Chromosomal instability in the cattle clones derived by somatic cell nuclear-transfer

Cytogenetic analysis was performed on peripheral lymphocytes collected from 20 cattle clones (19 showed no overt phenotypic abnormalities except for high birth weight while 1 exhibited left forelimb contracture), the donor cell cultures from which they were derived and lymphocytes from six insemination produced control cattle. All animals and cell cultures had a modal chromosome number of 60. The frequency of abnormal cells for donor cell cultures, clones, and controls was 6.68+/-0.30%, 5.30+/-5.49%, and 5.08+/-1.04%, respectively, and did not differ significantly among the groups. There were, however, two clones derived from different donor cell cultures with high incidences of 21.29% and 20.13%, of abnormal cells consisting of pseudodiploid (near-diploid), near-triploid and near-tetraploid, and tetraploid cells. Among these two clones, one had only a few endoreduplicated nuclei although further studies are necessary to precisely define the cytological origin and nature of the abnormal cells. The clones were evaluated at multiple time points for up to 20 months of age and the incidence of abnormal lymphocytes remained stable indicating that the chromosomally abnormal nuclei found in cloned animals was not a transient event. These results show that the majority of phenotypically normal clones have normal chromosomal make up but that instability of chromosome number can occur in clones that are phenotypically normal. Therefore, cytogenetical evaluation of peripheral lymphocytes and other tissues with follow up of the phenotypical consequences of these abnormalities is warranted even in phenotypically normal clones.     

Characteristics of genomic instability in clones of TK6 human lymphoblasts surviving exposure to 56Fe ions

Genomic instability in the human lymphoblast cell line TK6 was studied in clones surviving 36 generations after exposure to accelerated 56Fe ions. Clones were assayed for 20 characteristics, including chromosome aberrations, plating efficiency, apoptosis, cell cycle distribution, response to a second irradiation, and mutant frequency at two loci. The primary effect of the 56Fe-ion exposure on the surviving clones was a significant increase in the frequency of unstable chromosome aberrations compared to the very low spontaneous frequency, along with an increase in the phenotypic complexity of the unstable clones. The radiation-induced increase in the frequency of unstable chromosome aberrations was much greater than that observed previously in clones of the related cell line, WTK1, which in comparison to the TK6 cell line expresses an increased radiation resistance, a mutant TP53 protein, and an increased frequency of spontaneous unstable chromosome aberrations. The characteristics of the unstable clones of the two cell lines also differed. Most of the TK6 clones surviving exposure to 56Fe ions showed unstable cytogenetic abnormalities, while the phenotype of the WTK1 clones was more diverse. The results underscore the importance of genotype in the characteristics of instability after radiation exposure.     

An analysis of structural aberrations in human sperm chromosomes

We have analyzed structural aberrations in 5,000 sperm chromosome complements obtained from 20 men over a 5-yr period by fusion of human sperm with hamster eggs. Detailed data are presented on 366 abnormal cells with 379 analyzable breakpoints. The frequency of cells with structural aberrations ranged from 1.9% to 14.5% among donors; this interindividual variability was statistically significant (p less than 0.0001). In contrast, repeat samples from individual men showed no significant variation over time. The number of sperm chromosome sets processed per hamster egg had no effect on the frequency with which structural aberrations occurred, nor were sperm chromosome abnormalities altered by varying capacitation or culture conditions. The spectrum of structural aberrations observed in human sperm chromosomes and a chi-square analysis of breakpoints based on DNA content are presented. Although human sperm chromosome abnormalities were visualized with a cross-species system, we believe that they represent an inherent, biologically significant phenomenon.     

The chromosome changes in non-Burkitt lymphomas

Summary Cytogenetic analysis of 26 non-Burkitt lymphomas having abnormal clones, revealed non-random involvement of certain chromosomes in numerical and structural changes. In some cases, chromosome structural abnormalities could be correlated with histopathology of the tumours. A combined analysis of cases in the present series, and from the literature, indicates that the most common aberrations in the lymphoproliferative disorders involve structural changes of chromosome 14.To whom offprint requests should be sent     

Aberrations of chromosome 3. A marker of T-cell lymphomas?

