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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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Increased CSF1 expression induced in cells expressing the P72 variant of p53.A. Quantitative reverse transcription/polymerase chain reaction (QRT-PCR) analysis of RNA isolated from the thymuses of P72 and R72 Hupki mice, analyzed for the level of CSF1 and MDM2 following treatment of mice with 5 Gy gamma radiation after 4 hours. Values shown are the average of independent experiments in which a total of three homozygous P72 (P/P) and R72 (R/R) mice (n = 3 each genotype for treated, 1 each genotype for untreated) were analyzed following radiation; levels are normalized to cyclophilin A. Gy = Gray. Error bars mark standard error. B. Western analysis of lysates from H1299 cells containing tetracycline (tet) inducible p53 encoding either the P72 or the R72 variant for the levels of p53, GAPDH and p21 (CDKN1A). Cells were treated with 0.75 ug/ml doxycycline (doxy) for the indicated time points (hrs). C. QRT-PCR of RNA isolated from tet-inducible p53 P72 or R72 cells treated with doxycycline (doxy) for the indicated time points and analyzed for the level of CSF1 mRNA; values shown are the average of 4 independent experiments, normalized to GAPDH. The error bars mark standard deviation. D. QRT-PCR of RNA isolated from tet-inducible p53 P72 or R72 cells treated with doxycycline (doxy) for the indicated time points and analyzed for the level of BBC3 (PUMA); values shown are the average of four independent experiments, normalized to GAPDH. The error bars mark standard deviation. E. Enzyme-linked immunosorbent assay (ELISA) analysis of CSF1 protein level in lysates isolated from H1299 cells with tetracycline-inducible p53 (P72 or R72) untreated or treated with doxycycline (0.75 ug/mL) for the indicated time points. The data depicted are the averaged results from three independent experiments; error bars mark standard error.
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pone-0074297-g001: Increased CSF1 expression induced in cells expressing the P72 variant of p53.A. Quantitative reverse transcription/polymerase chain reaction (QRT-PCR) analysis of RNA isolated from the thymuses of P72 and R72 Hupki mice, analyzed for the level of CSF1 and MDM2 following treatment of mice with 5 Gy gamma radiation after 4 hours. Values shown are the average of independent experiments in which a total of three homozygous P72 (P/P) and R72 (R/R) mice (n = 3 each genotype for treated, 1 each genotype for untreated) were analyzed following radiation; levels are normalized to cyclophilin A. Gy = Gray. Error bars mark standard error. B. Western analysis of lysates from H1299 cells containing tetracycline (tet) inducible p53 encoding either the P72 or the R72 variant for the levels of p53, GAPDH and p21 (CDKN1A). Cells were treated with 0.75 ug/ml doxycycline (doxy) for the indicated time points (hrs). C. QRT-PCR of RNA isolated from tet-inducible p53 P72 or R72 cells treated with doxycycline (doxy) for the indicated time points and analyzed for the level of CSF1 mRNA; values shown are the average of 4 independent experiments, normalized to GAPDH. The error bars mark standard deviation. D. QRT-PCR of RNA isolated from tet-inducible p53 P72 or R72 cells treated with doxycycline (doxy) for the indicated time points and analyzed for the level of BBC3 (PUMA); values shown are the average of four independent experiments, normalized to GAPDH. The error bars mark standard deviation. E. Enzyme-linked immunosorbent assay (ELISA) analysis of CSF1 protein level in lysates isolated from H1299 cells with tetracycline-inducible p53 (P72 or R72) untreated or treated with doxycycline (0.75 ug/mL) for the indicated time points. The data depicted are the averaged results from three independent experiments; error bars mark standard error.

Mentions: We previously created humanized p53 knock-in mice for both codon 72 variants of p53, proline 72 (P72) and arginine 72 (R72), and showed that these variants functionally recapitulate human phenotypes in mice [15], [19]. In that same study, we performed a micro-array analysis to identify genes that were differentially regulated by the P72 and R72 variants of p53 in the irradiated mouse thymus. This analysis revealed that over 95% of p53 target genes displayed identical transactivation by the P72 and R72 polymorphic variants. However, we identified a subset of approximately a dozen genes that showed increased transactivation by the P72 variant of p53 [15]. One of these genes, macrophage colony stimulating factor 1, or CSF1, was selected here for further analysis. We first sought to confirm the micro-array data by analyzing CSF1 gene expression in age-matched littermate P72 and R72 Hupki mice generated from P/R heterozygous crosses. Three sibling mice homozygous for each genotype (PP or RR) were subjected to 5 Gy gamma radiation, and RNA was isolated from purified thymocytes 4 hours after radiation. Quantitative reverse-transcription/polymerase chain reaction (QRT-PCR) analyses indicated that, consistent with our previous findings, P72 mouse thymocytes expressed significantly higher levels of CSF1 mRNA relative to R72 after gamma irradiation (3.8+/−0.2 vs. 2.8+/−0.1, p = 0.0001). In contrast, transactivation of another p53 target gene, MDM2, showed no difference between the variants (Fig. 1A), nor did transactivation of several other p53 target genes (data not shown).


