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

2010 ◽  
Vol 84 (20) ◽  
pp. 10644-10652 ◽  
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.

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

2010 ◽  
Vol 84 (21) ◽  
pp. 10956-10964 ◽  
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.

scholarly journals NFX1 Interacts with mSin3A/Histone Deacetylase To Repress hTERT Transcription in Keratinocytes

2008 ◽  
Vol 28 (15) ◽  
pp. 4819-4828 ◽  
Author(s):  
Mei Xu ◽  
Weifeng Luo ◽  
David J. Elzi ◽  
Carla Grandori ◽  
Denise A. Galloway
Keyword(s):  
Human Papillomavirus ◽  
Histone Deacetylase ◽  
Ubiquitin Ligase ◽  
Chromatin Structure ◽  
Hairpin Rna ◽  
Hpv16 E6 ◽  
Human Papillomavirus Type 16 ◽  
Corepressor Complex ◽  
Htert Transcription ◽  
Short Hairpin

ABSTRACT Transcription of the catalytic subunit of telomerase (hTERT) in keratinocytes can be induced by human papillomavirus type 16 (HPV16) E6/E6AP ubiquitin ligase through degradation of the repressor, NFX1-91. Here, we demonstrate that NFX1-91 interacts with the corepressor complex mSin3A/histone deacetylase (HDAC) at the hTERT promoter. By degrading NFX1-91, E6/E6AP changes the chromatin structure at the hTERT promoter as indicated by enhanced acetylation of histones H3 and H4 as well as dimethylation of H3K4. Knockdown of NFX1-91 by short hairpin RNA (shRNA) mimics the effect of E6 and leads to acetylation of histones H3 and H4. Conversely, knockdown of E6AP by shRNA suppresses histone acetylation at the hTERT promoter. These data demonstrate that targeted degradation of NFX1-91 by E6/E6AP dissociates the mSin3A/HDAC complex from the hTERT promoter and induces hTERT transcription.

Retrovirus-mediated delivery of short hairpin RNA targeting human papillomavirus (HPV) 16 E6 and E7 oncogenes and induction of apoptosis in oropharyngeal squamous cell cancer (OSCC) cell line

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 6002-6002 ◽  
Author(s):  
T. Rampias ◽  
C. Sasaki ◽  
D. DiMaio ◽  
A. Psyrri
Keyword(s):  
Gene Therapy ◽  
Human Papillomavirus ◽  
Cell Cancer ◽  
Short Hairpin Rna ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Hairpin Rna ◽  
Hpv16 E6 ◽  
Short Hairpin ◽  
E6 And E7

6002 Background: Human papillomavirus type 16 is identified in almost 50% of the cases of OSCC. The E6 and E7 genes of HPVs encode oncoproteins that bind and degrade the tumor suppressor proteins p53 and Rb, respectively. We are exploring the potential use of short hairpin RNA (sh RNA) for gene therapy of HPV-positive OSCC. Methods: Small hairpin RNAs targeting E6 or E7 genes were delivered by a retrovirus vector to 93VU147T (bearing integrated HPV16 DNA) and 92VU040T (HPV negative) oropharyngeal cancer cell lines. Flow cytometry analysis was used to assess apoptosis after the retrovirus infection. The E6 and E7 mRNA downregulation was assessed by reverse transcription polymerase chain reaction (RT-PCR). At protein level p53 and Rb expression were evaluated with Western blotting analysis. Results: Apoptosis was seen in over 90% of 93VU147T cells 48 hours after infection whereas 92VU040T cells were not affected. RT-PCR demonstrated that HPV16 E6/E7 mRNA levels decreased significantly in infected 93VU147T cells. 93VU147T infected cells also showed a marked increase in p53 and Rb protein levels. Conclusions: Downregulation of E6/E7 gene expression in HPV16+ OSCC cells results in apoptosis and reactivation of p53 and Rb tumor suppression pathways. These results have significant implications in treating HPV-associated OSCC with HPV-targeted gene therapy. No significant financial relationships to disclose.

E6 and E7 Oncoproteins from Human Papillomavirus Type 16 Induce Activation of Human Transforming Growth Factorβ1Promoter throughout Sp1 Recognition Sequence

2006 ◽  
Vol 19 (3) ◽  
pp. 468-480 ◽  
Author(s):  
Oscar Peralta-Zaragoza ◽  
Víctor Bermúdez-Morales ◽  
Lourdes Gutiérrez-Xicotencatl ◽  
Juan Alcocer-González ◽  
Félix Recillas-Targa ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Recognition Sequence ◽  
Human Papillomavirus Type 16 ◽  
E6 And E7 Oncoproteins ◽  
E6 And E7

scholarly journals Intrabodies targeting human papillomavirus 16 E6 and E7 oncoproteins for therapy of established HPV-associated tumors

