The human papillomavirus type 16 E5 oncoprotein synergizes with EGF-receptor signaling to enhance cell cycle progression and the down-regulation of p27

E5 oncoprotein activity from high risk human papillomaviruses (HPVs) is associated with growth factor receptor signaling, but the function of this protein is not well understood. In this study, we investigated the role of HPV-16 E5 on the cell cycle progression during EGF-stimulation. Wild-type and NIH 3T3 cells over-expressing human EGF-receptor were transfected with HPV-16 E5 gene and the cell cycle progression was characterized. This analysis showed that the E5-expressing cells increased DNA synthesis (S-phase) by around 40%. Cell cycle protein analysis of E5-expressing cells showed a reduction in the half-life of p27^Kip1 protein as compared to control cells (18.4 vs. 12.7 h), an effect that was enhanced in EGF-stimulated cells (12.8 vs. 3.6 h). Blockage of EGF-receptor activity abrogated E5 signals as well as p27^Kip1 down-regulation. These results suggest that E5 and the EGF-receptor cooperate to enhance cell cycle entry and progression through regulating p27^Kip1 expression at protein level.     

The human papillomavirus type 58 E7 oncoprotein modulates cell cycle regulatory proteins and abrogates cell cycle checkpoints

HPV type 58 (HPV-58) is the third most common HPV type in cervical cancer from Eastern Asia, yet little is known about how it promotes carcinogenesis. In this study, we demonstrate that HPV-58 E7 significantly promoted the proliferation and extended the lifespan of primary human keratinocytes (PHKs). HPV-58 E7 abrogated the G1 and the postmitotic checkpoints, although less efficiently than HPV-16 E7. Consistent with these observations, HPV-58 E7 down-regulated the cellular tumor suppressor pRb to a lesser extent than HPV-16 E7. Similar to HPV-16 E7 expressing PHKs, Cdk2 remained active in HPV-58 E7 expressing PHKs despite the presence of elevated levels of p53 and p21. Interestingly, HPV-58 E7 down-regulated p130 more efficiently than HPV-16 E7. Our study demonstrates a correlation between the ability of down-regulating pRb/p130 and abrogating cell cycle checkpoints by HPV-58 E7, which also correlates with the biological risks of cervical cancer progression associated with HPV-58 infection.     

Effects of E5a and E7 genes of human papillomavirus type 11 on immortalized human epidermal keratinocytes and NIH 3T3 cells

Summary The E5a gene of HPV-11 expressed in NIH 3T3 cells led to tumorigenesis in nude mice; whereas when expressed in keratinocytes, E5a induced anchorage independent growth, but was nontumorigenic in nude mice. The E7 gene of HPV-11 expressed NIH 3T3 cells led to tumors in nude mice and morphological transformation, but not anchorage independent growth. Keratinocytes expressing the E7 gene induced colony formation in soft agarose, but not tumorigenesis in nude mice. Comparison of transforming activities of HPV-11 E5a and E7 genes of HPV-11 showed that the efficiency of cell transformation by E7 was weaker than that of E5a.     

Differential cell cycle response of nontumorigenic and tumorigenic human papillomavirus-positive keratinocytes towards transforming growth factor-β1

Human papillomaviruses (HPVs) are causative agents of a number of malignancies in humans, including cervical cancer. Their tumorigenic potential is linked to expression of the viral E6/E7 genes which can interfere with normal cell cycle control by targeting p53, p21WAF1, p27KIP1, and pRb. We show here that nontumorigenic and tumorigenic HPV-positive keratinocytes (HPK) exhibit striking differences in the response of cell cycle regulatory genes towards transforming growth factor beta-beta1. Treatment with this agent led to an efficient induction of p53 and the growth-inhibitory p15INK4 and p21WAF1 genes only in nontumorigenic HPKs and was linked to an efficient reduction in viral E6/E7 oncogene expression. This was associated with increased pRb levels, exhibiting sustained hypophosphorylation, and a permanent growth arrest in the G1 phase of the cell cycle. In contrast, tumorigenic HPKs exhibited only a modest rise in p53 protein levels and a substantially reduced induction of the p15INK4 and p21WAF1 genes, which was linked to a lesser degree of viral oncogene repression. In addition, tumorigenic HPKs rapidly resumed cell growth after a transient G1 arrest, concomitantly with the reappearance of hyperphosphorylated pRb. These results support the notion that the progression of HPV-positive cells to a malignant phenotype is associated with increased resistance to growth inhibition by transforming growth factor-beta1. This is linked in the tumorigenic cells to a lack of persistent G1 arrest, inefficient induction of several cell cycle control genes involved in growth inhibition, and inefficient repression of the growth-promoting viral E6/E7 oncogenes.     

