Satellite 2 demethylation induced by 5-azacytidine is associated with missegregation of chromosomes 1 and 16 in human somatic cells.

Satellite sequences are an important part of the pericentromeric regions in mammalian genomes; they play a relevant role in chromosome stability and DNA hypomethylation of these sequences has been reported in ICF syndrome and in some cancers that are closely associated with chromosomal abnormalities. Epigenetic modifications of satellite sequences and their consequences have not been extensively studied in human cells. In the present work, we evaluated satellite 2 methylation patterns in human lymphocytes exposed to 5-azacytidine (5-azaC) and assessed the relationship between these patterns and chromosome missegregation. Human lymphocytes were exposed to 10µM 5-azaC for 24, 48, and 72h. Segregation errors were evaluated in binucleate cells using FISH against pericentromeric regions of chromosomes 1, 9, and 16. DNA methylation patterns were evaluated by immunodetection, and by bisulfite plus urea conversion and sequencing. We have identified that 5-azaC induced missegregation of chromosomes 1 and 16, which have highly methylated satellite 2, after 72h of exposure. Chromosome methylation patterns showed a notable decrease in pericentromeric methylation. Bisulfite conversion and sequencing analysis demonstrated demethylation of satellite 2 associated to 5-azaC exposure, principally after 72h of treatment. This change occurred in a non-specific pattern. Our study demonstrates an association between loss of satellite 2 DNA methylation and chromosome loss in human lymphocytes.

Altered heterochromatin organization after perinatal exposure to zidovudine.

BACKGROUND: Zidovudine (3'-azido-3'-deoxythymidine, AZT), administered to pregnant women alone or in combination with other antiretroviral drugs, greatly reduces the mother-to-child transmission of HIV-1. The potential genotoxicity of these molecules is underestimated and wide-ranging evaluation of its biological and clinical consequences is required. METHODS: We investigated the nuclear organization of constitutive heterochromatin, a major domain participating in epigenetic regulation, in uninfected infants born to HIV-1-infected mothers treated with zidovudine and/or other nucleoside reverse transcriptase inhibitors (NRTIs) during pregnancy. We studied the organization of chromosome 1 heterochromatin (1q12) in peripheral leukocytes of 25 HIV-1-uninfected children (newborn to 9 years old): children born to HIV-1-infected mothers exposed to zidovudine and/or other NRTIs (n=15), children born to HIV-1-infected mothers not exposed to any NRTIs (n=6) and children born to HIV-1-uninfected mothers (n=4). RESULTS: Results differed significantly between NRTI-exposed and -unexposed children. By contrast, there was no difference between NRTI-unexposed children born to HIV-1-infected mothers and children born to HIV-uninfected mothers. The anomaly persisted in lymphocytes cultured for 48 h. There was no evidence of abnormal DNA methylation, a major feature of constitutive heterochromatin and associated with the loss of its structure. In a complementary sample of children, analysis of chromosome 11 and 16 heterochromatin suggests that the defect affects most of the other heterochromatic sites of the human genome. The heterochromatin defect persists long after the end of the exposure and appears in leukocytes of both myeloid and lymphoid lineages, suggesting that haematopoietic stem cells are affected.

Effects of chemotherapy on the cytogenetic constitution of Wilms' tumor.

The management of Wilms' tumors consists of a combination of surgery, chemotherapy, and possibly radiotherapy. To date, chemotherapy is being risk stratified according to histologic subtype and stage. Although the cytogenetic characteristics of Wilms' tumors are well established, the cytogenetic effects related to chemotherapy are widely unknown. We herein report on comparative genomic hybridization findings in 41 primary Wilms' tumors of blastemal type, of which 19 had received preoperative chemotherapy (PCT group) and 22 did not (non-PCT group). Overall, imbalances could be detected in 32 tumors, with +1q (17 cases), +7q (10 cases), +7p (6 cases), and -7p (6 cases) as the most common changes. Among these, +7q and -7p were both significantly associated with metastatic disease at the time of surgery (P = 0.002 and 0.007, respectively), and +7q was also associated with higher stage (stages III + IV; P = 0.003). There were significant differences in the cytogenetic constitution of tumors between the two treatment groups. As a trend, tumors in the preoperative-chemotherapy group had fewer changes (mean, 2.7) than those in the non-preoperative-chemotherapy group (mean, 3.8), and the frequencies of imbalances at 7p or +7q, respectively, were significantly lower compared with tumors in the non-preoperative-chemotherapy group (2 of 19 versus 10 of 22, P = 0.019; 1 of 19 versus 9 of 22, P = 0.011). In contrast, -1q was common in both the preop-CT group (10 of 19) and the non-preop-CT group (7 of 22). The results suggest that Wilms' tumor clones with +1q are not obliterated by preoperative chemotherapy, whereas cytogenetically more complex clones with +7q and/or imbalances at 7p seem more responsive and are more likely to be eliminated by chemotherapeutic treatment.

