WO2007062243A2 - Serum-free expansion of cells in culture - Google Patents
Serum-free expansion of cells in culture Download PDFInfo
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- WO2007062243A2 WO2007062243A2 PCT/US2006/045515 US2006045515W WO2007062243A2 WO 2007062243 A2 WO2007062243 A2 WO 2007062243A2 US 2006045515 W US2006045515 W US 2006045515W WO 2007062243 A2 WO2007062243 A2 WO 2007062243A2
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Definitions
- the present invention relates to compounds and methods for modulating the interaction between ⁇ -catenin or ⁇ -catenin and the coactivator protein CBP 5 or ⁇ -catenin or ⁇ -catenin and the coactivator protein p300, to promote proliferation/dedifferentiation or differentiation of stem/progenitor cells.
- Stem cells have received significant interest over the last few years due to their potential, under suitable cellular microenvironments, to differentiate and develop into a wide array of cell and tissue types.
- Several important biomedical applications would be enabled by the ability to generate sufficient pools of adult stem cells, including cell replacement therapy, gene therapy, and tissue engineering. According to the National Institutes of Health, the therapeutic use of stem cells will become a cornerstone of medicine within the next two decades:
- the invention relates to compounds and methods for modulating the interaction between ⁇ -catenin (or ⁇ -catenin) and the coactivator proteins CBP and p300 to either promote proliferation/dedifferentiation or differentiation of stem/progenitor cells.
- the agent increases the binding of ⁇ -catenin to CBP.
- the agent decreases the binding of p300 to ⁇ -catenin.
- the agent increases the binding of p300 to ⁇ -catenin, or the agent decreases the binding of CBP to ⁇ -catenin.
- the cell may be treated with the agent of the invention ex vivo and the cell may be a stem cell/progenitor cell.
- the agent is applied topically to a mammal comprising said cell.
- the agent modulates the interaction of Ser 89 phosphorylated p300 with a 14-3-3 protein, and the agent may be an analog of Fusicoccin, wherein the analog of Fusicoccin has the following general formula:
- the invention also relates to a method of modulating the interaction of ⁇ -catenin with CBP or p300 in a cell, wherein the agent modulates the interaction of prolyl isom erase (Pinl) with ⁇ -catenin, CBP or p300; in certain embodiments, the agent increases the association of Pinl with CBP.
- the agent modulates the interaction of prolyl isom erase (Pinl) with ⁇ -catenin, CBP or p300; in certain embodiments, the agent increases the association of Pinl with CBP.
- the agent may be incorporated into a biomaterial capable of supporting the growth of a stem cell; the stem cell may be a hematopoietic stem cell.
- the invention further relates to a method of enhancing the proliferation of a mammalian stem cell, comprising modulation of the interaction of ⁇ -catenin with CBP or p300; the agent may increase the binding of ⁇ -catenin to CBP; and the agent may decrease the binding of ⁇ - catenin to p300.
- the administration to the stem cell may be ex vivo, and the stem cells may be hematopoietic stem cells, hair cells, neural stem cells, pancreatic islet cells, or embryonic stem cells.
- the invention also relates to a method of maintaining embryonic stem cells in an undifferentiated state, comprising administering to the stem cell an agent, such as IQ-I, that selectively interacts with a differentially spliced regulatory subunit of PR72/130 of the serine/threonine protein phosphatase PP2A, where in the interaction of ⁇ -catenin/CBP is increased.
- an agent such as IQ-I
- the invention further relates to a method of maintaining embryonic stem cells in an undifferentiated state, comprising administering to the stem cell an agent that selectively interacts with a differentially spliced regulatory subunit of PR72/130 of the serine/threonine protein phosphatase PP2A, where in the interaction of ⁇ -catenin/p300 is decreased.
- the compound(s) inhibit the MAPK kinase pathway (MEK or ERK) and PKC in conjunction with stimulation of the canonical Wnt pathway, either with Wnt3a or a GSK3 inhibitor.
- MAPK kinase pathway MEK or ERK
- PKC PKC in conjunction with stimulation of the canonical Wnt pathway, either with Wnt3a or a GSK3 inhibitor.
- the invention further relates to assays for identifying compounds suitable for maintaining and/or promoting stem cell self-renewal.
- One such assay measures selective binding of a compound or agent to one or more of the PR72/130 subunits of the serine/threonine protein phosphatase PP2A.
- the agent will have activity similar to IQ-I .
- Figure IA-D shows that IQ-I maintains undifferentiated state of ESCs.
- Figure IA Structure of IQ-I.
- Figure IB IQ-I dose dependently maintains alkaline phosphatase activity.
- Figure 1C IQ-I, dose dependently, maintains SSEA-I expression.
- SSEA-I expression was analyzed 7 days after addition of IQ-I.
- Figure ID IQ-I enabled ESCs to proliferate in the undifferentiated state for at least 65 days, without MEF feeders or LIF.
- ESCs, in media supplemented with 4 ⁇ g/ml IQ-I were passaged 2-3 times every week at 1 X 10 5 -1 X 10 6 cells per 6cm dish and counted. All error bars represent mean ⁇ SD.
- FIG. 2A-C shows that IQ-I maintained ESCs self-renewal independently of LIF.
- Figure 2A IQ-I increases Nanog gene expression significantly compared to LIF. mRNA was isolated from ESCs, cultured under feeder-free system, and in the presence of either IQ-I (4 ⁇ g/ml) or LIF (1000 U/ml) for 21 hrs. Real-time RT-PCR for Nanog was performed. The control expression level of Nanog at day 0 was set at 1.
- Figure 2B removal of IQ-I decreases Nanog gene expression in ESCs. IQ-I was removed from ESCs which had been cultured under feeder- free system and in the presence of IQ-I (4 ⁇ g/ml), for three days.
- Figure 3A-C shows that IQ-I modulates Wnt signaling via interaction with PR72/130.
- Figure 3A affinity chromatography isolation of IQ-l's molecular target(s) were performed as described in Experimental Procedures. The two bands at 72 kDa and 130 kDa (labeled) were identified by mass spectral sequencing sequencing as the differentially spliced regulatory subunits PR72/130 of the serine/threonine protein phosphatase, PP2A.
- Figure 3B immunoblotting, using PR72/130 antisera, was performed to confirm the identity of the two bands.
- Figure 3C IQ-I causes developmental defects in zebrafish. 1-cell stage zebrafish embryos were treated with l ⁇ M IQ-I (Lower) or DMSO control (Upper) for 24 hrs. Results are representative of at least 10 embryos, from three independent experiments.
- Figure 4A-D shows that IQ-I maintenance of ESCs is Wnt/ ⁇ -catenin/CBP dependent.
- Figure 4A Wnt/ ⁇ -catenin Coactivator Switching Model.
- Figure 4B IQ-I increased the CBP/ ⁇ -catenin complex at the expense of the p300/ ⁇ -catenin complex.
- P19 cells were treated with Wnt3A supplemented with IQ-I, the CBP/ ⁇ -catenin ICG-OOl or DMSO control.
