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Inhibition of casein kinase 1-epsilon induces cancer-cell-selective, PERIOD2-dependent growth arrest.

Yang WS, Stockwell BR - Genome Biol. (2008)

Bottom Line: We employed a two-step screening strategy using human sarcoma cell lines and human fibroblast-derived isogenic cell lines, and found that short hairpin RNAs targeting CSNK1E, a clock gene that regulates circadian rhythms, can induce selective growth inhibition in engineered tumor cells.Treatment with IC261, a kinase domain inhibitor of casein kinase 1-epsilon (CK1epsilon), a protein product of CSNK1E, showed a similar degree of cancer-cell-selective growth inhibition.These data support the hypothesis that circadian clock genes can control the cell cycle and cell survival signaling, and emphasize a central role of CK1epsilon and PERIOD2 in linking these systems.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, Columbia University, Fairchild Center, Amsterdam Avenue, New York, NY 10027, USA.

ABSTRACT

Background: Kinases are under extensive investigation as targets for drug development. Discovering novel kinases whose inhibition induces cancer-cell-selective lethality would be of value. Recent advances in RNA interference have enabled the realization of this goal.

Results: We screened 5,760 short hairpin RNA clones targeting the human kinome to detect human kinases on which cancer cells are more dependent than normal cells. We employed a two-step screening strategy using human sarcoma cell lines and human fibroblast-derived isogenic cell lines, and found that short hairpin RNAs targeting CSNK1E, a clock gene that regulates circadian rhythms, can induce selective growth inhibition in engineered tumor cells. Analysis of gene-expression data revealed that CSNK1E is overexpressed in several cancer tissue samples examined compared to non-tumorigenic normal tissue, suggesting a positive role of CSNK1E in neogenesis or maintenance. Treatment with IC261, a kinase domain inhibitor of casein kinase 1-epsilon (CK1epsilon), a protein product of CSNK1E, showed a similar degree of cancer-cell-selective growth inhibition. In a search for substrates of CK1epsilon that mediate IC261-induced growth inhibition, we discovered that knocking down PER2, another clock gene involved in circadian rhythm control, rescues IC261-induced growth inhibition.

Conclusion: We identified CK1epsilon as a potential target for developing anticancer reagents with a high therapeutic index. These data support the hypothesis that circadian clock genes can control the cell cycle and cell survival signaling, and emphasize a central role of CK1epsilon and PERIOD2 in linking these systems.

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Knocking down CSNK1E induces G2/M cell cycle arrest and caspase-mediated apoptosis. (a) Two days after non-targeting shRNA or shCSNK1E treatment, HT1080 cells were fixed in methanol and stained with propidium iodide (Materials and methods). Flow cytometry of cells was performed on a FACSCalibur; calculation of cell cycle stages was performed using the cell cycle analysis program Modifit LT. Red area shows cell population in G1 or G2 cell cycle phase, while gray area shows dying cells. Label 'A' denotes apoptotic cell population. Insets show photographs of HT1080 cells treated with non-targeting shRNA or shCSNK1E. (b) Knocking down CSNK1E down-regulates CyclinB1 and CyclinA2. Cellular RNAs were prepared from HT1080 cells infected with either non-targeting shRNA (N.T.) or two different CSNK1E-targeting shRNAs (1834, 1837), and real-time PCR was performed with each gene-specific primer set. The expression levels of CyclinB1, CyclinA2 and CyclinD1 were first normalized to the level of an endogenous control (RPLPO), and then the relative expression level of each gene among the three cell lines was expressed as a ratio of transcripts in a cell line to those in non-targeted shRNA treated cells. Error bars indicate one standard deviation of triplicate data. (c) Knocking down CSNK1E induces caspase activation. Whole cell lysates from HT1080 cells infected with either non-targeting shRNA or shCSNK1E and cells treated with staurosporine were prepared. The cleavage of PARP1 or caspase-3 (Casp-3) in each sample was examined by western blotting using antibodies against PARP1 and cleaved caspase-3.
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Figure 4: Knocking down CSNK1E induces G2/M cell cycle arrest and caspase-mediated apoptosis. (a) Two days after non-targeting shRNA or shCSNK1E treatment, HT1080 cells were fixed in methanol and stained with propidium iodide (Materials and methods). Flow cytometry of cells was performed on a FACSCalibur; calculation of cell cycle stages was performed using the cell cycle analysis program Modifit LT. Red area shows cell population in G1 or G2 cell cycle phase, while gray area shows dying cells. Label 'A' denotes apoptotic cell population. Insets show photographs of HT1080 cells treated with non-targeting shRNA or shCSNK1E. (b) Knocking down CSNK1E down-regulates CyclinB1 and CyclinA2. Cellular RNAs were prepared from HT1080 cells infected with either non-targeting shRNA (N.T.) or two different CSNK1E-targeting shRNAs (1834, 1837), and real-time PCR was performed with each gene-specific primer set. The expression levels of CyclinB1, CyclinA2 and CyclinD1 were first normalized to the level of an endogenous control (RPLPO), and then the relative expression level of each gene among the three cell lines was expressed as a ratio of transcripts in a cell line to those in non-targeted shRNA treated cells. Error bars indicate one standard deviation of triplicate data. (c) Knocking down CSNK1E induces caspase activation. Whole cell lysates from HT1080 cells infected with either non-targeting shRNA or shCSNK1E and cells treated with staurosporine were prepared. The cleavage of PARP1 or caspase-3 (Casp-3) in each sample was examined by western blotting using antibodies against PARP1 and cleaved caspase-3.

