The human papillomavirus type 8 E2 protein induces skin tumors in transgenic mice

Transgenic mice expressing early genes of the cutaneous human papillomavirus 8 (HPV8) spontaneously develop skin papillomas, epidermal dysplasia, and squamous cell carcinoma (6%). As the HPV8 protein E2 revealed transforming capacity in vitro, we generated three epidermal specific HPV8-E2-transgenic FVB/N mouse lines to dissect its role in tumor development. The rate of tumor formation in the three lines correlated with the different E2-mRNA levels. More than 60% of heterozygous line 2 mice, but none of the HPV8-negative littermates, spontaneously developed ulcerous lesions of the skin over an observation period of up to 144 weeks, beginning on average 74+/-22 weeks after birth. Most lesions presented infundibular hyperplasia and acanthosis combined with low-grade dysplasia. Severe dysplasia of the epidermis occurred in 6%. Two carcinomas revealed a sharply demarcated spindle-cell component. Only 3 weeks after a single UV irradiation, 87% of heterozygous line 2 and 36% of line 35 mice developed skin tumors. A rapidly growing invasive tumor composed of spindle cells arose 10 weeks after irradiation of a line-35 animal. The histology of skin cancers in HPV8-E2 mice is reminiscent of a subset of highly aggressive squamous cell carcinoma in immunosuppressed transplant recipients with a massive spindle-cell component.     

Skin tumor formation in human papillomavirus 8 transgenic mice is associated with a deregulation of oncogenic miRNAs and their tumor suppressive targets

Background

Dysregulation of microRNA (miRNA) expression is regularly found in various types of cancer and contributes to tumorigenic processes. However, little is known about miRNA expression in non-melanoma skin cancer in which a pathogenic role of beta human papillomaviruses (HPV) is discussed. A carcinogenic potential of beta HPV8 could be demonstrated in a transgenic mouse model, expressing all early genes of HPV8 (HPV8-CER). A single UVA/B-dose induced oncogene expression and led to papilloma growth within three weeks.

Objective

Expression of miRNAs and their targets during HPV8-mediated tumor formation in mice.

Methods

Skin of untreated or UV-irradiated wild-type and HPV8-CER mice was analyzed for miRNA expression and localization by qPCR and in situ hybridization. MiRNA target protein expression was analyzed by immunohistochemical staining.

Results

Early steps in skin tumor formation in HPV8-CER mice were associated with an upregulation of the oncogenic miRNA-17-5p, -21 and -106a and a downregulation of the tumor-suppressive miRNA-155 and -206, which could be demonstrated by qPCR and in situ hybridization. The respective targets of miRNA-21 and -106a, the tumor suppressors PTEN, PDCD4 and Rb with their pivotal role in cell cycle regulation, apoptosis and proliferation were found to be downregulated.

Conclusion

This is the first report demonstrating that a cutaneous HPV type deregulates the expression of miRNAs. These deregulations are closely related to the UV-induced upregulation of HPV8 oncogene levels, which suggest a direct or indirect HPV8-specific effect on miRNA expression. These data presume that HPV8 interferes with the miRNA mediated gene regulation to induce tumorigenesis.     

Lymphocyte infiltration of the skin in transgenic mice carrying the human interleukin-2 gene

Inflammatory lesions of the skin such as erythema, depigmentation and hair loss were observed in C57/BL6(B6) transgenic mice that carried an intact human genomic interleukin-2 gene (gIL-2 transgenic mice). Accumulation of T lymphocytes in the perivascular and periadnexal areas of the dermis was the first change, followed by dermal papillary oedema, which occurred before the development of macroscopic skin lesions. In 3- or 4-week-old transgenic mice with slight erythema and depigmentation of the skin, there was an increase in the number of perivascular lymphocytes accompanied by the diffuse infiltration of neutrophils and monocytes in the damaged skin. These morphological skin changes were not observed in non-transgenic mice, which were bred together with transgenic litter mates. These findings suggest that lymphocyte infiltration of the perivascular space of the skin is a primary event of exogenously introduced human interleukin-2 gene, resulting in secondary cutaneous changes in gIL-2 transgenic mice.This work was presented in part at the 52nd Annual Meeting of the Society for Investigative Dermatology, Seattle, Wash., 1–3 May 1991     

