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CSF1 is a novel p53 target gene whose protein product functions in a feed-forward manner to suppress apoptosis and enhance p53-mediated growth arrest.

Azzam G, Wang X, Bell D, Murphy ME - PLoS ONE (2013)

Bottom Line: This leads to an increase in p53-mediated growth arrest, along with a concomitant decrease in apoptosis.We show that cells expressing CSF1 and CSF1R are indeed radio-resistant, and further, that this effect requires p53.Finally, these data may explain the association of the P72 variant and the CSF1/CSF1R pathway with increased senescence and radio-resistance in some epithelial tumor types.

View Article: PubMed Central - PubMed

Affiliation: Program in Molecular and Cellular Oncogenesis, The Wistar Institute, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
The p53 tumor suppressor gene has a common polymorphism at codon 72 that alters its function. We previously reported that the proline 72 polymorphic variant of p53 (P72) demonstrates increased ability to transactivate a subset of genes, relative to arginine 72 (R72); one of these genes is macrophage colony stimulating factor (CSF1). At present, the mechanism(s) underlying the increased transcriptional activity of P72 toward genes like CSF1 have not been completely elucidated. Additionally, the consequences of increased transcription of genes like CSF1 by the P72 variant to the downstream p53 pathway are unknown. In this report, we address these issues. We show that the CSF1 gene contains a conserved binding site for p53, and interestingly that the P72 variant shows increased ability to bind to this site. Moreover, we show that increased CSF1/CSF1R signaling in P72 cells feeds back on the p53 pathway to enhance p53 phosphorylation, levels, and transactivation of target genes, particularly the cyclin-dependent kinase inhibitor p21 (CDKN1A). This leads to an increase in p53-mediated growth arrest, along with a concomitant decrease in apoptosis. Notably, the CSF1/CSF1R signaling axis is overexpressed in several epithelial cancers, and there is clinical evidence that this pathway plays a role in radio-resistance of some cancers. We show that cells expressing CSF1 and CSF1R are indeed radio-resistant, and further, that this effect requires p53. These combined data are the first to implicate the CSF1/CSF1R pathway in the decision between p53-mediated growth arrest and apoptosis. They are also the first to highlight a cytokine as influential in cell fate determined by p53 in epithelial cells. Finally, these data may explain the association of the P72 variant and the CSF1/CSF1R pathway with increased senescence and radio-resistance in some epithelial tumor types.

