Biomedical subjects

G Packham

Publications and source records attributed to G Packham.

47 records · Page 3Linked to original sources

PubMed1994

The protein tyrosine kinase ZAP-70 can associate with the SH2 domain of proto-Vav.

Stimulation of the T cell antigen receptor (TCR) leads to tyrosine phosphorylation of a number of cellular proteins, including the vav proto-oncogene product. We now report the detection of several phosphotyrosine proteins (80, 74, and 70 kDa) from TCR-stimulated T cells that bind to the Src homology 2 (SH2) domain of proto-Vav (Vav-SH2) and co-immunoprecipitate with the proto-Vav product. Their binding to Vav-SH2 differs from that observed with SH2 domains from other proteins. None of the Vav-SH2-associated phosphoproteins bind to either of the Src homology 3 (SH3) domains of proto-Vav or to mutant Vav-SH2 proteins. The association of the phosphoproteins with Vav-SH2 requires induction of tyrosine phosphorylation of cellular proteins since proteins from lysates of herbimycin A-treated TCR-activated T cells fail to associate with Vav-SH2. Among the proteins from T cells that co-immunoprecipitate with the proto-Vav product and bind to its SH2 domain, specific antibodies identified the 70-kDa tyrosine phosphoprotein as ZAP-70, a protein tyrosine kinase (PTK) involved in TCR signal transduction. Binding of this PTK to Vav-SH2 is inhibited by a ZAP-70-specific synthetic tyrosine phosphopeptide. We suggest that ZAP-70 may function as a PTK for proto-Vav.

3T3 Cells↗

PubMed1994

Ornithine decarboxylase is a mediator of c-Myc-induced apoptosis.

c-Myc plays a central role in the regulation of cell cycle progression, differentiation, and apoptosis. However, the proteins which mediate c-Myc function(s) remain to be determined. Enforced c-myc expression rapidly induces apoptosis in interleukin-3 (IL-3)-dependent 32D.3 murine myeloid cells following IL-3 withdrawal, and this is associated with the constitutive, growth factor-independent expression of ornithine decarboxylase (ODC), a rate-limiting enzyme of polyamine biosynthesis. Here we have examined the role of ODC in c-Myc-induced apoptosis. Enforced expression of ODC, like c-myc, is sufficient to induce accelerated death following IL-3 withdrawal. ODC induced cell death in a dose-dependent fashion, and alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC enzyme activity, effectively blocked ODC-induced cell death. ODC-induced cell death was due to the induction of apoptosis. We also demonstrate that ODC is a mediator of c-Myc-induced apoptosis. 32D.3-derived c-myc clones have augmented levels of ODC enzyme activity, and their rates of death were also a function of their ODC enzyme levels. Importantly, the rates of death of c-myc clones were inhibited by treatment with DFMO. These findings demonstrate that ODC is an important mediator of c-Myc-induced apoptosis and suggest that ODC mediates other c-Myc functions.

Animals↗

PubMed1994

Position and orientation independent transactivation by c-Myc.

The c-myc oncogene c-Myc is commonly activated in cancer and transactivates gene expression by binding to CACGTG DNA sequences as a heterodimeric complex with Max. The ornithine decarboxylase (ODC), p53, prothymosin alpha and ECA39 promoters are transactivated by c-Myc, and are considered direct targets, as activation is mediated by CACGTG sequences. Interestingly, the c-Myc-responsive CACGTG sequences in the p53, prothymosin alpha, ECA39 and murine ODC genes are all downstream of the RNA CAP site, suggesting that downstream sequences are preferred c-Myc targets. Using a series of heterologous reporter constructs, we have tested the effects of position and orientation of c-Myc-responsive CACGTG sequences on c-Myc's ability to activate transcription. A single binding site conferred c-Myc-responsiveness independent of position and orientation, and over distances of 1.7 kbp. The extent of transactivation was not significantly influenced by position of the responsive elements. By contrast, the extent of transactivation was dependent upon the number of c-Myc binding sites. The results demonstrate that c-Myc activates transcription independent of position and orientation and that considerable flexibility exists in the interaction of c-Myc transactivation domains with the general transcription machinery.

