g2-chromatid breaks induced: Topics by Science.gov

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Dose--response of initial G2-chromatid breaks induced in normal human fibroblasts by heavy ions

NASA Technical Reports Server (NTRS)

Kawata, T.; Durante, M.; Furusawa, Y.; George, K.; Takai, N.; Wu, H.; Cucinotta, F. A.; Dicello, J. F. (Principal Investigator)

2001-01-01

PURPOSE: To investigate initial chromatid breaks in prematurely condensed G2 chromosomes following exposure to heavy ions of different LET. MATERIAL AND METHODS: Exponentially growing human fibroblast cells AG1522 were irradiated with gamma-rays, energetic carbon (13 keV/ microm, 80 keV/microm), silicon (55 keV/microm) and iron (140 keV/microm, 185keV/microm, 440keV/microm) ions. Chromosomes were prematurely condensed using calyculin-A. Initial chromatid-type and isochromatid breaks in G2 cells were scored. RESULTS: The dose response curves for total chromatid breaks were linear regardless of radiation type. The relative biological effectiveness (RBE) showed a LET-dependent increase, peaking around 2.7 at 55-80keV/microm and decreasing at higher LET. The dose response curves for isochromatid-type breaks were linear for high-LET radiations, but linear-quadratic for gamma-rays and 13 keV/microm carbon ions. The RBE for the induction of isochromatid breaks obtained from linear components increased rapidly between 13keV/microm (about 7) and 80keV/microm carbon (about 71), and decreased gradually until 440 keV/microm iron ions (about 66). CONCLUSIONS: High-LET radiations are more effective at inducing isochromatid breaks, while low-LET radiations are more effective at inducing chromatid-type breaks. The densely ionizing track structures of heavy ions and the proximity of sister chromatids in G2 cells result in an increase in isochromatid breaks.
A comparison of G2 phase radiation-induced chromatid break kinetics using calyculin-PCC with those obtained using colcemid block.

PubMed

Bryant, Peter E; Mozdarani, Hossein

2007-09-01

To study the possible influence of cell-cycle delay on cells reaching mitosis during conventional radiation-induced chromatid break experiments using colcemid as a blocking agent, we have compared the chromatid break kinetics following a single dose of gamma rays (0.75 Gy) in metaphase CHO cells using calyculin-induced premature chromosome condensation (PCC), with those using colcemid block. Calyculin-induced PCC causes very rapid condensation of G2 cell chromosomes without the need for a cell to progress to mitosis, hence eliminating any effect of cell-cycle checkpoint on chromatid break frequency. We found that the kinetics of the exponential first-order decrease in chromatid breaks with time after irradiation was similar (not significantly different) between the two methods of chromosome condensation. However, use of the calyculin-PCC technique resulted in a slightly increased rate of disappearance of chromatid breaks and thus higher frequencies of breaks at 1.5 and 2.5 h following irradiation. We also report on the effect of the nucleoside analogue ara A on chromatid break kinetics using the two chromosome condensation techniques. Ara A treatment of cells abrogated the decrease in chromatid breaks with time, both using the calyculin-PCC and colcemid methods. We conclude that cell-cycle delay may be a factor determining the absolute frequency of chromatid breaks at various times following irradiation of cells in G2 phase but that the first-order disappearance of chromatid breaks with time and its abrogation by ara A are not significantly influenced by the G2 checkpoint.
Induction and disappearance of G2 chromatid breaks in lymphocytes after low doses of low-LET gamma-rays and high-LET fast neutrons.

PubMed

Vral, A; Thierens, H; Baeyens, A; De Ridder, L

2002-04-01

To determine by means of the G2 assay the number of chromatid breaks induced by low-LET gamma-rays and high-LET neutrons, and to compare the kinetics of chromatid break rejoining for radiations of different quality. The G2 assay was performed on blood samples of four healthy donors who were irradiated with low-LET gamma-rays and high-LET neutrons. In a first set of experiments a dose-response curve for the formation of chromatid breaks was carried out for gamma-rays and neutrons with doses ranging between 0.1 and 0.5 Gy. In a second set of experiments, the kinetics of chromatid break formation and disappearance were investigated after a dose of 0.5 Gy using post-irradiation times ranging between 0.5 and 3.5 h. For the highest dose of 0.5 Gy, the number of isochromatid breaks was also scored. No significant differences in the number of chromatid breaks were observed between low-LET gamma-rays and high-LET neutrons for the four donors at any of the doses given. The dose-response curves for the formation of chromatid breaks are linear for both radiation qualities and RBEs = 1 were obtained. Scoring of isochromatid breaks at the highest dose of 0.5 Gy revealed that high-LET neutrons were, however, more effective at inducing isochromatid breaks (RBE = 6.2). The rejoining experiments further showed that the kinetics of disappearance of chromatid breaks following irradiation with low-LET gamma-rays or high-LET neutrons were not significantly different. Half-times of 0.92 h for gamma-rays and 0.84 h for neutrons were obtained. Applying the G2 assay, the results demonstrate that at low doses of irradiation, the induction as well as the disappearance of chromatid breaks is independent of the LET of the radiation qualities used (0.24 keV x microm(-1) 60Co gamma-rays and 20 keV x microm(-1) fast neutrons). As these radiation qualities produce the same initial number of double-strand breaks, the results support the signal model that proposes that chromatid breaks are the result
Kinetics of chromatid break repair in G2-human fibroblasts exposed to low- and high-LET radiations

NASA Technical Reports Server (NTRS)

Kawata, T.; Durante, M.; George, K.; Furusawa, Y.; Gotoh, E.; Takai, N.; Wu, H.; Cucinotta, F. A.

2001-01-01

The purpose of this study is to determine the kinetics of chromatid break rejoining following exposure to radiations of different quality. Exponentially growing human fibroblast cells AG1522 were irradiated with gamma-rays, energetic carbon (290 MeV/u), silicon (490 MeV/u) and iron (200 MeV/u, 600 MeV/u). Chromosomes were prematurely condensed using calyculin A. Prematurely condensed chromosomes were collected after several post-irradiation incubation times, ranging from 5 to 600 minutes, and the number of chromatid breaks and exchanges in G2 cells were scored. The relative biological effectiveness (RBE) for initial chromatid breaks per unit dose showed LET dependency having a peak at 55 keV/micrometers silicon (2.4) or 80 keV/micrometers carbon particles (2.4) and then decreased with increasing LET. The kinetics of chromatid break rejoining following low- or high-LET irradiation consisted of two exponential components. Chromatid breaks decreased rapidly after exposure, and then continued to decrease at a slower rate. The rejoining kinetics was similar for exposure to each type of radiation, although the rate of unrejoined breaks was higher for high-LET radiation. Chromatid exchanges were also formed quickly.
Rejoining of isochromatid breaks induced by heavy ions in G2-phase normal human fibroblasts

NASA Technical Reports Server (NTRS)

Kawata, T.; Durante, M.; Furusawa, Y.; George, K.; Ito, H.; Wu, H.; Cucinotta, F. A.

