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Selected Abstracts from the the 26th European Environmental Mutagen Society's Annual Meeting on Chromosome Instability and Cell Cycle Control

Fine structural analysis of DNA repair in mammalian cells

A.S. Balajee, A.May and V.A.Bohr Laboratory of Molecular Genetics, National Institute National Institute on Aging, Gerontology Research Center, 4940. Eastern Avenue, Baltimore MD 21224, USA.

Nucleotide excision repair of UV induced photolesions is heterogeneous across the genome. We have attempted to unravel the mechanistic basis for this repair heterogeneity by a number of immunological and biochemical approaches. Immunological labeling of the sites of repair and transcription simultaneously indicates that the early repair events are in close association with transcription sites in hamster cells. The initial events of repair occur at the site of nuclear matrix. Repair analysis in the loop and nuclear matrix associated DNA fractions indicate that the lesions are removed faster in nuclear matrix DNA as compared to loop fraction. Further, western blotting of nuclear matrix proteins from UV treated cells indicates the enrichment of a number of repair and transcription factors such as p21, p53, PCNA , XP-B and XP-D components of basal transcription factor, TFHH. This suggests that the nuclear matrix might be an important site for nucleotide excision repair. BrdU, labeling of UV induced repair sites in interphase nuclei at different post UV incubation times (0, 30 min, 2h, 4h and 8h) indicates the preferential enrichment of repair label at the periphery of the nuclear membrane in a time dependant fashion. Using antibodies specific for cyclobutane pyrimidine dimers and 6-4 photoproducts, we also found that the transcribing sequences located at the periphery of the nuclear membrane are efficient in the removal of lesions. This suggests that the nuclear membrane is an important component in excision repair. These results indicate that the nuclear architecture may play a critical role in intra genomic heterogeneity of UV induced DNA repair.

Spontaneous and chemically-induced aneuploidy in female germ cells

Ursula Eichenlaub-Ritter University of Bielfeld, Department of Biology, Institute for Gentechnology/ Microbiology, 33501 Bielefeld, Germany.

Trisomy is one of the major causes of genetic disease in the human. The extra chromosome appears predominantly derived from nondisjunction in female meiosis, but the origin of aneuploidy and the significance of environmental factors are still unknown. Previous in vivo studies on experimental animals showed that classical aneugens like the microtubule-depolymerizing drugs induce chromosome malsegregation in oocytes. However, in comparison to spermatogenesis, oogenesis does not appear to be more sensitive. We will discuss the reasons for sex-specific differences in spontaneous, age-related and chemically-induced aneuploidy, in mammalian germ cells. Evidence is provided indicating that disturbances in cell cycle progression and activity of kinases affecting checkpoint controls are responsible for age-effects. Moreover, conditions and chemicals were identified which influence oogenesis in a long-lasting way, cause spindle defects and mimic age-effects in disturbing the coordination between aspects of cytoplasmic and nuclear maturation. From these observations it is evident that further analysis is required i) to identify exposures and chemicals with similar aneugenic activities which may contribute to age-effects as well as to environmentally induced nondisjunction in mammalian oocytes and ii) to establish and further develop model systems for effective screening for such chemicals and their mode of action, most notably such interfering with cell cycle and check point control. (Supported by the EU, EV5V-CT94-0403, and the DFG, SFB 223).

The induction of multidrug resistance is inhibited by simultaneous treatment of tumor cells with adriamycin and an anti-recombinogenic agent

Rudolf Fahrig*, Angela Steinkamp-Zucht*, Andras Schaefer+ *Fraunhofer-lnstitut fur Toxikologie und Acrosolforschung, Hannovcr +Fraunhofer Arbeitsgruppe fur Toxikologie und Umweltmedizin. Hamburg