Expression of matrix metalloproteinase (MMP)-2, MMP-9, MMP-13, and MT1-MMP in skin tumors of human papillomavirus type 8 transgenic mice

Human papillomaviruses (HPV) are small DNA viruses that induce a wide variety of hyperproliferative lesions in cutaneous and mucosal epithelia. It is proposed that HPV is involved in non-melanoma skin cancer development. We have previously shown that HPV8 transgenic mice spontaneously develop papillomatous skin tumors. Histology revealed epidermal hyperplasia, acanthosis and hypergranulosis and in some cases squamous cell carcinomas (SCC). Zymographic and immunoblot analysis of normal skin extracts identified increased amounts of matrix metalloproteinase (MMP)-9, MMP-13 and MT1-MMP in HPV8-positive mice compared with HPV8-negative animals. In situ gelatin zymography of tumor specimens displayed a strong proteolytic activity in papillomas, and SCC putatively attributed to the increased amounts of activated MMP-9 found in tissue extracts. In addition, immunoblot analysis revealed increased amounts of active MMP-13 and MT1-MMP in tumor extracts as compared with control extracts. Immunohistochemical stainings of SCC specimens depicted MMP-13 to be specifically expressed in stromal fibroblasts neighboring the tumor islands, whereas MT1-MMP was detected both in tumor cells and in stromal cells. Taken together, these results implicate a role for MMPs in the development of HPV8-induced cutaneous tumors.     

Erythroplasia of queyrat: coinfection with cutaneous carcinogenic human papillomavirus type 8 and genital papillomaviruses in a carcinoma in situ

Erythroplasia of Queyrat is a carcinoma in situ that mainly occurs on the glans penis, the prepuce, or the urethral meatus of elderly males. Up to 30% progress to squamous cell carcinoma. The cause of erythroplasia of Queyrat is largely unknown. Human papillomavirus type 16 DNA has previously been detected only in very few distinctly characterized patients. We have investigated 12 paraffin-embedded biopsies from eight patients with penile erythroplasia of Queyrat and control biopsies of inflammatory penile lesions, of genital Bowen's disease, and of premalignant/malignant cervical or vulvar lesions by 10 different polymerase chain reaction protocols for the presence of cutaneous and genital/mucosal human papillomaviruses. Human papillomavirus typing was performed by sequencing (cloned) polymerase chain reaction products. Human papillomavirus DNA was detected in all erythroplasia of Queyrat patients and in none of the controls with inflammatory penile lesions. The rare cutaneous carcinogenic epidermodysplasia verruciformis-associated human papillomavirus type 8 was present in all erythroplasia of Queyrat patients and the genital high-risk human papillomavirus type 16 in seven of eight patients (88%). In addition to human papillomavirus type 8 and human papillomavirus type 16, four patients carried the genital carcinogenic human papillomavirus type 39 and/or type 51. All human papillomavirus type 8 sequences found in erythroplasia of Queyrat showed some polymorphism among each other and differed in specific nucleotide exchanges from the human papillomavirus type 8 reference sequence. Viral load determinations (human papillomavirus copies/beta-globin gene copies) by realtime polymerase chain reactions showed that the human papillomavirus type 16 levels in the erythroplasia of Queyrat biopsies were one to five orders of magnitude higher than the human papillomavirus type 8 levels. Human papillomavirus type 8 was not detected in cervical or vulvar precancerous and cancerous lesions and in Bowen's disease lesions that carried genital human papillomavirus types. The data suggest that in erythroplasia of Queyrat, in contrast to other genital neoplasias, a coinfection with human papillomavirus type 8 and carcinogenic genital human papillomavirus types occurs. The presence or absence of human papillomavirus type 8 might help to distinguish between penile erythroplasia of Queyrat and Bowen's diseases.     

