Genetically defined individual reference ranges for tryptase limit unnecessary procedures and unmask myeloid neoplasms.

Serum tryptase is a biomarker used to aid in the identification of certain myeloid neoplasms, most notably systemic mastocytosis, where basal serum tryptase (BST) levels >20 ng/mL are a minor criterion for diagnosis. Although clonal myeloid neoplasms are rare, the common cause for elevated BST levels is the genetic trait hereditary α-tryptasemia (HαT) caused by increased germline TPSAB1 copy number. To date, the precise structural variation and mechanism(s) underlying elevated BST in HαT and the general clinical utility of tryptase genotyping, remain undefined. Through cloning, long-read sequencing, and assembling of the human tryptase locus from an individual with HαT, and validating our findings in vitro and in silico, we demonstrate that BST elevations arise from overexpression of replicated TPSAB1 loci encoding canonical α-tryptase protein owing to coinheritance of a linked overactive promoter element. Modeling BST levels based on TPSAB1 replication number, we generate new individualized clinical reference values for the upper limit of normal. Using this personalized laboratory medicine approach, we demonstrate the clinical utility of tryptase genotyping, finding that in the absence of HαT, BST levels >11.4 ng/mL frequently identify indolent clonal mast cell disease. Moreover, substantial BST elevations (eg, >100 ng/mL), which would ordinarily prompt bone marrow biopsy, can result from TPSAB1 replications alone and thus be within normal limits for certain individuals with HαT.

Effects of 1 GeV/nucleon 56Fe particles on longevity, carcinogenesis and neuromotor ability in Atm-deficient mice.

As therapeutic uses of high-LET radiation become more prevalent and human space exploration continues to be a focus of NASA, it is important to understand the biological effects of high-LET radiation and the role of genetics in sensitivity to high-LET radiation. To study genetic susceptibility to radiation, we used mice deficient in Atm activity (AtmΔSRI). ATM is important in DNA repair, apoptosis and cell cycle regulation. Although homozygous mutations in ATM are rare, the prevalence of ATM heterozygosity is estimated to be 1% and results in an increased cancer risk. We found that the effects of 1 Gy 1 GeV/nucleon 56Fe particles on life span and tumorigenesis are genotype- and sex-specific. Significant effects of 1 Gy 1 GeV/nucleon 56Fe particles on incidence of non-cancer end points were seen; however, 2 Gy 1 GeV/nucleon 56Fe particles significantly affected neuromotor ability. Our results represent an extensive investigation into the late effects of high-LET radiation exposure in a sex- and genotype-dependent manner and provide a baseline for understanding the long-term risks of high-LET radiation.

Yeast DEL assay detects protection against radiation-induced cytotoxicity and genotoxicity: adaptation of a microtiter plate version.

The DEL assay in yeast detects DNA deletions that are inducible by many carcinogens. Here we use the colorimetric agent MTS to adapt the yeast DEL assay for microwell plate measurement of ionizing radiation-induced cell killing and DNA deletions. Using the microwell-based DEL assay, cell killing and genotoxic DNA deletions both increased with radiation dose between 0 and 2000 Gy. We used the microwell-based DEL assay to assess the effectiveness of varying concentrations of five different radioprotectors, N-acetyl-l-cysteine, l-ascorbic acid, DMSO, Tempol and Amifostine, and one radiosensitizer, 5-bromo-2-deoxyuridine. The microwell format of the DEL assay was able to successfully detect protection against and sensitization to both radiation-induced cytotoxicity and genotoxicity. Such radioprotection and sensitization detected by the microwell-based DEL assay was validated and compared with similar measurements made using the traditional agar-based assay format. The yeast DEL assay in microwell format is an effective tool for rapidly detecting chemical protectors and sensitizers to ionizing radiation and is automatable for chemical high-throughput screening purposes.

Cell cycle dependence of ionizing radiation-induced DNA deletions and antioxidant radioprotection in Saccharomyces cerevisiae.

The yeast DEL assay is an effective method for measuring intrachromosomal recombination events resulting in DNA deletions that when occurring in mammalian cells are often associated with genomic instability and carcinogenesis. Here we used the DEL assay to measure gamma-ray-induced DNA deletions throughout different phases of yeast culture growth. Whereas yeast survival differed by only up to twofold throughout the yeast growth phase, proliferating cells in lag and early exponential growth phases were tenfold more sensitive to ionizing radiation-induced DNA deletions than cells in stationary phase. Radiation-induced DNA deletion potential was found to correlate directly with the fraction of cells in S/G(2) phase. The ability of the antioxidants l-ascorbic acid and DMSO to protect against radiation-induced DNA deletions was also measured within the different phases of yeast culture growth. Yeast cells in lag and early exponential growth phases were uniquely protected by antioxidant treatment, whereas nondividing cells in stationary phase could not be protected against the induction of DNA deletions. These results are compared with those from mammalian cell studies, and the implications for radiation-induced carcinogenesis and radioprotection are discussed.