The induction of resistance to many structurally unrelated antineoplastic agents is the major cause of failure in cancer chemotherapy. In this way, tumours initially sensitive to chemotherapy often recur and survive exposure to multiple antineoplastic drugs. Resistant cells are, often characterized by the amplification of a small group of closely related genes responsible for multiple drug resistance (MDR). Amplification of genes mediating drug resistance has been documented not only in tumour cells in vitro but also in human tumours in vivo. Therefore, preventing the formation of gene amplification should lead to improvements in cancer chemotherapy. Virtually all proposed mechanisms for gene amplification involve recombination. Thus, recombination, and gene amplification may be causally related. To test this hypothesis, co-recominogens and anti-recombinogens were studied for their ability, to enhance or suppress the formation of gene amplification by an antineoplastic drug. In these experiments, an antirecombinogenic substance, that was able to prevent SV40 amplification in Chinese hamster cells, was selected. We observed that this anti-recombinogen, (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), inhibited the induction of multidrug resistance by adriamycin in tumor cells of mice and man.

Telomeres and cell division in Drosophila melanogaster

Maurizio Gatti, Dipartimento di Genetica e Biologia Molecolare, Universita' di Roma "La Sapienza" , Italy

Drosophila telomeres do not consist of tandemly repeated short sequences like most eukaryotes but contain multiple copies of telomere-specific retrotransposons. Despite their peculiar structure D. melanogaster telomeres exhibit several properties that are common to those of mammalian telomeres. For example, we have found that mutants in the UbcD1 gene exhibit frequent end-to-end associations of chromosomes through their telomeres. A similar cytological phenotype has been observed in senescent mammalian cells, in various types of tumors and ataxia telangiectasia cells. The UcbD1 gene encodes an ubiquitin conjugating (E2) enzyme. This suggests that ubiquitin-mediated proteolysis is normally needed to ensure proper telomere behaviour during Drosophila cell division. Notwithstanding the uniqueness of Drosophila telomere structure, similar functions required for dispersion of chromosome ends prior to rnetaphase, may occur in the cells of many kinds of organisms. Drosophila telorneres also act like mammalian chromosome ends in preventing the activation of cell cycle checkpoints. A series of data collected on various Drosophila mutants affecting mitotic chromosome stability indicate that in larval neuroblasts there is a process that functions in mitosis at, or prior to metaphase, to detect broken chromosomes. If cells with broken chromosomes are detected, they divide regularly but their daughter cells are prevented to progress to the next cell division. Chromosome breakage that occurs after metaphase (i.e. at anaphase) is not sensed until next cell division. Thus, cells containing chromosome breaks generated during anaphase proceed normally to the next division, but their daughters are not able to progress through the cell cycle. Together, these data strongly suggest that the cell cycle arrest caused by chromosome breakage is not related to gene dosage problems but it is the consequence of an active process that prevents the division of cells with broken chromosomes.

Mechanisms in loss of heterozygosity deduced from loss of HLA alleles

Giphart-Gassler M., De Nooij-van Dalen A.G., Cole J. Van der Keur M. and Lohman P.H.M. pt of Radiation Genetics and Chemical Mutagenesis. Leiden University, The Netherlands

The HLA genes of the human Major Histocompatibility Complex are extremely polymorphic and provide for a system of selectable markers with natural heterozygosity. Mechanisms involved in spontaneous or induced loss of heterozygosity (LOH) can be revealed by molecular and cytogenetic analysis of mutants that have lost the expression of alleles of one or several HLA loci. Such mutants have been isolated from cells of several lymphoblastoid cell lines by selection via immuno-cytoxicity or flow cytometry and cell sorting. The rate of spontaneous loss of HLA-A2 in vitro exceeded that of hprt mutation 10-50 fold. Only 10% of the spontaneous mutants had mutations which could be attributed to a limited DNA region spanning 100Kb to 1Mb. In the vast majority of the mutants, LOH extended to alleles of one, several or all microsatellite loci examined on chromosome 6. Gene dosage analysis of the remaining allele of a microsatellite close to HLA-A revealed the presence of two copies of that allele and FISH analysis revealed the presence of two intact chromosomes 6 in nearly all mutants. Our results show that the main contributing to LOH in vitro, somatic recombination and chromosome loss accompanied by chromosome duplication, occur frequently and can not be detected by cytogenetic analysis.

