E6* oncoprotein expression of human papillomavirus type-16 determines different ultraviolet sensitivity related to glutathione and glutathione peroxidase antioxidant defence

Clinical observations of non-melanoma skin cancer in immunocompromised patients, such as organ transplant recipients, suggest co-operative effects of human papillomavirus (HPV) and ultraviolet (UV) radiation. The aim of the present study is to evaluate UV sensitivity and DNA damage formation according to antioxidant status in HPV16-infected keratinocytes. We used SKv cell lines, infected with HPV16 and well characterized for their proliferative and tumorigenic capacities. We showed that SKv cell lines presented various E6* (a truncated form of E6) RNA levels. We demonstrated that the higher oncoprotein RNA expression level was associated with a higher resistance to solar-simulated radiation, more specifically to UVB radiation and to hydrogen peroxide. Moreover, this high resistance was associated with a low oxidative DNA damage formation after UV radiation and was related to high glutathione content and glutathione peroxidase activities. Therefore, the results of our study suggest that E6* levels could modulate the glutathione/glutathione peroxidase pathway providing a mechanism to protect HPV-infected keratinocytes against an environmental oxidative stress, such as UV radiation.     

p53 p21和PCNA在寻常疣中的过度表达

目的：研究寻常疣ｐ５３蛋白、ｐ２１蛋白和增殖细胞核抗原（ＰＣＮＡ）过度表达与人类乳头瘤病毒（ＨＰＶ）感染的关系。方法：采用免疫组化方法对石蜡包埋组织标本进行检测。结果：１５例寻常疣皮损中ｐ５３、ｐ２１以及ＰＣＮＡ阳性标本分别为６例（４０％）、５例（３３％）和９例（６６％）。ｐ５３阳性细胞多位于基底层，ｐ２１阳性细胞则多分布于棘细胞层的中下层，ＰＣＮＡ阳性细胞散在分布于整个增生的表皮。受ＨＰＶ感染的空泡样变性细胞核中均有ｐ５３、ｐ２１以及ＰＣＮＡ过度表达者４例。结论：证实了寻常疣中存在ｐ５３、ｐ２１和ＰＣＮＡ过度表达，并从原位上找到ＨＰＶ感染与ｐ５３、ｐ２１过度表达和细胞异常增生相关联的细胞学证据。     

Simultaneously detected aberrant p53 tumor suppressor protein and HPV-DNA localize mostly in separate keratinocytes in anogenital and common warts

Abstract The E6 oncoprotein of human papillomavirus (HPV) is known to inactivate the control function on cell cycle exerted by p53 tumor suppressor protein in vitro by binding to p53 and thus facilitating the degradation of p53. We have applied a simultaneous in situ demonstration method for detecting p53 protein and HPV-DNA on formalin-fixed tissue sections, and investigated the in vivo interrelationship of p53 protein and HPV-DNA. Immunohistochemical staining for p53 protein with polyclonal and monoclonal antibodies, recognizing both wild-type (wt) and mutated p53 protein, was performed first and in situ DNA hybridization (ISH) for HPV types 6/11 or 16/18 with digoxigenin-labelled probes thereafter. 47% (25/53) of 48 histologically confirmed primary or recurrent condylomata acuminata (CA), 2 Bowenoid papulosis (BP) and 3 common wart (CW) biopsies, positive for HPV 6/11 or HPV 16/18 DNA, showed keratinocytes immunopositive for p53 protein. Of these. 11 lesions with abundant numbers of p53-positive cells were further analyzed with the double method. Signals for abnormal p53 protein and HPV-DNA were detected in separate cell nuclei in all biopsies and, additionally, in the same cell nuclei in 3 biopsies (1 BP, 1 CA, 1 CW). Usually the p53 positivity localized more basally in the epidermis than HPV-DNA, although p53- and HPV-positive keratinocytes were always located closely. The findings were similar for HPV-types 6/11 and 16/18. Our finding of both p53 and HPV-6/11 signals in the same cell nuclei may indicate complexing of p53 and low-risk HPV's without degradation of p53. Our results show abnormal p53 expression in HPV-infected skin lesions, and suggest that p53 protein is susceptible to aberrations even in the cells in the vicinity of productive HPV infection. However, it is not yet fully understood how HPV interferes with p53 protein in these cells.     

