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Reactive oxygen species and p21Waf1/Cip1 are both essential for p53-mediated senescence of head and neck cancer cells.

Fitzgerald AL, Osman AA, Xie TX, Patel A, Skinner H, Sandulache V, Myers JN - Cell Death Dis (2015)

Bottom Line: The expression of p21 and production of ROS have been associated with the induction of cellular senescence, but the intricate relationship between p21 and ROS and how they work together to induce senescence remains elusive.We conclude that the level of ROS is crucial in initiating p53's transcription of p21 leading to senescence.Our data offer a rationale to consider the use of either ROS inducing agents or therapies that increase p21 expression in combination with radiation as approaches in cancer therapy and emphasizes the importance of considering TP53 status when selecting a patient's treatment options.

View Article: PubMed Central - PubMed

Affiliation: 1] Graduate School of Biomedical Sciences, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA [2] Department of Head and Neck Surgery, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA.

ABSTRACT
Treatment of head and neck squamous cell carcinoma, HNSCC, often requires multimodal therapy, including radiation therapy. The efficacy of radiotherapy in controlling locoregional recurrence, the most frequent cause of death from HNSCC, is critically important for patient survival. One potential biomarker to determine radioresistance is TP53 whose alterations are predictive of poor radiation response. DNA-damaging reactive oxygen species (ROS) are a by-product of ionizing radiation that lead to the activation of p53, transcription of p21(cip1/waf1) and, in the case of wild-type TP53 HNSCC cells, cause senescence. The expression of p21 and production of ROS have been associated with the induction of cellular senescence, but the intricate relationship between p21 and ROS and how they work together to induce senescence remains elusive. For the first time, we show that persistent exposure to low levels of the ROS, hydrogen peroxide, leads to the long-term expression of p21 in HNSCC cells with a partially functional TP53, resulting in senescence. We conclude that the level of ROS is crucial in initiating p53's transcription of p21 leading to senescence. It is p21's ability to sustain elevated levels of ROS, in turn, that allows for a long-term oxidative stress, and ensures an active p53-p21-ROS signaling loop. Our data offer a rationale to consider the use of either ROS inducing agents or therapies that increase p21 expression in combination with radiation as approaches in cancer therapy and emphasizes the importance of considering TP53 status when selecting a patient's treatment options.

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Long-term expression of p21Waf1/Cip1 is essential for radiation-induced senescence in HNSCC cells. (a) quantitative RT-PCR analysis of p21 mRNA expression in HNSCC cells treated with 4-Gy ionizing radiation and collected at indicated time points. Error bars represent S.D. of each sample performed in duplicate. (b) Western blot showing differences in p21 expression between wtp53 and mutp53 HNSCC cells when treated with 4-Gy ionizing radiation; protein lysates collected at the indicated time points. (c) Western blot results confirming lentiviral p21 knockdown in HN30 cells and their lack of response to ionizing radiation (d) Clonogenic assay showing resistance to ionizing radiation in HN30 cells with knockdown of p21 when compared with Lenti control. (e) SA-β-gal staining was performed 4 days after the cells were exposed to 4-Gy ionizing radiation. (f) Quantitation of SA-β-gal-positive senescent cells (those staining blue) from four randomly selected fields
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fig2: Long-term expression of p21Waf1/Cip1 is essential for radiation-induced senescence in HNSCC cells. (a) quantitative RT-PCR analysis of p21 mRNA expression in HNSCC cells treated with 4-Gy ionizing radiation and collected at indicated time points. Error bars represent S.D. of each sample performed in duplicate. (b) Western blot showing differences in p21 expression between wtp53 and mutp53 HNSCC cells when treated with 4-Gy ionizing radiation; protein lysates collected at the indicated time points. (c) Western blot results confirming lentiviral p21 knockdown in HN30 cells and their lack of response to ionizing radiation (d) Clonogenic assay showing resistance to ionizing radiation in HN30 cells with knockdown of p21 when compared with Lenti control. (e) SA-β-gal staining was performed 4 days after the cells were exposed to 4-Gy ionizing radiation. (f) Quantitation of SA-β-gal-positive senescent cells (those staining blue) from four randomly selected fields

Mentions: To assess whether the lack of radiation-induced senescence seen in and mutp53 HNSCC cells, relative to wtp53 HNSCC cells, was due to the loss of p53's transcriptional activity in the induction of p21, a protein well known to have an important role in mediating premature senescence, we measured the expression of the p21 gene by qRT-PCR after exposure to 4 Gy. As shown in Figure 2a, among the mutp53 bearing cells, only PCI-13 A161S had minimal, short-lived expression of the p21 gene compared with the enhanced, long-term expression seen in wtp53 cells. All other mutp53 or TP53 cell lines had no significant elevation in p21 mRNA. Western blot analysis demonstrated long-term expression of the p21 protein in wtp53 cells, with no expression in mutp53 and TP53 cells, but short-term expression in PCI-13 A161S cells (Figure 2b).


