Biomedical subjects

David P Lane

Publications and source records attributed to David P Lane.

13 recordsLinked to original sources

PubMed2003

Drug discovery and p53.

In the past two decades, the identification of commonly mutated oncogenes and tumour suppressor genes has driven an unprecedented growth in our understanding of the genetic basis of human cancer. Although oncogenes can clearly serve as classically defined drug targets whose inactivation by small molecules could place a brake on cancer cell proliferation, the restoration of mutated tumour suppressor gene activity by small molecules might appear on the surface to be unrealistic. However, there is a growing realization that many eukaryotic regulatory proteins are partially unfolded and such intrinsically disordered proteins acquire a folded structure after binding to their biological target. Molecular characterization of the p53 protein has shown that its conformational flexibility and intrinsic thermodynamic instability provide a foundation from which its conformation can be quickly post-translationally modified.

Allosteric Regulation↗

PubMed2003

Differences in the ubiquitination of p53 by Mdm2 and the HPV protein E6.

The human papillomavirus (HPV) protein E6 can promote the ubiquitination of the p53 tumour suppressor in vitro, providing an explanation for the ability of E6 to induce p53 degradation in vivo and contribute to the potential tumorigenic effect of the virus. Instead, in non-infected cells, p53 levels are primarily destabilised by the ubiquitin E3 ligase activity of the Mdm2 protein. Here we have compared the effects of E6 and Mdm2 on p53 ubiquitination in vivo. We show that whereas in the presence of Mdm2 proteasome inhibitors induce the accumulation of ubiquitinated forms of p53, this does not occur in the presence of E6. Accordingly, we confirm that the effect of E6 and p53 is independent of the six C-terminal lysine residues in p53, which have previously been described to play an important role for effective ubiquitination and degradation of 53 mediated by Mdm2. We also show that other yet unidentified residues in p53 are also susceptible to ubiquitination. These results indicate that E6 does not induce ubiquitination of p53 in the same way as Mdm2 in order to promote its degradation, suggesting important differences between the Mdm2 and E6 effects on p53 degradation.

Amino Acids↗

PubMed2003

Oligomerization of the human ARF tumor suppressor and its response to oxidative stress.

The tumor suppressor ARF plays an important role as an inhibitor of the Mdm2-mediated degradation of p53. Here we demonstrate that human ARF (p14ARF) can form homo-oligomers. The stability of the oligomers is favored by oxidizing agents in a reversible fashion and involves all three cysteine residues in p14ARF. Furthermore, the effect of p14ARF in clonogenic assays is moderately but reproducibly increased by the mutation of its cysteine residues. We also observed that altering the amino terminus of p14ARF resulted in the appearance of remarkably stable oligomers. This indicates that the amino terminus of p14ARF interferes with the ability of the protein to form multimeric complexes. These observations suggest that p14ARF activity may be linked to its oligomerization status and sensitive to the redox status of the cell.

Amino Acid Sequence↗

PubMed2003

Discovery of a novel family of CDK inhibitors with the program LIDAEUS: structural basis for ligand-induced disordering of the activation loop.

A family of 4-heteroaryl-2-amino-pyrimidine CDK2 inhibitor lead compounds was discovered with the new database-mining program LIDAEUS through in silico screening. Four compounds with IC(50) values ranging from 17 to 0.9 microM were selected for X-ray crystal analysis. Two distinct binding modes are observed, one of which resembles the hydrogen bonding pattern of bound ATP. In the second binding mode, the ligands trigger a conformational change in the activation T loop by inducing movement of Lys(33) and Asp(145) side chains. The family of molecules discovered provides an excellent starting point for the design and synthesis of tight binding inhibitors, which may lead to a new class of antiproliferative drugs.

Adenosine Triphosphate↗

PubMed2003

Exploiting the p53 pathway for cancer diagnosis and therapy.

Cancer research has reached an exciting phase of its evolution, as we now have an increasingly detailed molecular description of the genomic abnormalities and the biochemical pathways that drive the malignant progression of cancers. Investigation of the p53 tumour suppressor pathway, in particular, has become a key focus of current cancer research. This review describes how an increasing knowledge of the biochemistry of the p53 pathway is allowing us to develop novel strategies that rationally manipulate the function of this pathway in tumour cells for therapeutic benefit. Work in this field represents an outstanding illustration of how basic scientific research is being translated into clinical applications and the many challenges this involves.