The pol3-t hyperrecombination phenotype and DNA damage-induced recombination in Saccharomyces cerevisiae is RAD50 dependent.

The DNA polymerase delta (POL3/CDC2) allele pol3-t of Saccharomyces cerevisiae has previously been shown to be sensitive to methylmethanesulfonate (MMS) and has been proposed to be involved in base excision repair. Our results, however, show that the pol3-t mutation is synergistic for MMS sensitivity with MAG1, a known base excision repair gene, but it is epistatic with rad50Delta, suggesting that POL3 may be involved not only in base excision repair but also in a RAD50 dependent function. We further studied the interaction of pol3-t with rad50Delta by examining their effect on spontaneous, MMS-, UV-, and ionizing radiation-induced intrachromosomal recombination. We found that rad50Delta completely abolishes the elevated spontaneous frequency of intrachromosomal recombination in the pol3-t mutant and significantly decreases UV- and MMS-induced recombination in both POL3 and pol3-t strains. Interestingly, rad50Delta had no effect on gamma-ray-induced recombination in both backgrounds between 0 and 50 Gy. Finally, the deletion of RAD50 had no effect on the elevated frequency of homologous integration conferred by the pol3-t mutation. RAD50 is possibly involved in resolution of replication forks that are stalled by mutagen-induced external DNA damage, or internal DNA damage produced by growing the pol3-t mutant at the restrictive temperature.

Ionizing radiation induces microhomology-mediated end joining in trans in yeast and mammalian cells.

DNA double-strand breaks repaired through nonhomologous end joining require no extended sequence homology as a template for the repair. A subset of end-joining events, termed microhomology-mediated end joining, occur between a few base pairs of homology, and such pathways have been implicated in different human cancers and genetic diseases. Here we investigated the effect of exposure of yeast and mammalian cells to ionizing radiation on the frequency and mechanism of rejoining of transfected unirradiated linear plasmid DNA. Cells were exposed to gamma radiation prior to plasmid transfection; subsequently the rejoined plasmids were recovered and the junction sequences were analyzed. In irradiated yeast cells, 68% of recovered plasmids contained microhomologies, compared to only 30% from unirradiated cells. Among them 57% of events used>or=4 bp of microhomology compared to only 11% from unirradiated cells. In irradiated mammalian cells, 54% of plasmids used>or=4 bp of microhomology compared to none from unirradiated cells. We conclude that exposure of yeast and mammalian cells to radiation prior to plasmid transfection enhances the frequency of microhomology-mediated end-joining events in trans. If such events occur within genomic locations, they may be involved in the generation of large deletions and other chromosomal aberrations that occur in cancer cells.

Radiation-induced long-lived extracellular radicals do not contribute to measurement of intracellular reactive oxygen species using the dichlorofluorescein method.

The dichlorofluorescein method has become a standard technique for measuring reactive oxygen species (ROS) formed in cells by ionizing radiation. A recent report (Korystov et al., Radiat. Res. 168, 226-232, 2007) has suggested that the method is subject to an artifact in that it erroneously reports hydrogen peroxides generated in the extracellular medium as ROS formed intracellularly by ionizing radiation. It was hypothesized that radiation-induced extracellular peroxides enter cells in the minutes after radiation exposure and subsequently oxidize the intracellular dichlorofluorescin probe and that dichlorofluorescein fluorescence is not due to ROS formed intracellularly by ionizing radiation. We tested this hypothesis by measuring the contribution of long-lived radicals formed in medium by ionizing radiation on intracellular dichlorofluorescein fluorescence. We found no evidence that this artifact contributes significantly to intracellular dichlorofluorescein fluorescence. These results and those of Korystov et al. are discussed in view of cellular dichlorofluorescin leakage and radiation chemistry. We conclude that the dichlorofluorescein method is effective for quantifying intracellular ROS induced by ionizing radiation.

Refinement of the dichlorofluorescein assay for flow cytometric measurement of reactive oxygen species in irradiated and bystander cell populations.

