Effect of cytomegalovirus on cell cycle progression and formation of pathological mitoses in cultured human diploid fibroblasts

The effect of cytomegalovirus on the cell cycle was studied autoradiographically in an asynchronous culture of human diploid fibroblasts. The analysis of labeled mitosis showed that some cells infected in the S phase ceased to progress through the cell cycle at one of its phases (S, G2, or M); at the same time, at least part of infected cells remained capable of entering mitosis. Beginning from day 2 after infection by cytomegalovirus, the accumulation of pathological mitotic cells blocked at metaphase was observed in the culture. Approximately 50% of these cells contained 3H-thymidine label above chromosomes. This fact suggested the possibility of pathological mitosis in cells that were infected both at the S and other phases of the cell cycle. The detailed morphological analysis of chromosomes at different stages of infection demonstrated that the degree of their morphological changes increases from slight (stronger condensation) to severe pathology (fragmentation). In the aggregate, the results of the study suggested that abnormal chromosome morphology resulted from irreversible cell division arrest under the effect of cytomegalovirus.     

Effect of the Cytomegalovirus on Cell Cycle Progression and Pathological Mitoses in Cultured Human Diploid Fibroblasts

The effect of the cytomegalovirus on the cell cycle was studied autoradiographically in an asynchronous culture of human diploid fibroblasts. The analysis of labeled mitosis showed that some cells infected in the S phase ceased to progress through the cell cycle at one of its phases (S, G ₂, or M); at the same time, at least part of the infected cells remained capable of entering mitosis. Beginning from day 2 after infection by cytomegalovirus, the accumulation of pathological mitotic cells blocked at metaphase was observed in the culture. Approximately 50% of these cells contained ³H-thymidine label above chromosomes. This suggested the possibility of pathological mitosis in cells that were infected both at the S and other phases of the cell cycle. The detailed morphological analysis of chromosomes at different stages of infection demonstrated that the degree of their morphological changes increases from slight (stronger condensation) to severe pathology (fragmentation). In the aggregate, the results of the study suggested that abnormal chromosome morphology resulted from irreversible cell division arrest under the effect of the cytomegalovirus.     

Virus Deoxyribonucleic Acid Sequences in Subdiploid and Subtetraploid Revertants of Polyoma- Transformed Cells

Polyoma-transformed cells can revert in the properties characteristic of transformation, although they maintain the polyoma-specific T antigen. Transformed cells contain the same number of copies of polyoma virus deoxyribonucleic acid (DNA) per cell (eight) as revertants with a subdiploid or a subtetraploid chromosome number. The results indicate that the duplication of chromosomes in the subtetraploid revertants did not include the chromosomes that carry the viral genome. The virus DNA in both transformed and revertant cells was associated with high-molecular-weight cell DNA. Reversion of the properties of transformed cells was, therefore, not associated either with a decrease in number of virus DNA copies per cell or with a lack of association of the virus DNA with cell DNA.     

Localization polymorphism of EBV DNA genomes in the chromosomes of Burkitt lymphoma cell lines

The localization of Epstein-Barr virus (EBV) genomes in nuclei of the human lymphoblastoïd cell lines Raji, Jijoye, P₃HR-1, Daudi and Ramos was investigated by in situ hybridization with biotinylated EBV DNA probes. We found that all sites of hybridization were associated with the chromosomes. Only some of these sites were present on both chromatids and these had a non-random distribution; these sites could represent EBV sequences integrated at specific points on the chromosomes. The total mean site number corresponded with the number of viral DNA copies estimated in the different cell lines by other techniques, but the copy number was highly variable from cell to cell in a given line.     

Chromosome banding and genome compartmentalization in fishes

The diploid chromosome number of the Chinese raccoon dog varies from 54 (no B chromosomes) to 58 (4 B chromosomes). The B chromosomes are totally heterochromatic. An electron microscopic study was made of the synaptonemal complexes (SC) in spermatocytes of these animals. The SC karyotype consists of 27 regular chromosome pairs (autosomes and the sex chromosomes) plus the B chromosomes. The Bs pair effectively with one another at pachytene, but the SC axes of the B chromosomes are much denser than those of the A chromosomes. Depending on the number of Bs, both bivalents and multivalents have been observed. When three B chromosomes are present in a cell, parallel alignment of all three SCs can be seen. Formation of multivalents indicates high homology among these supernumerary heterochromatic chromosomes. Fusiform bulges are found along unpaired regions of all chromosomes which are particularly pronounced in diplotene.     

