Retinoblastoma Protein Prevents Staurosporine-Induced Cell Death in a Retinoblastoma-Defective Human Glioma Cell Line

Yamasaki, Fumiyuki; Kajiwara, Yoshinori; Hama, Seiji; Murakami, Taro; Hidaka, Toshikazu; Saito, Taiichi; Yoshioka, Hiroyuki; Sakamoto, Kazuhiko; Arita, Kazunori; Kurisu, Kaoru

doi:10.1159/000101048

Cited by 3 publications

(2 citation statements)

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“…Assays of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) uptake were done as described previously (21). Briefly, cells were seeded in 96-well plates at a density of 2,000 in 90 AL per well (except for A-431 cells at 200 cells in 90 AL) and cultured overnight, after which 10 AL of erlotinib solution at final concentrations of 0.03, 0.06, 0.1, 0.2, 0.3, 0.6, 1, 2, 3, 6, 10, or 20 Amol/L were added to the individual wells; plates were then incubated for 72 h at 37jC in a humidified atmosphere with 5% CO 2 .…”

Section: Introductionmentioning

confidence: 99%

Sensitivity of breast cancer cells to erlotinib depends on cyclin-dependent kinase 2 activity

Yamasaki

Zhang

Bartholomeusz

et al. 2007

Molecular Cancer Therapeutics

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Inhibitors of epidermal growth factor receptor (EGFR) tyrosine kinases, such as erlotinib and gefitinib, have not been very effective in the treatment of breast cancer although many breast cancer cells express EGFR. To address this apparent paradox, we examined possible predictors of the sensitivity of 10 breast cancer cell lines to erlotinib in light of cyclin-dependent kinase 2 (CDK2), considered the farthest downstream kinase that controls cell cycling in the EGFR signaling pathway. Expression of EGFR and HER2 were not associated with sensitivity to erlotinib. Expression of phosphorylated (p-)tyrosine, p-Akt, phosphorylated extracellular signal-regulated kinase (p-ERK) 1/ERK2 (p42/p44), and p27 after treatment of erlotinib was not associated with erlotinib sensitivity. However, suppression of CDK2 activity after erlotinib treatment correlated with erlotinib sensitivity (P < 0.0001). Restoration of CDK2 activity partially restored proliferation and induced erlotinib resistance in erlotinibsensitive cell lines, indicating that sensitivity to erlotinib in these breast cancer cells depends, at least in part, on CDK2 activity. p27, an inhibitor of CDK2, was not translocated into the nucleus in erlotinib-resistant cell lines. Knocking down p27 protein partially blocked erlotinib-induced cell death and cell cycle arrest. These findings indicate that the ability of erlotinib to suppress CDK2 activity is critical for cellular sensitivity to erlotinib, regardless of EGFR expression level, and that the presence of p27 in the cytoplasm also participates in erlotinib resistance.

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Section: Introductionmentioning

confidence: 99%

Sensitivity of breast cancer cells to erlotinib depends on cyclin-dependent kinase 2 activity

Yamasaki

Zhang

Bartholomeusz

et al. 2007

Molecular Cancer Therapeutics

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“…We treated this cell line with increasing concentrations of two different staurosporines (UCN-01 and staurosporine), two drugs that are known to inhibit cell proliferation of this and other cell lines with slightly different potencies [18]–[22]. Staurosporines potently inhibit a wide range of serine/threonine and tyrosine protein kinases, in particular protein kinase C (PKC) [20], [21], [23].…”

Section: Resultsmentioning

confidence: 99%

ColonyArea: An ImageJ Plugin to Automatically Quantify Colony Formation in Clonogenic Assays

et al. 2014

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The clonogenic or colony formation assay is a widely used method to study the number and size of cancer cell colonies that remain after irradiation or cytotoxic agent administration and serves as a measure for the anti-proliferative effect of these treatments. Alternatively, this assay is used to quantitate the transforming potential of cancer associated genes and chemical agents. Therefore, there is a need for a simplified and standardized analysis of colony formation assays for both routine laboratory use and for parallelized automated analysis. Here we describe the freely available ImageJ-plugin “ColonyArea”, which is optimized for rapid and quantitative analysis of focus formation assays conducted in 6- to 24-well dishes. ColonyArea processes image data of multi-well dishes, by separating, concentrically cropping and background correcting well images individually, before colony formation is quantitated. Instead of counting the number of colonies, ColonyArea determines the percentage of area covered by crystal violet stained cell colonies, also taking the intensity of the staining and therefore cell density into account. We demonstrate that these parameters alone or in combination allow for robust quantification of IC50 values of the cytotoxic effect of two staurosporines, UCN-01 and staurosporine (STS) on human glioblastoma cells (T98G). The relation between the potencies of the two compounds compared very well with that obtained from an absorbance based method to quantify colony growth and to published data. The ColonyArea ImageJ plugin provides a simple and efficient analysis routine to quantitate assay data of one of the most commonly used cellular assays. The bundle is freely available for download as supporting information. We expect that ColonyArea will be of broad utility for cancer biologists, as well as clinical radiation scientists.

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2008

Current Opinion in Ophthalmology

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Retinoblastoma Protein Prevents Staurosporine-Induced Cell Death in a Retinoblastoma-Defective Human Glioma Cell Line

Cited by 3 publications

References 93 publications

Sensitivity of breast cancer cells to erlotinib depends on cyclin-dependent kinase 2 activity

Sensitivity of breast cancer cells to erlotinib depends on cyclin-dependent kinase 2 activity

ColonyArea: An ImageJ Plugin to Automatically Quantify Colony Formation in Clonogenic Assays

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