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CSF1 is a novel p53 target gene.A. Left panel: Western blot analysis of the level of p53, GAPDH and p21 (CDKN1A) in lysates isolated from Hct116 cells (p53−/− or wild-type p53, +/+), untreated or treated with Etoposide (Etop, 50 uM) for the indicated time points. Right panel: QRT-PCR analysis of the level of CSF1 RNA in the samples depicted in the left panel; values shown are the average of 3 independent experiments normalized to control. Error bars mark standard deviation. B. Left panel: Western analysis of the level of p53, GAPDH and p21 in lysates isolated from Hep3B (p53−/−) or HepG2 (wt p53) untreated or treated with 2 uM Doxorubicin (Doxo) for the indicated time points. Right panel: QRT-PCR of RNA isolated from the samples in the left panel, untreated or treated with 2 uM Doxorubicin (Doxo) for the indicated time points (hours, hrs), analyzed for CSF1. Values shown are the average of 3 independent experiments normalized to control. Error bars mark standard deviation. C. Schematic of the Human CSF1 promoter, where the transcription start site (TSS) is denoted nucleotide +1, and the presence of canonical NF-kB and p53 response elements (RE) are denoted. Ex 1: exon 1. Ex 2: exon 2. D. Quantitative PCR (Q-PCR) analysis of Chromatin Immunoprecipitation (ChIP) eluates isolated from Hct 116 p53+/+ or −/− cells, untreated or treated with Etoposide (Etop, 50 uM) for 18 hours. ChIP was performed using antibody to p53 (Ab6, Calbiochem) or the equivalent amount of normal mouse Immunoglobulin G (IgG). Primer sets used for Q-PCR flank the predicted p53 response element in the CSF1 promoter depicted in (C), the positive control p53 response element in the p21 promoter (CDKN1A), and negative control primers (IGX1A, Qiagen), as indicated in the Materials and Methods. Values shown are the average of 2 independent experiments each repeated in duplicate, and are presented as the percent DNA bound normalized to input. E. Quantitative PCR (Q-PCR) analysis of Chromatin Immunoprecipitation (ChIP) eluates isolated from tetracycline-inducible H1299 cells, P72 or R72, untreated or treated with doxycycline (0.75 ug/ml) for 18 hours. ChIP was performed using antibody to p53 (fl393, Santa Cruz Biotechnology) or an equivalent amount of normal rabbit Immunoglobulin G (IgG). Primers used for Q-PCR analysis flank the predicted p53 response element on the CSF1 promoter, or the known p53 response element in the MDM2 promoter. Values shown are the average of 2 independent experiments each repeated in duplicate, and are presented as the relative DNA bound normalized to input.
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pone-0074297-g002: CSF1 is a novel p53 target gene.A. Left panel: Western blot analysis of the level of p53, GAPDH and p21 (CDKN1A) in lysates isolated from Hct116 cells (p53−/− or wild-type p53, +/+), untreated or treated with Etoposide (Etop, 50 uM) for the indicated time points. Right panel: QRT-PCR analysis of the level of CSF1 RNA in the samples depicted in the left panel; values shown are the average of 3 independent experiments normalized to control. Error bars mark standard deviation. B. Left panel: Western analysis of the level of p53, GAPDH and p21 in lysates isolated from Hep3B (p53−/−) or HepG2 (wt p53) untreated or treated with 2 uM Doxorubicin (Doxo) for the indicated time points. Right panel: QRT-PCR of RNA isolated from the samples in the left panel, untreated or treated with 2 uM Doxorubicin (Doxo) for the indicated time points (hours, hrs), analyzed for CSF1. Values shown are the average of 3 independent experiments normalized to control. Error bars mark standard deviation. C. Schematic of the Human CSF1 promoter, where the transcription start site (TSS) is denoted nucleotide +1, and the presence of canonical NF-kB and p53 response elements (RE) are denoted. Ex 1: exon 1. Ex 2: exon 2. D. Quantitative PCR (Q-PCR) analysis of Chromatin Immunoprecipitation (ChIP) eluates isolated from Hct 116 p53+/+ or −/− cells, untreated or treated with Etoposide (Etop, 50 uM) for 18 hours. ChIP was performed using antibody to p53 (Ab6, Calbiochem) or the equivalent amount of normal mouse Immunoglobulin G (IgG). Primer sets used for Q-PCR flank the predicted p53 response element in the CSF1 promoter depicted in (C), the positive control p53 response element in the p21 promoter (CDKN1A), and negative control primers (IGX1A, Qiagen), as indicated in the Materials and Methods. Values shown are the average of 2 independent experiments each repeated in duplicate, and are presented as the percent DNA bound normalized to input. E. Quantitative PCR (Q-PCR) analysis of Chromatin Immunoprecipitation (ChIP) eluates isolated from tetracycline-inducible H1299 cells, P72 or R72, untreated or treated with doxycycline (0.75 ug/ml) for 18 hours. ChIP was performed using antibody to p53 (fl393, Santa Cruz Biotechnology) or an equivalent amount of normal rabbit Immunoglobulin G (IgG). Primers used for Q-PCR analysis flank the predicted p53 response element on the CSF1 promoter, or the known p53 response element in the MDM2 promoter. Values shown are the average of 2 independent experiments each repeated in duplicate, and are presented as the relative DNA bound normalized to input.

