ABSTRACT
The WHO Classification of Tumours of Haematopoietic and Lymphoid Tissue notes instances of Burkitt lymphoma/leukemia (BL) with IG-MYC rearrangement displaying a B-cell precursor immunophenotype (termed herein "preBLL"). To characterize the molecular pathogenesis of preBLL, we investigated 13 preBLL cases (including 1 cell line), of which 12 were analyzable using genome, exome, and targeted sequencing, imbalance mapping, and DNA methylation profiling. In 5 patients with reads across the IG-MYC breakpoint junctions, we found evidence that the translocation derived from an aberrant VDJ recombination, as is typical for IG translocations arising in B-cell precursors. Genomic changes like biallelic IGH translocations or VDJ rearrangements combined with translocation into the VDJ region on the second allele, potentially preventing expression of a productive immunoglobulin, were detected in 6 of 13 cases. We did not detect mutations in genes frequently altered in BL, but instead found activating NRAS and/or KRAS mutations in 7 of 12 preBLLs. Gains on 1q, recurrent in BL and preB lymphoblastic leukemia/lymphoma (pB-ALL/LBL), were detected in 7 of 12 preBLLs. DNA methylation profiling showed preBLL to cluster with precursor B cells and pB-ALL/LBL, but apart from BL. We conclude that preBLL genetically and epigenetically resembles pB-ALL/LBL rather than BL. Therefore, we propose that preBLL be considered as a pB-ALL/LBL with recurrent genetic abnormalities.
Subject(s)
Burkitt Lymphoma/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cells, B-Lymphoid/pathology , Proto-Oncogene Proteins c-myc/genetics , V(D)J Recombination , Adolescent , Adult , Aged , Burkitt Lymphoma/diagnosis , Burkitt Lymphoma/pathology , Child , Child, Preschool , DNA Methylation , Female , Gene Rearrangement, B-Lymphocyte , Humans , Male , Middle Aged , Precursor Cell Lymphoblastic Leukemia-Lymphoma/diagnosis , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Precursor Cells, B-Lymphoid/metabolism , Retrospective Studies , Translocation, Genetic , Young AdultABSTRACT
The comet assay is frequently used in human biomonitoring for the detection of exposure to genotoxic agents. Peripheral blood samples are most frequently used and tested either as whole blood or after isolation of lymphocytes (i.e. peripheral blood mononuclear cells, PBMC). To investigate a potential impact of lymphocyte isolation on induced DNA damage in human blood samples, we exposed blood ex vivo to mutagens with different modes of genotoxic action. The comet assay was performed either directly with whole blood at the end of the exposure period or with lymphocytes isolated directly after exposure. In addition to the recommended standard protocol for lymphocyte isolation, a shortened protocol was established to optimise the isolation procedure. The results indicate that the effects of induced DNA strand breaks and alkali-labile sites induced by ionising radiation and alkylants, respectively, are significantly reduced in isolated lymphocytes. In contrast, oxidative DNA base damage (induced by potassium bromate) and stable bulky adducts (induced by benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide; BPDE) seem to be less affected. Our findings suggest that in vivo-induced DNA damage might also be reduced in isolated lymphocytes in comparison with the whole blood depending of the types of DNA damage induced. Because only small genotoxic effects can generally be expected in human biomonitoring studies with the comet assay after occupational and environmental exposure to genotoxic agents, any loss might be relevant and should be avoided. The possibility of such effects and their potential impact on variability of comet assay results in human biomonitoring should be considered when performing or evaluating such kind of studies.
