Radiation-induced cytogenetic damage in relation to changes in interphase chromosome conformation

The premature chromosome condensation (PCC) technique was used to study several factors that determine the yield of chromosome fragments as observed in interphase cells after irradiation. In addition to absorbed dose and the extent of chromosome condensation at the time of irradiation, changes in chromosome conformation as cells progressed through the cell cycle after irradiation affected dramatically the yield of chromosome fragments observed. As a test of the effect of chromosome decondensation, irradiated metaphase Chinese hamster ovary (CHO) cells were allowed to divide, and the prematurely condensed chromosomes in the daughter cells were analyzed in their G1 phase. The yield of chromosome fragments increased as the daughter cells progressed toward S phase and chromosome decondensation occurred. When early G1 CHO cells were irradiated and analyzed at later times in G1 phase, an increase in chromosome fragmentation again followed the gradual increase in chromosome decondensation. As a test of the effect of chromosome condensation, G0 human lymphocytes were irradiated and analyzed at various times after fusion with mitotic CHO cells, i.e., as condensation proceeded. The yield of fragments observed was directly related to the amount of chromosome condensation allowed to take place after irradiation and inversely related to the extent of chromosome condensation at the time of irradiation. It can be concluded that changes in chromosome conformation interfered with rejoining processes. In contrast, resting chromosomes (as in G0 lymphocytes irradiated before fusion) showed efficient rejoining. These results support the hypothesis that cytogenetic lesions become observable chromosome breaks when chromosome condensation or decondensation occurs during the cell cycle.     

The contribution of DNA and chromosome repair deficiencies to the radiosensitivity of ataxia-telangiectasia.

Cells derived from individuals with ataxia-telangiectasia (AT) are more sensitive to ionizing radiation and radiomimetic drugs, as evidenced by decreased survival and increased chromosome aberrations at mitosis when compared with normal cell lines. Our previous studies showed that, despite similar initial levels of DNA double-strand breaks (DSBs), AT cells express higher initial chromosome damage than do normal cells as demonstrated by the technique of premature chromosome condensation. However, this finding accounted for only a portion of the increased sensitivity (T. K. Pandita and W. N. Hittelman, Radiat. Res. 130, 94-103, 1992). The purpose of the study reported here was to examine the contribution of DNA and chromosome repair to the radiosensitivity of AT cells. Exponentially growing AT and normal lymphoblastoid cells were fractionated into cell cycle phase-enriched populations by centrifugal elutriation, and their DNA and chromosome repair characteristics were evaluated by DNA neutral filter elution (for DNA DSBs) and by premature chromosome condensation, respectively. AT cells exhibited a reduced fast-repair component in both G1- and G2-phase cells, as observed at the level of both DNA DSBs and the chromosome; however, S-phase cells showed nearly normal DNA DSB repair. The findings that AT cells exhibit an increased level of chromosome damage and a deficiency in the fast component (but not the slow component) of repair suggest that chromatin organization might play a major role in the observed sensitivity of AT cells. When survival was plotted as a function of the residual amount of chromosome damage in G1- and G2- phase cells after 90 min of repair, the curves for normal and AT cells approached each other but did not overlap. These results suggest that, although higher initial levels of chromosome damage and reduced chromosome repair capability can explain much of the radiosensitivity of AT cells, other differences in AT cells must also contribute to their sensitivity phenotype.     

Cytogenetic changes in human lymphoma U-937 induced to apoptosis by tumor necrosis factor

The frequency of cytogenetic abnormalities in cell populations of U-937 line in control group and after their exposure to tumor necrosis factor (TNF) was assayed. It has been found that the maximum effect of TNF is observed after 48-h exposure. It was exhibited as apoptosis induction, accumulation of cells with micronuclei and binuclear cells. An increased number of cells with premature chromosome condensation is an early marker of TNF action. Changes in distribution of the cells with various chromosome numbers may lead to the appearance of sublines with properties different from those of the original cell population.     

Initial chromosome damage but not DNA damage is greater in ataxia telangiectasia cells.

