Biomedical subjects

D P Lane

Publications and source records attributed to D P Lane.

At least 19 recordsLinked to original sources

PubMed1996

Epitope analysis of the murine p53 tumour suppressor protein.

The identification and characterisation of the p53 tumour suppressor has relied extensively on the use of immunological reagents. To facilitate further characterisation of the murine p53 protein (Mp53), and its interaction with other proteins, we have characterised the antigenic sites of Mp53 in fine detail. Using an overlapping Mp53 peptide library we report the identification by Pepscan ELISA of the epitopes of nine antibodies. We have also used this technique to determine whether corresponding epitopes were present in a human p53 (Hp53) peptide library. This comparison was extended to include polyclonal sera of mice immunized with either Mp53 or Hp53, to compare the overall range of antigenic sites. The range of antigenic sites identified by polyclonal sera is very similar, although the N-terminus of Mp53 is clearly not an immunodominant region, in contrast to the N-terminus of Hp53. However, the N-terminus of Mp53 is immunogenic in rabbits as demonstrated by the Pepscan ELISA of CM5 serum (a rabbit anti-Mp53 serum used in analysing p53 expression in mice). Since, very few new antigenic sites were identified in either Mp53 or Hp53, new approaches will have to be employed to identify novel immunological reagents against human and murine p53.

Animals↗

PubMed1996

Human and plant proliferating-cell nuclear antigen have a highly conserved binding site for the p53-inducible gene product p21WAF1.

The mechanism(s) whereby higher plants respond to environmental agents that damage their DNA, which leads to the arrest of cell division, is poorly understood. In mammalian cells, the tumour-suppressor protein p53 plays a central role in a DNA-damage-induced cell-cycle-checkpoint pathway by induction of transcription of a set of gene products that have a direct role in a DNA-damage-induced cell-cycle growth arrest. One such protein, p21WAF1, has been shown to be essential for radiation-induced growth arrest. There appear to be at least two cellular targets of p21WAF1 during checkpoint control, the G1-cyclin-dependent kinases (CDK) and proliferating-cell nuclear antigen (PCNA). The aim of the research reported here was to determine whether the interactions between the human growth inhibitor p21WAF1 and PCNA from plants and humans are conserved. If so, this would suggest that modulation of PCNA activity may play an important role in plant responses to DNA damage and would imply that functional homologue(s) of p21WAF1 exist in plants. We show that the p21WAF1-interaction domain of PCNA is conserved between humans and plants. A peptide that contains the site of human p21WAF1 that binds human PCNA has been used to precipitate PCNA from crude pea (Pisum savitum) extracts. We used the p21WAF1 peptide as an affinity matrix and showed that pea PCNA bound in a specific high-affinity manner. This finding was used to develop a purification protocol that allowed PCNA from plant tissue to be purified to homogeneity. Pure pea PCNA forms a stable complex with full-length human p21WAF1 and the specific amino acids of p21WAF1 required for the interaction have been identified. The critical residues were identical to those required for binding to human PCNA, which indicates that the interaction of human p21WAF1 with PCNA is highly conserved at each amino acid position between pea and human.

Amino Acid Sequence↗

PubMed1996

Two pathways for base excision repair in mammalian cells.

Abasic sites (apurinic/apyrimidinic, AP sites) are the most common DNA lesions generated by both spontaneous and induced base loss. In a previous study we have shown that circular plasmid molecules containing multiple AP sites are efficiently repaired by Chinese hamster extracts in an in vitro repair assay. An average patch size of 6.6 nucleotides for a single AP site was calculated. To define the exact repair patch, a circular DNA duplex with a single AP site was constructed. The repair synthesis carried out by hamster and human cell extracts was characterized by restriction endonuclease analysis of the area containing the lesion. The results indicate that, besides the repair events involving the incorporation of a single nucleotide at the lesion site, repair synthesis occurred also 3' to the AP site and involved a repair patch of approximately 7 nucleotides. This alternative repair pathway was completely inhibited by the presence in the repair reaction of a polyclonal antibody raised against human proliferating cell nuclear antigen. These data give the first evidence that mammalian cell extracts repair natural AP sites by two distinct pathways: a single nucleotide gap filling reaction targeted at the AP site and a proliferating cell nuclear antigen-dependent pathway that removes a short oligonucleotide containing the abasic site and 3'-flanking nucleotides.

Animals↗

PubMed1996

Modification of an N-terminal regulatory domain of T antigen restores p53-T antigen complex formation in the absence of an essential metal ion cofactor.

