A novel chemically directed route for the generation of definitive endoderm from human embryonic stem cells based on inhibition of GSK-3.

The use of small molecules to 'chemically direct' differentiation represents a powerful approach to promote specification of embryonic stem cells (ESCs) towards particular functional cell types for use in regenerative medicine and pharmaceutical applications. Here, we demonstrate a novel route for chemically directed differentiation of human ESCs (hESCs) into definitive endoderm (DE) exploiting a selective small-molecule inhibitor of glycogen synthase kinase 3 (GSK-3). This GSK-3 inhibitor, termed 1m, when used as the only supplement to a chemically defined feeder-free culture system, effectively promoted differentiation of ESC lines towards primitive streak (PS), mesoderm and DE. This contrasts with the role of GSK-3 in murine ESCs, where GSK-3 inhibition promotes pluripotency. Interestingly, 1m-mediated induction of differentiation involved transient NODAL expression and Nodal signalling. Prolonged treatment of hESCs with 1m resulted in the generation of a population of cells displaying hepatoblast characteristics, that is expressing α-fetoprotein and HNF4α. Furthermore, 1m-induced DE had the capacity to mature and generate hepatocyte-like cells capable of producing albumin. These findings describe, for the first time, the utility of GSK-3 inhibition, in a chemically directed approach, to a method of DE generation that is robust, potentially scalable and applicable to different hESC lines.

High basal γH2AX levels sustain self-renewal of mouse embryonic and induced pluripotent stem cells.

Phosphorylation of histone H2AX (γH2AX) is known to be the earliest indicator of DNA double-strand breaks. Recently, it has been shown that mouse embryonic stem cells (mESCs) have very high basal levels of γH2AX, even when they have not been exposed to genotoxic agents. As the specialized role of high basal γH2AX levels in pluripotent stem cells is still debated, we investigated whether H2AX phosphorylation is important in maintaining self-renewal of these cells. Here, we report that not only mESCs but also mouse-induced pluripotent stem cells (miPSCs), have high basal levels of γH2AX. We show that basal γH2AX levels decrease upon ESC and iPSC differentiation and increase when the cells are treated with self-renewal-enhancing small molecules. We observe that self-renewal activity is highly compromised in H2AX-/- cells and that it can be restored in these cells through reconstitution with a wild-type, but not a phospho-mutated, H2AX construct. Taken together, our findings suggest a novel function of H2AX that expands the knowledge of this histone variant beyond its role in DNA damage and into a new specialized biological function in mouse pluripotent stem cells.

The PI3K inhibitor arsenal: choose your weapon!

Owing to its widespread activation in inflammation and cancer, a growing appreciation of the therapeutic potential of inhibitors of the phosphoinositide 3-kinase (PI3K) pathway has stimulated intense interest in compounds with suitable pharmacological profiles. These are primarily directed toward PI3K itself. However, as class I PI3Ks are also essential for a range of normal physiological processes, broad spectrum PI3K inhibition could be poorly tolerated. In recent years, patents describing a new generation of PI3K inhibitors have started to appear, with a particular focus on the development of compounds with enhanced isoform selectivity for use as anti-cancer and anti-inflammatory therapies. However, challenges remain for the efforts to pharmacologically target this enzyme family in a successful manner.

Controlling embryonic stem cell proliferation and pluripotency: the role of PI3K- and GSK-3-dependent signalling.

ESCs (embryonic stem cells) are derived from the inner cell mass of pre-implantation embryos and are pluripotent, meaning they can differentiate into all of the cells that make up the adult organism. This property of pluripotency makes ESCs attractive as a model system for studying early development and for the generation of specific cell types for use in regenerative medicine and drug screening. In order to harness their potential, the molecular mechanisms regulating ESC pluripotency, proliferation and differentiation (i.e. cell fate) need to be understood so that pluripotency can be maintained during expansion, while differentiation to specific lineages can be induced accurately when required. The present review focuses on the potential roles that PI3K (phosphoinositide 3-kinase) and GSK-3 (glycogen synthase kinase 3)-dependent signalling play in the co-ordination and integration of mouse ESC pluripotency and proliferation and contrast this with our understanding of their functions in human ESCs.

Characterization of the phosphoinositide 3-kinase-dependent transcriptome in murine embryonic stem cells: identification of novel regulators of pluripotency.