- Nuclear lysates were co-immunoprecipitated with anti-CBP or anti-p300 antibody and immunoblotted for ⁇ -catenin.
- FIG. 4C phosphorylation of p300 Ser89, in a PKC dependent manner, increased the p300/ ⁇ -catenin interaction.
- wild type p300 (1-110 aa) and the mutant p300 (p300 S89A) were mixed with P19 lysates and co- immunoprecipitated using the HA-tag.
- Western blot analysis for p300 ⁇ Upper) or ⁇ -catenin loading control ⁇ Lower were performed.
- Lane 1 p300/ ⁇ -catenin binding, Lane 2, PKC ⁇ phosphorylated p300/ ⁇ -catenin binding, Lane 3, S89A p300/ ⁇ -catenin binding, Lane 4, PKC ⁇ phosphorylated S89A p300/ ⁇ -catenin binding.
- Figure 4D IQ-I decreased the phosphorylation of p300.
- P19 cells were treated with IQ-I or DMSO (control) and exposed to purified Wnt3A for 24hrs. Cell lysates were immunoblotted using antibodies specific for p300, or p300 phosphorylated at position Ser 89.
- Lane 1 negative control Lane 2, Wnt3a plus DMSO control, Top panel phospho Ser89 p300 immunoblot, Middle panel p300 immunoblot, Lane 3, Wnt3a plus IQ-I, Top panel phospho Ser89 p300 immunoblot, Middle panel p300 immunoblot.
- Figure 5 A-C shows the pluripotency of long term cultured ESCs.
- Figure 5A long term cultured ESCs were induced to form embryoid bodies in suspension cultures for 3 days. ESCs cultured in media in the presence of Wnt3A and IQ-I (4 ⁇ g/ml) for 48 days were able to form embryoid bodies ⁇ Left). ESCs lose their ability to form embryoid bodies after 3 days of culturing in the absence IQ-I ⁇ Right).
- Figure 5B IQ-I treated ESCs derived embryoid bodies were cultured (adherence culture) for 7-14 days to induce further differentiation.
- FIG. 6 shows TCF/ ⁇ -catenin reporter gene analysis.
- the TCF/ ⁇ -catenin reporter construct TopFlash was cotransfected with a constitutively active ⁇ -catenin into NIH- 3T3 cells - wt, CBP (+/-) and p300 (+/-) - in the presence or absence of IQ-I . There was no effect of IQ-I on the Fopflash reporter.
- Figure 7 shows long term culture of ESCs in feeder free system with serum- free media supplemented with IQ-I and purified Wnt3a.
- ESCs were cultured in 15% KSR, 4 ⁇ g/ml of IQ-I and 100ng/ml Wnt3a.
- ESCs were passaged 2-3 times per week at IXlO 5 -IXlO 6 cells per 6cm dish and cells were counted.
- Figure 8 shows that IQ-I maintenance of ESC proliferation and pluripotency was dependent on Wnt signaling in 15% KSR media. AP activity of ESCs cultured for 7days in IQ-I containing media supplemented with 15% FCS or 15% KSR. Addition of Wnt3a increases AP activity of ESCs cultured for 7days in 15% KSR containing media to the same level as 15% FCS containing media. (Error bars represent ⁇ S.D.)
- Figure 9 shows real time RT-PCR performed on ESCs cultured in 15% KSR supplemented with IQ-I and Wnt3a for 42 days without MEF feeders. Over the 42-day culture period, ESCs cultured in the media in the presence of IQ-I and Wnt3a maintain expression of the pluripotency markers, (a) Nanog, (b) Oct3/4 and (c) Rex-1. The expression level on day 0 was set as 1.
- Figure 10 is a schematic representation of two possible mechanisms of action of ⁇ -catenin, resulting from alternative interaction with CBP or p300 in the nucleus.
- Figure 11 illustrates that ICG-001 at lO ⁇ M concentration induced the differentiation of C2C12 myoblasts in growth medium, similar to differentiation media, and compared to growth medium alone.
- Figure 12 illustrates that differentiation of C2C12 myoblasts was induced by lO ⁇ M ICG-001 in growth medium, similar to differentiation medium.
- Stem cells are responsible for the regeneration and maintenance of tissues by balancing the processes of self-renewal (i.e., making new stem cells) and differentiation (i.e., generating cells committed to terminal differentiation). This balance results from integration of regulatory signals intrinsic to the stem cell, as well as extrinsic signals from the microenvironment. Perturbations in the balance between self-renewal and differentiation may result in disease, either as a result of stem cell depletion (e.g., aplastic anemia) or increased self-renewal (e.g., cancer). In neurogenesis, the transcription factors Soxl, Sox 2 and Sox3 play a role in maintaining neural cells in an undifferentiated state.
- stem cell depletion e.g., aplastic anemia
- self-renewal e.g., cancer
- the transcription factors Soxl, Sox 2 and Sox3 play a role in maintaining neural cells in an undifferentiated state.
- HSC hematopoietic stem cell
- CBP and p300 play important roles in HSC self-renewal and differentiation.
- CBP and p300 function as molecular integrators of various transcriptional signals. When recruited to promoters by transcription factors, they function as co-activators of transcription through multiple mechanisms, including chromatin remodeling, acetylation of associated proteins, and recruitment of the basal transcription machinery.
- CBP and p300 are highly homologous on a structural level, with up to 93% identity within certain protein-binding domains (SEQ ID NO: 1 and 2). For most functions, the two proteins appear to be functionally redundant.
- mouse genetic loss-of-function studies demonstrated a difference between p300 and CBP function in HSCs: loss of CBP results in defective HSC self-renewal, whereas loss of p300 results in defective hematopoietic differentiation.
- CBP and p300 have been previously shown to interact with many of the known transcription factors shown to be important in HSC regulation (e.g., HoxB4, ⁇ -catenin, Notch, AML- 1 , MLL). Earlier results suggest that within HSCs there may be transcription factors that are specifically co-activated by CBP that are critical for self-renewal, and others that are preferentially co-activated by p300 that are critically required for differentiation.
- One example of a signaling pathway that seems to utilize CBP and p300 differentially is the Wnt signaling pathway. The Wnt signaling pathway has been shown to play a pertinent role in the development and maintenance of various tissues, including blood, intestines, and skin.
- ⁇ -catenin (SEQ ID NO:3) is a vertebrate homolog of Drosophila gene armadillo, which functions in both cell adhesion and, as discussed herein, the Wnt signaling pathway, ⁇ -catenin (SEQ ID NO:4) is also a vertebrate homolog of armadillo, ⁇ -catenin and ⁇ -catenin have analogous structures and functions, and they have the ability to be regulated by the APC tumor suppressor.
- Activation of the Wnt signaling pathway requires the nuclear stabilization of TCF (T cell factor)/ ⁇ -catenin complexes and recruitment of transcriptional co-activators, such as CBP and p300.
