Targetting of the N-terminal domain of the human papillomavirus type 16 E6 oncoprotein with monomeric scFvs blocks the E6-mediated degradation of cellular p53

Giovane, Christine; Travé, Gilles; Briones, Amélie; Lutz, Yves; Wasylyk, Bohdan; Weiss, Étienne

doi:10.1002/(sici)1099-1352(199903/04)12:2<141::aid-jmr453>3.0.co;2-o

Cited by 38 publications

(46 citation statements)

References 45 publications

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“…Antibody Assays Monoclonal antibodies 4C6 (IgG1,n), 6F4 (IgG1,n), and 3F8 (IgG2a,n) were purified either by affinity chromatography with immobilized antigen as described (13) or by protein A chromatography (Amersham Biosciences) according to the manufacturer's instructions. Mouse polyclonal IgGs were obtained from Sigma-Aldrich.…”

Section: Methodsmentioning

confidence: 99%

“…All antibody preparations were dialyzed against 20 mmol/L HEPES (pH 7.5) and 150 mmol/L NaCl before use and kept at 4jC at a concentration of f3 mg/mL. The ability of antibodies to bind to E6 expressed in reticulocyte extracts was tested as described (13). Transduction experiments were carried out in 24-well plates or in four-well LabTek II chambered cover glasses (Nalge Nunc) with 1.5 Â 10 4 cells/mL seeded the day before.…”

Section: Methodsmentioning

confidence: 99%

“…Certain antibodies that bind to the NH 2 -terminal region of E6 were found to totally block the E6-mediated degradation of p53 in vitro (13,14). These antibodies were cloned as single-chain Fv fragments and accumulated as toxic aggregates when expressed in the cytoplasm (15).…”

Section: Introductionmentioning

confidence: 99%

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Suppression of cervical carcinoma cell growth by intracytoplasmic codelivery of anti-oncoprotein E6 antibody and small interfering RNA

Courtête¹,

Sibler²,

Zeder‐Lutz³

et al. 2007

Molecular Cancer Therapeutics

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Cervical cancer is caused by high-risk types of human papillomaviruses (HPV) that encode the E6 and E7 oncogenes. Silencing of E6 gene expression in HPVpositive cell lines by transfection of small interfering RNA (siRNA) with cationic lipids restores the dormant p53 tumor suppressor pathway. Because cationic lipids can also be used for intracytoplasmic delivery of proteins, we tested whether the delivery of monoclonal antibodies that bind to HPV16 E6 and neutralize its biological activity in vitro could restore p53 function in tumor cells. Here, we show that the 4C6 antibody is efficiently delivered into the cell cytoplasm using a lipidic reagent used for siRNA transfection. The delivery of 4C6 resulted in the nuclear accumulation of p53 protein in CaSki and SiHa cells but not in HeLa cells. Furthermore, the antibody-mediated p53 response was dramatically increased when a peptide corresponding to the 4C6 epitope and bearing a COOHterminal cysteine residue was added to the transduction mixture. We found that a fraction of the added peptides were dimers that allowed the formation of antibody polymers adsorbed onto the lipidic matrix. With this system, the proliferation of CaSki and SiHa cells was strongly diminished, but no apoptosis was detectable. Remarkably, cell growth was almost totally suppressed by the addition of E6-specific siRNA to the transduction complex. The results indicate that the activity of E6 oncoprotein can be down-regulated in vivo by lipidmediated antibody delivery and that antibodies and siRNA act synergistically when codelivered. This novel targeting strategy is simple to implement and may find therapeutic applications.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Suppression of cervical carcinoma cell growth by intracytoplasmic codelivery of anti-oncoprotein E6 antibody and small interfering RNA

Courtête¹,

Sibler²,

Zeder‐Lutz³

et al. 2007

Molecular Cancer Therapeutics

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“…Two antibodies, named 6F4 (described previously by Giovane et al, 1999) the peptide-coding regions of the retained phages were sequenced. In this case, no clear consensus was found, but most peptides contained an aromatic residue (F/Y) and an arginine interspaced by five residues (Fig.…”

mentioning

confidence: 99%

“…Two antibodies, named 6F4 (described previously by Giovane et al, 1999) and 4C6, were cloned. While 6F4 and 4C6 contained different CDR3-H and CDR3-L coding sequences, they reacted equally with SDS-denatured maltosebinding protein (MBP)-HPV-16 E6 fusion protein and did not recognize MBP-HPV-6 E6 or MBP-HPV-11 E6 fusion proteins, processed in parallel (not shown).…”

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confidence: 99%

Preferential nuclear localization of the human papillomavirus type 16 E6 oncoprotein in cervical carcinoma cells