Okadaic acid: chromosomal non-disjunction analysis in human lymphocytes and study of aneugenic pathway in CHO-K1 cells.

Okadaic acid (OA) is the main marine toxin implicated in the diarrhetic shellfish poisoning (DSP) in humans after consumption of contaminated bivalve molluscs. We have previously shown that OA was an in vitro aneugenic compound that induced chromosome loss via micronuclei formation in CHO-K1 cells. The aims of this study were to investigate the chromosomal non-disjunction (ND) potential of OA in human lymphocytes and the pathways involved for aneuploidy in CHO-K1 cells. Firstly, we analysed the formation of micronuclei and the non-disjunction for chromosomes 1 and 17 in binucleated human lymphocytes cells with the cytokinesis-blocked micronucleus (CBMN) assay coupled to a fluorescent in situ hybridization (FISH) technique with centromere-specific DNA probes. We showed that OA statistically increased the frequency of micronucleated lymphocytes in the dose range from 20 to 35 nM. However, FISH analysis did not reveal any increase in the non-disjunction for both chromosomes whatever the concentration between 2.5 and 35 nM. However, a significant increase in ND for the chromosome 17 was found at 1 nM. Secondly, in CHO-K1 cells, we investigated the dose and time dependent effects of OA: (i) on cell cycle progression, (ii) on mitotic-phase arrest and (ii) on mitotic spindle and centrosome abnormalities. The results showed that OA induced a progressive accumulation of mitotic CHO-K1 cells in prometaphase, an induction of multipolar mitotic spindle with centrosome amplification and the formation of multinucleated cells. We concluded that OA did not induce chromosome non-disjunction but should more likely induced chromosome loss in human lymphocytes. Moreover, our results obtained in CHO-K1 suggest that OA induced aneuploidy by preventing the chromosome attachment to the mitotic spindle and by amplifying the centrosome. The mode of action of the toxin in relation to its inhibition of protein phosphatases 1 (PP1) and 2A (PP2A) and the mitosis process is discussed.

Chromosome rearrangements in fumigant appliers: possible relationship to non-Hodgkin's lymphoma risk.

Appliers of pesticides (n = 18) who are exposed to the fumigant phosphine or who have a mixed exposure to other pesticides and phosphine demonstrate a significant increase in chromosome rearrangements in G-banded chromosomes from peripheral blood compared to control subjects (n = 26). Appliers who had discontinued using phosphine for at least 8 months prior to specimen collection (n = 5) do not demonstrate significant increases in chromosome rearrangements compared to controls. Breakpoint analysis of 6,138 metaphases from all subjects demonstrates 196 breaks per 3605 metaphases in exposed subjects and 102 breaks per 2,533 metaphases in control subjects. Bands with significantly more breaks than expected based on band length in all study subjects were 1q32, 3p14, 7p15, and 14q11. Three of these four bands had significantly more breaks than expected in the exposed group, and all four bands had a significant excess of breaks in the control group. There are four bands with a significant excess of breaks in the exposed group and no breaks in the control group; each of these occurs in a known protooncogene region. These are 1p13 (NRAS), 2p23 (NMYC), 14q32 (ELK2), and 21q12 (ETS-2). Most breaks at bands 1p13, 14q32, and 21q22 are associated with chromosome rearrangements and occurred in appliers who have a mixed exposure to phosphine and other pesticides. Cytogenetic abnormalities, i.e., rearrangements and/or deletions involving bands 1p13, 2p23, and 14q32, are associated with non-Hodgkin's lymphoma. We speculate that these findings could relate to the risk of evolution of a neoplastic clone in these workers. Epidemiological studies of similarly exposed workers indicate an excess of non-Hodgkin's lymphoma.