Quantitation of in situ hybridization of ribosomal ribonucleic acids to human diploid cells

The hybridization of 5S and 28S ribosomal RNAs to human fibroblast and leukocyte cells was used as a model system to quantitate the technique of in situ hybridization for human diploid cell types. Quantitation consisted of counting (scoring) the number of grains formed over both interphase nuclei and metaphase chromosomes on slides after various hybridization procedures. The average number of grains/nucleus per slide was then used to determine hybridization percentages. As with nitrocellulose filter hybridizations the kinetics of in situ hybridizations can be fit with a single first-order rate constant. However, the in situ hybridization rate was approximately 10 times slower than the corresponding filter hybridization rate. The efficiency of in situ hybridization was found to range between 5 and 15% for both leukocyte and fibroblast cell types and for both metaphase and interphase nuclei. Determination of the parameters of the in situ hybridization reaction of ribosomal RNAs to diploid chromosomes define the experimental conditions needed for the localization of single copy genes to diploid chromosomes.     

HBsAg重组DNA转化中国地鼠卵巢细胞二氢叶酸还原酶阴性突变株的细胞染色体变化及致瘤性

整合了含乙型肝炎病毒表面抗原(HBsAg)基因和dhfr基因的CHO-dhfr~-细胞,其染色体的畸变率和畸变类型都比亲代CHO-dhfr~-细胞高。但转化前后两系细胞的重要特性都未发生变异,即两者的染色体总数无差别,都是20条。两系细胞株接种裸鼠,均未发现有致瘤性。     

Nucleolar organizing regions of human chromosomes

Summary Silver-Stained cells from 49 parents with a history of several abortions were compared with cells from 35 parents with normal liveborn children. The modal and mean number of silver-stained NORs (Ag-NORs) observed on D- or G-group chromosomes was similar in both groups and between males and females. Ag-NORs were randomly distributed on all five acrocentric pairs. The distribution and size of Ag-NORs within an individual was not random and was fairly consistent from cell to cell.The mean number of associations per cell was similar in both males and females of the abortion group and was less than the number of associations in controls. The probability of D- or G-group chromosomes being associated was near the expected probability of 0.6 for D-association and 0.4 for G-association. The frequency of association of any chromosome combination did not differ statistically from the expected values, though the number of associations, 15/22, was higher than expected.     

Chromosome analysis on two long-term established human colorectal carcinoma cell lines

A chromosomal analysis was carried out on two colorectal carcinoma cell lines (WiDr and COLO 205), which were established 15-20 years ago in the US and were collected by the Cell Bank of the Veterans General Hospital in Taipei. Among the 200 cells counted, 65.5% of WiDr (male) had 68-73 chromosomes, while 74% of the COLO 205 (female) had 70-76 chromosomes per cell. The Y chromosome was absent in the 30 WiDr metaphases analyzed. None of the other chromosomes, including X and the autosomes of both WiDr and COLO 205, revealed such a numerical deficiency. Over half of the autosomes had an average number per cell above 2.0. The existence of 5 or 6 normal homologues for certain autosomes was not rare in either line. Numerous structural abnormal marker chromosomes were present in the cells. As compared with the original chromosome findings which were done over 10 years ago, we noted that the range of chromosome counts was wider and the number of marker chromosomes increased in these long-term cultivated cell lines.     