Subject(s)
Cell Separation/methods , DNA Damage , Lymphocytes/chemistry , Comet Assay , Environmental Exposure , Female , Humans , Lymphocytes/drug effects , Lymphocytes/radiation effects , Male , Mutagens/toxicityABSTRACT
The present study aims to further characterize benzo[a]pyrene diol-epoxide (BPDE)-induced comet assay effects. Therefore, we measured DNA effects by the comet assay and adduct levels by high-performance liquid chromatography (HPLC) in human lymphocytes and A549 cells exposed to (±)-anti-benzo[a]pyrene-7,8-diol 9,10-epoxide [(±)-anti-BPDE] or (+)-anti-benzo[a]pyrene-7,8-diol 9,10-epoxide [(+)-anti-BPDE]. Both, the racemic form and (+)-anti-BPDE, which is the most relevant metabolite with regard to mutagenicity and carcinogenicity, induced DNA migration in cultured lymphocytes in the same range of concentrations to a similar extent in the alkaline comet assay after exposure for 2h. Nevertheless, (+)-anti-BPDE induced significantly enhanced DNA migration after 16 and 18h post-cultivation which was not seen in response to (±)-anti-BPDE. Combination of the comet assay with the Fpg (formamidopyrimidine-DNA glycosylase) protein did not enhance BPDE-induced effects and thus indicated the absence of Fpg-sensitive sites (oxidized purines, N7-guanine adducts, AP-sites). The aphidicolin (APC)-modified comet assay suggested significant excision repair activity of cultured lymphocytes during the first 18h of culture after a 2 h-exposure to BPDE. In contrast to these repair-related effects measured by the comet assay, HPLC analysis of stable adducts did not reveal any significant removal of (+)-anti-BPDE-induced adducts from lymphocytes during the first 22h of culture. On the other hand, HPLC measurements indicated that A549 cells repaired about 70% of (+)-anti-BPDE-induced DNA-adducts within 22h of release. However, various experiments with the APC-modified comet assay did not indicate significant repair activity during this period in A549 cells. The conflicting results obtained with the comet assay and the HPLC-based adduct analysis question the real cause for BPDE-induced DNA migration in the comet assay and the reliability of the APC-modified comet assay for the determination of DNA excision repair activity in response to BPDE in different cell types.
Subject(s)
7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide/toxicity , Comet Assay , Mutagens/toxicity , Cell Line, Tumor , DNA Adducts , DNA Damage/drug effects , Environmental Pollutants/toxicity , Humans , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Lymphocytes/drug effects , Lymphocytes/metabolismABSTRACT
Repair of mutagen-induced DNA lesions during transportation, storage and cultivation of lymphocytes may have a significant impact on results obtained in human biomonitoring after occupational and environmental exposure of human populations to genotoxic chemicals. Using the comet assay in combination with the repair inhibitor aphidicolin and array gene expression analysis of 92 DNA repair genes, we investigated the repair of DNA lesions induced by methyl methanesulfonate (MMS) and benzo[a]pyrenediolepoxide (BPDE) in phytohaemagglutinin (PHA)-stimulated cultured human lymphocytes in the time segment before replication. The comet assay indicated fast repair of MMS-induced damage during the first hours of cultivation. In contrast, removal of BPDE-induced lesions was slower and significant amounts of damage seem to persist until S-phase. Gene expression analysis revealed that PHA stimulation had a clear effect on gene regulation in lymphocytes already during the first 18h of cultivation. Under the conditions of this study, genotoxic concentrations of MMS did not induce significant changes in gene expression. In contrast, exposure to BPDE led to altered expression of several genes in a time- and concentration-related manner. Of the significantly up-regulated genes, only two genes (XPA and XPC) were directly related to nucleotide excision repair. Our results suggest that PHA stimulation of human lymphocytes influences the expression of DNA repair genes in human lymphocytes. The effect of induced DNA damage on gene expression is comparatively low and depends on the mutagens used. PHA-stimulated lymphocytes repair induced DNA damage before they start to replicate but the repair activity during the first 18h of cultivation is not affected by changes in the expression of DNA repair genes during this period of time.
Subject(s)
DNA Repair , Lymphocytes/drug effects , Lymphocytes/metabolism , Mutagens/toxicity , Cells, Cultured , Comet Assay , DNA Damage/drug effects , Gene Expression Profiling , Gene Expression Regulation/drug effects , HumansABSTRACT
The low molecular weight DNA diffusion assay (LMW assay) has been recommended as a measure for cytotoxicity for the in vivo comet assay. To better understand the relationship between effects in the LMW assay, DNA migration in the comet assay and effects in established cytotoxicity tests, we performed in vitro experiments with cultured human cell lines (TK6, A549) and comparatively investigated five test substances (methyl methanesulfonate, (±)-benzo[a]pyrene diol epoxide, sodium dodecyl sulphate, menthol and sodium arsenite). We measured DNA migration (tail intensity) in the comet assay and the frequency of 'hedgehogs' (cells with almost all DNA in the tail), DNA diffusion in the LMW assay, cell viability (trypan blue and fluorescein diacetate/ethidium bromide staining) and inhibition of proliferation (relative cell counts). Our in vitro experiments indicate that effects in the LMW assay occur independently from DNA effects in the comet assay and are not related to the occurrence of hedgehogs. Results from the LMW assay are in good agreement with results from viability assays and seem to allow discriminating genotoxic from non-genotoxic substances when appropriate preparation times are considered. Measurements of cytotoxicity by these methods only at an early preparation time after exposure to genotoxic substances may lead to erroneous results.
