High catabolism of BrdU may explain unusual sister chromatid differentiation and replication banding patterns in cancer cells

The structure of harlequin-stained chromosomes following substitution with low levels of 5-bromodeoxyuridine (BrdUrd) over two cell cycles and high levels over the last part of one cycle (replication banding) was studied in Chinese hamster ovary (CHO) cells. By using correlative light (LM) and scanning electron microscopy (SEM), it was shown that the effects of both the ultraviolet light (u.v.) and hot SSC treatment steps of the harlequin staining procedure were necessary to obtain sister-chromatid differentiation (SCD) or replication banding. u.v. treatment alone resulted in dark Giemsa staining of both chromatids with SEM morphology of short compact protuberances and an overall flattened smooth appearance in both the unsubstituted and BrdUrd-substituted chromatids, a morphology essentially similar to that of untreated chromosomes. SSC alone on the other hand resulted in dark-staining chromatids with an SEM morphology of raised, loosely packed loops of fibres in both types of chromatids. u.v. and SSC treatment together resulted in differentiation, with dark-staining unifilarly (TB) chromatids in the LM corresponding to raised loosely packed loops in the SEM and pale bifilarly (BB) chromatids corresponding to the smooth compact flattened SEM appearance. Where the BrdUrd-substituted strand became the template (BT), or when the nascent strand TB contained high levels of BrdUrd substitution in replication banding, the chromatid stained pale and showed the compact smooth appearance in the SEM. The Giemsa staining ability and ultrastructural morphology of harlequin staining is discussed with respect to putative DNA loss and also in terms of preferential protein-protein, protein-DNA cross-linkage in BrdUrd-containing DNA. These changes are also compared with the ultrastructural morphology observed after other banding methods, where deterioration of protein and DNA-protein interaction resulting in aggregation of chromatin fibres appears to be the major mechanism.

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Replication banding patterns in the chromosomes of the rainbow trout

Journal of Heredity ◽

10.1093/oxfordjournals.jhered.a109979 ◽

1984 ◽

Vol 75 (6) ◽

pp. 431-434 ◽

Cited By ~ 29

Author(s):

Mary E. Delany ◽

Stephen E. Bloom

Keyword(s):

Rainbow Trout ◽

Banding Patterns ◽

Replication Banding

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The influence of demethylating agents on preimplantation development of mice

Zygote ◽

10.1017/s0967199498000318 ◽

1998 ◽

Vol 6 (4) ◽

pp. 351-358 ◽

Cited By ~ 9

Author(s):

E.L. Patkin ◽

M.E. Kustova ◽

P. Perticone

Keyword(s):

Sister Chromatid ◽

Cell Number ◽

Sister Chromatid Exchanges ◽

Preimplantation Development ◽

Preimplantation Embryos ◽

Cell Cycles ◽

Severe Reduction ◽

The Mean ◽

Sister Chromatid Differentiation ◽

Chromatid Exchanges

The effects of two demethylating drugs with a different mechanism of action (5-azacytidine (Aza) and L-ethionine (Eth)) on mouse preimplantation development were investigated. Preimplantation embryos were cultured for 24 h in the presence of the drug and for an additional 24 or 48 h (depending on the cleavage stage) in medium supplemented with bromodeoxyuridine to reveal sister chromatid exchanges (SCEs) and the number of cell cycles performed before harvesting. Striking differences between the two drugs were observed in their influence on proliferation of blastomeres, primary differentiation and sister chromatid differentiation (SCD), and in the pattern of DNA methylation and the frequency of SCEs per cell. At a final concentration of 1 μM Aza had no effects, whereas higher concentrations stopped development of all stages except the zygote. In contrast Eth treatments (5 mM) resulted in a severe reduction of the mean cell number per embryo in comparison with controls. Moreover both the absence of blastocyst formation and no effects on mitotic activity were detected. The most prominent effect of Eth was detected at the zygote and 4-cell stages. An unexpected decrease in SCE frequency in Eth-treated morulae and 4-cell embryos has been observed. Data are explained taking into account the different mechanisms of action of the agents.

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Chromosome Banding in Amphibia. XXXII. The Genus Xenopus (Anura, Pipidae)

Cytogenetic and Genome Research ◽

10.1159/000433481 ◽

2015 ◽

Vol 145 (3-4) ◽

pp. 201-217 ◽

Cited By ~ 7

Author(s):

Michael Schmid ◽

Claus Steinlein

Keyword(s):

