Data Analysis WorkbeNch (DAWN).

Synchrotron light source facilities worldwide generate terabytes of data in numerous incompatible data formats from a wide range of experiment types. The Data Analysis WorkbeNch (DAWN) was developed to address the challenge of providing a single visualization and analysis platform for data from any synchrotron experiment (including single-crystal and powder diffraction, tomography and spectroscopy), whilst also being sufficiently extensible for new specific use case analysis environments to be incorporated (e.g. ARPES, PEEM). In this work, the history and current state of DAWN are presented, with two case studies to demonstrate specific functionality. The first is an example of a data processing and reduction problem using the generic tools, whilst the second shows how these tools can be targeted to a specific scientific area.

Rab3a deletion reduces vesicle docking and transmitter release at the mouse diaphragm synapse.

Rab3a is a small GTP binding protein associated with presynaptic vesicles that is thought to regulate vesicle targeting to active zones. Although this rab3a function implies that vesicle docking and action potential-evoked release might be inhibited in rab3a gene-deleted synapses, such inhibition has never been demonstrated. To investigate vesicle docking at the neuromuscular junction of rab3a gene-deleted (rab3a(-)) mice, we performed electron microscopy analysis of the diaphragm slow-fatigue (type I) synapses. We found a significant (26%) reduction in the number of vesicles docked to the presynaptic membrane in rab3a(-) terminals, although intraterminal vesicles were not affected. Aiming to detect possible changes in quantal release due to rab3a gene deletion, we minimized the variability between preparations employing focal recordings of synaptic responses from visualized type I endplates. We found a significant decrease in both evoked (27% reduction in quantal content) and spontaneous (28% reduction in mini frequency) quantal release. The decrease in the evoked release produced by rab3a deletion was most pronounced at reduced extracellular Ca(2+) concentrations (over 50% decrease at 0.5 and 0.2 mM Ca(2+)). By manipulating extracellular calcium, we demonstrated that calcium cooperativity is not altered in rab3a(-) synapses, however calcium sensitivity of quantal release is affected. Thus, we demonstrated that rab3a positively regulates docking and basal quantal release at the mouse neuromuscular junction. This result is consistent with the proposed role of rab3a in trafficking and targeting vesicles to the active zones.

Spontaneous and ultraviolet light-induced direct repeat recombination in mammalian cells frequently results in repeat deletion.

Recombination is enhanced by transcription and by DNA damage caused by ultraviolet light (UV). Recombination between direct repeats can occur by gene conversion without an associated crossover, which maintains the gross repeat structure. There are several possible mechanisms that delete one repeat and the intervening sequences (gene conversion associated with a crossover, unequal sister chromatid exchange, and single-strand annealing). We examined transcription-enhanced spontaneous recombination, and UV-induced recombination between neomycin (neo) direct repeats. One neo gene was driven by the inducible MMTV promoter. Multiple (silent) markers in the second neo gene were used to map conversion tracts. These markers are thought to inhibit spontaneous recombination, and our data suggest that this inhibition is partially overcome by high level transcription. Recombination was stimulated by transcription and by UV doses of 6-12J/m(2), but not by 18J/m(2). About 70% of spontaneous and UV-induced products were deletions. In contrast, only 3% of DSB-induced products were deletions. We propose that these product spectra differ because spontaneous and UV-induced recombination is replication-dependent, whereas DSB-induced recombination is replication-independent.

Repair bias of large loop mismatches during recombination in mammalian cells depends on loop length and structure.

Repair of loop mismatches was investigated in wild-type and mismatch binding-defective Chinese hamster ovary (CHO) cells. Loop mismatches were formed in vivo during extrachromosomal recombination between heteroallelic plasmid substrates. Recombination was expected to occur primarily by single-strand annealing (SSA), yielding 12- or 26-base nonpalindromic loop mismatches, and 12-, 26-, or 40-base palindromic loop mismatches. Nonpalindromic loops were repaired efficiently and with bias toward loop loss. In contrast, the 12-base palindromic loop was repaired with bias toward loop retention, indicating that repair bias depends on loop structure. Among the palindromic loops, repair bias was dependent on loop length, with bias shifting from loop retention to loop loss with increasing loop size. For both palindromic and nonpalindromic loops, repair efficiencies and biases were independent of the general (MSH/MLH) mismatch repair pathway. These results are discussed with respect to the maintenance of large nonpalindromic insertions, and of small and large palindromes, in eukaryotic genomes.