Reactive oxygen species and p21Waf1/Cip1 are both essential for p53-mediated senescence of head and neck cancer cells.

Fitzgerald AL, Osman AA, Xie TX, Patel A, Skinner H, Sandulache V, Myers JN - Cell Death Dis (2015)

Long-term expression of p21Waf1/Cip1 is essential for radiation-induced senescence in HNSCC cells. (a) quantitative RT-PCR analysis of p21 mRNA expression in HNSCC cells treated with 4-Gy ionizing radiation and collected at indicated time points. Error bars represent S.D. of each sample performed in duplicate. (b) Western blot showing differences in p21 expression between wtp53 and mutp53 HNSCC cells when treated with 4-Gy ionizing radiation; protein lysates collected at the indicated time points. (c) Western blot results confirming lentiviral p21 knockdown in HN30 cells and their lack of response to ionizing radiation (d) Clonogenic assay showing resistance to ionizing radiation in HN30 cells with knockdown of p21 when compared with Lenti control. (e) SA-β-gal staining was performed 4 days after the cells were exposed to 4-Gy ionizing radiation. (f) Quantitation of SA-β-gal-positive senescent cells (those staining blue) from four randomly selected fields
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4385922&req=5

fig2: Long-term expression of p21Waf1/Cip1 is essential for radiation-induced senescence in HNSCC cells. (a) quantitative RT-PCR analysis of p21 mRNA expression in HNSCC cells treated with 4-Gy ionizing radiation and collected at indicated time points. Error bars represent S.D. of each sample performed in duplicate. (b) Western blot showing differences in p21 expression between wtp53 and mutp53 HNSCC cells when treated with 4-Gy ionizing radiation; protein lysates collected at the indicated time points. (c) Western blot results confirming lentiviral p21 knockdown in HN30 cells and their lack of response to ionizing radiation (d) Clonogenic assay showing resistance to ionizing radiation in HN30 cells with knockdown of p21 when compared with Lenti control. (e) SA-β-gal staining was performed 4 days after the cells were exposed to 4-Gy ionizing radiation. (f) Quantitation of SA-β-gal-positive senescent cells (those staining blue) from four randomly selected fields
Mentions: To assess whether the lack of radiation-induced senescence seen in and mutp53 HNSCC cells, relative to wtp53 HNSCC cells, was due to the loss of p53's transcriptional activity in the induction of p21, a protein well known to have an important role in mediating premature senescence, we measured the expression of the p21 gene by qRT-PCR after exposure to 4 Gy. As shown in Figure 2a, among the mutp53 bearing cells, only PCI-13 A161S had minimal, short-lived expression of the p21 gene compared with the enhanced, long-term expression seen in wtp53 cells. All other mutp53 or TP53 cell lines had no significant elevation in p21 mRNA. Western blot analysis demonstrated long-term expression of the p21 protein in wtp53 cells, with no expression in mutp53 and TP53 cells, but short-term expression in PCI-13 A161S cells (Figure 2b).

Bottom Line: The expression of p21 and production of ROS have been associated with the induction of cellular senescence, but the intricate relationship between p21 and ROS and how they work together to induce senescence remains elusive.We conclude that the level of ROS is crucial in initiating p53's transcription of p21 leading to senescence.Our data offer a rationale to consider the use of either ROS inducing agents or therapies that increase p21 expression in combination with radiation as approaches in cancer therapy and emphasizes the importance of considering TP53 status when selecting a patient's treatment options.

View Article: PubMed Central - PubMed

Affiliation: 1] Graduate School of Biomedical Sciences, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA [2] Department of Head and Neck Surgery, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA.

ABSTRACT
Treatment of head and neck squamous cell carcinoma, HNSCC, often requires multimodal therapy, including radiation therapy. The efficacy of radiotherapy in controlling locoregional recurrence, the most frequent cause of death from HNSCC, is critically important for patient survival. One potential biomarker to determine radioresistance is TP53 whose alterations are predictive of poor radiation response. DNA-damaging reactive oxygen species (ROS) are a by-product of ionizing radiation that lead to the activation of p53, transcription of p21(cip1/waf1) and, in the case of wild-type TP53 HNSCC cells, cause senescence. The expression of p21 and production of ROS have been associated with the induction of cellular senescence, but the intricate relationship between p21 and ROS and how they work together to induce senescence remains elusive. For the first time, we show that persistent exposure to low levels of the ROS, hydrogen peroxide, leads to the long-term expression of p21 in HNSCC cells with a partially functional TP53, resulting in senescence. We conclude that the level of ROS is crucial in initiating p53's transcription of p21 leading to senescence. It is p21's ability to sustain elevated levels of ROS, in turn, that allows for a long-term oxidative stress, and ensures an active p53-p21-ROS signaling loop. Our data offer a rationale to consider the use of either ROS inducing agents or therapies that increase p21 expression in combination with radiation as approaches in cancer therapy and emphasizes the importance of considering TP53 status when selecting a patient's treatment options.

Show MeSH
Related in: MedlinePlus