During aging, telomeres are gradually shortened, eventually leading to cellular senescence. By T/C-FISH (telomere/centromere-FISH), we investigated human telomere length differences on single chromosome arms of 205 individuals in different age groups and sexes. For all chromosome arms, we found a linear correlation between telomere length and donor age. Generally, males had shorter telomeres and higher attrition rates. Every chromosome arm had its individual age-specific telomere length and erosion pattern, resulting in an unexpected heterogeneity in chromosome-specific regression lines. This differential erosion pattern, however, does not seem to be accidental, since we found a correlation between average telomere length of single chromosome arms in newborns and their annual attrition rate. Apart from the above-mentioned sex-specific discrepancies, chromosome arm-specific telomere lengths were strikingly similar in men and women. This implies a mechanism that arm specifically regulates the telomere length independent of gender, thus leading to interchromosomal telomere variations.

Combined loss of heterozygosity (LOH) on 1p and 19q is reported in 50% to 90% of oligodendroglial tumors and has emerged as a strong and favorable prognostic factor. Fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) are the most widely used techniques. The aim of this study was to evaluate the reliability of FISH to predict LOH at 1p and 19q when performed on touch preparations from 40 oligodendroglial tumors, even if the majority of the nuclei showed chromosomal imbalance. PCR was used as the gold standard. The presence of none or one target signal was reported as FISH-LOH, whereas all other losses were defined as FISH-imbalance. The sum of nuclei with FISH-LOH and imbalance was calculated in each case (FISH-sum) and cut-off values were defined as the mean FISH-sum value in controls plus 3 standard deviations; 27.7% for 1p and 33.2% for 19q. These corresponded well with the optimal cut-off values for our data, calculated using the minimum error rate classification procedure (35.6% for 1p and 33.1% for 19q). Concurrent FISH and PCR results were encountered in 95% for 1p and 87.5% for 19q. FISH-sum was the best and simplest discriminating variable for correct classification of LOH status. Under these conditions, even a dominant population of nuclei showing FISH-imbalance represented an LOH status in the tumor cells. FISH on touch preparations is a quick and reliable method for 1p/19q testing, does not require normal DNA and can be easily performed in an immunohistochemistry unit.

Osteosarcoma (OS) is characterized by chromosomal instability and high copy number gene amplification. The breakage–fusion–bridge (BFB) cycle is a well-established mechanism of genome instability in tumors and in vitro models used to study the origins of complex chromosomal rearrangements and cancer genome amplification. To determine whether the BFB cycle could be increasing the de novo rate of formation of cytogenetic aberrations in OS, the frequency of anaphase bridge configurations and dicentric chromosomes in four OS cell lines was quantified. An increased level of anaphase bridges and dicentrics was observed in all the OS cell lines. There was also a strong association between the frequencies of anaphase bridges, dicentrics, centrosomal anomalies, and multipolar mitotic figures in all the OS cell lines, indicating a possible link in the mechanisms that led to the structural and numerical instabilities observed in OS. In summary, this study has provided strong support for the role of the BFB cycle in generating the extensive structural chromosome aberrations, as well as cell-to-cell cytogenetic variation observed in OS, thus conferring the genetic diversity for OS tumor progression.

Chromosome aberration analysis in astronauts has been used to provide direct, biologically motivated estimates of equivalent doses and risk associated to cosmic radiation exposure during space flight. However, the past studies concentrated on measurements of dicentrics and translocations, while chromosome intrachanges (inversions) have never been measured in astronauts' samples. Recent data reported in the literature suggest that densely ionizing radiation can induce a large fraction of intrachanges, thus leading to the suspicion that interchanges grossly underestimate the cosmic radiation-induced cytogenetic damage in astronauts. We have analyzed peripheral blood lymphocytes from 11 astronauts involved in short- or long-term space flights in low-Earth orbit using high-resolution multicolor banding to assess the frequency of intrachromosomal exchanges in both pre- and post-flight samples. We did not detect any inversions in chromosome 5 from a total of 2800 cells in astronauts' blood. In addition, no complex type exchanges were found in a total of 3590 astronauts' lymphocytes analyzed by multifluor fluorescence in situ hybridisation. We conclude that, within the statistical power of this study, the analysis of interchanges for biological dosimetry in astronauts does not significantly underestimate the space radiation-induced cytogenetic damage, and complex-type exchanges or intrachanges have limited practical use for biodosimetry at very low doses.

