PubMed Health⌕ Search

Biomedical subjects

G Packham

Publications and source records attributed to G Packham.

At least 37 records · Page 2Linked to original sources
PubMed1998

Infrequent bax gene mutations in B-cell lymphomas.

Mutation of the bax gene has been reported previously in lymphoid cell lines. In vitro experiments have shown that alterations in promoter and coding sequences of the gene abolish its apoptosis initiation function, which is considered crucial for tumour development. To assess bax gene mutations in lymphomagenesis, polymerase chain reaction-based single strand conformation polymorphism analysis (PCR-SSCP) and direct sequencing were used to detect altered sequences in the promoter region and all the six exons and their flanking sequences of the gene. Nodal and extranodal B-cell lymphomas (n = 112) including follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, splenic marginal zone B-cell lymphoma and low- and high-grade mucosa-associated lymphoid tissue (MALT) lymphomas were studied. Sequence alterations were found in 11 cases. Nine also showed the same altered sequences in corresponding non-tumour control tissue samples, indicating polymorphism. In the remaining two cases, sequence alterations (in exons 3 and 6) which altered the bax open reading frame were observed only in tumour tissues, indicating tumour-specific point mutation. These results suggest that inhibition of apoptosis through bax gene mutations is unlikely to be a common event in B-cell lymphoma, at least in the major types of nodal and extranodal B-cell lymphomas.

DNA, Neoplasm↗
PubMed1997

Mammalian cells express two differently localized Bag-1 isoforms generated by alternative translation initiation.

The Bcl-2 oncoprotein is a key regulator of apoptosis and the Bag-1 protein interacts with Bcl-2 and cooperates with Bcl-2 to suppress apoptosis. The human Bag-1 cDNA is essentially identical with a previously described cDNA encoding RAP46, which interacts with activated steroid hormone receptors. However, there is considerable confusion over the structure of Bag-1/RAP46 proteins and their relationship to endogenous Bag-1 proteins. Here we have characterized Bag-1 expression in mammalian cells. We demonstrate that, in addition to the previously identified 32 kDa murine and 36 kDa human Bag-1 proteins, cells express a second 50 kDa Bag-1 isoform. In some murine cell lines p50 is expressed at the same level as p32 Bag-1, and p50 and p32 Bag-1 proteins have distinct subcellular localizations, suggesting that they are functionally distinct. The published mouse Bag-1 cDNA is partial, and sequencing of additional murine Bag-1 RNA 5' sequences demonstrated that human and murine Bag-1 cDNAs contain longer open reading frames than originally suspected. We determined which open reading frames gave rise to the Bag-1 isoforms in human cells. Surprisingly, translation of neither protein initiated at the first in-frame methionine, and cells do not express Bag-1/RAP46 proteins with the previously proposed structures; p50 Bag-1 initiates at an upstream CUG codon, whereas p36 Bag-1 initiates at a downstream AUG codon. Therefore, cells express two differently localized Bag-1 isoforms generated by alternative translation initiation, and Bag-1 proteins may play a dual role in regulating apoptosis and steroid hormone-dependent transcription.

Amino Acid Sequence↗
PubMed1997

Induction of ornithine decarboxylase by IL-3 is mediated by sequential c-Myc-independent and c-Myc-dependent pathways.

Enforced c-Myc expression promotes continuous, growth factor-independent, cell cycle progression and activates expression of the ornithine decarboxylase (ODC) gene and its promoter. c-Myc-responsiveness of murine ODC is mediated by two conserved c-Myc:Max E-boxes in ODC intron 1. c-Myc and ODC are both required for cell growth and their expression is sequentially induced in G0/G1 cells stimulated with mitogens, yet their expression is not modulated by the cell cycle in proliferating cells. Here we demonstrate that regulation of ODC and its promoter by Interleukin-3 (IL-3) in murine myeloid cells is mediated in part by c-Myc, c-Myc induced ODC through the same transcription start site as IL-3 and, in asynchronously growing cells, maximal activity of the ODC promoter required the intronic c-Myc binding sites. However, induction of ODC following IL-3 stimulation of quiescent cells is mediated by at least two pathways. The first phase of this response was independent of the intronic c-Myc:Max E-boxes and de novo protein synthesis. Sustained induction of the ODC promoter however required the c-Myc:Max binding sites and protein synthesis. Accumulation of c-Myc following stimulation of quiescent cells with IL-3 correlated with the delayed phase of the response. Consistent with a two pathway model of ODC regulation, inducible overexpression of dominant negative form of c-Myc (In373-Myc), which specifically inhibits the c-Myc-Max network, inhibited the delayed, but not immediate, induction of ODC promoter activity in response to IL-3. Dominant negative c-Myc protein also effectively suppressed induction of the endogenous ODC gene by IL-3. Therefore, c-Myc functions as a direct and required-regulator of ODC. These results also suggest a model whereby c-Myc's role in regulating its targets may be to convert a transient, immediate-early, activation event into the persistent induction of gene expression.

