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

Abstract

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...

Gamma Interferon-Inducible Protein 10 Induces HeLa Cell Apoptosis through a p53-Dependent Pathway Initiated by Suppression of Human Papillomavirus Type 18 E6 and E7 Expression

“…We tried to authenticate these data by Western blot analysis; however we failed to obtain a satisfactory result. This is largely due to the low levels of E6 expression and poor reactivity of available E6 antibodies, which have been similarly reported by a number of publications (25,29,36).…”

Selective induction of apoptosis by leptomycin B in keratinocytes expressing HPV oncogenes

“…Indeed, a central function of the E6 protein is to inactivate pro-apoptotic proteins such as p53, 21 Bak 32 or Bax 33 proteolytically, thereby protecting against cell death. It has been demonstrated that the HPV 16 E6 protein preferentially localises to the nucleus of cervical cancer cells 34 and that HPV 18 E6 is co-exported with p53 from the nucleus to the cytoplasm. 35 LMB treatment of cells expressing Ôhigh riskÕ HPV E6 results in a block of p53 nuclear export and stabilisation of p53, 36 however this LMB-induced reduction in HPV 18 E6-mediated degradation was shown to be only partial, suggesting that both nuclear and cytoplasmic proteasomes can target p53 for degradation.…”

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

“…All antibodies used in this study were obtained from the following commercial sources, except for the 4C6 mouse monoclonal antibody to HPV16 E6 (5,28), generated by Etienne Weiss (University of Strasbourg) and provided through Arbor Vita Corporation (Sunnyvale, CA): actin (1501; Chemicon), E6AP (sc-25509; Santa Cruz), HA-horseradish peroxidase (Roche), IB␣ (9242; Cell Signaling), p53 (OP43; Calbiochem), ubiquitin (sc-9133; Santa Cruz), USP15 (H00009958-M01; Abnova), and Xpress (R910-25; Invitrogen).…”