Interexperimental and Interindividual Variations of DNA Repair Capacities After UV-B and UV-C Irradiations of Human Keratinocytes and Fibroblasts

Recent studies reported the association between increased risk of nonmelanoma skin cancers (NMSCs) and the use of hydrochlorothiazide (HCTZ), one of the most commonly prescribed diuretic, antihypertensive drug, over the world. Although HCTZ is known to be photosensitizing, the mechanisms involved in its potential prophotocarcinogenic effects remain unclear. Under acute exposure, therapeutically relevant concentrations of HCTZ (70, 140, and 370 ng/mL) amplified UVA-induced double-strand breaks, oxidative DNA, and protein damage in HaCaT human keratinocytes, and this effect was associated to a defective activity of the DNA repair enzyme, OGG1. Oxidative damage to DNA, but not that to proteins, was reversible within few hours. After chronic, combined exposure to HCTZ (70 ng/mL) and UVA (10 J/cm2), for 9 weeks, keratinocytes acquired a dysplastic-like phenotype characterized by a multilayered morphology and alterations in cell size, shape, and contacts. At the ultrastructural level, several atypical and enlarged nuclei and evident nucleoli were also observed. These transformed keratinocytes were apoptosis resistant, exhibited enhanced clonogenicity capacity, increased DNA damage and inflammation, defective DNA repair ability, and increased expression of the oncogene ΔNp63α and intranuclear β-catenin accumulation (a hallmark of Wnt pathway activation), compared to those treated with UVA alone. None of these molecular, morphological, or functional effects were observed in cells treated with HCTZ alone. All these features resemble in part those of preneoplastic lesions and NMSCs and provide evidence of a biological plausibility for the association among exposure to UVA, use of HCTZ, and increased risk of NMSCs. These results are of translational relevance since we used environmentally relevant UVA doses and tested HCTZ at concentrations that reflect the plasma levels of doses used in clinical practice. This study also highlights that drug safety data should be followed by experimental evaluations to clarify the mechanistic aspects of adverse events.

Download Full-text

Exploring the effect of UV-C radiation on earthworm and understanding its genomic integrity in the context of H2AX expression

Scientific Reports ◽

10.1038/s41598-020-77719-2 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Karthikeyan Subbiahanadar Chelladurai ◽

Jackson Durairaj Selvan Christyraj ◽

Ananthaselvam Azhagesan ◽

Vennila Devi Paulraj ◽

Muralidharan Jothimani ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Primary Response ◽

Coelomic Fluid ◽

Dependent Manner ◽

Perionyx Excavatus ◽

Dna Damages ◽

Highly Sensitive ◽

Evolutionarily Conserved ◽

Uv C

AbstractMaintaining genomic stability is inevitable for organism survival and it is challenged by mutagenic agents, which include ultraviolet (UV) radiation. Whenever DNA damage occurs, it is sensed by DNA-repairing proteins and thereby performing the DNA-repair mechanism. Specifically, in response to DNA damage, H2AX is a key protein involved in initiating the DNA-repair processes. In this present study, we investigate the effect of UV-C on earthworm, Perionyx excavatus and analyzed the DNA-damage response. Briefly, we expose the worms to different doses of UV-C and find that worms are highly sensitive to UV-C. As a primary response, earthworms produce coelomic fluid followed by autotomy. However, tissue inflammation followed by death is observed when we expose worm to increased doses of UV-C. In particular, UV-C promotes damages in skin layers and on the contrary, it mediates the chloragogen and epithelial outgrowth in intestinal tissues. Furthermore, UV-C promotes DNA damages followed by upregulation of H2AX on dose-dependent manner. Our finding confirms DNA damage caused by UV-C is directly proportional to the expression of H2AX. In short, we conclude that H2AX is present in the invertebrate earthworm, which plays an evolutionarily conserved role in DNA damage event as like that in higher animals.

Download Full-text

Molecular Signatures in the Prevention of Radiation Damage by the Synergistic Effect of N-Acetyl Cysteine and Qingre Liyan Decoction, a Traditional Chinese Medicine, Using a 3-Dimensional Cell Culture Model of Oral Mucositis

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2015/425760 ◽

2015 ◽

Vol 2015 ◽

pp. 1-10 ◽

Cited By ~ 4

Author(s):

Maria P. Lambros ◽

Lavanya Kondapalli ◽

Cyrus Parsa ◽

Hari Chandana Mulamalla ◽

Robert Orlando ◽

...