Aberrations of chromosome occur in different malignancies, but they are more frequent in lymphoproliferative ones than in the others. In this study here four out of five T-zone lymphomas had abnormalities of chromosome 3. This lead to the question of whether aberrations of chromosome 3 might be a marker of T-cell lymphomas. The conclusion which can be drawn from the cases described in the literature, the own unpublished cases, and these four T-zone lymphomas is that abnormalities of chromosome 3 cannot be regarded as a discriminative marker of T-cell derived malignancies. It is suggested that the relationship between chromosome aberrations and type of disease is not necessarily a direct one. One possible model of the genesis of chromosome aberrations in malignant diseases is presented.     

Studies on isolated cloned populations from irradiated human embryonic cell cultures.

The recovery of substrains with stable chromosome aberrations from irradiated fibroblast culture are reported. Four human fetal cell strains were exposed to 600 rad of gamma rays at 200 rad/min. The efficiency of recovering viable cloned subpopulations was approximately 87%, and the frequency of clones with abnormal chromosomes was 40/100 colonies. G-band chromosome analyses for 34 abnormal substrains are described. Karyotypes of some of the clones with complex rearrangements are also presented. Analyses of a total of 47 aberrant events in the 34 abnormal substrains revealed at 7:1 and a 9:1 translocation-inversion and translocation-deletion ratios, respectively. Five of the abnormal substrains were continuously cultured; all except one showed signs of sensecence toward the end of 44 +/- 10 doublings. Unusual prolonged proliferation capacity was observed in substrain FFS-1-9. The significance of this finding is discussed.     

Chromosomal Instability and Telomere Length Variations during the Life Span of Human Fibroblast Clones

Growth characteristics, karyotype changes, and telomere length variations were analyzed during the life span of 12 anchorage-independent clones isolated from a xeroderma pigmentosum fibroblast strain. After an initial period of comparable active growth, all the clones showed a decline in the growth rate and finally entered a phase of replicative senescence; however, the number of population doublings and the time required to enter senescence varied among the clones. Repeated cytogenetic analyses during culture propagation showed the appearance of chromosome anomalies, mainly telomeric association (tas) and unbalanced translocations. In all the clones the percentage of abnormal mitoses increased with culture passage, but reached different levels (from less than 10% to about 100%). This finding indicates that the replicative block may be associated with differently altered cytogenetic patterns. Specific chromosome arms (5p, 16q, 19q, and 20q) were preferentially involved intas,suggesting that alterations in chromosome ends may occur which predispose to fusion. In some clones it was possible to demonstrate the origin of marker chromosomes from the evolution oftas.Telomere length analysis by Southern blotting on DNA samples prepared from 7 clones and from the parental cell lines showed that the terminal restriction fragment (TRF) profiles were homogeneous in senescent parental cells and in the clones during the last part of their life in culture, regardless of the degree of karyotype abnormalities. The homogeneity of the TRF profiles supports the hypothesis of a critical telomere length at senescence.     

Chromosome studies in myelomatosis.

Cytogenetic studies of patients with multiple myeloma and plasma cell leukemia have shown that chromosome abnormalities occur in the bone marrow and/or the PHA-stimulated blood of at least half the patients. The abnormalities include numerical and structural aberrations and are highly variable. Hypodiploid modes occur fairly frequently. Addition of material to the long arm of the No. 14 chromosome (14q+ marker) occurs in about 17% of the small series of patients that have been studied with banding so far.     

The impact of chromosomal alteration on embryo development

Chromosome alterations, such as those affecting telomere erosion, predictably occur with each cell division, others, which involve changes to the expression and replication of the X-chromosome occur at particular stages of development, while those that involve loss or gain of chromosomes occur in a random and so far unpredictable manner. The production of embryos in vitro and by somatic cell nuclear transfer (SCNT) has been associated with altered expression of marker genes on the X-chromosome and an increased incidence of chromosomally abnormal cells during early development. In the case of SCNT embryos chromosome abnormalities may be associated with the nuclear donor cell. Telomere rebuilding subsequent to SCNT appears to vary according to species and type of donor cell used. It is speculated that the rate of telomere erosion and incidence of chromosome abnormalities affects developmental potential of early embryos and may be potential predictors of developmental outcome.     

