Article

Role of p53 in UVB-Induced Apoptosis in Human HaCaT Keratinocytes

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Abstract

Apoptosis represents an active form of cell death that is involved in the control of tissue homeostasis and in the deletion of DNA-damaged cells. Because the product of the tumor suppressor gene p53 has been demonstrated to be crucial for the induction of apoptosis in certain cell types, the present study was aimed at elucidating its role in ultraviolet-induced apoptosis in HaCaT keratinocytes. After in vitro ultraviolet B irradiation, p53 protein levels were noted to increase prior to the induction of apoptosis in a time- and concentration-dependent fashion. This increase could not be inhibited by the protein synthesis inhibitor cycloheximide. Because HaCaT keratinocytes are known to bear two p53 point mutations and because it is unclear whether p53 in HaCaT cells is still functional regarding induction of apoptosis, HaCaT cells were stably transfected with wild-type p53 cDNA inserted into the expression vector pCMV-Neo-Bam in sense (pC53-SN3) and anti-sense (pC53-ASN) direction. After selection with geniticin, growing colonies were screened for the presence of the transfected cDNA constructs by polymerase chain reaction. Cell clones bearing the anti-sense product were further analyzed for p53 expression by western blotting. Clones showing reduced p53 protein levels were irradiated with ultraviolet B light, and there was a clear reduction of apoptosis in the pC53-ASN bearing cell clones compared with the parental HaCaT cells. These studies demonstrate that blocking mutated p53 can partially block apoptosis in HaCaT keratinocytes and furthermore can confirm the key role for p53 in ultraviolet-induced apoptosis in human keratinocytes. Moreover, HaCaT keratinocytes and their p53-transfectants provide a convenient model that allows for further detailed analyses of apoptosis-associated biochemical and molecular events in human keratinocytes.

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... In spite of cytogenetic alterations and mutation within the the p53 gene as well as loss of various senescence genes [83], the HaCaT cell line exhibits non-tumorigenic growth [84] and has preserved the ability to express distinct differentiation markers, such as involucrin, filaggrin and keratins [76,81]. HaCaT cells are also sensitive to induction of apoptosis, similar to normal keratinocytes [85,86]. However, experimental results with HaCaT cell lines have to be viewed under the perspective that they do have genomic alterations for proteins that play a crucial role in cell cycle progression. ...
... Studies examining UV-B induced tumors of the skin [113], upregulation in the epidermis after UV-B exposure in vivo [114] and mediation of UV-B induced apoptosis in keratinocytes in vivo [115] and in vitro [85] suggest an important role for p53 in the UV-B stress response. A recent study postulates a repressor function of p53 for IEX-1 transcription. ...
Article
Cellular stress and damage response mechanisms play a crucial role in physiological defense against hyperproliferative diseases, genotoxic injury, mutations and malignancy. An important first step in the cellular stress response is upregulation of immediate early genes that initiate and coordinate subsequent cellular events. IEX-1 is a novel immediate early gene that has been shown to be induced by gamma irradiation, phorbol ester treatment and ultraviolet irradiation. Goal of this thesis was to more specifically characterize the role of IEX-1 in keratinocytes. Special emphasis was directed to elucidating the cellular response to stress and mitogenic stimulation and the role of IEX-1 in mediating apoptosis as well as localizing the protein within the cell during these events. By northern blot analysis, it could be shown that gamma irradiation of primary human keratinocytes results in a time dependent, rapid induction of IEX-1 expression followed by prompt downregulation, similar to previous findings in a squamous cell carcinoma cell line. In addition, UV-irradiation or treatment with the reactive oxygen species hydrogen peroxide also induced IEX-1 expression rapidly and transiently. Further incubation with the mitogenic factor for keratinocyte Epidermal Growth Factor (EGF) resulted in increased steady state levels of IEX-1 mRNA. Compared to normal keratinocytes, similar observations were made in the non-tumorigenic keratinocyte cell line HaCaT. Regulation of IEX-1 gene expression by the EGF-Receptor (EGFR) was investigated using an IEX-1-promoter-luciferase-reporter-gene assay and blockage of EGFR by the highly specific EGFR-tyrosine kinase inhibitor PD153035. Blockage of the receptor was followed by a marked decrease in IEX-1 promoter activity. Effects of IEX-1 overexpression in HaCaT cells were investigated. IEX-1 overexpression enhanced proliferation, as confirmed by [3H]-thymidine incorporation assay. To examine the effects of IEX-1 on apoptosis induced by various agents, IEX-1 over-expressing HaCaT were challenged with ultraviolet radiation, the DNA damaging agent camptothecin or serum-deprival. Cell survival, caspase 3-activity and nucleosome formation were measured to assess apoptosis. There was no difference observed in baseline apoptotic activity of cells cultured under non-stressed conditions, when comparing the IEX-1 overexpressing and the control cell line. However, upon stress-induction, the IEX-1 overexpressing cells showed markedly higher levels of apoptosis. Data characterizing the intracellular localization of IEX-1 were obtained by immunohistochemical staining as well as by molecular biological methods. IEX-1 was predominantly located within the cell nucleus, forming several intranuclear patches. Treatment with various stress-inducing agents did not significantly alter the localization or cause translocation of the IEX-1 protein. Distribution of IEX-1 within the skin and epidermis was assessed by immunocytochemistry of human skin specimens and revealed predominant localization of IEX-1 within the nucleus and cytoplasm of the basal epidermal and suprabasal layers. These findings implicate IEX-1 in the control of apoptosis upon cell stress as well as promotion of cell replication during favorable growth conditions. The function of IEX-1 seems to be closely linked to the differentiation status of the keratinocyte. As an immediate early gene product, IEX-1 is a novel regulator of keratinocyte growth and survival, similar to other critical cell cycle control proteins, such as p53, p21Waf1, c-myc and related proteins. This suggests that IEX-1 is another crucial element in the pattern of regulatory pathways of cell growth and defense against malignant transformation. Zelluläre Mechanismen als Antwort auf Zellstress oder Zellschäden spielen eine wesentliche Rolle in der physiologischen Verteidigungsstrategie gegen hyperproliferative Krankheiten, genotoxische Schäden, Mutationen oder maligne Entartung. Die Expression von Immediate Early Genen, die nachfolgende zelluläre Ereignisse initiieren und koordinieren, ist ein wichtiger erster Schritt der zellulären Stressantwort. IEX-1 ist ein vor kurzem entdecktes Immediate Early Gen, von dem gezeigt werden konnte, dass es durch die Einwirkung von Gammastrahlen, Phorbolestern und Ultraviolettstrahlung induziert wird. Ziel dieser Arbeit war es, die Funktion von IEX-1 in der Stressantwort von Keratinozyten genauer zu charakterisieren. Nicht nur die zelluläre Antwort auf Stress, sondern auch die mitogene Stimulation und die Rolle von IEX-1 in Bezug auf Apoptosereaktionswege sollten untersucht werden. Weiteres Interesse galt der Analyse der IEX-1-Proteinlokalisation während dieser Ereignisse. Mit Hilfe des Northern Blot Verfahrens konnte gezeigt werden, dass IEX-1 nach Gamma-Bestrahlung von primären menschlichen Keratinozyten innerhalb von Minuten induziert und dann sehr rasch wieder herunter geregelt wird, ähnlich wie schon zuvor mit einer Keratinozyten-Tumorzelllinie gezeigt wurde. Außerdem bewirkte UV-Strahlung und Behandlung mit reaktiven Sauerstoffradikalen eine rasche, aber vorübergehende Induktion von IEX-1. Zusätzlich rief die Inkubation der Keratinozyten mit epidermal growth factor (EGF) erhöhte steady state Spiegel von IEX-1 mRNA hervor. Ähnliche Beobachtungen konnten an der HaCaT-Zelllinie, einer nicht-tumorigenen Keratinozytenlinie gemacht werden. Die Regulation der IEX-1-Genexpression durch den epidermal growth factor-Rezeptor (EGFR) wurde mittels eines IEX-1-Promotor – Luciferase Assays untersucht. Dabei wurde der EGF-Rezeptor mit dem hochspezifischen EGFR-Tyrosinkinaseinhibitor PD153035 blockiert. Der Rezeptorblockade folgte ein starker Rückgang der IEX-1-Promotoraktivität. Die Auswirkungen der Überexpression von IEX-1 in HaCaT-Zellen wurde untersucht. Wachstumsstudien mit [3H]-Thymidin-Inkorporationsassays ergaben, dass IEX- Überexpression das Wachstum beschleunigt. Um die Auswirkungen von IEX-1 auf die Apoptose zu untersuchen, wurden IEX-1 überexprimierende HaCaT-Zellen mit ultravioletter Bestrahlung, dem DNA-toxischen Agens Captothecin oder Serumentzug behandelt. Die Apoptoserate wurde mittels Caspase-3-Enzymaktivität und Oligonucleosomenbildung im Zellplasma quantifiziert. Die entsprechenden Assays konnten erstmalig für adhärente Keratinozytenkulturen etabliert werden. Unter normalen Wachstumsbedingungen ohne Stress konnte kein Unterschied in der basalen Apoptoserate zwischen IEX-1-überexprimierenden Zellen und Kontrollzellen beobachtet werden. Wurden die Zellen jedoch Stress ausgesetzt, dann trat in den IEX-1-überexprimierenden Zellkulturen in erhöhtem Maße Apoptose auf. Das IEX-1-Protein konnte mit immunhistochemischen sowie molekularbiologischen Methoden vorwiegend im Zellkern lokalisiert werden, wo es sich in unterschiedlicher Verteilung zeigte. Stressinduzierende Behandlung der Zellen konnte keine Translokation des Proteines oder eine Veränderung im Verteilungsmuster bewirken. Untersuchung an menschlicher Epidermis mittels immunhistochemischer Färbung zeigte, dass IEX-1 hauptsächlich in den basalen und suprabasalen Schichten der Epidermis exprimiert wird. Mit diesen Ergebnissen konnte erstmals gezeigt werden, dass IEX-1 eine wichtige Rolle sowohl in der Kontrolle der Apoptose als Folge von Zellstress, als auch in der Vermittlung von Zellreplikation unter optimalen Wachstumsbedingungen zukommt. Die Wirkung von IEX-1 scheint dabei eng an den Differenzierungsstatus der Keratinozyten gebunden zu sein. Als Produkt eines Immediate early Gens spielt IEX-1 eine Rolle in der Vermittlung von Keratinozytenwachstum und Keratinozytenüberleben, ähnlich wie die wichtigen Zellzyklusproteine p53, p21Waf1,c-myc und andere verwandte Proteine. Damit zeichnet es sich als ein weiterer elementarer Baustein im Mosaik der Reaktionswege zur Regulierung von Zellwachstum und Bekämpfung von maligner Entartung ab.
... Furthermore, the application of OM-GL15 significantly protected keratinocytes from apoptosis and the apoptotic index of OM-GL15 at 1 μM was similar to that in the normal group. An increase in 8− OHdG levels is an important biomarker of DNA damage [28]. As illustrated in Fig. 7C, compared with the normal group, the level of 8− OHdG in the UVB group increased significantly, while application of OM-GL15 (100 nM, 1 μM) down-regulated the 8− OHdG level in mouse skins. ...
... The photoelectrons produced by UVB irradiation can be transferred to DNA or molecular oxygen, resulting in oxidative stress and DNA damage [39]. As a marker of DNA damage, 8− OHdG expression increases with cellular DNA damage, which then activates p53 to induce the expression of Bcl-2 family proteins (including anti-apoptotic protein Bcl-2 and apoptotic protein Bax) [28]. Based on TUNEL staining, we confirmed that OM-GL15 significantly inhibited mouse epidermal cell apoptosis (Fig. 7A, B). ...
Article
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Although the application potential of amphibian skin-derived active peptides in alleviating ultraviolet B (UVB)-induced damage has attracted increasing attention, research remains in its infancy. In this study, a new peptide (OM-GL15, GLLSGHYGRASPVAC) was identified from the skin of the green odorous frog (Odorrana margaretae). Results showed that OM-GL15 scavenged free radicals (2,2′-diazo-bis-3-ethylbenzothiazoline-6-sulfonic acid and 1,1-diphenyl-2-trinitrophenylhydrazine) and reduced Fe³⁺ to Fe²⁺. Moreover, topical administration of OM-GL15 significantly alleviated UVB-induced skin photodamage in mice. Exploration of the underlying mechanisms further showed that OM-GL15 exerted antioxidant potency. Specifically, the peptide reduced the levels of lipid peroxidation and malondialdehyde and protected epidermal cells from UVB-induced apoptosis by inhibiting DNA damage via down-regulation of p53, caspase-3, caspase-9, and Bax and up-regulation of Bcl-2. Our results highlight the potential application of amphibian skin-derived peptides in protection against UVB-induced photodamage and provide a novel peptide candidate for the development of anti-photodamage agents.
... Therefore, research on apoptosis using HaCaT cells was sometimes considered inappropriate to use this cell line because the apoptotic pathway would not function properly. However, Henseleit et al. (1997) showed that despite the mutations of the p53 gene in HaCaT cells, p53 can be involved in the UVB-induced apoptotic pathway in HaCaT keratinocytes [52]. After that, studies on apoptosis using UVB-induced HaCaT cells began to appear [53,54], and this study also attempted to evaluate whether ACC, the major substance, could impart an antiapoptotic effect to down-regulate pro-apoptotic molecules that begin to appear at a specific UVB dose. ...