Human papillomavirus infection in Bowen disease: Negative p53 expression,not p16INK4a overexpression,is correlated with human papillomavirus‐associated Bowen disease

Genital Bowen disease (BD) has been linked to the high‐risk types of human papillomavirus (HPV) infection. Recently, it has been recognized that HPV also can be associated with extragenital BD. HPV oncoproteins E6 and E7 interfere with the function of p53 and pRb, respectively, leading carcinogenesis. p16^INK4a overexpression induced by inactivation of pRb is recognized as a surrogate marker for HPV‐associated cervical cancer. In this study, we examined the presence of HPV DNA in 142 BD lesions by polymerase chain reaction (PCR), and determined the type of HPV by PCR restriction fragment length polymorphism or direct DNA sequencing. HPV DNA was detected in 66.7% of genital BD and 8.3% of extragenital BD. The types of HPV detected were HPV types 6, 16, 33, 52, 56, 58 and 59. We also investigated the expression of p16^INK4a, pRb and p53 by immunohistochemistry. Positive expression was detected in 88.6% for p16^INK4a, 25.2% for pRb, and 63.8% for p53. There was no significant difference in p16^INK4a and pRb expression between HPV‐positive and ‐negative BD. However, a strong correlation of HPV positivity with p53 negativity was found. A total of 66.7% of HPV‐positive BD showed no p53 expression, whereas the corresponding rate was 32.8% of HPV‐negative BD. This study demonstrated that HPV can participate in the development of BD, not only in the genital lesion, but also in extragenital lesion. p16^INK4a overexpression is not a marker for HPV infection in BD. Instead, negative p53 expression is correlated with HPV‐associated BD.     

细胞周期素E、p21、转录因子E2F-1在尖锐湿疣中的表达

目的　探讨细胞周期相关因子细胞周期素E(cyclinE)、p2 1(WAF1/CIP1)、核转录因子E2F 1在尖锐湿疣 (CA)皮损中的变化及其意义。方法　应用原位杂交技术检测HPV 6/ 11相关CA ,应用SP免疫组化技术检测 3 8例HPV 6/ 11阳性CA患者皮损和 13例正常人皮肤中cyclinE、p2 1、E2F 1与分布。 结果　①CA皮损中cyclinE、p2 1、E2F 1的表达均较正常皮肤增强 ,差异有显著性 (P <0 .0 1) ,阳性信号主要分布于表皮棘层和颗粒层。②CA皮损中cyclinE与p2 1的表达间存在正相关性 ,cyclinE与E2F 1、p2 1与E2F 1间无相关性。 结论　HPV 6/ 11CA可能通过细胞周期调控蛋白的相互协调与制约 ,引起角质形成细胞的过度增殖     

Accumulated p53 protein and UVA protection level of sunscreens

Nuclear p53 expression is a sensitive parameter for the detection of ultraviolet (UV)-induced skin damage, and it has been used as an endpoint to evaluate the effectiveness of sunscreens. In this study, we compared the protection provided by two sunscreens having identical sun protection factors (SPF) but different UVA protection factors (UVA-PF) measured by the persistent pigment darkening method (PPD). The SPF of the sunscreens was 7 and the UVA-PF were respectively 7 and 3. Nuclear p53 protein was quantified in human skin biopsies treated with sunscreens and exposed 8 times to 5 MED of solar simulated radiation (SSR). The results showed that both sunscreens offered only partial protection against the increased expression of nuclear p53 protein induced by repetitive SSR exposures. However, a significantly lower level of p53-positive cells was found in areas protected with the sunscreen having the higher UVA-PF compared to the other sunscreen protected areas. In order to verify whether the difference in efficacy of these products was due to the difference in UVA absorption capacity, we quantified epidermal p53 protein accumulation after 8 exposures to either UVA (320-400 nm) or UVA1 (340-400 nm). We showed that as with SSR, repetitive exposures to 12.5 and 25 J/cm2 of UVA or UVA1 induced a significant increase in p53-positive cells in the human epidermis. These results confirmed that SPF determined on the basis of an acute erythemal reaction does not predict the level of protection against cumulative damage. They also showed that the protection provided by two sunscreens with different UVA protection factors is different (based on nuclear p53 protein accumulation), and that the PPD method can distinguish varying levels of sunscreen efficacy against UVA-induced cell damage.     