Development of a nuclear polyhedrosis in cells of Rhynchosciara angelae (Diptera, Sciaridae) and patterns of DNA synthesis in the infected cells

An ultrastructural and autoradiographic study of the infection of cells of Rhynchosciara angelae by a nuclear polyhedrosis virus (RPV) is presented. RPV is a DNA virus and causes a dramatic increase in the volume of the infected cells and in the sizes of chromosomes and their DNA contents. The structure of the nucleoli changes with the infection and the changes are mainly related to an increase of DNA synthesis. The concentration of ribosomes increases in the cytoplasm of the infected cells. Autoradiographic study of the DNA synthesis showed that it varies with the infective process.Four patterns of DNA synthesis, in relation to the host chromosomes and the virus, were disclosed by means of tritiated thymidine incorporation in the infected nuclei. The patterns are: (1) incorporation mainly in the chromosomes, (2) incorporation in the chromosomes and in the nucleoplasm, (3) incorporation only in the nucleoplasm, and (4) incorporation mainly in the chromosomes in dissociation. There is indication of a succession 1→2 and 3→4. The succession of patterns indicates that the virus induces first the increase of synthesis of host cell DNA and RNA. The bulk of the synthesis of viral DNA is evident only after the host cell DNA and RNA machinery is amplified. The aspects of the formation of viral membrane indicate that it is a de novo process in which the membrane material is capable of self-assembly.     

Human tumor and rodent-human hybrid cells with an increased number of active human NORs.

A human fibrosarcoma line, HT1080-6TG, with a near diploid number of chromosomes, has an average of 7.3 chromosomes with an Ag-stained nucleolus organizer region (NOR). Cells of this line with an increased number of chromosomes have an increased number of Ag-stained NORs. This cell line has been used as the human parent in constructing mouse-human and rat-human hybrids that segregate rodent chromosomes. The hybrid ccell lines, which have 100 or more chromosomes per cell, show a proportionate increase in the number of Ag-stained NORs (means, 11.4--16.8). The frequency of association of acrocentric chromosomes increases in a similar fashion. There is no evidence of inactivation of human NORs in these cells.     

The karyotypic variability of mouse embryonic fibroblasts of 2 genotypes during spontaneous neoplastic transformation

Karyological analysis of 6 lines with distinct tumorigenic properties of mouse strains C3H/He and C57BL/6 has been carried out using a differential staining of chromosomes. The number of normal copies of chromosomes varied in all the investigated cell lines. The more and the less stable chromosomes different from line to line. All the cell lines were characterized by decreased numbers of copies of normal chromosome 7; a decreased number of normal copies of chromosome 2 and 16 was detected in the course of the cell spontaneous neoplastic evolution. The decreased number of normal copies of chromosomes 8, 12 and X, and the increased number of normal copies of chromosome 10 were specific of the cell lines with intermediate tumorigenicity. The maximum tumorigenic cell lines differed from all other lines by increased numbers of copies of chromosomes 4 and 5, and by a decrease in copy number of chromosome 6. The data obtained are discussed in terms of the search of the regularity of karyotypic changes in the course of the cell neoplastic evolution.     

Effect of laminin on numerical karyotype variability of kangaroo rat kidney cell lines

The numerical karyotypic variability has been investigated in "markerless" epithelial-like Rat kangaroo kidney cell lines NBL-3-11 and NBL-3-17 on cultivation on a laminin-2/4 coated surface. In cell line NBL-3-17, cultivated on the laminin-coated surface for 2, 4 and 12 days, the character of numerical karyotypic variability has changed. In 2 days the general character of cell distribution for the chromosome number did not change, but the frequency of cells with modal number of chromosomes decreases significantly, while that of cells with lower chromosome number show a tendency to increase. At a prolongation of cultivation time to 4 and 12 days, the numerical karyotypic heterogeneity in cell population increases due to a significant change in the general character of cell distribution for the chromosome number, which is caused by a significant decrease in the frequency of cells with the modal number of chromosomes, and by an increase in the frequency of cells with lower chromosome number. The analysis of distribution of individual chromosomes showed that the number of types of additional structural variants of the karyotype (SVK) increases significantly on cultivation on laminin for 2-12 days. In cell line NBL-3-11, cultivated on the laminin-coated surface for 2 and 4 days, the character of numerical karyotypic variability did not change compared to control variants. Possible reasons of the observed changes of numerical karyotypic variability in cell line NBL-3-17 is discussed. The reason of differences in the character of numerical karyotypic variability between cell lines NBL-3-11 and NBL-3-17 possibly consists in the change of gene expression, namely in a dose of certain functioning genes. The polymerase chain reaction with arbitrary primers revealed no differences between DNA patterns of cell lines NBL-3-17 and NBL-3-11. This can reflect a similarity in the primary DNA structure of both cell lines. Hence, these lines differ only in the number of homologous chromosomes (hypotriploid and hypodiploid).     

