WO1999031234A1 - Acide nucleique rad1 de l'homme, polypeptides, dosages, procedes et moyens therapeutiques - Google Patents

Acide nucleique rad1 de l'homme, polypeptides, dosages, procedes et moyens therapeutiques Download PDF

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Publication number
WO1999031234A1
WO1999031234A1 PCT/GB1998/003782 GB9803782W WO9931234A1 WO 1999031234 A1 WO1999031234 A1 WO 1999031234A1 GB 9803782 W GB9803782 W GB 9803782W WO 9931234 A1 WO9931234 A1 WO 9931234A1
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polypeptide
hradl
fragment
pcna
nucleic acid
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PCT/GB1998/003782
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English (en)
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Stephen Philip Jackson
Raimundo Freire
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Kudos Pharmaceuticals Limited
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Application filed by Kudos Pharmaceuticals Limited filed Critical Kudos Pharmaceuticals Limited
Priority to AU15721/99A priority Critical patent/AU1572199A/en
Priority to CA002313391A priority patent/CA2313391A1/fr
Priority to JP2000539134A priority patent/JP2002508180A/ja
Priority to EP98960037A priority patent/EP1037978A1/fr
Publication of WO1999031234A1 publication Critical patent/WO1999031234A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to screening methods, peptides, mimetics, and methods of use based on a newly cloned human gene ( "HRADI” ) encoding a homologue of Schizosaccharomyces pombe Radl, Saccharomyces cerevisiae Rad 17p, and Ustilago maydis Reel, and on the unexpected discovery and characterisation of an interaction between hRADl and PCNA (proliferating cell nuclear antigen) .
  • Various aspects of the present invention include those relating to the newly-cloned gene, its use, e.g. in production of the encoded polypeptide, and assays for molecules which intefere with interaction between hRadl and PCNA which, by virtue of the role of these proteins in DNA repair, cell-cycle control, and other cellular processes, have therapeutic potential.
  • Ataxia-telangiectasia is a human autosomal recessive disorder present at an incidence of around 1 in 100,000 in the general population.
  • A-T is characterised by a number of debilitating symptoms, including a progressive cerebellar degeneration, occulocutaneous telangiectasia, growth retardation, immune deficiencies and certain characteristics of premature ageing (reviewed in Jackson (1995) . Current Biology 5, 1210-1212; Meyn (1995) . Cancer Res . 55, 5991-6001; Shiloh (1995) . Eur. J. Hum . Genet . 3, 116-138) .
  • A-T patients exhibit an approximately 100-fold increased incidence of cancer, with patients being particularly predisposed to malignancies of lymphoid origin.
  • A-T heterozygotes which comprise -1% of the population, are reported to exhibit a higher incidence of breast cancer (Easton (1994) . International Journal of Radiation Biology 66, S177-S182; Meyn (1995). Cancer Res . 55, 5991-6001), although this remains controversial (Fitzgerald et al . , (1997) . Nature Genetics 15, 307-310; Bishop and Hopper (1997) . Nature Genetics 15, 226) .
  • A-T is characterised by a high degree of chromosomal instability, radio-resistant DNA synthesis, and hypersensitivity to ionising radiation (IR) and radiomimetic drugs.
  • A-T cells are defective in the radiation induced G ⁇ S, S, and G 2 -M cell cycle checkpoints that are thought to arrest the cell cycle in response to DNA damage in order to allow repair of the genome prior to DNA replication or mitosis (Beamish et al . , (1994). Radiat . Res . 138, S130-133; Beamish and Lavin (1994). Int . J. Radiat . Biol . 65, 175-184; Khanna et al . , (1995). Oncogene 11, 609-618; Barlow et al . , (1996). Cell 86, 159-171; Xu and Baltimore (1996) . Genes & Dev. 10, 2401- 2410) .
  • A-T cells exhibit deficient or severely delayed induction of the tumour suppressor protein p53 in response to IR (Kastan et al . , (1992) . Cell 71, 587-597; Khanna and Lavin (1993) . Oncogene 8, 3307-3312; Lu and Lane (1993). Cell 75, 765-778; Xu and Baltimore (1996) . Genes & Dev. 10, 2401-2410) .
  • p53 mediated transcriptional activation of p21/ AFl/CIPl and Gadd45, and the subsequent inhibition of G cyclin-dependent kinases are also defective in A-T cells following IR exposure (Artuso et al . , (1995).
  • the gene product that is defective in A-T therefore acts upstream of p53 in the IR- induced DNA damage signalling pathway.
  • ATM The gene mutated in A-T patients, termed ATM (A-T mutated) , has been mapped and its cDNA cloned (Savitsky et al . , (1995a). Science 268, 1749-1753; Savitsky et al . , (1995b). Hum . Mol . Genet . 4, 2025-2032). Sequence analyses reveal that the ATM gene encodes a -350 kDa polypeptide that is a member of the phosphatidylinositol (PI) 3-kinase family of proteins by virtue of a putative kinase domain in its carboxyl- terminal region.
  • PI phosphatidylinositol
  • PI 3-kinases such as PI 3-kinase itself, are involved in signal transduction and phosphorylate inositol lipids that act as intracellular second messengers (reviewed in Kapeller and Cantley (1994) . Bioessays 16, 565- 576) .
  • ATM bears most sequence similarity with a subset of the PI 3 -kinase protein family that comprises proteins which, like ATM, are involved in cell cycle control and/or in the detection and signalling of DNA damage (for reviews see Hunter (1995). Cell 83, 1-4; Keith and Schreiber (1995) . Science 270, 50-51; Zakian (1995) . Cell 82, 685-687; Jackson (1996) .
  • DNA-PK DNA dependent protein kinase
  • DNA-PK CS DNA dependent protein kinase catalytic subunit
  • S the set of proteins comprising S .
  • ATM and ATR have overlapping functions.
  • ATR-mediated cellular processes, pathways and effects may be modulated, so that reference to ATM should be taken also to include ATR unless context precludes.
  • the present invention relates to the identification of a novel component of the DNA replication arrest- and DNA damage-induced signalling pathway in humans and other animals.
  • the component is termed herein "hRadl", encoded by the "HRADI” gene. It is a homologue of S. pombe RAD1 .
  • the mouse homologue (“mRadl”, encoded by "MRAD1” ) has also been cloned. In therapeutic and diagnostic contexts hRadl is generally preferred for use in the present invention. However, references to “hRadl” and " HRADI” should be taken to include the possibility of use of the respective mouse homologue unless context precludes otherwise.
  • the mouse homologue may be used, for instance, in generation of appropriate animal models.
  • the present invention provides encoding nucleic acid, recombinant vectors and host cells, the encoded hRadl polypeptide, methods of making the polypeptide by expression from the encoding nucleic acid, and so on. Furthermore, the disclosure indicates how this protein functions and provides means, assays, and methods for the development of novel diagnostic, prophylactic and therapeutic agents for diseases such as cancer, infertility and degenerative disease states that may be associated with loss of function of this protein.
  • HRadl has also been shown by the inventors to interact with PCNA (see experiments described below) .
  • PCNA is a protein that interacts with a variety of other proteins and is implicated in various processes, including DNA replication, cell cycle control, nucleotide excision repair, post-replication mismatch repair, base excision repair, and apoptosis (for review, see Jonsson and Hubscher (1997) . BioEssays 19, 967-975) .
  • PCNA has been shown to interact with several other proteins, including DNA polymerase delta, DNA polymerase epsilon, replication factor C (RFC) , the nuclease Fenl, the cyclins and cyclin/cyclin- dependent protein kinase complexes that control cell cycle progression.
  • DNA polymerase delta DNA polymerase epsilon
  • RRC replication factor C
  • PCNA interacts with p21/Waf1/Cipl which is a strong inducer of Gl-phase cell cycle arrest and the protein Gadd45, which is induced in response to growth arrest and DNA damage.
  • Gadd45 Another protein with which PCNA interacts is MyDll ⁇ , a protein that is related to Gadd45 and which is induced upon terminal differentiation and apoptotic responses (for review of these various interactions with PCNA, see Jonsson and Hubscher (1997) . BioEssays 19, 967- 975)
  • hRadl or mRadl
  • PCNA may regulate the diversity of cellular processes that depend upon the ability of PCNA to interact with other proteins.
  • the interaction between hRadl and PCNA, together with the anticipated role of hRadl in DNA damage signalling, provides an indication that, particularly in response to DNA damage, hRadl directly or indirectly targets PCNA, thus causing cell cycle arrest.
  • Another possibility is that hRadl may activate PCNA, with DNA damage inhibiting such activation.
  • the present invention in various aspects provides for modulation of hRadl function and/or of interaction between hRadl and PCNA and/or other cellular components.
  • hRadl or hRadl and PCNA in screening methods and assays for agents which modulate hRadl function and/or interaction between hRadl and PCNA.
  • Such molecules may be identified by various means. For instance, information may be obtained about residues which are important for hRadl/PCNA interaction using alanine scanning and deletion analysis of hRadl and/or PCNA, and/or peptide fragments of either. When key residues are identified, computer sequence databases may be scanned for proteins including the same or similar pattern of residues, taking into account conservative variation in sequence (see below) as appropriate. Candidate molecules may then be used in one or more assays for interaction with hRadl.
  • hRadl/PCNA interaction may also be used in the design of peptide and non-peptidyl agents which modulate, particularly inhibit, such interaction as discussed further below.
  • Methods of obtaining agents able to modulate hRadl function and/or interaction between hRadl and PCNA include methods wherein a suitable end-point is used to assess interaction in the presence and absence of a test substance.
  • Assay systems may be used to determine activity of a hRadl-dependent pathway.
