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WO1999002704A2 - Phosphatase a double specificite et procedes d'utilisation - Google Patents

Phosphatase a double specificite et procedes d'utilisation Download PDF

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Publication number
WO1999002704A2
WO1999002704A2 PCT/US1998/014205 US9814205W WO9902704A2 WO 1999002704 A2 WO1999002704 A2 WO 1999002704A2 US 9814205 W US9814205 W US 9814205W WO 9902704 A2 WO9902704 A2 WO 9902704A2
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Prior art keywords
pten
phosphatase
alteration
nucleic acid
altered
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PCT/US1998/014205
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English (en)
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WO1999002704A3 (fr
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Nicholas K. Tonks
Michael P. Myers
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Cold Spring Harbor Laboratory
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Priority to AU84794/98A priority Critical patent/AU8479498A/en
Publication of WO1999002704A2 publication Critical patent/WO1999002704A2/fr
Publication of WO1999002704A3 publication Critical patent/WO1999002704A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • PTKs protein tyrosine kinases
  • cysteine residue in this motif is absolutely required for catalysis, as it acts as a nucleophile to attack the phosphorous atom in the phosphate moiety of its substrate, forming a thiol-phosphate intermediate (Barford, D., Flint, A.J. & Tonks, N.K. Science. 263:1397-1404 (1994)). Mutation of this cysteine to serine or alanine results in the complete loss of phosphatase activity (Flint, A.J. et al. Proc. Natl. Acad. Sci. USA.
  • the dual specificity phosphatases which catalyze the hydrolysis of phospho-seryl, -threonyl and -tyrosyl residues, also contain the canonical PTP catalytic motif (Sun, H., et al. Cell. 75:487-493 (1993)).
  • Prestical PTP catalytic motif does not guarantee that the protein is a protein phosphatase, as demonstrated by the discovery of CEL- 1 , an RNA capping enzyme (Takagi, T, et al. Cell. 89:867-873 (1997)).
  • PTPs exhibit a great deal of substrate specificity in vivo and are not simply unregulated antagonists of the signals mediated by the PTKs (Flint, A.J. et al. Proc. Natl. Acad. Sci. USA. 94:1680-1685 (1997), Garton, A.J., et al. Mol. Cell. Biol. 16:6408-6418 (1996)).
  • PTPs can exert both positive and negative effects on signaling pathways, indicating that they do not simply function as "off switches" (Hertog, J, et al. EMBOJ. 13:3020-3032 (1994)).
  • PTPs protein tyrosine phosphatases
  • the chromosomal locus 10q22-23 is deleted in a large number of tumors, especially glioblastoma (70%) and prostate tumors (30%) ((Steck, P.A., et al. Nature Genet. 15 :356-362 (1997), Li, J., et al. Science. 275: 1943-1946 (1997)).
  • PTEN is deleted in a large number of tumors, and germline mutations in PTEN give rise to a number of related neoplastic disorders, including Cowden's disease, an inherited disorder typified by the formation of multiple, benign tumors (hamartomas) in multiple tissues and an increased susceptibility to some forms of malignant cancer (Liaw, D. et al.
  • PTEN a candidate tumor suppressor gene identified on chromosome 10
  • cytoskeletal protein tensin a cytoskeletal protein tensin
  • purified PTEN showed a preference for acidic substrates.
  • Initial enzymatic characterization of PTEN revealed specificity for highly acidic substrates in vitro. This observation led to the consideration of signaling molecules with acidic characteristics as potential substrates and focused our attention on the phosphoinositides.
  • PTEN dephosphorylates phosphatidylinositol in vitro. It is also demonstrated that PTEN displays selectivity for the 3 position of the inositol ring, unlike the known lipid phosphatases which are specific for the 5 position. Furthermore, as also shown herein, PTEN is able to dephosphorylate serine, threonine and tyrosine residues when present in acidic substrates. Phosphatase activity of PTEN is necessary for its ability to function as a tumor suppressor or an apoptosis inducer. A variety of point mutations, including point mutations identified in tumor samples and Cowden's disease kindreds, ablated PTEN activity.
  • PTEN As a phosphatidylinositol phosphatase has been examined in a physiological context in work described herein. Firstly, expression of wild type PTEN in 293 cells suppresses the levels of phosphatidylinositol (Ptdlns (3,4,5)P ) whereas expression of a "substrate-trapping" mutant form of the enzyme leads to accumulation of the phospholipid. These data illustrate that PTEN recognizes phosphatidylinositol triphosphate as substrate in a cellular context.
  • ectopic expression of wild type PTEN results in suppression of the phosphorylation of PKB/Akt, a downstream target of the lipid products of phosphatidylinositol-3-OH kinase (PI 3 kinase). Furthermore, phosphorylation of the pro-apoptotic protein BAD, a substrate of PKB/Akt, was also suppressed in cells ectopically expressing wild type PTEN. Although it was possible to achieve expression of inactive mutant forms of PTEN in the prostate cancer cell line LnCaP, viable cells expressing the wild type enzyme could not be recovered.
  • the present invention relates to PTEN, and isolated nucleic acid sequences encoding PTEN, having phosphatase activity against phosphorylated non-proteinaceous substrates, tyrosine residues, phosphorylated serine residues, phosphorylated threonine residues, and combinations thereof.
  • PTEN dephosphorylates Ptdlns (3,4,5)P
  • PTEN dephosphorylates phosphorylated tyrosine, phosphorylated serine or phosphorylated threonine residues present in substrates containing acidic residues.
  • the invention also relates to altered forms of PTEN, and isolated nucleic acid sequences encoding the altered forms of
  • PTEN which have altered phosphatase activity against Ptdlns (3,4,5)P 3 phosphorylated tyrosine, phosphorylated serine or phosphorylated threonine residues.
  • the present invention further relates to use of the described nucleic acid sequences, their encoded gene products, altered forms of the nucleic acid sequences and encoded gene products, and antibodies that recognize or bind to PTEN or specific altered PTEN phosphatases.
  • These compositions can be used to assess the presence or activity of PTEN or altered PTEN as described herein.
  • These compositions can also be used in the diagnosis and treatment of disease, particularly in the diagnosis and treatment of conditions characterized by an alteration in PTEN which causes an alteration of phosphatase activity.
  • the alteration in PTEN results in an inhibition of phosphatase activity or an alteration (e.g., increase) in PTEN- induced apoptosis.
  • the alteration in PTEN results in an enhancement of phosphatase activity.
  • the present invention relates to an isolated nucleic acid sequence encoding PTEN phosphatase, wherein the encoded PTEN phosphatase dephosphorylates
  • the present invention also relates to an isolated nucleic acid sequence encoding PTEN phosphatase wherein the encoded PTEN phosphatase dephosphorylates phosphorylated tyrosine residues, phosphorylated serine residues, phosphorylated threonine residues and combinations thereof.
  • the present invention also pertains to an isolated nucleic acid sequence encoding PTEN phosphatase, wherein the encoded PTEN phosphatase possesses substrate sensitivity such that the phosphatase is active against acidic substrates or the activity is enhanced by presence of acidic amino acid residues in a substrate of the PTEN phosphatase.
  • the present invention also pertains to an isolated nucleic acid sequence encoding an altered phosphatase, wherein the phosphatase has an alteration in amino acid sequence such that phosphatase activity is inhibited (e.g., reduced or abolished).
  • the alteration in amino acid sequence is in the catalytic domain of the phosphatase.
  • the isolated nucleic acid sequence encoding the phosphatase can have an alteration wherein the alteration is a point mutation.
  • the alteration can be a point mutation resulting in alteration of histidine 123 to tyrosine, alteration of glycine 129 to arginine or alteration of glycine 129 to glutamic acid.