2001-01-01

We reported previously that exposure of normal human fibroblasts in G2 phase of the cell cycle to high-LET radiation produces a much higher frequency of isochromatid breaks than exposure to gamma rays. We concluded that an increase in the production of isochromatid breaks is a signature of initial high-LET radiation-induced G2-phase damage. In this paper, we report the repair kinetics of isochromatid breaks induced by high-LET radiation in normal G2-phase human fibroblasts. Exponentially growing human fibroblasts (AG1522) were irradiated with gamma rays or energetic carbon (290 MeV/nucleon), silicon (490 MeV/nucleon), or iron (200 MeV/nucleon) ions. Prematurely condensed chromosomes were induced by calyculin A after different postirradiation incubation times ranging from 0 to 600 min. Chromosomes were stained with Giemsa, and aberrations were scored in cells at G2 phase. G2-phase fragments, the result of the induction of isochromatid breaks, decreased quickly with incubation time. The curve for the kinetics of the rejoining of chromatid-type breaks showed a slight upward curvature with time after exposure to 440 keV/microm iron particles, probably due to isochromatid-isochromatid break rejoining. The formation of chromatid exchanges after exposure to high-LET radiation therefore appears to be underestimated, because isochromatid-isochromatid exchanges cannot be detected. Increased induction of isochromatid breaks and rejoining of isochromatid breaks affect the overall kinetics of chromatid-type break rejoining after exposure to high-LET radiation.
G2 Chromatid Damage and Repair Kinetics in Normal Human Fibroblast Cells Exposed to Low-or High-LET Radiation

NASA Technical Reports Server (NTRS)

Kawata, T.; Ito, H.; Uno, T.; Saito, M.; Yamamoto, S.; Furusawa, Y.; Durante, M.; George, K.; Wu, H.; Cucinotta, F. A.

2004-01-01

Radiation-induced chromosome damage can be measured in interphase using the Premature Chromosome Condensation (PCC) technique. With the introduction of a new PCC technique using the potent phosphatase inhibitor calyculin-A, chromosomes can be condensed within five minutes, and it is now possible to examine the early damage induced by radiation. Using this method, it has been shown that high-LET radiation induces a higher frequency of chromatid breaks and a much higher frequency of isochromatid breaks than low-LET radiation. The kinetics of chromatid break rejoining consists of two exponential components representing a rapid and a slow time constant, which appears to be similar for low- and high- LET radiations. However, after high-LET radiation exposures, the rejoining process for isochromatid breaks influences the repair kinetics of chromatid-type breaks, and this plays an important role in the assessment of chromatid break rejoining in the G2 phase of the cell cycle.
Induction by alkylating agents of sister chromatid exchanges and chromatid breaks in Fanconi's anemia.

PubMed

Latt, S A; Stetten, G; Juergens, L A; Buchanan, G R; Gerald, P S

1975-10-01

Sister chromatid exchanges, which may reflect chromosome repair in response to certain types of DNA damage, provide a means of investigating the increased chromosome fragility characteristic of Fanconi's anemia. By a recently developed technique using 33258 Hoechst and 5-bromodeoxyuridine, it was observed that the baseline frequency of sister chromatid exchanges in phytohemagglutinin-stimulated lymphocytes from four males with Fanconi's anemia differed little from that of normal lymphocytes. However, addition of the bifunctional alkylating agent mitomycin C (0.01 or 0.03 mug/ml) to the Fanconi's anemia cells during culture induces less than half of the increase in exchanges found in identically treated normal lymphocytes. This reduced increment in exchanges in accompanied by a partial suppression of mitosis and a marked increase in chromatid breaks and rearrangements. Many of these events occur at sites of incomplete chromatid interchange. The increase in sister chromatid exchanges induced in Fanconi's anemia lymphocytes by the monofunctional alkylating agent ethylmethane sulfonate (0.25 mg/ml) was slightly less than that in normal cells. Lymphocytes from two sets of parents of the patients with Fanconi's anemia exhibited a normal response to alkylating agents, while dermal fibroblasts from two different patients with Fanconi's anemia reacted to mitomycin C with an increase in chromatid breaks, but a nearly normal increment of sister chromatid exchanges. The results suggest that chromosomal breaks and rearrangements in Fanconi's anemia lymphocytes may result from a defect in a form of repair of DNA damage.
High-LET radiation-induced aberrations in prematurely condensed G2 chromosomes of human fibroblasts

NASA Technical Reports Server (NTRS)

Kawata, T.; Gotoh, E.; Durante, M.; Wu, H.; George, K.; Furusawa, Y.; Cucinotta, F. A.; Dicello, J. F. (Principal Investigator)

2000-01-01

PURPOSE: To determine the number of initial chromatid breaks induced by low- or high-LET irradiations, and to compare the kinetics of chromatid break rejoining for radiations of different quality. MATERIAL AND METHODS: Exponentially growing human fibroblast cells AG1522 were irradiated with gamma-rays, energetic carbon (290MeV/u), silicon (490MeV/u) and iron (200 and 600 MeV/u). Chromosomes were prematurely condensed using calyculin A. Chromatid breaks and exchanges in G2 cells were scored. PCC were collected after several post-irradiation incubation times, ranging from 5 to 600 min. RESULTS: The kinetics of chromatid break rejoining following low- or high-LET irradiation consisted of two exponential components representing a rapid and a slow time constant. Chromatid breaks decreased rapidly during the first 10min after exposure, then continued to decrease at a slower rate. The rejoining kinetics were similar for exposure to each type of radiation. Chromatid exchanges were also formed quickly. Compared to low-LET radiation, isochromatid breaks were produced more frequently and the proportion of unrejoined breaks was higher for high-LET radiation. CONCLUSIONS: Compared with gamma-rays, isochromatid breaks were observed more frequently in high-LET irradiated samples, suggesting that an increase in isochromatid breaks is a signature of high-LET radiation exposure.
The Rejoining Time of Chromatid Breaks Induced by Gamma Radiation in Vicia faba Root Tips at 3 °C

PubMed Central

Savage, J. R. K.; Neary, G. J.; Evans, H. J.

1960-01-01

The observation was made previously that the reduction in radiosensitivity in Vicia faba (as measured by postirradiation root growth) by prolonging the exposure time from about 10 minutes to 24 hours is much less marked at 3°C. than at 19°C. If chromosome damage is mainly responsible for the reduced root growth, this observation might be explained by a smaller drop in the "two-hit" aberration component, resulting from an increased time for which breaks are available for rejoining at 3°C. This hypothesis was tested by comparing chromatid aberration frequencies in root meristem cells produced by 105 rads of 60Co γ rays, given at dose rates of 19.4 and 0.073 rads per minute. Beans were maintained in aerated water at 2°C. prior to and during irradiation, and at this temperature the rate of development of cells was such that the two different exposure times both occupied a period during which the cell sensitivity was approximately constant. Immediately subsequent to irradiation, the roots were returned to 19°C. and examined cytologically. All chromatid aberrations were less frequent after low dose rate treatment, but only the chromatid interchange reduction was significant. The average time for which breaks are available for reunion, calculated from Lea's G function, was found to be 12 hours (95 per cent C.L. 6 to 24 hours). PMID:14442001
Progress towards understanding the nature of chromatid breakage.

