PTEN modulates cell cycle progression and cell survival by regulating phosphatidylinositol 3,4,5,-trisphosphate and Akt/protein kinase B signaling pathway

To investigate the molecular basis of PTEN-mediated tumor suppression, we introduced a null mutation into the mouse Pten gene by homologous recombination in embryonic stem (ES) cells. Pten-/- ES cells exhibited an increased growth rate and proliferated even in the absence of serum. ES cells lacking PTEN function also displayed advanced entry into S phase. This accelerated G1/S transition was accompanied by down-regulation of p27(KIP1), a major inhibitor for G1 cyclin-dependent kinases. Inactivation of PTEN in ES cells and in embryonic fibroblasts resulted in elevated levels of phosphatidylinositol 3,4,5,-trisphosphate, a product of phosphatidylinositol 3 kinase. Consequently, PTEN deficiency led to dosage-dependent increases in phosphorylation and activation of Akt/protein kinase B, a well-characterized target of the phosphatidylinositol 3 kinase signaling pathway. Akt activation increased Bad phosphorylation and promoted Pten-/- cell survival. Our studies suggest that PTEN regulates the phosphatidylinositol 3,4, 5,-trisphosphate and Akt signaling pathway and consequently modulates two critical cellular processes: cell cycle progression and cell survival.     

The PTEN/MMAC1 tumor suppressor phosphatase functions as a negative regulator of the phosphoinositide 3-kinase/Akt pathway

The PTEN/MMAC1 phosphatase is a tumor suppressor gene implicated in a wide range of human cancers. Here we provide biochemical and functional evidence that PTEN/MMAC1 acts a negative regulator of the phosphoinositide 3-kinase (PI3-kinase)/Akt pathway. PTEN/MMAC1 impairs activation of endogenous Akt in cells and inhibits phosphorylation of 4E-BP1, a downstream target of the PI3-kinase/Akt pathway involved in protein translation, whereas a catalytically inactive, dominant negative PTEN/MMAC1 mutant enhances 4E-BP1 phosphorylation. In addition, PTEN/MMAC1 represses gene expression in a manner that is rescued by Akt but not PI3-kinase. Finally, higher levels of Akt activation are observed in human prostate cancer cell lines and xenografts lacking PTEN/MMAC1 expression when compared with PTEN/MMAC1-positive prostate tumors or normal prostate tissue. Because constitutive activation of either PI3-kinase or Akt is known to induce cellular transformation, an increase in the activation of this pathway caused by mutations in PTEN/MMAC1 provides a potential mechanism for its tumor suppressor function.     

Photodynamic therapy results in induction of WAF1/CIP1/P21 leading to cell cycle arrest and apoptosis

Photodynamic therapy (PDT) is a promising new modality that utilizes a combination of a photosensitizing chemical and visible light for the management of a variety of solid malignancies. The mechanism of PDT-mediated cell killing is not well defined. We investigated the involvement of cell cycle regulatory events during silicon phthalocyanine (Pc4)-PDT-mediated apoptosis in human epidermoid carcinoma cells A431. PDT resulted in apoptosis, inhibition of cell growth, and G₀-G₁ phase arrest of the cell cycle, in a time-dependent fashion. Western blot analysis revealed that PDT results in an induction of the cyclin kinase inhibitor WAF1/CIP1/p21, and a down-regulation of cyclin D1 and cyclin E, and their catalytic subunits cyclin-dependent kinase (cdk) 2 and cdk6. The treatment also resulted in a decrease in kinase activities associated with all the cdks and cyclins examined. PDT also resulted in (i) an increase in the binding of cyclin D1 and cdk6 toward WAF1/CIP1/p21, and (ii) a decrease in the binding of cyclin D1 toward cdk2 and cdk6. The binding of cyclin E and cdk2 toward WAF1/CIP1/p21, and of cyclin E toward cdk2 did not change by the treatment. These data suggest that PDT-mediated induction of WAF1/CIP1/p21 results in an imposition of artificial checkpoint at G₁→S transition thereby resulting in an arrest of cells in G₀-G₁ phase of the cell cycle through inhibition in the cdk2, cdk6, cyclin D1, and cyclin E. We suggest that this arrest is an irreversible process and the cells, unable to repair the damages, ultimately undergo apoptosis.     