- TCF T cell factor
- ⁇ -catenin is constitutively produced in the cell, and inhibitory mechanisms exist to maintain ⁇ -catenin levels at below those that would lead to aberrant transcriptional activity in vivo, leading to pathological conditions such as cancer.
- Emami and colleagues PNAS 5 IOl, 12682-7, 2004
- ⁇ -catenin preferentially associates with CBP in cancer cells In one example of aberrant regulation, Emami and colleagues (PNAS 5 IOl, 12682-7, 2004) recently demonstrated that ⁇ -catenin preferentially associates with CBP in cancer cells.
- ⁇ -catenin when ⁇ -catenin was prevented from associating with CBP, by utilizing a ⁇ -catenin/CBP-specific inhibitor, ⁇ -catenin could bind to p300.
- the "alternative" binding of ⁇ -catenin to p300 was accompanied by the execution of a differentiative genetic program (Teo et al. PNAS submitted).
- ⁇ -catenin is thought to promote proliferation without differentiation by binding to and activating CBP, and to initiate differentiation with limited proliferation by binding to and activating p300. Perturbation of ⁇ - catenin interation with CBP and/or p300 is expected therefore to influence differentiation or proliferation.
- Stem cell therapy is based on the ability of human fetal or adult pluripotent stem cells to differentiate into a variety of cell types. Stem cells may be used to replace damaged cells as a treatment for many different diseases including cancer, Parkinson's disease, spinal cord injury, burns, diabetes, heart disease, rheumatoid arthritis, and osteoarthritis and for gene therapy (Lazic, S.E. et al. J Hematother Stem Cell Res, 12(6):635-642, Gafhi, Y. et al. Gene Ther, 11(4):417- 426). Stem cell therapy has long been an exciting potential medical breakthrough. The ability to inject normal stem cells into a patient, where they could generate organ-specific cells to potentially replace defective patient tissues, offers enormous potential.
- Embryonic stem cells represent an important research tool and a potential resource for regenerative medicine.
- ES cells are cocultured with a supportive feeder cell layer of murine embryonic fibroblasts (MEFs).
- MEFs murine embryonic fibroblasts
- the MEF feeder layer supplies factors which maintain the ES cell's capacity for self renewal and block spontaneous differentiation.
- IQ-I IQ-I prevents ⁇ -catenin from switching coactivator usage from CBP to p300.
- the increase in ⁇ -catenin/CBP mediated transcription at the expense of ⁇ -catenin/p300 mediated transcription is critical for the maintenance of stem cell pluripotency.
- ES cells derived from the inner cell mass of embryos can be cocultured on embryonic fibroblast cell layers. More recently, Xu et al. (Xu et al. Nat. Biotech. 2001, 19, 971) demonstrated that human ES cells can be grown under feeder free conditions utilizing MEF conditioned media. However variations in MEFs and MEF conditioned media, the lack of knowledge about what factors in conditioned media are important and concerns about zoonotic contamination emphasize the need for chemically defined conditions to expand ES cells. The ability to maintain adult skin stem cells in vitro has allowed engraftment of cultured skin onto bum victims (Green, H. (1991) SciAm, 265:96-102).
- BMT bone marrow transplantation
- PBSCT peripheral blood stem cell transplantation
- UCBSCT umbilical cord blood stem cell transplantation
- a unifying feature of all cancers is their capacity for unlimited self-renewal, which is also a defining characteristic of normal stem cells. Decades ago, it was discovered that the proliferative capacity of all cancer cells was not equivalent, and only a small minority of tumor cells were able to proliferate extensively (Hamburger, AW. et al. "(1977) Science,
- the Wnt/ ⁇ -catenin pathway initiates a signaling cascade critical in both normal development and the initiation and progression of cancer (Giles, RH et al. (2003) Biochim BiophysActa, 1653(l):l-24; Wodarz, A. et al. (1998) Amu Rev Cell Dev Biol, 14:59-88). Wnt signaling and in particular the nuclear functions of ⁇ -catenin have been shown to be important in the maintenance, proliferation as well as the differentiation of stem cells (Song, X. et al. (2003) Development, 130(14):3259-3268).
- the Wnt/ ⁇ -catenin pathway normally regulates expression of a range of genes involved in promoting both proliferation and differentiation. Activation of the Wnt pathway allows ⁇ -catenin to accumulate in the nucleus, bind to members of the TCF family of transcription factors, and form a transcriptionally active complex, by recruiting either the transcriptional coactivator CBP or its closely related homolog, p300. However, in greater than 85% of colon cancers, mutations in this pathway lead to constitutive activation and expression of target genes, e.g. c-myc, cyclin Dl and survivin, all of which are critical for rapid cell proliferation (Kolligs, FT. et al. (1999) MoI Cell Biol,
- HSC hematopoietic stem cells
- non-tumorigenic precursor cells e.g. C2C12 myoblasts (Figs. 11 and 12) and 3T3-L1 preadipocytes.
- the present invention is based on the premise that selectively inhibiting or down-modulating the ⁇ -catenin/p300 interaction (i.e. the right side of the pathway, Fig. 10) allows for proliferation without differentiation of pluripotent stem cells.
- the invention is also based in part on the discovery mat one mechanism involves the selective binding of a compound or agent to one or more of the PR72/130 subunits of the serine/threonine protein phosphatase PP2A, as disclosed in more detail in Example 1 herein.
- the agent will have activity similar to IQ- 1.
- the serine/threonine phosphatase PP2A is involved in regulating intracellular signaling, gene expression and cell cycle progression.
- a major function of PP2A is to regulate signaling cascades by opposing the activity of serine/threonine kinases (20).
- PP2A consists of a multisubunit complex. The core components of this trimeric complex are a 36 kDa catalytic, a 65 kDa regulatory (PR65) and a third variable subunit, one of which is PR72/130. PR72/130 represents tissue selective differentially spliced forms of the same gene (21).
- PR72/130 represents tissue selective differentially spliced forms of the same gene (21).
- PP2A regulates the Wnt signaling cascade at multiple levels (22, 23).
- PR72/130 has been shown to interact with the protein Naked cuticle (Nkd), a negative regulatory component of the Wnt signaling pathway (24), thereby modulating Wnt signaling (25).
- Nkd Naked cuticle
- PR72 like Nkd is a "negative" regulator of "canonical” Wnt/ ⁇ -catenin signaling and is involved in the switch from “canonical” Wnt signaling to "non-canonical” convergent extension (25).
- a preferable agent of the invention For maintenance of hematopoietic stem cell proliferation, a preferable agent of the invention "modulates" the proliferation of stem cells by affecting the post-translational modifications of any one of CBP, p300, or ⁇ -catenin, leading to a selective increase of ⁇ -catenin interaction with CBP or a selective decrease of ⁇ -catenin interaction with p300, wherein the agent does not directly bind to CBP or p300.
- “interact” and “interaction” is meant the normal biological relationship between two or more molecules, in this case ⁇ -catenin with CBP or p300.
- the agent increases the binding of ⁇ -catenin to CBP.
- the agent decreases the binding of ⁇ -catenin to p300.