  • the present invention is also drawn to alterations in amino acid sequence wherein the alteration occurs outside of the catalytic domain.
  • the alteration such as a point mutation, can occur within alpha helix 2 or alpha helix 7 of the phosphatase.
  • the point mutation can result in alteration of serine 170 to arginine, alteration of glycine 165 to arginine, or alteration of threonine 167 to proline.
  • the present invention further relates to an isolated PTEN phosphatase, wherein the phosphatase dephosphorylates acidic or lipid substrates such as PtdIns(3,4,5)P 3 .
  • the present invention also relates to an isolated PTEN phosphatase wherein the phosphatase dephosphorylates phosphorylated tyrosine residues, phosphorylated serine residues, phosphorylated threonine residues and combinations thereof.
  • the present invention relates to an isolated PTEN phosphatase wherein the phosphatase possesses substrate sensitivity such that phosphatase is active against acidic substrates or the activity is enhanced by presence of acidic amino acid residues in a substrate.
  • the present invention also pertains to an isolated altered phosphatase having an alteration in amino acid sequence such that phosphatase activity is inhibited.
  • the present invention also relates to an antibody that specifically binds to PTEN, or a portion or altered form of PTEN, as well as to antigen binding fragments of such antibodies.
  • the present invention further relates to methods of diagnosing or aiding in the diagnosis of a condition characterized by an alteration in PTEN which causes an alteration of phosphatase activity of PTEN in an individual, wherein nucleic acids in a sample of cells from the individual are combined under conditions appropriate for hybridization with a nucleic acid probe which hybridizes with nucleic acids encoding an altered PTEN or portion thereof, such as an alteration which causes an alteration of phosphatase activity of PTEN. Hybridization of nucleic acids in the sample of cells with the nucleic acid probe is detected, and hybridization of nucleic acids in the sample of cells with the nucleic acid probes is indicative of a condition characterized by an alteration in PTEN.
  • the nucleic acid probe detects an alteration in the catalytic domain, alpha helix 2 or alpha helix 7 of PTEN.
  • the alteration of phosphatase activity is inhibition of phosphatase activity.
  • the condition is tumorigenesis.
  • the present invention further relates to a method of diagnosing or aiding in the diagnosis of a condition characterized by an alteration in PTEN which causes an alteration of phosphatase activity of PTEN in an individual.
  • proteins in a sample of cells from the individual are contacted with an antibody which binds altered PTEN.
  • the altered PTEN contains at least one amino acid which is different from the wild-type or reference amino acid and has altered phosphatase activity.
  • Antibody binding to altered PTEN is detected, and binding of the antibody to altered PTEN is indicative of a condition characterized by an alteration in PTEN.
  • the specific antibody detects an altered PTEN wherein portions of the carboxy terminus of PTEN have been deleted, or wherein PTEN is altered in the catalytic domain, or wherein PTEN is altered in alpha helix 2 or alpha helix 7.
  • the alteration of phosphatase activity results in inhibition of phosphatase activity.
  • the condition is tumorigenesis.
  • the present invention also pertains to a method of diagnosing or aiding in the diagnosis of a condition characterized by an alteration in PTEN which causes an alteration of phosphatase activity of PTEN in an individual.
  • proteins from the individual are combined with a phosphorylated PTEN substrate, under conditions appropriate for dephosphorylation of the phosphorylated substrate by PTEN. Presence or absence of dephosphorylation of the phosphorylated PTEN substrate is detected, wherein absence (including reduction relative to an appropriate control) of dephosphorylation is indicative of a condition characterized by an alteration in PTEN phosphatase activity.
  • PTEN or altered PTEN is isolated from the proteins in a sample of cells from the individual and the phosphatase activity of the isolated PTEN or altered PTEN is measured.
  • proteins in a sample of cells from the individual can be contacted with a composition comprising specific antibody which binds PTEN or altered PTEN, under conditions appropriate for binding of the antibody to PTEN.
  • the antibody-bound PTEN or altered PTEN can be isolated using methods described.
  • the phosphorylated substrate is PtdIns(3,4,5)P 3 .
  • the phosphorylated substrate is composed of acidic amino acids.
  • the present invention also relates to a method of treating a condition in an individual in which enhancement of PTEN phosphatase activity is desirable.
  • PTEN or a nucleic acid molecule encoding PTEN is administered in an appropriate physiologically acceptable vehicle to an individual.
  • the condition can be hyperproliferative diseases including brain, prostate or breast cancers, as well as Cowden's disease.
  • the present invention also relates to a method of treating a condition in an individual in which reduction of PTEN phosphatase activity is desirable.
  • a condition in an individual in which reduction of PTEN phosphatase activity is desirable.
  • an altered form of PTEN or a nucleic acid molecule encoding an altered form of PTEN is administered in an appropriate physiologically acceptable vehicle to an individual.
  • the condition can be a degenerative disease including, but not limited to, Parkinson's and other neurodegenerative diseases.
  • Figures 1A-1C are the nucleotide sequence of the PTEN gene (SEQ ID NO: 1) and the predicted amino acid sequence (SEQ ID NO: 2). The predicted start codon for the amino acid sequence is at nucleotide position 490, generating a protein 403 amino acids in length.
  • Figures 2A-2C are graphic representations of results which show the proteinaceous substrate specificity of purified PTEN. PTEN was tested for protein phosphatase activity using the indicated tyrosine ( Figure 2A) or serine/threonine ( Figure 2B) substrates. Activity is expressed as pmol phosphate released.
  • PTENC124S A catalytically inactive mutant of PTEN (PTENC124S) was included as a control to rule out the possibility of contaminating bacterial phosphatases.
  • Figure 2C shows a comparison of PTEN and cdcl4 activities. PTEN and cdcl4 were assayed as above with maleylated lysozyme (RCML) or polyGlu 4 Tyr adhere and the activity is expressed as pmol phosphate released/min/mg.
  • FIG. 3 depicts the location of PTEN mutations.
  • the diagram of PTEN shows the locations of the point mutations, which are indicated by an "*", that were tested as described herein.
  • the predicted structural motif in which these mutations lie is indicated.
  • Figure 4 is a graphic representation of phosphatase activity assays (against proteinaceous substrates) which illustrates disruption of PTEN activity by point mutations found in tumor samples. The indicated point mutations were introduced into recombinant PTEN and their effects on phosphatase activity were determined. Assays were performed with polyGlu 4 Tyr, for 15 minutes. Activity is expressed as pmol of phosphate liberated/min mg of PTEN. Assays were performed in triplicate and are expressed as the mean +/- the standard deviation. The catalytically inactive mutant of PTEN (PTENC124S) was included as control to rule out the possibility of contaminating bacterial phosphatases.
  • Figure 5 A is a graph of the level of 32 P ; released by the indicated phosphatases.
  • Figure 5B is a graph of the rate of 32 P ; released from the indicated substrates by PTEN.
  • Figure 6 is a graph of the level of 32 P ( released by the indicated phosphatases transfected into HEK293 cells.
  • Figure 7 is a graph of the percentage of GFP positive LnCaP cells isolated after the cells were transfected with the indicated PTEN.
  • PTEN a tumor suppressor gene identified on chromosome 10
  • PTEN shares homology with the PTP family, as well as with the cytoskeletal protein tensin ((Steck, P. A., et al. Nature Genet. 15:356-362 (1997), Li, J., et al. Science. 275:1943-1946 (1997)).
  • PTEN was isolated from a locus on chromosome 10, 10q22-23, which is deleted in a large number of tumors.
  • Germline mutations in PTEN give rise to Cowden's disease and variants thereof, which are typified by the formation of multiple, benign tumors and an increased susceptibility to malignant cancers.
