Human Embryonic Stem Cells Suffer from Centrosomal Amplification

Holubcová, Zuzana; Matula, Pavel; Sedláčková, Miroslava; Vinarský, Vladimír; Doležalová, Dáša; Bárta, Tomáš; Dvořák, Petr; Hampl, Aleš

doi:10.1002/stem.549

Cited by 42 publications

(45 citation statements)

References 58 publications

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“…Suppression of CDK2 expression induces cell cycle arrest and cell proliferation inhibition in human cancer (33). Another research group reported that improved attachment to culture substratum inhibition of CDK2 diminished the frequency of multicentrosomal mitoses (34). In this study, we find that CDK2 displays a higher expression in the LSCC tissue than that in the adjacent normal mucosa.…”

Section: Discussionsupporting

confidence: 58%

NF-κB induces abnormal centrosome amplification by upregulation of CDK2 in laryngeal squamous cell cancer

Liu

Ma²,

Lu³

et al. 2011

Int J Oncol

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Abstract. Centrosome amplification can drive chromosomal instability (CIN) which is a major source of tumor initiation. The present study aimed to investigate the impact of nuclear factor kappa B (NF-κB) on centrosome amplification of Hep-2 cells. Immunofluorescence was performed to display centrosomes. BAY11-7082 was used as an inhibitor of NF-κB to assess the inhibition of centrosome amplification, and cyclin-dependent kinase 2 (CDK2), ensuring cell cycle cycle coordination with centrosome cycle was detected by Western blotting. Furthermore, a 1556-bp fragment of the CDK2 promoter was analyzed using the TRANSFAC-TESS software. Luciferase assay, including a series of truncated CDK2 promoters and site mutations, was carried out to determine NF-κB binding sites in the CDK2 promoter. Electrophoresis mobility shift and chromatin immunoprecipitation assays were applied to confirm whether NF-κB indeed binds to the 5'-promoter region of the CDK2 gene. To reveal the clinical significance of CDK2 expression in laryngeal squamous cell cancer, mRNA and protein levels were assessed by RT-PCR and Western blotting, respectively. We found that the transcription factor NF-κB plays a role in centrosome amplification in Hep-2 cells. Centrosome amplification is reduced by inhibition of the NF-κB pathway. Moreover, expression of the p65 subunit of NF-κB is sufficient to promote centrosome amplification and increase in CDK2 protein levels. We further identified a functional NF-κB binding site located in the CDK2 promoter. Single mutation of the NF-κB site III (construct mutIII) however resulted in 76±5% (p<0.01) luciferase activity reduction. Electromobility shift assays and chromatin immunoprecipitaton results suggest that NF-κB indeed binds to this responsive element associating with CDK2 expression and centrosome amplification. RT-PCR and Western blotting results revealed that both mRNA and protein levels of CDK2 were significantly higher in tumor tissues than those in paired adjacent normal laryngeal tissues.

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Section: Discussionsupporting

confidence: 58%

NF-κB induces abnormal centrosome amplification by upregulation of CDK2 in laryngeal squamous cell cancer

Liu

Ma²,

Lu³

et al. 2011

Int J Oncol

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“…hESCs were grown on gelatin-coated dishes in the presence of mitotically inactivated primary mouse embryonic fibroblasts (MEFs; mouse strain CF1; density 24,000 cells per square centimeter) as described previously [13,21]. For sample collection, hESC colonies were manually detached from the cell culture dish to avoid contamination with MEFs.…”

Section: Materials and Methods Hesc Culture And Uvc Irradiationmentioning

confidence: 99%

MicroRNAs Regulate p21Waf1/Cip1 Protein Expression and the DNA Damage Response in Human Embryonic Stem Cells

et al. 2012

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Studies of human embryonic stem cells (hESCs) commonly describe the nonfunctional p53-p21 axis of the G1/ S checkpoint pathway with subsequent relevance for cell cycle regulation and the DNA damage response (DDR). Importantly, p21 mRNA is clearly present and upregulated after the DDR in hESCs, but p21 protein is not detectable. In this article, we provide evidence that expression of p21 protein is directly regulated by the microRNA (miRNA) pathway under standard culture conditions and after DNA damage. The DDR in hESCs leads to upregulation of tens of miRNAs, including hESCspecific miRNAs such as those of the miR-302 family, miR-371-372 family, or C19MC miRNA cluster. Most importantly, we show that the hESC-enriched miRNA family miR-302 (miR-302a, miR-302b, miR-302c, and miR-302d) directly contributes to regulation of p21 expression in hESCs and, thus, demonstrate a novel function for miR-302s in hESCS. The described mechanism elucidates the role of miRNAs in regulation of important molecular pathway governing the G1/S transition checkpoint before as well as after DNA damage. STEM CELLS