Phosphoinositide 3-kinase (PI3K)-dependent signaling has been implicated in the regulation of embryonic stem (ES) cell fate. To gain further insight into the mechanisms regulated by PI3Ks in murine ES cells, we have performed expression profiling using Affymetrix GeneChips to characterize the transcriptional changes that arise as a result of inhibition of PI3K-dependent signaling. Using filtering of greater than 1.5-fold change in expression and an analysis of variance significance level of p < .05, we have defined a dataset comprising 646 probe sets that detect changes in transcript expression (469 down and 177 up) on inhibition of PI3Ks. Changes in expression of selected genes have been validated by quantitative reverse transcription polymerase chain reaction. Gene ontology analyses reveal significant over-representation of transcriptional regulators within our dataset. In addition, several known regulators of ES cell pluripotency, for example, Nanog, Esrrb, Tbx3, and Tcl-1, are among the downregulated genes. To evaluate the functional involvement of selected genes in regulation of ES cell self-renewal, we have used short interfering RNA-mediated knockdown. These studies identify genes not previously associated with control of ES cell fate that are involved in regulating ES cell pluripotency, including the protein tyrosine phosphatase Shp-1 and the Zscan4 family of zinc finger proteins. Further gain-of-function analyses demonstrate the importance of Zscan4c in regulation of ES cell pluripotency.

Three-dimensional culture systems for the expansion of pluripotent embryonic stem cells.

Mouse embryonic stem cell (ESC) lines, and more recently human ESC lines, have become valuable tools for studying early mammalian development. Increasing interest in ESCs and their differentiated progeny in drug discovery and as potential therapeutic agents has highlighted the fact that current two-dimensional (2D) static culturing techniques are inadequate for large-scale production. The culture of mammalian cells in three-dimensional (3D) agitated systems has been shown to overcome many of the restrictions of 2D and is therefore likely to be effective for ESC proliferation. Using murine ESCs as our initial model, we investigated the effectiveness of different 3D culture environments for the expansion of pluripotent ESCs. Solohill Collagen, Solohill FACT, and Cultispher-S microcarriers were employed and used in conjunction with stirred bioreactors. Initial seeding parameters, including cell number and agitation conditions, were found to be critical in promoting attachment to microcarriers and minimizing the size of aggregates formed. While all microcarriers supported the growth of undifferentiated mESCs, Cultispher-S out-performed the Solohill microcarriers. When cultured for successive passages on Cultispher-S microcarriers, mESCs maintained their pluripotency, demonstrated by self-renewal, expression of pluripotency markers and the ability to undergo multi-lineage differentiation. When these optimized conditions were applied to unweaned human ESCs, Cultispher-S microcarriers supported the growth of hESCs that retained expression of pluripotency markers including SSEA4, Tra-1-60, NANOG, and OCT-4. Our study highlights the importance of optimization of initial seeding parameters and provides proof-of-concept data demonstrating the utility of microcarriers and bioreactors for the expansion of hESCs.

The pleckstrin homology domain of Gab-2 is required for optimal interleukin-3 signalsome-mediated responses.

The adaptor protein Gab-2 coordinates the assembly of the IL-3 signalsome comprising Gab-2, Grb2, Shc, SHP-2 and PI3K. To investigate the role of the pleckstrin homology domain of Gab-2 in this process, epitope-tagged wild type Gab-2 (WTGab-2), Gab-2 lacking its PH domain (DeltaPHGab-2) and the Gab-2 PH domain alone (PHGab-2) were inducibly expressed in IL-3-dependent BaF/3 cells. Expression of DeltaPHGab-2 reduced IL-3-dependent proliferation and long-term activation of ERK1 and 2 and PKB by IL-3. While we demonstrate that the Gab-2 PH domain can bind PI(3,4,5)P3, it is dispensable for Gab-2 membrane localisation, tyrosine phosphorylation and signalsome formation. Rather, the proline-rich motifs of Gab-2 appear to contribute to the constitutive membrane localisation we observe, independently of tyrosine phosphorylation or the PH domain. Taken together, these findings suggest that once Gab-2 is tyrosine phosphorylated its PH domain is required for the optimal stabilisation of the signalsome, enabling full activation of downstream signals.

Novel octavalent cross-linker displays efficient trapping of protein-protein interactions.

A novel octavalent, resorcin[4]arene derived, cross-linker designed to overcome some of the limitations of commercially available reagents is significantly more efficient for covalent stabilisation of protein-protein interactions.

Isolation of C15: a novel antibody generated by phage display against mesenchymal stem cell-enriched fractions of adult human marrow.