Reactive oxygen species (ROS) have been implicated in many ionizing radiation-related phenomena, including bystander effects. The oxidation of 2'7'-dichlorofluorescin (DCFH) to fluorescent 2'7'-dichlorofluorescein (DCF) is commonly used for the detection of radiation-induced ROS. The DCF assay was adapted for efficient, systematic flow cytometry quantification of low-linear energy transfer (LET) gamma-radiation-induced ROS in vitro in Chinese hamster ovary (CHO) cells. This method is optimized for increased sensitivity to radiation-induced ROS and to discriminate against measurement of extracellular ROS. This method can detect a significant increase in ROS in cells exposed to gamma radiation at doses as low as 10 cGy. The antioxidants N-acetyl-cysteine and ascorbic acid (vitamin C) significantly reduced the amount of ROS measured in cells exposed to 5 Gy ionizing radiation. This method was used to measure the intracellular ROS in unirradiated CHO bystander cells co-cultured with low-LET-irradiated cells. No increase in ROS was measured in bystander cell populations co-cultured with the irradiated cells beginning 9 s after radiation exposure.

Development of a microtiter plate version of the yeast DEL assay amenable to high-throughput toxicity screening of chemical libraries.

The yeast plate-based deletion (DEL) assay has been previously shown to detect a wide range of carcinogens. Of 60 compounds of known carcinogenic activity, 92% were correctly detectable with the DEL assay whereas 62% were correctly detectable with the Ames assay [W.W. Ku, J. Aubrecht, R.J. Mauthe, R.H. Schiestl, A.J. Fornace Jr., Why not start with a single test: a transformational alternative to genotoxicity hazard and risk assessment, Toxicol. Sci. (2007)]. In this manuscript we describe a modification of the yeast DEL assay into a colorimetric assay using the MTS tetrazolium compound (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) to allow for efficient detection of chemical genotoxicity. It has been micro-scaled and can be performed in 96- or 384-well format. Chemicals previously characterized with the DEL plate-based assay were utilized to test the new well-based format, and a group of cross-linking agents, previously uncharacterized by the DEL assay, were scored for genotoxicity using this new assay format. These compounds induced a range of genotoxicity detectable with the well-based DEL assay, and a lack of sensitivity was found only at extremely low genotoxic levels determined by the plate-based DEL assay. We suggest this new well-based version of the DEL assay can be used as an economical alternative to the plate-based assay to screen large numbers of compounds, such as chemical libraries in a high-throughput screening setting.

Adaptive response to gamma radiation in mammalian cells proficient and deficient in components of nucleotide excision repair.

Cells preconditioned with low doses of low-linear energy transfer (LET) ionizing radiation become more resistant to later challenges of radiation. The mechanism(s) by which cells adaptively respond to radiation remains unclear, although it has been suggested that DNA repair induced by low doses of radiation increases cellular radioresistance. Recent gene expression profiles have consistently indicated that proteins involved in the nucleotide excision repair pathway are up-regulated after exposure to ionizing radiation. Here we test the role of the nucleotide excision repair pathway for adaptive response to gamma radiation in vitro. Wild-type CHO cells exhibited both greater survival and fewer HPRT mutations when preconditioned with a low dose of gamma rays before exposure to a later challenging dose. Cells mutated for ERCC1, ERCC3, ERCC4 or ERCC5 did not express either adaptive response to radiation; cells mutated for ERCC2 expressed a survival adaptive response but no mutation adaptive response. These results suggest that some components of the nucleotide excision repair pathway are required for phenotypic low-dose induction of resistance to gamma radiation in mammalian cells.

Point-source irradiation with eccentric rotation causes inhomogeneous dose distribution.

Radioactive point sources are regularly used for irradiating cell culture and other biological materials. Eccentric rotation is often used to minimize dose disparities that arise from irradiating samples that span a distance from the point source. Rotation provides a great improvement in dose homogeneity compared to inert irradiation yet still presents an obvious shortcoming for exposures in which the sample completes only partial rotation or fractional rotation. In such cases, certain areas of the sample have a closer average distance to the radiation source than other areas within the same sample. This obstacle can be partially overcome by adjusting rotation speed so the sample traverses a full rotation (or multiple thereof) throughout the total irradiation time. Here we investigate the effects of irradiation with eccentric rotation on dose homogeneity. We show that due to the inverse square law that governs dose, even exposures with full rotation result in inhomogeneous dose distributions. This dose inhomogeneity can be substantial, especially for large samples and small source- sample distances. We observed a 33% difference in survival across 100-mm dishes and a 400% difference for 150-mm dishes. The dose inhomogeneity inherent to eccentric rotation increases the actual average dose delivered across the sample compared to that delivered at sample center. We offer a table of correction factors that account for this dose increase and correct the dose delivered at center to the actual average dose delivered across the entire sample.