... Therefore, research on apoptosis using HaCaT cells was sometimes considered inappropriate to use this cell line because the apoptotic pathway would not function properly. However, Henseleit et al. (1997) showed that despite the mutations of the p53 gene in HaCaT cells, p53 can be involved in the UVB-induced apoptotic pathway in HaCaT keratinocytes [52]. After that, studies on apoptosis using UVB-induced HaCaT cells began to appear [53,54], and this study also attempted to evaluate whether ACC, the major substance, could impart an antiapoptotic effect to down-regulate pro-apoptotic molecules that begin to appear at a specific UVB dose. ...
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We recently demonstrated that advanced cooling composition (ACC) has effective ingredients that exhibit anti-inflammatory effects in RAW 264.7 cells stimulated with lipopolysaccharide (LPS) and exhibit strong antimicrobial effects on Pseudomonas aeruginosa, Staphylococcus aureus, MRSA (methicillin-resistant Staphylococcus aureus), Candida albicans, and Streptococcus mutans. To further investigate whether ACC has beneficial effects in ultraviolet B (UVB)-irradiated human keratinocytes (HaCaT cells), HaCaT cells were pretreated with ACC prior to UVB irradiation. Our data showed that ACC, which is effective at 100 µg/mL, is nontoxic and has an antioxidative effect against UVB-induced intracellular reactive oxygen species (ROS) in HaCaT cells. In addition, ACC exerts cytoprotective effects against UVB-induced cytotoxicity in HaCaT cells by inhibiting abnormal inflammation and apoptosis through the regulation of mitogen-activated protein kinase (MAPK) signals, such as jun-amino-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK). Therefore, these results indicate that ACC is a potentially beneficial raw material that possesses antioxidative, anti-inflammatory, and antiapoptotic effects against UVB-induced keratinocytes and may have applications in skin health.
... In addition to the mechanism in regulation of hdm2 splicing after UVB irradiation, our data showed that the alternative splicing of hdm2 might play a role in regulation of UVB-induced HaCaT cell death (Fig. 5). It was known that hdm2B can bind to hdm2-FL and inhibit the interaction of hdm2-FL with p53 and thus enhances the transcriptional activity of p53 (28), which regulates UVB-induced apoptosis (29,30). HaCaT cells carry a mutated p53 (31) that sensitize the cells to UVB irradiation (30). ...
... It was known that hdm2B can bind to hdm2-FL and inhibit the interaction of hdm2-FL with p53 and thus enhances the transcriptional activity of p53 (28), which regulates UVB-induced apoptosis (29,30). HaCaT cells carry a mutated p53 (31) that sensitize the cells to UVB irradiation (30). Our results suggest that the overexpressed hdm2-FL is able to bind to the mutated p53 and protect the cells from UVB-induced apoptosis. ...
Article
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Human homolog double minute 2 (hdm2), an oncoprotein, which binds to tumor suppressor p53 to facilitate its degradation, has been known to contribute to tumorigenesis. Its splicing variants are reported to be highly expressed in many cancers and can be induced by ultraviolet B light (UVB). However, the mechanisms of how UVB radiation induces hdm2 alternative splicing still remain unclear. In this study, we investigated the roles of two common splicing factors, heterogeneous nuclear ribonucleoproteins (hnRNP) A1 and serine/arginine-rich splicing factor 1 (SRSF1), in regulating UVB-induced hdm2 splicing. Our study indicated that while the expression of both hnRNP A1 and SRSF1 are induced, only hnRNP A1 is involved in hdm2 alternative splicing upon UVB irradiation. Overexpression of hnRNP A1 resulted in decrease of full-length hdm2 (hdm2-FL) and increase of hdm2B, one of hdm2 alternate-splicing forms; while down-regulated hnRNP A1 expression led to the decrease of the hdm2-FL and hdm2B in HaCaT cells. Protein-mRNA binding assay confirmed that UVB irradiation could increase the binding of hnRNP A1 to hdm2 pre-mRNA. In conclusion, we elucidated that UVB induces alternative splicing of hdm2 via increasing the expression and the binding of hnRNP A1 to hdm2 full-length mRNA. This article is protected by copyright. All rights reserved.
... HaCaT cells have 3 p53 point mutations; 1 in codon 179 of exon 5 on one allele, and 2 consecutive mutations in codons 281 and 282 of exon 8 on the other allele [74]. In spite of its mutations, data show that p53 in HaCaT cells remains functional with respect to inducing apoptosis [75]. In our hands they behave like wild-type cells with respect to bystander effect reporting. ...
Article
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The question of whether bystander and abscopal effects are the same is unclear. Our experimental system enables us to address this question by allowing irradiated organisms to partner with unexposed individuals. Organs from both animals and appropriate sham and scatter dose controls are tested for expression of several endpoints such as calcium flux, role of 5HT, reporter assay cell death and proteomic profile. The results show that membrane related functions of calcium and 5HT are critical for true bystander effect expression. Our original inter-animal experiments used fish species whole body irradiated with low doses of X-rays, which prevented us from addressing the abscopal effect question. Data which are much more relevant in radiotherapy are now available for rats which received high dose local irradiation to the implanted right brain glioma. The data were generated using quasi-parallel microbeams at the biomedical beamline at the European Synchrotron Radiation Facility in Grenoble France. This means we can directly compare abscopal and "true" bystander effects in a rodent tumour model. Analysis of right brain hemisphere, left brain and urinary bladder in the directly irradiated animals and their unirradiated partners strongly suggests that bystander effects (in partner animals) are not the same as abscopal effects (in the irradiated animal). Furthermore, the presence of a tumour in the right brain alters the magnitude of both abscopal and bystander effects in the tissues from the directly irradiated animal and in the unirradiated partners which did not contain tumours, meaning the type of signal was different. Copyright © 2015. Published by Elsevier Ltd.
... The line was developed from normal human skin that surrounded a melanoma and became immortal spontaneously (Boukamp et al. 1999). Although HaCaT cells have three p53 point mutations (Lehman et al. 1993), data show that the line remains functional with respect to inducing apoptosis and reproductive death and behaves as though wildtype p53 were present (Henseleit et al. 1997). In this laboratory, HaCaTs also behave like wild-type cells in terms of the bystander effect response. ...
Article
Out-of-field effects are of considerable interest in radiotherapy. The mechanisms are poorly understood but are thought to involve signaling processes, which induce responses in non-targeted cells and tissues. The immune response is thought to play a role. The goal of this research was to study the induction of abscopal effects in the bladders of NU-Foxn1nu mice after irradiating their brains using Pencil Beam (PB) or microbeam (MRT) irradiation at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. Athymic nude mice injected with F98 glioma cells into their right cerebral hemisphere 7 d earlier were treated with either MRT or PB. After recovery times of 2, 12, and 48 h, the urinary bladders were extracted and cultured as tissue explants for 24 h. The growth medium containing the potential signaling factors was harvested, filtered, and transferred to HaCaT reporter cells to assess their clonogenic survival and calcium signaling potential. The results show that in the tumor-free mice, both treatment modalities produce strong bystander/abscopal signals using the clonogenic reporter assay; however, the calcium data do not support a calcium channel mediated mechanism. The presence of a tumor reduces or reverses the effect. PB produced significantly stronger effects in the bladders of tumor-bearing animals. The authors conclude that immunocompromised mice produce signals, which can alter the response of unirradiated reporter cells; however, a novel mechanism appears to be involved.
... Increased level of p53 protein was detected in human keratinocytes after UV irradiation (Henseleit et al., 1997). Exposure of normal human keratinocytes to UV radiation induces the extrinsic apoptotic pathway, which is initiated by the cell surface death receptors, such as TNF 1 receptor (p55 or CD120a), Fas (CD95/APO-1), DR3 (Apo3 or WSL1), DR4 (TRAIL-R1) and DR5 (TRAIL-R2) (Haupt et al., 2003;Yan & Shi, 2005;Elmore, 2007). ...
Article
Apoptosis triggered by exogenous and endogenous stimuli such as ultraviolet radiation, oxidative stress, and genotoxic chemicals is a crucial phenomenon within biological systems. DNA damage activates and stabilizes p53 in nucleus and cytoplasm and regulates other proteins that stimulate intrinsic and extrinsic apoptotic pathways. Apoptosis is morphologically distinct from that of necrosis and both the phenomena depend on the types, developmental stages, physiological environment of tissues and the nature of death signal. Malfunctioning of apoptotic pathway may cause human diseases like cancer, neurodegenerative and autoimmune disorders. Recently, potent apoptosis-inducing compounds associated with human health have been recorded that prevent tumor promotion, progression, and the occurrence of cellular inflammatory responses. Certain photosensitizing drugs are being employed in photodynamic therapy to induce apoptosis for the treatment of cancer and non-cancerous cells. This review emphasizes the molecular mechanisms of apoptosis, associated diseases and certain therapeutic agents implicated in the elimination of malignant cells.
... Additionally, CLOCK-silenced HKCs experience a decrease in p53 levels compared to normal irradiated HKCs [26]. This shows that in primary keratinocyte cells, CLOCK can affect the expression of p53 and it could be possible that the point mutations in the p53 gene of HaCaT affects its interaction with CLOCK [37]. However, this possibility needs further confirmation. ...
Article
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Sunlight is an important factor in regulating the central circadian rhythm, including the modulation of our sleep/wake cycles. Sunlight had also been discovered to have a prominent influence on our skin’s circadian rhythm. Overexposure or prolonged exposure to the sun can cause skin photodamage, such as the formation of irregular pigmentation, collagen degradation, DNA damage, and even skin cancer. Hence, this review will be looking into the detrimental effects of sunlight on our skin, not only at the aspect of photoaging but also at its impact on the skin’s circadian rhythm. The growing market trend of natural-product-based cosmeceuticals as also caused us to question their potential to modulate the skin’s circadian rhythm. Questions about how the skin’s circadian rhythm could counteract photodamage and how best to maximize its biopotential will be discussed in this article. These discoveries regarding the skin’s circadian rhythm have opened up a completely new level of understanding of our skin’s molecular mechanism and may very well aid cosmeceutical companies, in the near future, to develop better products that not only suppress photoaging but remain effective and relevant throughout the day.
... However, one study demonstrated that blocking mutated p53 partially blocked UVB-induced apoptosis in HaCaT cell line and furthermore confirm the key role for p53 apoptosis in human keratinocytes. Moreover, these results clearly demonstrated that p53 must, at least in part, remain functional in spite of its mutation points in HaCaT [35]. Thus, our study demonstrates, for the first time in human cell line, results which indicate that the involvement of methylation in the P53 gene promoter motivated by oxidative stress in experimental model of acquisition resistance anoikis. ...
Article
Cell adhesion plays an important role in neoplastic transformation. Thus, anchorage-independent growth and epithelial-mesenchymal transition, which are features associated to anoikis-resistance, are vital steps in cancer progression and metastatic colonization. Cell attachment loss may induce intracellular oxidative stress, which triggers DNA damage as methylation changes. HaCaT lineage cells were submitted to periods of 1, 3, 5 and 24 h of anchorage blockage with the purpose of study of oxidative stress effect on changes in the DNA methylation pattern, derived from attachment blockade. Through this study, HaCaT anchorage blockage-induced oxidative stress was reported to mediate alterations in global DNA methylation changes and into TP53 gene promoter pattern during anoikis-resistance acquisition. Furthermore, at the first experimental time-periods (1, 3 and 5 h), genome hypermethylation was found; however, genome hypomethylation was observed in later time-periods (24 h) of attachment impediment. The TP 53 methylation analyses were performed after 24 h of replated anoikis-resistance cells and same methylation pattern was observed, occurring an early (1 and 3 h) hypermethylation that was followed by late (5 and 24 h) hypomethylation. However, LINE-1, a marker of genomic instability, was perceived in time-dependent hypomethylation. The mRNA levels of the DNMTs enzymes were influenced by cell attachment blockage, but non-conclusive results were obtained in order to match DNMTs transcription to pattern methylation results. In conclusion, DNA damage was found, leaded by oxidative stress that has come up from HaCaT anchorage blockade, which rises a global genome hypomethylation tendency as consequence, which might denote genomic instability.
... p53, a nuclear phosphoprotein first identified by Lane and Crawford in 1979, is encoded by a tumor suppressor gene on the short arm of chromosome 17 (21). It binds to DNA as a tetramer and activates the transcription of many genes involved in cell differentiation, proliferation, induction of DNA repair pathways and cell death (5,22,23). ...
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UVB irradiation has been shown to trigger a broad range of changes in gene expression in human skin; however, factors governing these events are still not well understood. In this study, we show that human constitutive photomorphogenic protein-1 (huCOP1), an E3 ligase, contributes to the orchestration of UVB response of keratinocytes. Accordingly, our data show that (i) huCOP1 protein is expressed both in the nucleus and in the cytoplasm of cultured keratinocytes, (ii) UVB reduces the levels of the huCOP1 mRNA and protein, and (iii) induces changes in the subcellular localization of huCOP1. Finally, we show that gene-specific silencing of huCOP1 induces the accumulation of the tumor suppressor p53 protein, which is further increased after UVB irradiation.