  • Assay systems may be used to determine activity of ATM/ATR sigalling pathways and effectors such as the p53 pathway.
  • HRadl is the human homologue of S. pombe Radl and S. cerevisiae Radl7p (as confirmed by the yeast complementation experiments described below) . These two latter factors work in concert with the S . pombe ATM homologue Rad3 and the S. cerevisiae ATM homologues Meclp and Tellp.
  • hRadl is likely to interact directly or indirectly with ATM and ATR (ATR is a human protein that is related to ATM and the yeast Meclp, Tellp and Rad3 factors (see above). According to genetic evidence, ATM and ATR have overlapping functions and so manipulation of hRadl may be used to manipulate both ATM- and ATR- dependent processes.) .
  • ATM has been shown to signal to p53 (Hawley and Friend (1996) . Genes . Dev. 10, 2383-2388; Kastan et al . , (1992). Cell 71, 587-597; Khanna and Lavin (1993) . Oncogene 8, 3307-3312; Lu and Lane (1993) . Cell 75, 765-778; Xu and Baltimore (1996) . Genes & Dev. 10, 2401-2410; Banin et al . , (1998). Science 281, 1674-1677; Canman et al . , (1998). Science 281, 1677-1679) and downstream targets of p53 (for example, Artuso et al . , (1995).
  • Radl interacts with PC ⁇ A, directly or indirectly.
  • PC ⁇ A is, as its name implies, associated with proliferating cells.
  • Such interaction (Radl/PC ⁇ A directly or via other factors) therefore provides a therapeutic target, e.g. via assays including two-hybrid screens, direct protein-protein interactions in vi tro, peptide-protein interactions in vi tro, and so on. Mimicking this reaction may be used in restraining cell growth.
  • inhibiting or perturbing this interaction may be used to prevent cells functioning in the appropriate way to DNA damage or stalled DNA replication forks.
  • inhibiting the interaction or otherwise inhibiting hRadl function may be used in radiosensitising tumours and other cells, to potentiate the killing effects of radiation and DNA damaging drugs in cancer radiotherapies and chemotherapies.
  • activators and inhibitors of HRadl activity may be identified and appropriate agents may be obtained, designed and used for any of a variety of purposes :
  • A-T Therapy Modulating hRadl to activate ATM function, or triggering ATM/ATR dependent signalling pathways, may be used in treating humans with A-T.
  • telomere length homeostasis Activators of hRadl which have an effect on ATM function may, therefore, find utility in treatment of individuals with AIDS through lengthening the telomeres of senescent lymphocytes in these individuals, thus allowing replenishment of the immune system.
  • ATM acts to regulate p53, so modulation of hRadl function will regulate p53, and may affect its interaction with one or more other components involved in the cell cycle, such as Mdm-2 (Kussi et al . , (1996). Science 274, 948-953; Picksley et al . , (1994). Oncogene 9, 2523-2529; Momand et al . , (1992). Cell 69, 1237-1245; Chen et al . , (1993). Mol . and Cell Biol . 13(7), 4107-4114 and references therein), CBP (Gu et al . , (1997). Nature 387, 819-822; Lill et al . , (1997).
  • An assay according to the present invention as discussed further below may determine the role of hRadl on any of these interactions and an agent found to be able to modulate such interaction may be used to disrupt or promote any of these interactions, e.g. in a therapeutic context.
  • telomere shortening (Metcalfe et al . , (1996). Nature
  • telomere length may be used to control regulators of hRadl which have an effect on ATM activity.
  • ATM does not appear to be part of the telomerease enzyme itself (Metcalfe et al . shows that telomerase levels are normal in A-T cells; also, there are data showing that A-T cells have somewhat shortened telomeres but do not have repressed levels of telomerase) .
  • ATM works not as part of telomerase but as part of a telomere length homeostatic mechanism. It is therefore likely that anti-hRadl drugs will work synergistically with anti-telomerase drugs.
  • Ageing A-T patients display enhanced rates of ageing, display a number of symptoms associated with increased age (neurological deterioration, cancers, immunological deficiencies etc) , and their cells show shortened lifespan in culture. Agents that modulate hRadl activity and have an effect on ATM activity may therefore be used to treat/prevent disease states associated with premature and normal ageing.
  • Drugs that modulate hRadl action may be used to treat A-T patients; treat cancer - through affecting cellular growth capacity by shortening cells telomeres; manipulate the immune system - A-T patients are somewhat immunodeficient ; treat cancer - radiosensitization of tumours etc (see below) .
  • hRadl modulators may be used to limit cell growth potential by affecting telomere length etc.
  • the linkage to p53 may allow p53 therapy, activating p53 in cancer cells, which may lead to cell growth arrest and/or cell death via apoptosis or another route.
  • hRadl activators may be used, for example, to inhibit cell proliferation by activating cell cycle checkpoint arrest in the absence of cellular damage, which may be used in the treatment of tumours, cancer, psoriasis, arteriosclerosis and other hyper-proliferative disorders.
  • Activators may be employed to activate p53 in cells without damaging the cells.
  • Cells of a patient may be treated so that normal cells (p53+) stop growing and are thus refractory to killing by administration of a drug that kills cells via interfering with cell division or DNA replication, while tumour cells (most of which are p53 negative) do not arrest and are consequently selectively killed by the aforementioned agents.
  • Cancer radiotherapy and chemotherapy may be augmented using agents in accordance with the present invention.
  • Ionising radiation (IR) and radiomimetic drugs are used commonly to treat cancers, and kill cancer cells predominantly by inflicting DNA damage.
  • Cells deficient in ATM are hypersensitive to ionising radiation and radiomimetics .
  • modulators of hRadl which inhibit the ATM pathway will hypersensitise cells to the killing effects of ionising radiation and chemotherapy.
  • S. pombe Radl and S . cerevisiae Radl7 mutants are hypersensitive to IR and other agents. Such active molecules may thus be used as adjuncts in cancer radiotherapy and chemotherapy.
  • telomere growth capacity may be modulated e.g. in treatment of cancer, ageing, and AIDS. It is established that ATM plays a crucial role in controlling the length of telomeric chromosomal ends (Metcalfe et al . , (1996). Nature Genetics 13, 350-353) . Telomeric ends in most normal cell types shorten at each cell division, and cells with excessively shortened telomeres are unable to divide. Indeed, AT cells have a limited growth capacity in vi tro (Metcalfe et al . ,
  • telomeres are thought to function as a "division counting apparatus" that limits the proliferative capacity of most normal mammalian cells. Modulators of hRadl function which inhibit ATM function may, therefore, have utility in preventing cancer progression by limiting the growth potential of cancerous or pre-cancerous cells.
  • Modulators of hRadl function which activate ATM may be used to release senescent cells from growth arrest and may thus have utility in treatments of aged individuals.
  • Modulators of hRadl function which activate ATM may, therefore, result in lengthening of the telomeres of such cells and restoring their proliferative capacity.
  • Interaction between hRadl and PCNA or other component may be inhibited by inhibition of the production of the relevant protein. For instance, production of one or more of these components may be inhibited by using appropriate nucleic acid to influence expression by antisense regulation.
  • anti-sense genes or partial gene sequences to down-regulate gene expression is now well-established. Double-stranded DNA is placed under the control of a promoter in a "reverse orientation" such that transcription of the "anti-sense" strand of the DNA yields RNA which is complementary to normal mRNA transcribed from the "sense" strand of the target gene.
  • the complementary anti-sense RNA sequence is thought then to bind with mRNA to form a duplex, inhibiting translation of the endogenous mRNA from the target gene into protein. Whether or not this is the actual mode of action is still uncertain. However, it is established fact that the technique works .
  • nucleic acid is used which on transcription produces a ribozyme, able to cut nucleic acid at a specific site - thus also useful in influencing gene expression.
  • Background references for ribozymes include
  • modulators which modulate (inhibit or potentiate) hRadl function and/or inhibit or potentiate interaction of hRadl and PCNA are provided as further aspects of the present invention.
  • the purpose of disruption, interference with or modulation of interaction between hRadl and PCNA may be to modulate any activity mediated by virtue of such interaction, as discussed above and further below.
  • hRadl (and mRadl) may function in concert with one or more additional peptides or polypeptides in addition to PCNA.
  • the present invention provides assays to detect these, for example making use of antibodies such as provided herein (or equivalents, or antisera or antibodies or monoclonal antibodies to different parts of the protein) .
  • Antibodies or other means available in the art may be used to study the purification of hRadl (or mRadl) from mammalian cell extracts. This may involve purifying or part-purifying to part or full purity and looking for associated factors, which may be cloned using protein micro-sequencing etc. Interacting molecules may be identified using, for example, a two-hybrid screen. Such other factors and/or their interactions with h/mRadl may be used to derive additional inhibitors/activators of h/mRadl-dependent signalling pathways .
  • Assays according to the present invention may be used in the identification of such additional polypeptides, for example by assaying for protein fractions that stimulate ATM and/or p53 activity.
  • the present invention also provides for the use of hRadl in identifying and/or obtaining such factors.
  • Protein or other co-factors of hRadl e.g. which enhance ATM and/or p53 activity, may be used in the design of inhibitors of this, providing another route for modulating the activity of interest. This may similarly be used to provide a route to deriving agents that activate hRadl, e.g. to affect ATM and/or p53 pathway activity.
  • nucleic acid encoding the amino acid sequence shown in SEQ ID NO. 2 for hRadl.
  • HRADI gene or "HRADI allele” includes normal alleles of the HRADI gene, and also alleles carrying one or more variations that are linked to a predisposition to a disorder as explained below. Alleles including such mutations are also known in the are as susceptibility alleles .
