Mutation of the murine Bloom's syndrome gene produces global genome destabilization

Bloom's syndrome (BS) is a genetic disorder characterized cellularly by increases in sister chromatid exchanges (SCEs) and numbers of micronuclei. BS is caused by mutation in the BLM DNA helicase gene and involves a greatly enhanced risk of developing the range of malignancies seen in the general population. With a mouse model for the disease, we set out to determine the relationship between genomic instability and neoplasia. We used a novel two-step analysis to investigate a panel of eight cell lines developed from mammary tumors that appeared in Blm conditional knockout mice. First, the panel of cell lines was examined for instability. High numbers of SCEs were uniformly seen in members of the panel, and several lines produced chromosomal instability (CIN) manifested by high numbers of chromosomal structural aberrations (CAs) and chromosome missegregation events. Second, to see if Blm mutation was responsible for the CIN, time-dependent analysis was conducted on a tumor line harboring a functional floxed Blm allele. The floxed allele was deleted in vitro, and mutant as well as control subclones were cultured for 100 passages. By passage 100, six of nine mutant subclones had acquired high CIN. Nine mutant subclones produced 50-fold more CAs than did nine control subclones. Finally, chromosome loss preceded the appearance of CIN, suggesting that this loss provides a potential mechanism for the induction of instability in mutant subclones. Such aneuploidy or CIN is a universal feature of neoplasia but has an uncertain function in oncogenesis. Our results show that Blm gene mutation produces this instability, strengthening a role for CIN in the development of human cancer.     

The chromosomal radiosensitivity of children whose parents were exposed to antitumor radiochemotherapy]

The effect of gamma radiation was studied on routine stained chromosomes from lymphocytes of children born to Hodgkin's disease patients after cancer therapy (CP) in comparison to children from healthy parents (HP). Irradiation (0, 0.25, 0.50, 1.00, 1.50 Gy) of the whole blood was performed in culture medium. Metaphases were obtained from 52-h cultures. Chromosomal aberrations were used as an endpoint. Aberrations of both chromosomal and chromatid types were scored in 150-200 metaphases for estimation of spontaneous level of cytogenetic injuries and in 100 metaphases of induced one. It is found that chromosomes of CP children are more radiosensitive than chromosomes of HP ones, the spontaneous frequency of chromosome aberrations being equal in both groups.     

Assessment of the genotoxicity of atenolol in human peripheral blood lymphocytes: Correlation between chromosomal fragility and content of micronuclei

The antihypertensive drug atenolol was found to induce chromosome loss, detected as micronuclei in the peripheral lymphocytes of treated patients. The fundamental question which chromosomes the micronuclei were derived from remains to be answered. Analysis of structural chromosomal aberrations (CAs) and expression of fragile sites (FS) were pursued in this study. They revealed a significantly higher incidence of chromosomal aberrations (chromatid and chromosome breaks) in patients compared with controls, where 10 FS emerged as specific. Also, the band 17q12–21, where known fragile sites have not been reported, was only expressed in atenolol-treated patients. Fluorescence in situ hybridization using chromosome-specific probes revealed the preferential involvement of chromosomes 7, 11, 17 and X in the micronuclei (MN) of patients. The results also suggest a correlation between chromosomal fragility and content of MN, and support the findings for a linkage between hypertension and a locus on chromosome 17.     

Liability to chromosome damage in lymphocytes of "cancer family" subjects: a study of spontaneous and induced chromosomal fragility

Spontaneous chromosomal fragility was detected in seven tumor patients and one healthy member from two families with a high recurrence of cancer. Major chromosome lesions, such as terminal deletions and rearranged chromosomes, were found at levels significantly higher than those reported for control individuals. The prevalence of these aberrations in comparison to minor ones (chromosome gaps and chromatid breaks) in this group of patients seems to indicate that the fragility observed is the end-point of a process of chromosomal instability, which may have already been brought to expression. Study of other parameters of genetic instability in the most unstable karyotypes showed that the chromosome damage observed was neither paralleled by abnormal SCE frequency nor sustained by defective DNA repair mechanisms or expression of inherited or constitutional fragile sites. As all the subjects investigated here had previously been shown to display intraindividual variations in the C-banded region of chromosome 1, it is possible that spontaneous fragility and acquired C-heterochromatin polymorphism may be markers that, combined with chromosomal instability, create genetic predisposition to cancer.     