Chromosome aberrations of clonal origin are present in astronauts' blood lymphocytes

Radiation-induced chromosome translocations remain in peripheral blood cells over many years, and can potentially be used to measure retrospective doses or prolonged low-dose rate exposures. However, several recent studies have indicated that some individuals possess clones of cells with balanced chromosome abnormalities, which can result in an overestimation of damage and, therefore, influence the accuracy of dose calculations. We carefully examined the patterns of chromosome damage found in the blood lymphocytes of twelve astronauts, and also applied statistical methods to screen for the presence of potential clones. Cells with clonal aberrations were identified in three of the twelve individuals. These clonal cells were present in samples collected both before and after space flight, and yields are higher than previously reported for healthy individuals in this age range (40-52 years of age). The frequency of clonal damage appears to be even greater in chromosomes prematurely condensed in interphase, when compared with equivalent analysis in metaphase cells. The individuals with clonal aberrations were followed-up over several months and the yields of all clones decreased during this period. Since clonal aberrations may be associated with increased risk of tumorigenesis, it is important to accurately identify cells containing clonal rearrangements for risk assessment as well as biodosimetry.     

Chromosome analysis in 31 cases of benign and malignant breast tumors: a study in Brazil

Cultures of 31 breast tumors, being 20 carcinomas and 11 benign lesions, were cytogenetically analysed. Clonal chromosome aberrations were detected in 16 carcinomas and in 4 benign lesions. Nine carcinomas and 2 benign lesions had multiple cytogenetically unrelated and related clones, whereas a single abnormal clone was observed in 7 carcinomas and in 2 benign lesions. Polyploid clones were found in 7 carcinomas and in 2 benign lesions. The presence of clonal chromosome aberrations and polyploid cells was not associated with the clinicopathologic parameters tested. Carcinomas had more clonal changes than benign lesions (p = 0.031), showing that cytogenetic features are of diagnostic value and that different chromosome anomalies might have different pathogenetic and prognostic significance.     

Paternally transmitted chromosomal aberrations in mouse zygotes determine their embryonic fate

The developmental consequences of chromosomal aberrations in embryos include spontaneous abortions, morphological defects, inborn abnormalities, and genetic/chromosomal diseases. Six germ-cell mutagens with different modes of action and spermatogenic stage sensitivities were used to investigate the relationship between the types of cytogenetic damage in zygotes with their subsequent risk of postimplantation death and of birth as a translocation carrier. Independent of the mutagen used, over 98% of paternally transmitted aberrations were chromosome type, rather than chromatid type, indicating that they were formed during the period between exposure of male germ cells and initiation of the first S phase after fertilization. There were consistent one-to-one agreements between the proportions of a) zygotes with unstable aberrations and the frequencies of dead embryos after implantation (slope = 0.87, confidence interval [CI]: 0.74, 1.16) and b) zygotes with reciprocal translocations and the frequency of translocation carriers at birth (slope = 0.74, CI: 0.48, 2.11). These findings suggest that chromosomal aberrations in zygotes are highly predictive of subsequent abnormal embryonic development and that development appears to proceed to implantation regardless of the presence of chromosomal abnormalities. Our findings support the hypothesis that, for paternally transmitted chromosomal aberrations, the fate of the embryo is already set by the end of G1 of the first cell cycle of development.     

Cytogenetic and molecular cytogenetic investigations in relapse of B-cell chronic lymphocytic leukemia

Сhromosomal abnormalities have been analyzed in bone marrow cells of 61 patients with relapse of B-cell chronic lymphocytic leukemia. The cytogenetic results have allowed the structural stratification of the obtained karyotypes into ten groups of clones: normal, normal/near tetraploid, abnormal/normal, abnormal/ near tetraploid/normal, evolution of clonal chromosome abnormalities; evolution of clonal chromosome abnormalities/normal, evolution of clonal chromosome abnormalities/near tetraploid/normal, independent clones, independent/normal clones; and independent/near tetraploid/normal clones. The identified structural rearrangements included translocations, deletions, insertions, and duplications; however, deletions with the involvement of bands 17p12, 13q12–q14, 11q14, and 11q23 dominated (63.8%). The application of i-FISH helped to show the presence of one to four abnormalities per karyotype. The identified cytogenetic and molecular cytogenetic rearrangements may signify a multilevel nature of the process underlying the development of resistant karyotypes. The results obtained under both methods have revealed the presence of a heterogenic cell population with possibly different levels of chemotherapy resistance.     