Efficient repair of all types of single-base mismatches in recombination intermediates in Chinese hamster ovary cells. Competition between long-patch and G-T glycosylase-mediated repair of G-T mismatches.

Repair of all 12 single-base mismatches in recombination intermediates was investigated in Chinese hamster ovary cells. Extrachromosomal recombination was stimulated by double-strand breaks in regions of shared homology. Recombination was predicted to occur via single-strand annealing, yielding heteroduplex DNA (hDNA) with a single mismatch. Nicks were expected on opposite strands flanking hDNA, equidistant from the mismatch. Unlike studies of covalently closed artificial hDNA substrates, all mismatches were efficiently repaired, consistent with a nick-driven repair process. The average repair efficiency for all mispairs was 92%, with no significant differences among mispairs. There was significant strand-independent repair of G-T --> G-C, with a slightly greater bias in a CpG context. Repair of C-A was also biased (toward C-G), but no A-C --> G-C bias was found, a possible sequence context effect. No other mismatches showed evidence of biased repair, but among hetero-mismatches, the trend was toward retention of C or G vs. A or T. Repair of both T-T and G-T mismatches was much less efficient in mismatch repair-deficient cells (approximately 25%), and the residual G-T repair was completely biased toward G-C. Our data indicate that single-base mismatches in recombination intermediates are substrates for at least two competing repair systems.

A role for p53 in DNA end rejoining by human cell extracts.

The tumor suppressor p53 is a major regulator in the response of human cells to DNA damage. In this study we assessed the role of p53 in the repair of DNA double-strand breaks in plasmid DNA using cell extracts from three human lymphoblastoid cell lines derived from the same donor. TK6, WI-L2-NS and TK6-E6-5e cells express wild-type, mutated and essentially no p53 protein, respectively. Total cellular extracts from TK6, WI-L2-NS and TK6-E6-5e cells were incubated with EcoRI linearized pUC19 DNA. Southern blot analysis of end-rejoined DNA indicated that the major products formed were linear multimers. There was approximately 2-fold greater end rejoining in WI-L2-NS and TK6-E6-5e extracts compared with TK6 extracts. Total DNA from end-rejoining reactions was purified and used to transform bacteria. Using the lacZ reporter gene as a measure of repair fidelity we found that misrepair, as indicated by white colonies, occurred at 4.1% to 6.5% of transformants, with no significant difference between the three cell lines. Gel analysis revealed that misrepair involved only deletions. Sequence analysis of 11 misrepaired products from each cell line showed 12 different deletions from 4 to 48 bp in length, but each cell line yielded similar product types. These results indicate that total cellular extracts from human lymphoblastoid cells lacking p53 or expressing mutated p53 have increased end-rejoining activity as compared with extracts from cells expressing wild-type p53. However, the p53 status does not influence the ratio of misrepair:correct repair, or the type of misrepair events.

Influence of X-rays on early response gene expression in rat astrocytes and brain tumour cell lines.

The effects of ionizing radiation on c-fos, c-jun and jun-B mRNA levels were determined in cultures of rat perinatal type 1 astrocytes and two rat brain tumour cell lines, 175A and 9L. In astrocyte cultures X-ray doses as low as 1 Gy induced the expression of c-fos and jun-B but had essentially no effect on c-jun. The maximum increase in expression was found 1 h after irradiation, which then rapidly returned to control levels. These findings suggest that astrocytes may play a role in mediating the radiation response of the central nervous system via X-ray-induced changes in gene expression. In contrast, doses of up to 20 Gy had no effect on c-fos, c-jun and jun-B mRNA levels in the two brain tumour cell lines. In addition, whereas 12-O-tetradecanoylphorbol-13-acetate induced the expression of these genes in astrocytes, it had little or no effect on fos or jun expression in 9L or 175A cells. These results suggest that the signal transduction pathways mediating radiation-induced gene expression may be different in normal astrocytes and brain tumour cells.

