scholarly journals The Global Transcriptional Effects of the Human Papillomavirus E6 Protein in Cervical Carcinoma Cell Lines Are Mediated by the E6AP Ubiquitin Ligase

2005 ◽  
Vol 79 (6) ◽  
pp. 3737-3747 ◽  
Author(s):  
Melissa L. Kelley ◽  
Kerri E. Keiger ◽  
Chan Jae Lee ◽  
Jon M. Huibregtse
Keyword(s):  
Human Papillomavirus ◽  
Cell Lines ◽  
Ubiquitin Ligase ◽  
Telomerase Activity ◽  
Transcriptional Program ◽  
Hpv E6 ◽  
Amplification Protocol ◽  
Independent Functions ◽  
Interfering Rna ◽  
E6 Protein

ABSTRACT The function of the human papillomavirus (HPV) E6 protein that is most clearly linked to carcinogenesis is the targeted degradation of p53, which is dependent on the E6AP ubiquitin ligase. Additional functions have been attributed to E6, including the stimulation of telomerase activity and the targeted degradation of other cellular proteins, but in most cases it is unclear whether these activities are also E6AP dependent. While E6 clearly influences the transcriptional program of HPV-positive cell lines through the inactivation of p53, it has been shown that at least a subset of its p53-independent functions are also reflected in the transcriptional program. For this study, we have determined the extent to which E6AP is involved in mediating the set of E6 functions that impact on the global transcriptional program of HPV-positive cell lines. The transcriptional profiles of ∼31,000 genes were characterized for three cell lines (HeLa, Caski, and SiHa cells) after small interfering RNA (siRNA)-mediated silencing of E6 or E6AP. We found that E6 and E6AP siRNAs elicited nearly identical alterations in the transcriptional profile of each cell line. Some of the expression alterations were apparent secondary effects of p53 stabilization, while the basis of most other changes was not reconcilable with previously proposed E6 functions. While expression changes of the TERT gene (telomerase catalytic subunit) were not revealed by the array, telomerase repeat amplification protocol assays showed that both E6 and E6AP knockouts resulted in a suppression of telomerase activity. Together, these results suggest that E6AP mediates a broad spectrum of E6 functions, including virtually all functions that impact on the transcriptional program of HPV-positive cell lines.

scholarly journals Metabolic rewiring is associated with HPV-specific profiles in cervical cancer cell lines

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kalliopi I. Pappa ◽  
George Daskalakis ◽  
Nicholas P. Anagnou
Keyword(s):  
Cervical Cancer ◽  
Cell Lines ◽  
Cancer Cell ◽  
High Resolution Mass Spectrometry ◽  
Cancer Cell Lines ◽  
Ultra Performance Liquid Chromatography ◽  
Hpv E6 ◽  
Cervical Cell ◽  
E6 Protein

AbstractBoth HPV-positive and HPV-negative cervical cancers are associated with aberrant metabolism, although the oncogenic drivers remain elusive. Here we show the assessment of the metabolomic profiles of four distinct cervical cell lines, a normal and three cancer cell lines, one HPV-negative (C33A) and two HPV-positive (SiHa HPV16+, HeLa HPV18+), employing an ultra performance liquid chromatography and a high resolution mass spectrometry. Out of the total 462 metabolites, 248 to 326 exhibited statistically significant differences, while Random Forests analysis identified unique molecules for each cell line. The two HPV+ cell lines exhibited features of Warburg metabolism, consistent with the role of the HPV E6 protein. SiHa and HeLa cells displayed purine salvage pathway activity, while C33A cells revealed synthesis of cytidine, via a novel mechanism. These data document a highly dynamic HPV-specific rewiring of metabolic pathways occurring in cervical cancer. Therefore, this approach can eventually provide novel mechanistic insights into cervical carcinogenesis.

scholarly journals NFX1-123 and Poly(A) Binding Proteins Synergistically Augment Activation of Telomerase in Human Papillomavirus Type 16 E6-Expressing Cells

