The human papillomavirus (HPV) E2 protein regulates viral gene expression and is also required for viral replication. HPV-transformed cells often contain chromosomally integrated copies of the HPV genome in which the viral E2 gene is disrupted. We have shown previously that re-expression of the HPV 16 E2 protein in HPV 16-transformed cells results in cell death via apoptosis. Here we show that the HPV 16 E2 protein can induce apoptosis in both HPV-transformed and non-HPV-transformed cell lines. E2-induced apoptosis is abrogated by a trans-dominant negative mutant of p53 or by overexpression of the HPV 16 E6 protein, but is increased by overexpression of wild-type p53. We show that mutations that block the DNA binding activity of E2 do not impair the ability of this protein to induce apoptosis. In contrast, removal of both N-terminal domains from the E2 dimer completely blocks E2-induced cell death. Heterodimers formed between wild-type E2 and N-terminally deleted E2 proteins also fail to induce cell death. Our data suggest that neither the DNA binding activity of E2 nor other HPV proteins are required for the induction of apoptosis by E2 and that E2-induced cell death occurs via a p53-dependent pathway.Papillomaviruses infect epithelial cells and generally induce the formation of benign hyperproliferative lesions. However, some papillomavirus types are associated with cancer. For example, human papillomavirus (HPV) 1 types 16 and 18 have been linked to cervical cancer in women (1) and bovine papillomavirus (BPV) types 2 and 4 have been linked to bladder cancer and cancer of the upper alimentary canal respectively, in cattle (2, 3). Human cervical cancers express the viral E6 and E7 oncogenes, and the products of these genes increase cell proliferation and promote cell immortalization (for a review, see Ref. 4). The human papillomavirus E2 gene, or lack thereof, is also thought to play a major role in the development of cervical cancer. Most cervical cancers contain chromosomally integrated copies of the HPV genome in which the viral E2 gene has been disrupted (5). Furthermore, mutations in the E2 gene increase the immortalization capacity of HPV 16 (6).The papillomavirus E2 genes encode sequence-specific DNAbinding proteins that regulate viral gene expression and are also required for viral DNA replication (reviewed in Ref. 7). The E2 proteins bind as dimers to multiple copies of an inverted repeat sequence found within the viral long control region. Depending on the particular virus and the particular E2 protein being studied, the binding of E2 to these sites can either activate or repress transcription of the E6 and E7 oncogenes. For example, the HPV 16 E2 protein activates transcription from the P97 promoter located at the 3Ј end of the HPV 16 long control region, whereas, under exactly the same conditions, the BPV1 E2 protein represses P97 promoter activity (8,9). Each subunit of the E2 dimer contains two domains separated by a flexible hinge: the N-terminal domain of each subunit mediates the regulation o...

The human papillomavirus (HPV) 16 E2 protein (hE2) binds to four sites present upstream of the P97 promoter and regulates transcription of the viral E6 and E7 oncogenes. We have determined the relative binding constants for the interaction of the full-length hE2 protein with these sites. Our results show that hE2 binds tightly to site 4, less tightly to sites 1 and 2, and weakly to site 3. Similar results have previously been obtained using a C-terminal fragment of the hE2 protein suggesting that the C-terminal domain is the sole determinant of DNA binding affinity and specificity. Using circular permutation assays we show that binding of the hE2 protein induces the formation of a significant DNA bend and that the hE2-induced DNA bend angle is the same at both tight and weak hE2-binding sites. An alignment of the four hE2-binding sites from the HPV 16 genome suggests that this protein recognizes an extended binding site when compared with the bovine papillomavirus E2 protein. Here we show that the hE2 protein binds tightly to sites containing an A:T or a G:C base pair at position 7 of its binding site but weakly to sites containing either C:G or T:A at this position. Using site-directed mutagenesis we demonstrate that an arginine at position 304 of the hE2 protein is responsible for the recognition of specific base pairs at this position.The recognition of specific DNA sequences by transcription factors is often the first step in the regulation of gene expression. An understanding of how these proteins recognize their target sequences, and the effect that this has on DNA conformation, is central to the question of how genes are controlled. We are studying the human papillomavirus E2 protein, a sequence-specific DNA-binding protein involved in the regulation of viral gene expression and DNA replication. Papillomaviruses infect epithelial cells and induce the formation of benign hyperproliferative lesions or warts. Over 70 distinct types of human papillomavirus (HPV) 1 have been described. Some of these viral types produce lesions that have the potential to undergo malignant transformation. HPV 16 and HPV 18, for example, are thought to play a primary role in the development of cervical cancer (for a review, see Ref. 1). The products of the viral E6 and E7 genes form complexes with the cellular tumor suppressor proteins p53 and Rb, respectively. These interactions bring about a change in cell growth rate and promote cell immortalization (reviewed in Ref.2). In HPV 16, transcription of the E6 and E7 genes is under the control of a single promoter (P97) that lies immediately upstream of the E6 gene (3). The activity of the P97 promoter is regulated by a variety of cellular transcription factors and by the viral E2 protein (4 -6).Much early work concentrated on the bovine papillomavirus (BPV) E2 protein. The BPV E2 protein (bE2) binds as a dimer to 12 inverted repeats (consensus sequence 5Ј-ACCGN 4 CGGT-3Ј) present upstream of the BPV early genes and activates transcription (7,8). Binding of bE2 protein to DNA is coo...

