Role of p21(Waf-1) in regulating the G1 and G2/M checkpoints in ultraviolet-irradiated keratinocytes

This study examines the role of p21(Waf-1) , a p53-dependent protein, in regulating mechanisms that protect keratinocytes against ultraviolet-B-induced cellular damage. Keratinocytes from p21(Waf-1) or p53-deficient mice were irradiated with ultraviolet B, and examined for DNA repair, cell cycle progression, and cell death. Both p21(Waf-1) -deficient and p53-deficient cells failed to maintain G2 arrest, and p21(Waf-1) -deficient cells, and to a lesser extent p53-deficient cells, also failed to undergo G1 arrest. After exposure to ultraviolet B, p53-deficient cells were more susceptible to cell death than wild-type cells. p21(Waf-1) -deficient cells did not undergo apoptotic cell death more often, however, but did have an increased frequency of nuclear abnormalities, suggesting mitotic catastrophe. TUNEL assay showed DNA fragmentation in the p53 +/+, p21(Waf-1) +/+, and p53 -/- cells, but not in p21(Waf-1) -/- cells. This result is consistent with the suggestion that p21(Waf-1) -deficient keratinocytes undergo mitotic cell death (catastrophe) after exposure to ultraviolet B irradiation in the system. Western analysis demonstrated that p21(Waf-1) expression was upregulated in p53-proficient and -deficient keratinocytes, supporting the notion that a p53-independent mechanism contributes to the response to ultraviolet B in keratinocytes. Finally, p21(Waf-1) -deficient cells had slightly less efficient nucleotide excision repair. In summary, this study suggests that p21(Waf-1) regulates the ultraviolet-B-induced G2/M checkpoint through p53, and the G1 checkpoint partially through p53. p21(Waf-1) does not significantly regulate DNA repair in ultraviolet-irradiated keratinocytes, however.     

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

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

DNA mismatch repair protein Msh6 is required for optimal levels of ultraviolet-B-induced apoptosis in primary mouse fibroblasts

Recent data support a role for DNA mismatch repair in the cellular response to some forms of exogenous DNA damage beyond that of DNA repair; cells with defective DNA mismatch repair have partial or complete failure to undergo apoptosis and/or G2M arrest following specific types of damage. We propose that the DNA mismatch repair Msh2/Msh6 heterodimer, responsible for the detection of DNA damage, promotes apoptosis in normal cells, thus protecting mammals from ultraviolet-induced malignant transformation. Using primary mouse embryonic fibroblasts derived from Msh6+/+ and Msh6-/- mice, we compare the response of DNA-mismatch repair-proficient and -deficient cells to ultraviolet B radiation. In the wild-type mouse embryonic fibroblasts, ultraviolet-B-induced increases in Msh6 protein levels were not dependent on p53. Msh6-/- mouse embryonic fibroblasts were significantly less sensitive to the cytotoxic effects of ultraviolet B radiation. Further comparison of the Msh6+/+ and Msh6-/- mouse embryonic fibroblasts revealed that Msh6-/- mouse embryonic fibroblasts undergo significantly less apoptosis following ultraviolet B irradiation, thus indicating that ultraviolet-B-induced apoptosis is partially Msh6 dependent. These data support a role for Msh6 in protective cellular responses of primary cells to ultraviolet-B-induced mutagenesis and, hence, the prevention of skin cancer.     

A low UVB dose, with the potential to trigger a protective p53-dependent gene program, increases the resilience of keratinocytes against future UVB insults

One protein central in the response of human keratinocytes to ultraviolet B damage is p53. By transactivating genes involved in either cell cycle arrest or DNA repair, p53 has a leading role in the recovery from this damage. Considering this role, we wished to investigate whether the triggering of a p53-dependent gene program by repetitive ultraviolet B (UVB) exposure can induce an adaptive response in human skin cells. In particular, we examined two p53-target genes, p21/WAF1 and p53R2, with a crucial role in p53-induced cell cycle arrest and p53-induced DNA repair respectively. Exposure to a mild UVB dose was able to induce an adaptive response in human keratinocytes, leading to increased survival of cells that maintain their capacity to repair DNA damage upon exposure to apoptotic doses of UVB. Our study indicates that this adaptation response is only achieved if the interval between subsequent UVB insults allows sufficient time for the p53-induced protective gene program to be induced. Our results also demonstrate that small but quickly recurring UVB exposures are as harmful as one intense, continual exposure to UVB irradiation. Future research will be oriented toward investigating alternative ways to induce an adaptive response without pre-exposing the cells to UV.     