携人乳头瘤病毒6型全基因细胞的组织工程皮片培养的初步研究

目的 建立人乳头瘤病毒6型（HPV6）全基因体外组织工程皮片培养模型,为进一步研究HPV病毒周期奠定基础。 方法 用电转的方法,将HPV6全长线性基因和质粒pEGFP-EGFP共转染hTERT细胞,G418抗性筛选,Southern印迹法检测细胞内HPV病毒含量;3T3 J2滋养层细胞、I型鼠尾胶原与含HPV6基因的hTERT细胞（HPV6.hTERT细胞）混合后,在金属网格上共同培养,逐渐形成皮片样结构。HE染色和免疫组化检测皮片的组织结构和HPV6 L1蛋白表达,电镜检查皮片病毒颗粒。 结果 HPV6全长线性基因成功转入hTERT细胞,Southern印迹法检测细胞内含HPV6 DNA;与3T3 J2细胞、I型鼠尾胶原共同培养的HPV6.hTERT细胞随时间而增殖分化,逐渐形成具有疣状增生外观的皮片。皮片HE染色显示,培养7 d即出现典型的皮肤分层结构;培养21 d皮片可见明显乳头瘤样增生、空泡细胞、角化过度、角化不全等HPV感染组织病理表现。免疫组化显示皮片上部有HPV6 L1蛋白表达。电镜检测发现皮片中存在HPV6病毒颗粒。 结论 HPV6全基因组织工程皮片培养模型为HPV的生物学研究提供了一个平台,但在应用上有一定的局限性。     

Distinctive distribution of human papillomavirus type 16 and type 20 DNA in the tonsillar and the skin carcinomas of a patient with epidermodysplasia verruciformis

BACKGROUND: Epidermodysplasia verruciformis (EV) is a rare skin disease characterized by disseminated pityriasis versicolor-like or flat wart-like lesions and by the development of skin carcinomas. It is well established that specific cutaneous human papillomaviruses (EV-HPVs) are associated with both benign and malignant skin lesions in EV patients. However, little is known of the relationship between HPV and the mucosal lesions of EV patients. OBJECTIVES: To detect and identify HPV types associated with skin and mucosal lesions of an EV patient. PATIENT/METHODS: We investigated the skin carcinoma and the coexisting tonsillar carcinoma of a 41-year-old man with EV. Histopathologically, both lesions were squamous cell carcinomas. We analysed these two lesions by immunohistochemistry, in situ hybridization, and by molecular virology. RESULTS: Neither skin nor tonsillar lesions exhibited positivity for HPV capsid antigen by immunohistochemistry. By Southern blot hybridization, however, the skin carcinoma harboured 'EV-specific' HPV20 DNA, while the tonsillar carcinoma harboured 'genital' HPV16 DNA. In addition, in situ hybridization localized the respective viral DNA in the corresponding lesion. CONCLUSIONS: The results indicate that EV-HPV could be responsible for the development of the skin carcinoma, but not the mucosal carcinoma in this patient.     

Detection of human papillomavirus type 2a DNA in verrucae vulgares by electron microscopic in situ hybridization

Electron microscopic in situ hybridization (EMISH) of common warts (verrucae vulgares) of the hands was performed using a biotinylated human papillomavirus type 2a (HPV-2a) DNA probe and immunogold labelling of ultrathin sections of 2% glutaraldehyde-fixed, Lowicryl K4M-embedded tissues. It was first established that the warts contained HPV-2a DNA by light microscopic in situ hybridization. The HPV-2a probe chiefly labelled cells in the horny, granular and upper spinous layers of the epidermis, and labelling decreased towards the basal cell layer. The gold particles were located precisely on the viral particles in the nuclei of granular cells. The lower limit of detection by EMISH was found to be the keratinocytes of the third cellular layer above the basal cells. These keratinocytes showed evidence of a viral cytopathic effect, suggesting that vegetative DNA replication in infected keratinocytes occurs at least as early as this level of the epidermis.Presented in part at the 91st Annual Meeting of the Japanese Dermatological Association, Chiba, April 1992     

