Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention provides methods for screening for substances which inhibit the interaction between FP prostanoid receptors and a compound having PGF 2ot activity, methods for inhibiting the interaction, methods of inhibiting signaling mediated by ⁇ -catenin, and methods of treating cancer.
• the primary amino acid sequences of the ovine FP A and FP B prostanoid receptor isoforms are the same throughout their amino termini and seven membrane spanning domains, but the FP B isoform is truncated and lacks the last 46 carboxyl terminal amino acids present in the FP A isoform (1).
• This is very similar to the EP 3 (2) and thromboxane A2 (3) prostanoid receptors in which alternative mRNA splicing gives rise to a variety of isoforms in humans and in other species (4).
• the physiological significance of these receptor isoforms is not clear, although differences have been shown to exist with respect to second messenger coupling and receptor desensitization.
• the inventor has discovered that the FP A and FP B receptor isoforms have similar pharmacological properties and that prostaglandin F 2 ⁇ (PGF j J 1 stimulates phosphoinositide turnover to a similar extent in cells expressing these isoforms (1).
• PGF j J 1 stimulates phosphoinositide turnover to a similar extent in cells expressing these isoforms (1).
• stimulation of FP A or FP B expressing cells with PGF 2 ⁇ activates Rho leading to the formation of actin stress fibers, phosphorylation of pl25 focal adhesion kinase and cell rounding (5).
• Cell rounding involves the retraction of filopodia and a change from an isolated dendritic appearance to one in which the cells are rounded and form small cobblestone-like aggregates (see Figure 1 A).
• FP A expressing cells return to their original dendritic morphology, but the FP B expressing cells do not and remain rounded (6).
• Tcf/ ⁇ -catenin mediated transcriptional activation is elevated 16 hours after an initial 1 hour treatment of FP B expressing cells with PGF 2 ⁇ . This is not observed in FP A expressing cells and suggests that FP B expressing cells remain rounded because of persistent activation of a Tcf/ ⁇ -catenin signaling pathway.
• FP prostanoid receptor antagonists In terms of this receptor-based mechanism, it can be predicted with virtual certainty that a FP prostanoid receptor antagonist would have the same functional consequence as selectively decreasing PGF 2 ⁇ Thus, there is a reasonable expectation that FP prostanoid receptor antagonists would be effective as drags in the treatment and prevention of colorectal cancer and possibly other cancers as well. It is also clearly evident that the use of recombinant FP prostanoid receptors in functional screens would be an effective means of discovering existing and novel substances that could be used as such drugs.
• NSAIDs block the key enzymes (cyclooxygenases) required for the biosynthesis of all prostanoid metabolites. Because so many metabolites are affected, it is actually very uncertain as to how the NSAIDs are producing their beneficial actions. In addition, the use of NSAIDs is associated with a number of adverse effects related to their widespread effects on prostanoid metabolite biosynthesis. Presently, as it concerns the treatment and prevention of cancer, there is no existing technology based on the pharmacological blockade of a specific prostanoid receptor subtype or isoform. Furthermore, there are no existing data that we are aware of that would even support such an approach if it were contemplated.
• One object of the present invention is a method of screening for substances which inhibit the interaction between a FP prostanoid receptors and a compound having PGF 2 ⁇ activity including contacting a ceil expressing the FP prostanoid receptors with the substance to be screened, contacting the cell with said PGF 2 ⁇ compound, and assaying the presence or absence of interaction between FP prostanoid receptors and said PGF 2 ⁇ compound, wherein the absence of an interaction between FP prostanoid receptors and said PGF 2 ⁇ compound indicates the substance inhibits the interaction.
• the FP prostanoid receptor is FP B and said PGF 2 ⁇ compound is PGF 2 ⁇ .
• Another object of the present invention is assaying with a detection method selected from RT- PCR, Northern blot, luciferase reporter gene, ⁇ -gal reporter gene, and other reporters.
• Another object of the present invention is where the inhibiting substance is an antibody.
• an antibody can bind to FP prostanoid receptors or said PGF 2 ⁇ compound.