中波紫外线辐射损伤角质形成细胞的p53信号传导通路研究

目的 探讨中波紫外线(UVB)辐射对角质形成细胞p53信号传导通路的影响.方法 以20、60和120mJ/cm²UVB辐射培养的取自健康儿童包皮的正常人表皮角质形成细胞,应用RT-PCR和免疫印迹方法,分别从mRNA水平和蛋白质水平,检测分析UVB辐射后2h、24h和48h,p53及其下游分子MDM2、p21、Bax和GADD45的表达.结果 不同剂量UVB辐射角质形成细胞后均可见p53表达水平持续性显著升高(P<0.05).低剂量UVB辐射角质形成细胞后,MDM2、p21、GADD45升高不明显(P>0.05),Bax不表达;UVB辐射剂量升至60mJ/cm²后,MDM2于辐射后2h短暂性显著升高,p21和Bax持续性显著升高.结论 UVB辐射激活了角质形成细胞的p53信号传导通路,p53下游分子的表达具有剂量依赖性和时相性.     

Effect of ultraviolet (UV) A, UVB or ionizing radiation on the cell cycle of human melanoma cells

BACKGROUND: One important component of the cellular response to irradiation is the activation of cell cycle checkpoints. It is known that both ultraviolet (UV) radiation and ionizing radiation (IR) can activate checkpoints at transitions from G(1) to S phase, from G(2) phase to mitosis and during DNA replication. OBJECTIVES: To evaluate the effects of irradiation with different wavelengths on cell cycle alterations. METHODS: p53-deficient IPC-298 melanoma cells were irradiated with 10 J cm(-2) UVA, 40 mJ cm(-2) UVB, or with 7.5 Gy IR. Cell cycle effects were then determined by DNA/5-bromodeoxyuridine dual-parameter flow cytometry. RESULTS: IPC-298 cells irradiated in G(1) with UVA were not arrested at the G(1)/S transition, but at the G(2)/M transition. Despite p53 deficiency, the cells showed a G(1) arrest after UVB exposure. Furthermore, IR did not affect G(1) or S phase, but induced G(2) phase arrest. Hence, the effects of UVA, but not of UVB, on the cell cycle in p53-deficient melanoma cells are comparable with those of IR. CONCLUSIONS: UVA and IR induce radical-mediated strand breaks and DNA lesions, and UVB essentially induces thymine dimers that lead to excision repair-related strand breaks. Different cell cycle effects may be a consequence of different types of DNA damage. The results showed that UVB-irradiated p53-deficient cells are arrested in G(1). Irradiation with the solar radiation component UVB can therefore result in a beneficial retardation of tumour promotion in human skin carrying p53-mutated cell clones.     

Enhanced epidermal ultraviolet responses in chronically sun-exposed skin are dependent on previous sun exposure

The p53 protein plays a key role in protecting cells from acquiring manifest mutations by inducing cell cycle arrest or apoptosis. The mechanisms for differences in epidermal responses to ultraviolet irradiation are unclear, although they have been shown to be related to both genetic events and environmental factors. In this study, we compared epidermal ultraviolet responses in chronically sun-exposed and non-sun-exposed skin using immunohistochemistry with antibodies recognizing thymine dimers and p53 protein. Six healthy volunteers were subjected to both artificial ultraviolet irradiation and natural sunlight, with and without photoprotection. A smaller number of thymine dimer-positive keratinocytes were detected 24 h after ultraviolet exposure in chronically sun-exposed skin compared to non-sun-exposed skin. Further, the p53 response was more variable in chronically sun-exposed skin. A significant correlation between total ultraviolet dose and number of p53-immunoreactive keratinocytes was found after natural sun exposure. Our findings suggest that repair of DNA damage is more efficient in chronically sun-exposed skin than in non-sun-exposed skin.     

