PubMed Health⌕ Search

Biomedical subjects

L Banks

Publications and source records attributed to L Banks.

At least 55 records · Page 3Linked to original sources
PubMed1996

HPV-16 E7 and adenovirus E1a complex formation with TATA box binding protein is enhanced by casein kinase II phosphorylation.

The major transforming protein of HPV-16 is encoded by the E7 gene. This has been shown to cooperate with EJ-ras in the immortalisation of primary rodent cells and with the viral E6 gene in the immortalisation of primary human keratinocytes. HPV-16 E7 protein has been shown to bind to a number of cellular proteins involved in the control of cell growth; including pRB, p107 and cyclin A. Loss of pRb or p107 binding results in the loss of transforming activity. In this paper we demonstrate that HPV-16 E7 can also complex with the core component of TFIID, the TATA Box Binding Protein (TBP). This interaction is partly dependent upon phosphorylation of the E7 protein by cellular casein kinase II (CKII), since phosphorylation of E7 by CKII increases the affinity with which E7 binds TBP. Similar results are also obtained with the Adenovirus Ela protein, indicating a conservation of function between these two viral oncoproteins. Mutation of the CKII site to two acidic amino acids significantly increases the affinity of E7 for TBP, indicating that the incorporation of two negative charges at this region of E7 is important in regulating the interaction with TBP.

Adenovirus E1A Proteins↗
PubMed1995

Conditional immortalization of primary cells by human papillomavirus type 18 E6 and EJ-ras defines an E6 activity in G0/G1 phase which can be substituted for mutations in p53.

The human papillomavirus (HPV) type 18 E6 gene cooperates with activated Ha-ras to immortalize primary mouse cells in culture. Using a plasmid where HPV18 E6 expression is regulated by the glucocorticoid inducible MMTV LTR, we have generated immortalized cell lines in which the continued expression of E6 was necessary for maintenance of the transformed phenotype. In the absence of exogenously added hormone these cells were found to arrest in G0/G1. Furthermore, we demonstrate that the effects of E6 were essentially p53 independent and therefore define a novel function by which E6 is able to modulate cell proliferation. In addition, when the E6 dependent cells were maintained under conditions of prolonged growth arrest by the removal of E6, revertant cells were isolated which were no longer dependent on E6 expression for continued proliferation. These revertant cells were found to have acquired a mutation in the cellular gene p53, suggesting that certain p53 mutations are dominant over an E6 requirement in this assay.

Animals↗
PubMed1995

HPV-18 E6 mediated inhibition of p53 DNA binding activity is independent of E6 induced degradation.

A key activity of the p53 protein during suppression of cell growth is its ability to stimulate transcription from promoters of cellular genes which contain a p53 responsive element. The E6 proteins from the oncogenic associated Human Papillomaviruses (HPVs) have been shown to inhibit specifically the p53 transcriptional activation and this has been proposed as a mechanism whereby the virus prevents the suppression of cell cycle progression and the induction of apoptosis. However, the mechanism by which E6 exercises this function is unknown, as is the ability of E6 to associate with different oligomeric forms of p53. In this study we demonstrate that E6 induces changes within the p53 protein which result both in inhibition of DNA binding and in dissociation of p53 protein previously bound to the DNA. These activities correlate exactly with the ability of E6 to inhibit p53 transcriptional activation and are independent of the ability of E6 to direct the degradation of the p53 protein. Further, we show that E6 labels wild type tetrameric and dimeric forms of p53 proteins for ubiquitin mediated degradation more readily than monomeric forms of the protein. However, in vivo analyses indicate that E6 is capable of inhibiting the transcriptional activation induced by the tetrameric, dimeric and monomeric forms of p53.

Base Sequence↗
PubMed1995

Mutations in the human papillomavirus type 16 E2 protein identify multiple regions of the protein involved in binding to E1.

Human papillomavirus type 16 (HPV-16) DNA replicates episomally and requires two virally expressed proteins, E1 and E2. The E1 protein has both helicase and ATPase activities and is absolutely required for viral DNA replication. The E2 protein is a potent transcriptional activator and greatly increases viral DNA replication by colocalizing E1 to the origin of replication. Recently, we characterized a region of the E2 protein essential for the binding to E1. In this study we have analysed in further detail the nature of the association between E1 and E2. Using an extensive set of E2 mutant proteins we have identified two widely separate regions of the E2 protein which are essential for binding to E1. Interestingly, two E2 mutants which fail to bind E1 also fail to activate gene expression, indicating the existence of multifunctional domains on the E2 protein. In addition, cotransfection of E1 with E2 significantly increases E2 transcriptional activity on an heterologous promoter.