Cells derived from individuals with ataxia telangiectasia (AT) exhibit increased sensitivity to ionizing radiation and certain drugs (e.g., bleomycin, neocarzinostatin, and etoposide) as evidenced by decreased survival and increased chromosome aberrations at mitosis when compared with normal cell lines. To understand better the basis of this sensitivity, three AT and two normal lymphoblastoid cell lines were fractionated into cell cycle phase-enriched populations by centrifugal elutriation and then examined for their survival and their relative initial levels of DNA damage (neutral DNA filter elution) and chromosome damage (premature chromosome condensation). AT cells exhibited decreased levels of survival in all phases of the cell cycle; however, AT cells in early G1 phase were especially sensitive compared with normal cells in G1 phase. While AT and normal cells exhibited similar levels of initial DNA double-strand breaks in exponential populations as well as throughout the cell cycle, AT cells showed nearly twofold higher initial levels of chromosome damage than normal control cells in G1 and G2 phase. These results suggest that there is a higher rate of conversion of DNA double-strand breaks into chromosome breaks in AT cells, perhaps due to a difference in chromatin organization or stability. Thus one determining component of cellular radiosensitivity might include chromatin structure.     

A cytogenetic investigation of DNA rereplication after hydroxyurea treatment: implications for gene amplification

Although the mechanisms leading to gene amplification are poorly understood, it has recently been proposed that the initial event of amplification is the rereplication of a variable, but relatively large, amount of the genome within a single cell cycle. We sought evidence for rereplication of DNA as a basis for gene amplification through two cytogenetic techniques: differential staining for sister-chromatid exchange analysis and premature chromosome condensation. Synchronized Chinese hamster ovary cells were incubated continuously with bromodeoxyuridine and treated with hydroxyurea (HU) when cells were approximately 2 h into the S phase. After 6 h exposure to HU, the drug was removed and at 3 h intervals thereafter metaphase cells were collected and the chromosomes were stained by the fluorescence-plus-Giemsa procedure. No staining patterns consistent with rereplication of DNA were observed. Since HU causes cytogenetic damage, the premature chromosome condensation technique was used to determine the kinetics of chromosome damage after removal of HU. Extensive G₂ chromosome damage within 1 h after removal of HU from the medium was found, although cesium chloride gradient analysis showed that there was no rereplication of DNA during this time. Contrary to a previous report, these results provide no evidence that incubation of cells with HU during S phase induces rereplication of DNA within a single cell cycle. The results observed are consistent with the hypothesis that drug-induced aberrations and the subsequent abnormal segregation of chromosomal fragments are the first steps in the process that leads to gene amplification in drug-treated mammalian cells.     

Chromatin dynamics during cell cycle mediate conversion of DNA damage into chromatid breaks and affect formation of chromosomal aberrations: biological and clinical significance

The formation of diverse chromosomal aberrations following irradiation and the variability in radiosensitivity at different cell-cycle stages remain a long standing controversy, probably because most of the studies have focused on elucidating the enzymatic mechanisms involved using simple DNA substrates. Yet, recognition, processing and repair of DNA damage occur within the nucleoprotein complex of chromatin which is dynamic in nature, capable of rapid unfolding, disassembling, assembling and refolding. The present work reviews experimental work designed to investigate the impact of chromatin dynamics and chromosome conformation changes during cell-cycle in the formation of chromosomal aberrations. Using conventional cytogenetics and premature chromosome condensation to visualize interphase chromatin, the data presented support the hypothesis that chromatin dynamic changes during cell-cycle are important determinants in the conversion of sub-microscopic DNA lesions into chromatid breaks. Consequently, the type and yield of radiation-induced chromosomal aberrations at a given cell-cycle-stage depends on the combined effect of DNA repair processes and chromatin dynamics, which is cell-cycle-regulated and subject to up- or down-regulation following radiation exposure or genetic alterations. This new hypothesis is used to explain the variability in radiosensitivity observed at various cell-cycle-stages, among mutant cells and cells of different origin, or among different individuals, and to revisit unresolved issues and unanswered questions. In addition, it is used to better understand hypersensitivity of AT cells and to provide an improved predictive G2-assay for evaluating radiosensitivity at individual level. Finally, experimental data at single cell level obtained using hybrid cells suggest that the proposed hypothesis applies only to the irradiated component of the hybrid.     

