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D J McCance

Publications and source records attributed to D J McCance.

At least 19 recordsLinked to original sources
PubMed2002

Down regulation of the interleukin-8 promoter by human papillomavirus type 16 E6 and E7 through effects on CREB binding protein/p300 and P/CAF.

Previously, we reported that human papillomavirus (HPV) type 16 E6 binds to C/H1, C/H3, and the C-terminal domains of coactivators p300 and CBP, causing the modulation of the transcription of certain genes controlled by NF-kappaB (p65 or relA) and p53. To establish the biological significance of these observations, we have focused on the transcriptional regulation of interleukin-8 (IL-8), a potent chemoattractant for T lymphocytes and neutrophils, which is also essential for the initiation of the local immune response. The IL-8 promoter is regulated by NF-kappaB/p65 in response to tumor necrosis factor alpha and requires the cooperation of the coactivators CBP/p300 and steroid receptor coactivator 1 (SRC-1) and the p300/CBP-associated factor (P/CAF) for optimal activation. Here we report that, in the presence of HPV-16 E6, the promoter activity of IL-8 was repressed. Moreover, from the mutational analysis of the IL-8 promoter, we found that E6 down-regulates the IL-8 promoter activity through the NF-kappaB/p65 binding site. This inhibition appears to result from the ability of HPV-16 E6 to compete with NF-kappaB/p65 and SRC-1 for binding to the N terminus and C terminus of CBP, respectively. Reporter data also showed that E7 represses IL-8 promoter activity, though to a lesser extent than E6 but, like E6, the repression by E7 is through the NF-kappaB/p65 binding site. E7 was shown for the first time to bind to P/CAF, and the binding was necessary for the down regulation of the IL-8 promoter. E6 and E7 together inhibited transcription of the IL-8 promoter to a greater extent than either alone. Finally, by RNase protection assay, we showed that the synthesis of endogenous IL-8 mRNA was repressed in keratinocytes stably expressing E6 and E7. Taken together, the results provide evidence that E6 and E7 can cooperatively disrupt IL-8 transcription through disruption of transcriptional active complexes, and this may have important consequences for immune responses in infected hosts.

Acetyltransferases↗
PubMed2001

The human papillomavirus type 16 E5 protein alters vacuolar H(+)-ATPase function and stability in Saccharomyces cerevisiae.

The human papillomavirus 16 (HPV-16) E5 oncoprotein is a small integral membrane protein that binds to the 16-kDa subunit of the vacuolar H(+)-ATPase (v-ATPase). Conservation within the family of v-ATPases prompted us to look to Saccharomyces cerevisiae as a potential model organism for E5 study. The E5 open reading frame, driven by a galactose-inducible promoter, was integrated into the yeast genome, and the resulting strain demonstrated a nearly complete growth arrest at neutral pH, consistent with defects associated with yeast v-ATPase mutants. Furthermore, this strain demonstrated a severe reduction in pH-dependent and v-ATPase-dependent vacuolar localization of fluorescent markers. Overexpression of the yeast 16-kDa subunit homolog partially suppressed E5-associated growth defects. E5 expression was correlated with a disassociation of the integral (V(o)) and peripheral (V(i)) v-ATPase sub-complexes, as well as a dramatic reduction of the steady-state levels of one mature V(o) subunit and the concomitant accumulation of its major proteolytic fragment, with unchanged levels of two V(i) subunits. Similar analyses of selected E5 mutants in yeast demonstrated a correlation between E5 biology and v-ATPase disruption. Our observations suggest that wild-type HPV-16 E5 acts during the assembly of the v-ATPase to inhibit, either directly or indirectly, V(o) stability and complex formation.

Enzyme Stability↗
PubMed2000

A mutagenic analysis of the E5 protein of human papillomavirus type 16 reveals that E5 binding to the vacuolar H+-ATPase is not sufficient for biological activity, using mammalian and yeast expression systems.

The E5 gene of human papillomavirus type 16 encodes a highly hydrophobic membrane protein previously shown to inhibit endosomal acidification, presumably by binding to the 16-kDa pore-forming subunit of the vacuolar H(+)-ATPase (v-ATPase). The role of this interaction in the disruption of v-ATPase activity was explored through extensive mutagenesis of E5 to identify residues that mediate binding to the 16-kDa subunit. Coimmunoprecipitations revealed that the hydrophobic span between residues 41 and 54 is primarily responsible for this interaction and can be replaced with random hydrophobic amino acids. Studies using mutated 16-kDa proteins indicated that the fourth transmembrane domain of the pore subunit mediates binding to E5. Analysis of the E5 mutants in a yeast expression system revealed that several mutants that retained the capacity to bind to the 16-kDa subunit in COS-1 cells failed to disrupt vacuolar acidification. These data argue that E5 binding to the pore subunit is not sufficient for the associated activity of disruption of v-ATPase function.