We have discovered that the ability of the tumor suppressor protein p53 to bind to the viral large T antigen (TAg) oncogene product is regulated by divalent cations. Both proteins were purified from an insect cell line infected with the appropriate baculovirus expression vector. In a two-site capture enzyme-linked immunosorbent assay, complex formation between the purified proteins is strictly dependent on the addition of specific concentrations of divalent metal ions, notably zinc, copper, cadmium, cobalt, manganese, and nickel. In the presence of zinc the pattern of proteolytic fragments obtained when TAg was subjected to proteolysis by endoproteinase Glu-C (V8) was strikingly different, supporting the idea that a conformational change in TAg associated with ion binding is required for it to complex with p53. Monoclonal antibody analysis provides supporting evidence for a conformational change. When TAg was captured onto an enzyme-linked immunosorbent assay plate coated with PAb 419 as opposed to many other anti-TAg antibodies, complex formation was completely independent of the presence of additional divalent cations. Our results suggest that the ability of p53 and TAg to form a stable complex in vitro is dependent upon a regulatory domain residing in the N terminus of TAg, zinc ions or the binding of a specific monoclonal antibody (PAb 419) provoking a conformational change in TAg that facilitates and supports complex formation.

Animals↗

PubMed1996

Allosteric regulation of the thermostability and DNA binding activity of human p53 by specific interacting proteins. CRC Cell Transformation Group.

Conformational stability is a prerequisite for the physiological activity of the tumor suppressor protein p53. p53 protein can be allosterically activated for DNA binding by phosphorylation or through noncovalent interaction with proteins such as DnaK, the Escherichia coli homologue of the heat shock protein Hsp70. We present in vitro evidence for a rapid temperature-dependent change in the conformation and tetrameric nature of wild-type p53 upon incubation at 37 degrees C, which correlates with a permanent loss in DNA binding activity. We show that p53 is allosterically regulated for stabilization of the wild-type conformation and DNA binding activity at 37 degrees C by binding of two classes of ligands to regulatory sites on the N and C terminus of the molecule through which an intrinsic instability of p53 is neutralized. Deletion of the domain conferring instability at the C terminus is sufficient to confer enhanced stability to the total protein. DnaK binding to the C terminus can profoundly protect p53 at 37 degrees C from a temperature-dependent loss of the DNA binding activity but does not renature or activate denatured p53. In contrast, another activator of the DNA binding activity of latent p53, the monoclonal antibody PAb421, which also interacts with the C terminus of the protein, is not able to protect p53 from thermal denaturation. Two monoclonal antibodies to the N terminus of p53, PAb1801 and DO-1, do not activate the latent DNA binding function of p53 but can protect the p53 wild-type conformation at 37 degrees C. Thus, activation of the DNA binding function of p53 is not synonymous with protection from thermal denaturation, and therefore, both of these pathways may be used in cells to control the physiological activity of p53. The protection of p53 conformation from heat denaturation by interacting proteins suggests a novel mechanism by which p53 function could be regulated in vivo.

Allosteric Regulation↗

PubMed1996

Inhibition of pRb phosphorylation and cell-cycle progression by a 20-residue peptide derived from p16CDKN2/INK4A.

BACKGROUND: The CDKN2/INK4A tumour suppressor gene is deleted or mutated in a large number of human cancers. Overexpression of its product, p16, has been shown to block the transition through the G1/S phase of the cell cycle in a pRb-dependent fashion by inhibiting the cyclin D-dependent kinases cdk4 and cdk6. Reconstitution of p16 function in transformed cells is therefore an attractive target for anti-cancer drug design. RESULTS: We have identified a 20-residue synthetic peptide--corresponding to amino acids 84-103 of p16--that interacts with cdk4 and cdk6, and inhibits the in vitro phosphorylation of pRb mediated by cdk4-cyclin D1. The amino-acid residues of p16 important for its interaction with cdk4 and cdk6 and for the inhibition of pRb phosphorylation were defined by an alanine substitution series of peptides. In normal proliferating human HaCaT cells and in cells released from serum starvation, entry into S phase was blocked by the p16-derived peptide when it was coupled to a small peptide carrier molecule and applied directly to the tissue culture medium. This cell-cycle block was associated with an inhibition of pRb phosphorylation in vivo. CONCLUSIONS: These results demonstrate that a p16-derived peptide can mediate three of the known functions of p16: firstly, it interacts with cdk4 and cdk6; secondly, it inhibits pRb phosphorylation in vitro and in vivo; and thirdly, it blocks entry into S phase. The fact that one small synthetic peptide can enter the cells directly from the tissue culture medium to inhibit pRb phosphorylation and block cell-cycle progression makes this an attractive approach for future peptidometic drug design. Our results suggest a novel and exciting means by which the function of the p16 suppressor gene can be restored in human tumours.

