共查询到20条相似文献,搜索用时 15 毫秒
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Mark A Merkley Ellen Hildebrandt Robert H Podolsky Hilal Arnouk Daron G Ferris William S Dynan Hubert Stöppler 《Proteome science》2009,7(1):29-15
Background
Infection with high-risk type human papilloma viruses (HPVs) is associated with cervical carcinomas and with a subset of head and neck squamous cell carcinomas. Viral E6 and E7 oncogenes cooperate to achieve cell immortalization by a mechanism that is not yet fully understood. Here, human keratinocytes were immortalized by long-term expression of HPV type 16 E6 or E7 oncoproteins, or both. Proteomic profiling was used to compare expression levels for 741 discrete protein features. 相似文献3.
Harms W Rothämel T Miller K Harste G Grassmann M Heim A 《Experimental cell research》2001,265(2):252-261
SMN, the affected protein in spinal muscular atrophy (SMA), is a cytoplasmic protein that also occurs in nuclear structures called "gems" and is involved in snRNP maturation. Coilin-p80 is a marker protein for nuclear Cajal bodies (coiled bodies; CBs) which are also involved in snRNP maturation, storage or transport. We now show that gems and CBs are present in all fetal tissues, even those that lack gems/CBs in the adult. Most gems and CBs occur as separate nuclear structures in fetal tissues, but their colocalization increases with fetal age and is almost complete in the adult. In adult tissues, up to half of all gems/CBs are inside the nucleolus, whereas in cultured cells they are almost exclusively nucleoplasmic. The nucleolar SMN is often more diffusely distributed, compared with nucleoplasmic gems. Up to 30% of cells in fetal tissues have SMN distributed throughout the nucleolus, instead of forming gems in the nucleoplasm. The results suggest a function for gems distinct from Cajal bodies in fetal nuclei and a nucleolar function for SMN. Spinal cord, the affected tissue in SMA, behaves differently in several respects. In both fetal and adult motor neurons, many gems/CBs occur as larger bodies closely associated with the nucleolar perimeter. Uniquely in motor neurons, gems/CBs are more numerous in adult than in fetal stages and colocalization of gems and CBs occurs earlier in development. These unusual features of motor neurons may relate to their special sensitivity to reduced SMN levels in SMA patients. 相似文献
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Chromosomal instability in human mesenchymal stem cells immortalized with human papilloma virus E6, E7, and hTERT genes 总被引:2,自引:0,他引:2
Takeuchi M Takeuchi K Kohara A Satoh M Shioda S Ozawa Y Ohtani A Morita K Hirano T Terai M Umezawa A Mizusawa H 《In vitro cellular & developmental biology. Animal》2007,43(3-4):129-138
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O. S. Ryzhakova T. A. Gureeva V. A. Zhurbitskaya N. I. Solovyeva 《Biochemistry (Moscow) Supplemental Series B: Biomedical Chemistry》2007,1(4):342-347
Matrix metalloproteinases (MMPs) play a critical role in tumor development and invasion. The aim of this study was to elucidate peculiarity of expression of interstitial collagenase (MMP-1) and its endogenous regulators during oncogenic transformation of fibroblasts by HPV-16 E7 gene. Papilloma virus types 16 and 18 are etiological factor of cervical cancer. We have studied expression of MT1-MMP, MMP-1, tissue inhibitor of these proteases, TIMP-1, and urokinase-like plasminogen activator (uPA), activating MMP-1 via plasmin. The study was carried out using fibroblasts immortalized by LT gene (IF) and transformed by E7 gene of HPV-16 fibroblasts (TF). Primary culture of Fisher rat embryo fibroblasts was used as a control (PF). mRNA expression, and enzymatic activity were studied by RT-PCR and by hydrolysis of fluorogenic type I collagen, respectively. Cell transformation was accompanied by: (a) 2–3 fold induction of MT1-MMP mRNA expression vs PF; (b) the decrease in mRNA level of TIMP-1 (1,5–2 fold); c) unchanged uPA expression. Cell immortalization is accompanied by: (a) the increase of MT1-MMP expression (1,5–2 fold); (b) unchanged TIMP-1 expression; (c) the increase of uPA expression (2–4 fold) vs PF and TF. MMP secreted activity and activity in lysates of TF increased but level of free endogenous MMP inhibitors decreased vs IF. Data on gene expression are consistent with enzymatic data on the collagenolytic activity. These results suggest changes in enzyme/inhibitor/activator ratio both TF and IF and significant enhancement of the destructive potential of the TF. 相似文献
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分别以卡介苗(BCG)基因组DNA及宫颈癌组织提取DNA为模板,通过PCR扩增得到19kD抗原的胞壁区及其上游调控元件(19ss)基因序列和HPV16型E7基因序列。先将19ss基因与大肠杆菌-分枝杆菌穿梭质粒pMV261重组,得到重组质粒pMCW。再将E7基因克隆至pMCW,得到重组质粒pMCW-E7。最后用PCR引物定点诱变法突变E7基因与转化有关的位点,得到重组质粒pMCW-mE7。对重组质粒pMCW-mE7用PCR扩增、双酶切及测序鉴定证实,19ss基因及突变的E7基因正确插入穿梭质粒pMV261。成功构建了重组穿梭质粒pMCW-mE7。 相似文献
