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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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Short-term treatment with H2O2 dramatically elevates ROS with differences in p21 expression and senescence varying by TP53 functional status. (a) DCFDA staining and flow cytometry of HN30 and HN31 cells treated with either 250 μM H2O2 for 1 h or 4 Gy, with cells collected at the indicated time points after treatment. Error bars represent S.D. of each sample performed in triplicate. (b) Western blot of HNSCC cells treated with 250 μM of H2O2 for 1  h with protein lysates collected at indicated time points. (c) Quantitation of SA-β-gal-positive senescent cells (those staining blue) from four randomly selected fields. (d) Western blot showing a time course of the difference in p21 protein stability and p53 activity in HN31 cells after treatment with either 250 uM for 1 hr or 4 Gy
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fig6: Short-term treatment with H2O2 dramatically elevates ROS with differences in p21 expression and senescence varying by TP53 functional status. (a) DCFDA staining and flow cytometry of HN30 and HN31 cells treated with either 250 μM H2O2 for 1 h or 4 Gy, with cells collected at the indicated time points after treatment. Error bars represent S.D. of each sample performed in triplicate. (b) Western blot of HNSCC cells treated with 250 μM of H2O2 for 1  h with protein lysates collected at indicated time points. (c) Quantitation of SA-β-gal-positive senescent cells (those staining blue) from four randomly selected fields. (d) Western blot showing a time course of the difference in p21 protein stability and p53 activity in HN31 cells after treatment with either 250 uM for 1 hr or 4 Gy

Mentions: In HN31 cells, direct treatment with 250 μM of H2O2 for 1 h elevated the overall level of ROS to an eightfold increase at 24 h, compared with just a 23% increase after 4 Gy (Figure 6a). By 96 h, although both HN30 and HN31 cells were able to eliminate a majority of the intracellular ROS, the level of ROS seen in HN30 cells with H2O2 treatment was almost equivalent to the level seen with exposure to 4 Gy, the level that is necessary to induce senescence. However, HN31 cells were able to compensate for their massive ROS burst by reducing the ROS back to their baseline level. Collectively, these results provide evidence that HNSCC cells, regardless of their p53 status, do maintain redox capabilities; yet, cells with a mutation of TP53 seem to have acquired the ability to reduce ROS to a level that will diminish the amount of permanent cell damage.


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)

Short-term treatment with H2O2 dramatically elevates ROS with differences in p21 expression and senescence varying by TP53 functional status. (a) DCFDA staining and flow cytometry of HN30 and HN31 cells treated with either 250 μM H2O2 for 1 h or 4 Gy, with cells collected at the indicated time points after treatment. Error bars represent S.D. of each sample performed in triplicate. (b) Western blot of HNSCC cells treated with 250 μM of H2O2 for 1  h with protein lysates collected at indicated time points. (c) Quantitation of SA-β-gal-positive senescent cells (those staining blue) from four randomly selected fields. (d) Western blot showing a time course of the difference in p21 protein stability and p53 activity in HN31 cells after treatment with either 250 uM for 1 hr or 4 Gy
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig6: Short-term treatment with H2O2 dramatically elevates ROS with differences in p21 expression and senescence varying by TP53 functional status. (a) DCFDA staining and flow cytometry of HN30 and HN31 cells treated with either 250 μM H2O2 for 1 h or 4 Gy, with cells collected at the indicated time points after treatment. Error bars represent S.D. of each sample performed in triplicate. (b) Western blot of HNSCC cells treated with 250 μM of H2O2 for 1  h with protein lysates collected at indicated time points. (c) Quantitation of SA-β-gal-positive senescent cells (those staining blue) from four randomly selected fields. (d) Western blot showing a time course of the difference in p21 protein stability and p53 activity in HN31 cells after treatment with either 250 uM for 1 hr or 4 Gy
Mentions: In HN31 cells, direct treatment with 250 μM of H2O2 for 1 h elevated the overall level of ROS to an eightfold increase at 24 h, compared with just a 23% increase after 4 Gy (Figure 6a). By 96 h, although both HN30 and HN31 cells were able to eliminate a majority of the intracellular ROS, the level of ROS seen in HN30 cells with H2O2 treatment was almost equivalent to the level seen with exposure to 4 Gy, the level that is necessary to induce senescence. However, HN31 cells were able to compensate for their massive ROS burst by reducing the ROS back to their baseline level. Collectively, these results provide evidence that HNSCC cells, regardless of their p53 status, do maintain redox capabilities; yet, cells with a mutation of TP53 seem to have acquired the ability to reduce ROS to a level that will diminish the amount of permanent cell damage.

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