Determination of the binding affinity of different human papillomavirus E7 proteins for the tumour suppressor pRb by a plate-binding assay.

A plate-binding assay was developed to quantify the affinity of the E7 oncoprotein from different human papillomavirus (HPV) types for the tumour suppressor pRb. The method is highly reproducible, sensitive and easy to handle. It could be easily adapted for the quantitative study of other interacting proteins and for screenings of inhibitors of protein/protein interactions. The pRb-binding affinity of six different E7 proteins has been quantified. The K(D) values vary from approximately 4.5x10(-9) M for HPV16 E7 to more than 1x10(-7) M for HPV10 and HPV48 E7. Point mutation C24G in the high affinity pRb-binding domain of HPV16 E7 results in a 3-fold affinity reduction. The data indicate that the high affinity pRb-binding domain of E7, LXCXE, is essential for the association between the viral and cellular proteins. However, other E7 domain(s), which appear(s) not to be present in all E7s, contribute to stabilize the E7-pRb association.     

西多福韦对人宫颈癌细胞CaSki内人乳头瘤病毒16抑制作用研究

目的从抗病毒角度探讨西多福韦对宫颈癌细胞 CaSki内人乳头瘤病毒16（HPV16）的抑制作用及对细胞周期的影响。方法用 MTT法检测西多福韦对细胞的毒性;用实时定量 PCR法检测其对病毒 E6、E7 mRNA水平的影响;用 Western blot方法检测其对病毒蛋白 E6、E7和细胞抑癌蛋白 p53、pRb表达水平的影响;用流式细胞法检测其对宫颈癌细胞周期的影响。结果西多福韦对宫颈癌细胞毒性较正常细胞大。可使HPV16阳性宫颈癌细胞 CaSki内 E6、E7 mRNA和蛋白水平降低,最大抑制率分别为（33.38±8.00）%、（28.32±2.73）%和98.92%、97.46%;可以使 p53、pRb蛋白水平升高,最大浓度时可以上调12.06和3.53倍;对 HPV16阴性宫颈癌细胞 C-33A p53蛋白表达无影响,但可提高 pRb蛋白水平;可导致 CaSki和 C-33A细胞发生 S期阻滞,最高浓度组细胞相对对照组 S期分别增加22.83%和67.64%。结论西多福韦可以在对细胞无毒的浓度下,抑制宫颈癌细胞内的 HPV16,诱导宫颈癌细胞发生 S期阻滞。     

宫颈癌组织中人乳头瘤病毒16型E7蛋白致癌机理初探

目的 研究宫颈癌组织中人乳头瘤病毒（ＨＰＶ）１６－Ｅ７蛋白对视网膜母细胞瘤基因（Ｒｅｔｉｎｏｂｌａｓｔｏｍａ）Ｒｂ蛋白及Ｅ２Ｆ－１的作用的机制,探讨ＨＰＶ１６－Ｅ７蛋白与宫颈癌发生的关系。方法 采用聚合酶链反应检测宫颈癌及正常宫颈组织中ＨＰＶ１６感染等,用蛋白印迹技术对ＨＰＶ１６ ＤＮＡ阳性的宫颈癌组织中是否存在ＨＰＶ１６－Ｅ７蛋白和Ｒ６蛋白－Ｅ２Ｆ－１形成的复合物进行检测。正常宫颈组织作为对照,     

