Unstable Chromosome Research Articles

Сравнительный анализ результатов цитогенетических обследований контрольных групп лиц в различных отечественных лабораториях

The incidence of unstable chromosome aberrations in peripheral blood lymphocytes from unirradiated control subjects was analyzed using cytogenetic data obtained from 9 cytogenetic laboratories located in Moscow, St.-Petersburg, Obninsk, and Dubna (Russia). The objective of this study was to estimate the level and spectrum of spontaneous chromosome aberrations in human lymphocytes. 1140 blood samples were taken from 1112 subjects (594 men and 546 women) aged 1 to 72. The total metaphase number was 466795. The uniform Giemsa method for peripheral blood lymphocyte cultures was used. After counting 466795 metaphases, 4288 chromosomal aberrations of various types were classified. The most frequent types of aberrations were acentrics and chromatid deletions. They made up 90% of the total number of aberrations. The remaining 10% were exchange aberrations. The number of chromosome exchanges (dicentrics and centric rings) was twice the number of chromatid exchanges. Overall, the portion ofcells with chromosomal or (and) chromatid aberrations was 0.89 +/- 0.01%; the frequency of acentrics was 0.29 +/- 0.01; the frequency of dicentrics was 0.046 +/- 0.003; the frequency of unstable chromosome aberrations was 0.35 +/- 0.01; and the frequency of chromatid aberrations was 0.57 +/- 0.01 per 100 cells.

Aneuploid human colonic epithelial cells are sensitive to AICAR-induced growth inhibition through EGFR degradation

Trisomy for chromosome 7 is frequently observed as an initiating event in sporadic colorectal cancer. Although unstable chromosome numbers and recurrent aneuploidies drive a large fraction of human cancers, targeted therapies selective to pre-neoplastic trisomic cells are non-existent. We have previously characterized a trisomy 7 cell line (1CT+7) spontaneously derived from normal diploid human colonic epithelial cells that aberrantly expresses the epidermal growth factor receptor (EGFR, chromosome 7p11). Recent studies identified AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside) as a pharmacological inhibitor of aneuploid murine fibroblast proliferation. Here, we report that AICAR induces profound cytostatic and metabolic effects on 1CT+7 cells, but not on their isogenic diploid counterpart. Dose-response experiments indicate that 1CT+7 cells are fourfold preferentially sensitive to AICAR compared to diploid cells. Unexpectedly, treatment of 1CT+7 cells with AICAR led to a reversible 3.5-fold reduction (P=0.0025) in EGFR overexpression. AICAR-induced depletion of EGFR protein can be abrogated through inhibition of the proteasome with MG132. AICAR also heavily promoted EGFR ubiquitination in cell-based immunoprecipitation assays, suggesting enhanced degradation of EGFR protein mediated by the proteasome. Moreover, treatment with AICAR reduced EGFR protein levels in a panel of human colorectal cancer cells in vitro and in xenograft tumors in vivo. Our data collectively support the pharmacological compound AICAR as a novel inhibitor of EGFR protein abundance and as a potential anticancer agent for aneuploidy-driven colorectal cancer.

Formation of bipolar spindles with two centrosomes in tetraploid cells established from normal human fibroblasts

Tetraploid cells with unstable chromosomes frequently arise as an early step in tumorigenesis and lead to the formation of aneuploid cells. The mechanisms responsible for the chromosome instability of polyploid cells are not fully understood, although the supernumerary centrosomes in polyploid cells have been considered the major cause of chromosomal instability. The aim of this study was to examine the integrity of mitotic spindles and centrosomes in proliferative polyploid cells established from normal human fibroblasts. TIG-1 human fibroblasts were treated with demecolcine (DC) for 4days to induce polyploidy, and the change in DNA content was monitored. Localization of centrosomes and mitotic spindles in polyploid mitotic cells was examined by immunohistochemistry and laser scanning cytometry. TIG-1 cells treated with DC became almost completely tetraploid at 2weeks after treatment and grew at the same rate as untreated diploid cells. Most mitotic cells with 8C DNA content had only two centrosomes with bipolar spindles in established tetraploid cells, although they had four or more centrosomes with multipolar spindles at 3days after DC treatment. The frequency of aneuploid cells increased as established tetraploid cells were propagated. These results indicate that tetraploid cells that form bipolar spindles with two centrosomes in mitosis can proliferate as diploid cells. These cells may serve as a useful model for studying the chromosome instability of polyploid cells.

