Jan J. W. A. Boei

Involvement of mouse Rev3 in tolerance of endogenous and exogenous DNA damage.

ABSTRACT The Rev3 gene of Saccharomyces cerevisiae encodes the catalytic subunit of DNA polymerase ζ that is implicated in mutagenic translesion synthesis of damaged DNA. To investigate the function of its mouse homologue, we have generated mouse embryonic stem cells and mice carrying a targeted disruption of Rev3. Although some strain-dependent variation was observed, Rev3−/− embryos died around midgestation, displaying retarded growth in the absence of consistent developmental abnormalities. Rev3−/− cell lines could not be established, indicating a cell-autonomous requirement of Rev3 for long-term viability. Histochemical analysis of Rev3−/− embryos did not reveal aberrant replication or cellular proliferation but demonstrated massive apoptosis in all embryonic lineages. Although increased levels of p53 are detected in Rev3−/− embryos, the embryonic phenotype was not rescued by the absence of p53. A significant increase in double-stranded DNA breaks as well as chromatid and chromosome aberrations was observed in cells from Rev3−/− embryos. The inner cell mass of cultured Rev3−/− blastocysts dies of a delayed apoptotic response after exposure to a low dose of N-acetoxy-2-acetylaminofluorene. These combined data are compatible with a model in which, in the absence of polymerase ζ, double-stranded DNA breaks accumulate at sites of unreplicated DNA damage, eliciting a p53-independent apoptotic response. Together, these data are consistent with involvement of polymerase ζ in translesion synthesis of endogenously and exogenously induced DNA lesions.

Analysis of radiation-induced chromosomal aberrations using telomeric and centromeric PNA probes.

Purpose : To generate dose-response curves for X-ray-induced chromosomal aberrations analysed in human blood lymphocytes using telomeric and centromeric peptide nucleic acid (PNA) probes. Materials and methods : Isolated human lymphocytes were X-irradiated with doses of 0, 1, 2, 3, 4 and 6 Gy. Aberrations were analysed in the first post-irradiation metaphases using telomeric and centromeric PNA probes. Results : Similar to the dose-response curves for the yield of dicentrics and centric rings, the dose-response curves for interstitial fragments and incomplete elements (derived from either terminal deletions or incomplete exchanges) follow a linear-quadratic function. Furthermore, it was estimated that 76% of excess acentric fragments originate from complete exchanges (interstitial deletions) and only 24% from incomplete exchanges or terminal deletions. Conclusions : Interstitial fragments form a major class of radiation-induced chromosomal aberrations. They are induced about half as frequently as dicentrics over the whole dose range investigated. The comparable trend of the dose-response curve for the different aberrations, including incomplete elements, indicates that all detected aberrations are formed by a similar underlying mechanism. It also suggests that the ratio between non- or incomplete repair (leading to open ends of broken chromosomes) and incorrect repair (leading to exchange aberrations) is independent of dose.PURPOSE To generate dose-response curves for X-ray-induced chromosomal aberrations analysed in human blood lymphocytes using telomeric and centromeric peptide nucleic acid (PNA) probes. MATERIALS AND METHODS Isolated human lymphocytes were X-irradiated with doses of 0, 1, 2, 3, 4 and 6 Gy. Aberrations were analysed in the first post-irradiation metaphases using telomeric and centromeric PNA probes. RESULTS Similar to the dose-response curves for the yield of dicentrics and centric rings, the dose-response curves for interstitial fragments and incomplete elements (derived from either terminal deletions or incomplete exchanges) follow a linear-quadratic function. Furthermore, it was estimated that 76% of excess acentric fragments originate from complete exchanges (interstitial deletions) and only 24% from incomplete exchanges or terminal deletions. CONCLUSIONS Interstitial fragments form a major class of radiation-induced chromosomal aberrations. They are induced about half as frequently as dicentrics over the whole dose range investigated. The comparable trend of the dose-response curve for the different aberrations, including incomplete elements, indicates that all detected aberrations are formed by a similar underlying mechanism. It also suggests that the ratio between non- or incomplete repair (leading to open ends of broken chromosomes) and incorrect repair (leading to exchange aberrations) is independent of dose.