A phosphatidylinositol 3-kinase/Akt/mTOR pathway mediates and PTEN antagonizes tumor necrosis factor inhibition of insulin signaling through insulin receptor substrate-1

Tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) by the insulin receptor permits this docking protein to interact with signaling proteins that promote insulin action. Serine phosphorylation uncouples IRS-1 from the insulin receptor, thereby inhibiting its tyrosine phosphorylation and insulin signaling. For this reason, there is great interest in identifying serine/threonine kinases for which IRS-1 is a substrate. Tumor necrosis factor (TNF) inhibited insulin-promoted tyrosine phosphorylation of IRS-1 and activated the Akt/protein kinase B serine-threonine kinase, a downstream target for phosphatidylinositol 3-kinase (PI 3-kinase). The effect of TNF on insulin-promoted tyrosine phosphorylation of IRS-1 was blocked by inhibition of PI 3-kinase and the PTEN tumor suppressor, which dephosphorylates the lipids that mediate PI 3-kinase functions, whereas constitutively active Akt impaired insulin-promoted IRS-1 tyrosine phosphorylation. Conversely, TNF inhibition of IRS-1 tyrosine phosphorylation was blocked by kinase dead Akt. Inhibition of IRS-1 tyrosine phosphorylation by TNF was blocked by rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), a downstream target of Akt. mTOR induced the serine phosphorylation of IRS-1 (Ser-636/639), and such phosphorylation was inhibited by rapamycin. These results suggest that TNF impairs insulin signaling through IRS-1 by activation of a PI 3-kinase/Akt/mTOR pathway, which is antagonized by PTEN.     

Human CUL-1 associates with the SKP1/SKP2 complex and regulates p21CIP1/WAF1 and cyclin D proteins

Deregulation of cell proliferation is a hallmark of cancer. In many transformed cells, the cyclin A/CDK2 complex that contains S-phase kinase associated proteins 1 and 2 (SKP1 and SKP2) is highly induced. To determine the roles of this complex in the cell cycle regulation and transformation, we have examined the composition of this complex. We report here that this complex contained an additional protein, human CUL-1, a member of the cullin/CDC53 family. The identification of CUL-1 as a member of the complex raises the possibility that the p19^SKP1/p45^SKP2/CUL-1 complex may function as the yeast SKP1-CDC53-F-box (SCF) protein complex that acts as a ubiquitin E3 ligase to regulate the G₁/S transition. In mammalian cells, cyclin D, p21^CIP1/WAF1, and p27^KIP1 are short-lived proteins that are controlled by ubiquitin-dependent proteolysis. To determine the potential in vivo targets of the p19^SKP1/p45^SKP2/CUL-1 complex, we have used the specific antisense oligodeoxynucleotides against either SKP1, SKP2, or CUL-1 RNA to inhibit their expression. Treatment of cells with these oligonucleotides caused the selective accumulation of p21 and cyclin D proteins. The protein level of p27 was not affected. These data suggest that the human p19^SKP1/p45^SKP2/CUL-1 complex is likely to function as an E3 ligase to selectively target cyclin D and p21 for the ubiquitin-dependent protein degradation. Aberrant expression of human p19^SKP1/p45^SKP2/CUL-1 complex thus may contribute to tumorigenesis by regulating the protein levels of G₁ cell cycle regulators.     

