WO1997025348A1 - Regulateur de proliferation cellulaire - Google Patents

Regulateur de proliferation cellulaire Download PDF

Info

Publication number
WO1997025348A1
WO1997025348A1 PCT/GB1997/000051 GB9700051W WO9725348A1 WO 1997025348 A1 WO1997025348 A1 WO 1997025348A1 GB 9700051 W GB9700051 W GB 9700051W WO 9725348 A1 WO9725348 A1 WO 9725348A1
Authority
WO
WIPO (PCT)
Prior art keywords
regulator
ink
antibody
cell
antigen
Prior art date
Application number
PCT/GB1997/000051
Other languages
English (en)
Inventor
George-Raoul Mazars
Parmjit Jat
Original Assignee
Ludwig Institute For Cancer Research
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ludwig Institute For Cancer Research filed Critical Ludwig Institute For Cancer Research
Priority to EP97900301A priority Critical patent/EP0873359A1/fr
Priority to AU13890/97A priority patent/AU1389097A/en
Publication of WO1997025348A1 publication Critical patent/WO1997025348A1/fr

Links

Classifications

    • 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
    • C07K14/4738—Cell cycle regulated proteins, e.g. cyclin, CDC, INK-CCR
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to a novel regulator of cell proliferation, hereinafter termed p24 INK ; a method for the isolation of p24 INK ; and uses thereof including, but not limited to, uses designed to affect the role of p24 INK in the cell cycle.
  • telomeric DNA and other essential sequences from the ends of chromosomes has been proposed to determine the finite proliferative potential; however, the processive loss of telomeric sequences has not been unequivocally demonstrated in rodent cells.
  • the genetic program hypothesis suggests that the finite mitotic potential is regulated by a biological clock that measures the finite life span so that upon its completion cells cease dividing and enter into the post mitotic state of senescence.
  • Cdks cyclin- dependant kinases
  • p27 ⁇ ipl inhibits cyclin E/cdk2 and cyclin A/cdk2 kinase activities and is induced in response to TGF- ⁇ and by contact inhibition.
  • p2l Wafl/CiP,/sdil another Cdk inhibitor, was identified as a gene that was transcriptionally upregulated by the tumour suppressor protein p53 and by virtue of its interaction with cdk2 in a yeast two hybrid system. Since transfection of p21 into cells inhibits DNA synthesis, it has been suggested that the growth inhibitory function of p53 may be mediated by p21.
  • P21 and p27 proteins share homology in the region involved in binding to cyclin/cdk complexes.
  • P21 also binds the proliferating cell nuclear antigen (PCNA); since PCNA is a component of DNA polymerse ⁇ , it has been suggested that this interaction may be involved in promoting DNA repair.
  • PCNA proliferating cell nuclear antigen
  • P21 mRNA was also isolated as a gene that was upregulated when human diploid fibroblasts undergo senescence and proposed to be a component of the mechanism that limits the finite proliferative potential.
  • p21 was similarly involved in the senescence of rodent embryonic fibroblasts.
  • our investigations have led us surprisingly to identify a novel cell cycle regulator which we have termed p24 INK .
  • p24 INK In addition, notwithstanding the fact that an antibody that binds to p24 1NK is described herein, the isolation of p24 INK also provides for the production of potentially more specific antibodies thereto, especially monoclonal antibodies. It will be understood that antibodies, monoclonal or otherwise, will be manufactured against the isolated and purified p24 INK using conventional techniques.
  • p24 INK can be used to selectively limit the lifespan of diseased tissue such as, but not limited to, cancer tissue, lymphoid hyperplasias, inflammatory regeneration, Keloid scarring of the skin and benign tumours.
  • diseased tissue such as, but not limited to, cancer tissue, lymphoid hyperplasias, inflammatory regeneration, Keloid scarring of the skin and benign tumours.
  • agents that bind p24 1NK such as T antigen, can be identified and the interaction between p24 INK and such agents can be blocked in diseased tissue in order to ensure that p24 INK activity is manifest.
  • T antigen agents that bind p24 1NK
  • T antigen can be identified and the interaction between p24 INK and such agents can be blocked in diseased tissue in order to ensure that p24 INK activity is manifest.
  • the expression of p24 INK could be manipulated by binding agents in situations where one might want to limit the number of times cells divide.
  • transplantation in stem cell transplantation or other cell transplantations, one may want to put back cells into individuals that are prevented from dividing too many times and consequently becoming cancerous.
  • transplanted cells do not exist for a long time and therefore repeated transplantations have been necessary. If one could manipulate the cells to be transplanted via, manipulation of p24 INK , to have a greater number of divisions before senescence then fewer transplantations might be necessary.
  • agents such as antagonists may be used to selectively bind to p24 INK and thus mask the effects of same.
  • reference made to p24 INK is intended to include homologues or analogues thereof and in particular agonists which behave in the same way as p24 ⁇ .
  • the invention has application in the manipulation of the promoter relating thereto so that enhanced levels of p24 INK may be expressed.