PubMed

Bryant, P E; Gray, L J; Peresse, N

2004-01-01

The wide range of sensitivities of stimulated T-cells from different individuals to radiation-induced chromatid breakage indicates the involvement of several low penetrance genes that appear to link elevated chromatid breakage to cancer susceptibility. The mechanisms of chromatid breakage are not yet fully understood. However, evidence is accumulating that suggests chromatid breaks are not simply expanded DNA double-strand breaks (DSB). Three models of chromatid breakage are considered. The classical breakage-first and the Revell "exchange" models do not accord with current evidence. Therefore a derivative of Revell's model has been proposed whereby both spontaneous and radiation-induced chromatid breaks result from DSB signaling and rearrangement processes from within large looped chromatin domains. Examples of such rearrangements can be observed by harlequin staining whereby an exchange of strands occurs immediately adjacent to the break site. However, these interchromatid rearrangements comprise less than 20% of the total breaks. The rest are thought to result from intrachromatid rearrangements, including a very small proportion involving complete excision of a looped domain. Work is in progress with the aim of revealing these rearrangements, which may involve the formation of inversions adjacent to the break sites. It is postulated that the disappearance of chromatid breaks with time results from the completion of such rearrangements, rather than from the rejoining of DSB. Elevated frequencies of chromatid breaks occur in irradiated cells with defects in both nonhomologous end-joining (NHEJ) and homologous recombination (HR) pathways, however there is little evidence of a correlation between reduced DSB rejoining and disappearance of chromatid breaks. Moreover, at least one treatment which abrogates the disappearance of chromatid breaks with time leaves DSB rejoining unaffected. The I-SceI DSB system holds considerable promise for the elucidation of these
RPA Mediates Recruitment of MRX to Forks and Double-Strand Breaks to Hold Sister Chromatids Together.

PubMed

Seeber, Andrew; Hegnauer, Anna Maria; Hustedt, Nicole; Deshpande, Ishan; Poli, Jérôme; Eglinger, Jan; Pasero, Philippe; Gut, Heinz; Shinohara, Miki; Hopfner, Karl-Peter; Shimada, Kenji; Gasser, Susan M

2016-12-01

The Mre11-Rad50-Xrs2 (MRX) complex is related to SMC complexes that form rings capable of holding two distinct DNA strands together. MRX functions at stalled replication forks and double-strand breaks (DSBs). A mutation in the N-terminal OB fold of the 70 kDa subunit of yeast replication protein A, rfa1-t11, abrogates MRX recruitment to both types of DNA damage. The rfa1 mutation is functionally epistatic with loss of any of the MRX subunits for survival of replication fork stress or DSB recovery, although it does not compromise end-resection. High-resolution imaging shows that either the rfa1-t11 or the rad50Δ mutation lets stalled replication forks collapse and allows the separation not only of opposing ends but of sister chromatids at breaks. Given that cohesin loss does not provoke visible sister separation as long as the RPA-MRX contacts are intact, we conclude that MRX also serves as a structural linchpin holding sister chromatids together at breaks. Copyright Â© 2016 Elsevier Inc. All rights reserved.
Replication-Dependent Sister Chromatid Recombination in Rad1 Mutants of Saccharomyces Cerevisiae

PubMed Central

Kadyk, L. C.; Hartwell, L. H.

1993-01-01

Homolog recombination and unequal sister chromatid recombination were monitored in rad1-1/rad1-1 diploid yeast cells deficient for excision repair, and in control cells, RAD1/rad1-1, after exposure to UV irradiation. In a rad1-1/rad1-1 diploid, UV irradiation stimulated much more sister chromatid recombination relative to homolog recombination when cells were irradiated in the G(1) or the G(2) phases of the cell cycle than was observed in RAD1/rad1-1 cells. Since sister chromatids are not present during G(1), this result suggested that unexcised lesions can stimulate sister chromatid recombination events during or subsequent to DNA replication. The results of mating rescue experiments suggest that unexcised UV dimers do not stimulate sister chromatid recombination during the G(2) phase, but only when they are present during DNA replication. We propose that there are two types of sister chromatid recombination in yeast. In the first type, unexcised UV dimers and other bulky lesions induce sister chromatid recombination during DNA replication as a mechanism to bypass lesions obstructing the passage of DNA polymerase, and this type is analogous to the type of sister chromatid exchange commonly observed cytologically in mammalian cells. In the second type, strand scissions created by X-irradiation or the excision of damaged bases create recombinogenic sites that result in sister chromatid recombination directly in G(2). Further support for the existence of two types of sister chromatid recombination is the fact that events induced in rad1-1/rad1-1 were due almost entirely to gene conversion, whereas those in RAD1/rad1-1 cells were due to a mixture of gene conversion and reciprocal recombination. PMID:8454200
Radiation induces premature chromatid separation via the miR-142-3p/Bod1 pathway in carcinoma cells.

PubMed

Pan, Dong; Du, Yarong; Ren, Zhenxin; Chen, Yaxiong; Li, Xiaoman; Wang, Jufang; Hu, Burong

2016-09-13

Radiation-induced genomic instability plays a vital role in carcinogenesis. Bod1 is required for proper chromosome biorientation, and Bod1 depletion increases premature chromatid separation. MiR-142-3p influences cell cycle progression and inhibits proliferation and invasion in cervical carcinoma cells. We found that radiation induced premature chromatid separation and altered miR-142-3p and Bod1 expression in 786-O and A549 cells. Overexpression of miR-142-3p increased premature chromatid separation and G2/M cell cycle arrest in 786-O cells by suppressing Bod1 expression. We also found that either overexpression of miR-142-3p or knockdown of Bod1 sensitized 786-O and A549 cells to X-ray radiation. Overexpression of Bod1 inhibited radiation- and miR-142-3p-induced premature chromatid separation and increased resistance to radiation in 786-O and A549 cells. Taken together, these results suggest that radiation alters miR-142-3p and Bod1 expression in carcinoma cells, and thus contributes to early stages of radiation-induced genomic instability. Combining ionizing radiation with epigenetic regulation may help improve cancer therapies.
Enhanced G2 chromatid radiosensitivity, an early stage in the neoplastic transformation of human epidermal keratinocytes in culture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gantt, R.; Sanford, K.K.; Parshad, R.

