Expression of human papillomavirus type 16 E7 gene induces DNA synthesis of rat 3Y1 cells

Human papillomavirus type 16 (HPV 16) open reading frames (ORF) E6, E7, and E6E7, placed under the control of dexamethasone-inducible mouse mammary tumor virus long terminal repeat, were introduced into rat 3Y1 cells, an immortalized fibroblast line, with the aid of neomycin-selection. The cell clones containing inducible HPV 16 ORFs were selected and examined for DNA synthesis. Following induction of HPV mRNA synthesis by the hormone, DNA synthesis was stimulated in the cells containing E7 or E6E7 ORFs. The data indicate that expression of the HPV 16 E7 gene is mitogenic for rat 3Y1 cells.     

The biological properties of E6 and E7 oncoproteins from human papillomaviruses

More than 100 different human papillomavirus (HPV) types have been isolated so far, and they can be sub-grouped in cutaneous or mucosal according to their ability to infect the skin or the mucosa of the genital or upper-respiratory tracts. A sub-group of human mucosal HPVs, referred to as high-risk HPV types, is responsible for approximately 5% of all human cancers, which represents one-third of all the tumours induced by viruses. Epidemiological and biological studies have shown that HPV16 is the most oncogenic type within the high-risk group. Emerging lines of evidence suggest that, in addition to the high-risk mucosal HPV types, certain cutaneous HPVs are involved in skin cancer. HPV-associated cancers are intimately linked to HPV persistence and the accumulation of chromosomal rearrangements. The products of the early genes, E6 and E7, of the high-risk mucosal HPV types play a key role in both events. Indeed, these proteins have developed a number of strategies to evade host immuno-surveillance allowing viral persistence, and to alter cell cycle and apoptosis control, facilitating the accumulation of DNA damage/mutations. Often, the two oncoproteins target the same cellular pathways with different mechanisms, showing a strong synergism in promoting cellular transformation and neutralizing the immune response. Here, we review most of the findings on the biological properties and molecular mechanisms of the oncoproteins E6 and E7 from mucosal and cutaneous HPV types.     

Molecular mechanisms of cervical carcinogenesis by high‐risk human papillomaviruses: novel functions of E6 and E7 oncoproteins

Over the last two decades, since the initial discovery of human papillomavirus (HPV) type 16 and 18 DNAs in cervical cancers by Dr. Harald zur Hausen (winner of the Nobel Prize in Physiology or Medicine, 2008), the HPVs have been well characterised as causative agents for cervical cancer. Viral DNA from a specific group of HPVs can be detected in at least 90% of all cervical cancers and two viral genes, E6 and E7, are invariably expressed in HPV‐positive cervical cancer cells. Their gene products are known to inactivate the major tumour suppressors, p53 and retinoblastoma protein (pRB), respectively. In addition, one function of E6 is to activate telomerase, and E6 and E7 cooperate to effectively immortalise human primary epithelial cells. Though expression of E6 and E7 is itself not sufficient for cancer development, it seems to be either directly or indirectly involved in every stage of multi‐step carcinogenesis. Epidemiological and biological studies suggest the potential efficacy of prophylactic vaccines to prevent genital HPV infection as an anti‐cancer strategy. However, given the widespread nature of HPV infection and unresolved issues about the duration and type specificity of the currently available HPV vaccines, it is crucial that molecular details of the natural history of HPV infection as well as the biological activities of the viral oncoproteins be elucidated in order to provide the basis for development of new therapeutic strategies against HPV‐associated malignancies. This review highlights novel functions of E6 and E7 as well as the molecular mechanisms of HPV‐induced carcinogenesis. Copyright © 2009 John Wiley & Sons, Ltd.     

Efficient transformation of rat 3Y1 cells by human adenovirus type 9

Infection with herpes simplex viruses (HSV) lead to a significant increase of the simian virus 40 (SV40) DNA content in the SV40-transformed hamster cell lines CO631 and Elona. Analysis of this gene-amplifying activity revealed (i) that it cosedimented with infectious herpesvirions in sucrose density gradients, (ii) that it was abolished by anti-HSV antibodies or (iii) by antiviral drugs acting on the HSV-induced DNA polymerase; and analysis of temperature-sensitive mutants showed that this DNA polymerase was an essential component of HSV-induced, gene-amplifying activity in SV40-transformed hamster cells.     