  • HRADI nucleic acid in accordance with the present invention includes a nucleic acid molecule which has a nucleotide sequence encoding a polypeptide which includes the amino acid sequence shown in SEQ ID NO. 2.
  • the coding sequence may be that shown in SEQ ID NO. 1 or it may be a mutant, variant, derivative or allele thereof.
  • the sequence may differ from that shown by a change which is one or more of addition, insertion, deletion and substitution of one or more nucleotides of the sequence shown. Changes to a nucleotide sequence may result in an amino acid change at the protein level, or not, as determined by the genetic code.
  • nucleic acid according to the present invention may include a sequence different from the sequence shown in SEQ ID NO. 1 yet encode a polypeptide with the same amino acid sequence.
  • the amino acid sequence of the complete hRadl polypeptide shown in SEQ ID NO. 2 consists of 282 residues.
  • the encoded polypeptide may comprise an amino acid sequence which differs by one or more amino acid residues from the amino acid sequence shown in SEQ ID NO. 2.
  • Nucleic acid encoding a polypeptide which is an amino acid sequence mutant, variant, derivative or allele of the sequence shown in SEQ ID NO. 2 is further provided by the present invention. Such polypeptides are discussed below.
  • Mutants, alleles, variants and derivatives of the specific sequences provided herein may have homology to the specific sequences in any of the terms described below in relation to use of peptides or polypeptides in different assays and other methods of the invention.
  • mouse homologue encoded by "MRAD1”
  • MRAD1 MRAD1
  • discussion in the present disclosure about uses to which "hRadl” and “HRADI” may be put should be taken to include the possibility of use of the respective mouse homologue, with reference to “mRad 1" (SEQ ID NO. 4) and “MRAD1 " (SEQ ID NO. 3) unless context precludes otherwise.
  • the present invention in various aspects also provides for modulating, interfering with or interrupting, increasing or potentiating hRadl activity and/or hRadl interacion with PCNA, using an appropriate agent .
  • the amino acid and nucleic acid sequences for PCNA are provided by the review by Jonsson and Hubscher (1997) . BioEssays 19, 967-975, and references cited therein.
  • Agents useful in accordance with the present invention may be identified by screening techniques which involve determining whether an agent under test binds or interacts with hRadl, and/or inhibits or disrupts the interaction of hRadl protein or a suitable fragment thereof with PCNA or a fragment thereof, or a suitable analogue, fragment or variant thereof.
  • Suitable fragments of hRadl or PCNA include those which include residues which interact with the counterpart protein. Smaller fragments, and analogues and variants of this fragment may similarly be employed, e.g. as identified using techniques such as deletion analysis or alanine scanning.
  • the present invention provides a peptide fragment of hRad 1 which is able to interact with PCNA and/or inhibit interaction between hRad 1 and PCNA, and provides a peptide fragment of PCNA which is able to interact with hRadl and/or inhibit interaction between PCNA and hRadl, such peptide fragments being obtainable by means of deletion analysis and/or alanine scanning of the relevant protein - making an appropriate mutation in sequence, bringing together a mutated fragment of one of the proteins with the other or a fragment thereof and determining interaction.
  • the peptide is short, as discussed below, and may be a minimal portion that is able to interact with the relevant counterpart protein and/or inhibit the relevant interaction.
  • One class of agents that can be used to affect hRadl activity and/or disrupt the interaction of hRadl and PCNA are peptides based on the sequence motifs of hRadl or PCNA that interact with counterpart PCNA or hRadl (as discussed already above) .
  • Such peptides tend to be short, and may be about 40 amino acids in length or less, preferably about 35 amino acids in length or less, more preferably about 30 amino acids in length, or less, more preferably about 25 amino acids or less, more preferably about 20 amino acids or less, more preferably about 15 amino acids or less, more preferably about 10 amino acids or less, or 9, 8, 7, 6, 5 or less in length.
  • the present invention also encompasses peptides which are sequence variants or derivatives of a wild type hRadl or PCNA sequence, but which retain ability to interact with PCNA or hRadl (respectively, as the case may be) and/or ability to modulate interaction between hRadl and PCNA.
  • a peptide or polypeptide may include an amino acid sequence which differs by one or more amino acid residues from the wild-type amino acid sequence, by one or more of addition, insertion, deletion and substitution of one or more amino acids.
  • variants, derivatives, alleles and mutants are included.
  • the amino acid sequence shares homology with a fragment of the relevant hRadl or PCNA fragment sequence, preferably at least about 30%, or 40%, or 50%, or 60%, or 70%, or 75%, or 80%, or 85%, 90% or 95% homology.
  • a peptide fragment of hRadl or PCNA may include 1, 2, 3, 4, 5, greater than 5, or greater than 10 amino acid alterations such as substitutions with respect to the wild-type sequence.
  • a derivative of a peptide for which the specific sequence is disclosed herein may be in certain embodiments the same length or shorter than the specific peptide.
  • the peptide sequence or a variant thereof may be included in a larger peptide, as discussed above, which may or may not include an additional portion of hRadl or PCNA.
  • 1, 2, 3, 4 or 5 or more additional amino acids, adjacent to the relevant specific peptide fragment in hRadl or PCNA, or heterologous thereto may be included at one end or both ends of the peptide.
  • homology at the amino acid level is generally in terms of amino acid similarity or identity. Similarity allows for "conservative variation”, i.e. substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine .
  • Similarity may be as defined and determined by the TBLASTN or other BLAST program, of Altschul et al . , (1990) J. Mol . Biol . 215, 403-10, which is in standard use in the art, or, and this may be preferred, either of the standard programs BestFit and GAP, which are part of the Wisconsin Package, Version 8, September 1994, (Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA, Wisconsin 53711) . BestFit makes an optimal alignment of the best segment of similarity between two sequences. Optimal alignments are found by inserting gaps to maximize the number of matches using the local homology algorithm of Smith and Waterman
  • Homology may be over the full-length of the relevant peptide or over a contiguous sequence of about 5, 10, 15, 20, 25, 30, 35, 50, 75, 100 or more amino acids, compared with the relevant wild-type amino acid sequence.
  • variant peptide sequences and peptide and non- peptide analogues and mimetics may be employed, as discussed further below.
  • a substance which may be a single molecule or a composition including two or more components, which includes a peptide fragment of hRadl, a peptide consisting essentially of such a sequence, a peptide including a variant, derivative or analogue sequence, or a non-peptide analogue or mimetic which has the ability to modulate hRadl activity, and/or interact with hRadl or PCNA and/or modulate, disrupt or interfere with interaction between hRadl and PCNA, directly or indirectly via one or more factors .
  • Variants include peptides in which individual amino acids can be substituted by other amino acids which are closely related as is understood in the art and indicated above.
  • Non-peptide mimetics of peptides are discussed further below.
  • a peptide according to the present invention and for use in various aspects of the present invention may include or consist essentially of a fragment of hRadl or PCNA as disclosed. Where one or more additional amino acids are included, such amino acids may be from hRadl or PCNA or may be heterologous or foreign to hRadl or PCNA. A peptide may also be included within a larger fusion protein, particularly where the peptide is fused to a non-hRadl or PCNA (i.e. heterologous or foreign) sequence, such as a polypeptide or protein domain.
  • the invention also includes derivatives of the peptides, including the peptide linked to a coupling partner, e.g. an effector molecule, a label, a drug, a toxin and/or a carrier or transport molecule, and/or a targeting molecule such as an antibody or binding fragment thereof or other ligand.
  • a coupling partner e.g. an effector molecule, a label, a drug, a toxin and/or a carrier or transport molecule, and/or a targeting molecule such as an antibody or binding fragment thereof or other ligand.
  • a coupling partner e.g. an effector molecule, a label, a drug, a toxin and/or a carrier or transport molecule, and/or a targeting molecule such as an antibody or binding fragment thereof or other ligand.
  • the carrier molecule is a 16 aa peptide sequence derived from the homeodomain of Antennapedia (e.g. as sold under the name "Pe
  • Peptides may be generated wholly or partly by chemical synthesis.
  • the compounds of the present invention can be readily prepared according to well-established, standard liquid or, preferably, solid-phase peptide synthesis methods, general descriptions of which are broadly available (see, for example, in J.M. Stewart and J.D. Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company, Rockford, Illinois (1984) , in M. Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis, Springer Verlag, New York (1984); and Applied Biosystems 430A Users Manual, ABI Inc., Foster City, California) , or they may be prepared in solution, by the liquid phase method or by any combination of solid-phase, liquid phase and solution chemistry, e.g. by first completing the respective peptide portion and then, if desired and appropriate, after removal of any protecting groups being present, by introduction of the residue X by reaction of the respective carbonic or sulfonic acid or a reactive derivative thereof.
  • Another convenient way of producing a peptidyl molecule according to the present invention is to express nucleic acid encoding it, by use of nucleic acid in an expression system.
  • the present invention also provides in various aspects nucleic acid encoding the peptides of the invention, which may be used for production of the encoded peptide.
  • nucleic acid is provided as an isolate, in isolated and/or purified form, or free or substantially free of material with which it is naturally associated, such as free or substantially free of nucleic acid flanking the gene in the human genome, except possibly one or more regulatory sequence (s) for expression.
  • Nucleic acid may be wholly or partially synthetic and may include genomic DNA, cDNA or RNA. Where nucleic acid according to the invention includes RNA, reference to the sequence shown should be construed as encompassing reference to the RNA equivalent, with U substituted for T.
  • Nucleic acid sequences encoding a polypeptide or peptide in accordance with the present invention can be readily prepared by the skilled person using the information and references contained herein and techniques known in the art (for example, see Sambrook, Fritsch and Maniatis, "Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), and Ausubel et al . , Current Protocols in Molecular Biology, John Wiley and Sons, (1992)), given the nucleic acid sequence and clones available. These techniques include (i) the use of the polymerase chain reaction (PCR) to amplify samples of such nucleic acid, e.g. from genomic sources, (ii) chemical synthesis, or (iii) preparing cDNA sequences.
