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D P Lane

Publications and source records attributed to D P Lane.

At least 37 records · Page 2Linked to original sources
PubMed2001

Use of peptides from p21 (Waf1/Cip1) to investigate PCNA function in Xenopus egg extracts.

Cell-free systems derived from unfertilized Xenopus eggs have been particularly informative in the study of the regulation and biochemistry of DNA replication. We have developed a Xenopus-based system to analyze proliferating cell nuclear antigen (PCNA)-specific effects on the functional properties of egg extracts. To do this, we have coupled peptides derived from p21 (Waf1/Cip1) to beads and used these to deplete PCNA from Xenopus egg extracts. The effect on various aspects of DNA replication can be analyzed after the readdition of PCNA and other purified proteins. Using this system, we have shown that replication of single-stranded M13 DNA is entirely dependent upon PCNA. By adding exogenous T7 DNA polymerase to PCNA-depleted extracts, we have uncoupled processive DNA replication from PCNA activity and so created an experimental system to analyze the dependence of postreplicative processes on PCNA function. We have shown that successful chromatin assembly is specifically dependent on PCNA. However, systems for analyzing the far more complex mechanisms required for the replication of nuclear double-stranded DNA have proved so far to be refractory to specific PCNA depletion.

Animals↗
PubMed2001

Tumour-specific therapeutic adenovirus vectors: repression of transgene expression in healthy cells by endogenous p53.

Approximately 50% of human tumours lack functional p53 suppressor protein. A promoter that is repressed by p53 in healthy cells could thus provide tumour-specific gene expression for a huge subset of tumours. In this report we describe a double recombinant adenovirus vector, 'Ad.p53R', encoding a therapeutic gene that is indirectly repressed by endogenous wild-type p53. Ad.p53R contains two independent expression cassettes; (1) the E. coli nitroreductase gene (NTR) driven by the human hsp70 promoter containing LacI binding sites (hsp70lacO-NTR) and (2) a p53-inducible lac repressor gene (tkGC3-lacI). In p53 null cells (Hep3B), Ad.p53R directed the same level of NTR expression as Ad.p53NR which lacks the tkGC3-lacI cassette. Moreover, injection of SW480 xenografts (mutated p53) with Ad.p53R resulted in a clear inhibition of growth in response to the prodrug CB1954. In cells retaining wt p53 (HepG2 and primary human endothelial cells), Ad.p53R expressed significantly less NTR (approximately 70%) than Ad.p53NR. Ad.p53R administered by i.v. injection also produced significantly less NTR than Ad.p53NR in normal tissues in vivo. Finally, adenovirus infection per se of cultured HepG2 cells at low MOI induced p53 stabilisation suggesting that adenovirus-mediated gene delivery may contribute to p53-based selectivity.

Adenoviridae↗
PubMed2000

Strategies for manipulating the p53 pathway in the treatment of human cancer.

Human cancer progression is driven in part by the mutation of oncogenes and tumour-suppressor genes which, under selective environmental pressures, give rise to evolving populations of biochemically altered cells with enhanced tumorigenic and metastatic potential. Given that human cancers are biologically and pathologically quite distinct, it has been quite surprising that a common event, perturbation of the p53 pathway, occurs in most if not all types of human cancers. The central role of p53 as a tumour-suppressor protein has fuelled interest in defining its mechanism of function and regulation, determining how its inactivation facilitates cancer progression, and exploring the possibility of restoring p53 function for therapeutic benefit. This review will highlight the key biochemical properties of p53 protein that affect its tumour-suppressor function and the experimental strategies that have been developed for the re-activation of the p53 pathway in cancers.

Animals↗
PubMed2000

Activation of p53 in cervical carcinoma cells by small molecules.

In over 90% of cervical cancers and cancer-derived cell lines, the p53 tumor suppressor pathway is disrupted by human papillomavirus (HPV). The HPV E6 protein promotes the degradation of p53 and thus inhibits the stabilization and activation of p53 that would normally occur in response to HPV E7 oncogene expression. Restoration of p53 function in these cells by blocking this pathway should promote a selective therapeutic affect. Here we show that treatment with the small molecule nuclear export inhibitor, leptomycin B, and actinomycin D leads to the accumulation of transcriptionally active p53 in the nucleus of HeLa, CaSki, and SiHa cells. Northern blot analyses showed that both actinomycin D and leptomycin B reduced the amount of HPV E6-E7 mRNA whereas combined treatment with the drugs showed almost complete disappearance of the viral mRNA. The combined treatment activated p53-dependant transcription, and increases in both p21(WAF1/CIP1) and Hdm2 mRNA were seen. The combined treatment resulted in apoptotic death in the cells, as evidenced by nuclear fragmentation and PARP-cleavage indicative of caspase 3 activity. These effects were greatly reduced by expressing a dominant negative p53 protein. The present study shows that small molecules can reactivate p53 in cervical carcinoma cells, and this reactivation is associated with an extensive biological response, including the induction of the apoptotic death of the cells.