Amino Acid Sequence↗
PubMed1999

The E6 protein of human papillomavirus type 16 binds to and inhibits co-activation by CBP and p300.

The co-activators CBP and p300 are important for normal cell differentiation and cell cycle progression and are the targets for viral proteins that dysregulate these cellular processes. We show here that the E6 protein from the oncogenic human papillomavirus type 16 (HPV-16) binds to three regions (C/H1, C/H3 and the C-terminus) of both CBP and p300. The interaction of E6 with CBP/p300 was direct and independent of proteins known to bind the co-activators, such as p53. The E6 protein from low-risk HPV type 6 did not interact with C/H3 or the C-terminus but associated with the C/H1 domain at 50% of the level of HPV-16. HPV-16 E6 inhibited the intrinsic transcriptional activity of CBP/p300 and decreased the ability of p300 to activate p53- and NF-kappaB-responsive promoter elements. Interestingly, some mutations in HPV-16 E6 abrogated C/H3-E6 interactions, but did not alter the ability of E6 to associate with the C/H1 domain, suggesting that these modified proteins could be used to delineate the functional significance of the C/H1 and C/H3 domains of CBP/p300.

Animals↗
PubMed1999

The E7 oncoprotein associates with Mi2 and histone deacetylase activity to promote cell growth.

E7 is the main transforming protein of human papilloma virus type 16 (HPV16) which is implicated in the formation of cervical cancer. The transforming activity of E7 has been attributed to its interaction with the retinoblastoma (Rb) tumour suppressor. However, Rb binding is not sufficient for transformation by E7. Mutations within a zinc finger domain, which is dispensable for Rb binding, also abolish E7 transformation functions. Here we show that HPV16 E7 associates with histone deacetylase in vitro and in vivo, via its zinc finger domain. Using a genetic screen, we identify Mi2beta, a component of the recently identified NURD histone deacetylase complex, as a protein that binds directly to the E7 zinc finger. A zinc finger point mutant which is unable to bind Mi2beta and histone deacetylase but is still able to bind Rb fails to overcome cell cycle arrest in osteosarcoma cells. Our results suggest that the binding to a histone deacetylase complex is an important parameter for the growthpromoting activity of the human papilloma virus E7 protein. This provides the first indication that viral oncoproteins control cell proliferation by targeting deacetylation pathways.

Adenosine Triphosphatases↗
PubMed1998

Rb binds c-Jun and activates transcription.

The retinoblastoma protein (Rb) acts as a critical cell-cycle regulator and loss of Rb function is associated with a variety of human cancer types. Here we report that Rb binds to members of the AP-1 family of transcription factors, including c-Jun, and stimulates c-Jun transcriptional activity from an AP-1 consensus sequence. The interaction involves the leucine zipper region of c-Jun and the B pocket of Rb as well as a C-terminal domain. We also present evidence that the complexes are found in terminally differentiating keratinocytes and cells entering the G1 phase of the cell cycle after release from serum starvation. The human papillomavirus type 16 E7 protein, which binds to both c-Jun and Rb, inhibits the ability of Rb to activate c-Jun. The results provide evidence of a role for Rb as a transcriptional activator in early G1 and as a potential modulator of c-Jun expression during keratinocyte differentiation.

Animals↗
PubMed1998

Retinoblastoma protein recruits histone deacetylase to repress transcription.

The retinoblastoma protein (Rb) silences specific genes that are active in the S phase of the cell cycle and which are regulated by E2F transcription factors. Rb binds to the activation domain of E2F and then actively represses the promoter by a mechanism that is poorly understood. Here we show that Rb associates with a histone deacetylase, HDAC1, through the Rb 'pocket' domain. Association with the deacetylase is reduced by naturally occurring mutations in the pocket and by binding of the human papilloma virus oncoprotein E7. We find that Rb can recruit histone deacetylase to E2F and that Rb cooperates with HDAC1 to repress the E2F-regulated promoter of the gene encoding the cell-cycle protein cyclin E. Inhibition of histone deacetylase activity by trichostatin A (TSA) inhibits Rb-mediated repression of a chromosomally integrated E2F-regulated promoter. Our results indicate that histone deacetylases are important for regulating the cell cycle and that active transcriptional repression by Rb may involve the modification of chromatin structure.