PreTect HPV-Proofer: real-time detection and typing of E6/E7 mRNA from carcinogenic human papillomaviruses

Monitoring human papillomavirus (HPV) E6/E7 mRNA expression may provide an accurate and informative diagnostic approach for detection of oncogene activity related to the development of severe dysplasia or cervical carcinoma. A multiplex nucleic acid sequence based amplification (NASBA) assay, utilizing molecular beacon probes for real-time detection was developed for the identification of E6/E7 mRNA from HPV types 16, 18, 31, 33 and 45. The assay is called PreTect HPV-Proofer and this report describes the development and the analytical performance of the assay. The reproducibility of PreTect HPV-Proofer with regard to a positive result was found to be between 96 and 100%, depending on HPV type. The melting temperature for the different molecular beacons was in the range of 48-55 degrees C, indicating conformational stability, i.e. the molecular beacons will not get activated by the 41 degrees C annealing temperature, but will be activated by the annealing to the target itself. The limit of detection for HPV 16 was ten SiHa or CaSki cells and for HPV 18 one HeLa cell. No cross reactivity was observed with E6/E7 mRNA from the other tested HPV types. mRNA from cervical cells was also successfully amplified after more than one year of storage. In conclusion, the PreTect HPV-Proofer assay, individually identifying E6/E7 mRNA expression from five carcinogenic HPV types, is a reproducible assay that may serve as a valuable tool in monitoring HPV infections producing proteins with a transforming potential.     

Menaquinone-7 regulates gene expression in osteoblastic MC3T3E1 cells

Previous study has shown that the vitamin K2 analog menaquinone-7 (MK-7) induces expression of the osteoblast-specific genes osteocalcin, osteoprotegerin, receptor activator of NFkappaB, and its ligand. Since MK-7 may also regulate osteoblast cell function, we examined the expression of osteoblast genes regulated by MK-7 administration. Differences between gene expression in control and MK-7-administered MC3T3E1 cells were analyzed using the suppression subtractive hybridization method. After 24 h of MK-7 administration, genes upregulated by MK-7 included tenascin C and BMP2. Genes downregulated by MK-7 administration included biglycan and butyrophilin. Real-time PCR showed a marked increase in tenascin C. When the protein level was examined using Western blot analysis, tenascin C was higher in MK-7-administered cells than in control cells. These results indicated that MK-7 affected the cellular function of osteoblastic MC3T3E1 cells. Considering BMP2 mRNA expression was higher in MK-7-administered cells than in control cells, the effect of MK-7 administration on the signal transduction system was examined. Western blot analysis showed that cells administered MK-7 displayed a higher phosphorylated Smad1 level than control cells. Because MC3T3E1 cells have a nuclear binding receptor for MK-7, this result might indicate an indirect effect of MK-7 through BMP2 production.     

Functional expression of rat brain cloned α1E calcium channels in COS-7 cells

 The properties of the rat brain α1E Ca²⁺ channel subunit and its modulation by accessory rat brain α2-δ and β1b subunits were studied by transient transfection in a mammalian cell line in order to attempt to reconcile the debate as to whether α1E forms a low-voltage-activated (LVA) or high-voltage-activated (HVA) Ca²⁺ channel and to examine its pharmacology in detail. α1E alone was capable of forming an ion-conducting pore in COS-7 cells. The properties of heteromultimeric α1E/α2-δ/β1b channels were largely dictated by the presence of the β1b subunit, which increased current density and tended to produce a hyperpolarizing shift in the voltage dependence of activation and inactivation. α1E/α2-δ/β1b channels did not appear to be regulated by Ca²⁺-induced inactivation. α1E was shown to exhibit a unique pharmacological profile. ω-Agatoxin IVA blocked the current in a dose-dependent manner with an IC₅₀ of approximately 50 nM and a maximum inhibition of about 80%, whilst ω-conotoxin MVIIC was without effect. The 1,4-dihydropyridine (DHP) antagonist nicardipine (1 μM) produced an inhibition of 51 ± 7%, whereas the DHP agonist S-(–)BAY K 8644 was without effect. Our findings suggest a re-evaluation of the classification of the α1E Ca²⁺ channel subunit; we propose that rat brain α1E forms a novel Ca²⁺ channel with properties more similar to a subtype of LVA than HVA Ca²⁺ current. Received: 30 August 1996 / Received after revision and accepted: 28 October 1996     