Binding Sites↗
PubMed1995

Mutations in the human papillomavirus type 16 E2 protein identify a region of the protein involved in binding to E1 protein.

Papillomavirus DNA replication is primarily dependent upon two viral gene products, E1 and E2. Work with bovine papillomavirus has shown that the E2 protein can bind directly to the E1 protein and enhance the binding of E1 to the viral origin of replication. However, little is known about the mechanism of interaction between E1 and E2 proteins. In this study we have analysed in detail the association between human papillomavirus type 16 (HPV-16) E1 and E2 proteins. Using a purified glutathione S-transferase-HPV-16 E1 fusion protein from Escherichia coli and E2 proteins produced by in vitro transcription-translation, we have developed a rapid and simple method for investigating the association between E1 and E2 in vitro. The binding of E2 to E1 was found to be dependent on sequences in the N-terminal activation domain of the E2 protein. Truncated forms of E2, including a putative repressor form of E2 encoding the DNA binding domain, failed to associate with E1 in this assay. The region of E2 required for efficient binding to E1 was then localized using mutants in the activation domain of E2. These results demonstrated that only a short region of E2 was required for association with E1. This region of E2 was found to be highly conserved amongst all papillomaviruses, suggesting a conservation of E2 function and a common mechanism of interaction between these virally encoded proteins.

Binding Sites↗
PubMed1995

The human papillomavirus (HPV)-6 and HPV-16 E5 proteins co-operate with HPV-16 E7 in the transformation of primary rodent cells.

E5 is the smallest transforming protein encoded by the human papillomaviruses (HPVs). It has been shown to promote anchorage-independent growth in established NIH 3T3 cells, an activity that is enhanced in the presence of epidermal growth factor (EGF). It is thought that this activity of E5 is brought about by an increase in the half-life of stimulated EGF receptors, possibly through the perturbation of receptor processing. Recent studies have also shown that E5 can co-operate with HPV-16 E7 to stimulate proliferation of primary rodent cells. Using haemagglutinin I epitope-tagged E5 proteins, we have compared the mitogenic activity of HPV-6 and HPV-16 E5. Both tagged proteins retain the ability to bind to the cellular 16 kDa H(+)-ATPase protein. In addition, both HPV-6 and HPV-16 E5 retain the ability to co-operate with E7 in primary rodent cells, although HPV-16 E5 is considerably more active than HPV-6 E5 in these mitogenic assays. Interestingly, transfection of a plasmid over-expressing c-Raf appears to be capable of functionally substituting for E5 in the co-mitogen assays. Polyclonal cell lines derived from baby rat kidney cells co-transfected with E7 and E5 genes continue to express both the E5 and E7 mRNA, although the level of E5 expression is very low and protein cannot be detected. These polyclonal lines appear to be immortal and in some cases demonstrate anchorage-independent growth, an activity which is enhanced by the addition of EGF.

Animals↗
PubMed1994

Characterization of the human papillomavirus E2 protein: evidence of trans-activation and trans-repression in cervical keratinocytes.

The major regulator of papillomavirus transcription is encoded by the viral E2 gene. The E2 gene has been well characterized in bovine papillomavirus (BPV) where it encodes at least three different polypeptides which differentially affect viral gene expression. In human papillomaviruses (HPVs) the E2 gene product is much less well characterized. In this study we have analysed the mechanism of action of the HPV-16, HPV-18 and BPV-1 E2 proteins in cervical keratinocytes. We show that the full length HPV E2 protein acts as a potent transcriptional activator of viral gene expression in both normal and immortalized keratinocytes. In contrast, the BPV-1 E2 protein produces transcriptional repression under identical conditions. A cDNA encoding the C-terminal half of the HPV-16 E2 protein in these assays weakly repressed viral gene expression. Further, co-transfection of this cDNA with the full length clone progressively abolishes the activation in trans by the full length HPV E2 protein. Gel retardation assays have defined a number of protein complexes between the long and short forms of E2 but with no evidence for preferential DNA binding. These results define two distinct activities for the HPV-16 E2 protein, indicate functional differences with the BPV E2 protein and suggest that splicing of the HPV E2 mRNA is a critical mechanism for controlling viral gene expression.