Keyword(s):

Dna Repair ◽

Chinese Medicine ◽

Traditional Chinese Medicine ◽

Three Dimensional ◽

Human Keratinocytes ◽

Transcriptional Level ◽

Tissue Cultures ◽

Primary Human Keratinocytes ◽

Microarray Gene Expression ◽

Acetyl Cysteine

Qingre Liyan decoction (QYD), a Traditional Chinese medicine, and N-acetyl cysteine (NAC) have been used to prevent radiation induced mucositis. This work evaluates the protective mechanisms of QYD, NAC, and their combination (NAC-QYD) at the cellular and transcriptional level. A validated organotypic model of oral mucosal consisting of a three-dimensional (3D) cell tissue-culture of primary human keratinocytes exposed to X-ray irradiation was used. Six hours after the irradiation, the tissues were evaluated by hematoxylin and eosin (H and E) and a TUNEL assay to assess histopathology and apoptosis, respectively. Total RNA was extracted and used for microarray gene expression profiling. The tissue-cultures treated with NAC-QYD preserved their integrity and showed no apoptosis. Microarray results revealed that the NAC-QYD caused the upregulation of genes encoding metallothioneins,HMOX1, and other components of the Nrf2 pathway, which protects against oxidative stress. DNA repair genes (XCP,GADD45G,RAD9, andXRCC1), protective genes (EGFRandPPARD), and genes of the NFκB pathway were upregulated. Finally, tissue-cultures treated prophylactically with NAC-QYD showed significant downregulation of apoptosis, cytokines and chemokines genes, and constrained damage-associated molecular patterns (DAMPs). NAC-QYD treatment involves the protective effect of Nrf2, NFκB, and DNA repair factors.

Download Full-text

UV Induces GADD45 in a p53-Dependent and -Independent Manner in Human Keratinocytes

Journal of Cutaneous Medicine and Surgery ◽

10.1177/120347540300700204 ◽

2003 ◽

Vol 7 (2) ◽

pp. 119-123 ◽

Cited By ~ 1

Author(s):

Tomoko Maeda ◽

Adrian B. Sim ◽

Duane A. Leedel ◽

Prescillia P. S. Chua ◽

Eugene G. Chomey ◽

...

Keyword(s):

Dna Repair ◽

P53 Protein ◽

Human Keratinocytes ◽

Cell Cycle Checkpoint ◽

Checkpoint Control ◽

Independent Manner ◽

Rnase Protection ◽

P53 Expression ◽

Multifunctional Protein ◽

Rna Protection

Background: GADD45 is a multifunctional protein involved in DNA repair and in cell cycle checkpoint control. p53 plays an important role in regulating DNA repair and in response to UVB in keratinocytes. Objective: GADD45 and p53 expression was examined and compared at the mRNA and protein level after exposure to UV irradiation. Methods: Human keratinocytes were exposed to increasing doses of UVB, and an RNA protection assay and a Western blot analysis were performed. Results: The RNase protection assays using human keratinocytes showed that GADD45 mRNA increases after 4 h and remains elevated for 24 h in cells irradiated at 100, 300, or 600 J/m2 UVB. The level of GADD45 protein increases after 8 h and remains elevated for 48 h, with maximal induction at 300 J/m2. p53 mRNA did not rise in concert with GADD45 at any dose used, and p53 protein was not up-regulated at the lower dose of 100 J/m2. Conclusion: GADD45 is regulated in both a p53-dependent and a p53-independent manner in keratinocytes after UV exposure.