An inhibitor of mTOR reduces neoplasia and normalizes p70/S6 kinase activity in Pten+/- mice

PTEN phosphatase acts as a tumor suppressor by negatively regulating the phosphoinositide 3-kinase (PI3K) signaling pathway. It is unclear which downstream components of this pathway are necessary for oncogenic transformation. In this report we show that transformed cells of PTEN^+/− mice have elevated levels of phosphorylated Akt and activated p70/S6 kinase associated with an increase in proliferation. Pharmacological inactivation of mTOR/RAFT/FRAP reduced neoplastic proliferation, tumor size, and p70/S6 kinase activity, but did not affect the status of Akt. These data suggest that p70/S6K and possibly other targets of mTOR contribute significantly to tumor development and that inhibition of these proteins may be therapeutic for cancer patients with deranged PI3K signaling.     

Loss of PTEN function may account for reduced proliferation pathway sensitivity to LY294002 in human prostate and bladder cancer cells

Purpose  Inhibition of phosphoinositide 3 (PI3)-kinase pathway is attractive for cancer treatment. To examine the role of the phosphatase and tensin homolog (PTEN) in the development of resistance to the treatment. Methods  We cultured human prostate cancer cells (DU145 and PC-3 cells) and bladder cancer cells (EJ-1 and UM-UC-3 cells) with a PI3-kinase inhibitor, LY294002 for more than 6 weeks and cell proliferation was studied. Activation of Akt1 and ERK was examined by immunoblotting. We introduced the wild type PTEN in UM-UC-3 cells and their proliferation along with the signaling pathways was also examined. Results  After 6 weeks, proliferation pathway sensitivity to LY294002 was reduced in cells expressing PTEN, but not in PTEN-null cells. PD98059, a MAPK/ERK kinase inhibitor, significantly inhibited proliferation of PTEN-expressing cells, but not PTEN-null cells. Stable PTEN expression in PTEN-null UM-UC-3 cells increased serum-induced ERK activation and sensitivity to PD98059-treatment, and reduced sensitivity to LY294002 after 6 weeks of exposure. Conclusions  Loss of PTEN function may protect against resistance to PI3-kinase inhibitors through an addiction to the PI3-kinase/Akt pathway.     

Truncating PREX2 mutations activate its GEF activity and alter gene expression regulation in NRAS-mutant melanoma

