Analysis of p53, p21(Waf1/Cip1) and TGF-beta(3) immunohistochemical staining in Bowen's disease

BACKGROUND: The p53 gene is one of a family of tumor suppressor genes that have been implicated in the genesis of a wide variety of malignant neoplasms including Bowen's disease. Its role in oncogenesis and tumor progression is thought to be of importance. Transforming growth factor beta (TGF-beta) is the most potent known inhibitor of the progression of normal epithelial cells through the cell cycle. p21(Waf1/Cip1) is thought to mediate p53 signaling induced by DNA-damaging agents to arrest the cell cycle. OBJECTIVE: The present study evaluates the expression of p53, p21(Waf1/Cip1) and TGF-beta(3) protein and speculates on their role in Bowen's disease. METHODS: Sixteen patients seen at our clinic between 1993 and 2000 were examined. We analyzed p21(Waf1/Cip1), p53 and TGF-beta(3) immunohistochemical staining in all specimens. RESULTS: In 7 of the Bowen's disease patients, overexpression of p53-positive cells was present in the middle and basal layers, and intense staining of p21(Waf1/Cip1) was observed in the upper spinous layers. In the other 9 Bowen's disease patients, we found positively stained cells for p21(Waf1/Cip1) but negative p53 immunostaining in the upper epidermal layer. Downregulated TGF-beta(3) expression was detected in all layers except the upper spinous layers. CONCLUSION: These observations suggest different roles for p21(Waf1/Cip1) and p53 within abnormal cells in Bowen's disease. p21(Waf1/Cip1) may induce terminal differentiation to the superficial layer in Bowen's disease via either a p53-independent or -dependent pathway. Moreover, downregualtion of TGF-beta(3) immunostaining provides relevant information concerning the pathogenesis of Bowen's disease.     

The expression of p53, p21, Bax and induction of apoptosis in normal volunteers in response to different doses of ultraviolet radiation

BACKGROUND: Ultraviolet radiation (UVR) damages keratinocytes. Direct DNA damage may undergo enzymatic repair followed by resumption of the normal cell cycle. Cells may also be eliminated without inflammation by the error-free process of programmed cell death or apoptosis. Necrosis of cells can occur after overwhelming damage. Failure of apoptosis leads to retention of cells with persistent mutations. OBJECTIVES: This study investigates p53-dependent apoptotic responses in normal skin following solar-simulated radiation (SSR). METHODS: Sun-protected buttock skin from normal volunteers with no history or clinical evidence of skin cancer was exposed to graded doses of SSR, 0.5, 1, 2 and 3 times the minimal erythema dose (MED). Biopsies taken at a range of time points (4.5, 9, 24, 33, 48 and 72 h) after UVR, quantified the time course and dose-response of apoptosis and the expression of the relevant proteins, p53, p21waf1/Cip1 and Bax, by single and double labelling techniques. RESULTS: Apoptosis was upregulated in a dose-dependent manner as was the expression of p53, p21waf1/Cip1 and Bax in response to SSR. Following exposure to 3 MEDs it was found that: (i) the maximum number of apoptotic cells occurred at 48 h; (ii) p53 protein expression was upregulated from 4 to 72 h preceding peak p21waf1/Cip1 protein expression (9-48 h) and peak Bax protein expression (33 h). CONCLUSIONS: These results suggest that, following SSR, normal human skin induces apoptosis by the p53, p21waf1/Cip1, Bax pathway in vivo. In addition, induction of apoptosis and expression of p53, p21waf1/Cip1 and Bax occurs in a dose-dependent manner.     

