A comprehensive evaluation of the genotoxic potential of chemicals requires the assessment of the ability to induce gene mutations and structural chromosome (clastogenic activity) and numerical chromosome (aneugenic activity) aberrations. Aneuploidy is a major cause of human reproductive failure and an important contributor to cancer and it is therefore important that any increase in its frequency due to chemical exposures should be recognized and controlled. The in vitro binucleate cell micronucleus assay provides a powerful tool to determine the ability of a chemical to induce chromosome damage. The application of an anti-kinetochore antibody to micronuclei allows their classification into kinetochore-positive and kinetochorenegative, indicating their origin by aneugenic or clastogenic mechanisms, respectively. The availability of chromosomespecific centromere probes allows the analysis of the segregation of chromosomes into the daughter nuclei of binucleate cells to evaluate chromosome non-disjunction. Quantitative relationships between the two major causes of aneuploidy, chromosome loss and non-disjunction, can be determined. The mechanisms leading to chromosome loss and non-disjunction can be investigated by the analysis of morphological and structural changes in the cell division apparatus by the application of specific stains and antibodies for various cell division components. We illustrate such analyses by the demonstration of the interaction of the monomer bisphenol-A with the centrosome of the mitotic spindle and the folic acid antagonist pyrimethamine with the centromeres of chromosomes. Both types of modifications lead to the induction of aneuploidy in exposed cells. Our studies also implicate the products of the p53 and XPD genes in the regulation of the fidelity of chromosome segregation at mitosis.

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Epidemiological studies indicate that benzene exposure is associated with an increased risk of human leukemia and lymphoma ( 1 , 2 ). Chromosomal translocations are thought to be initiating events in leukemia and lymphoma, and specific translocations are used as markers of diagnosis, disease progression, and relapse. As benzene is thought to act by producing chromosomal aberrations and altered cell differentiation ( 3 ), we investigated the possibility of using circulating levels of two acute myeloid leukemia (AML) -associated translocations, t(8;21) ( 4 ) and t(15;17) ( 5 ), and a follicular lymphoma -associated translocation, t(14;18) ( 6 ), as biomarkers of early effect for benzene. Evidence for the occurrence of these translocations in normal individuals is limited, with t(8;21) detected in 1 in 496 cord blood samples ( 7 ) and t(15;17) detected in healthy volunteers ( 8 ). As recently reviewed ( 9 ), the prevalence of t(14;18) in peripheral blood lymphocytes of healthy donors is relatively high at ~ 50% (range 8% -80% depending on the polymerase chain reaction (PCR) methodology employed and target cell population) ( 9 -13 ) and rises with smoking ( 14 ), but the correlation between frequency and age remains unclear ( 13 , 15 ). The biological consequences of these aberrations in healthy individuals are yet to be understood. One possibility is that the frequency of translocationpositive cells represents a biomarker of early effect for hematopoietic carcinogens which could correlate with the cumulative risk of developing t(14;18)-related follicular lymphoma and t(8;21)-and t(15;17)-related AML, and perhaps other cancers.The two principal technologies for detection of specifi c chromosome translocations are fl uorescence in situ hybridization (FISH) and real-time PCR. FISH distinguishes individual chromosomes by hybridization with sequence-specifi c, chromosomepainting probes labeled with spectrally nonoverlapping fl uorophores. In real-time quantitative PCR (qPCR), a fl uorescently labeled probe is hybridized to the amplifi cation target, and fl uorescent signal proportional to the input DNA/cDNA is generated during amplifi cation, thereby allowing quantifi cation. qPCR assays the whole peripheral blood mononuclear cell (PBMC) population in G 0 , whereas FISH examines a culture-modifi ed popula tion [consisting of mainly mature T lymphocytes stimulated by the mitogen phytohemagglutinin that have gone through two cell divi sions ( 16 )]. The two methods also generate different estimates of t(14;18) translocation levels with FISH detecting all possible t(14;18) breakpoints and the real-time assay described here only detecting breakpoints occurring in the major breakpoint region (MBR) of the BCL-2 gene. Although it was previously thought that the majority of BCL-2 breakpoints occur in the MBR ( 17 ), it has been shown more recently that many breakpoints occur outside of the MBR and the minor cluster region ( 18 ). The ability of FISH to detect more translocation breakpoints is offset by its lower relative sensit...