Mentions: We next sought to determine whether CSF1 is a direct target for transcriptional activation by p53. We first analyzed two pairs of cell lines that differ in p53 status for the level of CSF1 induced after exposure to DNA damaging agents. In the colorectal carcinoma Hct116 p53+/+ and −/− pair, etoposide treatment led to efficient stabilization of p53, induction of p21 (CDKN1A), and induction of CSF1 mRNA, all in a p53-dependent manner (Fig. 2A). Similarly, in Hep3B (p53 ) and HepG2 (wt p53) liver cell lines, CSF1 was induced by the DNA damaging agent doxorubicin in a p53-dependent manner (Fig. 2B). Analysis of the promoter and intronic regions of CSF1 using a position weight matrix (PWM) method [27] revealed the presence of two overlapping consensus p53 binding sites in intron 1 of this gene (Fig. 2C and Fig. S1). Consistent with this being an authentic p53 binding site, ChIP-seq data from lymphoblastoid cell lines treated with doxorubicin demonstrated a binding peak over this region for p53 (Fig. S1). Similarly, published p53 ChIP-seq data from U2OS cells treated with etoposide and Actinomycin D showed a weak but detectable binding peak in this region [28]. We performed chromatin immunoprecipitation in the Hct116 pair treated with etoposide. These experiments revealed that p53 antisera could consistently immunoprecipitate DNA surrounding this p53 binding site in Hct116 cells, but not in p53−/− cells (Fig. 2D). In contrast, oligonucleotides for IGX1A, which maps to a promoter desert, were negative for p53 binding, as were oligonucleotides for a region far downstream of the p53 binding site in CSF1 (Fig. 2D and data not shown). To begin to assess the mechanism whereby the P72 variant shows enhanced transactivation of CSF1 relative to R72, we next performed chromatin immunoprecipitation in tetracycline-inducible P72 and R72 cells using p53 antisera, and noted an approximately 2-fold increase in the ability of the P72 variant to ChIP the CSF1 consensus p53 binding site, relative to R72; in contrast, these two variants bound identically to the p53 binding site in the MDM2 promoter (Fig. 2E). Notably, increased binding of P72 was consistent using two different p53 antibodies (Fig. S2). The combined data support the conclusion that CSF1 is a direct p53 target gene, and that at least part of the mechanism for enhanced transactivation by P72 may involve increased DNA binding of this variant to this element in the CSF1 promoter.


CSF1 is a novel p53 target gene whose protein product functions in a feed-forward manner to suppress apoptosis and enhance p53-mediated growth arrest.

Azzam G, Wang X, Bell D, Murphy ME - PLoS ONE (2013)

