Specific sites for EBV association in the Namalwa Burkitt lymphoma cell line and in a lymphoblastoid line transformed in vitro with EBV

Localization of Epstein-Barr virus (EBV) DNA was studied by in situ hybridization on chromosomes from the Namalwa Burkitt lymphoma cell line and from a lymphoblastoid cell line transformed in vitro (ATL9/g). The five chromosome bands 1p32, 1q31, 5q21, 13q21, and 16p13 showed the presence of EBV DNA in both of the lines. Grain deposition at the site on chromosome 1q of the Burkitt line was particularly intense. It was also found that EBV DNA in the lymphoblastoid cell line co-localized with a stable achromatic gap at 1p32 whose presence seems to confer a proliferative advantage on the cells.     

Cytogenetic changes in the Namalwa cell line of human malignant lymphoma induced by inhibitors of DNA replication and synthesis

Namalwa cells originating from the malignant human lymphoma have been analyzed cytogenetically upon short-time exposure to subtoxic doses of inhibitors of DNA replication and synthesis, either etoposide or fludarabine. The intact cells were characterized by the modal class of the chromosomes within the diploid range with the proportion of the aberrant cells amounting to 16.0 +/- 0.5%. Upon exposure to etoposide the percentage of the aberrant cells increased amounting to 26.1 +/- 2.9 through 39.8 +/- 1.7% depending on the duration of the exposure and the dose of the drug. At the same time the number of the polyploid cells increased but the modal class retained within the diploid range. Upon exposure to fludarabine the percentage of the cells with the aberrant chromosomes increased to 57.1 +/- 2.9%. Two modal classes appeared--the first approaching the diploid number and the second being polyploid. The exposure to either etoposide or fludarabine resulted in increasing number of the chromatide aberrations with more frequent involvement of #1, #2, #5, #6, #7, #11, #13, #14, #16 and #17 chromosomes. The data obtained have shown the susceptibility of Namalwa cells to the subtoxic concentrations of the inhibitors of DNA synthesis and replication used in the study resulting in the survival of the novel clones resistant to the drugs.     

Nucleolar organizing regions of normal Chinese hamster and CHW cell line chromosomes

The chromosome preparations from fibroblasts of normal male and female Chinese hamsters and the cell line CHW were stained with AgNO3. The silver stain was usually localized at the telomeres of autosomes. The marker chromosome M1 in the CHW cell line has Ag-NOR near the centre of the long arm, which indicates that either the long arms of two number 5 chromosomes fused at the telomeres or the intact telomeric region of one chromosome fused with one with a deleted telomere. The variation of Ag-NORs' number per cell and Ag-heteromorphism in chromosome number 4 were observed. The Ag-NORs of chromosome number 4 and 5 are in approximately the same position as the positive C-bands and these may play a role in the preservation of heterochromatin.     

Mass characteristics of DNA obtained from chromosomes of a human carcinoma cell line

Sedimentation studies of DNA from chromosomes extracted from human mitotic cells showed that highmolecular-weight DNA can be obtained if cell hypotonic treatments and prolonged metaphase blocks are avoided. Two types of large double-stranded DNA were observed. One of these (M _r = 2.5×10⁸) appeared as a size class with characteristics reminiscent of the chromosomal DNA subunit hypothesis. However, this DNA is the decay product of larger molecules, whose minimum molecular weight is 6×10⁸.     

