WO2004074459A2 - Pten-deficient cells and their uses - Google Patents
Pten-deficient cells and their uses Download PDFInfo
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- WO2004074459A2 WO2004074459A2 PCT/US2004/005179 US2004005179W WO2004074459A2 WO 2004074459 A2 WO2004074459 A2 WO 2004074459A2 US 2004005179 W US2004005179 W US 2004005179W WO 2004074459 A2 WO2004074459 A2 WO 2004074459A2
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Classifications
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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)
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- 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
- C07K14/4747—Apoptosis related proteins
Definitions
- PTEN gene encodes a lipid dual-specificity phosphotase and is the major 3- phosphatase in the phosphoinositol-3-kinase (PI3K)/AKT pro-apoptotic pathway (Li and Sun, 1997, Maehama and Dixon, 1998; Stambolic et al. 1998).
- PI3K phosphoinositol-3-kinase
- PTEN Located on the short arm of chromosome 10 (10q23), PTEN is mutated in 40-50% of high grade gliomas, as well as many other tumor types, including those of the prostate, endometrium, breast, and lung (Li et al, Science 1997, Steck et al 1997, Maier et al 1998).
- PTEN is mutated in several rare autosomal dominant cancer predisposition syndromes, including Cowden disease, Lhermitte-Duclos disease and Bannayan-Zonana syndrome (Liaw et al 1997, Myers et al AJHG 1997, Maehama et al TCB 1999, Cantley and Neel 1999). Furthermore, the phenotype of PTEN- knockout mice revealed a requirement for PTEN in normal development and confirmed its role as a tumor suppressor (Podsypanina et al, 1999, Suzuki et al, 1998, Di Christofano et al, 1998).
- PTEN has been implicated in important cellular processes such as cell cycling, translation, and apoptosis.
- PTEN has been shown to regulate the phosphoinositol-3-kinase (PI3K)/AKT pro- apoptotic pathway (Li and Sun, 1997, Maehama and Dixon, 1998; Stambolic et al. 1998).
- PTEN has also been shown to regulate the mitogen-activated kinase (MAPK) pathway, which is critical for proliferation and differentiation (Ong et al. 2001; Takahashi-Tezuka et al. 1998; Yart et al. 2001).
- MAPK mitogen-activated kinase
- PTEN-def ⁇ cient human cells that are either heterozygous (PTEN “1” " ) or homozygous (PTEN " " ) for a PTEN mutation.
- PTEN-deficient cells result from targeted disruption of at least one PTEN gene via somatic cell gene targeting.
- the present invention relates to an isolated recombinant human somatic cell, and progeny thereof, in which at least one endogenous PTEN allele comprises a mutation that renders the PTEN allele nonfunctional.
- the invention encompasses cells heterozygous for such a PTEN mutation, as well as cells homozygous for such a PTEN mutation.
- PTEN mutations include both loss-of-function mutations, such as deletions, and gain-of-function mutations, such as dominant negative mutations and constitutive mutations.
- One embodiment of the present invention is a pair of isogenic human somatic cells in which one member is PTEN-deficient and the other member has at least two functional endogenous PTEN alleles, or otherwise possesses no non- functional PTEN alleles.
- the isogenic pair of cells of the invention offers the advantage of clonal homogeneity, ability to manipulate the external environment according to needs, and ease of biochemical investigations. Since PTEN is a tumor suppressor, the isogenic pair of the invention provides a well-defined and easily manipulated system to identify novel anti-cancer agents.
- the present invention also relates to a somatic cell gene targeting vector for mutating PTEN allele to produce a non-functional PTEN allele, and a gene targeting method for generating a human somatic cell with a mutation in at least one endogenous PTEN allele.
- the present invention also relates to methods of screening for anti-cancer agents using isogenic pairs of the invention. Since PTEN is a tumor suppressor gene, agents that selectively inhibit the growth of a PTEN-deficient cell but not its isogenic control cell can be valuable anti-cancer agents.
- the invention also contemplates a screening method for agents that selectively cause death of a PTEN- deficient cell but not an isogenic control cell.
- FIG. 1 PTEN gene targeting.
- A The targeting vector deletes ex on II of PTEN and replaces it with a promoterless, IRES-neo R gene flanked by LoxP sites. The positions of the EcoR I restriction sites (filled arrowheads) and PCR primers (arrows) used for screening of knockouts is indicated.
- B Confirmation of PTEN targeting by Southern blot analysis. Fragments corresponding to the wild-type allele and the targeted allele before and after cre-mediated excision of IRES- neo R are shown.
- C Western blot analysis of isogenic PTEN + + and PTEN " _ cells. Parental HCT116 cells and a neo R clone resulting from random integration of the targeting vector are compared to two independently-derived PTEN " " clones. Immunoblotting was performed with the antibodies indicated.
- FIG. 1 A PTEN-dependent, radiation-induced cell size checkpoint.
- A Light microscopy of isogenic PTEN 474" and PTEN 7" cells. Exponentially growing, unirradiated, PTEN " " cells (b) are morphologically indistinguishable from isogenic PTEN +/+ cells (a). Exposure to gamma radiation (IR) followed by six days of culture results in a dramatic increase in the size of PTEN " " cells (d) compared to identically treated PTEN 174" cells (c). Scale bars represent 100 ⁇ m.
- B Electron micrographs of irradiated PTEN 74" (a,b,c) and PTEN " cells (d,e,f). Scale bar represents 5 ⁇ m.
- Three PTEN 4"74" cell lines (parental HCT116 cells and two neoR clones generated by non-homologous integration of the targeting vector) and two independently-derived PTEN " clones (KO #1 and KO #2) were measured during exponential growth (a) and six days after irradiation (b).
- FIG. 3 Checkpoint restoration in PTEN 7" cells.
- PTEN 4" + and PTEN 7" cells were treated with 580 nM wortmannin (A) or 5 nM rapamycin (B), irradiated (6 Gy), cultured for six days, and measured.
- C LNCaP cells (PTEN 7" ) were treated with 10 ⁇ m LY294002, irradiated (6 Gy), cultured for three days, and measured.
- FIG. 4 Integrity of radiation-induced Gl and G2 checkpoints in PTEN "7" cells.
- A Flow cytometry of PTEN 4" + and PTEN 7" cells 24 hours after treatment with 6 Gy.
- B BrdU incorporation assays of untreated or irradiated (9 Gy) PTEN 474" and PTEN " " cells. The percentage of cells in S phase is indicated in the upper right corner of each panel.
- C Mitotic indices of HCT 116 PTEN 474" ( ⁇ ) and PTEN 7" cells ( ⁇ ) at the indicated times following 9 Gy, or 9 Gy plus nocodazole.
- FIG. 5 Radiosensitivity of PTEN " cells, and model.
- A Clonogenic survival assay of HCT116 PTEN 474" and PTEN 7" cells. Equivalent numbers of four PTEN 47 " clones ( ⁇ ) and four independently-derived PTEN 7" clones ( ⁇ ) were treated with various doses of gamma radiation. Each experiment was performed at least in triplicate, and the values of all four PTEN 4" 4" or PTEN " " cell lines were averaged after adjusting for plating efficiency. The mean number of colonies ⁇ SEM is shown.
- B Model of radiation-induced, PTEN-dependent cell size checkpoint. Treatment of human cells with radiation leads to Gl and G2 cell cycle arrests, and a simultaneous arrest in cell size. Deletion of p53 or p21 abolishes the Gl and G2 arrests, whereas deletion of PTEN abolishes the arrest in cell size.
- FIG. 1 Identification of PTEN knockouts by PCR. Homologous integration deletes an endogenous PCR priming site and moves it to a new location, causing the wild-type and targeted alleles of PTEN to generate PCR products of distinct sizes.
- FIG. 7 Crystal violet-stained colonies from the clonogenic survival assay shown in Fig. 5 A. PTEN 4"74" flasks (top row) have at least an order of magnitude more colonies than PTEN " " flasks (bottom row).
- the invention provides cells with mutation(s) in endogenous PTEN allele(s) introduced by gene targeting in the cells or ancestors thereof.
- the present invention encompasses recombinant cells produced as described herein (including any cells produced during gene targeting) and progeny thereof.
- the cells can be either homozygous (PTEN "7” ) or heterozygous (PTEN 4"7” ) for the PTEN mutation(s).
- a mutation can be any change in the sequence of the PTEN gene that alters PTEN function or activity.
- a mutation can be a deletion, an insertion, a frame shift or a point mutation.
- the invention relates to cells with PTEN mutations that render the PTEN allele non-functional by reducing or preventing PTEN expression.
- the reduction of PTEN activity can be either complete or partial.
- a non-functional PTEN allele may be a PTEN mutation that reduces PTEN expression, partially or completely, resulting in reduced levels of PTEN protein product.
- the reduction of PTEN expression can be at the transcriptional level and/or at the translational level.
- a non-functional PTEN allele can be a PTEN mutation that results in production of a PTEN protein with completely or partially reduced PTEN activity; such a protein can be expressed at normal or reduced levels.
- the cells of the present invention contain a deletion of all or a portion of an exon, such as exon 2, of the PTEN gene, resulting in reduced (partially or completely) PTEN protein expression level.
- cells comprise PTEN mutations that are gain-of-function mutations, such as a dominant negative mutation or a constitutive mutation.
- Cells used for gene targeting may be normal cells or tumor cells. They may be derived from a cell line or may be freshly isolated from a human or an animal.
- tumor cells used for gene targeting are selected from a group consisting of a glioblastoma multiforme cell, a colon adenocarcmoma cell, a malignant melanoma cell, an endometrial carcinoma cell, a prostate adenocarcmoma cell, a thyroid cancer cell and a breast cancer cell.
- primary colorectal tumor cells or colorectal tumor cell lines such as HCT 116, DLD1 and SW480, are used.
- the present invention also provides an isogenic pair of cells, which differ in their endogenous PTEN alleles.
- the first member of the pair referred to as the PTEN-deficient member or cell
- the second member referred to as the isogenic control member or cell
- the PTEN-deficient member has a deletion of all or a portion of an exon, such as exon 2, of the PTEN gene.
- the PTEN-deficient cell is homozygous for PTEN deletions. Any means known in the art to generate a cell line with targeted mutation can be used to produce cells of the present invention.
- PNS positive-negative selection
- the second method is a positive selection in genetic terms: it selects for recombination at the correct (homologous) locus by relying on the use of a positively selectable gene whose expression is made conditional on recombination at the homologous target site.
- the disclosure of Sedivy and Dutriaux is incorporated herein.
- the targeting vector comprises: (a) a first region of nucleic acids homologous to a region of PTEN gene, or to a region immediately upstream of the PTEN gene, of sufficient length to undergo homologous recombination with the region of an endogenous PTEN gene (referred to as the first homology arm); (b) nucleic acids encoding a selectable marker; and (c) a second region of nucleic acids homologous to a region of PTEN gene, or to a region immediately downstream of the PTEN gene, of sufficient length to undergo homologous recombination with the region of an endogenous PTEN gene (referred to as the second homology arm).
