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A W Braithwaite

Publications and source records attributed to A W Braithwaite.

At least 19 recordsLinked to original sources
PubMed2001

Induction of cell death by adenoviruses.

Adenoviruses have proved to be excellent tools for gaining insight into the regulation, and deregulation, of the mammalian cell cycle. With the widespread clinical use of gene therapy fast approaching, there comes a need for a better understanding of how the cell death process is regulated. A greater understanding will allow the development of therapeutic approaches that both maximise transgene expression while minimising cytotoxicity to the target cell. Consequently, much adenovirus research has centered on understanding the mechanisms governing adenovirus induced cell death or apoptosis. This review discusses recent advances in the field of adenovirus cell death regulation and evaluates the roles of implicated gene products and their respective data. The data suggest the existence of multiple virus gene products involved in cell death regulation and point towards several distinct, yet related, cell death pathways. A discussion of the shortcomings of current adenoviral research, along with a proposed model based upon the data is also given.

Adenoviridae↗
PubMed2001

IL-1 genotype and adult periodontitis among young New Zealanders.

Several recent studies have investigated the association between interleukin-1 genotype and periodontitis in clinical samples, where generalizability is an issue. The aim of this study was to investigate the association between adult periodontitis and IL-1 genotype in a population-based sample of 26-year-olds. Based on probing depth (PD) measurements, participants were divided into three disease groups: "Severe" (1+ teeth with 5+mm PD; N = 25), "Moderate" (2+ teeth with 4+mm PD; N = 36), and "Controls" (the remainder; N = 800). The "periodontitis-associated genotype" (PAG; Kornman et al., 1997) was present in 20.0% of the "Severe" group and in 34.8% of "Controls", whereas the IL-1A(+4845) [1,1]/IL-1B(+3953) [2,2] genotype was present in 12.0% and 0.9%, respectively. After controlling for sex, smoking status, and plaque levels, we found that those with IL-1B(+3953) [1,1]/IL-1A(+4845) [2,2] had 12.3 times the odds of being in the "Severe" group. Analysis of these data suggests that the IL-1A(+4845) [1,1]/IL-1B(+3953) [2,2] genotype is associated with periodontal disease in this young population. Future periodontal data collections as this cohort ages are required to confirm the predictive value of that genotype.

Adult↗
PubMed2000

Maf transcriptionally activates the mouse p53 promoter and causes a p53-dependent cell death.

An increase in the level of the tumor suppressor protein p53 can induce cell cycle arrest or cell death. Although mechanisms for regulating the life span of p53 have been described, there is growing evidence that transcriptional regulation of the p53 gene contributes significantly to controlling p53 protein levels and therefore the fate of a cell. However, the signal transduction pathways that lead to transcriptional activation of the p53 gene are poorly understood. The oncoprotein v-Maf and its cellular counterparts belong to the large combinatorially complex basic leucine zipper family of transcription factors, which include the AP1 family. To date few cellular targets of c-Maf have been identified. It is demonstrated here that v-Maf can bind as a homodimer to a variant Maf recognition element located between -66 and -54 upstream in the mouse p53 promoter. V-Maf and its cellular counterparts are shown to activate p53 expression through this site. The ability of v-Maf to activate p53 expression is modulated by AP1 family members. In addition, overexpression of v-Maf in primary cells leads to a p53-dependent cell death. Thus, Maf and members of the AP1 family are able to regulate p53 expression through this site in the p53 promoter.

Animals↗
PubMed2000

Efficient induction of cell death by adenoviruses requires binding of E1B55k and p53.

The use of an Elb55k-deficient adenovirus, ONYX-015, to selectively target tumor cells containing a mutated p53 gene has produced promising results. However, recent reports have questioned the selectivity of this virus, showing that ONYX-015 can replicate in cells containing a wild-type p53 and that p53 may actually be required for cell death. To address these apparent contradictions in the literature, we infected a number of mutant and wild-type p53-containing cell lines with ONYX-015 and wild-type adenovirus and observed their death profiles up to 10 days postinfection. We demonstrate that two distinct cell death phenotypes exist, one of which is rapid and dependent on the presence of p53 and one of which is p53 independent. Using adenoviruses expressing E1b55k proteins deficient in their ability to bind p53, we show that formation of a complex between p53 and the adenoviral Elb55k protein is necessary for the activation of the rapid cell death pathway. In the absence of p53 or the absence of complex formation between p53 and Elb55k, cell death is delayed considerably. These data suggest three things: that the selectivity of killing appears to be dependent on the presence of the E1b55k/p53 complex; that viruses lacking Elb55k (such as ONYX-015) kill cells in a delayed manner independent of p53; and that binding of E1b55k to p53 does not merely serve to inactivate p53, but rather is required for the induction of rapid cell death. The components of this complex that lead to rapid cell death remain to be determined.