• Another object of the present invention is a method of screening for substances which inhibit the interaction between a FP prostanoid receptors and a compound having PGF 2 ⁇ activity by introducing and expressing a polynucleotide which encodes the FP prostanoid receptors; contacting the cell expressing the FP prostanoid receptors with the substance to be screened; contacting said cell with said PGF 2 ⁇ compound; and assaying the presence or absence of interaction between FP prostanoid receptors and said PGF 2 ⁇ compound, wherein the absence of an interaction between FP prostanoid receptors and said PGF 2 ⁇ compound indicates the substance inhibits the interaction.
• Another object of the present invention is a method of inhibiting the interaction between FP prostanoid receptors and a compound having PGF 2 ⁇ activity ound by contacting said FP prostanoid receptors with a substance which is capable of inhibiting the interaction.
• Another object of the present invention is a method of inhibiting ⁇ -catenin signaling by contacting a cell expressing FP prostanoid receptors with a substance which is capable of inhibiting the interaction between FP prostanoid receptors and a compound having PGF 2 ⁇ activity.
• Another object of the present invention is a method of inhibiting G12 and G13 mediated signaling by contacting a cell expressing FP prostanoid receptors with a substance which is capable of inhibiting the interaction between FP prostanoid receptors and a compound having PGF 2 ⁇ activity.
• Another object of the present invention is a method of treating cancer by administering to a patient a substance which inhibits the interaction between FP prostanoid receptors and a compound having PGF 2 ⁇ activity in an amount sufficient to inhibit the interaction.
• Another object of the present invention is a method of screening for substances which inhibit ⁇ -catenin signaling by contacting a cell expressing FP prostanoid receptors with the substance to be screened; contacting said cell with a compound having PGF 2 ⁇ activity; assaying the signaling activity, phosphorylation and/or the subcellular localization of the ⁇ -catenin; wherein a change in one or more of these properties indicates the substance inhibits the interaction ⁇ -catenin signaling.
• Another object of the present invention is method of screening for a substance for their ability to inhibit cancer cell growth by contacting a cell expressing FP prostanoid receptors with the substance to be screened; contacting the cell with PGF 2 ⁇ ; assaying the change in cell growth, wherein a decrease in cell growth is indicative of the substance usefulness for the treatment of cancer.
• the inhibition of cancer cell growth includes screening for substances which are useful for treating cancer.
• FIG. 1 A, phase contrast microscopy (x225) of FP A and FP B expressing cells after treatment with either vehicle (panels a and c) or 1 ⁇ M PGF 2 ⁇ (panels b and d) for 1 hour at 37°C.
• B ⁇ - catenin FITC immunofluorescence (green) and nuclear DAPI fluorescence (blue) microscopy (x225) of FP A and FP B cells after the same treatment. Cells were labeled and prepared for microscopy as described in Experimental Procedures. The results are representative of more than three experiments.
• FIG. 1 Immunoblot of ⁇ -catenin in particulate and cytosolic fractions prepared from FP A and FP B expressing cells after treatment with either vehicle (lanes a and c) or 1 ⁇ M PGF 2cc (lanes b and d) for 1 hour at 37°C.
• B RT-PCR of ⁇ -catenin and control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA from FP A and FP B expressing cells after the same treatment. Immunoblotting (10 ⁇ g of protein per sample) and RT-PCR were done as described in Experimental Procedures. Results for both the immunoblotting and RT-PCR are representative of three independent experiments.
• GPDH glyceraldehyde-3-phosphate dehydrogenase
• FIG. 3 Immunoblot (IB) of ⁇ -catenin ( ⁇ -cat) in cytosolic fractions and nuclear extracts; and immunoblot of serine/threonine phosphorylated (PS/PT) cytosolic ⁇ -catenin, from FP A and FP B expressing cells after treatment with either vehicle (lanes a and c) or 1 ⁇ M PGF 2 ⁇ (lanes b and d) for 1 hour at 37°C.
• PS/PT serine/threonine phosphorylated
• Cytosolic fractions were prepared as described in Experimental Procedures and samples (100 ⁇ g protein) were immunoprecipitated (IP) with antibodies to ⁇ -catenin and were first probed with antibodies to phosphoserine and phosphothreonine (upper panel); and then were stripped and reprobed with antibodies to ⁇ -catenin (middle panel). Immunoblotting of nuclear extracts (lower panel) was done with 10 ⁇ g protein per sample without prior immunoprecipitation. Results are representative of three independent experiments.