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The in vitro study of mammalian hematopoiesis is hindered by the lack of immortalized human stromal cell lines that support hematopoiesis. We have immortalized human stromal vascular smooth muscle cells characterized by the expression of the alpha-smooth muscle (alpha-SM) actin. This marker is usually down-regulated as a result of oncogenic transformation. To correct this dedifferentiation, we placed the expression of human papilloma virus 16 E6/E7 oncogenes under the control of the tissue-specific alpha-SM actin promoter. The immortalization event is rare and requires polyclonal culture, but the corresponding established line retains alpha-SM actin expression. Moreover, when compared with other lines derived from the same cells from vectors made with the same oncogenes but driven by either an internal SV40 promoter or the viral long terminal repeat, this line is less transformed as shown by anchorage-independent growth assay. We show therefore that the use of a physiological promoter allows the production of human cell lines with a conserved phenotype. 相似文献
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Human papilloma virus E6/E7 genes can expand the lifespan of human corneal fibroblasts 总被引:2,自引:0,他引:2
Donna M. Peters Nathan Dowd Curtis Brandt Teresa Compton 《In vitro cellular & developmental biology. Animal》1996,32(5):279-284
Summary Human corneal fibroblasts were infected with a retroviral delivery vector containing the E6 and E7 genes from human Papilloma
virus type 16 in order to produce cell lines that have an expanded lifespan in culture. Morphologically, some of the trasfected
corneal fibroblast lines appeared to have the normal spindle-shape morphology of diploid fibroblasts, whereas other lines
appeared to have a more elongated morphology. All the cell lines were anchorage-dependent. Cells that had a normal morphology
grew at a rate similar to normal diploid human corneal fibroblasts and had a population doubling time of 48 h. All E6/E7 expressing
cell lines, regardless of morphology, produce types I, III, and V collagen, at levels similar to those observed in the parent
corneal diploid fibroblast. These corneal fibroblast lines will be a usefulin vitro system to study collagen expression and fibril formation, as well as normal stroma development. These results also demonstrate
that the use of E6/E7 genes to expand a cell’s lifespan can be a powerful tool because it does not appear to alter either
the growth rate of the cell or collagen expression. 相似文献
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The human papillomavirus type 16 E7 oncogene is required for the productive stage of the viral life cycle 总被引:8,自引:0,他引:8
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The production of the human papillomavirus type 16 (HPV-16) is intimately tied to the differentiation of the host epithelium that it infects. Infection occurs in the basal layer of the epithelium at a site of wounding, where the virus utilizes the host DNA replication machinery to establish itself as a low-copy-number episome. The productive stage of the HPV-16 life cycle occurs in the postmitotic suprabasal layers of the epithelium, where the virus amplifies its DNA to high copy number, synthesizes the capsid proteins (L1 and L2), encapsidates the HPV-16 genome, and releases virion particles as the upper layer of the epithelium is shed. Papillomaviruses are hypothesized to possess a mechanism to overcome the block in DNA synthesis that occurs in the differentiated epithelial cells, and the HPV-16 E7 oncoprotein has been suggested to play a role in this process. To determine whether E7 plays a role in the HPV-16 life cycle, an E7-deficient HPV-16 genome was created by inserting a translational termination linker (TTL) in the E7 gene of the full HPV-16 genome. This DNA was transfected into an immortalized human foreskin keratinocyte cell line shown previously to support the HPV-16 life cycle, and stable cell lines were obtained that harbored the E7-deficient HPV-16 genome episomally, the state of the genome found in normal infections. By culturing these cells under conditions which promote the differentiation of epithelial cells, we found E7 to be necessary for the productive stage of the HPV-16 life cycle. HPV-16 lacking E7 failed to amplify its DNA and expressed reduced amounts of the capsid protein L1, which is required for virus production. E7 appears to create a favorable environment for HPV-16 DNA synthesis by perturbing the keratinocyte differentiation program and inducing the host DNA replication machinery. These data demonstrate that E7 plays an essential role in the papillomavirus life cycle. 相似文献
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The p53 tumor suppressor protein can induce both cell cycle arrest and apoptosis in DNA-damaged cells. In human carcinoma cell lines expressing wild-type p53, expression of E7 allowed the continuation of full cell cycle progression following DNA damage, indicating that E7 can overcome both G1 and G2 blocks imposed by p53. E7 does not interfere with the initial steps of the p53 response, however, and E7 expressing cells showed enhanced expression of p21(waf1/cip1) and reductions in cyclin E- and A-associated kinase activities following DNA damage. One function of cyclin-dependent kinases is to phosphorylate pRB and activate E2F, thus allowing entry into DNA synthesis. Although E7 may substitute for this activity during cell division by directly targeting pRB, continued cell cycle progression in E7-expressing cells was associated with phosphorylation of pRB, suggesting that E7 permits the retention of some cyclin-dependent kinase activity. One source of this activity may be the E7-associated kinase, which was not inhibited following DNA damage. Despite allowing cell cycle progression, E7 was unable to protect cells from p53-induced apoptosis, and the elevated apoptotic response seen in these cells correlated with the reduction of cyclin A-associated kinase activity. It is possible that inefficient cyclin A-dependent inactivation of E2F at the end of DNA synthesis contributes to the enhanced apoptosis displayed by E7-expressing cells. 相似文献
