乳腺癌HPV16感染对iNOS、P53蛋白表达的影响

目的研究人乳头状瘤病毒（HPV16）在乳腺癌中的表达及作用机制,检测HPV16、诱导一氧化氮合酶（iNOS）及P53蛋白在乳腺癌的表达及其间的相关性。方法应用免疫组化SP法,共检测了52例乳腺癌和16例乳腺良性瘤HPV16、iNOS和P53蛋白的表达。结果HPV16和P53蛋白在乳腺癌组的阳性表达率均显著高于乳腺良性瘤组。统计分析表明,iNOS和P53蛋白表达阳性率与HPV感染率密切相关（P〈0．05）。结论HPV16感染参与了乳腺癌的发生,其致癌机制可能是通过诱导iNOS表达增加,产生NO,诱导p53基因突变,从而导致乳腺癌的发生。     

凋亡相关基因p53、bcl-2在乳腺导管非典型增生中表达的研究

为观察凋亡相关基因-p53,bcl-2在乳腺导管非典型增生及乳腺癌中的表达,探讨其与乳腺癌组织发生的关系。本实验应用原位杂交方法检测凋亡相关基因p53,bcl-2mRNA,应用免疫组织化学方法检测p53蛋白在44例乳腺导管非典型增生组织中的表达,并与6例乳腺导管单纯性增生及26例乳腺癌对比分析,实验结果为,p53mRNA在乳腺导管单纯性增生组织中呈较强表达（66.7%),在乳腺导自欺欺人这非典型增生组织中阳性表达为40%（轻度;55.6%,中度：41.7%,重度;26.1%)。在癌组织中的表达率为19.2%(导管内癌：21.4%,浸润性导管癌：16.7%),p53蛋白在导管单纯性增生组无表达,在导管非典型增生组阳性表达为24%（轻度：11.1%,中度;25%,重度;34.8%)。在癌组织中的阳性表达为38.4%(导管内癌：35.7%,浸润性导管癌：41.7%),bcl-2mRNA在单纯性增生组无表达,在非典型增生组中阳性表达为轻度：11.1%,中度：16.7%,重度：39.1%,在乳腺癌组织中阳性表达为导管内癌;78.6%,浸润性导管癌83.3%。实验结果表明,在乳腺导管重度非典型增生组织中可检测到p53基因有较高的表达缺失,突变及bcl-2mRNA过表达。     

宫颈癌中p53表达和病毒感染的研究

应用PCR对121例宫颈脱落细胞和组织分别检测HPV-16／18和HSV-2DNA。同时应用免疫组化S-P法检测76例宫颈组织中P53过度表达。结果发现,宫颈癌组织中HPV-16／18和HSV-2阳性率分别为61．3％和32．3％,慢性宫颈炎组HPV-16／18和HSV-2阳性率分别为22．5％和20．0％,与正常宫颈组比较均有显著性差异。宫颈癌中HPV-16／18和HSV-2混合感染率为16．1％。p53过度表达率在慢性宫颈炎、宫颈上皮内瘤变(CIN)、宫颈癌组织中呈梯度递增。另外,宫颈癌组织中P53过度表达与HPV-16／18、HSV-2的感染无相关性。提示：宫颈癌的发生与HPV-16／18关系密切,HSV-2可能与HPV-16／18协同作用导致宫颈癌的发生。宫颈组织中p53过度表达率与宫颈癌的进程有关,这在宫颈癌的防治方面有一定的临床意义。     

Multiple Functions of Human Papillomavirus Type 16 E6 Contribute to the Immortalization of Mammary Epithelial Cells

