人乳头瘤病毒58型E6蛋白对p53的作用研究

目的 研究HPV58 E6蛋白与p53蛋白的作用关系，以初步探讨其致癌机理。方法 先通过GST沉降试验和体外降解试验，体外研究HPV58 E6结合降解p53蛋白的作用，进而将表达质粒导人SAOS-2细胞，在细胞内研究HPV58 E6对p53诱导细胞凋亡活性的影响。结果 HPV58 E6能够有效结合p53蛋白并进一步诱导其降解，而且在细胞内，HPV58 E6具有抑制p53蛋白的诱导凋亡的功能。结论 p53蛋白的降解影响其细胞周期调控和诱导凋亡的功能，导致细胞发生恶性转化引发子宫颈癌等肿瘤。     

E6-p53和E7-pRb模式在人乳头瘤病毒致癌过程中的作用

在全球范围特别是发展中国家，宫颈癌的发病率居女性恶性肿瘤的第二位。目前认为人乳头瘤病毒（HPV）是宫颈癌的主要致病因子，而早期基因E6、E7是HPV致癌机制研究中的重点。体内体外实验均表明E6、E7是细胞转化所必需的，尤其是在高危型HPV感染机体时，E6结合并灭活肿瘤抑制因子p53、E7结合并降解抑制蛋白pRb，最终导致细胞无限分裂而不发生凋亡。E6、E7分别与抑癌蛋白p53、pRb相互作用的模式为人乳头瘤病毒致癌机制的进一步研究提供了基本的理论基础。     

HPV与宫颈癌关系及疫苗研究进展

流行病学和病原学研究表明,人乳头瘤病毒（HV）感染是妇女发生宫颈癌重要的原因之一.HPV是无包膜的小型双链环状DNA病毒,不同基因型病毒对细胞的转化能力不同,其中HPV-16、18与子宫颈癌关系最密切.HPV诱发官颈癌的主要机制,是其E6和E7蛋白基因在宫颈细胞中的表达增加,产生的E6和E7蛋白两个癌蛋白分别与抑癌蛋白p53和pRb结合而诱导后两者降解.HPV疫苗包括预防性疫苗和治疗性疫苗两大类.本文对HPV感染与宫颈癌发生的关系、疫苗研究进展进行了系统的阐述.     

p16蛋白与HPV mRNA检测在口咽部鳞状细胞癌诊断中的意义

目的 探讨p16蛋白与HPV mRNA检测在口咽部鳞状细胞癌(oropharyngeal squamous cell carcinoma, OPSCC)诊断中的意义。方法 收集2019年11月～2021年10月首都医科大学附属北京同仁医院诊治的105例OPSCC,采用免疫组化EnVision两步法检测p16、Ki-67、p53蛋白表达，并检测其中61例HPV16/18 mRNA的表达，分析其与临床病理特征的关系。结果 105例OPSCC中p16蛋白阳性58例(55.24%),阴性47例(44.76%);p16蛋白阳性患者比阴性患者的吸烟、饮酒史比例低(26/58 vs 37/47,16/58 vs 31/47),以扁桃体为常见发病部位(51/58 vs 18/47),易出现淋巴结转移(43/58 vs 23/47),Ki-67增殖指数较高(73.10%vs 47.45%),p53多为野生型(57/58 vs 16/47),形态学特征以非角化型鳞状细胞癌为主(29/58 vs 15/47),差异均有统计学意义(P<0.05)。61例HPV mRNA检测结果与p16蛋白表达完全一致(...     

牛乳头瘤病毒E6蛋白与p73蛋白的相互作用

为进一步探讨和阐明E6蛋白的致癌机制以及充分认识新发现的抑癌基因p73的作用，在体外通过免疫共沉淀法研究p73和牛乳头瘤病毒1型E6蛋白（BPI－1E6）的相互结合情况，后将表达p73和BPV－1E6的质粒导入SAOS－2细胞内，进一步研究细胞内E6蛋白对p73蛋白的诱导调亡和转录活化功能等生物学活性的影响。结果发现，BPV－1E6与p73蛋白结合较弱，且未检测到E6能诱导p73蛋白的降解。在SAOS－2细胞内，BPV－1E6蛋白确实能部分抑制p73蛋白的诱导细胞凋亡的功能，并且p73蛋白的转录激活作用也有影响，但这种影响作用颇弱。     