Download Full-text

The role of BRCA1 in DNA repair and chemosensitivity

Journal of Clinical Oncology ◽

10.1200/jco.2007.25.18_suppl.10606 ◽

2007 ◽

Vol 25 (18_suppl) ◽

pp. 10606-10606 ◽

Cited By ~ 1

Author(s):

V. B. Sharma ◽

A. W. Kurian ◽

A. Feldman ◽

J. M. Ford

Keyword(s):

Dna Repair ◽

Oxidative Dna Damage ◽

Excision Repair ◽

Brca1 Gene ◽

Brca1 Protein ◽

P53 Expression ◽

Increased Risk ◽

Uv C

10606 Introduction: The BRCA1 tumor suppressor protein has been implicated in many cellular processes, including several different DNA repair pathways. Inherited mutations in the BRCA1 gene result in an increased risk for developing breast and ovarian cancer. BRCA1 associated tumors are frequently estrogen receptor negative thus rendering them ineffective targets of hormonal manipulations. Therefore chemotherapy is the only treatment option available to patients with metastatic disease. Methods: Global genomic repair activity was detected by an immunoslotblot assay. UV-C and chemosensitivity was determined by the MTT assay. The comet assay was used to measure oxidative DNA damage (ODD). Results: Brca1 deficient cells demonstrate decreased global genomic repair independent of p53 expression. These cells are 3-fold more sensitive to UV-C radiation, 5-fold more sensitive to cisplatin, and 25-fold more sensitive to gemcitabine than Brca1 positive cells. The combination of cisplatin and gemcitabine is synergistic. This synergy is independent of Brca1 protein expression. In addition, the cholesterol lowering drug lovastatin is able mitigate the ODD caused by hydrogen peroxide. Reduced ODD is also observed in breast epithelial cells from high risk patients after 6 months of oral lovastatin use. Discussion: These results confirm our earlier findings that BRCA1 is involved in the nucleotide excision repair pathway. In addition, we demonstrate that this effect is independent of p53 expression. We also find that Brca1 deficient MMECs are more sensitive to UV-C, and the cytotoxic drugs cisplatin and gemcitabine. Cisplatin is known to cause DNA intra- and inter-strand crosslinks whereas gemcitabine is a nucleoside analog. The increased sensitivity of Brca1 deficient MMEC's to these agents supports the role of Brca1 in the different molecular pathways involved in their repair. In addition, we demonstrate that lovastatin can overcome ODD in vitro and in vivo and may be an effective agent to modify breast cancer risk for individuals at increased risk. No significant financial relationships to disclose.

Download Full-text

Short-term Exposure to UV-A, UV-B, and UV-C Irradiation Induces Alteration in Cytoskeleton and Autophagy in Human Keratinocytes

Ultrastructural Pathology ◽

10.3109/01913123.2012.756568 ◽

2013 ◽

Vol 37 (4) ◽

pp. 241-248 ◽

Cited By ~ 15

Author(s):

Milan Misovic ◽

Dusan Milenkovic ◽

Tamara Martinovic ◽

Darko Ciric ◽

Vladimir Bumbasirevic ◽

...

Keyword(s):

Human Keratinocytes ◽

Short Term ◽

Short Term Exposure ◽

Uv C

Download Full-text

miR-200a Modulates the Expression of the DNA Repair Protein OGG1 Playing a Role in Aging of Primary Human Keratinocytes

Oxidative Medicine and Cellular Longevity ◽

10.1155/2018/9147326 ◽

2018 ◽

Vol 2018 ◽

pp. 1-17 ◽

Cited By ~ 13

Author(s):

Lavinia Tinaburri ◽

Mariarosaria D’Errico ◽

Sara Sileno ◽

Riccardo Maurelli ◽

Paolo Degan ◽

...

Keyword(s):

