Perampanel, an antagonist of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, for the treatment of epilepsy: studies in human epileptic brain and nonepileptic brain and in rodent models.

Perampanel [Fycompa, 2-(2-oxo-1-phenyl-5-pyridin-2-yl-1,2-dihydropyridin-3-yl)benzonitrile hydrate 4:3; Eisai Inc., Woodcliff Lake, NJ] is an AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor antagonist used as an adjunctive treatment of partial-onset seizures. We asked whether perampanel has AMPA receptor antagonist activity in both the cerebral cortex and hippocampus associated with antiepileptic efficacy and also in the cerebellum associated with motor side effects in rodent and human brains. We also asked whether epileptic or nonepileptic human cortex is similarly responsive to AMPA receptor antagonism by perampanel. In rodent models, perampanel decreased epileptic-like activity in multiple seizure models. However, doses of perampanel that had anticonvulsant effects were within the same range as those engendering motor side effects. Perampanel inhibited native rat and human AMPA receptors from the hippocampus as well as the cerebellum that were reconstituted into Xenopus oocytes. In addition, with the same technique, we found that perampanel inhibited AMPA receptors from hippocampal tissue that had been removed from a patient who underwent surgical resection for refractory epilepsy. Perampanel inhibited AMPA receptor-mediated ion currents from all the tissues investigated with similar potency (IC50 values ranging from 2.6 to 7.0 µM). Cortical slices from the left temporal lobe derived from the same patient were studied in a 60-microelectrode array. Large field potentials were evoked on at least 45 channels of the array, and 10 µM perampanel decreased their amplitude and firing rate. Perampanel also produced a 33% reduction in the branching parameter, demonstrating the effects of perampanel at the network level. These data suggest that perampanel blocks AMPA receptors globally across the brain to account for both its antiepileptic and side-effect profile in rodents and epileptic patients.

Bistability dynamics in simulations of neural activity in high-extracellular-potassium conditions.

Modulation of extracellular potassium concentration ([K](o)) has a profound impact on the excitability of neurons and neuronal networks. In the CA3 region of the rat hippocampus synchronized epileptiform bursts occur in conditions of increased [K](o). The dynamic nature of spontaneous neuronal firing in high [K](o) is therefore of interest. One particular interest is the potential presence of bistable behaviors such as the coexistence of stable repetitive firing and fixed rest potential states generated in individual cells by the elevation of [K](o). The dynamics of repetitive activity generated by increased [K](o) is investigated in a 19-compartment hippocampal pyramidal cell (HPC) model and a related two-compartment reduced HPC model. Results are compared with those for the Hodgkin-Huxley equations in similar conditions. For neural models, [K](o) changes are simulated as a shift in the potassium reversal potential (E(K)). Using phase resetting and bifurcation analysis techniques, all three models are shown to have specific regions of E(K) that result in bistability. For activity in bistable parameter regions, stimulus parameters are identified that switch high-potassium model behavior from repetitive firing to a quiescent state. Bistability in the HPC models is limited to a very small parameter region. Consequently, our results suggest that it is likely some HPCs in networks exposed to high [K](o) continue to burst such that a stable, quiescent network state does not exist. In [K](o) ranges where HPCs are not bistable, the population may still exhibit bistable behaviors where synchronous population events are reversibly annihilated by phase resetting pulses, suggesting the existence of a nonsynchronous network attractor.

Positive Lyapunov exponents calculated from time series of strange nonchaotic attractors.

Time-series methods for estimating Lyapunov exponents may give a positive exponent when they are applied to the time series of strange nonchaotic systems. Strange nonchaotic systems are characterized by expanding and contracting regions in phase space that result in repeatedly expanding or contracting trajectories. Using time-series methods, the maximum time-series Lyapunov exponent is calculated as an average of the locally most expanding exponents that characterize the divergence of nearby trajectories following a reconstructed attractor over time. A positive exponent is reported by time-series methods for trajectories in an expanding region. While in a converging region, the most expanding dynamics are related to the quasiperiodic driving force. Statistically, a zero exponent related to the quasiperiodic force is obtained through time-series methods within converging regions. As a result, the calculated maximum Lyapunov exponent is positive.

Suppression of epileptiform activity by high frequency sinusoidal fields in rat hippocampal slices.