1987-03-01

A deficiency in DNA repair, manifest as enhanced chromatid radiosensitivity during the G2 phase of the cell cycle, together with a proliferative stimulus such as that provided by active oncogenes may be necessary and sufficient for the malignant neoplastic transformation of human keratinocytes in culture. Normal epidermal keratinocytes established as continuous cell lines by transfection with pSV3-neo or infection with adeno 12-SV40 hybrid virus developed enhanced G2 chromatid radiosensitivity after 18 passages in culture. In contrast to cells from primary or secondary culture, these cells could be transformed to malignant neoplastic cells by infection with Kirsten murine sarcoma virus containingmore » the Ki-ras oncogene or in one line by the chemical carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine; both of these agents produced a marked proliferative response. Cytological heterogeneity and karyotypic instability characterized the cells during their progression to neoplasia. These results are interpreted in terms of a mechanism for neoplastic transformation.« less
Induction of chromatin damage and distribution of isochromatid breaks in human fibroblast cells exposed to heavy ions

NASA Technical Reports Server (NTRS)

Kawata, Tetsuya; Ito, Hisao; Motoori, Ken; Ueda, Takuya; Shigematsu, Naoyuki; Furusawa, Yoshiya; Durante, Marco; George, Kerry; Wu, Honglu; Cucinotta, Francis A.

2002-01-01

The frequency of chromatid breaks and the distribution of isochromatid breaks were measured in G2-phase normal human fibroblasts prematurely condensed a short time after exposure to low- or high-LET radiations. The average number of isochromatid breaks from a single particle traversal increased with increasing LET values, while the average number of chromatid-type breaks appeared to reach a plateau. The distribution of isochromatid breaks after high-LET iron particles exposure was overdispersed compared to gamma-rays, indicating that a single iron particle traversal through a cell nucleus can produce multiple isochromatid breaks.
Induction of chromatin damage and distribution of isochromatid breaks in human fibroblast cells exposed to heavy ions.

PubMed

Kawata, Tetsuya; Ito, Hisao; Motoori, Ken; Ueda, Takuya; Shigematsu, Naoyuki; Furusawa, Yoshiya; Durante, Marco; George, Kerry; Wu, Honglu; Cucinotta, Francis A

2002-12-01

The frequency of chromatid breaks and the distribution of isochromatid breaks were measured in G2-phase normal human fibroblasts prematurely condensed a short time after exposure to low- or high-LET radiations. The average number of isochromatid breaks from a single particle traversal increased with increasing LET values, while the average number of chromatid-type breaks appeared to reach a plateau. The distribution of isochromatid breaks after high-LET iron particles exposure was overdispersed compared to gamma-rays, indicating that a single iron particle traversal through a cell nucleus can produce multiple isochromatid breaks.
Faithful anaphase is ensured by Mis4, a sister chromatid cohesion molecule required in S phase and not destroyed in G1 phase

PubMed Central

Furuya, Kanji; Takahashi, Kohta; Yanagida, Mitsuhiro

1998-01-01

The loss of sister chromatid cohesion triggers anaphase spindle movement. The budding yeast Mcd1/Scc1 protein, called cohesin, is required for associating chromatids, and proteins homologous to it exist in a variety of eukaryotes. Mcd1/Scc1 is removed from chromosomes in anaphase and degrades in G1. We show that the fission yeast protein, Mis4, which is required for equal sister chromatid separation in anaphase is a different chromatid cohesion molecule that behaves independent of cohesin and is conserved from yeast to human. Its inactivation in G1 results in cell lethality in S phase and subsequent premature sister chromatid separation. Inactivation in G2 leads to cell death in subsequent metaphase–anaphase progression but missegregation occurs only in the next round of mitosis. Mis4 is not essential for condensation, nor does it degrade in G1. Rather, it associates with chromosomes in a punctate fashion throughout the cell cycle. mis4 mutants are hypersensitive to hydroxyurea (HU) and UV irradiation but retain the ability to restrain cell cycle progression when damaged or sustaining a block to replication. The mis4 mutation results in synthetic lethality with a DNA ligase mutant. Mis4 may form a stable link between chromatids in S phase that is split rather than removed in anaphase. PMID:9808627
Histone H3 K79 methylation states play distinct roles in UV-induced sister chromatid exchange and cell cycle checkpoint arrest in Saccharomyces cerevisiae

PubMed Central

Rossodivita, Alyssa A.; Boudoures, Anna L.; Mecoli, Jonathan P.; Steenkiste, Elizabeth M.; Karl, Andrea L.; Vines, Eudora M.; Cole, Arron M.; Ansbro, Megan R.; Thompson, Jeffrey S.

2014-01-01

Histone post-translational modifications have been shown to contribute to DNA damage repair. Prior studies have suggested that specific H3K79 methylation states play distinct roles in the response to UV-induced DNA damage. To evaluate these observations, we examined the effect of altered H3K79 methylation patterns on UV-induced G1/S checkpoint response and sister chromatid exchange (SCE). We found that the di- and trimethylated states both contribute to activation of the G1/S checkpoint to varying degrees, depending on the synchronization method, although methylation is not required for checkpoint in response to high levels of UV damage. In contrast, UV-induced SCE is largely a product of the trimethylated state, which influences the usage of gene conversion versus popout mechanisms. Regulation of H3K79 methylation by H2BK123 ubiquitylation is important for both checkpoint function and SCE. H3K79 methylation is not required for the repair of double-stranded breaks caused by transient HO endonuclease expression, but does play a modest role in survival from continuous exposure. The overall results provide evidence for the participation of H3K79 methylation in UV-induced recombination repair and checkpoint activation, and further indicate that the di- and trimethylation states play distinct roles in these DNA damage response pathways. PMID:24748660
Complex chromatid-isochromatid exchanges following irradiation with heavy ions?

PubMed

Loucas, B D; Eberle, R L; Durante, M; Cornforth, M N

2004-01-01

We describe a peculiar and relatively rare type of chromosomal rearrangement induced in human peripheral lymphocytes that were ostensibly irradiated in G(0) phase of the cell cycle by accelerated heavy ions, and which, to the best of our knowledge, have not been previously described. The novel rearrangements which were detected using mFISH following exposure to 500 MeV/nucleon and 5 GeV/n 56Fe particles, but were not induced by either 137Cs gamma rays or 238Pu alpha particles, can alternatively be described as either complex chromatid-isochromatid or complex chromatid-chromosome exchanges. Different mechanisms potentially responsible for their formation are discussed. Copyright 2003 S. Karger AG, Basel
Cut2 proteolysis required for sister-chromatid seperation in fission yeast.