Subject(s)
Comet Assay/methods , DNA/chemistry , DNA/metabolism , 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide , Arsenites/pharmacology , Cell Death/drug effects , Cell Line, Tumor , Diffusion , Humans , Methyl Methanesulfonate/toxicity , Molecular Weight , Sodium Compounds/pharmacology , Sodium Dodecyl Sulfate/pharmacologyABSTRACT
The genotoxicity and mutagenicity of formaldehyde (FA) has been well-characterized during the last years. Besides its known direct DNA-damaging and mutagenic activity in sufficiently exposed cells, FA at low concentrations might also enhance the mutagenic and carcinogenic effects of other environmental mutagens by interfering with the repair of DNA lesions induced by these mutagens. To further assess potential co-mutagenic effects of FA, we exposed A549 human lung cells to FA in combination with various mutagens and measured the induction and removal of DNA damage by the comet assay and the production of chromosomal mutations by the cytokinesis-block micronucleus assay (CBMN assay). The mutagens tested were ionizing radiation (IR), (±)-anti-B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE), N-nitroso-N-methylurea (methyl nitrosourea; MNU) and methyl methanesulfonate (MMS). FA (10-75µM) did not enhance the genotoxic and mutagenic activity of these mutagens under the test conditions applied. FA alone and in combination with MNU or MMS did not affect the expression (mRNA level) of the gene of the O(6)-methylguanine-DNA methyltransferase (MGMT) in A549 cells. The results of these experiments do not support the assumption that low FA concentrations might interfere with the repair of DNA damage induced by other mutagens.
Subject(s)
Chromosome Aberrations/drug effects , Disinfectants/adverse effects , Drug Synergism , Formaldehyde/adverse effects , Lung Neoplasms/pathology , Mutagens/adverse effects , 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide/adverse effects , Alkylating Agents/adverse effects , Comet Assay , Drug Combinations , Gamma Rays/adverse effects , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Methyl Methanesulfonate/adverse effects , Methylnitrosourea/adverse effects , Micronucleus Tests , O(6)-Methylguanine-DNA Methyltransferase/genetics , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, CulturedABSTRACT
Recently, we have developed novel highly promising gene expression (GE) classifiers discriminating localized nodal (LFL) from systemic follicular lymphoma (SFL) with prognostic impact. However, few data are available in LFL especially concerning hotspot genetic alterations that are associated with the pathogenesis and prognosis of SFL. A total of 144 LFL and 527 SFL, enrolled in prospective clinical trials of the German Low Grade Lymphoma Study Group, were analyzed by fluorescence in situ hybridization to detect deletions in chromosomes 1p, 6q, and 17p as well as BCL2 translocations to determine their impact on clinical outcome of LFL patients. The frequency of chromosomal deletions in 1p and 17p was comparable between LFL and SFL, while 6q deletions and BCL2 translocations more frequently occurred in SFL. A higher proportion of 1p deletions was seen in BCL2-translocation-positive LFL, compared with BCL2-translocation-negative LFL. Deletions in chromosomes 1p, 6q, and 17p predicted clinical outcome of patients with SFL in the entire cohort, while only deletions in chromosome 1p retained its negative prognostic impact in R-CHOP-treated SFL. In contrast, no deletions in one of the investigated genetic loci predicted clinical outcome in LFL. Likewise, the presence or absence of BCL2 translocations had no prognostic impact in LFL. Despite representing a genetic portfolio closely resembling SFL, LFL showed some differences in deletion frequencies. BCL2 translocation and 6q deletion frequency differs between LFL and SFL and might contribute to distinct genetic profiles in LFL and SFL.