1. Sinusoidal high frequency (20-50 Hz) electric fields induced across rat hippocampal slices were found to suppress zero-Ca2+, low-Ca2+, picrotoxin, and high-K+ epileptiform activity for the duration of the stimulus and for up to several minutes following the stimulus. 2. Suppression of spontaneous activity by high frequency stimulation was found to be frequency (< 500 Hz) but not orientation or waveform dependent. 3. Potassium-sensitive microelectrodes showed that block of epileptiform activity was always coincident with a stimulus-induced rise in extracellular potassium concentration during stimulation. Post-stimulus inhibition was always associated with a decrease in extracellular potassium activity below baseline levels. 4. Intracellular recordings and optical imaging with voltage-sensitive dyes showed that during suppression neurons were depolarized yet did not fire action potentials. 5. Direct injection of sinusoidal current into individual pyramidal cells did not result in a tonic depolarization. Injection of large direct current (DC) depolarized neurons and suppressed action potential generation. 6. These findings suggest that high frequency stimulation suppresses epileptiform activity by inducing potassium efflux and depolarization block.

Differential effects of diethylstilbestrol and 2,3,7,8-tetrachlorodibenzo-p-dioxin on thymocyte differentiation, proliferation, and apoptosis in bcl-2 transgenic mouse fetal thymus organ culture.

Both the estrogenic drug diethylstilbestrol (DES) and the pervasive environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) inhibit thymocyte development. The mechanisms by which either agent induces thymic atrophy are still undetermined. We previously found that TCDD and DES inhibited C57BL/6 murine fetal thymocyte organ cultures (FTOC) at different stages of development. Now, using bcl-2 transgenic (TG) mice, we have further investigated their effects on FTOC proliferation, differentiation, maturation, and apoptosis. As with C57BL/6 mice, thymocyte development in C3H/bcl-2 FTOCs was inhibited by either TCDD (10 nM) or DES (20 microM) in both bcl-2 TG- and TG+ littermates. However, the percentage reduction of cell number induced by DES in bcl-2 TG+ FTOCs was significantly less than the level of inhibition in TG- FTOCs. There was no difference in the level of reduction from TCDD-exposed TG+ or TG- FTOC. Whereas TCDD increased production of mature CD8 cells in either strain, DES mainly yielded cells in the CD4(-)CD8(-)(DN) stage in TG- mice. The anti-apoptotic bcl-2 transgene overcame some DES blocking of DN thymocyte development, allowing more cells to differentiate into CD4 single-positive cells. Analysis of cell cycle showed that TCDD inhibited entry into S phase, whereas DES blocked cell cycling in the G2/M phase. TCDD did not induce detectable apoptosis in FTOC. However, unlike the effects of 17 beta-estradiol (E2) in vivo, DES induced apoptosis in the TG- FTOC, and these apoptotic cells were mainly in the DN subpopulation. This apoptosis could be prevented by the overexpression of bcl-2 in the TG+ mice. Our results demonstrate that, in addition to inhibition of fetal thymocytes at different stages of development by TCDD and DES, DES also induces thymic atrophy by both bcl-2-inhibitable apoptosis and by inducing cell cycle arrest in G2/M in the latest stage in the stem cell compartment. TCDD, on the other hand, does not induce apoptosis, but inhibits entry into cell cycle in the earliest stage in the stem cell compartment.

Subungual malignant melanoma mimicking a squamous cell carcinoma.

When a suspicious lower-extremity lesions is encountered, an appropriate biopsy should be performed. The lesion must then be excised with the proper margins and submitted for pathologic evaluation to ensure complete removal.

CpG methylation reduces genomic instability.

Hypomethylation of DNA in tumor cells is associated with genomic instability and has been suggested to be due to activation of mitotic recombination. We have studied the methylation patterns in two 650 kb double minute chromosomes present in two mouse tumor cell lines, resistant to methotrexate. Multiple copies of the double minute chromosomes amplifying the dihydrofolate reductase gene are present in both the cell lines. In one of the cell lines (Mut F), two unmethylated CpG islands in the double minute chromosomes are readily cleaved by methylation-sensitive rare-cutting restriction endonucleases. In the other cell line (Mut C), the cleavage sites in the double minute chromosomes are partially methylated and resistant to cleavage. The double minute chromosomes with the two unmethylated CpG islands undergo rapid dimerization, whereas the double minute chromosomes with the partially methylated CpG islands are unchanged in size for over a year in continuous culture. The partially methylated CpG islands can be demethylated by azacytidine treatment or naturally by extended time in culture, and become sensitive to cleavage with the rare-cutting restriction endonucleases. The Mut C double minute chromosomes, with the newly demethylated CpG islands, but not the double minute chromosomes with the partially methylated CpG islands, undergo deletions and dimerizations. These results suggest a role for CpG island methylation controlling mitotic recombination between and within large DNA molecules.