Overexpression of p2lWaf1/Cip1 immunohistochemical staining in Bowen's disease, but not in disseminated superficial porokeratosis

Disseminated superficial porokeratosis (DSP) consists of multiple small lesions of porokeratosis. Although the pathogenesis of DSP remains unclear, localized cloning of abnormal epidermis has been hypothesized. Malignant cutaneous neoplasms, especially Bowen's disease, have been frequently reported in DSP. Immunopositive p53 has been demonstrated in a variety of human malignant tumours, and its role in oncogenesis and tumour progression is thought to be important. p21Waf1/Cip1 is thought to mediate the signal of p53 induced by DNA damaging agents to arrest the cell cycle. To clarify the role of p53 and p21Waf1/Cip1 in Bowen's disease and DSP, we analysed 12 cases of Bowen's disease and eight cases of DSP by immunohistochemistry. In five of the 12 Bowen's disease patients and two of the eight DSP patients, positive p53 staining was detected. In contrast, whereas p21Waf1/Cip1 overexpression was detected in all Bowen's disease patients, it was not seen in DSP. The present data suggest that p53 immunostaining provides relevant information concerning the pathogenesis of Bowen's disease and DSP. Furthermore, high p21Waf1/Cip1 expression appears to be a useful indicator of tumour activity in Bowen's disease.     

Decreased growth inhibitory responses of squamous carcinoma cells to interferon-gamma involve failure to recruit cki proteins into cdk2 complexes.

Interferon-gamma induces an irreversible growth arrest and squamous differentiation in normal human epidermal keratinocytes. We present for the first time a careful biochemical analysis of the cell-cycle-related events that occur during interferon-gamma treatment of normal human epidermal keratinocytes. The interferon-gamma-induced irreversible growth arrest state is characterized by inhibition of cyclin-dependent kinases, prevention of Rb and p130 (Rb2) phosphorylation, and increases in p27(Kip1), p16(Ink4a), and p130 proteins, together with a transient increase in p21(Waf1/Cip1). Cells derived from squamous cell carcinomas are less responsive to interferon-gamma and do not terminally differentiate. We exploited these differences in response to interferon-gamma in order to identify the particular molecular defects in cell cycle control that promote carcinogenesis in squamous epithelia. In several squamous cell carcinoma cell lines as well as in interferon-gamma-insensitive HaCaT cells, interferon gamma was unable to significantly induce levels of p130 and/or p16 protein. In addition, p21 association with cdk2 complexes was undetectable in either the absence or the presence of interferon-gamma and, unlike normal human epidermal keratinocytes, p27 association with cdk2 did not increase with interferon-gamma treatment. These multiple defects appear to be intrinsic to the mechanisms of cell cycle regulation rather than due to defects in the interferon-gamma signaling pathway, as induction of several interferon-gamma-responsive genes including Stat 1, IRF-1, and p21 itself was normal. Interestingly, exogenous expression of p21 protein in the squamous cell carcinoma cell lines by adenovirus carrying wildtype p53 or p21 cDNA cooperated with interferon-gamma to produce a greater inhibition of growth than either agent alone, even though p21 protein could barely be detected in cdk2 complexes. We conclude that squamous cell carcinoma cells have intrinsic defects in their ability to regulate cdk-cki complexes in response to differentiation signals.     

羟氯喹及没食子酸酯对HaCaT细胞光照射的影响

目的 探讨羟氯喹和绿茶活性成分表没食子儿茶素没食子酸酯(EGCG)对中波紫外线(UVB)损伤永生化角质形成细胞株(HaCaT细胞)的保护作用及其机制。方法 采用UVB定时及定量照射培养的HaCaT细胞,分别加入羟氯喹和EGCG干预处理,以RT-PCR法检测各受试组p53、p21、c-fos基因表达水平。结果 UVB照射后可明显增加HaCaT细胞中p53,p21,c-fos mRNA表达,羟氯喹和EGCG可不同程度地下调上述基因表达水平。结论 羟氯喹和EGCG的光保护作用在HaCaT细胞可能与其抑制p53,p21,c-fos基因表达有关。     