Use of fluorescence in situ hybridization (FISH) to study chromosomal damage induced by radiation and bromodeoxyuridine in human colon cancer cells.

PURPOSE: Although the thymidine analog radiation sensitizer bromodeoxyuridine (BrdUrd) increases radiation-induced chromosomal aberrations, it is not known whether these aberrations are uniformly distributed among chromosomes. Using fluorescence in situ hybridization, we carried out a study to test the hypothesis that BrdUrd-induced radiosensitization may be mediated by nonuniform chromosomal damage. METHODS AND MATERIALS: Log phase HT29 human colon cancer cells were exposed to 10 microM BrdUrd (or media alone) for one cell cycle, and the G1 cells were separated by centrifugal elutriation. Half of the control and BrdUrd samples were irradiated with 8 Gy. Cells were then incubated for 24-28 h, and metaphase spreads were prepared. Fluorescence in situ hybridization was performed using paint probes for chromosomes 1 and 4. RESULTS: We found that radiation induced 0.20 aberrations per chromosome in chromosome 4. Based on the ratio of the relative lengths of chromosome 1-4 (1.34), it was predicted that chromosome 1 would have approximately 0.26 aberrations per chromosome. However, we observed 0.39 aberrations per chromosome 1, which was significantly greater than the predicted (p < 0.001 by chi-square). Incubation with BrdUrd prior to irradiation significantly increased the aberrations found in chromosome 1 (by a factor of 1.4) and chromosome 4 (by a factor of 1.9) compared to radiation alone (p < 0.001) for both chromosome 1 and 4). CONCLUSION: This study demonstrates that individual chromosomes in human colon cancer cells show significantly different rates of aberration after irradiation. Furthermore, the BrdUrd-mediated increase in radiation-induced chromosomal aberrations may not be uniform among chromosomes.

Differential involvement of chromosomes 1 and 4 in the formation of chromosomal aberrations in human lymphocytes after X-irradiation.

Whole blood samples from two healthy donors were cultured in the presence of 5-bromo-2'-deoxyuridine (BrdU) for a total of 107 h following in vitro X-irradiation with a dose of 2 Gy. Starting from 35 h after culture initiation, every subsequent 12 h a sample was taken from each culture and grown in the presence of demecolcine for another 12 h. At each sampling time, the aberrations involving chromosomes 1 and 4 were analysed using dual-colour fluorescence in situ hybridization (FISH) with chromosome-specific DNA libraries. Following differential staining of sister chromatids, the analysed cells were identified to be either in their first, second or third etc. mitosis after irradiation. Cells within the same postirradiation division contained higher frequencies of aberrations when derived from later sampling times, indicating a delay in progression of aberrant cells to mitosis. In contrast, when the aberration frequencies are calculated by sampling time (i.e. independent of the cell cycle) minimal effect of sampling time could be seen. This observation held true for all types of chromosomal aberrations. Analysis of about 2250 first-division cells for each donor (derived from all sampling times) indicates a relative overrepresentation of chromosome 4 in the formation of exchange aberrations/colour junctions. Whereas dicentric frequencies for chromosomes 1 and 4 were close to the expected values based on the DNA content of these chromosomes, frequencies of reciprocal translocations showed a clear overinvolvement of chromosome 4. This resulted in a distinct difference in the reciprocal translocation to dicentric ratio, being 1.12 for chromosome 1 and 2.09 for chromosome 4. These results indicate a non-DNA-proportional distribution of radiation-induced chromosome rearrangements in cultured human lymphocytes.

Smoking enhances asbestos-induced genotoxicity, relative involvement of chromosome 1: a study using multicolor FISH with tandem labeling.

Several experimental and epidermological studies have indicated augmentation of asbestos induced diseases by cigarette smoke by the mechanisms, which are still unknown. To determine whether smoking affects genetic system of the cells and further modifies asbestos induced genotoxicity, whole blood from non-smokers and smokers was exposed to asbestos fibres separately in vitro and micronucleus test was performed. The number of micronuclei was found to be significantly higher (P<0 05) in cases of smoker's lymphocytes, asbestos exposed non-smokers lymphocytes as well as asbestos exposed smokers lymphocytes, as compared with unexposed non-smokers lymphocytes. Further we investigated involvement of chromosome 1 in the damaging process using multicolor FISH technique. FISH is fast and reliable method, distinguishing both structural and numerical alterations. The centric/pericentric regions of chromosome 1 (cen-q12) were labeled, as the pericentric heterochromatin region 1 (q12) is quite large, highly repetitive and prone to breakage. Multicolor FISH assay suggested that the genetic damage by asbestos fibres mainly involve chromosome 1 but in case of cigarette smoking the damage is not strictly connected to chromosome 1 only, but also involves damage to other chromosomes. Further the study suggested that smoking makes genetic system of the cells more vulnerable to the deleterious effects of asbestos.