  • a novel, isolated regulator of cell proliferation having a molecular weight of 24 kilodaltons and a pattern of expression which is approximately maximal at the onset of cell senescence.
  • Reference herein to a novel regulator of cell proliferation, p24 INK is intended to include reference to any mammalian form of the protein given the high degree of inter-species homology and indeed the functional identity, and inter- changeability of the protein between species. This is demonstrated having regard to a review entitled Inhibitors of mammalian G, cyclin-dependent kinases (Genes & Development 9: 1149-1163, 1995 - Cold Spring Harbor Laboratory Press).
  • a recently identified cyclin-dependent kinase inhibitor termed p57 kip2 , and more particularly in vitro translated mouse p57 Wp2 , bound to cdk2, cdk3, cdk4 and cdk ⁇ in a cyclin-dependent manner.
  • this inhibitor was shown to inhibit the kinase activity of cyclin D-cdk4, D-cdk6, E-cdk2, E-cdk3, and A- cdk2 complexes in vitro; and its transfection into mink lung cells or into human Saos-2 osteosarcoma cells induced G] arrest.
  • p!5 INK4 which has been known to be induced in human epithelial (HaCaT) cells by growth factor has also been shown to be produced in many mouse tissues.
  • plo ⁇ 1 40 and pl9 INK4d are also expressed ubiquitously in proliferating culture cells and in normal mouse tissues. Then the invention relates to any mammalian p24 INK .
  • said regulator is a cyclin-dependant kinase inhibitor.
  • the expression of said inhibitor is independent of T antigen and/or independent of p53.
  • said regulator associates with T antigen.
  • T antigen associates with T antigen.
  • a novel regulator of cell proliferation, p24 INK comprising:
  • the method includes a further step of size-fractionation.
  • the method of the invention involves the use of an immunoaffinity column to which the said antibody is attached and exposure of at least said extract thereto under conditions which facilitate binding of p24 m ⁇ to said antibody; and then elution of said bound agent or antigen followed, optionally, depending upon the nature of the antibody, by a further separation technique which ideally involves a size-fractionation method.
  • the method for purification of the novel cell cycle regulator, p24 ⁇ may involve genetic techniques. These techniques involve screening an expression library such as an E.coli library prepared with mRNA extracted from a p24 INK expressing cell such as a tsa cell line using a p24 INK specific antibody such as the 471 antibody. Once a clone is identified it is used to screen a full length cDNA library. This full length clone is then over expressed in order to purify the protein in a large quantity. In order to check that the correct gene has been cloned antisera to the protein will be used.
  • an expression library such as an E.coli library prepared with mRNA extracted from a p24 INK expressing cell such as a tsa cell line using a p24 INK specific antibody such as the 471 antibody.
  • a PCR library is used. Since the sequence of the peptide that was used as an immunogen is known an oligonucleotide was designed which is used in combination with a vector specific primer to carry PCR on a cDNA library. This is done advantageously under fairly nonstringent conditions. As before, when a PCR fragment is generated it is used for screening a full length cDNA library. The isolated cDNA is then over expressed and used to generate antisera for determining the correct nature of the gene.
  • agents that selectively bind with p24 ,NK to affect the cell cycle.
  • p24 ⁇ to identify agents that selectively bind therewith so as to inhibit the effects thereof.
  • antibodies other than antibody 471 , with selectively bind with p24 INK , ideally, with a view to determining the level of expression thereof.
  • Figure 1 shows P21 expression:
  • A Northern blot analysis. 5 ⁇ g of total RNA prepared from tsa 129, SV4 and REF cells was loaded onto agarose gel, transferred on nitrocellulose membrane and probed with rat p21 cDNA probe labelled with 32p by random priming. Comparison of p21 mRNA expression in tsal29 cell line when shifted up from 33° C to 39.5° C for 3 days (lane 1, 2) and in normal Rat Embryo Fibroblasts (REFs) in early (lane 3) and late passages (lane 4). SV4 cell lines were used to verify that p21 mRNA expression is not dependant on temperature. The Blot was stripped and reprobed with a human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) probe for control of equal RNA loading.
  • GPDH human glyceraldehyde 3-phosphate dehydrogenase
  • Figure 2 shows passaging experiments at 33° C in normal Rat Embryo Fibroblasts from 13 day old Sprague-Dawley embryos in two separate experiments: same total cell extracts were analysed by Western blotting at 30 ⁇ g/lane with a variety of antisera against p21 (CP74, 472, 397), p24 INK (471) and p27 (528).
  • REFs stop dividing at passage 4 in experiment on the left panel and passage 3 in experiment on the right panel (to corresponds to cells before plating). Cells were called senescent when the number of cells counted after 4 days was lower than number of cells plated to extracts were prepared from cells before plating.
  • FIG. 3 shows the expression of p21 and p24 INK in passaging experiments using transgenic mouse embryo fibroblasts expressing thermolabile tsa58 T Ag (A).
  • the expression levels of p21 and p24 INK in normal (NMEFs) or transgenic (TMEFs) mouse embryo fibroblasts were compared.