The E6 proteins from cervical cancer-associated human papillomavirus (HPV) types such as HPV type 16 (HPV-16) induce proteolysis of the p53 tumor suppressor protein through interaction with E6-AP. We have previously shown that human mammary epithelial cells (MECs) immortalized by HPV-16 E6 display low levels of p53. HPV-16 E6 as well as other cancer-related papillomavirus E6 proteins also binds the cellular protein E6BP (ERC-55). To explore the potential functional significance of these interactions, we created and analyzed a series of E6 mutants for their ability to interact with E6-AP, p53, and E6BP in vitro. While there was a similar pattern of binding among these E6 targets, a subset of mutants differentiated E6-AP binding, p53 binding, and p53 degradation activities. These results demonstrated that E6 binding to E6-AP is not sufficient for binding to p53 and that E6 binding to p53 is not sufficient for inducing p53 degradation. The in vivo activity of these HPV-16 E6 mutants was tested in MECs. In agreement with the in vitro results, most of these p53 degradation-defective E6 mutants were unable to reduce the p53 level in early-passage MECs. Interestingly, several mutants that showed severely reduced ability for interacting with E6-AP, p53, and E6BP in vitro efficiently immortalized MECs. These immortalized cells exhibited low p53 levels at late passage. Furthermore, mutants defective for p53 degradation but able to immortalize MECs were also identified, and the immortal cells retained normal levels of p53 protein. These results imply that multiple functions of HPV-16 E6 contribute to MEC immortalization.     

cyclin D1, cyclin E 在乳腺良、恶性病变中的表达及其与p16,p21waf1及Rb 蛋白的关系

为探讨cyclinD1,cyclinE在乳腺癌发生发展中的作用及其与细胞周期调控相关基因蛋白的关系,采用免疫组化检测17例非增生乳腺导管上皮、19例不同程度增生的导管上皮及59例乳腺癌中cyclinD1,cyclinE,p16,p21waf1及Rb基因蛋白的表达.结果显示1.非增生乳腺导管上皮除1例cyclinE过表达外均无cyclinD1和cyclinE的过表达.乳腺癌的cyclinD1和cyclinE的过表达率均明显高于良性乳腺组织(P<0.05).2.乳腺癌cyclinD1过表达与淋巴结转移呈正相关(P<0.05),瘤体直径大于5cm者cyclinE过表达呈增加趋势,但差异无显著性意义.3.CyclinD1和cyclinE的过表达呈正相关(P<0.05).4.从非增生乳腺导管上皮到增生直至乳腺癌,p16,p21与cyclinD1,cyclinE含量的比值逐渐递减,而p21含量高于cyclinD1的乳腺癌体积小、淋巴结转移率低(P<0.05).p21阳性率与cyclinD1过表达呈正相关(P<0.01),也随cyclinE的过表达呈上升趋势.Rb基因蛋白的强表达与cyclinD1过表达呈正相关(P<0.01).结果表明CyclinD1和cyclinE蛋白过表达频发于乳腺癌早期,它们可能与p16、p21waf1、pRb共同参与了乳腺癌的发生发展.     

Enhanced degradation of p53 protein in HPV-6 and BPV-1 E6-immortalized human mammary epithelial cells.

Normal mammary epithelial cells are efficiently immortalized by the E6 gene of human papillomavirus (HPV)-16, a virus commonly associated with cervical cancers. Surprisingly, introduction of the E6 gene from HPV-6, which is rarely found in cervical cancer, or bovine papillomavirus (BPV)-1, into normal mammary cells resulted in the generation of immortal cell lines. The establishment of HPV-6 and BPV-1 E6-immortalized cells was less efficient and required a longer period in comparison to HPV-16 E6. These HPV-6- and BPV-1 E6-immortalized cells demonstrated dramatically reduced levels of p53 protein by immunoprecipitation. While the half-life of p53 protein in normal mammary epithelial cells was approximately 3 h, it was reduced to approximately 15 min in all the E6-immortalized cells. These results demonstrate that the E6 genes of both high-risk and low-risk papilloma viruses immortalize human mammary epithelial cells and induce a marked degradation of p53 protein in vivo.     

Presence of Human Papillomavirus DNA in Malignant Neoplasia and Non-Malignant Breast Disease

Breast cancer is the leading cause of cancer death among women worldwide. Multiple extrinsic and intrinsic factors are associated with this disease’s development. Various research groups worldwide have reported the presence of human papillomavirus (HPV) DNA in samples of malignant breast tumors. Although its role in mammary carcinogenesis is not fully understood, it is known that the HPV genome, once inserted into host cells, has oncogenic capabilities. The present study aimed to detect the presence of HPV DNA in 116 breast tissue biopsies and classify them according to their histology. It was found that 50.9% of the breast biopsies analyzed were malignant neoplasms, of which 74.6% were histologically classified as infiltrating ductal carcinoma. In biopsies with non-malignant breast disease, fibroadenoma was the most common benign neoplasm (39.1%). Detection of HPV DNA was performed through nested PCR using the external primer MY09/11 and the internal primer GP5+/6+. A hybridization assay genotyped HPV. HPV DNA was identified in 20.3% (12/59) of malignant neoplasms and 35% non-malignant breast disease (16/46). It was also detected in 27.3% (3/11) of breast tissue biopsies without alteration. However, there are no statistically significant differences between these groups and the existence of HPV DNA (p = 0.2521). Its presence was more frequent in non-malignant alterations than in malignant neoplasias. The most frequent genotypes in the HPV-positive samples were low-risk (LR) HPV-42 followed by high-risk (HR) HPV-31.     