... miR-125a-5p was previously reported to be expressed at significantly lower levels in UVB-irradiated mouse epidermis than in control epidermis (18), although the results of the present study demonstrated that the miRNA was significantly upregulated (2.74-fold). In addition, miR-1246 (4.23-fold downregulation in this study) was reported to be a target of the p53 transcription factor, which is significantly upregulated by UVB irradiation in keratinocytes (19), and miR-125a-3p (2.86-fold downregulation) has been demonstrated to reduce cell proliferation and migration by targeting Fyn kinase, which is activated by UVB irradiation in keratinocytes (20,21). In summary, the microarray data of the present study suggested that although the dysregulated miRNAs were specifically induced by arctiin treatment, they may be involved in the regulation of general UVB-mediated cellular transduction. ...
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Human keratinocytes are located in the outermost skin layer and thus particularly vulnerable to ultraviolet B (UVB) radiation exposure. Previous studies have focused on the cellular and molecular perspectives of UVB-induced keratinocyte damage. In the present study, it was demonstrated that pretreatment with the phytochemical arctiin, one of the lignin compounds, protects human HaCaT keratinocytes from UVB-mediated damage. Biochemical assays revealed that UVB-induced cytotoxicity and cell death were significantly reduced in arctiin-pretreated HaCaT cells. In addition, arctiin promoted the wound healing and DNA repair properties of keratinocytes. The photoprotective effects of arctiin were associated with changes in the expression levels of specific microRNAs (miRNAs) in HaCaT cells. A bioinformatics analysis demonstrated that the miRNAs were functionally involved in cancer, cell cycle, and Wnt and mitogen-activated protein kinase signaling pathways. In the present study, the results from the cellular and molecular assays demonstrated a novel role for arctiin in UVB protection in keratinocytes, which is mediated by miRNA responses and the suppression of UVB-induced cell death. Furthermore, arctiin is implicated as a potential chemopreventive agent through UVB protection of keratinocytes.
... Chemotherapy-induced alopecia (CIA) is considered to be the most traumatic side effect of cancer treatment, and stress caused by this can have a negative impact on the overall outcomes [38,39]. Cancer patients going through chemotherapy may lose their hair all over their bodies, but it is the most traumatic for patients to lose the hair on their heads. ...
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When it comes to cell proliferation, this is a process strictly controlled by extracellular signals, such as nutrients, growth factors, temperature, and more. Together, these establish the cell cycle control mechanism, which is the core of cell proliferation. Throughout the current review, a collection of the most pertinent background sources was built, in order to investigate the cytoprotective effect of cooling in opposition to toxicity brought on by chemotherapy, and subsequently another experimental approach was developed to comprehend the impacts of cooling on cell cycle arrest in cell culture. As a result, it is considered that this could show an approach for reaching selective toxicity of chemotherapeutic agents for cells, which in turn can provide a deeper comprehension of the molecular and cellular processes occurring within. With detailed information collected regarding the way that cell cycle events are controlled, it is considered that innovative ways of using this to protect for chemotherapy-induced alopecia will be established
... These curiosities became of potential importance for human skin cancer after the demonstration that individual sunburn cells contain the DNA double-strand breaks typical of cells undergoing apoptosis (Ziegler et al, 1994;Brash et al, 1996) and that Trp53 knockout mice exhibit an approximately 7-fold de®ciency in sunburn cell production (Ziegler et al, 1994). The effect of a point mutation on apoptosis depends on the particular amino acid substituted (Li et al, 1996;Rowan et al, 1996;Henseleit et al, 1997). The involvement of Trp53 in sunburn cell formation provides a connection to cancer, as TP53 mutations are present in most human nonmelanoma skin tumors or precancers (actinic keratoses) and Trp53 mutations are present in most murine skin squamous cell carcinomas induced by UVB (Brash et al, 1991(Brash et al, , 1996Nataraj et al, 1995;Dumaz et al, 1997). ...
Article
The stratum corneum and DNA repair do not completely protect keratinocytes from ultraviolet B. A third defense prevents cells with DNA photoproducts from becoming precancerous mutant cells: apoptosis of ultraviolet-damaged keratinocytes ("sunburn cells"). As signals for ultraviolet-induced apoptosis, some studies implicate DNA photoproducts in actively transcribed genes; other studies implicate non-nuclear signals. We traced and quantitated the in vivo DNA signal through several steps in the apoptosis-signaling pathway in haired mice. Homozygous inactivation of Xpa, Csb, or Xpc nucleotide excision repair genes directed the accumulation of DNA photoproducts to specific genome regions. Repair-defective Xpa–/– mice were 7–10-fold more sensitive to sunburn cell induction than wild-type mice, indicating that 86–90% of the ultraviolet B signal for keratinocyte apoptosis involved repairable photoproducts in DNA; the remainder involves unrepaired DNA lesions or nongenomic targets. Csb–/– mice, defective only in excising photoproducts from actively transcribed genes, were as sensitive as Xpa–/–, indicating that virtually all of the DNA signal originates from photoproducts in active genes. Conversely, Xpc–/– mice, defective in repairing the untranscribed majority of the genome, were as resistant to apoptosis as wild type. Sunburn cell formation requires the Trp53 tumor suppressor protein; 90–96% of the signal for its induction in vivo involved transcribed genes. Mdm2, which regulates the stability of Trp53 through degradation, was induced in vivo by low ultraviolet B doses but was suppressed at erythemal doses. DNA photoproducts in actively transcribed genes were involved in 89% of the Mdm2 response.Keywords: apoptosis, Cockayne syndrome, mdm2 protein, MeSH, protein p53, ultraviolet rays, xeroderma pigmentosum
... their p53 gene, resulting in p53 having an extended half-life (Lehman et al., 1993). However, p53 is still induced in HaCaTs following DNA damage suggesting it is still functional (Henseleit et al., 1997; Boswell et al., 2007). ...
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The human epidermis is a self-renewing, stratified epithelial tissue that provides the protective function of the skin. The principal cell type within the epidermis is the keratinocyte, and normal function of the epidermis requires that keratinocyte proliferation, differentiation and cell death be carefully controlled. There is clear evidence that signalling through adhesion receptors such as integrins and cadherins plays a key role in regulating epidermal function. Previous work has shown that Rho family GTPases regulate cadherin- and integrin-based adhesion structures and hence epidermal function. In this study, we show that a member of this family, Rnd3, regulates desmosomal cell-cell adhesion in that loss of Rnd3 expression leads to an increase in desmosomes at sites of cell-cell adhesion and altered colony morphology. Loss of Rnd3 expression is also associated with resistance to cisplatin-mediated apoptosis in keratinocytes and this resistance is mediated through the desmosomal protein plakoglobin. We propose a novel plakoglobin-dependent role for Rnd3 in the regulation of keratinocyte cell death.
... 9,39,40 In HaCaT cells with UV-type p53 mutations in both alleles, 41 the mutated p53 is stabilized 22 and has been suggested to play a role in apoptosis and differentiation. 42,43 In these cells, however, UV radiation fails to induce p21 expression, 22 which may be responsible for sustained p21 down-regulation. ...
Article
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Skin cancer is the most common cancer in the United States. Ultraviolet B (UVB) radiation in sunlight is the major environmental factor causing skin cancer. p21, a p53-inducible protein, plays an important role in cell cycle, DNA repair, and apoptosis. Here we have investigated the effect of UVB radiation on p21 and its molecular mechanisms and function in human HaCaT keratinocytes, which we used as a premalignant cellular model because normal skin harbors numerous clones of p53-mutated keratinocytes. We found that in human HaCaT keratinocytes UVB induces rapid p21 down-regulation via a proteasomal degradation mechanism. In p53-defective HaCaT cells, the p21 protein levels remain decreased at a later time post-UVB, but in normal human and mouse epidermal keratinocytes with wild-type p53 the p21 levels are initially reduced but later increase post-UVB. These findings indicate that loss of p53 function leads to sustained p21 down-regulation in response to UVB damage. Degradation of p21 following UVB radiation does not require ATR, ATM, or both, because either the ATR/ATM inhibitor caffeine or siRNA knockdown of ATR, ATM, or both failed to reverse p21 degradation. However, inhibiting MDM2 or GSK3β partially reduced UVB-induced p21 degradation, while inhibiting both enzymes completely prevented it. Restoring the p21 protein levels in UVB-irradiated keratinocytes reduced apoptosis. Although at the molecular level increasing p21 expression has no effect on the protein levels of the Bcl-2 family members, it enhances the activation of AKT, a critical survival pathway to protect cells from apoptosis. Our results suggest a distinct mechanism of p21 degradation in keratinocytes by UVB, and this p21 degradation may significantly enhance UVB-induced apoptosis of premalignant keratinocytes with a p53 defect to eliminate damaged cells and therefore prevent skin cancer development.
... Increased level of p53 protein was detected in human keratinocytes after UV irradiation (Henseleit et al., 1997). Exposure of normal human keratinocytes to UV radiation induces the extrinsic apoptotic pathway, which is initiated by the cell surface death receptors, such as TNF 1 receptor (p55 or CD120a), Fas (CD95/APO-1), DR3 (Apo3 or WSL1), DR4 (TRAIL-R1) and DR5 (TRAIL-R2) (Haupt et al., 2003; Yan & Shi, 2005; Elmore, 2007). ...
Article
Apoptosis triggered by exogenous and endogenous stimuli such as ultraviolet radiation, oxidative stress, and genotoxic chemicals is a crucial phenomenon within biological systems. DNA damage activates and stabilizes p53 in nucleus and cytoplasm and regulates other proteins that stimulate intrinsic and extrinsic apoptotic pathways. Apoptosis is morphologically distinct from that of necrosis and both the phenomena depend on the types, developmental stages, physiological environment of tissues and the nature of death signal. Malfunctioning of apoptotic pathway may cause human diseases like cancer, neurodegenerative and autoimmune disorders. Recently, potent apoptosis-inducing compounds associated with human health have been recorded that prevent tumor promotion, progression, and the occurrence of cellular inflammatory responses. Certain photosensitizing drugs are being employed in photodynamic therapy to induce apoptosis for the treatment of cancer and non-cancerous cells. This review emphasizes the molecular mechanisms of apoptosis, associated diseases and certain therapeutic agents implicated in the elimination of malignant cells.
... Certainly HaCaT cells are capable of p53-independent apoptosis, since irradiation in the presence or absence of E6 and E7 led to apoptotic cell death without any change in p53 or p21 levels (Magal et al., 1998). However, blocking HaCaT p53 can partially block apoptosis (Henseleit et al., 1997), suggesting that HaCaT p53 retains some activity, but that both p53-independent and -dependent apoptosis pathways are present. Our data indicate that induction of apoptosis and then differentiation was resident in the C-terminus of the HPV-16 E2 protein, the region that interacts with p53 (Parish et al., 2006). ...
Article
Expression of the HPV E2 open reading frame in cervical cancer cells has been shown to affect the expression of both viral and cellular genes. We have examined the phenotypic effects of the expression of human papillomavirus 16 E2 open reading frame in the human keratinocyte cell line HaCaT. Increased levels of apoptotic cell death were seen within 24h of the transfection of HPV-16 E2 expression constructs. However, in those cells which survived selection and retained the intact E2 ORF, long-term stable expression of E2, as detected by RT-PCR, produced cells which developed phenotypes typical of terminally differentiated cells. These included characteristic morphological changes and expression of involucrin, filaggrin and senescence markers. This provides the first evidence of a role for E2 in stimulation of the normal epithelial differentiation programme, which would promote the progression of the HPV life cycle.
... To observe the presence of DNA double-strand breaks repair system, the 53BP1 visualization was performed (Fig.4). It has to be pointed out that due to the method of their immortalization, HaCaT cells have a tendency for the appearance of a certain number of spontaneous DSB [46]. The cells treated with UVB doses 25 and 50 mJ/cm 2 reveal 53BP1 staining signal comparable to the control (Fig.4Aa,b,c and B), whereas very low signal was detected in cells radiated with UVB 100 mJ/cm 2 dose ( Fig.4Ad and B). ...
Article
The usage of active compounds of dietary phytochemicals in prevention of UV-induced skin diseases is increasingly gaining attention in the development of skin care products. The purpose of this study was to measure the influence of delphinidin (as a botanical agent) on the cell mechanical properties evaluated by the atomic force microscopy (AFM) technique in the immortalized human keratinocyte cell line (HaCaT) exposed to UVB radiation. The cells were treated with various doses of UVB radiation with and without pre and post-treatment with selected concentrations of delphinidin. The measurements of the elastic properties revealed that the exposure of HaCaT cells to high dose of the UVB radiation (100 mJ/cm2) caused a decrease in the cell elastic modulus. It was accompanied by the decrease of metabolic activity, rearrangement of actin cytoskeleton and disappearance of the cell repair marker 53BP1. Both pre-treatment and post-treatment with delphinidin at non-cytotoxic concentrations (5 or 10 μM), restored the elastic modulus of irradiated keratinocytes. A direct AFM analysis showed that the UVB-mediated decrease of the cell stiffness was restored more effectively when cells were treated with delphinidin after the UVB irradiation. The results demonstrate the regenerative effect of delphinidin on the mechanical properties of cells exposed to UVB radiation (100 mJ/cm2), which may be due to antioxidant and inhibitory effect on matrix metalloproteinases activation.
... Some of the cell lines analysed in this study have shown significant differences in cytotoxicity compared to the overall cell viability results. Interestingly, a decrease or increase in cell viability in those cell types after MSN exposure did not show any correlation with either the presence of p53 mutation in those cell lines (HaCaT-L, Caco-2, U937, DMSCs and Daudi cells) or the absence (C2C12, LS174T) [96,[114][115][116][117][118][119][120], highlighting the role of other features in influencing the cytotoxicity results. ...