Abstract 3863: Aneuploid human colonic epithelial cells are sensitive to AICAR-induced growth inhibition through EGFR degradation

Abstract Trisomy for chromosome 7 is frequently observed as an initiating event in sporadic colorectal cancer. Although unstable chromosome numbers and recurrent aneuploidies drive a large fraction of human cancers, targeted therapies selective to pre-neoplastic trisomic cells are non-existent. We have previously characterized a trisomy 7 cell line (1CT+7) spontaneously derived from normal diploid human colonic epithelial cells that aberrantly express the epidermal growth factor receptor (EGFR, located on 7p11). Recent studies identified AICAR as a pharmacological inhibitor of aneuploid murine fibroblast proliferation. Here, we report that AICAR induces profound cytostatic and metabolic effects on 1CT+7 cells but not their isogenic diploid counterpart. Dose-response assays demonstrate that 1CT+7 cells are four-fold preferentially sensitive to AICAR compared to diploid cells. Unexpectedly, treatment of 1CT+7 cells with AICAR led to a reversible 3.5-fold reduction (p=0.0025) in EGFR overexpression. AICAR-induced depletion of EGFR protein can be abrogated through inhibition of the proteasome with MG132. AICAR also heavily promoted EGFR ubiquitination in cell-based immunoprecipitation assays, suggesting enhanced degradation of EGFR protein mediated by the proteasome. Moreover, treatment with AICAR reduced EGFR protein levels in a panel of human colorectal cancer cells in vitro and in xenograft tumors in vivo. Our data collectively supports the pharmacological compound AICAR as a novel inhibitor of EGFR protein abundance and as a potential therapeutic agent for aneuploidy-driven colorectal cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3863. doi:1538-7445.AM2012-3863

442: Mechanisms of telomere shortening in placentas from pregnancies complicated with intrauterine growth restriction

pregnancies complicated with intrauterine growth restriction Tal Biron-Shental, Yudith Sharon, Dvora Kidron, Moshe D. Fejgin, Aliza Amiel Meir Medical Center, Tel Aviv University, Obstetrics and Gynecology, Kfar Saba, Israel, Meir Medical Center, Genetic Institute, Kfar Saba, Israel, Meir Medical Center, Tel Aviv University, Pathology, Kfar Saba, Israel, Meir Medical Center, Faculty of Life Sciences, Bar Ilan University, Genetic Institute, Kfar Saba, Israel OBJECTIVE: Telomeres are nucleoprotein structures sited at the termini of the chromosomes that protect them from end to end fusion and degradation. Intrauterine Growth Restriction (IUGR) correlates with placental telomere shortening and with senescence. Senescence is associated with changes in the nuclear chromatin known as senescence-associated heterochromatin foci (SAHFs). Shortened and therefore unstable chromosomes can be stabilized by transfer of telomeres from other chromosomes. This process is termed telomere capture. Our aim was to assess mechanisms of telomere shortening in placentas from pregnancies complicated with IUGR. STUDY DESIGN: Placental biopsies derived from 13 pregnancies complicated with IUGR and from 12 gestational-age matched uncomplicated pregnancies. Clinical and histopathological characteristics were collected to ensure IUGR was secondary to placental insufficiency. We estimated the percentage of trophoblasts with SAHFs and the expression of the telomere capture mechanism using Dapi staining and FISH technique. RESULTS: Significantly increased expression of SAHFs (p 0.013) and significantly increased appearance of the telomere capture mechanism (p 0.023) were observed in placental biopsies from pregnancies complicated with IUGR compared to the control samples. (table). CONCLUSION: These results support and explain the observations of telomere shortening in IUGR placentas. We suggest that these findings may play a role in the pathophysiology of IUGR, perhaps by promoting senescence in trophoblasts of IUGR placentas.