HPV-16、EB病毒对细胞增殖的影响及与子宫颈癌发病的关系

目的 了解宫颈癌患者人乳头瘤病毒16（HPV 16）、EB病毒（EBV）感染情况,探讨HPV-16、EBV对细胞核增殖性抗原（PCNA）表达的影响及在子宫颈癌发病中的意义。方法 免疫组织化学SP法检测59例宫颈癌和20例非癌性子宫颈上皮细胞中HPV-16、EBV蛋白表达和PCNA表达的情况,并分析它们的表达与病理参数的关系。结果 子宫颈癌和非癌性子宫颈上皮细胞中HPV-16、EBV及PCNA的阳性表达率依次分别为69．49％、57．63％及77．97％和30％、25％及15％;子宫颈癌和非癌性子宫颈上皮细胞中HPV-16、EBV的共同阳性表达率分别为35．59％和0％,子宫颈癌中HPV-16、EBV及PCNA的阳性表达率均高于非癌性子宫颈上皮（P〈0．05）。病理Ⅰ级、Ⅱ级和Ⅲ级子宫颈癌中HPV-16、EBV、PCNA的阳性表达率分别为65．00％、55．00％、60．00％,69．57％、60．87％、82．61％和75．OO％、56．25％、93．75％。各级子宫颈癌间HPV-16、EBV的阳性表达率差异无显著性,但PCNA的表达率随病理分级的增加显著上升（P〈0．05）。不同期别子宫颈癌问HPV-16、EBV及PCNA的阳性表达率差异无显著性。HPV-16阳性与阴性组子宫颈上皮PCNA的阳性表达率分别为82．98％（39／47）与43．75％（14／32）,两者差异有显著性（P〈0．05）。EBV阳性及阴性组子宫颈上皮PCNA的阳性表达率分别为71．79％（28／39）及42．50％（17／40）,两者差异有显著性（P〈0．05）。HPV-16和EBV共同阳性表达的21例子宫颈癌PCNA阳性表达率均为100％。结论 HPV-16、EBV通过增加PCNA表达的促细胞增生作用可能是子宫颈癌的发生机制之一。     

Immunohistochemical analysis of p53 and HER-2/neu proteins in human tumors.

We examined samples of tumors of human breast, ovary, and colon of various degrees of malignancy for the expression of p53 protein, using a panel of anti-p53 antibodies and peroxidase immunohistochemistry. Of 66 tumor cases (24 cases of ovarian carcinoma, 23 cases of colon adenocarcinoma, and 19 cases of breast carcinoma), 36 (53%) showed high levels of expression of p53 using a human-specific antibody, and 16 (24%) showed high expression of a mutant form of p53. In the mutant p53-positive breast tumor samples, six (86%) were positive for HER-2/neu reactivity, compared with colon (0/4) and ovarian tumors (1/5). The pattern of p53 intracellular localization and tissue distribution, and the relationship between the expression of mutant p53 and cell differentiation, were also examined; poorly differentiated cells showed either overexpression of p53 or higher levels of mutant p53 in comparison with more normal cells.     