DNA demethylation and pericentromeric rearrangements of chromosome 1.

Rearrangements in the vicinity of the centromere of chromosome 1 are over-represented in many types of human cancer and are a characteristic feature of a rare genetic disease called ICF (immunodeficiency, centromeric heterochromatin instability, and facial anomalies). Evidence is presented that implicates DNA hypomethylation in the formation of these pericentromeric chromosomal anomalies. The DNA methylation inhibitors 5-azadeoxycytidine and 5-azacytidine, but not other tested genotoxins, induced the preferential formation of pericentromeric rearrangements of chromosome 1 at a very high frequency in a pro-B-cell line (FLEB14) and at a lower frequency in a mature B-cell line (AHH-1). These abnormal chromosomes appear identical to the diagnostic chromosomal aberrations in the ICF syndrome. A major component of the pericentromeric DNA in chromosome 1, satellite 2, was shown to be hypomethylated in an ICF B-cell line, although DNA from this cell line did not display detectable overall hypomethylation. It is hypothesized that demethylation in certain DNA regions, including in pericentromeric satellite DNA, helps lead to pericentromeric chromosomal rearrangements in lymphocytes from ICF patients and in normal lymphoblastoid cells incubated in vitro with DNA demethylating agents.

Chromosome damage and aneuploidy detected by interphase multicolour FISH in benzene-exposed shale oil workers.

A multicolour tandem-labelling fluorescence in situ hybridization (FISH) procedure was used to detect chromosome alterations in peripheral blood cells of a group of Estonian petrochemistry workers. Twelve workers employed in benzene production and five cokery workers, together with eight unexposed rural controls, were enrolled in the study. The methodology employed, based on the in situ hybridization of adjacent centromeric and pericentromeric regions, allowed the simultaneous detection of both chromosome breakage, involving damage-prone pericentromeric regions, and hyperploidy in interphase cells. Blood smears from all subjects were hybridized with chromosome 1 specific probes, in order to detect genotoxic damage in circulating lymphocytes and granulocytes. Moreover, lymphocyte cultures were established, harvested 48 h following mitogen stimulation and hybridized with the tandem chromosomes 1 and 9 probes. No significant difference in the incidence of breakage was detected in the nucleated cells of blood smears of exposed vs. control subjects. In contrast, modest but significantly increased frequencies of breakage affecting both chromosomes 1 and 9 were observed in the cultured lymphocytes of the benzene-exposed workers compared to the unexposed controls, suggesting an expression of premutagenic lesions during the S-phase in vitro. Across the entire study group, the frequencies of breakage affecting chromosomes 1 and 9 in the stimulated lymphocytes were highly intercorrelated (p < 0.001). No significant difference was found in the incidence of hyperploidy among the study groups, although a tendency to higher values was observed in benzene-exposed workers. Although the relatively small size of the study groups does not allow firm conclusions on the role of occupational exposure, the observed patterns are suggestive of effects in the benzene-exposed workers. This work also shows that tandem labelling FISH can be usefully applied in human biomonitoring, allowing the simultaneous detection of both hyperploidy and chromosome breakage at interphase in different cell types.

Persistence of chromosomal alterations affecting the 1cen-q12 region in a human lymphoblastoid cell line exposed to diepoxybutane and mitomycin C.