PREX2 (phosphatidylinositol-3,4,5-triphosphate-dependent Rac-exchange factor 2) is a PTEN (phosphatase and tensin homolog deleted on chromosome 10) binding protein that is significantly mutated in cutaneous melanoma and pancreatic ductal adenocarcinoma. Here, genetic and biochemical analyses were conducted to elucidate the nature and mechanistic basis of PREX2 mutation in melanoma development. By generating an inducible transgenic mouse model we showed an oncogenic role for a truncating PREX2 mutation (PREX2^E824*) in vivo in the context of mutant NRAS. Using integrative cross-species gene expression analysis, we identified deregulated cell cycle and cytoskeleton organization as significantly perturbed biological pathways in PREX2 mutant tumors. Mechanistically, truncation of PREX2 activated its Rac1 guanine nucleotide exchange factor activity, abolished binding to PTEN and activated the PI3K (phosphatidyl inositol 3 kinase)/Akt signaling pathway. We further showed that PREX2 truncating mutations or PTEN deletion induces down-regulation of the tumor suppressor and cell cycle regulator CDKN1C (also known as p57^KIP2). This down-regulation occurs, at least partially, through DNA hypomethylation of a differentially methylated region in chromosome 11 that is a known regulatory region for expression of the CDKN1C gene. Together, these findings identify PREX2 as a mediator of NRAS-mutant melanoma development that acts through the PI3K/PTEN/Akt pathway to regulate gene expression of a cell cycle regulator.Recent large-scale multidimensional genomic analyses of many cancers have established a framework in which biological functions and genetic interactions of established and novel cancer genes can be explored (1, 2). We initially identified PREX2 (phosphatidylinositol-3,4,5-triphosphate-dependent Rac-exchange factor 2) as being significantly mutated in human melanomas (3), an observation that was corroborated by the recently completed TCGA melanoma study (4). PREX2 is a guanine nucleotide exchanger (GEF) for Rac1 (5, 6) and is known to bind to the tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) (7). PREX2 has recently been shown to regulate insulin signaling and glucose homeostasis through modulation of the PI3K (phosphatidyl inositol 3 kinase) pathway, and also to regulate Rac1 mediated cellular invasion in a manner that cross-talks with PTEN signaling (8). Further expanding the significance of genetic perturbations of PREX2 in cancer, a recent report by the International Cancer Genome Consortium (ICGC) described the identification PREX2 as a significantly mutated gene in pancreatic ductal adenocarcinoma (PDAC) (9). Interestingly, PREX2 harbors a wide spectrum of mutations including missense and truncating mutations in PDAC, similar to observations in melanoma (3, 9).To date, the most obvious connection between PREX2 and cancer relevant pathways is through its physical interaction with PTEN (7). PTEN catalyzes the conversion of phosphatidylinositol-3,4,5-trisphosphate to phosphatidylinositol-4,5 bisphosphate. PTEN acts as a tumor suppressor and plays important roles in multiple cellular processes primarily by antagonizing PI3-kinase-AKT signaling (10–13). Pathologically, PTEN is inactivated via multiple mechanisms in about a third of melanoma tumors resulting in activation of the downstream PI3K/Akt signaling pathway (14, 15). Despite these connections to cancer signaling pathways, the exact mechanism of tumorigenesis by PREX2 mutations remains unknown. Here, we elucidated a previously unidentified mechanism of action of PREX2 mutations in melanoma pathogenesis.To study PREX2 mutations in vivo, we generated an inducible transgenic mouse model that expresses one of the truncating PREX2 mutants observed in melanoma patients, and showed that melanoma development was accelerated in this genetic context. Using integrated gene expression analysis, we identified several cellular pathways including cell cycle regulation and cytoskeletal organization to be deregulated in PREX2 mutant tumors. Biochemically, we showed that truncating PREX2 mutations increase its Rac1 guanine nucleotide exchange (GEF) activity, abolish binding to the tumor suppressor PTEN and activate the PI3K/Akt signaling pathway. Finally, we showed that PREX2 mutation or PTEN deletion induces DNA hypomethylation and down-regulation of expression of CDKN1C (p57^KIP2), a critical cell cycle regulator. In conclusion, this study demonstrates the oncogenic capability of PREX2 truncations in vivo and identifies a mechanistic link to an established oncogenic signaling pathway and downstream regulation of a tumor suppressor to impact melanoma pathogenesis.     

Recombinant human prothrombin kringle-2 induces bovine capillary endothelial cell cycle arrest at G0–G1 phase through inhibition of cyclin D1/CDK4 complex: Modulation of reactive oxygen species generation and up-regulation of cyclin-dependent kinase inhibitors

Prothrombin is a plasma glycoprotein involved in blood coagulation and, as we have previously reported, prothrombin kringles inhibit BCE (bovine capillary endothelial) cell proliferation. To reveal the mechanism, we investigated the influence of rk-2 (recombinant human prothrombin kringle-2) on the BCE cell cycle progression and ROS (reactive oxygen species) generation using FACS (fluorescence-activated cell sorter) analysis. Cell cycle analysis showed a decrease of G₁ phase cells in cells treated with bFGF (basic fibroblast growth factor) and an increase in cells treated with rk-2, as compared with the control cells. But, the portion of the S phase was reversed. In Western blot analysis, bFGF induced cytoplasmic translocation of p21^Waf1/Cip1 and p27^Kip1 and phosphorylation of p27^Kip1 but rk-2 treatment inhibited translocation of p21^Waf1/Cip1 and p27^Kip1 from nucleus to cytoplasm and phosphorylation of p27^Kip1. Also, rk-2 induced up-regulation of p53 and nuclear p21^Waf1/Cip1 and inhibited the cyclin D1/CDK4 (cyclin-dependent kinase 4) complex. The ROS level of rk-2-treated BCE cells was increased 2-fold when compared with the control, but treatment with NAC (N-Acetyl-L-cysteine), an anti-oxidant, decreased ROS generation about 55% as compared with the rk-2 treatment. NAC treatment also restored cell cycle progression inhibited by rk-2 and down-regulated p53 and nuclear p21^Waf1/Cip1 expression induced by rk-2.These data suggest that rk-2 induces the BCE cell cycle arrest at G₀–G₁ phase through inhibition of the cyclin D1/CDK4 complex caused by increase of ROS generation and nuclear cyclin-dependent kinase inhibitors.     