  • PTEN possesses the signature motif that defines the PTP family of enzymes
  • PtdIns(3,4,5)P 3 as a second messenger in signaling events controlling cell proliferation suggested that PTEN may recognize this acidic phospholipid as a physiological.
  • phosphatase activity of PTEN is necessary for its ability to function as a tumor suppressor, because a variety of point mutations, which are known to occur in tumors and certain Cowden's disease kindreds, ablated PTEN activity.
  • a key step in understanding the function of PTEN is to identify its physiological substrates. Work described herein indicates that the lipid second messenger molecule phosphatidyl inositol phosphate, which has been implicated in signaling events associated with cell growth, is a physiological substrate of PTEN.
  • Glioblastoma is one of the most common and malignant forms of cancer, and is often characterized by the constitutive activation of EGF-dependent signaling pathways due to the amplification of members of the EGF receptor family of protein tyrosine kinases (PTKs).
  • PTKs protein tyrosine kinases
  • the products of tumor suppressor genes can attenuate these signaling pathways and their loss through deletion or mutation contributes to tumor progression.
  • the 10q23 region of human chromosome 10 is frequently deleted or mutated in a wide variety of tumor types, most frequently in glioblastoma, endometrial cancer and prostate cancer, indicating the presence of a tumor suppressor gene at this locus. Recently, mutated PTEN has been identified in a number of tumor cell lines (Steck, P. A.
  • PTEN appears to be preferentially lost in advanced cancers, suggesting that its deletion is not the transforming event but that PTEN is inhibiting other cellular functions necessary for tumor progression (Whang, Y.E. et al. Proc. Natl. Acad. Sci. USA 95:5246-5250 (1998), Rasheed, B.K. et al. Cancer Res. 57:4187-4190 (1997)).
  • PTEN has been shown to be disrupted in a large number of breast and prostate tumor samples (Steck, P. A., et al. Nature Genet. 15:356-362 (1997), Li, J., et al. Science. 275:1943-1946 (1997)).
  • germline mutations in PTEN give rise to Cowden's disease, a disorder characterized by the formation of multiple benign tumors (hamartomas), as well as an increased susceptibility to malignant cancers of the ovary, breast, and thyroid (Mallory, S.B. Derm.
  • the term “substrate specificity” describes the minimum level of interaction between phosphatase and substrate, such that 32 P is released at a minimum of 50 pmol/min/mg phosphatase under conditions described in the Examples.
  • the term “acidic substrate” is intended to include polypeptides comprised of amino acids having acidic functional groups in addition to serine, threonine or tyrosine residues, such that the acidic functional groups are localized near the phosphate group in the three dimensional structure of the substrate, and non- proteinaceous substrates, such as PtdIns(3,4,5)P 3 .
  • PTEN was shown herein to exhibit activity against serine/threonine, as well as tyrosine phosphorylated proteins (the Table and Figures 2A and 2B). PTEN showed a preference for substrates containing acidic residues. Even the best serine/threonine substrates, the ETE (RRREEETEEE, SEQ ID NO: 6) and DSD (RRRDDDSDDD, SEQ ID NO: 5) peptides, were not as efficiently dephosphorylated as polyGlu 4 Tyr, (the Table and Figure 2A).
  • Casein phosphorylated by casein kinase II, was dephosphorylated to a lesser extent by PTEN ( Figure 2B), suggesting that PTEN may require multiple acidic residues positioned both - and C-terminally to the phosphorylated residue.
  • PTEN dephosphorylates PtdIns(3,4,5)P 3 as efficiently as polyGluTyr
  • PTENC124S a mutant PTEN in which the cysteine from the signature catalytic motif is replaced with serine
  • Mutations that occur in PTEN during tumorigenesis typically fall into three general classes: (i) genomic deletions encompassing all or most of PTEN, (ii) frame shift mutations resulting in the production of truncated PTEN proteins and (iii) point mutations resulting in at least one substitution of an amino acid for another (Steck, P. A., et al. Nature Genet. 15:356-362 (1997), Li, J., et al. Science. 275:1943-1946 (1997)). As described herein, a variety of mutations found in tumor samples have been introduced into PTEN. Comparisons of PTEN with other phosphatases, whose crystal structures have been solved, aids in predicting how these point mutations might disrupt PTEN activity.
  • H123Y histidine 123 to tyrosine
  • mutation of glycine 129 to arginine isolated from glioblastoma
  • glycine 129 is not as highly conserved as others found in the catalytic motif, the substitution of the large, charged side chain of arginine, a mutation found in a glioblastoma cell line, for the much smaller glycine, is likely to have a deleterious effect on the overall structure of the phosphate binding loop and is likely to impede the binding of phosphate.
  • Table 1 Specific activity of PTEN measured with tyrosine, serine and threonine phosphorylated substrates.
  • the G165R and the S170R mutations result in substitution of relatively small, uncharged amino acids for a much larger positively charged residue, potentially disrupting important interactions between this ⁇ -helix and surrounding structures. Moreover, the substitution of threonine 167 for proline is also likely to disrupt these interactions by interrupting the proper folding of this ⁇ -helix. These data indicate that this conserved helix ( ⁇ 7 in YopH and VHR) is a required motif in the dual specificity phosphatases.
  • PTEN was tested for its ability to dephosphorylate the MAP kinase ERK2. However, as described herein, PTEN was incapable of dephosphorylating ERK in vitro, strongly suggesting that the MAP kinases are not regulated by PTEN in vivo.
  • PTENC124S A catalytically- inactive mutant of PTEN (PTENC124S), in which the essential, catalytic cysteine from the signature motif was replaced with serine, was unable to dephosphorylate PtdIns(3,4,5)P 3 indicating that the lipid phosphatase activity was not due to a bacterial contaminant.
  • cdcl4 a dual specificity phosphatase closely related to PTEN, was also unable to dephosphorylate PtdIns(3,4,5)P 3 , demonstrating that recognition of this phospholipid substrate is not a general property of other, even closely related, dual specificity phosphatases.
  • PTEN also dephosphorylated inositol(l, 3,4,5) tetrakisphosphate, the polar headgroup of PtdIns(3,4,5)P 3 but at a slower rate.
  • lipid substrate labeled exclusively in the 3-position with 32 P was incubated with PTEN or SHIP, a well characterized 5-phosphatase (Lioubin, M.N. et al. Genes Dev. 70:1084-1095 (1996)). The products of these reactions were analyzed by thin layer chromatography.
  • PTEN-G129E PTP signature motif
  • PTEN-G129R Another mutation (PTEN-G129R), isolated from a glioblastoma, in which the same glycine residue is changed to arginine, exhibited reduced activity when assayed with either PtdIns(3,4,5)P 3 or polyGluTyr.
  • PtdIns(3,4,5)P 3 in response to expression of PTEN-C124S is likely due to the ability of this form of PTEN to behave as a "substrate trapping" mutant, which yields a stable complex with the lipid substrate and protects it from dephosphorylation by endogenous phosphatases (Sun, H. et al. Cell 75:487-493 (1993)).
  • the accumulation of PtdIns(3,4,5)P 3 in the presence of a substrate trapping mutant confirms that PtdIns(3,4,5)P 3 is a physiological target of PTEN ((Sun, H. et al. Cell 75:487-493 (1993), (Garton, A.J. et al. Mol Cell. Biol.
  • PtdIns(3,4,5)P 3 Signaling downstream of PtdIns(3,4,5)P 3 was also assessed by determining the phosphorylation status of PKB/Akt, a kinase whose activation is dependent upon the generation of PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 (Bos, J.L. Trends Biochem. Sci. 20:441- 442 (1995)).