Mutation Linked to Cancers, Unstable Chromosomes

Mutation Linked to Cancers, Unstable Chromosomes

Anchoring the dog to its relatives reveals new evolutionary breakpoints across 11 species of the Canidae and provides new clues for the role of B chromosomes

The emergence of genome-integrated molecular cytogenetic resources allows for comprehensive comparative analysis of gross karyotype architecture across related species. The identification of evolutionarily conserved chromosome segment (ECCS) boundaries provides deeper insight into the process of chromosome evolution associated with speciation. We evaluated the genome-wide distribution and relative orientation of ECCSs in three wild canid species with diverse karyotypes (red fox, Chinese raccoon dog, and gray fox). Chromosome-specific panels of dog genome-integrated bacterial artificial chromosome (BAC) clones spaced at ∼10-Mb intervals were used in fluorescence in situ hybridization analysis to construct integrated physical genome maps of these three species. Conserved evolutionary breakpoint regions (EBRs) shared between their karyotypes were refined across these and eight additional wild canid species using targeted BAC panels spaced at ∼1-Mb intervals. Our findings suggest that the EBRs associated with speciation in the Canidae are compatible with recent phylogenetic groupings and provide evidence that these breakpoints are also recurrently associated with spontaneous canine cancers. We identified several regions of domestic dog sequence that share homology with canid B chromosomes, including additional cancer-associated genes, suggesting that these supernumerary elements may represent more than inert passengers within the cell. We propose that the complex karyotype rearrangements associated with speciation of the Canidae reflect unstable chromosome regions described by the fragile breakage model.

A Genetic and Structural Study of Genome Rearrangements Mediated by High Copy Repeat Ty1 Elements

Ty elements are high copy number, dispersed repeated sequences in the Saccharomyces cerevisiae genome known to mediate gross chromosomal rearrangements (GCRs). Here we found that introduction of Ty912, a previously identified Ty1 element, onto the non-essential terminal region of the left arm of chromosome V led to a 380-fold increase in the rate of accumulating GCRs in a wild-type strain. A survey of 48 different mutations identified those that either increased or decreased the rate of Ty-mediated GCRs and demonstrated that suppression of Ty-mediated GCRs differs from that of both low copy repeat sequence- and single copy sequence-mediated GCRs. The majority of the Ty912-mediated GCRs observed were monocentric nonreciprocal translocations mediated by RAD52-dependent homologous recombination (HR) between Ty912 and a Ty element on another chromosome arm. The remaining Ty912-mediated GCRs appeared to involve Ty912-mediated formation of unstable dicentric translocation chromosomes that were resolved by one or more Ty-mediated breakage-fusion-bridge cycles. Overall, the results demonstrate that the Ty912-mediated GCR assay is an excellent model for understanding mechanisms and pathways that suppress genome rearrangements mediated by high copy number repeat sequences, as well as the mechanisms by which such rearrangements occur.

Estimation of relative biological effectiveness of tritium according to chromosome aberration frequency in human blood lymphocytes

The goal of this work was to determine the relative biological effectiveness (RBE) of tritium β-radiation according to the chromosome aberration frequency in the peripheral blood lymphocytes after in vitro and in vivo radiation exposures. The experimental RBE assessment of tritium β-radiation relative to 60Co γ-radiation according to unstable chromosome aberration frequency in the peripheral blood lymphocytes under particular conditions is described. It has been demonstrated that tritium β-radiation is, in general, more effective in the dose range of up to 1 Gy, which is most pronounced at low doses. The RBE value of tritium β-radiation at minimum doses reached 2.2 and decreased at higher doses (1 Gy) to 1.25. The data on comparative analysis of the frequency of stable chromosome aberrations in the blood lymphocytes of professional nuclear workers (Sarov, Russia) after long-term chronic exposure to tritium β-radiation, as compared with γ-irradiation, are reported for the first time. The higher biological effectiveness of tritium β-radiation was demonstrated and was estimated as 2.5.