Animals↗
PubMed1994

The human papillomavirus type 16 E5 gene cooperates with the E7 gene to stimulate proliferation of primary cells and increases viral gene expression.

The E5 gene from HPV-16 has recently been shown to stimulate anchorage-independent growth of murine 3T3 cells and this phenotype was enhanced in the presence of epidermal growth factor (EGF). Since EGF is capable of stimulating cellular signal transduction, we have compared levels of EGF-induced c-fos and c-jun mRNA in E5-expressing 3T3 cells. We present data showing that the expression of c-fos and c-jun was higher in E5-expressing 3T3 cells than in control cells. Complexes of c-fos/c-jun constitute the AP1 transcription factor and the HPV-16 promoter/enhancer contains AP1 enhancer elements. HPV-16 promoter activity was therefore examined in cells transfected with the E5 gene and data are presented which reveal that the viral enhancer is more active in E5-expressing cells. Since the viral E7 gene product has been shown to cooperate with v-fos and certain growth factors for transformation and stimulation of DNA synthesis, we investigated the possible cooperation between E5 and E7 to induce cell proliferation. Transfection of E5 and E7 genes into primary rodent epithelial cells produced a potent mitogenic response which was enhanced in the presence of EGF. These results suggest that E5 may cooperate with the E7 gene to stimulate cell proliferation in vivo.

3T3 Cells↗
PubMed1994

Mutational analysis of HPV-18 E6 identifies domains required for p53 degradation in vitro, abolition of p53 transactivation in vivo and immortalisation of primary BMK cells.

The two major transforming proteins of oncogenic human papillomaviruses are encoded by the E6 and E7 oncogenes. Both viral proteins interact specifically with the products of cellular human tumour suppressor genes; E6 with p53 and E7 with Rb. However, the mechanism of action of E6 is still poorly understood in comparison with that of E7. Although extensive in vitro studies have been done with mutant E6 proteins, very little is known about the activities of E6 in vivo. In this study we have analysed the structure-function relationships of HPV-18 E6 in in vitro analyses and we correlate this with in vivo activity. These studies define a number of domains on the E6 molecule which are involved in the ability of E6 to target p53 for degradation in vitro. This analysis demonstrates that domains previously shown to be important in HPV-16 E6 (Crook et al., 1991; Mietz et al., 1992) are also conserved in HPV-18 and also reconciles the differences between these reports. A series of in vivo studies demonstrate that E6 mediated degradation of p53 in vitro is irrelevant both for cell transformation and for the ability of E6 to abolish p53 transcriptional activation. In addition, we show that at least four distinct regions of the E6 protein are involved in the p53 association in vivo.

3T3 Cells↗
PubMed1994

DNA damage induced p53 mediated transcription is inhibited by human papillomavirus type 18 E6.

Cervical cancer is similar to other human cancers in that it develops through a multistep process. However, infection with oncogenic human papillomaviruses (HPVs) is believed to be essential for the initiation of this disease. Although HPV may play a central role in the early stages of neoplasia, the accumulation of mutations in an assortment of genes precedes the development of malignant cervical carcinoma. The mechanisms by which abnormalities accumulate are various, but it is possible that viral proteins are involved. In particular, the viral E6 oncoprotein has been shown to interact with the cellular tumour suppressor protein p53, which is involved in DNA damage repair pathways. Hence, E6 may contribute to the genomic instability through this interaction with p53. We have tested this hypothesis by monitoring the effects of E6 upon DNA damage induced p53 transcriptional activity. This study shows that HPV-18 E6 inhibits p53 transcriptional activity following genotoxic stress with UV radiation. No effect was observed when a mutant E6 unable to direct the degradation of p53 was included in this assay. These results suggest that continued E6 expression may contribute to the accumulation of DNA damage associated with the progression of cervical cancer.