Antibiotics, Antineoplastic↗
PubMed2000

Rapid induction of apoptosis mediated by peptides that bind initiation factor eIF4E.

Overexpression of the translation initiation factor eIF4E leads to cell transformation and occurs in a number of human cancers [1]. mRNA translation and cell growth can be regulated through the availability of eIF4E to form initiation complexes by binding to eIF4G. The availability of eIF4E is blocked through the binding of members of a family of eIF4E-binding proteins (4E-BPs) [2] [3]. Indeed, cell transformation caused by the overexpression of eIF4E can be reversed by the overexpression of 4E-BPs [4] [5] [6] [7] [8]. To study the role of eIF4E in cell transformation, we developed a series of peptides based on the conserved eIF4E-binding motifs in 4E-BPs and eIF4G [9] linked to the penetratin peptide-carrier sequence, which mediates the rapid transport of peptides across cell membranes. Surprisingly, introduction of these eIF4E-binding peptides into MRC5 cells led to rapid, dose-dependent cell death, with characteristics of apoptosis. Single alanine substitutions at key positions in the peptides impair their binding to eIF4E and markedly reduce their ability to induce apoptosis. A triple alanine substitution, which abolishes binding to eIF4E, renders the peptide unable to induce apoptosis. Our data provide strong evidence that the peptides induce apoptosis through binding to eIF4E. They do not induce apoptosis through inhibition of protein synthesis, as chemical inhibitors of translation did not induce apoptosis or affect peptide-induced cell death. Thus these new data indicate that eIF4E has a direct role in controlling cell survival that is not linked to its known role in mRNA translation.

Adaptor Proteins, Signal Transducing↗
PubMed2000

A quantitative study of the in vitro binding of the C-terminal domain of p21 to PCNA: affinity, stoichiometry, and thermodynamics.

Proliferating cell nuclear antigen (PCNA) plays an essential role in DNA replication, repair, and control of cell proliferation, and its activity can be modulated by interaction with p21(Waf1/Cip1) [Cox, L. S., (1997) Trends Cell Biol. 7, 493-497]. This protein-protein interaction provides a particularly good model target for designing therapeutic agents to treat proliferative disorders such as cancer. In this study, the formation of complexes between PCNA and peptides derived from the C-terminus of p21 has been investigated at the molecular level and quantified using a competitive PCNA binding assay and isothermal titration calorimetry (ITC). The affinity constant for the interaction between p21 (141-160) peptide and PCNA has been determined to be 1.14 x 10(7) M(-)(1), corresponding to a K(d) of 87.7 nM. Measurement of the interaction of truncation and substitution analogues based on the p21 (141-160) sequence with PCNA revealed that the N-terminal part (residues 141-152) of the above peptide is the minimum recognition motif, required for PCNA binding. Truncation of the C-terminal region p21 (153-160), though, inhibited significantly the ability of the peptides to compete with the full-length p21 (141-160) for binding to PCNA. Alanine mutation of Met 147 or Asp 149 completely abolished or significantly decreased, respectively, the level of the PCNA binding and the inhibition of SV40 DNA replication. Comparison of the data obtained by the competitive PCNA binding assay and the ITC measurements demonstrated the usefulness of this assay for screening for compounds that could modulate the PCNA-p21 interaction. Using this assay, we have screened rationally designed peptides for binding to PCNA and interruption of the PCNA-p21 (141-160) complex. As a result of this screening, we have identified a 16-residue peptide (consensus motif 1 peptide) with the following sequence: SAVLQKKITDYFHPKK. Consensus motif 1 peptide and p21 (141-160) have similar affinities for binding PCNA and abilities to inhibit in vitro replication of DNA originated from SV40. Such peptides could prove useful in assessing p21-mimetic strategies for cancer treatment.

Amino Acid Sequence↗
PubMed2000

An N-terminal p14ARF peptide blocks Mdm2-dependent ubiquitination in vitro and can activate p53 in vivo.