  • NMEFs and TMEFs were serially cultivated every 4 days in presence of 10 units per ml of recombinant mouse ⁇ -INF obtained from Gibco-BRL at 33° C until the NMEFs stopped proliferating. Total cell extracts were prepared 2 days after replating. In this experiment, cells stop dividing at passage 6.
  • FIG. 4 shows coprecipitation of T antigen.
  • A An equal amount RIPA cell lysate prepared from tsa 129 cells cultured at 33° C was immunoprecipitated using antibodies specific for p21(SX118, 397), p27 (528) or p53 (421). Monoclonal antibody M73 specific for the Adeno virus EIA protein was used a negative control(). The immunoprecipitated proteins were fractionated and analysed for the presence of T antigen by immunoblotting using a mixture of monoclonal antibodies 419 and 416.
  • B RIPA cell lysate prepared from tsal29 cells at 33° C was immunoprecipitated with 471 in the presence of increasing amounts of cold peptide used to raise this antiserum. IX corresponds to an equal amount of peptide whereas 10X is ten-fold higher amount of peptide. The immunoprecipitates were first analysed for the presence T antigen and then reprobed for p21 using SX118.
  • tsa cell lines such as tsa 129 were derived by immortalising REFs with the thermolabile large T antigen encoded by the SV40 early region mutant tsA58 and thus grow continuously at the permissive temperature (33° ) but rapidly undergo growth arrest upon inactivation of T antigen at the nonpermissive temperature (39.5°). Isolation and characterisation of these cell lines has been previously described (Jat and Sharp 1989).
  • 471 can detect a fusion protein of glutathione-S-transferase-p21 (GST-p21) which was used as the positive control in all immunoblotting experiments and 471 is also capable of immunoprecipitating p21 (Figure 4), thus confirming its specificity.
  • GST-p21 glutathione-S-transferase-p21
  • mice prepared from the H-2K b tsA58 mice (Jat et al, 1991). These mice harbour the thermolabile f_?A58 T antigen under the control of the ⁇ -interferon ( ⁇ -INF) inducible H-2K b promoter and thus allow the preparation of fibroblast populations in which the expression of T antigen can be modulated in every cell by manipulating the growth conditions.
  • ⁇ -INF ⁇ -interferon
  • Fibroblast cultures were prepared from 12/13-day old mouse embryos from transgenic homozygous males x normal females (TMEFs) and normal males x normal females (NMEFs) and serially cultivated in the presence of 10 units per ml ⁇ -interferon at 33° C ( ⁇ -INF) until the NMEFs ceased dividing. Under these permissive growth conditions TMEFs yield immortal cultures that can be passaged indefinitely whereas NMEFs stop dividing; ⁇ -INF was included in both cultures to ensure that the cultures were directly comparable. At the indicated passages protein extracts were prepared and analysed.
  • Protein extracts prepared from p21-/- fibroblasts were analysed. Results presented in Figure 3D show that although p21 level was undetectable in p21- /- extracts, approximately the same amount of p24 INK protein was present in these cells compared to TMEFs. Thus the p24 INK protein is not a product of the p21 Waf, Cip, SdU gene.
  • p24 INK was essentially extracted using immunoaffmity purification. Techniques relating to immunoaffinity purification are described in the laboratory text book "Antibodies: A Laboratory Manual” by Ed Harlow and David Lane, Cold Spring Harbour Laboratories, 1988, and in particular page 449 describing cell lysis and pages 511-552 describing large scale immunoaffinity purification.
  • the method involves three basic steps. 1. Preparation of an antibody -matrix.
  • monoclonal antibodies or affinity-purified polyclonal antibodies are used.
  • the 471 antibody we have used the 471 antibody and covalently attached it to a solid-phase matrix that is a sepharose column. Any contaminating macro-molecules were removed by washing.
  • the nature of the antibody used determined the amount of cross-binding.
  • 471 antibody may bind p21 as well as p24 INK and therefore following use of the immunoaffinity column, and elution of the bound antigen, p21 and p24 INK , using conventional techniques a further separation technique is required.
  • a sizing column was used to separate the two proteins such as an FPLC superose 12 or superose 75 column to size-fractionate the proteins.
  • p24 INK a protein whose expression increases when rodent embryonic fibroblasts are serially passaged in vitro. This protein clearly shares some homology to p2l Wafl/c ' P1/sdil since it was identified using a peptide affinity purified antibody prepared against a p21 peptide.
  • p 21 af,/Ci P 1 ' Sdil gene was also upregulated during serial passaging of rodent embryonic fibroblasts but the pattern of accumulation does not correlate with the loss of proliferative potential; it also does not accumulate to the maximal level in the presence of T antigen which was not surprising since T antigen complexes with p53 and p53 is known to upregulate p21.
  • p24 INK was upregulated when rodent embryo fibroblasts are serially passaged in vitro and undergo senescence; the upregulation occurs at the same rate in the presence or absence of T antigen and is thus independent of p53.
  • p24 INK is an inhibitor of growth, and so our results suggests that the biological clock that measures the finite life span involves the accumulation of ⁇ 24 INK and that its growth inhibitory effects can be overcome by association with T antigen.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Toxicology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

