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High-content, high-throughput analysis of cell cycle perturbations induced by the HSP90 inhibitor XL888.

Lyman SK, Crawley SC, Gong R, Adamkewicz JI, McGrath G, Chew JY, Choi J, Holst CR, Goon LH, Detmer SA, Vaclavikova J, Gerritsen ME, Blake RA - PLoS ONE (2011)

Bottom Line: We additionally observed unexpected complexity in the response of the cell cycle-associated client PLK1 to HSP90 inhibition, and we suggest that inhibitor-induced PLK1 depletion may contribute to the striking metaphase arrest phenotype seen in many of the M-arrested cell lines.M-phase arrest correlated with the presence of TP53 mutations, while G2 or G1 arrest was more commonly seen in cells bearing wt TP53.We draw upon previous literature to suggest an integrated model that accounts for these varying observations.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Pharmacology, Exelixis, Inc., South San Francisco, California, United States of America. xl888.mail@gmail.com

ABSTRACT

Background: Many proteins that are dysregulated or mutated in cancer cells rely on the molecular chaperone HSP90 for their proper folding and activity, which has led to considerable interest in HSP90 as a cancer drug target. The diverse array of HSP90 client proteins encompasses oncogenic drivers, cell cycle components, and a variety of regulatory factors, so inhibition of HSP90 perturbs multiple cellular processes, including mitogenic signaling and cell cycle control. Although many reports have investigated HSP90 inhibition in the context of the cell cycle, no large-scale studies have examined potential correlations between cell genotype and the cell cycle phenotypes of HSP90 inhibition.

Methodology/principal findings: To address this question, we developed a novel high-content, high-throughput cell cycle assay and profiled the effects of two distinct small molecule HSP90 inhibitors (XL888 and 17-AAG [17-allylamino-17-demethoxygeldanamycin]) in a large, genetically diverse panel of cancer cell lines. The cell cycle phenotypes of both inhibitors were strikingly similar and fell into three classes: accumulation in M-phase, G2-phase, or G1-phase. Accumulation in M-phase was the most prominent phenotype and notably, was also correlated with TP53 mutant status. We additionally observed unexpected complexity in the response of the cell cycle-associated client PLK1 to HSP90 inhibition, and we suggest that inhibitor-induced PLK1 depletion may contribute to the striking metaphase arrest phenotype seen in many of the M-arrested cell lines.

Conclusions/significance: Our analysis of the cell cycle phenotypes induced by HSP90 inhibition in 25 cancer cell lines revealed that the phenotypic response was highly dependent on cellular genotype as well as on the concentration of HSP90 inhibitor and the time of treatment. M-phase arrest correlated with the presence of TP53 mutations, while G2 or G1 arrest was more commonly seen in cells bearing wt TP53. We draw upon previous literature to suggest an integrated model that accounts for these varying observations.

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Timelapse analysis of XL888-treated cells.(A) Timelapse movie frames showing CHL-1 cells treated with DMSO, 123 nM XL888, or 100 nM taxol (paclitaxel) for 18 h. Note the different morphology of metaphase-arrested XL888-treated cells vs. prometaphase-arrested taxol-treated cells. (B) Timelapse movie frames showing CHL1 cells treated with DMSO or 123 nM XL888 for the indicated times. In this panel (B) as well as in panels (C) and (D), the same microscope field is shown at successive timepoints. In the 8 h and 18 h panels, some examples of XL888-treated cells displaying the linear, metaphase-like morphology are highlighted with white arrowheads; in the 32 h panel some examples of dead or dying cells are highlighted with yellow arrowheads. (C) Timelapse movie frames showing A549 cells treated with DMSO or 370 nM XL888 for the indicated times. In the 18 h panels showing XL888-treated cells, some examples of probable G2-arrested cells (based on cell size and lack of division) are highlighted with white arrowheads; in the 32 h panel, some examples of dead or dying cells are highlighted with yellow arrowheads (D) Timelapse movie frames showing A375 cells treated with DMSO or 123 nM XL888 for the indicated times. In the XL888-treated cells, some examples of the “linear quasi-metaphase” morphology are highlighted with white arrowheads (8 h, 14 h); some examples of dead or dying cells are highlighted with yellow arrowheads(32 h). For all three cell lines, 17-AAG effects were similar to those of XL888 (data not shown). Experiments were performed at least two times, and results from independent trials were consistent.
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pone-0017692-g006: Timelapse analysis of XL888-treated cells.(A) Timelapse movie frames showing CHL-1 cells treated with DMSO, 123 nM XL888, or 100 nM taxol (paclitaxel) for 18 h. Note the different morphology of metaphase-arrested XL888-treated cells vs. prometaphase-arrested taxol-treated cells. (B) Timelapse movie frames showing CHL1 cells treated with DMSO or 123 nM XL888 for the indicated times. In this panel (B) as well as in panels (C) and (D), the same microscope field is shown at successive timepoints. In the 8 h and 18 h panels, some examples of XL888-treated cells displaying the linear, metaphase-like morphology are highlighted with white arrowheads; in the 32 h panel some examples of dead or dying cells are highlighted with yellow arrowheads. (C) Timelapse movie frames showing A549 cells treated with DMSO or 370 nM XL888 for the indicated times. In the 18 h panels showing XL888-treated cells, some examples of probable G2-arrested cells (based on cell size and lack of division) are highlighted with white arrowheads; in the 32 h panel, some examples of dead or dying cells are highlighted with yellow arrowheads (D) Timelapse movie frames showing A375 cells treated with DMSO or 123 nM XL888 for the indicated times. In the XL888-treated cells, some examples of the “linear quasi-metaphase” morphology are highlighted with white arrowheads (8 h, 14 h); some examples of dead or dying cells are highlighted with yellow arrowheads(32 h). For all three cell lines, 17-AAG effects were similar to those of XL888 (data not shown). Experiments were performed at least two times, and results from independent trials were consistent.