生殖器上皮HPV感染皮损中p53蛋白过度表达的检测

采用免疫组化方法对６例鲍温样丘疹病和９例尖锐湿疣生殖器上皮人类乳头瘤病毒（ＨＰＶ）感染皮损进行了ｐ５３蛋白、ｐ２１蛋白和增殖细胞核抗原（ＰＣＮＡ）表达的检测。结果ｐ５３阳性率为：鲍温样丘疹病（３／６）、尖锐湿疣（６／９），ｐ５３阳性细胞多位于基底层以及棘细胞层的中下层，同时发现受ＨＰＶ感染的空泡样变性细胞核中有ｐ５３蛋白和ＰＣＮＡ的过度表达，说明ｐ５３蛋白表达与ＨＰＶ感染及角朊细胞异常增生有关。     

Molecular events associated with apoptosis and proliferation induced by ultraviolet-B radiation in the skin of hairless mice

BACKGROUND: It is recognized that UV radiation produced apoptotic cells (sun burn cells) in the epidermis of mice. However, the relationship between apoptosis and cell proliferation after UV exposure in the skin of hairless mice are still unclear. OBJECTIVE: To investigate the effects of ultraviolet (UV) radiation on molecular events associated with apoptosis and proliferation in SKH1-hr mouse skin. METHODS: Mice were irradiated with daily UVB exposure of 0.1 or 0.25 J/cm(2) for 14 days. The skin tissues were analyzed at 2 and 24 h after the end irradiation for the presence of apoptotic cells and Bromodeoxyuridine (BrdU)-positive cells. We measured the expression of p53, p21, bcl-2, bax and E2F-1. RESULTS: The results indicated that UVB irradiation caused to increase apoptotic cells in the epidermis of mice. The expression of p53 and p21 was increased at 2 and 24 h after irradiation compared with the control. UV radiation induced high levels of bax at 2 and 24 h after irradiation with a concomitant decrease in bcl-2 expression. The expression of E2F-1 in the skin was also increased at 2 and 24 h after irradiation. Coinciding with these changes, BrdU positive cells increased at 2 and 24 h after UVB exposure at the epidermis of hairless mice, which observed the apoptotic expression. CONCLUSION: These results suggest that UVB irradiation of mouse skin induces apoptosis and is mediated by the p53/p21/E2F-1/bax pathway and that the dead cells are replaced by hyperproliferative cells, leading to epidermal hyperplasia.     

Ultraviolet-B irradiation alters the cell cycle machinery in murine epidermis in vivo.

Ultraviolet radiation of mouse skin leads to epidermal hyperplasia, inflammation, and subsequent tumor development. In this study we determined to what extent the cell cycle machinery is altered during epidermal proliferation after ultraviolet B radiation. A minimal erythema dose, 90 mJ per cm2, increased the protein expression of the G1 phase cyclins, cyclin D1 and E, by 12 h. The majority of epidermal cells entered S phase between 18 and 24 h as determined by 5'-bromo-2'-deoxyuridine incorporation, proliferating cell nuclear antigen, and cyclin A immunohistochemistry. An increase in cyclin-dependent kinase 2 (cdk-2) protein expression occurred after 12 h, but no changes in cdk-4 or cdk-6 protein levels were observed. The increase in cyclin D1, E, and A protein expression was associated with an increase in cyclin D1-cdk-4, cyclin E-cdk-2, and cyclin A-cdk-2 complex formation. p53 protein expression was elevated through 48 h, and the cdk inhibitor protein p21(Cip1/WAF1) was elevated 6-fold to 7.5-fold between 12 and 24 h. The elevated p21(Cip1/WAF1) protein contributed to an enhanced association with cdk-2 and cdk-4 at 3-24 h and 6-24 h post-ultraviolet B irradiation, respectively. These data indicate that 90 mJ per cm2 of ultraviolet B irradiation induces a DNA damage response, by increasing p53 and p21(Cip1/WAF1) protein expression, but also induces a rapid and sustained increase in S phase by 18 h.     

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.     