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)

Increased CSF1 expression induced in cells expressing the P72 variant of p53.A. Quantitative reverse transcription/polymerase chain reaction (QRT-PCR) analysis of RNA isolated from the thymuses of P72 and R72 Hupki mice, analyzed for the level of CSF1 and MDM2 following treatment of mice with 5 Gy gamma radiation after 4 hours. Values shown are the average of independent experiments in which a total of three homozygous P72 (P/P) and R72 (R/R) mice (n = 3 each genotype for treated, 1 each genotype for untreated) were analyzed following radiation; levels are normalized to cyclophilin A. Gy = Gray. Error bars mark standard error. B. Western analysis of lysates from H1299 cells containing tetracycline (tet) inducible p53 encoding either the P72 or the R72 variant for the levels of p53, GAPDH and p21 (CDKN1A). Cells were treated with 0.75 ug/ml doxycycline (doxy) for the indicated time points (hrs). C. QRT-PCR of RNA isolated from tet-inducible p53 P72 or R72 cells treated with doxycycline (doxy) for the indicated time points and analyzed for the level of CSF1 mRNA; values shown are the average of 4 independent experiments, normalized to GAPDH. The error bars mark standard deviation. D. QRT-PCR of RNA isolated from tet-inducible p53 P72 or R72 cells treated with doxycycline (doxy) for the indicated time points and analyzed for the level of BBC3 (PUMA); values shown are the average of four independent experiments, normalized to GAPDH. The error bars mark standard deviation. E. Enzyme-linked immunosorbent assay (ELISA) analysis of CSF1 protein level in lysates isolated from H1299 cells with tetracycline-inducible p53 (P72 or R72) untreated or treated with doxycycline (0.75 ug/mL) for the indicated time points. The data depicted are the averaged results from three independent experiments; error bars mark standard error.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3760869&req=5

pone-0074297-g001: Increased CSF1 expression induced in cells expressing the P72 variant of p53.A. Quantitative reverse transcription/polymerase chain reaction (QRT-PCR) analysis of RNA isolated from the thymuses of P72 and R72 Hupki mice, analyzed for the level of CSF1 and MDM2 following treatment of mice with 5 Gy gamma radiation after 4 hours. Values shown are the average of independent experiments in which a total of three homozygous P72 (P/P) and R72 (R/R) mice (n = 3 each genotype for treated, 1 each genotype for untreated) were analyzed following radiation; levels are normalized to cyclophilin A. Gy = Gray. Error bars mark standard error. B. Western analysis of lysates from H1299 cells containing tetracycline (tet) inducible p53 encoding either the P72 or the R72 variant for the levels of p53, GAPDH and p21 (CDKN1A). Cells were treated with 0.75 ug/ml doxycycline (doxy) for the indicated time points (hrs). C. QRT-PCR of RNA isolated from tet-inducible p53 P72 or R72 cells treated with doxycycline (doxy) for the indicated time points and analyzed for the level of CSF1 mRNA; values shown are the average of 4 independent experiments, normalized to GAPDH. The error bars mark standard deviation. D. QRT-PCR of RNA isolated from tet-inducible p53 P72 or R72 cells treated with doxycycline (doxy) for the indicated time points and analyzed for the level of BBC3 (PUMA); values shown are the average of four independent experiments, normalized to GAPDH. The error bars mark standard deviation. E. Enzyme-linked immunosorbent assay (ELISA) analysis of CSF1 protein level in lysates isolated from H1299 cells with tetracycline-inducible p53 (P72 or R72) untreated or treated with doxycycline (0.75 ug/mL) for the indicated time points. The data depicted are the averaged results from three independent experiments; error bars mark standard error.
Mentions: We previously created humanized p53 knock-in mice for both codon 72 variants of p53, proline 72 (P72) and arginine 72 (R72), and showed that these variants functionally recapitulate human phenotypes in mice [15], [19]. In that same study, we performed a micro-array analysis to identify genes that were differentially regulated by the P72 and R72 variants of p53 in the irradiated mouse thymus. This analysis revealed that over 95% of p53 target genes displayed identical transactivation by the P72 and R72 polymorphic variants. However, we identified a subset of approximately a dozen genes that showed increased transactivation by the P72 variant of p53 [15]. One of these genes, macrophage colony stimulating factor 1, or CSF1, was selected here for further analysis. We first sought to confirm the micro-array data by analyzing CSF1 gene expression in age-matched littermate P72 and R72 Hupki mice generated from P/R heterozygous crosses. Three sibling mice homozygous for each genotype (PP or RR) were subjected to 5 Gy gamma radiation, and RNA was isolated from purified thymocytes 4 hours after radiation. Quantitative reverse-transcription/polymerase chain reaction (QRT-PCR) analyses indicated that, consistent with our previous findings, P72 mouse thymocytes expressed significantly higher levels of CSF1 mRNA relative to R72 after gamma irradiation (3.8+/−0.2 vs. 2.8+/−0.1, p = 0.0001). In contrast, transactivation of another p53 target gene, MDM2, showed no difference between the variants (Fig. 1A), nor did transactivation of several other p53 target genes (data not shown).

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