Genomic instability in the offspring of testicular tumour patients

S.Gundy National Institute of Oncology, Dept. Onco-Cytogenetics, Budapest, 1122,Hungary

Genomic instability seems to be a driving force behind multistep carcinogenesis. In former examinations we found a four-fold increase of chromosomal aberration rate in peripheral blood lymphocytes of more than one hundred untreated testicular cancer patients above the level of age- and occupation-matched healthy controls. These data suggested the existence of some genetic instability expressed as chromosome instability of testicular tumour patients, which may be a factor in the development of testicular malignancy as well. To find out whether genomic instability exists in the offspring of testicular cancer patients, we studied the frequencies of congenital abnormalities (CA), childhood tumors (CT) and chromosomal aberrations (CAb) in a case-control examination. Two-hundred children fathered by men untreated or treated for testicular carcinoma were investigated. Neither the tumour per se, nor the treatment of the fathers affected the incidence of CAs compared to the controls. However, 5 tumour cases (Wilms' tumour, brain tumour, leukaemia, neuroblastoma and histiocytosis) occurred in index children, irrespective of conception before or after father's treatment. (The incidence of childhood tumours in age of 0-15 years in Hungary is 15.05/100 000). This finding is extremely interesting and suggests that the testicular cancer of a father may increase the susceptibility to tumourigenesis in the offspring. In further studies the role of the environmental component was investigated for genetic instability in the offspring. One hundred index children (In) were analysed for chromosomal aberrations, and nearly two-hundred children from environmentally contaminated (Ec) and non-contaminated (Nc) areas as controls. We did not find significant difference between children of Ec and Nc groups. This age-group indicates probably less clastogenic response to environmental factors than the adults, however, index children showed a two-fold increase of spontaneous chromosomal aberration frequency. Although the number of children investigated here is quite limited, the study population has been the largest one so far. Because of the high prevalence of childhood tumours and the elevated level of chromosomal aberrations we do suspect the existence of genomic instability in the offspring of testicular tumour patients, similarly to the patients of Li-Fraumeni syndrome. To clarify the role of genetic and environmental components of genomic instability in this risk group, further epidemiologic and molecular studies are required. This work was supported by the grant of US-Hungarian Science and Technology Joint Fund under project JFNO-391.

Genome instability and resistance to DNA damage

Karran P., Branch P., Hampson R., Yamada M., Aquilina G., Ceccotti S. and Bignami M. Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Herts, UK and Istituto Superiore di Sanita, Rome. Italy

Microsatellite instability is observed as multiple alterations in regions of repeated di - or mononucleotides throughout the genome of tumors. It occurs in essentially all tumors that are associated with the familial predisposition to colorectal and other carcinomas in -Hereditary Nonpolyposis Colorectal Cancer (HNPCC). Microsatellite instability is also seen in 10-20% of apparently sporadic colorectal cancers as well as numerous other tumors in which there is no overt genetic predisposition. The genetics of HNPCC has identified four genes involved in a human mismatch repair pathway that corrects DNA replication errors. In sporadic and familial tumors which exhibit microsatellite instability, both alleles of one of these genes are inactivated by mutation. One surprising consequence of mismatch repair deficiency is a resistance to the cytotoxic effects of some DNA damaging agents. To date, mismatch repair deficiencies have been associated with resistance to methylating agents such as N methyl-N-nitrosourea and temozolomide, which is used in chemotherapy for melanoma, and to cisplatin which is particularly effective against ovarian and testicular tumors. The clinical effectiveness of both temozolomide and cisplatin is compromised by the emergence of resistant tumors. Mismatch recognition proteins apparently initiate attempts at processing certain base pairs containing methylated or platinated DNA bases. These processing attempts are unsuccessful and lead to the death of the cell. Models for how this resistance might arise will be presented together with speculation about the possible role that endogenous sources of DNA damage might play in the genesis of mismatch repair-defective tumors.