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“…The embryonic stem cells were treated according to the procedure described in (12). Kinetochores are protein structures on chromatids, where the spindle fibers attach during cell division to pull sister chromatids apart.…”

Section: Data Setsmentioning

confidence: 99%

“…The sequences of nucleic acids can be labeled by Fluorescence In-Situ Hybridization (FISH), which allows us to visualize parts of chromosomes such as telomeres or centromeres or even individual genes (3). It is known that the spatial distribution of subcellular objects is related to their function, and thus reliable 3D spot detection is of paramount importance in many biological applications, for example, in particle tracking (4), studies on the spatial arrangement of genes (5)(6)(7), in telomere studies (8)(9)(10)(11), kinetochore studies (12), and in co-localization studies (13). Reliable spot detection is also essential in the Photoactivation Localization Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM) imaging methods (14,15).…”

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confidence: 99%

Performance and sensitivity evaluation of 3D spot detection methods in confocal microscopy

Štěpka

Matula

et al. 2015

Cytometry Pt A

Self Cite

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Reliable 3D detection of diffraction-limited spots in fluorescence microscopy images is an important task in subcellular observation. Generally, fluorescence microscopy images are heavily degraded by noise and non-specifically stained background, making reliable detection a challenging task. In this work, we have studied the performance and parameter sensitivity of eight recent methods for 3D spot detection. The study is based on both 3D synthetic image data and 3D real confocal microscopy images. The synthetic images were generated using a simulator modeling the complete imaging setup, including the optical path as well as the image acquisition process. We studied the detection performance and parameter sensitivity under different noise levels and under the influence of uneven background signal. To evaluate the parameter sensitivity, we propose a novel measure based on the gradient magnitude of the F 1 score. We measured the success rate of the individual methods for different types of the image data and found that the type of image degradation is an important factor. Using the F 1 score and the newly proposed sensitivity measure, we found that the parameter sensitivity is not necessarily proportional to the success rate of a method. This also provided an explanation why the best performing method for synthetic data was outperformed by other methods when applied to the real microscopy images. On the basis of the results obtained, we conclude with the recommendation of the HDome method for data with relatively low variations in quality, or the Sorokin method for image sets in which the quality varies more. We also provide alternative recommendations for high-quality images, and for situations in which detailed parameter tuning might be deemed expensive. V C 2015 International Society for Advancement of Cytometry Key terms fluorescence microscopy; 3D imaging; diffraction-limited spot detection; parameter sensitivity RELIABLE spot detection in 3D confocal microscopy is an important task in cell observation. The objects of interest such as proteins or other biomolecules (e.g., sequences of nucleic acids) are typically fluorescently tagged and they appear as bright, diffraction-limited particles in 3D images. For example, the proteins of interest can be genetically modified to become fluorescent while keeping their function (1), or fluorescent antibodies can be attached to proteins (2). The sequences of nucleic acids can be labeled by Fluorescence In-Situ Hybridization (FISH), which allows us to visualize parts of chromosomes such as telomeres or centromeres or even individual genes (3). It is known that the spatial distribution of subcellular objects is related to their function, and thus reliable 3D spot detection is of paramount importance in many biological applications, for example, in particle tracking (4), studies on the spatial arrangement of genes (5-7), in telomere studies (8-11), kinetochore studies (12), and in co-localization studies (13). Reliable spot detection is also essential in the Photoac...

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Human Embryonic Stem Cells Suffer from Centrosomal Amplification

Cited by 42 publications

References 58 publications

NF-κB induces abnormal centrosome amplification by upregulation of CDK2 in laryngeal squamous cell cancer

NF-κB induces abnormal centrosome amplification by upregulation of CDK2 in laryngeal squamous cell cancer

MicroRNAs Regulate p21Waf1/Cip1 Protein Expression and the DNA Damage Response in Human Embryonic Stem Cells

Performance and sensitivity evaluation of 3D spot detection methods in confocal microscopy

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