Adult bone marrow stroma contains a source of mesenchymal stem cells (MSC) that have the capacity to self-renew and differentiate into multiple stromal lineages. These rare cells can be visualised indirectly by the formation of heterogeneous colonies, containing stem cells and their differentiated progeny in long-term culture. If MSC and their associated progenitor and precursor populations are to reach their full therapeutic potential, markers will be required to identify and characterize specific bone marrow stromal subsets. We sought to use phage display to generate antibodies against bone marrow mononuclear cells (BMMNC) enriched for colony forming cells. Initially, we identified our target cell population by comparing the colony forming efficiency (CFE) of CD49a-positive, STRO-1-positive and CD45-negative BMMNC subpopulations with unseparated BMMNC. Selection with anti-CD49a gave the greatest enrichment (19-fold) of colony forming cells and in light of these findings, we generated phage antibodies against CD49a-positive BMMNC by simultaneous positive/negative selection. A dominant clone (C15), generated after 3 rounds of selection, has been isolated and sequenced, then characterized for cell and tissue specificity. Sequence analysis showed that the V(H) and V(L) gene segments of C15 aligned most closely to the VH26/DP-47 and IGLV3S1/DPL16 germline V segments found in the synthetic repertoire. C15 bound to 4% of freshly isolated BMMNC and localized to osteoblastic cells and proximal marrow cells in areas of active bone formation in sections of osteophyte. C15 binding was upregulated in cultured bone marrow stromal cells (BMSC) and was also detected on bone-derived cell lines. This report demonstrates that phage display is a powerful tool for the isolation of antibodies against rare cell populations, and provides a platform for the future application of this technology in the search for antigens on MSC and other rare cell populations.

Phosphoinositide 3-kinases can act independently of p27Kip1 to regulate optimal IL-3-dependent cell cycle progression and proliferation.

We have examined the role of phosphoinositide 3-kinases (PI3K) in interleukin (IL)-3-dependent cell cycle progression and compared the effects of LY294002 with expression of a dominant negative form of p85, termed Deltap85, which more specifically inhibits class I(A) PI3Ks. Inhibition of PI3Ks in BaF/3 led to accumulation of cells in G1 and extension of cell cycle transit times. Biochemically, both LY294002 and Deltap85 decreased levels of p107 and cyclins D2, D3 and E and reduced retinoblastoma protein (pRb) phosphorylation. Significantly, only LY294002 treatment increased expression of p27(Kip1). Interestingly, LY294002 decreased IL-3-induced proliferation of primary bone marrow-derived mast cells (BMMC) derived from both wild-type and p27(Kip1)-deficient mice and importantly, LY294002 treatment failed to upregulate p27(Kip1) in wild-type BMMC. These data support a role for class I(A) PI3K in regulating optimal cell cycle progression in response to IL-3 and demonstrate that upregulation of p27(Kip1) is not essential for attenuation of the cell cycle resulting from PI3K inhibition.

Molecular interactions of SHP1 and SHP2 in IL-3-signalling.

SHP1 and SHP2 tyrosine phosphatases have both been implicated in signalling pathways downstream of the interleukin-3 (IL-3) receptor. We have investigated the co-association of SHP1 and SHP2 with tyrosine-phosphorylated proteins in IL-3-dependent BaF/3 cells. We demonstrate that both SHP1 and SHP2 associate with Aic2A (beta chain of the IL-3 receptor), Gab2 and the paired inhibitory receptor B (PIR-B). The individual SH2 domains of SHP2 can independently bind Gab2, potentially important for the adapter function of SHP2. Association of both phosphatases with Aic2A and Gab2 increases upon IL-3 treatment. Recruitment of SHP1 to PIR-B also increases in response to IL-3, suggesting a functional link between inhibitory and cytokine receptor signalling. Aic2A is a rapid target for dephosphorylation following IL-3 stimulation and substrate-trapping versions of both phosphatases identify Aic2A and Gab2 as substrates for SHP1 and SHP2. These studies suggest that SH2-domain interactions are important for targetting these phosphatases to their substrates.

Regulation of interleukin-3-induced substrate phosphorylation and cell survival by SHP-2 (Src-homology protein tyrosine phosphatase 2).

