Human papillomavirus type 16 E6 and E7 oncogenes abrogate radiation-induced DNA damage responses in vivo through p53-dependent and p53-independent pathways

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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Studies on in Vivo Induction of Cytotoxic T Lymphocyte Responses by Synthetic Peptides from E6 and E7 Oncoproteins of Human Papillomavirus Type 16

Viral Immunology ◽

10.1089/vim.1995.8.165 ◽

1995 ◽

Vol 8 (3) ◽

pp. 165-174 ◽

Cited By ~ 19

Author(s):

ASIS K. SARKAR ◽

GUILLERMO TORTOLERO-LUNA ◽

PRAMOD N. NEHETE ◽

RALPH B. ARLINGHAUS ◽

MICHELE FOLLEN MITCHELL ◽

...

Keyword(s):

Human Papillomavirus ◽

T Lymphocyte ◽

Synthetic Peptides ◽

Cytotoxic T Lymphocyte ◽

Human Papillomavirus Type 16 ◽

E6 And E7 Oncoproteins ◽

E6 And E7 ◽

Lymphocyte Responses ◽

In Vivo Induction

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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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MicroRNA 203 Expression in Keratinocytes Is Dependent on Regulation of p53 Levels by E6

Journal of Virology ◽

10.1128/jvi.00703-10 ◽

2010 ◽

Vol 84 (20) ◽

pp. 10644-10652 ◽

Cited By ~ 53

Author(s):

Declan J. McKenna ◽

Simon S. McDade ◽

Daksha Patel ◽

Dennis J. McCance

Keyword(s):

Dna Damage ◽

Human Papillomavirus ◽

Cell Biology ◽

Viral Oncoprotein ◽

Hpv16 E6 ◽

Human Papillomavirus Type 16 ◽

Proliferation And Differentiation ◽

Short Hairpin ◽

E6 And E7 ◽

Short Hairpin Rnas

ABSTRACT A screen of microRNA (miRNA) expression following differentiation in human foreskin keratinocytes (HFKs) identified changes in several miRNAs, including miRNA 203 (miR-203), which has previously been shown to play an important role in epithelial cell biology by regulating p63 levels. We investigated how expression of human papillomavirus type 16 (HPV16) oncoproteins E6 and E7 affected miR-203 expression during proliferation and differentiation of HFKs. We demonstrated that miR-203 expression is reduced in HFKs where p53 function is compromised, either by the viral oncoprotein E6 or by knockout of p53 using short hairpin RNAs (p53i). We show that the induction of miR-203 observed during calcium-induced differentiation of HFKs is significantly reduced in HFKs expressing E6 and in p53i HFKs. Induction of miR-203 in response to DNA damage is also reduced in the absence of p53. We report that proliferation of HFKs is dependent on the level of miR-203 expression and that overexpression of miR-203 can reduce overproliferation in E6/E7-expressing and p53i HFKs. In summary, these results indicate that expression of miR-203 is dependent on p53, which may explain how expression of HPV16 E6 can disrupt the balance between proliferation and differentiation, as well as the response to DNA damage, in keratinocytes.

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Human papillomavirus type 16 E6 and E7 oncoproteins interact with the nuclear p53-binding protein 1 in an in vitro reconstructed 3D epithelium: new insights for the virus-induced DNA damage response

Virology Journal ◽

10.1186/s12985-018-1086-4 ◽

2018 ◽

Vol 15 (1) ◽

Cited By ~ 4

Author(s):

Diletta Francesca Squarzanti ◽

Rita Sorrentino ◽

Manuela Miriam Landini ◽

Andrea Chiesa ◽

Sabrina Pinato ◽

...

Keyword(s):

Dna Damage ◽

Human Papillomavirus ◽

Dna Damage Response ◽

Binding Protein ◽

Human Papillomavirus Type 16 ◽

Damage Response ◽

E6 And E7 Oncoproteins ◽

E6 And E7

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Targeted expression of the E6 and E7 oncogenes of human papillomavirus type 16 in the epidermis of transgenic mice elicits generalized epidermal hyperplasia involving autocrine factors.