Article
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Mesoporous Silica Nanoparticles (MSNs) have received increasing attention in biomedical applications due to their tuneable pore size, surface area, size, surface chemistry, and thermal stability. The biocompatibility of MSNs, although generally believed to be satisfactory, is unclear. Physicochemical properties of MSNs, such as diameter size, morphology, and surface charge, control their biological interactions and toxicity. Experimental conditions also play an essential role in influencing toxicological results. Therefore, the present study includes studies from the last five years to statistically analyse the effect of various physicochemical features on MSN-induced in-vitro cytotoxicity profiles. Due to non-normally distributed data and the presence of outliers, a Kruskal–Wallis H test was conducted on different physicochemical characteristics, including diameter sizes, zeta-potential measurements, and functionalisation of MSNs, based on the viability results, and statistical differences were obtained. Subsequently, pairwise comparisons were performed using Dunn’s procedure with a Bonferroni correction for multiple comparisons. Other experimental parameters, such as type of cell line used, cell viability measurement assay, and incubation time, were also explored and analysed for statistically significant results.
... L'immortalisation des kératinocytes qui a servi à établir la lignée de cellules HaCaT a été réalisée par l'intermédiaire du virus SV40. La lignée ainsi transformée a donné naissance à une lignée immortalisée, mutée sur P53 (Henseleit et al., 1997) mais encore capable de différenciation épidermique spontanée. Cette lignée cellulaire HaCaT est évidemment immortelle (> 140 passages), mais non tumorigène. ...
Thesis
Le manque de thérapies innovantes en chimiothérapie humaine incite la communauté scientifique à s'intéresser à de nouvelles sources de composés bioactifs. Nous pouvons citer les métabolites secondaires de plantes, auxquels appartiennent les acides hydroxy pentacycliques triterpénoiques (AHPTs) et plus particulièrement les Acides Ursolique (AU), Oléanolique (AO) et Bétulinique (AB). Ces molécules font l'objet de nombreuses études qui tendent à démontrer leurs propriétés : anti-infectieuses, anticancéreuses, antiprolifératives, anti-inflammatoires, hépatoprotectrices. Le principal obstacle à leur utilisation à des fins thérapeutiques, reste l'insolubilité de ces AHPTs dans l'eau. L'objectif de ce travail a donc été d'augmenter leur hydrosolubilité. Dans un premier temps, en accord avec les recommandations et/ou normes existantes, nous avons démontré que le spectre d'activité antibactérienne de l'AU et de l'AO se limitait aux bactéries à Gram positif. Aucun AHPT n'a montré d'activité antifongique. Seul l'AB a montré une activité intéressante sur le Cytomégalovirus humain (hCMV) ; aucune activité antivirale n'ayant été retrouvée sur le Poliovirus. Enfin, l'AB, mais encore plus l'AU ont montré une activité anticancéreuse à l'encontre de cellules modèles de leucémie myéloïde chronique (LMC). Dans un deuxième temps, nous avons procédé à la fabrication et à l'étude de complexes entre les AHPTs et des cyclodextrines. Nous avons retenu la gamma-cyclodextrine (gamma-CD), qui présentait l'avantage de complexer les 3 AHPTs avec une constante de formation « moyenne » à « élevée ». Ces complexes AHPTs :gamma-CD ont été caractérisés en utilisant diverses techniques : chromatographiques, thermiques et spectrométriques. Nous avons conclu à l'obtention de complexes d'inclusion qui ont permis d'augmenter la solubilité des AHPTs. Dans une dernière partie, nous avons évalué les activités biologiques des complexes AHPTs : gamma-CD. Les résultats montrent que les complexes AU : gamma-CD et AO : gamma-CD restent actifs à l'encontre des bactéries à Gram positif (mais avec une efficacité plus faible) ; tandis que le complexe AB : gamma-CD se révèle être actif sur certaines bactéries. Le complexe AB : gamma-CD, et de façon surprenante le complexe AU : gamma-CD présentent une activité antivirale à l'encontre du hCMV. Enfin, la diminution de la cytotoxicité liée à la complexation des AHPTs accroit l'intérêt des molécules d'AU et d'AB sur les cellules de LMC
... Of the tested stimuli including interferon gamma, tumor necrosis factor alpha, interleukin-4 and muramyl dipeptide, only interferon gamma induced HaCaT cell apoptosis [85,86]. This may be explained by spontaneous mutations in both alleles of pro-apoptotic protein p53 in immortalized HaCaT cells that affect the apoptosis pathway [87,88] and may also increase this cell line resistance to the G3-BCL construct. ...
Article
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Glioblastoma multiforme (GBM) is the most malignant type of central nervous system tumor that is resistant to all currently used forms of therapy. Thus, more effective GBM treatment strategies are being investigated, including combined therapies with drugs that may cross the blood brain barrier (BBB). Another important issue considers the decrease of deleterious side effects of therapy. It has been shown that nanocarrier conjugates with biotin can penetrate BBB. In this study, biotinylated PAMAM G3 dendrimers substituted with the recognized anticancer agents cyclooxygenase-2 (COX-2) inhibitor celecoxib and peroxisome proliferator-activated receptor γ (PPARγ) agonist Fmoc-L-Leucine (G3-BCL) were tested in vitro on human cell lines with different p53 status: glioblastoma (U-118 MG), normal fibroblasts (BJ) and immortalized keratinocytes (HaCaT). G3-BCL penetrated efficiently into the lysosomal and mitochondrial compartments of U-118 MG cells and induced death of U-118 MG cells via apoptosis and inhibited proliferation and migration at low IC50 = 1.25 µM concentration, considerably lower than either drug applied alone. Comparison of the effects of G3-BCL on expression of COX-2 and PPARγ protein and PGE2 production of three different investigated cell line phenotypes revealed that the anti-glioma effect of the conjugate was realized by other mechanisms other than influencing PPAR-γ expression and regardless of p53 cell status, it was dependent on COX-2 protein level and high PGE2 production. Similar G3-BCL cytotoxicity was seen in normal fibroblasts (IC50 = 1.29 µM) and higher resistance in HaCaT cells (IC50 = 4.49 µM). Thus, G3-BCL might be a good candidate for the targeted, local glioma therapy with limited site effects.
... The HaCaT cell line was developed by spontaneous immortalization of human keratinocytes cultured continuously with low Ca 2+ at 38.5°C (Boukamp et al. 1988;Colombo et al. 2017). These cells lack the SV40 T-antigen or human papillomavirus genomic sequences (Boukamp et al. 1988) but do contain two point mutations in the TP53 gene (Henseleit et al. 1997). However, arsenic exposure inhibits the functions of TP53 (Ganapathy et al. 2016;Shen et al. 2008), thus alleviating concerns about a nonfunctional TP53 in this cell line with respect to arsenic-induced cSCC. ...
Article
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Background: Chronic arsenic exposure via drinking water is associated with an increased risk of developing cancer and noncancer chronic diseases. Pre-mRNAs are often subject to alternative splicing, generating mRNA isoforms encoding functionally distinct protein isoforms. The resulting imbalance in isoform species can result in pathogenic changes in critical signaling pathways. Alternative splicing as a mechanism of arsenic-induced toxicity and carcinogenicity is understudied. Objective: This study aimed to accurately profile differential alternative splicing events in human keratinocytes induced by chronic arsenic exposure that might play a role in carcinogenesis. Methods: Independent quadruplicate cultures of immortalized human keratinocytes (HaCaT) were maintained continuously for 28 wk with 0 or 100 nM sodium arsenite. RNA-sequencing (RNA-Seq) was performed with poly(A) RNA isolated from cells harvested at 7, 19, and 28 wk with subsequent replicate multivariate analysis of transcript splicing (rMATS) analysis to detect and quantify differential alternative splicing events. Reverse transcriptase-polymerase chain reaction (RT-PCR) for selected alternative splicing events was performed to validate RNA-Seq predictions. Functional enrichment was performed by gene ontology (GO) analysis of the differential alternative splicing event data set at each time point. Results: At least 600 differential alternative splicing events were detected at each time point tested, comprising all the five main types of alternative splicing and occurring in both open reading frames (ORFs) and untranslated regions (UTRs). Based on functional relevance ELK4, SHC1, and XRRA1 were selected for validation of predicted alternative splicing events at 7 wk by RT-PCR. Densitometric analysis of RT-PCR data corroborated the rMATS predicted alternative splicing for all three events. Protein expression validation of the selected alternative splicing events was challenging given that very few isoform-specific antibodies are available. GO analysis demonstrated that the enriched terms in differential alternatively spliced mRNAs changed dynamically with the time of exposure. Notably, RNA metabolism and splicing regulation pathways were enriched at the 7-wk time point, when the greatest number of differentially alternatively spliced mRNAs are detected. Our preliminary proteomic analysis demonstrated that the expression of the canonical isoforms of the splice regulators DDX42, RMB25, and SRRM2 were induced upon chronic arsenic exposure, corroborating the splicing predictions. Discussion: These results using cultures of HaCaT cells suggest that arsenic exposure disrupted an alternative splice factor network and induced time-dependent genome-wide differential alternative splicing that likely contributed to the changing proteomic landscape in arsenic-induced carcinogenesis. However, significant challenges remain in corroborating alternative splicing data at the proteomic level. https://doi.org/10.1289/EHP9676.
... The molecular mechanisms of UVB-induced apoptosis also involve the tumor suppressor protein p53, which has been found to be mutated in HaCaT cells and most skin tumor cells. Blocking the mutated p53 proteins could partially block UVB-induced apoptosis in HaCaT cells [62]. Hence, other signaling pathways influence the homeostasis between cell survival and the induction of apoptosis in HaCaT cells. ...
Article
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Ultraviolet B (UVB) exposure is the primary risk factor for the deadliest type of skin cancer—melanoma. Incorporating natural antioxidants in skin protection products is currently a favored research theme. For this study, we selected Phyllanthus emblica L. fruit extract (PE) to assess its potential use in dermal protection against UVB-induced keratinocyte inflammation and apoptosis. High-performance liquid chromatography (HPLC) was used to investigate PE’s phytochemical constituents (ascorbic acid, ellagic acid, gallic acid, chlorogenic acid, and quercetin), while ferric reducing antioxidant power (FRAP), oxygen radical absorbance capacity (ORAC), total ROS, OH•, O2•−, and H2O2-scavenging activities were used to determine the antioxidant properties. PE significantly increased the cell viability (MTT assay) and reduced apoptosis (Hoechst staining) in HaCaT cells exposed to UVB (40 mJ/cm2). PE abolished oxidative stress by reducing the production of intracellular ROS, O2•− and H2O2 production. Catalase activity (but not superoxide dismutase or glutathione peroxidase activity) was enhanced in keratinocytes incubated with PE prior to UVB exposure. Western blot analysis suggested that PE inhibited cytochrome c release and inhibited the dysregulation of PI3K/Akt without any impact on p38 activation. PE attenuated the inflammatory response to UVB irradiation by inhibiting AP-1, NF-κB, and the mediator PGE2. Thus, PE is a candidate with great potential for use as an active ingredient in skin care products.
... Since both TP53 alleles are mutated, there is no wild type p53 protein in the HaCaT keratinocytes [57]. However, the mutated p53 in HaCaT cells might at least be functional despite its point mutations [58]. Indeed, the mutated p53 in HaCaT cells can undergo apoptosis through activation by phosphorylating at its Ser15 residue after UVB irradiation [59]. ...
Article
A dysfunction in the mitochondrial-lysosomal axis of cellular homeostasis is proposed to cause cells to age quicker and to accumulate lipofuscin. Typical protocols to mediate lipofuscinogenesis are based on the induction of the senescent phenotype either by allowing many consecutive cycles of cell division or by treating cells with physical/chemical agents such as ultraviolet (UV) light or hydrogen peroxide. Due to a direct connection with the physiopathology of age-related macular degeneration, lipofuscin that accumulates in retinal pigment epithelium (RPE) cells have been extensively studied, and the photochemical properties of RPE lipofuscin are considered as standard for this pigment. Yet, many other tissues such as the brain and the skin may prompt lipofuscinogenesis, and the properties of lipofuscin granules accumulated in these tissues are not necessarily the same as those of RPE lipofuscin. Here, we present a light-induced protocol that accelerates cell aging as judged by lipofuscinogenesis and maximizes this lipofuscinogenesis. Photosensitization of cells previously incubated with nanomolar concentrations of 1,9-dimethyl methylene blue (DMMB), severely and specifically damages mitochondria and lysosomes, leading to a lipofuscin-related senescent phenotype. By applying this protocol in human immortalized non-malignant keratinocytes (HaCaT) cells, we observed a 2.5-fold higher level of lipofuscin accumulation compared to the level of lipofuscin accumulation in cells treated with a typical UV protocol. Lipofuscin accumulated in keratinocytes exhibited the typical red light emission, with excitation maximum in the blue wavelength region (∼450 nm). Fluorescence lifetime image microscopy data showed that the keratinocyte lipofuscin has an emission lifetime of ∼ 1.7 ns. Lipofuscin-loaded cells (but not control cells) generated a substantial amount of singlet oxygen (¹O2) when irradiated with blue light (420 nm), but there was no ¹O2 generation when excitation was performed with a green light (532 nm). These characteristics were compared with those of RPE cells, considering that keratinocyte lipofuscin lacks the bisretinoids derivatives present in RPE lipofuscin. Additionally, we showed that lipofuscin-loaded keratinocytes irradiated with visible light presented critical DNA damages, such as double-strand breaks and Fpg-sensitive sites. We propose that the DMMB protocol is an efficient way to disturb the mitochondrial-lysosomal axis of cellular homeostasis, and consequently, it can be used to accelerate aging and to induce lipofuscinogenesis. We also discuss the consequences of the lipofuscin-induced genotoxicity of visible light in keratinocytes.