• FIG. 4 A, phase contrast microscopy (x225) and B, stimulation of Tcf/Lef responsive luciferase reporter gene activity after FP A and FP B expressing cells were treated with either vehicle or 1 ⁇ M PGF 2 ⁇ for 1 hour and were washed extensively drug-free media and incubated for an additional 16 hours at 37 °C in drug-free media.
• the transfection conditions, drug washout, and luciferase assay are provided in Experimental Procedures. Luciferase data are normalized to the vehicle treated FP A cells and are the means +/- the standard errors of three independent experiments each performed in duplicate.
• PGF 2 ⁇ is understood to mean prostaglandin F 2 ⁇ , analogues of PGF 2 ⁇ , and substances which mimic the action of PGF 2 ⁇ at the FP prostanoid receptor;
• GPCR is understood to mean G-protein coupled receptor; Tcf is understood to mean T-cell factor; Lef is understood to mean lymphoid enhancer factor; FITC is understood to mean fluorescein isothiocyanate; DAPI is understood to mean 4',6-diamidino-2-phenylindole; RT is understood to mean reverse transcription; PCR is understood to mean polymerase chain reaction;
• GSK-3 ⁇ is understood to mean glycogen synthase kinase-3 ⁇ ; and APC is understood to mean adenomatous polyposis coli.
• This invention provides a method for identifying substances potentially useful for the treatment and prevention of pre-cancerous and cancerous lesions in mammals.
• FP prostanoid receptors In performing the present method use of cells either endogenously or exogenously expressing the FP prostanoid receptors can be used.
• a suitable vector carrying the gene which encodes the FP A receptor can be introduced into the cell by procedure known in the art.
• the vector should be suitably constructed so as to facilitate expression of the FP prostanoid receptor gene upon introduction.
• the gene may be maintained episomally or may be integrated into the cellular chromosomes using methods known in the art.
• the FP prostanoid receptor gene which can be used in accordance with the present methods are those which are isolated from mammalian species, particularly, mouse, rat, human, sheep, cow and the like.
• the methods of screening substances can be performed in vitro or in vivo.
• Types of assays which are embodied within the present invention include analyzing the morphology of the cell; wherein said morphology is selected from the group consisting of cell rounding, loss of filopodia, and formation of cell aggregates; wherein an absence of a change in cell morphology compared to a cell not contacted with PGF 2 ⁇ indicates inhibition of the interaction; measuring apoptosis in said cell; assaying comprises measuring the transcription activity of a Tcf/Lef responsive promoter; or measuring the level of phosphorylation of ⁇ -catenin in the cell, wherein a increased level of phosphorylation compared to the ⁇ -catenin in a cell not contacted with said substance indicates the inhibition of the interaction.
• Additional detection methods for determining whether a substance successfully inhibits FP prostanoid receptors and PGF 2 ⁇ embodied within the present invention include inositol phosphate stimulation, activation of Rho, stress fiber formation, and phosphorylation of P125 (see reference 6).
• the cells are transfected with a reporter construct as are known in the art.
• reporter constructs are preferably sensitive to changes in -catenin signaling efficacy; typically by including a responsive promoter, e.g., a Tcf/Lef promoter.
• a responsive promoter e.g., a Tcf/Lef promoter.
• Levels of activity from the reporter construct can be determined by measuring changes in transcript levels, e.g, using Northern blots, dot-blots, primer extensions, RNase protections, RT-PCR and the like.
• the responsive promoter is functionally linked to a reporter gene whereby the levels of activity are measured by assaying changes in enzymatic, fluorescence or colorimetric activity of the reporter gene.
• reporter genes are known in the art and some examples include ⁇ -galactosidase, luciferase, green fluorescence protein and the like. These and other methods, genes, and vectors are described in, for example, Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1982) and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989) and the various references cited therein.
• the inhibition of the interaction between FP prostanoid receptors and PGF 2 ⁇ also inhibits signaling mediated by the G-proteins G12 and G13.