Impact of radiation quality on the spectrum of induced chromosome exchange aberrations.

Purpose: To study the impact of radiation quality on the spectrum of chromosome exchange aberrations in human lymphocytes using chromosome arm-specific and telomeric probes. The analysis is focused on: (1) incomplete exchanges, (2) interstitial fragments, (3) interarm intrachanges and (4) the complexity of the aberration patterns. The present data after neutron exposure are compared with previously obtained data after X-irradiation. Materials and methods: Isolated human lymphocytes from three donors were irradiated with 1 MeV fast neutrons (0.25, 0.5, 1.0, 1.5, 2.0Gy). Analysis was performed on first post-irradiation metaphases with arm-specific probes for chromosome 1 in combination with a pan-centromeric probe, or with telomeric and centromeric PNA probes. Results: In comparison with X-rays, exposure to neutrons leads to: (1) similar frequencies of incomplete exchanges or terminal deletions, (2) a significantly higher induction of both inter- and intraarm intrachanges, (3) a higher proportion of complex aberrations, and (4) aberrations with a higher degree of complexity, i.e. derived from more chromosome breaks which interact more frequently in a non-reciprocal fashion. Essentially no dose dependence was found for the yield ratios between the various types of chromosomal aberrations. Conclusions: Despite the reduced rejoining eyciency of DNA double-strand breaks induced by high-LET radiation, exposure to neutrons does not lead to enhanced levels of unrejoined chromosome breaks that can be observed as incomplete exchanges in cells that have reached mitosis. Proximity effects are more pronounced after densely ionizing radiation than after sparsely ionizing radiation. Clustered damage produced by neutron tracks results in a high proportion of complex aberrations and in non-reciprocal interactions of chromosome breaks. Most of the exchanges occur within one neutron track and little interaction seems to take place between the breaks formed in different tracks.

Different Sets of Translesion Synthesis DNA Polymerases Protect From Genome Instability Induced by Distinct Food-Derived Genotoxins

DNA lesions, induced by genotoxic compounds, block the processive replication fork but can be bypassed by specialized translesion synthesis (TLS) DNA polymerases (Pols). TLS safeguards the completion of replication, albeit at the expense of nucleotide substitution mutations. We studied the in vivo role of individual TLS Pols in cellular responses to benzo[a]pyrene diolepoxide (BPDE), a polycyclic aromatic hydrocarbon, and 4-hydroxynonenal (4-HNE), a product of lipid peroxidation. To this aim, we used mouse embryonic fibroblasts with targeted disruptions in the TLS-associated Pols η, ι, κ, and Rev1 as well as in Rev3, the catalytic subunit of TLS Polζ. After exposure, cellular survival, replication fork progression, DNA damage responses (DDR), and the induction of micronuclei were investigated. The results demonstrate that Rev1, Rev3, and, to a lesser extent, Polη are involved in TLS and the prevention of DDR and of DNA breaks, in response to both agents. Conversely, Polκ and the N-terminal BRCT domain of Rev1 are specifically involved in TLS of BPDE-induced DNA damage. We furthermore describe a novel role of Polι in TLS of 4-HNE-induced DNA damage in vivo. We hypothesize that different sets of TLS polymerases act on structurally different genotoxic DNA lesions in vivo, thereby suppressing genomic instability associated with cancer. Our experimental approach may provide a significant contribution in delineating the molecular bases of the genotoxicity in vivo of different classes of DNA-damaging agents.

Intrachanges as part of complex chromosome-type exchange aberrations

The chromosome-type exchange aberrations induced by ionizing radiation during the G(0)/G(1) phase of the cell cycle are believed to be the result of illegitimate rejoining of chromosome breaks. From numerous studies using chromosome painting, it has emerged that even after a moderate dose of radiation, a substantial fraction of these exchanges is complex. Most of them are derived from the free interaction between the ends of three or more breaks. Other studies have demonstrated that chromosomes occupy distinct territories in the interphase nucleus. Since breaks that are in close proximity have an enhanced interaction probability, it seems likely that after ionizing radiation many of the interacting breaks will be present within one chromosome or chromosome arm. Unfortunately, the majority of these intrachanges remain undetected, even when sophisticated molecular cytogenetic detection methods (i.e. mFISH) are applied to paint all chromosome pairs in distinct colors. In the present paper, we evaluate the limitations of full-color painting for the detection of complex exchanges and the correct interpretations of break interactions.