The human papillomavirus E7 oncoprotein

The human papillomavirus (HPV) E7 oncoprotein shares functional similarities with such proteins as adenovirus E1A and SV40 large tumor antigen. As one of only two viral proteins always expressed in HPV-associated cancers, E7 plays a central role in both the viral life cycle and carcinogenic transformation. In the HPV viral life cycle, E7 disrupts the intimate association between cellular differentiation and proliferation in normal epithelium, allowing for viral replication in cells that would no longer be in the dividing population. This function is directly reflected in the transforming activities of E7, including tumor initiation and induction of genomic instability.     

Epithelial cells immortalized by human papillomaviruses have premalignant characteristics in organotypic culture.

Three HPV-16--and four HPV-18--immortalized human foreskin keratinocyte cell lines were analyzed on organotypic epidermal raft cultures at various passage levels. This culture system allowed normal cultured keratinocytes to stratify and differentiate in a manner similar to normal epidermis. All seven HPV-immortalized cell lines displayed epidermal morphologies on organotypic cultures, which were clearly abnormal and resembled premalignant lesions in vivo. Features of premalignant lesions that were shared by all of the HPV-immortalized cell lines included disorganized tissue architecture, mitotic cells present throughout the living layers of the epidermal sheet, abnormal mitoses, enlarged nuclei, and variable cell size and shape. Most HPV-immortalized cell lines were stable in terms of epidermal morphology with long-term passage in culture. Two of the HPV-18--immortalized cell lines, however, lost all morphologically apparent terminal squamous differentiation potential after long-term passage in monolayer culture. These results strongly support the idea that immortalization of squamous epithelial cells in culture by HPV-transforming genes generates a morphologically premalignant cell.     

人细胞因子受体样因子3在真核及原核细胞中的表达

目的:观察CRLF3蛋白在293T细胞中的表达及亚细胞定位,并在大肠杆菌中表达和纯化了重组CRLF3蛋白.方法:将真核表达载体pCMV-myc-CRLF3瞬时转染293T细胞,转染24 h和48 h后免疫荧光染色,共聚焦显微镜观察蛋白表达及定位;构建原核表达质粒pGEX-4T-1-CRLF3,实现插入基因的融合表达,经GST亲合层析纯化蛋白.结果:CRLF3蛋白在293T细胞中高效表达,主要分布在细胞质和细胞膜;成功构建了表达CRLF3融合蛋白的原核表达载体,并在大肠杆菌内高效表达,纯化后得到Mr约为74 000的融合蛋白.结论:在真核细胞中过表达的CRLF3蛋白主要定位在细胞质和细胞膜;获得了重组GST-CRLF3融合蛋白,为进一步探讨CRLF3的功能奠定基础.     

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.     

Detection of human papillomaviruses in exfoliated cervicovaginal cells by in situ DNA hybridization analysis.