• This invention relates to a novel method for screening test substances for their ability to treat and prevent neoplasia, especially pre-cancerous lesions, safely.
• the present invention provides a method for identifying test substances that can be used to treat and prevent neoplasia, including precancerous lesions, with minimal side effects associated with inhibition and other non-specific interactions.
• the method of this invention is useful to identify substances that can be used to treat or prevent neoplasms, and which are not characterized by the serious side effects of conventional NSAIDs.
• Cancer and precancer may be thought of as diseases that involve unregulated cell growth.
• Cell growth involves a number of different factors. One factor is how rapidly cells proliferate, and another involves how rapidly cells die. Cells can die either by necrosis or apoptosis depending on the type of environmental stimuli. Cell differentiation is yet another factor that influences tumor growth kinetics. Resolving which of the many aspects of cell growth is affected by a test substance is important to the discovery of a relevant target for pharmaceutical therapy. Screening assays based on this selectivity can be combined with tests to determine which substances having growth inhibiting activity.
• Precancerous lesion includes syndromes represented by abnormal neoplastic, including dysplastic, changes of tissue. Examples include dysplastic growths in colonic, breast, prostate or lung tissues, or conditions such as dysplastic nevus syndrome, a precursor to malignant melanoma of the skin. Examples also include, in addition to dysplastic nevus syndromes, polyposis syndromes, colonic polyps, precancerous lesions of the cervix (i.e., cervical dysplasia), esophagus, lung, prostatic dysplasia, prostatic intraneoplasia, breast and/or skin and related conditions (e.g., actinic keraosis), whether the lesions are clinically identifiable or not.
• dysplastic growths in colonic, breast, prostate or lung tissues or conditions such as dysplastic nevus syndrome, a precursor to malignant melanoma of the skin. Examples also include, in addition to dysplastic nevus syndromes, polyposis syndromes, colonic polyps, precancerous
• Carcinoma or “cancer” refers to lesions which are cancerous. Examples include malignant melanomas, breast cancer, prostate cancer and colon cancer. As used herein, the terms “neoplasia” and “neoplasms” refer to both cancerous and pre-cancerous lesions.
• the screening method of the present invention involves further determining whether the substance reduces the growth of tumor cells.
• Various cell lines can be used in the sample depending on the tissue to be tested.
• these cell lines include: colonic adenocarcinoma; lung adenocarcinoma carcinoma; breast adenocarcinoma; melanoma line; keratinocytes; prostrate carcinoma and other cancer model cell lines commonly used in the art. Cytotoxicity data obtained using these cell lines are indicative of an inhibitory effect on neoplastic lesions.
• These and other cell lines are well characterized, and are used commonly used in the art for screening for new anti-cancer drugs.
• One embodiment of the present method of screening for substances which is useful for selecting substances for the treatment of cancer include the tumor progression model.
• This model includes the induction of cells into a cancerous state by applying TPA. The subsequent or concurrent administration of the tested substance and reduction in tumor progression would be indicative of the successful inhibition of the interaction between the FP prostanoid receptor and PGF 2 ⁇ .
• an IC 50 value is determined and used for comparative purposes. This value is equivalent to the concentration of drug needed to inhibit tumor cell growth by 50% relative to the control. Preferably, the IC 50 value should be less than 100 ⁇ M for the substance to be considered further for potential use for treating neoplastic lesions.
• One measure of successful inhibition is to assay the presence or absence of apoptosis in the cell carrying the FP prostanoid receptor. Methods of detecting apoptosis include the TUNEL assay and ELISA assay. These and other methods are disclosed in Tomei, L. D.
• Examples of useful substances capable of inhibiting the interaction between FP prostanoid receptors and PGF 2 ⁇ are antibodies that bind to either FP prostanoid receptors or PGF 2 ⁇ .
• antibodies binding to the FP prostanoid receptors bind to a extracellular potion of the receptor.
• Such antibodies are readily obtainable by one of skill in the art using conventional antibody isolation and production methods. Such methods are described in Harlow and Lane. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988, which is hereby incorporated by reference.
• compositions suitable for administration to a animal, preferably a human.
• pharmaceutical compositions typically include pharmaceutically acceptable carriers.
