G1 and G2 cell-cycle arrest following microtubule depolymerization in human breast cancer cells

Blajeski, April L.; Phan, Vy; Kottke, Timothy; Kaufmann, Scott H.

doi:10.1172/jci200213275

Cited by 31 publications

(20 citation statements)

References 42 publications

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“…4B). Presence of cyclin D3-positive and tetraploid cells in the G1/S interphase confirms nocodazole effect on MCF-7 cell cycle [24].…”

Section: In Vitro Drug Effect Profiling By Imcmentioning

confidence: 54%

“…276855). Final concentrations for each compound were selected from previously published data [24][25][26] by premixing the required initial drug stock volume with full growth media and then transferring 0.5 mL volume to corresponding cell chambers (Table S1): 10 lM of etoposide (Cell Signaling Technology Ò , Danvers, MA, USA; Cat. No.…”

Section: In Vitro Drug Treatmentmentioning

confidence: 99%

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Multidimensional profiling of drug‐treated cells by Imaging Mass Cytometry

2019

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In pharmaceutical research, high‐content screening is an integral part of lead candidate development. Measuring drug response in vitro by examining over 40 parameters, including biomarkers, signaling molecules, cell morphological changes, proliferation indices, and toxicity in a single sample, could significantly enhance discovery of new therapeutics. As a proof of concept, we present here a workflow for multidimensional Imaging Mass Cytometry™ (IMC™) and data processing with open source computational tools. CellProfiler was used to identify single cells through establishing cellular boundaries, followed by histoCAT™ (histology topography cytometry analysis toolbox) for extracting single‐cell quantitative information visualized as t‐SNE plots and heatmaps. Human breast cancer‐derived cell lines SKBR3, HCC1143, and MCF‐7 were screened for expression of cellular markers to generate digital images with a resolution comparable to conventional fluorescence microscopy. Predicted pharmacodynamic effects were measured in MCF‐7 cells dosed with three target‐specific compounds: growth stimulatory EGF, microtubule depolymerization agent nocodazole, and genotoxic chemotherapeutic drug etoposide. We show strong pairwise correlation between nuclear markers pHistone3 S28 , Ki‐67, and p4E‐BP1 T37/T46 in classified mitotic cells and anticorrelation with cell surface markers. Our study demonstrates that IMC data expand the number of measured parameters in single cells and brings higher‐dimension analysis to the field of cell‐based screening in early lead compound discovery.

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“…4B). Presence of cyclin D3-positive and tetraploid cells in the G1/S interphase confirms nocodazole effect on MCF-7 cell cycle [24].…”

Section: In Vitro Drug Effect Profiling By Imcmentioning

confidence: 54%

Section: In Vitro Drug Treatmentmentioning

confidence: 99%

Multidimensional profiling of drug‐treated cells by Imaging Mass Cytometry

2019

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“…MSC cell cycle has been shown to be only partially affected by nocodazole (Polioudaki, Kastrinaki, Papadaki, & Theodoropoulos, 2009), whereas cancer cells enter cell cycle arrest post-nocodazole treatment (Blajeski, Phan, Kottke, & Kaufmann, 2002), and hence there may be secondary effects on other processes such as mitosis that may explain the differential effects of nocodazole on confined migration between hMSCs and cancer cells. Future investigations may elucidate the cause for discrepancies between these cell types.…”

Section: Discussionmentioning

confidence: 99%

Physical confinement alters cytoskeletal contributions towards human mesenchymal stem cell migration

Doolin

Stroka

2018

Cytoskeleton

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The in vivo microenvironment is critical for providing physico-chemical signaling cues which ultimately regulate human mesenchymal stem cell (hMSC) behavior in clinically-relevant applications. hMSCs experience mechanical confinement of the cell body and nucleus in three dimensional (3D) tissues during homing and in porous tissue engineered scaffolds, yet the effects of this mechanical cue on hMSC migration are not known. Here, we use a microchannel device to systematically examine the effect of confinement on hMSC migration and cytoskeletal organization. Notably, we show that hMSC actin and microtubules change from filamentous in unconfined spaces to a more diffuse network in confinement, and that confinement abrogates the presence of paxillin-rich focal adhesions seen in 2D. Furthermore, several morphological parameters of the hMSC body are altered in confinement. Interestingly, hMSC speed displays a biphasic trend as a function of confinement, and increasing hMSC passage number decreases speed in all but the narrowest microchannels. Confinement also alters the relative contributions of cytoskeletal (i.e., actin and microtubule) and contractile (i.e., myosin II and Rho kinase) machinery in hMSC migration in unconfined and confined spaces. These results provide an improved understanding of how hMSCs navigate mechanical confinement, which is a central component of complicated 3D microenvironments. Hence, this work may provide insight towards more effective control of hMSC localization in porous tissue engineered scaffolds and mobilization to distinct tissue sites during homing after clinical therapy.

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“…Stabilization of MTs by paclitaxel does not slow progression through interphase, but rather blocks cells in mitosis [Uetake and Sluder, 2007]. Others have shown that MT depolymerization slows cell cycle progression, particularly during G2 [Rieder and Cole, 2000;Blajeski et al, 2002;Balestra and Jimenez, 2008], but Uetake and Sluder [2007] have argued that an interphase MT integrity checkpoint does not exist; rather it is the length of the previous mitosis that matters. Taken together, these data suggest that a drug-induced change in MT stability does not alter cell cycle progression outside of M phase, raising the possibility that stathmin depletion does not slow G2 via increased MT stability.…”

Section: Introductionmentioning

confidence: 99%

The microtubule cytoskeleton is required for a G2 cell cycle delay in cancer cells lacking stathmin and p53

2012

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In several cancer cell lines, depleting the microtubule-destabilizing protein stathmin/oncoprotein18 leads to a G2 cell cycle delay and apoptosis. These phenotypes are observed only in synergy with low levels of p53, but the pathway(s) activated by stathmin depletion to delay the cell cycle are unknown. We found that stathmin depletion caused greater microtubule stability in synergy with loss of p53, measured by the levels of acetylated α-tubulin and the rate of centrosomal microtubule nucleation. Nocodazole or vinblastine-induced microtubule depolymerization abrogated the stathmin-depletion induced G2 delay, measured by the percentage of cells staining positive for several markers (TPX2, CDK1 with inhibitory phosphorylation), indicating that microtubules are required to lengthen G2. Live cell imaging showed that stathmin depletion increased time in G2 without an impact on the duration of mitosis, indicating that the longer interphase duration is not simply a consequence of a previous slowed mitosis. In contrast, stabilization of microtubules with paclitaxel (8 nM) slowed mitosis without lengthening the duration of interphase, demonstrating that increased microtubule stability alone is not sufficient to delay cells in G2.

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G1 and G2 cell-cycle arrest following microtubule depolymerization in human breast cancer cells

Cited by 31 publications

References 42 publications

Multidimensional profiling of drug‐treated cells by Imaging Mass Cytometry

Multidimensional profiling of drug‐treated cells by Imaging Mass Cytometry

Physical confinement alters cytoskeletal contributions towards human mesenchymal stem cell migration

The microtubule cytoskeleton is required for a G2 cell cycle delay in cancer cells lacking stathmin and p53

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