Apoptosis↗

PubMed2003

The p53-Mdm2 pathway: targets for the development of new anticancer therapeutics.

The tumour suppressor p53 is at the centre of a network of regulatory pathways that guard over the continued integrity of the living cell and its progeny after exposure to different forms of stress, particularly those capable of inducing DNA damage. Tumour cells very frequently circumvent this control by disabling the function of p53, or other proteins in the p53 network, through mutation. Here we review the different therapeutic strategies that have been adopted to exploit common neoplastic aberrations in the p53 pathways. We emphasise in particular those approaches where modulation with pharmaceutical agents has already shown some promise, including pharmacological rescue of mutant p53, modulation of the protein-protein interaction between p53 and one of its negative regulators, Mdm2, as well as interference with downstream targets.

Antineoplastic Agents↗

PubMed2003

Peptidomimetic design of CDK inhibitors targeting the recruitment site of the cyclin subunit.

The recognition of cyclin-dependent kinase (CDK)/cyclin complexes by various cell-cycle regulatory proteins, including certain tumour suppressors and transcription factors, occurs at least in part through a protein-protein interaction with a binding groove on the cyclin subunit. Since CDK function is generally deregulated in tumour cells, blocking of this recruitment site prevents recognition and subsequent phosphorylation of CDK substrates and offers a therapeutic approach towards restoration of checkpoint control in transformed cells. Here we discuss the finding that peptides derived from such cyclin-interacting proteins, and rendered permeable through conjugation to cellular delivery vectors, can apparently induce tumour cells to undergo apoptosis selectively. We review the current status of 3D-structural information available on cyclin-peptide interactions and we summarise our extensive peptide structure-activity relationship studies in light of this information. We also show how a combination of molecular modelling and introduction into synthetic peptides of peptidomimetic elements, such as non-natural amino acid residues and conformational constraints, is being used hopefully to arrive at drug candidates capable of modulating CDK function in a selective mechanism-based approach rather than through ATP antagonism.

Animals↗

PubMed2002

In vitro and in vivo antitumor properties of the cyclin dependent kinase inhibitor CYC202 (R-roscovitine).

CDK2 inhibitors have been proposed as effective anti-cancer therapeutics. We show here that CYC202 (R-roscovitine) is a potent inhibitor of recombinant CDK2/cyclin E kinase activity (IC(50) = 0.10 microM) with an average cytotoxic IC(50) of 15.2 microM in a panel of 19 human tumour cell lines, and we also demonstrate selectivity for rapidly proliferating cells over non-proliferating cells. A study of the cell cycle effects of CYC202 in Lovo colorectal carcinoma cells showed that the major effect was not the predicted arrest in one part of the cycle, but rather an induction of cell death from all compartments of the cell cycle. The maximum tolerated dose given intravenously to mice was in excess of 20 mg/kg. Doses up to 2,000 mg/kg were tolerated when administered orally in mice. Following repeated intraperitoneal administration (3 times daily for 5 days) of 100 mg/kg to nude mice bearing the Lovo human colorectal tumour, CYC202 induced a significant antitumour effect with a 45% reduction in tumour growth compared to controls. A second experiment using the human uterine xenograft MESSA-DX5 treated with orally administered CYC202 (500 mg/kg 3 times daily for 4 days) also exhibited a significant reduction in the rate of growth of the tumour (62%). These data, showing enzyme and cellular potency together with antitumour activity, confirm the potential of CDK2 inhibitors such as CYC202 as anticancer drugs.

Animals↗

PubMed2002

Cell type-specific responses of human cells to inhibition of replication licensing.