Effect of chromosomal instability on the mutation-selection balance in unicellular populations

This paper develops a mathematical model describing the evolutionary dynamics of a unicellular, asexually replicating population exhibiting chromosomal instability. Chromosomal instability is a form of genetic instability characterized by the gain or loss of entire chromosomes during cell division. We assume that the cellular genome is divided into several homologous groups of chromosomes, and that a single functional chromosome per homologous group is required for the cell to have the wild-type fitness. If the fitness is unaffected by the total number of chromosomes in the cell, our model is analytically solvable, and yields a mean fitness at mutation-selection balance that is identical to the mean fitness when there is no chromosomal instability. If this assumption is relaxed and the total number of chromosomes in the cell is not allowed to increase without bound, then chromosomal instability leads to a reduction in mean fitness. The results of this paper provide a useful baseline that can inform both future theoretial and experimental studies of chromosomal instability.     

Induction of tumorigenesis and chromosomal abnormalities in human amniocytes infected with simian virus 40 and kirstein sarcoma virus

Summary Cell cultures of epithelial-like human amniocytes were infected with simian virus 40 (SV40) and Kirsten sarcoma virus (KSV) in various sequential orders, and tested for agar growth, chromosome abnormalities, and tumorigenesis in the nude mouse assay. We observed that regardless of the order in which the viruses were introduced, the doubly infected cells always exhibited the typical SV40 premalignantly transformed phenotype before changing to the malignant phenotype. All doubly transformed cells from different cell donors produced tumors in adult and suckling nude athymic mice, classified as poorly differentiated sarcomas. Infection with SV40 alone conferred extended life span and accelerated growth without, the malignant capability of tumor production. Kirsten sarcoma virus alone produced only focal cell alteration with no change in cell longevity or tumorigenesis. Chromosome studies of the premalignant and malignant cells from one cell donor did not reveal any significant clonal development for marker chromosomes in either cell line. Chromosome 12, which carries the homologous cellular oncogene to KSV, had no increase in aberrations in the malignant cells. Chromosome 8 was most often involved in aberrations, and the most frequent aberration for both series was dicentric chromosomes due to telomere fusion. For other translocations the breakpoints were almost exclusively in the centromere regions. The vulnerability of telomere and centromere regions to the free virus present in these precrisis cells is discussed, and similarities in regard to types of aberrations in transfection experiments are noted.     

Cytogenetic and immunological specificity of mammalian hybrid cell lines

Karyotypes of the hybrid cell lines NS-RL-3 (TK- -sheep kidney cells and rabbit lymphocytes) and betaCR-NS (TK- -rabbit beta-cells and TK- -sheep kidney cells) were investigated. It was shown that both hybrid cell lines were characterized by presence of both sheep and rabbit chromosomes, which number and structure varies depending on the cell type and the number of passages. In some cases the aberrant chromosomes were identified. It was observed, that 40-50% of the NS-RL-3 cells survived in culture in the presence of the human blood serum, and also were identified during 7-28 days after their introduction into the organism of the animal. Thus, the partial immunological tolerance of the hybrid cell lines has been suggested.     

