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Induction of the CLOCK gene by E2-ERα signaling promotes the proliferation of breast cancer cells.

Xiao L, Chang AK, Zang MX, Bi H, Li S, Wang M, Xing X, Wu H - PLoS ONE (2014)

Bottom Line: In this study, we investigated the regulation of CLOCK expression by ERα and its roles in cell proliferation.In these cells, E2 promoted the binding of ERα to the EREs (estrogen-response elements) of CLOCK promoter, thereby up-regulating the transcription of CLOCK.Taken together, these results demonstrated that CLOCK could be an important gene that mediates cell proliferation in breast cancer cells.

View Article: PubMed Central - PubMed

Affiliation: School of Life Science and Biotechnology, Dalian University of Technology, Dalian, China.

ABSTRACT
Growing genetic and epidemiological evidence suggests a direct connection between the disruption of circadian rhythm and breast cancer. Moreover, the expression of several molecular components constituting the circadian clock machinery has been found to be modulated by estrogen-estrogen receptor α (E2-ERα) signaling in ERα-positive breast cancer cells. In this study, we investigated the regulation of CLOCK expression by ERα and its roles in cell proliferation. Immunohistochemical analysis of human breast tumor samples revealed high expression of CLOCK in ERα-positive breast tumor samples. Subsequent experiments using ERα-positive human breast cancer cell lines showed that both protein and mRNA levels of CLOCK were up-regulated by E2 and ERα. In these cells, E2 promoted the binding of ERα to the EREs (estrogen-response elements) of CLOCK promoter, thereby up-regulating the transcription of CLOCK. Knockdown of CLOCK attenuated cell proliferation in ERα-positive breast cancer cells. Taken together, these results demonstrated that CLOCK could be an important gene that mediates cell proliferation in breast cancer cells.

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ERα ligands regulate the expression of CLOCK at the transcription level.Analysis of CLOCK mRNA levels in MCF-7 cells by real-time PCR. MCF-7 cells were cultured in phenol red free medium and charcoal striped FCS medium for 2 days before being treated with E2 or ICI and the expression of CLOCK was then analyzed by real-time PCR. Expression of CLOCK was normalized against GAPDH mRNA level (internal control). A, Cells treated with different concentrations of E2 (10−10 to 10−6 M) for 8 h. B, Cells treated with 1 µM E2 for different periods of time. C, Cells treated with 1 µM E2 or 0.1 µM ICI for 12 h. D, MCF-7 cells transfected with empty vector for ERα (pcDNA3), ERα, shCon (control for shERα) or shERα#1 construct. E, MCF-7 cells were cultured in phenol red-free medium and charcoal-striped FCS medium for 2 days before being treated with E2, Act D or CHX and the expression of CLOCK was then analyzed by real-time PCR. Cells treated with 0.5 µg/ml Act D, 10 µg/ml CHX alone or in combination with 1 µM E2 for 12 h. A-E, Relative levels were calculated by giving an arbitrary value of 1 to the control. CLOCK transcript levels were normalized to GAPDH transcript level and expressed as arbitrary units relative to the vehicle control (set as 1). Each experiment was performed in triplicate and repeated at least three times. Data shown are the means ± SDs. P value was determined by ANOVA with Bonferroni test (*, P<0.05. ns, not significant).
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pone-0095878-g003: ERα ligands regulate the expression of CLOCK at the transcription level.Analysis of CLOCK mRNA levels in MCF-7 cells by real-time PCR. MCF-7 cells were cultured in phenol red free medium and charcoal striped FCS medium for 2 days before being treated with E2 or ICI and the expression of CLOCK was then analyzed by real-time PCR. Expression of CLOCK was normalized against GAPDH mRNA level (internal control). A, Cells treated with different concentrations of E2 (10−10 to 10−6 M) for 8 h. B, Cells treated with 1 µM E2 for different periods of time. C, Cells treated with 1 µM E2 or 0.1 µM ICI for 12 h. D, MCF-7 cells transfected with empty vector for ERα (pcDNA3), ERα, shCon (control for shERα) or shERα#1 construct. E, MCF-7 cells were cultured in phenol red-free medium and charcoal-striped FCS medium for 2 days before being treated with E2, Act D or CHX and the expression of CLOCK was then analyzed by real-time PCR. Cells treated with 0.5 µg/ml Act D, 10 µg/ml CHX alone or in combination with 1 µM E2 for 12 h. A-E, Relative levels were calculated by giving an arbitrary value of 1 to the control. CLOCK transcript levels were normalized to GAPDH transcript level and expressed as arbitrary units relative to the vehicle control (set as 1). Each experiment was performed in triplicate and repeated at least three times. Data shown are the means ± SDs. P value was determined by ANOVA with Bonferroni test (*, P<0.05. ns, not significant).