Amino Acid Sequence↗

PubMed1996

Oxidative stress is involved in the UV activation of p53.

In many vertebrate cells exposure to ultraviolet light lead to a dramatic increase in the cellular levels of the tumour suppressor protein p53, followed by a biological response of either growth arrest or programmed cell death. Ultraviolet light can be absorbed directly by cellular macromolecules, leading to photochemical modification of DNA and proteins. Additionally, it also causes free radical formation, resulting in oxidative stress. Whereas ultraviolet light and ionizing radiation both induce DNA lesions which trigger an activation of the p53 pathway, the magnitude of the p53 response elicited by ionizing radiation is comparatively low. Following irradiation with ultraviolet light two populations of p53-reactive cells are induced: a population accumulating high levels of p53 protein and a population with comparatively low levels of p53, similar in magnitude to the p53 response following ionizing radiation. Pretreatment of cells with N-acetylcysteine, an agent known to counteract oxidative stress, attenuates the cellular p53 response to ultraviolet light by reducing the number of cells with high p53 levels but does not affect the response to ionizing radiation. We demonstrate that N-acetylcysteine pretreatment does not prevent the inflicted DNA damage and therefore conclude that oxidative stress is a causative agent in the ultraviolet light activation of the p53 pathway.

3T3 Cells↗

PubMed1996

The p53-MDM2 interaction in a cancer-prone family, and the identification of a novel therapeutic target.

One approach to developing novel anti-cancer agents is to identify and characterise targets that directly regulate cell growth and are dysfunctional in the disease state. One such target is the interaction between the p53 tumour suppressor and the oncogene product of the murine double minute gene, MDM2. MDM2 is known to bind wild-type p53 and block the transcriptional activation of p53-dependent genes. We have previously described a cancer-prone family with elevated levels of wild-type p53, and now show that MDM2 is also over-expressed in the proband from this family. Interestingly, the overexpression of MDM2 is independent of other p53-regulated genes such as p21WAF1. The present work and a review of recent insights into the p53-MDM2 interaction, and p53 transcriptional activity, identify a new target site for the rational development of novel anti-cancer agents.

Antineoplastic Agents↗

PubMed1995

Small peptides activate the latent sequence-specific DNA binding function of p53.

Normal cells contain p53 protein in a latent state that can be activated for sequence-specific transcription by low levels of UV radiation without an increase in protein levels. Microinjection of cells with an antibody specific to the C-terminal negative regulatory domain can activate the function of p53 as a specific transcription factor in the absence of irradiation damage, suggesting that posttranslational modification of a negative regulatory domain in vivo is a rate-limiting step for p53 activation. Small peptides derived from the negative regulatory domain of p53 have been used as biochemical tools to distinguish between allosteric and steric mechanisms of negative regulation of p53 tetramer activity. Presented is the development of a highly specific peptide activation system that is consistent with an allosteric mechanism of negative regulation and that forms a precedent for the synthesis of novel low molecular mass modifiers of the p53 response.

Allosteric Regulation↗

PubMed1995

Two distinct signaling pathways activate the latent DNA binding function of p53 in a casein kinase II-independent manner.

Post-translational modification of a carboxyl-terminal negative regulatory domain in vitro by either casein kinase II or protein kinase C allosterically activates the latent sequence-specific DNA binding function of p53. Reported here is a biochemical approach to determine the types of signaling pathways and enzymes that are involved in p53 activation in cells. Using a novel chromatographic method, we have been able to separate three distinct biochemical forms of p53 that have been synthesized in vivo; two are in an activated state, and one is in a latent state for sequence-specific DNA binding. The two activated forms of p53 appear to be controlled individually by either a constitutive or a UV-inducible signaling pathway. p53 lacking the COOH-terminal casein kinase II site (p53 delta 4) was characterized biochemically and used to determine the affects of deletion of the casein kinase II motif on the production of the two activated forms of p53 in vivo. As observed with full-length p53, the production of two distinct chromatographic forms of activated p53 delta 4 occurs in vivo, indicating that p53 activation can occur through a casein kinase II-independent pathway and suggesting that two other factors are involved in activation of p53 in vivo.

Amino Acid Sequence↗

PubMed1995

p53-dependent growth arrest following calcium phosphate-mediated transfection of murine fibroblasts.