Gadd45a activation protects melanoma cells from ultraviolet B-induced apoptosis

Epidemiological and biological studies indicate that solar UVB radiation is involved in cutaneous malignant melanoma etiology. Indeed, melanocytes are very frequently exposed to solar UV radiation, which induces cell damage and may promote cell transformation. We previously showed that melanocytes and melanoma cells exposed to UVB radiation activates a p53-independent pathway involving Gadd45a and, more recently, that Gadd45a plays a critical role in UVB-induced G2 cell cycle arrest of melanoma cells. In this study, we demonstrate that the inhibition of UV-induced Gadd45a overexpression by RNA interference results in a dramatic increase of cell death. We identify this cell death as apoptosis, with activation of Caspase-3 and a decrease in Bcl-x(L) expression. Furthermore, we show that inhibition of UV-induced Gadd45a overexpression also leads to increased sensitivity of melanoma cells to therapeutic agents such as DTIC and Cisplatin. We conclude that UVB-induced Gadd45a overexpression protects melanoma cells from apoptosis, both by causing a G2 cell cycle arrest and by inhibiting the mitochondrial apoptotic pathway. These observations suggest that Gadd45a inactivation could be a useful way to sensitize melanoma cells to chemotherapy. JID journal club article: For questions, answers, and open discussion about this article please go to http://network.nature.com/group/jidclub     

Ultraviolet responses of Gorlin syndrome primary skin cells

Background  Gorlin syndrome, or naevoid basal cell carcinoma syndrome (NBCCS), is an autosomal dominant disorder associated with mutations in the PTCH1 gene, which encodes the receptor of SONIC HEDGEHOG. In addition to developmental abnormalities, patients with NBCCS are prone to basal cell carcinoma (BCC), the most frequent type of nonmelanoma skin cancer in humans.
Objectives  As ultraviolet (UV) exposure plays a prominent role in the development of sporadic BCC, we aimed to determine whether primary NBCCS skin cells exhibit differential responses to UV exposure compared with wild-type (WT) skin cells.
Methods  Primary fibroblast and keratinocyte strains were isolated from nonlesional skin biopsies of 10 patients with characteristic NBCCS traits. After identification of PTCH1 mutations, capacities of NBCCS cells to repair UV-induced DNA lesions and to survive after UV irradiation, as well as p53 responses, were compared with those of WT skin cells.
Results  The c1763insG PTCH1 mutation is described for the first time. DNA repair and cell survival analyses following UV irradiation revealed no obvious differences between responses of NBCCS and WT fibroblasts and keratinocytes. However, p53 accumulation after UV irradiation was abnormally persistent in all NBCCS primary keratinocyte strains compared with WT keratinocytes.
Conclusions  Our observations that NBCCS cells harbour normal DNA repair and survival capacities following UV irradiation better explain that BCC proneness of patients with NBCCS does not solely concern body areas exposed to sunlight and suggest rather that it might be due to cell cycle alterations.     

Molecular responses to photogenotoxic stress induced by the antibiotic lomefloxacin in human skin cells: from DNA damage to apoptosis

Photo-unstable chemicals sometimes behave as phototoxins in skin, inducing untoward clinical side-effects when exposed to sunlight. Some drugs, such as psoralens or fluoroquinolones, can damage genomic DNA, thus increasing the risk of photocarcinogenesis. Here, lomefloxacin, an antibiotic from the fluoroquinolone family known to be involved in skin tumor development in photoexposed mice, was studied using normal human skin cells in culture: fibroblasts, keratinocytes, and Caucasian melanocytes. When treated cells were exposed to simulated solar ultraviolet A (320-400 nm), lomefloxacin induced damage such as strand breaks and pyrimidine dimers in genomic DNA. Lomefloxacin also triggered various stress responses: heme-oxygenase-1 expression in fibroblasts, changes in p53 status as shown by the accumulation of p53 and p21 proteins or the induction of MDM2 and GADD45 genes, and stimulation of melanogenesis by increasing the tyrosinase activity in melanocytes. Lomefloxacin could also lead to apoptosis in keratinocytes exposed to ultraviolet A: caspase-3 was activated and FAS-L gene was induced. Moreover, keratinocytes were shown to be the most sensitive cell type to lomefloxacin phototoxic effects, in spite of the well-established effectiveness of their antioxidant equipment. These data show that the phototoxicity of a given drug can be driven by different mechanisms and that its biologic impact varies according to cell type.     