  • PCR polymerase chain reaction
  • DNA encoding hRadl or PCNA fragments may be generated and used in any suitable way known to those of skill in the art, including by taking encoding DNA, identifying suitable restriction enzyme recognition sites either side of the portion to be expressed, and cutting out said portion from the DNA. The portion may then be operably linked to a suitable promoter in a standard commercially available expression system. Another recombinant approach is to amplify the relevant portion of the DNA with suitable PCR primers. Modifications to the relevant sequence may be made, e.g. using site directed mutagenesis, to lead to the expression of modified peptide or to take account of codon preference in the host cells used to express the nucleic acid.
  • the sequences may be incorporated in a vector having one or more control sequences operably linked to the nucleic acid to control its expression.
  • the vectors may include other sequences such as promoters or enhancers to drive the expression of the inserted nucleic acid, nucleic acid sequences so that the polypeptide or peptide is produced as a fusion and/or nucleic acid encoding secretion signals so that the polypeptide produced in the host cell is secreted from the cell.
  • Polypeptide can then be obtained by transforming the vectors into host cells in which the vector is functional, culturing the host cells so that the polypeptide is produced and recovering the polypeptide from the host cells or the surrounding medium.
  • Prokaryotic and eukaryotic cells are used for this purpose in the art, including strains of E. coli, yeast, and eukaryotic cells such as COS or CHO cells .
  • the present invention also encompasses a method of making a polypeptide or peptide (as disclosed) , the method including expression from nucleic acid encoding the polypeptide or peptide (generally nucleic acid according to the invention) .
  • This may conveniently be achieved by growing a host cell in culture, containing such a vector, under appropriate conditions which cause or allow expression of the polypeptide.
  • Polypeptides and peptides may also be expressed in in vitro systems, such as reticulocyte lysate.
  • Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, eukaryotic cells such as mammalian and yeast, and baculovirus systems.
  • Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, COS cells and many others.
  • a common, preferred bacterial host is E. coli
  • Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • Vectors may be plasmids, viral e.g. 'phage, or phagemid, as appropriate.
  • plasmids viral e.g. 'phage, or phagemid, as appropriate.
  • Many known techniques and protocols for manipulation of nucleic acid for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, Ausubel et al . eds., John Wiley & Sons, 1992.
  • a further aspect of the present invention provides a host cell containing heterologous nucleic acid as disclosed herein.
  • the nucleic acid of the invention may be integrated into the genome (e.g. chromosome) of the host cell. Integration may be promoted by inclusion of sequences which promote recombination with the genome, in accordance with standard techniques.
  • the nucleic acid may be on an extra-chromosomal vector within the cell, or otherwise identifiably heterologous or foreign to the cell.
  • a still further aspect provides a method which includes introducing the nucleic acid into a host cell.
  • the introduction which may (particularly for in vi tro introduction) be generally referred to without limitation as "transformation", may employ any available technique.
  • suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia or, for insect cells, baculovirus.
  • suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage.
  • direct injection of the nucleic acid could be employed.
  • Marker genes such as antibiotic resistance or sensitivity genes may be used in identifying clones containing nucleic acid of interest, as is well known in the art.
  • the introduction may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells (which may include cells actually transformed although more likely the cells will be descendants of the transformed cells) under conditions for expression of the gene, so that the encoded polypeptide (or peptide) is produced. If the polypeptide is expressed coupled to an appropriate signal leader peptide it may be secreted from the cell into the culture medium.
  • a polypeptide or peptide may be isolated and/or purified from the host cell and/or culture medium, as the case may be, and subsequently used as desired, e.g. in the formulation of a composition which may include one or more additional components, such as a pharmaceutical composition which includes one or more pharmaceutically acceptable excipients, vehicles or carriers (e.g. see below).
  • nucleic acid encoding a peptidyl molecule according to the present invention may take place in vivo by way of gene therapy, to affect, disrupt or interfere with hRadl function, such as interaction between hRadl and PCNA.
  • a host cell containing nucleic acid according to the present invention may be comprised (e.g. in the soma) within an organism which is an animal, particularly a mammal, which may be human or non-human, such as rabbit, guinea pig, rat, mouse or other rodent, cat, dog, pig, sheep, goat, cattle or horse, or which is a bird, such as a chicken. Genetically modified or transgenic animals or birds comprising such a cell are also provided as further aspects of the present invention.
  • mice may be used to test for radiosensitivity, enhanced cancer predisposition, genomic instability, impaired p53 induction, infertility, immune system dysfunction and/or neurodegenerative features (Barlow et al . , (1996). Cell 86, 159-171; Xu and Baltimore (1996). Genes & Dev. 10, 2401-2410) .
  • vi tro that is within host cells or in cell-free systems.
  • those cells or cells of the same or similar type may be grafted into an appropriate host animal for in vivo testing.
  • hRadl function or activity may be measured in an animal system such as a tumour model, e.g. involving a xenograft, relying on active hRadl.
  • the animal may be subject to radio- or chemo-therapy and a test substance administered.
  • An augmentation of the reaction in the animal to the radio- or chemo-therapy may be indicative of an appropriate effect.
  • hRadl function may be assayed indirectly by consideration of ATM and/or p53 pathway activity.
  • Suitable screening methods are conventional in the art. They include techniques such as radioimmunosassay, scintillation proximetry assay and ELISA methods. For instance, in assaying for substances able to modulate an interaction between proteins, one of the relevant proteins or a fragment, or an analogue, derivative, variant or functional mimetic thereof, is immobilised whereupon the other is applied in the presence of the agents under test.
  • a scintillation proximetry assay a biotinylated protein fragment may be bound to streptavidin coated scintillant - impregnated beads (produced by Amersham) . Binding of radiolabelled peptide is then measured by determination of radioactivity induced scintillation as the radioactive peptide binds to the immobilized fragment. Agents which intercept this are thus inhibitors of the interaction.
  • the present invention provides an assay method for a substance with ability to modulate, e.g. disrupt or interfere with interaction between hRadl and PCNA, the method including : (a) bringing into contact a substance according to the invention including a peptide fragment of hRadl, or a derivative, variant or analogue thereof as disclosed, a substance including the relevant fragment of PCNA or a variant, derivative or analogue thereof.
  • a test compound which disrupts, reduces, interferes with or wholly or partially abolishes interaction between said substances e.g. including a fragment of either protein
  • said substances e.g. including a fragment of either protein
  • which may modulate hRadl and/or PCNA activity may thus be identified.
  • Agents which increase or potentiate interaction between the two substances may be identified using conditions which, in the absence of a positively-testing agent, prevent the substances interacting.
  • Another general aspect of the present invention provides an assay method for a substance able to interact with the relevant region of hRadl or PCNA as the case may be, the method including:
  • a test compound found to interact with the relevant portion of hRadl may be tested for ability to modulate, e.g. disrupt or interfere with, hRadl interaction with PCNA and/or ability to affect p53 and/or ATM activity or other activity mediated by hRad las discussed already above.
  • test compound found to interact with the relevant portion of PCNA may be tested for abiliy to modulate, e.g. disrupt or interfere with, hRadl activity and/or hRadl interaction with PCNA and/or ability to affect ATM and/or p53 activity or other activity mediated by hRadl and/or PCNA as discussed elsewhere herein.
  • Another general aspect of the present invention provides an assay method for a substance able to affect hRadl activity, the method including:
  • hRadl activity may be determined in the presence and absence of PCNA to allow for an effect of a test compound on activity to be attributed to an effect on interaction between hRadl and PCNA, as disclosed.
  • hRadl may interact with PCNA indirectly and/or interact with one or more other components of interest in the ATM and/or p53 pathway or other pathway of interest to DNA repair and/or cell cycle control, so that references to PCNA in relation to assays and other aspects of the present invention may equally be taken to refer to one or more of such other components .
  • p53 activities which may be determined include induction of expression of a protein such as p21 (Wafl/Cipl) , cellular sensitivity to ionizing radiation, p53 -induced apoptosis activity, p53 -induced anti-proliferative activity, p53- induced senescence of cells.
  • a protein such as p21 (Wafl/Cipl)
  • cellular sensitivity to ionizing radiation p53 -induced apoptosis activity
  • p53 -induced anti-proliferative activity p53-induced induced senescence of cells.
  • Preliminary assays in vi tro may be followed by, or run in parallel with, in vivo assays.
  • Performance of an assay method according to the present invention may be followed by isolation and/or manufacture and/or use of a compound, substance or molecule which tests positive for ability to modulate hRadl activity and/or modulate interaction between hRadl and PCNA and/or modulate ATM or p53 activity or any hRadl-, ATM- and/or p53- mediated activity.
  • the precise format of an assay of the invention may be varied by those of skill in the art using routine skill and knowledge. For example, interaction between substances may be studied in vi tro by labelling one with a detectable label and bringing it into contact with the other which has been immobilised on a solid support.
  • Suitable detectable labels, especially for peptidyl substances include 35 S-methionine which may be incorporated into recombinantly produced peptides and polypeptides.
  • Recombinantly produced peptides and polypeptides may also be expressed as a fusion protein containing an epitope which can be labelled with an antibody.
  • the protein which is immobilized on a solid support may be immobilized using an antibody against that protein bound to a solid support or via other technologies which are known per se .
  • a preferred in vi tro interaction may utilise a fusion protein including glutathione-S-transferase (GST) . This may be immobilized on glutathione agarose beads.