... immortalized human keratinocytes harboring p53 and multiple further mutations (Henseleit et al. 1997). In these cells, no phenotypic changes were observed upon TAgs expression underlining again the importance of the cellular context. ...
Thesis
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Merkel cell carcinoma (MCC) is a rare and aggressive skin cancer. In approximately 80% of cases, genomic integration of the Merkel cell polyomavirus (MCPyV) is observed and overexpression of the two MCPyV T antigens (TAgs) is regarded as the main oncogenic determinant of MCPyV-positive MCC cases. However, the nature of the cells from which MCC arises is unknown. Therefore, the goal of the present work was to determine the cell of origin of MCC. First, we characterized MCC patients’ tumors and demonstrated a high similarity of MCPyV- negative MCC with extracutaneous neuroendocrine carcinoma while MCPyV-positive MCC differs from these two groups with respect to morphology, immunohistochemical profile, genetics, origin and behavior. Based on the analysis of a trichoblastoma/MCC combined tumor, we demonstrated that a MCPyV-positive MCC can arise following MCPyV integration in an epithelial cell. In addition, the high similarity between trichoblastoma cells and Merkel cell (MC) progenitors of the hair follicle suggests that these hair follicle cells may represent a general start point for the development of MCPyV-positive MCC. A contribution of the viral TAgs to the development of the characteristic Merkel cell-like MCC phenotype is suggested by experiments demonstrating induction of Merkel cell markers upon TAg expression in human primary keratinocytes or hair follicle cells. As potential mechanisms mediating these phenotypic changes, we identified the capability of MCPyV LT to repress degradation of master regulator of MC development, i.e. the transcription factor ATOH1. To conclude, our work suggests that MCPyV integration in epithelial cells of the hair follicle may represent an important path for MCC development.
... Following DNA damage, p53 is activated to induce transcription of the Bcl-2 family of proteins, including the antiapoptotic protein Bcl-2 and the pro-apoptotic protein Bax. 34 Caspases, a family of cysteine proteases, are the central regulators of apoptosis. 35 Once activated, the initiator caspases (including caspase-9, playing a key role in the mitochondrialcontrolled apoptosis signaling pathway) cleave and activate downstream effector caspases (including caspase-3), which in turn execute apoptosis cascades. ...
... Pro-apoptotic stimuli inducing p53 further aggravate mitochondrial pathway-mediated apoptosis. p53 plays a crucial role in ultraviolet-induced apoptosis in HaCaT keratinocytes [26]. Under physiological conditions, p53 is maintained at a low concentration, inhibiting its transcriptional activity. ...
Article
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Ultraviolet B (UVB) radiation-induced oxidative skin cell damage is a major cause of photoaging. In the present study, a low molecular weight fucoidan fraction (SHC4) was obtained from Sargassum horneri by Celluclast-assisted extraction, followed by step gradient ethanol precipitation. The protective effect of SHC4 was investigated in human keratinocytes against UVB-induced oxidative stress. The purified fucoidan was characterized by Fourier-transform infrared spectroscopy (FTIR), 1H nuclear magnetic resonance (NMR), agarose gel-based molecular weight analysis and monosaccharide composition analysis. SHC4 had a mean molecular weight of 60 kDa, with 37.43% fucose and 28.01 ± 0.50% sulfate content. The structure was mainly composed of α-L-Fucp-(1→4) linked fucose units. SHC4 treatment dose-dependently reduced intracellular reactive oxygen species (ROS) levels and increased the cell viability of UVB exposed HaCaT keratinocytes. Moreover, SHC4 dose-dependently inhibited UVB-induced apoptotic body formation, sub-G1 accumulation of cells and DNA damage. Inhibition of apoptosis was mediated via the mitochondria-mediated pathway, re-establishing the loss of mitochondrial membrane potential. The UVB protective effect of SHC4 was facilitated by enhancing intracellular antioxidant defense via nuclear factor erythroid 2–related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling. Further studies may promote the use of SHC4 as an active ingredient in cosmetics and nutricosmetics.
... Other than the aforementioned mediators, the mitochondrial outer membrane permeabilization causes numerous proteins to be released antagonizing the activation of caspases. p53 is another molecular mediator that plays a critical role during UVinduced apoptosis in HaCaT cells [31]. Under normal conditions, a low concentration of p53 is maintained in the cytosol. ...
Article
Ultraviolet B (UVB) can induce oxidative damage to outermost layers of skin causing suntans, sunburns, and, in severe cases, blisters leading to photoaging. Low molecular weight (MW) fucoidan is renowned for possessing enhanced antioxidant activities. The present study discloses the use of step gradient ethanol precipitation in refining fucoidan fractions (SSQC1-SSQC4) from Sargassum siliquastrum and evaluation of their UVB-protective effects in human HaCaT keratinocytes. Among the fractions, SSQC4 indicated the best bioactive effects. ¹H NMR, FTIR, monosaccharide composition by HPAEC-PAD analysis, MW estimation by agarose gel electrophoresis were used to characterize the fractions. SSQC4 was comprising of fucoidan, with an estimated MW distribution of 8–25 kDa. Exposure of UVB increased intracellular ROS, DNA damage, loss of mitochondrial membrane potential, apoptotic body formation causing cell death through the mitochondria-mediated apoptosis pathway. SSQC4 treatment could dose-dependently attenuate the ROS levels and suppress mitochondria-mediated apoptosis in UVB exposed keratinocytes. SSQC4 treatment enhanced cellular antioxidant defense by increasing Nrf2 mediated HO-1 generation, which was identified as the cause of observed bioactivities. The safety and stability of SSQC4 could be further evaluated to promote its use as a bioactive natural ingredient in UV-protective cosmetics.
... The objective of this study was to further elucidate and characterize the role of 5-HT and 5-HTR in the amplification of CD in HaCaT keratinocytes following IR exposure. HaCaT is an important keratinocyte model for human health in exposure to environmental radioactivity because (1) the cell line was immortalized spontaneously and not by mutagenic or viral transformation (Boukamp et al., 1998), (2) HaCaT is non-tumorigenic and exhibits differentiated keratinocyte characteristics as in human skin in vivo (Boukamp et al., 1998); this cell line has functional p53 (Henseleit et al., 1997;Le et al., 2017;Lehman et al., 1993) and is radio-responsive to RIBEs (Curtis et al., 2018;Le et al., 2017). One major shortcoming of current literature is the lack of a comprehensive pharmacological profile in relation to the effects of 5-HT on keratinocyte cell survival. ...
Article
Ionizing radiation (IR) is an environmental carcinogen and the biological damages it elicits are mechanistically distinct between high and low doses. Non-targeted effects occurring in nonirradiated cells such as the radiation-induced bystander effect predominate at low doses of IR. However, the role of non-targeted effects in environmental radiation protection is often overlooked because the governing mechanisms are complex and multifactorial. An improved understanding of the signaling molecules and their capacity to sensitize specific cell types are essential in establishing environmental IR risks. In particular, serotonin (5-HT) has been identified to exacerbate both direct irradiation and bystander-induced cell death (CD) in certain cell types, although not all cell types are responsive to 5-HT in this respect. In this study, we further characterize the role of 5-HT and 5-HT receptors (5-HTR) in the amplification of CD following IR exposure in human keratinocytes. We examined the survival of HaCaT cells treated with 5-HT and the 5-HTR antagonists ketanserin (5-HT2A) and ondansetron (5-HT3) following exposure to direct IR and irradiated cell condition medium (ICCM). Nonirradiated cell survival was consistent with the vehicle control among 5-HT concentrations ranging from 0.001 to 100 μM. Significant 5-HT concentration-dependent increases in CD occurred following direct IR exposure. Nonirradiated ICCM-recipient CD was not altered by 5-HT (0.001-100 μM) when present during donor cell irradiation among all IR doses. Increases in direct irradiation CD evoked by 5-HT were significantly attenuated by ondansetron, blocking the effect of 5-HT, whereas ketanserin did not alter CD. Western blotting of these target 5-HTRs revealed protein expression of the 5-HT3 receptor, while the 5-HT2A receptor was not detected. We have demonstrated a definitive role for 5-HT in the exacerbation of CD following direct IR exposure and identified the 5-HT3 receptor as a potential target for ameliorating radiation damage in keratinocytes.
... In HaCaT cells the cytotoxicity of construct G3 1B16C15L was the lowest with IC 50 = 2.88 μM. It has been shown that HaCaT cells had point mutations in both alleles of tumor suppressor, pro-apoptotic protein p53, that effect apoptosis pathway and may increase this cell line resistance to celecoxib (Henseleit et al., 1997;Lehman et al., 1993). Similar phenomenon was observed in wild type p53 LNCaP cells treated with celecoxib, that inhibited cellular growth and proliferation more efficient as compared to p53-mutated DU145 cells (Katkoori et al., 2013). ...
Article
Tumors still remain one of the main causes of mortality due to the lack of effective anti-cancer therapy. Recently it has been shown, that overexpression of inducible cyclooxygenase-2 (COX-2) and decrease of peroxisome proliferator-activated receptor γ (PPARγ) expression accompany many malignances, therefore, it has been proposed, that COX-2 inhibitors and PPARγ agonists are potential candidates for anticancer therapy and their synergistic, antineoplastic action has been described. In the present study a COX-2 inhibitor (celecoxib) and/or PPARγ agonist (Fmoc-l-Leucine) were conjugated with the biotinylated G3 PAMAM dendrimer to form a three different constructs targeted to cells with increased biotin uptake. All conjugates were characterized by the NMR spectroscopy. Investigation of three types of human cells: normal skin fibroblasts (BJ), immortalized keratinocytes (HaCaT) and cancer lines: glioblastoma (U-118 MG) and squamous cell carcinoma (SCC-15) revealed similar biotin labeled ATTO590 accumulation (after 24 h), except for SCC-15 with significantly lower loading. Constitutive expression of COX-2 protein was confirmed in all tested cells with significantly higher levels (2-2.5 times) in both cancer lines. Comparison of cytotoxicity of the new synthetized dendrimers clearly documented the highest cytotoxicity of the G31B16C15L dendrimer conjugated with both drugs (1: 1) as compared with drugs alone and single conjugates. Additive effects of construct with both compounds were shown for fibroblasts and both cancer cell lines in the order BJ > U-118 MG > SCC-15 with IC50 in the range: 0.69, 1.44 and 2.22 μM, respectively and lowest cytotoxicity in HaCaT cells (IC50 = 2.88). Our results showed, that biotinylated G3 PAMAM dendrimers substituted with COX-2 inhibitor, celecoxib, and PPARγ agonist, Fmoc-l-Leucine (1:1) may be a good candidate for local therapy of glioblastoma but not a skin cancer. Since the effect of PPARγ agonists on COX-2 expression vary depending upon the cell type, specificity of used agonist and the presence of other environmental factors, it is necessary to carefully evaluate the response of chosen drugs on the target cells.
... High correlation between cancer suppressor protein p53 and apoptosis regulation in psoriasis patients was found in immunostaining results of psoriasis skin biopsies (Moorchung et al. 2015). Moreover, it was widely demonstrated that UV exposure can either stimulate the p53 expression (Henseleit et al. 1997;Qin et al. 2002) which implies the potential DNA damage (Schürer et al. 1993;Herzinger et al. 1995;Takasawa et al. 2005;Aitken et al. 2007). Since p53 mutations were identified in psoriasis patients, relevant studies have been conducted investigating the mutations caused by UV-based phototherapy (Nataraj et al. 1997;Seidl et al. 2001;Mudigonda et al. 2012). ...
Article
Application of high-dosage UVB irradiation in phototherapeutic dermatological treatments present health concerns attributed to UV-exposure. In assessing UV-induced photobiological damage, we investigated dose-dependent effects of UVB irradiation on human keratinocyte cells (HaCaT). Our study implemented survival and apoptosis assays and revealed an unexpected dose response wherein higher UVB-dosage induced higher viability. Established inhibitors, such as AKT− (LY294002), PKC− (Gö6976, and Rottlerin), ERK− (PD98059), P38 MAPK− (SB203580), and JNK− (SP600125), were assessed to investigate UV-induced apoptotic pathways. Despite unobvious contributions of known signaling pathways in dose-response mediation, microarray analysis identified transcriptional expression of UVB-response genes related to the respiratory-chain. Observed correlation of ROS-production with UVB irradiation potentiated ROS as the underlying mechanism for observed dose responses. Inability of established pathways to explain such responses suggests the complex nature underlying UVB-phototherapy response.
... First, that the mechanisms of cytotoxicity induced by AT is different from that of the others tested exracts, at least for this particular cell line. Second, HaCaT cells contain two heterozygous p53 mutations (exons 5 and 8) [3] and has a more extended protein half-life than the wild type p53, and despite the mutations the p53 protein is still functional and regulates cell cycle at the G0/G1 check-point [18]. The present finding confirms that HaCaT indeed has a functional p53 protein that arrest the cell cycle at G0/G1 to allow for repair of the damage, and that AT can interfere with regulation of the cell cycle for this particular cell line. ...