- the first homology arm comprises at least 800 nucleotides homologous to a region of the second intron of the PTEN gene and the second homology arm comprises at least 800 nucleotides homologous to a region of the third intron of the PTEN gene.
- the targeting vector comprises: (a) at least 800 nucleotides homologous to a region of the second intron of the PTEN gene; (b) a first lox site; (c) an internal ribosome entry site (IRES); (d) nucleic acids encoding a selectable marker; (e) a second lox site; and (f) at least 800 nucleotides homologous to a region of the third intron of the PTEN gene.
- the first and second lox sites may be loxP sites or derivatives thereof. This kind of targeting vector is referred to as a lox-containing targeting vector.
- the selectable marker can be any antibiotic resistance gene, such as a neomycin resistance gene.
- the first homology arm comprises 1665 nucleotides homologous to a region immediately upstream of the second exon of the PTEN gene
- the second homology arm comprises 2549 nucleotides homologous to a region beginning at nucleotide 409 of the third intron the PTEN gene.
- the length of the homology arms can be varied depending on, for example, the region(s) of the PTEN gene to be deleted.
- the arms can be the same length or different.
- the arm can be 800-1200, 1200-1600, 1600-2000, 2000- 2400, 2400-2800, or 2800-3200 nucleotides in length.
- Another embodiment of the invention relates to methods of generating targeted mutations in at least one endogenous PTEN allele by gene targeting.
- the method comprises: (a) transfecting human somatic cells with a targeting vector of the present invention, thereby producing transfected human somatic cells; and (b) maintaining the transfected cells under conditions appropriate for integration of the targeting vector into endogenous PTEN allele(s) in the transfected cells, thereby producing cells in which the targeting vector is integrated in at least one endogenous PTEN allele.
- the cells thus produced can be subjected to another round of gene targeting using a second targeting vector with a selectable marker different from the marker in the first targeting vector, to produce cells having targeting vectors integrated in both endogenous PTEN alleles.
- the targeting method of the present invention makes use of Cre-lox system.
- the method comprises: (a) transfecting human somatic cells with a lo)t-containing targeting vector of the invention thereby producing transfected human somatic cells; (b) maintaining the transfected cells thus produced under conditions appropriate for integration of the targeting vector into an endogenous PTEN allele, thereby producing cells having the lox targeting vector integrated in one endogenous PTEN allele; (c) providing the cells having the lox targeting vector integrated in one endogenous PTEN allele with Cre; (d) maintaining the Cre-containing cells produced by step (c) under conditions appropriate for Cre to excise one of the two lox sites and nucleic acids encoding the selectable marker, thereby producing PTEN 1"7" cells comprising mutation in one endogenous PTEN allele; (e) transfecting PTEN 4"7” cells produced in (d) with the lox-containing targeting vector; and (f) maintaining PTEN 4"7" transfected cells produced in (e) under conditions appropriate for integration of
- the method may further comprise excising with Cre one of the two lox sites and the selectable marker from the second integrated PTEN allele.
- PTEN " cells thus produced comprise one lox site in each of the two endogenous PTEN alleles.
- Cre may be provided by infecting cells with Cre adenovirus, or alternatively, by transfecting cells with a Cre-expressing vector, such as pGK-Cre (Li and Hendrickson, 2002)
- the Cre may be derived from bacteriophage PI, and the first and second lox sites may be loxP or derivatives thereof.
- the present invention encompasses cells produced by gene targeting methods as described herein, including those cells produced during any intermediate steps, and any progeny thereof.
- the invention includes PTEN 4"7" cells with a targeting vector integrated in one endogenous PTEN allele, as well as PTEN 4"7" cells with a mutation in one endogenous PTEN allele and a lox site integrated in the same PTEN allele.
- the invention further includes PTEN 7" cells in which one endogenous PTEN allele comprises an integrated targeting vector, and another PTEN allele comprises a mutation and a lox site.
- the invention also includes PTEN " " cells in which each of the two endogenous PTEN alleles comprises a mutation and a lox site.
- the mutation may be a deletion of an exon, such as exon 2, of the PTEN gene.
- One embodiment of the invention relates to methods of identifying anti- cancer agents using the cells of the present invention.
- the method comprises: (1) contacting the isogenic pair of the present invention with a candidate agent (or a mixture of candidate agents), which is a molecule or compound to be assessed for its ability to selectively kill PTEN-deficient cells; and (2) determine viability of the cells of the isogenic pair. If the candidate agent selectively causes death of PTEN- deficient cells as compared to isogenic control cells, the candidate agent is an anti- cancer agent.
- the methods of the invention can be used to screen for novel anti- cancer agents. Alternatively, these methods are applicable for assessing the anti- cancer potentials of known agents.
- Another aspect of the invention relates to methods of identifying anti-cancer agents using the cells of the present invention.
- the method comprises: (1) contacting PTEN-deficient cells of the present invention with a candidate agent (or a mixture of candidate agents); and (2) determine viability of the cells. If the candidate agent causes death of PTEN-deficient cells, the agent is an anti-cancer agent.
- Viability of a cell can be determined by contacting the cell with a dye and viewing it under microscope. Viable cells can be observed to have an intact membrane and do not stain, whereas dying or dead cells having "leaky” membranes do stain. Incorporation of the dye by the cell indicates the death of the cell.
- a dye useful for this purpose is trypan blue.
- a candidate agent can induce apoptosis, a specific mode of cell death, in PTEN-deficient cells.
- the invention also includes agents that preferentially induce apoptosis in PTEN-deficient cells, as compared to the isogenic control cells.
- Apoptosis is recognized by a characteristic pattern of morphological, biochemical and molecular changes. Cells going through apoptosis appear shrunken and rounded. They also can be observed to become detached from a culture dish in which they are maintained. The morphological changes involve a characteristic pattern of condensation of chromatin and cytoplasm which can be readily identified by microscopy.
- apoptotic cells When stained with a DNA-binding dye, e.g., H33258, apoptotic cells display classic condensed and punctuate nuclei instead of homogenous and round nuclei.
- a hallmark of apoptosis is endonucleolysis, a molecular change in which nuclear DNA is initially degraded at the linker sections of nucleosomes to give rise to fragments equivalent to single and multiple nucleosomes.
- these DNA fragments When these DNA fragments are subjected to gel electrophoresis, they reveal a series of DNA bands which are positioned approximately equally distant from each other on the gel. The size difference between the two bands next to each other is about the length of one nucleosome, i.e., 120 base pairs.
- DNA bands This characteristic display of the DNA bands is called a DNA ladder and it indicates apoptosis of the cell.
- Apoptotic cells can also be identified by flow cytometric methods based on measurement of cellular DNA content, increased sensitivity of DNA to denaturation, or altered light scattering properties. These methods are well known in the art. Additional assays for cell viability are described in Chapter 15 of Handbook of Fluorescent Probes and Research Products (Molecular .Probes Handbook), which is incorporated in its entirety herein.
- an anti-cancer agent can be identified by assessing growth of the isogenic pair in the presence of a candidate agent.
- This embodiment comprises: (a) maintaining isogenic pairs of cells in the presence of a candidate agent (or a mixture of candidate agents); and (b) comparing growth of PTEN-deficient cells with growth of isogenic control cell. If the candidate agent preferentially inhibits the growth of PTEN-deficient cells as compared to isogenic control cells, the agent is an anti-cancer agent.
- the growth inhibition of PTEN-deficient cells caused by a candidate agent can be either partial (slowing down cell growth) or complete inhibition (i.e., arresting cells at a certain point in cell cycle). Cell growth can be measured by any techniques known in the art.
- Such techniques include, for example, MTT assay (based on reduction of the tetrazolium salt 3, [4,5- dimethylthiazol-2-yl]-2,5-diphenytetrazolium bromide), and PicoGreen assay using the DNA-binding dye Picogreen, both of which are described in Torrance, et al., Nat. Biotech. 19:940-945 (2001), incorporated herein in its entirety.
- MTT assay based on reduction of the tetrazolium salt 3, [4,5- dimethylthiazol-2-yl]-2,5-diphenytetrazolium bromide
- PicoGreen assay using the DNA-binding dye Picogreen both of which are described in Torrance, et al., Nat. Biotech. 19:940-945 (2001), incorporated herein in its entirety.
- Other assays for cell proliferation/growth are described in Chapter 15 of Handbook of Fluorescent Probes and Research Products (Molecular Probes Handbook).
- the cells of the present invention can be further
- Suitable markers include GFP and its variants, including, EGFP, YFP, BFP and ECFP. These and other GFP variants were described in Hadjantonakis and Nagy, Histochem Cell Biol. 115: 49-58 (2001), published online: December 21, 2000, which is incorporated herein.
- the PTEN-deficient cells of the present invention can be modified to include a GFP marker, whereas the isogenic control cells can be modified to include a BFP marker. Fluorometry can be used to monitor the cell growth in this example.
- the present invention encompasses methods of generating targeted disruption of PTEN allele(s) in normal human cells, as well as the cells thus generated. Since PTEN is a tumor suppressor gene, homozygous disruption of PTEN gene in a normal cell may conceivably lead to transformation from a normal cell to a cancerous cell. This transformation process provides a useful system to study how to prevent or slow down transformation. Accordingly, a further aspect of the invention pertains to another method of identifying anti-cancer agents using the cells of the present invention.
- the method comprises: (1) contacting a PTEN-/- cell resulted from targeted disruption of PTEN alleles in a normal cell with a candidate agent; and (2) assessing the ability of the agent to prevent or slow down transformation from a normal to a cancerous cell. If the candidate agent prevents or slows down the transformation process, then the agent is an anti-cancer agent. Transformation can be assayed by means well known in the art.
- a further aspect of the invention relates to methods of identifying agents that modulate PTEN activity.
- the method comprises: (1) comparing gene expression in a PTEN-deficient cell with that in a isogenic control cell; (2) identifying a gene whose expression is altered in the PTEN-deficient cell; (3) contacting the isogenic pair with a candidate agent; and (4) assessing the ability of the agent to restore the altered expression of the gene identified in step (2) in the PTEN-deficient cell to the level of expression in the isogenic control cell. If the agent restores the gene expression, then the agent is an agent that modulates at least one of PTEN activities.
- Another aspect of the present invention relates to methods for identifying candidate agents that restore a PTEN function in PTEN-deficient cells.
- the method comprises: (1) contacting the isogenic pair of the present invention with a candidate agent; (2) assessing a function of PTEN in the isogenic pair; and (3) comparing the function of PTEN in the PTEN-deficient cell to the isogenic control cell to determine if the agent is capable of restoring PTEN function.
- Any one or more of the cellular functions of PTEN may be assessed, including, for example, angiogenesis, cellular migration, immunoreceptor modulation, p53 signaling and apoptotic cell death, PI3 and AKT signaling.
- exemplary assays include those which assess alterations in activated AKT levels, alterations in microvessel formation, alterations in TSP1 levels, alterations in VEGF levels, alterations in TIMP3 levels, alterations in MMP9 activation and subcellular localization of FOXO1 a protein.