Telomerase is the reverse transcriptase responsible for the maintenance of telomeric repeat sequences in most species that have been studied. Inactivation of telomerase causes telomere shortening and results in the loss of the telomere's protective function, which in mammals leads to cell-cycle arrest and apoptosis. Experiments performed on Arabidopsis thaliana mutants lacking telomerase activity revealed their unusually high tolerance for genome instability. Here we present molecular and cytogenetic analysis of two cell lines (A and B) derived from seeds of late-generation telomerase-deficient A. thaliana. These cultures have survived for about 3 years and are still viable. However, neither culture has adapted mechanisms to maintain terminal telomeric repeats. One culture (B) suffers from severe growth irregularities and a high degree of mortality. Karyological analysis revealed dramatic genomic rearrangements, a large variation in ploidy, and an extremely high percentage of anaphase bridges. The second cell line (A) survived an apparent crisis and phenotypically appears wild-type with respect to growth and morphology. Despite these indications of genome stabilization, a high percentage of anaphase bridges was observed in the A line. We conclude that the restructured chromosome termini provide the A line with partial protection from end-joining repair activities, thus allowing normal growth.

In order to address the problem of the action of cytostatics on chromosome ends, telomere length was measured in human lymphocyte cultures exposed to mitomycin C (MMC) and bleomycin (BLM). Telomere-specific PNA probes were used for the quantitative estimation of the relative telomere length of each individual chromosome by fluorescence in situ hybridization. A high inter-cellular and inter-individual variability of relative telomere lengths was found throughout all experiments. Different responses could be observed with respect to the action of the examined mutagens: The total average fluorescence intensity of labeled telomere repeats was decreased under the action of MMC in two of the experiments, while two revealed no significant alteration. BLM caused no significant change of total average telomeric signal intensity in four, a clear decrease in one of the six experiments, and an increase in another. Although all chromosome ends contributed to the observed trends, single telomeres were affected in a very distinct way. The highest concentration of MMC (1 microg/ml) induced significant shortening of telomeres of the chromosome arms; 2q, 3p, 5q, 7p, 10q, 11p, 13q, 17p, 18p&q, and 21q in two independent experiments. In one BLM experiment with 8 microg/ml, the most distinct decrease (p< or =0.005) of telomeric fluorescence was found at the ends of chromosome arms; 1q, 6p, 17p, 20p&q, and 22q. The increase of telomeric signal intensity affected the telomeres of some individual chromosome arms more than others, e.g. 4q, 6p, 7p, 8p, 13p, and 18q. Although the telomere length of the individual chromosome arms varied widely, clear trends could be observed with respect to the rank which was occupied by telomeric length of the various chromosome arms. The telomeres of the 1p, 3p, 4q, 5p, 12q, and 13q chromosome arms throughout all experiments were among the longest; and those of 13p, 15p, 21p, and 22p were among the shortest telomeres of the karyotype. From these data, it can be concluded that MMC affects the telomeric repeat area of chromosomes more than BLM, which mostly had no significant effect on telomere length in the performed experiments.

Damage to telomeres induced by cytostatic therapy theoretically could generate telomere shortening and, subsequently, induce an additional genomic instability in neoplastic cells. Model experiments were carried out to examine this hypothesis. Cells of the T-ALL derived cell line CCRF-CEM were exposed to various different concentrations of Bleomycin (BLM) or Mitomycin C (MMC) for various times. Telomere lengths of metaphase chromosomes of the exposed cells were compared with those without this exposure (controls). In addition, telomerase activity was determined with a TRAP assay under the given conditions using the BLM experiments as a model. Although slight changes of total telomere length could be found in single experiments, the differences between exposed and non-exposed cells were not significant. Also, a considerable telomerase activity was shown which, however, did not substantially differ between exposed and non-exposed cells. From these data it may be concluded that, at least in the examined cell line, telomeres are not a preferential target for this kind of mutagenic attack.

In this study, 107 children with acute lymphoblastic leukemia (ALL) were analysed for the presence of hyperdiploidy by cytogenetics and interphase fluorescence in situ hybridisation (I-FISH). Structural aberrations in hyperdiploid cells were investigated by multiple colour FISH (mFISH). Clones with high hyperdiploidy (>50 chromosomes) (HeH) were found in 46 patients (43%). In nine of these (20%), the abnormal clone was present in <20% of the total cell population. There was no significant difference in EFS between those patients with HeH in 2.5-20% or >20% of cells. Structural rearrangements in the HeH clone were found in 10 patients (22%). In this study, HeH karyotypes containing structural aberrations were an indication of a poor prognosis in childhood ALL.

