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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. |