De novo ceramide synthesis participates in the ultraviolet B irradiation-induced apoptosis in undifferentiated cultured human keratinocytes

Ultraviolet irradiation is a major environmental cause of skin cancers, whereas ultraviolet-induced DNA repair and apoptosis are defense mechanisms that rescue and/or protect keratinocytes from this risk. Multiple pathways are involved in ultraviolet-induced keratinocyte apoptosis, including activation of p38-mitogen-activated protein kinase, protein kinase C, and CD95, each of which are associated with caspase activation. Alternatively, ceramides could serve as ultraviolet-induced, second messenger lipids, because they induce cell cycle arrest and apoptosis in a variety of cell types, including keratinocytes. We investigated the role of ceramide versus caspase, and the responsible pathway for ceramide generation in ultraviolet B-induced apoptosis of cultured normal human keratinocytes maintained in low calcium (0.07 mm) medium. Ultraviolet B (40 mJ per cm2) significantly inhibited cultured normal human keratinocyte proliferation, assessed as [3H-methyl]thymidine-thymidine incorporation into DNA, 2 h after irradiation. Terminal nick deoxynucleotide end-labeling-positive apoptotic cells (14.8% at 24 h and 34.4% at 48 h) and trypan blue-positive apoptotic cells (8.4% at 24 h and 28.6% at 48 h) became evident in a time-dependent manner after ultraviolet B irradiation, in parallel with activation of caspase-3. The ceramide content of irradiated cultured normal human keratinocytes increased significantly by 8 h, whereas glucosylceramide only modestly increased, and sphingomyelin content remained unaltered. Metabolic studies with radiolabeled serine, palmitic acid, and phosphorylcholine revealed that the ultraviolet B-induced increase in ceramide results primarily from increased de novo synthesis rather than accelerated sphingomyelin hydrolysis. Increased ceramide synthesis, in turn, could be attributed to increased activity of ceramide synthase (i.e., 1.7-fold increase 8 h after ultraviolet B irradiation), whereas serine palmitoyltransferase activity did not change. Both fumonisin B1, an inhibitor of ceramide synthase, and ISP-1, myriocin an inhibitor of serine palmitoyltransferase, significantly attenuated the ultraviolet B-induced apoptosis in a caspase-3-independent fashion, whereas co-incubation with a caspase-3 inhibitor (Ac-DEVD-chloromethyl-ketone) further attenuated the ultraviolet B-induced apoptosis. Thus, increased de novo ceramide synthesis signals ultraviolet B-induced apoptosis, by a pathway independent of, but in concert with, caspase-3 activation.     

Keratinocyte G2/M growth arrest by 1,25-dihydroxyvitamin D3 is caused by Cdc2 phosphorylation through Wee1 and Myt1 regulation

1,25-dihydroxyvitamin D3 (1,25[OH]2VD3) has an antiproliferative effect on keratinocyte growth, and its derivatives are used for the treatment of psoriasis. It was reported previously that 1,25[OH]2VD3 induced cell cycle arrest not only at the G0/G1 phase but also at the G2/M phase. However, the mechanism of 1,25[OH]2VD3-induced G2/M phase arrest in keratinocytes has not been fully understood. The addition of 10(-8) to 10(-6) M 1,25[OH]2VD3 to cultured normal human keratinocytes enhanced the expression of Myt1 mRNA preceding Wee1 mRNA; 10(-6) M 1,25[OH]2VD3 unregulated Myt1 mRNA from 6 h to 24 h and Wee1 mRNA from 12 to 48 h. Interestingly, the levels of phosphorylated Cdc2 were increased between 6 h and 48 h after 1,25[OH]2VD3 treatment, although the expression levels of Cdc2 mRNA and its protein production were reduced. 1,25[OH]2VD3 also decreased the expression of cyclin B1, which forms a complex with Cdc2. These data indicated that the increase of Myt1 and Wee1 induced the phosphorylation of Cdc2 leading to G2/M arrest. In conclusion, the induction of Cdc2 phosphorylation due to the increase of Wee1 and Myt1 as well as the reduction of Cdc2 and cyclin B1 are involved in 1,25[OH]2VD3-induced G2/M arrest of keratinocytes.     