Influence of DNA repair capacity and cell differentiation on UV-induced gene expression.

Terminally differentiated (TD) 3T3-T cells have a reduced capacity to repair ultraviolet (UV)-induced DNA damage, as compared with the repair capabilities of growth-arrested and cycling (stem) 3T3-T cells. In this study UV-induced expression of the immediate early response genes c-fos and c-jun and the secondary response gene type IV collagenase in TD, growth-arrested and stem 3T3-T cells was investigated by northern blot analysis. At each UV dose (0-10 J/m2) there was an increase in c-fos and, to a lesser extent, c-jun expression 0.5 h after irradiation in each 3T3-T phenotype as compared with unirradiated controls. Maximum induction of c-fos was reached at 0.5-1 J/m2 in TD and growth-arrested cells, which was 10-fold greater than in stem cells. The induction of c-jun in TD and growth-arrested cells reached maximums at 0.5 J/m2; at this dose each was greater than in stem cells. The increases in c-fos and c-jun expression were transient in each phenotype reaching maximums from 0.5 to 1 h after irradiation. The expression of type IV collagenase was increased in the non-cycling phenotypes at 2-8 h after irradiation. However, collagenase expression was not detected in unirradiated or irradiated stem cells. These results suggest that growth arrest, not differentiation or DNA repair capacity, is the primary influence on gene induction after UV irradiation.

Enhancement of tumor cell killing in vitro by pre- and post-irradiation exposure to aclacinomycin A.

Aclacinomycin A (ACM), a potent inducer of leukemic cell differentiation, significantly enhances the radiosensitivity of a human colon tumor cell line (Clone A) when cultures are exposed to 15-nM concentrations for 3 days before irradiation. We now demonstrate that incubation with ACM after irradiation can also enhance Clone A cell killing. The maximum increase in cell killing, based on colony-forming ability, occurred when Clone A cells were exposed for 1 h to 5 microM ACM added 1 or 2 h after irradiation. The post-irradiation ACM protocol reduced the terminal slope (as reflected by D0) of the radiation cell survival curve with no change in the low-dose, shoulder region of the curve (Dq value). In contrast, for pre-irradiation treatment with ACM (15 nM, 3 days), the shoulder region of the curve was reduced with no change in the terminal slope. For pre- and post-irradiation ACM treatment the dose enhancement factors at 0.10 survival were 1.22 and 1.28, respectively. When ACM was given both before and after irradiation both the shoulder and terminal slope values decreased to produce a dose enhancement factor at a surviving fraction of 0.10 of 1.50. These data suggest that the enhanced cell killing produced by pre- and post-irradiation treatment with ACM is achieved through different mechanisms.

Decreased repair of radiation-induced DNA double-strand breaks with cellular differentiation.

Although the majority of mammalian cells in situ are terminally differentiated, most DNA repair studies have used proliferating cells. In an attempt to understand better the relationship between differentiation and DNA repair, we have used the murine 3T3-T proadipocyte cell line. In this model system, proliferating (stem) cells undergo growth arrest (GD cells) and subsequently terminally differentiate into adipocytes when exposed to media containing platelet-depleted human plasma. Pulsed-field gel electrophoresis was used to evaluate the induction and repair of DNA double-strand breaks (DSBs) after ionizing radiation. The levels of radiation-induced DSBs in GD and terminally differentiated cells were similar, but in both cases greater than those found in stem cells at each radiation dose tested (0 to 40 Gy); these differences appear to be due to growth arrest in G1 phase. DNA DSBs were repaired with biphasic kinetics for each cell type. For terminally differentiated cells 25% of DNA DSBs remained unrejoined compared with < 10% for GD and stem cells after a repair time of 4 h. These data indicate that terminal differentiation of 3T3-T cells is associated with a reduction in the repair of ionizing radiation-induced DNA DSBs.

Radiation-induced apoptosis of oligodendrocytes in vitro.