Multicolor fluorescence in situ hybridization (FISH) with tandem-labeling probes for the 1cen-q12 region is a potential biomarker for the detection of structural chromosomal aberrations (CAs) in human cells. To determine the suitability of this technique for biomonitoring humans exposed to 1,3-butadiene (BD) and to characterize the alterations induced as well as their stability over time, the human lymphoblastoid cell line AZH-1 was treated with 5 microM diepoxybutane (DEB) or the positive control mitomycin C (MMC; 0.1 microM) for 24 h. Following the removal of the test chemicals, cell cultures were grown for an additional 19 days in the absence of the test compound. Using the tandem FISH technique, aliquots from the main cultures were examined for the induction of CAs affecting the 1cen-q12 region at various intervals. A significant increase in chromosomal breakage/exchanges affecting the 1cen-q12 region was seen in both the DEB- and MMC-treated interphase and metaphase cells. The damage peaked at approximately 48 h following the addition of the test compound and declined with time. However, at day 20, the frequency of aberrant cells was still significantly higher than the control levels. For comparison, the frequency of micronuclei (MN) formed and their origin was determined using the cytochalasin B-modified MN assay and FISH with a pancentromeric probe. Showing a similar pattern, the frequency of centronere-negative MN peaked at 48 h, but however was not significantly elevated above control levels at 20 days. At early time points, aberrations detected using the FISH assay consisted of nearly equal proportions of unstable- and stable-type aberrations, while at the later time points, translocations were the predominant aberration type. In addition, the use of tandem-label FISH in combination with BrdU-immunfluorescence staining, showed that almost identical frequencies of structural aberrations could be seen in actively replicating and non-replicating cell populations. These studies indicate that a small but significant proportion of the alterations detected using this FISH technique persists over time and that this technique may be valuable for biomonitoring chromosomal alterations in BD-exposed populations.

Comparison of spontaneous and idoxuridine-induced micronuclei by chromosome painting.

Fluorescence in situ hybridisation (FISH) technique with chromosome specific library (CSL) DNA probes for all human chromosomes were used to study about 9000 micronuclei (MN) in normal and idoxuridine (IUdR)-treated lymphocyte cultures of female and male donors. In addition, MN rates and structural chromosome aberrations were scored in Giemsa-stained chromosome spreads of these cultures. IUdR treatment (40 microg/ml) induced on the average a 12-fold increase of the MN rate. Metaphase analysis revealed no distinct increase of chromosome breaks but a preferential decondensation at chromosome 9q12 (28-79%) and to a lower extend at 1q12 (8-21%). Application of FISH technique with CSL probes to one male and one female untreated proband showed that all human chromosomes except chromosome 12 (and to a striking high frequency chromosomes 9, X and Y) occurred in spontaneous MN. In cultures containing IUdR, the chromosomal spectrum found in MN was reduced to 10 chromosomes in the male and 13 in the female proband. Eight chromosomes (2, 6, 12, 13, 14, 15, 17 and 18) did not occur in MN of both probands. On the contrary chromosomes 1 and especially 9 were found much more frequently in the MN of IUdR-treated cultures than in MN of control cultures. DAPI-staining revealed heterochromatin signals in most of the IUdR-induced MN. In an additional study, spontaneous and IUdR-induced MN were investigated in lymphocytes of another female donor using CSL probes only for chromosomes 1, 6, 9, 15, 16 and X. The results confirmed the previous finding that chromosomes 1 and 9 occur very often in MN after IUdR-treatment. The results indicate that decondensation of heterochromatic regions on chromosomes 1 and 9 caused by IUdR treatment strongly correlates with MN formation by these chromosomes.

Expression of folate sensitive and aphidicolin induced chromosomal fragile sites in familial neuroblastoma.

The expression of folate sensitive and aphidicolin induced fragile sites in the blood lymphocyte chromosomes of affected and unaffected members from 2 neuroblastoma families were studied. The subjects included 4 neuroblastoma patients, and 9 of their clinically healthy first degree relatives and corresponding number of age and sex matched controls. Lymphocytes cultured in folate deprived culture medium showed rare fragile sites at band p13.1 of chromosome 1, in a frequency of 3%-5% in all the 4 neuroblastoma patients. In aphidicolin treated cultures, the patients and unaffected members in neuroblastoma families, showed hypersensitivity to aphidicolin, as evidenced by the significant increase in percentage of aberration/cell (ab/c) and damaged cells (dc), over that of controls (P < 0.01). Aphidicolin induced fragile sites were more pronounced in chromosomes 1 and 2. A larger number of subjects have to be studied to prove whether altered fragile site expression may be a cytogenetic evidence for an individual or familial cancer predisposing genetic constitution.