Genetic control of mitosis. Adaptive modifications of v158 mutation expression

Cytogenetic parameters of mitosis were studied in the neural ganglions and imaginal disks of the third-instar Drosophila larvae of the marker lines ry506 and w; Cy/L; D/Sb; two wild-type lines Lausenne and Hikone-AW; and the v158 line mutant for the cell-cycle gene in the 85F locus. The control lines and their various tissues differ in a number of mitotic traits, which are believed to be the natural modifications of chromosome condensation and segregation and do not disturb homeostasis of the developing ry506, Lausenne, and Hikone-AW flies. Mutation v158 affects centromere disjunction. In imaginal disks, this results in arrest of either mitosis or anaphase initiation, whereas, in the neural ganglions, chromosomes integrate into a monopolar spindle at prophase and unipolar cells appear in anaphase. Different effects of the mutation in various tissues are assumed to be caused by different activity of the checkpoint system. When the mutation was maintained heterozygous for a long time, adaptive modification of its expression was observed. A comparison of the rates of two major and parallel mitotic processes, spindle formation and chromosome condensation, showed that adaptive modification can proceed via the adjustment of these rates.     

Chromosome condensation may enhance X-ray-related cell lethality in a temperature-sensitive mutant (tsBN2) of baby hamster kidney cells (BHK21)

In the tsBN2 cell line, which has a temperature-sensitive defect in the regulatory mechanism for chromosome condensation, the lethal effect of X rays was enhanced by incubating the cells at a nonpermissive temperature (40 degrees C) following X irradiation. This enhancement was suppressed in the presence of cycloheximide, which inhibits induction of premature chromosome condensation. The findings obtained in the case of delayed incubation at 40 degrees C and in synchronized cells indicate that X-ray-related potentially lethal damage, which can be expressed by chromosome condensation, is produced in the cells at any stage of the cell cycle, but it is repairable for all cells except those at around the late G2-M phase, where chromosome condensation occurs at a permissive temperature (33.5 degrees C). These observations suggest that the high sensitivity of late G2-M cells to X rays is caused by the events associated with chromosome condensation.     

Cell proliferation in chorionic villi at different gestational ages, as analyzed by premature chromosome condensation

The technique of premature chromosome condensation (PCC) was adapted to human first-trimester chorionic villi cells to analyze the cell-cycle kinetics of interphase chromatin. Uncultured cells of the cytotrophoblast (CT) and the mesenchymal core (MC) were obtained by a two-step digestion. PCC was induced by fusion of the chorionic interphase cells with mitotic Chinese hamster ovary or HeLa cells. Cells showing PCC in G1 (classes 1-6), S, and G2 were found. To analyze further the proliferation stages of chorionic G1 interphases, the proliferation potential index (PPI) of 34 placentae recovered between the 8th and 12th week of gestation was determined. The mean PPI found in the CT and MC cells ranged from 18% to 73%, values similar to those described for intensely proliferating tissues. The highest mean PPI value (73%) was observed in CT cells from placentae recovered at the 9th week of gestation, indicating a high specific proliferative activity of CT cells at this developmental stage.     

A genomewide linkage study of 1,933 families affected by premature coronary artery disease: The British Heart Foundation (BHF) Family Heart Study

Coronary artery disease (CAD) and its most important complication, myocardial infarction (MI), are the leading cause of premature death in the Western world. CAD has a substantial genetic basis, especially when it occurs early. We investigated the genetic determinants of premature CAD by performing a genomewide linkage analysis of 4,175 affected subjects from 1,933 families recruited throughout the United Kingdom. Each family had at least two available siblings with CAD, with validated onset before age 66 years. Linkage analysis was performed using 416 microsatellite markers. We observed suggestive linkage, for both CAD and MI, to a region on chromosome 2. For CAD, a LOD score of 1.86 was observed at marker D2S2271, which, in an ordered subset analysis, increased to 2.70 in families (n=1,698) with a minimum age at diagnosis of 56 years or younger. For MI, an overlapping peak with a LOD score of 1.15 was observed at marker D2S2216, which increased to 2.1 in families (n=801) with a minimum age at diagnosis of 59 years or younger. Exclusion mapping showed that 100% of the autosomal genome could be excluded for locus-specific sibling relative risks of 1.5 and 1.6 for CAD and MI, respectively. The region identified on chromosome 2 overlaps linked regions observed in two other smaller genome scans for CAD. Together, these findings strongly suggest that there is a locus on chromosome 2 that influences coronary atherosclerosis risk. The exclusion of a common locus that increases risk of CAD to siblings by >50% has important implications for strategies for further defining the genetic basis of CAD.     