Replication origins are 'licensed' for a single initiation event by loading Mcm2-7 complexes during late mitosis and G1. Licensing is blocked at other cell cycle stages by the activity of cyclin-dependent kinases and a small protein called geminin. Here, we describe the effects of over-expressing a non-degradable form of geminin in various cell lines. Geminin expression reduced the quantity of Mcm2 bound to chromatin and blocked cell proliferation. U2OS (p53+/Rb+) cells showed an early S phase arrest with high cyclin E and undetectable cyclin A levels, consistent with the activation of an intra-S checkpoint. Saos2 (p53-/Rb-) cells showed an accumulation of cells in late S and G2/M with approximately normal levels of cyclin A, consistent with loss of this intra-S phase checkpoint. Geminin also induced apoptosis in both these cell lines. In contrast, IMR90 primary fibroblasts over-expressing geminin arrested in G1 with reduced cyclin E levels and no detectable apoptosis. A 'licensing checkpoint' may therefore act in primary cells to prevent passage into S phase in the absence of sufficient origin licensing. These results suggest that inhibition of the licensing system may cause cancer-specific cell killing and therefore represent a novel anti-cancer target.

Adenoviridae↗

PubMed2002

P14ARF promotes accumulation of SUMO-1 conjugated (H)Mdm2.

p14ARF tumour suppressor stabilises and activates p53 by directly interacting with (H)Mdm2 [(human) murine double minute 2 homologue] and inhibiting its E3 ubiquitin ligase activity. Here we demonstrate that p14ARF promotes accumulation of (H)Mdm2 conjugated to the small ubiquitin-like protein SUMO-1. Mutational analysis demonstrated that the N-terminus of Mdm2 is a target for p14ARF-mediated SUMO conjugation. SUMO modification requires residues 2-14 in p14ARF that interact with (H)Mdm2 and residues 82-101 in exon 2 involved in nucleolar localisation of p14ARF. These data suggest a novel role for p14ARF as a regulator of activity of (H)Mdm2, which could be related to its tumour suppressing activities.

Animals↗

PubMed2002

'Mind the gap'.

Explore the source record for details and available documents.

Biomedical Research↗

PubMed2002

Therapeutic exploitation of the p53 pathway.

Analysis of the gene encoding p53 could serve to evaluate the effectiveness of a cancer treatment. Mutations in this gene occur in half of all human cancers, and regulation of the protein is defective in a variety of others. Novel strategies that exploit our knowledge of the function and regulation of p53 are being actively investigated. Strategies directed at treating tumours that have p53 mutations include gene therapy, viruses that only replicate in p53 deficient cells, and the search for small molecules that reactivate mutant p53. Potentiating the function of p53 in a non-genotoxic way in tumours that express wildtype protein can be achieved by inhibiting the expression and function of Mdm2 or viral oncoproteins.

Adenoviridae↗

PubMed2002

Hypophosphorylation of Mdm2 augments p53 stability.

The Mdm2 protein mediates ubiquitylation and degradation of p53 and is a key regulator of this tumor suppressor. More recently, it has been shown that Mdm2 is highly phosphorylated within its central acidic domain. In order to address the issue of how these modifications might regulate Mdm2 function, putative phosphorylation sites within this domain were substituted, individually or in pairs, with alanine residues. Mutants with serine-to-alanine substitutions between residues 244 and 260 abolished or at least reduced the capacity of Mdm2 to promote p53 degradation. In each case, loss of degradation function was independent of the ability to bind to p53 or p14ARF. Moreover, each of the Mdm2 mutants completely retained the capacity to act as a ubiquitin ligase in vivo. Thus, ubiquitylation and degradation can be uncoupled. Two-dimensional phosphopeptide mapping coupled with the use of phospho-specific antibodies revealed that Mdm2 is phosphorylated physiologically at several sites within this region, consistent with the idea that phosphorylation is important for Mdm2 activity. Strikingly, treatment of cells with ionizing radiation resulted in a significant decrease in the phosphorylation of residues that are important for p53 turnover. This hypophosphorylation preceded p53 accumulation. These findings indicate that Mdm2 contributes an additional function toward the degradation of p53 that is distinct from its ubiquitin ligase activity and is regulated by phosphorylation. Our model suggests that hypophosphorylation of Mdm2 in response to ionizing irradiation inactivates this novel function, thereby contributing to p53 stabilization.

Amino Acid Sequence↗