Mentions: The effect of ERα on the level of CLOCK expression was further investigated by determining the changes in the level of CLOCK mRNA in MCF-7 cells in response to E2 or ICI after 24 h of treatment. CLOCK mRNA level was up-regulated in response to E2 in a dose-dependent manner in the range of 10−10 to 10−6 M (Fig. 3A). Thus 10−6 M E2 was chosen for subsequent studies. CLOCK mRNA level was increased 4 h after E2 treatment (Fig. 3B). However, when the cells were treated with ICI, the level of CLOCK mRNA was reduced compared to that of the control (Fig. 3C). The effect of ERα on the modulation of CLOCK transcription in response to E2 was further confirmed by overexpressing ERα in MCF-7 cells and knocking down ERα with shERα. As expected, ERα ectopic expression up-regulated CLOCK transcription, while knockdown of ERα down-regulated CLOCK transcription (Fig. 3D). In addition, Act D repressed the basal expression of CLOCK and abolished E2-induced up-regulation of CLOCK. Although Act D globally represses gene transcription, including the transcription of GAPDH, it inhibited the transcription of CLOCK more than that of GAPDH. In contrast, cycloheximide (CHX, a translation inhibitor) had no effect on E2-induced up-regulation of CLOCK (Fig. 3E), indicating that CLOCK is a primary ERα transcriptional target because the effect of E2 does not require the synthesis of new proteins since all necessary factors are preexisting in the cells.


Induction of the CLOCK gene by E2-ERα signaling promotes the proliferation of breast cancer cells.

Xiao L, Chang AK, Zang MX, Bi H, Li S, Wang M, Xing X, Wu H - PLoS ONE (2014)