E6 and E7 of human papillomavirus type 18 and UVB irradiation corporately regulate interleukin‐6 and interleukin‐8 expressions in basal cell carcinoma

The lack of a human papillomavirus (HPV)‐infected skin cancer cell line has hampered the investigation of the interaction of UV and HPV in skin carcinogenesis. We identified a human basal cell carcinoma (BCC‐1/KMC) cell line integrated with E6 and E7 genes of high‐risk HPV type 18 and demonstrated that repression of E6 and E7 results in proliferation inhibition. Sublethal ultraviolet‐B (UVB) irradiation induced the expressions of interleukin‐6 (IL‐6) and interleukin‐8 (IL‐8), as well as viral E6 and E7 genes, in BCC‐1/KMC cells. When E6 and E7 expressions were inhibited, IL‐6/IL‐8 expressions were repressed. Furthermore, IL‐6/IL‐8 remained inducible by UVB irradiation when E6 and E7 were inhibited. These results indicated that IL‐6 and IL‐8 can be upregulated by viral E6 and E7 proteins without UVB irradiation. Moreover, chronic exposure to UVB upregulates IL‐6 and IL‐8 when E6/E7 is induced by UVB.     

HPV16E6蛋白与hDaxx在HeLa细胞的定位及对凋亡的影响

目的 探讨外源人乳头瘤病毒16型E6蛋白(HPV16 E6)与人Daxx蛋白(hDaxx)在HeLa细胞内的亚细胞定位及诱导HeLa细胞凋亡的影响.方法 Western印迹法检测融合蛋白DsRed-HPV 16 E6和EGFP-hDaxx的表达.激光共聚焦显微镜观察HPV16 E6和hDaxx的亚细胞定位.将HeLa细胞分为对照组、TNF-α处理组、转染空载体组、转染HPV16 E6组、共转染HPV16 E6和hDaxx组.后4组均经TNF-α诱导.用流式细胞仪测定细胞凋亡率,分光光度法检测Caspase-8与Caspase-3的相对活性.结果 融合蛋白DsRed-HPV16E6和EGFP-hDaxx在细胞内表达并分布于胞质与胞核,出现共定位现象,且部分hDaxx从胞核转移至胞质.转染E6组凋亡率(21.4％±1.1％)低于转染空载体组(27.0％±0.9％,P＜ 0.01);与转染E6组相比较,共转染组凋亡率(32.5％±2.1％)显著升高(P＜0.01).转染E6组Caspase-8和Caspase-3相对活性分别为0.057±0.003、0.054±0.006,均低于空载体组(0.092±0.012、0.093±0.005,均P＜0.01);与转染E6组相比较,共转染组Caspase-8和Caspase-3相对活性(0.109±0.013、0.110±0.004)均显著升高(P值均＜ 0.01).结论 HPV16 E6使部分hDaxx从胞核转位至胞质,二者发生共定位.HPV16E6蛋白可抑制TNF-α诱导的HeLa细胞凋亡,bDaxx高表达可下调HPV16 E6蛋白这种作用.     

血管内皮生长因子165基因转染小鼠硬皮病皮损的实验研究

目的:探讨硬皮病基因治疗的新方法。方法:建立小鼠硬皮病动物模型后,应用电穿孔技术将血管内皮生长因子(VEGF)165真核表达质粒导入硬皮病小鼠硬化的皮下;再利用免疫组化、原位杂交法检测小鼠硬皮病皮损VEGF165的表达及观察组织病理改变。结果:①转基因后硬皮病鼠毛发生长明显增多,而未转基因硬皮病鼠毛发未见生长。②组织病理检查示转基因鼠毛囊明显增生,毛囊数每一低倍视野平均为(12.0±1.6)个,显著高于未转基因硬皮病鼠组(P<0.05)。真皮厚度增加,新生血管增加。③免疫组化、原位杂交法检测示转基因鼠表皮和真皮细胞及间质VEGF165表达明显增加。结论:电穿孔技术可将外源性基因转入小鼠硬化的皮肤,使其高表达。外源性VEGF165基因转染后,可明显促进小鼠硬化的皮肤毛发生长,已萎缩的真皮组织增生。     