Cytogenetic study of the in vitro evolution of lymphoblastoid cell lines obtained from the lymphocytes of patients suffering from chromosome instability syndromes

D. Lefrancois, F. Apiou, B. Malfoy and B. Dutrillaux Institut Curie, UMR 147 CNRS, Paris, France.

Ten lymphoblastoid cell lines were maintained in vitro for more than two years and were regularly checked for possible chromosomes abnormalities. Three lines were originated from lymphocytes of normal healthy control subjects, three lines from patients suffering from Fanconi anemia, three lines from patients suffering from Ataxia telangiectasia and one line from the lymphocytes of a patient suffering from ICS. The evolution of the karyotypes of these ten lines when passaged in vitro is described : chromosome abnormalities could be evidenced in the three pathological cases. In the case of the three Ataxia telangiectasia patients, chromosomes 11, 14, 17 and 18 were significantly involved. Control lymphoblastoid cell lines remained stable.

Interferonogenesis, DNA repair and cytogenetic alterations in the blood of people exposed to radiation as a result of the accident at the Siberian chemical plant (April 6, 1993).

Nicolai N.Llyinskikh, Vechaslav V.Novitskyi, Olumpiada A.Vasilyeva, Natali N Naidyonova, Ekaterina N.Ilyinskikh, Igor I.Ivaitchuk Siberian medical university 634050, Tomsk-50, a/ya 808, Russia.

It has been found that the residents of Samus, a settlement exposed to radiation as a result of the accident at the Siberian chemical plant on April 6, 1993, have changes in the level of cells with cytogenetic disorders, excision DNA repair, interferon production of blood lymphocytes as well as in the number and reactivity of T- and B lymphocytes. It has been suggested that the disorders in the genetic apparatus of lymphoid cells caused by radiation lead to a decrease in their interferon productivity. The blast transformation capability of blood T-lymphocytes under the influence of PHA (phytohemagglutinin) in the residents of Samus was much lower than in the control. The same is true about their ability for spontaneous rosette formation with ram erythrocytes (E-RFC). At the same time, the B-Lymphocyte ability for complement rosette formation (EACRFC) was considerably higher than the norm. When examining the inhabitants of Samus we found a negative correlation between T-lymphocyte immunoreactivity (blast transformation to PHA and spontaneous rosette formation) and the number of T-lymphocytes with cytogenetic disorders. In other cases, the correlation between was not observed. The lymphocytes of the residents of Samus produced less interferon of both kinds and had a reduced excision DNA repair than the lymphocytes of the residents in the control settlement. It has been suggested that the disorders in the genetic apparatus of lymphoid cells caused by radiation lead to a decrease in their interferon productivity.

Mitotic Checkpoint Control of Chromosome Segregation and Cell Cleavage

Margolis R.L., Martineau S. and Andreassen P.R. Institut de Biologic Structurale-J.P.Ebel, 41 avenue des Martyrs., 38027 Grenoble Cedex 01, France.

Cyclin B degradation, which normally occurs at the onset of anaphase, is required for mitotic exit in mammalian cells. The protease inhibitor acetylleucylleucylnorleucinal (ALLN) inhibits the degradation of cyclin B in mitotic BHK cells and blocks mitotic exit. A subset of cells that are presynchronized in prophase of mitosis with nocodazole progress to and arrest at the completion of anaphase following release from nocodazole into ALLN. Cyclin B degradation which normally occurs at the onset of anaphase is therefore not necessary for anaphase separation of chromosomes in mammalian cells. ALLN thus permits dissection of those anaphase events that require cyclin B degradation. Both cyclin B and p34cdc2 are normally found associated with kinetochores until the onset of anaphase when cyclin B degrades and p34cdc2 delocalizes. In ALLN arrested anaphase cells, both cyclin B and p34cdc2 remain kinetochore associated. A family of proteins, designated passenger proteins, normally dissociate from the kinetochore at the onset of anaphase, become associated with the mitotic spindle and migrate toward the spindle equator where they concentrate in late anaphase. We find that the passenger proteins, 'I'D-60 and CENP-E, which normally exhibit this anaphase behavior, remain kinetochore associated in ALLN arrested cells. We conclude that degradation of cyclin B is required both for the inactivation and delocalization of p34cdc2 at the onset of anaphase, and may be of functional significance to kinetochore behaviour. Inactivation of p34 cdc2 may be required to alter the behaviour and subcellular distribution of kinetochore components at the onset of anaphase. As the failure of p34cdc2 and of the passenger proteins, to delocalize does not impede chromosome progression in anaphase ,I reqtilrcnient for the migration of passenger proteins may have meaning in the context of proper completion of events subsequent to anaphase, such as cell cleavage.