2007 ◽  
Vol 81 (8) ◽  
pp. 3786-3796 ◽  
Author(s):  
Rachel A. Katzenellenbogen ◽  
Erin M. Egelkrout ◽  
Portia Vliet-Gregg ◽  
Lindy C. Gewin ◽  
Philip R. Gafken ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Epithelial Cells ◽  
Binding Proteins ◽  
Telomerase Activity ◽  
Protein Domain ◽  
Mrna Levels ◽  
Hpv16 E6 ◽  
Hpv E6 ◽  
Protein Partners ◽  
Reporter Assays

ABSTRACT Overcoming senescence signals in somatic cells is critical to cellular immortalization and carcinogenesis. High-risk human papillomavirus (HPV) can immortalize epithelial cells in culture through degradation of the retinoblastoma protein by HPV E7 and activation of hTERT transcription, the catalytic subunit of telomerase, by the heterodimer HPV E6/E6-associated protein (E6AP). Recent work in our laboratory identified a novel repressor of hTERT transcription, NFX1-91, which is targeted for ubiquitin-mediated degradation by HPV type 16 (HPV16) E6/E6AP. In contrast, NFX1-123, a splice variant NFX1, increased expression from an hTERT promoter that was activated by HPV16 E6/E6AP. Here, we show that HPV16 E6 bound both NFX1-91 and NFX1-123 through the common central domain of NFX1 in the absence of E6AP. NFX1-123 positively regulated hTERT expression, as its knockdown decreased hTERT mRNA levels and telomerase activity and its overexpression increased telomerase activity. We identified new protein partners of NFX1-123, including several cytoplasmic poly(A) binding proteins (PABPCs) that interacted with NFX1-123 through its N-terminal PAM2 motif, a protein domain characteristic of other PABPC protein partners. Furthermore, NFX1-123 and PABPCs together had a synergistic stimulatory effect on hTERT-regulated reporter assays. The data suggest that NFX1-123 is integral to hTERT regulation in HPV16 E6-expressing epithelial cells and that the interaction between NFX1-123 and PABPCs is critical to hTERT activity.

scholarly journals The Effect of Human Papillomavirus E6 Oncogene on the Radiosensitivity of Non-Oropharyngeal Cancer Cells

2017 ◽  
Vol 6 (2) ◽  
pp. 7
Author(s):  
Angela Hong ◽  
Xiaoying Zhang ◽  
Xiao Mei Zhang ◽  
Barbara Rose
Keyword(s):  
Lung Cancer ◽  
Human Papillomavirus ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Oropharyngeal Cancer ◽  
Colorectal Adenocarcinoma ◽  
Large Cell ◽  
Hpv E6 ◽  
Large Cell Lung Cancer

Background:We have previously shown that stable transfection of the human papillomavirus (HPV) E6*I oncogene can sensitize two HPV negative oropharyngeal cancer (OSCC) cell lines to radiation. In the current study, we extended our work on OSCC to determine whether the HPV E6 oncogene can enhance the radiosensitivity of non-OSCC cell lines.Methods:Three non-OSCC cell lines (melanoma, colorectal adenocarcinoma and large cell lung cancer) were stably transfection with the HPV E6 oncogene (E6 total, E6*I and E6*II) and treated with different doses of radiation. Clonogenic assays were used to measure the radiation survival.Results:Following transfection, there was a reduction in the survival of the melanoma cell line after 2 Gy (SF2) from 0.401 (untransfected) to 0.219 (Melanoma-E6 total). This reduction was not evident at higher doses of radiation. There was no significant change in the SF2 of melanoma-E6*I (0.303) and melanoma-E6*II (0.414). The SF2 colorectal adenocarcinoma and large cell lung cancer cell lines did not change significantly after transfection.Conclusions: The radiosensitizing effect of HPV E6 oncogene is cell line specific. We found no clear evidence of a radiosensitising effect of E6 in these three non-OSCC cancer cell lines.

scholarly journals Regulation of O-Linked N-Acetyl Glucosamine Transferase (OGT) through E6 Stimulation of the Ubiquitin Ligase Activity of E6AP