  • GST glutathione-S-transferase
  • a test compound can be assayed by determining its ability to diminish the amount of labelled peptide or polypeptide which binds to the immobilized GST-fusion polypeptide. This may be determined by fractionating the glutathione-agarose beads by SDS- polyacrylamide gel electrophoresis.
  • the beads may be rinsed to remove unbound protein and the amount of protein which has bound can be determined by counting the amount of label present in, for example, a suitable scintillation counter.
  • An assay according to the present invention may also take the form of an in vivo assay.
  • the in vivo assay may be performed in a cell line such as a yeast strain or mammalian cell line in which the relevant polypeptides or peptides are expressed from one or more vectors introduced into the cell.
  • test compound to modulate interaction between hRadl and another protein, such as PCNA, may be determined using a so-called two-hybrid assay.
  • a polypeptide or peptide containing a fragment of hRadl or PCNA as the case may be, or a peptidyl analogue or variant thereof as disclosed may be fused to a DNA binding domain such as that of the yeast transcription factor GAL 4.
  • the GAL 4 transcription factor includes two functional domains . These domains are the DNA binding domain (GAL4DBD) and the GAL4 transcriptional activation domain (GAL4TAD) .
  • GAL4DBD DNA binding domain
  • GAL4TAD GAL4 transcriptional activation domain
  • interaction of the polypeptides or peptides may be measured by the use of a reporter gene probably linked to a GAL 4 DNA binding site which is capable of activating transcription of said reporter gene.
  • This assay format is described by Fields and Song, 1989, Nature 340; 245-246. This type of assay format can be used in both mammalian cells and in yeast. Other combinations of DNA binding domain and transcriptional activation domain are available in the art and may be preferred, such as the LexA DNA binding domain and the VP60 transcriptional activation domain .
  • the hRadl or PCNA polypeptide or peptide may be employed as a fusion with (e.g.) the LexA DNA binding domain, and the counterpart
  • a third expression cassette which may be on a separate expression vector, from which a peptide or a library of peptides of diverse and/or random sequence may be expressed.
  • a reduction in reporter gene expression results from the presence of a peptide which disrupts the hRadl/PCNA (for example) interaction, which interaction is required for transcriptional activation of the ⁇ - galactosidase gene.
  • a test substance is not peptidyl and may not be expressed from encoding nucleic acid within a said third expression cassette, a similar system may be employed with the test substance supplied exogenously.
  • test substance or compound which may be added to an assay of the invention will normally be determined by trial and error depending upon the type of compound used. Typically, from about 0.001 nM to ImM or more concentrations of putative inhibitor compound may be used, for example from 0.01 nM to lOO ⁇ M, e.g. 0.1 to 50 ⁇ M, such as about 10 ⁇ M. Greater concentrations may be used when a peptide is the test substance. Even a molecule which has a weak effect may be a useful lead compound for further investigation and development .
  • Compounds which may be used may be natural or synthetic chemical compounds used in drug screening programmes. Extracts of plants which contain several characterised or uncharacterised components may also be used.
  • Combinatorial library technology provides an efficient way of testing a potentially vast number of different substances for ability to modulate an interaction with and/or activity of a polypeptide.
  • Such libraries and their use are known in the art, for all manner of natural products, small molecules and peptides, among others.
  • the use of peptide libraries may be preferred in certain circumstances.
  • Antibodies directed to a site on hRadl form a further class of putative inhibitor compounds, which also includes antibodies against a site on another polypeptide or peptide which interacts with hRadl (e.g. PCNA).
  • Candidate inhibitor antibodies may be characterised and their binding regions determined to provide single chain antibodies and fragments thereof which are responsible for disrupting the interaction.
  • Antibodies may be obtained using techniques which are standard in the art . Methods of producing antibodies include immunising a mammal (e.g. mouse, rat, rabbit, horse, goat, sheep or monkey) with the protein or a fragment thereof. Antibodies may be obtained from immunised animals using any of a variety of techniques known in the art, and screened, preferably using binding of antibody to antigen of interest. For instance, Western blotting techniques or immunoprecipitation may be used (Armitage et al . , (1992) Nature 357, 80-82) . Isolation of antibodies and/or antibody- producing cells from an animal may be accompanied by a step of sacrificing the animal.
  • an antibody specific for a protein may be obtained from a recombinantly produced library of expressed immunoglobulin variable domains, e.g. using lambda bacteriophage or filamentous bacteriophage which display functional immunoglobulin binding domains on their surfaces; for instance see WO92/01047.
  • the library may be naive, that is constructed from sequences obtained from an organism which has not been immunised with any of the proteins (or fragments) , or may be one constructed using sequences obtained from an organism which has been exposed to the antigen of interest.
  • Antibodies according to the present invention may be modified in a number of ways. Indeed the term “antibody” should be construed as covering any binding substance having a binding domain with the required specificity. Thus the invention covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including synthetic molecules and molecules whose shape mimicks that of an antibody enabling it to bind an antigen or epitope.
  • Example antibody fragments capable of binding an antigen or other binding partner are the Fab fragment consisting of the VL, VH, Cl and CHI domains; the Fd fragment consisting of the VH and CHI domains; the Fv fragment consisting of the VL and VH domains of a single arm of an antibody; the dAb fragment which consists of a VH domain; isolated CDR regions and F(ab')2 fragments, a bivalent fragment including two Fab fragments linked by a disulphide bridge at the hinge region. Single chain Fv fragments are also included.
  • a hybridoma producing a monoclonal antibody according to the present invention may be subject to genetic mutation or other changes. It will further be understood by those skilled in the art that a monoclonal antibody can be subjected to the techniques of recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody. Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the complementarity determining regions (CDRs) , of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin. See, for instance, EP184187A, GB 2188638A or EP-A-0239400. Cloning and expression of chimeric antibodies are described in EP-A- 0120694 and EP-A-0125023.
  • Hybridomas capable of producing antibody with desired binding characteristics are within the scope of the present invention, as are host cells, eukaryotic or prokaryotic, containing nucleic acid encoding antibodies (including antibody fragments) and capable of their expression.
  • the invention also provides methods of production of the antibodies including growing a cell capable of producing the antibody under conditions in which the antibody is produced, and preferably secreted.
  • the reactivities of antibodies on a sample may be determined by any appropriate means. Tagging with individual reporter molecules is one possibility.
  • the reporter molecules may directly or indirectly generate detectable, and preferably measurable, signals.
  • the linkage of reporter molecules may be directly or indirectly, covalently, e.g. via a peptide bond or non-covalently. Linkage via a peptide bond may be as a result of recombinant expression of a gene fusion encoding antibody and reporter molecule.
  • the mode of determining binding is not a feature of the present invention and those skilled in the art are able to choose a suitable mode according to their preference and general knowledge.
  • Antibodies may also be used in purifying and/or isolating a polypeptide or peptide according to the present invention, for instance following production of the polypeptide or peptide by expression from encoding nucleic acid therefor.
  • Antibodies may be useful in a therapeutic context (which may include prophylaxis) to disrupt hRadl/PCNA interaction with a view to inhibiting their activity.
  • Antibodies can for instance be micro-injected into cells, e.g. at a tumour site, subject to radio- and/or chemo-therapy (as discussed already above) .
  • Antibodies may be employed in accordance with the present invention for other therapeutic and non-therapeutic purposes which are discussed elsewhere herein.
  • candidate inhibitor compounds may be based on modelling the 3-dimensional structure of a polypeptide or peptide fragment and using rational drug design to provide potential inhibitor compounds with particular molecular shape, size and charge characteristics.
  • a compound found to have the ability to affect hRadl activity, such as via interaction with PCNA and/or via ATM and/or p53 activity has therapeutic and other potential in a number of contexts, as discussed.
  • such a compound may be used in combination with any other active substance, e.g. for anti-tumour therapy another anti- tumour compound or therapy, such as radiotherapy or chemotherapy.
  • the assay of the invention when conducted in vivo, need not measure the degree of modulation of interaction between, for example, hRadl and PCNA (or appropriate fragment, variant or derivative thereof) or of modulation of ATM and/or p53 activity caused by the compound being tested.
  • the effect on DNA repair, homologous recombination, cell viability, cell killing (e.g. in the presence and absence of radio- and/or chemo-therapy) , and so on, may be measured. It may be that such a modified assay is run in parallel with or subsequent to the main assay of the invention in order to confirm that any such effect is as a result of the modulation of hRadl function, such as inhibition of interaction between hRadl and PCNA, caused by said inhibitor compound and not merely a general toxic effect.
  • an agent identified using one or more primary screens e.g. in a cell-free system as having ability to interact with hRadl and/or PCNA and/or modulate activity of hRadl, e.g. via ATM and/or p53 activity, may be assessed further using one or more secondary screens.
  • a secondary screen may involve testing for cellular radiosensitisation and/or sensitisation to radiomimetic drugs, effect on chromosome telomere length, direct killing of cells whose survival is dependent on Radl function, inducing or preventing cell -cycle arrest following irradiation or other cellular insult, an effect of p53 induction following ionising radiation or other cellular insult, or induction of p21 or other downstream p53 target .
  • the substance or agent may be investigated further. Furthermore, it may be manufactured and/or used in preparation, i.e. manufacture or formulation, of a composition such as a medicament, pharmaceutical composition or drug. These may be administered to individuals, e.g. for any of the purposes discussed elsewhere herein.
  • the agent may be peptidyl, e.g. a peptide which includes a sequence as recited above, or may be a functional analogue of such a peptide.
  • the expression "functional analogue” relates to peptide variants or organic compounds having the same functional activity as the peptide in question, which may interfere with the interaction between hRad 1 and another cellular component such as PCNA.