Rapamycin specifically interferes with GM-CSF signaling in human dendritic cells,leading to apoptosis via increased p27KIP1 expression

The longevity of dendritic cells (DCs) is a critical regulatory factor influencing the outcome of immune responses. Recently, we demonstrated that the immunosuppressive drug rapamycin (Rapa) specifically induces apoptosis in DCs but not in other myeloid cell types. The present study unraveled the mechanism used by Rapa to induce apoptosis in human monocyte-derived DCs. Our data demonstrate that granulocyte-macrophage colony-stimulating factor (GM-CSF) preserves DC survival specifically via the phosphatidylinositol-3 lipid kinase/mammalian target of rapamycin (PI3K/mTOR) signaling pathway, which is abrogated by Rapa at the level of mTOR. Disruption of this GM-CSF signaling pathway induced loss of mitochondrial membrane potential, phosphatidyl-serine exposure, and nuclear changes. Apoptosis of these nonproliferating DCs was preceded by an up-regulation of the cell cycle inhibitor p27(KIP1). Overexpression of p27(KIP1) in DCs using adenoviral gene transduction revealed that apoptosis is directly regulated by p27(KIP1). Furthermore, both overexpression of p27(KIP1) and disruption of the GM-CSF/PI3K/mTOR signaling pathway decreased the expression of the antiapoptotic protein mcl-1. This mTOR/p27(KIP1)/mcl-1 survival seems unique for DCs and may provide novel opportunities to influence immune responses by specific interference with the life span of these cells.     

Transduction of interleukin-2 antiapoptotic and proliferative signals via Akt protein kinase

The interleukin-2 (IL-2) receptor (IL-2R) is composed of three subunits. Of these, IL-2Rα is required for high-affinity IL-2 binding, while IL-2Rβ and IL-2Rγ_c are required for the transduction of IL-2-generated signals. Signals transduced via the S region of the IL-2Rβ (amino acids 267–322) in BAF/3 cells activate the phosphatidylinositol 3-kinase (PI3-kinase) and induce the expression of Bcl-2 and c-myc. Through the induction of Bcl-2, IL-2 inhibits apoptosis and through the combination of Bcl-2 and c-myc it stimulates progression through the cell cycle. Here we show that the protein kinase encoded by the Akt proto-oncogene is activated by IL-2. Akt activation by IL-2 depends on PI3-kinase signals transduced via the S region of the IL-2Rβ and is linked to the translocation of Akt to the cell membrane. Expression of catalytically active Akt mutants in BAF/3 cells expressing IL-2Rβ[A0]ΔS promotes the expression of Bcl-2 and c-myc, inhibits apoptosis induced by IL-3 deprivation or staurosporine, and stimulates cell cycle progression. The same mutants also stimulate cell cycle progression in 2780a, an IL-2-dependent T cell line that undergoes G₁ arrest rather than apoptosis after IL-2 deprivation. The activation of Akt by IL-2 via the PI3-kinase and the rescue of the PI3-kinase-mediated antiapoptotic and proliferative IL-2 signals by catalytically active Akt indicate that these signals are transduced by Akt.     

