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p53 Signaling and Cancer Cell Response to Genotoxic Stress: Beyond Cell Cycle Checkpoints and Apoptosis

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Biology".

Deadline for manuscript submissions: closed (31 December 2020) | Viewed by 16496

Special Issue Editor

Prof. Dr. Razmik Mirzayans

E-Mail Website
Guest Editor
Department of Oncology, Cross Cancer Institute, University of Alberta, Edmonton, AB T6G 1Z2, Canada
Interests: DNA damage response; p53 signaling; senescence; polyploidy; multinucleation; radioresistance; chemoresistance
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

The landscape of the DNA damage response has changed. Notably, the biological outputs orchestrated by the p53 tumor suppressor extends far beyond conventional cell cycle arrest and/or apoptosis. Under physiological conditions (e.g., the absence of ectopic p53 gene expression), the activation of the p53 signaling pathway following exposure to ionizing radiation and chemotherapeutic agents serves to prevent death through apoptosis and other modes of cell death, and to induce a senescence-like proliferation arrest. The latter cells exhibit a highly enlarged morphology, remain dormant, secrete a myriad of growth promoting factors, and have the potential of giving rise to tumor repopulating progeny. Furthermore, cancer cells exhibiting apoptotic features following exposure to genotoxic agents can undergo a reversal process (anastasis), ultimately resulting in tumor re-population.

The purpose of this Special Issue is to bring together research/review articles on the growing complexity surrounding p53 in general, and the long-term biological outputs controlled by p53 and its key downstream effectors (e.g., CDKN1A) in particular. Articles on tumor heterogeneity, advances in single-cell detection methodologies to study cancer cell responses to genotoxic stress, and novel therapeutic approaches by targeting proliferation-arrested (dormant) cancer cells are particularly welcomed.

Prof. Dr. Razmik Mirzayans
Guest Editor

Manuscript Submission Information

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Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. International Journal of Molecular Sciences is an international peer-reviewed open access semimonthly journal published by MDPI.

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Keywords

  • wild-type p53 signaling
  • mutant p53 signaling
  • cell cycle checkpoints
  • apoptosis
  • anastasis (apoptosis reversal)
  • therapy-induced senescence
  • tumor heterogeneity

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Published Papers (2 papers)