  • Many tumor cell lines have been characterized which have lost PTEN expression through deletion or mutation of the endogenous gene (Steck, P.A. et al. Nature Genetics 15:356-362 (1997), Li, J. et al. Science 275:1943-1946 (1997)).
  • PKB/Akt death effector protein BAD
  • Phosphorylation of BAD by PKB/Akt promotes its dissociation from the BCL ⁇ and its association with 14-3-3, thus blocking the ability of BAD to induce apoptosis. Therefore, phosphorylation of BAD promotes survival in cells expressing BCL ⁇ (Datta, S.R. et al. Cell 91:231-241 (1997)).
  • PTEN is a functional antagonist of signaling events induced by PI 3 -kinase.
  • PI 3 -kinase The activation of PI 3 -kinase has been shown to be required for tumor cell invasion (Shaw, L.M. et al. Cell 97:949-960 (1997)) and both PI 3-kinase and PKB/Akt have been isolated as transforming oncogenes in retroviruses (Bellacosa, A. et al. Science 254: 274-277(1991), Chang, H.W. et al. Science 275:1848-1850 (1997)). These data indicate that PI 3 -kinase and PKB/Akt function in a growth promoting signaling pathway. Our data demonstrates that PTEN functions to suppress these growth promoting signals by dephosphorylating the phospholipid products of PI 3-kinase.
  • the present invention relates to an isolated nucleic acid sequence encoding a PTEN, which dephosphorylates phosphorylated tyrosine residues, phosphorylated serine residues, phosphorylated threonine residues and combinations thereof, and which possesses substrate specificity such that the phosphatase activity is enhanced by the presence of acidic amino acid residues in a substrate.
  • the invention pertains to an isolated nucleic acid sequence which encodes a dual specificity phosphatase PTEN having an alteration in amino acid sequence such that the phosphatase activity is inhibited, and having homology to tensin.
  • the invention also relates to a nucleic acid sequence that encodes a phosphatase capable of removing phospate from the 3-position of inositol and phosphatidylinositol.
  • the invention further relates to nucleic acid sequences which hybridize to nucleic acid sequence encoding PTEN or altered PTEN.
  • the invention also relates to PTEN or altered PTEN encoded by the isolated nucleic acid sequences described herein.
  • alteration is intended to mean any change in nucleic acid sequence that results in, but is not limited to, missense, nonsense, frame shift, addition or deletion mutations or combinations thereof, in the wild type nucleic acid sequence. Alterations in either the nucleotide sequence or the amino acid sequence of the phosphatase include additions, substitutions and deletions of one or more nucleotides or amino acid residues.
  • the terms “dual specificity phosphatase” or “dual specific phosphatase” refer to an enzyme which removes phosphate from phosphorylated tyrosine, phosphorylated serine, phosphorylated threonine and combinations thereof.
  • isolated is used herein to indicate that the material in question (e.g., DNA) exists in a physical milieu distinct from that in which it occurs in nature.
  • the isolated protein of the invention may be substantially isolated with respect to the complex cellular milieu in which it naturally occurs.
  • isolated gene or nucleotide sequence also includes a gene or nucleotide sequence which is synthesized chemically or by recombinant means.
  • recombinant DNA contained in a vector is included in the definition of "isolated”, as used herein.
  • isolated nucleotide sequences include recombinant DNA molecules in heterologous host cells, as well as partially or substantially purified DNA molecules in solution.
  • RNA transcripts of the DNA molecules of the present invention are also encompassed by "isolated" nucleotide sequences.
  • isolated nucleotide sequences are useful in the manufacture of the encoded protein, as probes for isolating homologous sequences (e.g., from other mammalian species), for gene mapping (e.g., by in situ hybridization with chromosomes), or for detecting expression of the PTEN or altered PTEN gene in tissue (e.g., human tissue), such as by Northern blot analysis.
  • Methods of isolating nucleic acid, manufacturing the encoded protein, gene mapping and nucleic acid hybridization are well known in the art.
  • the present invention also pertains to nucleotide sequences which are not necessarily found in nature but encode PTEN or altered PTEN.
  • DNA molecules which comprise a sequence which is different from the naturally-occurring PTEN- encoding or altered PTEN-encoding nucleotide sequence but which, due to the degeneracy of the genetic code, encode PTEN or altered PTEN of the present invention, are the subject of this invention.
  • the invention also encompasses nucleotide sequences which encode portions, analogues or derivatives of PTEN or altered PTEN. These nucleotide sequences can be naturally-occurring, such as in the case of allelic variation, or non-naturally-occurring, such as those induced by various mutagens and mutagenic processes.
  • Suitable variations include, but are not limited to, addition, deletion and substitution of one or more nucleotides which can result in conservative or non- conservative amino acid changes, including additions and deletions.
  • the nucleotide or amino acid variations can be silent or conserved; such that they do not alter the characteristics or activity of PTEN or altered PTEN.
  • nucleic acid sequence is intended to include both double-stranded and single-stranded RNA and DNA polymers having a length sufficient to hybridize to PTEN or altered forms thereof via the sense or anti-sense strand.
  • the nucleic acid molecule comprises at least about 25 nucleotides, more preferably at least about 50 nucleotides, and even more preferably at least about 200 nucleotides.
  • the nucleotide sequence can be only that which encodes at least a fragment of the amino acid sequence of the PTEN protein or altered PTEN protein; alternatively, the nucleotide sequence can include at least a fragment of the PTEN or altered PTEN amino acid coding sequence along with additional non-coding sequences, such as introns and non-coding 3' and 5' sequences (including regulatory sequences, for example). Additionally, the nucleotide sequence can be fused to a marker sequence, for example, a sequence which encodes a polypeptide to assist in isolation or purification of the polypeptide.
  • Such sequences include, but are not limited to, those which encode a glutathione-S-transferase (GST) fusion protein, a hemaglutin A (HA) peptide marker from influenza, a Myc peptide marker from the myc oncogene and a polyhistidine tag.
  • GST glutathione-S-transferase
  • HA hemaglutin A
  • the altered PTEN phosphatase has an alteration within the catalytic domain.
  • the alteration is a point mutation, such as a substitution of tyrosine to histidine at amino acid 123 or a substitution of arginine to glycine at amino acid 129.
  • the encoded PTEN phosphatase has an alteration in alpha helix 2 or in alpha helix 7, wherein alpha helices 2 and 7 are structural motifs defined by the crystal structure of vaccinia HI -related phosphatase (VHR) (Yuvaniyama, J et al Science 272:1328-1331 (1996)).
  • the alteration can be a point mutation, such as a substitution of arginine for serine at amino acid 170, a substitution of arginine for glycine at amino acid 165, and a substitution of proline for threonine at amino acid 167.
  • the altered nucleic acid or amino acid sequences can be obtained by mutagenesis of sequences from natarally-occurring sources, or can be recombinantly produced or synthesized using techniques well known in the art, e.g. by chemical or other methods.
  • the present invention further pertains to antibodies or antigen-binding fragments thereof which specifically bind to PTEN or altered PTEN as described herein, and to nucleic acid probes which hybridize to nucleic acid sequences encoding the altered PTEN phosphatase as described herein.
  • the invention also relates to use of the compositions described herein, such as in assays to measure phosphatase activity.
  • Compositions and methods described herein can be used in the diagnosis, therapy or prophylaxis of a condition characterized by an alteration in PTEN which causes an alteration of phosphatase activity of PTEN in an individual.
  • the method comprises combining a biological sample to be tested (e.g., cells, tissue or bodily fluids) with appropriate reagents for detecting an alteration in PTEN.
  • a biological sample to be tested e.g., cells, tissue or bodily fluids
  • alteration of phosphatase activity is intended to include both inhibition and enhancement of catalytic activity.
