Basal cell skin cancer after total-body irradiation and hematopoietic cell transplantation.

Previous studies identified radiation therapy as a key modifier of basal cell carcinoma (BCC) risk in survivors of hematopoietic cell transplantation (HCT). In the present analysis, risk of BCC was analyzed in relation to age at transplant, attained age, race, total-body irradiation (TBI), and radiation fractionation in 6,306 patients who received HCT at ages 0-65 years after conditioning regimens with (n = 3870) or without (n = 2436) TBI, and who were followed from 100 days to 36.2 years after HCT. While age-specific BCC rates in the unirradiated patient population were higher than those reported for two non-patient populations, the general characteristics were similar; rates increased with attained age, were eightfold lower for non-white patients, and were higher in more recent birth cohorts. After adjusting for these effects, risk in unirradiated patients did not vary significantly with age at HCT. The additional BCC risk associated with radiation exposure was largest for the youngest ages at exposure to radiation, with relative risks exceeding 20 for those transplanted at ages less than 10 years, and decreased with increasing age at exposure until age 40 years, above which no excess risk was identified. Relative risk in the irradiated population did not vary significantly with attained age, dose fractionation or race. Risks per unit dose in HCT patients were similar to other populations exposed under clinical settings to similar radiation doses and were more than 10-fold lower than seen in the atomic bomb survivors, 97% of whom were exposed to doses <1 Sv.

DNA copy-number instability in low-dose gamma-irradiated TK6 lymphoblastoid clones.

Genomic instability that might occur early during low-dose, fractionated radiation exposures may be traceable in radiogenic compared to spontaneous cancers. Using a human 18K cDNA microarray-based comparative genome hybridization protocol, we measured changes in DNA copy number at over 14,000 loci in nine low-dose (137)Cs gamma-irradiated (acute exposure to 10 cGy/day x 21 days) and nine unirradiated TK6 clones and estimated locus-specific copy-number differences between them. Radiation induced copy-number hypervariability at thousands of loci across all chromosomes, with a sevenfold increase in low-level, randomly positioned DNA gains. Recurrent gains at 40 loci occurred among irradiated clones and were distributed nonrandomly across the genome, with the highest densities in 3q, 13q and 20q at sites that were hypodiploid without irradiation. Another nonrandomly distributed set of 94 loci exhibited relative recurrent gains from a hypodiploid state to a diploid state, suggesting hemizygous-to-homozygous transitions. Frequently recurring losses at 57 loci were concentrated on the single X-chromosome but were sparsely distributed at 0-2 loci per autosome. These results suggest induced mitotic homologous recombination as a possible mechanism of low-dose radiation-induced genomic instability. Genomic instability induced in TK6 cells resembled that seen in radiogenic tumors and suggests a way that radiation could induce genomic instability in preneoplastic cells.

A role for endogenous and radiation-induced DNA double-strand breaks in p53-dependent apoptosis during cortical neurogenesis.

Prenatal exposure to low-dose radiation increases the risk of microcephaly and/or mental retardation. Microcephaly is also associated with genetic mutations that affect the non-homologous end-joining pathway of DNA double-strand break repair. To examine the link between these two causal factors, we characterized the neural developmental effects of acute radiation exposure in mouse littermate embryos harboring mutations in the Ku70 and p53 genes. Both low-dose radiation exposure and Ku70 deficiency induced morphologically indistinguishable cortical neuronal apoptosis. Irradiated Ku70-deficient embryos displayed anatomical damage indicative of increased radiosensitivity in the developing cerebral cortex. Deleting the p53 gene not only rescued cortical neuronal apoptosis at all levels but also restored the in vitro growth of Ku70-deficient embryonic fibroblasts despite the presence of unrepaired DNA/chromosomal breaks. The results confirm the role of DNA double-strand breaks as a common causative agent of apoptosis in the developing cerebral cortex. Furthermore, the findings suggest a disease mechanism by which the presence of endogenous DNA double-strand breaks in the newly generated cortical neurons becomes radiomimetic when DNA end joining is defective. This in turn activates p53-dependent neuronal apoptosis and leads to microcephaly and mental retardation.

Induction and loss of a TP53-dependent radioadaptive response in the human lymphoblastoid cell model TK6 and its abrogation by BCL2 over-expression.

