Activation of PDK-1 maintains mouse embryonic stem cell self-renewal in a PKB-dependent manner.

The phosphatidylinositol 3' kinase (PI3K) pathway is involved in many cellular processes including cell proliferation, survival and glucose transport, and is implicated in various disease states, such as cancer and diabetes. Although there have been numerous studies dissecting the role of PI3K signaling in different cell types and disease models, the mechanism by which PI3K signaling regulates embryonic stem (ES) cell fate remains unclear. It is believed that in addition to proliferation and tumorigenesis, PI3K activity may also be important for ES cell self-renewal. Paling et al. reported that the inhibition of PI3K led to a reduction in the ability of leukemia inhibitory factor to maintain self-renewal, causing cells to differentiate. Studies in our lab have revealed that ES cells completely lacking glycogen synthase kinase-3 (GSK-3) remain undifferentiated compared with wild-type ES cells. GSK-3 is negatively regulated by PI3K, suggesting that PI3K may have a vital role in maintaining pluripotency in ES cells through GSK-3. By using a modified Flp recombinase system, we expressed activated alleles of 3-phosphoinositide-dependent protein kinase-1 and protein kinase B to create stable, isogenic ES cell lines to further study the role of the PI3K signaling pathway in stem cell fate determination. In vitro characterization of the transgenic cell lines revealed a strong tendency toward the maintenance of pluripotency, and this phenotype was found to be independent of canonical Wnt signal transduction. In summary, PI3K signaling is sufficient to maintain the self-renewal and survival of stem cells. As this pathway is frequently mutationally activated in cancers, its effect on suppressing differentiation may contribute to its oncogenicity.

Platelet-derived growth factor mediated altered expression and regulation of ornithine decarboxylase in H-ras-transformed cell lines.

This study demonstrates a novel link between alterations in platelet-derived growth factor (PDGF) regulation of ornithine decarboxylase (ODC) expression during malignant conversion. H-ras-transformed cell lines exhibited PDGF-mediated alterations in ODC gene expression. These alterations involved transcriptional, posttranscriptional, and cycloheximide-mediated events. PDGF-mediated alterations in ODC gene expression in NR3 cells (capable of only benign tumour formation) were ras-dependent, involved a tyrosine kinase activity and mitogen-activated protein (MAP) kinase-mediated signalling events, and were independent of both protein kinase C (PKC) events and pertussis toxin-sensitive (PTS) G-protein-mediated signalling. PDGF-mediated alterations in ODC gene expression in C2 cells [capable of malignant progression (metastasis formation)] were ras-dependent, required a tyrosine kinase activity, involved both MAP kinase-mediated events and phosphatidylinositol-3-kinase (PI-3-kinase)-mediated events, and were dependent upon PTS G-protein-mediated signalling but independent of PKC-mediated events. PDGF-mediated regulation of ODC gene expression changes in response to H-ras-mediated cellular transformation and malignant progression.

Rescue of the early vascular defects in Tek/Tie2 null mice reveals an essential survival function.

Disruption of the signaling pathways mediated by the receptor tyrosine kinase Tek/Tie2 has shown that this receptor plays a pivotal role in vascularization of the developing embryo. In this report, we have utilized the tetracycline-responsive binary transgenic system to overcome the early lethal cardiovascular defects associated with the tekDeltasp null allele in order to investigate the role of Tek in later stages of vessel growth. We show for the first time in vivo that synchronized loss of tek expression correlates with rapid endothelial cell apoptosis in hemorrhagic regions of the embryo, demonstrating an ongoing requirement for Tek-mediated signal transduction in vascular maintenance.

Tumour promoter mediated altered expression and regulation of ornithine decarboxylase and S-adenosylmethionine decarboxylase in H-ras-transformed fibrosarcoma cell lines.