- the overall result biases the ⁇ -catenin pathway towards "proliferative/non- differentiative program" of the target cells, which according to the invention are adult stem cells, such as hematopoietic stem cells, neural stem cells, or skin stem cells.
- An agent will "modulate" the proliferation of stem cells if the stem cells undergo more proliferation and/or less differentiation that in the absence of the agent.
- preferential binding of ⁇ -catenin to CBP is associated with maintaining hematopoietic stem cells in an undifferentiated state wherein they undergo continuous proliferation, resulting in enhanced numbers of undifferentiated cells useful for repopulating the hematopoietic system of a mammal, such as a human, in need of such treatment.
- modulation can be measured using, for example, assays described in the Examples herein.
- Agents suitable for use according to the invention can be screened using co- immunoprecipitation methods as described in Emami et al. PNAS, 101, 12682-7, 2004. Briefly, target cells, in this case HSC, are transfected with full-length ⁇ -catenin or with full-length p300. Nuclear lysates are treated with a radiolabeled test agent alone, or with cold test agent. Unbound radiolabeled test agent is removed, and incorporation of the radiolabeled test agent is measured. The results indicate whether the test agent specifically interacts with p300.
- Unbound proteins are removed by washing, then the specific interactions between ⁇ -catenin and p300, and ⁇ -catenin and CBP, are challenged using the test agent, for testing the compounds which directly bind to CBP or phosphor Ser89 p300.
- Agents that either increase the binding of ⁇ -catenin to CBP or decrease the binding of ⁇ -catenin to p300 are further tested in vitro using a suitable model of hematopoietic stem cell proliferation/differentiation.
- a suitable model of hematopoietic stem cell proliferation/differentiation One such model is described in Rebel, V. I. et al., PNAS 99:14789- 14794, 2002.
- Agents according to the invention may achieve the desired biological effects through one of several mechanisms.
- the agent may increase the binding of ⁇ -catenin to the amino-terminal 110 amino acids of CBP, or it may decrease the binding of ⁇ -catenin to the amino-terminal 110 amino acids of p300.
- the decrease in binding of ⁇ -catenin to p300 may be achieved by inhibiting the phosphorylation of Ser 89 of p300, wherein the phosphorylation is catalyzed by protein kinese C-epsilon (PKC), calcium/calmodulin-dependent protein kinase (CaMK), protease-activated receptor-4 (PAR-4), protease-activated receptor- 1 (PAR-I), or other serine/threonine protein kinase either directly or indirectly via a kinase cascade.
- PLC protein kinese C-epsilon
- CaMK calcium/calmodulin-dependent protein kinase
- PAR-4 protease-activated receptor-4
- PAR-I protease-activated receptor- 1
- the decreased phosphorylation of Ser 89 of p300 may be achieved by increasing the phosphorylation of Ser 90, for example by mitogen-activated protein kinase 4 (MAPK), cyclin- dependent kinase (CDK), or other serine/threonine protein kinase.
- mitogen-activated protein kinase 4 MAPK
- CDK cyclin- dependent kinase
- serine/threonine protein kinase for example by mitogen-activated protein kinase 4 (MAPK), cyclin- dependent kinase (CDK), or other serine/threonine protein kinase.
- a preferable agent of the invention may modulate the interaction of Ser 89- phosphorylated p300 with 14-3-3 proteins.
- Such agents may be analogs of Fusicoccin.
- Fusicoccin is a fungal toxin that is used to study H+-ATPase activation. The mechanism involves inducing an irreversible bond between the C-terminal portion of H+- ATPase, and 14- 3-3 protein. (Svennilid, F. et al., Plant Cell 11:2379-2392, 1999.) As a result, the C-terminal auto-inhibiting domain is displaced.
- analogs of Fusicoccin may modulate the interaction of Ser-89 phosphorylated p300 with 14-3-3 proteins, resulting in the decreased interaction of p300 with ⁇ -catenin.
- the agent modulates the interaction of Pin 1 with ⁇ -catenin or with CBP or p300. In one embodiment of the invention, the agent increases the association of Pin 1 with ⁇ -Catenin/CBP.
- Pinl prolyl isomerase
- Pinl overexpression has been reported to occur in human breast cancer. (Ryo, A. et al., Nat. Cell Biol. 3:793-801 (2001)). Pinl has also been implicated in normal spermatogenesis. Atchison, F. W. et al. (Biol. Reprod. 69: 1989-1997, 2003) reported that adult Pinl -deficient mice exhibited evidence of accelerated exhaustion of stem cell potential, and possible bias towards the differentiation pathway in the absence of Pin 1.
- Phosphorylation affects the conformation of proteins and creates conditions for binding of signal transducers to certain suitable domains capable of recognizing the phosphorylated residue or residues.
- Pinl specifically recognizes phosphorylated S/T-P bonds (Ser/Thr-Pro motifs). For example, Pinl directly binds a phosphorylated Ser-Pro motif (Ser 246-Pro) next to the APC-binding site in ⁇ -catenin, inhibits ⁇ -catenin interaction with adenomatous polyposis coli protein (APC), and thereby increases its translocation into the nucleus.
- APC adenomatous polyposis coli protein
- Pinl can also affect coactivator interactions with transcription factors.
- P73 is a transcription factor related to the tumor suppressor p53. Pinl -modified p73 displayed a higher affinity for p300 than unmodified p73. (Montovani, F. et al., MoI. Cell 14:625-636, 2004.)
- Pinl binding to phosphorylated ⁇ -catenin can increase the ⁇ -catenin/CBP interaction and thereby ⁇ -catenin/CBP dependent gene transcription promoting proliferation at the expense of differentiation.
- the agents of the invention can be incorporated into biomaterials on which hematopoietic stem cells are grown. Examples are disclosed in Horak et al., Biomaterials 25, 5249-60, 2004 and Harrison et al. Biomaterials 25, 4977-86, 2004.
- hematopoietic stem cells are disclosed herein as an embodiment of a target for the methods of the invention, the methods are applicable to any adult mammalian stem cells (or ES cells) that can be used for tissue regeneration.
- Adult stem cells constitute an undifferentiated population of cells that retain the ability to proliferate throughout postnatal life and to differentiate into specialized cells to replace cells that become diseased, die or are lost. (Agrawal, S. et al; Trends in Biotechnology 23:78-83, 2005.)
- stem cells suitable for use according to the invention include neural stem cells, skin stem cells, muscle stem cells, and pancreatic islet cells.
- pancreas-derived multipotent precursors Other stem cells may be induced to direct their differentiation toward the ⁇ cell.
- the methods and agents of the invention are suitable for inducing proliferation and limiting differentiation, in order to achieve a suitable number of cells for therapeutic use.
- Adult neural stem cells can differentiate into neurons, astrocytes, and oligodendrocytes, which are the three major lineages of the adult nervous system. For such applications of the invention, it may be appropriate to manipulate adult neural stem cells in situ in order to achieve neurogeneration in vivo.