Article
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In vitro cell proliferation, cell cycle arrest and induction of apoptosis were investigated, using three human head and neck squamous cell carcinoma (HNSCC) cell lines (OSCC-3, SCC-61, and SQ-20B). Aqueous extracts of Camellia sinensis, Ilex paraguariensis, and Ardisia compressa were tested and (−) epigallocatechin-3-gallate (EGCG) was used for comparison. For EGCG the IC50 values were between 80 and 166μM and for the extracts among 75 and 505μM eq. (+) catechin, with C. sinensis demonstrating dominant cytotoxicity. There was not a correlation between antioxidant capacity and cytotoxicity. Flow cytometry analysis revealed similarities in response for EGCG and C. sinensis. The A. compressa extract altered DNA distribution (P<0.05) and was the most effective in induction of apoptosis via caspases (P<0.05). Not all HNSCC cells tested responded to the same preventive agents. The fact that A. compressa inhibits HNSCC cell proliferation makes this aqueous extract a potential source of chemopreventive agents.
... In particular, exposure of hepatoma (HepG2) cells to PZDHA was shown to induce growth arrest in G0/G1 phase [18], whereas PZDHA exposure to breast carcinoma (MDA-MB-231) cells caused growth arrest in G2/M phase [20]. In addition, the fact that both A375 and A431 cells have a wild-type p53 status [37,38] while HaCaT cells have a p53-mutated one [39] suggests that such observed differences in cell cycle distribution could, perhaps, be attributed to p53 status. Finally, although PZDHA-induced stimulation of oxidative stress was evident in all three cell lines, there was a differential response in ROS production based on the cell type itself and in the context of being either a melanoma (A375) or a non-melanoma (A431) or a keratinocyte (HaCaT) one ( Figure 5A-C). ...
Article
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Skin cancer is among the most common cancer types accompanied by rapidly increasing incidence rates, thus making the development of more efficient therapeutic approaches a necessity. Recent studies have revealed the potential role of decosahexaenoic acid ester of phloridzin (PZDHA) in suppressing proliferation of liver, breast, and blood cancer cell lines. In the present study, we investigated the cytotoxic potential of PZDHA in an in vitro model of skin cancer consisting of melanoma (A375), epidermoid carcinoma (A431), and non-tumorigenic (HaCaT) cell lines. Decosahexaenoic acid ester of phloridzin led to increased cytotoxicity in all cell lines as revealed by cell viability assays. However, growth inhibition and induction of both apoptosis and necrosis was more evident in melanoma (A375) and epidermoid carcinoma (A431) cells, whereas non-tumorigenic keratinocytes (HaCaT) appeared to be more resistant as detected by flow cytometry. More specifically, PZDHA-induced cell cycle growth arrest at the G2/M phase in A375 and A431 cells in contrast to HaCaT cells, which were growth arrested at the G0/G1 phase. Elevated intracellular generation of reactive oxygen species ROS was detected in all cell lines. Overall, our findings support the potential of PZDHA as a novel therapeutic means against human skin cancer.
Article
Ultraviolet light B (UVB), contained in sunlight, induces damaging effects on skin by impairing cells in the epidermis and dermis. In particular, keratinocytes in the epidermis are those cells which are mainly affected by UVB light. UVB radiation induces cell death, growth arrest, DNA damage and restricts cell migration. Various phytochemicals have been shown to alleviate UVB-induced cellular damage. Troxerutin is a natural flavonoid rutin mainly found in extracts of Sophora japonica, and is a well-known antioxidant and anti-inflammatory compound used in experimental mouse models. In this study, we examined the effects of troxerutin on UVB-induced damage in HaCaT cells. HaCaT cells were pre-treated with troxerutin (0-10 µM) and then exposed to UVB radiation (50 mJ/cm2). Cell viability, cell cycle and migration assays were performed to determine the protective effects of troxerutin on the cells. DNA repair activity was also measured. Troxerutin protected the cells against UVB-induced damage, such as cell death, growth arrest, restriction of cell migration and decreased DNA repair activity in HaCaT cells. Analyses of microRNA (miRNA) expression demonstrated that the protective effects of troxerutin correlated with alterations in miRNA expression, as indicated by Gene Ontology analyses of putative target genes. Overall, our data demonstrate that troxerutin exerts protective effects against UVB-induced damage by regulating miRNA expression.
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Beta-papillomaviruses (beta-HPV) have been linked to the development of skin cancer in humans. Because both E6 and E7 proteins from beta-HPV have been involved in the potential carcinogenicity of these viruses, we investigated their role on UVB-induced apoptosis in HaCaT cell line. HaCaT cells have been transduced with both E6/E7 using a retroviral system and treated with PRIMA-1. Apoptosis was assessed by flow cytometry to measure mitochondrial membrane potential and DNA fragmentation. HaCat keratinocytes transduced with both E6 and E7 genes of seven beta-HPV types (HPV5, HPV8, HPV14, HPV24, HPV36, HPV38 and HPV49) did not demonstrate any inhibition of UVB-induced apoptosis, even after p53 reactivation through PRIMA-1. Our data suggest that the expression of E6 and E7 exert different modulatory effects on UVB-induced apoptosis according to beta-HPV types and to the cellular genetic context.
Article
Protein kinase C delta (PKC-delta) protein levels are frequently low in chemically and UV-induced mouse skin tumors as well as in human cutaneous squamous cell carcinomas (SCCs). Furthermore, overexpression of PKC-delta in human SCC lines and mouse epidermis is sufficient to induce apoptosis and suppress tumorigenicity, making PKC-delta a potential tumor suppressor gene for SCCs. Here we report that PKC-delta is lost in human SCCs at the transcriptional level. We used laser capture microdissection to isolate cells from three normal human epidermis and 14 human SCCs with low PKC-delta protein. Analysis by quantitative reverse transcription-PCR revealed that PKC-delta RNA was reduced an average of 90% in the SCCs tested, consistent with PKC-delta down-regulation at the protein level. Analysis of DNA from nine of the same tumors revealed that PKC-delta gene was deleted in only one tumor. In addition, Ras-transformed human keratinocytes, which have selective down-regulation of PKC-delta at both protein and mRNA levels, had significantly repressed human PKC-delta promoter activity. Together, these results indicate that PKC-delta gene expression is suppressed in human SCCs, probably via transcription repression. Our results have implications for the development of topical therapeutic strategies to trigger the re-expression of pro-apoptotic PKC-delta to induce apoptosis in SCCs.
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A diverse array of biological processes are under circadian controls. In mouse skin, ultraviolet ray (UVR)‐induced apoptosis and DNA damage responses are time‐of‐day dependent, which are controlled by core clock proteins. This study investigates the roles of clock proteins in regulating UVB responses in human keratinocytes (HKCs). We found that the messenger RNA expression of brain and muscle ARNT‐like 1 (BMAL1) and circadian locomotor output cycles kaput (CLOCK) genes is altered by low doses (5 mJ/cm²) of UVB in the immortalized HaCat HKCs cell line. Although depletion of BMAL1 or CLOCK has no effect on the activation of Rad3‐related protein kinases–checkpoint kinase 1–p53 mediated DNA damage checkpoints, it leads to suppression of UVB‐stimulated apoptotic responses, and downregulation of UVB‐elevated expression of DNA damage marker γ‐H2AX and cell cycle inhibitor p21. Diminished apoptotic responses are also observed in primary HKCs depleted of BMAL1 or CLOCK after UVB irradiation. While CLOCK depletion shows a suppressive effect on UVB‐induced p53 protein accumulation, depletion of either clock gene triggers early keratinocyte differentiation of HKCs at their steady state. These results suggest that UVB‐induced apoptosis and DNA damage responses are controlled by clock proteins, but via different mechanisms in the immortalized human adult low calcium temperature and primary HKCs. Given the implication of UVB in photoaging and photocarcinogenesis, mechanistic elucidation of circadian controls on UVB effects in human skin will be critical and beneficial for prevention and treatment of skin cancers and other skin‐related diseases.
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Solar UV radiation consists of both UVA and UVB. The wavelength-specific molecular responses to UV radiation have been studied, but the interaction between UVA and UVB has not been well understood. In this study, we found that long-wavelength UVA, UVA1, augmented UVB-induced cell death, and examined the underlying mechanisms. Human keratinocytes HaCaT were exposed to UVA1, followed by UVB irradiation. Irradiation by UVA1 alone showed no effect on cell survival, whereas the UVA1 pre-irradiation remarkably enhanced UVB-induced cell death. UVA1 delayed the repair of pyrimidine dimers formed by UVB and the accumulation of nucleotide excision repair (NER) proteins to damaged sites. Gap synthesis during NER was also decreased, suggesting that UVA1 delayed NER, and unrepaired pyrimidine dimers and single-strand breaks generated in the process of NER were left behind. Accumulation of this unrepaired DNA damage might have led to the formation of DNA double-strand breaks (DSBs), as was detected using gel electrophoresis analysis and phosphorylated histone H2AX assay. Combined exposure enhanced the ATM–Chk2 signaling pathway, but not the ATR–Chk1 pathway, confirming the enhanced formation of DSBs. Moreover, UVA1 suppressed the UVB-induced phosphorylation of Akt, a survival signal pathway. These results indicated that UVA1 influenced the repair of UVB-induced DNA damage, which resulted in the formation of DSBs and enhanced cell death, suggesting the risk of simultaneous exposure to high doses of UVA1 and UVB.Graphic abstract
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Effective medical treatment and preventive measures for chemical warfare agent sulfur mustard (HD)-caused incapacitating skin toxicity are lacking, because of limited knowledge of its mechanism of action. The proliferating basal epidermal cells are primary major sites of attack during HD-caused skin injury. Therefore, employing mouse JB6 and human HaCaT epidermal cells, here, we investigated the molecular mechanism of HD analogue 2-chloroethyl ethyl sulfide (CEES)-induced skin cytotoxicity. As compared to the control, up to 1 mM CEES treatment of these cells for 2, 4, and 24 h caused dose-dependent decreases in cell viability and proliferation as measured by DNA synthesis, together with S and G2-M phase arrest in cell cycle progression. Mechanistic studies showed phosphorylation of DNA damage sensors and checkpoint kinases, ataxia telangiectasia-mutated (ATM) at ser1981 and ataxia telangiectasia-Rad3-related (ATR) at ser428 within 30 min of CEES exposure, and modulation of S and G2-M phase-associated cell cycle regulatory proteins, which are downstream targets of ATM and ATR kinases. Hoechst-propidium iodide staining demonstrated that CEES-induced cell death was both necrotic and apoptotic in nature, and the latter was induced at 4 and 24 h of CEES treatment in HaCaT and JB6 cells, respectively. An increase in caspase-3 activity and both caspase-3 and poly(ADP-ribose)polymerase (PARP) cleavage coinciding with CEES-caused apoptosis in both cell lines suggested the involvement of the caspase pathway. Together, our findings suggest a DNA-damaging effect of CEES that activates ATM/ATR cell cycle checkpoint signaling as well as caspase-PARP pathways, leading to cell cycle arrest and apoptosis/necrosis in both JB6 and HaCaT cells. The identified molecular targets, quantitative biomarkers, and epidermal cell models in this study have the potential and usefulness in rapid development of effective prophylactic and therapeutic interventions against HD-induced skin toxicity.
Thesis
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This thesis demonstrates the relevance of bystander effect mechanisms after exposure to two Synchrotron modalities – Microbeam Radiation Therapy and Pencilbeam – that are currently at the preclinical stage but aim to treat brain tumours. We elucidate the relationship between the hyper-radiosensitivity phenomenon and radiation-induced bystander effects by studying the dose response of three glioma cell lines. The relevance of these low-dose effects for both Synchrotron modalities is because the tissue exposed to low valley-doses is predicted to be where hyper-radiosensitivity and bystander effects might be expected to predominate. In vivo experiments were conducted in the European Synchrotron radiation Facility in Grenoble, France and also in the University of Freiburg’s Hospital in Freiburg, Germany. Experiments conducted in vitro were performed at McMaster University. The most relevant results of this thesis revealed that the low-dose hyper-radiosensitivity phenomenon can coexist with radiation-induced bystander effects and evidence points towards bystander signalling mechanisms as the primary cause of cell killing during hyper-radiosensitivity. Bystander and abscopal effects can occur in rats and even in immune-compromised nude mice after exposure to Synchrotron Microbeam Radiation and Pencilbeam. Bystander effects can be communicated from irradiated rats to healthy unirradiated cage mate rats and the presence of a tumour modulates both the bystander and abscopal responses. The γ-H2AX biomarker can successfully be used for the detection of DNA damage in the brain of rodents after Synchrotron Radiation. In conclusion, this thesis considerably expands the understanding of the role of bystander effects in cells lines, tissues, and animals exposed to Synchrotron radiation. It is suggested that further exploration of the role of bystander effects and hyper-radiosensitivity during Synchrotron treatments could identify new targets leading to better tumour control.
