P16INK4A is implicated in both the immediate and adaptative response of human keratinocytes to UVB irradiation (original) (raw)
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Journal of Investigative Dermatology, 2009
While the precise mechanisms of melanoma development are unknown, recent in vivo studies have revealed that the p16 Ink4a /Rb pathway is disrupted in melanomagenesis. Here, we characterize the role of p16/Rb in coordinating the early events in UVB-irradiated skin. Foreskins and melanoma cell cultures were irradiated with low and high acute UVB doses and examined for cell-cycle-and apoptosis-associated genes. In melanoma cells, low UVB dose upregulated p16, p53, and p21 expression levels in Malme-3M, and high UVB dose accentuated the expression of p53 and p21 Cip1/Waf1 , in particular; however, in SkMel-28 cells only p16 expression was upregulated in response to UV irradiation. In HaCaT cells, high UVB dose caused dramatic increase in p53 expression followed by upregulation of p21 Cip1/Waf1 and Bax, and downregulation of Bcl-2 leading to apoptosis. In HaCaT cells, reinstatement of p16 pathway restored cell-cycle arrest in response to low dose. Foreskin organ culture experiments confirmed our in vitro cell results. These data indicate that the p53 and p16 pathways respond independently to UVB insult. The p16 pathway is favored at low doses and results in cell-cycle arrest; the p53 pathway is more responsive to higher doses and induces apoptosis depending on p53 mutation status.
UVB-induced Decrease of p16/CDKN2A Expression in Skin Cancer Patients
Pigment Cell Research, 2001
The UVB-induced decline of p16/ The lack of p16 expression has been shown in cultured melanoma cells, however contradictory evidence for p16 ex-CDKN2A in skin cancer patients might offer new insights into photocarcinogenesis. The putative sequence of events could pression in melanoma tissues exist. Ultraviolet (UV) C and UVB have been shown to affect p16 expression, which impairs start with a down-regulation of p16/CDKN2A expression, which would lead to impaired cell cycle regulation. Altered cell cycle regulation in vitro and in vivo. In this study, expression patterns of p16/CDKN2A following UVB expo-p16/CDKN2A gene expression was determined by reverse transcription polymerase chain reaction in seven skin cancer sure could be of value for identifying people with an increased risk of UV-induced skin cancer. patients, in one dysplastic nevus patient and in seven healthy individuals, prior to UVB exposure and at various times after application of one minimal erythema dose (MED). Five of the Key words: p16, Melanoma, Squamous cell carcinoma, Human skin, RT-PCR, UVB, MED seven skin cancer patients showed a down-regulation of p16/ CDKN2A expression after UVB exposure, while controls other studies have shown that p16 inactivation is not frequent in primary melanoma tumors (7). Loss of heterozygosity of 9p21 markers are found in some metastatic melanomas, which suggests that p16 may be involved in the progression rather than in the initiation of melanomas (8). Studies of nevus and melanoma tissues demonstrated a decline or loss of p16 expression following the status of dedifferentiation from nevus via primary melanoma to melanoma metastases (9-12). Those studies demonstrate that the loss of p16/CDKN2A gene expression is a frequent event in sporadic melanoma tumorigenesis. Additionally, the p16/CDKN2A gene is likely to be an important genetic contributor to melanoma susceptibility. Moreover, UV radiation (UVR) has often been discussed as an exogenous
P16 UV mutations in human skin epithelial tumors
Oncogene, 1999
The p16 gene expresses two alternative transcripts (p16a and p16b) involved in tumor suppression via the retinoblastoma (Rb) or p53 pathways. Disruption of these pathways can occur through inactivation of p16 or p53, or activating mutations of cyclin dependant kinase 4 gene (Cdk4). We searched for p16, Cdk4 and p53 gene mutations in 20 squamous cell carcinomas (SSCs), 1 actinic keratosis (AK), and 28 basal cell carcinomas (BCCs), using PCR-SSCP. A deletion and methylation analysis of p16 was also performed. Six dierent mutations (12%) were detected in exon 2 of p16 (common to p16a and p16b), in ®ve out of 21 squamous lesions (24%) (one AK and four SCCs) and one out of 28 BCCs (3.5%). These included four (66%) ultraviolet (UV)-type mutations (two tandems CC : GG to TT : AA transitions and two C : G to T : A transitions at dipyrimidic site) and two transversions. P53 mutations were present in 18 samples (37%), mostly of UV type. Of these, only two (one BCC and one AK) harboured simultaneously mutations of p16, but with no consequence on p16b transcript. Our data demonstrate for the ®rst time the presence of p16 UV induced mutations in non melanoma skin cancer, particularly in the most aggressive SCC type, and support that p16 and p53 are involved in two independent pathways in skin carcinogenesis.
