Analysis of novel human papillomavirus type 16 late mRNAs in differentiated W12 cervical epithelial cells

Loss of telomeres has been hypothesized to be important in cellular senescence and may play a role in carcinogenesis. In this study, we have measured telomere length in association with the immortalization and transformation of human cervical and foreskin epithelial cells by the human papillomavirus type 16 or 18 E6 and E7 open reading frames. By using a telomeric TTAGGG repeat probe, it was shown that the telomeres of precrisis normal and E6-, E7-, and E6/E7-expressing cells gradually shortened with passaging (30 to 100 bp per population doubling). Cells that expressed both E6 and E7 went through a crisis period and gave rise to immortalized lines. In contrast to precrisis cells, E6/E7-immortalized cells generally showed an increase in telomere length as they were passaged in culture, with some later passage lines having telomeres that were similar to or longer than the earliest-passage precrisis cells examined. No consistent association could be made between telomere length and tumorigenicity of cells in nude mice. However, of the three cell lines that grew in vivo, two had long telomeres, thus arguing against the hypothesis that cancer cells favor shortened telomeres. Our results indicate that arrest of telomere shortening may be important in human papillomavirus-associated immortalization and that restoration of telomere length may be advantageous to cells with regard to their ability to proliferate.

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Restoration of telomeres in human papillomavirus-immortalized human anogenital epithelial cells.

Molecular and Cellular Biology ◽

10.1128/mcb.14.2.961 ◽

1994 ◽

Vol 14 (2) ◽

pp. 961-969 ◽

Cited By ~ 109

Author(s):

A J Klingelhutz ◽

S A Barber ◽

P P Smith ◽

K Dyer ◽

J K McDougall

Keyword(s):

Human Papillomavirus ◽

Epithelial Cells ◽

Telomere Length ◽

Open Reading Frames ◽

Population Doubling ◽

Telomere Shortening ◽

Human Papillomavirus Type 16 ◽

Immortalized Cells ◽

E6 And E7

Loss of telomeres has been hypothesized to be important in cellular senescence and may play a role in carcinogenesis. In this study, we have measured telomere length in association with the immortalization and transformation of human cervical and foreskin epithelial cells by the human papillomavirus type 16 or 18 E6 and E7 open reading frames. By using a telomeric TTAGGG repeat probe, it was shown that the telomeres of precrisis normal and E6-, E7-, and E6/E7-expressing cells gradually shortened with passaging (30 to 100 bp per population doubling). Cells that expressed both E6 and E7 went through a crisis period and gave rise to immortalized lines. In contrast to precrisis cells, E6/E7-immortalized cells generally showed an increase in telomere length as they were passaged in culture, with some later passage lines having telomeres that were similar to or longer than the earliest-passage precrisis cells examined. No consistent association could be made between telomere length and tumorigenicity of cells in nude mice. However, of the three cell lines that grew in vivo, two had long telomeres, thus arguing against the hypothesis that cancer cells favor shortened telomeres. Our results indicate that arrest of telomere shortening may be important in human papillomavirus-associated immortalization and that restoration of telomere length may be advantageous to cells with regard to their ability to proliferate.

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Characterization of Human Bronchial Epithelial Cells Immortalized by the E6 and E7 Genes of Human Papillomavirus Type 16

Experimental Cell Research ◽

10.1006/excr.1994.1115 ◽

1994 ◽

Vol 212 (1) ◽

pp. 36-41 ◽

Cited By ~ 36

Author(s):

Jean Viallet ◽

Chi Liu ◽

Jacqueline Edmond ◽

Ming-Sound Tsao

Keyword(s):

Human Papillomavirus ◽

Epithelial Cells ◽

Bronchial Epithelial Cells ◽

Human Bronchial Epithelial Cells ◽

Bronchial Epithelial ◽

Human Papillomavirus Type 16 ◽

E6 And E7

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SF2/ASF Binds the Human Papillomavirus Type 16 Late RNA Control Element and Is Regulated during Differentiation of Virus-Infected Epithelial Cells

Journal of Virology ◽

10.1128/jvi.78.19.10598-10605.2004 ◽

2004 ◽

Vol 78 (19) ◽

pp. 10598-10605 ◽

Cited By ~ 47

Author(s):

Maria G. McPhillips ◽

Thanaporn Veerapraditsin ◽

Sarah A. Cumming ◽

Dimitra Karali ◽

Steven G. Milligan ◽

...

Keyword(s):

Human Papillomavirus ◽

Epithelial Cells ◽

Posttranscriptional Regulation ◽

Regulatory Element ◽

Sr Proteins ◽

Control Element ◽

Splice Sites ◽

Late Gene Expression ◽

Hpv 16 ◽

Human Papillomavirus Type 16

ABSTRACT Pre-mRNA splicing occurs in the spliceosome, which is composed of small ribonucleoprotein particles (snRNPs) and many non-snRNP components. SR proteins, so called because of their C-terminal arginine- and serine-rich domains (RS domains), are essential members of this class. Recruitment of snRNPs to 5′ and 3′ splice sites is mediated and promoted by SR proteins. SR proteins also bridge splicing factors across exons to help to define these units and have a central role in alternative and enhancer-dependent splicing. Here, we show that the SR protein SF2/ASF is part of a complex that forms upon the 79-nucleotide negative regulatory element (NRE) that is thought to be pivotal in posttranscriptional regulation of late gene expression in human papillomavirus type 16 (HPV-16). However, the NRE does not contain any active splice sites, is located in the viral late 3′ untranslated region, and regulates RNA-processing events other than splicing. The level of expression and extent of phosphorylation of SF2/ASF are upregulated with epithelial differentiation, as is subcellular distribution, specifically in HPV-16-infected epithelial cells, and expression levels are controlled, at least in part, by the virus transcription regulator E2.

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The Human Papillomavirus Type 16 E7 Oncogene Is Required for the Productive Stage of the Viral Life Cycle

Journal of Virology ◽

10.1128/jvi.74.14.6622-6631.2000 ◽

2000 ◽

Vol 74 (14) ◽

pp. 6622-6631 ◽

Cited By ~ 139

Author(s):

Elsa R. Flores ◽

B. Lynn Allen-Hoffmann ◽

Denis Lee ◽

Paul F. Lambert

Keyword(s):

Human Papillomavirus ◽

Life Cycle ◽

Dna Replication ◽

Epithelial Cells ◽

Dna Synthesis ◽

Copy Number ◽

Keratinocyte Differentiation ◽

Replication Machinery ◽

Hpv 16 ◽

Human Papillomavirus Type 16

ABSTRACT 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.

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