Amino Acid Sequence↗
PubMed1998

Active domains of human papillomavirus type 11 E1 protein for origin replication.

Viral proteins E1 and E2 are essential for transient human papillomavirus (HPV) DNA replication. E1 is a multifunctional protein which can bind DNA and complex with E2, has ATPase and helicase activities, and interacts with DNA polymerase alpha-primase. E2 is a transactivator-repressor protein, playing an important role in replication and transcriptional regulation. A series of deletion mutants of HPV-11 E1 were constructed and tested in functional assays to define those domains of HPV-11 E1 which are important for binding to the origin DNA and E2. The domain of HPV-11 E1 involved in binding to the origin was located between aa 186 and 649, and that for binding to E2 was between aa 346 and 649. Since E1 binds to the origin more efficiently in the presence of E2, we also mapped the DNA binding domain of E1 in the presence of E2, and found that when binding was enhanced, the region of E1 involved in binding was similar to that observed with E1 alone. The same deletion mutation constructs of E1 were subcloned into an expression vector for use in transient replication assays to study the effect of the deletions on the replication of the origin DNA in vivo and the data suggest that the C-terminal domain contains important functions for replication.

Binding Sites↗
PubMed1997

Sequence variation in the early genes E1E4, E6 and E7 of human papilloma virus type 6.

The majority of condylomata acuminata (anogenital warts) are caused by infection with Human Papilloma Virus type 6 (HPV-6). We have sequenced the HPV-6 early genes, E1-E4, E6 and E7 from wart biopsy DNA samples sourced from the UK and USA and derived a consensus sequence for these genes and the proteins they encode. When compared to the prototype HPV-6b sequence, published over 12 years ago, the E1-E4 consensus sequence showed 3/91 (3.3%) amino acid changes, the E6 consensus sequence showed 1/150 (0.7%) changes and the E7 consensus sequence showed 1/98 (1.0%) changes. Since many of the early gene sequences from biopsy material were more similar to the HPV-6a subtype than HPV-6b, this data supports the use of HPV-6a as the HPV-6 prototype.

Condylomata Acuminata↗
PubMed1996

The E5 gene of HPV-16 enhances keratinocyte immortalization by full-length DNA.

Human papillomavirus type 16 (HPV-16) is the most common papillomavirus genome found in human cervical cancers and its DNA is capable of immortalizing primary keratinocytes in vitro. When expressed by their native promoter, two separate HPV-16 oncogenes, E6 and E7, cooperate to immortalize primary human keratinocytes. Early HPV cervical lesions express abundant amounts of E5-specific RNA, and using a quantitative keratinocyte immortalization assay, we demonstrate here that E5 can act in cis to increase (4-10 fold) the efficiency of cellular immortalization by E6/E7. These results suggest that the expression of the HPV-16 E5 gene may play a role in the pathogenesis of early HPV infections by potentiating the effects of E6/E7.

3T3 Cells↗
PubMed1996

The human papillomavirus type 16 E7 gene product interacts with and trans-activates the AP1 family of transcription factors.

The E7 gene product of human papillomavirus type 16 (HPV16) binds to the retinoblastoma gene product (pRb) and dissociates pRb-E2F complexes. However, the observation that the ability of E7 to bind pRb is not required for the HPV16-induced immortalization of primary keratinocytes prompted a search for other cellular factors bound by E7. Using a glutathione-S-transferase (GST) fusion protein system, we show that E7 complexes with AP1 transcription factors including c-Jun, JunB, JunD and c-Fos. The ability of E7 to complex with c-Jun in vivo is demonstrated by co-immunoprecipitation and the yeast two-hybrid system. An analysis of E7 point mutants in the GST system indicates that the E7 zinc-finger motif, but not the pRb binding domain, is involved in these interactions. Using c-Jun deletion mutants, E7 binding maps between amino acids 224 and 286 of c-Jun. E7 trans-activates c-Jun-induced transcription from a Jun responsive promoter, and this activity correlates with the ability of E7 mutants to bind Jun proteins. Finally, a transcriptionally inactive c-Jun deletion, which can bind E7, interferes with the E7-induced transformation of rat embryo fibroblasts in cooperation with an activated ras, indicating that the Jun-E7 interaction is physiologically relevant and that Jun factors may be targeted in the E7 transformation pathway.