PubMed

Funabiki, H; Yamano, H; Kumada, K; Nagao, K; Hunt, T; Yanagida, M

1996-05-30

Although mitotic cyclins are well-known substrates for ubiquitin-mediated proteolysis at the metaphase-anaphase transition, their degradation is not essential for separation of sister chromatids; several lines of evidence suggest that proteolysis of other protein(s) is required, however. Here we report the anaphase-specific proteolysis of the Schizosaccharomyces pombe Cut2 protein, which is essential for sister-chromatid separation. Cut2 is located in the nucleus, where it is concentrated along the short metaphase spindle. The rapid degradation of Cut2 at anaphase requires its amino-terminal region and the activity of Cut9 (ref. 14), a component of the 20S cyclosome/anaphase-promoting complex (APC), which is necessary for cyclin destruction. Expression of non-degradable Cut2 blocks sister-chromatid separation but not cell-cycle progression. This defect can be overcome by grafting the N terminus of cyclin B onto the truncated Cut2, demonstrating that the regulated proteolysis of Cut2 is essential for sister-chromatid separation.

Ultraviolet-induced sister chromatid exchanges in V-79 cells with normal and BrdUrd-substituted DNA and the influence of intercalating substances and cysteine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Speit, G.; Mehnert, K.; Wolf, M.

1982-06-01

The influence of intercalating substances (proflavine, ethidium bromide) and of an SH compound (L-cysteine) on uv-induced sister chromatid exchanges (SCEs) was investigated in V-79 cells with normal and BrdUrd-substituted DNA. The results are discussed in relation to the primary damages leading to SCE induction produced by uv irradiation. The data indicate that neither the pyrimidine dimers nor DNA single-strand breaks are the primary cause of SCE induction, and that the damages leading to SCEs by uv irradiation differ from those which cause chromosome aberrations.
Double strand breaks and cell-cycle arrest induced by the cyanobacterial toxin cylindrospermopsin in HepG2 cells.

PubMed

Alja, Štraser; Filipič, Metka; Novak, Matjaž; Žegura, Bojana

2013-08-21

The newly emerging cyanobacterial cytotoxin cylindrospermopsin (CYN) is increasingly found in surface freshwaters, worldwide. It poses a potential threat to humans after chronic exposure as it was shown to be genotoxic in a range of test systems and is potentially carcinogenic. However, the mechanisms of CYN toxicity and genotoxicity are not well understood. In the present study CYN induced formation of DNA double strand breaks (DSBs), after prolonged exposure (72 h), in human hepatoma cells, HepG2. CYN (0.1-0.5 µg/mL, 24-96 h) induced morphological changes and reduced cell viability in a dose and time dependent manner. No significant increase in lactate dehydrogenase (LDH) leakage could be observed after CYN exposure, indicating that the reduction in cell number was due to decreased cell proliferation and not due to cytotoxicity. This was confirmed by imunocytochemical analysis of the cell-proliferation marker Ki67. Analysis of the cell-cycle using flow-cytometry showed that CYN has an impact on the cell cycle, indicating G0/G1 arrest after 24 h and S-phase arrest after longer exposure (72 and 96 h). Our results provide new evidence that CYN is a direct acting genotoxin, causing DSBs, and these facts need to be considered in the human health risk assessment.
Chromosomal Integrity after UV Irradiation Requires FANCD2-Mediated Repair of Double Strand Breaks.

PubMed

Federico, María Belén; Vallerga, María Belén; Radl, Analía; Paviolo, Natalia Soledad; Bocco, José Luis; Di Giorgio, Marina; Soria, Gastón; Gottifredi, Vanesa

2016-01-01

Fanconi Anemia (FA) is a rare autosomal recessive disorder characterized by hypersensitivity to inter-strand crosslinks (ICLs). FANCD2, a central factor of the FA pathway, is essential for the repair of double strand breaks (DSBs) generated during fork collapse at ICLs. While lesions different from ICLs can also trigger fork collapse, the contribution of FANCD2 to the resolution of replication-coupled DSBs generated independently from ICLs is unknown. Intriguingly, FANCD2 is readily activated after UV irradiation, a DNA-damaging agent that generates predominantly intra-strand crosslinks but not ICLs. Hence, UV irradiation is an ideal tool to explore the contribution of FANCD2 to the DNA damage response triggered by DNA lesions other than ICL repair. Here we show that, in contrast to ICL-causing agents, UV radiation compromises cell survival independently from FANCD2. In agreement, FANCD2 depletion does not increase the amount of DSBs generated during the replication of UV-damaged DNA and is dispensable for UV-induced checkpoint activation. Remarkably however, FANCD2 protects UV-dependent, replication-coupled DSBs from aberrant processing by non-homologous end joining, preventing the accumulation of micronuclei and chromatid aberrations including non-homologous chromatid exchanges. Hence, while dispensable for cell survival, FANCD2 selectively safeguards chromosomal stability after UV-triggered replication stress.
Genes on chromosomes 1 and 4 in the mouse are associated with repair of radiation-induced chromatin damage.

PubMed

Potter, M; Sanford, K K; Parshad, R; Tarone, R E; Price, F M; Mock, B; Huppi, K

1988-04-01

Early-passage skin fibroblasts from different inbred and congenic strains of mice were X-irradiated (1 Gy), and the number of chromatid breaks was determined at 2.0 h after irradiation. The cells from DBA/2N, C3H/HeN, STS/A, C57BL/6N, BALB/cJ, and AKR/N had 25 to 42 chromatid breaks per 100 metaphase cells (efficient repair phenotype). NZB/NJ had greater than 78 and BALB/cAn had 87 to 110 chromatid breaks per 100 cells (inefficient repair phenotype). Differences between BALB/cAn and BALB/c. DBA/2 congenic strains which carry less than 1% of the DBA/2 genome indicate that two genes, one on chromosome 1 linked to bcl-2-Pep-3 and the other on chromosome 4 closely linked to Fv-1, affect the efficiency with which the cells repair radiation-induced chromatin damage.
Mitomycin C-induced pairing of heterochromatin reflects initiation of DNA repair and chromatid exchange formation.

PubMed

Abdel-Halim, H I; Natarajan, A T; Mullenders, L H F; Boei, J J W A

2005-04-15

Chromatid interchanges induced by the DNA cross-linking agent mitomycin C (MMC) are over-represented in human chromosomes containing large heterochromatic regions. We found that nearly all exchange breakpoints of chromosome 9 are located within the paracentromeric heterochromatin and over 70% of exchanges involving chromosome 9 are between its homologues. We provide evidence that the required pairing of chromosome 9 heterochromatic regions occurs in G(0)/G(1) and S-phase cells as a result of an active cellular process initiated upon MMC treatment. By contrast, no pairing was observed for a euchromatic paracentromeric region of the equal-sized chromosome 8. The MMC-induced pairing of chromosome 9 heterochromatin is observed in a subset of cells; its percentage closely mimics the frequency of homologous interchanges found at metaphase. Moreover, the absence of pairing in cells derived from XPF patients correlates with an altered spectrum of MMC-induced exchanges. Together, the data suggest that the heterochromatin-specific pairing following MMC treatment reflects the initiation of DNA cross-link repair and the formation of exchanges.
INDUCTION OF DNA STRAND BREAKS BY TRIHALOMETHANES IN PRIMARY HUMAN LUNG EPITHELIAL CELLS

EPA Science Inventory

Abstract

Trihalomethanes (TEMs) are disinfection by-products and suspected human carcinogens present in chlorinated drinking water. Previous studies have shown that many THMs induce sister chromatid exchanges and DNA strand breaks in human peripheral blood lymphocyte...
Genes on chromosomes 1 and 4 in the mouse are associated with repair of radiation-induced chromatin damage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Potter, M.; Sanford, K.K.; Parshad, R.