食管鳞状细胞癌癌变过程中HPV16/18 E6蛋白与PIK3CA、PIK3CB突变的相关性及其临床意义

目的 探讨HPV感染与PIK3CA、PIK3CB突变在食管鳞状细胞癌(esophageal squamous cell carcinoma, ESCC)癌变过程中的表达差异及其临床意义。方法 采用免疫组化EnVision法检测32例食管正常黏膜、35例食管低级别异型增生(low-grade intraepithelial neoplasia, LGIN)、34例食管高级别异型增生(high-grade intraepithelial neoplasia, HGIN)及137例ESCC中HPV16/18 E6蛋白、p53、PIK3CA、PIK3CB的表达，并分析蛋白表达之间的相关性及与临床病理特征的关系。结果 HPV16/18 E6蛋白在食管正常黏膜组、LGIN组、HGIN组、ESCC组的阳性率分别为0、8.6%、26.5%、29.9%,HPV16/18 E6蛋白在ESCC和HGIN中的表达高于食管正常黏膜和LGIN(P<0.05);p53蛋白在食管正常黏膜组、LGIN组、HGIN组、ESCC组的阳性率分别为9.4%、28.6%、50.0%、68.6%,p53在HGIN中的表达高于...     

HPV与宫颈癌关系及疫苗研究进展

流行病学和病原学研究表明,人乳头瘤病毒(HV)感染是妇女发生宫颈癌重要的原因之一.HPV是无包膜的小型双链环状DNA病毒,不同基因型病毒对细胞的转化能力不同,其中HPV-16、18与子宫颈癌关系最密切.HPV诱发官颈癌的主要机制,是其E6和E7蛋白基因在宫颈细胞中的表达增加,产生的E6和E7蛋白两个癌蛋白分别与抑癌蛋白p53和pRb结合而诱导后两者降解.HPV疫苗包括预防性疫苗和治疗性疫苗两大类.本文对HPV感染与宫颈癌发生的关系、疫苗研究进展进行了系统的阐述.     

人乳头瘤病毒58型衣壳蛋白的表达及其抗体制备

目的：构建人乳头瘤病毒58型(hpv58)衣壳蛋白L1的杆状病毒，昆虫细胞表达系统。方法：用杆状病毒，昆虫细胞表达系统大量表达HPV58L1蛋白，并用HPV58L1病毒相似颗粒(VLP)免疫BALB／c小鼠制备特异性抗血清。结果：HPV58L1蛋白在SF9细胞中被高效表达，其相对分子质量为55kD；CsCI超速离心结合蔗糖密度超速离心可纯化此蛋白。在电镜下可见纯化的病毒衣壳蛋白L1形成VLP。用其免疫小鼠制备的相应抗体能与HPV58L1特异性结合。结论：成功表达了HPV58衣壳蛋白L1，并制备出高滴度的抗体。     

稳定表达人乳头瘤病毒(HPV)58型E6E7融合基因的B16细胞系的建立

目的:构建pIRES-neo-HPV58E6E7真核表达载体,稳定转染入小鼠黑色素瘤B16细胞,建立稳定表达HPV58E6E7基因的B16细胞系。方法:采用PCR方法扩增出HPV58E6E7融合基因的全长序列,利用DNA重组技术将其定向插入到真核表达载体pIRES-neo中,并加入酶切位点和6×his标签,构建重组真核表达质粒pIRES-neo-HPV58E6E7。利用阳离子脂质体介导法将其转染入小鼠黑色素瘤B16细胞,经G418加压筛选出稳定转染的阳性克隆。利用Western blot、流式细胞术、免疫荧光等检测方法验证HPV58E6E7融合基因在稳定转染的B16细胞株中的表达。结果:经PCR、限制性内切酶鉴定及测序分析,pIRES-neo-HPV58E6E7重组质粒构建正确,转染B16细胞株后,经过Western blot、流式细胞术和免疫荧光等检测显示B16细胞株能够稳定、高效表达HPV58E6E7融合基因,表明B16-HPV58E6E7稳定转染细胞系构建成功。结论:成功构建了pIRES-neo-HPV58E6E7真核表达载体,建立了可以高效、稳定表达HPV58E6E7融合基因的B16细胞系。该稳定转染细胞系的建立为进一步研究HPV58治疗性基因疫苗的功能提供了良好的靶细胞,为其在肿瘤免疫治疗中的应用奠定了研究基础。     