Chromosomal changes induced by chrysotile fibres or benzo-3,4-pyrene in rat pleural mesothelial cells

The induction of chromosomal aberrations in rat pleural mesothelial cells (RPMC) following in vitro treatment with chrysotile fibres has been demonstrated. The production of chromosomal aberrations was also observed after treatment of the cells with benzo-3,4-pyrene (BP). The yield of abnormal metaphases was dose-dependent and reached 58% at a BP dose of 2 micrograms/ml. Chrysotile fibres at 7 micrograms/ml induced 21% abnormal metaphases and the frequency decreased with further increases in fibre concentration. Their decline is possibly related to a lethal effect. Chrysotile-induced chromosomal aberrations were primarily of the chromatid type and included breaks and fragments. BP induced chromosome exchanges which were not seen following chrysotile treatment. Minutes and double minutes were detected in BP-treated RPMC and occasionally found after chrysotile application. These results confirm that chrysotile fibres are clastogenic for some cultured cells and demonstrate that the fibres induce chromosome damage in target RPMC.     

Role of the Fancg gene in protecting cells from particulate chromate-induced chromosome instability

Particulate hexavalent chromium (Cr(VI)) is a known human lung carcinogen. Cr(VI)-induced tumors exhibit chromosome instability (CIN), but the mechanisms underlying these effects are unknown. We investigated a possible role for the Fanconi anemia (FA) pathway in particulate Cr(VI)-induced chromosomal damage by focusing on the Fancg gene, which plays an important role in cellular resistance to DNA interstrand crosslinks. We used the isogenic Chinese hamster ovary (CHO) KO40 fancg mutant compared with parental and gene-complemented cells. We found that fancg cells treated with lead chromate had lower intracellular Cr ion levels than control cell lines. Accounting for differences of Cr ion levels between cell lines, we discovered that fancg cells treated with lead chromate had increased cytotoxicity and chromosomal aberrations, which was not observed after restoring the Fancg gene. Chromosomal damage was manifest as increased total chromosome damage and percent metaphases with damage, specifically an increase in chromatid and isochromatid breaks. We conclude that Fancg protects cells from particulate Cr(VI)-induced cytotoxicity and chromosome damage, which is consistent with the known sensitivity of fancg cells to crosslinking damage and the ability of Cr(VI) to produce crosslinks.     

Chromosomal instability in breast cancer patients

Summary We have carried out cytogenetic studies, using the G-banding technique, in peripheral blood lymphocytes of 10 patients affected by breast carcinoma. The frequency of aberrant metaphases (7.36%) is significantly different from that of our laboratory controls (3.76% of aberrant metaphases) but not from that detected in patients suffering from bladder cancer (10.64%) and Hodgkin's disease (11.03%), two conditions that have previously been described as chromosomally unstable. Our results suggest that breast carcinoma patients show a degree of chromosomal instability that could be related to a predisposition to neoplastic disease.     

Chromosomal changes: induction, detection methods and applicability in human biomonitoring

The objective of this state of the art paper is to review the mechanisms of induction, the fate, the methodology, the sensitivity/specificity and predictivity of two major cytogenetic endpoints applied for genotoxicity studies and biomonitoring purposes: chromosome aberrations and micronuclei. Chromosomal aberrations (CAs) are changes in normal chromosome structure or number that can occur spontaneously or as a result of chemical/radiation treatment. Structural CAs in peripheral blood lymphocytes (PBLs), as assessed by the chromosome aberration (CA) assay, have been used for over 30 years in occupational and environmental settings as a biomarker of early effects of genotoxic carcinogens. A high frequency of structural CAs in lymphocytes (reporter tissue) is predictive of increased cancer risk, irrespective of the cause of the initial CA increase. Micronuclei (MN) are small, extranuclear bodies that arise in dividing cells from acentric chromosome/chromatid fragments or whole chromosomes/chromatids that lag behind in anaphase and are not included in the daughter nuclei in telophase. The cytokinesis-block micronucleus (CBMN) assay is the most extensively used method for measuring MN in human lymphocytes, and can be considered as a "cytome" assay covering cell proliferation, cell death and chromosomal changes. The key advantages of the CBMN assay lie in its ability to detect both clastogenic and aneugenic events and to identify cells which divided once in culture. Evaluation of the mechanistic origin of individual MN by centromere and kinetochore identification contributes to the high sensitivity of the method. A number of findings support the hypothesis of a predictive association between the frequency of MN in cytokinesis-blocked lymphocytes and cancer development. Recent advances in fluorescence in situ hybridization (FISH) and microarray technologies are modifying the nature of cytogenetics, allowing chromosome and gene identification on metaphase as well as in interphase. Automated scoring by flow cytometry and/or image analysis will enhance their applicability.     