PURPOSE: To characterize the radioadaptive response in the human lymphoblastoid cell model TK6, and determine: (i) Whether repeated low dose exposures are more effective than single acute exposures in inducing resistance, (ii) the time-course for induction and loss of resistance following chronic exposures, and (iii) the effect of TP53 deletion or BCL2 over-expression on the induction of an adaptive response. MATERIALS AND METHODS: TK6, a human B-lymphoblastoid cell line, TK6-BCL2, a TK6 line that over-expresses BCL2 and is resistant to radiation-induced apoptosis, and NH32, a TP53 knockout of TK6 that is also resistant to apoptosis were studied. Cells were exposed to chronic, daily doses of 10 cGy given over 1 -21 days before being challenged with 1 -5 Gy exposures. Cell survival and chromatid break induction following high dose challenge were used to evaluate adaptive radiation responses. RESULTS: Exposure to 10 cGy gamma rays induced resistance to killing and chromosome break induction in TK6 cells, but not in either TK6-BCL2 or NH32 cells. Resistance in TK6 was observed 4 h after exposure, and cells remained resistant for about 48 h. Maximal resistance was induced by a single 10 cGy dose. Repeated 10 cGy exposures had no additional effect on radiation sensitivity, except to maintain the induced radioresistance. CONCLUSION: An adaptive response is maximally and rapidly induced by a single low dose exposure in TK6 cells, and it has a limited lifespan. Induction of an adaptive response in TK6 cells can be abrogated by either TP53 loss or BCL2 over-expression. The characteristics of induced resistance in TK6 cells suggest that alterations in TP53-dependent apoptotic responses may be one mechanism for resistance.

Variability: the common factor linking low dose-induced genomic instability, adaptation and bystander effects.

The characteristics of low dose radiation-induced genomic instability, adaptive responses, and bystander effects were compared in order to probe possible underlying mechanisms, and develop models for predicting response to in vivo low dose radiation exposures. While there are some features that are common to all three (e.g., absence of a true dose-response, the multiple endpoints affected by each), other characteristics appear to distinguish one from the other (e.g., TP53 involvement, LET response, influence of DNA repair). Each of the responses is also highly variable; not all cell and tissue models show the same response and there is much interindividual variation in response. Most of these studies have employed in vitro cell culture or tissue explant models, and understanding underlying mechanisms and the biological significance of these low dose-responses will require study of tissue-specific in vivo endpoints. The in vitro studies strongly suggest that modeling low dose radiation effects will be a complex process, and will likely require separate study of each of these low dose phenomena. Knowledge of instability responses, for example, may not aid in predicting other low dose effects in the same tissue.

Selenium and GPx-1 overexpression protect mammalian cells against UV-induced DNA damage.

Supplementation of the culture media of human MCF-7 breast carcinoma cells or mouse fibroblasts with low levels of selenium (30 nM) provided as sodium selenite was shown to protect these cells from ultraviolet (UV)-induced chromosome damage, as quantified by micronucleus assay. Selenium supplementation was also effective in reducing UV-induced gene mutations as measured in the lacI shuttle vector model. Protection was dependent on functional BRCA1 activity, a protein implicated in breast cancer risk and DNA damage repair. In addition, overexpression of GPx-1, a selenoprotein with antioxidant activity, also attenuated UV induced micronuclei formation in the absence of selenium supplementation. Combining selenium supplementation with GPx-1 overexpression further reduced UV-induced micronucleus frequency. These data provide evidence that the benefits of selenium supplementation might be either through the prevention or repair of DNA damage, and they implicate at least one selenoprotein (GPx-1) in the process.

Environmental mutagenesis and radiation biology: The legacy of William Morgan.

A symposium entitled Environmental Mutagenesis and Radiation Biology was held on September 27, 2016 to honor the memory of Dr. William F. Morgan who passed away unexpectedly on November 13, 2015. The speakers presented the latest reviews on homologous recombination repair, induced genetic instability, bystander effects, and risk estimate development. Their presentations are presented following the introduction.

Atm haploinsufficiency does not affect ionizing radiation mutagenesis in solid mouse tissues.