A human papillomavirus 8 E7 protein produced in plants is able to trigger the mouse immune system and delay the development of skin lesions

We investigated the potential of Nicotiana benthamiana to express the E7 protein of human papillomavirus 8 (HPV-8), a paradigm genotype among cutaneous HPVs. The protein, modified in its putative pRb-binding domain (E7(QGD)), was transiently expressed in leaves following infiltration with agrobacteria carrying either a binary vector combined with silencing suppressor constructs or replicating tobacco mosaic virus (TMV)-based vectors with different targeting signals. HPV-8 E7(QGD) yields ranged from 250?ng to 4.6?mg per gram of fresh leaf tissue. The highest yields were obtained with TMV-based vectors targeting the antigen to the apoplast. HPV8-CER (H2(q)) mice transformed with the complete early region of HPV-8 showed a delay in the onset of skin papillomatous lesions and produced E7-specific immunoglobulins G when inoculated subcutaneously with leaf extracts expressing E7(QGD). Furthermore, we demonstrated that the plant-made HPV-8 E7(QGD) induced a specific cytotoxic response in C57BL/6 (H2(b)) mice.     

Analysis of centrosome overduplication in correlation to cell division errors in high-risk human papillomavirus (HPV)-associated anal neoplasms

High-risk HPV-associated anal neoplasms are difficult to treat and biomarkers of malignant progression are needed. A hallmark of carcinogenic progression is genomic instability, which is frequently associated with cell division errors and aneuploidy. The HPV-16 E7 oncoprotein has been previously shown to rapidly induce centriole and centrosome overduplication and to cooperate with HPV-16 E6 in the induction of abnormal multipolar mitoses. Based on this function, it has been suggested that HPV-16 E7 may act as a driving force for chromosomal instability. However, a detailed analysis of centrosome overduplication in primary HPV-associated neoplasms has not been performed so far. Here, we determined the frequency of centrosome overduplication in HPV-associated anal lesions using a recently identified marker for mature maternal centrioles, Cep170. We detected centrosome overduplication in a small but significant fraction of cells. Remarkably, centrosome overduplication, but not aberrant centrosome numbers per se or centrosome accumulation, correlated significantly with the presence of cell division errors. In addition, our experiments revealed that in particular pseudo-bipolar mitoses may play a role in the propagation of chromosomal instability in high-risk HPV-associated tumors. These results provide new insights into the role of centrosome aberrations in cell division errors and encourage further studies on centrosome overduplication as a predictive biomarker of malignant progression in HPV-associated anal lesions.     

Are transforming properties of the bovine papillomavirus E5 protein shared by E5 from high-risk human papillomavirus type 16?

The E5 proteins of bovine papillomavirus type 1 (BPV-1) and human papillomavirus type 16 (HPV-16) are small (44-83 amino acids), hydrophobic polypeptides that localize to membranes of the Golgi apparatus and endoplasmic reticulum, respectively. While the oncogenic properties of BPV-1 E5 have been characterized in detail, less is known about HPV-16 E5 due to its low expression in mammalian cells. Using codon-optimized HPV-16 E5 DNA, we have generated stable fibroblast cell lines that express equivalent levels of epitope-tagged BPV-1 and HPV-16 E5 proteins. In contrast to BPV-1 E5, HPV-16 E5 does not activate growth factor receptors, phosphoinositide 3-kinase or c-Src, and fails to induce focus formation, although it does promote anchorage-independent growth in soft agar. These variant activities are apparently unrelated to differences in intracellular localization of the E5 proteins since retargeting HPV-16 E5 to the Golgi apparatus does not induce focus formation.     