ABSTRACT
Chromosomal breakpoints involving the MYC gene locus, frequently referred to as MYC rearrangements (MYC - R+), are a diagnostic hallmark of Burkitt lymphoma and recurrent in many other subtypes of B-cell lymphomas including follicular lymphoma, diffuse large B-cell lymphoma and other high-grade B-cell lymphomas and are associated with an aggressive clinical course. In remarkable contrast, in MCL, only few MYC - R+ cases have yet been described. In the current study, we have retrospectively analysed 16 samples (MYC - R+, n = 15, MYC - R-, n = 1) from 13 patients and describe their morphological, immunophenotypic and (molecular) genetic features and clonal evolution patterns. Thirteen out of fifteen MYC - R+ samples showed a non-classical cytology including pleomorphic (centroblastic, immunoblastic), anaplastic or blastoid. MYC translocation partners were IG-loci in 4/11 and non-IG loci in 7/11 analysed cases. The involved IG-loci included IGH in 3 cases and IGL in one case. PAX5 was the non-IG partner in 2/7 patients. The MYC - R+ MCL reported herein frequently displayed characteristics associated with an aggressive clinical course including high genomic-complexity (6/7 samples), frequent deletions involving the CDKN2A locus (7/10 samples), high Ki-67 proliferation index (12/13 samples) and frequent P53 expression (13/13 samples). Of note, in 4/14 samples, SOX11 was not or only focally expressed and 3/13 samples showed focal or diffuse TdT-positivity presenting a diagnostic challenge as these features could point to a differential diagnosis of diffuse large B-cell lymphoma and/or lymphoblastic lymphoma/leukaemia.
Subject(s)
Biomarkers, Tumor/genetics , Chromosome Breakpoints , Cyclin D1/genetics , Gene Rearrangement , Lymphoma, Mantle-Cell/genetics , Proto-Oncogene Proteins c-myc/genetics , Aged , Aged, 80 and over , Biomarkers, Tumor/analysis , Child, Preschool , Clonal Evolution , Comparative Genomic Hybridization , Cytogenetic Analysis , DNA Nucleotidylexotransferase/analysis , Diagnosis, Differential , Female , Genetic Predisposition to Disease , Humans , Immunohistochemistry , Immunophenotyping , Lymphoma, Mantle-Cell/immunology , Lymphoma, Mantle-Cell/pathology , Male , Middle Aged , Neoplasm Grading , Phenotype , Predictive Value of TestsABSTRACT
Burkitt lymphoma (BL) is the most common B-cell lymphoma in children. Within the International Cancer Genome Consortium (ICGC), we performed whole genome and transcriptome sequencing of 39 sporadic BL. Here, we unravel interaction of structural, mutational, and transcriptional changes, which contribute to MYC oncogene dysregulation together with the pathognomonic IG-MYC translocation. Moreover, by mapping IGH translocation breakpoints, we provide evidence that the precursor of at least a subset of BL is a B-cell poised to express IGHA. We describe the landscape of mutations, structural variants, and mutational processes, and identified a series of driver genes in the pathogenesis of BL, which can be targeted by various mechanisms, including IG-non MYC translocations, germline and somatic mutations, fusion transcripts, and alternative splicing.
Subject(s)
Burkitt Lymphoma/genetics , Genome, Human , Transcriptome/genetics , Adolescent , Alternative Splicing/genetics , Amino Acid Sequence , Basic Helix-Loop-Helix Transcription Factors/chemistry , Basic Helix-Loop-Helix Transcription Factors/metabolism , Child , Child, Preschool , Chromosome Breakpoints , Cohort Studies , DNA Methylation/genetics , DNA Mutational Analysis , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , INDEL Mutation/genetics , Male , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Open Reading Frames/genetics , Polymorphism, Single Nucleotide/genetics , Proto-Oncogene Proteins c-myc/genetics , Translocation, Genetic , Whole Genome SequencingABSTRACT
Mutations in genes coding for proteins involved in DNA damage response (DDR) and repair, such as C9orf72 and FUS (Fused in Sarcoma), are associated with neurodegenerative diseases and lead to amyotrophic lateral sclerosis (ALS). Heterozygous loss-of-function mutations in NEK1 (NIMA-related kinase 1) have also been recently found to cause ALS. NEK1 codes for a multifunctional protein, crucially involved in mitotic checkpoint control and DDR. To resolve pathological alterations associated with NEK1 mutation, we compared hiPSC-derived motoneurons carrying a NEK1 mutation with mutant C9orf72 and wild type neurons at basal level and after DNA damage induction. Motoneurons carrying a C9orf72 mutation exhibited cell specific signs of increased DNA damage. This phenotype was even more severe in NEK1c.2434A>T neurons that showed significantly increased DNA damage at basal level and impaired DDR after induction of DNA damage in an maturation-dependent manner. Our results provide first mechanistic insight in pathophysiological alterations induced by NEK1 mutations and point to a converging pathomechanism of different gene mutations causative for ALS. Therefore, our study contributes to the development of novel therapeutic strategies to reduce DNA damage accumulation in neurodegenerative diseases and ALS.