- Active stem cells exist in adult brain in the dentate gyrus region of the hippocampus and the subventricular zone of the forebrain, and these stem cells can differentiate into neurons, astrocytes and oligodendrocytes.
- quiescent stem cell pools exist in the spinal cord, substantia nigra, optic nerve, and hypothalamus. (Agrawal, S. et al., 2005). Thus, defined pools of neural stem cells are available for modulation according to the invention.
- Skin stem cells may be induced to proliferate in vivo in order to enhance or restore hair growth. Recent evidence suggests that the Wnt pathway is involved in the ability of skin epithelial cells to acquire and/or maintain characteristics of multipotent stem cells. (Alonso, L. et al.; PNAS 100:11830-11835, 2003). Multipotent stem cells in skin receive Wnt signals before they commit to form hair follicles. In transgenic mouse skin in which ⁇ -catenin is constitutively stabilized, adult interfollicular epidermis takes on characteristics of embryonic skin, and may have the capacity to develop into hair follicles. (Gat, V., Cell 95:605-614, 1998).
- agents and methods of the invention are suitable for enhancing the proliferation of multipotent stem cells in the skin, to provide a reservoir of cells capable of forming hair follicles in order to increase or replace lost h air growth, including in fly applications, for example by topical use.
- U.S. Patent No. 6,419,913 discloses compositions suitable for topical delivery of therapeutic agents including agents for treatment of hair loss.
- U.S. Patent No. 6,680,344 also discloses topical delivery of agents for treating hair loss.
- the invention provides methods to enhance the proliferation of mammalian stem cells expressing an exogenous gene, prior to administration of the cells for therapeutic use.
- the gene therapy may also be conducted in vivo, for example, to alter the differentiation potential of neural stem cells.
- OCT4 is a known marker of the undifferentiated stem/progenitor cell state, and the promoter region can be functionally linked to a reporter gene such as EGFP (enhanced green fluorescent protein) as described in Gerrard, L. et al., Stem Cells 23:124-133 (2005)), or luciferase.
- EGFP enhanced green fluorescent protein
- Methods for testing the effect of small molecules on stem cells in vitro include those described by Chen, J.K. et al., P.N.A.S. 99:14701-14076 (2002) and Frank-Kamenetsky, M. et al., J. Biol 1:10 (2002).
- Embryonic stem cells represent an important tool for research and in principle, a potential resource for regenerative medicine (Hori Y et al Proc. Natl. Acad. Sci. USA 2002, 99, 16105, Kim JH et al. Nature, 2002, 418, 50, Lanza RP et al, Nat Med, 1999, 5, 975).
- Murine ES (mES) cells derived from the pluripotent inner cell mass, can be grown in the absence of feeder cells in media supplemented with serum and LIF. The ability of LIF to maintain mES cell pluripotency, requires activation of the STAT3 signaling pathway. However, LIF does not maintain the undifferentiated state of hES cells despite the fact that it activates the STAT3 signaling pathway (Humphrey R and Beattie G Stem Cells 2004, Daheron L and Opitz S Stem Cells 2004).
- IQ-I dose dependently maintained ES cell proliferation and pluripotency.
- IQ-I could maintain ES cell proliferation in the absence of serum if the media was supplemented with Wnt3a.
- Wnt3a plus IQ-I was sufficient to maintain ES cell proliferation and pluripotency for extended periods of time in culture (at least for 48 days).
- Wnt3a plus IQ-I increased the expression of Oct4 and Sox2 and decreased the expression of c-myc in P19 embryonic carcinoma cells.
- Boyer et al. Cell 122, 947, 2005
- Oct4 and Sox2 co-occupy the promoters/enhancers of a substantial portion of the genes required for the maintenance of human ES cells.
- c-myc appears to be a critical player in the balance between stem cell self renewal and differentiation and increased upon differentiation (Wilson A et al Genes Dev 18, 2747, 2004).
- Fig. 5 This model highlights the distinct roles of the coactivators CBP and p300 in the Wni/ ⁇ -catenin signaling pathway (Emami et al 2004, Ma et al 2005, Teo et al 2005, McMillan and Kahn 2005).
- the critical feature of the model is that TCF/ ⁇ -catenin/CBP mediated transcription is critical for stem cell/progenitor cell proliferation, whereas a switch to TCF/ ⁇ -catenin/p300 mediated transcription, whether induced chemogenomically with ICG-001 or endogenously, is critical to initiate a differentiative program with a more limited proliferative capacity.
- IQ-I selectively promoted the ⁇ -catenin/CBP interaction at the expense of the corresponding p300/ ⁇ -catenin interaction.
- affinity chromatography approach it is shown herein that the molecular targets of IQ-I are the differentially spliced regulatory subunits PR72/PR130 of the protein phosphatase PP2A (Bernards R 2004 and in press).
- Wnt signaling pathway clearly plays an important role in the expansion of "stem/progenitor" populations (Reya T et al. Nature, 2005.; 434, 843).
- Wnt signaling is also critical in the differentiation processes and development of cells, tissues and organs. These divergent behaviors of Wnt signaling are apparently controlled via selective usage of the coactivator proteins CBP or p300 (Ma et al., Teo et al. and McMillan and Kahn 2005). The endogenous choice of coactivator usage appears to be controlled by a complex array of differential post-translational modifications.
- Additional compounds useful for practicing the methods described and claimed herein include compounds disclosed and taught in Japanese patent publication JP2006/180763 A2, filed December 27, 2004, which is incorporated by reference herein.
- Compounds of the patent publication include those designated as compounds with the structures as disclosed in JP2006/180763 A2; examples are shown below.
- Compounds include the following, designated below as formula (compound) numbers.
- R 1 , R 2 , R 3 and R 4 may be the same or different; and represent an electron attractive group, an electron donating group or a hydrogen atom.
- Ring A indicates a 5 to 8 member ring containing inside the ring at least one hetero atom.
- X indicates an alkylene group with 0 to 10 atoms on the main chain. An alkylene group with 0 atoms indicates a single bond.
- the alkylene group may have at least one electron attractive group, electron donating group or hydrogen atom as a substitution group.
- G is an aromatic group which may have an electron attractive group, electron donating group or hydrogen group.
- Said ring A may have at least one electron attractive group and / or electron donating group as a substitution group instead of a -XG group.
- R 1 , R 2 , R 3 and R 4 , X and G are the same as defined in Compound 1 above.
- R 5 , R 6 , R 7 , R 8 and R 9 may be the same or they may be different and they indicate an electron attractive group, an electron donating group or a hydrogen atom.
- R 1 , R 2 , R 3 , R 4 , X and G are the same as defined in Compound 1 above.
- R 5 and R 6 may be the same or different and they indicate an electron attractive group, an electron donating group or a hydrogen atom.
- R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 and R 9 are the same as defined above.
- R 10 , R 1 ', R 12 and R 13 may be the same or different and represent an electron attractive group, an electron donating group or a hydrogen atom.