Nek1 kinase functions in DNA damage response and checkpoint control through a pathway independent of ATM and ATR

Never-in-mitosis A related protein kinase 1 (Nek1) is involved early in a DNA damage sensing/repair pathway. We have previously shown that cells without functional Nek1 fail to activate the more distal kinases Chk1 and Chk2 and fail to arrest properly at G1/S or M-phase checkpoints in response to DNA damage. As a consequence, foci of damaged DNA in Nek1 null cells persist long after the instigating insult, and Nek1 null cells develop unstable chromosomes at a rate much higher than identically cultured wild type cells. Here we show that Nek1 functions independently of canonical DNA damage responses requiring the PI3 kinase-like proteins ATM and ATR. Chemical inhibitors of ATM/ATR or mutation of the genes that encode them fail to alter the kinase activity of Nek1 or its localization to nuclear foci of DNA damage. Moreover ATM and ATR activities, including the localization of the proteins to DNA damage sites and phosphorylation of early DNA damage response substrates, are intact in Nek1 -/- murine cells and in human cells with Nek1 expression silenced by siRNA. Our results demonstrate that Nek1 is important for proper checkpoint control and characterize for the first time a DNA damage response that does not directly involve one of the known upstream mediator kinases, ATM or ATR.

Ring chromosomes: vicious circles at the end and beginning of life

May 2001 Summary Many human malignant tumours exhibit abnormal chromosomal segregation at cell division. It is believed that these anomalies play a role in tumorigenesis by increasing the rate of chromosome mutations, including deletion and amplification of genes involved in cellular proliferation and/or survival. In vitro experiments have also shown that mitotic instability may be a mechanism for developing resistance to cytotoxic drugs. Abnormal mitotic mechanisms may result in numerical or structural aberrations in the daughter cells. Numerical aberrations can be caused either by the loss of chromosomes at metaphase/anaphase or by multipolar divisions associated with abnormal number or structure of centrosomes. Structural rearrangements have been associated with chromosomal breakage-fusion-bridge (BFB) cycles that can be initiated by telomeric dysfunction, giving rise to unstable dicentric or ring chromosomes. In most tumours exhibiting chromosomal instability, including highgrade malignant pancreatic, ovarian, and head- and neck carcinomas, as well as osteo- and soft tissue sarcomas, the two types of instability occur together. However, in some low-grade mesenchymal and neuroglial tumours, rarely showing numerical changes, BFB events involving telomeric associations and ring chromosomes dominate the mitotic process. At progression towards higher malignancy in these tumours, complex structural and numerical aberrations become more frequent. This may be explained by a process initiated by telomeric dysfunction and anaphase bridging, which, in turn, may give rise to an increased frequency of multinucleated cells through failure of cytokinesis. These cells will contain an abnormal number of centrosomes leading to multipolar mitotic figures at the next cell division. Further understanding of these events may lead to novel strategies for detection and treatment of malignancy.

Different outcomes of telomere-dependent anaphase bridges

Chromosomal instability occurs early in the development of cancer and may represent an important step in promoting the multiple genetic changes required for the initiation and/or progression of the disease. Telomere erosion is one of the factors that contribute to chromosome instability through end-to-end chromosome fusions entering BFB (breakage-fusion-bridge) cycles. Uncapped chromosomes with short dysfunctional telomeres represent an initiating substrate for both pre- and post-replicative joining, which leads to unstable chromosome rearrangements prone to bridge at mitotic anaphase. Resolution of chromatin bridge intermediates is likely to contribute greatly to the generation of segmental chromosome amplification events, unbalanced chromosome rearrangements and whole chromosome aneuploidy. Accordingly, telomere-driven instability generates highly unstable genomes that could promote cell immortalization and the acquisition of a tumour phenotype.