Molecular and Cellular Biology ◽

10.1128/mcb.14.12.8250 ◽

1994 ◽

Vol 14 (12) ◽

pp. 8250-8258 ◽

Cited By ~ 32

Author(s):

P Auewarakul ◽

L Gissmann ◽

A Cid-Arregui

Keyword(s):

Human Papillomavirus ◽

Growth Factor ◽

Epithelial Cells ◽

Transgenic Mice ◽

Transforming Growth Factor ◽

Basal Layer ◽

Epidermal Hyperplasia ◽

Human Papillomavirus Type 16 ◽

E6 And E7

The E6 and E7 early genes of human papillomavirus type 16 have been shown in vitro to play a central role in the transforming capability of this virus. To explore their effects on differentiating epithelial cells in vivo, we used a bovine cytokeratin 10 (K10) promoter to target the expression of E6 and E7 to the suprabasal layers of the epidermis of transgenic mice. In two different lines of mice efficiently expressing the transgene, animals displayed generalized epidermal hyperplasia, hyperkeratosis and parakeratosis in the skin and the forestomach, both known to be sites of K10 expression. Northern (RNA) blot analysis revealed high levels of E6 and E7 transcripts, and in situ hybridizations localized these transcripts to the suprabasal strata of epidermis. In vivo labeling of proliferating cells showed two distinct effects of E6 and E7 expression in the epidermis: (i) an increase in the number of growing cells in the undifferentiated basal layer and (ii) abnormal proliferation of differentiated cells in the suprabasal strata. The expression of c-myc in the skin of transgenics was higher than that in control animals. The induction of c-myc transcription by topical application of tetradecanoyl phorbol acetate was prevented by simultaneous treatment with transforming growth factor beta 1 in nontransgenic skin but not in transgenic skin. In addition, transforming growth factor alpha was found to be overexpressed in the suprabasal layers of the transgenic epidermis. These findings suggest that autocrine mechanisms are involved in the development and maintenance of epidermal hyperplasia. Animals of both lines developed papillomas in skin sites exposed to mechanical irritation and wounding, suggesting that secondary events are necessary for progression to neoplasia. Collectively, these results provide new insights into the tumor promoter activities of human papillomavirus type 16 in epithelial cells in vivo.

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Compromised Spindle Assembly Checkpoint due to Altered Expression of Ubch10 and Cdc20 in Human Papillomavirus Type 16 E6- and E7-Expressing Keratinocytes

Journal of Virology ◽

10.1128/jvi.00259-10 ◽

2010 ◽

Vol 84 (21) ◽

pp. 10956-10964 ◽

Cited By ~ 22

Author(s):

Daksha Patel ◽

Dennis J. McCance

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Human Papillomavirus ◽

Spindle Assembly Checkpoint ◽

Apoptotic Cell ◽

Cyclin B ◽

Mitotic Catastrophe ◽

Altered Expression ◽

Human Papillomavirus Type 16 ◽

E6 And E7

ABSTRACT Cells expressing human papillomavirus type 16 (HPV-16) E6 and E7 proteins exhibit deregulation of G2/M genes, allowing bypass of DNA damage arrest signals. Normally, cells with DNA damage that override the G2 damage checkpoint would precociously enter mitosis and ultimately face mitotic catastrophe and apoptotic cell death. However, E6/E7-expressing cells (E6/E7 cells) have the ability to enter and exit mitosis in the presence of DNA damage and continue with the next round of the cell cycle. Little is known about the mechanism that allows these cells to gain entry into and exit from mitosis. Here, we show that in the presence of DNA damage, E6/E7 cells have elevated levels of cyclin B, which would allow entry into mitosis. Also, as required for exit from mitosis, cyclin B is degraded in these cells, permitting initiation of the next round of DNA synthesis and cell cycle progression. Proteasomal degradation of cyclin B by anaphase-promoting complex/cyclosome (APC/C) is, in part, due to elevated levels of the E2-conjugating enzyme, Ubch10, and the substrate recognition protein, Cdc20, of APC/C. Also, in E6/E7 cells with DNA damage, while Cdc20 is complexed with BubR1, indicating an active checkpoint, it is also present in complexes free of BubR1, presumably allowing APC/C activity and slippage through the checkpoint.

