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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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Timecourse analysis of cell cycle profiles and PLK1 levels.Cells were treated with the indicated concentrations of XL888 (14, 41, 123, 370, 1110, 1670 nM) at time = 0. Plates were then fixed at 4, 8, 12, 24, or 36 h and stained for a modified version of HC cell cycle analysis that allows for simultaneous detection of cell cycle phenotypes and of an additional marker protein, PLK1. For a given cell line, profiles of %G1 and %S were nearly identical to the %G1 and %S data shown in Figure 5, so those data are not displayed here; see Figure 5 for reference. See text for discussion. (A) CHL-1 (B) EBC-1 (C) A549 (D) A375. 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-g007: Timecourse analysis of cell cycle profiles and PLK1 levels.Cells were treated with the indicated concentrations of XL888 (14, 41, 123, 370, 1110, 1670 nM) at time = 0. Plates were then fixed at 4, 8, 12, 24, or 36 h and stained for a modified version of HC cell cycle analysis that allows for simultaneous detection of cell cycle phenotypes and of an additional marker protein, PLK1. For a given cell line, profiles of %G1 and %S were nearly identical to the %G1 and %S data shown in Figure 5, so those data are not displayed here; see Figure 5 for reference. See text for discussion. (A) CHL-1 (B) EBC-1 (C) A549 (D) A375. 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 postulated that depletion of the client protein PLK1 could be contributing to the metaphase arrest phenotype. PLK1 is involved in entry into M, mitotic exit, and cytokinesis, and its depletion has been shown to result in an inability to complete mitosis [23], [26], [27]. To determine if reduced PLK1 levels correlated with M-phase accumulation, we used a modified version of the cell cycle assay (Figure 7) to simultaneously track PLK1 levels and cell cycle phase (G1, S, and G2/M) in a timecourse analysis of two M-class lines (CHL-1, EBC-1), one G2-class line (A549), and one G1-class line (A375). Because the G1 and S profiles in cells analyzed with this modified version of the assay were extremely similar to those in the standard assay (Figure 5), Figure 7 shows only PLK1 levels and the combined G2/M profile, rather than the complete G1-S-G2/M data set.


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)

Timecourse analysis of cell cycle profiles and PLK1 levels.Cells were treated with the indicated concentrations of XL888 (14, 41, 123, 370, 1110, 1670 nM) at time = 0. Plates were then fixed at 4, 8, 12, 24, or 36 h and stained for a modified version of HC cell cycle analysis that allows for simultaneous detection of cell cycle phenotypes and of an additional marker protein, PLK1. For a given cell line, profiles of %G1 and %S were nearly identical to the %G1 and %S data shown in Figure 5, so those data are not displayed here; see Figure 5 for reference. See text for discussion. (A) CHL-1 (B) EBC-1 (C) A549 (D) A375. 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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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3049797&req=5

pone-0017692-g007: Timecourse analysis of cell cycle profiles and PLK1 levels.Cells were treated with the indicated concentrations of XL888 (14, 41, 123, 370, 1110, 1670 nM) at time = 0. Plates were then fixed at 4, 8, 12, 24, or 36 h and stained for a modified version of HC cell cycle analysis that allows for simultaneous detection of cell cycle phenotypes and of an additional marker protein, PLK1. For a given cell line, profiles of %G1 and %S were nearly identical to the %G1 and %S data shown in Figure 5, so those data are not displayed here; see Figure 5 for reference. See text for discussion. (A) CHL-1 (B) EBC-1 (C) A549 (D) A375. 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 postulated that depletion of the client protein PLK1 could be contributing to the metaphase arrest phenotype. PLK1 is involved in entry into M, mitotic exit, and cytokinesis, and its depletion has been shown to result in an inability to complete mitosis [23], [26], [27]. To determine if reduced PLK1 levels correlated with M-phase accumulation, we used a modified version of the cell cycle assay (Figure 7) to simultaneously track PLK1 levels and cell cycle phase (G1, S, and G2/M) in a timecourse analysis of two M-class lines (CHL-1, EBC-1), one G2-class line (A549), and one G1-class line (A375). Because the G1 and S profiles in cells analyzed with this modified version of the assay were extremely similar to those in the standard assay (Figure 5), Figure 7 shows only PLK1 levels and the combined G2/M profile, rather than the complete G1-S-G2/M data set.

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