Animals↗
PubMed1996

Mapping of HPV-11 E1 binding site and determination of other important cis elements for replication of the origin.

The viral proteins E1 and E2 are essential for the replication of the HPV-11 origin. We have now mapped the E1 binding site (E1BS) and show by mutation analysis that the E1BS and an adjoining poly(A)-rich region are necessary for efficient replication of the origin. We have also shown, using suboptimal levels of E1 protein, that enhancement of E1 binding by E2 partially protects the E1BS. Finally, we show that E1 can enhance the binding of E2 even in the absence of the E1BS.

Base Sequence↗
PubMed1995

Poly-L-ornithine-mediated transfection of human keratinocytes.

Human keratinocytes are notoriously difficult to transfect. We have optimized a method for introducing plasmid DNA into keratinocytes that pairs the polycation poly-L-ornithine with a dimethylsulfoxide (DMSO) shock. The optimum poly-L-ornithine conditions for keratinocyte transfection entailed incubating the cells with 12 micrograms/ml poly-L-ornithine and 10 micrograms DNA for 6 h, followed by a 4-min 25% DMSO shock. Based on kinetic studies, 1 h is enough time to produce 10% positive cells in transient transfections, which increases up to an average of 20% after 6 h. Transfected cells survive passaging, and marker plasmids and selection can be used to yield stable transfectants at a rate twofold higher than in cells transfected with polybrene and DMSO. Transient transfection rates were significantly higher using poly-L-ornithine/DMSO than with the polybrene/DMSO or polybrene/glycerol methods previously reported. Overall, transfection mediated by poly-L-ornithine provides an efficient and inexpensive means of transiently or stably introducing DNA into keratinocytes.

Cell Survival↗
PubMed1995

The E5 oncoprotein of human papillomavirus type 16 inhibits the acidification of endosomes in human keratinocytes.

The human papillomavirus type 16 E5 oncoprotein possesses mitogenic activity that acts synergistically with epidermal growth factor (EGF) in human keratinocytes and inhibits the degradation of the EGF receptor in endosomal compartments after ligand-stimulated endocytosis. One potential explanation for these observations is that E5 inhibits the acidification of endosomes. This may be mediated through the 16-kDa component of the vacuolar proton-ATPase, since animal and human papillomavirus E5 proteins bind this subunit protein. Using a ratio-imaging technique to determine endosomal pH, we found that the acidification of endosomes in E5-expressing keratinocytes was delayed at least fourfold compared with normal human keratinocytes and endosomes in some cells never completely acidified. Furthermore, E5 expression increased the resistance of keratinocytes to protein synthesis inhibition by diphtheria toxin, a process dependent on efficient endosomal acidification. Finally, artificially inhibiting endosomal acidification with chloroquine during the endocytosis of EGF receptors in keratinocytes demonstrated many of the same effects as the expression of human papillomavirus type 16 E5, including prolonged retention of undegraded EGF receptors in intracellular vesicles.

Cells, Cultured↗
PubMed1994

Gene safety.

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Adenoviridae↗
PubMed1994

Serological differentiation of human papillomavirus types 11, 16 and 18 using recombinant virus-like particles.

The L1 major capsid protein-coding sequences of human papillomavirus (HPV) types 11, 16 and 18 were expressed in the baculovirus system. Virus-like particles (VLPs) were purified from recombinant-infected Spodoptera frugiperda Sf9 cells and cell-free culture supernatants. Rabbits immunized with purified VLPs developed antibodies that reacted only with the specific VLP type used as the immunogen. In addition, rabbit antibodies raised against infectious HPV-11 virions only reacted with HPV-11 L1 VLPs and not with VLPs derived from either HPV-16 or HPV-18. These results suggest that HPV-11, HPV-16 and HPV-18 virions are antigenically distinct from one another. This observation should be considered in future studies of immune responses to HPV.

Animals↗
PubMed1994

Human papillomaviruses.

This article concentrates on those human papillomaviruses (HPVs) involved in premalignant and malignant disease of the lower genital tract. There is discussion of the role of HPVs in disease, the frequency of infection and disease in different age groups, and the methods of testing for HPV DNA in cervical cells or biopsies. Included is an appendix that briefly describes the virally coded proteins and what is known of their function.

Adult↗