Phosphorylation of nonhistone proteins during premature chromosome condensation in a temperature-sensitive mutant, tsBN2

In tsBN2 cells, a temperature-sensitive (ts) mutant of the BHK21 cell line, with a ts-defect in its regulatory system for chromosome condensation, antigens that react with mitotic specific mouse monoclonal antibody MPM-2 were produced when premature chromosome condensation (PCC) was induced by a temperature shift. The polypeptides of antigens recognized by MPM-2 in tsBN2 cells with PCC were identical to those of antigens in mitotic cells. These antigens appeared concomitantly with chromosome condensation, which suggests that these mitotic-specific antigens may be related to chromosome condensation. As the production of mitotic-specific antigens was inhibited by W-7, a specific and potent antagonist of calmodulin, calmodulin may function in the mitotic phosphorylation of nonhistone protein.     

Analysis of X-ray induced aberrations in mammalian chromosomes by electrofusion induced premature chromosome condensation

Summary Premature chromosome condensation (PCC) was induced by electrofusion of metaphase cells of an Ehrlich ascites tumor cell line with interphase cells of a Muntjac cell line or of a Chinese Hamster subline. Electrofusion was performed by cell alignment in a weakly inhomogeneous a.c. field of 200 V/cm amplitude (peak-to-peak value) and of 1.7 MHz frequency, followed by the application of a series of breakdown (fusion) pulses of 5 kV/cm strength and 15 µs duration. Most of the PCC's were of the G2 type despite the large proportion of G1 and S cells in the suspension. The number of chromatid aberrations observed in electrofused cells which had not been subjected to irradiation was not significantly above the spontaneous level. This indicates that electrofusion, at least as used here, did not lead to lesions expressed as structural aberrations. When interphase cells were irradiated by X-ray doses below 3 Gy before electrofusion PCC analysis showed chromosome damage consisting mainly of breaks and gaps. The frequency of aberrations recorded by PCC was 6 to 40 fold larger than that seen in conventional metaphase analysis. This large increase probably arose because of an effective suppression of the G2 repair of chromosomal lesions by the fast condensation process which took place within about 30 min. This assumption was supported by PCC experiments in which the time between X-irradiation and fusion with subsequent chromosome condensation was varied. The results demonstrated that G2 repair of chromosomal lesions was not detectable until 20 min after fusion with a half-time of the repair kinetics of about 1.5 h. The selectivity of premature chromosome condensation in G2 cells is discussed in terms of the differences between electrofusion and chemically or virally induced fusion. It is assumed that the concentration and the transfer rate of the chromosome condensation factor from the metaphase to the interphase cell are the limiting factors in achieving PCC. This is because the localised permeabilisation of the membrane and the dominance of two-cell fusions are characteristic of electrofusion.     

Consanguinity analysis in Israeli mental retardates.

Consanguinity rates were analyzed in 904 families of retardates studied in 11 Israeli Jewish ethnic groups. It was estimated that the representative recessive gene frequency is .00518, implying that a gene equilibrium maintained by mutation alone is improbable and that some other hypothesis should be considered. The proportions of homozygotes among the following idiopathic subgroups are estimated as follows: 18%-19% homozygotes among severe idiopathic retardates with nonconsanguineous parents and no affected siblings; 74%-76% homozygotes among severe idiopathic retardates with first-cousin parents and no affected siblings; 5% homozygotes among mild idiopathic and idiopathic-familial retardates with nonconsanguineous parents; and 41% homozygotes among mild idiopathic and idiopathic-familial retardates with first-cousin parents. The estimated number of major gene loci within ethnic groups is 17-21 for severe idiopathic retardation and 43-61 for mild idiopathic retardation. These findings provide a basis for genetic counseling of families with single retardates of unknown cause. They can also be useful in epidemiologic studies of nongenetic factors. The great prevalence of common gene defects causing retardation, coupled with the rarity of disorders of amino acid metabolism in the same series, seem to indicate that further emphasis on amino acid metabolism may be nonproductive in the scientific study of retardation and that other biochemical approaches should be encouraged.     