Masson

Hindelang

Sibler

et al. 2003

Journal of General Virology

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The E6 protein of the high-risk human papillomavirus type 16 (HPV-16) is involved in the tumorigenesis of human cervical cells by targeting numerous cellular proteins. We characterized new anti-E6 monoclonal antibodies and used them for precise localization of the E6 oncoprotein within carcinoma cells. Overexpressed E6 protein was predominantly detected in the nucleus of transiently transfected HaCaT cells. While mostly localized at the periphery of condensed chromatin, E6 was also associated with nuclear ribonucleoproteic ultrastructures and with some ribosomal areas in the cytoplasm of SiHa and CaSki cells. The chimeric b-galactosidase-E6 protein expressed in transfected HeLa cells was essentially localized in the nuclear compartment. Together, these data indicate that the E6 sequence of HPV-16 may encode a nuclear localization signal. The preferential nuclear distribution of this viral oncoprotein in HPV-transformed cells correlates with its activities at the transcriptional level.High-risk strains of human papillomaviruses (HPVs) such as HPV-16 are associated with lesions that can progress to cervical cancer (zur Hausen, 1999). The genome of these viruses encodes an oncoprotein, E6, which mediates degradation of the cellular p53 tumour suppressor protein in vitro (Scheffner et al., 1992) and in vivo (Lechner et al., 1992). Although this particular property of E6 remains the main explanation of its transforming activity, recent findings indicate that E6 is a multifunctional protein with p53-independent activities. It has been shown that E6 activates transcription of the telomerase gene (hTERT; Klingelhutz et al., 1996) and acts as a DNA-binding protein with high affinity for four-way DNA junctions (Ristriani et al., 2000(Ristriani et al., , 2001. In addition to p53, E6 interacts with a variety of cellular proteins, of which a significant number act at the transcriptional level (Mantovani & Banks, 2001).Previous studies on the localization of HPV-16 E6 within human carcinoma cells have led to contradictory results, most probably due to the low level of endogenous E6 protein and the poor reactivity of the available anti-E6 antibodies. E6 protein was localized in the cytoplasmic perinuclear region of SiHa cells (Daniels et al., 1998) and co-localized with p53 in the cytoplasm of CaSki cells (Liang et al., 1993). In transfected COS1 cells, overexpressed HPV-16 E6 protein is essentially found in the nuclear compartment (Kanda et al., 1991;Schwalbach et al., 2000;Sherman & Schlegel, 1996), while in transfected human cells, E6 of HPV-18, another malignant strain, is evenly distributed in the cytoplasm and the nucleus (Guccione et al., 2002). We generated monoclonal antibodies that specifically bind to the recombinant E6 protein of HPV-16. The aim of this study was to determine the precise intracellular localization of the E6 protein in transformed cells in order to establish whether the cellular distribution of this oncoprotein correlates with its recently described p53-independent biological functions. Here, we h...

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Escape of p53 protein from E6‐mediated degradation in HeLa cells after cisplatin therapy

Węsierska‐Gądek¹,

Schloffer

Kotala

et al. 2002

Intl Journal of Cancer

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We previously reported that therapy of human cervical carcinoma HeLa cells with CP induced segregation of nucleoli and changes of nuclei characteristic of apoptosis. We raised the question of whether p53 can be reactivated by chemotherapy in HeLa cells despite the presence of HPVencoded E6 activity. Cellular levels of p53 protein increased after CP treatment, reaching a maximum after 6 hr. p53 protein accumulated preferentially in the nucleoli, with a peak after 15 hr. CP-induced nucleolar targeting of p53 appears to be selective because p73, another member of the p53 gene family, accumulated primarily in nuclei in response to CP. Monitoring of the intranuclear distribution of Hdm-2, a negative regulator of p53, revealed this protein in the nucleoli of untreated controls translocated into chromatin during CP therapy. Interestingly, p14ARF showed an inverse intranuclear redistribution. Proteasome inhibitors were not able to mimic the effect of CP on p53 levels. Since the reduced stability of wild-type p53 protein in HeLa cells is a consequence of its enhanced ubiquitination by virally encoded E6 protein, resulting in its accelerated degradation, we checked the cellular level of E6 during CP therapy. Six hours after application of CP, E6 protein expression was markedly reduced. This coincided with the increase of cellular p53 and preceded the nucleolar accumulation of p53 protein, indicating that repression of virally coded E6 protein by CP contributes to the restoration of p53 expression.

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Targetting of the N-terminal domain of the human papillomavirus type 16 E6 oncoprotein with monomeric scFvs blocks the E6-mediated degradation of cellular p53

Cited by 38 publications

References 45 publications

Suppression of cervical carcinoma cell growth by intracytoplasmic codelivery of anti-oncoprotein E6 antibody and small interfering RNA

Suppression of cervical carcinoma cell growth by intracytoplasmic codelivery of anti-oncoprotein E6 antibody and small interfering RNA

Preferential nuclear localization of the human papillomavirus type 16 E6 oncoprotein in cervical carcinoma cells

Escape of p53 protein from E6‐mediated degradation in HeLa cells after cisplatin therapy

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