Genotoxicity assessment in patients with thalassemia minor

Thalassemia is an inherited blood disorder that affects both genders and results in reduced synthesis of hemoglobin, and thus causing anemia. Previous studies have shown that the severe form of this disease, thalassemia major, is associated with genotoxicity. This includes increases in the level of sister chromatid exchange (SCEs), chromosomal aberrations (CAs) and micronuclei. In this study, we assessed genotoxicity in the lymphocytes of thalassemia minor subjects using sister chromatid exchange (SCE) and chromosomal aberration (CA) assays. In addition, we investigated the level of oxidative DNA damage by measuring 8-hydroxy-2'-deoxyguanosine (8OHdG) biomarker in urine samples. Eighteen thalassemia minor subjects and eighteen matched normal healthy controls were volunteered in the study. In addition, seven thalassemia major patients were recruited as positive controls. The results showed increases in the frequency of SCEs (P<0.05) in thalassemia minor compared to healthy controls. However, no difference in CAs frequency was detected between thalassemia minor and controls (P>0.05). Both SECs and CAs in thalassemia major patients were significantly higher compared to other groups (P<0.05). Regarding urine 8OHdG levels, the result showed a slight increase in thalassemia minor compared to healthy controls but the difference was not significant (P>0.05). In conclusion, our results showed that thalassemia minor is associated with genotoxicity to blood lymphocytes as indicated by SCEs assay.     

Genomic Aberrations in an African American Colorectal Cancer Cohort Reveals a MSI-Specific Profile and Chromosome X Amplification in Male Patients

Objective

DNA aberrations that cause colorectal cancer (CRC) occur in multiple steps that involve microsatellite instability (MSI) and chromosomal instability (CIN). Herein, we studied CRCs from AA patients for their CIN and MSI status.

Experimental Design

Array CGH was performed on 30 AA colon tumors. The MSI status was established. The CGH data from AA were compared to published lists of 41 TSG and oncogenes in Caucasians and 68 cancer genes, proposed via systematic sequencing for somatic mutations in colon and breast tumors. The patient-by-patient CGH profiles were organized into a maximum parsimony cladogram to give insights into the tumors'' aberrations lineage.

Results

The CGH analysis revealed that CIN was independent of age, gender, stage or location. However, both the number and nature of aberrations seem to depend on the MSI status. MSI-H tumors clustered together in the cladogram. The chromosomes with the highest rates of CGH aberrations were 3, 5, 7, 8, 20 and X. Chromosome X was primarily amplified in male patients. A comparison with Caucasians revealed an overall similar aberration profile with few exceptions for the following genes; THRB, RAF1, LPL, DCC, XIST, PCNT, STS and genes on the 20q12-q13 cytoband. Among the 68 CAN genes, all showed some level of alteration in our cohort.

Conclusion

Chromosome X amplification in male patients with CRC merits follow-up. The observed CIN may play a distinctive role in CRC in AAs. The clustering of MSI-H tumors in global CGH data analysis suggests that chromosomal aberrations are not random.     

Effects of CCNU therapy on human chromosomes

Peripheral blood lymphocytes of 9 patients under CCNU therapy were examined for frequency of sister-chromatid exchanges (SCEs) and chromosomal aberrations (CAs). 7 out of 9 patients were treated with only CCNU, whereas the remaining 2 were treated with other chemotherapeutic agents in combination with CCNU. Compared to normal individuals, a significantly increased frequency of SCE was observed in the patients before starting anticancer therapy (P less than 0.001). Increased incidences of structural changes in chromosomes were observed in cells from all the treated patients. The most frequent aberrations were of chromatid type. After administration of a single dose of CCNU, an increase in SCE frequencies was observed which remained elevated even after 6 weeks. It was concluded that increases in SCEs and CAs in lymphocytes were caused by CCNU treatment. Further studies are needed to elucidate whether any CAs observed in the present study could participate in the induction of second neoplasm.     