It has been suggested that glial cells and/or their progenitors are the primary target cells for radiation-induced demyelination. Cultures of terminally differentiated oligodendrocytes, immature oligodendrocytes, and O-2A progenitor cells were generated from the cerebral cortex and spinal cord of perinatal rat pups. Irradiation of cultures of terminally differentiated oligodendrocytes resulted in a significant increase in the percentage of apoptotic cells from 15% in control to 30% in irradiated samples, with the maximum increase induced by 10 Gy. This increase in apoptosis could be observed by 1 h after irradiation with the maximum level reached at 3-6 h. Apoptotic cells were not detected before or after irradiation of cultures of O-2A progenitor cells or immature oligodendrocytes. These data suggest that radiation-induced apoptosis of terminally differentiated oligodendrocytes may be involved in early demyelination.

Enhancement of radiation-induced cell killing and DNA double-strand breaks in a human tumor cell line using nanomolar concentrations of aclacinomycin A.

Several agents that induce differentiation have previously been shown to induce the terminal differentiation of leukemic cells and enhance the radiosensitivity of certain solid tumor cell lines in vitro using millimolar concentrations. We now report that aclacinomycin A (ACM), a potent inducer of leukemic cell differentiation in vitro, can significantly enhance the radiosensitivity of a human colon tumor cell line (Clone A) at a concentration of 10 nM. Based on colony-forming efficiency, the maximum increase in radiosensitivity was found using 15 nM ACM for 3 days with a dose enhancement factor of 1.4 at a surviving fraction of 0.10. This treatment increased cell doubling time, but had no effect on cell-cycle phase distribution. To gain insight into the mechanisms responsible for this radiosensitization, gamma-ray-induced DNA single- and double-strand breaks were examined. Aclacinomycin A had no effect on the induction of DNA single-strand breaks but significantly enhanced the formation of gamma-ray-induced DNA double-strand breaks. The rate or extent of repair of the induced double-strand breaks was not influenced by ACM treatment. These data suggest that ACM, at achievable plasma concentrations, can enhance the radiosensitivity of a human tumor cell line by increasing the initial level of radiation-induced DNA double-strand breaks.

Loss of intragenomic DNA repair heterogeneity with cellular differentiation.

The influence of terminal differentiation on UV-induced DNA damage and its repair in transcriptionally active and inactive genomic sequences was investigated using the murine 3T3-T proadipocyte cell culture system. Actively cycling 3T3-T cells terminally differentiate into adipocytes after exposure to media containing platelet-depleted human plasma. Suitable DNA fragments were analyzed from four genes: beta-actin, adenosine deaminase, dihydrofolate reductase, and lipoprotein lipase. As a result of 3T3-T cell differentiation, lipoprotein lipase and beta-actin expression was modified, whereas adenosine deaminase and dihydrofolate reductase expression was not affected. A DNA fragment representing the transcriptionally inactive locus 70-38 was also evaluated. UV-induced cyclobutane pyrimidine dimers, detected as UV-specific endonuclease-sensitive sites, in each fragment increased linearly as a function of UV dose (0-20 J/m2) independently of gene expression or differentiation. Sequence-specific repair of dimers was measured in stem and terminally differentiated 3T3-T cells after UV irradiation (10 J/m2). For undifferentiated stem cells, the rate and extent of dimer repair was higher in the actively transcribed adenosine deaminase and dihydrofolate reductase genes than in the inactive lipoprotein lipase or 70-38 fragments, the greater difference being observed in the first 8 h post-UV irradiation. In contrast, similar dimer repair rates were found for each DNA fragment in terminally differentiated 3T3-T cells. These data suggest that cellular differentiation is accompanied by a loss of heterogeneity in intragenomic DNA repair.

Enhancement of the radiosensitivity of two human tumour cell lines by hexamethylene bisacetamide.