Chromosome Pair ◽

Repetitive Sequences ◽

5S Rdna ◽

Mitotic Chromosomes ◽

Banding Patterns ◽

Single Chromosome ◽

Replication Banding ◽

Rdna Sequences ◽

High Degree ◽

Replication Bands

Mitotic chromosomes of 16 species of the frog genus Xenopus were prepared from kidney and lung cell cultures. In the chromosomes of 7 species, high-resolution replication banding patterns could be induced by treating the cultures with 5-bromodeoxyuridine (BrdU) and deoxythymidine (dT) in succession, and in 6 of these species the BrdU/dT-banded chromosomes could be arranged into karyotypes. In the 3 species of the clade with 2n = 20 and 4n = 40 chromosomes (X. tropicalis, X. epitropicalis, X. new tetraploid 1), as well as in the 3 species with 4n = 36 chromosomes (X. laevis, X. borealis, X. muelleri), the BrdU/dT-banded karyotypes show a high degree of homoeology, though differences were detected between these groups. Translocations, inversions, insertions or sex-specific replication bands were not observed. Minor replication asynchronies found between chromosomes probably involve heterochromatic regions. BrdU/dT replication banding of Xenopus chromosomes provides the landmarks necessary for the exact physical mapping of genes and repetitive sequences. FISH with an X. laevis 5S rDNA probe detected multiple hybridization sites at or near the long-arm telomeric regions in most chromosomes of X. laevis and X. borealis, whereas in X. muelleri, the 5S rDNA sequences are located exclusively at the long-arm telomeres of a single chromosome pair. Staining with the AT base pair-specific fluorochrome quinacrine mustard revealed brightly fluorescing heterochromatic regions in the majority of X. borealis chromosomes which are absent in other Xenopus species.

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Improved sister-chromatid differentiation using paraffin-coated bromodeoxyuridine tablets in mice

Mutation Research Letters ◽

10.1016/0165-7992(83)90042-8 ◽

1983 ◽

Vol 119 (1) ◽

pp. 83-88 ◽

Cited By ~ 79

Author(s):

Alfred F. McFee ◽

Karen W. Lowe ◽

Juan R. San Sebastian

Keyword(s):

Sister Chromatid ◽

Sister Chromatid Differentiation

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Sister chromatid differentiation after in situ detection of ultraviolet-induced DNA breaks under electron microscopy

Biology of the Cell ◽

10.1016/0248-4900(94)90063-9 ◽

1994 ◽

Vol 82 (1) ◽

pp. 33-37

Author(s):

Joséluis Fernández ◽

Asunción Campos ◽

Vicente Goyanes ◽

Ismael Buño ◽

Jaime Gosálvez

Keyword(s):

Electron Microscopy ◽

Sister Chromatid ◽

Dna Breaks ◽

Sister Chromatid Differentiation ◽

In Situ Detection

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Distinct roles of cohesin-SA1 and cohesin-SA2 in 3D chromosome organization

10.1101/166264 ◽

2017 ◽

Author(s):

Aleksandar Kojic ◽

Ana Cuadrado ◽

Magali De Koninck ◽

Miriam Rodríguez-Corsino ◽

Gonzalo Gómez-López ◽

...

Keyword(s):

Cancer Cells ◽

Sister Chromatid ◽

Cell Types ◽

Dna Looping ◽

Chromosome Organization ◽

Genome Architecture ◽

Cell Type ◽

Chromatid Cohesion ◽

Cell Type Specific ◽

Specific Contribution

AbstractIn addition to mediating sister chromatid cohesion, cohesin plays a central role in DNA looping and segmentation of the genome into contact domains (TADs). Two variant cohesin complexes that contain either STAG/SA1 or SA2 are present in all cell types. Here we addressed their specific contribution to genome architecture in non-transformed human cells. We found that cohesin-SA1 drives stacking of cohesin rings at CTCF-bound sites and thereby contributes to the stabilization and preservation of TAD boundaries. In contrast, a more dynamic cohesin-SA2 promotes cell type-specific contacts between enhancers and promoters within TADs independently of CTCF. SA2 loss, a condition frequently observed in cancer cells, results in increased intra-TAD interactions, likely altering the expression of key cell identity genes.

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Hardwired synthetic lethality within the cohesin complex in human cancer cells

10.1101/125708 ◽

2017 ◽

Author(s):

Petra van der Lelij ◽

Simone Lieb ◽

Julian Jude ◽

Gordana Wutz ◽

Catarina P. Santos ◽

...

Keyword(s):

Bladder Cancer ◽

Cancer Cells ◽

Sister Chromatid ◽

Sister Chromatid Cohesion ◽

Cohesin Complex ◽

Wild Type ◽

Synthetic Lethal ◽

Recurrent Mutations ◽

Wide Range ◽

Chromatid Cohesion

AbstractRecent genome analyses have identified recurrent mutations in the cohesin complex in a wide range of human cancers. Here we demonstrate that the most frequently mutated subunit of the cohesin complex, STAG2, displays a strong synthetic lethal interaction with its paralog STAG1. Mechanistically, STAG1 loss abrogates sister chromatid cohesion in STAG2 mutated but not in wild-type cells leading to mitotic catastrophe, defective cell division and apoptosis. STAG1 inactivation inhibits the proliferation of STAG2 mutated but not wild-type bladder cancer and Ewing sarcoma cell lines. Restoration of STAG2 expression in a mutated bladder cancer model alleviates the dependency on STAG1. Thus, STAG1 and STAG2 act redundantly to support sister chromatid cohesion and cell survival. STAG1 represents a hardwired, context independent vulnerability of cancer cells carrying mutations in the major emerging tumor suppressor STAG2. Exploiting synthetic lethal interactions to target recurrent cohesin mutations in cancer, e.g. by inhibiting STAG1, holds the promise for the development of selective therapeutics.

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