The Dose Response of Chromosome Aberrations in Human Lymphocytes InducedIn Vitroby Very Low-Dose γ Rays

This paper considers the dose-effect relationship for unstable chromosome aberration yields in human lymphocytes in very low-dose range. Data are presented for (60)Co γ-ray doses of 0, 10, 20, 40 and 1000 mGy. More than 5,000 metaphases were scored for each data point at the very low doses, and each cell was double-checked using a semi-automated metaphase finding/relocation system. Aberration yields of dicentrics plus centric rings followed an excellent linear dose response down to zero dose; the yields were significantly above the control frequency from 20 mGy.

FOLLOW-UP STUDY BY CHROMOSOME ABERRATION ANALYSIS AND MICRONUCLEUS ASSAYS IN VICTIMS ACCIDENTALLY EXPOSED TO 60Co RADIATION

The goal of this study was to assess the persistence of chromosomal aberrations and micronuclei of three victims 2 y after accidental radiation exposure to Co gamma rays. Traditional chromosome aberration analysis was performed by scoring the dicentric chromosomes (dic) and rings (r) in peripheral blood lymphocytes. Micronuclei were detected using the cytokinesis block micronucleus assay. G-banding and semi-automatic karyotype analysis was used to record translocations (t), inversions (inv) and deletions (del). The frequency of unstable chromosomal aberrations (dicentrics and rings) remained at high levels 6 mo after the accident. Two years after exposure, the frequency was reduced to 4-11% in the three victims. However, stable chromosome aberrations, which were detected by G-banding and included t, inv, and del, remained at a high level and have an obvious dose-dependent relationship even 2 y post-exposure. The frequency of micronuclei decreased faster than that of chromosome aberrations, reaching almost a normal level two years after the accident, especially for the child victim. Unstable chromosome aberrations reduced gradually, but the stable aberration remained at a high level along with the time-lapse. The micronucleus assay was less valuable for assessing long-term effects after high dose irradiation.

Establishment and characterization of novel human uterine leiomyosarcoma cell lines

Patients with unresectable advanced uterine leiomyosarcoma have a very poor prognosis because no effective chemotherapeutic protocols exist. There are currently few established primary human uterine leiomyosarcoma cell lines that can be used to investigate effective therapies. To overcome this problem, we carried out long-term in vitro cell culture and/or nude mouse transplantation and successfully established novel human uterine leiomyosarcoma cell lines from extrauterine and intrauterine tumors that were surgically excised from a single patient. The established cells were characterized by flow cytometry, anticancer drug-sensitivity assays and karyotyping analyses. All the established cell lines showed unstable multiple chromosome abnormalities. Since the cells can proliferate in vitro and in vivo, they will be useful for developing new therapeutic strategies for advanced uterine leiomyosarcoma patients.

The Hows and Ys of Genome Integrity

Much of the Y chromosome consists of large palindromic arrays harboring genes that are critical for spermatogenesis. In this issue, Lange et al. (2009) show that although gene conversion within these arrays maintains their integrity, it also permits rare unequal sister chromatid-exchange events within palindromes that create unstable dicentric chromosomes, resulting in infertility, sex reversal, and Turner syndrome.