  • analogues include chemical compounds which are modelled to resemble the three dimensional structure of the relevant domain in the contact area, and in particular the arrangement of the key amino acid residues as they appear in in the protein.
  • the present invention provides the use of the above substances in methods of designing or screening for mimetics of the substances.
  • the present invention provides a method of designing mimetics of hRadl having the biological activity of hRadl, of hRadl or PCNA binding or inhibition, the activity of allosteric inhibition of hRadl and/or the activity of modulating, e.g. inhibiting, hRadl interaction with another cellular component such as PCNA, said method comprising:
  • Suitable modelling techniques are known in the art. This includes the design of so-called “mimetics” which involves the study of the functional interactions fluorogenic oligonucleotide the molecules and the design of compounds which contain functional groups arranged in such a manner that they could reproduced those interactions.
  • the designing of mimetics to a known pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a "lead" compound. This might be desirable where the active compound is difficult or expensive to synthesise or where it is unsuitable for a particular method of administration, e.g. peptides are not well suited as active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal. Mimetic design, synthesis and testing may be used to avoid randomly screening large number of molecules for a target property.
  • the pharmacophore Once the pharmacophore has been found, its structure is modelled to according its physical properties, e.g. stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g. spectroscopic techniques, X-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modelling process. In a variant of this approach, the three-dimensional structure of the ligand and its binding partner are modelled. This can be especially useful where the ligand and/or binding partner change conformation on binding, allowing the model to take account of this the design of the mimetic.
  • a template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted.
  • the template molecule and the chemical groups grafted on to it can conveniently be selected so that the mimetic is easy to synthesise, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound.
  • the mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it. Further optimisation or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.
  • the mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it. Further optimisation or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.
  • the present invention further provides the use of a peptide which includes a sequence as disclosed, or a derivative, active portion, analogue, variant or mimetic, thereof able to interact with hRadl or PCNA and/or modulate, e.g. inhibit, interaction between hRadl and another cellular component such as PCNA and/or modulate, e.g inhibit, hRadl activity, e.g. ATM and/or p53 activity, in screening for a substance able to interact with hRadl and/or other cellular component which interacts with hRadl, such as PCNA, and/or modulate, e.g. inhibit, interaction between hRadl and such other cellular component, and/or inhibit hRadl activity.
  • a peptide which includes a sequence as disclosed, or a derivative, active portion, analogue, variant or mimetic, thereof able to interact with hRadl or PCNA and/or modulate, e
  • such a substance, e.g. inhibitor, according to the present invention is provided in an isolated and/or purified form, i.e. substantially pure. This may include being in a composition where it represents at least about 90% active ingredient, more preferably at least about 95%, more preferably at least about 98%. Such a composition may, however, include inert carrier materials or other pharmaceutically and physiologicaly acceptable excipients. As noted below, a composition according to the present invention may include in addition to an inhibitor compound as disclosed, one or more other molecules of therapeutic use, such as an anti-tumour agent.
  • the present invention extends in various aspects not only to a substance identified as a modulator of hRadl and PCNA interaction and/or hRadl- mediated activity, property or pathway, such as an activity, property or pathway mediated via ATM or p53 , in accordance with what is disclosed herein, but also a pharmaceutical composition, medicament, drug or other composition comprising such a substance, a method comprising administration of such a composition to a patient, e.g. for a purpose discussed elsewhere herein, which may include preventative treatment, use of such a substance in manufacture of a composition for administration, e.g. for a purpose discussed elsewhere herein, and a method of making a pharmaceutical composition comprising admixing such a substance with a pharmaceutically acceptable excipient, vehicle or carrier, and optionally other ingredients.
  • a substance according to the present invention such as an inhibitor of hRadl and PCNA interaction may be provided for use in a method of treatment of the human or animal body by therapy which affects an hRadl-mediated activity in cells, e.g. tumour cells.
  • therapy which affects an hRadl-mediated activity in cells, e.g. tumour cells.
  • Other purposes of a method of treatment employing a substance in accordance with the present invention are discussed elsewhere herein.
  • the invention further provides a method of modulating an hRadl activity, such as mediated via an ATM and/or p53 pathway, e.g. for a purpose discussed elsewhere herein, which includes administering an agent which modulates, inhibits or blocks the interaction of hRadl with another cellular component, such as PCNA, such a method being useful in treatment where such modulation, inhibition or blocking is desirable, or an agent which increase, potentiates or strengthens interaction of hRadl with such cellular component, useful in treatment where this is desirable.
  • the invention further provides a method of treatment which includes administering to a patient an agent which interferes with the interaction of hRadl and another cellular component, such as PCNA. Exemplary purposes of such treatment are discussed elsewhere herein.
  • administration is preferably in a "prophylactically effective amount” or a "therapeutically effective amount” as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual.
  • prophylaxis may be considered therapy
  • the actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated.
  • compositions may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
  • Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention may include, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous or intravenous.
  • compositions for oral administration may be in tablet, capsule, powder or liquid form.
  • a tablet may include a solid carrier such as gelatin or an adjuvant.
  • Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
  • the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection.
  • Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
  • Liposomes particularly cationic liposomes, may be used in carrier formulations.
  • the agent may be administered in a localised manner to a tumour site or other desired site or may be delivered in a manner in which it targets tumour or other cells.
  • Targeting therapies may be used to deliver the active agent more specifically to certain types of cell, by the use of targeting systems such as antibody or cell specific ligands. Targeting may be desirable for a variety of reasons, for example if the agent is unacceptably toxic, or if it would otherwise require too high a dosage, or if it would not otherwise be able to enter the target cells.
  • these agents may be produced in the target cells by expression from an encoding gene introduced into the cells, eg in a viral vector (a variant of the VDEPT technique - see below) .
  • the vector may targeted to the specific cells to be treated, or it may contain regulatory elements which are switched on more or less selectively by the target cells.
  • the agent e.g. small molecule, mimetic
  • the agent may be administered in a precursor form, for conversion to the active form by an activating agent produced in, or targeted to, the cells to be treated.
  • an activating agent produced in, or targeted to, the cells to be treated.
  • This type of approach is sometimes known as ADEPT or VDEPT, the former involving targeting the activator to the cells by conjugation to a cell-specific antibody, while the latter involves producing the activator, e.g. an enzyme, in a vector by expression from encoding DNA in a viral vector (see for example, EP-A-415731 and WO 90/07936) .
  • An agent may be administered in a form which is inactive but which is converted to an active form in the body.
  • the agent may be phosphorylated (e.g. to improve solubility) with the phosphate being cleaved to provide an active form of the agent in the body.
  • a composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated, such as cancer, virus infection or any other condition in which a hRadl- mediated effect is desirable.
  • Nucleic acid according to the present invention encoding a polypeptide or peptide able to modulate, e.g. interfere with, hRadl, and/or induce or modulate activity of other hRadl- mediated cellular pathway or function, may be used in methods of gene therapy, for instance in treatment of individuals, e.g. with the aim of preventing or curing (wholly or partially) a disorder or for another purpose as discussed elsewhere herein.
  • Vectors such as viral vectors have been used in the prior art to introduce nucleic acid into a wide variety of different target cells. Typically the vectors are exposed to the target cells so that transfection can take place in a sufficient proportion of the cells to provide a useful therapeutic or prophylactic effect from the expression of the desired polypeptide.
  • the transfected nucleic acid may be permanently incorporated into the genome of each of the targeted cells, providing long lasting effect, or alternatively the treatment may have to be repeated periodically.
  • viruses have been used as gene transfer vectors, including papovaviruses, such as SV40, vaccinia virus, herpesviruses, including HSV and EBV, and retroviruses .
  • papovaviruses such as SV40
  • vaccinia virus vaccinia virus
  • herpesviruses including HSV and EBV
  • retroviruses retroviruses .
  • Many gene therapy protocols in the prior art have used disabled murine retroviruses.
  • nucleic acid into cells includes electroporation, calcium phosphate co-precipitation, mechanical techniques such as microinjection, transfer mediated by liposomes and direct DNA uptake and receptor- mediated DNA transfer.
  • Receptor-mediated gene transfer in which the nucleic acid is linked to a protein ligand via polylysine, with the ligand being specific for a receptor present on the surface of the target cells, is an example of a technique for specifically targeting nucleic acid to particular cells.
  • a polypeptide, peptide or other substance able to modlate or interfere with the interaction of the relevant polypeptide, peptide or other substance as disclosed herein, or a nucleic acid molecule encoding a peptidyl such molecule may be provided in a kit, e.g. sealed in a suitable container which protects its contents from the external environment.
  • a kit may include instructions for use.
  • the present invention provides for the provision of purified hRadl.
  • Purified hRadl for instance about 10% pure, more preferably about 20% pure, more preferably about 30% pure, more preferably about 40% pure, more preferably about 50% pure, more preferably about 60% pure, more preferably about 70% pure, more preferably about 80% pure, more preferably about 90% pure, more preferably about 95% pure, or substantially pure hRadl is obtainable, e.g. using antibodies as provided herein.
  • Individuals with defects in HRADI may have increased cancer risk, elevated risk of neurodegenerative diseases and other degenerative diseases, and/or may be hypersensitive to various DNA damaging agents. Indeed, studies using the GENEBRIDGE cell hybrid panel (Gyapay et al . , (1996). Hum .
  • the HRADI gene may be used in diagnostic and prognostic contexts, e.g. testing individuals to see if they are predisposed to cancer or testing cancer patients to see if their disease involves HRADI and/or hRadl dysfunction. Patients may be tested before delivering radiotherapies to optimise the amount of radiation delivered or to decide whether radiotherapy is of is not appropriate.
  • a number of methods are known in the art for analysing biological samples from individuals to determine whether the individual carries a HRADI allele predisposing them to any particular disorder, such as one or more of the disorders discussed above.