Mitomycin C-induced pairing of heterochromatin reflects initiation of DNA repair and chromatid exchange formation

Chromatid interchanges induced by the DNA cross-linking agent mitomycin C (MMC) are over-represented in human chromosomes containing large heterochromatic regions. We found that nearly all exchange breakpoints of chromosome 9 are located within the paracentromeric heterochromatin and over 70% of exchanges involving chromosome 9 are between its homologues. We provide evidence that the required pairing of chromosome 9 heterochromatic regions occurs in G0/G1 and S-phase cells as a result of an active cellular process initiated upon MMC treatment. By contrast, no pairing was observed for a euchromatic paracentromeric region of the equal-sized chromosome 8. The MMC-induced pairing of chromosome 9 heterochromatin is observed in a subset of cells; its percentage closely mimics the frequency of homologous interchanges found at metaphase. Moreover, the absence of pairing in cells derived from XPF patients correlates with an altered spectrum of MMC-induced exchanges. Together, the data suggest that the heterochromatin-specific pairing following MMC treatment reflects the initiation of DNA cross-link repair and the formation of exchanges.

No Threshold for the Induction of Chromosomal Damage at Clinically Relevant Low Doses of X Rays

The recent steep increase in population dose from radiation-based medical diagnostics, such as computed tomography (CT) scans, requires insight into human health risks, especially in terms of cancer development. Since the induction of genetic damage is considered a prominent cause underlying the carcinogenic potential of ionizing radiation, we quantified the induction of micronuclei and loss of heterozygosity events in human cells after exposure to clinically relevant low doses of X rays. A linear dose-response relationship for induction of micronuclei was observed in human fibroblasts with significantly increased frequencies at doses as low as 20 mGy. Strikingly, cells exposed during S-phase displayed the highest induction, whereas non S-phase cells showed no significant induction below 100 mGy. Similarly, the induction of loss of heterozygosity in human lymphoblastoid cells quantified at HLA loci, was linear with dose and reached significance at 50 mGy. Together the findings favor a linear-no-threshold model for genetic damage induced by acute exposure to ionizing radiation. We speculate that the higher radiosensitivity of S-phase cells might relate to the excessive cancer risk observed in highly proliferative tissues in radiation exposed organisms.

Xenobiotic metabolism in differentiated human bronchial epithelial cells

Differentiated human bronchial epithelial cells in air liquid interface cultures (ALI-PBEC) represent a promising alternative for inhalation studies with rodents as these 3D airway epithelial tissue cultures recapitulate the human airway in multiple aspects, including morphology, cell type composition, gene expression and xenobiotic metabolism. We performed a detailed longitudinal gene expression analysis during the differentiation of submerged primary human bronchial epithelial cells into ALI-PBEC to assess the reproducibility and inter-individual variability of changes in transcriptional activity during this process. We generated ALI-PBEC cultures from four donors and focussed our analysis on the expression levels of 362 genes involved in biotransformation, which are of primary importance for toxicological studies. Expression of various of these genes (e.g., GSTA1, ADH1C, ALDH1A1, CYP2B6, CYP2F1, CYP4B1, CYP4X1 and CYP4Z1) was elevated following the mucociliary differentiation of airway epithelial cells into a pseudo-stratified epithelial layer. Although a substantial number of genes were differentially expressed between donors, the differences in fold changes were generally small. Metabolic activity measurements applying a variety of different cytochrome p450 substrates indicated that epithelial cultures at the early stages of differentiation are incapable of biotransformation. In contrast, mature ALI-PBEC cultures were proficient in the metabolic conversion of a variety of substrates albeit with considerable variation between donors. In summary, our data indicate a distinct increase in biotransformation capacity during differentiation of PBECs at the air–liquid interface and that the generation of biotransformation competent ALI-PBEC cultures is a reproducible process with little variability between cultures derived from four different donors.