Cette invention concerne un nouveau régulateur de prolifération cellulaire appelé p24INK. Cette protéine participe à la régulation du cycle cellulaire et, d'une manière plus précise, l'horloge biologique qui mesure la durée de vie finie d'une cellule implique une accumulation de p24INK. L'isolation de cette protéine permet ainsi d'obtenir un moyen de régulation du cycle cellulaire, tant à des fins de recherche que cliniques.
PCT/GB1997/000051 1996-01-11 1997-01-09 Regulateur de proliferation cellulaire WO1997025348A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP97900301A EP0873359A1 (fr) 1996-01-11 1997-01-09 Regulateur de proliferation cellulaire
AU13890/97A AU1389097A (en) 1996-01-11 1997-01-09 Regulator of cell proliferation

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9600518.6 1996-01-11
GBGB9600518.6A GB9600518D0 (en) 1996-01-11 1996-01-11 Regulator of cell proliferation

Publications (1)

Publication Number Publication Date
WO1997025348A1 true WO1997025348A1 (fr) 1997-07-17

Family

ID=10786876

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB1997/000051 WO1997025348A1 (fr) 1996-01-11 1997-01-09 Regulateur de proliferation cellulaire

Country Status (4)

Country Link
EP (1) EP0873359A1 (fr)
AU (1) AU1389097A (fr)
GB (1) GB9600518D0 (fr)
WO (1) WO1997025348A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5302706A (en) * 1991-12-16 1994-04-12 Baylor College Of Medicine Senescent cell derived inhibitors of DNA synthesis
WO1995035115A1 (fr) * 1994-06-21 1995-12-28 The University Of North Carolina At Chapel Hill Adn codant une proteine de 18 kd inhibant la kinase cyclinodependante

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5302706A (en) * 1991-12-16 1994-04-12 Baylor College Of Medicine Senescent cell derived inhibitors of DNA synthesis
WO1995035115A1 (fr) * 1994-06-21 1995-12-28 The University Of North Carolina At Chapel Hill Adn codant une proteine de 18 kd inhibant la kinase cyclinodependante