The p53 tumour suppressor protein is down-regulated by the action of Mdm2, which targets p53 for rapid degradation by the ubiquitin-proteasome pathway. The p14ARF protein is also a potent tumour suppressor that acts by binding to Mdm2 and blocking Mdm2-dependent p53 degradation and transcriptional silencing. We have screened a series of overlapping synthetic peptides derived from the p14ARF protein sequence and found that a peptide corresponding to the first 20 amino acids of ARF (Peptide 3) could bind human Mdm2. The binding site for Peptide 3 on Mdm2 was determined by deletion mapping and lies adjacent to the binding site of the anti-Mdm2 antibody 2A10, which on microinjection into cells can activate p53-dependent transactivation of a reporter plasmid. To determine whether Peptide 3 could similarly activate p53, we expressed a fusion of green fluorescent protein and Peptide 3 in MCF7 and U-2 OS cells and were able to demonstrate induction of p53 protein and p53-dependent transcription. Peptide 3 was able to block in vitro ubiquitination of p53 mediated by Mdm2. Small peptides which are sufficient to block degradation of p53 could provide therapeutic agents able to restore p53-dependent cell death pathways in tumours that retain wild-type p53 expression.

Amino Acid Sequence↗
PubMed2000

Mdm2 binding to a conformationally sensitive domain on p53 can be modulated by RNA.

Biochemical characterisation of the interaction of mdm2 protein with p53 protein has demonstrated that full-length mdm2 does not bind stably to p53-DNA complexes, contrasting with C-terminal truncations of mdm2 which do bind stably to p53-DNA complexes. In addition, tetrameric forms of the p53His175 mutant protein in the PAb1620+ conformation are reduced in binding to mdm2 protein. These data suggest that the mdm2 binding site in the BOX-I domain of p53 becomes concealed when either p53 binds to DNA or when the core domain of p53 is unfolded by missense mutation. This further suggests that the C-terminus of mdm2 protein contains a negative regulatory domain that affects mdm2 protein binding to a second, conformationally sensitive interaction site in the core domain of p53. We investigated whether there was a second docking site on p53 for mdm2 protein by examining the interaction of full-length mdm2 with p53 lacking the BOX-I domain. Although mdm2 protein did bind very weakly to p53 protein lacking the BOX-I domain, addition of RNA activated mdm2 protein binding to this truncated form of p53. These data provide evidence for three previously undefined regulatory stages in the p53-mdm2 binding reaction: (1) conformational changes in p53 protein due to DNA binding or point mutation conceals a secondary docking site of mdm2 protein; (2) the C-terminus of mdm2 is the primary determinant which confers this property upon mdm2 protein; and (3) mdm2 protein binding to this secondary interaction site within p53 can be stabilised by RNA.

Blotting, Western↗
PubMed2000

p53 is phosphorylated at the carboxyl terminus and promotes the differentiation of human HaCaT keratinocytes.

The p53 phosphoprotein acts as a tumor-suppressor gene product through the inhibition of cell growth and induction of apoptosis in a transcription-dependent manner. These functions require p53 activation through different biochemical postranslational modifications. Given the relevance of this protein in ultraviolet light-induced carcinogenesis, whose targets are primarily skin keratinocytes, we studied the functions of p53 in epidermal cell differentiation. We selected HaCaT cells, a human keratinocyte cell line bearing point-mutated, transcriptionally inactive, but highly stable p53, which facilitates immunochemical and biochemical analysis. In addition, a reliable in vitro differentiation system has been developed with these cells (Paramio et al. Oncogene 17:949, 1998). We report that during HaCaT differentiation there is a loss of immunoreactivity of p53 against antibodies that specifically recognize epitopes located at the carboxyl terminus of the protein. Because treatment with phosphatase restores this immunoreactivity, we conclude that p53 is phosphorylated at the carboxyl terminus during keratinocyte differentiation. This biochemical modification has been associated with the transcriptional activation of the molecule, and because p53 is involved in differentiation processes in other cell types, we investigated the potential functions of p53 during epidermal differentiation. To this end, we generated HaCaT clones expressing a murine temperature-sensitive p53 (Mp53ts) by transfection because the endogenous p53 is not functional even with phosphorylation. We characterized the expression and effects of the transfected protein in different selected clones. The ultraviolet-light response of these clones was restored, demonstrating the functionality of Mp53ts in these cells. We also observed that, with induction of differentiation, Mp53ts transfected cells differentiate faster than the parental or vector-transfected control cells, demonstrating that p53 promotes epidermal differentiation. The sustained expression of p53 in differentiating cells leads to massive cell death and detachment, a phenomenon that may be similar to epidermal terminal differentiation. In addition, we observed that the expression of p53-dependent genes such as p21waf/cip1 and mdm2 (which are known to participate in epidermal differentiation) increases during HaCaT differentiation, i.e., in a p53-independent manner.

