Inmaculada Domínguez

SPONTANEOUS AND RADIATION-INDUCED CHROMOSOMAL BREAKAGE AT INTERSTITIAL TELOMERIC SITES

The Chinese hamster genome contains a total of 18 cytologically detectable arrays of interstitial telometic sequences. A combination of G-banding and twocolour fluorescence in situ hybridization revealed that 25 out of 27 (93%) breakpoints of spontaneously occurring terminal deletions in four immortalized Chinese hamster cell lines were located in chromosomal regions containing interstitial telomeric sequences. Each of the four immortalized Chinese hamster cell lines expressed telomerase. Radiation experiments revealed the sensitivity of interstitial telomeric sequences to radiation-induced chromosomal breakage in all telomerase-positive cell lines. However, radiation-induced chromosomal breakage at interstitial telomeric sites in non-transformed, primary Chinese hamster cells was almost non-existent. Telomerase activity in primary Chinese hamster cells was not detected. These results indirectly suggest that interstitial telomeric sites represent a favourable substrate for chromosomal healing.

Mechanisms of induction of chromosomal aberrations and their detection by fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) technique using chromosome specific probes has revolutionized the field of radiation cytogenetics in the last few years. Some of the new insights on the origins of radiation induced chromosome aberrations in human, mouse and Chinese hamster, using FISH are reviewed in this paper.

Roles of DNA topoisomerases in chromosome segregation and mitosis.

DNA topoisomerases are highly specialized nuclear enzymes that perform topological changes in the DNA molecule in a very precise and unique fashion. Taking into account their fundamental roles in many events during DNA metabolism such as replication, transcription, recombination, condensation or segregation, it is no wonder that the last decade has witnessed an exponential interest on topoisomerases, mainly after the discovery of their potential role as targets in novel antitumor therapy. The difficulty of the lack of topoisomerase II mutants in higher eukaryotes has been partly overcome by the availability of drugs that act as either poisons or true catalytic inhibitors of the enzyme. These chemical tools have provided strong evidence that accurate performance of topoisomerase II is essential for chromosome segregation before anaphase, and this in turn constitutes a prerequisite for the development of normal mitosis. In the absence of cytokinesis, cells become polyploid or endoreduplicated.

Adaptive response to radiation damage in human lymphocytes conditioned with hydrogen peroxide as measured by the cytokinesis-block micronucleus technique

We have made use of the cytokinesis-block micronucleus method to evaluate the adaptive response in human lymphocytes from two donors conditioned with low doses of hydrogen peroxide before irradiation with either 1.5 or 3.0 Gy of X-rays. A protective effect of pre-exposure to H2O2 against radiation damage detected as micronuclei in binucleate cells was evident in cells allowed to recover for different periods after X-ray exposure, though cells challenged with 3.0 Gy of X-rays showed the adaptive response at later fixation time than those exposed to the lower dose (1.5 Gy). We propose this protocol as an interesting alternative to the single fixation method to score chromosomal aberrations at metaphase for the study of the adaptive response.

A high yield of translocations parallels the high yield of sister chromatid exchanges in the CHO mutant EM9

The fluorescence plus Giemsa (FPG) and fluorescence in situ hybridization (FISH) techniques have been used to determine, respectively, the frequencies of sister chromatid exchanges (SCEs) and stable chromosome aberrations (translocations) induced by different concentrations of BrdU in the Chinese hamster ovary cell mutant EM9 and its parental line AA8. The results indicate that BrdU induced a high frequency of SCEs and translocations in EM9 as compared with AA8, and that the translocation/dicentric ratio was also higher in the mutant cell line than in the parental cell line in both untreated and BrdU-treated cultures. These observations may indicate a possible relationship between the molecular mechanisms involved in the formation of SCEs and translocations.

Analysis of radiation-induced chromosome aberrations in Chinese hamster cells by FISH using chromosome-specific DNA libraries

The frequencies of chromosome aberrations induced by different doses of X-rays were determined in both splenocytes and primary lung fibroblasts of Chinese hamster by bi-colour FISH using a combination of four chromosome-specific DNA libraries. The results indicate that the X-rays induced more translocations than dicentrics in Chinese hamster cells, in which the karyotype is comprised of both metacentric and acrocentric chromosomes. These results are similar to those reported in human lymphocytes, in which the karyotype contains many metacentric chromosomes. On the contrary, in mouse, which is characterized by acrocentric chromosomes only, the frequencies of translocations and dicentrics are induced in nearly equal proportions by X-rays. The ratio of translocations to dicentrics obtained in Chinese hamster cells was approximately 1.4-1.5, which supports the importance of the karyotypic features of a species in the relative induction of translocations to dicentrics. An analysis was also made on the yield of translocations and dicentrics involving individual chromosomes and the results indicate a non-random involvement of these chromosomes in the formation of aberrations.

The PARP inhibitor Olaparib disrupts base excision repair of 5-aza-2'-deoxycytidine lesions.