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
C.J. SHERR AND J.M. ROBERTS: "Inhibitors of mammalian G1 cyclin-dependent kinases", GENES & DEVELOPMENT, vol. 9, no. 10, 15 May 1995 (1995-05-15), pages 1149 - 1163, XP000673236 *
Y. XIONG ET AL.: "p21 is a universal inhibitor of cyclin kinases", NATURE, vol. 366, 16 December 1993 (1993-12-16), LONDON GB, pages 701 - 704, XP002030423 *

Also Published As

Publication number Publication date
GB9600518D0 (en) 1996-03-13
AU1389097A (en) 1997-08-01
EP0873359A1 (fr) 1998-10-28

Similar Documents

Publication Publication Date Title
Chen et al. Inactivation of the type II receptor reveals two receptor pathways for the diverse TGF-β activities
Horii et al. A novel oncogene, ost, encodes a guanine nucleotide exchange factor that potentially links Rho and Rac signaling pathways.
Ohtsubo et al. Human cyclin E, a nuclear protein essential for the G1-to-S phase transition
Chou et al. Intermediate filament reorganization during mitosis is mediated by p34cdc2 phosphorylation of vimentin
Zhu et al. Characterization of a novel 350-kilodalton nuclear phosphoprotein that is specifically involved in mitotic-phase progression
Porter et al. Human Speedy: a novel cell cycle regulator that enhances proliferation through activation of Cdk2
Shirodkar et al. The transcription factor E2F interacts with the retinoblastoma product and a p107-cyclin A complex in a cell cycle-regulated manner
Cheng et al. TANK, a co-inducer with TRAF2 of TNF-and CD 40L-mediated NF-kappaB activation.
Lukas et al. DNA tumor virus oncoproteins and retinoblastoma gene mutations share the ability to relieve the cell's requirement for cyclin D1 function in G1.
Yamaguchi et al. XIAP, a cellular member of the inhibitor of apoptosis protein family, links the receptors to TAB1–TAK1 in the BMP signaling pathway
Turner et al. Paxillin LD4 motif binds PAK and PIX through a novel 95-kD ankyrin repeat, ARF–GAP protein: a role in cytoskeletal remodeling
Datto et al. Transforming growth factor beta induces the cyclin-dependent kinase inhibitor p21 through a p53-independent mechanism.
Dimri et al. Inhibition of E2F activity by the cyclin-dependent protein kinase inhibitor p21 in cells expressing or lacking a functional retinoblastoma protein
Dahia et al. Mutation and expression analysis of the p27/kip1 gene in corticotrophin-secreting tumours
Montagnoli et al. Overexpression of the nerve growth factor-inducible PC3 immediate early gene is associated with growth inhibition
Mosialos et al. Epstein-Barr virus infection induces expression in B lymphocytes of a novel gene encoding an evolutionarily conserved 55-kilodalton actin-bundling protein
Nakamura et al. Cyclin I: a new cyclin encoded by a gene isolated from human brain
JP3064012B2 (ja) 哺乳動物細胞周期蛋白質
CA2220753C (fr) Nouveaux peptides et nouvelles compositions modulant l'apoptose
Auvinen et al. Ornithine decarboxylase-and ras-induced cell transformations: reversal by protein tyrosine kinase inhibitors and role of pp130CAS
CA2180570C (fr) Proteine p27 isolee et molecules d'acide nucleique codant cette proteine
US5821082A (en) Anti-proliferation domain of a human Bcl-2 and DNA encoding the same
Seong et al. Phosphorylation of a novel zinc-finger-like protein, ZPR9, by murine protein serine/threonine kinase 38 (MPK38)
US6280955B1 (en) Interleukin-1 receptor accessory proteins, nucleic acids and methods
Tchang et al. Stabilization and expression of high levels of p53 during early development in Xenopus laevis

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE HU IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TR TT UA UG US UZ VN AM AZ BY KG KZ MD RU TJ TM

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): KE LS MW SD SZ UG AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 1997900301

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1997900301

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

NENP Non-entry into the national phase

Ref country code: JP

Ref document number: 97524980

Format of ref document f/p: F

WWW Wipo information: withdrawn in national office

Ref document number: 1997900301

Country of ref document: EP