The Mammalian Centromere: A Biological Enigma.

Mitchell A.R., Kipling D. Jeppesen P. MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh, EH4 2XU, UK

The centromere in mammalian chromosomes is recognised by cytologists as a narrowing of chromatin (the primary, constriction) which appears to be at a specific position in the chromosome. Loss of centromere function through a chromosome breakage event or by a deletion leads to the formation of an unstable acentric fragment. 'This acentric fragment has lost the ability to segregate correctly during cell division. Thus loss of centromere function leads to loss of genetic information to daughter cells. The molecular mechanisms which govern the function of the centromere have yet to be completely unravelled although some of the individual components which contribute to its structure have been identified. Some DNA sequences localised to centromeric chromatin have been well characterised. This is particularly the case with human and mouse centromeric DNAs where different families of repeated DNAs have been identified and mapped in relation to each other. Centromeric protein have been identified and their interactions with specific sequence motifs within some centromeric DNAs have led to model predictions for the structure of centromeric chromatin. This talk will look at our present understanding of the mammalian centromere in the light of these recent developments.

Deregulated Apoptosis Is A Hallmark in the Two Chromosome Instability Syndromes Fanconi Anemia and Ataxia Telangiectasia.

Moustacchi E., Ridet A., Guillouf C., Duchaud E., Cundari E., Rosselli F. Institut Curie - Recherele, Paris, France.

Fanconi anemia (FA) and ataxia telanglectasia (AT) are human genetic disorders associated with specific cell cycle perturbations, spontaneous and induced chromosomal anomalies, hypersensitivity to specific genotoxic agents and predisposition to cancer. Evidence is presented that necrosis rather than apoptosis is the cellular death pathway responsible of FA hypersensibility to mitomycin C (MMC) and of AT to ionizing radiations. FA and AT lymphoblastoid cell lines as well as lymphocytes from patients demonstrate higher frequencies of apoptic cells than cells from normal donors. Cells from both syndromes are deficient in y-rayinduced apoptosis. In FA, the alteration in the control of apoptosis concerns also pathways independent of DNA damage such as that initiated by Fas-activation. It is also shown that ectopic expression of the wild-type FAC gene corrects the MMC hypersensitivity, and the anomalies in apoptosis and in cell cycle response observed in FA cells. All together, our findings strongly involve the FA and the ATM genes as playing a major role in the control of apoptosis and may, explain some of the phenotypic characteristics of the diseases.

Rosselli F., Ridet A., Soussi T., Duchaud E., Alapetite C. and Moustacchi E. p53-dependent pathway of radio-induced apoptosis is altered in Fanconi anemia. Oncogene, 10, 9-17 (1995).

Duchaud E., Ridet A., Stoppa-Lyonnet D., Janin N., Moustacchi and Rosselli F. Deregulated apoptosis in ataxia telangiectasia. Association with clinical stigmata and radiosensitivity,. Cancer Res., 56, 1400- 1406 (1996).

Centromeric association in mammalian cells.

Musio Antonio1, Turchi Gino1, Sbrana Isabella2 1 J.R.C. ECVAM TP580 Ispra Italy; 2 Dip. Scienze dell'Ambiente e del Territorio Universita di Pisa Italy.