Animals↗
PubMed1997

High level expression of p27(kip1) and cyclin D1 in some human breast cancer cells: inverse correlation between the expression of p27(kip1) and degree of malignancy in human breast and colorectal cancers.

The expression of cyclin-dependent kinase inhibitor p27(kip1) in human tumors and normal tissues was investigated using a panel of novel anti-p27(kip1) mAbs. An inverse correlation between expression of p27(kip1) and cell proliferation was generally observed after analyzing its expression in 25 different normal human tissues. In some highly proliferative human breast cancer cells, however, high level p27(kip1) expression was seen, indicating the existence of a mechanism by which some growing tumor cells may tolerate this inhibitor of cell cycle progression. Detailed studies demonstrated a correlation between the high level expression of p27(kip1) and cyclin D1 in human breast cancer cells. There was also an inverse correlation between the expression of p27(kip1) and the degree of tumor malignancy in human breast and colorectal cancers, indicating that p27(kip1) may be a useful prognostic marker in these cancers.

Biomarkers, Tumor↗
PubMed1996

c-Myc induces apoptosis and cell cycle progression by separable, yet overlapping, pathways.

Enforced c-Myc expression promotes inappropriate cell cycle progression of growth factor deprived cells and triggers concomitant apoptosis. However, it is not clear what role dysregulation of the cell cycle plays in c-Myc-induced apoptosis. Ornithine decarboxylase (ODC) is a transcriptional target of c-Myc and contributes to c-Myc induced apoptosis. Here we have established that high levels of ODC overexpression in interleukin-3 (IL-3)-dependent 32D.3 myeloid cells induces apoptosis at rates comparable to those induced by enforced c-Myc expression. However, ODC-induced apoptosis was not accompanied by dysregulation of cell cycle controls, indicating that cell death was not triggered by inappropriate cell cycle progression. Nonetheless, ODC was required downstream of c-Myc for myeloid cell growth. These results suggested that c-Myc-induced pathways leading to cell cycle progression and apoptosis are separable, yet that they share common mediators. In agreement with this concept, treatment of cells over-expressing c-Myc with the growth inhibitory agent dibutyryl cyclic AMP (Bt2cAMP) arrested these cells G1, without inducing apoptosis. However, c-Myc retained the ability to induce apoptosis of Bt2cAMP-arrested cells following removal of IL-3, demonstrating that Bt2cAMP selectively inhibits c-Myc-induced pathways promoting cell cycle progression but not apoptosis. These results suggest a "multiple effectors' model in which c-Myc regulates the expression of mediators which alone are sufficient to induce apoptosis in the absence of survival factors, yet are required in concert to promote cell cycle progression.

Animals↗
PubMed1996

Cytokines suppress apoptosis independent of increases in reactive oxygen levels.

Antioxidants suppress apoptosis induced by diverse stimuli in many cells, including immune cells, suggesting that reactive oxygen species (ROS) are common mediators of apoptosis. We evaluated the potential role for ROS in the apoptosis of myeloid progenitors following withdrawal of survival factors and in the apoptosis triggered by enforced c-myc expression, two model systems of programmed cell death. ROS are potential mediators of these cell deaths, as low concentrations of H2O2 (0.1-0.2 mM) or menadione (less than = 10 microM) induced myeloid cell apoptosis, and cytokines effectively suppressed this cell death. Apoptosis following IL-3 withdrawal and c-Myc-induced cell death was also effectively suppressed by the antioxidants pyrrolidine dithiocarbamate and glutathione peroxidase. Moreover, measurements of intracellular ROS in cells treated with oxidants or antioxidants showed a correlation between levels of reactive oxygen and the induction or suppression of apoptosis. However, apoptosis following IL-3 withdrawal and c-Myc-induced cell death was not associated with increased reactive oxygen. Therefore, myeloid programmed cell death is not associated with increases in ROS.