  • inhibition includes any detectable reduction in the indicated measure of interest, including reduction of activity and complete loss of activity.
  • Enhancement includes any increase in activity.
  • a sample to be tested can be any biological sample, such as blood, lymph, urine, tissue extracts or cellular extracts.
  • a sample of cells can be bodily fluids, tissues, cell culture samples and purified or non- purified protein samples.
  • the invention relates to a method of diagnosing or aiding in the diagnosis of a condition characterized by an alteration in PTEN which causes an alteration of phosphatase activity of PTEN in an individual.
  • the occurrence (presence or absence and/or quantity) of DNA or RNA encoding PTEN or altered PTEN is detected using hybridization-based techniques.
  • This embodiment of the method comprises the steps of rendering nucleic acids in a sample of cells from the individual available for hybridization with complementary nucleic acids; combining the resulting product with a nucleic acid probe which detects an alteration in nucleic acid sequences encoding an altered form of PTEN which causes an alteration of phosphatase activity of PTEN, under conditions appropriate for hybridization of complementary nucleic acids in the sample of cells with the nucleic acid probe; and detecting hybridization of nucleic acids in the sample of cells with the nucleic acid probe.
  • Appropriate nucleic acid probes are those which hybridize to the altered PTEN sequence and do not hybridize to the wild type PTEN sequence.
  • Hybridization of nucleic acids in the sample of cells with the nucleic acid probes is indicative of a condition characterized by an alteration in PTEN which causes an alteration of phosphatase activity of PTEN.
  • the nucleic acid probe detects an alteration in the catalytic domain, alpha helix 2 or alpha helix 7 of PTEN.
  • the condition is tumorigenesis.
  • nucleic acid probes provided by the present invention can be used to screen by single strand conformation polymorphism (SSCP) (Orita, M. et al. Proc. Natl. Acad. Sci. USA 86:2766-2770 (1989), Suzuki, Y., et al. Oncogene 5:1037-1043 (1990), Murakami, Y., et al Oncogene 6:37-42 (1991)), hybridization, sequencing or PCR for specific mutations which lead to an inhibition of phosphatase activity.
  • SSCP single strand conformation polymorphism
  • the probe can be labeled in a suitable manner for detection of single nucleotide substitution by mobility shift analysis, hybridization between the probe and nucleic acids derived from a suitable sample or for direct sequencing of nucleic acids derived from a suitable sample. Furthermore, PCR products may be detected by standard electrophoresis techniques. These probes can vary in length from 10 nucleotides to more than 200 nucleotides, and will correspond to unique sequences present in PTEN or altered forms thereof.
  • the occurrence of PTEN and/or altered PTEN is detected using antibody techniques well known in the art.
  • This embodiment of the method comprises rendering proteins in a sample of cells from the individual available for binding with antibodies, e.g., by disrupting the cells with an appropriate detergent in a suitable buffer solution; combining the resulting product with an antibody that binds altered PTEN, under conditions appropriate for binding of the antibody to proteins; and detecting antibody binding to altered PTEN in the sample.
  • Binding of the antibody to altered PTEN is indicative of a condition characterized by an alteration in PTEN which causes an alteration of phosphatase activity of PTEN.
  • a composition comprising an antibody is intended to include both the antibody itself and source which express the desired antibody, including any source of monoclonal or polyclonal antibodies that interact specifically with PTEN or altered forms thereof. Sources which expresses the desired antibody, include, but are not limited to, serum, ascites fluid, primary culture, hybridomas, and conditioned media derived from primary culture or hybridomas.
  • Specific antibodies can be used to detect the presence of PTEN or altered forms of the phosphatase with inhibited phosphatase activity, using standard enzyme-linked immunosorbant assay, radioimmunoassay and immunoblot analysis.
  • Specific antibodies of the present invention can also be used for immuno-cytochemistry on cells or tissues.
  • polyclonal and monoclonal antibodies including non-human and human antibodies, humanized antibodies, chimeric antibodies and antigen-binding fragments thereof (Current Protocols in Immunology, John Wiley & Sons, N.Y. (1994); EP Application 173,494 (Morrison); International Patent Application WO86/01533 (Neuberger); and U.S. Patent No.
  • a mammal such as a mouse, rat, hamster or rabbit, can be immunized with an immunogenic form of PTEN or altered PTEN (e.g., PTEN, altered PTEN, an antigenic fragment of PTEN, or an antigenic fragment of altered PTEN comprising the altered portion of the phosphatase, which are capable of eliciting an antibody response).
  • Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or other techniques well known in the art.
  • the protein or polypeptide can be administered in the presence of an adjuvant.
  • the progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibody.
  • antibody as used herein is intended to include fragments thereof, such as Fab and F(ab) 2 .
  • Antibodies described herein can be used to inhibit the activity of the phosphatase described herein, particularly in vitro and in cell extracts, using methods known in the art. Additionally, such antibodies, in conjunction with a label, such as a radioactive label, can be used to assay for the presence of the expressed protein in a cell from, e.g., a tissue sample, and can be used in an immunoabsorption process, such as an ELISA, to isolate PTEN or altered PTEN. Tissue samples which can be assayed include human tissues, e.g., differentiated and non-differentiated cells. Examples include bone marrow, thymus, kidney, liver, brain, pancreas, fibroblasts and epithelium.
  • PTEN486 a peptide fragment of PTEN, PTEN Glu-388-Val403, was used to generate polyclonal, anti-PTEN antiserum in a rabbit.
  • the resultant antibody is designated PTEN486.
  • the invention also pertains to an anti-PTEN antibody, particularly PTEN486, which binds PTEN at the C-terminal region. This antibody can be used to identify and/or isolate both wild type PTEN and altered PTEN, provided that the altered PTEN is not altered such that the C-terminal 16 amino acid residues are deleted.
  • phosphatase activity assays are well known in the art.
  • This embodiment of the method comprises rendering proteins in a sample of cells from the individual available for assessment of phosphatase activity; combining the resulting product with a phosphorylated PTEN substrate, under conditions appropriate for dephosphorylation of the phosphorylated substrate by PTEN; and detecting dephosphorylation of the phosphorylated substrate.
  • PTEN is isolated (i.e., purified) from a crude (non-purified) sample using specific antibody, and utilized in the phosphatase assay as described.
  • a specific substrate of PTEN is utilized in the phosphatase assay described in conjunction with either purified PTEN or a non-purified sample containing PTEN.
  • Inhibition of dephosphorylation relative to an appropriate control or reference sample is indicative of a condition characterized by an alteration in PTEN which causes an alteration of phosphatase activity of PTEN.
  • An appropriate control or reference sample is the amount of dephosphorylation in a cell sample known to have normal (e.g., wild type) PTEN activity. The control or reference sample activity can be determined simultaneously with, prior to or after the assessment of the sample being tested.
  • the present invention provides a method to be used in the diagnosis of cancers, e.g., brain, prostate and breast cancers and Cowden's disease, or in the determination of pre-disposition thereto.
  • cancers e.g., brain, prostate and breast cancers and Cowden's disease
  • condition is intended to include active disorders, e.g., disorders which have manifested their symptoms, and predisposition to a disorder (e.g., the genetic tendency toward a disorder which has not yet manifested itself symptomatically).
  • This invention also provides a method to diagnose conditions involving altered forms of PTEN, for example using phosphatase assays which combine suitable substrates, such as PtdIns(3,4,5)P 3 or phosphorylated polyGlu graspTyr ⁇ , with tissue, cellular, or protein samples in an appropriate assay as described herein, wherein inhibition of phosphatase activity is indicative of an alteration in PTEN.
  • suitable substrates such as PtdIns(3,4,5)P 3 or phosphorylated polyGlu swipeTyr ⁇
  • the present invention may be used in the treatment of conditions in which enhancement or inhibition of PTEN phosphatase activity is desirable.