黑果枸杞水提取物抑制UVB辐射后HaCaT细胞的凋亡及p16、p53蛋白表达

目的：明确黑果枸杞水提取物对中波紫外线(UVB)辐射引起的HaCaT细胞凋亡及p16、p53蛋白表达的影响。方法： 体外培养HaCaT细胞,分为对照组、UVB组、UVB+黑果枸杞水提取物组,UVB照射剂量为30 mJ/cm2 UVB,黑果枸杞水提取物浓度为2 mg/mL。流式细胞仪检测各组UVB照射后24 h细胞凋亡率,Western blot检测各组HaCaT细胞p16、p53蛋白的表达水平。结果：与对照组（6.12±1.19）%比较,UVB组HaCaT细胞凋亡率为(74.89±3.90)%、UVB+黑枸杞水提取物组为（57.52±2.93）%,差异有统计学意义（P<0.05）。对照组p16和p53蛋白水平为0.1±0.03和0.21±0.07,UVB组为0.28±0.06和0.5±0.04、UVB+黑枸杞水提取物组为0.15±0.025和0.25±0.01,差异均有统计学意义（Ps<0.05）。结论：黑果枸杞水提物可抑制UVB引起的HaCaT细胞凋亡以及p16、p53蛋白表达。     

Coexpression of p21Waf1/Cip1 and p53 in sun-exposed normal epidermis, but not in neoplastic epidermis

Summary Wild-type p53 accumulation induced by DNA damaging agents such as ultraviolet (UV) radiation. γ-irradiation and drugs, may arrest the cell cycle until DNA damage is repaired. p21^Waf1/Cip1 is a cyclin-dependent kinase (CDK) inhibitor induced by wild-type p53. CDK is activated by cyclin and progresses the cell cycle. On the other hand. CDK inhibitors inhibit CDK activity to arrest the cell cycle. Thus, p21^Waf1/Cip1 is thought to mediate the signal of p53 induced by DNA damaging agents to arrest the cell cycle. p21^Waf1/Cip1 is induced by wild-type, but not mutant p53. To investigate p21^Waf1/Cip1 regulation by p53 in epidermis in vivo , immunohistochemical staining of p21^Waf1/Cip1 and p53 were conducted in chronically sun-exposed normal epidermis and in neoplastic epidermis. p21^Waf1/Cip1 expression was found to be coincident with the p53-positive regions or not coincident with the p53-positive regions in chronically sun-exposed normal epidermis, whereas there was only low or undetectable p21^Waf1/Cip1 expression in any regions including the p53-positive regions of solar keratosis and squamous cell carcinoma of the skin. This suggests that wild-type p53 and p21^Waf1/Cip1 may play a part in chronically sun-exposed normal epidermis response to UV exposure, whereas p21^Waf1/Cip1 cannot be induced by mutated p53 in solar keratosis and squamous cell carcinoma of the skin.     

Loss of CDKN2A and p14ARF expression occurs frequently in human nonmelanoma skin cancers.

BACKGROUND: The CDKN2A locus on human chromosome 9p21 encodes two proteins named p16INK4a and p14ARF, known to function as tumour suppressors via the retinoblastoma (Rb) or the p53 pathway. The p53 tumour suppressor gene is the most commonly mutated gene in human and mouse cancers. Disruption of the p53 and Rb pathways is a fundamental trend of most human cancer cells. Recent studies have shown that the CDKN2A gene plays an active role in the p53 and Rb tumour suppressor pathways. Genetic abnormalities in CDKN2A have been well documented in human melanoma, but their involvement in nonmelanoma skin cancer (NMSC) is less clear. OBJECTIVES: To determine whether genetic abnormalities in CDKN2A and p53 genes play a role in the development of NMSC. METHODS: We analysed 40 primary NMSCs in 40 patients (21 squamous cell carcinomas, 17 basal cell carcinomas and two actinic keratoses) for p16INK4a and p14ARF protein expression and for genetic alterations in exons 1alpha, 1beta and 2 of CDKN2A. RESULTS: Immunohistochemical analysis revealed loss of expression of p16INK4a and p14ARF proteins in 38 and 39 of 40 NMSCs, respectively. Amplification of genomic DNA by polymerase chain reaction revealed homozygous deletion of exon 1beta in 20% of tumours and of exon 2 in 82.5% of tumours. Of 22 NMSCs with p53 mutations, 13 (59%) had ultraviolet (UV) signature mutations in the p53 gene; all of them were strongly positive for p53 immunostaining. CONCLUSIONS: In addition to mutations in the p53 gene, loss of expression of CDKN2A via deletion also plays an important role in the pathogenesis of human NMSC. While p53 mutations are induced by UVB, deletions in CDKN2A could arise spontaneously, perhaps during tumour progression.     