Mentions: We used live-cell timelapse analysis to further characterize the cell cycle perturbations induced by HSP90 inhibition. CHL-1 (M-class), A549 (G2-class), and A375 (G1-class) cells were stably transfected with a histone-H2B-GFP plasmid to fluorescently mark chromatin, then treated with XL888 and imaged every 30 min for 36–48 h to track cell fate. Timelapse analysis (Figure 6A–B) revealed that XL888-treated CHL-1 cells arrested in M with highly organized chromosomes in a linear, metaphase-like configuration (as did other M-class cells; data not shown). It is notable that this metaphase-like phenotype was very different from the disorganized chromatin and prometaphase arrest that typically result from treatment with checkpoint-activating agents such as taxol (paclitaxel). The distinctive linear chromosome configuration in XL888-treated cells persisted for up to 16–18 h, although with increasing time, it became somewhat more disorganized, and some lagging chromosomes began to appear. Eventually, after prolonged M-arrest, CHL-1 cells underwent cell death without exiting from mitosis.


High-content, high-throughput analysis of cell cycle perturbations induced by the HSP90 inhibitor XL888.

Lyman SK, Crawley SC, Gong R, Adamkewicz JI, McGrath G, Chew JY, Choi J, Holst CR, Goon LH, Detmer SA, Vaclavikova J, Gerritsen ME, Blake RA - PLoS ONE (2011)

Timelapse analysis of XL888-treated cells.(A) Timelapse movie frames showing CHL-1 cells treated with DMSO, 123 nM XL888, or 100 nM taxol (paclitaxel) for 18 h. Note the different morphology of metaphase-arrested XL888-treated cells vs. prometaphase-arrested taxol-treated cells. (B) Timelapse movie frames showing CHL1 cells treated with DMSO or 123 nM XL888 for the indicated times. In this panel (B) as well as in panels (C) and (D), the same microscope field is shown at successive timepoints. In the 8 h and 18 h panels, some examples of XL888-treated cells displaying the linear, metaphase-like morphology are highlighted with white arrowheads; in the 32 h panel some examples of dead or dying cells are highlighted with yellow arrowheads. (C) Timelapse movie frames showing A549 cells treated with DMSO or 370 nM XL888 for the indicated times. In the 18 h panels showing XL888-treated cells, some examples of probable G2-arrested cells (based on cell size and lack of division) are highlighted with white arrowheads; in the 32 h panel, some examples of dead or dying cells are highlighted with yellow arrowheads (D) Timelapse movie frames showing A375 cells treated with DMSO or 123 nM XL888 for the indicated times. In the XL888-treated cells, some examples of the “linear quasi-metaphase” morphology are highlighted with white arrowheads (8 h, 14 h); some examples of dead or dying cells are highlighted with yellow arrowheads(32 h). For all three cell lines, 17-AAG effects were similar to those of XL888 (data not shown). Experiments were performed at least two times, and results from independent trials were consistent.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3049797&req=5