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Research

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16 pages, 2917 KiB  
Article
Loss of p53 Sensitizes Cells to Palmitic Acid-Induced Apoptosis by Reactive Oxygen Species Accumulation
by Guowu Yu, Hongwei Luo, Na Zhang, Yongbin Wang, Yangping Li, Huanhuan Huang, Yinghong Liu, Yufeng Hu, Hanmei Liu, Junjie Zhang, Yi Tang and Yubi Huang
Int. J. Mol. Sci. 2019, 20(24), 6268; https://doi.org/10.3390/ijms20246268 - 12 Dec 2019
Cited by 65 | Viewed by 9560
Abstract
Palmitic acid, the most common saturated free fatty acid, can lead to lipotoxicity and apoptosis when overloaded in non-fat cells. Palmitic acid accumulation can induce pancreatic β-cell dysfunction and cardiac myocyte apoptosis. Under various cellular stresses, the activation of p53 signaling can lead [...] Read more.
Palmitic acid, the most common saturated free fatty acid, can lead to lipotoxicity and apoptosis when overloaded in non-fat cells. Palmitic acid accumulation can induce pancreatic β-cell dysfunction and cardiac myocyte apoptosis. Under various cellular stresses, the activation of p53 signaling can lead to cell cycle arrest, DNA repair, senescence, or apoptosis, depending on the severity/type of stress. Nonetheless, the precise role of p53 in lipotoxicity induced by palmitic acid is not clear. Here, our results show that palmitic acid induces p53 activation in a dose- and time-dependent manner. Furthermore, loss of p53 makes cells sensitive to palmitic acid-induced apoptosis. These results were demonstrated in human colon carcinoma cells (HCT116) and primary mouse embryo fibroblasts (MEF) through analysis of DNA fragmentation, flow cytometry, colony formation, and Western blots. In the HCT116 p53−/− cell line, palmitic acid induced greater reactive oxygen species formation compared to the p53+/+ cell line. The reactive oxygen species (ROS) scavengers N-acetyl cysteine (NAC) and reduced glutathione (GSH) partially attenuated apoptosis in the HCT116 p53−/− cell line but had no obvious effect on the p53+/+ cell line. Furthermore, p53 induced the expression of its downstream target genes, p21 and Sesn2, in response to ROS induced by palmitic acid. Loss of p21 also leads to more palmitic acid-induced cell apoptosis in the HCT116 cell line compared with HCT116 p53+/+ and HCT116 p53−/−. In a mouse model of obesity, glucose tolerance test assays showed higher glucose levels in p53−/− mice that received a high fat diet compared to wild type mice that received the same diet. There were no obvious differences between p53−/− and p53+/+ mice that received a regular diet. We conclude that p53 may provide some protection against palmitic acid- induced apoptosis in cells by targeting its downstream genes in response to this stress. Full article
(This article belongs to the Special Issue p53 Signaling and Cancer Cell Response to Genotoxic Stress: Beyond Cell Cycle Checkpoints and Apoptosis)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Palmitic acid induces p53 expression in human colon carcinoma cells (HCT116) in a dose- and time-dependent manner. (<b>A</b>) HCT116 p53<sup>+/+</sup> and p53<sup>−/−</sup> cell lines were treated with the indicated dose of palmitic acid. A total of 40 µg of total protein extract was resolved on SDS-PAGE. Immunoblotting was performed using p53 and p21 antibodies, and β-actin was used as a loading control. (<b>B</b>) Quantitative analysis of (A) by ImageJ software. (<b>C</b>) HCT116 cells were treated with 250 µM palmitic acid at indicated time points; 40 µg of total protein extract was resolved on SDS-PAGE. Immunoblotting was performed using p53 and p21 antibodies, and β-actin was used as a loading control. (<b>D</b>) Quantitative analysis of (C) by ImageJ software. (<b>E</b>) Palmitic acid, not oleic acid, specifically activated p53 expression. HCT116 cells were treated with 250 µM oleic acid and 250 µM palmitic acid for 24 h. Immunoblotting was performed using p53 and p21 antibodies. (<b>F</b>) Quantitative analysis of E by ImageJ software. The data are expressed as mean ± SE of three independent experiments.</p> Full article ">Figure 2
<p>Distinct apoptosis effects induced by palmitic acid in HCT116 p53<sup>+/+</sup> and p53<sup>−/−</sup> cell lines. (<b>A</b>) HCT116 cells were treated with 250 µM oleic acid or 250 µM palmitic acid for 24 h. Crystal violet staining was performed to show all cells (blue) in the plate. (<b>B</b>) After 24 h, HCT116 p53<sup>+/+</sup> and p53<sup>−/−</sup> cells were treated with 250 µM palmitic acid for the indicated times. Cells were harvested, and genomic DNA was extracted. A total of 5 µg of DNA was loaded for each sample on a 1% agarose gel to detect DNA laddering. (<b>C</b>) Apoptotic cells (Sub-G1) were quantified by flow cytometry analysis after treating with 500 µM palmitic acid for the indicated time. Cells were stained with 2 µg/mL propidium iodide. DNA content analysis was performed. Values indicate the percentage of sub-G1 cells. (<b>D</b>) Percentages of apoptotic cells were quantified as the mean ± SE of three independent experiments. Asterisks represent significant differences between HCT116 p53<sup>+/+</sup> and p53<sup>−/−</sup> (<span class="html-italic">p</span> &lt; 0.01) at indicated PA treatment time points.</p> Full article ">Figure 3