2021 ◽  
Vol 22 (19) ◽  
pp. 10286
Author(s):  
Kangli Peng ◽  
Ruochuan Liu ◽  
Caiwei Jia ◽  
Yiyang Wang ◽  
Geon H. Jeong ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Ubiquitin Ligase ◽  
Hek293 Cells ◽  
Cellular Proteins ◽  
Cellular Processes ◽  
Endogenous Expression ◽  
Protein Ubiquitination ◽  
Ubiquitin Ligase Activity ◽  
E6 Protein ◽  
Stimulation Of

Glycosyltransferase OGT catalyzes the conjugation of O-linked β-D-N-acetylglucosamine (O-GlcNAc) to Ser and Thr residues of the cellular proteins and regulates many key processes in the cell. Here, we report the identification of OGT as a ubiquitination target of HECT-type E3 ubiquitin (UB) ligase E6AP, whose overexpression in HEK293 cells would induce the degradation of OGT. We also found that the expression of E6AP in HeLa cells with the endogenous expression of the E6 protein of the human papillomavirus (HPV) would accelerate OGT degradation by the proteasome and suppress O-GlcNAc modification of OGT substrates in the cell. Overall, our study establishes a new mechanism of OGT regulation by the ubiquitin­–proteasome system (UPS) that mediates the crosstalk between protein ubiquitination and O-GlcNAcylation pathways underlying diverse cellular processes.

scholarly journals Telomerase Activity Is Sufficient To Allow Transformed Cells To Escape from Crisis

1999 ◽  
Vol 19 (3) ◽  
pp. 1864-1870 ◽  
Author(s):  
Tanya L. Halvorsen ◽  
Gil Leibowitz ◽  
Fred Levine
Keyword(s):  
Cell Lines ◽  
Simian Virus 40 ◽  
Telomerase Activity ◽  
Simian Virus ◽  
T Antigen ◽  
Transformed Cells ◽  
Pancreatic Cell ◽  
Amplification Protocol ◽  
Late Passage ◽  
Population Doublings

ABSTRACT The introduction of simian virus 40 large T antigen (SVLT) into human primary cells enables them to proliferate beyond their normal replicative life span. In most cases, this temporary escape from senescence eventually ends in a second proliferative block known as “crisis,” during which the cells cease growing or die. Rare immortalization events in which cells escape crisis are frequently correlated with the presence of telomerase activity. We tested the hypothesis that telomerase activation is the critical step in the immortalization process by studying the effects of telomerase activity in two mortal SVLT-Rasval12-transformed human pancreatic cell lines, TRM-6 and βlox5. The telomerase catalytic subunit, hTRT, was introduced into late-passage cells via retroviral gene transfer. Telomerase activity was successfully induced in infected cells, as demonstrated by a telomerase repeat amplification protocol assay. In each of nine independent infections, telomerase-positive cells formed rapidly dividing cell lines while control cells entered crisis. Telomere lengths initially increased, but telomeres were then maintained at their new lengths for at least 20 population doublings. These results demonstrate that telomerase activity is sufficient to enable transformed cells to escape crisis and that telomere elongation in these cells occurs in a tightly regulated manner.

scholarly journals Human Papillomavirus 16 (HPV-16), HPV-18, and HPV-31 E6 Override the Normal Phosphoregulation of E6AP Enzymatic Activity

2017 ◽  
Vol 91 (22) ◽  
Author(s):  
Jayashree Thatte ◽  
Lawrence Banks
Keyword(s):  
Human Papillomavirus ◽  
Enzymatic Activity ◽  
Ubiquitin Ligase ◽  
Subcellular Distribution ◽  
Hpv 16 ◽  
Human Papillomavirus 16 ◽  
Hpv E6 ◽  
Ubiquitin Ligase Activity ◽  
Hpv 18 ◽  
Stimulation Of