Mosaic Analysis Gives an Estimate of the Extent of Genomic Involvement in the Development of the Visual System in Drosophila Melanogaster

To investigate the role of vital loci in the development of the visual system of Drosophila, we induced mitotic recombination in individuals heterozygous for recessive organismal lethals and selected for analysis the resulting mosaics with homozygous mutant eye clones. Heads bearing clones were serially sectioned, silver-stained and examined for aberrations in the ommatidia and the neural structures to which they project. In our screen of 68 lines bearing diepoxybutane-induced X-linked lethals, 26 yielded few or no homozygous mutant clones (putative cell-lethals). Of the rest, 20 lines produced individuals with morphologically abnormal eye clones showing various degrees of aberrations in the ommatidial architecture. In 14 of these 20, the laminar cartridges innervated by the mutant clones were also disorganized. Clones with normal structure were found in 18 of the lines, and three lines were resistant to the induction of mitotic recombination. In a single line, comparatively normal clones in the eye projected to a lamina with subtle but consistent abnormalities. To the extent that we have a representative sample, these results suggest that about two-thirds of all vital genes may be essential for the normal assembly and neural connectivity of the eye. This points to a high degree of pleiotropy in the manner in which information in the genome of the fly is used in development.     

Chromosome Studies in Leukemia

This review discusses the significance of chromosomal abnormalities found in leukemia with the bias of belief that these have a primary role or are the mechanism of action of leukemogenic agents. The Philadelphia chromosome (Ph¹) is present in marrow cells examined without culture at any stage of most patients with chronic granulocytic leukemia (CGL). the presence of this chromosome is of diagnostic and prognostic value.Varied chromosomal abnormalities have been found in acute leukemia. Each abnormality, which may be unique, is absent in remission, found again at relapse and is seldom changed by therapy. Abnormalities may be of number of chromosomes (aneuploid) or structural rearrangements resulting in “marker” chromosomes, Ranges of abnormal numbers of chromosomes, when present, usually have related patterns which suggest origin of several cell types from one initial cell. Cells from patients with increased risk of leukemia owing to genetic factors have a high incidence of chromosome breakage and structural rearrangements suggesting a mechanism for production of clones of abnormal, possibly leukemic, cells.     

High frequency of common DNA copy number abnormalities detected by bacterial artificial chromosome array comparative genomic hybridization in 24 breast cancer cell lines

Breast cancer is a widespread disease in Japan and across the world. Breast cancer cells, as well as most other types of cancer cells, have diverse chromosomal aberrations. Clarifying the character of these chromosomal aberrations should contribute to the development of more suitable therapies, along with the predictions of metastasis and prognosis. Twenty-four breast cancer cell lines were analyzed by bacterial artificial chromosome (BAC) array comparative genomic hybridization (CGH). The array slide contained duplicate spots of 4030 BAC clone DNAs covering the entire human genome with 1 Mbp resolution. In all 24 breast cancer cell lines, frequent and significant amplifications as well as deletions were detected by BAC array CGH. Common DNA copy number gains, detected in 60% (above 15 cell lines) of the 24 breast cancer cell lines were found in 76 BAC clones, located at 1q, 5p, 8q, 9p, 16p, 17q, and 20q. Moreover, common DNA copy number loss was detected in 136 BAC clones, located at 1q, 2q, 3p, 4p, 6q, 8p, 9p, 11p, 13q, 17p, 18q, 19p, Xp, and Xq. The DNA copy number abnormalities found included abnormality of the well-known oncogene cMYC (8q24.21); however, most of them were not reported to relate to breast cancer. BAC array CGH has great potential to detect DNA copy number abnormalities, and has revealed that breast cancer cell lines have substantial heterogeneity.     