- the feasibility of this approach has been demonstrated recently in Kau et al., 2003. In this paper, the authors were able to restore at least one function of PTEN using small organic molecules in PTEN-deficient cells.
- the screening methods mentioned above are based on assays performed on cells. These cell-based assays may be performed in a high throughput screening (HTS) format, which has been described in the art. For example, Stockwell et al.
- the candidate agents used in the invention may be pharmacologic agents already known in the art or may be agents previously unknown to have any pharmacological activity.
- the agents may be naturally arising or designed in the laboratory. They may be isolated from microorganisms, animals, or plants, or may be produced recombinantly, or synthesized by chemical methods known in the art.
- candidate agents are identified from small chemical libraries, peptide libraries or collections of natural products using the methods of the present invention. For example, Tan et al. described a library with over two million synthetic compounds that is compatible with miniaturized cell-based assays (Tan et al., 1998). It is within the scope of the present invention that such a library may be used to screen for anti-cancer agents using the methods of the invention.
- ChemBridge DiverSet library ChemBrdige Corporation, San Diego, CA. Libraries are also available from academic investigators, such as NCI's Developmental Therapeutics Program (Bethesda, MD), including NCT Structural Diversity Set and NCI marine extracts.
- the present invention also relates to treatment methods for cancers using the anti-cancer agents of the present invention.
- the methods comprise administering the anti-cancer agents of the invention to cancer patients.
- treatment methods of the invention are suitable to treat cancers that include, but not limited to, colon adenocarcmoma, endometrial cancer, glioma, prostate adenocarcmoma, malignant melanoma, thyroid cancer and brain cancer.
- the therapeutic agents identified by a method of the present invention may also be used in the treatment of a PTEN-associated condition, in which enhancement or inhibition (modulation) of PTEN activity is desirable.
- 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).
- the present invention also pertains to pharmaceutical compositions comprising therapeutic agents identified by methods described herein.
- a therapeutic agent 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 introducing therapeutic agents 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 rechargeable or biodegradable devices and slow release polymeric devices.
- the pharmaceutical compositions of this invention can also be administered as part of a combinatorial therapy with other agents, or with other treatment methods, h the context of cancer, such other treatment methods include radiation therapy, chemotherapy and surgery.
- a high efficiency promoterless PTEN targeting vector was created using homologous recombination in Saccharomyces cerevisiae.
- an 8-kb BsmBI- Sphl fragment containing exon II of PTEN was cloned from BAG 106D15A into yeast shuttle vector YEp24 and sequenced.
- a PCR product containing an 8-kb BsmBI- Sphl fragment containing exon II of PTEN was cloned from BAG 106D15A into yeast shuttle vector YEp24 and sequenced.
- a PCR product containing an 8-kb BsmBI- Sphl fragment containing exon II of PTEN was cloned from BAG 106D15A into yeast shuttle vector YEp24 and sequenced.
- a PCR product containing an 8-kb BsmBI- Sphl fragment containing exon II of PTEN was cloned from BAG 106D15A into yeast shuttle vector YEp24
- IRES-neo R gene flanked by LoxP sites a priming site for PCR-based identification of knockouts, restriction sites for Southern blot-based identification of knockouts, and 50 nucleotides of homology to the subcloned PTEN genomic fragment was co- transformed into S. cerevisiae with the linearized recombinant yeast shuttle vector. Successful recombinants were identified by whole cell PCR. Recombinant plasmids were then shuttled into Escherichia coli, and their integrity confirmed via restriction analysis and DNA sequencing. Completed targeting vectors were linearized and transfected into HCT116 cells.
- HCT116 and LNCaP cells were obtained from ATCC.
- HCT116 cells were grown in McCoy's 5 A media containing 10% fetal bovine serum (FBS) and penicillin/streptomycin.
- FBS fetal bovine serum
- Stably transfected putative knockouts clones were selected in 0.6 mg/mL G418.
- LNCaP cells were grown in RPMI 1640 containing 10% FBS and penicillin/streptomycin.
- Lysates derived from equivalent numbers of cells were separated by SDS-PAGE on either 4-12% Bis-Tris or 3-8% Tris-Acetate gels, transferred to PVDF membranes, probed with primary and horseradish-peroxidase coupled secondary antibodies, and visualized by ECL (Amersham).
- Antibodies were obtained from Cascade Bioscience (PTEN clone 6H2.1); Cell Signaling Technologies (total Akt, phospho-Akt (S473), phospho-Akt (T308), total tuberin, phospho-tuberin (T1462)); and BD Biosciences (HJF-l ⁇ ).
- Cells were trypsinized in 0.5 mL, added to 0.5 mL serum-containing media, and further diluted in 10 mL Isoton® II. Cell diameters and volumes were dete ⁇ nined using a MultisizerTM 3 Coulter Counter (Beckman Coulter). At least 10,000 cells were counted per measurement.
- Flow cytometry Cells were fixed in 70% ethanol and stained in phosphate-buffered saline (PBS) containing 0.1% Triton X-100, 50 ⁇ g/mL RNAse, and 50 ⁇ g/mL propidium iodide. DNA content was measured on a FACSort flow cytometer (Becton Dickinson), and data were analysed using ModFit software (Verity Software House) on both linear and log scales. At least 20,000 cells were analyzed per sample. BrdU incorporation assay Cells were pulsed with 10 ⁇ M BrdU for 1 or 2 hours, trypsinized, and centrifuged.
- PBS phosphate-buffered saline
- Cells were treated with 0.2 ⁇ g/mL nocodazole and immediately irradiated. At various time points, cells were collected by trypsinization, centrifuged, and simultaneously fixed and stained in a solution containing 3.7% formaldehyde, 0.5% Nonidet P-40, and 10 ⁇ g/mL Hoechst 33258 in PBS. Nuclei were visualized by fluorescence microscopy. Nuclei with condensed, evenly staining chromosomes were scored as mitotic. At least 300 cells were counted for each determination.
- HCT116 PTEN 4" + and PTEN 7" cells were treated with a single dose of gamma radiation using a 137 Cs source. 24 hours following irradiation the cells were trypsinized, counted, and plated at various dilutions in T25s. Colonies were allowed to grow undisturbed for 12 days, stained with crystal violet, and counted in a blinded fashion.
- Somatic cell gene targeting was used to create an isogenic set of human cancer cell lines appropriate for functional genetic analysis of PTEN. Though approaches based on small interfering RNAs were technically appealing, we chose gene targeting because it is the only technique available for the creation of a non- leaky, completely null allele. HCT116 cells were selected since they are suitable for somatic cell gene targeting; have well-defined, intact checkpoint responses; and have two wild-type alleles of PTEN (Waldman et al. 1995; Li et al. 1998). Of note, mutational inactivation of PTEN is found in approximately 14% of RER+ colorectal carcinomas (Zhou et al. 2002). The targeting strategy is depicted in Fig. 1 A and described in Materials and methods.
- PTEN 4" + cells enlarged slightly and then growth-arrested
- PTEN " " cells enlarged dramatically, increasing nine times in volume by post-irradiation day 6 (Figs. 2A-D). Similar results were obtained after treatment with 9 Gy (data not shown).
- four independently-derived PTEN 4"74" cell lines parental HCTl 16 cells and three clonal cell lines resulting from non-homologous integration of the targeting vector
- four independently-derived homozygous knockout PTEN " cell lines were studied.
- Each of the four PTEN 7" cell lines (and none of the four PTEN 4"74" cell lines) enlarged dramatically following radiation (data not shown).
- irradiated PTEN 4"74" and PTEN 7" cells were stained with the vital dye Hoechst 33342 (10 ⁇ g/mL), flow sorted in a FACSAria high speed cell sorter (Becton Dickinson) to separate the Gl and G2/M populations, and measured.
- PTEN can also dephosphorylate protein substrates such as FAK and She (Tamura et al. 1998).
- PI3K inhibitors were employed to test whether abrogation of the cell size checkpoint was due to the specific loss of the PTEN lipid phosphatase activity.
- PTEN "7" cells were pre-treated for one hour with doses of wortmannin known to inactivate Akt through the reduction of cellular PtdIns(3,4,5)P 3 levels. The cells were then irradiated, cultured for six days in the presence of the inhibitor, and measured (Fig. 3 A). Wortmannin was able to efficiently restore the cell size checkpoint, resulting in a post-irradiation size increase only 6% that of untreated irradiated cells.
- LNCaP cells enlarged, as though they were deficient in the PTEN-dependent cell size checkpoint (Fig. 3C). Although it was not technically feasible to restore functional, normally-regulated PTEN to these cells, we mimicked restoration of PTEN through the application of LY294002. As in PTEN 7" HCTl 16 cells, treatment of LNCAP cells with LY294002 restored the cell size checkpoint, enabling irradiated LNCaP cells to maintain their normal size. Importantly, LY294002 did not affect the size of unirradiated LNCaP cells.
- PTEN 7" cells were up to an order of magnitude more sensitive to radiation than isogenic PTEN 4" + cells. These results are consistent with a recent report that treatment of the PTEN-null human A 172 glioblastoma cell line with wortmannin leads to radioresistance, but are inconsistent with other reports suggesting that inhibition of PI3K causes radiosensitivity (Okaichi et al. 2002; McKenna and Muschel 2003). The factors governing radiosensitivity are clearly complex, and the role of PtdIns(3,4,5)P 3 in the cellular response to radiation is a subject of some controversy.
- the PTEN knockout cells represent, to our knowledge, the only isogenic set of human cells that differ only in the presence or the complete absence of endogenous wild-type PTEN. As such they may prove useful not only for studying the role of PTEN in cancer pathogenesis, but also for anticancer drug discovery targeting the PTEN pathway (Torrance et al. 2001).
- TSC2 regulates VEGF through mTOR-dependent and -independent pathways. Cancer Cell. 4:147-158.
- PTEN affects cell size, cell proliferation and apoptosis during Drosophila eye development. Development. 126:5365-5372.
- Akt/protein kinase B overcomes a G(2)/m cell cycle checkpoint induced by DNA damage. Mol. Cell. Biol. 22:7831- 7841.
- Kau T.R., et al., A Chemical Genetic Screen Identifies Inhibitors of Regulated Nuclear Export of a Forkhead Transcription Factor in PTEN-deficient Tumor Cells, Cancer Cell, 463-476 (Dec. 2003).
- PTE ⁇ /MMAC1/TEP1 suppresses the tumorigenicity and induces Gl cell cycle arrest in human ghoblastoma cells. Proc. Natl. Acad. Sci. U.S.A. 95:15406-15411.
- the PTE ⁇ /MMAC1 tumor suppressor induces cell death that is rescued by the AKT/protein kinase B oncogene. Cancer Res. 58:5667-5672.
- Podsypanina K., Lee, R.T., Politis, C, Hennessy, I., Crane, A., Puc, J., Neshat, M., Wang, H., Yang, L., Gibbons, J., Frost, P., Dreisbach, V., Blenis, J., Gaciong, Z., Fisher, P., Sawyers, C, Hedrick-Ellenson, L., and Parsons, R. 2001.