Ataxia telangiectasia (AT) is a hereditary disease with autosomal recessive inheritance of ATM (ataxia telangiectasia mutation) alleles. AT is associated with severe sensitivity to ionizing radiation and a strong predisposition to develop cancer. A modest increase in cancer, particularly for the breast, has been shown for ATM carriers (i.e. heterozygotes), and a modest increase in radiation sensitivity has also been shown for those patients and their cells. However, the extent of these effects is unclear. Based on the well-established relationship between cancer and mutation, we used a mouse model for Atm haploinsufficiency to ask whether partial loss of Atm function could lead to an increased mutagenic response for solid tissues of mice exposed to radiation. The autosomal mouse Aprt gene was used as the mutational target and kidney and ear as the target tissues in B6D2F1 hybrids. Although induction of autosomal mutations was readily demonstrated in both tissues, a comparison of these data with those from an identical study performed with B6D2F1 mice that were wild-type for Atm (Cancer Res. 62, 1518-1523, 2002) revealed that Atm haploinsufficiency did not alter the radiation mutagenic response for the cells of either tissue. Moreover, no effect of Atm haploinsufficiency on reduced cellular viability due to radiation exposure was observed. The results demonstrate that Atm haploinsufficiency does not alter the radiation mutagenic response or decrease viability for normally quiescent cells in solid tissues of the mouse.

Ill-posed problem and regularization in reconstruction of radiobiological parameters from serial tumor imaging data.

The main objective of this article is to improve the stability of reconstruction algorithms for estimation of radiobiological parameters using serial tumor imaging data acquired during radiation therapy. Serial images of tumor response to radiation therapy represent a complex summation of several exponential processes as treatment induced cell inactivation, tumor growth rates, and the rate of cell loss. Accurate assessment of treatment response would require separation of these processes because they define radiobiological determinants of treatment response and, correspondingly, tumor control probability. However, the estimation of radiobiological parameters using imaging data can be considered an inverse ill-posed problem because a sum of several exponentials would produce the Fredholm integral equation of the first kind which is ill posed. Therefore, the stability of reconstruction of radiobiological parameters presents a problem even for the simplest models of tumor response. To study stability of the parameter reconstruction problem, we used a set of serial CT imaging data for head and neck cancer and a simplest case of a two-level cell population model of tumor response. Inverse reconstruction was performed using a simulated annealing algorithm to minimize a least squared objective function. Results show that the reconstructed values of cell surviving fractions and cell doubling time exhibit significant nonphysical fluctuations if no stabilization algorithms are applied. However, after applying a stabilization algorithm based on variational regularization, the reconstruction produces statistical distributions for survival fractions and doubling time that are comparable to published in vitro data. This algorithm is an advance over our previous work where only cell surviving fractions were reconstructed. We conclude that variational regularization allows for an increase in the number of free parameters in our model which enables development of more-advanced parameter reconstruction algorithms.

In vitro analysis of transport and metabolism of 4'-thiothymidine in human tumor cells.

INTRODUCTION: The use of thymidine (TdR) and thymidine analogs such as 3'-fluoro-3'-deoxythymidine (FLT) as positron emission tomography (PET)-based proliferation markers can provide information on tumor response to treatment. Studies on another TdR analog, 4'-thiothymidine (4DST), suggest that it might be a better PET-based proliferation tracer than either TdR or FLT. 4DST is resistant to the catabolism that complicates analysis of TdR in PET studies, but unlike FLT, 4DST is incorporated into DNA. METHODS: To further evaluate 4DST, the kinetics of 4DST transport and metabolism were determined and compared to FLT and TdR. Transport and metabolism of FLT, TdR and 4DST were examined in the human adenocarcinoma cell line A549 under exponential-growth conditions. Single cell suspensions were incubated in buffer supplemented with radiolabeled tracer in the presence or absence of nitrobenzylmercaptopurine ribonucleoside (NBMPR), an inhibitor of equilibrative nucleoside transporters (ENT). Kinetics of tracer uptake was determined in whole cells and tracer metabolism measured by high performance liquid chromatography of cell lysates. RESULTS: TdR and 4DST were qualitatively similar in terms of ENT-dependent transport, shapes of uptake curves, and relative levels of DNA incorporation. FLT did not incorporate into DNA, showed a significant temperature effect for uptake, and its transport had a significant NBMPR-resistant component. Overall 4DST metabolism was significantly slower than either TdR or FLT. CONCLUSIONS: 4DST provides a good alternative for TdR in PET and has advantages over FLT in proliferation measurement. However, slow 4DST metabolism and the short half-life of the (11)C label might limit widespread use in PET.

Validation of FLT uptake as a measure of thymidine kinase-1 activity in A549 carcinoma cells.

