Bruno Morolli

Targeted Inactivation of Mouse RAD52 Reduces Homologous Recombination but Not Resistance to Ionizing Radiation

ABSTRACT The RAD52 epistasis group is required for recombinational repair of double-strand breaks (DSBs) and shows strong evolutionary conservation. In Saccharomyces cerevisiae, RAD52 is one of the key members in this pathway. Strains with mutations in this gene show strong hypersensitivity to DNA-damaging agents and defects in recombination. Inactivation of the mouse homologue of RAD52in embryonic stem (ES) cells resulted in a reduced frequency of homologous recombination. Unlike the yeast Scrad52 mutant,MmRAD52−/− ES cells were not hypersensitive to agents that induce DSBs. MmRAD52 null mutant mice showed no abnormalities in viability, fertility, and the immune system. These results show that, as in S. cerevisiae, MmRAD52is involved in recombination, although the repair of DNA damage is not affected upon inactivation, indicating that MmRAD52 may be involved in certain types of DSB repair processes and not in others. The effect of inactivating MmRAD52 suggests the presence of genes functionally related to MmRAD52, which can partly compensate for the absence of MmRad52 protein.

Identification of RUNX1/AML1 as a classical tumor suppressor gene

Based on our previous results indicating the presence of a tumor suppressor gene (TSG), chromosome 21 was analysed for loss of heterozygosity (LOH) in 18 patients with acute myeloid leukemia (17, AML-M0; one, AML-M1). Allelotyping at polymorphic loci was performed on purified material, allowing unequivocal detection of allelic loss and homozygous deletions. Six AML-M0 patients shared a common region of LOH harboring a single gene: RUNX1 (AML1), the most frequent site of translocations in acute leukemia and a well-known fusion oncogene. Fluorescence in situ hybridization allowed the identification of deletions with breakpoints within RUNX1 in two patients as the cause of LOH. In the four others the LOH pattern and the presence of two karyotypically normal chromosomes 21 were in line with mitotic recombination. Further molecular and cytogenetic analyses showed that this caused homozygosity of primary RUNX1 mutations: two point mutations, a partial deletion and, most significantly, a complete deletion of RUNX1. These findings identify RUNX1 as a classical TSG: both alleles are mutated or absent in cancer cells from four of the 17 AML-M0 patients examined. In contrast to AML-M0, the AML-M1 patient was trisomic for chromosome 21 and has two mutated and one normal RUNX1 allele, suggesting that the order of mutagenic events leading to leukemia may influence the predominant tumor type.

XR-C1, a new CHO cell mutant which is defective in DNA-PKcs, is impaired in both V(D)J coding and signal joint formation

DNA-dependent protein kinase (DNA-PK) plays an important role in DNA double-strand break (DSB) repair and V(D)J recombination. We have isolated a new X-ray-sensitive CHO cell line, XR-C1, which is impaired in DSB repair and which was assigned to complementation group 7, the group that is defective in the XRCC7 / SCID ( Prkdc ) gene encoding the catalytic subunit of DNA-PK (DNA-PKcs). Consistent with this complementation analysis, XR-C1 cells lackeddetectable DNA-PKcs protein, did not display DNA-PK catalytic activity and were complemented by the introduction of a single human chromosome 8 (providing the Prkdc gene). The impact of the XR-C1 mutation on V(D)J recombination was quite different from that found in most rodent cells defective in DNA-PKcs, which are preferentially blocked in coding joint formation, whereas XR-C1 cells were defective in forming both coding and signal joints. These results suggest that DNA-PKcs is required for both coding and signal joint formation during V(D)J recombination and that the XR-C1 mutant cell line may prove to be a useful tool in understanding this pathway.

The ToxTracker assay: novel GFP reporter systems that provide mechanistic insight into the genotoxic properties of chemicals.

People are exposed to an ever-increasing number of chemical compounds that are developed by industry for a wide range of applications. These compounds may harmfully react with different cellular components and activate specific defense mechanisms that provide protection against the toxic, mutagenic, and possibly oncogenic consequences of exposure. Monitoring the activation of specific cellular signaling pathways upon exposure may therefore allow reliable and mechanism-based assessment of potential (geno)toxic properties of chemicals, while providing insight into their primary mode of toxicity. By whole-genome transcription profiling of mouse embryonic stem cells, we identified genes that were transcriptionally activated upon exposure to either genotoxic compounds or pro-oxidants. For selected biomarker genes, we constructed reporters encoding C-terminal green fluorescent protein (GFP)-tagged fusion proteins. GFP reporter genes were located on bacterial artificial chromosomes, thereby enabling transcriptional regulation of the reporters by their own physiological promoter. The Bscl2-GFP reporter is selectively activated after exposure to genotoxic agents and its induction is associated with inhibition of DNA replication and activation of the ataxia telangiectasia and Rad3-related protein signaling pathway. The Srxn1-GFP reporter is preferentially induced upon oxidative stress and is part of the nuclear factor (erythroid-derived 2)-like 2-antioxidant response pathway. The novel (geno)toxicity assay (ToxTracker) that utilize the differential responsiveness of various reporter cell lines will enable prediction of the primary reactive properties of known and unknown chemicals.