The cytosolic SHP-2 (Src homology protein tyrosine phosphatase 2) has previously been implicated in IL-3 (interleukin-3) signalling [Bone, Dechert, Jirik, Schrader and Welham (1997) J. Biol. Chem. 272, 14470 -14476; Craddock and Welham (1997) J. Biol. Chem. 272, 29281-29289; Welham, Dechert, Leslie, Jirik and Schrader (1994) J. Biol. Chem. 269, 23764-23768; Qu, Nguyen, Chen and Feng (2001) Blood 97, 911-914]. To investigate the role of SHP-2 in IL-3 signalling in greater detail, we have inducibly expressed WT (wild-type) or two potentially substrate-trapping mutant forms of SHP-2, generated by mutation of Asp-425 to Ala (D425A) or Cyst-459 to Ser (C459S), in IL-3-dependent BaF/3 cells. Effects on IL-3-induced tyrosine phosphorylation, signal transduction and functional responses were examined. Expression of C459S SHP-2 protected the beta-chain of the murine IL-3R (IL-3 receptor), the adaptor protein Gab2 (Grb2-associated binder 2), and a cytosolic protein of 48 kDa from tyrosine dephosphorylation, consistent with them being bona fide substrates of SHP-2 in IL-3 signalling. The tyrosine phosphorylation of a 135 kDa transmembrane protein was also protected upon expression of C459S SHP-2. We have identified the inhibitory immunoreceptor PECAM-1 (platelet endothelial cell adhesion molecule-1)/CD31 (cluster determinant 31) as a component of this 135 kDa substrate and also show that IL-3 can induce tyrosine phosphorylation of PECAM-1. Expression of WT, C459S and D425A forms of SHP-2 had little effect on IL-3-driven proliferation or STAT5 (signal transduction and activators of transcription) phosphorylation or activation of protein kinase B. However, expression of WT SHP-2 increased ERK (extracellular-signal-regulated kinase) activation. Interestingly, expression of C459S SHP-2 decreased ERK activation at later times after IL-3 stimulation, but potentiated IL-3-induced activation of Jun N-terminal kinases. In addition, expression of C459S SHP-2 decreased cell survival in suboptimal IL-3 and upon IL-3 withdrawal. These findings indicate that SHP-2 plays an important role in mediating the anti-apoptotic effect of IL-3 and raises the possibility that PECAM-1 participates in the modulation of cytokine-induced signals.

MicroRNA-122: a novel hepatocyte-enriched in vitro marker of drug-induced cellular toxicity.

Emerging hepatic models for the study of drug-induced toxicity include pluripotent stem cell-derived hepatocyte-like cells (HLCs) and complex hepatocyte-non-parenchymal cellular coculture to mimic the complex multicellular interactions that recapitulate the niche environment in the human liver. However, a specific marker of hepatocyte perturbation, required to discriminate hepatocyte damage from non-specific cellular toxicity contributed by non-hepatocyte cell types or immature differentiated cells is currently lacking, as the cytotoxicity assays routinely used in in vitro toxicology research depend on intracellular molecules which are ubiquitously present in all eukaryotic cell types. In this study, we demonstrate that microRNA-122 (miR-122) detection in cell culture media can be used as a hepatocyte-enriched in vitro marker of drug-induced toxicity in homogeneous cultures of hepatic cells, and a cell-specific marker of toxicity of hepatic cells in heterogeneous cultures such as HLCs generated from various differentiation protocols and pluripotent stem cell lines, where conventional cytotoxicity assays using generic cellular markers may not be appropriate. We show that the sensitivity of the miR-122 cytotoxicity assay is similar to conventional assays that measure lactate dehydrogenase activity and intracellular adenosine triphosphate when applied in hepatic models with high levels of intracellular miR-122, and can be multiplexed with other assays. MiR-122 as a biomarker also has the potential to bridge results in in vitro experiments to in vivo animal models and human samples using the same assay, and to link findings from clinical studies in determining the relevance of in vitro models being developed for the study of drug-induced liver injury.

Formation of a polarised primitive endoderm layer in embryoid bodies requires fgfr/erk signalling.