  • the cells are contacted with a nucleic acid molecule encoding PTEN on an altered form thereof.
  • contacting means adding the PTEN expressing construct to the extracellular space of the cells, transfecting the cells with a PTEN expressing nucleic acid construct or targeting said construct to specific cell types, such as PTEN deficient cells. Methods of adding, transfecting and targeting are well known in the art.
  • wild type PTEN or a nucleic acid encoding wild type PTEN, or a mimic thereof may be administered in an appropriate vehicle, with an optional physiological composition to an individual having a condition characterized by lack of PTEN phosphatase activity (leading to a condition such as a hyper-proliferative condition).
  • the condition is alleviated due to the activity of the introduced PTEN.
  • the hyperproliferative condition may include brain, prostate or breast cancers in addition to Cowden's disease and other hyperproliferative diseases involving reduced PTEN phosphatase activity.
  • altered forms of PTEN, or a nucleic acid sequence encoding altered forms of PTEN may be administered in an appropriate vehicle with an optional physiological composition to an individual in whom an increase in PTEN phosphatase activity is desirable, such as, for example, to induce apoptosis in proliferative or hyperproliferative cells. Methods of measuring apoptosis are well known in the art.
  • altered forms of PTEN, or a nucleic acid sequence encoding altered forms of PTEN may be administered in an appropriate vehicle with an optional physiological composition to an individual in whom reduction or attenuation of PTEN phosphatase activity is desirable. Administration alleviates the condition, which can include disorders such as degenerative conditions.
  • the altered forms of PTEN or nucleic acids encoding the altered forms of PTEN may derived from the altered forms described herein, including a "substrate trapping" form of PTEN (as described in U.S. patent application Serial No. 08/685,992, filed July 25, 1996).
  • the altered PTEN is active in cells whereby the endogenous phosphatase and the introduced, substrate trapping PTEN share a common substrate, such that the overall activity of the phosphatase is reduced due to the sequestration of the substrate.
  • the degenerative condition may include Parkinson's and other neurodegenerative diseases which may involve PTEN phosphatase activity.
  • the present invention also pertains to pharmaceutical compositions comprising polypeptides described herein.
  • a polypeptide or protein, or product thereof, of the present invention can be formulated with a physiologically acceptable medium to prepare a pharmaceutical composition.
  • the particular physiological medium may include, but is not limited to, water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol) and dextrose solutions.
  • the optimum concentration of the active ingredient(s) in the chosen medium can be determined empirically, according to well known procedures, and will depend on the ultimate pharmaceutical formulation desired.
  • Methods of introduction of exogenous peptides at the site of treatment include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, oral and intranasal. Other suitable methods of introduction can also include gene therapy, rechargeable or biodegradable devices and slow release polymeric devices.
  • the present invention is related to use of PTEN or a nucleic acid molecule encoding PTEN, for the manufacture of a medicament for use in a method of treating a condition in an individual in which enhancement of PTEN phosphatase activity is desirable.
  • the present invention is also related to use of an altered form of PTEN or a nucleic acid molecule encoding an altered form of PTEN, for the manufacture of a medicament for use in a method of treating a condition in an individual in which reduction of PTEN phosphatase activity is desirable.
  • the present invention is further related to use of PTEN or a nucleic acid molecule encoding PTEN, for the manufacture of a medicament for use in a method of treating prostate cancer.
  • the pharmaceutical compositions of this invention can also be administered as part of a combinatorial therapy with other agents.
  • Example 1 Isolation and Purification of PTEN.
  • a full length PTEN cDNA was generated by ligating the Notl-Bglll fragment from EST264611 with the Bglll-EcoRl fragment of EST365465 into pBluescript digested with Notl -EcoRI.
  • the resulting full length PTEN cDNA was amplified by PCR using pfu polymerase (Stratagene) and primers that add a 5' BamHI site (5'CGCGGATCCATGACAGCCATCATCAAAGAGATCGTTAGC) (SEQ ID NO: 3) and a 3' EcoRI site (5'CGCGAATTCTCAGACTTTTGTAATTTGTGTATGC) (SEQ ID NO: 4).
  • the resulting fragment was subcloned into pGEX2T (Pharmacia) and the sequence verified by automated sequencing.
  • the frozen pellets were resuspended in 5 ml of ice cold 20 mM Tris, 150 mM NaCl and 5 mM EDTA, pH 8.0 supplemented with lysozyme (1 mg/ml), aprotinin ( ⁇ g/ml), leupeptin (5 ⁇ g/ml), and benzamidine (1 mM) and incubated on ice for 15 minutes.
  • the bacteria were lysed by sonicating 3 times for 1 minute each with a Branson Model 450 sonifier, power setting 4, 70% duty cycle.
  • the lysate was cleared by centrifugation at 30,000 x g for 10 minutes, diluted with an equal volume of HBS (50 mM Hepes, 150 mM NaCl, pH 7.4).
  • Glutathione-Sepharose 4B 300 ⁇ L was added, and the resulting slurry was incubated at 4 °C on a rocking platform for 1-2 hours.
  • the glutathione-Sepharose was washed 5 times, each with 10 ml of ice cold HBS and the washed fusion proteins were eluted with a solution containing 20 mM glutathione, 50 mM Hepes and 30% glycerol, pH 8.0.
  • Protein concentrations were determined by the method of Bradford, using BSA as a standard, and the integrity of the fusion proteins were verified by SDS-polyacrylamide gel electrophoresis. Mutations identified from tumor samples and cell lines (4,5 and Dr. L. Hedrick (Johns Hopkins University) personal communication) were introduced into pGEX2T-PTEN using the Quickchange mutagenesis kit as described by the manufacturer (Stratagene). For all mutations, the entire PTEN open reading frame was sequenced to confirm that no other mutations had been introduced. The DNA sequence and translation are shown in Figures 1 A-IC.
  • tyrosine phosphorylated substrates were phosphorylated with the cytoplasmic fragment of the ⁇ subunit of the insulin receptor kinase ( ⁇ IRK) and purified as described (Flint, A.J. et al. Embo J. 12:1937-1946 (1993)).
  • Serine phosphorylated substrates were phosphorylated with recombinant protein kinase A (New England Biolabs) or with recombinant casein kinase II (a gift from Dr. D.
  • Protein substrates were precipitated by the addition of ammonium sulfate to 80%, incubated on ice for 30 minutes and harvested by centrifugation. the precipitated proteins were washed 3 times with 80% ammonium sulfate and then resuspended in 500 ⁇ l of 1 M Hepes, pH 7.5. The solubilized proteins were dialyzed against several changes of 50 mM Imidizole, pH 7.2. Peptide substrates were purified using a Sep-pak C18 reverse phase cartridge (Waters) as described (Myers, MP et al. Mol. Cell Biol. 14:6954-6961 (1994)).
  • the purified peptides were lyophilized to dryness and resuspended in 50 mM Imidizole, pH 7.2. Random copolymers of glutamate and tyrosine, with a 4:1 ratio of glutamate to tyrosine or with a 1 :1 ratio (polyGluiTyr,), were purchased from Sigma and phosphorylated with ⁇ IRK at a final polymer concentration of 1 mg/ml and purified, as described, using Sep-pak C18 reverse phase chromatography. Phosphorylated ERK2 was produced in E. coli by co-expression of activated MEK and was a gift from Dr. D. Barford (Oxford University). Phosphatase Assays
  • the standard phosphatase assay contained 10 ⁇ M substrate, 50 mM Hepes, pH 7.0, 10 mM MgCl 2 , 10 mM DTT.