• the pharmaceutically acceptable carrier which can be used in the present invention is not limited particularly and includes an excipient, a binder, a lubricant, a colorant, a disintegrant, a buffer, an isotonic agent, a preservative, an anesthetic, and the like which can be used in a medical field.
• the pharmaceutical composition can be applied by any suitable administration method depending on the purpose of treatment and selected from injection (subcutaneous, intracutaneous, intravenous, intraperitoneal, etc.), eye dropping, instillation, percutaneous administration, oral administration, inhalation, and the like.
• injection subcutaneous, intracutaneous, intravenous, intraperitoneal, etc.
• eye dropping eye dropping, instillation, percutaneous administration, oral administration, inhalation, and the like.
• the dosage form such as injectable preparations (solutions, suspensions, emulsions, solids to be dissolved when used, etc.), tablets, capsules, granules, powders, liquids, liposome inclusions, ointments, gels, external powders, sprays, inhalating powders, eye drops, eye ointments, suppositories, pessaries, and the like can be selected appropriately depending on the administration method, and the inhibiting substance of the present invention can be accordingly formulated.
• Formulation in general is described in Chapter 25.2 of Comprehensive Medicinal Chemistry, Volume 5, Editor Hansch et al, Pergamon Press 1990.
• the dose of the medicine of the present invention should be set up individually depending on the purpose of administration (prevention, maintenance (prevention of aggravation), alleviation (improvement of symptom) or cure); the kind of disease; the symptom, sexuality and age of patient; the administration method and the like and is not limited particularly.
• HEK-293 cells stably expressing the ovine FP A and FP B prostanoid receptor isoforms (5) were split and grown in six-well plates containing 22-mm round glass cover slips for 3-4 days.
• Cells were treated with either vehicle (sodium carbonate, 0.002% final) or 1 ⁇ M PGF 2 ⁇ and were rapidly washed, fixed, and incubated with a 1 : 1000 dilution of a mouse monoclonal antibody to ⁇ -catenin (Transduction Laboratories). They were then washed and incubated with a 1 :4000 dilution of an FITC-conjugated goat anti-mouse secondary antibody (Sigma). Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI, Sigma). Cells were visualized by phase-contrast and epifluorescence microscopy as previously described (6).
• samples were rotated for 2 hours at 4°C with antibodies to ⁇ -catenin, followed by addition of protein-G sepharose (Amersham) and rotation for another hour.
• the sepharose was washed with lysis buffer and then resuspended with SDS-PAGE sample buffer and boiled.
• Samples were electrophoresed on 7.5% SDS- polyacrylamide gels, transferred to nitrocellulose membranes, and incubated with either antibodies to ⁇ -catenin or with a mixture of mouse monoclonal antibodies to phosphoserine (Sigma) and phosphothreonine (Sigma).
• RT-PCR was done using the Superscript Preamplification System (Life Technologies) and 1 ⁇ g of RNA/sample that had been pretreated with DNase I. This was followed by PCR using an initial incubation at 94°C for 5 minutes, followed by 20 cycles of 94°C, 60°C, and 68°C each for 2 minutes, and a final incubation at 68°C for 10 minutes.
• the human ⁇ -catenin and GAPDH primer pairs were exactly according to Rezvani & Liew (8).
• Product sizes were 521 bp for ⁇ -catenin and 737 bp for GAPDH and were resolved by electrophoresis on 1.5% agarose gels. Preliminary experiments were done to find the optimal conditions for quantitative amplification of ⁇ -catenin and GAPDH mRNA.
• Tcf/Lef Reporter Gene Assay Cells were split into 10-cm dishes and the next day were transiently transfected using FuGENE-6 (Roche) and either 10 ⁇ g/dish of the wildtype Tcf/Lef reporter plasmid, TOPflash, or the mutant plasmid, FOPflash. FOPflash differs from TOPflash by mutation of its Tcf binding sites and serves to differentiate Tcf/ ⁇ -catenin mediated signaling from background (Upstate Biotechnology). Cells were incubated overnight and were treated for 1 hour at 37°C with either vehicle or 1 ⁇ M PGF 2 ⁇ .