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A promoter within the E6 ORF of human papillomavirus type 16 contributes to the expression of the E7 oncoprotein from a monocistronic mRNA

Journal of General Virology ◽

10.1099/vir.0.19250-0 ◽

2003 ◽

Vol 84 (12) ◽

pp. 3429-3441 ◽

Cited By ~ 19

Author(s):

Jacob A. Glahder ◽

Christina N. Hansen ◽

Jeppe Vinther ◽

Birger S. Madsen ◽

Bodil Norrild

Keyword(s):

Human Papillomavirus ◽

Transcription Initiation ◽

Constitutive Expression ◽

Initiation Site ◽

Transcription Initiation Site ◽

Hpv 16 ◽

Reading Frame ◽

Human Papillomavirus Type 16 ◽

E6 And E7

Human papillomavirus type 16 (HPV-16) has the capacity to transform human primary keratinocytes. Maintenance of the transformed phenotype requires constitutive expression of the oncoproteins E6 and E7. The low-risk HPV types express E7 from monocistronic mRNA, but for the high-risk types, no mRNA that encodes E7 as the first open reading frame (ORF) has been identified. We recently identified a transcription initiation site within the E6 ORF of HPV-16 at nt 542. In the present study we have characterized the P542 promoter, which putatively controls monocistronic expression of E7. The monocistronic mRNA is not very abundant, but we have shown that an E7–luciferase fusion protein can be expressed in SiHa cells from a monocistronic HPV-16 transcript initiated at nt 542. The monocistronic mRNA expresses E7–luciferase more efficiently than the most abundant in vivo-like mRNA E6*IE7, initiated by P97 and spliced from nt 226 to 409. Furthermore, the translation initiation of E7 is most abundant from the monocistronic mRNA. We have also shown that the P542 promoter is downregulated by the transcription factor activator protein 4 (AP-4) and the differentiation-dependent factor hSkn-1a, both binding downstream of the transcription initiation site. In conclusion, we have found that P542 is a relatively weak promoter compared with P97 and may be downregulated in differentiated epithelial cells.

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Targeted expression of the E6 and E7 oncogenes of human papillomavirus type 16 in the epidermis of transgenic mice elicits generalized epidermal hyperplasia involving autocrine factors

Molecular and Cellular Biology ◽

10.1128/mcb.14.12.8250-8258.1994 ◽

1994 ◽

Vol 14 (12) ◽

pp. 8250-8258

Author(s):

P Auewarakul ◽

L Gissmann ◽

A Cid-Arregui

Keyword(s):

Human Papillomavirus ◽

Growth Factor ◽

Epithelial Cells ◽

Transgenic Mice ◽

Transforming Growth Factor ◽

Basal Layer ◽

Epidermal Hyperplasia ◽

Human Papillomavirus Type 16 ◽

E6 And E7

The E6 and E7 early genes of human papillomavirus type 16 have been shown in vitro to play a central role in the transforming capability of this virus. To explore their effects on differentiating epithelial cells in vivo, we used a bovine cytokeratin 10 (K10) promoter to target the expression of E6 and E7 to the suprabasal layers of the epidermis of transgenic mice. In two different lines of mice efficiently expressing the transgene, animals displayed generalized epidermal hyperplasia, hyperkeratosis and parakeratosis in the skin and the forestomach, both known to be sites of K10 expression. Northern (RNA) blot analysis revealed high levels of E6 and E7 transcripts, and in situ hybridizations localized these transcripts to the suprabasal strata of epidermis. In vivo labeling of proliferating cells showed two distinct effects of E6 and E7 expression in the epidermis: (i) an increase in the number of growing cells in the undifferentiated basal layer and (ii) abnormal proliferation of differentiated cells in the suprabasal strata. The expression of c-myc in the skin of transgenics was higher than that in control animals. The induction of c-myc transcription by topical application of tetradecanoyl phorbol acetate was prevented by simultaneous treatment with transforming growth factor beta 1 in nontransgenic skin but not in transgenic skin. In addition, transforming growth factor alpha was found to be overexpressed in the suprabasal layers of the transgenic epidermis. These findings suggest that autocrine mechanisms are involved in the development and maintenance of epidermal hyperplasia. Animals of both lines developed papillomas in skin sites exposed to mechanical irritation and wounding, suggesting that secondary events are necessary for progression to neoplasia. Collectively, these results provide new insights into the tumor promoter activities of human papillomavirus type 16 in epithelial cells in vivo.

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