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Tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine produced in the skin in response to ultraviolet B radiation (UVB). TNF-α facilitates UVB-induced apoptosis and probably contributes to removal of damaged cells. Surprisingly, murine TNF-α-knockout models have demonstrated that TNF-α is necessary for the early stages of skin carcinogenesis and development of squamous cell carcinoma. In the present PhD thesis, we examined the effects of TNF-α on DNA repair and cell cycle regulation in UVB-irradiated keratinocytes. In the model of premalignant keratinocytes (HaCaT), TNF-α abolished the UVB-induced G2/M checkpoint and diminished the DNA repair despite induction of apoptosis. TNF-α activated the protein kinase B/Akt and regulation of its downstream targets, mTOR, Bad and FoxO3a. This effect was dependent on atypical protein kinase C species (aPKC) since a specific peptide blocking the activity of the PKCξ and ι/λ abrogated the activation of Akt by TNF-α. The aPKC-Akt axis was likely to be responsible for the TNF-α-induced decrease in DNA repair since blocking of Akt activity restored DNA repair. Since anti-TNF-α approaches are increasingly used in the therapy of autoimmune diseases and one of the safety concerns is the potential enhancement of skin carcinogenesis, we investigated the effect of the chimeric monoclonal anti-TNF-α antibody infliximab on UVB-irradiated HaCaT cells. Cells treated with infliximab had significantly increased levels of DNA damage despite enhanced G2/M checkpoint arrest, increased apoptosis and inhibition of Akt. In conclusion, we identified a possible novel mechanism by which TNF-α promotes UVB-induced skin carcinogenesis. This depends on aPKC-Akt activation and inhibition of DNA repair. TNF-α-treated cells are prone to escape checkpoint control and are possibly more likely to accumulate mutations, which may constitute a relevant mechanism enhancing tumor development. The effect of anti-TNF-α therapy on skin carcinogenesis warrants further investigation as our study indicates that, in contrast to what had been expected, infliximab may impair DNA repair.
Article
Camptothecin (CPT), a DNA topoisomerase I inhibitor, was originally isolated from the fruits of the Chinese Camptotheca acuminata tree. CPT and its derivatives have been used in the treatment of psoriasis and cancer in China for decades. It is well known that tumor necrosis factor-α (TNF-α) is a key proinflammatory cytokine in the pathogenesis of psoriasis. In this study, we investigated the effect of CPT on TNF-α-treated HaCaT cells. The results indicated that CPT in the concentration range of 0.5-2.0 μg·ml(-1) failed to show any proapoptotic effect in HaCaT cells. It was found that both CPT and TNF-α up-regulated the expression of TRAIL receptor 1/2 but not TRAIL in HaCaT cells. Furthermore, the expression of antiapoptotic proteins (IAP1, IAP2, and Bcl-X(L)) was up-regulated by TNF-α and suppressed by CPT in HaCaT cells. Because these gene products are known to be regulated by nuclear factor-kappa B (NF-κB), we examined the role of CPT on NF-κB activation. It was found that CPT not only failed to inhibit TNF-α-induced NF-κB activation but also contributed to NF-κB activation. In addition to these effects, CPT also promoted the production of interleukin-6, similar to TNF-α, in HaCaT cells. In conclusion, despite ample evidence supporting CPT-induced carcinoma cell apoptosis, our study clearly shows that CPT fails to show any proapoptotic effects in HaCaT cells, even though it enhanced TRAIL receptor 1/2 expression and inhibited the expression of TNF-α-induced antiapoptotic proteins. Taken together, this study demonstrates that CPT fails to block the activity of TNF-α. With respect to the NF-κB-activating role of CPT, we suggest that the benefit of CPT in the treatment of psoriasis should be reevaluated.
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The wild type p53 inducible phosphatase (Wip1) plays an important role in modulating not only stress responses by various environmental stresses, but when overexpressed it also impairs the intrinsic tumor surveillance networks that are frequently found in a number of cancers including skin cancers. As a result, using a pharmacological inhibitor of Wip1 has been suggested to be a novel chemotherapeutic approach to recover the innate tumor surveillance in a variety of cancers. We studied the effect of a pharmacological inhibitor of Wip1 in skin keratinocytes, under a ultra-violet (UV) stress condition. A human keratinocyte cell line or human epidermal keratinocytes were exposed to UV, with or without the sole commercially available chemical inhibitor of Wip1, CCT007093; subsequently, we determined the diverse stress responses, including apoptosis and the activation of stress signaling. We demonstrate that the Wip1 inhibitor unexpectedly attenuated the UV-mediated apoptotic response in skin keratinocytes, as a consequence of attenuated JNK activation and reduced H2AX phosphorylation in both, skin keratinocytes and a Wip1-null cell model. On the other hand, the loss of Wip1 expression, either by knockout or knockdown in mice or human keratinocytes respectively, promoted apoptosis and potentiated H2AX phosphorylation following UV treatment. Of note, CCT007093 treatment appeared to promote apoptosis in breast cancer cells and skin transformed keratinocytes that ectopically expressed Wip1, demonstrating that the effect of CCT007093 differs based on the level of Wip1 expression. Thus, our studies suggest that the development of a more potent and specific Wip1 inhibitor is necessary to achieve the desired chemotherapeutic potential and to avoid off-target effects.
Article
Dihydroxyacetone (DHA), the active substance in sunless tanning lotions reacts with the amino groups of proteins to form a brown-colored complex. This non-enzymatic glycation, known as the Maillard reaction, can also occur with free amino groups in DNA, raising the possibility that DHA may be genotoxic. To address this issue we investigated the effects of DHA on cell survival and proliferation of a human keratinocyte cell line, HaCaT. Dose- and time-dependent morphological changes, chromatin condensation, cytoplasmic budding and cell detachment were seen in cells treated with DHA. Several dead cells were observed after long-time (24 h) incubation with 25 mM DHA or more. Furthermore, an extensive decline in proliferation was observed 1 day after DHA exposure for 24 h. When applied in different concentrations (5–50 mM) and for different time periods (1, 3 or 24 h) DHA caused a G2/M block after the cyclin B1 restriction point. Exit from this cell-cycle block was associated with massive apoptosis, as revealed by a clonogenic assay, TUNEL staining and electron microscopy. Furthermore, DHA caused DNA damage as revealed by the alkaline comet assay. Preincubation with antioxidants prevented the formation of DNA strand breaks. The DHA toxicity may be caused by direct redox reactions, with formation of ROS as the crucial intermediates. The genotoxic capacity of DHA raises a question about the long-term clinical consequences of treatment of the skin with this commonly used compound.
Article
Ultraviolet B (UVB) irradiation is known to induce activation of cellular stress response pathways in cultured cells or intact human skin, as demonstrated by phosphorylation of MAP kinase family members and up- or down-stream targets, using biochemical assays. This study demonstrates by immunohistochemistry that low-dose UVB irradiation of normal human skin induces rapid and reversible phosphorylation of c-jun (a target of c-jun N-terminal kinase) and p38 mitogen activated protein kinase (p38 MAP kinase). Phosphorylation was maximal at 4–8 h and returned to normal levels at 48 h after irradiation. Nuclear localization of these phosphorylated substrates was found using antisera against the epitope containing the phosphorylated serine-73 of c-jun, and the dually phosphorylated epitope (threonine-180 and tyrosine-182) of p38 MAP kinase. Nearly all epidermal cells were positive for c-jun phosphorylation, whereas p38 phosphorylation was seen predominantly in the differentiated layers. In contrast to the massive activation of c-jun and p38, only a small population of the suprabasal cells showed nuclear translocation of nuclear factor kappa B (NFκB), and a few scattered cells became apoptotic, as determined by TUNEL (TdT mediated dUTP nick end labelling) staining. The expression of involucrin and skin-derived anti-leukoproteinase (SKALP)/elafin, two genes putatively under control of the c-jun and p38 pathways, was found to be increased. These findings establish the first cellular localization of UVB-induced protein phosphorylation of stress response proteins in human epidermis, thereby providing a link between cellular activation and gene expression in defined cell populations. Copyright
Chapter
Basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) are the most frequent tumors in the Caucasian population. The formation of these tumors is a consequence of long term UV-exposure of the skin. UV-light induces DNA damage in cells. If the damaged DNA cannot be repaired or the DNA damaged cell is not eliminated by apoptosis (so-called sunburn cells), cell transformation and tumor development can be the outcome. Fas-ligand (FasL), a member of the tumor necrosis superfamily, is a key molecule involved in the elimination of sunburn cells. FasL is expressed in normal skin epidermis, preferentially in the basal layer. Regulation of FasL expression has a dual effect on cancerogenesis. On the one hand, FasL expression is down regulated in skin epidermis by UV irradiation leading to the loss of its sensor function and thereby increasing the risk of cell transformation and skin tumor development. On the other hand, once BCC or SCC have developed, FasL is strongly up-regulated. High expression of FasL may now serve to protect the tumor from the attack of immune effector cells. To prove the immune escape hypothesis in vivo, the prevention or downregulation of FasL expression in tumor tissue is required. Two approaches were successfully applied to silence the FasL gene in BCC tissues ex vivo, the antisense technology and RNA interference with small interfering RNA duplexes. With both techniques FasL expression can be efficiently downregulated in BCC tissues pathing the way to test the immune escape hypothesis in vivo.
Article
Background Vitiligo is a common disease of unknown cause that produces disfiguring white patches of depigmentation. Previous studies have suggested the effectiveness of UV-B radiation in generalized vitiligo (GV) therapy, but there was no evidence to support the same role for segmental vitiligo (SV).Objective The purpose of this study was to use UV-B radiation exclusively on vitiligo patches of individuals affected by S V to evaluate the effectiveness of this therapy.Subjects and methodsEight individuals with SV were treated for 6 months with a new device called BIOSKIN® that can produce a focused beam of UV-B (microphoto-therapy) on vitiligo patches only. Photographs of the subjects were taken at the beginning of the therapy and once a month thereafter for 6 months. The response to treatment was estimated in two comparable photographs using planimetry. A control group of eight individuals matched for sex and age was treated with placebo, using the same device but not releasing any kind of detectable light.ResultsAfter 6 months of microphototherapy five subjects of the eight studied achieved normal pigmentation on more than 75% of the treated areas. In particular, three of these were totally repigmented. Two individuals achieved 50–75% pigmentation of the treated areas, and only one showed less than 50% repigmentation (Table 3). In the control group only one patient showed moderate repigmentation (less than 50%) (Table 3) (Fig. 3).ConclusionUV-B microphototherapy seems highly effective in restoring pigmentation in patients affected by vitiligo. As no side-effects have been observed, this could represent the treatment of choice in the limited (segmental) forms of vitiligo.
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Lasers generating predominantly thermal energy are used in medicine and research for a variety of purposes including surgical excision, pan retinal photocoagulation for treating diabetic retinopathy, cornea shape remodeling, treatment of photoaged skin, and hair removal. Not surprisingly, there has been an increase in the number of laser injuries, especially eye injuries, due to laser misuse or accidents over the last four decades. When sufficient energy is provided, most visible and near infrared wavelength laser systems will damage the retinal pigment epithelium (RPE). This damage is generally due to thermal injury. Of particular concern is thermal laser injury to the macular region of the retina, which may result in a blinding trauma that produces an immediate psychological and physical debilitation. To provide rational treatments for laser-induced injury, a better understanding of the nature of this injury is required. To this end, we established methods for studying laser-induced injury with in vitro models utilizing cultured human cells.
Article
PURPOSE: To determine and compare the effects of pre-conditioning and post-conditioning towards gamma radiation responses in human cancer cells and keratinocytes MATERIALS AND METHODS: The clonogenic survival of glioblastoma cells (T98G), keratinocytes (HaCaT), and colorectal carcinoma cells (HCT116 p53+/+ and p53-/-) was assessed following gamma ray exposure from a Cs-137 source. The priming dose preceded the challenge dose in pre-conditioning whereas the priming dose followed the challenge dose in post-conditioning. The priming dose was either 5 mGy or 0.1 Gy. The challenge dose was 0.5 – 5 Gy. RESULTS: In both pre- and post-conditioning where the priming dose was 0.1 Gy and the challenge dose was 4 Gy, RAR developed in T98G but not in HaCaT cells. In HCT116 p53+/+, pre-conditioning had either no effect or a radiosensitizing effect and whereas post-conditioning induced either radiosensitizing or radioadaptive effect. The different observed outcomes were dependent on dose, the time interval between the priming and challenge dose, and the time before the first irradiation. Post-conditioning effects could occur with a priming dose as low as 5 mGy in HCT116 p53+/+ cells. When HCT116 cells had no p53 protein expression, the radiosensitizing or radioadaptive response by the conditioning effect was abolished. CONCLUSIONS: The results suggest that radiation conditioning responses are complex and depend on at least the following factors: the magnitude of priming/challenge dose, the time interval between priming and challenge dose, p53 status, cell seeding time prior to the first radiation treatment. This work is the first parallel comparison demonstrating the potential outcomes of pre- and post-conditioning in different human cell types using environmentally and medically relevant radiation doses.
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The implication of radiation-induced bystander effect (RIBE) for both radiation protection and radiotherapy has attracted significant attention, but a key question is how to modulate the RIBE. The present study found that, when a fraction of glioblastoma cells in T98G population were individually targeted with precise helium particles through their nucleus, micronucleus (MN) were induced and its yield increased non-linearly with radiation dose. After co-culturing with irradiated cells, additional MN could be induced in the non-irradiated bystander cells and its yield was independent of irradiation dose, giving direct evidence of a RIBE. Further results showed that the RIBE could be eliminated by pifithrin-α (p53 inhibitor) but enhanced by wortmannin (PI3K inhibitor). Moreover, it was found that nitric oxide (NO) contributed to this RIBE, and the levels of NO of both irradiated cells and bystander cells could be extensively diminished by pifithrin-α but insignificantly reduced by wortmannin. Our results indicate that RIBE can be modulated by p53 and PI3K through a NO-dependent and NO-independent pathway, respectively.