• Figure 1 A shows phase contrast microscopy of HEK cells stably expressing either the ovine FP A prostanoid receptor (panels a and b) or the ovine FP B prostanoid receptor (panels c and d) following 1 hour treatment with either vehicle (panels a and c) or 1 ⁇ M PGF 2 ⁇ (panels b and d).
• ovine FP A prostanoid receptor panels a and b
• ovine FP B prostanoid receptor panels c and d
• Figure IB shows that PGF 2 ⁇ treatment resulted in a marked accumulation of ⁇ -catenin in regions of cell-to-cell contact in FP B expressing cells (panels c and d), but not in FP A expressing cells (panels a and b). Both cells lines, however, showed agonist dependent cell rounding following treatment with PGF 2 ⁇ ( Figure 1 A) indicating that the process of cell rounding itself was not responsible for the increased contiguous accumulation of ⁇ -catenin in the FP B expressing cells.
• ⁇ -catenin is well recognized as a signaling molecule that undergoes stimulus dependent translocation from the cytosol to the nucleus where it is involved in the regulation of Tcf/Lef mediated gene transcription (9-11).
• Figure 2A shows that the expression of ⁇ -catenin is higher in both the particulate and cytosolic fractions from FP B expressing cells as compared with FP A expressing cells.
• Serine/threonine phosphorylation of ⁇ -catenin by glycogen synthase kinase-3 ⁇ marks ⁇ -catenin for degradation and is a critical factor in the regulation of its signaling activity (12,13).
• cytosolic ⁇ -catenin is phosphorylated leading to an association with the tumor suppressor protein, adenomatous polyposis coli (APC), and the scaffolding protein, axin, which is then followed by ubiquitination and proteasomal degradation (14).
• APC adenomatous polyposis coli
• FIG. 3 shows that in FP A expressing cells the vehicle control levels of cytosolic ⁇ - catenin are very low and there is no detectable phosphorylation (lane a). Following treatment with PGF 2 ⁇ the levels of cytosolic ⁇ -catenin increase and there is a marked increase in phosphorylation (lane b). In FP B expressing cells the vehicle control levels of cytosolic ⁇ -catenin are already elevated and so is phosphorylation (lane c). This probably reflects endogenous GSK-3 ⁇ activity and tight coupling to the elevated levels of cytoplasmic ⁇ -catenin.
• ⁇ -catenin is known to interact with members of the Tcf/Lef family of transcription factors (15), which in many instances serves as a switch for cellular differentiation and transformation. Because of this signaling potential, we were interested in the possibility that the failure of FP B expressing cells to return to their original dendritic morphology following removal of PGF 2 ⁇ might represent a transformation event induced by a ⁇ - catenin mediated switch in gene expression. To examine this we transiently transfected either FP A or FP B expressing cells with a Tcf/Lef responsive reporter plasmid (16) and measured luciferase reporter gene activity following treatment with 1 ⁇ M PGF 2 ⁇ .
• FP B expressing cells differ in several important regards from FP A expressing cells in terms of their potential for activation of Tcf/ ⁇ -catenin mediated signaling.
• First FP B expressing cells show PGF 2 ⁇ stimulated accumulation of ⁇ -catenin at their contiguous cell boundaries that is not evident in FP A expressing cells.
• Second, while both FP A and FP B expressing cells show PGF 2 ⁇ stimulated increases in cytosolic ⁇ -catenin, in FP A expressing cells this is accompanied by increased ⁇ -catenin phosphorylation and in FP B expressing cells by decreased ⁇ -catenin phosphorylation.
• FP B expressing cells show a profound stimulation of Tcf/Lef reporter gene activity 16 hours after agonist removal that is essentially absent in FP A expressing cells.
• a key control point could be in the differential phosphorylation of ⁇ -catenin.
• the agonist stimulated accumulation of ⁇ -catenin at the contiguous cell boundaries of FP B cells results in enhanced adhesive interactions with E-cadherin.
• this could initiate E-cadherin outside-in signaling leading to the sequential activation of phosphatidylinositol 3-kinase and Akt kinase (17).
• This is potentially meaningful because phosphorylation of GSK-3 ⁇ by Akt kinase is inhibitory (18) and could lead to the decreased phosphorylation of ⁇ -catenin found in agonist treated FP B cells.

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