scholarly journals Deciphering the Genome Protection Roles of Autophagy in Primary Human Dermal Fibroblasts (HDFs) against Ultraviolet-(B) –Induced Skin Photodamage

2020 ◽  
Author(s):  
Sheikh Ahmad Umar ◽  
Sheikh Abdullah Tasduq
Keyword(s):  
Dna Damage ◽  
Stress Response ◽  
Er Stress ◽  
Dna Damage Response ◽  
Dermal Fibroblasts ◽  
Ultraviolet B ◽  
Great Promise ◽  
Er Stress Response ◽  
Damage Response ◽  
Photo Damage

AbstractUltraviolet-B (UV-B) exposure to skin causes photo-damage and acts as the primary etiological agent in photo-carcinogenesis. UV-B exposure induces photodamage in epidermal cells and is the major factor that challenges skin homeostasis. Autophagy allows fundamental adaptation of cells to metabolic needs and stresses. Cellular dysfunction is observed in aged tissues and in toxic insults to cells that undergo through stress. Conversely, promising anti-aging strategies aimed at inhibiting the mTOR pathway has been found to significantly improve the aging related disorders. Recently, autophagy has been found to positively regulate skin homeostasis by enhancing DNA damage recognition. Here we investigated the Geno-protective roles of autophagy in UV-B exposed primary HDFs. We found that improving autophagy levels in HDFs regulates UV-B mediated cellular stress by decreasing the formation of DNA photo adducts, alleviates oxidative and ER stress response and by regulating the expression levels of cell cycle regulatory proteins P21 and P27. Autophagy also prevents HDFs from UV-B -induced nuclear damage as is evident from Tunnel assay and Acridine Orange/Ethidium Bromide co-staining. Salubrinal, (an eIf2α inhibitor) significantly decreases the DNA damage response in HDFs. P62 silenced HDFs show enhanced DNA damage response and disturbs the tumor suppressor axis PTEN/pAKT towards damage whereas ATG7 silenced HDFs reveal an unexpected consequence by decreasing the UV-B -induced DNA damage compared to UV-B treated HDFs. Together, our results suggest that autophagy is essential in protecting skin cells from UV-B radiation -induced photo-damage and holds great promise in devising it as a suitable therapeutic strategy against skin photo-damage.HighlightsAutophagy is an immediate molecular event induced following exposure of primary HDFs to UV-B –irradiationAutophagy offers pro-survival capacity to HDFs under UV-B induced genotoxic stressAutophagy regulates DNA Damage Response via regulation of oxidative and ER stress in UV-B exposed HDFsRelieving ER stress response offers significant protection to primary HDFs from UV-B by decreasing the DNA damageAutophagy deprivation to HDFs via P62 silencing potentiates UV-B -induced DNA damage responseATG7 silencing in UV-B exposed HDFs unexpectedly alleviates the DNA Damage Response in primary HDFs

scholarly journals Pharmacological Activation of Autophagy Restores Cellular Homeostasis in Ultraviolet-(B)-Induced Skin Photodamage

2021 ◽  
Vol 11 ◽  
Author(s):  
Sheikh Ahmad Umar ◽  
Naikoo Hussain Shahid ◽  
Lone Ahmad Nazir ◽  
Malik Ahmad Tanveer ◽  
Gupta Divya ◽  
...  
Keyword(s):  
Dna Damage ◽  
Er Stress ◽  
Stress Responses ◽  
Repair Process ◽  
Dermal Fibroblasts ◽  
Ultraviolet B ◽  
Cellular Damage ◽  
Great Promise ◽  
Independent Manner ◽  
Photo Damage