The elimination and differentiation of chromosomes in the germ line of sciara

The germ line chromosomes of S. coprophila have been followed from the time of origin of the germ cells up to the time of meiosis in the male and up to first larval molt in the female. The mechanism which prevents the accumulation of L (limited) chromosomes in the germ line is a unique process of chromosome elimination: it occurs in male and female embryos after the germ cells have migrated from the pole plasm to the definitive gonad site, and it involves the movement of whole L chromosomes through the nuclear membrane into the cytoplasm. The extra paternal X chromosome is eliminated from the germ cells at the same time and in the same manner. Following this elimination there is a cytological differentiation of the chromosomes remaining inside the nucleus. First, the 4 paternal homologues of the regular complement undergo a loosening of coils and become light-staining whereas the maternal homologues remain condensed like the L's. Next, the L chromosomes undergo a process of extreme attenuation and dispersion following which they return to the condensed state. H³-thymidine autoradiography on gonial and premeiotic cells in the testis reveals that the L chromosomes undergo DNA replication at the end of the S period, also that there are asynchronies in DNA synthesis among the regular chromosomes. The phenomena of differential chromosome staining and asynchronous DNA replication are considered in the light of current theory regarding heterochromatization and gene inactivation, also in relation to the phenomenon of chromosome imprinting encountered in this genus.The studies reported here were supported by the National Science Foundation grants GB-42 and GB-2857, and in part by Contract No. AT-(40-1)-2690 under the Division of Biology and Medicine, U.S. Atomic Energy Commission.Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, in the Faculty of Pure Science, Department of Botany, Columbia University. This work was carried out in the laboratory of Professor J. Herbert Taylor and has been supported in part by U.S. Public Health Training Grant No. 2 T 1-GM-216-05. Grateful acknowledgement is made to Professor Spencer W. Brown, Department of Genetics, University of California, Berkeley, in whose laboratory the final studies were completed.     

Replication of the dihydrofolate reductase genes on double minute chromosomes in a murine cell line

The purpose of this study is to determine the kinetics of the replication of intrachromosomal versus extrachromosomal amplified dihydrofolate reductase (DHFR) genes. Previous studies reported that the DHFR gene, when carried intrachromosomally on a homogeneously staining region, replicates (as a unit) within the first 2 h of the S phase of the cell cycle. We wished to determine if the extrachromosomal location of the amplified genes carried on double minute chromosomes effects the timing of their replication. Equilibrium cesium chloride ultracentrifugation was used to separate newly replicated (BUdR-labeled) DNA from bulk DNA in a synchronized cell population. Hybridization with the cDNA for the DHFR gene allowed us to determine the period of time within the cell cycle in which the DHFR DNA sequences were replicated. We found that, in contrast to intrachromosomal dihydrofolate reductase genes that uniformly replicate as a unit at the beginning of the S phase of the cell cycle, dihydrofolate reductase genes carried on double minute chromosomes (DMs) replicate throughout the S phase of the cell cycle. These results suggest that control of replication of extrachromosomal DNA sequences may differ from intrachromosomal sequences.     

Sequence of centromere separation: characterization of multicentric chromosomes in a rat cell line

The B₁ cell line of rat cerebral endothelium origin exhibits several dicentric and multicentric chromosomes. These chromosomes, unlike multicentrics in mouse (Vig and Zinkowski 1986) do not show premature centromere separation. All centromeres deposit kinetochore proteins and appear to be functional. Even the centromeres which fail to migrate to the poles during anaphase and make side arm bridges bind to spindle microtubules. Some multicentric chromosomes show kinetochores spaced apart with intervening stretches of euchromatin while others are located adjacent to each other thus exhibiting tandem repeats and forming a compound kinetochore (Brinkeley et al. 1984). Also, unlike mouse multicentric chromosomes in which different pericentric regions and the centromeres replicate at different times, the rat chromosomes appear to replicate all pericentric and centric regions in a given multicentric simultaneously. The present studies indicate that centromeres in rat and mouse replicate during the last part of the S-phase and in continuation with the pericentric heterochromatin.     

Uptake and early fate of metaphase chromosomes ingested by the Wi-L2 human lymphoid cell line

Aspects of the ingestion and early intracellular fate of homologous. [3H]-thymidine-labeled chromosomes (donor) were studied in recipient Wi-L2 cells in the absence of reutilized radioactivity. As much as 67% of the cell-associated radioactivity was resistant to hydrolysis by DNase I after 4 h of incubation. Cell fractionation and electron microscope autoradiography indicated that chromosome uptake was rapid, into both cytoplasmic and nuclear fractions and was facilitator and dose dependent. Sedimentation analysis demonstrated that at 4 h donor DNA of approximate single-strand mol wt of 1--6 X 10(6), as compared to 6--12 X 10(6) for chromosomal DNA, was recoverable in cell fractions. By 6 h, a significant portion of the nucleus-associated donor DNA was converted into material of higher mol wt, although no evidence was found for integration into recipient DNA. Cytoplasmic donor DNA continued to be degraded. An average number of chromosome equivalents of nucleus-associated donor DNA to recipient cell nuclei of 1--4 was obtained and its relationship to the lower frequency of chromosome-mediated gene transfer is discussed.     