[The characteristics of the localization of induced ruptures in human chromosomes]. / Osobennosti lokalizatsii indutsirovannykh razryvov v khromosomakh cheloveka.

Levels of spontaneous and of C-mitomycine and cyclophosphane-induced chromosome aberrations are determined among the persons not subject to professionally industrial hazards. It has been revealed that in all the experimental series the aberration rate in those, who carried extreme polymorphic variants, was much higher compared to those without these variants in their karyotypes. Among the persons with the above extreme versions, the impact of chromosome ruptures is stronger in the vicinity of the structural heterochromatin. Possible causes of the discovered phenomenon are discussed.

[The determination of chromosome fragile sites in the peripheral blood lymphocytes of patients with colorectal cancer taking into account the cancer pathology predisposition in the familial anamnesis]. / Opredelenie khromosomnoi sait-lomkosti v limfotsitakh perifericheskoi krovi bol'nykh kolorektal'nym rakom s uchetom otiagoshchennosti semeinogo anamneza po onkopatologii.

Results of comparative study of spontaneous and 5-bromdeoxyuridine-induced fragility of peripheral blood lymphocytes chromosomes in 9 patients with colorectal adenocarcinoma were presented. It was shown the increase of average spontaneous level of chromosomal fragility in patients with tumor aggregation in family as well as without it to 4.5 +/- 1.0 and 5.3 +/- 1.1 per 100 tested cells, accordingly. The increase of average level of damaged chromosomes in spectrum of rare sites to 12.5 +/- 2.6 in the patients with tumor aggregation in pedigree comparing to the patients without oncopathology in family 8.0 +/- 1.7 was observed. The most number of rare fragile sites was observed in 1q21 site of the chromosome 1. Possible connection between fragile sites of chromosomes in normal cells and malignant processes in the patients with colorectal cancer is discussed.

Impact of genomic aberrations including chromosome 1 abnormalities on the outcome of patients with relapsed or refractory multiple myeloma treated with lenalidomide and dexamethasone.

Previous literature suggests that cytogenetics may be used for risk-adapted therapy in patients with relapsed/refractory multiple myeloma (MM) treated with lenalidomide and dexamethasone. However, the significance of each abnormality is still unclear, and chromosome 1 abnormalities have yet to be studied in this population. We therefore evaluated genetic risk factors including chromosome 1q gain and 1p loss by cIg-FISH in 143 patients with relapsed/refractory MM treated with lenalidomide and dexamethasone, and correlated the genomic aberrations with patient clinical outcomes. Patients had a median of two (range 1-7) previous therapies in this cohort. A total of 119 out of 143 (83%) patients had an objective response, with median time to progression (TTP) and overall survival (OS) of 11 and 28 months, respectively. Patients with del(1p21) or del(17p) (p53) deletions had a significantly shorter TTP. OS was shorter in patients with 1p21 or 17p deletions, but did not reach statistical significance. Prior bortezomib or thalidomide treatment was associated with shorter TTP and OS. Multivariate analysis identified del(17p), del(1p21), and prior bortezomib or thalidomide therapy as independent risk factors for shorter TTP. Our data suggest that chromosome 17p and 1p21 deletions adversely impact the outcome of lenalidomide and dexamethasone treated patients with relapsed/refractory MM. Improved therapeutic strategies are required for these patients.

Preferential induction of chromosome 1 multibranched figures and whole-arm deletions in a human pro-B cell line treated with 5-azacytidine or 5-azadeoxycytidine.

5-Azacytidine (azaCR) causes genomic demethylation and decondensation of juxtacentromeric heterochromatin in chromosomes 1, 9, and 16. We determined the karyotypes of a pro-B cell line (FLEB14) treated with azaCR or its deoxynucleoside analog (azaCdR). About 80% of the induced rearrangements were in chromosome 1, and almost 90% of these involved its pericentromeric region. Multibranched figures with up to seven chromosome 1 arms, as well as whole-arm deletions of this chromosome, were the predominant anomalies, often with one normal homolog of chromosome 1 present. Isochromosomes 1 and fusions in the pericentromeric regions of chromosomes 1 and 16 or chromosomes 1 and 9 were also seen. The overlap of the spectrum of chromosomal rearrangements in azaCR- or azaCdR-treated FLEB14 cells and in mitogen-stimulated lymphocytes from patients with a rare genetic disease (ICF) associated with localized DNA hypomethylation supports the hypothesis that the DNA demethylating activity of azaCR is essential for the induction of these pericentromeric rearrangements. These studies may help elucidate the overrepresentation of chromosome 1 pericentromeric rearrangements in many types of cancer cells.