  • the purpose of such analysis may be used for diagnosis or prognosis, and serve to detect the presence of an existing cancer or other disorder, to help identify the type of disorder, to assist a physician in determining the severity or likely course of the disorder and/or to optimise treatment of it.
  • the methods can be used to detect HRADI alleles that are statistically associated with a susceptibility to a disorder, such as cancer, in the future, identifying individuals who would benefit from regular screening to provide early diagnosis.
  • the disclosure herein paves the way for aspects of the present invention to provide the use of materials and methods, such as are disclosed and discussed above, for establishing the presence or absence in a test sample of an variant form of the gene, in particular an allele or variant specifically associated with a disorder such as cancer.
  • This may be for diagnosing a predisposition of an individual to a disorder or disease. It may be for diagnosing a disorder or disease a patient with the disease as being associated with HRADI mutation. Determination of the presence or absence of wild-type or a particular allele or variant sequence may be used in a pharmacogenomics context, assessing susceptibility of patients or populations of patients to one or more available treatments.
  • the methods divide into those screening for the presence of nucleic acid sequences and those that rely on detecting the presence or absence of polypeptide.
  • the methods make use of biological samples from individuals that may or are suspected to contain the nucleic acid sequences or polypeptide. Examples of biological samples include blood, plasma, serum, tissue samples, tumour samples, saliva and urine .
  • Exemplary approaches for detecting nucleic acid or polypeptides include:
  • a specific binding member capable of binding to a HRADI nucleic acid sequence (either a normal sequence or a known mutated sequence) , the specific binding member comprising nucleic acid hybridisable with the HRADI sequence, or substances comprising an antibody domain with specificity for a native or mutated HRADI nucleic acid sequence or the polypeptide encoded by it, the specific binding member being labelled so that binding of the specific binding member to its binding partner is detectable; or,
  • the HRADI nucleic acid in the sample will initially be amplified, e.g. using PCR, to increase the amount of the analyte as compared to other sequences present in the sample . This allows the target sequences to be detected with a high degree of sensitivity if they are present in the sample.
  • Tests may be carried out on preparations containing genomic DNA, cDNA and/or mRNA.
  • Testing cDNA or mRNA has the advantage of the complexity of the nucleic acid being reduced by the absence of intron sequences, but the possible disadvantage of extra time and effort being required in making the preparations.
  • RNA is more difficult to manipulate than DNA because of the wide-spread occurrence of RN'ases.
  • Nucleic acid in a test sample may be sequenced and the sequence compared with the sequence shown in SEQ ID NO. 1 to determine whether or not a difference is present. If so, the difference can be compared with known susceptibility alleles to determine whether the test nucleic acid contains one or more of the variations indicated, or the difference can be investigated for association with a particular disorder such as cancer.
  • nucleic acid for testing may be prepared from nucleic acid removed from cells or in a library using a variety of other techniques such as restriction enz me digest and electrophoresis .
  • Nucleic acid may be screened using a variant- or allele-specific probe.
  • a probe corresponds in sequence to a region of the HRADI gene, or its complement, containing a sequence alteration known to be associated with disease susceptibility.
  • specific hybridisation of such a probe to test nucleic acid is indicative of the presence of the sequence alteration in the test nucleic acid.
  • more than one probe may be used on the same test sample .
  • Allele- or variant-specific oligonucleotides may similarly be used in PCR to specifically amplify particular sequences if present in a test sample.
  • Assessment of whether a PCR band contains a gene variant may be carried out in a number of ways familiar to those skilled in the art.
  • the PCR product may for instance be treated in a way that enables one to display the mutation or polymorphism on a denaturing polyacrylamide DNA sequencing gel, with specific bands that are linked to the gene variants being selected.
  • An alternative or supplement to looking for the presence of variant sequences in a test sample is to look for the presence of the normal sequence, e.g. using a suitably specific oligonucleotide probe or primer.
  • RN'ase A cleaves at the site of a mis-match. Cleavage can be detected by electrophoresing test nucleic acid to which the relevant probe or probe has annealed and looking for smaller molecules (i.e. molecules with higher electrophoretic mobility) than the full length probe/test hybrid.
  • Other approaches rely on the use of enzymes such as resolvases or endonucleases.
  • an oligonucleotide probe that has the sequence of a region of the normal HRADI gene (either sense or anti-sense strand) in which mutations associated with disease susceptibility are known to occur may be annealed to test nucleic acid and the presence or absence of a mis-match determined. Detection of the presence of a mis-match may indicate the presence in the test nucleic acid of a mutation associated with disease susceptibility.
  • an oligonucleotide probe that has the sequence of a region of the HRADI gene including a mutation associated with disease susceptibility may be annealed to test nucleic acid and the presence or absence of a mis-match determined. The presence of a mis-match may indicate that the nucleic acid in the test sample has the normal sequence. In either case, a battery of probes to different regions of the gene may be employed.
  • the presence of differences in sequence of nucleic acid molecules may be detected by means of restriction enzyme digestion, such as in a method of DNA fingerprinting where the restriction pattern produced when one or more restriction enzymes are used to cut a sample of nucleic acid is compared with the pattern obtained when a sample containing the normal gene or a variant or allele is digested with the same enzyme or enzymes .
  • a sample may be tested for the presence of a binding partner for a specific binding member such as an antibody (or mixture of antibodies) , specific for one or more particular variants of the polypeptide shown in SEQ ID NO. 2.
  • a binding partner for a specific binding member such as an antibody (or mixture of antibodies) , specific for one or more particular variants of the polypeptide shown in SEQ ID NO. 2.
  • a sample may be tested for the presence of a binding partner for a specific binding member such as an antibody (or mixture of antibodies) , specific for the polypeptide shown in SEQ ID NO. 2.
  • a binding partner for a specific binding member such as an antibody (or mixture of antibodies) , specific for the polypeptide shown in SEQ ID NO. 2.
  • the sample may be tested by being contacted with a specific binding member such as an antibody under appropriate conditions for specific binding, before binding is determined, for instance using a suitable reporter system.
  • a specific binding member such as an antibody under appropriate conditions for specific binding
  • different reporting labels may be employed for each antibody so that binding of each can be determined.
  • a specific binding member such as an antibody may be used to isolate and/or purify its binding partner polypeptide from a test sample, to allow for sequence and/or biochemical analysis of the polypeptide to determine whether it has the sequence and/or properties of the polypeptide whose sequence is shown in SEQ ID NO. 2, or if it is a mutant or variant form.
  • Amino acid sequence is routine in the art using automated sequencing machines .
  • Nucleic acid according to the present invention such as a full-length coding sequence or oligonucleotide probe or primer, may be provided as part of a kit, e.g. in a suitable container such as a vial in which the contents are protected from the external environment.
  • the kit may include instructions for use of the nucleic acid, e.g. in PCR and/or a method for determining the presence of nucleic acid of interest in a test sample.
  • a kit wherein the nucleic acid is intended for use in PCR may include one or more other reagents required for the reaction, such as polymerase, nucleosides, buffer solution etc.
  • the nucleic acid may be labelled.
  • a kit for use in determining the presence or absence of nucleic acid of interest may include one or more articles and/or reagents for performance of the method, such as means for providing the test sample itself, e.g. a swab for removing cells from the buccal cavity or a syringe for removing a blood sample (such components generally being sterile) .
  • the present invention provides an apparatus for screening HRADI nucleic acid, the apparatus comprising storage means including a nucleic acid sequence as set out herein, the stored sequence being used to compare the sequence of the test nucleic acid to determine the presence of mutations.
  • PCR- polymerase chain reaction-
  • oligonucleotides worked in the PCR. We cloned and sequenced this PCR fragment and discovered by DNA sequencing followed by sequence analyses that it encoded the mouse homolog of S . pombe Radl (mouse Radl; mRadl) .
  • MRADl the mouse gene and its product are referred to as MRADl and mRadl, respectively.
  • SEQ ID NO. 3 the sequence of its protein product, mRadl, is shown in SEQ ID NO. 4.
  • hRadl cDNA comprising amino acid residues 1 to 282 as a hexa-histidine tagged fusion in E. coli .
  • This protein was purified by Ni2 + chelate affinity chromatography and then used to raise polyclonal antisera in rabbits.
  • Use of the resulting sera in Western immunoblot assays revealed that two of the antisera were capable of recognising the recombinant hRadl protein with very high specificity and selectivity (less than 1 ng of protein being easily detectable) .
  • these two reactive antisera were found to be able to detect a protein of -30 kDa in crude human or mouse cell extracts.
  • pre-blocking of the antisera with recombinant hRadl abrogated their ability to recognise the -30 kDa protein. Since this endogenous protein is approximately the size predicted from the hRadl (or mRadl) cDNA and since it migrates in a manner consistent with it being 1-3 kDa smaller than the recombinant hRadl protein (which contains a -1 kDa tag) , we conclude that the antisera are indeed able to recognise mammalian Radl in extracts of human or mouse cells.
  • HRADI is a functional homologue of S. pombe RADl and plays roles in the signalling of DNA damage and stalled replication complexes was obtained by demonstration that the human gene complements substantially both the radiation sensitivity and cell cycle checkpoint phenotypes of a spradl mutant strain (Freire et al . , (1998). Genes Dev. 12, 2560-2573) .
  • the S. pombe radl mutant strain is sensitive to DNA damaging agents such as UV- and ⁇ -radiation, at least in part because it is unable to delay progress through the cell cycle when the DNA is damaged.
  • HRADI performs similar functions to the yeast gene
  • the HRADI cDNA was expressed in the S . pombe radl mutant strain and in the parental wild- type (WT) strain.