Studies performed on mammalian mitotic metaphases have shown that chromosomal instability involved the association between specific chromosomal regions such as nucleolus organizer regions, constitutive heterocromatic regions, telomeric regions and somatic pairing of homologous chromosomes. The best-known association is represented by association between nucleolus organizer regions of acrocentric autosomes which results from a functional relationship of the structural genes for ribosomal RNA in the nucleoli in the interphase nucleus. So far, very few cases of centromeric association have been described suggesting that it represents a rare constitutional change in mammalian karyotype. Now, we report a further case of spontaneous centromeric association in a immortalized Chinese hamster epithelial liver (CHEL) cell line. The most remarkable property observed in normal metaphases was the nonrandom position of a microchromosome: it was situated near or in close proximity to a centromere of a metacentric chromosome. It was noteworthy that the frequency of this association was significantly enhanced by treatment with two environmental contaminants such as chrysene 1,2-diol and phenanthrene 1,2 quinone. These observations suggest that centromeric association may confer a selective advantage to cells therefore to be causally related to in vitro immortalization and transformation of CHEL cells. We tried to identify the origin and the nature of the microchromosome through the use of conventional banding techniques and fluorescence in situ hybridization with (TTAGGG)n telomeric sequence. With these techniques neither differential centromere staining nor telomeric signals were detected and therefore other studies are required to understand the nature of the microchrmosome.

Mechanisms of formation of chromosome aberrations

A. T. Natarajan Department of Radiation Genetics & Chemical Mutagenesis, Leiden University, The Netherlands

Fluorescent in situ hybridization (FISH) technique using chromosome specific DNA probes has revolutionized the field of radiation cytogenetics. In addition to available human chromosome specific DNA libraries, we have generated painting probes for mouse and Chinese hamster chromosomes. Combining these probes with centromeric and telomeric probes it is possible to analyse chromosomal aberrations in great detail. Some of the new insights on the origin of radiation and chemically induced chromosome aberrations will be presented and discussed. Emphasis will be made on the non-random induction of aberrations among and within the chromosomes. Possible factors responsible for this heterogeneity will be discussed.

The study of the aneugenic activity of trichlorfon detected by centromere-specific probes in human lymphoblastoid cell lines

J.M. Parry, A.T. Doherty and E.M. Parry School of Biological Sciences, University of Wales. Swansea, Singleton Park, Swansea, SA2 8PP. UK

Trichlorfon an acetylcholinesterase inhibitor and has human exposure due to its use as an anthelminth. Excessive use of Triclilorfon in fish farming in Hungary has been associated with a large increase in congenital abnormalities (73% of live births) in one village in the years 1989-90 (Czeizel et al., 1993). The human lymphoblastoid cell line AHH-1 and the metabolically competent derivative MCL-5 (which expresses CYP 1 A2,2A6,3A4,2E 1 and epoxide hydrolase), have demonstrated their ability to detect a variety of pro-genotoxins in the in vitro micronucleus assay, (Crofton-Sleigh et al., 1993; Doherty et al., 1996). Non-disjunction has been detected by, three colour F.I.S.H. using the centromeric probes for human chromosomes 2,7 & 18. The potential of the pesticide trichlorfon to induce mitotic aneuploidy has been investigated in genetically engineered human lymphoblastoid cell lines. Trichlorfon failed to induce micronuclei in the AHH-1 and MCL-5 cell lines when treated in media at normal cell culture pH (pH 6.3). Under a treatment pH of 5.5, trichlorfon exposures resulted in the induction of both chromosome loss and chromosome non-disjunction as measured by, fluorescence in situ hybridisation (FISH) using a pan-centromeric probe for all human centromeres and centromere probes specific for chromosomes 2, 7 and 18. At treatment concentrations greater than 20ug/ml, trichlorfon also induced chromosome structural damage resulting in the production of centromere negative micronuclei.

References Crofton-Sleigh et al. (1993) Mutagenesis, 8, 363-73 Czcizel et al. (1993) Lancet, 341, 539-42 Doherty et al. (1996) Mutagenesis, 11 247-274

Dicuran - induced mutagenicity in S. typhimurium and V79 CHO Cells

S. Ramljak and B. Nagy Department of Molecular Biology, Faculty of Science, University of Zagreb, Rooseveltov trg 6, HR-10000 Zagreb, Croatia.