Localization of the human NCAM gene to band q23 of chromosome 11: the third gene coding for a cell interaction molecule mapped to the distal portion of the long arm of chromosome 11

cDNA clones containing sequences coding for the murine neural cell adhesion molecule (N-CAM) were used in Southern hybridizations on human genomic DNA and demonstrated approximately 90% homology between human and murine NCAM genes. In situ hybridization with one of these clones was performed on human metaphase chromosomes and allowed the localization of the human NCAM gene to band q23 of chromosome 11. The genes for two other cell surface molecules believed to be involved in cell-cell interactions, Thy-1 and the delta chain of the T3-T cell receptor complex, have recently been localized to the same region of chromosome 11 in man. Moreover, this region of the human chromosome 11 appears to be syntenic to a region of murine chromosome 9 that also contains the staggerer locus: staggerer mice show abnormal neurological features which may be related to abnormalities in the conversion of the embryonic to the adult forms of the N-CAM molecule.     

Cytogenetic analysis of human spermatozoa using intracytoplasmic sperm injection into mouse oocytes

The chromosome complement of human spermatozoa has been analyzed after their intracytoplasmic injection into unfertilized mouse oocytes. A total of 427 metaphase plates have been obtained, including 176 metaphase plates from spermatozoa with normal head morphology (108 and 68 spermatozoa from patients with normal (the control group) and abnormal spermogram parameters, respectively), and 251 metaphase plates from spermatozoa with abnormal heads (76, 91, 67, and 17 spermatozoa with large, amorphous, elongated, and round heads, respectively). The frequency of chromosome abnormalities in the control group is 26.1%, with hyperploidy, hypoploidy, and structural aberrations accounting for 7.4, 12.3, and 6.4% of the abnormalities, respectively. In none of the groups did the ratio between the numbers of X- and Y-bearing spermatozoa significantly differ from 1 : 1. The diploidy frequency was significantly higher in spermatozoa with large and amorphous heads compared to the control group (2.36, 3.29, and 0%, respectively). None of the groups of spermatozoa differed from the control group with respect to the frequency of structural aberrations. The type of the abnormal head morphology has been found to be correlated with the sperm chromosome complement.     

UP1 extends life of primary porcine fetal fibroblasts in culture

Genetic modification of somatic cell nuclei and subsequent nuclear transfer has opened an opportunity to create gene-targeted animals. However, somatic cells have a limited life span in culture and it is not possible to introduce precise genetic changes in both alleles in this narrow time window. To increase the life span of somatic cell in culture, both genetic and chemical approaches have been tried with varying success. Here, we report the effect of two anti-oxidants, glutathione and n-t-butyl hydroxylamine, and of the expression of UP1, a shortened derivative of heterogeneous nuclear riboprotein (hnRNP)A1, on the life extension of primary porcine fibroblasts in culture. Under our experimental conditions, the use of anti-oxidants did not result in any prolongation of the life span. In contrast, UP1 expression increased the life span significantly. While most control cells stopped growing by PDL 20, and none survived beyond PDL 35, 100% of UP1-expressing clones reached PDL50, and 40% made it to PDL65. The five UP1-expressing clones were karyotyped at PDL 50. While all of them had a range of numerical chromosomal abnormalities, two clones retained 30-40% normal cells, all the cells in other three clones had abnormal chromosome numbers. Thus, expression of UP1 may be useful in extending the life span of somatic cells in culture. This, in turn, will facilitate the process of gene targeting in this cell type.     

Possible correlations between cell killing, chromosome damage and DNA repair after X-irradiation

To gain information about the possible pathway from primary DNA damage to cell killing via the formation of chromosome aberrations, we have examined the effects of the DNA synthesis inhibitor ara A on survival, on the occurrence of chromosome abnormalities and on the repair of DNA strand breaks. Our results are not inconsistent with the idea that the increased expression or 'fixation' of PLD measured after treatment with ara A is a reflection of an increase in the formation of chromosome damage comprising both exchange type and deletion type aberrations. These aberrations may arise from unrepaired or misrepaired dsb in the DNA. Treatment of irradiated cells with ara A results in a larger number of residual dsb which may be partly the reason for the increase in the frequency of acentric chromosome fragments. The reasons for the increase also in the frequency of exchange aberrations in the presence of ara A are not known but one possibility is that the probability of interaction between two dsb remains high during treatment with ara A due to the strong inhibition of dsb repair, whereas in untreated controls this probability decreases steeply with time after irradiation.