3T3 Cells↗

PubMed1994

v-raf suppresses apoptosis and promotes growth of interleukin-3-dependent myeloid cells.

Interleukin-3 (IL-3) is required for the proliferation, survival and differentiation of myeloid progenitors. In the absence of IL-3, murine myeloid 32D.3 cells accumulate in the G1 phase of the cell cycle and subsequently undergo programmed cell death, or apoptosis. Here we demonstrate that enforced expression of the v-raf oncogene suppresses apoptosis of myeloid 32D.3 cells following the withdrawal of IL-3. Surprisingly, steady state levels of Bcl-2, an oncogene known to suppress apoptosis, were not dependent upon IL-3 in 32D.3 cells and its levels were not augmented in v-raf clones. This suggests that ability of v-raf to suppress apoptosis in the absence of ligand is either Bcl-2 independent or that v-raf kinase promotes Bcl-2 function. v-raf also promoted growth of these cells in the presence of IL-3. v-raf clones proliferated at an increased rate due to a shortened G1 phase and had decreased requirements for IL-3 for growth. Therefore, transformation of myeloid cells by v-raf involves signaling pathways which promote both cell cycle progression and cell survival.

Animals↗

PubMed1993

The ornithine decarboxylase gene is a transcriptional target of c-Myc.

Constitutive c-myc expression suppresses cell cycle arrest, promotes entry into S phase, and results in the growth factor-independent expression of ornithine decarboxylase (ODC; EC 4.1.1.17). The ODC gene contains a conserved repeat of the Myc binding site, CACGTG, in intron 1. In this report, we demonstrate that c-Myc is a potent transactivator of ODC promoter-reporter gene constructs in fibroblasts that requires the CACGTG repeat. These sites conferred Myc responsiveness on heterologous promoter constructs, suggesting that ODC is regulated by Myc at the level of transcription initiation. Analysis of deletion and point mutants of c-myc revealed that domains required for transactivation of the ODC promoter did not include the leucine zipper of the Myc protein. This suggests that Myc may interact with transcription factors other than Max to transactivate the ODC gene.

3T3 Cells↗

PubMed1993

Strain variation in Epstein-Barr virus immediate early genes.

RNase protection analysis of mRNAs for Epstein-Barr virus (EBV) genes activated early in the induction of the productive cycle in Akata cells has confirmed that BZLF1 and BRLF1 behave as immediate early genes. These are likely to be the only virus immediate early genes since analysis of three other transcripts previously suggested to behave as immediate early genes in this system shows that they are, in fact, early genes. Western blotting of BRLF1 and BZLF1 demonstrates expression of these proteins early in induction of the productive cycle and reveals that the BZLF1 protein of Akata virus migrates more slowly on SDS gels than the BZLF1 protein of the prototypic virus strain B95-8. The nucleotide sequence of the BZLF1 gene and the immediate early promoters of Akata EBV have been determined and compared to other EBV strains. RFLP analysis of sequence variation in the BZLF1 gene of EBV strains from many different parts of the world shows a distribution of BZLF1 types which are not simply related to the established A and B EBV types.

Amino Acid Sequence↗

PubMed1992

Differential splicing of Epstein-Barr virus immediate-early RNA.

A variant form of splicing of RNA crossing the Epstein-Barr virus (EBV) BZLF1 gene was observed in the late productive cycle of EBV. This splice omits the middle exon of BZLF1 and joins the outer two exons of BZLF1 in frame, but the shortened form of BZLF1 protein (Z delta) could not be detected in natural EBV infections.

Blotting, Western↗

PubMed1991

Differentiation-associated expression of the Epstein-Barr virus BZLF1 transactivator protein in oral hairy leukoplakia.