UNLABELLED: The thymidine analog (18)F-3'-deoxy-3' -fluorothymidine (FLT) is being used clinically for PET imaging of tumor proliferation. Appropriate use of this tracer requires validating the mechanisms by which it accumulates in dividing cells. We tested the accuracy with which FLT uptake predicted the activity of cytosolic thymidine kinase-1 (TK(1)), an enzyme that is upregulated before and during DNA synthesis. METHODS: Cultured A549 human lung carcinoma cells were manipulated to a range of proliferation rates from actively dividing to growth arrested. Uptake of radiolabeled FLT was compared with cell cycle activity, which was expressed as the percentage of cells in S phase, and with activity of cytosolic TK(1). We also compared uptake of FLT and deoxyglucose. We genetically manipulated A549 cells by transfecting them with human papillomavirus type 16 E6 (designated A549-E6) to abrogate function of the tumor suppressor gene, p53. Although radiation typically inhibits progression of mammalian cells through the cell cycle, abrogation of p53 function eliminates this inhibition. We then compared FLT uptake with the percentage of cells in S phase and TK(1) activity in irradiated A549-E6 cells and in irradiated control cells having normal p53 function and the expected radiation-induced growth delay. RESULTS: A549 cells with only 3%-5% cells in S phase took up little FLT and had low levels of TK(1) activity. When cells were stimulated to grow by being placed into fresh medium, we observed a strong correlation between increased FLT uptake and increased TK(1) activity. As expected, FLT uptake varied much more as a function of growth than did uptake of deoxyglucose. Nonproliferating A549 cells did not enter the cell cycle if they were irradiated before being placed into fresh medium, and they did not accumulate FLT or show elevated TK(1) activity. In contrast, radiation did not inhibit the cell cycle progression of A549-E6 cells. When subcultured, they began to grow and showed increased uptake of FLT commensurate with greater TK(1) activity. CONCLUSION: In cultured A549 cells FLT uptake is positively correlated with cell growth and TK(1) activity. Inhibition of cell cycle progression prevents FLT uptake and increased TK(1) activity. These results suggest that FLT images reflect TK(1) activity and the percentage of cells in S phase.

Monitoring tumor cell proliferation by targeting DNA synthetic processes with thymidine and thymidine analogs.

UNLABELLED: The use of radiolabeled thymidine (TdR) and thymidine analogs as PET-based tracers of tumor growth rate is based on the assumption that measurement of uptake of these nucleosides, a function primarily of thymidine kinase-1 (TK(1)) activity, provides an accurate measure of active cell proliferation in tumors. The goal of this study was to test this hypothesis and determine how well these tracers track changes in proliferation of tumor cells. METHODS: TK(1) activity; S-phase fraction; and uptake of TdR, 3'-deoxy-3'-fluorothymidine (FLT), and 2'-fluoro-5-methyl-1-(beta-D-2-arabino-furanosyl) uracil (FMAU) were determined in plateau-phase and exponentially growing cultures of 3 human and 3 murine tumor cell lines. RESULTS: TK(1) activity and S-phase fraction increased in all cell lines as cells moved from plateau-phase conditions to exponential growth. Some cell lines had relatively large TK(1) activities and S-phase fractions under plateau-phase conditions, consistent with a loss of normal cell cycle checkpoint control in these cells. There were also 2 cell lines in which TK(1) activity changed little as cells moved from the plateau phase to exponential growth, suggesting that in these cell lines, de novo nucleotide synthesis pathways predominate over salvage pathways. Both TdR and FLT detected changes in TK(1) activity. The slope of the relationship between TdR uptake and TK(1) activity was nearly twice that for FLT and more than 40-fold that for FMAU. CONCLUSION: Although not all tumors show a strong TK(1) dependence of proliferation, in all cell lines for which proliferation is highly TK(1) dependent, phosphorylation of TdR or FLT accurately reflects changes in TK(1) enzyme activity.

The TP53 dependence of radiation-induced chromosome instability in human lymphoblastoid cells.