<p>Distinct PA-induced apoptosis effects in mouse embryo fibroblasts (MEF) p53<sup>+/+</sup> and p53<sup>−/−</sup> cell lines. (<b>A</b>) Primary MEF cells were treated with palmitic acid for the indicated time periods (0–48 h). Genomic DNA was isolated from primary MEF after treatment with palmitic acid. A total of 5 µg of DNA was loaded for each sample on a 1% agarose gel to detect DNA laddering. (<b>B</b>) Primary MEF cells were treated with palmitic acid for the indicated time. A total of 40 µg of total protein extract was resolved on SDS-PAGE. p53, cleaved caspase-3, PARP, and cleaved PARP were detected by Western blotting. β-actin was used as a loading control. (<b>C</b>) FACS analysis of primary MEF p53<sup>+/+</sup> and p53<sup>−/−</sup> cells treated with palmitic acid for the indicated time. Cells were fixed with cold methanol, stained with propidium iodide, and subjected to DNA content analysis by flow cytometry. (<b>D</b>) Primary MEF p53<sup>−/−</sup> cells showed greater apoptosis than primary MEF p53<sup>+/+</sup> cells following 24 h of palmitic acid treatment, visualized by staining with Annexin V-FITC for 15 min at RT. An Advanced Microscopy Group (AMG) Evos f1 microscope was used for imaging. The scale is 25 µm.</p> Full article ">Figure 3 Cont.
<p>Distinct PA-induced apoptosis effects in mouse embryo fibroblasts (MEF) p53<sup>+/+</sup> and p53<sup>−/−</sup> cell lines. (<b>A</b>) Primary MEF cells were treated with palmitic acid for the indicated time periods (0–48 h). Genomic DNA was isolated from primary MEF after treatment with palmitic acid. A total of 5 µg of DNA was loaded for each sample on a 1% agarose gel to detect DNA laddering. (<b>B</b>) Primary MEF cells were treated with palmitic acid for the indicated time. A total of 40 µg of total protein extract was resolved on SDS-PAGE. p53, cleaved caspase-3, PARP, and cleaved PARP were detected by Western blotting. β-actin was used as a loading control. (<b>C</b>) FACS analysis of primary MEF p53<sup>+/+</sup> and p53<sup>−/−</sup> cells treated with palmitic acid for the indicated time. Cells were fixed with cold methanol, stained with propidium iodide, and subjected to DNA content analysis by flow cytometry. (<b>D</b>) Primary MEF p53<sup>−/−</sup> cells showed greater apoptosis than primary MEF p53<sup>+/+</sup> cells following 24 h of palmitic acid treatment, visualized by staining with Annexin V-FITC for 15 min at RT. An Advanced Microscopy Group (AMG) Evos f1 microscope was used for imaging. The scale is 25 µm.</p> Full article ">Figure 4
<p>HCT116 p53<sup>−/−</sup> cells accumulate more reactive oxygen species (ROS) than p53<sup>+/+</sup> cells after palmitic acid treatment. (<b>A</b>) Palmitic acid induces ROS in a dose-dependent manner after 24 h of treatment. ROS were analyzed by flow cytometry as described in Materials and Methods. HCT116 p53<sup>+/+</sup> and p53<sup>−/−</sup> cells were treated with 0, 250, and 500 μM palmitic acid for 24 h. (<b>B</b>) Quantification of ROS induced by palmitic acid in HCT116 p53<sup>+/+</sup> and p53<sup>−/−</sup> cell lines. The data are expressed as mean ± SE of three independent experiments (<span class="html-italic">n</span> = 3). Asterisks represent significant differences between HCT116 p53<sup>+/+</sup> and p53<sup>−/−</sup> cells (<span class="html-italic">p</span> &lt; 0.01). (<b>C</b>) Relative ROS levels of primary MEF p53<sup>+/+</sup> and p53<sup>−/−</sup> cells. The data are expressed as mean ± SE of three independent experiments (<span class="html-italic">n</span> = 3). Asterisks represent significant differences between primary MEF p53<sup>+/+</sup> and p53<sup>−/−</sup> cells (<span class="html-italic">p</span> &lt; 0.01). (<b>D</b>) A 24 h treatment with the antioxidants glutathione (GSH, 20 µg/mL) or N-Acetyl cysteine (NAC, 200 µg/mL) attenuated primary MEF cell apoptosis caused by palmitic acid in MEF p53<sup>−/−</sup> cells. A total of 5 µg of DNA was loaded for each sample on a 1% agarose gel to detect DNA laddering.</p> Full article ">Figure 5
<p>Expression of <span class="html-italic">p21</span> and <span class="html-italic">Sesn2</span> genes in p53<sup>+/+</sup> and p53<sup>−/−</sup> cells under PA stress. (<b>A</b>) Relative mRNA expression of <span class="html-italic">p21</span> under 250 µM PA stress at indicated time points in HCT116 p53<sup>+/+</sup> and p53<sup>−/−</sup> cell lines. ** indicates <span class="html-italic">p</span> &lt; 0.01, <span class="html-italic">n</span> = 6. (<b>B</b>) Relative mRNA expression of <span class="html-italic">Sesn2</span> under 250 µM PA stress at indicated time points in HCT116 p53<sup>+/+</sup> and p53<sup>−/−</sup> cell lines. * indicates <span class="html-italic">p</span> &lt; 0.05, <span class="html-italic">n</span> = 6. (<b>C</b>) Relative mRNA expression of <span class="html-italic">p21</span> under 250 µM PA stress at indicated time points in MEF p53<sup>+/+</sup> and p53<sup>−/−</sup> cell lines. ** indicates <span class="html-italic">p</span> &lt; 0.01, <span class="html-italic">n</span> = 6. (<b>D</b>) Relative mRNA expression of <span class="html-italic">Sesn2</span> under 250 µM PA stress at indicated time points in MEF p53<sup>+/+</sup> and p53<sup>−/−</sup> cell lines. ** indicates <span class="html-italic">p</span> &lt; 0.01, <span class="html-italic">n</span> = 6. (<b>E</b>) Apoptotic cells (Sub-G1) were quantified by flow cytometry analysis after treating with 500 µM palmitic acid for the indicated time in HCT116 p53<sup>+/+</sup>, HCT116 p53<sup>−/−</sup>, and p53 p21<sup>−/−</sup> cell lines. Cells were stained with 2 µg/mL propidium iodide. DNA content analysis was performed. Values indicate the percentage of sub-G1 cells.</p> Full article ">Figure 6
<p>Biological effects of PA stress on mice feed a high fat diet. (<b>A</b>) Growth curves of mouse weight under a high fat diet and a regular diet. ** indicates <span class="html-italic">p</span> &lt; 0.01, <span class="html-italic">n</span> = 5. (<b>B</b>) Glucose tolerance test results at indicated time points after glucose injection. ** indicates <span class="html-italic">p</span> &lt; 0.01, <span class="html-italic">n</span> ≥ 3.</p> Full article ">