Amino Acid Sequence↗
PubMed1993

Human mouse chimeric CD7 monoclonal antibody (SDZCHH380) for the prophylaxis of kidney transplant rejection.

mAb directed against CD7 have been shown to inhibit T cell proliferation in the allogeneic mixed lymphocyte reaction suggesting that CD7 may be an appropriate target for in vivo immunotherapy. We performed a prospective randomized clinical trial with a human-mouse chimeric CD7 mAb (SDZCHH380) and compared it with murine OKT3 for the prophylaxis of kidney transplant rejection. Twenty recipients of first cadaveric renal allografts were randomized to receive either SDZCHH380 or OKT3. SDZCHH380 was well tolerated. Rejection was delayed to day 35. No patients were sensitized to SDZCHH380. In contrast 7/10 OKT3 patients made anti-OKT3 antibodies. SDZCHH380 coated peripheral blood and lymph node T cells and, in contrast to OKT3, induced minimal release of IL-2, IL-6, TNF-alpha, and IFN-gamma. In addition, we showed that CD7-negative T cells mediated rejection in one of the SDZCHH380-treated patients. We conclude that the human-mouse chimeric CD7 mAb SDZCHH380 is well tolerated, is not immunogenic, and merits further study in the prophylaxis of transplant rejection.

Adolescent↗
PubMed1993

Diagnostic iodised oil embolisation of liver tumours--the Hammersmith experience.

Oily embolisation of the liver as a diagnostic technique in the management of liver tumours has not proved reliable in our hands. In our study of 20 patients we found: (1) The technique was not risk-free, with complications in four patients, one of whom required emergency surgery. (2) The Bruneton classification is far too simplistic to be relied upon. (3) Residual Lipiodol, particularly in the left lobe of the liver is problematic, making it difficult to distinguish normal from diseased liver. (4) The technique did not contribute to decisions about surgical management in any one of our 20 cases though we accept it might occasionally do so. Our experience was disappointing and in marked contradistinction to enthusiastic reports by some other authors.

Carcinoma, Hepatocellular↗
PubMed1993

Characterization of factors involved in human papillomavirus type 16-mediated immortalization of oral keratinocytes.

We have examined intrinsic and external factors that influence human papillomavirus type 16 (HPV-16)-mediated immortalization of oral keratinocytes. The efficiency with which HPV can immortalize human oral keratinocytes was quantified and a considerable difference in the transfection and immortalization competence of the cells was detected. The ability of HPV-16 to immortalize oral cells appeared to be linked to the age of the culture upon transfection. The addition of dexamethasone to the transfected cultures increased the efficiency of immortalization, possibly indicating a role for a critical level of HPV gene expression in initial outgrowth of immortalized colonies. We also document in detail the changes in the oral keratinocyte induced by HPV-16 immortalization. These include alterations associated with crisis and feeder independence as well as basic changes in keratin expression and differentiation.

Cell Differentiation↗
PubMed1993

Human papillomavirus type 16 E6 gene cooperates with EJ-ras to immortalize primary mouse cells.

Human papillomaviruses (HPVs) are small DNA tumor viruses, a subset of which is closely associated with the development of cervical cancer. The viral E6 and E7 open reading frames encode multifunctional proteins that bind respectively to the p53 protein and to the product of the retinoblastoma tumor-suppressor gene. In this study we demonstrate that the HPV-16 E6 gene cooperates with EJ-ras to immortalize primary cultures of mouse kidney epithelial cells. HPV-16-immortalized cell lines expressing E6 but not E7 contained low levels of wild-type p53 protein. In contrast, those cells immortalized by EJ-ras alone contained elevated p53 protein levels, and were shown to contain a mutation in the gene. These results suggest that activating mutations in the p53 gene can functionally substitute for HPV-16 E6 in transforming primary cells.

Animals↗
PubMed1992

The E5 gene from human papillomavirus type 16 is an oncogene which enhances growth factor-mediated signal transduction to the nucleus.

Although human papillomavirus type 16 (HPV-16) is believed to be a major etiological agent in the development of cervical cancer, the biological function of several of its early genes remains to be established. In the present study, we have defined some of the biological properties of the E5 gene from HPV-16. Expression of the HPV-16 E5 gene in 3T3-A31 cells induced transformation to anchorage-independent growth (colony formation in soft agar). Addition of epidermal growth factor (EGF) to the soft-agar medium caused the E5-expressing cells to form larger colonies than those formed in the absence of EGF. Parental 3T3-A31 cells did not form colonies in soft agar either in the presence or in the absence of EGF. Analysis of clones expressing high levels of E5 mRNA revealed that these cells also expressed higher levels of c-fos mRNA in response to serum, EGF and platelet-derived growth factor (PDGF) than did the parental 3T3-A31 cells. Cells expressing the E5 gene were also capable of accelerated growth in low serum and were more tumorigenic in nude mice than were control cells. We conclude that the E5 gene from HPV-16 is an oncogene which transforms cells in part through enhancing signal transduction from growth factors to the nucleus.

3T3 Cells↗