Adenoviridae↗
PubMed1999

Inhibition of SV40 large T antigen induced apoptosis by small T antigen.

It is well established that the expression of simian virus 40 (SV40) early gene products causes oncogenic transformation of rodent cells. An important aspect of this process is the inactivation of the p53 and retinoblastoma (pRb) tumour suppressor proteins through interaction with the SV40 large tumour antigen (LT). In addition, the SV40 small tumour antigen (ST) may enhance LT induced transformation. Here we show that LT induces apoptotic cell death in rat embryo fibroblast (REF) cells and that ST functions to inhibit this effect by a mechanism which is different from other known anti-apoptotic proteins. Mutational analysis of LT indicates that mutants defective in the pRb-binding domain are unable to induce apoptosis whereas LT mutants defective in the p53-binding domain are still competent to induce apoptosis. Thus, interaction between LT and one or more pRb family members must occur for induction of apoptosis and that binding of p53 by LT is insufficient to inhibit LT induced apoptosis in REFs. The data presented herein suggest that the anti-apoptotic function of ST may explain, at least in part, how ST contributes to SV40 early region induced transformation of REF cells.

Animals↗
PubMed1999

The adenovirus oncoprotein E1a stimulates binding of transcription factor ETF to transcriptionally activate the p53 gene.

Expression of the tumor suppressor protein p53 plays an important role in regulating the cellular response to DNA damage. During adenovirus infection, levels of p53 protein also increase. It has been shown that this increase is due not only to increased stability of the p53 protein but to the transcriptional activation of the p53 gene during infection. We demonstrate here that the E1a proteins of adenovirus are responsible for activating the mouse p53 gene and that both major E1a proteins, 243R and 289R, are required for complete activation. E1a brings about the binding of two cellular transcription factors to the mouse p53 promoter. One of these, ETF, binds to three upstream sites in the p53 promoter and one downstream site, whereas E2F binds to one upstream site in the presence of E1a. Our studies indicate that E2F binding is not essential for activation of the p53 promoter but that ETF is. Our data indicate the ETF site located downstream of the start site of transcription is the key site in conferring E1a responsiveness on the p53 promoter.

Adenovirus E1A Proteins↗
PubMed1998

p53-dependent cell death/apoptosis is required for a productive adenovirus infection.

The p53 tumor suppressor protein binds to both cellular and viral proteins, which influence its biological activity. One such protein is the large E1b tumor antigen (E1b58kDa) from adenoviruses (Ads), which abrogates the ability of p53 to transactivate various promoters. This inactivation of p53 function is believed to be the mechanism by which E1b58kDa contributes to the cell transformation process. Although the p53-E1b58kDa complex occurs during infection and is conserved among different serotypes, there are limited data demonstrating that it has a role in virus replication. However, loss of p53 expression occurs after adenovirus infection of human cells and an E1b58kDa deletion mutant (Onyx-015, also called dl 1520) selectively replicates in p53-defective cells. These (and other) data indicate a plausible hypothesis is that loss of p53 function may be conducive to efficient adenovirus replication. However, wild-type (wt) Ad5 grows more efficiently in cells expressing a wt p53 protein. These studies indicate that the hypothesis may be an oversimplification. Here, we show that cells expressing wt p53, as well as p53-defective cells, allow adenovirus replication, but only cells expressing wt p53 show evidence of virus-induced cytopathic effect. This correlates with the ability of adenovirus to induce cell death. Our data indicate that p53 plays a necessary part in mediating cellular destruction to allow a productive adenovirus infection. In contrast, p53-deficient cells are less sensitive to the cytolytic effects of adenovirus and as such raise questions about the use of E1b58kDa-deficient adenoviruses in tumor therapy.

Adenoviruses, Human↗
PubMed1997

p53/E1b58kDa complex regulates adenovirus replication.

