Differences in the Expression of Human Papillomavirus Type 16 (HPV-16) E6 Oncogene mRNA in SiHa Cell Line Inoculated with CMV, HSV or Ureaplasmas

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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Cloning and sequencing of the murine farnesyltransferase α-encoding cDNA from a cell line which expresses the human papillomavirus type-16 E6 gene

Gene ◽

10.1016/0378-1119(95)00445-c ◽

1995 ◽

Vol 164 (2) ◽

pp. 373-374 ◽

Cited By ~ 1

Author(s):

Hiroshi Shirasawa ◽

Tomoaki Kinoshita ◽

Yuji Shino ◽

Kohji Mori ◽

Kumiko Shimizu ◽

...

Keyword(s):

Human Papillomavirus ◽

Cell Line ◽

Cloning And Sequencing ◽

Human Papillomavirus Type 16 ◽

E6 Gene ◽

A Cell

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Detection of Class-Specific Antibodies to Baculovirus-Derived Human Papillomavirus Type 16 (HPV-16) Capsid Proteins

Immunology of Human Papillomaviruses ◽

10.1007/978-1-4615-2449-6_26 ◽

1994 ◽

pp. 155-160 ◽

Cited By ~ 3

Author(s):

John Cason ◽

Parminder K. Kambo ◽

Bhavneet Shergill ◽

John Bible ◽

Barbara Kell ◽

...

Keyword(s):

Human Papillomavirus ◽

Capsid Proteins ◽

Hpv 16 ◽

Specific Antibodies ◽

Human Papillomavirus Type 16

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Human Papillomavirus Type 16 E7 Maintains Elevated Levels of the cdc25A Tyrosine Phosphatase during Deregulation of Cell Cycle Arrest

Journal of Virology ◽

10.1128/jvi.76.2.619-632.2002 ◽

2002 ◽

Vol 76 (2) ◽

pp. 619-632 ◽

Cited By ~ 57

Author(s):

Don X. Nguyen ◽

Thomas F. Westbrook ◽

Dennis J. McCance

Keyword(s):

Cell Cycle ◽

Human Papillomavirus ◽

Cell Cycle Arrest ◽

Cell Cycle Progression ◽

Tyrosine Phosphatase ◽

Early Gene ◽

Cycle Progression ◽

Hpv 16 ◽

Human Papillomavirus Type 16 ◽

Cycle Arrest

ABSTRACT Essential to the oncogenic properties of human papillomavirus type 16 (HPV-16) are the activities encoded by the early gene product E7. HPV-16 E7 (E7.16) binds to cellular factors involved in cell cycle regulation and differentiation. These include the retinoblastoma tumor suppressor protein (Rb) and histone deacetylase (HDAC) complexes. While the biological significance of these interactions remains unclear, E7 is believed to help maintain cells in a proliferative state, thus establishing an environment that is conducive to viral replication. Most pathways that govern cell growth converge on downstream effectors. Among these is the cdc25A tyrosine phosphatase. cdc25A is required for G1/S transition, and its deregulation is associated with carcinogenesis. Considering the importance of cdc25A in cell cycle progression, it represents a relevant target for viral oncoproteins. Accordingly, the present study focuses on the putative deregulation of cdc25A by E7.16. Our results indicate that E7.16 can impede growth arrest induced during serum starvation and keratinocyte differentiation. Importantly, these E7-specific phenotypes correlate with elevated cdc25A steady-state levels. Reporter assays performed with NIH 3T3 cell lines and human keratinocytes indicate that E7 can transactivate the cdc25A promoter. In addition, transcriptional activation by E7.16 requires the distal E2F site within the cdc25A promoter. We further demonstrate that the ability of E7 to abrogate cell cycle arrest, activate cdc25A transcription, and increase cdc25A protein levels requires intact Rb and HDAC-1 binding domains. Finally, by using the cdk inhibitor roscovitine, we reveal that E7 activates the cdc25A promoter independently of cell cycle progression and cdk activity. Consequently, we propose that E7.16 can directly target cdc25A transcription and maintains cdc25A gene expression by disrupting Rb/E2F/HDAC-1 repressor complexes.