Oncogene, 2003
To gain insight into the transformation of epidermal cells into squamous carcinoma cells (SCC), we compared the response to ultraviolet B radiation (UVB) of normal human epidermal keratinocytes (NHEK) versus their transformed counterpart, SCC, using biological and molecular profiling. DNA microarray analyses (Affymetrix s , B12 000 genes) indicated that the major group of upregulated genes in keratinocytes fall into three categories: (i) antiapoptotic and cell survival factors, including chemokines of the CXC/CC subfamilies (e.g. IL-8, GRO-1,-2,-3, SCYA20), growth factors (e.g. HB-EGF, CTGF, INSL-4), and proinflammatory mediators (e.g. COX-2, S100A9), (ii) DNA repair-related genes (e.g. GADD45, ERCC, BTG-1, Histones), and (iii) ECM proteases (MMP-1,-10). The major downregulated genes are DNp63 and PUMILIO, two potential markers for the maintenance of keratinocyte stem cells. NHEK were found to be more resistant than SCC to UVB-induced apoptosis and this resistance was mainly because of the protection from cell death by secreted survival factors, since it can be transferred from NHEK to SCC cultures by the conditioned medium. Whereas the response of keratinocytes to UVB involved regulation of key checkpoint genes (p53, MDM2, p21 Cip1 , DNp63), as well as antiapoptotic and DNA repair-related genes-no or little regulation of these genes was observed in SCC. The effect of UVB on NHEK and SCC resulted in upregulation of 251 and 127 genes, respectively, and downregulation of 322 genes in NHEK and 117 genes in SCC. To further analyse these changes, we used a novel unsupervised coupled two-way clustering method that allowed the identification of groups of genes that clearly partitioned keratinocytes from SCC, including a group of genes whose constitutive expression levels were similar before UVB. This allowed the identification of discriminating genes not otherwise revealed by simple static comparison in the absence of UVB irradiation. The implication of the changes in gene profile in keratinocytes for epithelial cancer is discussed.
Photochemical & Photobiological Sciences, 2012
UVA-radiation (315-400 nm) has been demonstrated to be capable of inducing DNA damage and is regarded as a carcinogen. While chromosomal aberrations found in UVA-irradiated cells and skin tumors provided evidence of the genetic involvement in UVA-carcinogenesis, its epigenetic participation is still illusive. We thus analysed the epigenetic patterns of 5 specific genes that are involved in stem cell fate (KLF4, NANOG), telomere maintenance (hTERT) and tumor suppression in cell cycle control (P16 INK4a , P21 WAFI/CIPI) in chronically UVA-irradiated HaCaT human keratinocytes. A striking reduction of the permissive histone mark H3K4me3 has been detected in the promoter of P16 INK4a (4-fold and 9-fold reduction for 10 and 15 weeks UVA-irradiated cells, respectively), which has often been found deregulated in skin cancers. This alteration in histone modification together with a severe promoter hypermethylation strongly impaired the transcription of P16 INK4a (20-fold and 40-fold for 10 weeks and 15 weeks UVA-irradiation, respectively). Analysis of the skin tumor-derived cells revealed the same severe impairment of the P16 INK4a transcription attributed to promoter hypermethylation and enrichment of the heterochromatin histone mark H3K9me3 and the repressive mark H3K27me3. Less pronounced UVA-induced epigenetic alterations were also detected for the other genes, demonstrating for the first time that UVA is able to modify transcription of skin cancer associated genes by means of epigenetic DNA and histone alterations.
Photochemistry and Photobiology, 2007
In cultured human keratinocytes, the tumor suppressor p53 acts as a control element in the protective response to UVB radiation and is affected by a variety of factors linked to cellular adhesion and differentiation. Because keratinocytes within the epidermis are not a homogeneous population but differ in their proliferative capacity and differentiation status, we compared the UVB responsiveness of primary keratinocyte populations isolated from various skin biopsies using p53 expression as a marker for their sensitivity to UVB. Besides keratinocytes exhibiting a UVB dose-and time-dependent upregulation of p53, keratinocyte populations were detected with high p53 expression levels even without irradiation. Such keratinocytes did not regulate p53 expression in response to UVB. Furthermore their p53-mediated UVB response was influenced by cocultivation with human dermal fibroblasts (HDF) but not with cell cycle-arrested human normal keratinocytes or HaCaT keratinocytes. When these cells were cultivated together with arrested HDF, they did not only reveal increased p53 expression levels after UVB treatment but also a more pronounced transcriptional activation of the p53 downstream target gene p21. These findings indicate that the UVB response of keratinocytes, specifically the activation of the tumor suppressor p53, is heterogeneous and can be affected by growth conditions.