The DNA damage signal for Mdm2 regulation, Trp53 induction, and sunburn cell formation in vivo originates from actively transcribed genes.

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 approximately 89% of the Mdm2 response.     

Infliximab inhibits DNA repair in ultraviolet B-irradiated premalignant keratinocytes

Anti-tumor necrosis factor-alpha (TNFalpha) approaches are increasingly used in the therapy of autoimmune diseases. One of the safety concerns is the potential enhancement of skin carcinogenesis. The aim of this study was to investigate if the TNFalpha neutralizing antibody, infliximab, directly affects the cell cycle and DNA repair in premalignant human keratinocytes after ultraviolet-B (UVB) irradiation. We found that infliximab-treated cells exhibited an enhanced G2/M cell cycle arrest and increased apoptosis after 10-20 mJ/cm(2) UVB. In spite of this, the level of cyclobutane pyrimidine dimers (CPD) in infliximab-treated cells was significantly increased at both 24 and 48 h after irradiation with 10 mJ/cm(2) UVB. As we have recently shown that protein kinase B/Akt is involved in the TNFalpha signalling pathway and promotes cell survival and skin carcinogenesis, we measured activatory phosphorylations of Akt (Ser-473 and Thr-308) and the signalling via related pathways Erk 1/2, p38 and p70-S6K. Infliximab inhibited Akt and its downstream targets p70-S6K and Erk 1/2, and stimulated p38 both in sham-irradiated and UVB-irradiated cells. In conclusion, despite the fact that infliximab blocks Akt and stimulates the G2/M checkpoint and apoptosis in UVB-irradiated keratinocytes, the repair of CPD is impaired. It is conceivable that anti-TNFalpha treatments may contribute to the accumulation of mutagenic lesions in the epidermis and enhance the early stages of skin carcinogenesis.     

FRAP DNA-dependent protein kinase mediates a late signal transduced from ultraviolet-induced DNA damage

Ultraviolet radiation induces signal transduction at both early (<6 h) and late (>6 h) times after exposure. The inflammatory and immunosuppressive cytokine tumor necrosis factor alpha is induced at late times, and is induced by ultraviolet-induced DNA damage, as defects in DNA repair increase, and enhanced photoproduct repair reduces, tumor necrosis factor alpha expression. Here we show that late tumor necrosis factor alpha gene expression is sensitive to rapamycin, implicating FKBP12-rapamycin-associated protein, a member of the DNA protein kinase family, as a signal transducer of ultraviolet-induced DNA damage. FKBP12-rapamycin-associated protein was localized in the nucleus of keratinocytes and its level was increased following ultraviolet irradiation. Immuno- precipitated FKBP12-rapamycin-associated protein was stimulated by ultraviolet-irradiated DNA to phosphorylate p53 in vitro, and in vivo rapamycin reduced ultraviolet induction of p53 by 20%. Rapamycin further inhibited the ultraviolet-induced phosphorylation of the FKBP12-rapamycin-associated protein downstream target kinase p70S6K. In mice, topical application of rapamycin before ultraviolet exposure protected against suppression of the contact hypersensitivity that is a hallmark of ultraviolet-induced cytokine gene expression. These results demonstrate that the FKBP12-rapamycin-associated DNA protein kinase transduces the signal of ultraviolet-induced DNA damage into production of immunosuppressive cytokines at late times after ultraviolet irradiation.     