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Adeno-Associated Virus Major Rep78 Protein Disrupts Binding of TATA-Binding Protein to the p97 Promoter of Human Papillomavirus Type 16

Journal of Virology ◽

10.1128/jvi.74.5.2459-2465.2000 ◽

2000 ◽

Vol 74 (5) ◽

pp. 2459-2465 ◽

Cited By ~ 17

Author(s):

Pei-Fen Su ◽

Shu-Yuan Chiang ◽

Cheng-Wen Wu ◽

Felicia Y.-H. Wu

Keyword(s):

Human Papillomavirus ◽

Binding Protein ◽

Core Promoter ◽

Tata Binding Protein ◽

Tata Box ◽

Dependent Manner ◽

Hpv 16 ◽

Adeno Associated Virus ◽

Human Papillomavirus Type 16 ◽

E6 And E7

ABSTRACT Adeno-associated virus type 2 (AAV) is known to inhibit the promoter activities of several oncogenes and viral genes, including the human papillomavirus type 16 (HPV-16) E6 and E7 transforming genes. However, the target elements of AAV on the long control region (LCR) upstream of E6 and E7 oncogenes are elusive. A chloramphenicol acetyltransferase assay was performed to study the effect of AAV on the transcription activity of the HPV-16 LCR in SiHa (HPV-positive) and C-33A (HPV-negative) cells. The results reveal that (i) AAV inhibited HPV-16 LCR activity in a dose-dependent manner, (ii) AAV-mediated inhibition did not require the HPV gene products, and (iii) the AAV replication gene product Rep78 was involved in the inhibition. Deletion mutation analyses of the HPV-16 LCR showed that regulatory elements outside the core promoter region of the LCR may not be direct targets of AAV-mediated inhibition. Further study with the electrophoretic mobility shift assay demonstrated that Rep78 interfered with the binding of TATA-binding protein (TBP) to the TATA box of the p97 core promoter more significantly than it disrupted the preformed TBP-TATA complex. These data thus suggest that Rep78 may inhibit transcription initiation of the HPV-16 LCR by disrupting the interaction between TBP and the TATA box of the p97 core promoter.

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Transcriptional Activation of the Telomerase hTERT Gene by Human Papillomavirus Type 16 E6 Oncoprotein

Journal of Virology ◽

10.1128/jvi.75.9.4467-4472.2001 ◽

2001 ◽

Vol 75 (9) ◽

pp. 4467-4472 ◽

Cited By ~ 177

Author(s):

Tim Veldman ◽

Izumi Horikawa ◽

J. Carl Barrett ◽

Richard Schlegel

Keyword(s):

Human Papillomavirus ◽

Transcriptional Activation ◽

Transcriptional Control ◽

Regulatory Region ◽

Rnase Protection ◽

Hpv 16 ◽

Htert Gene ◽

Human Papillomavirus Type 16 ◽

E6 Gene

ABSTRACT The E6 and E7 oncogenes of human papillomavirus type 16 (HPV-16) are sufficient for the immortalization of human genital keratinocytes in vitro. The products of these viral genes associate with p53 and pRb tumor suppressor proteins, respectively, and interfere with their normal growth-regulatory functions. The HPV-16 E6 protein has also been shown to increase the telomerase enzyme activity in primary epithelial cells by an unknown mechanism. We report here that a study using reverse transcription-PCR and RNase protection assays in transduced primary human foreskin keratinocytes (HFKs) shows that the E6 gene (but not the E7 gene) increases telomerase hTERT gene transcription coordinately with E6-induced telomerase activity. In these same cells, the E6 gene induces a 6.5-fold increase in the activity of a 1,165-bp 5′ promoter/regulatory region of the hTERT gene, and this induction is attributable to a minimal 251-bp sequence (−211 to +40). Furthermore, there is a 35-bp region (+5 to +40) within this minimal E6-responsive promoter that is responsible for 60% of E6 activity. Although the minimal hTERT promoter contains Myc-responsive E-box elements and recent studies have suggested a role for Myc protein in hTERT transcriptional control, we found no alterations in the abundance of either c-Myc or c-Mad in E6-transduced HFKs, suggesting that there are other or additional transcription factors critical for regulating hTERT expression.