Early-passage skin fibroblasts from different inbred and congenic strains of mice were X-irradiated (1 Gy), and the number of chromatid breaks was determined at 2.0 h after irradiation. The cells from DBA/2N, C3H/HeN, STS/A, C57BL/6N, BALB/cJ, and AKR/N had 25 to 42 chromatid breaks per 100 metaphase cells (efficient repair phenotype). NZB/NJ had greater than 78 and BALB/cAn had 87 to 110 chromatid breaks per 100 cells (inefficient repair phenotype). Differences between BALB/cAn and BALB/c. DBA/2 congenic strains which carry less than 1% of the DBA/2 genome indicate that two genes, one on chromosome 1 linked to bcl-2-Pep-3 and themore » other on chromosome 4 closely linked to Fv-1, affect the efficiency with which the cells repair radiation-induced chromatin damage.« less
"Breaking up is hard to do": the formation and resolution of sister chromatid intertwines.

PubMed

Baxter, Jonathan

2015-02-13

The absolute necessity to resolve every intertwine between the two strands of the DNA double helix provides a massive challenge to the cellular processes that duplicate and segregate chromosomes. Although the overwhelming majority of intertwines between the parental DNA strands are resolved during DNA replication, there are numerous chromosomal contexts where some intertwining is maintained into mitosis. These mitotic sister chromatid intertwines (SCIs) can be found as; short regions of unreplicated DNA, fully replicated and intertwined sister chromatids--commonly referred to as DNA catenation--and as sister chromatid linkages generated by homologous recombination-associated processes. Several overlapping mechanisms, including intra-chromosomal compaction, topoisomerase action and Holliday junction resolvases, ensure that all SCIs are removed before they can prevent normal chromosome segregation. Here, I discuss why some DNA intertwines persist into mitosis and review our current knowledge of the SCI resolution mechanisms that are employed in both prokaryotes and eukaryotes, including how deregulating SCI formation during DNA replication or disrupting the resolution processes may contribute to aneuploidy in cancer. Copyright © 2014 Elsevier Ltd. All rights reserved.
Chromosomal Integrity after UV Irradiation Requires FANCD2-Mediated Repair of Double Strand Breaks

PubMed Central

Federico, María Belén; Vallerga, María Belén; Radl, Analía; Paviolo, Natalia Soledad; Bocco, José Luis; Di Giorgio, Marina; Soria, Gastón; Gottifredi, Vanesa

2016-01-01

Fanconi Anemia (FA) is a rare autosomal recessive disorder characterized by hypersensitivity to inter-strand crosslinks (ICLs). FANCD2, a central factor of the FA pathway, is essential for the repair of double strand breaks (DSBs) generated during fork collapse at ICLs. While lesions different from ICLs can also trigger fork collapse, the contribution of FANCD2 to the resolution of replication-coupled DSBs generated independently from ICLs is unknown. Intriguingly, FANCD2 is readily activated after UV irradiation, a DNA-damaging agent that generates predominantly intra-strand crosslinks but not ICLs. Hence, UV irradiation is an ideal tool to explore the contribution of FANCD2 to the DNA damage response triggered by DNA lesions other than ICL repair. Here we show that, in contrast to ICL-causing agents, UV radiation compromises cell survival independently from FANCD2. In agreement, FANCD2 depletion does not increase the amount of DSBs generated during the replication of UV-damaged DNA and is dispensable for UV-induced checkpoint activation. Remarkably however, FANCD2 protects UV-dependent, replication-coupled DSBs from aberrant processing by non-homologous end joining, preventing the accumulation of micronuclei and chromatid aberrations including non-homologous chromatid exchanges. Hence, while dispensable for cell survival, FANCD2 selectively safeguards chromosomal stability after UV-triggered replication stress. PMID:26765540
Effect of borax on immune cell proliferation and sister chromatid exchange in human chromosomes

PubMed Central

Pongsavee, Malinee

2009-01-01

Background Borax is used as a food additive. It becomes toxic when accumulated in the body. It causes vomiting, fatigue and renal failure. Methods The heparinized blood samples from 40 healthy men were studied for the impact of borax toxicity on immune cell proliferation (lymphocyte proliferation) and sister chromatid exchange in human chromosomes. The MTT assay and Sister Chromatid Exchange (SCE) technic were used in this experiment with the borax concentrations of 0.1, 0.15, 0.2, 0.3 and 0.6 mg/ml. Results It showed that the immune cell proliferation (lymphocyte proliferation) was decreased when the concentrations of borax increased. The borax concentration of 0.6 mg/ml had the most effectiveness to the lymphocyte proliferation and had the highest cytotoxicity index (CI). The borax concentrations of 0.15, 0.2, 0.3 and 0.6 mg/ml significantly induced sister chromatid exchange in human chromosomes (P < 0.05). Conclusion Borax had effects on immune cell proliferation (lymphocyte proliferation) and induced sister chromatid exchange in human chromosomes. Toxicity of borax may lead to cellular toxicity and genetic defect in human. PMID:19878537
Sister chromatid exchanges induced by inhaled anesthetics

DOE Office of Scientific and Technical Information (OSTI.GOV)

White,A.E.; Takehisa, S.; Eger II, E.I.

1970-05-01

There is sufficient evidence that anesthetics may cause cancer to justify a test of their carcinogenic potential. Baden et al., using the Ames test, a rapid and inexpensive genetic indicator of carcinogenicity, have shown that among currently used anesthetics fluorxene alone caused bacterial mutations. The authors used the sister chromatid exchange (SCE) technique, another rapid assay of mutagenic-carcinogenic potential. The frequency of sister chromatid exchanges in Chinese hamster ovary cells increases when the cell cultures are exposed to mutagen-carcinogens, particulary in the presence of a metabolic activating system. With this test system a one-hour exposure to 1 MAC nitrous oxide,more » diethyl ether, trichloroethylene, halothane, enflurane, isoflurane, methoxyflurane, or chloroform did not increase SCE values. Divinyl ether, fluroxene and ethyl vinyl ether increased SCE values in the same circumstances. Results of this study of mammalian cells suggest that no currently used anesthetic is a mutagen-carcinogen. The results also suggest that anesthetics containing a vinyl moiety may be mutagen-carcinogens.« less
Zinc Chromate Induces Chromosome Instability and DNA Double Strand Breaks in Human Lung Cells