Hutchinson-Gilford Progeria Syndrome (HGPS) is an extremely rare genetic disorder characterized by premature ageing in childhood and serves as a valuable model for the human ageing process in general. Most recently, point mutations in the lamin A (LMNA) gene on chromosome 1q have been associated with the disease, however how these mutations relate to the complex phenotype of HGPS remains to be established. It has been shown that fibroblasts from HGPS patients are frequently resistant to immortalization with telomerase (hTERT), consistent with the idea that the loss of a dominant acting HGPS gene is a pre-requisite for immortalization. In this study we report the first detailed cytogenetic analysis of hTERT-immortalised HGPS cell lines from three patients and one corresponding primary fibroblast culture. Our results provide evidence for a cytogenetic mosaicism in HGPS with a distinctive pattern of chromosome aberrations in all the HGP clones. Chromosome 11 alterations were observed at a high frequency in each immortalised HGPS cell line but were also present at a lower frequency in the corresponding primary cells. Moreover, we were able to identify the 11q13→q23 region as a potential site of breakage. Our results are therefore consistent with a role of chromosome 11 alterations in the escape from senescence observed in HGPS cells. In addition to this defined rearrangement, we consistently observed complex chromosomal rearrangements, suggesting that HGPS displays features of chromosomal instability.

Canine oral melanoma (COM) is the most frequent tumour with oral localization in dogs. Copy number gains and amplifications of CCND1, a gene coding for Cyclin D1, are the most frequent chromosomal aberrations described in human non-UV induced melanomas. Twenty-eight cases of COM were retrieved from paraffin-blocks archives. A total of 4 μm thick sections were immunostained with an antibody against human Cyclin D1 and Ki-67. Cyclin D1 and Ki-67 expressions were scored through two counting methods. DNA was extracted from 20 μm thick sections of formalin-fixed paraffin-embedded blocks. Pathological and surrounding healthy tissue was extracted independently. Cyclin D1 immunolabelling was detected in 69% (18/26) while Ki-67 was present in 88.5% (23/26) of cases. Statistical analysis revealed correlation between two counting methods for Cyclin D1 (r = 0.54; P = .004) and Ki-67 (r = 0.56; P = .003). The correlation found between Ki-67 and Cyclin D1 indexes in 16/26 cases labelled by both antibodies (r = 0.7947; P = .0002) suggests a possible use of Cyclin D1 index as prognostic marker. Polymerase chain reaction analysis on CCND1 coding sequence revealed the presence of nine somatic mutations in seven samples producing synonymous, missense and stop codons. Since none of the single-nucleotide polymorphisms was found to be recurrent, it is suggested that overexpression of Cyclin D1 may be the consequence of alterations of CCND1 upstream regions or other genetic aberrations not detectable with the methodology used in this study. Future studies are needed to verify the potential use of Cyclin D1 index as prognostic indicator and to highlight the molecular events responsible for Cyclin D1 overexpression in COMs. K E Y W O R D S CCND1, Cyclin D1, dog, immunohistochemistry, melanoma

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AHH-1 TK ϩ/-is a human B cell-derived lymphoblastoid cell line that constitutively expresses a high level of the cytochrome CYP1A1. The MCL-5 cell line was developed by transfection of AHH-1 with cDNAs encoding the human cytochrome P450s, CYP1A2, CYP2A6, CYP2E1, CYP3A4 and microsomal epoxide hydrolase carried in plasmids. The metabolic components of these cell lines make them a useful screening tool for use in mutagenicity studies. Although AHH-1 and MCL-5 are closely related, the two cell lines show differences which cannot be attributed to transfection. In the present study both cell lines were investigated for chromosome stability by comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) using whole chromosome probes and telomeric probes. Amplification in chromosomes 4q, 3q and 9p was observed in both cell lines. To compare the cell lines directly, AHH-1 and MCL-5 DNAs were co-hybridized on the same metaphases using a modified CGH technique. The only difference observed between AHH-1 and MCL-5 was the degree of amplification involving the subtelomeric region of chromosome 4; the additional telomeric region (4q) was translocated onto chromosome 11 and/or chromosome X. FISH was use to show the presence of isochromosomes 3q and 9p in both cell lines with a chromosome number of 48 or higher. These data demonstrate that CGH and FISH with chromosome-specific probes are able to resolve complex karyotypes and to highlight subchromosomal regions involved in rearrangements and potential chromosome fragile sites. Analyses such as those described here may be of considerable value in the determination of the stability of a variety of the cell lines used in the mutagenicity testing of chemicals.

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