Hypo-phosphorylation of the retinoblastoma protein (pRb) by cyclin D:Cdk4/6 complexes results in active pRb

In cycling cells, the retinoblastoma protein (pRb) is un- and/or hypo-phosphorylated in early G₁ and becomes hyper-phosphorylated in late G₁. The role of hypo-phosphorylation and identity of the relevant kinase(s) remains unknown. We show here that hypo-phosphorylated pRb associates with E2F in vivo and is therefore active. Increasing the intracellular concentration of the Cdk4/6 specific inhibitor p15^INK4b by transforming growth factor β treatment of keratinocytes results in G₁ arrest and loss of hypo-phosphorylated pRb with an increase in unphosphorylated pRb. Conversely, p15^INK4b-independent transforming growth factor β-mediated G₁ arrest of hepatocellular carcinoma cells results in loss of Cdk2 kinase activity with continued Cdk6 kinase activity and pRb remains only hypo-phosphorylated. Introduction of the Cdk4/6 inhibitor p16^INK4a protein into cells by fusion to a protein transduction domain also prevents pRb hypo-phosphorylation with an increase in unphosphorylated pRb. We conclude that cyclin D:Cdk4/6 complexes hypo-phosphorylate pRb in early G₁ allowing continued E2F binding.     

KSHV viral cyclin binds to p27KIP1 in primary effusion lymphomas

Primary effusion lymphomas (PELs) represent a unique non-Hodgkin lymphoma that is consistently infected by Kaposi sarcoma herpesvirus (KSHV). PEL cells express high levels of the cell cycle inhibitor p27(KIP1) and yet proliferate actively. KSHV genome encodes a viral cyclin homolog, v-cyclin, which has previously been implicated in down-regulation of p27(KIP1) levels. To address how PEL cells can tolerate high p27(KIP1) levels, we investigated functional interactions between v-cyclin and p27(KIP1) using PEL-derived cell lines as a model system. Here we demonstrate that v-cyclin and p27(KIP1) stably associate in PEL cells in vivo suggesting an attractive model by which p27(KIP1) is inactivated in the actively proliferating PEL cells. Moreover, we show that v-cyclin and cyclin-dependent kinase 6 (CDK6) form an active kinase without p27(KIP1) and that CDK6 is the in vivo catalytic subunit of v-cyclin in PEL cells. These findings suggest that KSHV may promote oncogenesis in PEL by expressing v-cyclin, which both overrides negative cell cycle controls present in the PEL precursor cells and induces a strong proliferative signal via CDK6 kinase activity.     

DAB2IP coordinates both PI3K-Akt and ASK1 pathways for cell survival and apoptosis

In metastatic prostate cancer (PCa) cells, imbalance between cell survival and death signals such as constitutive activation of phosphatidylinositol 3-kinase (PI3K)-Akt and inactivation of apoptosis-stimulated kinase (ASK1)-JNK pathways is often detected. Here, we show that DAB2IP protein, often down-regulated in PCa, is a potent growth inhibitor by inducing G₀/G₁ cell cycle arrest and is proapoptotic in response to stress. Gain of function study showed that DAB2IP can suppress the PI3K-Akt pathway and enhance ASK1 activation leading to cell apoptosis, whereas loss of DAB2IP expression resulted in PI3K-Akt activation and ASK1-JNK inactivation leading to accelerated PCa growth in vivo. Moreover, glandular epithelia from DAB2IP^−/− animal exhibited hyperplasia and apoptotic defect. Structural functional analyses of DAB2IP protein indicate that both proline-rich (PR) and PERIOD-like (PER) domains, in addition to the critical role of C2 domain in ASK1 activity, are important for modulating PI3K-Akt activity. Thus, DAB2IP is a scaffold protein capable of bridging both survival and death signal molecules, which implies its role in maintaining cell homeostasis.     