- the double single sided broken line indicates a single bond or a double bond. When the double single sided broken line indicates a double bond, a geometrical isomer is present for the wavy line part. There are no particular restrictions on where these geometrical isotropes are disposed and they may be independent of one another, or they may be E bodies or Z bodies.
- A may be a hydrogen atom or Formula 6:
- R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 and R 9 are the same as defined above.
- R 10 , R 1 ', R 12 may be the same or different and indicate an electron attractive group, an electron donating group or a hydrogen atom.
- R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are the same as defined above.
- R 7 , R 8 and R 9 may be the same or different and indicate an electron attractive group, an electron donating group or a hydrogen atom.
- R 1 , R 2 , R 3 and R 4 may be the same or different and represent an electron attractive group, an electron donating group or a hydrogen atom.
- electron donating group is meant a substitution group which can donate electrons to a benzene ring; by “electron attractive group” is addition, the electron donating group is defined as ⁇ ⁇ o and the electron attractive group is defined as ⁇ > o using Hammett's substitution group constant ⁇ . (Fundamental Organic Reaction Theory, Hashimoto Yasunobu, et al., Sankyo Publishing, 1997).
- R 1 , R 2 , R 3 , R 4 and X and G are defined as above.
- R 5 , R 6 , R 7 , R 8 and R 9 may be the same or different and indicate an electron attractive group, an electron donating group or a hydrogen atom.
- R 1 , R 2 , R 3 , R 4 and X and G are defined as above.
- R 5 and R 6 may be the same or different and represent an electron attractive group, an electron donating group or a hydrogen atom.
- A is a hydrogen atom or Formula 13:
- R 1 through R 13 may be the same or different and they represent an electron attractive group, an electron donating group or a hydrogen atom.
- R 1 through R 9 are electron attractive group, an electron donating group or a hydrogen atom which may be the same or different.
- R 1 through R 9 should be a group or an atom selected from a group made up of an alkyl group, an alkoxy group, a hydroxyl group, a nitro group, a nitrile group, an acetoxy group, an acetoxy alkyl group, a cyclic alkyl amino alkyl group which may include an oxygen atom, a dialkyl aminoalkyl group, a dialkyl amino vinyl group, a hydroxy alkyl amino alkyl group, an aryl aminovinyl group, an alkoxy carbonyl group, a halogen atom and a hydrogen atom.
- R 1 and R 2 should be a hydrogen atom
- R 3 should be a hydroxy group or an acetoxy group
- R should be an acetoxy alkyl group, a cyclic alkyl aminoalkyl group which may contain an oxygen atom, a di-lower alkyl amino lower alkyl group, a hydroxy lower alkyl amino lower alkyl group or a hydrogen atom
- R 5 should be a lower alkyl group, a di-lower alkyl amino vinyl group or an aryl amino vinyl group
- R 6 should be a nitro group
- R 7 , R 8 and R 9 should be a lower alkyl group, a lower alkoxy group or a hydrogen atom which may be the same or different.
- the compound examples include: 2-methyl-3-nitro- 1 -phenyl- lH-indole-6-ol l-(4-methoxy-phenyl)-2-methyl-3-nitro-lH-indole-6-ol 2-methyl-3 -nitro- 1 -p-tolyl- lH-indole-6-ol
- Methyl is especially suitable.
- Examples of the lower alkoxy group represented by R are as follows: methoxy, ethoxy, propoxy, isopropoxy, butoxy, sec-butoxy, tert-butoxy, pentoxy, hexyloxy, heptyloxy, octyloxy and the like. Methoxy is especially suitable.
- Examples of the halogen atom represented by R are as follows: fluorine, chlorine, bromine, iodine and the like but chlorine or bromine are especially suitable.
- Examples of the lower acyl group represented by R are as follows: formyl, acetyl, propionyl, butyryl and the like and acetyl is especially suitable.
- Examples of the lower alkyl group which may form the cyclic structure represented by R are as follows: cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and the like. Cyclopentyl, cyclohexyl and cycloheptyl are especially suitable.
- Examples of the lower alkoxy carbonyl represented by R are as follows: methoxy carbonyl, ethoxy carbonyl, propoxy carbonyl and the like, with methoxy carbonyl or ethoxy carbonyl being especially suitable.
- Examples of the amino carbonyl represented by R are as follows: -CONR 2 (R is a hydrogen atom which may be the same or different; it represents the lower alkyl group illustrated previously, and a phenyl group which may have a substitution group).
- Examples of the secondary aminocarbonyl group represented by R are -CONHR (indicates a lower alkyl group illustrated previously and a phenyl group which may have a substitution group).
- the tertiary amino carbonyl group may be -CONR 2 (R represents the lower alkyl group which was illustrated previously which may be the same or different, and a phenyl group which may have a substitution group).
- Examples of the amino alkyl group represented by R are: - (CH 2 ),, -NR 2 (where n is an integer from 1 to 8 and preferably 1).
- R is a hydrogen atom, a lower alkyl group, or a lower alkyl group which may form a cyclic structure which may be the same or different (1 to 3 hetero atoms may be included in the nitrogen and oxygen in the cyclic structure), and a phenyl group which may have a substitution group) and the like.
- acetoxy alkyl represented by R is: -(CH 2 ) n -Oac (where n is an integer from 1 to 8) and n is preferably 1.
- FBS ⁇ nvitrogen
- ImM MEM nonessential amino acids Invitrogen
- ImM 2-mercaptoethanol Sigma
- 2mM L-glutamine Invitrogen
- 1,000 U/ml LIF CHEMICON
- ESCs was determined. ESCs were plated into 96-well tissue culture plates (Falcon) at a density of 316-1,000 cells per well in ESC medium without LIF. Compounds were added at a final concentration of 4 ⁇ g/ml. Cells were treated with compounds for 7 days and test cell populations were washed with PBS and lOO ⁇ l of p-nitrophenyl phosphate solution (MOSS Inc.) was added. The absorbance at 405nm was measured by spectrophotometer (Spectramax, Molecular Devices).
- NIH-3T3 cells wt
- Real-time RT-PCR Total RNA was isolated and reverse-transcribed using Superscript III (Invitrogen).