A total of 851 specimens of exfoliated cervicovaginal cells and 27 specimens of male urethral smears obtained from 706 individuals with various clinical findings were examined for the presence of human papillomavirus (HPV) types 6, 11, 16, 18, 31, and 33 by in situ DNA hybridization analysis. The nonradioactive DNA in situ hybridization method used in this study showed no detectable cross-hybridization either among different types of HPV (except between types 6 and 11) or between HPV DNA and human cellular DNA. Furthermore, this system was found to be more sensitive than the Southern blotting method in detecting HPV. HPV was found in 233 of 276 (84.4%) and in 34 of 47 (72.3%) samples of cervicovaginal cells from patients with urogenital condylomata and cervical dysplasia, respectively. HPV was also detected in 6 of 39 (15.4%) women with normal cytological findings who were also symptom-free. Young women who were at low risk but were infected with HPV showed significantly reduced ratios of helper-inducer T lymphocytes to suppressor-cytotoxic T lymphocytes compared with those of uninfected normal controls (1.28 +/- 0.31 versus 2.47 +/- 0.64; P less than 0.001). This in situ DNA hybridization method can have broad application to the screening of HPV in early lesions and in normal-looking tissues and may be used to identify patients at risk of more serious or possibly malignant progression.     

A new PCR-based assay amplifies the E6-E7 genes of most mucosal human papillomaviruses (HPV)

We established a new assay to detect the E6-E7 DNA of mucosal human papillomaviruses (HPV) by a PCR-based method using four pairs of degenerate LCR and E7 primers (LCR-E7 PCR). This assay amplifies the full length of E6 and the N-terminal part of E7. HPV typing was performed using restriction-fragment-length polymorphism (RFLP), and by analyzing the sequences of cloned PCR products. We compared this assay with the first generation hybrid captured assay (HCA-I) and the MY09/11-PCR method. LCR-E7 PCR was able to detect more than 34 mucosal HPV types and theoretically should detect two additional types. LCR-157 PCR and HCA-I detected HPV DNA in 70% (69/99) and 55% (54/99) of low-grade cervical intraepithelial lesions (LSIL), 89% (105/118) and 76% (90/118) of high-grade cervical intraepithelial lesions (HSIL), and 90% (56/62) and 79% (49/62) of invasive squamous cell carcinomas (SCC), respectively. LCR-E7 PCR was more sensitive than the HCA-1 test. Discordant results between the LCR-E7 and MY 11/09-PCR tests were observed in one of 185 (0.5%) normal samples, seven of 85 (8.2%) LSIL samples, seven of 82 (8.5%) HSIL samples, and four of 72 (5.6%) SCC samples. The discordant results were mostly observed in samples with a low-copy number of the HPV genome or with multiple HPV infection. The sensitivity of LCR-E7 PCR was equivalent to that of MY 11/09 ECR, and false positives were less frequent in LCR-E7 PCR. LCR-E7 PCR may be useful for determining the biological activity of detected HPV types, since this method amplifies the entire E6 gene.     

A major transcript of human papillomavirus type 16 in transformed NIH 3T3 cells contains polycistronic mRNA encoding E7, E5, and E1^E4 fusion gene

We have cloned cDNA of the major 1.8 kb mRNA from HPV 16-transformed NIH 3T3 cells (PM3T3). The entire nucleotide sequences of this cDNA were determined and compared with prototype HPV 16 genomic DNA sequences. The 5'-end of the cDNA was flanked by approximately 300 bp of cellular sequences, and the 3'-end of the cDNA sequences contained poly A residues following at nt 4230. HPV 16 sequences began at nt 124, downstream of a major viral p97 promoter, within the E6 open reading frame (ORF). The first splice donor site was at nt 226 and the splice acceptor site was at nt 409, suggesting that the E6 gene is inert. Second splice donor and acceptor sites were located at nt 880 and at nt 3357, respectively. This mRNA was thus shown to consist of three exons, resulting in polycistronic mRNA containing three potentially functional virus early genes--E7, E1--E4, and E5--actively transcribed in the transformant.     

The prostaglandin E1 analogue, misoprostol, regulates inflammatory cytokines and immune functions in vitro like the natural prostaglandins E1, E2 and E3.

