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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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Related in: MedlinePlus

Development of the high-content (HC) cell cycle assay.(A) Markers used in the HC cell cycle assay and their distribution during the cell cycle. As defined by the HC cell cycle assay, G1 phase formally includes both G0 and G1 phases—however, for the sake of simplicity, we refer to it as “G1” rather than “G0/G1”. See text for discussion of assay development and validation. (B) Images of A549 cells stained for HC cell cycle analysis with Hoechst 33342, cyclin A, EdU, and pH3. Top panel, a field of asynchronous cycling cells; bottom panel, examples of G1, S, G2, and M cells. Bottom panels: the G2 panel shows one G2 cell (white arrowhead), one G1 cell, and one S cell; the M panel shows two M cells (white arrowheads) and three G1 cells. The G1 and S panels show exclusively G1 or S cells, respectively. The inset table summarizes the Boolean logic used to identify cell cycle phases when images are analyzed with the Cellomics Target Activation algorithm. (C) A DNA distribution plot of data derived from HC cell cycle analysis of DMSO-treated Calu-6 cells. This plot combines aspects of FACS (DNA content, as measured by total nuclear intensity of Hoechst 33342 staining) with the image-based cell cycle phase assignment, and demonstrates that the phase assignments correlate well with the DNA content expected for a given phase (i.e. G1 lies primarily at 2N; S lies between 2N and 4N, G2+M lies primarily at 4N). Note that complex karyotypes in some cell lines can contribute to a complex distribution, such that each phase is not completely contained within discrete boundaries of 2N-4N DNA content.
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pone-0017692-g001: Development of the high-content (HC) cell cycle assay.(A) Markers used in the HC cell cycle assay and their distribution during the cell cycle. As defined by the HC cell cycle assay, G1 phase formally includes both G0 and G1 phases—however, for the sake of simplicity, we refer to it as “G1” rather than “G0/G1”. See text for discussion of assay development and validation. (B) Images of A549 cells stained for HC cell cycle analysis with Hoechst 33342, cyclin A, EdU, and pH3. Top panel, a field of asynchronous cycling cells; bottom panel, examples of G1, S, G2, and M cells. Bottom panels: the G2 panel shows one G2 cell (white arrowhead), one G1 cell, and one S cell; the M panel shows two M cells (white arrowheads) and three G1 cells. The G1 and S panels show exclusively G1 or S cells, respectively. The inset table summarizes the Boolean logic used to identify cell cycle phases when images are analyzed with the Cellomics Target Activation algorithm. (C) A DNA distribution plot of data derived from HC cell cycle analysis of DMSO-treated Calu-6 cells. This plot combines aspects of FACS (DNA content, as measured by total nuclear intensity of Hoechst 33342 staining) with the image-based cell cycle phase assignment, and demonstrates that the phase assignments correlate well with the DNA content expected for a given phase (i.e. G1 lies primarily at 2N; S lies between 2N and 4N, G2+M lies primarily at 4N). Note that complex karyotypes in some cell lines can contribute to a complex distribution, such that each phase is not completely contained within discrete boundaries of 2N-4N DNA content.

Mentions: We developed a high-throughput, high-content, image-based cell cycle analysis method (Figure 1A–B) in which S-phase cells are defined by incorporation of the thymidine analog EdU (5-ethynyl-2′-deoxyuridine) into DNA, and M-phase cells are defined by immunostaining for the mitotic marker phospho-histone H3 (pH3) [24]. Immunostaining for cyclin A, which is present in S, G2, and M [25], allowed us to derive G1 and G2 phase assignments: G2 cells were defined as positive for cyclin A staining but negative for EdU and negative for pH3, while G1 cells were defined as negative for EdU, cyclin A, and pH3. To evaluate the accuracy of the phase designations, HeLa and A549 cells were synchronized with a double-thymidine block and released at timed intervals to create populations enriched for G1/S or G2/M. High-content cell cycle analysis showed the expected phase enrichments in these synchronized cells, as well as in asynchronous cells that were treated with taxol (paclitaxel) or hydroxyurea to enrich for M or for G1/S (data not shown). We also generated a “DNA distribution plot” histogram that combines a FACS-like display of DNA content with an overlay of cell cycle phase assignments (Figure 1C), and demonstrated that phase assignments in DMSO-treated Calu-6 cells were consistent with the expected 2N vs. 4N DNA content. DNA distribution patterns varied in different cell lines according to their degree of aneuploidy and heterogeneity, but the majority exhibited distinguishable 2N and 4N populations. (See Dataset S1 for an example of the custom Excel macro used to generate DNA distribution plots from raw cell cycle data output.)