Chromosomal damage in peripheral blood lymphocytes of patients treated for testicular cancer

Summary We describe the presence of a large number of chromosome aberrations in lymphocytes of 50 patients with testicular cancer. These chromosomal aberrations were not only found in patients treated with chemotherapy but also in untreated patients or in patients after surgery alone. Our results suggest a role for genetic instability in the pathogenesis of testicular cancer. This instability might be a risk factor for the development of secondary malignancies.     

Association of ionizing radiation-induced foci of NBS1 with chromosomal instability and breast cancer susceptibility

NBS1, a protein essential for DNA double-strand break repair, relocalizes into subnuclear structures upon induction of DNA damage by ionizing radiation, forming ionizing radiation-induced foci. We compared radiation-induced NBS1 foci in peripheral blood lymphocytes (PBLs) from 46 sporadic breast cancer patients and 30 healthy cancer-free volunteers. The number of persistent radiation-induced NBS1 foci per nucleus at 24 h after irradiation for patients with invasive cancer was significantly higher than for normal healthy volunteers. The frequency of spontaneous chromosome aberration increased as the number of persistent radiation-induced NBS1 foci increased, indicating that the number of persistent radiation-induced NBS1 foci might be associated with chromosome instability. There was also an inverse correlation between the number of radiation-induced NBS1 foci and the activity of DNA-dependent protein kinase (DNA-PK), which plays an important role in the nonhomologous end-joining (NHEJ) pathway, another mechanism of DNA DSB repair, indicating a close interrelationship between homologous recombination (HR) and NHEJ in DNA DSB repair. In conclusion, the number of persistent radiation-induced NBS1 foci is associated with chromosomal instability and risk of sporadic breast cancer and hence might be used to select individuals for whom a detailed examination is necessary because of their increased susceptibility to breast cancer, although refinement of the techniques for technical simplicity and accuracy will be required for clinical use.     

The effects of increasing dosages of X-radiation on the chromosomes of the central mudminnow, Umbra limi (Kirtland) (Salmoniformes: Umbridae)

Fish subjected to 350 R, 660 R and 990 R of X-radiation showed chromosomal aberrations such as chromatid breaks and gaps, and chromatid exchanges between several chromosomes. The frequency of aberrations/metaphase increased with radiation dosage. Likewise, the percentage of aberrant cells increased with increased irradiation. The countable metaphases fish was lower for higher doses of radiation. At lower doses single chromatid breaks accounted for most of the aberrations whereas complex aberrations involving the breakage and exchange of fragments between several chromosomes were more frequent in fish subjected to 990 R. Gill tissue yielded three times as many countable metaphases as did spleen tissue.     

Cytogenetic abnormalities in a patient with hypercalcemia and papillary thyroid carcinoma

Summary Cytogenetic examinations on multiple peripheral blood cultures of a patient with papillary thyroid carcinoma and hypercalcemia revealed the following features: (1) The average frequency of cells with aberrations was 11.6%, considerably higher than in controls. Among metaphases with chromosomal abnormalities, 4.5% had chromosome-type aberrations. (2) One homolog of chromosome 11 showed a fragile site in the proximal end of the long arm, and in three metaphases the segment distal to the fragile site showed branched morphology. (3) The rate of sister chromatid exchanges was within normal limits (8.78/metaphase). (4) The patient's two sons showed 7.0% and 5.0% abnormal metaphases, in the high normal range.     

Chromosomal instability and human hepatocarcinogenesis

Recently, many studies have identified losses and gains of several chromosomal loci in human hepatocellular carcinoma (HCC) with fine microsatellite analysis and comparative genomic hybridization. Although distribution of aberrant chromosomal arms differs among HCCs, loss of 1p, 4q, 6q, 8p, 9p, 10q, 13q, 16q and 17p, and gain of 1q, 6p, 8q, 17q and 20q have been recurrently reported, and loss of 4q and 16q seems to occur preferentially in hepatitis B virus-related HCCs. Accumulation of these aberrant chromosomal regions is associated with tumor progression, and some chromosomal aberrations, such as loss of 1p, are frequently identified in well-differentiated HCCs and also detected even in dysplastic nodule and cirrhotic nodule. This evidence suggests that chromosomal instability (CIN) emerges at an early stage during hepatocarcinogenesis and is successively inherent to tumor cells, resulting in acquisition of malignant phenotype. The molecular basis of CIN is beginning to be explored; however, several mechanisms may be involved for CIN of HCC.     