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Protein domains governing interactions between E2F, the retinoblastoma gene product, and human papillomavirus type 16 E7 protein. 总被引:11,自引:5,他引:11
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P S Huang D R Patrick G Edwards P J Goodhart H E Huber L Miles V M Garsky A Oliff D C Heimbrook 《Molecular and cellular biology》1993,13(2):953-960
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Raffaella R Gioia D De Andrea M Cappello P Giovarelli M Marconi P Manservigi R Gariglio M Landolfo S 《Experimental cell research》2004,293(2):331-345
Immunohistochemical analysis has demonstrated that the human IFI16 gene, in addition to the hematopoietic tissues, is highly expressed in endothelial cells and squamous stratified epithelia. In this study, we have developed a reliable HSV-derived replication-defective vector (TO-IFI16) to efficiently transduce IFI16 into primary human umbilical vein endothelial cells (HUVEC), which are usually poorly transfectable. HUVEC infection with TO-IFI16 virus suppressed endothelial migration, invasion and formation of capillary-like structures in vitro. In parallel, sustained IFI16 expression inhibited HUVEC cell cycle progression, accompanied by significant induction of p53, p21, and hypophosphorylated pRb. Further support for the involvement of these pathways in IFI16 activity came from the finding that infection with TO-IFI16 virus does not impair the in vitro angiogenic activity and cell cycle progression of HUVEC immortalized by HPV16 E6/E7 oncogenes, which are known to inactivate both p53 and pRb systems. This use of a reliable viral system for gene delivery into primary human endothelial cells assigns a potent angiostatic activity to an IFN-inducible gene, namely IFI16, and thus throws further light on antiangiogenic therapy employing IFNs. 相似文献
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Quantitative role of the human papillomavirus type 16 E5 gene during the productive stage of the viral life cycle 总被引:6,自引:0,他引:6
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Genther SM Sterling S Duensing S Münger K Sattler C Lambert PF 《Journal of virology》2003,77(5):2832-2842
Human papillomaviruses (HPVs) are small circular DNA viruses that cause warts. Infection with high-risk anogenital HPVs, such as HPV type 16 (HPV16), is associated with human cancers, specifically cervical cancer. The life cycle of HPVs is intimately tied to the differentiation status of the host epithelium and has two distinct stages: the nonproductive stage and the productive stage. In the nonproductive stage, which arises in the poorly differentiated basal epithelial compartment of a wart, the virus maintains itself as a low-copy-number nuclear plasmid. In the productive stage, which arises as the host cell undergoes terminal differentiation, viral DNA is amplified; the capsid genes, L1 and L2, are expressed; and progeny virions are produced. This stage of the viral life cycle relies on the ability of the virus to reprogram the differentiated cells to support DNA synthesis. Papillomaviruses encode multiple oncoproteins, E5, E6, and E7. In the present study, we analyze the role of one of these viral oncogenes, E5, in the viral life cycle. To assess the role of E5 in the HPV16 life cycle, we introduced wild-type (WT) or E5 mutant HPV16 genomes into NIKS, a keratinocyte cell line that supports the papillomavirus life cycle. By culturing these cells under conditions that allow them to remain undifferentiated, a state similar to that of basal epithelial cells, we determined that E5 does not play an essential role in the nonproductive stage of the HPV16 life cycle. To determine if E5 plays a role in the productive stage of the viral life cycle, we cultured keratinocyte populations in organotypic raft cultures, which promote the differentiation and stratification of epithelial cells. We found that cells harboring E5 mutant genomes displayed a quantitative reduction in the percentage of suprabasal cells undergoing DNA synthesis, compared to cells containing WT HPV16 DNA. This reduction in DNA synthesis, however, did not prevent amplification of viral DNA in the differentiated cellular compartment. Likewise, late viral gene expression and the perturbation of normal keratinocyte differentiation were retained in cells harboring E5 mutant genomes. These data demonstrate that E5 plays a subtle role during the productive stage of the HPV16 life cycle. 相似文献