细胞周期蛋白E和p27在外生殖器表皮肿瘤发病中的作用

目的探讨细胞周期蛋白E和p27在外生殖器表皮良、恶性增殖病变中的意义。方法采用聚合酶链反应-限制性片段长度多态性分析方法,对99例外生殖器尖锐湿疣、原位鳞状细胞癌和浸润性鳞癌进行HPVDNA检测和分型,应用免疫组化方法检测上述病变中细胞周期蛋白E和p27表达。结果①上述病变中细胞周期蛋白E的表达较正常表皮显著升高,且HPV阳性组高于阴性组;比较HPV阳性的3种病变中细胞周期蛋白E表达,尖锐湿疣<原位鳞癌<浸润性鳞癌。②尖锐湿疣中p27表达较正常表皮略升高,而原位鳞癌和浸润性鳞癌中则显著下降,HPV阳性组低于阴性组,比较HPV阳性的3种病变中p27的表达,尖锐湿疣>原位鳞癌>浸润性鳞癌。③上述病变中细胞周期蛋白E与p27的表达有相关性。结论上述病变中HPV感染与细胞周期蛋白E和p27的表达有相关性,HPV可能通过影响细胞周期蛋白E和p27的表达及二者的相互作用引起感染表皮的增殖及恶变。     

p53 mutation in metastatic melanomas and primary melanomas from sun-exposed and sun-protected sites

Background p53 mutation has been observed in many human malignancies, including skin cancers. However, the data in melanoma has been conflicting. Material and methods We have examined, by immunohistochemistry, using the DO7 and CM1 antibodies, the frequency of p53 overexpression in 14 metastatic melanomas and 61 primary melanomas, of which 30 were from sun-protected sites and 31 from sun-exposed sites. Results Ten of 14 metastatic melanomas showed p53 overexpression compared to only eight of 61 primary melanomas (P < 0.004). No significant difference in p53 expression was found between primary melanomas from sun-protected (2/30) and sun-exposed sites (6/31). We have also examined p53 mutation by single-strand conformation polymorphism (SSCP) analysis of four melanoma cell lines. One cell line established from a primary melanoma from a sun-protected site showed evidence of an altered migration pattern in exon 7. Sequencing analysis of this region confirmed a point mutation in codon 244, showing a G to C transversion. This mutation is unlikely to be due to ultraviolet (UV) radiation since mutations caused by UV radiation are predominantly CC → TT or C → T transitions. Conclusions In summary, p53 mutation in primary melanoma is uncommon and does not appear to be related to UV radiation. p53 mutation is more common in metastatic melanomas suggesting that it may be involved as a late event in melanoma progression.     

Cyclobutane pyrimidine dimer formation and p53 production in human skin after repeated UV irradiation

Substantial differences in DNA damage caused by a single UV irradiation were found in our previous study on skin with different levels of constitutive pigmentation. In this study, we assessed whether facultative pigmentation induced by repeated UV irradiation is photoprotective. Three sites on the backs of 21 healthy subjects with type II-III skin were irradiated at 100-600 J/m(2) every 2-7 days over a 4- to 5-week period. The three sites received different cumulative doses of UV (1900, 2900 or 4200 J/m(2)) and were biopsied 1 day after the last irradiation. Biomarkers examined included pigment content assessed by Fontana-Masson staining, melanocyte function by expression of melanocyte-specific markers, DNA damage as cyclobutane pyrimidine dimers (CPD), nuclear accumulation of p53, apoptosis determined by TUNEL assay, and levels of p21 and Ser46-phosphorylated p53. Increases in melanocyte function and density, and in levels of apoptosis were similar among the 3 study sites irradiated with different cumulative UV doses. Levels of CPD decreased while the number of p53-positive cells increased as the cumulative dose of UV increased. These results suggest that pigmentation induced in skin by repeated UV irradiation protects against subsequent UV-induced DNA damage but not as effectively as constitutive pigmentation.     