We have explored a role for the adenovirus (Ad5) E1b58kDa/p53 protein complex in adenovirus replication. This was done by using virus mutants containing different defects in the E1b58kDa gene and cell lines that express either a wild-type p53 protein or a mutant p53 protein. We find that infection of wild-type p53-containing cells with wild-type Ad5 causes a shutoff of p53 and alpha-actin protein synthesis by distinct mechanisms, but neither occurs in mutant p53 cells. Our data also indicate that the shutoff is dependent on formation of the p53/E1b complex and may also involve another virus protein, E4ORF6. Following from these observations we asked whether failure to form the complex resulted in impaired adenovirus replication. Our experiments showed that neither wild-type Ad5 nor the E1b mutant dl338 could replicate in cells expressing a mutant p53 protein, but that wild-type adenovirus replicated well in wild-type p53-expressing cells. Collectively, our data suggest that the interaction between p53 and the E1b58kDa protein is necessary for efficient adenovirus replication. This is the first time such a direct link between the complex and virus replication has been demonstrated. These data raise serious questions about the usefulness of E1b-defective viruses in tumor therapy.

Adenoviridae↗
PubMed1996

The growth-inhibitory function of p53 is separable from transactivation, apoptosis and suppression of transformation by E1a and Ras.

p53 is known to suppress oncogenic cell transformation, inhibit cell growth, induce apoptosis and activate and repress gene transcription. To investigate the relationships between these functions, we have examined various mutant forms of p53 for their abilities to perform each activity. This study has shown that growth inhibition is not a prerequisite for apoptotic cell death as these two functions are separate and alternative activities of p53. Additionally, we have demonstrated that the ability of p53 to suppress transformation (by adenovirus E1a and activated Ras) correlates with its ability to induce apoptosis and not with its ability to inhibit cell growth. Although p53 is thought to inhibit growth through the transactivation of p21WAFI, our study has demonstrated that transcriptional activation and repression are neither sufficient nor necessary for growth inhibition. This indicates that p53 has more than one mechanism for inhibiting cell growth and that another type of biochemical function must be involved. Furthermore, we have shown that transcriptional activation and repression may each be necessary, and the combination of these activities may even be sufficient, for p53-dependent apoptosis. In summary, our results have provided new information about the cellular and biochemical mechanisms through which p53 acts as a tumor suppressor.

Adenovirus E1A Proteins↗
PubMed1995

Involvement of RB-1, p53, p16INK4 and telomerase in immortalisation of human cells.

Involvement of the retinoblastoma susceptibility (RB-1), p16INK4, p53 and telomerase genes in immortalisation was examined by determining their status in 15 human cell lines representing four immortalisation complementation groups. No abnormalities of RB-1, p53 and p16INK4 were detected in cell lines containing DNA tumour virus proteins known to bind to the protein products of the RB-1 and p53 genes. In contrast, in all other cell lines from each of the four groups either RB-1 was mutant or p16INK4 protein was undetectable and there were cell lines containing p53 mutations in three of the groups. Telomerase activity was detected in 12/15 lines, including some of the virally immortalised lines and in some lines from each group. Since none of these changes correlated with complementation group, other genetic changes must be required for immortalisation.

Base Sequence↗
PubMed1995

Identification of an upstream region of the mouse p53 promoter critical for transcriptional expression.

We have investigated the transcription factor requirements for basal expression of the mouse p53 promoter by using a combination of reporter and electrophoretic mobility shift assays (EMSAs). We have found that only four regions of the promoter bind transcription factors in EMSAs, suggesting that these are the only important factors for basal transcription. These factors are NF1, USF, ETF-like and a novel factor which we have called PF2. Construction of promoter deletion mutants has shown that the absence of the PF2 site completely inactivates the promoter, whereas deletion of other sites, whilst reducing promoter activity, does not. We suggest that this novel transcription factor (PF2) is critical for expression of the mouse p53 promoter.

Animals↗
PubMed1994

Down-regulation of human adenovirus E1a by E3 gene products: evidence for translational control of E1a by E3 14.5K and/or E3 10.4K products.

The mechanism for down-regulation of E1a expression by products encoded in the E3 transcription unit of human adenovirus types 2 and 5, that occurs in infected L929 cells, has been investigated further. We show that the phenomenon occurs in different mouse cells and also in some human cells suggesting that the observations have relevance to natural human infections. We also provide evidence that probably all viral proteins are down-regulated by E3 products, although to different extents, but that host proteins are unaffected. Whereas E1a protein levels and synthesis are reduced in the presence of E3 products, E1a protein half-life and polysomal E1a RNA levels and size distribution are not. These data suggest that E3 products down-regulate E1a protein levels by interfering with the translation of E1a-specific mRNA. Studies were additionally carried out with mutant adenoviruses containing different defects in the E3 transcription unit. Based on these studies it seems likely that the E3 14.5K and 10.4K proteins are crucially involved in E1a down-regulation. Our data are discussed in terms of strategies for immune evasion by group C human adenoviruses.