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Wild-type p53 protein has many properties consistent with its being the product of a tumour suppressor gene. Although the normal roles of tumour suppressor genes are still largely unknown, it seems that they could be involved in promoting cell differentiation as well as in mediating growth arrest by growth-inhibitory cytokines. Hence, the abrogation of wild-type p53 expression, which is a common feature of many tumours, could eliminate these activities. We have now tested this notion by restoring the expression of p53 in a murine myeloid leukaemic cell line that normally lacks p53. The use of a temperature-sensitive p53 mutant allowed us to analyse cells in which the introduced p53 had either wild-type or mutant properties. Although there seemed to be no effect on differentiation, the introduction of wild-type p53 resulted in rapid loss of cell viability in a way characteristic of apoptosis (programmed cell death). The effect of wild-type p53 was counteracted by interleukin-6. Thus products of tumour suppressor genes could be involved in restricting precursor cell populations by mediating apoptosis.
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In contrast to mouse epidermal cells, human skin keratinocytes are rather resistant to transformation in vitro. Immortalization has been achieved by SV40 but has resulted in cell lines with altered differentiation. We have established a spontaneously transformed human epithelial cell line from adult skin, which maintains full epidermal differentiation capacity. This HaCaT cell line is obviously immortal (greater than 140 passages), has a transformed phenotype in vitro (clonogenic on plastic and in agar) but remains nontumorigenic. Despite the altered and unlimited growth potential, HaCaT cells, similar to normal keratinocytes, reform an orderly structured and differentiated epidermal tissue when transplanted onto nude mice. Differentiation-specific keratins (Nos. 1 and 10) and other markers (involucrin and filaggrin) are expressed and regularly located. Thus, HaCaT is the first permanent epithelial cell line from adult human skin that exhibits normal differentiation and provides a promising tool for studying regulation of keratinization in human cells. On karyotyping this line is aneuploid (initially hypodiploid) with unique stable marker chromosomes indicating monoclonal origin. The identity of the HaCaT line with the tissue of origin was proven by DNA fingerprinting using hypervariable minisatellite probes. This is the first demonstration that the DNA fingerprint pattern is unaffected by long-term cultivation, transformation, and multiple chromosomal alterations, thereby offering a unique possibility for unequivocal identification of human cell lines. The characteristics of the HaCaT cell line clearly document that spontaneous transformation of human adult keratinocytes can occur in vitro and is associated with sequential chromosomal alterations, though not obligatorily linked to major defects in differentiation.
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The p53 tumor suppressor protein is a transcriptional activator, which can mediate apoptotic cell death in a variety of cell types. To determine whether sequence-specific trans-activation is a prerequisite for the induction of apoptosis by p53, the apoptotic effects of various p53 deletion mutants were monitored in an assay based on the transient transfection of HeLa cells. A truncated protein (p53dl214), containing only the first 214 amino-terminal residues of murine p53, induced extensive apoptosis, albeit at a slower rate than trans-activation-competent wild-type p53. p53dl214 also suppressed the transformation of rat fibroblasts by several oncogene combinations and particularly by myc plus ras and HPV E7 plus ras. p53dl214 lacks a major portion of the DNA-binding domain and cannot activate p53-responsive promoters. Moreover, a human p53 protein carrying mutations in residues 22 and 23 also triggered HeLa cell apoptosis, despite failing to induce significant activation of relevant p53 target promoters. These data suggest the existence of two p53-dependent apoptotic pathways--one requiring activation of specific target genes, and the other independent of sequence-specific trans-activation. The latter pathway may actually be totally uncoupled from the binding of p53 to its consensus DNA sites. The relative contribution of trans-activation-independent apoptosis to tumor suppression by p53 may be dictated by the specific genetic lesions present in the particular tumor.
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The DNA from a wide variety of human tumors has sustained mutations within the conserved p53 coding regions. We have purified wild-type and tumor-derived mutant p53 proteins expressed from baculovirus vectors and examined their interactions with SV40 DNA. Using DNAase I footprinting assays, we observed that both human and murine wild-type p53 proteins bind specifically to sequences adjacent to the late border of the viral replication origin. By contrast, mutant p53 proteins failed to bind specifically to these sequences. SV40 T antigen prevented wild-type p53 from interacting with this region. These data show that normal but not oncogenic forms of p53 are capable of sequence-specific interactions with viral DNA. Furthermore, they provide insights into the mechanisms by which viral proteins might regulate the control of viral growth and cell division.
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Mammalian epithelium is a tissue with a very high turnover rate. It consists of a rapidly proliferating compartment comprising basal and suprabasal keratinocytes, from which cells move upwards while differentiating into granular keratinocytes. The end product is shed as an enucleate corneocyte, which has a mechanically rigid, chemically resistant cross-linked keratinous envelope. The loss of the nucleus occurs specifically in the granular keratinocyte layer; here, cells with the classical apoptotic morphology of clumped and marginated condensed chromatin may be observed. This morphology is characteristic of "programmed" cell death in other systems, of which the lymphocyte has been most extensively studied, and is associated with the cleavage of nuclear DNA into nucleosome-sized fragments. In the present investigation we separated newborn mouse skin into basal and granular keratinocyte fractions and examined the state of the DNA in each fraction. Our results indicate that cells in the basal layer, while their DNA is perfectly intact, are preparing to die. DNA fragmentation is initiated in the granular keratinocyte layer and is identical in pattern to that seen in other examples of programmed cell death.
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Mutations in the evolutionarily conserved codons of the p53 tumor suppressor gene are common in diverse types of human cancer. The p53 mutational spectrum differs among cancers of the colon, lung, esophagus, breast, liver, brain, reticuloendothelial tissues, and hemopoietic tissues. Analysis of these mutations can provide clues to the etiology of these diverse tumors and to the function of specific regions of p53. Transitions predominate in colon, brain, and lymphoid malignancies, whereas G:C to T:A transversions are the most frequent substitutions observed in cancers of the lung and liver. Mutations at A:T base pairs are seen more frequently in esophageal carcinomas than in other solid tumors. Most transitions in colorectal carcinomas, brain tumors, leukemias, and lymphomas are at CpG dinucleotide mutational hot spots. G to T transversions in lung, breast, and esophageal carcinomas are dispersed among numerous codons. In liver tumors in persons from geographic areas in which both aflatoxin B1 and hepatitis B virus are cancer risk factors, most mutations are at one nucleotide pair of codon 249. These differences may reflect the etiological contributions of both exogenous and endogenous factors to human carcinogenesis.
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The cell cycle is composed of a series of steps which can be negatively or positively regulated by various factors. Chief among the negative regulators is the p53 protein. Alteration or inactivation of p53 by mutation, or by its interactions with oncogene products of DNA tumour viruses, can lead to cancer. These mutations seem to be the most common genetic change in human cancers.
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The tumor-suppressor gene p53 is altered by missense mutation in numerous human malignancies. However, the biochemical properties of p53 and the effect of mutation on these properties are unclear. A human DNA sequence was identified that binds specifically to wild-type human p53 protein in vitro. As few as 33 base pairs were sufficient to confer specific binding. Certain guanines within this 33-base pair region were critical, as methylation of these guanines or their substitution with thymine-abrogated binding. Human p53 proteins containing either of two missense mutations commonly found in human tumors were unable to bind significantly to this sequence. These data suggest that a function of p53 may be mediated by its ability to bind to specific DNA sequences in the human genome, and that this activity is altered by mutations that occur in human tumors.
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Mutations of the p53 gene occur commonly in colorectal carcinomas and the wild-type p53 allele is often concomitantly deleted. These findings suggest that the wild-type gene may act as a suppressor of colorectal carcinoma cell growth. To test this hypothesis, wild-type or mutant human p53 genes were transfected into human colorectal carcinoma cell lines. Cells transfected with the wild-type gene formed colonies five- to tenfold less efficiently than those transfected with a mutant p53 gene. In those colonies that did form after wild-type gene transfection, the p53 sequences were found to be deleted or rearranged, or both, and no exogenous p53 messenger RNA expression was observed. In contrast, transfection with the wild-type gene had no apparent effect on the growth of epithelial cells derived from a benign colorectal tumor that had only wild-type p53 alleles. Immunocytochemical techniques demonstrated that carcinoma cells expressing the wild-type gene did not progress through the cell cycle, as evidenced by their failure to incorporate thymidine into DNA. These studies show that the wild-type gene can specifically suppress the growth of human colorectal carcinoma cells in vitro and that an in vivo-derived mutation resulting in a single conservative amino acid substitution in the p53 gene product abrogates this suppressive ability.
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Apoptosis is regarded as a suicidal cell response since the dying cell appears to be an active participant. Previous studies have shown that apoptosis of various murine cell types, induced by a variety of stimuli, required RNA and/or protein synthesis. However, when human promyelocytic leukemia HL-60 cells were induced to undergo apoptosis by treatment with the calcium ionophore A23187 or microtubule-disrupting agents, in the presence of inhibitors of macromolecular synthesis, apoptosis of these cells was neither abrogated nor delayed. Furthermore, the presence of either cycloheximide, an inhibitor of protein synthesis, or actinomycin D, an RNA synthesis inhibitor, alone was found to induce large scale apoptosis of these cells. Apoptosis in these cells was characterized by cell and chromatin condensation followed by nuclear and DNA fragmentation. In common with many other studies, this DNA fragmentation was found to have an approximately 200-bp multiple pattern, which is consistent with the activation of an endogenous endonuclease which cleaves at internucleosomal sites. Calcium-dependent endonuclease activity of this type was also detected in the isolated nuclei of untreated HL-60 cells. The morphologic and biochemical changes characteristic of apoptosis were found to precede cell death, as measured by trypan blue uptake and were completely distinct from death caused by toxic stimuli such as azide, ethanol, or heat treatment. Similar experiments with six other human cell lines confirmed that this phenomenon was not peculiar to the HL-60 cell line. These results suggest that certain dividing cell populations do not require RNA or protein synthesis to undergo apoptosis and further, that continuous transcription and translation of some regulatory protein(s) may be required to maintain control over the apoptotic "machinery" of such cells.
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The E6 protein encoded by the oncogenic human papillomavirus types 16 and 18 is one of two viral products expressed in HPV-associated cancers. E6 is an oncoprotein which cooperates with E7 to immortalize primary human keratinocytes. Insight into the mechanism by which E6 functions in oncogenesis is provided by the observation that the E6 protein encoded by HPV-16 and HPV-18 can complex the wild-type p53 protein in vitro. Wild-type p53 gene has tumor suppressor properties, and is a target for several of the oncoproteins encoded by DNA tumor viruses. In this study we demonstrate that the E6 proteins of the oncogenic HPVs that bind p53 stimulate the degradation of p53. The E6-promoted degradation of p53 is ATP dependent and involves the ubiquitin-dependent protease system. Selective degradation of cellular proteins such as p53 with negative regulatory functions provides a novel mechanism of action for dominant-acting oncoproteins.
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We introduced a mouse IL-2 cDNA expression vector into an IL-2-dependent mouse helper T cell line HT-2. Transfected cells secreted substantial amounts of IL-2, to which they themselves responded by proliferating without further requirement for exogenous IL-2. The proliferation was a direct function of the cell density and was inhibitable by antibodies against IL-2 or IL-2-R, indicating the autocrine nature of the proliferation. Those producing higher amounts of IL-2 were found to be tumorigenic when inoculated into nude mice. The latency period of tumor development correlated inversely with the level of IL-2 secreted. Tumor cells proliferated in vitro in an IL-2 autocrine fashion indistinguishable from that of the inoculated cells. We thus provide evidence that the aberrant activation of the IL-2 autocrine circuit can lead T cells to malignant transformation.
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Cosmid and lambda clones containing the human p53 gene were isolated and characterized in detail. The gene is 20 kilobases (kb) long and has 11 exons, the first and second exons being separated by an intron of 10 kb. Restriction fragments upstream of sequences known to be within the first identified exon were tested for promoter activity by cloning them in front of the chloramphenicol acetyltransferase gene and transfecting the resulting constructs into HeLa cells. A 0.35-kb DNA fragment was identified that had promoter activity. Results of primer extension experiments indicated that the mRNA cap site falls within this fragment, as expected. Analysis of the sequence upstream of the presumptive cap site indicated that the human p53 promoter may be of an unusual type.
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Two new methods were used to establish a rapid and highly sensitive prenatal diagnostic test for sickle cell anemia. The first involves the primer-mediated enzymatic amplification of specific beta-globin target sequences in genomic DNA, resulting in the exponential increase (220,000 times) of target DNA copies. In the second technique, the presence of the beta A and beta S alleles is determined by restriction endonuclease digestion of an end-labeled oligonucleotide probe hybridized in solution to the amplified beta-globin sequences. The beta-globin genotype can be determined in less than 1 day on samples containing significantly less than 1 microgram of genomic DNA.
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An adult patient with multiple unusual histiocytic tumors of the skin is described. As shown by immunohistologic study, electron microscopy, and immunoelectron microscopy, the tumors represent circumscribed proliferations of the Langerhans cell-related indeterminate dendritic cells of the skin. This distinct cutaneous histiocytosis may represent a paraneoplastic syndrome.