Amino Acid Sequence↗
PubMed2000

Structure-activity relationship of truncated and substituted analogues of the intracellular delivery vector Penetratin.

Peptides derived from the third alpha-helix of the homeodomain (residues 43-58; Penetratin) of Antennapedia, a Drosophila homeoprotein, were prepared by simultaneous multiple synthesis. Sets of N- and C-terminally truncated peptides, as well as a series of alanine substitution analogues, were studied. Cell penetration assays using human cell cultures with these peptides revealed that the C-terminal segment 52Arg-Arg-Met-Lys-Trp-Lys-Lys58 of the parent sequence was necessary and sufficient for efficient cell membrane translocation. Individual Ala substitutions of the peptide's basic residues led to markedly decreased cell internalization ability, whereas replacement of hydrophobic residues was tolerated surprisingly well. Subcellular localization was seen to be affected by substitutions, with analogues being addressed preferentially to the cytosol or to the nucleus. Conformational constriction of the Penetratin sequence through placement and oxidation of flanking cysteine residues afforded a cyclic disulfide peptide which had lost most of its membrane translocation capacity.

Antennapedia Homeodomain Protein↗
PubMed2000

Defective nuclear localization of p53 protein in a Chinese hamster cell line is associated with the formation of stable cytoplasmic protein multimers in cells with gene amplification.

Many p53 functions require p53 transport into the nucleus. Mutant p53 also generally accumulates in the nucleus of transformed or neoplastic cells. However, examples of cytoplasmic accumulation of wild-type or mutant p53 have also been reported. Various explanations have been provided for defective nuclear localization. Here we propose a novel example of cytoplasmic p53 localization which occurs in cells showing gene amplification and appears to be due to the formation of stable p53 multimers. We studied a methotrexate-resistant Chinese hamster cell line (MTX M) carrying amplified dihydrofolate reductase genes and derived from a cell line with p53 nuclear accumulation. MTX M showed cytoplasmic p53 localization and, on immunoblots, several extra bands in the high molecular weight region, besides the expected 53 kDa band. p53 localization and the appearance of high molecular weight bands appeared to be correlated with the degree of DNA amplification. However, amplification of dihydrofolate reductase itself was not involved. Changing the p53 phosphorylation status quantitatively influenced the formation of high molecular weight bands. Cell fusion experiments demonstrated that p53 cytoplasmic localization in MTX M is a dominant phenotype. This result suggests that the defect causing lack of nuclear localization in this cell line does not reside in the nucleus. In the cytoplasm of MTX M and of wild-type/MTX M heterodikaryons p53 gives rise to protein complexes that are unable to re-enter the nucleus. The formation of such protein complexes is dependent on the amplification of an unknown gene product.

3T3 Cells↗
PubMed2000

Multiple C-terminal lysine residues target p53 for ubiquitin-proteasome-mediated degradation.

In normal cells, p53 is maintained at a low level by ubiquitin-mediated proteolysis, but after genotoxic insult this process is inhibited and p53 levels rise dramatically. Ubiquitination of p53 requires the ubiquitin-activating enzyme Ubc5 as a ubiquitin conjugation enzyme and Mdm2, which acts as a ubiquitin protein ligase. In addition to the N-terminal region, which is required for interaction with Mdm2, the C-terminal domain of p53 modulates the susceptibility of p53 to Mdm2-mediated degradation. To analyze the role of the C-terminal domain in p53 ubiquitination, we have generated p53 molecules containing single and multiple lysine-to-arginine changes between residues 370 and 386. Although wild-type (WT) and mutant molecules show similar subcellular distributions, the mutants display a higher transcriptional activity than WT p53. Simultaneous mutation of lysine residues 370, 372, 373, 381, 382, and 386 to arginine residues (6KR p53 mutant) generates a p53 molecule with potent transcriptional activity that is resistant to Mdm2-induced degradation and is refractory to Mdm2-mediated ubiquitination. In contrast to WT p53, transcriptional activity directed by the 6KR p53 mutant fails to be negatively regulated by Mdm2. Those differences are also manifest in HeLa cells which express the human papillomavirus E6 protein, suggesting that p53 C-terminal lysine residues are also implicated in E6-AP-mediated ubiquitination. These data suggest that p53 C-terminal lysine residues are the main sites of ubiquitin ligation, which target p53 for proteasome-mediated degradation.

Amino Acid Substitution↗
PubMed2000

Inhibitors of cyclin-dependent kinases as anti-cancer therapeutics.