The loss of a single telomere can result in instability of multiple chromosomes in a human tumor cell line

Spontaneous telomere loss has been proposed as an important mechanism for initiating the chromosome instability commonly found in cancer cells. We have previously shown that spontaneous telomere loss in a human cancer cell line initiates breakage/fusion/bridge (B/F/B) cycles that continue for many cell generations, resulting in DNA amplification and translocations on the chromosome that lost its telomere. We have now extended these studies to determine the effect of the loss of a single telomere on the stability of other chromosomes. Our study showed that telomere acquisition during B/F/B cycles occurred mainly through translocations involving either the nonreciprocal transfer or duplication of the arms of other chromosomes. Telomere acquisition also occurred through small duplications involving the subtelomeric region of the other end of the same chromosome. Although all of these mechanisms stabilized the chromosome that lost its telomere, they differed in their consequences for the stability of the genome as a whole. Telomere acquisition involving nonreciprocal translocations resulted in the loss of a telomere on the donor chromosome, which consequently underwent additional translocations, isochromosome formation, or complete loss. In contrast, telomere acquisition involving duplications stabilized the genome, although the large duplications created substantial allelic imbalances. Thus, the loss of a single telomere can generate a variety of chromosome alterations commonly associated with human cancer, not only on a chromosome that loses its telomere but also on other chromosomes. Factors promoting telomere loss are therefore likely to have an important role in generating the karyotype evolution associated with human cancer.     

Distribution of sugar-binding sites within interphase nuclei and mitotic chromosomes of a human cell line

Using gold labelled neoglycoproteins containing either alpha-D-glucose, N-acetyl-beta-D-glucosamine, alpha-D-mannose, 6-phospho-alpha-D-mannose, and alpha-L-fucose (BSA), we investigated their intranuclear binding sites in the TG human cell line. Although gold-labelled BSA did not give any noticeable labelling, the presence of 1% free BSA in the medium containing the gold labelled neoglycoproteins was revealed to be a key factor of the labelling. During interphase in the presence of free BSA most of the labelling was detected in the nucleoplasm. The border of the condensed chromatin, known to be the site of hnRNA synthesis as well as the interchromatin areas enriched in RNPs were labelled. Condensed chromatin also contained binding-sites. The nucleolus was seen to present low labelling in comparison with the labelling observed over the nucleoplasm. These nucleolar binding sites were located both in the dense fibrillar and granular components. No labelling could be detected over the fibrillar centers which are very conspicuous in this cell line. During mitosis sugar-binding sites were observed over the chromosomes. Data reported here show for the first time that lectin-like proteins and chromatin components are colocalized both during interphase and mitosis. In addition, within the nucleolus the presence of sugar-binding proteins was seen to be restricted to the dense fibrillar and granular components.     

Characterization of human heteroploid cell line J-111: Reverse banding patterns of marker chromosomes

The karyotype of the human cell line, J-111, has been studied employing R-banding by fluorescence using acridine orange technique (RFA). The model chromosome number of this line was 112. All human chromosomes except the Y were present in each metaphase. Twenty-one marker chromosomes were distinguished and their possible origins were investigated. Of these, twelve were consistently present in all cells. Nine markers were highly variable. Four typical marker chromosomes of HeLa cells were found and their origins were identified, indicating that the line is a HeLa contaminant. The reverse banding patterns of all marker chromosomes are presented and the value of the RFA technique is discussed.     