  • the reaction was initiated by the addition of enzyme, typically 1-2 ⁇ g, to prewarmed (30°C) substrate mix, resulting in a final volume of 60 ⁇ l.
  • the reactions were allowed to proceed at 30 °C for the indicated times and stopped by the addition of a suspension of activated charcoal in 900 mM HC1, 90 mM NaPPi and 2 mM NaPi (Zhang, S.-H., et al. J. Biol. Chem. 270:20067-20072 (1995)).
  • PTEN was assayed against a number of tyrosine phosphorylated proteins and peptides, including reduced carboxyamidomethylated and maleylated lysozyme (RCML), mylelin basic protein (MBP), polyGluTyr, and polyGlujTyr, as well as the peptide EDNDYINASL.
  • RCML reduced carboxyamidomethylated and maleylated lysozyme
  • MBP mylelin basic protein
  • polyGluTyr polyGluTyr
  • polyGlujTyr polyGlujTyr
  • PTEN activity was assayed using a number of proteins and peptides phosphorylated on serine and threonine residues. Similar to the findings with tyrosine phosphorylated substrates, PTEN dephosphorylated serine/threonine residues in substrates that had a preponderance of acidic residues. Specifically, PTEN dephosphorylated two peptide substrates (DSD and ETE) with the highest efficiency (Table and Figure 2B).
  • PTEN showed specificity even amongst acidic serine/threonine substrates, exhibiting a reduced activity when casein, phosphorylated by casein kinase II or protein kinase A, was used as substrate (Table and Figure 2B). As might be anticipated in light of these properties, PTEN exhibited almost undetectable activity when assayed with polybasic substrates, such as MBP or Kemptide (LRRASLG). The finding that PTEN dephosphorylated two peptide substrates of casein kinase II also indicates that the inability of PTEN to dephosphorylate EDNDYINASL does not simply reflect its inability to dephosphorylate small peptide substrates.
  • PTEN Glu388-Val403 (Cyj-Glu-Asn-Glu-Pro-Phe-Asp-Glu- Asp-Gln-His-Thr-Gln-Thr-Lys-Val)
  • PTEN Lys6-Aspl9 (Lys-Glu-Ile-Val-Ser-Arg- Asn-Lys-Arg-Arg-Tyr-Gln-Glu-Asp-Cy )
  • PTEN Ser226-Lys237 (Cy -Ser-Ser-Asn- Ser-Gly-Pro-Thr-Arg-Arg-Glu-Asp-Lys)
  • PTEN Thr321-Lys332 (Cy_s-Thr-Lys-Asn- Asp-Leu-Asp-Lys-Ala-Asn-Lys-Asp-Lys).
  • PTEN486 Rabbit 486, which was injected with PTEN Glu388-Val403 produced antibodies that recognized endogenous PTEN on immunoblots and in immunoprecipitations (this antibody will be referred to as PTEN486). All other rabbits failed to produce antibodies that could recognize PTEN on immunoblots or in immunoprecipitations.
  • the pooled upper phases (containing inorganic phosphate) were removed, dried down and resuspended in a 1M TCA, 1% ammonium molybdate solution. Following extraction with 2 volumes of toluene ⁇ sobutylalchohol (1 : 1) the upper phase was removed and counted. Site selectively was determined by incubating recombinant PTEN or SHIP (a gift from C. Erneux, Free University, Brussels) with radiolabeled PtdIns(3,4,5)P 3 and the lipid products from these reactions were analyzed by thin layer chromatography or HPLC.
  • the phospholipids were extracted by performing a Bligh and Dyer extraction and the pooled lower phases were dried down and resuspended in 20 ⁇ l of chloroform:methanol (2:1) and applied to an oxalate activated silica gel TLC plate. Plates were developed in methanol chlorofo ⁇ n/ water/ammonia (100:75:25:15) and the phospholipids were detected by autoradiography. PtdIns(3,4,5)P 3 mass assays were performed as described (van der Kaay, J. etal. J. Biol. Chem. 272: 5477-5481 (1997)).
  • Figure 5 demonstrates that PTEN dephosphorylates PtdIns(3,4,5)P 3 .
  • panel A recombinant PTEN was incubated with radiolabeled polyGluTyr and PtdIns(3,4,5)P 3 and the release of 32 P j was measured as described.
  • panel B PtdIns(3,4,5)P 3 , PtdIns(3,4,)P 2 , or PtdIns(3)P were incubated with PTEN and the release of 32 P, was determined as described.
  • HEK293 cells were transfected via calcium phosphate co-precipitation with 20 ⁇ g of DNA per 10 cm dish. The calcium phosphate:DNA co-precipitate was removed by washing with PBS 16 hours after addition and the cells were returned to growth medium for 36 hours before harvesting. Co-transfection of PTEN and pi 10 constructs were performed using 9.5 ⁇ g of each plasmid DNA. Transfection efficiency was determined by including a GFP expression plasmid (1 ⁇ g) in all transfections.
  • Figure 6 demonstrates that expression of PTEN antagonizes PI 3-kinase.
  • HEK293 cells were transfected with PTEN or PTENC124S in combination with pi 10 PI 3 -kinase or an activated, membrane bound PI-3 kinase (pi 10-CAAX).
  • the resulting levels of PtdIns(3,4,5)P 3 were determined as described and were normalized to total protein. Data is expressed as pmol PtdIns(3,4,5)P 3 per mg protein.
  • Example 8 Expression of PTEN in Glioblastoma Cell Lines Decreases the Amount of Activated PKB/Akt
  • PTEN retroviral expression vectors were constructed in pBabePuro. Following transfection into packaging lines, the viral supernatants were harvested, diluted with growth media (described above) and incubated with U87 MG or U373 MG cells for 16 hours at 30° C. Infected cells were selected with puromycin (1.5 ⁇ g/ml) and drug resistant colonies were expanded to generate clonal cell lines.
  • PTEN expression was reconstituted in glioblastoma cell lines U87 MG, or U373 MB, by infecting with recombinant retrovirus.
  • Confluent dishes were either left untreated, stimulated with insulin (lO ⁇ g/ml for 10 minutes), stimulated with PDGF (50 ng/ml for 10 minutes) or pretreated with Wormtannin (150 nM for 30 minutes) and then stimulated with both insulin (lO ⁇ g/ml) and PDGF (50 ng/ml) for 10 minutes and then lysed.
  • LnCaP cells a PTEN deficient prostate cancer cell line
  • cationic lipids Transfast, Promega
  • DNA:lipid ratio 1.
  • Cells were incubated with the DNA:lipid complexes for 12 hours and then transfected cells were identified based on the expression of a co-transfected GFP vector or by performing immunofluorescence using antibodies to the HA-epitope tag located at the N-terminus of PTEN essentially as described (Tiganis, T. et l. Mol. Cell Biol. 75:1622-1634 (1998)). Apoptotic cells were detected by staining with Annexin V (Clontech).
  • Cells were washed with binding buffer (20 mM Hepes, pH 7.4, 137 mM NaCl, 2 mM CaCl 2 ) and then incubated for 30 minutes with Annexin V (500 ng/ml) in the same buffer. Cells were washed twice with binding buffer and then fixed with 4% paraformaldehyde (in binding buffer) for 30 minutes at 4° C and then processed for immunofluorescence.
  • binding buffer 20 mM Hepes, pH 7.4, 137 mM NaCl, 2 mM CaCl 2
  • Annexin V 500 ng/ml
  • FIG. 7 demonstrates that expression of PTEN induces apoptosis in LnCaP cells.
  • PTEN, PTENC124S, or PTENG129E were co-transfected with a green flourescent protein (GFP) expression vector into LnCaP cells.
  • the co-transfections also included either empty vector or a constitutively active PKB/Akt expression vector.