中波紫外线对HaCaT细胞核因子-κB和p53表达的影响

目的研究中波紫外线(UVB)对HaCaT细胞核因子-κB(NF-κB)和p53表达的影响。方法以60mJ/cm2的UVB照射HaCaT细胞后8h、16h和24h,采用real time PCR和western blot等技术检测NF-κB和p53的表达情况。结果 1与未处理组相比,UVB照射HaCaT细胞8h、16h和24h后,NF-κB mRNA水平升高程度分别为27%、32%和42%,p53 mRNA水平升高程度分别为13%、20%和28%;2未处理组HaCaT细胞NF-κB和p53蛋白的表达水平分别为0.3989±0.04802和0.3018±0.03605,UVB照射8h、16h和24h后,二者的表达均随时间延长而升高,NF-κB为0.4283±0.0195、0.5976±0.0401和0.8255±0.0214,p53为0.3925±0.0244、0.4595±0.0362和0.5811±0.0482,差异有统计学意义(P0.05)。结论 UVB可致HaCat细胞中NF-κB和p53表达水平升高。     

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.     

中波紫外线通过EGFR/PI3K/AKT途径上调HaCaT细胞转铁蛋白受体的表达

目的 探讨中波紫外线辐射对HaCaT转铁蛋白受体表达的影响及其信号途径。方法 流式细胞仪检测HaCaT细胞转铁蛋白受体的表达。实时PCR检测转铁蛋白受体mRNA表达。结果 UVB辐射可上调HaCaT细胞转铁蛋白受体mRNA及其蛋白的表达，并在一定范围内（10 mJ/cm2 ~ 30 mJ/cm2）呈剂量依赖性。表皮生长因子受体（EGFR）抑制因子PD153035、磷脂酰肌醇3激酶（PI3K）抑制因子LY294002和渥曼青霉素（wortmannin）均可显著抑制UVB辐射对转铁蛋白受体的上调作用（P < 0.01）。结论 紫外线可通过EGFR/PI3K/AKT途径上调HaCaT 细胞转铁蛋白受体的表达。     

Long-term growth arrest of PUVA-treated fibroblasts in G2/M in the absence of p16(INK4a) p21(CIP1) or p53

Premature aging of the skin is a prominent side-effect of psoralen photoactivation, a therapy used for different skin disorders. Recently, we demonstrated that treatment of fibroblasts with 8-methoxypsoralen and ultraviolet A irradiation resulted in growth arrest with morphological and functional changes reminiscent of replicative senescence. To further elucidate the underlying molecular mechanisms, we analysed the cell-cycle phases of the growth-arrested fibroblasts. After PUVA treatment, fibroblasts arrested in G2/M, in contrast to spontaneously senesced fibroblasts arresting in a cell-cycle phase with many features similar to G1. To address the role of the cell-cycle controlling genes p16(INK4a), p21(CIP1) and p53, we analysed the expression of these genes. p16(INK4a), p21(CIP1) and p53 protein levels increased substantially with different time kinetics in growth-arrested fibroblasts. Because p16(INK4a), p21(CIP1) and p53 are involved in replicative senescence, we applied the PUVA regimen to fibroblasts deficient in either of these genes. p16(INK4a), p21(CIP1) and p53 null mutant fibroblast strains underwent growth arrest with a senescent phenotype similar to wild-type human fibroblasts. Based on these results, we propose that redundant or alternate pathways are involved in the response of dermal fibroblasts to PUVA treatment resulting in a phenocopy of replicative senescence in vitro.     