The chromosomes of CHO,an aneuploid Chinese hamster cell line: G-band,C-band,and autoradiographic analyses

Chinese hamster ovary cells (line CHO) have been used extensively for metabolic, genetic, and radiobiological studies with only a superficial appreciation for the degree of aneuploidy characteristic of the line. A thorough karyologic analysis of CHO chromosomes using autoradiographic replication patterns, as well as centromere band (C-band) and Giemsa band (G-band) analysis, is presented. Our results demonstrate that only 8 of the 21 CHO chromosomes are normal when compared with euploid Chinese hamster chromosomes. In the 13 altered chromosomes, we found evidence of translocations, deletions, and pericentric inversions. These altered chromosomes have been characterized with respect to both origin and destination of translocated material. With the exception of the X₂ chromosome, essentially all of the euploid chromatin is present in CHO cells. Autoradiographic replication patterns show that the normal sequence of chromosomal DNA synthesis is altered. Some sites which replicate late in euploid cells replicate early in CHO, and several late-replicating chromosomes in CHO cells replicate in early- or mid-S in euploid material. These studies may serve to elucidate the observed differences in mutagenic behavior between euploid fibroblasts and CHO cells.     

The radiosensitivity of the chromosomes of the cells of human squamous cell carcinoma cell lines.

Measurement of the radiation sensitivity of chromosomes was used to address the influence of cell cycle distribution and of DNA content and ploidy on radiation responses in seven human squamous cell carcinoma cell lines. The cell lines varied about twofold in DNA content and chromosome number, and the X-ray sensitivities (D0) of the lines ranged from 1.1 to 2.7 Gy. The more resistant cell lines (D0 greater than 1.8 Gy) had faster growth rates and larger proportions of cells in S phase in asynchronous cultures. Aberration frequencies were measured in cells irradiated in G1 and G2 phase. The more resistant lines had fewer induced aberrations in both phases than did sensitive lines, implying that they were more resistant to radiation in both of these cell cycle phases. Therefore, while the larger S-phase population seen in the resistant cell lines probably contributes to the resistant phenotype, it cannot explain all of the intrinsic differences in radiation sensitivity. There was no relationship between DNA content and radiation sensitivity as measured by the cell survival assay or the induction of chromosome aberrations, although cells with larger DNA contents tended to have more chromosome damage per cell at equitoxic doses.     

Characterization of 10 marker chromosomes in a prostatic cancer cell line by in situ hybridization.

Marker chromosomes contain potentially valuable information about breakpoints in cancer. However, routine banding procedures, by themselves, provide only limited information about the identity of marker chromosomes. In this study, the use of fluorescence in situ hybridization (FISH) with chromosome-specific centromeric probes and whole-chromosome-specific DNA libraries greatly enhanced the identification of 10 marker chromosomes in the primary prostatic cancer cell line PPC-1. Centromeric probes for chromosomes 1, 2, 3, 4, 10, 12, and 17 and whole-chromosome paint libraries for chromosomes 1, 2, 3, 4, 8, and 12, in conjunction with analysis of G-banded metaphases, allowed the major portion(s) of these 10 PPC-1 marker chromosomes to be defined. The results increase the number of identifiable chromosomal breakpoints in this cell line from 9 to 28 sites.     

Germ line specific DNA and chromosomes of the ciliate Stylonychia lemnae

The variation in DNA content of the micronucleus (germinal nucleus) of Stylonychia lemnae and its relation to the number of chromosomes was examined. Different populations possess similar amounts of micronuclear DNA but there are differences of ±30% between clones of the same population. However, the DNA content varies by about 100% in the micronuclei during the lifetime of a clone. The haploid micronucleus contains 35 or 36 chromosomes which persist in the developing macronucleus anlagen and grow to giant chromosomes. Besides this remaining subset, the micronucleus contains a variable number of germ line restricted chromosomes (mean about 140; range between 100 and 180). The somatic macronucleus eliminates these elements early in its development. The varying number of the germ line restricted chromosomes is responsible for the variation in the micronuclear DNA content.