Differential sensitivity of double minute chromosomes to hydroxyurea treatment in cultured methotrexate-resistant mouse cells.

Treating mammalian cells with continuous sub-lethal doses of Hydroxyurea (HU) causes the loss of double minute chromosomes (DMs) containing amplified oncogenes in culture. Recently, we have shown that treating glioblastoma multiforme cells in culture with low doses of HU causes the loss of DMs containing epidermal growth factor receptor genes. Loss of amplified EGFR genes was accompanied by cessation of growth, and greatly decreased tumorigenicity. To further study HU-induced elimination of DMs we have now followed the fate of dihydrofolate reductase gene (DHFR) amplifying DMs in methotrexate-resistant mouse cells during simultaneous treatment with both MTX and HU. We report that in the presence of both HU and MTX, the amplified genes decreased to 25% of starting levels in the first week of treatment, but that ultimately the cells become resistant to HU and reamplify the DHFR gene. We also report that some DHFR amplifying DMs are much more sensitive to HU than others. This study demonstrates that HU does not simply increase the rate of passive loss of DMs.

No detectable misrejoining in double-minute chromosomes.

Pulsed-field gel electrophoresis combined with Southern hybridization and rare-cutting restriction endonuclease digestion has been used recently to quantify misrejoining of DNA double-strand breaks (DSBs) resulting from exposure to ionizing radiation. Measurements are made 24 h after a high dose of radiation. These studies have suggested that a large fraction of DSBs are misrejoined to result in gross rearrangements. In the experiments described here, we show that elimination of broken DNA also eliminates "misrejoined" DNA. Mouse cells resistant to high levels of methotrexate by virtue of 100-fold amplification of the dyhydrofolate reductase (Dhfr) gene were treated with 50 and 100 Gy of ionizing radiation. The cells were allowed to repair the damage for 24 h. After the repair period, the cells were immobilized in agarose. Aliquots of each sample were pre-electrophoresed to remove linear DNA molecules smaller than 6 Mbp resulting from apoptosis or necrosis. The samples repairing damage from 50 or 100 Gy that did not receive the pre-electrophoresis showed high levels of label in a region of the lane that could be due to misrejoining DNA molecules. However, when the DNA from cells undergoing apoptosis or necrosis was removed from these samples, the levels of "misrejoined" DNA were reduced to levels far below those of unirradiated controls. These results suggest that other radiation-induced effects present 24 h after irradiation with 50 or 100 Gy are more significant than misrejoining for altering hybridization to regions of the lane outside the specific bands. Measurements of misrejoining using PFGE, rare-cutting restriction endonucleases, and Southern hybridization are likely to be compromised by nonspecific hybridization to broken and difficult-to-digest DNA resulting from apoptosis or necrosis.

Polydactyly of the foot. Overview with case presentations.

The authors offer two unique case presentations of pedal polydactyly as well as a general discussion of this congenital anomaly. This presentation demonstrates how a common condition has very individualized treatment plans that are based on communication between the patient and the podiatric surgeon.

Chromosome breakpoints near CpG islands in double minutes.

Double minute chromosomes (DMs) are the principal genetic vehicles for amplifying oncogenes in human tumors and drug resistance genes in cultured mouse cells. Mouse EMT-6 cells resistant to methotrexate (MTX) generally contain circular DMs, approximately 1 megabase (Mb) in size, that amplify the dihydrofolate reductase (DHFR) gene. The 1 Mb DMs generally have CpG islands located 500 kb upstream of the DHFR gene. The purpose of this study was to determine the relationship between CpG islands and chromosomal breakpoints giving rise to the DM. We show that EMT-6 cells growing in very low levels of MTX that do not yet contain the 1 Mb DHFR-amplifying DM, develop a NotI/EagI site 500 kb upstream of the DHFR gene. This NotI site is close to, if not identical with, one of the chromosomal breakpoints giving rise to the DM. We show that 500 kb of DM DNA from upstream of the DHFR gene is derived from 500 kb of chromosomal DNA upstream of the chromosomal DHFR gene. The downstream breakpoint maps to a region approximately 200 kb downstream of the DHFR gene near a chromosomal SstII/EagI site. Therefore, approximately 700 kb of DM DNA was derived from the genomic region surrounding the DHFR gene. To confirm the organization of the DM DNA, we isolated DNA probes from the 1 Mb DM. Using pulsed field gel electrophoresis and Southern hybridization, we determined the approximate location of each probe with respect to the CpG island in both the DM and the chromosome. Approximately 300 kb of chimeric DNA from a region unrelated to the DHFR gene was incorporated during DM formation. Implications for the mechanism of DM formation are discussed.