- An inhibitor of mTOR reduces neoplasia and normalizes p70/S6 kinase activity in Pten+/- mice. Proc. Natl. Acad. Sci. U.S.A. 98:10320-10325.
- the PTEN/MMAC1 tumor suppressor phosphatase functions as a negative regulator of the phosphoinositide 3-kinase/Akt pathway. Proc. Natl. Acad. Sci. U.S.A. 95:15587- 15591.
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Abstract
The present invention provides isogenic pairs of human cells that differ only in their PTEN alleles. Also provided are targeting vectors and methods for generating human somatic cells deficient in the PTEN gene. These isogenic pairs of cells are useful in screening assays to identify anti-cancer agents and agents that modulate PTEN activity.
Description
PTEN-DEFICIENT CELLS AND THEIR USES
Cross-Reference to Related Applications
The present application claims the benefit of U.S. Provisional Application No. 60/448,799, filed February 19, 2003, the specification of which is incorporated herein by reference in its entirety.
Funding
Work described herein was funded, in whole or in part, by Grant No. KOI CA87828 from National Cancer Institute, NIH. The United States government has certain rights in the invention.
Background of the Invention
PTEN gene encodes a lipid dual-specificity phosphotase and is the major 3- phosphatase in the phosphoinositol-3-kinase (PI3K)/AKT pro-apoptotic pathway (Li and Sun, 1997, Maehama and Dixon, 1998; Stambolic et al. 1998). Located on the short arm of chromosome 10 (10q23), PTEN is mutated in 40-50% of high grade gliomas, as well as many other tumor types, including those of the prostate, endometrium, breast, and lung (Li et al, Science 1997, Steck et al 1997, Maier et al 1998). In addition, PTEN is mutated in several rare autosomal dominant cancer predisposition syndromes, including Cowden disease, Lhermitte-Duclos disease and Bannayan-Zonana syndrome (Liaw et al 1997, Myers et al AJHG 1997, Maehama et al TCB 1999, Cantley and Neel 1999). Furthermore, the phenotype of PTEN- knockout mice revealed a requirement for PTEN in normal development and confirmed its role as a tumor suppressor (Podsypanina et al, 1999, Suzuki et al, 1998, Di Christofano et al, 1998). In addition to its role in cancers, PTEN has been implicated in important cellular processes such as cell cycling, translation, and apoptosis. For example, PTEN has been shown to regulate the phosphoinositol-3-kinase (PI3K)/AKT pro- apoptotic pathway (Li and Sun, 1997, Maehama and Dixon, 1998; Stambolic et al.
1998). PTEN has also been shown to regulate the mitogen-activated kinase (MAPK) pathway, which is critical for proliferation and differentiation (Ong et al. 2001; Takahashi-Tezuka et al. 1998; Yart et al. 2001).
Given the critical roles of PTEN in cancer and in other important cellular processes, it is important to study how PTEN functions in order to facilitate the design and screening of effective drags for treating cancer and other PTEN- associated conditions. Cells or animal models in which the PTEN gene is disrupted can provide valuable systems for such studies. To this end, three groups have reported the creation of PTEN knockout mice. However, the PTEN homozygous disruption results in embryonic lethal phenotype in mice, making a mouse model that recapitulates human biology unfeasible ((Podsypanina et al, 1999, Suzuki et al, 1998, Di Christofano et al, 1998). The lack of animal models makes human cells with targeted PTEN disruption especially desirable. Furthermore, drugs that selectively kill cancer cells, but do not affect normal cells, are desirable for cancer treatment. There is therefore a need for well-controlled screening methods to identify such drugs.
Summary of the Invention Described herein are PTEN-defϊcient human cells that are either heterozygous (PTEN"1" ") or homozygous (PTEN" ") for a PTEN mutation. In one embodiment, PTEN-deficient cells result from targeted disruption of at least one PTEN gene via somatic cell gene targeting.
Accordingly, one embodiment the present invention relates to an isolated recombinant human somatic cell, and progeny thereof, in which at least one endogenous PTEN allele comprises a mutation that renders the PTEN allele nonfunctional. The invention encompasses cells heterozygous for such a PTEN mutation, as well as cells homozygous for such a PTEN mutation. PTEN mutations include both loss-of-function mutations, such as deletions, and gain-of-function mutations, such as dominant negative mutations and constitutive mutations. One embodiment of the present invention is a pair of isogenic human somatic cells in which one member is PTEN-deficient and the other member has at least two functional endogenous PTEN alleles, or otherwise possesses no non-
functional PTEN alleles. The isogenic pair of cells of the invention offers the advantage of clonal homogeneity, ability to manipulate the external environment according to needs, and ease of biochemical investigations. Since PTEN is a tumor suppressor, the isogenic pair of the invention provides a well-defined and easily manipulated system to identify novel anti-cancer agents.
The present invention also relates to a somatic cell gene targeting vector for mutating PTEN allele to produce a non-functional PTEN allele, and a gene targeting method for generating a human somatic cell with a mutation in at least one endogenous PTEN allele. The present invention also relates to methods of screening for anti-cancer agents using isogenic pairs of the invention. Since PTEN is a tumor suppressor gene, agents that selectively inhibit the growth of a PTEN-deficient cell but not its isogenic control cell can be valuable anti-cancer agents. The invention also contemplates a screening method for agents that selectively cause death of a PTEN- deficient cell but not an isogenic control cell.
Brief Description of the Drawings
Figure 1. PTEN gene targeting. (A) The targeting vector deletes ex on II of PTEN and replaces it with a promoterless, IRES-neoR gene flanked by LoxP sites. The positions of the EcoR I restriction sites (filled arrowheads) and PCR primers (arrows) used for screening of knockouts is indicated. (B) Confirmation of PTEN targeting by Southern blot analysis. Fragments corresponding to the wild-type allele and the targeted allele before and after cre-mediated excision of IRES- neoR are shown. (C) Western blot analysis of isogenic PTEN+ + and PTEN" _ cells. Parental HCT116 cells and a neoR clone resulting from random integration of the targeting vector are compared to two independently-derived PTEN" " clones. Immunoblotting was performed with the antibodies indicated.
Figure 2. A PTEN-dependent, radiation-induced cell size checkpoint. (A) Light
microscopy of isogenic PTEN474" and PTEN7" cells. Exponentially growing, unirradiated, PTEN" " cells (b) are morphologically indistinguishable from isogenic PTEN+/+ cells (a). Exposure to gamma radiation (IR) followed by six days of culture results in a dramatic increase in the size of PTEN" " cells (d) compared to identically treated PTEN174" cells (c). Scale bars represent 100 μm. (B) Electron micrographs of irradiated PTEN 74" (a,b,c) and PTEN " cells (d,e,f). Scale bar represents 5 μm. (C) Cell size histograms of untreated and irradiated isogenic PTEN+/+ and PTEN"7" cells. Three PTEN4"74" cell lines (parental HCT116 cells and two neoR clones generated by non-homologous integration of the targeting vector) and two independently-derived PTEN " clones (KO #1 and KO #2) were measured during exponential growth (a) and six days after irradiation (b). (D) Time course of PTEN474" (•) and PTEN" " (■) cell size following IR. Each measurement represents the average size for the cell lines described in (C). All cell lines were measured at least in triplicate. The average mode ± SEM are shown.
Figure 3. Checkpoint restoration in PTEN7" cells. PTEN4" + and PTEN7" cells were treated with 580 nM wortmannin (A) or 5 nM rapamycin (B), irradiated (6 Gy), cultured for six days, and measured. (C), LNCaP cells (PTEN7") were treated with 10 μm LY294002, irradiated (6 Gy), cultured for three days, and measured.
Figure 4. Integrity of radiation-induced Gl and G2 checkpoints in PTEN"7" cells. (A) Flow cytometry of PTEN4" + and PTEN7" cells 24 hours after treatment with 6 Gy. (B) BrdU incorporation assays of untreated or irradiated (9 Gy) PTEN474" and PTEN" " cells. The percentage of cells in S phase is indicated in the upper right corner of each panel. (C) Mitotic indices of HCT 116 PTEN474" (♦) and PTEN7" cells (■) at the indicated times following 9 Gy, or 9 Gy plus nocodazole.
Figure 5. Radiosensitivity of PTEN" cells, and model. (A) Clonogenic survival assay of HCT116 PTEN474" and PTEN7" cells. Equivalent numbers of four PTEN47 " clones (♦) and four independently-derived PTEN7" clones (■) were treated with various doses of gamma radiation. Each experiment was performed at least in triplicate, and the values of all four PTEN4" 4" or PTEN" " cell lines were averaged
after adjusting for plating efficiency. The mean number of colonies ± SEM is shown. (B) Model of radiation-induced, PTEN-dependent cell size checkpoint. Treatment of human cells with radiation leads to Gl and G2 cell cycle arrests, and a simultaneous arrest in cell size. Deletion of p53 or p21 abolishes the Gl and G2 arrests, whereas deletion of PTEN abolishes the arrest in cell size.
Figure 6. Identification of PTEN knockouts by PCR. Homologous integration deletes an endogenous PCR priming site and moves it to a new location, causing the wild-type and targeted alleles of PTEN to generate PCR products of distinct sizes.
Figure 7. Crystal violet-stained colonies from the clonogenic survival assay shown in Fig. 5 A. PTEN4"74" flasks (top row) have at least an order of magnitude more colonies than PTEN" " flasks (bottom row).
Detailed Description of the Invention
The invention provides cells with mutation(s) in endogenous PTEN allele(s) introduced by gene targeting in the cells or ancestors thereof. Thus, the present invention encompasses recombinant cells produced as described herein (including any cells produced during gene targeting) and progeny thereof. The cells can be either homozygous (PTEN"7") or heterozygous (PTEN4"7") for the PTEN mutation(s). A mutation can be any change in the sequence of the PTEN gene that alters PTEN function or activity. For example, a mutation can be a deletion, an insertion, a frame shift or a point mutation.
In one embodiment, the invention relates to cells with PTEN mutations that render the PTEN allele non-functional by reducing or preventing PTEN expression. The reduction of PTEN activity can be either complete or partial. For example, a non- functional PTEN allele may be a PTEN mutation that reduces PTEN expression, partially or completely, resulting in reduced levels of PTEN protein product. The reduction of PTEN expression can be at the transcriptional level and/or at the translational level. Alternatively, a non-functional PTEN allele can be
a PTEN mutation that results in production of a PTEN protein with completely or partially reduced PTEN activity; such a protein can be expressed at normal or reduced levels. In a particular embodiment, the cells of the present invention contain a deletion of all or a portion of an exon, such as exon 2, of the PTEN gene, resulting in reduced (partially or completely) PTEN protein expression level.
In another embodiment of the invention, cells comprise PTEN mutations that are gain-of-function mutations, such as a dominant negative mutation or a constitutive mutation.