Review

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24 pages, 2632 KiB  
Review
Cellular Responses to Platinum-Based Anticancer Drugs and UVC: Role of p53 and Implications for Cancer Therapy
by David Murray and Razmik Mirzayans
Int. J. Mol. Sci. 2020, 21(16), 5766; https://doi.org/10.3390/ijms21165766 - 11 Aug 2020
Cited by 31 | Viewed by 4720
Abstract
Chemotherapy is intended to induce cancer cell death through apoptosis and other avenues. Unfortunately, as discussed in this article, moderate doses of genotoxic drugs such as cisplatin typical of those achieved in the clinic often invoke a cytostatic/dormancy rather than cytotoxic/apoptosis response in [...] Read more.
Chemotherapy is intended to induce cancer cell death through apoptosis and other avenues. Unfortunately, as discussed in this article, moderate doses of genotoxic drugs such as cisplatin typical of those achieved in the clinic often invoke a cytostatic/dormancy rather than cytotoxic/apoptosis response in solid tumour-derived cell lines. This is commonly manifested by an extended apoptotic threshold, with extensive apoptosis only being seen after very high/supralethal doses of such agents. The dormancy response can be associated with senescence-like features, polyploidy and/or multinucleation, depending in part on the p53 status of the cells. In most solid tumour-derived cells, dormancy represents a long-term survival mechanism, ultimately contributing to disease recurrence. This review highlights the nonlinearity of key aspects of the molecular and cellular responses to bulky DNA lesions in human cells treated with chemotherapeutic drugs (e.g., cisplatin) or ultraviolet light-C (a widely used tool for unraveling details of the DNA damage-response) as a function of the level of genotoxic stress. Such data highlight the growing realization that targeting dormant cancer cells, which frequently emerge following conventional anticancer treatments, may represent a novel strategy to prevent or, at least, significantly suppress cancer recurrence. Full article
(This article belongs to the Special Issue p53 Signaling and Cancer Cell Response to Genotoxic Stress: Beyond Cell Cycle Checkpoints and Apoptosis)
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Figure 1