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Immune Responses and Therapeutic Antitumor Effects of an Experimental DNA Vaccine Encoding Human Papillomavirus Type 16 Oncoproteins Genetically Fused to Herpesvirus Glycoprotein D

Clinical and Vaccine Immunology ◽

10.1128/cvi.00264-10 ◽

2010 ◽

Vol 17 (10) ◽

pp. 1576-1583 ◽

Cited By ~ 17

Author(s):

Mariana O. Diniz ◽

Marcio O. Lasaro ◽

Hildegund C. Ertl ◽

Luís C. S. Ferreira

Keyword(s):

Human Papillomavirus ◽

T Cell ◽

Immune Responses ◽

Dna Vaccine ◽

T Cell Responses ◽

Glycoprotein D ◽

Antitumor Effects ◽

Hpv 16 ◽

Human Papillomavirus Type 16 ◽

Cell Responses

ABSTRACT Recombinant adenovirus or DNA vaccines encoding herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) genetically fused to human papillomavirus type 16 (HPV-16) oncoproteins (E5, E6, and E7) induce antigen-specific CD8+ T-cell responses and confer preventive resistance to transplantable murine tumor cells (TC-1 cells). In the present report, we characterized some previously uncovered aspects concerning the induction of CD8+ T-cell responses and the therapeutic anticancer effects achieved in C57BL/6 mice immunized with pgD-E7E6E5 previously challenged with TC-1 cells. Concerning the characterization of the immune responses elicited in mice vaccinated with pgD-E7E6E5, we determined the effect of the CD4+ T-cell requirement, longevity, and dose-dependent activation on the E7-specific CD8+ T-cell responses. In addition, we determined the priming/boosting properties of pgD-E7E6E5 when used in combination with a recombinant serotype 68 adenovirus (AdC68) vector encoding the same chimeric antigen. Mice challenged with TC-1 cells and then immunized with three doses of pgD-E7E6E5 elicited CD8+ T-cell responses, measured by intracellular gamma interferon (IFN-γ) and CD107a accumulation, to the three HPV-16 oncoproteins and displayed in vivo antigen-specific cytolytic activity, as demonstrated with carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled target cells pulsed with oligopeptides corresponding to the H-2Db -restricted immunodominant epitopes of the E7, E6, or E5 oncoprotein. Up to 70% of the mice challenged with 5 × 105 TC-1 cells and immunized with pgD-E7E6E5 controlled tumor development even after 3 days of tumor cell challenge. In addition, coadministration of pgD-E7E6E5 with DNA vectors encoding pGM-CSF or interleukin-12 (IL-12) enhanced the therapeutic antitumor effects for all mice challenged with TC-1 cells. In conclusion, the present results expand our previous knowledge on the immune modulation properties of the pgD-E7E6E5 vector and demonstrate, for the first time, the strong antitumor effects of the DNA vaccine, raising promising perspectives regarding the development of immunotherapeutic reagents for the control of HPV-16-associated tumors.

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The interaction between steroid hormones, human papillomavirus type 16, E6 oncogene expression, and cervical cancer

International Journal of Gynecological Cancer ◽

10.1136/ijgc-00009577-200311000-00015 ◽

2003 ◽

Vol 13 (6) ◽

pp. 834-842 ◽

Cited By ~ 2

Author(s):

M. Moodley ◽

S. Sewart ◽

C. S. Herrington ◽

R. Chetty ◽

R. Pegoraro ◽

...

Keyword(s):

Cervical Cancer ◽

Human Papillomavirus ◽

Steroid Hormones ◽

Housekeeping Gene ◽

Hpv 16 ◽

Tissue Samples ◽

Hpv Dna ◽

Human Papillomavirus Type 16 ◽

Transforming Proteins

Various risk factors have been implicated in the causation of cervical cancer including human papillomavirus (HPV), the early genes (E6 and E7) of which encode the main transforming proteins. Studies have suggested that steroid hormones may enhance the expression of these genes leading to loss of p53 gene-mediated cell apoptosis. A total of 120 cervical tissue samples were obtained from patients with proven cervical cancer. Patients who used depo-medroxyprogesterone acetate steroid contraception were recruited as part of the steroid arm. Only HPV DNA type 16 samples were used for the study. Controls included three cell lines (CaSki, SiHa, & C33A) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal housekeeping gene. Of 120 patients, there were 111 patients with HPV type 16 identified. Of this number, RNA was present in 63 samples. There were 30 women (30/63) who used steroid contraception. In relation to patients who used contraception, HPV 16 E6 gene expression was present in 79% (n = 23) and 88% (n = 30) of steroid users compared to nonusers, respectively. In total there were 25 patients (40%) with expression of the HPV 16 E6*I gene and 30 patients with expression of the E6*II gene. There were 57% of steroid users (n = 17) who had expression of the E6*I/E6*II gene, compared to 52% (n = 17) of nonusers (P = 0.800). From a molecular level, this study does not confirm the role of injectable progesterones in cervical carcinogenesis.

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