Dicuran is known as a selective herbicide which is widely used for grass weed control in the wheat and barley fields. Since atypical spikes and cytogenetical abnormalities were found in the wheat field treated with Dicuran our aim was to determine its mutagenicity. Two in vitro test systems were used for mutagenic evaluation of Dicuran on bacterial cells and V79 Chinese hamster cells. For detection of reverse mutations standard plate incorporation test with preincubation was used on Salmonella typhimurium,strains TA100 and TA98 in the absence of S9 mix. Preliminary results show a significant increase in number of revertants in base-substitution sensitive strain TA100 after treatment with following concentrations: 0.05, 0.1, 0.25 and 0.5 uM. The drug did not reverted frameshift mutation in strain TA98. The effect of Dicuran on the formation of hypoxanthine guanine phosphoribosyl transferase (HPRT) locus was assessed in cultured V79 cells. Mutation frequencies were determined with a 6-day expression time and corrected for the number of spontaneous background mutants. Although mutation induction was linear as a function of Dicuran dose it was reduced at the very toxic dose. These results suggest that the mutagenic effect of Dicuran on studied bacterial and plant cells is also present in mammalian cells, and should be of important consideration in its frequent use.

Ataxia-telangiectasia and the ATM gene: new insights into cell cycle response to DNA damage.

Department of Human Genetics, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel.

The autosomal recessive disorder ataxia-telangiectasia (A-T) is highly pleiotropic. A-T is characterized by degeneration of the Purkinje cells in the cerebellum leading to progressive neuromotor deterioration, immunodeficiency, developmental defects in specific tissues, profound predisposition to malignancy, and acute sensitivity to ionizing radiation. A-T cells show chromosomal instability, premature senescence, and radiosensitivity. Several radiation-induced pathways that regulate the cell cycle seem to be defective in A-T cells, at least one of which is mediated by p53. The responsible gene, ATM, has recently been cloned in our laboratory using a positional cloning approach and shown to encode a high molecular weight protein containing a carboxy-terminal region similar to the catalytic domain of PI 3-kinases. This protein is a member of an expanding family of large proteins in various organisms, which contain a PI 3-kinase -like region and are involved in cellular responses to DNA damaging agents and/or cell cycle regulation. Structural analysis of normal and mutant ATM alleles suggests several levels of regulation of this gene, and is encouraging a search for ATM mutations in disorders other than A-T. Purification of the ATM protein and analysis of its enzymatic activity are in progress. Several lines of evidence, including the nature of the agents that elicit the hypersensitivity of A-T cells, point to the possibility of a defect in cellular responses to damage induced by oxidative stress, which affects various cellular macromolecules. The ATM protein might therefore play a role in such defense mechanisms. This hypothesis broadens the previous concept of the A-T defect, and explains several aspects of the A-T phenotype which cannot be accounted for by defective processing of DNA damage.

Genotoxicity of not directly DNA damaging compounds

H. Stopper Department of Toxicology, University of Wurzburg, Wurzburg, FRG.

An interaction of compounds with DNA in a not directly damaging way can still result in genotoxic effects. Some of such compounds cause a change in DNA conformation. We hypothesize that the interaction of proteins with that DNA may be impaired as a consequence. In the case of proteins that are essential for a normal course of mitosis - like the kinetochore complex or topoisomerase II - this can lead to the loss of chromatin in the cell. Our data from rnicronucleus experiments and molecular analysis strongly suggest such a mechanism for the DNA intercalating 1,8-dihydroxyanthraquinones and the hypomethylating agent 5-azacytidine. In addition, increased DNA strand breakage by proteins from the DNA replication and repair machinery may also be due to an impairment of the DNA-protein interaction by that same mechanism. We assume that this indirect pathway of genotoxicity will lead to a spectrum of mutational lesions (gene mutation, translocation, mitotic recombination, deletion, chromosome loss) different from that of directly damaging DNA agents. To be able to investigate the kind of lesions in mutant colonies we have established a combination of two methods in the LS178Y mouse lymphoma cell system. We analyze loss of heterozygozity using polymorphic microsatellites and combine the results with those from whole chromosome in situ hybridization with a chromosome specific "painting" probe. Due to a size difference between the centromeres from the two alleles of the chromosome harbouring the selectable gene all above mentioned types of mutational lesions can be distinguished from each other. Our goal is to provide evidence for a chain of events from the chemicals activity to mutation.