Seropositivity for human papillomavirus and incidence of subsequent squamous cell and basal cell carcinomas of the skin in patients with a previous nonmelanoma skin cancer

Background Infection with human papillomaviruses (HPVs) is a risk factor for several epithelial cancers, but its relationship with keratinocyte tumours has not yet been established. Objective In this prospective study we investigated the possible role of different HPVs in the incidence of a subsequent nonmelanoma skin cancer (NMSC). Methods One hundred and fifty‐three patients with squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) enrolled in a previous case–control study were re‐contacted, and a follow‐up visit was offered. Demographic and clinical data, date of first NMSC presentation, Fitzpatrick skin type and history of NMSC during the follow‐up period were ascertained. Recurrences and new second cancers were considered together as ‘outcomes’ in time‐to‐event analyses and in Cox proportional hazard models. Results Clinical data were obtained in 107 patients. HPV seropositivity at baseline was strongly associated with the risk of developing a second SCC after 5 years for a number of beta and gamma HPV types. For example, HPV‐24‐seropositive patients with an SCC at baseline had a 4‐fold increased risk of developing a subsequent SCC (hazard ratio 4·35, 95% confidence interval 1·2–15·6, P = 0·024). No association between serological status for any HPV type tested and an increased risk of BCC was found. Conclusions We observed a consistent pattern of a positive association between seropositivity for beta and gamma HPV types and the risk of a subsequent SCC in patients with a previous SCC. Our data corroborate the results of previous case–control studies and may spur further prospective studies on the causal role of HPVs in NMSC.     

The neurofibromatosis type 1 (Nf1) tumor suppressor is a modifier of carcinogen-induced pigmentation and papilloma formation in C57BL/6 mice

There is increasing evidence implicating the human NF1 gene in epithelial carcinogenesis. To test if NF1 can play a part in skin tumor formation, we analyzed effects of the skin cancer initiator dimethylbenz-anthracene and/or the tumor promoter 12-O-tetradecanoyl-13-acetylphorbol on mice heterozygous for null mutations in Nf1 (Nf1+/-). Mice were on the C57BL/6 background, noted for resistance to chemical carcinogens. Nf1+/- mice (18 of 24) developed papillomas after treatment with dimethylbenzanthracene and 12-O-tetradecanoyl-13-acetylphorbol; papillomas did not develop in wild-type C57BL/6 mice nor Nf1+/- mice treated with 12-O-tetradecanoyl-13-acetylphorbol alone. All papillomas analyzed (six of six) had mutations in codon 61 of H-ras, demonstrating strong cooperation between the Nf1 GTPase activating protein for Ras, neurofibromin, and Ras-GTP. After exposure to 12-O-tetradecanoyl-13-acetylphorbol, Nf1+/- keratinocytes showed significant, sustained, increases in proliferation, implicating Nf1 in phorbol ester responsive pathways. Thus, Nf1 levels regulate the response of keratinocytes to 12-O-tetradecanoyl-13-acetylphorbol. Nf1+/- mice also showed a 2-fold increase in the development of pigmented skin patches stimulated by dimethylbenzanthracene; patches were characterized by hair follicles in anagen phase, implicating keratinocytes in the aberrant hyperpigmentation. Our results show that mutation in the Nf1 gene causes abnormal keratinocyte proliferation that can be revealed by environmental assaults such as carcinogen exposure. The data support a plausible role for NF1 mutation in human epithelial carcinogenesis.     