Animals↗
PubMed1995

Vav and Ras induce fibroblast transformation by overlapping signaling pathways which require c-Myc function.

Recent evidence has suggested that the Vav oncoprotein may function as a hematopoietic-specific GTP exchange factor for the Ras superfamily of proteins. However, transformation of NIH3T3 fibroblast cells by Vav is morphologically distinct from that induced by activated Ras oncogenes, suggesting that the two oncoproteins induce separate signal transduction pathways which promote transformation. To address this issue, the effects of dominant negative mutants of H-ras and proto-Vav (proto-VavR695L, a mutation in the VavSH2 domain) were tested on Vav- and Ras-induced transformation. These mutants partially inhibited both Vav- and Ras-induced transformation, suggesting that they may induce a common downstream signaling pathway which potentiates transformation. As an independent measure of Vav function we also tested the ability of the purified protein encoded by VavSH2 to influence Germinal Vesicle Breakdown (GVBD) during Xenopus oocyte maturation. Microinjection of the VavSH2 protein alone, but not mutant VavR695L SH2 protein, was sufficient to induce GVBD and accelerated maturation induced by normal Ras, suggesting that in this system as well Vav and Ras signals overlap through a common effector. A key target of multiple signalling pathways is c-Myc. Dominant negative versions of c-Myc totally abolished morphologic transformation of NIH3T3 cells by both Vav and Ras oncogenes. These results suggest that distinct, but overlapping, signalling pathways are induced by Vav and Ras and that fibroblast cell transformation by either oncogene requires c-Myc functions.

3T3 Cells↗
PubMed1995

The role of ornithine decarboxylase in c-Myc-induced apoptosis.

Translocations or amplifications which activate members of the myc gene family are common in human cancer. c-Myc is a central regulator of normal cell proliferation and differentiation (Luscher and Eisenman, 1991; Marcu et al. 1992) and can induce apoptosis (Askew et al. 1991, 1993; Evan et al. 1992; Bissonette et al. 1992; Shi et al. 1992). c-Myc is a transcription factor (Amati et al. 1992; Kretzner et al. 1992), however, the targets regulated by c-Myc which mediate its activities are not known. Here, we discuss the role of the ornithine decarboxylase (ODC) gene as a direct transcriptional target of c-Myc and demonstrate a role for ODC enzyme activity in c-Myc-induced apoptosis.

Animals↗
PubMed1995

E2F-1:DP-1 induces p53 and overrides survival factors to trigger apoptosis.

The E2F DNA binding activity consists of a heterodimer between E2F and DP family proteins, and these interactions are required for association of E2F proteins with pRb and the pRb-related proteins p107 and p130, which modulate E2F transcriptional activities. E2F-1 expression is sufficient to release fibroblasts from G0 and induce entry into S phase, yet it also initiates apoptosis. To investigate the mechanisms of E2F-induced apoptosis, we utilized interleukin-3 (IL-3)-dependent 32D.3 myeloid cells, a model of hematopoietic progenitor programmed cell death. In the absence of IL-3, E2F-1 alone was sufficient to induce apoptosis, and p53 levels were diminished. DP-1 alone was not sufficient to induce cell cycle progression or alter rates of death following IL-3 withdrawal. However, overexpression of both E2F-1 and DP-1 led to the rapid death of cells even in the presence of survival factors. In the presence of IL-3, levels of endogenous wild-type p53 increased in response to E2F-1, and coexpression of DP-1 further augmented p53 levels. These results provide evidence that E2F is a functional link between the tumor suppressors p53 and pRb. However, induction of p53 alone was not sufficient to trigger apoptosis, suggesting that the ability of E2F to override survival factors involves additional effectors.

Animals↗
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↗