Mentions: The proliferation-rate-dependent action of shCSNK1E (Figure 2b) raises the possibility that shRNA treatment induces cell cycle arrest; thus, fast growing cells have greater growth inhibition. To test this hypothesis, we stained the DNA of shRNA-treated cells with propidium-iodide and analyzed the cell cycle distribution by flow cytometry. The cell cycle distribution profile indicates that, after expression of shCSNK1E, HT1080 cells were arrested in the second gap (G2) phase of the cell cycle, with a concomitant increase in the population of cells harboring less than the normal diploid DNA content (that is, sub-G1), implying apoptosis had occurred (Figure 4a).


Inhibition of casein kinase 1-epsilon induces cancer-cell-selective, PERIOD2-dependent growth arrest.

Yang WS, Stockwell BR - Genome Biol. (2008)

Knocking down CSNK1E induces G2/M cell cycle arrest and caspase-mediated apoptosis. (a) Two days after non-targeting shRNA or shCSNK1E treatment, HT1080 cells were fixed in methanol and stained with propidium iodide (Materials and methods). Flow cytometry of cells was performed on a FACSCalibur; calculation of cell cycle stages was performed using the cell cycle analysis program Modifit LT. Red area shows cell population in G1 or G2 cell cycle phase, while gray area shows dying cells. Label 'A' denotes apoptotic cell population. Insets show photographs of HT1080 cells treated with non-targeting shRNA or shCSNK1E. (b) Knocking down CSNK1E down-regulates CyclinB1 and CyclinA2. Cellular RNAs were prepared from HT1080 cells infected with either non-targeting shRNA (N.T.) or two different CSNK1E-targeting shRNAs (1834, 1837), and real-time PCR was performed with each gene-specific primer set. The expression levels of CyclinB1, CyclinA2 and CyclinD1 were first normalized to the level of an endogenous control (RPLPO), and then the relative expression level of each gene among the three cell lines was expressed as a ratio of transcripts in a cell line to those in non-targeted shRNA treated cells. Error bars indicate one standard deviation of triplicate data. (c) Knocking down CSNK1E induces caspase activation. Whole cell lysates from HT1080 cells infected with either non-targeting shRNA or shCSNK1E and cells treated with staurosporine were prepared. The cleavage of PARP1 or caspase-3 (Casp-3) in each sample was examined by western blotting using antibodies against PARP1 and cleaved caspase-3.
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Figure 4: Knocking down CSNK1E induces G2/M cell cycle arrest and caspase-mediated apoptosis. (a) Two days after non-targeting shRNA or shCSNK1E treatment, HT1080 cells were fixed in methanol and stained with propidium iodide (Materials and methods). Flow cytometry of cells was performed on a FACSCalibur; calculation of cell cycle stages was performed using the cell cycle analysis program Modifit LT. Red area shows cell population in G1 or G2 cell cycle phase, while gray area shows dying cells. Label 'A' denotes apoptotic cell population. Insets show photographs of HT1080 cells treated with non-targeting shRNA or shCSNK1E. (b) Knocking down CSNK1E down-regulates CyclinB1 and CyclinA2. Cellular RNAs were prepared from HT1080 cells infected with either non-targeting shRNA (N.T.) or two different CSNK1E-targeting shRNAs (1834, 1837), and real-time PCR was performed with each gene-specific primer set. The expression levels of CyclinB1, CyclinA2 and CyclinD1 were first normalized to the level of an endogenous control (RPLPO), and then the relative expression level of each gene among the three cell lines was expressed as a ratio of transcripts in a cell line to those in non-targeted shRNA treated cells. Error bars indicate one standard deviation of triplicate data. (c) Knocking down CSNK1E induces caspase activation. Whole cell lysates from HT1080 cells infected with either non-targeting shRNA or shCSNK1E and cells treated with staurosporine were prepared. The cleavage of PARP1 or caspase-3 (Casp-3) in each sample was examined by western blotting using antibodies against PARP1 and cleaved caspase-3.
Mentions: The proliferation-rate-dependent action of shCSNK1E (Figure 2b) raises the possibility that shRNA treatment induces cell cycle arrest; thus, fast growing cells have greater growth inhibition. To test this hypothesis, we stained the DNA of shRNA-treated cells with propidium-iodide and analyzed the cell cycle distribution by flow cytometry. The cell cycle distribution profile indicates that, after expression of shCSNK1E, HT1080 cells were arrested in the second gap (G2) phase of the cell cycle, with a concomitant increase in the population of cells harboring less than the normal diploid DNA content (that is, sub-G1), implying apoptosis had occurred (Figure 4a).