稳定表达人乳头瘤病毒58型(HPV58)E6E7融合基因的人宫颈癌细胞株C33A的构建及验证

为构建稳定表达人乳头瘤病毒(HPV)58型E6E7融合基因的人宫颈癌C33A细胞系,将实验前期构建好并实现真核表达的重组慢病毒颗粒LV-HPV58E6E7转染入HPV(–)的人宫颈癌C33A细胞,经流式细胞仪分选出稳定转染的阳性克隆,利用四唑盐比色(MTT)法检测转染后细胞的生长情况以及流式细胞术检测细胞周期,并将稳定表达HPV58E6E7融合基因的C33A细胞LV-HPV58E6E7/C33A接种于裸鼠左腋下成瘤,用荧光定量PCR(q RT-PCR)、Western blot检测瘤组织中HPV58型E6E7融合基因的转录和表达。结果显示HPV58E6E7融合基因可促进C33A细胞的增殖;LV-HPV58E6E7/C33A细胞株能在裸鼠体内稳定转录及表达HPV58型E6E7融合基因。这表明我们成功建立了能稳定表达HPV58E6E7融合基因的人宫颈癌C33A细胞系LV-HPV58E6E7/C33A,为HPV58型治疗性疫苗的免疫效果检测提供了抗原细胞来源。     

Degradation of p53 only is not sufficient for the growth stimulatory effect of human papillomavirus 16 E6 oncoprotein in human embryonic fibroblasts

Certain types of human papillomavirus (HPV), such as types 16 and 18, are thought to be responsible for the development of cervical carcinomas. The E6 and E7 genes of these viruses have transforming activities in various cultured cells and their mRNAs and proteins are expressed in almost all cervical carcinoma cells. Inactivation of the tumor suppressor p53 protein by the E6 gene is believed to be critical for transformation by these oncogenic HPVs. To determine whether degradation of the p53 protein is, in fact, sufficient for cellular transformation by the E6 gene, the E6 gene of HPV16 was introduced into human embryonic fibroblasts (HEF) using recombinant murine retrovirus and examined whether reduction of the p53 protein could substitute for the E6 function. It was found that HEF cells transfected with the E6 gene showed an increased saturation density and degraded the p53 protein. However, when expression of the p53 protein in normal HEF cells was suppressed by the antisense oligonucleotide of the p53 gene, growth stimulation was not observed. These results show that the E6 gene stimulates growth of HEF cells, but that this activity involves some other E6 gene-mediated functions than degradation of the p53 protein. © 1994 Wiley-Liss, Inc.     

Increased angiogenesis in the uterine cervix associated with human papillomavirus infection

We studied the relationship between angiogenesis (using the CD34 antibody), the presence of human papilloma virus (HPV) infection, HPV E6 protein expression and the accumulation of p53 protein at various phases of tumour progression in the uterine cervix. Expression of CD34, p53 and HPV E6 protein was evaluated by immunocytochemistry. Presence of the mutant p53 was detected using a mutant specific ELISA, and the type of HPV was determined by the Polymerase Chain Reaction. A total of 230 cervical tissue samples were analyzed and included 40 cases of apparently normal cervical epithelium, 37 low grade squamous intraepithelial lesions (SILs), 43 high grade SILs, 36 well-differentiated squamous cell carcinomas (DSCC), 31 moderately differentiated (MDSCC) and 43 poorly differentiated carcinomas (PDSCC). There was an excellent correlation between the extent of angiogenesis and histological abnormality (r = 0.912, p = 0.000004). The least extent of angiogenesis was seen in normal cervical tissue and low grade SILs where the mean (low power) intra lesional vascular density (ILVD) was 12 +/- 1.13 and 25.66 +/- 5.20, respectively. In high grade squamous intraepithelial lesions (SILs), the mean ILVD value was 80.84 +/- 25.57. In well-differentiated squamous cell carcinomas (WDSCC's) the mean value was 144.22 +/- 28.67 while in moderately differentiated squamous cell carcinomas (MDSCC's) the mean value was 166.29 +/- 34.95 and in poorly differentiated tumours (PDSCC's) 192.42 +/- 27.98. The extent of angiogenesis also correlated to presence of HPV (r = 0.505, p = 0.00001). Increased CD34 expression was associated with the presence of HPV types 16 and 18. A similar correlation was also evident in HPV, 16/18 infected cases expressing the E6 protein (r = 0.612, p = 0.000001). CD34 expression also correlated well with p53 accumulation (r = 0.859, p = 0.000002). Presence of HPV infection significantly correlated with the extent of histological abnormality (r = 0.467, p = 0.00001). Expression of E6 also showed this significant correlation (r = 0.644, p = 0.00002). Accumulation of p53 was significantly more elevated in HPV 16-infected lesions (r = 0.518, p = 0.00001) and E6-expressing cells (r = 0.650, p = 0.000004). Only 12 of the 230 cases analyzed showed presence of the mutant p53 protein. Angiogenesis appears to increase with histological abnormality in the uterine cervix. Angiogenesis also appears to be influenced by high risk HPV infection, the expression of the E6 transforming protein and the p53 tumour suppressor protein.     