Interaction of the S phase regulator Cdc18 with cyclin-dependent kinase in fission yeast

The fission yeast gene cdc18⁺ is required for entry into S phase and for coupling mitosis to the successful completion of S phase. Cdc18 is a highly unstable protein that is expressed only once per cell cycle at the G₁/S boundary. Overexpression of Cdc18 causes a mitotic delay and reinitiation of DNA replication, suggesting that the inactivation of Cdc18 plays a role in preventing rereplication within a given cell cycle. In this paper, we present evidence that Cdc18 is associated with active cyclin-dependent kinase in vivo. We have expressed Cdc18 as a glutathione S-transferase fusion in fission yeast and demonstrated that the fusion protein is functional in vivo. We find that the Cdc18 fusion protein copurifies with a kinase activity capable of phosphorylating histone H1 and Cdc18. The activity was identified by a variety of methods as the cyclin-dependent kinase containing the product of the cdc2⁺ gene. The amino terminus of Cdc18 is required for association with cyclin-dependent kinase, but the association does not require the consensus cyclin-dependent kinase phosphorylation sites in this region. Additionally, both G₁/S and mitotic forms of cyclin-dependent kinase phosphorylate and interact with Cdc18. These interactions between Cdc18 and cyclin-dependent kinases suggest mechanisms by which cyclin-dependent kinases could activate the initiation of DNA replication and could prevent rereplication.     

Cardioprotective stimuli mediate phosphoinositide 3-kinase and phosphoinositide dependent kinase 1 nuclear accumulation in cardiomyocytes

The phosphoinositide 3-kinase (PI3K)/phosphoinositide dependent kinase 1 (PDK1) signaling pathway exerts cardioprotective effects in the myocardium through activation of key proteins including Akt. Activated Akt accumulates in nuclei of cardiomyocytes suggesting that biologically relevant targets are located in that subcellular compartment. Nuclear Akt activity could be potentiated in both intensity and duration by the presence of a nuclear-associated PI3K/PDK1 signaling cascade as has been described in other non-myocyte cell types. PI3K/PDK1 distribution was determined in vitro and in vivo by immunostaining and nuclear extraction of cultured rat neonatal cardiomyocytes or transgenic mouse hearts. Results show that PI3K and PDK1 are present at a basal level in cardiomyocytes nuclei and that cardioprotective stimulation with atrial natriuretic peptide (ANP) increases their nuclear localization. In comparison, overexpression of nuclear-targeted Akt does not mediate increased translocation of either PI3K or PDK1 indicating that accumulation of Akt does not drive PI3K or PDK1 into the nuclear compartment. Furthermore, PI3K and phospho-Akt⁴⁷³ show parallel temporal accumulation in the nucleus following (MI) infarction challenge. These findings demonstrate the presence of a dynamically regulated nuclear-associated signaling cascade involving PI3K and PDK that presumably influences nuclear Akt activation.     

Inhibition of vascular smooth muscle cell proliferation,migration, and survival by the tumor suppressor protein PTEN

Phosphatidylinositol (PI) 3-kinase signaling regulates numerous cellular processes, including proliferation, migration, and survival, which are required for neointimal hyperplasia and restenosis. The effectors of PI 3-kinase are activated by the phospholipid products of PI 3-kinase. In this report, we investigated the hypothesis that overexpression of the tumor suppressor protein PTEN, an inositol phosphatase specific for the products of PI 3-kinase, would inhibit the vascular smooth muscle cell (VSMC) responses necessary for neointimal hyperplasia and restenosis. Effects of PTEN were assessed in primary rabbit VSMCs after overexpression with a recombinant adenovirus and compared with uninfected or control virus-infected cells. PTEN was expressed endogenously in VSMCs, and PTEN overexpression inhibited PDGF-induced phosphorylation of p70(s6k), Akt, and glycogen synthase kinase-3-alpha and -beta but not ERK1 or -2. Overexpression of PTEN significantly inhibited both basal and PDGF-mediated VSMC proliferation and migration, the latter possibly due in part to downregulation of focal adhesion kinase. Moreover, PTEN overexpression induced cleavage of caspase-3 and significantly increased apoptosis compared with control cells. Taken together, these results demonstrate that PTEN overexpression potently inhibits the VSMC responses required for neointimal hyperplasia and restenosis. Adenovirus-expressed PTEN may therefore provide a useful tool for the local treatment of these and other vascular proliferative disorders.     