不同剂量中波紫外线对HaCaT细胞p62及自噬体形成关键基因Beclin-1、Atg12和Atg3的调控效应研究

【摘要】 目的 探讨不同剂量中波紫外线（UVB）照射培养的HaCaT细胞后不同时间点对p62、Beclin-1、Atg12和Atg3蛋白表达水平的调控效应。 方法 使用4.5、10和50 mJ/cm2 UVB照射HaCaT细胞,照射后加入新鲜培养基继续培养4 h和12 h,同时设相同处理但不照射UVB的细胞为对照细胞。另设观察组,在照射后立即（包括对照细胞）,使用含蛋白酶抑制剂E64D（10 μg/L）和胃酶抑素（10 μg/L）的培养基孵育4 h和12 h,以阻断对p62的降解。使用Western印迹法测定HaCaT细胞p62、Beclin-1、Atg12和Atg3蛋白的表达水平。 结果 50 mJ/cm2 UVB照射HaCaT细胞后4 h,p62表达水平（p62与内参的比值为0.473 ± 0.022）较对照细胞（0.246 ± 0.038）升高（t = 15.27,P < 0.05）;阻断溶酶体后,细胞新生p62的水平（0.445 ± 0.035）与阻断溶酶体的对照（0.244 ± 0.016）相比,仍为上调（t = 7.62,P < 0.05）。在4.5 mJ/cm2 UVB照射细胞后12 h,与对照（0.254 ± 0.035）相比,HaCaT细胞p62表达上调（0.497 ± 0.047,t = 22.89,P < 0.05）;阻断溶酶体后,与阻断溶酶体的对照（0.257 ± 0.025）相比,p62表达水平仍然上调（0.548 ± 0.051,t = 17.42,P < 0.05）。4.5 mJ/cm2和50 mJ/cm2 UVB照射后4 h和12 h,对照细胞和照射细胞间的Beclin-1、Atg12和Atg3的表达差异均无统计学意义（P > 0.05）,阻止溶酶体对自噬体内容物的降解也未能发现Beclin-1、Atg12和Atg3的表达差异（P > 0.05）。 结论 p62表达在不同剂量UVB照射和照射后不同时间存在差异调控,并且这种调控效应与自噬体形成可能没有生物学联系。     

Effect of ultraviolet radiation on the expression of pp38MAPK and furin in human keratinocyte-derived cell lines

Background: Ultraviolet radiation (UVR) is known to induce the activation of stress‐inflammation signal transduction pathways, and to induce the activity of many proteases in skin cells. It is unknown whether the activation of proteases such as furin is related to changes in the phosphorylation status of p38MAPK. Methods: The effect of UVR on immortalized keratinocyte (HaCaT) and squamous cell carcinoma (Colo16) cells was investigated with respect to cell survival, phosphorylation of p38MAPK, and the proprotein convertase, furin. The cells were exposed to either a low or a high dose of UVA and/or UVB and the viability was monitored over 48 h, along with changes in the intracellular expression of p38MAPK and furin. Results: Low‐dose UVA (2 kJ/m²) and/or UVB (0.2 kJ/m²) radiation had no effect on cell viability, except in UVA‐irradiated Colo16 cells. High UVA (20 kJ/m²) caused a loss of cell viability in HaCaT cells, but not in Colo16 cells. The opposite effect was seen in cells exposed to a high UVB dose (2 kJ/m²). The viability of both cell cultures decreased when exposed to high‐dose UVA+B radiation. UV irradiation downregulated the expression of phosphorylated p38 (pp38) in HaCaT cells irrespective of the UV dose and type. In Colo16 cells, UV radiation induced pp38 expression in the cells following exposure, with the highest increase in cells exposed to high‐dose UVA. The expression of furin in UV‐irradiated HaCaT cells was similar to that seen for pp38 expression. In Colo16 cells, UV radiation induced furin expression, with the highest increase seen in cells 24 h after exposure to both high‐dose UVB and UVA+B radiation. Conclusion: The results show that there are differences between the effect of UV types and doses on cell function in the keratinocyte‐derived cell lines examined in this study. The level of furin expression in Colo16 cells correlated to changes in pp38 levels in the cells following exposure to UV radiation, but not in HaCaT cells. From an improved understanding of the signalling pathways and their downstream events and how these may differ as a result of tumorigenesis, it may enable the development of inhibitors, which may have therapeutic applications.