Immunohistochemical detection of p53 protein in cutaneous lesions from transplant recipients harbouring human papillomavirus DNA

Human papillomaviruses (HPV) are thought to be involved in the malignant evolution of cutaneous lesions from transplant recipients. As E6 proteins from potentially oncogenic HPV types degradep53 tumour suppressor gene product in vitro, we analysed p53 protein status in benign, premalignant and malignant skin lesions from grafted patients, to determine whether HPV may interfere with p53 function. With immunohistochemistry, p53 protein accumulation was detected in 70% of skin lesions from grafted patients. p53 immunoreactivity was confined to basal keratinocytes in benign lesions (warts, condylomas), while suprabasal keratinocytes were also stained in premalignant and malignant skin lesions (precancerous keratoses, squamous cell carcinomas). Multiple HPV carriage was detected with in situ hybridization in benign and malignant skin lesions from transplant recipients: low risk HPV types 1, 2, 6, 11 and potentially oncogenic HPV types 5, 16, 18 were frequently found. There was no clear correlation between p53 detection and the presence of the HPV types under study. The frequent detection of p53 protein in cutaneous lesions from grafted patients is suggestive of p53 protein accumulation interfering with normal function. Our results may reflect the presence of mutated p53 proteins due to the mutagenic effect of ultra-violet (UV), or wild-type p53 protein accumulation in response to UV-induced DNA damage, or may be produced by the interaction with HPV-encoded E6 proteins.     

The role of TP53 in Cervical carcinogenesis

Functional loss of the tumor suppressor p53 by alterations in its TP53 gene is a frequent event in cancers of different anatomical regions. Cervical cancer is strongly linked to infection by high-risk human papillomavirus (HPV) types. The viral oncoprotein E6 has the ability to associate with and neutralize the function of p53. E6 interacts with a 100-kDa cellular protein, termed E6 associated protein (E6AP; also called ubiquitin-protein ligase E3A or UBE3A), which functions as an ubiquitin protein ligase. The dimeric complex then binds p53 and E6AP catalyzes multi-ubiquitination and degradation of p53. The ability to promote p53 degradation is an exclusive property of E6 from the high-risk HPV types. Indeed, the low-risk E6 proteins lack this activity, although they can bind p53. Consistent with the E6 function of the high-risk HPV types, the majority of cervical cancer cells have a wild-type p53 gene, but the protein levels are strongly decreased. Several independent studies have shown that in a small percentage of cervical tumors the p53 gene is mutated. However, this event appears to be unrelated to the presence or absence of HPV infection and the nature of the tumor.     

p53 gene mutations and MDM2 amplification are uncommon in primary carcinomas of the uterine cervix.

The p53 gene is the most frequently altered gene known thus far in a wide variety of human cancers. Inactivation of p53, either through mutation or through interaction with the human papillomavirus (HPV) E6 oncoprotein, is a characteristic feature of all cervical carcinoma cell lines that have been studied. These findings suggest that p53 inactivation is required for cervical carcinoma development and that HPV infection and p53 mutation may be mutually exclusive. We have studied the p53 gene in 35 primary cervical carcinomas. DNA sequence and single strand conformational polymorphism analyses were used to evaluate p53 in 27 squamous carcinomas (25 HPV-positive) and eight adenocarcinomas (four HPV-positive). A missense mutation of p53 was observed in one HPV 16-positive squamous carcinoma, demonstrating that p53 mutations can occur in combination with HPV infection. The HPV-negative tumors all lacked p53 gene mutations. The absence of p53 mutations in HPV-negative cases prompted an assessment of tumors for MDM2 gene amplification. The MDM2 gene encodes a p53 binding protein and has been found to be amplified in some human tumors lacking p53 mutations. MDM2 amplification was not identified in any of the tumors we examined, including four HPV-negative cases. Our findings show that HPV infection and p53 gene mutation are not mutually exclusive and suggest that many HPV-negative carcinomas may arise via a pathway independent of p53 inactivation.     

Expression of p16, Rb, and p53 proteins in squamous cell carcinomas of the anorectal region harboring human papillomavirus DNA.