Subject(s)
Amyotrophic Lateral Sclerosis/genetics , DNA Damage/genetics , Motor Neurons/metabolism , NIMA-Related Kinase 1/genetics , Amyotrophic Lateral Sclerosis/pathology , Humans , Mutation , TransfectionABSTRACT
The comet assay in combination with the polymerase inhibitor aphidicolin (APC) has been used to measure DNA excision repair activity, DNA repair kinetics and individual DNA repair capacity. Since APC can enhance genotoxic effects of mutagens measured by the comet assay, this approach has been proposed for increasing the sensitivity of the comet assay in human biomonitoring. The APC-modified comet assay has mainly been performed with human blood and it was shown that it not only enhances the detection of DNA damage repaired by nucleotide excision repair (NER) but also damage typically repaired by base excision repair (BER). Recently, we reported that in contrast to blood leukocytes, A549 cells (a human lung adenocarcinoma cell line) seem to be insensitive towards the repair-inhibiting action of APC. To further elucidate the general usefulness of the APC-modified comet assay for studying repair in cultured mammalian cells, we comparatively investigated further cell lines (HeLa, TK6, V79). DNA damage was induced by BPDE (benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide) and MMS (methyl methanesulfonate) in the absence and presence of APC (3 or 15µM). APC was either added for 2h together with the mutagen or cells were pre-incubated for 30min with APC before the mutagen was added. The results indicate that the cell lines tested differ fundamentally with regard to their sensitivity and specificity towards the repair-inhibiting effect of APC. The actual cause for these differences is still unclear but potential molecular explanations are discussed. Irrespective of the underlying mechanism(s), our study revealed practical limitations of the use of the APC-modified comet assay.
Subject(s)
Aphidicolin/toxicity , Comet Assay , DNA/drug effects , Cell Line , DNA Damage/drug effects , DNA Damage/genetics , DNA Polymerase II/metabolism , DNA Polymerase III/metabolism , Humans , Mutagens/toxicityABSTRACT
Mouse mutants with an impaired DNA damage response frequently exhibit a set of remarkably similar defects in the HSPC compartment that are of largely unknown molecular basis. Using Mixed-Lineage-Leukemia-5 (Mll5)-deficient mice as prototypical examples, we have identified a mechanistic pathway linking DNA damage and HSPC malfunction. We show that Mll5 deficiency results in accumulation of DNA damage and reactive oxygen species (ROS) in HSPCs. Reduction of ROS efficiently reverses hematopoietic defects, establishing ROS as a major cause of impaired HSPC function. The Ink4a/Arf locus also contributes to HSPC phenotypes, at least in part via promotion of ROS. Strikingly, toxic ROS levels in Mll5-/- mice are critically dependent on type 1 interferon (IFN-1) signaling, which triggers mitochondrial accumulation of full-length Bid. Genetic inactivation of Bid diminishes ROS levels and reverses HSPC defects in Mll5-/- mice. Overall, therefore, our findings highlight an unexpected IFN-1 > Bid > ROS pathway underlying DNA damage-associated HSPC malfunction.
Subject(s)
BH3 Interacting Domain Death Agonist Protein/metabolism , DNA Damage , Hematopoietic Stem Cells/metabolism , Interferon Type I/metabolism , Reactive Oxygen Species/metabolism , Signal Transduction , Acetylcysteine/administration & dosage , Acetylcysteine/pharmacology , Administration, Oral , Animals , Animals, Newborn , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Genetic Loci , Hematopoiesis/drug effects , Hematopoietic Stem Cells/drug effects , Histone-Lysine N-Methyltransferase , Intracellular Space/metabolism , Mice , Poly I-C/pharmacology , Protein Transport/drug effects , Receptors, Interferon/metabolism , Signal Transduction/drug effects , Survival AnalysisABSTRACT
Mutations within the FUS gene (Fused in Sarcoma) are known to cause Amyotrophic Lateral Sclerosis (ALS), a neurodegenerative disease affecting upper and lower motoneurons. The FUS gene codes for a multifunctional RNA/DNA-binding protein that is primarily localized in the nucleus and is involved in cellular processes such as splicing, translation, mRNA transport and DNA damage response. In this study, we analyzed pathophysiological alterations associated with ALS related FUS mutations (mFUS) in human induced pluripotent stem cells (hiPSCs) and hiPSC derived motoneurons. To that end, we compared cells carrying a mild or severe mFUS in physiological- and/or stress conditions as well as after induced DNA damage. Following hyperosmolar stress or irradiation, mFUS hiPS cells recruited significantly more cytoplasmatic FUS into stress granules accompanied by impaired DNA-damage repair. In motoneurons wild-type FUS was localized in the nucleus but also deposited as small punctae within neurites. In motoneurons expressing mFUS the protein was additionally detected in the cytoplasm and a significantly increased number of large, densely packed FUS positive stress granules were seen along neurites. The amount of FUS mislocalization correlated positively with both the onset of the human disease (the earlier the onset the higher the FUS mislocalization) and the maturation status of the motoneurons. Moreover, even in non-stressed post-mitotic mFUS motoneurons clear signs of DNA-damage could be detected. In summary, we found that the susceptibility to cell stress was higher in mFUS hiPSCs and hiPSC derived motoneurons than in controls and the degree of FUS mislocalization correlated well with the clinical severity of the underlying ALS related mFUS. The accumulation of DNA damage and the cellular response to DNA damage stressors was more pronounced in post-mitotic mFUS motoneurons than in dividing hiPSCs suggesting that mFUS motoneurons accumulate foci of DNA damage, which in turn might be directly linked to neurodegeneration.