CSF1 is a novel p53 target gene.A. Left panel: Western blot analysis of the level of p53, GAPDH and p21 (CDKN1A) in lysates isolated from Hct116 cells (p53−/− or wild-type p53, +/+), untreated or treated with Etoposide (Etop, 50 uM) for the indicated time points. Right panel: QRT-PCR analysis of the level of CSF1 RNA in the samples depicted in the left panel; values shown are the average of 3 independent experiments normalized to control. Error bars mark standard deviation. B. Left panel: Western analysis of the level of p53, GAPDH and p21 in lysates isolated from Hep3B (p53−/−) or HepG2 (wt p53) untreated or treated with 2 uM Doxorubicin (Doxo) for the indicated time points. Right panel: QRT-PCR of RNA isolated from the samples in the left panel, untreated or treated with 2 uM Doxorubicin (Doxo) for the indicated time points (hours, hrs), analyzed for CSF1. Values shown are the average of 3 independent experiments normalized to control. Error bars mark standard deviation. C. Schematic of the Human CSF1 promoter, where the transcription start site (TSS) is denoted nucleotide +1, and the presence of canonical NF-kB and p53 response elements (RE) are denoted. Ex 1: exon 1. Ex 2: exon 2. D. Quantitative PCR (Q-PCR) analysis of Chromatin Immunoprecipitation (ChIP) eluates isolated from Hct 116 p53+/+ or −/− cells, untreated or treated with Etoposide (Etop, 50 uM) for 18 hours. ChIP was performed using antibody to p53 (Ab6, Calbiochem) or the equivalent amount of normal mouse Immunoglobulin G (IgG). Primer sets used for Q-PCR flank the predicted p53 response element in the CSF1 promoter depicted in (C), the positive control p53 response element in the p21 promoter (CDKN1A), and negative control primers (IGX1A, Qiagen), as indicated in the Materials and Methods. Values shown are the average of 2 independent experiments each repeated in duplicate, and are presented as the percent DNA bound normalized to input. E. Quantitative PCR (Q-PCR) analysis of Chromatin Immunoprecipitation (ChIP) eluates isolated from tetracycline-inducible H1299 cells, P72 or R72, untreated or treated with doxycycline (0.75 ug/ml) for 18 hours. ChIP was performed using antibody to p53 (fl393, Santa Cruz Biotechnology) or an equivalent amount of normal rabbit Immunoglobulin G (IgG). Primers used for Q-PCR analysis flank the predicted p53 response element on the CSF1 promoter, or the known p53 response element in the MDM2 promoter. Values shown are the average of 2 independent experiments each repeated in duplicate, and are presented as the relative DNA bound normalized to input.
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pone-0074297-g002: CSF1 is a novel p53 target gene.A. Left panel: Western blot analysis of the level of p53, GAPDH and p21 (CDKN1A) in lysates isolated from Hct116 cells (p53−/− or wild-type p53, +/+), untreated or treated with Etoposide (Etop, 50 uM) for the indicated time points. Right panel: QRT-PCR analysis of the level of CSF1 RNA in the samples depicted in the left panel; values shown are the average of 3 independent experiments normalized to control. Error bars mark standard deviation. B. Left panel: Western analysis of the level of p53, GAPDH and p21 in lysates isolated from Hep3B (p53−/−) or HepG2 (wt p53) untreated or treated with 2 uM Doxorubicin (Doxo) for the indicated time points. Right panel: QRT-PCR of RNA isolated from the samples in the left panel, untreated or treated with 2 uM Doxorubicin (Doxo) for the indicated time points (hours, hrs), analyzed for CSF1. Values shown are the average of 3 independent experiments normalized to control. Error bars mark standard deviation. C. Schematic of the Human CSF1 promoter, where the transcription start site (TSS) is denoted nucleotide +1, and the presence of canonical NF-kB and p53 response elements (RE) are denoted. Ex 1: exon 1. Ex 2: exon 2. D. Quantitative PCR (Q-PCR) analysis of Chromatin Immunoprecipitation (ChIP) eluates isolated from Hct 116 p53+/+ or −/− cells, untreated or treated with Etoposide (Etop, 50 uM) for 18 hours. ChIP was performed using antibody to p53 (Ab6, Calbiochem) or the equivalent amount of normal mouse Immunoglobulin G (IgG). Primer sets used for Q-PCR flank the predicted p53 response element in the CSF1 promoter depicted in (C), the positive control p53 response element in the p21 promoter (CDKN1A), and negative control primers (IGX1A, Qiagen), as indicated in the Materials and Methods. Values shown are the average of 2 independent experiments each repeated in duplicate, and are presented as the percent DNA bound normalized to input. E. Quantitative PCR (Q-PCR) analysis of Chromatin Immunoprecipitation (ChIP) eluates isolated from tetracycline-inducible H1299 cells, P72 or R72, untreated or treated with doxycycline (0.75 ug/ml) for 18 hours. ChIP was performed using antibody to p53 (fl393, Santa Cruz Biotechnology) or an equivalent amount of normal rabbit Immunoglobulin G (IgG). Primers used for Q-PCR analysis flank the predicted p53 response element on the CSF1 promoter, or the known p53 response element in the MDM2 promoter. Values shown are the average of 2 independent experiments each repeated in duplicate, and are presented as the relative DNA bound normalized to input.
Mentions: We next sought to determine whether CSF1 is a direct target for transcriptional activation by p53. We first analyzed two pairs of cell lines that differ in p53 status for the level of CSF1 induced after exposure to DNA damaging agents. In the colorectal carcinoma Hct116 p53+/+ and −/− pair, etoposide treatment led to efficient stabilization of p53, induction of p21 (CDKN1A), and induction of CSF1 mRNA, all in a p53-dependent manner (Fig. 2A). Similarly, in Hep3B (p53 ) and HepG2 (wt p53) liver cell lines, CSF1 was induced by the DNA damaging agent doxorubicin in a p53-dependent manner (Fig. 2B). Analysis of the promoter and intronic regions of CSF1 using a position weight matrix (PWM) method [27] revealed the presence of two overlapping consensus p53 binding sites in intron 1 of this gene (Fig. 2C and Fig. S1). Consistent with this being an authentic p53 binding site, ChIP-seq data from lymphoblastoid cell lines treated with doxorubicin demonstrated a binding peak over this region for p53 (Fig. S1). Similarly, published p53 ChIP-seq data from U2OS cells treated with etoposide and Actinomycin D showed a weak but detectable binding peak in this region [28]. We performed chromatin immunoprecipitation in the Hct116 pair treated with etoposide. These experiments revealed that p53 antisera could consistently immunoprecipitate DNA surrounding this p53 binding site in Hct116 cells, but not in p53−/− cells (Fig. 2D). In contrast, oligonucleotides for IGX1A, which maps to a promoter desert, were negative for p53 binding, as were oligonucleotides for a region far downstream of the p53 binding site in CSF1 (Fig. 2D and data not shown). To begin to assess the mechanism whereby the P72 variant shows enhanced transactivation of CSF1 relative to R72, we next performed chromatin immunoprecipitation in tetracycline-inducible P72 and R72 cells using p53 antisera, and noted an approximately 2-fold increase in the ability of the P72 variant to ChIP the CSF1 consensus p53 binding site, relative to R72; in contrast, these two variants bound identically to the p53 binding site in the MDM2 promoter (Fig. 2E). Notably, increased binding of P72 was consistent using two different p53 antibodies (Fig. S2). The combined data support the conclusion that CSF1 is a direct p53 target gene, and that at least part of the mechanism for enhanced transactivation by P72 may involve increased DNA binding of this variant to this element in the CSF1 promoter.