Cytogenetic analysis of Chernobyl clean up workers determined by chromosome painting

J.D. Tucker1, M.J. Ramsey1, D.O. Nelson1, D.H. Moore II2 , R.H. Jensen2, P.G. Pleshanov3, I. Vorobtsova4, 1Lawrence Livermore Nat'l. Lab., 2U. Calif. San Francisco, 3Ministry of Health and Medical Industry, Russia, 4Central Res. Inst. of Roentgenology and Radiology, Russia.

The Chernobyl reactor accident in 1986 was the largest non-military single release of radioactivity in history. This disaster provided an opportunity, to study low dose chronic (or sub-acute) ionizing radiation exposures to a large population. Soviet citizens were sent to the site of the accident to contain and clean up the radioactive contamination. Their exposure to radiation was intentionally kept low, in the range of 5 and 25 cagy for most individuals. To examine the biological effects of this exposure on these individuals, we are performing chromosome painting of metaphase lymphocytes to quantify the frequencies of stable aberrations (translocations and insertions. To date, we have analyzed samples from 290 subjects by painting chromosomes 1, 2 and 4 simultaneously, and examining a minimum of 500 cell equilvalents per subject. Age and smoking status are available at this time for 205 subjects, including 51 controls and 154 clean up workers. Our data indicate a highly significant difference in frequency of stable aberrations between controls and exposed workers (P<0.00 1). This difference is explainable by, invoking an average dose of radiation to the cleanup workers of 12 + 4 cagy (mean + S.E.) of radiation, in excellent agreement with the intended doses. A strong effect of age is also seen on the translocation frequencies among smokers (P<0.0001). These observations generally agree with our published work on the American population. This work was performed in part under the auspices of the US DOE by LLNL tinder contract No. W7405-ENG-48.

Drug-induced telomeric association in human lymphocytes

Wootton. A. Stemp,G and Gatehouse, D. Genetic and Reproductive Toxicology Dept. GlaxoWellcome Research and Development Ltd., Park Rd. Ware. HERTS. UK.

During routine screening for potential genotoxic activity, a new drug candidate GW TE 001 was found to induce a high percentage of dicentric, tricentric and multicentric chromosomes in cultured human lymphocytes. These effects were seen both in the absence and presence of S9-mix but were more pronounced in the latter. Further analyses of these chromosomal changes using specific chromosome paints (for chromosomes l, 2.and 3) and telomeric probes demonstrated that the multicentric chromosomes had arisen from telomeric association and not through breakage and reunion of the chromosomes. This was corroborated by the absence of discernible acentric fragments in the metaphase preparations. In addition it was found that a possible polymorphism existed with regards to the sensitivity of different human lymphocyte donors towards the drug. Around 50% of the donors examined were insensitive to in vitro treatment and dicentric/multicentric chromosomes were not observed. Furthermore it was not possible to induce significant levels of dicentric/multicentric chromosomes in cultured human lymphoblastoid 131A cells using identical treatment conditions. It is possible that the target for drug activity, is lost or modified during the process of transformation in these cells. Using a modified PCR-based TRAP assay procedure it was found that the drug had no effect on the ribonucleoprotein enzyme, telomerase. This was not unexpected since this enzyme is either absent or present at extremely low levels, in cultured lymphocytes. It is likely, that the drug is affecting the telomeres directly in such a way that association occurs. At the present time the mechanism of action of this drug is not known, and -additional studies both in vitro and in vivo, are required. However this molecule may prove to be all extremely useful tool in studies on telomere function and cell ageing.


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