A variety of genotoxic agents can induce an accumulation of p53 protein in the nuclei of mammalian cells and lead to either growth arrest or apoptosis in a p53-dependent manner. Recently, the induction of the p53 pathway has also been reported for non-genotoxic agents such as heat shock and hypoxia, rendering it likely that p53 activity might be triggered by a wider range of cellular stress factors. Here we report the effect of calcium phosphate-mediated transfection, a technique commonly used in studies of transient gene expression in mammalian cells, on fibroblasts containing wild-type p53. Incubation with a calcium phosphate precipitate results in a transient growth arrest in the presence or absence of plasmid DNA. The effect is accompanied by a rise in p53 protein levels and increased transcription of a p53-dependent reporter construct and is not observed in fibroblasts derived from p53 knockout mice.

3T3 Cells↗

PubMed1995

A direct effect of activated human p53 on nuclear DNA replication.

p53 is a transcriptional activator and repressor, but recent evidence suggests that some of its many biological functions may not be dependent on transcription. To determine whether p53 exerts a direct influence on nuclear DNA replication, purified human p53 was added to a transcription-free DNA replication extract from Xenopus eggs. Full-length human p53 that inhibits SV40 DNA replication in vitro had no effect on nuclear DNA synthesis in the Xenopus system. In contrast, a C-terminal truncated form of p53 (p53 delta 30), which is constitutively active for DNA binding and similar to an alternately spliced form found in vivo, showed a concentration-dependent inhibition of DNA replication in both the soluble SV40 system and eukaryotic nuclei. This inhibition occurred primarily at initiation of DNA synthesis. Oxidation of p53 delta 30, which eliminates DNA binding activity, also abrogated the protein's ability to inhibit nuclear DNA synthesis. The p53 binding DNA consensus sequence enhanced rather than competed away inhibitory activity of p53 delta 30. Therefore, p53 that is constitutively active for DNA binding can inhibit nuclear DNA replication in the absence of transcription. This inhibition may require binding of p53 to DNA, in addition to interactions between p53 and proteins of the replication complex.

Animals↗

PubMed1995

Characterisation of epitopes on human p53 using phage-displayed peptide libraries: insights into antibody-peptide interactions.

We previously described the use of a phage-displayed library of random hexapeptides to define and localise the epitope on the human tumor suppressor protein p53 recognised by the monoclonal antibody PAb240. Here we have extended these results to a further eight anti-p53 monoclonal antibodies and to two further libraries, which display 12-mer and 20-mer peptides, respectively. First, we showed that selection of PAb240 binding clones from the 12-mer and 20-mer libraries gives essentially identical results to those obtained by screening the 6-mer library. Second, we used the 6-mer and 12-mer libraries to define the detailed specificity profiles of six antibodies (DO-1, DO-2, DO-7, Bp53-11, Bp53-12 and Bp53-19), which recognise the same short, highly immunogenic N-terminal segment of p53. Finally, we employed all three libraries to reveal the distinct mechanisms by which PAb421 and PAb122, two monoclonal antibodies that allosterically activate sequence-specific DNA binding by p53, react specifically with the same positively-charged C-terminal segment. In each case the epitope locations inferred from the selected sequences were confirmed by probing an array of overlapping synthetic peptides representing the primary sequence of p53. The results emphasise the consequences for epitope mapping of screening random, as opposed to antigen-derived, peptide libraries; specifically (1) that comparison of selected sequences reveals the contribution of individual residues to binding energy and specificity; (2) that heteroclitic reactions can lead to definition of a consensus that is related to but distinct from the immunising epitope and (3) that isolation of non-immunogen-homologous "mimotope" sequences reveals discrete, alternative ligand structures. The results with PAb421 and PAb122 provide examples where, while selection from the 12-mer and 20-mer libraries leads to isolation of immunogen-homologous sequences, selection from the 6-mer library results in the isolation either of no binding clones (PAb122) or solely of "mimotope" sequences with no discernible homology to the original antigen (PAb421). In addition the results with PAb421 reveal that linear epitopes can be longer than previously thought and can be formally discontinuous, consisting of independent contact motifs, which show promiscuous relative positioning.

Amino Acid Sequence↗

PubMed1995

A small peptide inhibitor of DNA replication defines the site of interaction between the cyclin-dependent kinase inhibitor p21WAF1 and proliferating cell nuclear antigen.