Expression Profiling of UVB Response in Melanocytes Identifies a Set of p53-Target Genes
Journal of Investigative Dermatology, 2006
Epidermal melanocytes execute specific physiological programs in response to UV radiation (UVR) at the cutaneous interface. Many melanocytic responses, including increased dendrite formation, enhanced melanogenesis/melanization, and cell cycle arrest impact the ability of melanocytes to survive and to attenuate the UVR insult. Although some of the molecules that underlie these UVR programs are known, a coherent view of UVR-induced transcriptional changes is lacking. Using primary melanocyte cultures, we assessed for UVR-mediated alterations in over 47,000 transcripts using Affymetrix Human Genome U133 Plus 2.0 microarrays. From the 100 most statistically robust changes in transcript level, there were 84 genes that were suppressed 42.0-fold by UVR; among these transcripts, the identities of 48 of these genes were known. Similarly, there were 99 genes that were induced 42.0-fold by UVR; the identity of 57 of these genes were known. We then subjected these top 100 changes to the Ingenuity Pathway analysis program and identified a group of p53 targets including the cell cycle regulator CDKN1A (p21CIP), the WNT pathway regulator DKK1 (dickkopf homolog 1), the receptor tyrosine kinase EPHA2, growth factor GDF15, ferrodoxin reductase (FDXR), p53-inducible protein TP53I3, transcription factor ATF3, DNA repair enzyme DDB2, and the b-adrenergic receptor ADBR2. These genes were also found to be consistently elevated by UVR in six independent melanocyte lines, although there were interindividual variations in magnitude. WWOX, whose protein product interacts and regulates p53 and p73, was found to be consistently suppressed by UVR. There was also a subgroup of neurite/axonal developmental genes that were altered in response to UVR, suggesting that melanocytic and neuronal arborization may share similar mechanisms. When compared to melanomas, the basal levels of many of these p53-responsive genes were greatly dysregulated. Three genes -CDKN1A, DDB2 and ADRB2 -exhibited a trend towards loss of expression in melanomas thereby raising the possibility of a linked role in tumorigenesis. These expression data provide a global view of UVR-induced changes in melanocytes and, more importantly, generate novel hypotheses regarding melanocyte physiology.
p16 Gene Expression in Basal Cell Carcinoma
Archives of Medical Research, 2008
Basal cell carcinoma (BCC) develops predominantly in sun-exposed skin in fair-skinned individuals prone to sunburn. BCC typically occurs in adults. High exposure to ultraviolet (UV) radiation increases rate of developing BCC, a slowly growing tumor that occurs in hair-growing squamous epithelium and rarely metastasizes. In genetic studies, BCC patients have cell-cycle abnormalities of different parts of the signaling pathway. Retinoblastoma regulatory pathway is important in cell cycle arrest. In this pathway, p16INK4a, an inhibitor of Rb pathway, binds to CDK4 and CDK6 competitively with cyclin D1 to prevent phosphorylation of tumor suppressor pRB gene. Alteration of this pathway contributes to development of human cancers and also is effective in skin cancers. In this study, we analyzed mRNA expression using in situ RT-PCR and the role of immunohistochemical expression of p16INK4a in BCC.Expression of p16 in ten samples of Iranian paraffin-embedded skin BCC were studied using in situ RT-PCR and immunohistochemistry on p16INK4a gene.Nuclear and cytoplasmic staining intensity of samples within tumor cells and normal skin tissue illustrates different mRNA and protein expression of p16 gene. mRNA of p16 gene and the expressed protein induce cell cycle proliferation and involve both tumor tissue as well as normal skin tissue. However, in this study it was found that there is significant protein and mRNA expression in BCC cells when compared to normal skin tissue (p <0.05).p16 gene is involved in the pathogenesis of human skin BCC in view of increased p16 mRNA and expressed protein within tumor cells.
The p16INK4a tumor suppressor controls p21WAF1 induction in response to ultraviolet light
Nucleic Acids Research, 2006
p16 INK4a and p21 WAF1 , two major cyclin-dependent kinase inhibitors, are the products of two tumor suppressor genes that play important roles in various cellular metabolic pathways. p21 WAF1 is upregulated in response to different DNA damaging agents. While the activation of p21 WAF1 is p53dependent following grays , the effect of ultraviolet (UV) light on p21 WAF1 protein level is still unclear. In the present report, we show that the level of the p21 WAF1 protein augments in response to low UVC fluences in different mammalian cells. This upregulation is mediated through the stabilization of p21 WAF1 mRNA in a p16 INK4a-dependent manner in both human and mouse cells. Furthermore, using p16-siRNA treated human skin fibroblast; we have shown that p16 controls the UV-dependent cytoplasmic accumulation of the mRNA binding HuR protein. In addition, HuR immunoprecipitations showed that UV-dependent binding of HuR to p21 mRNA is p16-related. This suggests that p16 induces p21 by enabling the relocalization of HuR from the nucleus to the cytoplasm. Accordingly, we have also shown that p16 is necessary for efficient UV-dependent p53 up-regulation, which also requires HuR. These results indicate that, in addition to its role in cell proliferation, p16 INK4a is also an important regulator of the cellular response to UV damage.