ABSTRACT The human papillomavirus (HPV) E6 oncoproteins recruit the cellular ubiquitin ligase E6AP/UBE3A to target cellular substrates for proteasome-mediated degradation, and one consequence of this activity is the E6 stimulation of E6AP autoubiquitination and degradation. Recent studies identified an autism-linked mutation within E6AP at T485, which was identified as a protein kinase A phosphoacceptor site and which could directly regulate E6AP ubiquitin ligase activity. In this study, we have analyzed how T485-mediated regulation of E6AP might affect E6 targeting of some of its known substrates. We show that modulation of T485 has no effect on the ability of E6 to direct either p53 or Dlg for degradation. Furthermore, T485 regulation has no effect on HPV-16 or HPV-31 E6-induced autodegradation of E6AP but does affect HPV-18 E6-induced autodegradation of E6AP. In cells derived from cervical cancers, we find low levels of both phosphorylated and nonphosphorylated E6AP in the nucleus. However, ablation of E6 results in a dramatic accumulation of phospho-E6AP in the cytoplasm, whereas nonphosphorylated E6AP accumulates primarily in the nucleus. Interestingly, E6AP phosphorylation at T485 confers association with 14-3-3 proteins, and this interaction seems to be important, in part, for the ability of E6 to recruit phospho-E6AP into the nucleus. These results demonstrate that HPV E6 overrides the normal phosphoregulation of E6AP, both in terms of its enzymatic activity and its subcellular distribution. IMPORTANCE Recent reports demonstrate the importance of phosphoregulation of E6AP for its normal enzymatic activity. Here, we show that HPV E6 is capable of overriding this regulation and can promote degradation of p53 and Dlg regardless of the phosphorylation status of E6AP. Furthermore, E6 interaction with E6AP also significantly alters how E6AP is subject to autodegradation and suggests that this is not a simple stimulation of an already-existing activity but rather a redirection of E6AP activity toward itself. Furthermore, E6-mediated regulation of the subcellular distribution of phospho-E6AP appears to be dependent, in part, upon the 14-3-3 family of proteins.

scholarly journals The Ubiquitin-Specific Peptidase USP15 Regulates Human Papillomavirus Type 16 E6 Protein Stability

2009 ◽  
Vol 83 (17) ◽  
pp. 8885-8892 ◽  
Author(s):  
Robin M. Vos ◽  
Jennifer Altreuter ◽  
Elizabeth A. White ◽  
Peter M. Howley
Keyword(s):  
Human Papillomavirus ◽  
Protein Stability ◽  
Affinity Purification ◽  
Peptidase Activity ◽  
Hpv16 E6 ◽  
Protein Levels ◽  
Human Papillomavirus Type 16 ◽  
Associated Proteins ◽  
Interfering Rna ◽  
E6 Protein

ABSTRACT Proteomic identification of human papillomavirus type 16 (HPV16) E6-interacting proteins revealed several proteins involved in ubiquitin-mediated proteolysis. In addition to the well-characterized E6AP ubiquitin-protein ligase, a second HECT domain protein (HERC2) and a deubiquitylating enzyme (USP15) were identified by tandem affinity purification of HPV16 E6-associated proteins. This study focuses on the functional consequences of the interaction of E6 with USP15. Overexpression of USP15 resulted in increased levels of the E6 protein, and the small interfering RNA-mediated knockdown of USP15 decreased E6 protein levels. These results implicate USP15 directly in the regulation of E6 protein stability and suggest that ubiquitylated E6 could be a substrate for USP15 ubiquitin peptidase activity. It remains possible that E6 could affect the activity of USP15 on specific cellular substrates, a hypothesis that can be tested as more is learned about the substrates and pathways controlled by USP15.

scholarly journals E6 Proteins from Multiple Human Betapapillomavirus Types Degrade Bak and Protect Keratinocytes from Apoptosis after UVB Irradiation