pone-0017692-g006: Timelapse analysis of XL888-treated cells.(A) Timelapse movie frames showing CHL-1 cells treated with DMSO, 123 nM XL888, or 100 nM taxol (paclitaxel) for 18 h. Note the different morphology of metaphase-arrested XL888-treated cells vs. prometaphase-arrested taxol-treated cells. (B) Timelapse movie frames showing CHL1 cells treated with DMSO or 123 nM XL888 for the indicated times. In this panel (B) as well as in panels (C) and (D), the same microscope field is shown at successive timepoints. In the 8 h and 18 h panels, some examples of XL888-treated cells displaying the linear, metaphase-like morphology are highlighted with white arrowheads; in the 32 h panel some examples of dead or dying cells are highlighted with yellow arrowheads. (C) Timelapse movie frames showing A549 cells treated with DMSO or 370 nM XL888 for the indicated times. In the 18 h panels showing XL888-treated cells, some examples of probable G2-arrested cells (based on cell size and lack of division) are highlighted with white arrowheads; in the 32 h panel, some examples of dead or dying cells are highlighted with yellow arrowheads (D) Timelapse movie frames showing A375 cells treated with DMSO or 123 nM XL888 for the indicated times. In the XL888-treated cells, some examples of the “linear quasi-metaphase” morphology are highlighted with white arrowheads (8 h, 14 h); some examples of dead or dying cells are highlighted with yellow arrowheads(32 h). For all three cell lines, 17-AAG effects were similar to those of XL888 (data not shown). Experiments were performed at least two times, and results from independent trials were consistent.
Mentions: We used live-cell timelapse analysis to further characterize the cell cycle perturbations induced by HSP90 inhibition. CHL-1 (M-class), A549 (G2-class), and A375 (G1-class) cells were stably transfected with a histone-H2B-GFP plasmid to fluorescently mark chromatin, then treated with XL888 and imaged every 30 min for 36–48 h to track cell fate. Timelapse analysis (Figure 6A–B) revealed that XL888-treated CHL-1 cells arrested in M with highly organized chromosomes in a linear, metaphase-like configuration (as did other M-class cells; data not shown). It is notable that this metaphase-like phenotype was very different from the disorganized chromatin and prometaphase arrest that typically result from treatment with checkpoint-activating agents such as taxol (paclitaxel). The distinctive linear chromosome configuration in XL888-treated cells persisted for up to 16–18 h, although with increasing time, it became somewhat more disorganized, and some lagging chromosomes began to appear. Eventually, after prolonged M-arrest, CHL-1 cells underwent cell death without exiting from mitosis.

Bottom Line: We additionally observed unexpected complexity in the response of the cell cycle-associated client PLK1 to HSP90 inhibition, and we suggest that inhibitor-induced PLK1 depletion may contribute to the striking metaphase arrest phenotype seen in many of the M-arrested cell lines.M-phase arrest correlated with the presence of TP53 mutations, while G2 or G1 arrest was more commonly seen in cells bearing wt TP53.We draw upon previous literature to suggest an integrated model that accounts for these varying observations.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Pharmacology, Exelixis, Inc., South San Francisco, California, United States of America. xl888.mail@gmail.com

ABSTRACT

Background: Many proteins that are dysregulated or mutated in cancer cells rely on the molecular chaperone HSP90 for their proper folding and activity, which has led to considerable interest in HSP90 as a cancer drug target. The diverse array of HSP90 client proteins encompasses oncogenic drivers, cell cycle components, and a variety of regulatory factors, so inhibition of HSP90 perturbs multiple cellular processes, including mitogenic signaling and cell cycle control. Although many reports have investigated HSP90 inhibition in the context of the cell cycle, no large-scale studies have examined potential correlations between cell genotype and the cell cycle phenotypes of HSP90 inhibition.

Methodology/principal findings: To address this question, we developed a novel high-content, high-throughput cell cycle assay and profiled the effects of two distinct small molecule HSP90 inhibitors (XL888 and 17-AAG [17-allylamino-17-demethoxygeldanamycin]) in a large, genetically diverse panel of cancer cell lines. The cell cycle phenotypes of both inhibitors were strikingly similar and fell into three classes: accumulation in M-phase, G2-phase, or G1-phase. Accumulation in M-phase was the most prominent phenotype and notably, was also correlated with TP53 mutant status. We additionally observed unexpected complexity in the response of the cell cycle-associated client PLK1 to HSP90 inhibition, and we suggest that inhibitor-induced PLK1 depletion may contribute to the striking metaphase arrest phenotype seen in many of the M-arrested cell lines.

Conclusions/significance: Our analysis of the cell cycle phenotypes induced by HSP90 inhibition in 25 cancer cell lines revealed that the phenotypic response was highly dependent on cellular genotype as well as on the concentration of HSP90 inhibitor and the time of treatment. M-phase arrest correlated with the presence of TP53 mutations, while G2 or G1 arrest was more commonly seen in cells bearing wt TP53. We draw upon previous literature to suggest an integrated model that accounts for these varying observations.

Show MeSH
Related in: MedlinePlus