Dna Repair ◽

Oxidative Dna Damage ◽

Human Keratinocytes ◽

Primary Human Keratinocytes ◽

Human Keratinocyte ◽

Age Related ◽

Dna Repair Protein ◽

Repair Activity ◽

Dna Strand ◽

Base Lesion

Oxidative DNA damage accumulation may induce cellular senescence. Notably, senescent cells accumulate in aged tissues and are present at the sites of age-related pathologies. Although the signaling of DNA strand breaks has been extensively studied, the role of oxidative base lesions has not fully investigated in primary human keratinocyte aging. In this study, we show that primary human keratinocytes from elderly donors are characterized by a significant accumulation of the oxidative base lesion 8-OH-dG, impairment of oxidative DNA repair, and increase of miR-200a levels. Notably, OGG1-2a, a critical enzyme for 8-OH-dG repair, is a direct target of miR-200a and its expression levels significantly decrease in aged keratinocytes. The 8-OH-dG accumulation displays a significant linear relationship with the aging biomarker p16 expression during keratinocyte senescence. Interestingly, we found that miR-200a overexpression down-modulates its putative target Bmi-1, a well-known p16 repressor, and up-regulates p16 itself. miR-200a overexpression also up-regulates the NLRP3 inflammasome and IL-1β expression. Of note, primary keratinocytes from elderly donors are characterized by NRPL3 activation and IL-1β secretion. These findings point to miR-200a as key player in primary human keratinocyte aging since it is able to reduce oxidative DNA repair activity and may induce several senescence features through p16 and IL-1β up-regulation.

Download Full-text

Effect of Retinoic Acid on Apoptosis and DNA Repair in Human Keratinocytes after UVB Irradiation

Journal of Cutaneous Medicine and Surgery ◽

10.1177/120347540000400102 ◽

2000 ◽

Vol 4 (1) ◽

pp. 2-7 ◽

Cited By ~ 9

Author(s):

Gang Li ◽

Jason A. Bush ◽

Vincent C. Ho

Keyword(s):

Dna Repair ◽

Retinoic Acid ◽

Skin Cancer ◽

Human Keratinocytes ◽

Epidemiological Studies ◽

Skin Carcinogenesis ◽

Apoptosis Assay ◽

Initiation Stage ◽

Induced Apoptosis ◽

Damaged Dna

Background: Skin cancer is extremely common. Epidemiological studies indicated that ultraviolet radiation (UV) is the primary cause for skin cancers, and that retinoic acid (RA) is able to inhibit this UV-induced skin carcinogenesis; however, the molecular mechanism of the anti-UV action of RA is unclear. Objective: The purpose of this study is to investigate if RA enhances the removal of UV-induced DNA damage. Methods: The effect of RA on UV-induced apoptosis and DNA repair was investigated by ELISA apoptosis assay and CAT assay. Results: Both all-trans-RA and 9-cis-RA did not promote UV-induced apoptosis nor the repair of UV-damaged DNA in human keratinocytes. Furthermore, RA did not induce the expression of p53. Conclusion: The inhibition of RA on skin carcinogenesis is not due to enhanced removal of UV-damaged DNA. Therefore, RA does not inhibit skin cancer development at the initiation stage, but possibly at the promotion and progression stages.

Download Full-text

The Tardigrade Damage Suppressor Protein Promotes Transcription Factor Activation and Expression of DNA Repair Genes in Human Cells in Response to Hydroxyl Radicals and UV-C Exposure

10.20944/preprints202107.0333.v1 ◽

2021 ◽

Author(s):

Claudia Ricci ◽

Giulia Riolo ◽

Carlotta Marzocchi ◽

Jlenia Brunetti ◽

Alessandro Pini ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Expression Patterns ◽

Dna Damage Repair ◽

Human Cells ◽

H2o2 Treatment ◽

Damage Repair ◽

Transcription Factor Activation ◽

Uv C ◽

Repair Genes

The Ramazzottius varieornatus tardigrade is an extremotolerant terrestrial invertebrate belonging to the phylum of Tardigrada. At a length of 0.1-1.0 mm, tardigrades are small animals with an exceptional tolerance to extreme conditions such as high pressure, chemicals and irradia-tion. These properties have been attributed to the recently-discovered Dsup protein. Dsup is a nucleosome-binding protein that prevents DNA damage against X-ray and oxidative stress without impairing cell life, also in Dsup-transfected animal and plant cells. However, the precise “protective” role of this protein is still under study. We performed experiments on human cells and shows that, as compared to control cells, Dsup+ cells are more resistant to UV-C exposure and H2O2. Real-time PCR identified different expression patterns of endogenous genes involved in apoptosis, cell survival and DNA damage repair in Dsup+ cells in response to H2O2 and UV-C. While H2O2 treatment in Dsup+ cells only marginally involved the activation of pathways responsible for DNA repair reinforcing the idea of a direct protective effect of the protein on DNA, in UV-C exposed cells, Dsup efficiently upregulates DNA damage repair genes. In conclusion, our data may help to delineate the different mechanisms by which the Dsup protein operates in response to different insults.

Download Full-text