PubMed Central

Xie, Hong; Holmes, Amie L.; Young, Jamie L.; Qin, Qin; Joyce, Kellie; Pelsue, Stephen C.; Peng, Cheng; Wise, Sandra S.; Jeevarajan, Antony S.; Wallace, William T.; Hammond, Dianne; Wise, John Pierce

2014-01-01

Hexavalent chromium Cr(VI) is a respiratory toxicant and carcinogen, with solubility playing an important role in its carcinogenic potential. Zinc chromate, a water insoluble or ‘particulate’ Cr(VI) compound, has been shown to be carcinogenic in epidemiology studies and to induce tumors in experimental animals, but its genotoxicity is poorly understood. Our study shows that zinc chromate induced concentration-dependent increases in cytotoxicity, chromosome damage and DNA double strand breaks in human lung cells. In response to zinc chromate-induced breaks, MRE11 expression was increased and ATM and ATR were phosphorylated, indicating that the DNA double strand break repair system was initiated in the cells. In addition, our data show that zinc chromate-induced double strand breaks were only observed in the G2/M phase population, with no significant amount of double strand breaks observed in G1 and S phase cells. These data will aid in understanding the mechanisms of zinc chromate toxicity and carcinogenesis. PMID:19027772
Genotoxic assessment in peripheral blood lymphocytes of post-polio individuals using sister chromatid exchange analysis and micronucleus assay.

PubMed

Bhattacharya, Saurabh Kumar; Saraswathy, Radha; Sivakumar, E

2011-07-01

Environmental pollution is a complex issue because of the diversity of anthropogenic agents, both chemical and physical, that have been detected and catalogued. The consequences to biota from exposure to genotoxic agents present an additional problem because of the potential for these agents to produce adverse change at the cellular and organism levels. Past studies in virus have focused on structural damage to the DNA of environmental species that may occur after exposure to genotoxic agents and the use of this information to document exposure and to monitor remediation. In an effort to predict effects at the population, community and ecosystem levels, in the present study, we attempt to characterize damage occurring through genotoxic agents like 5-bromo-2-deoxyuridine, BrdU, using sister chromatid exchange technique and the formation of micronuclei (MN) in the peripheral lymphocytes of the post-polio syndrome sequelae affected by poliovirus. Analysis of structural chromosomal aberrations (CAs) and involvement of the specific chromosome break were pursued in this study. They revealed a significantly higher incidence of CAs (chromatid and chromosome breaks) in patients compared with controls, where the specific chromosome break has emerged as specific. Also, the maximum numbers of breaks were found to be in chromosome 1 at the position 1p36.1. The results also suggest a correlation between CAs and content of MN.
6-gingerol prevents patulin-induced genotoxicity in HepG2 cells.

PubMed

Yang, Guang; Zhong, Laifu; Jiang, Liping; Geng, Chengyan; Cao, Jun; Sun, Xiance; Liu, Xiaofang; Chen, Min; Ma, Yufang

2011-10-01

Patulin (PAT) is a mycotoxin produced by several Penicillium, Aspergillus and Byssochlamys species. Since PAT is a potent genotoxic compound, and PAT contamination is common in fruits and fruit products, the search for newer, better agents for protection against genotoxicity of PAT is required. In this study, the chemoprotective effect of 6-gingerol against PAT-induced genotoxicity in HepG2 cells was investigated. The comet assay and micronucleus test (MNT) were used to monitor genotoxic effects. To further elucidate the underlying mechanisms, the intracellular generation of reactive oxygen species (ROS) and level of reduced glutathione (GSH) were tested. In addition, the level of oxidative DNA damage was evaluated by immunocytochemical analysis of 8-hydroxydeoxyguanosine (8-OHdG). The results showed that 6-gingerol significantly reduced the DNA strand breaks and micronuclei formation caused by PAT. Moreover, 6-gingerol effectively suppressed PAT-induced intracellular ROS formation and 8-OHdG level. The GSH depletion induced by PAT in HepG2 cells was also attenuated by 6-gingerol pretreatment. These findings suggest that 6-gingerol has a strong protective ability against the genotoxicity caused by PAT, and the antioxidant activity of 6-gingerol may play an important part in attenuating the genotoxicity of PAT. Copyright © 2011 John Wiley & Sons, Ltd.
Defects in the Fanconi Anemia Pathway and Chromatid Cohesion in Head and Neck Cancer.

PubMed

Stoepker, Chantal; Ameziane, Najim; van der Lelij, Petra; Kooi, Irsan E; Oostra, Anneke B; Rooimans, Martin A; van Mil, Saskia E; Brink, Arjen; Dietrich, Ralf; Balk, Jesper A; Ylstra, Bauke; Joenje, Hans; Feller, Stephan M; Brakenhoff, Ruud H

2015-09-01

Failure to repair DNA damage or defective sister chromatid cohesion, a process essential for correct chromosome segregation, can be causative of chromosomal instability (CIN), which is a hallmark of many types of cancers. We investigated how frequent this occurs in head and neck squamous cell carcinoma (HNSCC) and whether specific mechanisms or genes could be linked to these phenotypes. The genomic instability syndrome Fanconi anemia is caused by mutations in any of at least 16 genes regulating DNA interstrand crosslink (ICL) repair. Since patients with Fanconi anemia have a high risk to develop HNSCC, we investigated whether and to which extent Fanconi anemia pathway inactivation underlies CIN in HNSCC of non-Fanconi anemia individuals. We observed ICL-induced chromosomal breakage in 9 of 17 (53%) HNSCC cell lines derived from patients without Fanconi anemia. In addition, defective sister chromatid cohesion was observed in five HNSCC cell lines. Inactivation of FANCM was responsible for chromosomal breakage in one cell line, whereas in two other cell lines, somatic mutations in PDS5A or STAG2 resulted in inadequate sister chromatid cohesion. In addition, FANCF methylation was found in one cell line by screening an additional panel of 39 HNSCC cell lines. Our data demonstrate that CIN in terms of ICL-induced chromosomal breakage and defective chromatid cohesion is frequently observed in HNSCC. Inactivation of known Fanconi anemia and chromatid cohesion genes does explain CIN in the minority of cases. These findings point to phenotypes that may be highly relevant in treatment response of HNSCC. ©2015 American Association for Cancer Research.
Frequency of sister chromatid exchange and chromosomal aberrations in asbestos cement workers.

PubMed

Fatma, N; Jain, A K; Rahman, Q

1991-02-01

Exposure to asbestos minerals has been associated with a wide variety of adverse health effects including lung cancer, pleural mesothelioma, and cancer of other organs. It was shown previously that asbestos samples collected from a local asbestos factory enhanced sister chromatid exchanges (SCEs) and chromosomal aberrations in vitro using human lymphocytes. In the present study, 22 workers from the same factory and 12 controls were further investigated. Controls were matched for age, sex, and socioeconomic state. The peripheral blood lymphocytes were cultured and harvested at 48 hours for studies of chromosomal aberrations and at 72 hours for SCE frequency determinations. Asbestos workers had a raised mean SCE rate and increased numbers of chromosomal aberrations compared with a control population. Most of the chromosomal aberrations were chromatid gap and break types.
Histone hyperacetylation during meiosis interferes with large-scale chromatin remodeling, axial chromatid condensation and sister chromatid separation in the mammalian oocyte.