Bottom Line: We employed a two-step screening strategy using human sarcoma cell lines and human fibroblast-derived isogenic cell lines, and found that short hairpin RNAs targeting CSNK1E, a clock gene that regulates circadian rhythms, can induce selective growth inhibition in engineered tumor cells.Treatment with IC261, a kinase domain inhibitor of casein kinase 1-epsilon (CK1epsilon), a protein product of CSNK1E, showed a similar degree of cancer-cell-selective growth inhibition.These data support the hypothesis that circadian clock genes can control the cell cycle and cell survival signaling, and emphasize a central role of CK1epsilon and PERIOD2 in linking these systems.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, Columbia University, Fairchild Center, Amsterdam Avenue, New York, NY 10027, USA.

ABSTRACT

Background: Kinases are under extensive investigation as targets for drug development. Discovering novel kinases whose inhibition induces cancer-cell-selective lethality would be of value. Recent advances in RNA interference have enabled the realization of this goal.

Results: We screened 5,760 short hairpin RNA clones targeting the human kinome to detect human kinases on which cancer cells are more dependent than normal cells. We employed a two-step screening strategy using human sarcoma cell lines and human fibroblast-derived isogenic cell lines, and found that short hairpin RNAs targeting CSNK1E, a clock gene that regulates circadian rhythms, can induce selective growth inhibition in engineered tumor cells. Analysis of gene-expression data revealed that CSNK1E is overexpressed in several cancer tissue samples examined compared to non-tumorigenic normal tissue, suggesting a positive role of CSNK1E in neogenesis or maintenance. Treatment with IC261, a kinase domain inhibitor of casein kinase 1-epsilon (CK1epsilon), a protein product of CSNK1E, showed a similar degree of cancer-cell-selective growth inhibition. In a search for substrates of CK1epsilon that mediate IC261-induced growth inhibition, we discovered that knocking down PER2, another clock gene involved in circadian rhythm control, rescues IC261-induced growth inhibition.

Conclusion: We identified CK1epsilon as a potential target for developing anticancer reagents with a high therapeutic index. These data support the hypothesis that circadian clock genes can control the cell cycle and cell survival signaling, and emphasize a central role of CK1epsilon and PERIOD2 in linking these systems.

Show MeSH
Related in: MedlinePlus