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)

Development of the high-content (HC) cell cycle assay.(A) Markers used in the HC cell cycle assay and their distribution during the cell cycle. As defined by the HC cell cycle assay, G1 phase formally includes both G0 and G1 phases—however, for the sake of simplicity, we refer to it as “G1” rather than “G0/G1”. See text for discussion of assay development and validation. (B) Images of A549 cells stained for HC cell cycle analysis with Hoechst 33342, cyclin A, EdU, and pH3. Top panel, a field of asynchronous cycling cells; bottom panel, examples of G1, S, G2, and M cells. Bottom panels: the G2 panel shows one G2 cell (white arrowhead), one G1 cell, and one S cell; the M panel shows two M cells (white arrowheads) and three G1 cells. The G1 and S panels show exclusively G1 or S cells, respectively. The inset table summarizes the Boolean logic used to identify cell cycle phases when images are analyzed with the Cellomics Target Activation algorithm. (C) A DNA distribution plot of data derived from HC cell cycle analysis of DMSO-treated Calu-6 cells. This plot combines aspects of FACS (DNA content, as measured by total nuclear intensity of Hoechst 33342 staining) with the image-based cell cycle phase assignment, and demonstrates that the phase assignments correlate well with the DNA content expected for a given phase (i.e. G1 lies primarily at 2N; S lies between 2N and 4N, G2+M lies primarily at 4N). Note that complex karyotypes in some cell lines can contribute to a complex distribution, such that each phase is not completely contained within discrete boundaries of 2N-4N DNA content.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3049797&req=5

pone-0017692-g001: Development of the high-content (HC) cell cycle assay.(A) Markers used in the HC cell cycle assay and their distribution during the cell cycle. As defined by the HC cell cycle assay, G1 phase formally includes both G0 and G1 phases—however, for the sake of simplicity, we refer to it as “G1” rather than “G0/G1”. See text for discussion of assay development and validation. (B) Images of A549 cells stained for HC cell cycle analysis with Hoechst 33342, cyclin A, EdU, and pH3. Top panel, a field of asynchronous cycling cells; bottom panel, examples of G1, S, G2, and M cells. Bottom panels: the G2 panel shows one G2 cell (white arrowhead), one G1 cell, and one S cell; the M panel shows two M cells (white arrowheads) and three G1 cells. The G1 and S panels show exclusively G1 or S cells, respectively. The inset table summarizes the Boolean logic used to identify cell cycle phases when images are analyzed with the Cellomics Target Activation algorithm. (C) A DNA distribution plot of data derived from HC cell cycle analysis of DMSO-treated Calu-6 cells. This plot combines aspects of FACS (DNA content, as measured by total nuclear intensity of Hoechst 33342 staining) with the image-based cell cycle phase assignment, and demonstrates that the phase assignments correlate well with the DNA content expected for a given phase (i.e. G1 lies primarily at 2N; S lies between 2N and 4N, G2+M lies primarily at 4N). Note that complex karyotypes in some cell lines can contribute to a complex distribution, such that each phase is not completely contained within discrete boundaries of 2N-4N DNA content.
Mentions: We developed a high-throughput, high-content, image-based cell cycle analysis method (Figure 1A–B) in which S-phase cells are defined by incorporation of the thymidine analog EdU (5-ethynyl-2′-deoxyuridine) into DNA, and M-phase cells are defined by immunostaining for the mitotic marker phospho-histone H3 (pH3) [24]. Immunostaining for cyclin A, which is present in S, G2, and M [25], allowed us to derive G1 and G2 phase assignments: G2 cells were defined as positive for cyclin A staining but negative for EdU and negative for pH3, while G1 cells were defined as negative for EdU, cyclin A, and pH3. To evaluate the accuracy of the phase designations, HeLa and A549 cells were synchronized with a double-thymidine block and released at timed intervals to create populations enriched for G1/S or G2/M. High-content cell cycle analysis showed the expected phase enrichments in these synchronized cells, as well as in asynchronous cells that were treated with taxol (paclitaxel) or hydroxyurea to enrich for M or for G1/S (data not shown). We also generated a “DNA distribution plot” histogram that combines a FACS-like display of DNA content with an overlay of cell cycle phase assignments (Figure 1C), and demonstrated that phase assignments in DMSO-treated Calu-6 cells were consistent with the expected 2N vs. 4N DNA content. DNA distribution patterns varied in different cell lines according to their degree of aneuploidy and heterogeneity, but the majority exhibited distinguishable 2N and 4N populations. (See Dataset S1 for an example of the custom Excel macro used to generate DNA distribution plots from raw cell cycle data output.)

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