Cells used for gene targeting may be normal cells or tumor cells. They may be derived from a cell line or may be freshly isolated from a human or an animal. In one embodiment, tumor cells used for gene targeting are selected from a group consisting of a glioblastoma multiforme cell, a colon adenocarcmoma cell, a malignant melanoma cell, an endometrial carcinoma cell, a prostate adenocarcmoma cell, a thyroid cancer cell and a breast cancer cell. In a particular embodiment, primary colorectal tumor cells or colorectal tumor cell lines, such as HCT 116, DLD1 and SW480, are used.
The present invention also provides an isogenic pair of cells, which differ in their endogenous PTEN alleles. The first member of the pair, referred to as the PTEN-deficient member or cell, has a mutation in at least one endogenous PTEN allele. The second member, referred to as the isogenic control member or cell, has at least two functional endogenous PTEN alleles, or otherwise possesses no nonfunctional PTEN alleles. In one embodiment, the PTEN-deficient member has a deletion of all or a portion of an exon, such as exon 2, of the PTEN gene. In a further embodiment, the PTEN-deficient cell is homozygous for PTEN deletions. Any means known in the art to generate a cell line with targeted mutation can be used to produce cells of the present invention. In mammalian cells, homologous recombination occurs at much lower frequency compared to non- homologous recombination. To facilitate the selection of homologous recombination events over the non-homologous recombination events, at least two enrichment methods have been developed: the positive-negative selection (PNS) method and the "promoterless" selection method (Sedivy and Dutriaux, 1999). Briefly, PNS, the first method, is in genetic terms a negative selection: it selects
against recombination at the incorrect (non-homologous) loci by relying on the use of a negatively selectable gene that is placed on the flanks of a targeting vector. On the other hand, the second method, the "promoterless" selection, is a positive selection in genetic terms: it selects for recombination at the correct (homologous) locus by relying on the use of a positively selectable gene whose expression is made conditional on recombination at the homologous target site. The disclosure of Sedivy and Dutriaux is incorporated herein.
Accordingly, one embodiment of the present invention relates to targeting vectors and targeting methods for creating mutations in endogenous PTEN allele(s). In one embodiment, the targeting vector comprises: (a) a first region of nucleic acids homologous to a region of PTEN gene, or to a region immediately upstream of the PTEN gene, of sufficient length to undergo homologous recombination with the region of an endogenous PTEN gene (referred to as the first homology arm); (b) nucleic acids encoding a selectable marker; and (c) a second region of nucleic acids homologous to a region of PTEN gene, or to a region immediately downstream of the PTEN gene, of sufficient length to undergo homologous recombination with the region of an endogenous PTEN gene (referred to as the second homology arm). In a further embodiment, the first homology arm comprises at least 800 nucleotides homologous to a region of the second intron of the PTEN gene and the second homology arm comprises at least 800 nucleotides homologous to a region of the third intron of the PTEN gene.
In a particular embodiment, the targeting vector comprises: (a) at least 800 nucleotides homologous to a region of the second intron of the PTEN gene; (b) a first lox site; (c) an internal ribosome entry site (IRES); (d) nucleic acids encoding a selectable marker; (e) a second lox site; and (f) at least 800 nucleotides homologous to a region of the third intron of the PTEN gene. The first and second lox sites may be loxP sites or derivatives thereof. This kind of targeting vector is referred to as a lox-containing targeting vector. The selectable marker can be any antibiotic resistance gene, such as a neomycin resistance gene. In a specific embodiment, the first homology arm comprises 1665 nucleotides homologous to a region immediately upstream of the second exon of the PTEN gene, and the second
homology arm comprises 2549 nucleotides homologous to a region beginning at nucleotide 409 of the third intron the PTEN gene.
The length of the homology arms can be varied depending on, for example, the region(s) of the PTEN gene to be deleted. The arms can be the same length or different. For example, the arm can be 800-1200, 1200-1600, 1600-2000, 2000- 2400, 2400-2800, or 2800-3200 nucleotides in length.
Another embodiment of the invention relates to methods of generating targeted mutations in at least one endogenous PTEN allele by gene targeting. The method comprises: (a) transfecting human somatic cells with a targeting vector of the present invention, thereby producing transfected human somatic cells; and (b) maintaining the transfected cells under conditions appropriate for integration of the targeting vector into endogenous PTEN allele(s) in the transfected cells, thereby producing cells in which the targeting vector is integrated in at least one endogenous PTEN allele. The cells thus produced can be subjected to another round of gene targeting using a second targeting vector with a selectable marker different from the marker in the first targeting vector, to produce cells having targeting vectors integrated in both endogenous PTEN alleles.
In a particular embodiment, the targeting method of the present invention makes use of Cre-lox system. The method comprises: (a) transfecting human somatic cells with a lo)t-containing targeting vector of the invention thereby producing transfected human somatic cells; (b) maintaining the transfected cells thus produced under conditions appropriate for integration of the targeting vector into an endogenous PTEN allele, thereby producing cells having the lox targeting vector integrated in one endogenous PTEN allele; (c) providing the cells having the lox targeting vector integrated in one endogenous PTEN allele with Cre; (d) maintaining the Cre-containing cells produced by step (c) under conditions appropriate for Cre to excise one of the two lox sites and nucleic acids encoding the selectable marker, thereby producing PTEN1"7" cells comprising mutation in one endogenous PTEN allele; (e) transfecting PTEN4"7" cells produced in (d) with the lox-containing targeting vector; and (f) maintaining PTEN4"7" transfected cells produced in (e) under conditions appropriate for integration of the targeting vector into a second PTEN allele, thus producing PTEN7"cells comprising mutations in both endogenous PTEN
alleles. The method may further comprise excising with Cre one of the two lox sites and the selectable marker from the second integrated PTEN allele. In this embodiment, PTEN " cells thus produced comprise one lox site in each of the two endogenous PTEN alleles. Cre may be provided by infecting cells with Cre adenovirus, or alternatively, by transfecting cells with a Cre-expressing vector, such as pGK-Cre (Li and Hendrickson, 2002) The Cre may be derived from bacteriophage PI, and the first and second lox sites may be loxP or derivatives thereof.
The present invention encompasses cells produced by gene targeting methods as described herein, including those cells produced during any intermediate steps, and any progeny thereof. In particular, the invention includes PTEN4"7" cells with a targeting vector integrated in one endogenous PTEN allele, as well as PTEN4"7" cells with a mutation in one endogenous PTEN allele and a lox site integrated in the same PTEN allele. The invention further includes PTEN7" cells in which one endogenous PTEN allele comprises an integrated targeting vector, and another PTEN allele comprises a mutation and a lox site. The invention also includes PTEN" " cells in which each of the two endogenous PTEN alleles comprises a mutation and a lox site. The mutation may be a deletion of an exon, such as exon 2, of the PTEN gene. One embodiment of the invention relates to methods of identifying anti- cancer agents using the cells of the present invention. The method comprises: (1) contacting the isogenic pair of the present invention with a candidate agent (or a mixture of candidate agents), which is a molecule or compound to be assessed for its ability to selectively kill PTEN-deficient cells; and (2) determine viability of the cells of the isogenic pair. If the candidate agent selectively causes death of PTEN- deficient cells as compared to isogenic control cells, the candidate agent is an anti- cancer agent. The methods of the invention can be used to screen for novel anti- cancer agents. Alternatively, these methods are applicable for assessing the anti- cancer potentials of known agents. Another aspect of the invention relates to methods of identifying anti-cancer agents using the cells of the present invention. The method comprises: (1) contacting PTEN-deficient cells of the present invention with a candidate agent (or a
mixture of candidate agents); and (2) determine viability of the cells. If the candidate agent causes death of PTEN-deficient cells, the agent is an anti-cancer agent.
Viability of a cell can be determined by contacting the cell with a dye and viewing it under microscope. Viable cells can be observed to have an intact membrane and do not stain, whereas dying or dead cells having "leaky" membranes do stain. Incorporation of the dye by the cell indicates the death of the cell. A dye useful for this purpose is trypan blue.
A candidate agent can induce apoptosis, a specific mode of cell death, in PTEN-deficient cells. The invention also includes agents that preferentially induce apoptosis in PTEN-deficient cells, as compared to the isogenic control cells. Apoptosis is recognized by a characteristic pattern of morphological, biochemical and molecular changes. Cells going through apoptosis appear shrunken and rounded. They also can be observed to become detached from a culture dish in which they are maintained. The morphological changes involve a characteristic pattern of condensation of chromatin and cytoplasm which can be readily identified by microscopy. When stained with a DNA-binding dye, e.g., H33258, apoptotic cells display classic condensed and punctuate nuclei instead of homogenous and round nuclei. A hallmark of apoptosis is endonucleolysis, a molecular change in which nuclear DNA is initially degraded at the linker sections of nucleosomes to give rise to fragments equivalent to single and multiple nucleosomes. When these DNA fragments are subjected to gel electrophoresis, they reveal a series of DNA bands which are positioned approximately equally distant from each other on the gel. The size difference between the two bands next to each other is about the length of one nucleosome, i.e., 120 base pairs. This characteristic display of the DNA bands is called a DNA ladder and it indicates apoptosis of the cell. Apoptotic cells can also be identified by flow cytometric methods based on measurement of cellular DNA content, increased sensitivity of DNA to denaturation, or altered light scattering properties. These methods are well known in the art.
Additional assays for cell viability are described in Chapter 15 of Handbook of Fluorescent Probes and Research Products (Molecular .Probes Handbook), which is incorporated in its entirety herein.
Alternatively, an anti-cancer agent can be identified by assessing growth of the isogenic pair in the presence of a candidate agent. This embodiment comprises: (a) maintaining isogenic pairs of cells in the presence of a candidate agent (or a mixture of candidate agents); and (b) comparing growth of PTEN-deficient cells with growth of isogenic control cell. If the candidate agent preferentially inhibits the growth of PTEN-deficient cells as compared to isogenic control cells, the agent is an anti-cancer agent. The growth inhibition of PTEN-deficient cells caused by a candidate agent can be either partial (slowing down cell growth) or complete inhibition (i.e., arresting cells at a certain point in cell cycle). Cell growth can be measured by any techniques known in the art. Such techniques include, for example, MTT assay (based on reduction of the tetrazolium salt 3, [4,5- dimethylthiazol-2-yl]-2,5-diphenytetrazolium bromide), and PicoGreen assay using the DNA-binding dye Picogreen, both of which are described in Torrance, et al., Nat. Biotech. 19:940-945 (2001), incorporated herein in its entirety. Other assays for cell proliferation/growth are described in Chapter 15 of Handbook of Fluorescent Probes and Research Products (Molecular Probes Handbook). In one embodiment, the cells of the present invention can be further modified to include a marker to facilitate the monitoring of cell growth. Suitable markers include GFP and its variants, including, EGFP, YFP, BFP and ECFP. These and other GFP variants were described in Hadjantonakis and Nagy, Histochem Cell Biol. 115: 49-58 (2001), published online: December 21, 2000, which is incorporated herein.