The BZLF1 protein of Epstein-Barr virus (EBV) is a key immediate-early protein which has been shown to disrupt virus latency in EBV-infected B cells. We have generated a monoclonal antibody, BZ1, to BZLF1 which reacts in immunohistology, immunoblotting, and immunoprecipitation and which recognizes both the active, dimeric form and the inactive, monomeric form of the protein. Biopsies of oral hairy leukoplakia, an AIDS-associated lesion characterized by high-level EBV replication, were examined by immunohistochemistry using the BZ1 monoclonal antibody. A differentiation-associated pattern of BZLF1 expression was observed, BZ1 reacting with nuclei of the upper spinous layer of the lesion. This finding suggests that the BZLF1 promoter may be regulated by the degree of squamous differentiation. A comparison of in situ hybridization to EBV DNA and viral capsid antigen staining with BZ1 reactivity suggested that BZLF1 expression precedes rampant virus replication. The inability to detect EBV in the lower epithelial layers of oral hairy leukoplakia raises questions concerning the nature of EBV latency and persistence in stratified squamous epithelium.

Antibodies, Monoclonal↗

PubMed1991

The BZLF1 protein of EBV has a coiled coil dimerisation domain without a heptad leucine repeat but with homology to the C/EBP leucine zipper.

The EBV transactivator protein BZLF1 can bind many sites in the EBV genome, most of which have homology to a consensus AP-1 site, the binding site for the fos/jun family of transcription factors. Here we present evidence that BZLF1 can also recognise the binding site for the CCAAT/enhancer binding protein C/EBP and that a BZLF1 binding site within the BZLF1 promoter is recognised by the C/EBP protein. Analysis of the BZLF1 DNA binding domain suggests that the BZLF1 protein binds to DNA as a dimer using sequences adjacent to a basic DNA binding motif. The BZLF1 dimerisation domain does not have a heptad repeat of leucine residues common to leucine zipper proteins but does have characteristics of a coiled coil structure, as judged by site directed mutagenesis. We therefore propose that the dimerisation domain of BZLF1 is structurally related to the coiled coil structure of leucine zippers but lacks the highly conserved leucine repeat. We show that the PZLF1 dimerisation domain has residues in common with the C/EBP leucine zipper and discuss the possible implications of this relationship.

Amino Acid Sequence↗

PubMed1990

Structure and function of the Epstein-Barr virus BZLF1 protein.

Five DNA-binding sites for the Epstein-Barr virus BZLF1 protein have been identified within three of the early viral promoters, and four of these binding sites contain a consensus AP-1 site. The part of the BZLF1 protein required for sequence-specific DNA binding to one of these AP-1-like sites was identified by deletion mapping. Site-directed mutagenesis of this DNA target suggests that BZLF1 may work partly by overcoming a cellular repressor of viral transcription.

Amino Acid Sequence↗

PubMedDate not supplied

Inhibition of superoxide dismutase by 2-methoxyoestradiol analogues and oestrogen derivatives: structure-activity relationships.

Superoxide dismutases catalyse the dismutation of highly reactive superoxide ions to produce hydrogen peroxide and several lines of evidence suggest that these enzymes play important roles in the development and response to treatment of human cancers. For example, Mn-containing superoxide dismutase is frequently overexpressed in various cancer types and can contribute to resistance to apoptosis. 2-Methoxyoestradiol is a naturally occurring metabolic product of 17beta-oestradiol that inhibits tubulin polymerization and possesses growth inhibitory and cytotoxic activity in vitro and in vivo. More recently 2-methoxyoestradiol has also been shown to inhibit superoxide dismutase (SOD) in a tetrazolium salt based enzyme assay, suggesting that oestrogen derivatives might be useful starting points for the development of effective, non-toxic enzyme inhibitors. Here we have tested the SOD inhibiting activity of a range of oestrogen derivatives to determine structural features important for enzyme inhibition.

2-Methoxyestradiol↗