Decitabine (5-aza-2′-deoxycytidine, 5-azadC) is used in the treatment of Myelodysplatic syndrome (MDS) and Acute Myeloid Leukemia (AML). Its mechanism of action is thought to involve reactivation of genes implicated in differentiation and transformation, as well as induction of DNA damage by trapping DNA methyltranferases (DNMT) to DNA. We demonstrate for the first time that base excision repair (BER) recognizes 5-azadC-induced lesions in DNA and mediates repair. We find that BER (XRCC1) deficient cells are sensitive to 5-azadC and display an increased amount of DNA single- and double-strand breaks. The XRCC1 protein co-localizes with DNMT1 foci after 5-azadC treatment, suggesting a novel and specific role of XRCC1 in the repair of trapped DNMT1. 5-azadC-induced DNMT foci persist in XRCC1 defective cells, demonstrating a role for XRCC1 in repair of 5-azadC-induced DNA lesions. Poly (ADP-ribose) polymerase (PARP) inhibition prevents XRCC1 relocation to DNA damage sites, disrupts XRCC1–DNMT1 co-localization and thereby efficient BER. In a panel of AML cell lines, combining 5-azadC and Olaparib cause synthetic lethality. These data suggest that PARP inhibitors can be used in combination with 5-azadC to improve treatment of MDS and AML.

Immunofluorescent analysis of the organization of telomeric DNA sequences and their involvement in chromosomal aberrations in hamster cells

We have investigated the organization of telomeric TTAGGG)n repeats in the extended DNA loops of chromatin of human and hamster cells by immunofluorescent technique. In humans, telomeric repeats which are predominantly localized at the termini of all the chromosomes, have been found associated with nuclear matrix. This distribution pattern did not alter, even after the removal of 90% of the DNA from the nuclear halos by EcoRI digestion. This suggests that the telomeric sequences are tightly associated with nuclear matrix and hence cannot be solubilized by nucleases. In contrast, in Chinese hamster cells (CHO B11), a major proportion of interstitial telomeric repeats are found in the loop regions, like beads on a string, with attachments to the periphery of the nuclear matrix. Unlike in human cells, EcoRI digestion removed most of the telomeric repeats from the loop regions of Chinese hamster cells. This indicates that intrachromosomal sequences are not associated with nuclear matrix, and this finding has been further substantiated by Southern hybridization of matrix associated and loop DNA fractions of hamster cells with the (TTAGGG)n probe. The organizational differences in the telomeric repeat sequences of Chinese hamster and human cells might be due to their chromosomal location as well as their interaction with nucleoprotein complexes specific for the termini of the eukaryotic chromosomes. Furthermore, the interstitial (TTAGGG)n sequences were found to be more frequently involved in the chromosomal aberrations induced by restriction enzymes. This suggests that the intrachromosomal sites of telomeric sequences behave as hot spots for DNA damage.

Construction of Chinese hamster chromosome specific DNA libraries and their use in the analysis of spontaneous chromosome rearrangements in different cell lines

Chinese hamster (Cricetulus griseus) and a wide variety of cell lines derived from it have been extensively used for radiobiological and genotoxicity studies. In this report, we describe the development of chromosome-specific DNA libraries for Chinese hamster chromosomes 1, 2, 3, 5, 6, 7 and 8 by the linker/adaptor method. The specificity of these probes have been checked by FISH on primary embryonic fibroblast (CHE) metaphase chromosomes. The diploid number of chromosomes in different cell lines of CHO often varies from 19 to 21 and karyotyping is very difficult owing to many spontaneous rearrangements. Using Chinese hamster chromosome-specific DNA libraries, we have analysed the spontaneous chromosome rearrangements in three different Chinese hamster transformed cell lines, V79, CHO-KI and CHO-9. The results indicate that one of each of the first and fifth pair of chromosomes are involved in reciprocal translocations in both CHO-9 and CHO-K1 cells. The hybridisation patterns obtained with chromosome-specific probes 2, 3, 5, 6, 7 and 8 indicated rearrangements have occurred involving terminal and interstitial translocations in V79, CHO-KI and CHO-9 cells.

5-Aza-2′-deoxycytidine causes replication lesions that require Fanconi anemia-dependent homologous recombination for repair

5-Aza-2′-deoxycytidine (5-azadC) is a DNA methyltransferase (DNMT) inhibitor increasingly used in treatments of hematological diseases and works by being incorporated into DNA and trapping DNMT. It is unclear what DNA lesions are caused by 5-azadC and if such are substrates for DNA repair. Here, we identify that 5-azadC induces DNA damage as measured by γ-H2AX and 53BP1 foci. Furthermore, 5-azadC induces radial chromosomes and chromatid breaks that depend on active replication, which altogether suggest that trapped DNMT collapses oncoming replication forks into double-strand breaks. We demonstrate that RAD51-mediated homologous recombination (HR) is activated to repair 5-azadC collapsed replication forks. Fanconi anemia (FA) is a rare autosomal recessive disorder, and deaths are often associated with leukemia. Here, we show that FANCG-deficient cells fail to trigger HR-mediated repair of 5-azadC-induced lesions, leading to accumulation of chromatid breaks and inter-chromosomal radial fusions as well as hypersensitivity to the cytotoxic effects of 5-azadC. These data demonstrate that the FA pathway is important to protect from 5-azadC-induced toxicity. Altogether, our data demonstrate that cytotoxicity of the epigenetic drug 5-azadC can, at least in part, be explained by collapsed replication forks requiring FA-mediated HR for repair.