SUMMARY: BACKGROUND: Secretory carcinoma (SC) of the breast is a rare and indolent tumor. Although originally described in children, it is now known to occur in adults of both sexes. Recently, the tumor was associated with the ETV6-NTRK3 gene translocation. CASE PRESENTATION: A 52-year-old male was diagnosed with secretory breast carcinoma and underwent a modified radical mastectomy. At 18 months the tumor recurred at the chest wall and the patient developed lung metastases. He was treated concurrently with radiation and chemotherapy without response. His tumor showed the ETV6-NTRK3 translocation as demonstrated by fluorescent in situ hybridization (FISH). CONCLUSION: SC is a rare slow-growing tumor best treated surgically. There are insufficient data to support the use of adjuvant radiation or chemotherapy. Its association with the ETV6-NTRK3 fusion gene gives some clues for the better understanding of this neoplasm and eventually, the development of specific therapies.

Conventional genetic theories have failed to explain why cancer (1) is not heritable and thus extremely rare in newborns, (2) is caused by non-mutagenic carcinogens, (3) develops only years to decades after initiation by carcinogens, (4) follows pre-neoplastic aneuploidy, (5) is aneuploid, (6) is chromosomally and phenotypically "unstable", (7) carries specific aneusomies, (8) generates much more complex phenotypes than conventional mutation such as multidrug resistance, (9) generates nonselective phenotypes such as metastasis (no benefit at native site) and "immortality" (not necessary for tumorigenesis), and (10) does not contain carcinogenic mutations. We propose, instead, that cancer is a chromosomal disease. Accordingly carcinogenesis is initiated by random aneuploidies, which are induced by carcinogens or spontaneously. Since aneuploidy unbalances 1000s of genes, it corrupts teams of proteins that segregate, synthesize and repair chromosomes. Aneuploidy is therefore a steady source of chromosomal variations from which, in classical Darwinian terms, selection encourages the evolution and malignant progression of cancer cells. The rates of specific chromosomal variations can exceed conventional mutations by 4-11 orders of magnitude, depending on the degrees of aneuploidy. Based on their chromosomal constitution cancer cells are new cell "species" with specific aneusomies, but unstable karyotypes. The cancer-specific aneusomies generate complex, malignant phenotypes through the abnormal dosages of 1000s of genes, just as trisomy 21 generates Down syndrome. In sum, cancer is caused by chromosomal disorganization, which increases karyotypic entropy. Thus, cancer is a chromosomal rather than a genetic disease. The chromosomal theory explains (1) non-heritable cancer because aneuploidy is not heritable, (2) non-mutagenic carcinogens as aneuploidogens, (3) long neoplastic latencies by the low probability of evolving new species, (4) nonselective phenotypes via genes hitchhiking with selective chromosomes, and (5) immortality because, through their cellular heterogeneity, cancers survive negative mutations and cytotoxic drugs via resistant subspecies.

Long-lived, sensitive, and specific biomarkers of particular mutagenic agents are much sought after and potentially have broad applications in the fields of cancer biology, epidemiology, and prevention. Many clastogens induce a spectrum of chromosome aberrations, and some of them can be exploited as biomarkers of exposure. Densely ionizing radiation, for example, alpha particle radiation (from radon or plutonium) and neutron radiation, preferentially induces complex chromosome aberrations, which can be detected by the 24-color multifluor fluorescence in situ hybridization (mFISH) technique. We report the detection and quantification of stable complex chromosome aberrations in lymphocytes of healthy former nuclear-weapons workers, who were exposed many years ago to plutonium, gamma rays, or both, at the Mayak weapons complex in Russia. We analyzed peripheral-blood lymphocytes from these individuals for the presence of persistent complex chromosome aberrations. A significantly elevated frequency of complex chromosome translocations was detected in the highly exposed plutonium workers but not in the group exposed only to high doses of gamma radiation. No such differences were found for simple chromosomal aberrations. The results suggest that stable complex chromosomal translocations represent a long-lived, quantitative, low-background biomarker of densely ionizing radiation for human populations exposed many years ago.