CpG islands and double-minute chromosomes.

Double-minute chromosomes (DMs) amplify oncogenes in human tumors. The organization of genomic DNA in four independently isolated DMs amplifying the DHFR (dihydrofolate reductase) gene has been compared by mapping locations of CpG islands. When cleaved with methylation-sensitive rare-cutting restriction endonucleases, three hypomethylated GC-rich DNA sequences were frequently found in specific regions in these DMs. One such zone was in the CpG island containing the divergently transcribed promoter separating the DHFR and the Rep-3 genes. The other two sites were approximately 500 kb upstream and 300 kb downstream of the DHFR gene. An approximately 800-kb amplified core genomic region containing the DHFR gene using DM-specific probes has been identified in this study. All the DMs consisted of the core amplified region combined with additional DNA fragments. These additional fragments are different for each DM. Therefore, while the DNAs in each of the DMs are different, they have common hypomethylated regions in similar locations. These results suggest a role for the location of hypomethylated GC-rich sites such as the CpG islands in genesis of DMs.

The hydroxyurea-induced loss of double-minute chromosomes containing amplified epidermal growth factor receptor genes reduces the tumorigenicity and growth of human glioblastoma multiforme.

OBJECTIVE: We investigated whether the hydroxyurea-induced loss of double-minute chromosomes containing amplified epidermal growth factor receptor (EGFR) genes would lead to a loss of tumorigenicity of a glioblastoma multiforme cell line. METHODS: Glioblastoma multiforme cells were treated in vitro with 0 (HU0) or 100 micromol/L (HU100) hydroxyurea and then injected into the flanks of nude mice. Survival and tumor volumes were evaluated. Pulsed-field gel electrophoresis, Southern blot hybridization, and slot-blot analysis were used to determine EGFR amplification levels. Flow cytometry and immunofluorescent staining were used for cell-cycle analysis and EGFR protein expression. RESULTS: Prior to injection, HU100 cells lost 95% of their amplified EGFR genes and developed into tumors 6 weeks after injection versus 3 weeks for HU0 cells. Mice with HU100 tumors had a median survival of 62 days versus 43 days for control mice with HU0 tumors. Pulse-field gel electrophoresis analysis showed that HU100 tumors had reamplified the EGFR gene as double-minute chromosomes of the same size as those originally present before hydroxyurea treatment. When HU100 cells were cultured in the absence of hydroxyurea, the EGFR gene also reamplified. HU100 cells grew at less than half the rate of untreated HU0 control cells in culture and showed a decreased number of cells entering the cell cycle. Immunofluorescent staining of HU150 (150 micromol/L) cells showed decreased EGFR protein expression. CONCLUSION: The EGFR gene is important for tumorigenicity in mice and growth in culture. Hydroxyurea induces the loss of double-minute chromosome-amplified EGFR genes against a selection gradient and significantly delays the onset of tumors. These results support the potential use of low-dose hydroxyurea for the treatment of human glioblastoma multiforme.

The scid defect results in much slower repair of DNA double-strand breaks but not high levels of residual breaks.

Severe combined immune deficiency (scid) mice fail to produce mature B and T cells and are sensitive to ionizing radiation. They contain a mutation in the 460-kDa catalytic subunit of the DNA-dependent protein kinase that is involved in both V(D)J rejoining and DNA double-strand break (DSB) repair. The kinetics of DSB rejoining was quantified in both scid cells and the parental C.B-17 cells after three different doses of X irradiation: 3, 7.5 and 10 Gy. Repair of DNA DSBs was determined using pulsed-field gel electrophoresis, Southern hybridization and phosphor image analysis. After X irradiation, the cells were allowed to repair at 37 degrees C for up to 1 h or up to 24 h. The most profound difference between the two cell lines was the greatly reduced rate of the slow component of DSB repair in scid cells. C.B-17 cells repaired most of the damage within 1 h, whereas scid cells required 4 to 6 h to reach a similar level after the same dose. No residual or unrepairable DSBs were detected in either cell line 24 h after doses as high as 10 Gy. The scid cells subjected to two doses of 1.5 Gy separated by increasing amounts of time showed no ability to repair sublethal damage between doses, whereas C.B-17 cells receiving two doses of 3.75 Gy separated by increasing periods did show increased levels of survival. These results indicate that scid cells can repair radiation-induced DNA DSBs, although at a reduced rate, but they lack the ability to undergo repair of sublethal damage.