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Francesca Paolini ◽  
Carla Amici ◽  
Mariantonia Carosi ◽  
Claudia Bonomo ◽  
Paola Di Bonito ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Antitumor Activity ◽  
Tumor Growth ◽  
Tumor Progression ◽  
Cellular Transformation ◽  
Neoplastic Progression ◽  
Single Chain ◽  
Hpv16 E6 ◽  
Associated Lesions ◽  
E6 And E7

Abstract Background The oncogenic activity of the high risk human papillomavirus type 16 (HPV16) is fully dependent on the E6 and E7 viral oncoproteins produced during viral infection. The oncoproteins interfere with cellular homeostasis by promoting proliferation, inhibiting apoptosis and blocking epithelial differentiation, driving the infected cells towards neoplastic progression. The causal relationship between expression of E6/E7 and cellular transformation allows inhibiting the oncogenic process by hindering the activity of the two oncoproteins. We previously developed and characterized some antibodies in single-chain format (scFvs) against the HPV16 E6 and E7 proteins, and demonstrated both in vitro and in vivo their antitumor activity consisting of protective efficacy against tumor progression of HPV16-positive cells. Methods Envisioning clinical application of the best characterized anti-HPV16 E6 and –HPV16 E7 scFvs, we verified their activity in the therapeutic setting, on already implanted tumors. Recombinant plasmids expressing the anti-HPV16 E6 scFvI7 with nuclear targeting sequence, or the anti-HPV16 E7 scFv43M2 with endoplasmic reticulum targeting sequence were delivered by injection followed by electroporation to three different preclinical models using C57/BL6 mice, and their effect on tumor growth was investigated. In the first model, the HPV16+ TC-1 Luc cells were used to implant tumors in mice, and tumor growth was measured by luciferase activity; in the second model, a fourfold number of TC-1 cells was used to obtain more aggressively growing tumors; in the third model, the HPV16+ C3 cells where used to rise tumors in mice. To highlight the scFv possible mechanism of action, H&E and caspase-3 staining of tumor section were performed. Results We showed that both the anti-HPV16 E6 and HPV16 E7 scFvs tested were efficacious in delaying tumor progression in the three experimental models and that their antitumor activity seems to rely on driving tumor cells towards the apoptotic pathway. Conclusion Based on our study, two scFvs have been identified that could represent a safe and effective treatment for the therapy of HPV16-associated lesions. The mechanism underlying the scFv effectiveness appears to be leading cells towards death by apoptosis. Furthermore, the validity of electroporation, a methodology allowed for human treatment, to deliver scFvs to tumors was confirmed.

scholarly journals Multiple ASF/SF2 Sites in the Human Papillomavirus Type 16 (HPV-16) E4-Coding Region Promote Splicing to the Most Commonly Used 3′-Splice Site on the HPV-16 Genome

2010 ◽  
Vol 84 (16) ◽  
pp. 8219-8230 ◽  
Author(s):  
Monika Somberg ◽  
Stefan Schwartz
Keyword(s):  
Cervical Cancer ◽  
Human Papillomavirus ◽  
Splice Site ◽  
Binding Sites ◽  
Mrna Levels ◽  
Coding Region ◽  
Cervical Lesions ◽  
Hpv 16 ◽  
Human Papillomavirus Type 16 ◽  
E6 And E7

ABSTRACT Our results presented here demonstrate that the most abundant human papillomavirus type 16 (HPV-16) mRNAs expressing the viral oncogenes E6 and E7 are regulated by cellular ASF/SF2, itself defined as a proto-oncogene and overexpressed in cervical cancer cells. We show that the most frequently used 3′-splice site on the HPV-16 genome, site SA3358, which is used to produce primarily E4, E6, and E7 mRNAs, is regulated by ASF/SF2. Splice site SA3358 is immediately followed by 15 potential binding sites for the splicing factor ASF/SF2. Recombinant ASF/SF2 binds to the cluster of ASF/SF2 sites. Mutational inactivation of all 15 sites abolished splicing to SA3358 and redirected splicing to the downstream-located, late 3′-splice site SA5639. Overexpression of a mutant ASF/SF2 protein that lacks the RS domain, also totally inhibited the usage of SA3358 and redirected splicing to the late 3′-splice site SA5639. The 15 ASF/SF2 binding sites could be replaced by an ASF/SF2-dependent, HIV-1-derived splicing enhancer named GAR. This enhancer was also inhibited by the mutant ASF/SF2 protein that lacks the RS domain. Finally, silencer RNA (siRNA)-mediated knockdown of ASF/SF2 caused a reduction in spliced HPV-16 mRNA levels. Taken together, our results demonstrate that the major HPV-16 3′-splice site SA3358 is dependent on ASF/SF2. SA3358 is used by the most abundantly expressed HPV-16 mRNAs, including those encoding E6 and E7. High levels of ASF/SF2 may therefore be a requirement for progression to cervical cancer. This is supported by our earlier findings that ASF/SF2 is overexpressed in high-grade cervical lesions and cervical cancer.

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