Lack of genetic and epigenetic changes in meningiomas without NF2 loss.

Approximately 60% of sporadic meningiomas are caused by inactivation of the NF2 tumour suppressor gene. The causative gene for the remaining meningiomas is unknown. Previous studies have shown that these tumours have no recurrent karyotypic abnormalities. They differ from their NF2‐related counterparts in that they are more often of the meningothelial subtype and are located preferentially in the anterior skull base. To gain more insight into the aetiology of these tumours, we studied genetic and epigenetic alterations in 25 meningiomas without NF2 involvement. We first established a genome‐wide allelotype using 3 microsatellite markers per chromosome arm. Loss of heterozygosity (LOH) was detected at a low frequency and no indication for the location of putative tumour suppressor genes could be established. We next screened the subtelomeric regions by using 2–3 polymorphic markers close to each telomere. Again no evidence for LOH of a particular chromosome arm was obtained, and no LOH was found in the genomic regions containing the NF2‐related ERM family members ezrin and radixin, DAL‐1, protein 4.1R, and TSLC1. Mutations in the X‐chromosome based family member, moesin, were analysed by SSCP and were not detected. Microsatellite instability was studied using 6 commonly used markers but none of these was altered in any meningioma. Methylation was detected in 5 of 16 genes (NF2, p14ARF, CDH1, BRCA1, RB1) previously shown to be silenced in a variety of tumour types. However, methylation percentages for these genes were generally higher in a group of NF2‐related meningiomas, with the exception of the BRCA1 gene. The NF2 gene was methylated in only 1 of 21 tumours. In conclusion, meningiomas with an intact NF2 gene have a normal karyotype and no obvious genetic or epigenetic aberrations, suggesting that the gene(s) involved in the pathogenesis of these tumours are altered by smaller events than can be detected with the techniques used in our study. Copyright

A novel type of X-ray-sensitive Chinese hamster cell mutant with radioresistant DNA synthesis and hampered DNA double-strand break repair

It has been shown that the Chinese hamster cell mutant V-C8 is sensitive to different DNA damaging agents, such as mitomycin C (MMC), alkylating agents, UV light, and X-rays. We found that V-C8 is also sensitive to the following radiomimetic agents: bleomycin (approximately 2-fold, based on D10 values), H2O2 (approximately 2-fold), streptonigrin (approximately 11-fold), and etoposide (approximately 8-fold). Two independent spontaneous MMC-resistant revertants isolated from V-C8 cells show a level of cell killing by X-rays, EMS, and UV light which is similar to that of wild-type cells, suggesting that the observed pattern of cross-sensitivity of V-C8 cells to a wide spectrum of DNA damaging agents results from a single mutation. V-C8 cells also display radioresistant DNA synthesis following gamma-irradiation which, however, remained almost unchanged in the V-C8 revertants. The measurement of the level and rate of repair of DNA single- and double-strand breaks (SSBs and DSBs, respectively) by the DNA elution technique showed that the V-C8 mutant has a slower repair of DSBs induced by gamma-rays. The described unique phenotype of V-C8 cells suggested that V-C8 represents a novel type of mutant amongst X-ray-sensitive hamster cell mutants. To confirm this, complementation analysis with other X-ray-sensitive mutants was performed. V-C8 cells were fused with EM9, XR-1, xrs5, sxi-1, V-3, V-E5, irs3, and BLM2 mutant cells, representing different complementation groups. All the obtained hybrids regained X-ray resistance (or bleomycin resistance in the case of V-C8/BLM2 hybrids) similar to that of wild-type cells, indicating that V-C8 represents a new complementation group. The results presented indicate that V-C8 is defective in a gene involved in a pathway operating in the responses to different DNA damaging agents in mammalian cells.

The extended ToxTracker assay discriminates between induction of DNA damage, oxidative stress and protein misfolding.

Chemical exposure of cells may damage biomolecules, cellular structures, and organelles thereby jeopardizing cellular homeostasis. A multitude of defense mechanisms have evolved that can recognize specific types of damaged molecules and will initiate distinct cellular programs aiming to remove the damage inflicted and prevent cellular havoc. As a consequence, quantitative assessment of the activity of the cellular stress responses may serve as a sensitive reporter for the induction of specific types of damage. We have previously developed the ToxTracker assay, a mammalian stem cell-based genotoxicity assay employing two green fluorescent protein reporters specific for DNA damage and oxidative stress. We have now expanded the ToxTracker assay with an additional four reporter cell lines to include monitoring of additional stress signaling pathways. This panel of six green fluorescent protein reporters is able to discriminate between different primary reactivity of chemicals being their ability to react with DNA and block DNA replication, induce oxidative stress, activate the unfolded protein response, or cause a general P53-dependent cellular stress response. Extensive validation using the compound library suggested by the European Centre for the Validation of Alternative Methods (ECVAM) and a large panel of reference chemicals shows that the ToxTracker assay has an outstanding sensitivity and specificity. In addition, we developed Toxplot, a dedicated software tool for automated data analysis and graphical representation of the test results. Rapid and reliable identification by the ToxTracker assay of specific biological reactivity can significantly improve in vitro human hazard assessment of chemicals.