细胞周期蛋白E和p27在鲍温样丘疹病和外生殖器表皮原位癌中的表达

目的：探讨细胞周期相关因子细胞周期蛋白E及其依赖性蛋白激酶抑制蛋白p27，在高危型人乳头瘤病毒(HPV)相关的鲍温样丘疹病及外生殖器表皮原位癌中的变化及意义。方法：采用通用型引物聚合酶链反应-限制性片段长度多态性(PCR—RFLP)方法对51例外生殖器部位上述病变进行HPVDNA的检测和分型，应用免疫组化方法检测病变中细胞周期蛋白E和p27的表达。结果：40例鲍温样丘疹病、5例生殖器鲍温病及6例Queyrat增殖性红斑HPVDNA的阳性率分别为55．0％、100．0％和33．3％，HPV16为主要型别；高危型HPV相关的病变中细胞周期蛋白E的表达明显高于正常上皮，且HPV阳性组明显高于HPV阴性组，而p27表达明显低于正常上皮，且HPV阳性组明显低于HPV阴性组。上述疾病中细胞周期蛋白E与p27的表达呈负相关。结论：HPV16感染与鲍温样丘疹病及外生殖器表皮原位癌的发生有密切关系，并可能通过影响细胞周期蛋白E和p27表达及两者的相互作用引起感染上皮的增生及恶变。     

CD1a+, CD3+, CD4+, CD8+, CD68+ and cutaneous lymphocyte-associated antigen-positive cells in Bowen's disease

BACKGROUND: Bowen's disease (BD) is a squamous cell carcinoma in situ that rarely invades into the underlying dermis. However, little is known about its immunohistology. Objectives To evaluate the relationship between the cytological properties of the tumour cells in BD and the host immune response. METHODS: We examined the expression of p53, proliferating cell nuclear antigen (PCNA) and Ki67 antigen, and the number of mitotic cells, together with the number of intratumoral and dermal infiltrating CD1a+, CD3+, CD4+, CD8+, CD68+ and cutaneous lymphocyte-associated antigen (CLA)+ cells in 18 cases of genital BD. RESULTS: When compared with normal genital skin (n = 10), there was a significantly higher number of mitotic cells as well as higher expression of p53+, PCNA+ and Ki67+ cells in BD. There was significant mutual correlation between CD3+, CD4+ and CD68+ cells in the tumoral epidermis. The number of CD1a+ Langerhans cells significantly decreased in BD epidermis; however, dermal CD1a+ cells were increased. Interestingly, numbers of dermal CD1a+ cells significantly correlated with those of intratumoral CD3+, CD4+ and CD68+ cells. In situ hybridization for human papillomavirus (HPV) demonstrated that HPV-infected BD had significantly less infiltration of intratumoral CD3+ cells and CLA+ cells. CONCLUSIONS: The present data suggest that dermal CD1a+ cells may participate in the immune surveillance and that HPV infection may interfere with the intratumoral infiltration of CLA+ cells in BD.     

The HPV16 E6 oncoprotein and UVB irradiation inhibit the tumor suppressor TGFβ pathway in the epidermis of the K14E6 transgenic mouse

High‐risk human papillomaviruses (HR‐HPVs) are the causative agents of cervical cancer, and they are also associated with a subset of head and neck squamous cell carcinomas. In addition, HPVs have also been postulated in the development of non‐melanoma skin cancers (NMSC). In these cancers, the oncogene E6 is best known for its ability to inactivate the tumor suppressor p53 protein. Interestingly, in transgenic mice for HPV16 E6 (K14E6), it was reported that E6 alone induced epithelial hyperplasia and delay in differentiation in skin epidermis independently of p53 inactivation. Transforming growth factor β (TGFβ) is an important regulator of cell growth/differentiation and apoptosis, and this pathway is often lost during tumorigenesis. Ultraviolet radiation B (UVB) exposure activates diverse cellular responses, including DNA damage and apoptosis. In this study, we investigated whether the E6 oncogene alone or in combination with UVB dysregulate some components of the TGFβ pathway in the epidermis of K14E6 mice. We used 8‐day‐old K14E6 and non‐transgenic mice irradiated and unirradiated with a single dose of UVB. We found that the E6 oncogene and UVB irradiation impair the TGFβ pathway in epidermis of K14E6 mice by downregulation of the TGFβ type II receptor (TβRII). This loss of TβRII prevents downstream activation of Smad2 and target genes as p15, an important regulator of cell cycle progression. In summary, the TGFβ signalling in cells of the epidermis is downregulated in our mouse model by both the E6 oncoprotein and the UVB irradiation.