The primitive endoderm arises from the inner cell mass during mammalian pre-implantation development. It faces the blastocoel cavity and later gives rise to the extraembryonic parietal and visceral endoderm. Here, we investigate a key step in primitive endoderm development, the acquisition of apico-basolateral polarity and epithelial characteristics by the non-epithelial inner cell mass cells. Embryoid bodies, formed from mouse embryonic stem cells, were used as a model to study this transition. The outer cells of these embryoid bodies were found to gradually acquire the hallmarks of polarised epithelial cells and express markers of primitive endoderm cell fate. Fgf receptor/Erk signalling is known to be required for specification of the primitive endoderm, but its role in polarisation of this tissue is less well understood. To investigate the function of this pathway in the primitive endoderm, embryoid bodies were cultured in the presence of a small molecule inhibitor of Mek. This inhibitor caused a loss of expression of markers of primitive endoderm cell fate and maintenance of the pluripotency marker Nanog. In addition, a mislocalisation of apico-basolateral markers and disruption of the epithelial barrier, which normally blocks free diffusion across the epithelial cell layer, occurred. Two inhibitors of the Fgf receptor elicited similar phenotypes, suggesting that Fgf receptor signalling promotes Erk-mediated polarisation. This data shows that primitive endoderm cells of the outer layer of embryoid bodies gradually polarise, and formation of a polarised primitive endoderm layer requires the Fgf receptor/Erk signalling pathway.

Zscan4 is regulated by PI3-kinase and DNA-damaging agents and directly interacts with the transcriptional repressors LSD1 and CtBP2 in mouse embryonic stem cells.

The Zscan4 family of genes, encoding SCAN-domain and zinc finger-containing proteins, has been implicated in the control of early mammalian embryogenesis as well as the regulation of pluripotency and maintenance of genome integrity in mouse embryonic stem cells. However, many features of this enigmatic family of genes are poorly understood. Here we show that undifferentiated mouse embryonic stem cell (ESC) lines simultaneously express multiple members of the Zscan4 gene family, with Zscan4c, Zscan4f and Zscan4-ps2 consistently being the most abundant. Despite this, between only 0.1 and 0.7% of undifferentiated mouse pluripotent stem cells express Zscan4 protein at a given time, consistent with a very restricted pattern of Zscan4 transcripts reported previously. Herein we demonstrate that Zscan4 expression is regulated by the p110α catalytic isoform of phosphoinositide 3-kinases and is induced following exposure to a sub-class of DNA-damage-inducing agents, including Zeocin and Cisplatin. Furthermore, we observe that Zscan4 protein expression peaks during the G2 phase of the cell cycle, suggesting that it may play a critical role at this checkpoint. Studies with GAL4-fusion proteins suggest a role for Zscan4 in transcriptional regulation, further supported by the fact that protein interaction analyses demonstrate that Zscan4 interacts with both LSD1 and CtBP2 in ESC nuclei. This study advances and extends our understanding of Zscan4 expression, regulation and mechanism of action. Based on our data we propose that Zscan4 may regulate gene transcription in mouse ES cells through interaction with LSD1 and CtBP2.

Glycogen synthase kinase-3 inhibition enhances translation of pluripotency-associated transcription factors to contribute to maintenance of mouse embryonic stem cell self-renewal.

Maintenance of embryonic stem cell (ESC) self-renewal and pluripotency are controlled by extrinsic factors, molecular signaling pathways and transcriptional regulators. While many of the key players have been studied in depth, how the molecular signals interact with transcription factors of the pluripotency network to regulate their action remains less well understood. Inhibition of glycogen synthase kinase 3 (Gsk-3) has been implicated in the maintenance of mouse ESC pluripotency, although there is contradictory data on its role, with enhancement of cell survival and metabolism, stabilisation of c-Myc and activation of Wnt signalling proposed as potential mechanisms. We have discovered that suppression of Gsk-3 activity leads to enhanced protein levels of key transcriptional regulators of the pluripotency network, notably Nanog, Tbx3 and c-Myc. Protein stability was unchanged following Gsk-3 inhibition, although interestingly, Nanog and Tbx3 proteins were found to have half-lives of 1-3 h, while that of Oct4 protein was longer, at 6 h. We demonstrate that the effects on protein levels seen following inhibition of Gsk-3 are due to both enhanced de novo synthesis of Nanog protein and increases in the proportion of Nanog and Tbx3 RNAs bound to polysomes, findings consistent with Gsk-3 regulating translation of these factors. These effects were not due to changes in regulators of general translation initiation machinery nor mediated via the 5' or 3' UTR sequences of Nanog alone. The data we present provide both new conceptual insight into the mechanisms regulated by Gsk-3 that may contribute to ESC self-renewal and, importantly, establish control of protein translation as an additional mechanism involved in modulation of ESC pluripotency.

Differential coupling of self-renewal signaling pathways in murine induced pluripotent stem cells.