- Real-time RT-PCR (Sybr Green; BioRad) was performed using with the gene- specific primers: Nanog-F agggtctgctactgagatgctctg (SEQ ID NO:5) Nanog-R caaccactggtttttctgccaccg (SEQ ID NO:6) GAPDH-F ggtgaaggtcggtgtgaacgga (SEQ ID NO:7) GAPDH-R tgttagtggggtctcgctctg (SEQ ID NO:8) Superscript III RT-PCR system (Invitrogen) according to the manufacturer' s protocol. 1 ⁇ 1 of cDNA was amplified by PCR with one or more of the gene-specific primers of SEQ ID NOs:5-
- Rex- 1 -R aaagctcttctcgcagccat (SEQ ID NO : 12)
- Sox-2-F gcatgtcctactcgcagcag (SEQ ID NO: 13) Sox-2-R gctgatcatgtcccggaggt (SEQ ID NO: 14)
- ESCs were cultured in 96-well cell culture dishes coated with a 0.1% aqueous gelatin solution and transfected with 0.2 ⁇ g/well of pSTAT3-TA-
- Luc (CLONTECH), using Lipofectamine 2000 (Invitrogen). After 6 hours, cells were washed and exposed to either IQ-I at the indicated doses, or LIF, for 24 hours. Transfection efficiencies were normalized with pRL-null luciferase plasmid. Luciferase assays were performed with the
- NIH-3T3 cells wt
- NIH-3T3 CBP(+/-) cells NIH-3T3 p300(+/-) cells were transfected as described in (26). Briefly, cells were transfected with
- Control DMSO was 0.025%. Cells were incubated for 24 hours. At the end of this incubation period, cells were washed, lysed and subjected to SDS-PAGE. CBP and p300 were detected using the rabbit polyclonal antibody (A-22)(l:5000); and (C-20)(l:5000) (Santa Cruz), respectively. Phospho-serine89-p300 was detected using custom antisera at a dilution of 1:100. Co-immunoprecipitation of ⁇ -catenin with CBP or p300 in P19 cells. Co- iimunoprecipitation was performed as described in (27). Briefly, P19 cells were treated with washed and lysed using.
- Nuclear fraction was isolated and precleared with antibodies to CBP(A- 22) or p300(N-15).
- ⁇ -catenin was detected using a mouse monoclonal antibody (Becton Dickinson) at a dilution of 1:5000.
- Recombinant p300 ( 1 - 110 aa) fused to an HA-tag were incubated with PKC ⁇ in kinase buffer (20 niM Hepes, pH 7.4, 10 mM magnesium acetate, 1 mM dithiothreitol, 100 ⁇ M ATP).
- Co-immunoprecipitation was carried out in Pl 9 lysates mixed with the in vitro phosphorylated p300 using an HA-tag antibody.
- p300 was detected using the rabbit polyclonal antibody (C20), 1:5000.
- ⁇ -catenin was detected using a mouse monoclonal antibody (Becton Dickinson) at a dilution of 1 :5000
- P19 cells were cultured to 90-100% confluency. Cells were lysed in protein-binding buffer [PBB, 20 mM Hepes, pH 7.9/10OmM NaCL/0.5 mM EDTA/0.5% Nonidet P-40/6mM MgC12/5 mM 2-mercaptoethanol/One tablet of Complete protease inhibitor mixture (Roche Molecular Biochemicals)]. Biotinylated IQ-I was bound overnight at room temperature to a 50% slurry of streptavidin-agarose beads (Amersham Pharmacia) in buffer containing 50% DMSO and 50% PBB. Beads were washed to remove unbound IQ-I and then incubated with whole cell lysates. Proteins eluted by boiling in SDS were Coomassie stained to detect target proteins, or immunoblotted.
- PBB protein-binding buffer
- Biotinylated IQ-I was bound overnight at room temperature to a 50% slurry of streptavidin-agarose beads (A
- Zebrafish experiments Wild-type (AB) zebrafish strain at 1-cell stage were treated with IQ-I at a final concentration of 1 ⁇ M in embryo medium for 24h. Control zebrafish embryos were incubated at an equivalent concentration of DMSO. At the end of this 24h treatment, embryos were manually dechorionated and imaged. Zebrafish embryos were maintained at 28 0 C. Results shown are representative of those obtained from at least 10 embryos for each group, from 2 independent experiments. Immunocytochemistry and Antibodies. Cells were fixed for 20 min with 4% paraformaldehyde in PBS. Immunostaining was carried out using standard protocols.
- IQ-I (Asahi Kasei Pharma, Figure 1) selectively increased ⁇ - catenin's usage of CBP as a coactivator at the expense of the use of p300, thereby maintaining the embryonic (ES) cells in an undifferentiated state.
- the combination of Wnt3a and IQ-I allowed for proliferation and maintenance of pluripotency as judged by the expression of Oct4, Nanog and Rexl, whereas IQ-I alone was not sufficient to cause proliferation and maintain pluripotency of ES cells.
- the present example relates to the identification of the molecular target of IQ-I.
- whole cell lysates from Pl 9 embryonic carcinoma cells were treated with biotinylated IQ-I.
- biotinylated IQ-I selectively bound three proteins (Fig. 3 A, lane 3).
- the two bands at 72 and 130 kDa were identified by mass spectral sequencing as the differentially spliced regulatory subunits PR72/130 of the serine/threonine protein phosphatase, PP2A. This was subsequently confirmed by immunoblotting (Fig. 3B).
- cytokine LIF by activating the Stat3 signal transduction pathway, maintains murine ESC symmetrical self-renewal and blocks differentiation (13, 14, 15). LIF did not maintain human ESC self-renewal (16, 17). Nanog is a divergent homeoprotein pluripotency sustaining factor for ESCs (18, 19). Nanog has been shown to act in parallel with LIF-driven stimulation of Stat3 to drive ESC self-renewal. Additionally, elevated expression of Nanog is sufficient for clonal expansion of ESCs and maintenance of expression of the key stem cell transcription factor Oct4, in the absence of Stat3 activation (19).
- IQ-I 5 To further investigate the mechanism of action of IQ-I 5 the effects of IQ-I on Nanog expression were determined. Whereas feeder-free ESCs treated with LIF only slightly increased Nanog levels, IQ-I significantly increased and maintained Nanog expression in culture as judged by real time RT-PCR (Fig. 2A). Removal of IQ-I from culture led to a precipitous drop in Nanog level (Fig. 2B). Loss of Nanog expression has been previously correlated with ESC differentiation (18, 19). To further confirm that the effects of IQ-I were not mediated via Stat3 signaling, a Stat3/luciferase reporter gene construct was utilized.
- IQ-I MODULATES WNT SIGNALING VIA INTERACTION WITH PR72/130 This example was performed to examine the effects of IQ-I on "non-canonical" Wnt signaling by looking at convergent extension during zebrafish (danio rerio) development. Zebraf ⁇ sh embryos treated with IuM IQ-I showed significant developmental defects (Fig. 3C). In particular a shortened tail, consistent with inhibition of convergent extension and similar to the effects of PR72 morpholinos, was observed (25).
- Example I 5 From this Example and Example I 5 it can r ⁇ /Z/I JU results in the disruption ot the PR72/13 ⁇ /Nkd complex, and inhibits "negative" regulation of canonical Wnt/ ⁇ -catenin signaling and a concurrent switch to "non-canonical" convergent extension.
- Wnt/ ⁇ -catenin signaling has been demonstrated to inhibit neuronal differentiation and maintain pluripotency in stem cells (1-3) and is critical for the expansion of progenitors (4).
- Wnt/ ⁇ -catenin signaling is also required for neural differentiation of ESCs and neural stem cells (5-6).
- ICG-OOl a recently characterized specific antagonist of the ⁇ - catenin/CBP interaction
- a model was developed to explain the divergent activities of Wnt/ ⁇ - catenin signaling (7, 8, 26, 27).