Adenovirus E1A Proteins↗
PubMed1994

Functional characterization of Xenopus laevis p53: evidence of temperature-sensitive transactivation but not of repression.

We have investigated the effect of Xenopus laevis p53 (Xp53) on transcription from a variety of promoters which are regulated by mouse p53 using a chloramphenicol acetyltransferase reporter system. Although Xp53 transactivated promoters that are up-regulated by mouse p53, it was unable to cause repression. This ability to transactivate gene expression was dependent on a temperature of 32 degrees C, and activity was lost at 37 degrees C. Temperature-sensitive transactivation was correlated with temperature-dependent binding of Xp53 to the adenovirus E1B58K protein. Despite the marked loss of transcriptional activation and binding to E1B58K at 37 degrees C, Xp53 was still capable of binding simian virus 40 large T antigen and inhibiting simian virus 40 origin-dependent DNA replication. These data show that Xp53 is temperature sensitive for N-terminal activities and suggest that the transactivation and repression "domains" of p53 are distinct.

Adenovirus E1B Proteins↗
PubMed1993

Wild-type mouse p53 down-regulates transcription from different virus enhancer/promoters.

The protein encoded by the tumour-suppressor gene p53 can complex with SV40 virus large T antigen, the adenovirus E1B 58-kDa protein and the E6 protein of human papillomavirus type 16. The functions of these complexes are unclear, but there is some evidence to suggest that binding of p53 to these viral proteins may inactivate p53 function. Recent reports have shown that p53 is involved in regulation of transcription. We have considered the possibility that p53 may regulate transcription of viral genes important for virus replication and/or transformation. Inactivation of p53 function by formation of such complexes might then permit correct expression of these viral genes. Since p53 can bind to the SV40 virus enhancer/promoter, we have investigated the effect of p53 on transcription from this promoter and report here that mouse p53 is a potent repressor of the SV40 enhancer/promoter. Mutations within p53 severely inhibited this activity and provided some evidence to show that the N-terminus of p53 contains residues essential for this function. We also show that mouse p53 represses transcription from the promoters of viruses that do not express proteins that complex with p53: the human cytomegalovirus early promoter and the Rous sarcoma virus long terminal repeat. By studying the effect of p53 on transcription in different cell lines, we show that the effects of p53 on promoters may be cell type specific.

Animals↗
PubMed1993

p53 confers a selective advantage on transfected HeLa cells.

The p53 gene, which is frequently mutated in various tumors, encodes a phosphoprotein thought to have a key role in the regulation of cell proliferation. To explore their biological effects, the HeLa carcinoma line, which does not express p53, was co-transfected with plasmid constructs expressing wild-type or mutant p53 proteins, or unrelated proteins, along with a plasmid conferring resistance to a neomycin-kanamycin antibiotic analog (G418). Both wild-type and mutant forms of p53 stimulated the number of G418-resistant colonies between 5- and 36-fold. Further investigation of colony development revealed that p53 enhanced cell survival, leading to increased colony numbers, but did not stimulate cell growth. Nonetheless, we suggest that an initial slowing of cell growth caused by expression of the unintegrated p53 plasmids renders the transfectants resistant to selection with G418, thus causing a higher frequency of G418-resistant colonies. p53 constructs were found to be expressed transiently in HeLa cells as expected, but the G418-resistant colonies frequently failed to express p53. This loss of p53 expression may be due to negative regulatory effects of p53 on the cytomegalovirus promoter that drives the selection marker.

Animals↗
PubMed1992

Down-regulation of E1a expression by E3 gene products in group C adenoviruses.

Mutant group C adenoviruses defective in expression of the E3 transcription unit were found to overexpress E1a proteins relative to wild-type adenoviruses. This result suggests that one or more proteins encoded in the E3 region (present in wild-type viruses) down-regulate E1a expression. This interpretation was confirmed by transfection experiments in which a plasmid expressing the E3 region reduced expression of E1a in 293 cells. Experiments to examine the molecular basis of this down-regulation of E1a suggest that E3 protein products interfere with the translation of viral mRNA molecules.

Adenovirus Early Proteins↗