The effect of the maturation-inducing polar solvent, hexamethylene bisacetamide (HMBA), on the radiosensitivity of two human tumour cell lines (clone A, a colon carcinoma; and EJ, a bladder carcinoma) was investigated. Exposure of clone A or EJ cells to HMBA resulted in a concentration-dependent increase in doubling time, a decreased plating efficiency and changes in cell morphology, which are consistent with the formation of a better-differentiated phenotype. Growth of clone A cells in 2 or 3 mM HMBA, followed by irradiation and plating into HMBA-free medium, resulted in a significant enhancement in radiosensitivity, as determined by colony-forming ability. A similar increase in radiosensitivity was detected for EJ cells; however, for these cells a concentration of 7 mM HMBA was required. The increased radiosensitivity caused by HMBA was observed primarily in the low-dose, shoulder region of the gamma-ray cell survival curves for both cell lines, which is reflected by an increase in the alpha component of the survival curve with essentially no effect on beta. These data indicate that HMBA can radiosensitise human tumour cells at concentrations and for exposure periods that can be achieved in the clinic.

Enhancement of radiation-induced DNA double-strand breaks and micronuclei in human colon carcinoma cells by N-methylformamide.

The differentiation-inducing agent N-methylformamide (NMF) enhances the sensitivity of some cell lines to ionizing radiation. To elucidate the mechanism of NMF-mediated radiosensitization, we examined the effects of this agent on gamma-ray-induced DNA double-strand breaks and micronuclei in two cell lines, clone A (human colon carcinoma) and HCA-1 (murine hepatocarcinoma). Both cell lines form a better differentiated phenotype upon exposure to NMF, yet only clone A is radiosensitized. The neutral (pH 9.6) elution assay was used to evaluate the effects of this maturational agent on radiation-induced double-strand breaks in these cell lines. Exposure of HCA-1 cells to NMF had no effect on the level of DNA double-strand breaks induced by gamma rays. In clone A cells, however, exposure to NMF enhanced the initial formation of gamma-ray-induced double-strand breaks at each dose tested. The repair of double-strand breaks in both cell lines was not influenced by NMF. As a measure of chromosome fragmentation after irradiation, we evaluated micronuclei using the cytokinesis block method. Exposure to NMF had no effect on radiation-induced micronuclei formation in HCA-1 cells yet significantly enhanced the frequency of micronuclei induced by radiation in clone A cells. In clone A cells, the increases in radiation-induced double-strand breaks and micronuclei as a function of NMF exposure time reached maximums by approximately 72 h. These data suggest that NMF-mediated radiosensitization is the result of an increase in the initial level of radiation-induced DNA double-strand breaks.

Effects of N-methylformamide on the growth, cell cycle, and glutathione status of murine TLX5 lymphoma cells.

The growth of the murine TLX5 lymphoma is inhibited in vivo by administration of N-methylformamide (Gescher, A., et al., Br. J. Cancer, 45: 843-850, 1982). Continuous incubation of TLX5 murine lymphoma cells in vitro with N-methylformamide for 72 h, at concentrations of between 43 and 170 mM (0.25 and 1% v/v), brought about a concentration-dependent decrease in growth rate (50% inhibitory concentration = 68 mM) and viability. Cell replication was decreased by 37% after 48 h exposure to 106 mM N-methylformamide, while viability was maintained at 82%. Analysis of the distribution of these cells in the cell cycle by flow cytofluorimetry showed a 23% increase in the proportion of G1 cells and a fall in the proportion of cells in the S and G2/M phases. As the drug concentration and time of exposure to N-methylformamide were increased, with an associated reduction in cell replication and viability, the proportion of G1 cells rose. When TLX5 cells were washed free of N-methylformamide after an exposure to 106 mM for 48 h and cultured in drug-free medium, the cells returned to exponential growth and to a normal cell cycle distribution. Clonogenic assays showed that the recovery of proliferation, after removal of the drug, was due to that of all those cells which, in a parallel experiment, excluded the dye trypan blue. It is concluded that the cessation of replication and the accumulation of cells in G1 of the cell cycle, after treatment with N-methylformamide, are probably not events representative of terminal differentiation but rather of cytostasis, which was accompanied by rapid cell death. Coincident with the reduction of TLX5 cell proliferation caused by N-methylformamide and the accumulation of cells in G1, cellular glutathione concentrations fell by 80%. A similar fall was induced by treatment of the cells with D,L-buthionine[S,R]-sulfoximine (5 microM) for 48 h, but this treatment had no effect on cell growth.