  • WT parental wild- type
  • HRADI was expressed in a S. pombe expression vector in which HRADI was under the control of the thiamine repressible nmt promoter (Maundrell (1990) . J. Biol . Chem . 265, 10857-10864) .
  • Expression of hRadl protein was verified by Western blotting using a hRadl-specific polyclonal antibody.
  • HRADI reproducibly rescued the UV sensitivity of spradl mutant cells and, at higher UV doses, the viability of the strain expressing HRADI was around 15- fold greater than that of the spradl-deficient strain. However, only around 3 -fold complementation of the ionizing radiation sensitivity of the spradl mutant strain was obtained.
  • spradl is also involved in the S-M phase checkpoint that causes cell cycle arrest in S phase when unreplicated DNA is present. This involvement is demonstrated by the hypersensitivity of spradl mutants to hydroxyurea, which blocks DNA replication by inhibiting the de novo synthesis of deoxyribonucleotides .
  • hRadl is mainly nuclear and is not induced by DNA damaging agents
  • hRadl affinity-purified anti-hRadl antibodies were used in immunofluorescence microscopy studies. This revealed that, despite the absence of any obvious nuclear localization sequences, the endogenous hRadl protein is located mainly in the nucleus of HeLa cells but appears to be excluded from the nucleoli. A low but significant signal was also detected in the cytoplasm, however, suggesting that a small proportion of hRadl may be cytoplasmic. Similar staining with anti-Radl antibody was also obtained when other human cells were analysed. In agreement with these results, Western blotting of nuclear and cytoplasmic fractions confirmed that hRadl is predominantly a nuclear protein.
  • hRadl The levels of some proteins implicated in cell cycle checkpoint control are regulated in response to genotoxic insults and/or throughout the cell cycle, and this was tested for hRadl. No marked variation in hRadl levels was observed at various times after exposing cells to UV or ionizing radiation. By contrast, the well -characterized induction of p53 by these agents was clearly evident. Similarly, no significant variation of hRadl protein levels or hRadl electrophoretic mobility on SDS-polyacrylamide gels was detected. These results, together with the nuclear localization of hRadl, are in accord with hRadl functioning as part of the DNA damage detection machinery rather than as an inducible downstream effector of DNA damage signalling pathways.
  • hRadl does not appear to possess inherent nuclease activi ty
  • hRadl is localised to si tes of meiotic recombination
  • Radl is localised very specifically with paired homologous chromosomes, and is localised specifically to the synaptie elements between these chromosome pairs. This therefore reinforces the notion that Radl is crucially involved in chromosomal synapsis and the associated process of meiotic recombination.
  • the mammalian Radl expression profile (spatially and temporally) is distinct from that of human ATM and the ATM-related protein termed ATR (Keegan et al . , (1996) . Genes & Dev. 10, 2423-2437; Hawley and Friend (1996) . Genes . Dev. 10, 2383-2388) , indicating that the function of hRadl in meiotic recombination and associated events is distinct from those of ATM and ATR.
  • hRadl levels are regulated during spermatogenesis
  • Radl protein is associated with discrete foci on the chromosomes of mouse spermatocytes undergoing meiotic prophase I, indicating that mammalian Radl plays a key role in regulating meiotic progression and that defects in this protein will lead to defects in gametogenesis and, hence, infertility.
  • Developing rat spermatocytes were fractionated by size through the use of centrifugal elutriation. This yielded five fractions containing cells at various stages of meiotic prophase I, which were then analyzed by Western blotting.
  • hRadl was found in relatively large amounts in the early stages of prophase I, corresponding mainly to late zygotene and early pachytene stage cells, whereas its levels declined in subsequent stages of prophase I, until it was almost undetectable in fraction 5, which contained late pachytene and early diplotene stage cells. This suggested that Radl might perform a crucial role in meiotic progression.
  • hRadl interacts wi th PCNA
  • yeast 2 -hybrid screen was employed.
  • hRadl Full-length hRadl was expressed in S. cerevisiae as a fusion to the DNA binding domain of lexA and this yeast strain was then used to screen a human B cell cDNA library tagged with the transcriptional activation domain of GAL4.
  • Several overlapping clones were isolated for the human protein PCNA (proliferating cell nuclear antigen; review by
  • NVINKIILQS EGLREAFSEL DMTSEVLQIT M ⁇ PDKPYFRL STFGNAGSSH LDYPKDSDLM EAFHCNQTQV NRYKISLLKP STKALVLSCK VSIRTDNRGF LSLQYMIRNE DGQICFVEYY CCPDEEVPES ES
  • ATGCCTCTCC TAACCCAGTA CAATGAAGAG GAGTACGAAC AGTACTGCTT AGTGGCCAGC CTTGACAACG TTAGGAATCT CTCCACTGTC TTGAAAGCCA TTCATTTCAG AGAACACGCC ACGTGTTTTG CTACCAAAAA CGGAATCAAG GTTACAGTGG AGAATGCAAA GTGTGTGCAA GCAAATGCCT TTATTCAGGC TGACGTGTTT CAGGAATTTG TCATTCAGGA AGAATCTGTT ACTTTTCGAA TTAACTTAAC TATCCTTTTA GACTGTTTAT CTATTTTTGG ATCAAGTCCT ACACCAGGGA CTTTGACTGC GCTTCGGATG TGTTACCAAG GTTATGGTCA CCCACTGATG CTATTTCTAG AAGAAGGAGG AGTGGTGACG GTCTGCAAAA TTACCACTCA GGAGCCTGAG GAGACACTGG ATTTTGATTT CTGCAGCACC AATGTTATGA ATAAAATTAT CCTGCA

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Abstract

L'invention concerne le clonage des homologues de S. pombe Rad1 et S. cerevisiae Rad17 chez l'homme et la souris. Le HRAD1 est particulièrement utile pour identifier et obtenir des molécules permettant de contrôler la prolifération cellulaire, et la sensibilisation des cellules à la radiothérapie. Des dosages peuvent être effectués à partir de l'interaction du HRad1 avec un antigène nucléaire des cellules de prolifération afin de rechercher des agents présentant un potentiel thérapeutique.
PCT/GB1998/003782 1997-12-16 1998-12-16 Acide nucleique rad1 de l'homme, polypeptides, dosages, procedes et moyens therapeutiques WO1999031234A1 (fr)

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AU15721/99A AU1572199A (en) 1997-12-16 1998-12-16 Human RADl nucleic acid, polypeptides, assays, therapeutic methods and means
CA002313391A CA2313391A1 (fr) 1997-12-16 1998-12-16 Acide nucleique rad1 de l'homme, polypeptides, dosages, procedes et moyens therapeutiques
JP2000539134A JP2002508180A (ja) 1997-12-16 1998-12-16 ヒトｒａｄ１核酸、ポリペプチド、検定、治療方法および手段
EP98960037A EP1037978A1 (fr) 1997-12-16 1998-12-16 Acide nucleique rad1 de l'homme, polypeptides, dosages, procedes et moyens therapeutiques

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WO1999049042A1 (fr) * 1998-03-27 1999-09-30 Icos Corporation POINT DE CONTROLE DU CYCLE CELLULAIRE DE Rad1 D'ORIGINE HUMAINE
WO2001021784A2 (fr) * 1999-09-23 2001-03-29 Rigel Pharmaceuticals, Inc. Nouvelles proteines du cycle cellulaire associees a l'antigene pcna, compositions et procedes d'utilisation

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WO1997042209A1 (fr) * 1996-05-03 1997-11-13 Human Genome Sciences, Inc. Polypeptides humains de type rad

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WO1997042209A1 (fr) * 1996-05-03 1997-11-13 Human Genome Sciences, Inc. Polypeptides humains de type rad

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DATABASE EMEST 11 E.M.B.L. Databases; 27 June 1997 (1997-06-27), HILLIER L ET AL: "Homo sapiens cDNA clone 842769 5' similar to SW:RAD1_SCHPO P22193 DNA REPAIR PROTEIN RAD1", XP002098403 *
DATABASE EMEST 15 E.M.B.L. Databases; 20 August 1996 (1996-08-20), HILLIER L ET AL: "Homo sapiens cDNA clone 470124 3' similar to SW:RAD1_SCHPO P22193 DNA REPAIR PROTEIN RAD1", XP002098404 *
DATABASE EMEST 18 E.M.B.L. Databases; 25 June 1997 (1997-06-25), MARRA M ET AL: "Mus musculus cDNA clone 789687 5' similar to SW:RAD1_SCHPO P22193 DNA REPAIR PROTEIN RAD1.", XP002098402 *
FREIRE R ET AL: "Human and mouse homologs of Schizosaccharomyces pombe rad1+ and Saccharomyces cerevisiae RAD17: linkage to checkpoint control and mammalian meiosis", GENES AND DEVELOPMENT, vol. 12, 15 August 1998 (1998-08-15), pages 2560 - 2573, XP002098400 *
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999049042A1 (fr) * 1998-03-27 1999-09-30 Icos Corporation POINT DE CONTROLE DU CYCLE CELLULAIRE DE Rad1 D'ORIGINE HUMAINE
WO2001021784A2 (fr) * 1999-09-23 2001-03-29 Rigel Pharmaceuticals, Inc. Nouvelles proteines du cycle cellulaire associees a l'antigene pcna, compositions et procedes d'utilisation
WO2001021784A3 (fr) * 1999-09-23 2001-10-04 Rigel Pharmaceuticals Inc Nouvelles proteines du cycle cellulaire associees a l'antigene pcna, compositions et procedes d'utilisation
US6387658B1 (en) 1999-09-23 2002-05-14 Rigel Pharmaceuticals, Inc. PCNA-associated cell cycle proteins, compositions and methods of use

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