特异siRNA抑制宫颈癌细胞中人乳头状瘤病毒16型E6表达的研究

目的 优化HPV-16 E6癌基因特异的U6质粒表达的siRNA，抑制HPV癌基因表达及其对子宫颈癌细胞生长繁殖的影响。方法 选择4个分别针对HPV-16 E6 mRNA外显子和内含子序列为靶序列，合成DNA链，构建表达HPV-16 E6短发卡样dsRNA的重组pSilencer1．0-U6载体，导入HPV-16DNA阳性的宫颈癌细胞株CaSki中，观察该细胞中HPV-16 E6、E7基因表达水平及其蛋白含量的变化，并观察细胞生长被抑制的情况。结果 4种HPV-16 E6 siRNA均能降低宫颈癌细胞CaSki的生长速率。通过细胞生长曲线观察到HPV-16 E6 shRNA表达质粒导入细胞0-96h内，可降低细胞生长速度。荧光定量RT-PCR检测HPV-16 E6 siRNA可使宫颈癌细胞株CaSki中HPV-16 E6、E7基因转录的mRNA水平降低，其中针对E6 mRNA内含子的重组shRNA只抑制E6基因的表达水平。Western blot分析表明，4个HPV-16 E6 siRNA作用72h后，未能检测到宫颈癌细胞中HPV-16 E6蛋白。结论 HPV-16 E6 siRNA能使宫颈癌细胞CaSki生长缓慢；选择针对E6内含子的siRNA作用位点，特异性抑制E6表达；而针对E6外显子的siRNA作用位点，可抑制E6和E7基因的表达，是用于治疗HPV阳性宫颈癌细胞的理想靶位。     

Molecular basis for the lack of T cell proliferation induced by an altered peptide ligand

In this report, we explore the mechanisms underlying cell cycle progression in T cells stimulated with an altered peptide ligand (APL) versus wild-type peptide. APL stimulation did not induce proliferation compared to wild-type peptide stimulation. To determine the point at which cell cycle progression is blocked, we have examined molecules responsible for regulating the retinoblastoma tumor suppressor gene product, pRb, which in its active state prevents G1/S progression. The majority of cells stimulated with an APL did not progress beyond G1; however, a small population did make the G1/S transition. These few cells passed the late G1 restriction point, divided and subsequently arrested at the next G1 phase. The lack of sustained signaling events following stimulation with an APL failed to induce cyclin E:cdk2 activity, a regulator which hyper-phosphorylates and inactivates pRb. Exogenous IL-2 addition did not compensate for the lack of proliferation following APL stimulation. Furthermore, the inability of the cells to enter S phase during partial T cell activation cannot be accounted for by p27Kip1 inhibition of cyclin E:cdk2 complexes. Upon APL stimulation, an increase in association of p27Kip1 with cyclin E:cdk2 complex was not observed, suggesting that instead, decreased cyclin E:cdk complex formation might contribute to the failure to progress from G1/S. Therefore, while for a majority of cells, wild-type stimulation results in cell cycle progression, APL stimulation is not sufficient to drive cells beyond G1.      

Dual expression of Epstein-Barr virus,latent membrane protein-1 and human papillomavirus-16 E6 transform primary mouse embryonic fibroblasts through NF-κB signaling

The prevalence of Epstein-Barr virus (EBV) and high-risk human papilloma virus (HPV) infections in patients with oral cancer in Okinawa, southwest islands of Japan, has led to the hypothesis that carcinogenesis is related to EBV and HPV co-infection. To explore the mechanisms of transformation induced by EBV and HPV co-infection, we analyzed the transformation of primary mouse embryonic fibroblasts (MEFs) expressing EBV and HPV-16 genes, alone or in combination. Expression of EBV latent membrane protein-1 (LMP-1) alone or in combination with HPV-16 E6 increased cell proliferation and decreased apoptosis, whereas single expression of EBV nuclear antigen-1 (EBNA-1), or HPV-16 E6 did not. Co-expression of LMP-1 and E6 induced anchorage-independent growth and tumor formation in nude mice, whereas expression of LMP-1 alone did not. Although the singular expression of these viral genes showed increased DNA damage and DNA damage response (DDR), co-expression of LMP-1 and E6 did not induce DDR, which is frequently seen in cancer cells. Furthermore, co-expression of LMP-1 with E6 increased NF-κB signaling, and the knockdown of LMP-1 or E6 in co-expressing cells decreased cell proliferation, anchorage independent growth, and NF-κB activation. These data suggested that expression of individual viral genes is insufficient for inducing transformation and that co-expression of LMP-1 and E6, which is associated with suppression of DDR and increased NF-κB activity, lead to transformation. Our findings demonstrate the synergistic effect by the interaction of oncogenes from different viruses on the transformation of primary MEFs.     