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Abstract

L'invention concerne des protéines PTEN, des protéines PTEN modifiées et des molécules d'acides nucléiques codant ces protéines ou ces protéines modifiées. L'invention se rapporte en outre à des procédés de diagnostic et de traitement, par exemple, du cancer de la prostate, qui utilisent des compostions renfermant des PTEN, des PTEN modifiées, ou bien des molécules d'acides nucléiques codant ces PTEN ou ces PTEN modifiées.
PCT/US1998/014205 1997-07-08 1998-07-08 Phosphatase a double specificite et procedes d'utilisation WO1999002704A2 (fr)

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WO2000060092A3 (fr) * 1999-04-07 2001-01-04 Ceptyr Inc Phosphatase a specificite double dsp-3
WO2001002582A1 (fr) * 1999-07-02 2001-01-11 Ceptyr, Inc. Phosphatase dsp-3 a specificite double
US6248327B1 (en) 1998-09-11 2001-06-19 Vanderbilt University Modulation of endothelial cell surface receptor activity in the regulation of angiogenesis
US6284538B1 (en) 1999-07-21 2001-09-04 Isis Pharmaceuticals, Inc. Antisense inhibition of PTEN expression
WO2001012819A3 (fr) * 1999-08-13 2002-01-24 Sugen Inc Nouvelles phosphatases de proteines et diagnostic et traitement des troubles lies a la phosphatase
WO2001073060A3 (fr) * 2000-03-24 2002-04-04 Millennium Pharm Inc 18221, nouveau phosphatase a specificite double et ses utilisations
EP1289472A2 (fr) * 2000-05-30 2003-03-12 Advanced Research & Technology Institute Compositions et methodes d'identification d'agents modulateurs de la fonction pten et des mecanismes de la pi-3 kinase
KR20040018598A (ko) * 2002-08-23 2004-03-04 코웰창업투자(주) Ｔａｔ-ＰＴＥＮ 융합 단백질, 이의 제조 방법 및 용도
WO2004074459A2 (fr) * 2003-02-19 2004-09-02 Georgetown University Cellules deficientes en pten et utilisations de celles-ci
EP1567860A2 (fr) * 2002-11-05 2005-08-31 The Regents Of The University Of California Procedes, substances et materiel permettant d'examiner des voies associees a la progression de glioblastome
US7078210B2 (en) 1999-07-02 2006-07-18 Ceptyr, Inc. DSP-3 dual-specificity phosphatase
EP2181704A2 (fr) 2002-12-30 2010-05-05 Angiotech International Ag Liberation de medicaments a partir d'une compostion polymere a gelification rapide
WO2011044701A1 (fr) * 2009-10-16 2011-04-21 The University Of British Columbia Compositions d'inhibiteurs de phosphatase et d'homologue de tensine (pten), utilisations et procédés

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NZ320425A (en) * 1995-10-23 2001-03-30 Imp Cancer Res Tech Diagnosis of susceptibility to cancer using nucleic acid probes
US6262242B1 (en) * 1997-01-30 2001-07-17 Board Of Regents, The University Of Texas System Tumor suppressor designated TS10Q23.3
CA2280097A1 (fr) * 1997-02-07 1998-08-13 The Trustees Of Columbia University In The City Of New York Utilisation du gene suppresseur de tumeur p-ten dans le diagnostic et le traitement de cancer

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US6248327B1 (en) 1998-09-11 2001-06-19 Vanderbilt University Modulation of endothelial cell surface receptor activity in the regulation of angiogenesis
WO2000060092A3 (fr) * 1999-04-07 2001-01-04 Ceptyr Inc Phosphatase a specificite double dsp-3
US7078210B2 (en) 1999-07-02 2006-07-18 Ceptyr, Inc. DSP-3 dual-specificity phosphatase
WO2001002582A1 (fr) * 1999-07-02 2001-01-11 Ceptyr, Inc. Phosphatase dsp-3 a specificite double
WO2001002581A1 (fr) * 1999-07-02 2001-01-11 Ceptyr, Inc. Phosphatase dsp-3 a double specificite
EP1772517A3 (fr) * 1999-07-02 2007-04-18 Ceptyr, Inc. Phosphatase DSP-3, à double spécificité
EP1772517A2 (fr) * 1999-07-02 2007-04-11 Ceptyr, Inc. Phosphatase DSP-3, à double spécificité
US6284538B1 (en) 1999-07-21 2001-09-04 Isis Pharmaceuticals, Inc. Antisense inhibition of PTEN expression
WO2001012819A3 (fr) * 1999-08-13 2002-01-24 Sugen Inc Nouvelles phosphatases de proteines et diagnostic et traitement des troubles lies a la phosphatase
WO2001073060A3 (fr) * 2000-03-24 2002-04-04 Millennium Pharm Inc 18221, nouveau phosphatase a specificite double et ses utilisations
US6664089B2 (en) 2000-03-24 2003-12-16 Millennium Pharmaceuticals, Inc. 38692 and 21117, novel dual specificity phosphatase molecules and uses therefor
WO2001073059A3 (fr) * 2000-03-24 2002-06-20 Millennium Pharm Inc 38692 et 21117, nouvelles molecules de phosphatase a double specificite et leurs utilisations
EP1289472A4 (fr) * 2000-05-30 2004-09-08 Advanced Res & Tech Inst Compositions et methodes d'identification d'agents modulateurs de la fonction pten et des mecanismes de la pi-3 kinase
EP1289472A2 (fr) * 2000-05-30 2003-03-12 Advanced Research & Technology Institute Compositions et methodes d'identification d'agents modulateurs de la fonction pten et des mecanismes de la pi-3 kinase
KR20040018598A (ko) * 2002-08-23 2004-03-04 코웰창업투자(주) Ｔａｔ-ＰＴＥＮ 융합 단백질, 이의 제조 방법 및 용도
EP1567860A4 (fr) * 2002-11-05 2006-05-10 Univ California Procedes, substances et materiel permettant d'examiner des voies associees a la progression de glioblastome
EP1567860A2 (fr) * 2002-11-05 2005-08-31 The Regents Of The University Of California Procedes, substances et materiel permettant d'examiner des voies associees a la progression de glioblastome
EP2181704A2 (fr) 2002-12-30 2010-05-05 Angiotech International Ag Liberation de medicaments a partir d'une compostion polymere a gelification rapide
WO2004074459A3 (fr) * 2003-02-19 2006-05-26 Univ Georgetown Cellules deficientes en pten et utilisations de celles-ci
WO2004074459A2 (fr) * 2003-02-19 2004-09-02 Georgetown University Cellules deficientes en pten et utilisations de celles-ci
WO2011044701A1 (fr) * 2009-10-16 2011-04-21 The University Of British Columbia Compositions d'inhibiteurs de phosphatase et d'homologue de tensine (pten), utilisations et procédés
CN102666845A (zh) * 2009-10-16 2012-09-12 不列颠哥伦比亚大学 磷酸酶和张力蛋白同系物（pten）抑制剂组合物，用途以及方法
CN102666845B (zh) * 2009-10-16 2015-06-10 不列颠哥伦比亚大学 磷酸酶和张力蛋白同系物(pten)抑制剂组合物,用途以及方法
CN104740614A (zh) * 2009-10-16 2015-07-01 不列颠哥伦比亚大学 磷酸酶和张力蛋白同系物(pten)抑制剂组合物,用途以及方法
US9127079B2 (en) 2009-10-16 2015-09-08 The University Of British Columbia Inhibitors of phosphatase and tensin homolog (PTEN) conjugates
US10258666B2 (en) 2009-10-16 2019-04-16 The University Of British Columbia Inhibitors of phosphatase and tensin homolog (PTEN) compositions, uses and methods

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