Visualization of Telomere Integrity and Function In Vitro and In Vivo Using Immunofluorescence Techniques

Cesare, Anthony J.; Heaphy, Christopher M.; O’Sullivan, Roderick J.

doi:10.1002/0471142956.cy1240s73

Cited by 47 publications

(57 citation statements)

References 40 publications

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“…Yet, we predicted that they were also found at other sites across the genome. In order to distinguish between telomere and non-telomere RPA foci, we performed telomere FISH combined with IF to measure the percentage of RPA foci colocalizing with telomeric DNA (36). As expected, we observed colocalization of RPA foci with telomeres ( Figure 3A and C).…”

Section: Deletion Of Ctc1 Leads To Increased Telomeric But Not Genomesupporting

confidence: 73%

“…For IF-FISH, IF was performed for gH2AX or chromatin-bound RPA32, as described above. Telomere FISH was then performed, as previously described (36). Briefly, after the last wash, following secondary antibody incubation, the coverslips were fixed with 2% formaldehyde in 1x PBS for 10 min at RT.…”

Section: Immunofluorescence (If) and If-fluorescence In Situ Hybridizmentioning

confidence: 99%

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Human CTC1 primarily functions in telomere maintenance/protection and promotes CHK1 phosphorylation in response to global replication stress

Ackerson

Gable

Stewart

2020

Preprint

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CST (CTC1-STN1-TEN1) is a heterotrimeric, RPA-like protein that binds to single stranded DNA (ssDNA) and functions in the replication of telomeric and non-telomeric DNA. Previous studies have shown that deletion of CTC1 results in decreased cell proliferation and telomeric DNA damage signaling. However, a detailed analysis of the consequences of conditional CTC1 knockout (KO) have not been fully elucidated. Here, we investigated the effects of CTC1 KO on cell cycle progression, genome-wide replication and activation of the DNA damage response. We find that CTC1 KO results in p53-mediated G2 arrest and increased apoptosis, but not genome-wide replication defects or DNA damage. Instead, the G2 arrest is dependent on the accumulation of telomeric RPA following CTC1 KO, suggesting that the primary function of CST is in telomere end protection and maintenance not genome-wide replication.However, despite increased RPA-ssDNA, global CHK1 phosphorylation was not detected in CTC1 KO cells. Further analysis revealed that CTC1 KO significantly inhibits CHK1 phosphorylation following hydroxyurea-induced replication stress, due to decreased levels of the ATR activator TopBP1. Overall, our results identify that telomere not genome-wide DNA damaging signaling leads to decrease proliferation following CTC1 deletion and that CST promotes ATR-CHK1 signaling through the regulation of TopBP1.

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Section: Deletion Of Ctc1 Leads To Increased Telomeric But Not Genomesupporting

confidence: 73%

Section: Immunofluorescence (If) and If-fluorescence In Situ Hybridizmentioning

confidence: 99%

Human CTC1 primarily functions in telomere maintenance/protection and promotes CHK1 phosphorylation in response to global replication stress

Ackerson

Gable

Stewart

2020

Preprint

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“…The telomere‐specific fluorescence in situ hybridization (FISH) was performed as previously described . Briefly, cleared and deparaffinized sections were boiled in 10 mM citrate solution (pH 6) for 20 minutes.…”

Section: Methodsmentioning

confidence: 99%

“…The telomere-specific fluorescence in situ hybridization (FISH) was performed as previously described. 14 weak-nuclear staining was present in >50% of cells or strong nuclear staining >10% of cells as previously described.…”

Section: Introductionmentioning

confidence: 99%

Assessment of ARX expression, a novel biomarker for metastatic risk in pancreatic neuroendocrine tumors, in endoscopic ultrasound fine‐needle aspiration

Hackeng

Morsink

Moons

et al. 2019

Diagnostic Cytopathology

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Background The transcription factors ARX and PDX1, and alternative lengthening of telomeres (ALT) were recently described as prognostic markers for resected non‐functional pancreatic neuroendocrine tumors (PanNETs). ALT positive tumors with ARX expression relapse most often. Currently, tumor size is the only preoperative marker used to decide whether or not to operate, thus additional preoperative prognostic markers are needed. Therefore, it is critical to assess the performance of these biomarkers on preoperative cytologic specimens. Methods Endoscopic fine‐needle aspiration cellblock material and corresponding surgical specimens of 13 patients with PanNETs were assessed for histology, immunohistochemical staining of ARX, PDX1, Synaptophysin, Ki67, and telomere‐specific fluorescence in situ hybridization to detect ALT, and then associated with clinicopathological features. Scoring for ARX and PDX1 was performed blinded by two independent observers. Results Of the 13 surgical specimens, 8 were ARX+/PDX1−, 2 ARX−/PDX1+, and 3 ARX+/PDX1+. Concordance between cytologic and surgical specimens for ARX protein expression was 100%, whereas concordance for PDX1, ALT, and WHO tumor grade was 85%, 91%, and 73%, respectively. There was a perfect inter‐observer agreement in ARX and PDX1 scoring. Conclusion ARX can reliably be determined in cytologic specimens and has low inter‐observer variability. For cytology, false‐positive PDX1 expression was observed, possibly due to contamination or sampling, while ALT had a false‐negative case due to incomplete sampling. As previously observed, tumor grade is underestimated in cytologic specimens. Thus, ARX and ALT are the most promising markers to predict metastatic behavior in PanNETs, thereby warranting further validation in larger studies.

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“…Conventional fluorescent in situ hybridization was performed as described previously [49] with 537 modifications as described below. Briefly, cells were exposed to 100 ng mL −1 colcemid for 2 hr 538 and fixed in 3:1 Methanol/Acetic acid for 6 minutes.…”

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confidence: 99%

A Single Defined Sister Chromatid Fusion Destabilizes Cell Cycle through Micronuclei Formation

Kagaya

Noma

Yamamoto

et al. 2019

Preprint

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Chromosome fusion is deleterious among oncogenic chromosome rearrangements, and has been proposed to cause multiple tumor-driving abnormalities. Conventional methodologies, however, lack the strictness of the experimental controls on the fusion such as the exact timing, the number and the types of fusion in a given cell. Here, we developed a human cell-based sister chromatid fusion visualization system (FuVis), in which a single defined sister chromatid fusion is induced by CRISPR/Cas9 concomitantly with mCitrine expression. Fused chromosome developed numerical and structural abnormalities, including chromosome fragmentation, an indicative of eventual chromothripsis. Live cell imaging and hierarchical Bayesian modeling indicated that micronucleus (MN) is generated in the first few cell cycle, and that cells with MN tend to possess cell cycle abnormalities. These results demonstrate that, although most cells can tolerate a single fusion, even a single sister chromatid fusion destabilizes cell cycle through MN formation.

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Visualization of Telomere Integrity and Function In Vitro and In Vivo Using Immunofluorescence Techniques

Cited by 47 publications

References 40 publications

Human CTC1 primarily functions in telomere maintenance/protection and promotes CHK1 phosphorylation in response to global replication stress

Human CTC1 primarily functions in telomere maintenance/protection and promotes CHK1 phosphorylation in response to global replication stress

Assessment of ARX expression, a novel biomarker for metastatic risk in pancreatic neuroendocrine tumors, in endoscopic ultrasound fine‐needle aspiration

A Single Defined Sister Chromatid Fusion Destabilizes Cell Cycle through Micronuclei Formation

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