- ⁇ -catenin/CBP-mediated transcription is critical for "stem/progenitor" cell proliferation, whereas a switch to ⁇ - catenin/p300-mediated transcription is critical to initiate a differentiative program with a more limited proliferative capacity.
- the Wnt/ ⁇ -catenin reporter constructs TOPFLASH and FOPFLASH in wild-type NIH- 3T3 cells (wt), NIH-3T3 CBP(+/-) cells and NIH-3T3 p300(+/-) cells were used to determine whether IQ-I, by targeting the PR72/130 subunit with PP2A, was selectively increasing ⁇ - catenin's usage of CBP as a coactivator at the expense of p300, thereby maintaining the ESCs in the undifferentiated state (Fig. 4A).
- a point mutant constitutively translocating ⁇ -catenin pt- mut ⁇ -cat was used to stimulate reporter activity (28).
- IQ-I did not affect TOPFLASH or FOPFLASH activity in either the wt or CBP (+/-) cells, a 2-3 fold increase in TOPFLASH activity was observed in the p300 (+/-) cells (Fig. 6). This suggests that the effects of IQ-I are coactivator specific and dependent on the expression level of p300.
- IQ-I by increasing Wnt/ ⁇ -catenin/CBP-dependent signaling and preventing the switch to Wnt/ ⁇ - catenin/p300 mediated transcription was sufficient to maintain long term murine ESC pluripotency.
- IQ-I the differentially spliced regulatory subunits PR72/PR130 of the protein phosphatase PP2A. This is extremely interesting in that PR72 interacts with the protein Nkd (25), a Wnt-inducible antagonist of "canonical” Wnt/ ⁇ -catenin signaling (24). Similar to the results observed with PR72 morpholinos (25), IQ-I had dramatic effects on "non-canonical” two splice variants PR72 and PRl 3 O in the switch from "canonical” to "non-canonical” wnt signaling appears to be quite complex. Recently, Bernards et al.
- PR130 can antagonize some of the effects of Nkd in a variety of assays.
- PR130 morphilinos affected somite development and tail formation in xenopus embryos (34). Therefore, the effects of IQ-I on zebrafish embryonic development are consistent with potentially inhibiting both splice variants. Without being bound by a particular mechanism, the results are consistent with a model in which the interaction of IQ-I with PR72/130 results in the disruption of the PR72/130 Nkd complex thereby modulating Wnt signaling.
- IQ-I selectively promoted the ⁇ -catenin/CBP interaction at the expense of the corresponding ⁇ -catenin/p300 interaction.
- IQ-I by enhancing Wnt/ ⁇ -catenin/CBP-mediated transcription and preventing the switch to Wnt/ ⁇ -catenin/p300-mediated transcription allows for the long term expansion of murine ESCs while maintaining pluripotency without MEFs or serum.
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Cited By (9)
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GB2436737B (en) * | 2006-03-30 | 2008-07-09 | Univ Edinburgh | Culture medium containing kinase inhibitors,and uses thereof |
WO2009101407A2 (en) * | 2008-02-11 | 2009-08-20 | Cambridge Enterprise Limited | Improved reprogramming of mammalian cells, and the cells obtained |
WO2009157562A1 (en) * | 2008-06-27 | 2009-12-30 | 独立行政法人産業技術総合研究所 | Method for establishment of adult pancreatic stem cell, and method for differentiation of adult pancreatic stem cell |
WO2010139982A1 (en) * | 2009-06-02 | 2010-12-09 | The University Of Sheffield | Indole derivatives for the stimulation of stem cell proliferation |
WO2011096440A1 (en) | 2010-02-03 | 2011-08-11 | PRISM BioLab株式会社 | Compound capable of binding to naturally occurring denatured protein, and method for screening for the compound |
WO2012135845A1 (en) | 2011-04-01 | 2012-10-04 | Qiagen | Gene expression signature for wnt/b-catenin signaling pathway and use thereof |
US8389699B2 (en) | 2008-03-31 | 2013-03-05 | Applied Biosystems, Llc | Nucleic acid terminators incorporating a cationic moiety and methods for their use |
US8431395B2 (en) | 2006-08-01 | 2013-04-30 | The University Court Of The University Of Edinburgh | Pluripotent cells from rat and other species |
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US7576084B2 (en) | 2001-10-12 | 2009-08-18 | Choongwae Pharma Corporation | Reverse-turn mimetics and method relating thereto |
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Cited By (13)
Publication number | Priority date | Publication date | Assignee | Title |
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GB2436737B (en) * | 2006-03-30 | 2008-07-09 | Univ Edinburgh | Culture medium containing kinase inhibitors,and uses thereof |
US9074180B2 (en) | 2006-03-30 | 2015-07-07 | The University Court Of The University Of Edinburgh | Culture medium containing kinase inhibitors, and uses thereof |
US8431395B2 (en) | 2006-08-01 | 2013-04-30 | The University Court Of The University Of Edinburgh | Pluripotent cells from rat and other species |
WO2009101407A2 (en) * | 2008-02-11 | 2009-08-20 | Cambridge Enterprise Limited | Improved reprogramming of mammalian cells, and the cells obtained |
WO2009101407A3 (en) * | 2008-02-11 | 2009-10-22 | Cambridge Enterprise Limited | Improved reprogramming of mammalian cells, and the cells obtained |
US8389699B2 (en) | 2008-03-31 | 2013-03-05 | Applied Biosystems, Llc | Nucleic acid terminators incorporating a cationic moiety and methods for their use |
USRE46683E1 (en) | 2008-03-31 | 2018-01-23 | Applied Biosystems, Llc | Nucleic acid terminators incorporating a cationic moiety and methods for their use |
WO2009157562A1 (en) * | 2008-06-27 | 2009-12-30 | 独立行政法人産業技術総合研究所 | Method for establishment of adult pancreatic stem cell, and method for differentiation of adult pancreatic stem cell |
WO2010139982A1 (en) * | 2009-06-02 | 2010-12-09 | The University Of Sheffield | Indole derivatives for the stimulation of stem cell proliferation |
WO2011096440A1 (en) | 2010-02-03 | 2011-08-11 | PRISM BioLab株式会社 | Compound capable of binding to naturally occurring denatured protein, and method for screening for the compound |
WO2012135845A1 (en) | 2011-04-01 | 2012-10-04 | Qiagen | Gene expression signature for wnt/b-catenin signaling pathway and use thereof |
CN109988760A (en) * | 2017-12-29 | 2019-07-09 | 中国人民武装警察部队总医院 | A kind of inhibition hepatoma cell proliferation and invade active mRNAi and its application |
CN109988760B (en) * | 2017-12-29 | 2021-03-30 | 中国人民武装警察部队总医院 | mRNAi for inhibiting liver cancer cell proliferation and invasion activity and application thereof |
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AU2006318327B2 (en) | 2011-08-18 |
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EP1960777B1 (en) | 2009-10-21 |
KR20080073774A (en) | 2008-08-11 |
JP2009517085A (en) | 2009-04-30 |
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