2008 ◽  
Vol 82 (21) ◽  
pp. 10408-10417 ◽  
Author(s):  
Michael P. Underbrink ◽  
Heather L. Howie ◽  
Kristin M. Bedard ◽  
Jennifer I. Koop ◽  
Denise A. Galloway
Keyword(s):  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Intrinsic Apoptotic Pathway ◽  
Uv Damage ◽  
Uv Treatment ◽  
Apoptotic Pathway ◽  
Hpv16 E6 ◽  
Hpv E6 ◽  
Hpv Types ◽  
E6 Protein

ABSTRACT Human papillomavirus (HPV) types from the beta genus (beta-HPVs) have been implicated in the development of skin cancer. A potentially important aspect of their carcinogenic role is the ability of the E6 protein to degrade the proapoptotic family member Bak, which gives cells the ability to survive UV damage. However, it is unknown if the ability to degrade Bak is limited to certain beta-HPV types or whether E6 expression in keratinocytes affects other proteins important for apoptosis signaling. We tested the abilities of E6 proteins from several representative members of the beta-HPVs to degrade Bak and protect UV-treated keratinocytes from apoptosis. The E6 proteins of the beta-HPV type 5 (HPV5), -8, -20, -22, -38, -76, -92, and -96, as well as the alpha genus HPV HPV16, all degraded Bak or prevented its accumulation following UV treatment but did not degrade Bak constitutively. In addition, when tested using HPV16 E6 (16E6) and 8E6 as representative E6 proteins from the alpha and beta genera, respectively, Bak degradation was dependent on the E3 ubiquitin ligase, E6AP. Other important regulators of apoptotic signaling were examined and found to be unperturbed by the expression of the beta-HPV E6 proteins. Importantly, the expression of beta-HPV E6 proteins protected keratinocytes from apoptosis to the same extent as 16E6-expressing cells. In conclusion, several of the beta-HPV types possess the ability to protect UV-treated keratinocytes from apoptosis by reducing levels of Bak in those cells, thus blocking the intrinsic apoptotic pathway.

scholarly journals Telomerase Activity in Reactive and Neoplastic Lymphoid Tissues: Infrequent Detection of Activity in Hodgkin's Disease

Blood ◽  
1997 ◽  
Vol 89 (1) ◽  
pp. 26-31 ◽  
Author(s):  
Pierre Brousset ◽  
Talal Al Saati ◽  
Nadia Chaouche ◽  
Reine-Claude Zenou ◽  
Daniel Schlaifer ◽  
...  
Keyword(s):  
Lymph Nodes ◽  
Cell Lines ◽  
Hodgkin's Disease ◽  
Hodgkin’S Disease ◽  
Telomerase Activity ◽  
Lymphoma Cell ◽  
Lymphoid Tissues ◽  
Telomerase Inhibitor ◽  
Amplification Protocol ◽  
Detection Assay

We used the recently described sensitive and rapid detection assay called telomeric repeat amplification protocol (TRAP) to detect telomerase activity in lymphoblastoid (n = 5) and lymphoma cell lines (n = 7), hyperplastic lymph nodes (n = 6) and tonsils (n = 5), and tissues involved by non-Hodgkin's lymphoma (NHL) (n = 43) and Hodgkin's disease (HD) (n = 14). Clearly evident telomerase activity was found in all lymphoblastoid and lymphoma cell lines, and in 34 of 43 cases (80%) of NHL. These results were expected because of the proliferative and immortal nature of the cell lines and most malignant cells. However, positive results were obtained with the TRAP assay in all hyperplastic lymph nodes and tonsils, which raises the issue of derepression of telomerase activity during an immune response. Telomerase activity alone therefore does not distinguish malignant lymphoid proliferations from reactive states. Telomerase activity was detected in only 1 of 14 cases (7%) of lymphoid tissues involved by HD. Eight of the 13 negative cases were considered to be interpretable because of the lack (3 of 13 cases) or low level (5 of 13 cases) of telomerase inhibition. The five remaining cases could not be evaluated because of their telomerase inhibitor content. The findings imply either transient or very low levels of telomerase activity in HD or that HD for the greater part is a telomerase-independent neoplasm. Microdissection studies are needed to identify subsets of cells carrying telomerase activity in both reactive and neoplastic lymphoid tissues.

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