Growth hormone exerts antiapoptotic and proliferative effects through two different pathways involving nuclear factor-kappaB and phosphatidylinositol 3-kinase

Dependence of murine pro-B Ba/F3 cells on interleukin-3 can be substituted by GH when cells are stably transfected with the GH receptor (GHR) complementary DNA. Recently, we demonstrated that Ba/F3 cells produce GH, which is responsible for the survival of cells expressing the GHR. This GH effect involves the activation of nuclear factor-kappaB (NF-kappaB). Here, we examined the signaling pathways mediating proliferation of growth factor-deprived Ba/F3 GHR cells. Exogenous GH stimulation of Ba/F3 GHR cells induced cyclins E and A and the cyclin-dependent kinase inhibitor p21(waf1/cip1) and repressed cyclin-dependent kinase inhibitor p27(kip1). The presence of the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor Ly 294002 abolished proliferation induced by GH, arresting Ba/F3 GHR cells at the G(1)/S boundary, but did not promote apoptosis. Thus, the proliferative effect of GH is closely related to PI 3-kinase activation, whereas PI 3-kinase is not essential for GH-induced cell survival. Addition of Ly 294002 resulted in a moderate decrease in NF-kappaB activation by GH, suggesting a possible link between PI 3-kinase and NF-kappaB signaling by GH. Expression of c-myc was also induced by GH in Ba/F3 GHR cells, and inactivation of either PI 3-kinase or NF-kappaB reduced this induction. Overexpression of the dominant negative repressor mutant c-Myc-RX resulted in an inhibition of the GH proliferative effect, suggesting the involvement of c-myc in GH-induced proliferation. Taken together, these results suggest that the effects of GH on cell survival and proliferation are mediated through two different signaling pathways, NF-kappaB and PI 3-kinase, respectively; although cross-talk between them has not been excluded. NF-kappaB, which has been shown to be responsible for the antiapoptotic effect of GH, could also participate in GH-induced proliferation, as c-myc expression is promoted by PI 3-kinase, in an NF-kappaB-dependent and -independent manner.     

Inhibition of the phosphatidylinositol-3 kinase/Akt promotes G1 cell cycle arrest and apoptosis in Hodgkin lymphoma

Activation of the phosphatidylinositol 3-kinase (PI(3)K) pathway has been linked with tumour cell growth, survival and resistance to therapy in several cancer types. The active, phosphorylated form of Akt (pAkt) was found to be aberrantly expressed in Hodgkin lymphoma (HL)-derived cell lines and in Hodgkin-Reed-Sternberg (HRS) cells in 27 of 42 (64.3%) of primary lymph node sections of HL, indicative of PI(3)K activity. Akt phosphorylation was not associated with loss of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) expression, but with its phosphorylation in HL-cell lines, suggesting that its biological function is impaired. Akt phosphorylation was further induced by CD30 ligand (CD30L), CD40L and receptor activator of nuclear factor kappa B (RANK) ligand. The PI(3)K inhibitor LY294002 demonstrated antiproliferative effects in a dose- and time-dependent manner, which was associated with Akt dephosphorylation on Thr308 and Ser473 sites and dephosphorylation of the downstream ribosomal protein S6. LY209002 induced cell cycle arrest in the G0/G1 phase and apoptosis, which were associated with upregulation of MDM2, downregulation of cyclin D1, activation of caspase 9 and poly-ADP-ribose polymerase cleavage. The Akt inhibitor QLT394 also demonstrated antiproliferative effects in a dose- and time-dependent manner, dephosphorylated ribosomal S6 and cleaved caspase 9. Collectively, these data suggest that the aberrant activation of the PI(3)K/Akt survival pathway in HRS cells is not because of loss of PTEN expression. Our data suggest that PTEN phosphorylation and activation of CD30, CD40 and RANK may play a role in activating Akt in HRS cells.