Analysis of proteins associated with chromosome condensation in baby hamster kidney cells

To identify proteins concerned with chromosome condensation processes, we used a temperature-sensitive mutant, tsBN2 derived from BHK21, in which premature chromosome condensation occurred at high temperature. When the proteins synthesized in tsBN2 during the induction of premature chromosome condensation were analyzed by two-dimensional gel electrophoresis, we found that an acidic protein with a molecular weight of 35,000 was specifically associated with chromosome condensation. In the normal cell cycle, this protein was synthesized from the G2 through the M phase. The protein was located mainly in the chromosome fraction and was phosphorylated.     

The analysis of chromatin remodeling and the staining for DNA methylation and histone acetylation do not provide definitive indicators of the developmental ability of inter-species cloned embryos

The restricted supply of oocytes in the domestic dog limits the development of reproductive biotechnologies in this species. Inter-species somatic cell nuclear transfer could be an alternative for cloning animals whose oocytes are difficult to obtain. In this study, the possibility of cloning dog embryos using pig oocytes was investigated by evaluating nuclear remodeling. Chromatin remodeling, assessed by premature chromosome condensation, pseudo-pronuclei formation, DNA methylation and histone acetylation, along with the developmental ability was compared between intra- and inter-species cloned embryos. The incidence of premature chromosome condensation was significantly higher in intra-species cloned embryos relative to inter-species cloned embryos (87.2% vs. 61.7%; P<0.05), but comparable pseudo-pronuclei formation was observed in both (85.3% vs. 75.8%). None of the inter-species cloned embryos developed beyond the 8-cell stage while 18.3% of intra-species cloned embryos developed to the blastocyst stage. The relative level of both DNA methylation and histone acetylation was similar between intra- and inter-species cloned embryos at all times examined. These results suggest that although partial chromatin remodeling occurs, further investigation is needed to be able to use pig oocytes as recipient oocytes in dog cloning.     

Bends in human mitotic metaphase chromosomes, including a bend marking the X-inactivation center.

Bends in mitotic metaphase chromosomes are not distributed randomly throughout the karyotype. The frequency of bends at centromeres is positively correlated with the relative length of the chromosomes and negatively correlated with the centromere index (more bends in metacentrics, fewer in acrocentrics). The frequency of bends in the noncentromeric regions (except at Xq13-Xq21) is positively correlated with the relative length of chromosome arms. A bend at Xq13.3 to Xq21.1 was more frequent than a bend in any other region of the karyotype, centromeric or noncentromeric. It was observed in one member of the X-chromosome pair in 63% of 46,XX cells. In contrast, it was observed in only 2% of 46,XY cells. RBG-staining showed that this specific bend is confined to the lyonized X chromosome. These observations in cells from normal subjects were confirmed using G-banding and RBG-staining on cells from nine subjects with different X-chromosome abnormalities and on metaphases from amniotic fluid cell and lymphocyte cultures. The "center for Barr body condensation" has been localized to the region between Xq11.2 and Xq21.1. The functional and structural relationship is unclear, but we believe this highly specific bend may represent a visible manifestation of the condensation process; it could represent the first folded (and last unfolded) position, upon or around which the rest of the chromosome condenses. The late replication of this region may also be a factor. The smallest region of overlap (SRO) for the X-chromosome inactivation center and the specific chromosome bend is Xq13.3 to Xq21.1.     