An assay for quantifying DNA double-strand break repair that is suitable for small numbers of unlabeled cells.

A system based on pulsed-field gel electrophoresis (PFGE) is described which measures the induction and repair of DNA double-strand breaks (DSBs) in a biologically relevant X-ray dose range (below 10 Gy) using as few as 125 cells per time. This system was used to measure repair in cells of a freshly obtained human glioblastoma multiforme tumor. No prelabeling of the cells is required, and many different cell types can be studied using this system. Under the pulsed-field conditions used, DNA in the range of 2 to 6 Mb enters the PFGE gel and forms an upper compression zone directly under each well. To quantify the DSBs after electrophoresis, the DNA was transferred to nylon membranes and hybridized with 32P-labeled chromosomal DNA. Phosphor screens were exposed to the membranes and scanned on a phosphor imager. The kinetics of induction and repair was determine by measuring the amount of DNA in the compression zones compared to the amount in the wells. EMT-6 cells were used to demonstrate this method. Induction of DSBs by doses of 0-7.5 Gy X rays was assayed using approximately 12,500 cells per dose and was shown to be linear. Double-strand breaks from 1 Gy were detected above background. To determine a lower limit of the number of cells that could be used to measure DSB repair, cells were embedded in agarose at decreasing concentrations per plug, exposed to 7.5 Gy X irradiation and allowed to repair at 37 degrees C for up to 60 min. DNA from approximately 12,500, 1,250 and 125 cells per time was loaded and subjected to PFGE. The average fast-repair half-time was 3 min and the slow-repair half-time was 35 min. The kinetics of DSB repair in glioblastoma multiforme cells was also determined using this system. Agarose plugs were prepared from a cell suspension, irradiated with 7.5 Gy X rays and allowed to repair for up to 90 min. DNA from approximately 1,250 tumor cells was electrophoresed and analyzed as described above for EMT-6 cells. For this particular tumor, approximately 75% of the induced DSBs were repaired after 90 min. Data presented show that this PFGE-based system is an extremely sensitive method for measuring DSB induction and repair after low doses of X rays using very few cells.

The use of high-throughput synthesis and purification in the preparation of a directed library of adrenergic agents.

A library of potential agonists and antagonists for adrenergic receptors was prepared using high-throughput solution-phase parallel synthesis. Traditional solution-phase reductive amination reactions followed by rapid purification by ion exchange chromatography yielded products with near-analytical purity. An array of ketones and amines, arranged in an 8 x 12 matrix, were combined to form 96 individual compounds.

Hydroxyurea accelerates the loss of epidermal growth factor receptor genes amplified as double-minute chromosomes in human glioblastoma multiforme.

OBJECTIVE: We sought to determine whether hydroxyurea could accelerate the loss of amplified epidermal growth factor receptor (EGFR) genes from glioblastoma multiforme (GBM). There is good reason to think that elimination of amplified EGFR genes from GBMs will negatively impact tumor growth. Hydroxyurea has previously been shown to induce the loss of amplified genes from extrachromosomal double minutes (dmin) but not from chromosomal homogeneously staining regions. METHODS: Pulsed-field gel electrophoresis and Southern blot hybridization were used to demonstrate EGFR genes amplified as dmin. Giemsa-stained metaphase spreads were prepared in an attempt to visualize dmin. A GBM cell line containing amplified EGFR genes was treated continuously in vitro with 0 to 150 mumol/L hydroxyurea, and slot blot analysis was used to show the loss of amplified EGFR genes. RESULTS: Amplified EGFR genes were found on dmin in 4 of 11 (36%) fresh human GBM biopsy specimens. None of the GBMs contained EGFR genes amplified as homogeneously staining regions. Amplified dmin were not microscopically visible when stained with Giemsa because of their small size. Slot blot analysis showed that these low doses of hydroxyurea accelerated the loss of amplified EGFR genes in a dose- and time-dependent fashion. Pulsed-field gel electrophoresis and Southern blot analysis confirmed that EGFR gene loss was accompanied by amplified dmin loss in a dose-dependent fashion. CONCLUSION: These studies suggest the potential use of low-dose hydroxyurea in the treatment of GBMs.