人乳头瘤病毒6bL1双顺反子载体的构建及其在哺乳动物细胞内的表达

目的构建尖锐湿疣患者人乳头瘤病毒6b型晚期基因(HPV6b L1)的双顺反子表达载体,并使其在哺乳动物细胞内表达,以期建立含有HPV6b L1的细胞模型.方法表达质粒pEGFP-HPV6bL1经双酶切纯化后与经过相同双酶切的真核表达质粒pIRES2-EGFP连接,酶切鉴定,挑选阳性克隆进行测序.重组质粒pIRES2-HPV6bL1-EGFP转染进小鼠成纤维细胞(NIH3T3),荧光显微镜下观察EGFP蛋白的表达.RT-PCR检测HPV6b L1 mRNA的生成.结果成功构建含HPV6b L1的重组质粒pIRES2-HPV6bL1-EGFP.重组体成功转染进NIH3T3细胞,并用G418筛选.同时荧光倒置显微镜下可观察到细胞内有绿色荧光蛋白的表达.进一步进行RT-PCR,检测到HPV6b L1 mRNA的生成.结论成功构建携带HPV6b L1的重组体pIRES2-HPV6bL1-EGFP并转染入NIH3T3细胞.经荧光倒置显微镜观察及RT-PCR方法检测证明HPV6b L1在NIH3T3细胞内成功表达.     

人乳头瘤病毒6bL1双顺反子载体的构建及其在哺乳动物细胞内的表达

目的 构建尖锐湿疣患者人乳头瘤病毒6b型晚期基因（HPV6b L1）的双顺反子表达载体,并使其在哺乳动物细胞内表达,以期建立含有HPV6b L1的细胞模型。方法 表达质粒pEGFP-HPV6bL1经双酶切纯化后与经过相同双酶切的真核表达质粒pIRES2-EGFP连接,酶切鉴定,挑选阳性克隆进行测序。重组质粒pIRES2-HPV6bL1-EGFP转染进小鼠成纤维细胞（NIH3T3）,荧光显微镜下观察EGFP蛋白的表达,RT-PCR检测HPV6b L1 mRNA的生成。结果 成功构建含HPV6b L1的重组质粒pIRES2-HPV6bL1-EGFP。重组体成功转染进NIH3T3细胞,并用G418筛选。同时荧光倒置显微镜下可观察到细胞内有绿色荧光蛋白的表达。进一步进行RT-PCR,检测到HPV6b L1 mRNA的生成。结论 成功构建携带HPV6b L1的重组体pIRES2-HPV6bL1-EGFP并转染入NIH3T3细胞。经荧光倒置显微镜观察及RT-PCR方法检测证明HPV6b L1在NIH3T3细胞内成功表达。     

人乳头瘤病毒6bL1双顺反子载体的构建及其在哺乳动物细胞内的表达

目的构建尖锐湿疣患者人乳头瘤病毒6b型晚期基因(HPV6b L1)的双顺反子表达载体,并使其在哺乳动物细胞内表达,以期建立含有HPV6b L1的细胞模型.方法表达质粒pEGFP-HPV6bL1经双酶切纯化后与经过相同双酶切的真核表达质粒pIRES2-EGFP连接,酶切鉴定,挑选阳性克隆进行测序.重组质粒pIRES2-HPV6bL1-EGFP转染进小鼠成纤维细胞(NIH3T3),荧光显微镜下观察EGFP蛋白的表达.RT-PCR检测HPV6b L1 mRNA的生成.结果成功构建含HPV6b L1的重组质粒pIRES2-HPV6bL1-EGFP.重组体成功转染进NIH3T3细胞,并用G418筛选.同时荧光倒置显微镜下可观察到细胞内有绿色荧光蛋白的表达.进一步进行RT-PCR,检测到HPV6b L1 mRNA的生成.结论成功构建携带HPV6b L1的重组体pIRES2-HPV6bL1-EGFP并转染入NIH3T3细胞.经荧光倒置显微镜观察及RT-PCR方法检测证明HPV6b L1在NIH3T3细胞内成功表达.