Ultraviolet-B-induced G1 arrest is mediated by downregulation of cyclin-dependent kinase 4 in transformed keratinocytes lacking functional p53

In order to identify potential novel targets for ultraviolet-B-induced skin tumorigenesis, we assessed the effect of ultraviolet-B exposure on cell cycle progression of transformed keratinocytes with mutant p53. We show that ultraviolet-B exposure of human epidermoid carcinoma A431 cells results in G1 cell cycle arrest in both asynchronously growing and synchronized cells. A significant increase in G1 cell population was observed following exposure to doses of ultraviolet-B as low as 10 mJ per cm2. When irradiated with ultraviolet-B, cells synchronized in G1 with mimosine did not exit G1. G1 cell cycle arrest was associated with a decrease in the hyperphosphorylated forms of retinoblastoma protein that was detectable within 4 h and gradually disappeared by 12 h. We also observed a decrease in cyclins D1, D2, and D3, and cyclin-dependent kinase 4 proteins, and a concomitant decrease in cyclin-dependent kinase 4/cyclin D1 associated kinase activity, whereas ultraviolet-B exposure had no effect on cyclin-dependent kinase 2 and cyclin-dependent kinase 6 levels during this time period. Incubation of cells with proteasome inhibitors MG-115 and MG-132 prevented the decrease in cyclin D1, D2, and D3, and cyclin-dependent kinase 4 protein. Taken together, our results suggest that ultraviolet-B-induced cell cycle arrest in A431 cells is mediated by cyclin-dependent kinase 4 downregulation. This identifies a novel pathway for G1 cell cycle arrest in transformed keratinocytes following ultraviolet-B irradiation.     

UVB对Jurkat细胞Gadd45a表达和DNA甲基化水平的影响

目的 探讨中波紫外线（UVB）对Jurkat细胞Gadd45a表达和DNA甲基化水平的影响。方法 分别以UVB 1.0 J/cm2和1.5 J/cm2照射Jurkat细胞后,于6、12、24、48 h收集细胞,采用实时定量 RT-PCR法检测Gadd45a mRNA水平和甲基化敏感基因 CD11a、CD70 mRNA水平,同时检测Jurkat细胞基因组DNA总体甲基化水平的变化。结果 ①和照射前Jurkat细胞比较,UVB 1.0 J/cm2照射后6、12、24、48 h Jurkat细胞Gadd45a mRNA水平升高,以照射后6、12 h差异有统计学意义（P < 0.01,P < 0.05）。UVB 1.0 J/cm2照射后,甲基化敏感基因CD11a、CD70 mRNA水平也升高,以照射后12 h差异有统计学意义（P值均 < 0.05）。②以UVB 1.5 J/cm2照射Jurkat细胞,和照射前Jurkat细胞比较,照射后6、12、24、48 h,Jurkat细胞Gadd45a、CD11a、CD70 mRNA水平均显著升高（P < 0.05或P < 0.01）。③和照射前Jurkat细胞比较,Jurkat细胞经UVB 1.0 J/cm2照射后6、12、24、48 h总体甲基化水平降低,照射后6、12 h甲基化水平差异有统计学意义（P < 0.05,P < 0.01）。Jurkat细胞经UVB 1.5 J/cm2照射后6、12、24、48 h的总体甲基化水平显著降低,差异均有统计学意义（P值分别 < 0.01、< 0.01、< 0.01、< 0.05）。④UVB照射后Jurkat细胞的Gadd45a mRNA和DNA总体甲基化水平呈显著负相关（r = -0.395,P < 0.05）。结论 UVB能诱导Jurkat细胞Gadd45a表达上调,同时Jurkat细胞基因组DNA发生低甲基化。     

Epidermal growth factor receptor pathway mitigates UVA-induced G2/M arrest in keratinocyte cells

UVA irradiation contributes largely to photocarcinogenesis. In the process of keratinocyte transformation, the activation of EGFR by UV is now considered as a critical event. However, the mechanism that links the EGFR pathway and photocarcinogenesis is not totally understood. In this study, we report that the EGFR/Akt pathway mitigated G2/M arrest in human HaCaT keratinocytes and normal human keratinocytes treated with low doses of UVA irradiation. EGFR-mediated Akt activation resulted in increased level of checkpoint 1 kinase (Chk1) inhibitory phosphorylation (Ser280). In contrast, EGFR/Akt pathway inhibition resulted in the abrogation of Ser280 Chk1 phosphorylation, increased level of Chk1 stimulatory phosphorylation (Ser345), and restoration of G2/M arrest. Altogether, these results suggest that, after UVA exposure, the EGFR/Akt pathway subverts the G2/M checkpoint. This effect may have serious implications in photocarcinogenesis by allowing damaged cells to transit through the cell cycle.