The dose and TP53 dependence for the induction of chromosome instability were examined in cells of three human lymphoblastoid cell lines derived from WIL2 cells: TK6, a TP53-normal cell line, NH32, a TP53-knockout created from TK6, and WTK1, a WIL2-derived cell line that spontaneously developed a TP53 mutation. Cells of each cell line were exposed to (137)Cs gamma rays, and then surviving clones were isolated and expanded in culture for approximately 35 generations before the frequency and characteristics of the instability were analyzed. The presence of dicentric chromosomes, formed by end-to-end fusions, served as a marker of chromosomal instability. Unexposed TK6 cells had low levels of chromosomal instability (0.002 +/- 0.001 dicentrics/cell). Exposure of TK6 cells to doses as low as 5 cGy gamma rays increased chromosome instability levels nearly 10-fold to 0.019 +/- 0.008 dicentrics/cell. There was no further increase in instability levels beyond 5 cGy. In contrast to TK6 cells, unexposed cultures of WTK1 and NH32 cells had much higher levels of chromosome instability of 0.034 +/- 0.007 and 0.041 +/- 0.009, respectively, but showed little if any effect of radiation on levels of chromosome instability. The results suggest that radiation exposure alters the normal TP53-dependent cell cycle checkpoint controls that recognize alterations in telomere structure and activate apoptosis.

Induction of genomic instability in TK6 human lymphoblasts exposed to 137Cs gamma radiation: comparison to the induction by exposure to accelerated 56Fe particles.

The induction of genomic instability in TK6 human lymphoblasts by exposure to (137)Cs gamma radiation was investigated by measuring the frequency and characteristics of unstable clones isolated approximately 36 generations after exposure. Clones surviving irradiation and control clones were analyzed for 17 characteristics including chromosomal aberrations, growth defects, alterations in response to a second irradiation, and mutant frequencies at the thymidine kinase and Na(+)/K(+) ATPase loci. Putative unstable clones were defined as those that exhibited a significant alteration in one or more characteristics compared to the controls. The frequency and characteristics of the unstable clones were compared in clones exposed to (137)Cs gamma rays or (56)Fe particles. The majority of the unstable clones isolated after exposure to either gamma rays or (56)Fe particles exhibited chromosomal instability. Alterations in growth characteristics, radiation response and mutant frequencies occurred much less often than cytogenetic alterations in these unstable clones. The frequency and complexity of the unstable clones were greater after exposure to (56)Fe particles than to gamma rays. Unstable clones that survived 36 generations after exposure to gamma rays exhibited increases in the incidence of dicentric chromosomes but not of chromatid breaks, whereas unstable clones that survived 36 generations after exposure to (56)Fe particles exhibited increases in both chromatid and chromosome aberrations.

Characteristics of genomic instability in clones of TK6 human lymphoblasts surviving exposure to 56Fe ions.

Genomic instability in the human lymphoblast cell line TK6 was studied in clones surviving 36 generations after exposure to accelerated 56Fe ions. Clones were assayed for 20 characteristics, including chromosome aberrations, plating efficiency, apoptosis, cell cycle distribution, response to a second irradiation, and mutant frequency at two loci. The primary effect of the 56Fe-ion exposure on the surviving clones was a significant increase in the frequency of unstable chromosome aberrations compared to the very low spontaneous frequency, along with an increase in the phenotypic complexity of the unstable clones. The radiation-induced increase in the frequency of unstable chromosome aberrations was much greater than that observed previously in clones of the related cell line, WTK1, which in comparison to the TK6 cell line expresses an increased radiation resistance, a mutant TP53 protein, and an increased frequency of spontaneous unstable chromosome aberrations. The characteristics of the unstable clones of the two cell lines also differed. Most of the TK6 clones surviving exposure to 56Fe ions showed unstable cytogenetic abnormalities, while the phenotype of the WTK1 clones was more diverse. The results underscore the importance of genotype in the characteristics of instability after radiation exposure.

Persistence of chromatid aberrations in the cells of solid mouse tissues exposed to 137Cs gamma radiation.

Primary mouse ear and kidney cultures were established for determination of cytogenetic aberrations at short (3 days to 1 month) and long (12-23 months) times after exposure of their right sides to 7.5 Gy of (137)Cs gamma radiation. In every case, higher levels of aberrations were observed in primary cultures established from the irradiated tissues than in those established from the contralateral tissues. The most common aberrations in the contralateral tissues and those from nonirradiated mice were chromatid and isochromatid breaks and small chromatid fragments. Primary cells from irradiated tissues removed from animals within a month of exposure displayed a variety of unstable chromosome-type aberrations characteristic of recent exposure to ionizing radiation including rings, dicentrics, double minutes, and large acentric fragments. The percentages of cells exhibiting chromatid breaks and small chromatid fragments were also markedly elevated. Although the levels of chromosome-type aberrations found in primary cells from irradiated tissues dropped to near background levels a year or more after exposure, chromatid-type aberrations remained elevated. These results are consistent with long-term persistence of damage in the genomes of ionizing radiation-exposed cells in solid tissues and the induction of genomic instability in vivo.