Ultraviolet (UV) exposure to the skin causes photo-damage and acts as the primary etiological agent in photo-carcinogenesis. UV-B exposure induces cellular damage and is the major factor challenging skin homeostasis. Autophagy allows the fundamental adaptation of cells to metabolic and oxidative stress. Cellular dysfunction has been observed in aged tissues and in toxic insults to cells undergoing stress. Conversely, promising anti-aging strategies aimed at inhibiting the mTOR pathway have been found to significantly improve the aging-related disorders. Recently, autophagy has been found to positively regulate skin homeostasis by enhancing DNA damage recognition. Here, we investigated the geno-protective roles of autophagy in UV-B-exposed primary human dermal fibroblasts (HDFs). We found that UV-B irradiation to HDFs impairs the autophagy response in a time- and intensity-independent manner. However, improving autophagy levels in HDFs with pharmacological activators regulates the UV-B-induced cellular stress by decreasing the induction of DNA photo-adducts, promoting the DNA repair process, alleviating oxidative and ER stress responses, and regulating the expression levels of key cell cycle regulatory proteins. Autophagy also prevents HDFs from UV-B-induced nuclear damage as is evident in TUNEL assay and Acridine Orange/Ethidium Bromide co-staining. Salubrinal (an eIF2α phosphatase inhibitor) relieves ER stress response in cells and also significantly alleviates DNA damage and promotes the repair process in UV-B-exposed HDFs. P62-silenced HDFs show enhanced DNA damage response and also disturb the tumor suppressor PTEN/pAKT signaling axis in UV-B-exposed HDFs whereas Atg7-silenced HDFs reveal an unexpected consequence by decreasing the UV-B-induced DNA damage. Taken together, these results suggest that interventional autophagy offers significant protection against UV-B radiation-induced photo-damage and holds great promise in devising it as a suitable therapeutic strategy against skin pathological disorders.

scholarly journals Phosphorylation of PLK3 Is Controlled by Protein Phosphatase 6

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1506 ◽  
Author(s):  
Cecilia Aquino Perez ◽  
Matous Palek ◽  
Lenka Stolarova ◽  
Patrick von Morgen ◽  
Libor Macurek
Keyword(s):  
Dna Damage ◽  
Stress Response ◽  
Osmotic Stress ◽  
Dna Damage Response ◽  
Protein Phosphatase ◽  
Gene Editing ◽  
Kinase Activity ◽  
Cell Cycle Control ◽  
Damage Response ◽  
Transformed Cell Lines

Polo-like kinases play essential roles in cell cycle control and mitosis. In contrast to other members of this kinase family, PLK3 has been reported to be activated upon cellular stress including DNA damage, hypoxia and osmotic stress. Here we knocked out PLK3 in human non-transformed RPE cells using CRISPR/Cas9-mediated gene editing. Surprisingly, we find that loss of PLK3 does not impair stabilization of HIF1α after hypoxia, phosphorylation of the c-Jun after osmotic stress and dynamics of DNA damage response after exposure to ionizing radiation. Similarly, RNAi-mediated depletion of PLK3 did not impair stress response in human transformed cell lines. Exposure of cells to various forms of stress also did not affect kinase activity of purified EGFP-PLK3. We conclude that PLK3 is largely dispensable for stress response in human cells. Using mass spectrometry, we identify protein phosphatase 6 as a new interacting partner of PLK3. Polo box domain of PLK3 mediates the interaction with the PP6 complex. Finally, we find that PLK3 is phosphorylated at Thr219 in the T-loop and that PP6 constantly dephosphorylates this residue. However, in contrast to PLK1, phosphorylation of Thr219 does not upregulate enzymatic activity of PLK3, suggesting that activation of both kinases is regulated by distinct mechanisms.

Replication Associated Stress in the Fetal Stem Cell Pool of FA Mice Can be Rescued By Pharmacologic Inhibition of Constitutively Activated P38 MAPK

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2411-2411
Author(s):  
Youngme Yoon ◽  
Ashley N. Kamimae-Lanning ◽  
Kelsie Storm ◽  
Natalya A Goloviznina ◽  
Peter Kurre
Keyword(s):  
Dna Damage ◽  
Stress Response ◽  
P38 Mapk ◽  
Dna Damage Response ◽  
Colony Formation ◽  
Model Systems ◽  
Donor Chimerism ◽  
Damage Response ◽  
Physiologic Role