ERα ligands regulate the expression of CLOCK at the transcription level.Analysis of CLOCK mRNA levels in MCF-7 cells by real-time PCR. MCF-7 cells were cultured in phenol red free medium and charcoal striped FCS medium for 2 days before being treated with E2 or ICI and the expression of CLOCK was then analyzed by real-time PCR. Expression of CLOCK was normalized against GAPDH mRNA level (internal control). A, Cells treated with different concentrations of E2 (10−10 to 10−6 M) for 8 h. B, Cells treated with 1 µM E2 for different periods of time. C, Cells treated with 1 µM E2 or 0.1 µM ICI for 12 h. D, MCF-7 cells transfected with empty vector for ERα (pcDNA3), ERα, shCon (control for shERα) or shERα#1 construct. E, MCF-7 cells were cultured in phenol red-free medium and charcoal-striped FCS medium for 2 days before being treated with E2, Act D or CHX and the expression of CLOCK was then analyzed by real-time PCR. Cells treated with 0.5 µg/ml Act D, 10 µg/ml CHX alone or in combination with 1 µM E2 for 12 h. A-E, Relative levels were calculated by giving an arbitrary value of 1 to the control. CLOCK transcript levels were normalized to GAPDH transcript level and expressed as arbitrary units relative to the vehicle control (set as 1). Each experiment was performed in triplicate and repeated at least three times. Data shown are the means ± SDs. P value was determined by ANOVA with Bonferroni test (*, P<0.05. ns, not significant).
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pone-0095878-g003: ERα ligands regulate the expression of CLOCK at the transcription level.Analysis of CLOCK mRNA levels in MCF-7 cells by real-time PCR. MCF-7 cells were cultured in phenol red free medium and charcoal striped FCS medium for 2 days before being treated with E2 or ICI and the expression of CLOCK was then analyzed by real-time PCR. Expression of CLOCK was normalized against GAPDH mRNA level (internal control). A, Cells treated with different concentrations of E2 (10−10 to 10−6 M) for 8 h. B, Cells treated with 1 µM E2 for different periods of time. C, Cells treated with 1 µM E2 or 0.1 µM ICI for 12 h. D, MCF-7 cells transfected with empty vector for ERα (pcDNA3), ERα, shCon (control for shERα) or shERα#1 construct. E, MCF-7 cells were cultured in phenol red-free medium and charcoal-striped FCS medium for 2 days before being treated with E2, Act D or CHX and the expression of CLOCK was then analyzed by real-time PCR. Cells treated with 0.5 µg/ml Act D, 10 µg/ml CHX alone or in combination with 1 µM E2 for 12 h. A-E, Relative levels were calculated by giving an arbitrary value of 1 to the control. CLOCK transcript levels were normalized to GAPDH transcript level and expressed as arbitrary units relative to the vehicle control (set as 1). Each experiment was performed in triplicate and repeated at least three times. Data shown are the means ± SDs. P value was determined by ANOVA with Bonferroni test (*, P<0.05. ns, not significant).
Mentions: The effect of ERα on the level of CLOCK expression was further investigated by determining the changes in the level of CLOCK mRNA in MCF-7 cells in response to E2 or ICI after 24 h of treatment. CLOCK mRNA level was up-regulated in response to E2 in a dose-dependent manner in the range of 10−10 to 10−6 M (Fig. 3A). Thus 10−6 M E2 was chosen for subsequent studies. CLOCK mRNA level was increased 4 h after E2 treatment (Fig. 3B). However, when the cells were treated with ICI, the level of CLOCK mRNA was reduced compared to that of the control (Fig. 3C). The effect of ERα on the modulation of CLOCK transcription in response to E2 was further confirmed by overexpressing ERα in MCF-7 cells and knocking down ERα with shERα. As expected, ERα ectopic expression up-regulated CLOCK transcription, while knockdown of ERα down-regulated CLOCK transcription (Fig. 3D). In addition, Act D repressed the basal expression of CLOCK and abolished E2-induced up-regulation of CLOCK. Although Act D globally represses gene transcription, including the transcription of GAPDH, it inhibited the transcription of CLOCK more than that of GAPDH. In contrast, cycloheximide (CHX, a translation inhibitor) had no effect on E2-induced up-regulation of CLOCK (Fig. 3E), indicating that CLOCK is a primary ERα transcriptional target because the effect of E2 does not require the synthesis of new proteins since all necessary factors are preexisting in the cells.

Bottom Line: In this study, we investigated the regulation of CLOCK expression by ERα and its roles in cell proliferation.In these cells, E2 promoted the binding of ERα to the EREs (estrogen-response elements) of CLOCK promoter, thereby up-regulating the transcription of CLOCK.Taken together, these results demonstrated that CLOCK could be an important gene that mediates cell proliferation in breast cancer cells.

View Article: PubMed Central - PubMed

Affiliation: School of Life Science and Biotechnology, Dalian University of Technology, Dalian, China.

ABSTRACT
Growing genetic and epidemiological evidence suggests a direct connection between the disruption of circadian rhythm and breast cancer. Moreover, the expression of several molecular components constituting the circadian clock machinery has been found to be modulated by estrogen-estrogen receptor α (E2-ERα) signaling in ERα-positive breast cancer cells. In this study, we investigated the regulation of CLOCK expression by ERα and its roles in cell proliferation. Immunohistochemical analysis of human breast tumor samples revealed high expression of CLOCK in ERα-positive breast tumor samples. Subsequent experiments using ERα-positive human breast cancer cell lines showed that both protein and mRNA levels of CLOCK were up-regulated by E2 and ERα. In these cells, E2 promoted the binding of ERα to the EREs (estrogen-response elements) of CLOCK promoter, thereby up-regulating the transcription of CLOCK. Knockdown of CLOCK attenuated cell proliferation in ERα-positive breast cancer cells. Taken together, these results demonstrated that CLOCK could be an important gene that mediates cell proliferation in breast cancer cells.

Show MeSH
Related in: MedlinePlus