Human papillomavirus (HPV) has been implicated as an etiologic agent for the development of squamous cell carcinoma of the anorectal region. It has been shown that the HPV E6 and E7 oncoproteins are able to inactivate the tumor suppressor functions of p53 and Rb. In cervical and head and neck cancers, HPV infection is also associated with an overexpression of p16, a cyclin-dependent kinase inhibitor. The expression of these cell cycle regulators in squamous cell carcinomas of the anorectal region has not been well studied. In the current study, 29 cases of squamous cell carcinoma of the anorectal region were immunohistochemically examined for the expression of p16, Rb, and p53 proteins. Tumor cell DNA was also extracted from paraffin blocks and subjected to broad-spectrum HPV DNA testing and typing. The results show that the tumor cells exhibited a strong and diffuse nuclear stain (with some cytoplasmic positivity) for p16 in all 29 cases (100%). The adjacent nonneoplastic squamous epithelium or colonic mucosa, in contrast, was completely negative. Loss of Rb nuclear staining in tumor cells was observed in 20 cases (69%). The p53 protein was essentially undetectable, with only 6 cases containing <10% positive cells. HPV DNA was detected in every case (100%), with 25 cases (86%) harboring Type 16. In addition, almost identical results were obtained in 12 HPV-positive squamous cell carcinomas of the upper aerodigestive tract. This was in marked contrast to those of HPV-negative tumors, where positive p16 staining and loss of Rb expression were seen in only 2/21 (10%) and 1/21 (5%) cases, respectively. These observations indicate that overexpression of p16 and loss of Rb nuclear staining are commonly associated with high-risk HPV infection, which may serve as useful surrogate biomarkers for identifying squamous cell carcinomas harboring HPV DNA.     

HPV 16 E6 blocks TNF-mediated apoptosis in mouse fibroblast LM cells.

The interaction between hosts and the viruses that infect them is a dynamic one, and a growing literature documents the fact that many viruses have developed mechanisms designed to avoid elimination by the host immune system. One of the immune strategies used by the host and targeted by virus proteins is apoptosis triggered by the cytokine tumor necrosis factor (TNF). Mouse fibroblast LM cells are spontaneously sensitive to TNF. When the wild-type E6 protein from the human papillomavirus type 16 (HPV 16) was expressed in LM cells, the cells became resistant to TNF. This resistance was examined by several means, including cell morphology, the dose- and time-independent response to TNF in a cell death ELISA, trypan blue exclusion, and cell proliferation. The level of p53 did not rise in TNF-treated cells prior to apoptosis, suggesting a p53-independent mechanism. Significant, though not complete, resistance to TNF was also observed following transfection of a plasmid expressing a mutant E6 protein, which is unable to mediate rapid degradation of the p53 tumor suppressor. These results indicate that the HPV 16 E6 protein can protect LM cells from TNF-triggered apoptosis and likely does so by a mechanism other than mediation of p53 degradation.     

Cellular steady-state levels of "high risk" but not "low risk" human papillomavirus (HPV) E6 proteins are increased by inhibition of proteasome-dependent degradation independent of their p53- and E6AP-binding capabilities

The group of mucosal epithelia-infecting human papillomaviruses (HPV) can be subdivided in "low" and "high risk" HPV types. Both types induce benign neoplasia (condyloma), but only the infection with a "high risk" HPV type is causally associated with an increased risk of developing anogenital tumors. The oncogenic potential of high risk HPVs resides at least partially in the viral E6 protein. The E6 protein targets the cellular p53 protein for proteasome-dependent degradation, which is associated with the immortalizing and transforming functions of these viruses. Recently the E6-dependent proteasome-mediated destabilization of additional cellular proteins (E6TP1, c-myc, Bak, hMCM7, human scribble, E6AP, MAGI-1) has been described, but the cellular mechanisms controlling the viral E6 protein stability itself have been so far not analyzed. In this study, we transiently expressed the E6 genes of the high risk HPV type 16, the low risk HPV types 6a and 11, and the cutaneous epithelia-infecting HPV types 5 and 8 from a eucaryotic expression vector and compared the cellular steady-state levels of the expressed E6 proteins. We demonstrated that the high risk HPV 16 E6 protein possesses the lowest steady-state level in comparison to the low risk HPV type E6 proteins and the cutaneous epithelia-infecting HPV type E6 proteins. Inhibition of cellular proteasome-dependent protein degradation led to an increase in steady-state levels of high risk but not of low risk E6 proteins. Analysis of functionally deficient HPV 16 E6 proteins in p53 null- and p53 wild-type-expressing cell lines revealed that the cellular steady-state level of this protein is influenced neither by its p53- nor its E6AP-binding abilities.