Initiation, progression, and completion of the cell cycle are regulated by various cyclin-dependent kinases (CDKs), which are thus critical for cell growth. Tumour development is closely associated with genetic alteration and deregulation of CDKs and their regulators, suggesting that inhibitors of CDKs may be useful anti-cancer therapeutics. Indeed, early results suggest that transformed and normal cells differ in their requirement for e.g. cyclin/CDK2 and that it may be possible to develop novel antineoplastic agents devoid of the general host toxicity observed with conventional cystostatic drugs. Numerous active-site inhibitors of CDKs have been studied; the main limitation with these ATP antagonists is kinase specificity for CDKs. However, screening of compound collections, as well as rational design based on enzyme-ligand complex crystal structures, are now yielding pre-clinical candidates, particularly certain purine and flavonoid analogues, with impressive potency and selectivity. Natural CDK inhibitors (CKIs), e.g. the tumour suppressor gene products p16(INK4), p21(WAF1), and p27(KIP1), form the starting point for the design of mechanism-based CDK inhibitors. A number of these small proteins have been dissected and inhibitory lead peptides amenable to peptidomimetic development have been identified. Conversion of these peptides into pharmaceutically useful molecules is greatly aided by the recent elucidation of CKI/CDK crystal and solution structures. Additional interaction sites on CDKs being exploited for the purposes of inhibitor design include: phosphorylation/dephosphorylation sites, macromolecular substrate binding site, CKS regulatory subunit binding sites, cyclin-binding site, cellular localisation domain, and destruction box. Finally, progress has recently been made in the application of antisense technology in order to target CDK activity.

Amino Acid Sequence↗
PubMed1999

Biochemical characterization of different conformational states of the Sf9 cell-purified p53His175 mutant protein.

In this study, we expressed and purified the p53 mutant encoded by the His175 allele (p53His175) in a baculovirus expression system in order to study the folding and the DNA binding activity of the protein. A two-site ELISA revealed that purified p53His175 protein preferentially displayed a PAb1620 conformation, which appeared to be not sufficient to interact specifically with DNA. The cryptic DNA binding activity of this mutant was then investigated by electrophoretic mobility shift assay in the presence of anti-p53 antibodies, and shown to be refractory to significant activation by PAb421 (a potent allosteric activator of wild-type p53's DNA binding activity). Nevertheless, p53His175 DNA binding was regulated by antibodies targeting the N-terminal region of the protein. Furthermore, while the protein preferentially displayed a PAb1620 conformation, our data suggested the existence of an equilibrium between at least two folding states of the protein (PAb1620 and PAb240 conformations). A model rationalizing the conformation, antibody-interacting ability and DNA binding regulation potential of p53His175 is presented.

Antibodies, Monoclonal↗
PubMed1999

Effects on normal fibroblasts and neuroblastoma cells of the activation of the p53 response by the nuclear export inhibitor leptomycin B.

p53 tumour suppressor protein levels and p53-dependent transcriptional activity have been recently shown to increase in cells treated with leptomycin B (LMB), an inhibitor of nuclear export. Experiments presented here show that LMB treatment leads to growth arrest and a senescence-like phenotype in human normal fibroblast cultures. This effect is reversible after removal of the drug and further passage by trypsinization. Instead, LMB has a strong cytotoxic effect on human neuroblastoma cell lines even at nanomolar concentrations. In both these cell types the effects of LMB are attenuated when the activity of the endogenous wild type p53 protein is abrogated by overexpression of a dominant negative p53 mutant. We conclude that the induction of the p53 response by LMB plays an important role in the effects of this drug on cultured cells.

Antibiotics, Antineoplastic↗
PubMed1999

SUMO-1 modification activates the transcriptional response of p53.

The p53 tumour suppressor protein is regulated by ubiquitin-mediated proteasomal degradation. In normal cells p53 is constitutively ubiquitylated by the Mdm2 ubiquitin ligase. When the p53 response is activated by stress signals p53 levels rise due to inhibition of this degradative pathway. Here we show that p53 is modified by the small ubiquitin-like protein SUMO-1 at a single site, K386, in the C-terminus of the protein. Modification in vitro requires only SUMO-1, the SUMO-1 activating enzyme and ubc9. SUMO-1 and ubiquitin modification do not compete for the same lysine acceptor sites in p53. Overexpression of SUMO-1 activates the transcriptional activity of wild-type p53, but not K386R p53 where the SUMO-1 acceptor site has been mutated. The SUMO-1 modification pathway therefore acts as a potential regulator of the p53 response and may represent a novel target for the development of therapeutically useful modulators of the p53 response.

Binding Sites↗