Abstract Fanconi Anemia (FA) is a rare, recessively heritable disorder with prominent failure of hematopoiesis. The physiologic role of FA proteins has not been fully resolved to date. While several existing model systems delineate its role in DNA damage response caused by alkylating agents, aldehydes, and inflammatory cytokines, all rely on experimental induction. We previously demonstrated the in utero onset of hematopoietic failure in mice with genetic disruption of Fancc. Herein, we found significant deficits in the fetal liver (FL) hematopoietic stem and progenitor cell (HSPC) pool in Fancd2 mice. Both AA4.1+ Sca-1+ Lin- expressing progenitors (ASL) and CD48- CD150+ Lin- Sca-1+ (SLAM) cells were decreased in frequency in Fancd2-/- versus WT FL. Similarly, we observed a significant decrease in progenitor colony formation and deficits in primary and secondary transplantation among Fancd2-/- FL compared to WT. Fancd2-/- FL cells were characteristically sensitive to mitomycin C and had significantly fewer SLAM cells in the G0 phase of cell cycle and elevated p21 expression, indicating canonical P53 activation. Consistent with prior reports by other groups on embryonic stem cells and our own Fancc-/- FL studies, we found neither exaggerated frequency of apoptotic cells, nor transcriptional induction of Puma or Noxa. We hypothesized that the observed deficits in developmental HSPC pool expansion reflect replication-associated stress. At the transcriptional level, we found activation of the DNA damage response via Rad51 and Prkdc, corroborated by immunofluorescent imaging of Rad51 foci as well as comet assays in FL cells. Next, we tested P38 MAPK as a stress response previously found to confer repopulation deficits in postnatal BM failure among Fancc and Fanca mice; here, our experiments revealed baseline (unprovoked) activation of phospho-p38 and rescue of Fancd2-/- progenitor colony formation using a pharmacological inhibitor, SB203580. Results were further strengthened by transplantation, revealing increased Fancd2-/- donor chimerism after in vivo administration of SB203580. The gains in donor chimerism persisted even after cessation of drug administration. These results suggest that replication-associated stress in the rapidly cycling fetal Fancd2-/- HSPC pool evokes a cellular stress response that constrains physiological expansion. Our work emphasizes the prenatal onset of hematopoietic failure and reveals pharmacological rescue by inhibition of constitutively active P38 MAPK. Furthermore, FA fetal hematopoiesis is an original model of unprovoked hematopoietic failure that allows the study of physiologic role of FA proteins in HSPC. Disclosures No relevant conflicts of interest to declare.

scholarly journals Adaptation to Endoplasmic Reticulum Stress Enhances Resistance of Oral Cancer Cells to Cisplatin by Up-Regulating Polymerase η and Increasing DNA Repair Efficiency

2020 ◽  
Vol 22 (1) ◽  
pp. 355
Author(s):  
Cho-Yi Chen ◽  
Masaoki Kawasumi ◽  
Tien-Yun Lan ◽  
Chi-Lam Poon ◽  
Yi-Sian Lin ◽  
...  
Keyword(s):  
Dna Damage ◽  
Endoplasmic Reticulum ◽  
Dna Repair ◽  
Stress Response ◽  
Er Stress ◽  
Cancer Progression ◽  
Late Stage ◽  
Cisplatin Resistance ◽  
Nuclear Antigen ◽  
Er Stress Response

Endoplasmic reticulum (ER) stress response is an adaptive program to cope with cellular stress that disturbs the function and homeostasis of ER, which commonly occurs during cancer progression to late stage. Late-stage cancers, mostly requiring chemotherapy, often develop treatment resistance. Chemoresistance has been linked to ER stress response; however, most of the evidence has come from studies that correlate the expression of stress markers with poor prognosis or demonstrate proapoptosis by the knockdown of stress-responsive genes. Since ER stress in cancers usually persists and is essentially not induced by genetic manipulations, we used low doses of ER stress inducers at levels that allowed cell adaptation to occur in order to investigate the effect of stress response on chemoresistance. We found that prolonged tolerable ER stress promotes mesenchymal–epithelial transition, slows cell-cycle progression, and delays the S-phase exit. Consequently, cisplatin-induced apoptosis was significantly decreased in stress-adapted cells, implying their acquisition of cisplatin resistance. Molecularly, we found that proliferating cell nuclear antigen (PCNA) ubiquitination and the expression of polymerase η, the main polymerase responsible for translesion synthesis across cisplatin-DNA damage, were up-regulated in ER stress-adaptive cells, and their enhanced cisplatin resistance was abrogated by the knockout of polymerase η. We also found that a fraction of p53 in stress-adapted cells was translocated to the nucleus, and that these cells exhibited a significant decline in the level of cisplatin-DNA damage. Consistently, we showed that the nuclear p53 coincided with strong positivity of glucose-related protein 78 (GRP78) on immunostaining of clinical biopsies, and the cisplatin-based chemotherapy was less effective for patients with high levels of ER stress. Taken together, this study uncovers that adaptation to ER stress enhances DNA repair and damage tolerance, with which stressed cells gain resistance to chemotherapeutics.

scholarly journals DNA Damage Response and Cell Cycle Regulation in Pluripotent Stem Cells

Genes ◽  
2021 ◽  
Vol 12 (10) ◽  
pp. 1548
Author(s):  
Andy Chun Hang Chen ◽  
Qian Peng ◽  
Sze Wan Fong ◽  
Kai Chuen Lee ◽  
William Shu Biu Yeung ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Dna Damage ◽  
Dna Damage Response ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Genomic Stability ◽  
Great Promise ◽  
Cell Based Therapy ◽  
Damage Response