For example, the PTEN-deficient cells of the present invention can be modified to include a GFP marker, whereas the isogenic control cells can be modified to include a BFP marker. Fluorometry can be used to monitor the cell growth in this example. As mentioned above, the present invention encompasses methods of generating targeted disruption of PTEN allele(s) in normal human cells, as well as the cells thus generated. Since PTEN is a tumor suppressor gene, homozygous disruption of PTEN gene in a normal cell may conceivably lead to transformation
from a normal cell to a cancerous cell. This transformation process provides a useful system to study how to prevent or slow down transformation. Accordingly, a further aspect of the invention pertains to another method of identifying anti-cancer agents using the cells of the present invention. The method comprises: (1) contacting a PTEN-/- cell resulted from targeted disruption of PTEN alleles in a normal cell with a candidate agent; and (2) assessing the ability of the agent to prevent or slow down transformation from a normal to a cancerous cell. If the candidate agent prevents or slows down the transformation process, then the agent is an anti-cancer agent. Transformation can be assayed by means well known in the art.
Using gene expression profiling of PTEN-deficient cells and isogenic control cells, the present inventors have identified genes whose expression is altered (either increased or reduced) in PTEN-deficient cells. Accordingly, a further aspect of the invention relates to methods of identifying agents that modulate PTEN activity. The method comprises: (1) comparing gene expression in a PTEN-deficient cell with that in a isogenic control cell; (2) identifying a gene whose expression is altered in the PTEN-deficient cell; (3) contacting the isogenic pair with a candidate agent; and (4) assessing the ability of the agent to restore the altered expression of the gene identified in step (2) in the PTEN-deficient cell to the level of expression in the isogenic control cell. If the agent restores the gene expression, then the agent is an agent that modulates at least one of PTEN activities.
Another aspect of the present invention relates to methods for identifying candidate agents that restore a PTEN function in PTEN-deficient cells. The method comprises: (1) contacting the isogenic pair of the present invention with a candidate agent; (2) assessing a function of PTEN in the isogenic pair; and (3) comparing the function of PTEN in the PTEN-deficient cell to the isogenic control cell to determine if the agent is capable of restoring PTEN function. Any one or more of the cellular functions of PTEN may be assessed, including, for example, angiogenesis, cellular migration, immunoreceptor modulation, p53 signaling and apoptotic cell death, PI3 and AKT signaling. Specifically, exemplary assays include those which assess alterations in activated AKT levels, alterations in microvessel formation, alterations in TSP1 levels, alterations in VEGF levels, alterations in
TIMP3 levels, alterations in MMP9 activation and subcellular localization of FOXO1 a protein. The feasibility of this approach has been demonstrated recently in Kau et al., 2003. In this paper, the authors were able to restore at least one function of PTEN using small organic molecules in PTEN-deficient cells. The screening methods mentioned above are based on assays performed on cells. These cell-based assays may be performed in a high throughput screening (HTS) format, which has been described in the art. For example, Stockwell et al. described a high-throughput screening of small molecules in miniaturized mammalian cell-based assays involving post-translational modifications (Stockwell et al., 1999). Likewise, Qian et al. described a leukemia cell-based assay for high- throughput screening for anti-cancer agents (Qian et al., 2001). Both references are incorporated herein in their entirety.
The candidate agents used in the invention may be pharmacologic agents already known in the art or may be agents previously unknown to have any pharmacological activity. The agents may be naturally arising or designed in the laboratory. They may be isolated from microorganisms, animals, or plants, or may be produced recombinantly, or synthesized by chemical methods known in the art. In some embodiments, candidate agents are identified from small chemical libraries, peptide libraries or collections of natural products using the methods of the present invention. For example, Tan et al. described a library with over two million synthetic compounds that is compatible with miniaturized cell-based assays (Tan et al., 1998). It is within the scope of the present invention that such a library may be used to screen for anti-cancer agents using the methods of the invention. There are numerous commercially available compound libraries, such as the ChemBridge DiverSet library (ChemBrdige Corporation, San Diego, CA). Libraries are also available from academic investigators, such as NCI's Developmental Therapeutics Program (Bethesda, MD), including NCT Structural Diversity Set and NCI marine extracts.
The present invention also relates to treatment methods for cancers using the anti-cancer agents of the present invention. The methods comprise administering the anti-cancer agents of the invention to cancer patients. In one embodiment, treatment methods of the invention are suitable to treat cancers that include, but not
limited to, colon adenocarcmoma, endometrial cancer, glioma, prostate adenocarcmoma, malignant melanoma, thyroid cancer and brain cancer.
The therapeutic agents identified by a method of the present invention may also be used in the treatment of a PTEN-associated condition, in which enhancement or inhibition (modulation) of PTEN activity is desirable. The term "condition", as used herein, 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). The present invention also pertains to pharmaceutical compositions comprising therapeutic agents identified by methods described herein. A therapeutic agent 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 introducing therapeutic agents 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 rechargeable or biodegradable devices and slow release polymeric devices. The pharmaceutical compositions of this invention can also be administered as part of a combinatorial therapy with other agents, or with other treatment methods, h the context of cancer, such other treatment methods include radiation therapy, chemotherapy and surgery.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, virology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are described in the literature. See, for example, Molecular Cloning: A Laboratory Manual, 3rd Ed., ed. by
Sambrook and Russell (Cold Spring Harbor Laboratory Press: 2001); the treatise, Methods In Enzymology (Academic Press, Inc., N. Y.); Using Antibodies, Second
Edition by Harlow and Lane, Cold Spring Harbor Press, New York, 1999; Current Protocols in Cell Biology, ed. by Bonifacino, Dasso, Lippincott-Schwartz, Harford, and Yamada, John Wiley and Sons, Inc., New York, 1999. All patents, patent applications and references cited herein are incorporated in their entirety by reference.
Exemplification The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.
Materials and methods
Human somatic cell gene targeting
A high efficiency promoterless PTEN targeting vector was created using homologous recombination in Saccharomyces cerevisiae. In brief, an 8-kb BsmBI- Sphl fragment containing exon II of PTEN was cloned from BAG 106D15A into yeast shuttle vector YEp24 and sequenced. Next, a PCR product containing an
IRES-neoR gene flanked by LoxP sites, a priming site for PCR-based identification of knockouts, restriction sites for Southern blot-based identification of knockouts, and 50 nucleotides of homology to the subcloned PTEN genomic fragment was co- transformed into S. cerevisiae with the linearized recombinant yeast shuttle vector. Successful recombinants were identified by whole cell PCR. Recombinant plasmids were then shuttled into Escherichia coli, and their integrity confirmed via restriction analysis and DNA sequencing. Completed targeting vectors were linearized and transfected into HCT116 cells. Individual G418-resistant colonies were obtained, expanded, cryopreserved, and tested via PCR for the presence of a heterozygous knockout. 20% of G418 resistant clones were knockouts. After excision of the neoR gene with adeno-cre, heterozygous knockout clones were then re-transfected with the linearized PTEN targeting vector for deletion of the remaining allele. G418-
resistant clones were then expanded, cryopreserved, and tested via PCR and Southern blot for the presence of a homozygous knockout. Additional technical details of human somatic cell gene targeting are discussed elsewhere (Waldman et al. 2003). Cell culture
HCT116 and LNCaP cells were obtained from ATCC. HCT116 cells were grown in McCoy's 5 A media containing 10% fetal bovine serum (FBS) and penicillin/streptomycin. Stably transfected putative knockouts clones were selected in 0.6 mg/mL G418. LNCaP cells were grown in RPMI 1640 containing 10% FBS and penicillin/streptomycin.
Western blot analysis
Lysates derived from equivalent numbers of cells were separated by SDS-PAGE on either 4-12% Bis-Tris or 3-8% Tris-Acetate gels, transferred to PVDF membranes, probed with primary and horseradish-peroxidase coupled secondary antibodies, and visualized by ECL (Amersham). Antibodies were obtained from Cascade Bioscience (PTEN clone 6H2.1); Cell Signaling Technologies (total Akt, phospho-Akt (S473), phospho-Akt (T308), total tuberin, phospho-tuberin (T1462)); and BD Biosciences (HJF-lα).
Measurement of cell size Cells were trypsinized in 0.5 mL, added to 0.5 mL serum-containing media, and further diluted in 10 mL Isoton® II. Cell diameters and volumes were deteπnined using a Multisizer™ 3 Coulter Counter (Beckman Coulter). At least 10,000 cells were counted per measurement.
Flow cytometry Cells were fixed in 70% ethanol and stained in phosphate-buffered saline (PBS) containing 0.1% Triton X-100, 50 μg/mL RNAse, and 50 μg/mL propidium iodide. DNA content was measured on a FACSort flow cytometer (Becton Dickinson), and data were analysed using ModFit software (Verity Software House) on both linear and log scales. At least 20,000 cells were analyzed per sample. BrdU incorporation assay
Cells were pulsed with 10 μM BrdU for 1 or 2 hours, trypsinized, and centrifuged. Using the BrdU Flow Kit (BD Pharmingen), cells were then fixed and permeabilized, treated with DNAse to expose BrdU epitopes, and stained with FITC-conjugated anti-BrdU antibodies. Cells were then counterstained with propidium iodide and analyzed by flow cytometry.
Mitotic trap assay
Cells were treated with 0.2 μg/mL nocodazole and immediately irradiated. At various time points, cells were collected by trypsinization, centrifuged, and simultaneously fixed and stained in a solution containing 3.7% formaldehyde, 0.5% Nonidet P-40, and 10 μg/mL Hoechst 33258 in PBS. Nuclei were visualized by fluorescence microscopy. Nuclei with condensed, evenly staining chromosomes were scored as mitotic. At least 300 cells were counted for each determination.
Clonogenic survival assay
Equivalent numbers of exponentially growing HCT116 PTEN4" + and PTEN7" cells were treated with a single dose of gamma radiation using a 137Cs source. 24 hours following irradiation the cells were trypsinized, counted, and plated at various dilutions in T25s. Colonies were allowed to grow undisturbed for 12 days, stained with crystal violet, and counted in a blinded fashion.
Results and Discussions
Targeted deletion of PTEN in human cancer cells
Somatic cell gene targeting was used to create an isogenic set of human cancer cell lines appropriate for functional genetic analysis of PTEN. Though approaches based on small interfering RNAs were technically appealing, we chose gene targeting because it is the only technique available for the creation of a non- leaky, completely null allele. HCT116 cells were selected since they are suitable for somatic cell gene targeting; have well-defined, intact checkpoint responses; and have two wild-type alleles of PTEN (Waldman et al. 1995; Li et al. 1998). Of note, mutational inactivation of PTEN is found in approximately 14% of RER+ colorectal carcinomas (Zhou et al. 2002). The targeting strategy is depicted in Fig. 1 A and
described in Materials and methods. Homozygous deletion of PTEN in multiple, independently-derived clonal cell lines was confirmed by PCR, Southern blot, and Western blot analysis (Fig. IB, C and Figure 6). Isogenic PTEN474' and PTEN7" cells were morphologically indistinguishable (Fig. 2A, panels a,b). PTEN is known to regulate Akt and therefore modulate the phosphorylation state of Akt effectors. To determine if deletion of PTEN led to these expected biochemical consequences, Western blots with phospho-specific antibodies were performed (Fig. 1C). Deletion of PTEN resulted in Akt activation, as measured by an increase in the phosphorylation of critical residues S473 and T308 (Fig. 1C and data not shown). Inactivation of PTEN also led to the concomitant phosphorylation of the Akt substrate tuberin (TSC2) on T1462. Additionally, PTEN deletion led to a modest increase in the TSC2 -regulated gene HIF-lα (Brugarolas et al. 2003). These data demonstrate that deletion of PTEN in HCTl 16 cells leads to effective modulation of the PTEN signaling pathway. PTEN controls a radiation-induced size checkpoint in human cells
During exponential growth, PTEN-proficient and deficient human cells were similar in size (13.7 ± 0.7 μm and 14.5 + 1.5 μm for PTEN474" and PTEN"7" cells, respectively) (Fig. 2A, panels a,b; Fig. 2C, panel a). To determine whether the reported effects of PTEN on cell size might be the result of an unrecognized aberrant checkpoint, cells were measured after treatment with 6 Gy gamma radiation.