Aberrant cell cycle regulation in cervical carcinoma

Carcinoma of the uterine cervix is one of the most common malignancies among women worldwide. Human papillomaviruses (HPV) have been identified as the major etiological factor in cervical carcinogenesis. However, the time lag between HPV infection and the diagnosis of cancer indicates that multiple steps, as well as multiple factors, may be necessary for the development of cervical cancer. The development and progression of cervical carcinoma have been shown to be dependent on various genetic and epigenetic events, especially alterations in the cell cycle checkpoint machinery. In mammalian cells, control of the cell cycle is regulated by the activity of cyclin-dependent kinases (CDKs) and their essential activating coenzymes, the cyclins. Generally, CDKs, cyclins, and CDK inhibitors function within several pathways, including the p16(INK4A)-cyclin D1-CDK4/6-pRb-E2F, p21(WAF1)- p27(KIP1)-cyclinE-CDK2, and p14(ARF)-MDM2-p53 pathways. The results from several studies showed aberrant regulation of several cell cycle proteins, such as cyclin D, cyclin E, p16(INK4A), p21(WAF1), and p27(KIP1), as characteristic features of HPV- infected and HPV E6/E7 oncogene-expressing cervical carcinomas and their precursors. These data suggested further that interactions of viral proteins with host cellular proteins, particularly cell cycle proteins, are involved in the activation or repression of cell cycle progression in cervical carcinogenesis.     

Differences in transformation activity between HPV-18 and HPV-16 map to the viral LCR-E6-E7 region.

Homologous, subgenomic fragments of the viral LCR and E6/E7 transforming genes of HPV-18 and HPV-16 were amplified from several primary cervical, penile, and vulvar tumors and cloned into a pUC-18-derived vector. When assayed by a quantitative transformation assay using primary human keratinocytes, the subgenomic regions of HPV-16 and HPV-18 exhibited transforming activities similar to that of the full-length, prototype HPV genomes. More importantly, the HPV-18 LCR-E6-E7 region was approximately 10- to 50-fold more active than that of HPV-16. These studies demonstrate (1) that the transforming activity differences previously observed between prototype HPV-16 and HPV-18 map to the LCR-E6-E7 region, and (2) that individual and independent isolates of HPV-16 and HPV-18 exhibit consistent differences in transforming potential, even when isolated from different anatomic sites.     

Constitutive expression of thymidylate synthase from LCDV-C induces a transformed phenotype in fish cells

Thymidylate synthase (TS), an essential enzyme in DNA synthesis and repair, plays a key role in the events of cell cycle regulation and tumor formation. Here, an investigation was presented about subcellular location and biological function of viral TS from lymphocystis disease virus from China (LCDV-C) in fish cells. Fluorescence microscopy revealed that LCDV-C TS was predominantly localized in the cytoplasm in fish cells. Cell cycle analysis demonstrated that LCDV-C TS promoted cell cycle progression into S and G2/M phase in the constitutive expressed cells. As a result, the cells have a faster growth rate compared with the control cells as revealed by cell growth curves. For foci assay, the TS-expressed cells gave rise to foci 4-5 weeks after incubation. Microscopic examination of the TS-induced foci revealed multilayered growth and crisscross morphology characteristic of transformed cells. Moreover, LCDV-C TS predisposed the transfected cells to acquire an anchorage-independent phenotype and could grow in 0.3% soft agar. So the data reveal LCDV-C TS is sufficient to induce a transformed phenotype in fish cells in vitro and exhibits its potential ability in cell transformation. To our knowledge, it is the first report on viral TS sequences associated with transforming activity.