Karyotype analysis utilizing differentially stained constitutive heterochromatin of human and murine chromosomes

Constitutive heterochromatin of chromosomes can be visualized utilizing a new differential staining technique which was originally developed by Gall and Pardue (1971). The method facilitates the more certain identification of specific chromosomes within and between cell populations of different origins. Marker chromosomes can be identified in established cell lines over many months of serial passage. Chromosomes of similar morphology within karyotypes of man and mouse can be distinguished in a number of instances. For example, the Y chromosomes of both mouse and man can now be easily detected. The hetero-chromatic staining method also permits discrimination between mouse and human chromosomes in somatic cell hybrids, thus facilitating the assignment of gene markers to chromosomes in somatic cell genetics systems. Instances of translocation of centric heterochromatin to other parts of chromosomes in established tissue culture cell lines are described. An instance of the inheritance of a polymorphic variation in autosomal heterochromatin in man is reported. It is postulated that polymorphisms in the centric heterochromatin may account largely for small heritable chromosome length variations previously described in human populations and termed minor chromosome variants.     

Changes in the karyotype of the cell line,DSIR-HA-1179, and a comparison with that of its parent insect,Heteronychus arator (F.) (Coleoptera: Scarabaeidae)

Abstract

Eighteen months after establishment of the primary cell culture, 36 metaphase nuclei of cell line DSIR-HA-1179, derived from the black beetle, Heteronychus arator (F.), had between 32 and 43 chromosomes; one cell contained 67 chromosomes. Structural abnormalities such as double minute chromosomes, ring and di- and tri-centric chromosomes were observed. Thirty-six months after establishment the chromosomes appeared to be similar, but the number of chromosomes per cell was much more variable (range, 12–73; 42 metaphase cells). The modal chromosome number of testicular tissues from H. arator was 2n = 20 — including an Xy pair — which is characteristic of the Scarabaeidae. The karyotype of DSIR-HA-1179 cells is unstable, with the number of chromosomes becoming more variable with continued propagation.     

The E1 replication protein of bovine papillomavirus type 1 contains an extended nuclear localization signal that includes a p34cdc2 phosphorylation site.

Bovine papillomavirus (BPV) DNA replication occurs in the nucleus of infected cells. Most enzymatic activities are carried out by host cell proteins, with the viral E1 and E2 proteins required for the assembly of an initiation complex at the replication origin. In latently infected cells, viral DNA replication occurs in synchrony with the host cell chromosomes, maintaining a constant average copy number of BPV genomes per infected cell. By analyzing a series of mutants of the amino-terminal region of the E1 protein, we have identified the signal for transport of this protein to the cell nucleus. The E1 nuclear transport motif is highly conserved in the animal and human papillomaviruses and is encoded in a similar region in the related E1 genes. The signal is extended relative to the simple nuclear localization signals and contains two short amino acid sequences which contribute to nuclear transport, located between amino acids 85 and 108 of the BPV-1 E1 protein. Mutations in either basic region reduce nuclear transport of E1 protein and interfere with viral DNA replication. Mutations in both sequences simultaneously prevent any observable accumulation of the protein and reduce replication in transient assays to barely detectable levels. Surprisingly, these mutations had no effect on the ability of viral genomes to morphologically transform cells, although the plasmid DNA in the transformed cells was maintained at a very low copy number. Between these two basic amino acid blocks in the nuclear transport signal, at threonine 102, is a putative site for phosphorylation by the cell cycle regulated kinase p34cdc2. Utilizing an E1 protein purified from either a baculovirus vector system or Escherichia coli, we have shown that the E1 protein is a substrate for this kinase. An E1 gene mutant at threonine 102 encodes for a protein which is no longer a substrate for the p34cdc2 kinase. Mutation of this threonine to isoleucine had no observable effect on either nuclear localization of E1 or DNA replication of the intact viral genome.     

A karyotype study of the Vero cell line cultured long term in a monolayer by static and roller methods

A cytogenetic investigation of Vero cells, before and after adaptation to the medium containing a cattle serum, was carried out by methods of differential chromosome staining. Under these conditions, both the modal number of chromosomes (from 58 to 55) and the karyotype structure, namely the copy number of normal chromosomes and the marker composition were shown to change. The Vero cell karyotype stability was studied in the continued culture by the static (50 passages) and roll-bottle (37 passages) methods. The quantitative changes (the rising percentage of diploid cells, and the change of cell fraction involving the modal number of chromosomes) were shown to occur in spite of the chromosome composition stability, which limits the time of using Vero cells as a substrate for preparation of vaccines.