乳腺癌及良性乳腺组织p185和p16蛋白表达的免疫组织化学研究

应用免疫组化S P法检测了 37例良性乳腺组织 (非上皮增生组 17例、上皮增生组 2 0例 )和 5 9例乳腺癌组织中癌基因蛋白 p185和抑癌基因 p16蛋白的表达状况。结果显示非上皮增生组、上皮增生组和癌的p185阳性率分别为 0 %、15 %和 47%(p <0 0 1) ;p16阳性率分别为 41%、30 %和 34%。p185和p16的表达无明显相关性。乳腺癌早期的 p185过表达和p16失表达率高于浸润性导管癌。两者的阳性率均随组织学级别的增高和瘤体的增大而呈上升趋势 ,但 p >0 0 5。淋巴结转移组的p185阳性率 ( 6 4%)明显高于无淋巴结转移者 ( 32 %) ,p <0 0 5。表明 p185过表达和p16失表达在乳腺癌的发生发展中各自发挥独立的作用。p185是乳腺癌重要的肿瘤标志物。     

E6/E7 expression of human papillomavirus type 20 (HPV-20) and HPV-27 influences proliferation and differentiation of the skin in UV-irradiated SKH-hr1 transgenic mice

The functional role of UV irradiation, in combination with the E6 and E7 proteins of the cutaneous human papillomavirus (HPV) types in the malignant conversion of benign papillomatous lesions, has not been elucidated. Transgenic SKH-hr1 hairless mice expressing HPV-20 and HPV-27 E6 and E7 proteins in the suprabasal compartment were generated and exposed to chronic UV irradiation. Histological and immunohistochemical examination of skin samples revealed enhanced proliferation of the epidermal layers and papilloma formation in both transgenic strains in comparison to what was observed with nontransgenic mice. Squamous cell carcinoma developed in the HPV-20 E6/E7 transgenic line as well as in the HPV-27 E6/E7 transgenic line. Several weeks after cessation of UV-B exposure, enhanced proliferation, as measured by BrdU incorporation, was maintained only in HPV-20 transgenic skin. Keratin 6 expression was increased in the transgenic mice throughout all cell layers. Expression of the differentiation markers involucrin and loricrin was reduced and disturbed. p63alpha expression was differentially regulated with high levels of cytoplasmic expression in clusters of cells in the granular layer of the skin in the transgenic lines 20 weeks after cessation of UV-B exposure, in contrast to uninterrupted staining in the nontransgenic lines. p53 was expressed in clusters of cells in nontransgenic and HPV-27 transgenic mice, in contrast to an even distribution in a higher number of cells in HPV-20 transgenic animals.     

Human papillomavirus type 16 E6 and HPV-16 E6/E7 sensitize human keratinocytes to apoptosis induced by chemotherapeutic agents: roles of p53 and caspase activation

We and others have previously reported that human papillomavirus (HPV)-16 E6 protein expression sensitizes certain cell types to apoptosis. To confirm that this sensitization occurred in HPV's natural host cells, and to explore the mechanism(s) of sensitization, we infected human keratinocytes (HKCs) with retroviruses containing HPV-6 E6, HPV-16 E6, HPV-16 E7, or HPV-16 E6/E7. Apoptosis was monitored by DNA fragmentation gel analysis and direct observation of nuclei in cells stained with DAPI. Exposure of HKCs to etoposide, cisplatin, mitomycin C (MMC), atractyloside, and sodium butyrate, resulted in a time and dose-dependent induction of apoptosis. Expression of HPV-16 E6 and HPV-16 E6/E7, but not HPV-6 E6 or HPV-16 E7, enhanced the sensitivity of HKCs to cisplatin-, etoposide- and MMC-, but not atractyloside- or sodium butyrate-induced apoptosis. Expression of both HPV-16 E6 and HPV-16 E6/E7 decreased, but did not abolish, p53 protein levels relative to normal HKCs, and resulted in cytoplasmic localization of wt p53. P53 induction occurred in HPV-16 E6 and HPV-16 E6/E7 expressing cells after exposure to cisplatin or MMC, though never to levels found in normal untreated HKCs. P21 levels were decreased in HPV-16 E6 and HPV-16 E6/E7 expressing HKCs, and no induction of p21 was seen in these cells following exposure to cisplatin or MMC. Caspase-3 activity was found to be elevated in HPV-16 E6-expressing HKCs following exposure to cisplatin and MMC as documented by fluorometric and Western Blot analysis. Expression of wt CrmA or treatment of HPV-16 E6 expressing HKCs with the caspase-3 inhibitor DEVD.fmk prevented HPV-16 E6-induced sensitization in HKCs. These results suggest that HPV-16 E6 and HPV-16 E6/E7 expression sensitizes HKCs to apoptosis caused by cisplatin, etoposide and MMC, but not atractyloside or sodium butyrate. The data also suggest that wt p53 and caspase-3 activity are required for HPV-16 E6 and HPV-16 E6/E7-induced sensitization of HKCs to DNA damaging agents.     