<p>Genomic fingerprints of mutagenic agents would have wide applications in the field of cancer biology, epidemiology and prevention. The differential spectra of chromosomal aberrations induced by different clastogens suggest that ratios of specific aberrations can be exploited as biomarkers of carcinogen exposure. We have tested this hypothesis using the novel technique of multicolor banding in situ hybridization (mBAND) in human peripheral blood lymphocytes exposed in vitro to X rays, neutrons, heavy ions, or the restriction endonuclease AluI. In the heavy-ion-irradiated cells, we further analyzed aberrations in chromosome 5 using multicolor FISH (mFISH). Contrary to the expectations of biophysical models, our results do not support the use of the ratios of inter-/intrachromosomal exchanges or intra-/interarm intrachanges as fingerprints of exposure to densely ionizing radiation. However, our data point to measurable differences in the ratio of complex/simple interchanges after exposure to different clastogens. These data should be considered in current biophysical models of radiation action in living cells.</p>

The aim of the present study was to estimate the genotoxicity of desflurane, applied as a volatile anaesthetic. The potential genotoxicity was determined by the comet assay as the extent of DNA fragmentation in human peripheral blood lymphocytes in vitro. The comet assay detects DNA strand breaks induced directly by genotoxic agents as well as DNA fragmentation due to cell death. Another anaesthetic, halothane, already proved to be a genotoxic agent, was used as a positive control. Both analysed drugs were capable of increasing DNA migration in a dose-dependent manner under experimental conditions applied. The results of the study demonstrated that the genotoxicity of desflurane was comparable with that of halothane. However, considering the pharmacodynamics of both drugs, the genotoxic activity of desflurane may be connected with a less harmful effect on the exposed patients or medical staff.

OBJECTIVE: To study chromosome analysis by comparative genomic hybridization (CGH) compared with the conventional technique in early amniocentesis. MATERIAL AND METHOD: Cross-sectional descriptive study design was performed in 32 singleton pregnant women with gestational age between 12-15 weeks. Transabdominal amniocentesis was carried out under ultrasound guidance. The amniotic fluid samples were simultaneously investigated using CGH and the conventional cytogenetics study as a gold standard. RESULTS: Amniocentesis were done for advanced maternal age in all cases. The mean maternal age was 35.8 years (35-42 years). The mean gestational age was 13.7 weeks (12-15 weeks). The chromosome analysis by CGH technique of uncultured amniocyte showed 17 normal female chromosomes (53.1%) and 15 normal male chromosomes (46.9%). This finding was the same as the conventional cytogenetics method. The mean duration of the CGH method was 6 days and that of the conventional cytogenetics method was 13.7 days (10-23 days). CONCLUSION: The CGH technique is a reliable technique for a rapid prenatal diagnosis of chromosome study in early gestation.

PURPOSE: Uveal melanoma is a highly malignant disease with a mortality rate of 50% at 10 to 15 years. Previous studies have shown that chromosomal changes are associated with decreased survival of the patient. However, in these studies the small number of tumors analyzed did not allow robust statistical analysis. In the present study, the independent numerical changes in chromosomes 1, 3, 6, and 8 on disease-free survival (DFS) was assessed in a large series of patients with uveal melanoma. METHODS: One hundred twenty tumors from patients with uveal melanoma were analyzed for numerical changes in chromosomes 1, 3, 6, and 8, with cytogenetic analysis, fluorescent in situ hybridization, and/or comparative genomic hybridization. Data were correlated with disease outcome in univariate and multivariate analyses, by Kaplan-Meier and Cox regression analyses. RESULTS: At a mean follow-up time of 45 months, 42 patients had died or had metastatic disease. In the univariate analysis, loss of chromosome 3, gain of 8q, largest tumor diameter, or the presence of epithelioid cells was associated with a decreased DFS. In the multivariate analysis, the effect of monosomy 3 on survival was largely modified by changes in 1p36. Regarding all chromosomal changes, only the concurrent loss of the short arm of chromosome 1 and all of chromosome 3 was an independent prognostic parameter for disease-free survival (P < 0.001). CONCLUSIONS: In uveal melanoma, concurrent loss of the short arm of chromosome 1 and all of chromosome 3 is an independent predictor of decreased DFS.

<p>Recurrent chromosome breakpoints in tumour cells may point to cancer genes, but not many have been molecularly characterised. We have used multicolour-banding fluorescence in situ hybridisation (mbanding-FISH) on breast tumour cell lines to identify regions of chromosome break created by inversions, duplications, insertions and translocations on chromosomes 1, 5, 8, 12 and 17. We delineate a total of 136 regions of break, some of them occurring with high frequency. We further describe two examples of dual-colour FISH characterisation of breakpoints, which target the 1p36 and 5p11-12 regions. Both breaks involve genes whose function is unknown to date. The mbanding-FISH strategy constitutes an efficient first step in the search for potential cancer genes.</p>