Chronic sun exposure alters both the content and distribution of dermal glycosaminoglycans.

Chronic sun exposure leads to structural and functional alterations in exposed skin. Photoageing is a process distinct from the changes taking place due to chronological ageing. Unique alterations in the dermal extracellular matrix occur as a result of photoageing and are responsible for many of these physiological changes taking place in sun-damaged skin. Accompanying the deposition of abnormal elastic tissue, or solar elastosis, are significant alterations in dermal glycosaminoglycans (GAGs). Accumulation of GAGs as a result of photoageing, as demonstrated in both humans and animal models of photoageing, seems almost paradoxical in view of the large amounts of GAGs present in the skin of newborns, making their skin well hydrated and supple, in sharp contrast to the weathered appearance of photoaged skin. We investigate the relative GAG content of photoaged skin using immunoperoxidase stains specific for hyaluronic acid and chondroitin sulphate, and determine the location of these GAGs using confocal laser scanning microscopy. Our results demonstrate significant increases in GAG staining in sun-damaged vs. sun-protected skin from the same individuals, as measured by computer-based image analysis. Furthermore, confocal laser scanning microscopy reveals that the increased dermal GAGs in sun-damaged skin are deposited on the elastotic material of the superficial dermis of photodamaged skin, and not between collagen and elastic fibres as in normal skin. The abnormal location of GAGs on these fibres may explain the apparent paradoxical weathered appearance of photodamaged skin despite increased GAGs.

Long-term sun exposure alters the collagen of the papillary dermis. Comparison of sun-protected and photoaged skin by northern analysis, immunohistochemical staining, and confocal laser scanning microscopy.

BACKGROUND: Long-term solar irradiation produces both morphologic and functional changes in affected skin. Because collagen is the major structural component of skin, any alteration in its production or degradation could have profound effects on cutaneous functional integrity. OBJECTIVE: Our purpose was to investigate alterations in the production and morphology of collagen fibers brought about by long-term sun exposure. METHODS: We compared collagen and collagenase gene expression and collagen immunohistochemical staining and used confocal laser scanning microscopy for morphologic examination of dermal collagen fibers in photodamaged compared with sun-protected skin from the same persons. RESULTS: Despite a large increase in elastin messenger RNA in sun-damaged skin, collagen and collagenase gene expression remained essentially unchanged. However, striking alterations in the papillary dermis of photoaged skin were found, which revealed large, abnormally clumped elastic fibers and deformed collagen fibers of various diameters, replacing the normal architecture of the papillary dermis. CONCLUSION: Our data provide evidence for normal collagen gene expression in sun-damaged skin and suggest that degradation and remodeling of collagen take place in the papillary dermis accompanied by deposition of other matrix components, predominantly abnormal elastic fibers.

Induction and repair of DNA double-strand breaks in the same dose range as the shoulder of the survival curve.

We have used pulsed-field gel electrophoresis (PFGE) to test two hypotheses that have been proposed to explain the survival curves with shoulders which are characteristic of low-LET ionizing radiation: (1) Neutral elution studies of the induction of double-strand breaks (DSBs) have suggested that ionizing radiation might induce DSBs in a nonlinear fashion at low doses. (2) Based on analogies to enzyme kinetics, DSB repair might be saturating in the shoulder region. We quantified DSB induction and survival resulting from doses between 0 and 5 Gy spanning the shoulder region of the survival curve. We found that DSB induction was linear at all doses tested down to 0.5 Gy, the limits of sensitivity. Therefore, nonlinear DSB induction cannot account for the shape of the survival curve. To determine whether the DSB repair system was saturated in the shoulder region, we quantified the rate of DSB repair as a function of dose of X rays between 1.25 and 20 Gy. The repair of DSBs was exponential with half-times of repair constant for doses below 10 Gy, and averaged 28 min. We determined the initial rate of repair from the exponential repair kinetics for each dose. The initial rate of repair after radiation treatment increased linearly with dose up to at least 10 Gy. Therefore, saturating DSB repair cannot explain the shoulder of the survival curve.