PubMed

Yang, Feikun; Baumann, Claudia; Viveiros, Maria M; De La Fuente, Rabindranath

2012-01-01

Histone acetylation regulates higher-order chromatin structure and function and is critical for the control of gene expression. Histone deacetylase inhibitors (HDACi) are currently under investigation as novel cancer therapeutic drugs. Here, we show that female germ cells are extremely susceptible to chromatin changes induced by HDACi. Our results indicate that exposure to trichostatin A (TSA) at nanomolar levels interferes with major chromatin remodeling events in the mammalian oocyte leading to chromosome instability. High resolution analysis of chromatin structure and live-cell imaging revealed a striking euchromatin decondensation associated with histone H4 hyperacetylation following exposure to 15 nM TSA in >90% of pre-ovulatory oocytes. Dynamic changes in large-scale chromatin structure were detected after 2 h of exposure and result in the formation of misaligned chromosomes in >75% (P<0.05) of in vitro matured oocytes showing chromosome lagging as well as abnormal sister chromatid separation at anaphase I. Abnormal axial chromatid condensation during meiosis results in the formation of elongated chromosomes exhibiting hyperacetylation of histone H4 at lysine 5 and lysine 16 at interstitial chromosome segments, but not pericentric heterochromatin, while highly decondensed bivalents exhibit prominent histone H3 phosphorylation at centromeric domains. Notably, no changes were observed in the chromosomal localization of the condensin protein SMC4. These results indicate that HDAC activity is required for proper chromosome condensation in the mammalian oocyte and that HDACi may induce abnormal chromosome segregation by interfering with both chromosome-microtubule interactions, as well as sister chromatid separation. Thus, HDACi, proposed for cancer therapy, may disrupt the epigenetic status of female germ cells, predisposing oocytes to aneuploidy at previously unrecognized low doses.
Colchicine promotes a change in chromosome structure without loss of sister chromatid cohesion in prometaphase I-arrested bivalents.

PubMed

Rodríguez, E M; Parra, M T; Rufas, J S; Suja, J A

2001-12-01

In somatic cells colchicine promotes the arrest of cell division at prometaphase, and chromosomes show a sequential loss of sister chromatid arm and centromere cohesion. In this study we used colchicine to analyse possible changes in chromosome structure and sister chromatid cohesion in prometaphase I-arrested bivalents of the katydid Pycnogaster cucullata. After silver staining we observed that in colchicine-arrested prometaphase I bivalents, and in contrast to what was found in control bivalents, sister kinetochores appeared individualised and sister chromatid axes were completely separated all along their length. However, this change in chromosome structure occurred without loss of sister chromatid arm cohesion. We also employed the MPM-2 monoclonal antibody against mitotic phosphoproteins on control and colchicine-treated spermatocytes. In control metaphase I bivalents this antibody labelled the tightly associated sister kinetochores and the interchromatid domain. By contrast, in colchicine-treated prometaphase I bivalents individualised sister kinetochores appeared labelled, but the interchromatid domain did not show labelling. These results support the notion that MPM-2 phosphoproteins, probably DNA topoisomerase IIalpha, located in the interchromatid domain act as "chromosomal staples" associating sister chromatid axes in metaphase I bivalents. The disappearance of these chromosomal staples would induce a change in chromosome structure, as reflected by the separation of sister kinetochores and sister axes, but without a concomitant loss of sister chromatid cohesion.
Cyclophosphamide induced in vivo sister chromatid exchanges (SCE) in Mus musculus. I: Strain differences and empirical association with relative chromosome size.

PubMed

Reimer, D L; Singh, S M

1982-01-01

The inducibility of sister chromatid exchanges (SCEs) by cyclophosphamide (CP) in bone marrow cells was evaluated in vivo in the three genetic strains of mice (C3H/s, C57BL/6J, and Balb/c). Female mice (10 to 12 wks old, mean = 22.9g, SD = 3.2g) were administered with nine hourly injections of 214.19 mg/kg 5-Bromo-2' deoxyuridine (BrdU) followed by 0, 0.048, 0.449, 4.585 or 46.93 mg/kg CP and 4 mg/kg colcemid. SCEs were evaluated following differential staining procedures of Perry and Wolff (1974). The base-line SCEs were similar in all strains with about ten SCEs/cell. Increasing CP concentrations yielded an increased level of SCEs. Most cells showed extensive damage in CP doses exceeding 4.55 mg/kg. No SCE evaluation was possible beyond this concentration. Strain differences were evident at every dose of CP, and Balb/c was the least susceptible strain to SCE induction. F1 hybrids involving C3H/s female and Balb/c male showed SCE values closer to Balb/c. Data on the association between chromosome length and frequency of SCEs are provided. They empirically establish a positive correlation (r = 0.90) between the two features. Most induced SCEs were interstitially located rather than terminally positioned on the chromosome.
Cell killing and chromatid damage in primary human bronchial epithelial cells irradiated with accelerated 56Fe ions

NASA Technical Reports Server (NTRS)

Suzuki, M.; Piao, C.; Hall, E. J.; Hei, T. K.

2001-01-01

We examined cell killing and chromatid damage in primary human bronchial epithelial cells irradiated with high-energy 56Fe ions. Cells were irradiated with graded doses of 56Fe ions (1 GeV/nucleon) accelerated with the Alternating Gradient Synchrotron at Brookhaven National Laboratory. The survival curves for cells plated 1 h after irradiation (immediate plating) showed little or no shoulder. However, the survival curves for cells plated 24 h after irradiation (delayed plating) had a small initial shoulder. The RBE for 56Fe ions compared to 137Cs gamma rays was 1.99 for immediate plating and 2.73 for delayed plating at the D10. The repair ratio (delayed plating/immediate plating) was 1.67 for 137Cs gamma rays and 1.22 for 56Fe ions. The dose-response curves for initially measured and residual chromatid fragments detected by the Calyculin A-mediated premature chromosome condensation technique showed a linear response. The results indicated that the induction frequency for initially measured fragments was the same for 137Cs gamma rays and 56Fe ions. On the other hand, approximately 85% of the fragments induced by 137Cs gamma rays had rejoined after 24 h of postirradiation incubation; the corresponding amount for 56Fe ions was 37%. Furthermore, the frequency of chromatid exchanges induced by gamma rays measured 24 h after irradiation was higher than that induced by 56Fe ions. No difference in the amount of chromatid damage induced by the two types of radiations was detected when assayed 1 h after irradiation. The results suggest that high-energy 56Fe ions induce a higher frequency of complex, unrepairable damage at both the cellular and chromosomal levels than 137Cs gamma rays in the target cells for radiation-induced lung cancers.