Chromosome painting reveals specific patterns of chromosome occurrence in mitomycin C- and diethylstilboestrol-induced micronuclei.

Cultures of human blood lymphocytes from three subjects were incubated with the clastogen mitomycin C (MMC, 500 ng/ml) and the aneugen diethylstilboestrol (DES, 80 microM) 23 h before harvesting, to induce formation of micronuclei (MN) and numerical and structural alterations in metaphase chromosomes. We used fluorescence in situ hybridization (FISH) with painting probes for all human chromosomes to determine which chromosomes had contributed material to the induced MN. MMC treatment induced an approximately 18-fold increase in MN and led to a significant increase in hypodiploidy and structural chromosome aberrations in metaphase preparations. Undercondensation of pericentromeric heterochromatin of chromosomes 9 and 1 occurred in 20-75% of metaphases and FISH disclosed an abundance of material from these chromosomes in induced MN (62-69% from chromosome 9 and 7-12% from chromosome 1). DES treatment of lymphocytes induced a seven-fold increase in MN frequency and four-fold increase in the frequency of numerical aberrations; structural aberrations were not significantly increased. FISH analysis showed that material from all chromosomes was present in DES-induced MN, with material from chromosome 1 present in 16% of MN and material from each other chromosomes being present in 2-10% of MN. Material from chromosomes 14, 19 and 21 was significantly more frequent material from chromosome Y significantly less frequent in DES-treated cells than in controls. The findings of the MMC studies indicate that the heterochromatin block of chromosome 9 is a specific target for MMC-induced undercondensation, which induces a preferential occurrence of chromosome 9 material in MN. DES, in contrast, does not trigger heterochromatin decondensation and fails to induce such a significant appearance of material of particular chromosomes in MN.

Mechanically stretched chromosomes as targets for high-resolution FISH mapping.

When used with metaphase chromosomes, fluorescence in situ hybridization (FISH) makes it possible to localize probes to individual chromosome bands and to establish the order of probes separated by > or = 2-3 Mb in dual-color hybridizations. We evaluated the use of mechanically stretched chromosomes as hybridization targets for increased mapping resolution. Mapping resolution was tested by pair-wise hybridizations with probes from the 1p32-p33 region, spanning distances from 20 to approximately 1500 kb. Probes separated by > or = 170 kb could be ordered relative to one another and to the centromere-telomere axis of the chromosome. The advantages of the technique are the simple procedure for preparing the slides, the straightforward interpretation of the results, and the ability to score the predominant order from < 10 stretched chromosomes. However, because of the variability of stretching from one sample to another, the calculation of actual physical distances between probes is not possible. To illustrate the utility of this method, we showed that the gene for receptor tyrosine kinase TIE lies centromeric to COL9A2, RLF, and L-MYC genes at 1p32. The use of mechanically stretched chromosomes provides < or = 10-fold increased mapping resolution as compared with conventional metaphase FISH. Thus, the technique effectively bridges the gap between metaphase mapping and ultra-high-resolution mapping (1-300 kb) techniques, such as the DNA fiber FISH.

Aneugenic activity in human cultured lymphocytes. An overall study with colchicine using the micronucleus assay and fluorescence in situ hybridization techniques.

The effects induced by aneugenic agents on chromosome segregation are manifold. The biological relevance of these effects has led to the development of assays specifically detecting aneugens. In this context, the micronucleus (MN) assay in binucleated human lymphocytes along with FISH has been considered a pertinent tool for detecting aneugenic and clastogenic activity. However, the MN assay is insensitive in detecting aneugenic effects other than chromosome loss. By using the aneugenic model compound colchicine and X chromosome centromere-specific FISH, we have shown that besides chromosome loss in binucleated cells, other effects such as MN in mononucleated cells, cells arrested at metaphase, polyploidy and non-disjunction are also consistently induced by aneugenic agents. A chromosome 1 centromeric probe was used simultaneously with X chromosome centromeric labeling in mononucleated cells in order to distinguish polysomy from polyploidy. It is concluded that all these effects should be considered for a comprehensive evaluation of aneugenic activity.