Modification of 2-deoxy-D-glucose on radiation-and chemotherapeutic drug-induced chromosomal aberrations

Chemotherapy is the treatment of cancer with drugs, often used as either adjuvant or neoadjuvant or in conjunction with radiation and surgery. Unfortunately, majority of the drugs are toxic to normal tissues, the toxicity being resulting from multidrug protocol used to induce remissions and achieve tumor care. While it has been demonstrated for compounds like the 2-deoxy-glucose (2-DG) used as a modulator for radiation-induced damages, such studies were rarely reported for chemotherapeutic drugs. To study the effect of 2-DG on radiation-and chemotherapeutic drug-induced chromosomal aberrations in normal and tumor cells exposed in vitro. The peripheral blood lymphocytes (PBLs) and BMG-1 cells were exposed to radiation and chemotherapeutic drugs (bleomycin and mitomycin-C) in the presence and absence of 2-DG. The treated cells were cultured for various durations, arrested at either metaphase or cytokinesis stage of the cell cycle. The stable and unstable aberrations were recorded using Giemsa staining and FISH technique. The cell cycle kinetics was studied using fluorescence plus Giemsa (FPG) staining. The presence of 2-DG reduced stable and unstable chromosome aberrations (CA) significantly (P < 0.001), in PBLs induced by radiation, bleomycin and mitomycin-C, when compared to cells treated with radiation or the drugs and increased significantly in BMG cells (P < 0.001). Furthermore, the presence of 2-DG altered the cell cycle kinetics in the PBLs and BMG-1 cells. Thus the overall results showed protection effect on the normal cell damages induced by radiation and chemotherapeutic drugs, while sensitizes the tumor cell. The obtained results suggest that 2-DG in combination with radiotherapy/chemotherapy could lead to an improvement in tumor therapy by sensitizing the tumor cells while protecting the normal cells.

Influence of hOGG1 genotype on the frequencies of stable and unstable chromosome aberrations in radiation-exposed individuals

Influence of hOGG1 genotype on the frequencies of stable and unstable chromosome aberrations in radiation-exposed individuals

Mechanism of adaptive response: Estimation of human lymphocyte adaptive response to radiation using various criteria

Blood lymphocytes of 15 healthy donors have been investigated for their ability to reduce radiosensitivity after low-dose irradiation-radio-induced adaptive response (AR). The frequency of unstable chromosome aberrations was used to evaluate cell radiosensitivity after the irradiation of cells in low adaptive (5 cGy) and high challenge (1 Gy) doses in comparison with the effect of challenge irradiation only. Three indexes have been used, i.e., (A) the frequency of cells with aberrations per total analyzed cell, (B) the number of chromosome aberrations per one cell, (C) and the number of chromosome aberrations per one aberrant cell. It was found that the donors can be divided in the four following groups: 1. AR was not estimated any of the indexes used; 2. AR was estimated with indexes A and B, but not C; 3. AR was shown by indexes B and C; 4. AR was evident with all three indexes. The generally accepted AR repair model only explains the appearance of group-3 and-4 donors, but not group-2. For the purpose of understanding the AR mechanisms and the difference in AR estimations with various criteria, the metaphase distribution by the number of chromosome aberrations has been analyzed for each donor. It was shown that, in group-2 donors, the number of cells without aberrations after adaptive and challenge irradiations was significantly higher than after irradiation with a challenge dose only. Thus, in this group, AR is formed as a result of the changed frequency of cells in the 0 class (population shift). A similar shift is observed in the metaphase distribution in the donors of group 4, but not in group 3. The data obtained show that AR is probably a result of several processes, including the activation of the reparation of premutational genome damages, population shifts evident in the frequency of undamaged cells, and, possibly, the activation of apoptotic cell death. The complex character of AR is reflected to different degrees in each criterion of radiosensitivity.

On the XO sex chromosome system of Aulacoscelis melanocera Stal (Aulacoscelinae: Chrysomelidae: Coleoptera)

Aulascoscelinae, a small, neotropical subfamily of Chrysomelidae (Coleoptera), is almost unknown cytologi-cally. Although most male Aulacoscelis melanocera have 11+XO meioformula (corresponding to 2n = 23; X), with “nucleolus” attached to X, one male exhibited a condition best interpreted as ll + Xyp, with an unstable sex chromosome association.