Pluripotent stem cells (PSCs) hold great promise in cell-based therapy because of their pluripotent property and the ability to proliferate indefinitely. Embryonic stem cells (ESCs) derived from inner cell mass (ICM) possess unique cell cycle control with shortened G1 phase. In addition, ESCs have high expression of homologous recombination (HR)-related proteins, which repair double-strand breaks (DSBs) through HR or the non-homologous end joining (NHEJ) pathway. On the other hand, the generation of induced pluripotent stem cells (iPSCs) by forced expression of transcription factors (Oct4, Sox2, Klf4, c-Myc) is accompanied by oxidative stress and DNA damage. The DNA repair mechanism of DSBs is therefore critical in determining the genomic stability and efficiency of iPSCs generation. Maintaining genomic stability in PSCs plays a pivotal role in the proliferation and pluripotency of PSCs. In terms of therapeutic application, genomic stability is the key to reducing the risks of cancer development due to abnormal cell replication. Over the years, we and other groups have identified important regulators of DNA damage response in PSCs, including FOXM1, SIRT1 and PUMA. They function through transcription regulation of downstream targets (P53, CDK1) that are involved in cell cycle regulations. Here, we review the fundamental links between the PSC-specific HR process and DNA damage response, with a focus on the roles of FOXM1 and SIRT1 on maintaining genomic integrity.

Differential effects of selenium (Se) on normal and malignant cells treated with chemotherapy (CT) and radiation (RT).

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e13583-e13583
Author(s):  
Michael B. Jameson ◽  
Richard J Lobb ◽  
Gregory M Jacobson ◽  
Ray T Cursons
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Stress Response ◽  
Comet Assay ◽  
Er Stress ◽  
Stress Responses ◽  
Mononuclear Cells ◽  
Malignant Cells ◽  
Er Stress Response ◽  
Therapeutic Selectivity

e13583 Background: Preclinical work has demonstrated that Se compounds potentiate anticancer effects of CT and RT while reducing normal tissue toxicities. The molecular basis for the therapeutic selectivity has yet to be fully elucidated but includes modulation of intracellular glutathione (GSH) concentrations, endoplasmic reticulum (ER) stress responses, DNA repair, induction of apoptosis and cellular resistance to CT and RT. Our aim was to evaluate the dose-response relationship of the Se compound methylseleninic acid (MSA) on molecular pathways involved in the response of normal and malignant cells to CT and RT. Methods: Peripheral blood mononuclear cells (PBMC) obtained from healthy blood donors and malignant THP-1 human monocytic leukaemia cells were exposed in vitro to MSA 2.5, 5 or 15 µM in varying combinations with MSA, RT, cisplatin (Pt), doxorubicin (Dox) and cytosine arabinoside (Ara-C). GSH concentration was measured by ELISA, DNA damage and repair by COMET assay, cell viability by MTT assay and ER stress response protein expression by western blotting. Results: MSA was selectively toxic to THP-1 cells and induced a protective increase in GSH in PBMC but a decrease in high concentrations within THP-1 cells. DNA damage induced by Ara-C or Dox in the COMET assay was significantly reduced by MSA in PBMC but increased in THP-1 cells. Cell death after 2 Gy RT was increased by all doses of MSA in THP-1 cells but only by the highest dose in PBMC. The cytotoxicity of Dox and Ara-C at sublethal doses was significantly enhanced by MSA in THP-1 cells and to a lesser extent in PBMC, but MSA increased cell death from Pt only in THP-1 cells. MSA induced a protective ER stress response in PBMC exposed to Ara-C but an apoptotic response in THP-1 cells. Conclusions: MSA at clinically-relevant concentrations had a differential effect on cell survival and death responses to RT and CT with relative protection of PBMC and enhanced death of THP-1 cells. Several mechanisms mediated this therapeutic selectivity and the dose-dependence of the Se effect varied between malignant and normal cells. These assays could potentially be used in clinical trials to evaluate pharmacodynamic markers of Se effects in conjunction with CT and/or RT.

scholarly journals Iron Chelation Reinforces DNA Damage Response and Leads to G2/M Checkpoint Activation and Autophagy in Myeloid Bone Marrow Cells of Preleukemia Mouse Model