BACKGROUND: p21WAF1 is a potent inhibitor of the cell-cycle regulatory cyclin-dependent kinases (Cdks). It acts on Cdks in the G1 and S phases of the cell cycle, and also binds to proliferating cell nuclear antigen (PCNA), blocking DNA replication in vitro. Transcription of p21WAF1 can be induced by the human tumour suppressor protein p53, suggesting that the action of p21WAF1 may be important in cancer prevention. We have investigated the interaction between p21WAF1 and PCNA using a genetic two-hybrid screen and with arrays of synthetic peptides derived from the p21WAF1 protein sequence. RESULTS: We have established that the carboxy-terminal region of p21WAF1 interacts with PCNA in a yeast two-hybrid screen. Interaction with p21WAF1 involves the central loop of PCNA, which connects the two domains of the PCNA monomer. The interaction was finely mapped using peptides derived from the entire sequence of the p21WAF1 protein, and the critical residues were found to be QTSMTDFY (amino acids 144-151 of p21WAF1). Remarkably, a 20-residue peptide containing this sequence inhibited replication of simian virus 40 (SV40) DNA in vitro and could capture PCNA from whole cell extracts, demonstrating that small molecules can retain the biological activity characteristic of the whole protein. Sequential alanine-scan mutations of the peptide demonstrated that its ability to block replication correlates with its affinity for binding PCNA. CONCLUSIONS: We have shown that PCNA and the cell-cycle regulator p21WAF1 interact in vivo, and that this interaction requires the central loop of PCNA and an eight amino-acid motif from the carboxyl terminus of p21WAF1.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1995

On the regulation of the p53 tumour suppressor, and its role in the cellular response to DNA damage.

The p53 gene is required for the normal apoptotic response of mammalian cells to DNA damage caused by ionizing radiation and DNA damaging drugs. DNA damage results in the accumulation of biologically active p53. This response is potentially lethal and is therefore highly regulated. By using both biochemical and cell biological approaches a number of discrete control pathways have been identified. These include analysis of cellular and viral proteins that bind to p53 to inactivate its function, the discovery of cells with defects in the p53 activation pathway and the analysis of an allosteric regulation of p53 function controlled by phosphorylation.

Amino Acid Sequence↗

PubMed1995

Conformational changes in p53 analysed using new antibodies to the core DNA binding domain of the protein.

The p53 protein contains a protease resistant core section that binds to DNA in a sequence specific manner and whose crystal structure has been determined. This core is flanked at the N-terminus by the transcriptional transactivation domain and at the C-terminus by sequences involved in the oligomerisation of the protein. Extensive immunochemical analysis of p53 has shown that dominant antigenic sites lie within these N- and C-terminal domains while few antibodies to the central core have been identified. One of these, PAb240, has been extensively characterised as its epitope is cryptic in the native DNA binding core structure but is exposed by denaturation. This epitope is also exposed on many p53 proteins that contain point mutations in the core domain suggesting that these mutations may have a common affect on the structure of the core. To investigate this further we have generated several new antibodies to novel sites on p53 and mapped their epitopes using synthetic peptides. We find that antibodies to two other discrete sites in the core can also, like PAb240, recognize cryptic epitopes and distinguish mutant from wild-type conformations implying that the point mutations found in p53 in human tumours have widespread effects on the folding pattern of the DNA binding domain.

Amino Acid Sequence↗

PubMed1995

Tumour suppressors, kinases and clamps: how p53 regulates the cell cycle in response to DNA damage.

The human tumour suppressor protein p53 is critical for regulation of the cell cycle on genotoxic insult. When DNA is damaged by radiation, chemicals or viral infection, cells respond rapidly by arresting the cell cycle. A G1 arrest requires the activity of wild-type p53, as it is not observed in cells lacking functionally wild-type protein, and at least some component of S phase and G2/M arrests is also thought to be p53-dependent. p53 functions as a transcription factor which binds specific DNA sequences, and recently major downstream targets have been identified, including p21Cip1, an inhibitor of the cell cycle kinases that also blocks the replicative but not the repair function of DNA polymerase delta auxiliary factor, PCNA. Current interest focuses on developing novel cancer therapies based on our knowledge of the activity of p53 and p21Cip1 in the cell cycle.

Alternative Splicing↗

PubMed1995

Absence of p53 autoantibodies in a significant proportion of breast cancer patients.

We analysed antibodies specific for human p53 in sera from primary breast cancer patients using three different immunoassays and we related these results to the p53 level in tumour tissue detected by immunohistochemistry. Only 44% (11/25) of apparently enzyme-linked immunosorbent assay (ELISA)-positive sera were from patients with a high level of p53 protein in more than 50% of their tumour cells. Surprisingly, 36% (9/25) of the sera originated from patients with no detectable p53 protein at all. Immunoprecipitation data suggested that the reason for this discrepancy is that at least some of the antibodies detected as positive in the ELISA in these sera from patients with clinical stage I and stage II breast cancers may be induced by immunogens other than p53 protein. Many of these reactions give apparently positive signals in a variety of p53 assays, and very stringent analysis is required to avoid possible misinterpretation of these responses as a p53-specific B-cell response in human cancer patients.

Autoantibodies↗