Adipose tissue plays a crucial endocrine role in controlling whole body glucose homeostasis and insulin sensitivity. Given the substantial rise in obesity and obesity-related diseases such as diabetes, it is important to understand the molecular basis of adipocyte differentiation and its control. Many studies have successfully exploited gene array technology to monitor changes in the profile of expressed genes during adipocyte differentiation, although this method only measures changes at the level of individual mRNA species. Using two-dimensional polyacrylamide gel electrophoresis, high-throughput image analysis, and candidate picking coupled with sequencing mass spectrometry, we have followed the changes in protein expression profile that occur during the differentiation of 3T3-L1 fibroblasts into adipocytes in response to dexamethasone, isobutyl methyl xanthine and insulin, or to the PPARgamma agonist, ciglitazone. Using this technique we have found alterations in the profile of over 2000 protein species during adipogenesis. Our studies reveal previously unknown alterations during adipogenesis in the expression or mobility (on sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of coactosin, which promotes actin filament destabilization, several signalling molecules, including RhoGDI-1, RhoGDI-2 and EHD1, and NEDD5 a protein involved in cytokinesis.

Human papillomavirus (HPV) type 16 infects the genital tract and is generally acknowledged to be a causative agent of cervical cancer. HPV infection alone is not sufficient to induce cervical cancer and other factors such as steroid hormones are thought to play a role in the establishment and/or progression of this disease. The HPV-16 E2 protein is required for virus replication and modulates viral gene expression whereas the HPV-16 E7 protein is required for cell transformation. We and others have shown that both the E2 and E7 proteins can induce apoptotic cell death in HPVtransformed and non-HPV transformed cell lines. Here we show that the steroid hormones oestrogen and progesterone can both increase the levels of E2-and E7-induced apoptosis. The oestrogen metabolite 16α-hydroxyoestrone also increases E2-and E7-induced cell death and the dietary component indole-3-carbinol, which reduces the formation of 16α-hydroxyoestrone from oestrogen, blocks the effects of oestrogen. Thus the metabolism of oestrogen to 16α-hydroxyoestrone appears to be required for the effects of this hormone on E2-and E7-induced cell death. We also show that the oestrogen receptor antagonist 3-hydroxytamoxifen blocks the effects of oestrogen on E2-and E7-induced cell death, whereas the anti-progesterone RU486 blocks the effects of both progesterone and oestrogen. We discuss these results in terms of the origin and progression of cervical cancer.

Insulin regulates the activity of the AP-1 (activator protein-1) transcriptional complex in several cell types. One component of the AP-1 complex is the transcription factor Fra-1 (fos-related antigen-1), and we have demonstrated previously that insulin stimulates the expression of Fra-1 mRNA in CHO.T cells [Griffiths, Black, Culbert, Dickens, Shaw, Gillespie and Tavaré (1998) Biochem. J. 335, 19-26]. Here we demonstrate that insulin stimulates the activity of a fra-1 promoter linked to a luciferase reporter gene, indicating that the ability of insulin to induce expression of Fra-1 mRNA is due, at least in part, to an increase in gene transcription. Furthermore, we found that insulin induces the serine phosphorylation of Fra-1 and reduces its mobility during SDS/PAGE as a result of phosphorylation. The ability of insulin to induce the accumulation of Fra-1 mRNA, stimulate the fra-1 promoter and stimulate phosphorylation of Fra-1 all require the mitogen-activated protein (MAP) kinase cascade, which leads to the activation of extracellular-signal-regulated kinase (Erk) 1/2. Consequently, our results demonstrate that the Erk cascade plays a dual role in the co-ordinated regulation of the transcription and the phosphorylation of Fra-1 by insulin.