Second malignant neoplasms following hematopoietic stem cell transplantation.

Hematopoietic stem cell transplantation is being successfully used to treat a variety of malignant and nonmalignant disorders. This therapy has resulted in an increasing number of survivors who are at risk for adverse long-term outcomes, including the development of second and subsequent malignant neoplasms. We review the incidence and spectrum of posttransplantation malignancies and discuss risk factors and future directions for research.

Effect of p53 activation on cell growth, thymidine kinase-1 activity, and 3'-deoxy-3'fluorothymidine uptake.

The use of thymidine (TdR) and thymidine analogs such as 3'-deoxy-3'-fluorothymidine (FLT) as positron emission tomography (PET)-based tracers of tumor proliferation rate is based on the hypothesis that measurement of uptake of these nucleosides, a function primarily of thymidine kinase-1 (TK(1)) activity, provides an accurate measure of cell proliferation in tumors. Tumor growth is influenced by many factors including the oxygen concentration within tumors and whether tumor cells have been exposed to cytotoxic therapies. The p53 gene plays an important role in regulating growth under both of these conditions. The goal of this study was to investigate the influence of p53 activation on cell growth, TK(1) activity, and FLT uptake. To accomplish this, TK(1) activity, S phase fraction, and the uptake of FLT were determined in plateau-phase and exponentially growing cultures of an isogenic pair of human tumor cell lines in which p53 expression was normal or inactivated by human papilloma virus type 16 E6 expression. Ionizing radiation exposure was used to stimulate p53 activity and to induce alterations in cell cycle progression. We found that exposure of cells to ionizing radiation induced dose-dependent changes in cell cycle progression in both cell lines. The relationship between S phase percentage, TK(1) activity, and FLT uptake were essentially unchanged in the p53-normal cell line. In contrast, TK(1) activity and FLT uptake remained high in the p53-deficient variant even when S phase percentage was low due to a p53-dependent G2 arrest. We conclude that a functional p53 response is required to maintain the normal relationship between TK1 activity and S phase percentage following radiation exposure.

Enhancing the biological activity of immobilized osteopontin using a type-1 collagen affinity coating.

The covalent attachment of biomolecules onto surfaces represents a step toward the improvement of biomaterial properties by providing relevant biological signals of interest to the cell culture or tissue environment. The chemistries involved, however, often attach proteins to the surface in a random fashion, rather than the conformation or orientation most easily recognized by cells and other proteins both in vitro and in vivo. An alternative approach is to take advantage of natural interactions to both bind and orient a biomolecule "naturally," thereby enhancing its biological activity. Type 1 collagen has been shown to bind to osteopontin (OPN), a protein implicated in processes such as wound healing, endothelial cell survival, and angiogenesis. This study seeks to characterize, quantify, and exploit this interaction in order to present a more naturally recognized form of OPN to the environment surrounding a biomaterial. Binding of OPN to type 1 collagen was confirmed using Surface Plasmon Resonance (SPR). Radio-iodination of OPN showed that binding to collagen was dose-dependent and maximal in basic conditions. Principal component analysis of Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) data identified differences in OPN immobilized via different techniques. Adhesion of bovine aortic endothelial cells on OPN immobilized using the affinity coating was also significantly enhanced compared to controls. Investigation into the in vivo relevance of this immobilization method is currently underway.

Radiation biology in the context of changing patterns of radiotherapy.

The last decade has witnessed a revolution in the clinical application of high-dose "ablative" radiation therapy. Initially this approach was limited to the treatment of brain tumors, but more recently we have seen its successful extension to tumors outside the brain, e.g., for small lung nodules. These advances have been driven largely by improvements in image-guided inverse treatment planning that allow the dose per fraction to the tumor to be increased over the conventional 2 Gy dose while keeping the late normal tissue complications at an acceptable level by dose limitation. Despite initial concerns about excessive late complications, as might be expected based on dose extrapolations using the linear-quadratic equation, these approaches have shown considerable clinical promise. Our knowledge of the biological consequences of high-doses of ionizing radiation in normal and cancerous tissues has lagged behind these clinical advances. Our intent here is to survey recent experimental findings from the perspective of better understanding the biological effects of high-dose therapy and whether they are truly different from conventional doses. We will also consider the implications of this knowledge for further refining and improving these approaches on the basis of underlying mechanisms.