Conventional mutation-selection theories have failed to explain (i) how cancer cells become spontaneously resistant against cytotoxic drugs at rates of up to 10(-3) per cell generation, orders higher than gene mutation, even in cancer cells; (ii) why resistance far exceeds a challenging drug-a state termed multidrug resistance; (iii) why resistance is associated with chromosomal alterations and proportional to their numbers; and (iv) why resistance is totally dependent on aneuploidy. We propose here that cancer-specific aneuploidy generates drug resistance via chromosomal alterations. According to this mechanism, aneuploidy varies the numbers and structures of chromosomes automatically, because it corrupts the many teams of proteins that segregate, synthesize, and repair chromosomes. Aneuploidy is thus a steady source of chromosomal variation from which, in classical Darwinian terms, resistance-specific aneusomies are selected in the presence of chemotherapeutic drugs. Some of the thousands of unselected genes that hitchhike with resistance-specific aneusomies can thus generate multidrug resistance. To test this hypothesis, we determined the rates of chromosomal alterations in clonal cultures of human breast and colon cancer lines by dividing the fraction of nonclonal karyotypes by the number of generations of the clone. These rates were about 10(-2) per cell generation, orders higher than mutation. Chromosome numbers and structures were determined in metaphases hybridized with color-coded chromosome-specific DNA probes. Further, we tested puromycin-resistant subclones of these lines for resistance-specific aneusomies. Resistant subclones differed from parental lines in four to seven specific aneusomies, of which different subclones shared some. The degree of resistance was roughly proportional to the number of these aneusomies. Thus, aneuploidy is the primary cause of the high rates and wide ranges of drug resistance in cancer cells.

PURPOSE: To investigate if deviations from DNA-proportional distribution of radiation-induced chromosomal aberrations are individually variable. MATERIALS AND METHODS: Peripheral blood lymphocytes were collected from seven healthy donors and exposed to different doses of gamma rays. Chromosomes 2, 8 and 14 were painted in different colors and aberrations scored with the help of an image-analysis system. RESULTS: Chromosome 2 was generally less sensitive than expected on the basis of DNA-proportional distribution and the extent of inter-donor variability was minimal. A higher than expected frequency of aberrations was found in chromosome 14 of five donors, while a higher than expected frequency of aberrations was found in chromosome 8 of two donors. CONCLUSIONS: Inter-donor variability may explain some of the controversies regarding the inter-chromosomal distribution of radiation-induced aberrations.

Previous studies have documented that Tumor necrosis factor alpha (TNFalpha) is a potent negative regulator of normal hematopoiesis. However, the mechanism by which TNFalpha acts at the cellular level is not totally understood. Although apoptotic cell killing appears to be the most common cellular effect of TNFalpha, other studies suggest that this cytokine may elicit other cellular responses such as prolonged growth inhibition. In this context, we have investigated whether TNFalpha may induce senescence in hematopoietic cells, which display intrinsic defect in the apoptotic machinery. The present study described that, in the leukemic KG1 cells, TNFalpha induced no apoptosis but a senescence state characterized by prolonged growth arrest, increased beta-galactosidase activity, p21WAF-1 induction, decreased telomerase activity, telomeric disturbances (shortening, losses, fusions), and additional chromosomal aberrations. Telomerase inhibition correlated with reduced levels of hTERT transcripts. GM-CSF prevented TNFalpha effects and allowed leukemic cells to recover growth capacity. Finally, our study shows for the first time that, at least in some hematopoietic cells, TNFalpha may induce senescence with important functional consequences, including sustained growth inhibition and genetic instability, and that this cellular response is efficiently regulated by hematopoietic growth factors.

A number of genetic abnormalities have been detected in multiple myeloma (MM) using cytogenetic techniques. The prominent abnormalities are deletions of 13q and translocations affecting the IgH locus on 14q32. The recurrence of chromosomal abnormalities in MM suggests a specific role for them concerning its pathogenesis. We performed comparative genomic hybridization (CGH) on samples from 53 patients with MM and 4 with monoclonal gammopathies of undetermined significance. In 31 cases (54%), normal ratio profiles were found, whereas 26 cases (46%) had aberrant profiles. The most common aberrations were gains of 9p (n = 14), 11 (n = 9), and 21q (n = 5) and loss of 22 (n = 7). In earlier reports on cytogenetics of lymphomas, gains of 9p are described as characteristic of primary mediastinal B-cell lymphoma, but the consensus region is smaller than in the present study (9p23pter vs 9p13pter). Therefore, we suggest a stronger genetic affinity between MM and primary mediastinal B-cell lymphoma than MM and other B-cell lymphomas. To support this suggestion, more molecular cytogenetic techniques and expression analyses have to be performed.

Digital object identifier (DOI): 10.1309/5KWK-P6UK-GNXX-HMYH