TSHR蛋白在宫颈癌的表达及其与HPV-16感染的关系

目的研究促甲状腺激素受体（TSHR）在子宫颈癌组织的表达及其与乳头瘤病毒（HPV－16）的关系。方法应用免疫组织化学链霉菌抗生物素过氧化物酶（SP）法检测79例子宫颈癌和30例子宫颈炎组织HPV－16与TSHR蛋白表达。79例癌症患者中病理分级〈Ⅱ级33例,≥Ⅱ级46例;病理分期〈Ⅱ期56例,≥Ⅱ期23例;无淋巴结转移66例,有淋巴结转移13例;肿瘤大小〈3cm44例,肿瘤大小≥3cm35例。结果HPV－16在子宫颈癌表达率55．70％明显高于宫颈炎5％（P〈0．05）,TSHR在子宫颈癌表达率68．35％明显高于宫颈炎26．67％（P〈0．05）。HPV－16表达与肿瘤的大小、肿瘤分级、分期、淋巴结转移不相关。TSHR表达与肿瘤的大小呈正相关,P〈0．05,与肿瘤分级、分期及淋巴结转移不相关。HPV－16与TSHR在宫颈癌表达呈正相关。结论HPV感染对宫颈癌病变起到强烈的预警作用。TSHR不仅在甲状腺滤泡上皮细胞表达,在子宫颈癌细胞也表达,TSHR过表达能促进宫颈细胞的异常增殖,其异常功能可能是恶性肿瘤特定的临床表型。HPV与TSHR在子宫颈癌变过程中起协同作用。     

p16和cyclinD1在乳腺导管非典型增生癌变过程中的表达

目的 探讨乳腺导管非典型增生癌变过程中p16和cyclinD1的变化及其相互关系。方法 应用免疫组织化学方法检溯p16、cyclin成在乳腺各组病变中的蛋白表达情况。结果 乳腺导管单纯性增生、非典型增生组织中p16蛋白表达率明显高于乳腺癌组织,差异有显性。cyclinD1在中,重度非典型增生组表达最为明显,且与乳腺导管单纯性增生组及轻度非典型增生组比较,差异均有显性。p16、cyclinD1蛋白在乳腺各组病变中表达呈负相关关系。结论 p16、cyclinDl在乳腺增生性病变中呈现不同程度表达,其表达强度在一定程度上与细胞的恶性倾向有关,检测其表达水平可作为乳腺导管非典型增生组织恶性转化的一个客观检测指标。其中,cyclinD1蛋白可能是乳腺癌发生过程中的早期分子事件,可作为临床早期发现乳腺癌的免疫学指标。     

Productive replication of adeno-associated virus can occur in human papillomavirus type 16 (HPV-16) episome-containing keratinocytes and is augmented by the HPV-16 E2 protein

We used a sensitive assay to test whether an adeno-associated virus (AAV) productive replication cycle can occur in immortalized human keratinocytes carrying episomal human papillomavirus type 16 (HPV-16) DNA. Following transfection with cloned AAV DNA, infectious AAV was produced, and the infectivity was blocked by anti-AAV antiserum. The HPV-16 E2 protein substantially increased the yield of AAV. Other HPV early proteins did not, in our experiments, show this ability. E2 has been shown to be able to affect p53 levels and to block cell cycle progression at mitosis. We tested the effect of changes in p53 expression on AAV replication and found that large differences in the level of p53 did not alter AAV DNA replication. In extension of this, we found that cellular help for AAV in response to stress was also independent of p53. To test if a mitotic block could trigger AAV DNA replication, we treated the cells with the mitotic inhibitor nocodazole. AAV DNA replication was stimulated by the presence of nocodazole in these and a number of other cell types tested. Yields of infectious virus, however, were not increased by this treatment. We conclude that the HPV-16 E2 protein stimulates AAV multiplication in these cells and propose that this occurs independently of the effects of E2 on p53 and cell cycle progression. Since the effect of E2 was not seen in keratinocytes lacking the HPV-16 episome, we suggest that E2 can help AAV by working in concert with other HPV-16 proteins.