Whereas PTEN4" + cells enlarged slightly and then growth-arrested, PTEN" " cells enlarged dramatically, increasing nine times in volume by post-irradiation day 6 (Figs. 2A-D). Similar results were obtained after treatment with 9 Gy (data not shown). To exclude the possibility that this phenotype was a clone-specific artifact unrelated to PTEN, four independently-derived PTEN4"74" cell lines (parental HCTl 16 cells and three clonal cell lines resulting from non-homologous integration of the targeting vector) and four independently-derived homozygous knockout PTEN " cell lines were studied. Each of the four PTEN7" cell lines (and none of the four PTEN4"74" cell lines) enlarged dramatically following radiation (data not shown). Time course studies indicated that the PTEN7" cells enlarged continuously following treatment, rather than growth-arresting after an initial increase in size (Fig. 2D). These data
pointed to the presence of a radiation-induced cell size checkpoint in HCTl 16 cells that was dependent on PTEN.
Next, we sought to rule out the possibility that the size difference in irradiated PTEN4"74" and PTEN7" cells might be secondary to polyploidization. To test this, irradiated PTEN4"74" and PTEN7" cells were stained with the vital dye Hoechst 33342 (10 μg/mL), flow sorted in a FACSAria high speed cell sorter (Becton Dickinson) to separate the Gl and G2/M populations, and measured. Irradiated PTEN7" cells in both the Gl and G2/M phases of the cell cycle were dramatically larger than their isogenic, flow sorted PTEN4" 4" counterparts (Gl = 13.0 and 21.5 μm for PTEN4" 4" and PTEN" " cells, respectively; G2/M = 13.4 and 29.4 μm for PTEN4"74" and PTEN" " cells, respectively). Furthermore, flow cytometry demonstrated that PTEN" " cells showed no increased propensity to undergo post-irradiation polyploidization (data not shown). These experiments demonstrated that abrogation of the cell size checkpoint is independent of ploidy. Pharmacological inhibition ofPBK or tnTOR in PTE " cells leads to checkpoint restoration hi addition to functioning as a lipid phosphatase, PTEN can also dephosphorylate protein substrates such as FAK and She (Tamura et al. 1998). PI3K inhibitors were employed to test whether abrogation of the cell size checkpoint was due to the specific loss of the PTEN lipid phosphatase activity. To do this, PTEN"7" cells were pre-treated for one hour with doses of wortmannin known to inactivate Akt through the reduction of cellular PtdIns(3,4,5)P3 levels. The cells were then irradiated, cultured for six days in the presence of the inhibitor, and measured (Fig. 3 A). Wortmannin was able to efficiently restore the cell size checkpoint, resulting in a post-irradiation size increase only 6% that of untreated irradiated cells.
Importantly, the size of irradiated PTEN4" + cells was virtually unaffected by wortmannin treatment. Similar results were obtained with LY294002 (data not shown). This experiment demonstrated that the elevated levels of PtdIns(3,4,5)P caused by PTEN inactivation were responsible for abrogation of the PTEN- dependent, radiation-induced cell size checkpoint.
PTEN inactivation can also lead to mTOR activation. It has recently been demonstrated that pharmacological inhibition of mTOR can ameliorate the cell size increase seen in PTEN" " neurons in conditional PTEN knockout mice, and that mTOR inhibition can slow the neoplastic proliferation of tumors in PTEN4"7" mice (Kwon et al. 2003, Podsypanina et al. 2001 ; Neshat et al. 2001). To test if mTOR activation was similarly necessary for abrogation of the radiation-induced cell size checkpoint, PTEN4"74" and PTEN7" cells were pre-treated with rapamycin, irradiated, cultured for six days in the presence of rapamycin, and measured. As depicted in Fig. 3B, treatment with rapamycin led to partial restoration of the cell size checkpoint, resulting in a post-irradiation size increase only 43% that of untreated irradiated cells. Rapamycin was non-toxic at the doses tested in this experiment (1- 500 nM). Importantly, treatment with rapamycin had little or no effect on the size of irradiated PTEN4" + cells. These data indicate that mTOR plays a significant role in modulating the PTEN-dependent radiation-induced cell size checkpoint, and suggest that other as-of-yet unidentified effectors play an important role as well.
A PtdIns(3,4,5)P3-dependent, radiation-induced cell size checkpoint exists in LNCaP cells
Next, we attempted to determine if the radiation-induced cell size checkpoint was identifiable in LNCaP cells, a genetically unmodified human prostate cancer cell line with homozygous mutational inactivation of PTEN (Li et al. 1997).
Following irradiation, LNCaP cells enlarged, as though they were deficient in the PTEN-dependent cell size checkpoint (Fig. 3C). Although it was not technically feasible to restore functional, normally-regulated PTEN to these cells, we mimicked restoration of PTEN through the application of LY294002. As in PTEN7" HCTl 16 cells, treatment of LNCAP cells with LY294002 restored the cell size checkpoint, enabling irradiated LNCaP cells to maintain their normal size. Importantly, LY294002 did not affect the size of unirradiated LNCaP cells. These data are particularly interesting in light of the recent findings of Fingar et al, who demonstrated that pre-treatment with LY294002 blocks the ability of pl6INK4A- arrested rat cells to grow to an increased size. Our experiment demonstrates that a human cancer cell line other than HCTl 16, with naturally-occurring PTEN
inactivation, also displays an aberrant, PtdIns(3,4,5)P3-regulated, radiation-induced cell size checkpoint.
Maintenance of the radiation-induced Gl and G2 checkpoints in PTEN cells
Next, a variety of experimental approaches were employed to determine whether abrogation of the radiation-induced cell size checkpoint was accompanied by aberrations in the conventional radiation-induced Gl and G2 arrests. This was a particularly important point, since it remained a formal possibility that the abrogation in cell size control might be secondary to a more primary defect in Gl or G2 checkpoint control. Of note, HCTl 16 cells have wild-type p53 and have been previously shown to have intact radiation-induced Gl and G2 checkpoints
(Waldman et al. 1995; Bunz et al. 1998). Three methods were employed to measure the state of the Gl and G2 checkpoints in PTEN " cells. First, flow cytometry was performed to measure the percentage of cells in Gl (2N) and G2/M (4N) following irradiation. There was no consistent difference in the cell cycle distributions correlating either with PTEN status or with the magnitude of size increase following irradiation (Fig. 4A). Second, BrdU incorporation assays were performed to directly measure the ability of the Gl checkpoint to prohibit entry of Gl cells into S phase following irradiation. The radiation-induced Gl checkpoint was completely intact in both PTEN4" 4" and PTEN" " cells (Fig. 4B). Third, mitotic trap assays were performed to directly measure the ability/ of the G2 checkpoint to prohibit entry of G2 cells into mitosis following irradiation. There was no measurable defect in the radiation- induced G2 checkpoint in PTEN"7" cells (Fig. 4C). Interestingly, Kandel et al. have recently reported attenuation of the radiation-induced G2 checkpoint in HCTl 16 cells that ectopically express a myristolated, activated form of Akt. The discrepancies between the data described in Kandel et al. and the data described herein are likely due to differences in the means used to achieve activated Akt. Taken together, our study revealed no aberrations in the radiation-induced Gl and G2 checkpoints in PTEN"7" cells. As such, the role for PTEN in controlling a radiation-induced cell size checkpoint appears to be a primary defect. Radios ensitivity in PTE " cells
Hartwell has suggested that abrogation of checkpoints may underlie the radio sensitivity and chemosensitivity found in many types of human cancer (Hartwell and Kastan 1994). As such, we hypothesized that abrogation of the PTEN- dependent cell size checkpoint would confer sensitivity to radiation therapy. To test this, eight isogenic PTEIN and PTEN" " cell lines were treated with various doses of gamma radiation and studied via clonogenic survival assay. As depicted in Fig. 5 A and Fig. 7, PTEN7" cells were up to an order of magnitude more sensitive to radiation than isogenic PTEN4" + cells. These results are consistent with a recent report that treatment of the PTEN-null human A 172 glioblastoma cell line with wortmannin leads to radioresistance, but are inconsistent with other reports suggesting that inhibition of PI3K causes radiosensitivity (Okaichi et al. 2002; McKenna and Muschel 2003). The factors governing radiosensitivity are clearly complex, and the role of PtdIns(3,4,5)P3 in the cellular response to radiation is a subject of some controversy. However, unlike virtually all other studies that address this point, our results were obtained without the use of pharmacological inhibitors, and therefore without their attendant non-specific toxicities. Further genetic studies are clearly warranted and will likely provide additional clarification. In summary, these data provide experimental confirmation for the proposed relationship between checkpoint control and radiosensitivity in human cancer, and are consistent with the clinical observation that some cancers commonly harboring mutations in PTEN (i.e., endometrial adenocarcmoma) are treatable with radiation therapy.
Conclusions
A common theme in cancer research has been the role of tumor suppressor genes such as p53 in control of the DNA damage-induced Gl and G2 checkpoints. Here we build on that theme by identifying a separate radiation-induced size checkpoint in human cells, and by demonstrating its dependence on PTEN, a commonly mutated tumor suppressor gene (Fig. 5B). To our knowledge, the existence of such a DNA damage-inducible size checkpoint has neither been postulated nor demonstrated in any organism. We show that this checkpoint is dependent on PtdIns(3,4,5)P and partially dependent on mTOR.
A number of recent studies performed in D. melanogaster and in mammalian systems have examined a related point - the biochemical basis of the coordination of cell cycle progression with cell growth (Fingar et al. 2002; Conlon and Raff 2003). The conclusion of these studies - that cell cycle progression and cell growth are coordinated but genetically separable - is consistent with the data presented herein.
This new role for PTEN in controlling cell size during radiation-induced cell cycle arrest is likely related to the recently-demonstrated role for PTEN in regulating the size of both cardiomyocytes and neurons (but not mouse embryo fibroblasts or stem cells) from PTEN knockout mice. It is intriguing to us that the two stimuli currently known to induce a size increase in PTEN" " cells (radiation and terminal differentiation) are similar to two of the most potent inducers of p53 responses (DNA damage and senescence) (Kastan et al. 1991; Serrano et al. 1997). Further study of such potential relationships seems warranted.