ABSTRACT
The comet assay is widely used in human biomonitoring to measure DNA damage in whole blood or isolated peripheral blood mononuclear cells (PBMC) as a marker of exposure to genotoxic agents. Cytogenetic assays with phytohemagglutinin (PHA)-stimulated cultured T lymphocytes are also frequently performed in human biomonitoring. Cytogenetic effects (micronuclei, chromosome aberrations, sister chromatid exchanges) may be induced in vivo but also occur ex vivo during the cultivation of lymphocytes as a consequence of DNA damage present in lymphocytes at the time of sampling. To better understand whether DNA damage measured by the comet assay in PBMC is representative for DNA damage in T cells, we comparatively investigated DNA damage and its repair in PBMC and T cells obtained by immunomagnetic cell sorting. PBMC cultures and T cell cultures were exposed to mutagens with different modes of genotoxic action and DNA damage was measured by the comet assay after the end of a 2h exposure and after 18h post-incubation. The mutagens tested were methyl methanesulfonate (MMS), (±)-anti-B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE), 4-nitroquinoline-1-oxide (4NQO), styrene oxide and potassium bromate. MMS and potassium bromate were also tested by the modified comet assay with formamido pyrimidine glycosylase (FPG) protein. The results indicate that the mutagens tested induce DNA damage in PBMC and T cells in the same range of concentrations and removal of induced DNA lesions occurs to a comparable extent. Based on these results, we conclude that the comet assay with PBMC is suited to predict DNA damage and its removal in T cells.
Subject(s)
Comet Assay/methods , DNA Repair , Mutagenesis , T-Lymphocytes/cytology , T-Lymphocytes/metabolism , 4-Nitroquinoline-1-oxide/toxicity , 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide/toxicity , Bromates/toxicity , Cells, Cultured , DNA Repair/drug effects , DNA Repair/immunology , Epoxy Compounds/toxicity , Humans , Immunomagnetic Separation , Male , Methyl Methanesulfonate/toxicity , Mutagenesis/drug effects , Mutagenesis/immunology , Mutagenicity Tests , Mutagens/toxicity , T-Lymphocytes/drug effectsABSTRACT
Few data are available regarding the reliability of fluorescence in-situ hybridization (FISH), especially for chromosomal deletions, in high-throughput settings using tissue microarrays (TMAs). We performed a comprehensive FISH study for the detection of chromosomal translocations and deletions in formalin-fixed and paraffin-embedded (FFPE) tumor specimens arranged in TMA format. We analyzed 46 B-cell lymphoma (B-NHL) specimens with known karyotypes for translocations of IGH-, BCL2-, BCL6- and MYC-genes. Locus-specific DNA probes were used for the detection of deletions in chromosome bands 6q21 and 9p21 in 62 follicular lymphomas (FL) and six malignant mesothelioma (MM) samples, respectively. To test for aberrant signals generated by truncation of nuclei following sectioning of FFPE tissue samples, cell line dilutions with 9p21-deletions were embedded into paraffin blocks. The overall TMA hybridization efficiency was 94%. FISH results regarding translocations matched karyotyping data in 93%. As for chromosomal deletions, sectioning artefacts occurred in 17% to 25% of cells, suggesting that the proportion of cells showing deletions should exceed 25% to be reliably detectable. In conclusion, FISH represents a robust tool for the detection of structural as well as numerical aberrations in FFPE tissue samples in a TMA-based high-throughput setting, when rigorous cut-off values and appropriate controls are maintained, and, of note, was superior to quantitative PCR approaches.