Dynamic Monitoring of Cellular Remodeling Induced by the Transforming Growth Factor-β1

Staršíchová, Andrea; Kubala, Lukáš; Lincová, Eva; Pernicová, Zuzana; Kozubı́k, Alois; Souček, Karel

doi:10.1007/s12575-009-9017-9

Cited by 13 publications

(22 citation statements)

References 10 publications

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“…These observations are in agreement with a previously published study by Jacksch et al demonstrating the higher colony-forming capacity of CD133 high Caco2 colon cancer cells (22). Label-free RTCA based on the detection of cellular impedance had great potential in both basic and applied research (16,17,23,24). In this study, we revealed for the first time that it is possible to combine the direct deposition of a limited number of immunolabeled cells with long-term, real-time, label-free analysis of cell proliferation.…”

Section: Discussionsupporting

confidence: 91%

Automatic cell cloning assay for determining the clonogenic capacity of cancer and cancer stem‐like cells

et al. 2013

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The clonogenic assay is a well-established in vitro method for testing the survival and proliferative capability of cells. It can be used to determine the cytotoxic effects of various treatments including chemotherapeutics and ionizing radiation. However, this approach can also characterize cells with different phenotypes and biological properties, such as stem cells or cancer stem cells. In this study, we implemented a faster and more precise method for assessing the cloning efficiency of cancer stem-like cells that were characterized and separated using a high-speed cell sorter. Cell plating onto a microplate using an automatic cell deposition unit was performed in a single-cell or dilution rank mode by the fluorescence-activated cell sorting method. We tested the new automatic cell-cloning assay (ACCA) on selected cancer cell lines and compared it with the manual approach. The obtained results were also compared with the results of the limiting dilution assay for different cell lines. We applied the ACCA to analyze the cloning capacity of different subpopulations of prostate and colon cancer cells based on the expression of the characteristic markers of stem (CD44 and CD133) and cancer stem cells (TROP-2, CD49f, and CD44). Our results revealed that the novel ACCA is a straightforward approach for determining the clonogenic capacity of cancer stem-like cells identified in both cell lines and patient samples. ' 2013 International Society for Advancement of Cytometry

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Section: Discussionsupporting

confidence: 91%

Automatic cell cloning assay for determining the clonogenic capacity of cancer and cancer stem‐like cells

et al. 2013

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“…12,13 Such hypothesis was confirmed in Figure 6e as we could observe an induction of Figure S4d). 43 Altogether, these data clearly suggest that the absence of TAp73 impairs TGF-β signaling toward the tumor suppressing SMAD4-dependent pathway.…”

Section: Resultsmentioning

confidence: 81%

TAp73 loss favors Smad-independent TGF-β signaling that drives EMT in pancreatic ductal adenocarcinoma

et al. 2016

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Advances made in pancreatic cancer therapy have been far from sufficient and have allowed only a slight improvement in global survival of patients with pancreatic ductal adenocarcinoma (PDA). Recent progresses in chemotherapy have offered some hope for an otherwise gloomy outlook, however, only a limited number of patients are eligible because of important cytotoxicity. In this context, enhancing our knowledge on PDA initiation and evolution is crucial to highlight certain weaknesses on which to specifically target therapy. We found that loss of transcriptionally active p73 (TAp73), a p53 family member, impacted PDA development. In two relevant and specific engineered pancreatic cancer mouse models, we observed that TAp73 deficiency reduced survival and enhanced epithelial-to-mesenchymal transition (EMT). Through proteomic analysis of conditioned media from TAp73 wild-type (WT) and deficient pancreatic tumor cells, we identified a secreted protein, biglycan (BGN), which is necessary and sufficient to mediate this pro-EMT effect. Interestingly, BGN is modulated by and modulates the transforming growth factor-β (TGF-β) pathway, a key regulator of the EMT process. We further examined this link and revealed that TAp73 impacts the TGF-β pathway by direct regulation of BGN expression and Sma and Mad-related proteins (SMADs) expression/ activity. Absence of TAp73 leads to activation of TGF-β signaling through a SMAD-independent pathway, favoring oncogenic TGF-β effects and EMT. Altogether, our data highlight the implication of TAp73 in the aggressiveness of pancreatic carcinogenesis through modulation of the TGF-β signaling. By suggesting TAp73 as a predictive marker for response to TGF-β inhibitors, our study could improve the classification of PDA patients with a view to offering combined therapy involving TGF-β inhibitors.

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“…Growth factor-induced morphological changes can be quantified in cells by measuring impedance (36,37), which provides a real-time, noninvasive analysis suitable for measuring the kinetics of short-and long-term cellular responses. Changes in cellular impedance triggered by agents such as EGF and insulin have been shown to correlate with those measured by conventional means [e.g., autophosphorylation of the EGF receptor as measured by ELISA (36)], and impedance now is commonly used for assessing morphological changes such as membrane ruffling or formation of lamellipodia in a quantita- tive fashion.…”

Section: Resultsmentioning

confidence: 99%

Phosphodiesterase-8A binds to and regulates Raf-1 kinase

Brown¹,

Day²,

Huston³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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V-raf-1 murine leukemia viral oncogene homolog 1 (Raf-1) is a key activator of the ERK pathway and is a target for cross-regulation of this pathway by the cAMP signaling system. The cAMP-activated protein kinase, PKA, inhibits Raf-1 by phosphorylation on S259. Here, we show that the cAMP-degrading phosphodiesterase-8A (PDE8A) associates with Raf-1 to protect it from inhibitory phosphorylation by PKA, thereby enhancing Raf-1's ability to stimulate ERK signaling. PDE8A binds to Raf-1 with high (picomolar) affinity. Mapping of the interaction domain on PDE8A using peptide array technology identified amino acids 454-465 as the main binding site, which could be disrupted by mutation. A cell-permeable peptide corresponding to this region disrupted the PDE8A/Raf-1 interaction in cells, thereby reducing ERK activation and the cellular response to EGF. Overexpression of a catalytically inactive PDE8A in cells displayed a dominant negative phenotype on ERK activation. These effects were recapitulated at the organism level in genetically modified (PDE8A −/− ) mice. Similarly, PDE8 deletion in Drosophila melanogaster reduced basal ERK activation and sensitized flies to stress-induced death. We propose that PDE8A is a physiological regulator of Raf-1 signaling in some cells.

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Dynamic Monitoring of Cellular Remodeling Induced by the Transforming Growth Factor-β1

Cited by 13 publications

References 10 publications

Automatic cell cloning assay for determining the clonogenic capacity of cancer and cancer stem‐like cells

Automatic cell cloning assay for determining the clonogenic capacity of cancer and cancer stem‐like cells

TAp73 loss favors Smad-independent TGF-β signaling that drives EMT in pancreatic ductal adenocarcinoma

Phosphodiesterase-8A binds to and regulates Raf-1 kinase

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