In addition to these findings, the cell system described herein will likely prove useful for studying other aspects of PTEN biology in human cells. The PTEN knockout cells represent, to our knowledge, the only isogenic set of human cells that differ only in the presence or the complete absence of endogenous wild-type PTEN. As such they may prove useful not only for studying the role of PTEN in cancer pathogenesis, but also for anticancer drug discovery targeting the PTEN pathway (Torrance et al. 2001).
In conclusion, here we identify a genetic mechanism that enforces an arrest in cell size during cell cycle arrest (modulation of PtdIns(3,4,5)P3 signaling by PTEN), and demonstrate that it is distinct from the mechanisms that enforce DNA damage-inducible Gl and G2 arrests. We suggest that abrogation of the DNA damage-induced size checkpoint may contribute to the pathogenesis of human cancer.
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Claims
1. An isolated recombinant human somatic cell, wherein at least one endogenous PTEN allele comprises a mutation that renders the PTEN allele non-functional.
2. The human somatic cell of claim 1, wherein the cell is a human cancer cell.
3. The human cancer cell of claim 2, wherein the cell is selected from a group consisting of: ghoblastoma multiforme cell, a colon adenocarcmoma cell, a malignant melanoma cell, an endometrial carcinoma cell, a prostate adenocarcinoma cell, a thyroid cancer cell and a breast cancer cell.
4. The cell of claim 3, wherein the mutation is a deletion of all or a portion of exon 2 of the PTEN allele.
5. The cell of claim 4, wherein both endogenous PTEN alleles comprise a deletion of all or a portion of exon 2 of the PTEN gene.
6. An isolated recombinant human somatic cell, wherein at least one endogenous PTEN allele comprises a mutation.
7. The human somatic cell of claim 6, wherein the mutation is a dominant negative PTEN mutation.
8. The human somatic cell of claim 6, wherein the mutation is a constitutive PTEN mutation.
9. An isogenic pair of human cancer cells, wherein members of the pair differ in the endogenous PTEN alleles and wherein a first member, referred to as the PTEN-deficient cell, comprises a mutation in at least one endogenous PTEN allele, and a second member, referred to as the isogenic control cell, comprises two normal endogenous PTEN alleles.
10. The isogenic pair of claim 9, wherein the first member comprises a mutation in both endogenous PTEN alleles.
11. The isogenic pair of claim 10, wherein the mutation is a deletion of all or a portion of exon 2 of the PTEN gene.
12. The isogenic pair of any one of claims 9-11, wherein the cell is a human cancer cell selected from the group consisting of: ghoblastoma multiforme cell, a colon adenocarcinoma cell, a malignant melanoma cell, an endometrial carcinoma cell, a prostate adenocarcinoma cell, a thyroid cancer cell and a breast cancer cell.
13. A somatic cell gene targeting vector comprising, in the following order:
(a) at least 800 nucleotides homologous to a region of the second intron of a PTEN gene;
(b) nucleic acid encoding a selectable marker; and
(c) at least 800 nucleotides homologous to a region of the third intron of a PTEN gene.
14. The somatic cell gene targeting vector of claim 13, wherein the selectable marker is an antibiotic resistance gene.
15. The somatic cell gene targeting vector of claim 14, wherein the selectable marker is a neomycin resistance gene.
16. A somatic cell gene targeting vector comprising, in the following order:
(a) at least 800 nucleotides, referred to as the first homology arm, homologous to a region of the second intron of a PTEN gene;
(b) a first lox site;
(c) an internal ribosome entry site (IRES); (d) nucleic acids encoding a selectable marker;
(e) a second lox site situated in the same orientation as the first lox site; and
(f) at least 800 nucleotides, referred to as the second homology arm, homologous to a region of the third intron of a PTEN gene.
17. The targeting vector of claim 16, wherein the first and second lox sites are loxP sites.
18. The somatic cell gene targeting vector of claim 17, wherein the first homology arm comprises 1665 nucleotides immediately upstream of the second exon of the PTEN gene, and the second homology arm comprises
2549 nucleotides beginning at nucleotide 409 of the third intron of the PTEN gene.
' 19. The somatic cell gene targeting vector of claim 17, wherein the selectable marker is an antibiotic resistance gene.
20. The somatic cell gene targeting vector of claim 18, wherein the selectable marker is a neomycin resistance gene.
21. A method for generating a human somatic cell comprising a mutation in at least one endogenous PTEN allele by gene targeting, comprising:
(a) transfecting human somatic cells with the targeting vector of any of claims 13-19, thereby producing transfected human somatic cells; and
(b) maintaining transfected cells produced in (a) under conditions appropriate for integration of the targeting vector into the endogenous PTEN allele(s) in the transfected cells, thereby producing cells having the targeting vector integrated in at least one endogenous PTEN allele.
22. The method of claim 20, further comprising:
(a) transfecting cells having a targeting vector integrated in a first endogenous PTEN allele produced by a method of claim 20 with a second targeting vector of any of claims 13-19, wherein the second targeting vector has a selectable marker different from the first integrated targeting vector; and
(b) maintaining transfected cells produced in (a) under conditions appropriate for integration of the second targeting vector into a second endogenous PTEN allele in the transfected cells, thereby producing cells having targeting vectors integrated in both endogenous PTEN alleles.
23. A method for generating a human somatic cell comprising a mutation in both endogenous PTEN alleles by gene targeting, comprising:
(a) transfecting human somatic cells with the targeting vector of any of claims 16-19, thereby producing transfected human somatic cells;
(b) maintaining transfected cells produced in (a) under conditions appropriate for integration of the targeting vector into the endogenous PTEN allele in the transfected cells, thereby producing cells having the targeting vector integrated in a first endogenous PTEN allele;
(c) providing cells having the targeting vector integrated in one endogenous PTEN allele with Cre, thereby producing Cre-containing cells; and
(d) maintaining the Cre-containing cells under conditions appropriate for Cre to excise one of the two lox sites and nucleic acids encoding a selectable marker, thereby producing PTEN4"7" cells comprising deletion in one endogenous PTEN allele. (e) transfecting PTEN4"7" cells produced in (d) with the targeting vector of any of claims 16-19,
(f) , maintaining transfected cells produced in (e) under conditions appropriate for integration of the targeting vector into a second PTEN allele in the transfected cells, thereby producing PTEN" " cells comprising deletion in both endogenous PTEN alleles.
24. The method of claim 22, wherein providing cells with Cre in step (c) comprises infecting the cells with Cre adenovirus.
25. The method of claim 22, wherein providing cells with Cre in step (c) comprises transfecting the cells with a Cre-expressing vector.
26. A method for identifying an anti-cancer agent, comprising:
(a) contacting an isogenic pair of any of claims 9-12 with a candidate agent; and
(b) assessing the growth of the isogenic pair, wherein if the agent preferentially slows the growth of the PTEN-deficient cell as compared to the isogenic control cell, the agent is an anti-cancer agent.
27. A method of identifying an anti-cancer agent, comprising:
(a) contacting an isogenic pair of any of claims 9-12 with a candidate agent; and
(b) determining viability of the isogenic pair, wherein if the agent preferentially causes death in the PTEN-deficient cell as compared to the isogenic control cell, the agent is an anti-cancer agent.
28. A method of identifying an anti-cancer agent, comprising:
(a) incubating a cell of any of claims 1 -8 in the presence of a candidate agent; and (b) determining viability of the PTEN-deficient cell, wherein if the agent causes cell death in the PTEN-deficient cell, the agent is an anti-cancer agent.
29. The method of claim 27 or 28, wherein the viability of the cells is determined by applying a dye to the cell, assessing the incorporation of the dye by the cell, wherein the incorporation of the dye by the cell indicating death of the cell.
30. The method of claim 29, wherein the dye is trypan blue.
31. An isolated recombinant human somatic cell, wherein both endogenous PTEN alleles comprise a deletion of all or a portion of exon 2 of the PTEN gene, resulting in a cell which undergoes transformation to turn into a cancer cell.
32. A method of identifying anti-cancer agents, comprising:
(a) contacting the cell of claim 31 with a candidate agent; and
(b) assessing the ability of the agent to prevent transformation resulted from the deletion of both endogenous PTEN alleles, wherein if the candidate agent prevents or slows down the transformation, the agent is an anti-cancer agent.
33. A method of treating cancer in a subject in need thereof, comprising administering to the subject an anti-cancer agent identified by a method of any one of claims 26-30 and 32.
34. A packaged pharmaceutical composition, which composition includes an amount of an anti-cancer agent identified by a method of any one of claims 26-30 and 32 sufficient for use in treating cancer.
35. The packaged pharmaceutical of claim 34, further comprising a label and/or instructions for use of the pharmaceutical composition in the treatment of cancer.
36. Use of an anti-cancer agent identified by a method of any one of claims 26- 30 and 32 in the manufacture of a medicament for the treatment of cancer.
37. A method of identifying an agent that modulates PTEN activity, comprising:
(a) comparing gene expression in a PTEN-deficient cell with gene expression in the isogenic control cell of an isogenic pair of any of claims 9-12;
(b) identifying a gene whose expression is different between the two members of the isogenic pair;
(c) contacting the isogenic pair with a candidate agent; and
(d) assessing the ability of the agent to restore the altered expression of the gene identified in step (b) in a PTEN-deficient cell to the level of expression in an isogenic control cell, wherein if the agent restores the altered expression of the gene identified in step (b) in a PTEN-deficient cell to the level of expression in an isogenic control cell, the agent is an agent that modulates PTEN activity.
38. A method of modulating PTEN activity in a subj ect with a PTEN- associated condition, comprising administering to the subject an agent identified by a method of claim 37.
39. The method of claim 38, wherein the PTEN-associated condition is cancer and agent is an anti-cancer agent.
40. A packaged pharmaceutical composition, which composition includes an amount of a PTEN-modulating agent identified by a method, of claim 39 sufficient for use in treating a PTEN-associated condition.
41. The packaged pharmaceutical of claim 40, further comprising a label and/or instructions for use of the pharmaceutical composition in the treatment of a
PTEN-associated condition.
42. Use of an anti-cancer agent identified by a method of claim 39 in the manufacture of a medicament for the treatment of a PTEN-associated condition
43. Phenotypic data associated with an isogenic pair of any of claims 9-12, wherein the phenotypic data is in an electronic database.
44. A method for identifying a candidate agent that restores at least one PTEN function in PTEN-deficient cells, comprising:
(a) contacting the isogenic pair of any of claims 9-12 with a candidate agent;
(b) assessing at least one function of PTEN in the isogenic pair; and
(c) comparing the at least one function of PTEN in the PTEN-deficient cell to the isogenic control cell,
wherein if the at least one function of PTEN in the PTEN-deficient cell is comparable to that in the isogenic control cell, the candidate agent is an agent that restores the at least one PTEN function.
45. The method of claim 44, wherein the at least one function of PTEN is assessed by measuring subcellular localization of FOXO la protein.
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