Figure 1
<p>A partial schematic of the DNA damage response network illustrating the importance of both the positive and negative regulatory activities of p53 and its downstream target p21 on proteins involved in cytoprotective/antioxidant and cytotoxic/apoptotic pathways. The arrows indicate stimulation, and the T-shaped lines indicate inhibition; the black lines/text refer to responses seen following exposure of cells to moderate concentrations/doses of DNA-damaging agents typical of those where the progressive loss of colony-forming ability is seen; and the red lines/text refer to responses to high/supralethal exposures. The green arrow represents the strategy discussed in <a href="#sec5-ijms-21-05766" class="html-sec">Section 5</a> whereby pharmacological inhibitors of antiapoptotic proteins such as BCL-2 and BCL-XL might be clinically useful for transitioning chemotherapy-induced dormant cancer cells (which have the potential to cause tumour recurrence/metastasis via the generation of para-diploid cancer stem cell (CSC)-like tumour repopulating cells) into an apoptotic cell-death pathway from which they will not return. TCS, therapy-induced cell senescence; PGCC, polyploid giant cancer cell. The protein abbreviations are provided in the abbreviation list.</p> Full article ">Figure 2
<p>Dose-response curves for loss of colony-forming ability, apoptosis and therapy-induced cell senescence (TCS) in normal human fibroblasts following exposure to increasing doses of ultraviolet light-C (UVC) (254 nm): (■) Colony-forming assay (CFA) data for GM38 cells [<a href="#B49-ijms-21-05766" class="html-bibr">49</a>]; (<span style="color:blue">■</span>) UVC-induced TCS in GM38 cells at 7 days after UVC exposure based on the number of cells that retained viability, exhibited flattened and highly enlarged morphology, and were positive for senescence-associated β-galactosidase activity [<a href="#B62-ijms-21-05766" class="html-bibr">62</a>]; (<span style="color:red">●</span>) UVC-induced apoptosis in 2525T cells based on the flow cytometric sub-diploid DNA content assay [<a href="#B49-ijms-21-05766" class="html-bibr">49</a>]; (<span style="color:red">■</span>) UVC-induced apoptosis in GM38 cells based on flow cytometric determination of Annexin V-positive (phosphatidylserine externalized) cells [<a href="#B62-ijms-21-05766" class="html-bibr">62</a>]; (<span style="color:red">♦</span>) UVC-induced apoptosis in primary 293T fibroblasts based on the Annexin V assay [<a href="#B65-ijms-21-05766" class="html-bibr">65</a>]; and (<span style="color:red">▲</span>) UVC-induced apoptosis in AG1522 fibroblasts based on sub-diploid DNA content [<a href="#B64-ijms-21-05766" class="html-bibr">64</a>]. All apoptosis data were assessed at 72 h after UVC exposure.</p> Full article ">Figure 3
<p>Dose-response curves for loss of colony-forming ability and for the induction of apoptosis and therapy-induced cell senescence (TCS) in HCT116 (colon carcinoma) and 224 (melanoma) p53-<span class="html-italic">WT</span> human tumour cells treated with increasing concentrations of cisplatin for 6 h. (▲) colony-forming ability assayed at 10 days posttreatment, (<span style="color:red">●</span>) apoptosis based on caspase-3 activation assayed at 14 h posttreatment, and (<span style="color:blue">■</span>) TCS based on a combination of senescence-associated β-galactosidase activity and PKH2 staining assayed at 6 days posttreatment. Data from Berndtsson et al. [<a href="#B69-ijms-21-05766" class="html-bibr">69</a>].</p> Full article ">Figure 4
<p>Dose-response curves for cisplatin-induced apoptosis and loss of proliferative potential in exponentially-growing cultures of the MCF7 and HCT116 p53-<span class="html-italic">WT</span> human cancer cell lines. (<span style="color:red">●</span>) apoptosis data for cells exposed to increasing concentrations of cisplatin for 2 h. After 4 days, adherent and floating cells were combined, stained with Hoechst 33342 and propidium iodide and evaluated under a fluorescence microscope. Cells that had lost membrane integrity (i.e., propidium iodide-stained cells) as well as those exhibiting nuclear fragmentation were scored as apoptotic. From Mirzayans and Murray [<a href="#B63-ijms-21-05766" class="html-bibr">63</a>]. (●) Our unpublished data were obtained using the 3 day proliferation inhibition assay which involves direct cell counting. The experiments were performed as described [<a href="#B72-ijms-21-05766" class="html-bibr">72</a>] except that cells were incubated with cisplatin for 24 h followed by incubation in drug-free medium for 48 h.</p> Full article ">Figure 5
<p>Cartoon illustrating the generation and fate of polyploid giant cancer cells (PGCCs). Cancer cells (top, left) stressed by factors such as anticancer agents or hypoxia can undergo a complex series of adaptations, including endoreduplication and cell fusion, that result in the emergence of PGCCs with either a highly enlarged nucleus or multiple nuclei. PGCCs may contribute to tumour repopulation following cancer therapy by at least three mechanisms: (i) depolyploidization through undergoing a complex genome reduction process, mediated by key regulators of mitosis, meiosis and self-renewal, ultimately giving rise to para-diploid progeny (i.e., containing a near-diploid number of chromosomes) that exhibit recovery of proliferative capability; (ii) depolyploidization by amitotic processes (e.g., neosis), involving budding and bursting (nuclear fragmentation) similar to prokaryotes and unicellular eukaryotes, to generate tumour initiating cells with cancer stem cell (CSC)-like properties; and (iii) secretion of factors that create a permissive tissue microenvironment for tumour growth and progression. Some features of PGCCs are listed in the left lower box. For details, please consult [<a href="#B85-ijms-21-05766" class="html-bibr">85</a>,<a href="#B87-ijms-21-05766" class="html-bibr">87</a>,<a href="#B88-ijms-21-05766" class="html-bibr">88</a>].</p> Full article ">
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