The ability to reprogram somatic cells to induced pluripotent stem cells (iPSCs), exhibiting properties similar to those of embryonic stem cells (ESCs), has attracted much attention, with many studies focused on improving efficiency of derivation and unraveling the mechanisms of reprogramming. Despite this widespread interest, our knowledge of the molecular signaling pathways that are active in iPSCs and that play a role in controlling their fate have not been studied in detail. To address this shortfall, we have characterized the influence of different signals on the behavior of a model mouse iPSC line. We demonstrate significant responses of this iPSC line to the presence of serum, which leads to profoundly enhanced proliferation and, depending on the medium used, a reduction in the capacity of the iPSCs to self-renew. Surprisingly, this iPSC line was less sensitive to withdrawal of LIF compared to ESCs, exemplified by maintenance of expression of a Nanog-GFP reporter and enhanced self-renewal in the absence of LIF. While inhibition of phosphoinositide-3 kinase (PI3K) signaling decreased iPSC self-renewal, inhibition of Gsk-3 promoted it, even in the absence of LIF. High passages of this iPSC line displayed altered characteristics, including genetic instability and a reduced ability to self-renew. However, this second feature could be restored upon inhibition of Gsk-3. Collectively, our data suggest modulation of Gsk-3 activity plays a key role in the control of iPSC fate. We propose that more careful consideration should be given to characterization of the molecular pathways that control the fate of different iPSC lines, since perturbations from those observed in naïve pluripotent ESCs could render iPSCs and their derivatives susceptible to aberrant and potentially undesirable behaviors.

A novel role for P2X7 receptor signalling in the survival of mouse embryonic stem cells.

The growth of a pluripotent embryonic stem (ES) cell population is dependent on cell survival, proliferation and self-renewal. The nucleotide ATP represents an important extracellular signalling molecule that regulates the survival of differentiated cells, however, its role is largely undefined in embryonic stem cells. Here we report a role for ATP-gated P2X7 receptors in ES cell survival. The functional expression of P2X7 receptors in undifferentiated mouse ES cells is demonstrated using a selective P2X7 antagonist and small interfering RNA knockdown of these receptors. Our data illustrate a key role for the P2X7 receptor as an essential pro-survival signal required for optimal ES cell colony growth in the presence of leukemia inhibitor factor (LIF). However, chronic exposure to exogenous ATP leads to rapid P2X7-dependent cell death via necrosis. Together, these data demonstrate a novel role for P2X7 receptors in regulation of ES cell behaviour where they can mediate either a pro-survival or pro-death signal depending on the mode of activation.

Blocking phosphoinositide 3-kinase activity in colorectal cancer cells reduces proliferation but does not increase apoptosis alone or in combination with cytotoxic drugs.

In response to growth factors, class IA phosphoinositide 3-kinases (PI3K) phosphorylate phosphatidylinositol-4,5-bisphosphate, converting it to phosphatidylinositol-3,4,5-trisphosphate to activate protein kinase B/Akt. This is widely reported to promote tumorigenesis via increased cell survival, proliferation, migration, and invasion, and many tumor types, including colorectal cancer, exhibit increased PI3K signaling. To investigate the effect of inhibiting PI3K and as an alternative to the use of small molecular inhibitors of PI3K with varying degrees of selectivity, HT29 and HCT116 colorectal cancer cells bearing mutant PIK3CA were generated that could be induced with doxycycline to express synchronously a dominant negative subunit of PI3K, Deltap85alpha. On induction, decreased levels of phosphorylated protein kinase B were detected, confirming PI3K signaling impairment. Induction of Deltap85alpha in vitro reduced cell number via accumulation in G(0)-G(1) phase of the cell cycle in the absence of increased apoptosis. These effects were recapitulated in vivo. HT29 cells expressing Deltap85alpha and grown as tumor xenografts had a significantly slower growth rate on administration of doxycycline with reduced Ki67 staining without increased levels of apoptotic tissue biomarkers. Furthermore, in vitro Deltap85alpha expression did not sensitize HT29 cells to oxaliplatin- or etoposide-induced apoptosis, irrespective of drug treatment schedule. Further analysis comparing isogenic HCT116 cells with and without mutation in PIK3CA showed no effect of the mutation in either proliferative or apoptotic response to PI3K inhibition. These data show in colorectal cancer cells that PI3K inhibition does not provoke apoptosis per se nor enhance oxaliplatin- or etoposide-induced cell death.