Cell death mechanisms associated with G2 radiosensitivity in patients with prostate cancer and benign prostatic hyperplasia

Cells respond to genotoxic insults such as ionizing radiation by halting in the G2 phase of the cell cycle. Delayed cell death (mitotic death) can occur when the cell is released from G2, and specific spindle defects form endopolyploid cells (endoreduplication/tetraploidy). Enhanced G2 chromosomal radiosensitivity has been observed in many cancers and genomic instability syndromes, and it is manifested by radiation-induced chromatid aberrations observed in lymphocytes of patients. Here we compare the G2 chromosomal radiosensitivity in prostate patients with benign prostatic hyperplasia (BPH) or prostate cancer with disease-free controls. We also investigated whether there is a correlation between G2 chromosomal radiosensitivity and aneuploidy (tetraploidy and endoreduplication), which are indicative of mitotic cell death. The G2 assay was carried out on all human blood samples. Metaphase analysis was conducted on the harvested chromosomes by counting the number of aberrations and the mitotic errors (endoreduplication/tetraploidy) separately per 100 metaphases. A total of 1/14 of the controls were radiosensitive in G2 compared to 6/15 of the BPH patients and 15/17 of the prostate cancer patients. Radiation-induced mitotic inhibition was assessed to determine the efficacy of G2 checkpoint control in the prostate patients. There was no significant correlation of G2 radiosensitivity scores and mitotic inhibition in BPH patients (P = 0.057), in contrast to prostate cancer patients, who showed a small but significant positive correlation (P = 0.029). Furthermore, there was no significant correlation between G2 radiosensitivity scores of BPH patients and endoreduplication/ tetraploidy (P = 0.136), which contrasted with an extremely significant correlation observed in prostate cancer patients (P < 0.0001). In conclusion, cells from prostate cancer patients show increased sensitivity to the induction of G2 aberrations from ionizing radiation exposure but paradoxically show reduced mitotic indices and aneuploidy as a function of aberration frequency.     

Frequency and distribution of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced structural chromosome aberrations in fibroblasts from sarcoma and non-Hodgkin's lymphoma patients

The frequency and distribution of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced structural aberrations, i.e., chromatid and chromosome gaps, breaks, and exchanges, were studied in fibroblasts from 16 patients with sarcoma, 15 with non-Hodgkin's lymphoma (NHL), and 14 controls. The mean frequencies of aberrant cells, and gap, break, and gap + break events per 100 cells were 22.9, 5.1, 28.6, and 33.7 in the sarcoma group; 19.1, 5.0, 22.5, and 27.5 in the NHL group; and 23.5, 6.1, 33.5, and 39.6 in the control group. None of the differences between the groups were statistically significant. The distribution of MNNG-induced aberrations was non-random (P less than 0.001) in all 3 groups. Eight, 11, and 17 chromosome bands in the sarcoma, NHL, and control groups, respectively, were particularly break-prone. Only 2 hot spots in the sarcoma group (1p32, 11q23), and 3 in the NHL group (1p36, 3q25, 6p21), coincided with the 25 and 60 bands known to be involved in primary rearrangements in sarcomas and NHL. We conclude that neither the frequency nor the distribution of MNNG-induced chromosomal aberrations indicates any latent chromosomal instability in sarcoma and NHL patients.     

Linear Model of Colon Cancer Initiation

Cancer results if regulatory mechanisms of cell birth and death are disrupted. Colorectal tumorigenesis is initiated by somatic or inherited mutations in the APC tumor suppressor gene pathway. Several additional genetic hits in other tumor suppressor genes and oncogenes drive the progression from polyps to malignant, invasive cancer. The majority of colorectal cancers present chromosomal instability, CIN, which is caused by mutations in genes that are required to maintain chromosomal stability. A major question in cancer genetics is whether CIN is an early event and thus a driving force of tumor progression. We present a new mathematical model of colon cancer initiation assuming a linear flow from stem cells to differentiated cells to apoptosis. We study the consequences of mutations in different cell types and calculate the conditions for CIN to precede APC inactivation. We find that early emergence of CIN is very likely in colorectal tumorigenesis.