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3351-3351 ◽  
Author(s):  
Leona Raskova Kafkova ◽  
Zuzana Somikova ◽  
Jana Kucerova ◽  
Lenka Calabkova ◽  
Pavla Luzna ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Damage ◽  
Er Stress ◽  
Dna Damage Response ◽  
Chelation Therapy ◽  
Bone Marrow Cells ◽  
Iron Chelation ◽  
S Phase ◽  
Damage Response ◽  
P38mapk Pathway

Abstract Iron chelation therapy is commonly used in patients with myelodysplastic syndrome (MDS), to prevent majorcomplications of iron overload. Besides effects on maintaining control of iron stores and preventing iron-induced cardiac disease, the impact of chelation therapy on overall survival and leukemia-free survival in MDS has been documented, but not well understood. Since MDS bone marrow cells are known to activate DNA damage response (DDR) signaling and iron chelators target cancer cells through multiple stress-response mechanisms (endoplasmic reticulum (ER) stress, autophagy), we hypothesized that iron chelation could reinforce DDR signaling and could thus support tumor-suppressing role of DDR. Also nucleotide deficiency was shown to contribute to DDR, and iron chelation is known to inhibit ribonucleotide reductase (RR), an iron-dependent enzyme, which supplies cells with deoxyribonucleotides (dNTPs). Here, we tested the effects of lysosomotropic iron chelator deferoxamine mesylate (DFO) in a preleukemia mouse model, wherein epigenetic oncogene-induced leukemogenesis is preceded with a long-lasting preleukemia stage (Takacova S, et al. Cancer Cell. 2012;21(4):517-31.). Preleukemic, aberrantly proliferating myeloid cells in this model activate a replication checkpoint and ATR-Chk1-mediated DDR (consistent with oncogene-induced replication stress) and attain hallmarks of senescence (with a long latency), resulting in the inhibition of leukemia progression. A group of 10 preleukemia mice and a group of 10 control mice aged 7 month were treated twice daily with DFO doses adjusted to 88,8 mg/kg (i.p. injection) in order to mimic serum concentrations of the drug achieved in patients. After 6 weeks of chelator administration, the treatment lead to the activation of Chk1(S345) in the bone marrow (BM) of control mice, but did not result in accumulation of γH2AX, a marker of DNA damage, in BM of these mice. In contrast, in preleukemia mice, with already activated threshold of ATR-Chk1 signaling (marker of ongoing oncogene-induced replication stress), Chk1(S345) remained unchanged after DFO treatment. However, we observed significant accumulation of γH2AX foci in oncogene-positive BM cells. These data suggested that iron removal may induce Chk1 activation in vivo, and, in addition, may reinforce activation of DDR in preleukemia cells perhaps due to synthetic effect of iron chelation with oncogene activation resulting in increased levels in DDR signaling (assessment of oxidative DNA damage (8-oxoguanine staining) is ongoing). Next, we analyzed whether iron chelation in both groups of mice influences DNA replication, in which the limiting step is the availability of dNTPs. The RR activity was significantly decreased in the BM of both groups of DFO-treated mice, however, with no impact on the concentration of BM dNTPs; in fact, dNTPs have accumulated in BM of these mice. We revealed that this was a consequence of the activation of S-phase checkpoint in control mice, and of a decrease of actively replicating myeloid cells and activation of G2/M checkpoint in preleukemia mice. Cellular iron depletion was shown to activate p38MAPK pathway (Yu Y, Richardson DR. J Biol Chem. 2011;286(17):15413-27.). p38MAPK pathway, and its component MK2, establishes intra-S-phase cell cycle checkpoint and activates G2/M checkpoint (as a part of DDR, in parallel to Chk1 activation (Reinhardt HC, et al. Curr Opin Cell Biol. 2009;21:245-55.)). Indeed, our preliminary result revealed phosphorylated MK2 specifically in preleukemia mouse BM treated with DFO. Since we did not detect increased apoptosis in BM of DFO treated mice, and because p38MAPK pathway is involved in the activation of ER stress and autophagy, we tested whether markers of ER stress and autophagy are detectable in the mice upon DFO treatment. MyD116 (marker of recovery from ER stress) and LC3-II (marker of autophagy), were specifically induced in preleukemia cells upon DFO treatment. Collectively, these data demonstrate that preleukemia cells exposed to DFO activate distinct but functionally overlapping signaling pathways, resulting in reinforced DDR. Whether this mechanism could increase a barrier against leukemia transformation of chelated MDS patients remains to be investigated. Authorship: LRK and ZS: equal credit as first authors. Acknowledgment: Supported by the Czech Science Foundation (P301/12/1503) and by IGA_LF_2015_015. Disclosures No relevant conflicts of interest to declare.

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