ETV6 mutations and loss in AML-M0

ETV6 (ETS translocation-variant gene 6, located on chromosome 12p), also known as TEL, encodes a transcription repressor belonging to the E26 transforming specific (ETS) family of DNA-binding proteins. ETV6 is known as a proto-oncogene involved in translocation with over 40 partners.1 In acute myeloid leukemia (AML) only a few rare translocations result in transforming fusion proteins,1 indicating that the oncogenic role of ETV6 does not play a major part in AML. However, abnormalities of the short arm of chromosome 12 (12p) are found in about 5% of AML and myelodysplastic syndromes. Most abnormalities consist of total or partial loss of 12p usually affecting ETV6 and CDKN1B, implicating these genes as tumor-suppressor genes.1, 2, 3, 4 Recently, heterozygous mutations of ETV6, resulting in loss of repressor activity, were found in AML, adding to the view that ETV6 might have tumor-suppressor characteristics.5

Sensitive DsRed fluorescence-based reporter cell systems for genotoxicity and oxidative stress assessment

Various in vitro test systems have been developed for genotoxic risk assessment in early drug development. However, these genotoxicity tests often show limited specificity, and provide limited insights into the mode of toxicity of the tested compounds. To identify genes that could serve as specific biomarkers for genotoxicity or oxidative stress, we exposed mouse embryonic stem (ES) cells to various genotoxic and oxidative stress-inducing compounds and performed genome-wide expression profiling. Differentially expressed genes were classified based on the fold-change of expression and their specificity for either genotoxic or oxidative stress. Promoter regions of four selected genes (Ephx1, Btg2, Cbr3 and Perp) were fused to a DsRed fluorescent reporter gene and stably integrated in mouse ES cells. Established stable reporter cell lines displayed significant induction of DsRed expression upon exposure to different classes of genotoxic and oxidative stress-inducing compounds. In contrast, exposure to non-genotoxic carcinogenic compounds did not induce DsRed expression even at cytotoxic doses. Expression of the Cbr3-DsRed reporter was more responsive to compounds that induce oxidative stress while the other three DsRed reporters reacted more specific to direct-acting genotoxic agents. Therefore, the differential response of the Btg2- and Cbr3-DsRed reporters can serve as indicator for the main action mechanism of genotoxic and oxidative stress-inducing compounds. In addition, we provide evidence that inhibition of DNA replication results in preferential activation of the Btg2-DsRed genotoxicity reporter. In conclusion, we have generated sensitive mouse ES cell reporter systems that allow detection of genotoxic and oxidative stress-inducing properties of chemical compounds and can be used in high-throughput assays.

Genome wide molecular analysis of minimally differentiated acute myeloid leukemia

This study used single nucleotide polymorphism (SNP)-array technology to study copy number changes and to determine regions of loss of heterozygosity in minimally differentiated acute myeloid leukemia. Several chromosomal regions were found to be deleted or duplicated, and mutations in 163gene were the most frequent mutations detected. Background Minimally differentiated acute myeloid leukemia is heterogeneous in karyotype and is defined by immature morphological and molecular characteristics. This originally French-American-British classification is still used in the new World Health Organization classification when other criteria are not met. Apart from RUNX1 mutation, no characteristic molecular aberrations are recognized. Design and Methods We performed whole genome single nucleotide polymorphism analysis and extensive molecular analysis in a cohort of 52 patients with minimally differentiated acute myeloid leukemia. Results Many recurring and potentially relevant regions of loss of heterozygosity were revealed. These point towards a variety of candidate genes that could contribute to the pathogenesis of minimally differentiated acute myeloid leukemia, including the tumor suppressor genes TP53 and NF1, and reinforced the importance of RUNX1 in this leukemia. Furthermore, for the first time in this minimally differentiated form of leukemia we detected mutations in the transactivation domain of RUNX1. Mutations in other acute myeloid leukemia associated transcriptions factors were infrequent. In contrast, FLT3, RAS, PTPN11 and JAK2 were often mutated. Irrespective of the RUNX1 mutation status, our results show that RAS signaling is the most important pathway for proliferation in minimally differentiated acute myeloid leukemia. Importantly, we found that high terminal deoxynucleotidyl transferase expression is closely associated with RUNX1 mutation, which could allow an easier diagnosis of RUNX1 mutation in this hematologic malignancy. Conclusions Our results suggest that in patients without RUNX1 mutation, several other molecular aberrations, separately or in combination, contribute to a common minimally differentiated phenotype.