We examined whether some immune functions related to the action and production of cytokines could be regulated by the natural prostaglandins E (PGE) and the PGE1 (ester) analogue, Misoprostol. PGE1,2,3 and Misoprostol inhibited: (1) the mitogenic activity of interleukin-1 (IL-1) for mouse thymocytes; (2) spreading of mouse macrophages on glass; (3) tumour necrosis factor (TNF) (alpha and beta) production by human peripheral blood mononuclear cells and rat macrophages; (4) IL-1 production by rat and mouse peritoneal macrophages; and (5) interferon-gamma (IFN-gamma) production by human peripheral blood mononuclear cells. These PGE had little effect on IL-1 production by human monocytes. By contrast, they all enhanced IL-6 production by rat and mouse macrophages and human monocytes. These effects were noted at concentrations below 500 nM (even as low as 10 nM). The relative potency of the prostanoids tested for both inhibitory and stimulatory effects was PGE1 = PGE2 = or greater than PGE3 greater than Misoprostol greater than PGA2 much greater than PGF1-alpha = PGF2-alpha = PGD2 (no effect). There is strong evidence that PGE1,2,3 and Misoprostol bind to the same receptor(s) and trigger the second messenger, cAMP, since dibutyryl cAMP (a lipophilic analogue of cAMP) had the same effects as the PGE. These PGE also induced elevated intracellular cAMP levels in and competed with [3H]PGE2 for binding to human and rat cells with the same relative potencies as described above.     

Phylogeny and evolution of papillomaviruses based on the E1 and E2 proteins

Papillomaviridae are a family of small double-stranded DNA viruses that infect stratified squamous epithelia in vertebrates. Members of this family are causative agents of malignant tumours, such as cervical cancer while others are associated with benign proliferative lesions. So far, Papillomaviruses (PVs) are classified according to the sequence identity in the capsid gene L1. However, evidence has accumulated indicating a discontinuity in the evolutionary history of the L1 and L2 genes of many PVs, giving rise to differences in the phylogenetic reconstructions of the early and of the late genes. Neither the oncogenes E5, E6 and E7 nor the upstream regulatory region are suitable for phylogenetic inference due to the poor conservation along the Papillomaviridae family. We have analysed here the evolutionary relationships of the PVs with respect to the E1 and E2 proteins, and the results provide both phylogeny and biologic behaviour of the viruses. The hierarchical taxonomic relationships can be structured as an alternative classification system in which mucosal high-risk viruses, mucosal low-risk viruses and viruses associated with cutaneous lesions are grouped separately and do not appear intermingled. Some important trends are also observed: first, evolution of the PVs has not been homogeneous, even in viruses that infect the same host, and second mucosal human PVs have evolved faster than their cutaneous counterparts. The evolutionary analysis based on the E1 and E2 proteins will allow us to better understand the generation of the diversity of the PVs and the development of malignancy associated with these viruses.     

Specific functions of human NK cells.

Since natural killer (NK) cells lack both CD3/TCR molecules and surface Ig, it is generally thought that they are unable to recognize antigens. However, CD3-CD16+ cells were found to respond in MLC against irradiated allogeneic mononuclear cells and to lyse normal PHA blasts derived from the stimulating donor, but not autologous cells or cells derived from most allogeneic donors. A similar pattern was obtained with cloned NK cells, thus indicating that the ability to specifically recognize given normal allogeneic cells is a clonally distributed function. Moreover, analysis of NK clones for their ability to lyse either tumor cells or normal PHA blasts (both derived from individual cancer patients) indicated that the two phenomena are distinct. Analysis of a large number of NK clones derived from a given individual for their ability to lyse a panel of allogeneic donors allowed the identification of at least four groups of clones characterized by unique patterns of reactivity ("specificities"). We further studied the mode of inheritance of the various NK-defined specificities: all the characters "susceptibility to lysis" by NK clones (displaying one or another specificity) segregated independently, were inherited in an autosomic recessive manner and were carried by chromosome 6. The finding of clonally distributed specific functions in NK cells suggested the existence of clonally distributed receptor molecules. Along this line mAbs were raised against NK clones, and screened for their ability to trigger the immunizing clones: two mAbs (termed GL183 and EB6) were directed against a novel family of 58-kDa surface molecules.(ABSTRACT TRUNCATED AT 250 WORDS)