamental link between the upstream DNA damage-sensing protein ATM and downstream checkpoint effector proteins Rb and HDAC1.
87
Exposure to Low Dose (1-10 cGy) Ionizing Radiation: Assessment of Effects in Humans and Relevance to Cancer
Z. Goldberg1, C.W. Schwietert1, R.L. Stern1, M. Arnold2, C.L. Hartmann Siantar2, R. Cary3, M. Descalle2, B.E. Lehnert3 1 Dept. of Radiation Oncology, University of California Davis, Sacramento, CA, 2Laurence Livermore National Laboratory, Livermore, CA, 3Los Alamos National Laboratory, Los Alamos, NM Purpose/Objective: Significant biological effects can occur in animals, animal cells, immortalized human cell lines and primary human cells after exposure to doses of ionizing radiation (IR) in the ⬍1-10 cGy range. However, it is unknown how these observations mimic or even relate to actual human responses when exposed to radiation in vivo. Yet this information is crucial in determining safe exposure levels for health and regulatory purposes. Thus, human translational data must be obtained with which to correlate in vitro experimental findings and evaluate their applicability to “real life”. Materials/Methods: Using human skin samples, irradiated in vivo during therapeutic radiation, gene expression changes are assessed by microarray hybridization technology. Preliminary studies, for system validation, have been completed on tissue samples obtained from aesthetic surgery. Core biopsies of 3 mm diameter are obtained and subject to mock irradiation (IR), 1 cGy, 10 cGy or 100 cGy. RNA is extracted at multiple time points, out to 24 hours post IR, allowing for a temporal profile of gene expression changes to be determined and the stability of message in unirradiated samples to be verified. The RNA is hybridized to a 25,000 cDNA microarray. The physics components of the study use film and ion chamber measurements to validate the PEREGRINE Monte Carlo simulations that determine the biopsy site for the actual patient sample collection. Results: Both depth dose and profile curves have been measured experimentally and simulated using the PEREGRINE 3D Monte Carlo code. PEREGRINE simulations can model dose delivered in areas beyond the field edge, underneath blocks, collimator jaws, or both within 5-15% of measured values. Depth doses show an initial rapid rise in dose, which peaks at less than 2 cm depth, followed by a sharp decrease in dose to a 30-40% of dmax at a depth of 5 cm. Beyond 5 cm, the depth dose curves increase minimally with depth. The biologic components of the model have been successfully tested. RNA can be extracted from small tissue samples and amplified with fidelity. Tissues from different areas of the body have been sampled and compared within a given individual to examine homogeneity of the skin response. Interarray variability has been examined and quantified within our system. Preliminary gene expression microarray hybridization data have suggested that as many as 116 genes have altered expression of at least a 2-fold extent following exposure to as little as 1 cGy. Conclusions: PEREGRINE is capable of determining within 5-15% the actual site of 1-10 cGy dose delivery to skin in areas outside of the treatment beam. Towards minimizing uncertainties in the final analysis, exit-surface collection of the samples is planned. Coupled with real time TLD measurements, we anticipate overall dosimetric uncertainties of 5%. We have validated the feasibility of testing the biologic components of the model, using an ex vivo irradiation model, as well as by showing gene changes in skin at 1 cGy. Skin samples exposed to IR in vivo can now be obtained from patients undergoing therapeutic IR to begin to define the cellular response to low dose IR in a complex biologic system.
88
Glioblastoma Cell Line Killing by Fractionated Radiation Does Not Follow the Assumption of Equal Effect Per Fraction
M. Sarvi, L. Sappelsa, E.R. Blazek Department of Radiation Oncology, Rush University, Chicago, IL Purpose/Objective: The survival of cells exposed to fractionated irradiation is termed effective survival. It is generally assumed that the effective surviving fraction (ESF) can be calculated using the linear-quadratic (LQ) model for the survival from one dose. This model assumes that n fractions of dose d are separated sufficiently for complete repair of sublethal damage, and that successive fractions have equal effect: ESFLQ(n ⫻ d) ⫽ [exp(-␣d–d2)]n (1) The aim of this study was to measure ESF curves for two glioblastoma multiforme (GBM) cell lines, U87MG and T98G, and to compare these curves to those expected from Eq. 1 using values of ␣ and determined from our single-fraction survival curves. Materials/Methods: T98G and U87MG obtained from the ATCC were harvested using trypsin-EDTA, and single-cell suspensions were counted by hemacytometer. Suspensions were serially diluted into 96-well culture plates at different densities for each dose and fractionation schedule, and irradiations were performed with a 137Cs irradiator at 0.8 Gy/min at room temperature. For single-dose experiments, duplicate plates received 2-18 Gy at 2-Gy increments. Fractionated irradiations were performed daily (including weekends) as follows: Duplicate plates received either 1-30 fractions of 2 Gy/d, 1-15 fractions of 4 Gy/d, or 1-10 fractions of 6 Gy/d. Four weeks after the last radiation fraction, cells were stained with crystal violet. In the dilution clonogenic assay, the well control fraction WCF is the fraction of all wells without any colonies of at least 50 cells.
53