Bottom Line: This leads to an increase in p53-mediated growth arrest, along with a concomitant decrease in apoptosis.We show that cells expressing CSF1 and CSF1R are indeed radio-resistant, and further, that this effect requires p53.Finally, these data may explain the association of the P72 variant and the CSF1/CSF1R pathway with increased senescence and radio-resistance in some epithelial tumor types.

View Article: PubMed Central - PubMed

Affiliation: Program in Molecular and Cellular Oncogenesis, The Wistar Institute, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
The p53 tumor suppressor gene has a common polymorphism at codon 72 that alters its function. We previously reported that the proline 72 polymorphic variant of p53 (P72) demonstrates increased ability to transactivate a subset of genes, relative to arginine 72 (R72); one of these genes is macrophage colony stimulating factor (CSF1). At present, the mechanism(s) underlying the increased transcriptional activity of P72 toward genes like CSF1 have not been completely elucidated. Additionally, the consequences of increased transcription of genes like CSF1 by the P72 variant to the downstream p53 pathway are unknown. In this report, we address these issues. We show that the CSF1 gene contains a conserved binding site for p53, and interestingly that the P72 variant shows increased ability to bind to this site. Moreover, we show that increased CSF1/CSF1R signaling in P72 cells feeds back on the p53 pathway to enhance p53 phosphorylation, levels, and transactivation of target genes, particularly the cyclin-dependent kinase inhibitor p21 (CDKN1A). This leads to an increase in p53-mediated growth arrest, along with a concomitant decrease in apoptosis. Notably, the CSF1/CSF1R signaling axis is overexpressed in several epithelial cancers, and there is clinical evidence that this pathway plays a role in radio-resistance of some cancers. We show that cells expressing CSF1 and CSF1R are indeed radio-resistant, and further, that this effect requires p53. These combined data are the first to implicate the CSF1/CSF1R pathway in the decision between p53-mediated growth arrest and apoptosis. They are also the first to highlight a cytokine as influential in cell fate determined by p53 in epithelial cells. Finally, these data may explain the association of the P72 variant and the CSF1/CSF1R pathway with increased senescence and radio-resistance in some epithelial tumor types.

Show MeSH
Related in: MedlinePlus