草鱼染色体的包装（间期—中期）

以草鱼ＺＣ７９０１细胞株为材料,观察鱼类细胞从间期染色质到中期染色体的包装过程。主要通过（１）分裂期与间期细胞融合,诱导染色体早熟凝集;（２）染色体“伸长”处理;（３）培养细胞的低渗处理;（４）染色质辅展等方法,制作染色体标本,进行扫描和透射电镜观察。观察表明,鱼类染色质的基本结构与哺乳类细胞相同,也是直径约１０ｎｍ的核丝。染色体的色装有两种形式：一种是多级螺旋化形成直径约３００ｎｍ的染色单体,     

Initiation of DNA synthesis in the prematurely condensed chromosomes of G1 cells

The objective of this study was to investigate whether G1 cells could enter S phase after premature chromosome condensation resulting from fusion with mitotic cells. HeLa cell synchronized in early G1, mid-G1, late G1, and G2 and human diploid fibroblasts synchronized in G0 and G1 phases were separately fused by use of UV-inactivated Sendai virus with mitotic HeLa cells. After cell fusion and premature chromosome condensation, the fused cells were incubated in culture medium containing Colcemid (0.05 micrograms/ml) and [3H]thymidine ([3H]ThdR) (0.5 microCi/ml; sp act, 6.7 Ci/mM). At 0, 2, 4, and 6 h after fusion, cell samples were taken to determine the initation of DNA synthesis in the prematurely condensed chromosomes (PCC) on the basis of their morphology and labeling index. The results of this study indicate that PCC from G0, G1, and G2 cells reach the maximum degree of compaction or condensation at 2 h after PCC induction. In addition, the G1-PCC from normal and transformed cells initiated DNA synthesis, as indicated by their "pulverized" appearance and incorporation of [3H]ThdR. Further, the initiation of DNA synthesis in G1-PCC occurred significantly earlier than in the mononucleate G1 cells. Neither pulverization nor incorporation of label was observed in the PCC of G0 and G2 cells. These findings suggest that chromosome decondensation, although not controlling the timing of a cell's entry into S phase, is an important step for the initiation of DNA synthesis. These data also suggest that the entry of a S phase may be regulated by cell cycle phase-specific changes in the permeability of the nuclear envelope to the inducers of DNA synthesis present in the cytoplasm.     

Comparison of the rates of aneuploidy occurrence in quiescent and dividing cells of humans exposed to adverse environmental factors

Fluorescence in situ hybridization (FISH) was used to compare aneuploidy rates in four autosomes and two sex chromosomes in interphase nuclei of noncultivated (quiescent) and cultivated (induced to divide with phytohemagglutinin (PHA)) leukocytes in people engaged in nuclear-chemical industry and in a control group of people not exposed to mutagenic factors occupationally or at home. The overall rates of numerical chromosome aberrations for all of the six chromosomes studied showed little difference, although a higher rate of loss of the X- and Y-chromosomes was observed in the exposed group. In individuals exposed to several adverse environmental factors, the overall rate of numerical chromosome aberrations in cultivated cells after at least one DNA replication cycle exceeded that in noncultivated cells by 52% (P = 0.01), whereas only a trend for its increase was observed in the control group (23%, P = 0.25). Thus, the effect of adverse environmental factors in humans caused more than a twofold increase in the difference between the rates of aneuploid cells in cultivated and non-cultivated leukocytes in the exposed group as compared to control. It is conjectured that cell division is accompanied by the expression of potential damage of mitotic chromatid segregation apparatus accumulated in vivo. These defects, realized during cell division, bring about numerical chromosome aberrations.     

Premature chromosome condensation. II. The nature of chromosome gaps produced by alkylating agents and ultraviolet light

Chinese hamster ovary (CHO) cells were treated with ultraviolet radiation or the alkylating agents, nitrogen mustard or trenimon, and chromosome damage to G2 phase cells were scored by the premature chromosome condensation (PCC) method or the metotic chromosome method. Treatment with these agents produced gaps but not chromatid breaks or exchanges. After UV treatment, the gap frequency observed in G2-PCC was higher than in the mitotic chromosomes, while the reverse trend was observed after treatment with nitrogen mustard or trenimon. These results suggest that two types of chromosome gaps exist, both of which are observable in mitotic chromosomes while only one type is observable in PCC due to differences in the stages of condensation between PCC and mitotic chromosomes.