Alterations in cellular growth are important in the progression of malignant disease. Cell growth regulation by tumour promoters can be complex. The present study demonstrates a novel link between alterations in phorbol ester tumour promoter mediated regulation during malignant conversion and the expression of ornithine decarboxylase and S-adenosylmethionine decarboxylase, key rate-limiting and regulatory activities in the biosynthesis of polyamines. H-ras-transformed mouse 10 T 1/2 cell lines exhibiting increasing malignant potential were investigated for possible phorbol ester tumour promoter mediated changes in ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) gene expression. Selective induction of ODC and SAMDC gene expression was observed, since in contrast to nontransformed parental 10 T1/2 cells, ras-transformed cells capable of benign tumour formation (NR3 cells) and ras-transformed cells capable of metastasis formation (C2 cells) exhibited marked alterations in the levels of ODC and SAMDC gene expression. Increased ODC gene and SAMDC gene expression in response to phorbol-12-myristate-13-acetate (PMA) treatment was found to involve transcriptional events in both NR3 cells and in C2 cells. Post-transcriptional events also played a role in the regulation of ODC gene expression in NR3 cells and in C2 cells, and in the regulation of SAMDC gene expression in C2 cells but not in NR3 cells. In NR3 cells, alterations in ODC and in SAMDC gene expression was an event requiring de novo protein synthesis, whereas in highly malignant C2 cells, protein synthesis inhibition following cycloheximide treatment in cooperation with PMA resulted in an augmentation of both ODC and SAMDC gene expression. Evidence is presented to suggest that the PMA-mediated alterations in ODC and in SAMDC gene expression in NR3 cells and in C2 cells involved protein kinase C - mediated events. The status of the cellular polyamine levels was also an important determinant of the PMA-mediated alterations that occurred in ODC and in SAMDC expression in these H-ras transformed cells. Collectively, these results suggest that PMA can modulate ODC and SAMDC expression in H-ras transformed cells and that the mechanisms involved in the PMA- mediated regulation of ODC and SAMDC gene expression changes as a function of H-ras mediated cellular transformation and malignant progression. This study further suggests a mechanism of PMA stimulation of transformed cells wherein early alterations in the regulatory control of ODC and SAMDC gene expression are important and critical.

Transformation by H-ras can result in aberrant regulation of ornithine decarboxylase gene expression by transforming growth factor-beta(1).

Inhibition of DNA synthesis and cell proliferation is frequently lost during malignant transformation and occasionally, tumour cell proliferation is actually stimulated by transforming growth factor beta(1) (TGF-beta(1)). The present study demonstrates a novel link between alterations in TGF-beta(1) regulation during cellular transformation and malignant conversion and the expression of ornithine decarboxylase (ODC) which is a key rate limiting activity in the biosynthesis of polyamines and which is an enzyme that plays an important role in cell growth and differentiation. H-ras transformed mouse 10T(1/2) cell lines of varying degrees of malignant potential were examined for possible TGF-beta(1)-mediated alterations in ODC expression. Selective induction of ODC gene expression occurred. This induction was dependent upon the cellular phenotype expressed and the mechanisms responsible for the regulation of the TGF-beta(1)-mediated alterations in ODC expression varied as a function of malignant potential. The TGF-beta(1)-mediated alterations in ODC gene expression involves de novo protein synthesis, transcriptional, and post-transcriptional events. Evidence is also presented to suggest a possible role for protein kinase C-mediated events, protein phosphatases, and G-protein-coupled events in the TGF-beta(1)-mediated regulation of ODC expression in H-ras transformed cells. Evidence is also presented to suggest a possible role for cellular polyamines in the TGF-beta(1)-mediated alterations in ODC expression in H-ras transformed cells. Additionally, alterations in cellular polyamines were shown to influence TGF-beta(1) gene expression in H-ras transformed cells and that these alterations occurred, in part, through post-transcriptional events. The TGF-beta(1)-mediated regulation of ODC expression in H-ras transformed cells of varying degrees of malignant potential appears to be complex, multifaceted, and interactive. This study illustrates the importance of TGF-beta(1)-mediated regulation of ODC expression as a result of H-ras mediated cellular transformation and malignant progression, and further suggests that this TGF-beta(1)-mediated regulation constitutes an integral part of an altered growth regulatory program.