Vivian Lee

PRAS40 is a target for mammalian target of rapamycin complex 1 and is required for signaling downstream of this complex

Signaling through the mammalian target of rapamycin complex 1 (mTORC1) is positively regulated by amino acids and insulin. PRAS40 associates with mTORC1 (which contains raptor) but not mTORC2. PRAS40 interacts with raptor, and this requires an intact TOR-signaling (TOS) motif in PRAS40. Like TOS motif-containing proteins such as eIF4E-binding protein 1 (4E-BP1), PRAS40 is a substrate for phosphorylation by mTORC1. Consistent with this, starvation of cells of amino acids or treatment with rapamycin alters the phosphorylation of PRAS40. PRAS40 binds 14-3-3 proteins, and this requires both amino acids and insulin. Binding of PRAS40 to 14-3-3 proteins is inhibited by TSC1/2 (negative regulators of mTORC1) and stimulated by Rheb in a rapamycin-sensitive manner. This confirms that PRAS40 is a target for regulation by mTORC1. Small interfering RNA-mediated knockdown of PRAS40 impairs both the amino acid- and insulin-stimulated phosphorylation of 4E-BP1 and the phosphorylation of S6. However, this has no effect on the phosphorylation of Akt or TSC2 (an Akt substrate). These data place PRAS40 downstream of mTORC1 but upstream of its effectors, such as S6K1 and 4E-BP1.

β1-Integrin–collagen interaction reduces chondrocyte apoptosis

We have observed that the spent culture media in suspended chondrocyte cultures is essential for the survival of the cells, since complete change of the spent media induces severe programmed cell death (apoptosis). Moreover, we showed that extracellular matrix (ECM) molecules in the culture media provide vital chondrocyte–matrix interactions; when media are changed, cells are deprived of matrix molecules and undergo apoptosis. In this paper we report that interaction with collagen, a ubiquitous extracellular matrix molecule, is essential for chondrocyte survival. Such an interaction causes chondrocyte aggregation and reduces the level of chondrocyte apoptosis. Hyaluronan, an abundant ECM molecule, can influence the effects of collagen by preventing chondrocyte aggregation. Degradation of hyaluronan with hyaluronidase results in chondrocyte aggregation, and this reduces the level of chondrocyte apoptosis. Experiments with an antibody to integrin β1 suggest that the collagen–chondrocyte interactions are mediated through integrin β1, and these interactions may protect chondrocytes from apoptosis. We hypothesize that hyaluronan binds aggrecan and link protein, forming stable ternary complexes, which interact with the chondrocyte surface, perhaps via CD44, and thus maintains a stable chondrocyte–matrix network.

Identification of the Motif in Versican G3 Domain That Plays a Dominant-negative Effect on Astrocytoma Cell Proliferation through Inhibiting Versican Secretion and Binding

This study was designed to investigate the mechanisms by which mutant versican constructs play a dominant-negative effect on astrocytoma cell proliferation. Although a mini-versican or a versican G3 construct promoted growth of U87 astrocytoma cells, a mini-versican lacking epidermal growth factor (EGF) motifs (versicanΔEGF) and a G3 mutant (G3ΔEGF) exerted a dominant-negative effect on cell proliferation. G3ΔEGF-transfected cells formed smaller colonies, arrested cell cycle at G1 phase, inhibited expression of cell cycle proteins cdk4 and cyclin D1, and contained multiple nucleoli. In cell surface binding assays, G3 products expressed in COS-7 cells and bacteria bound to U87 cell surface. G3ΔEGF products exhibited decreased binding activity, but higher levels of G3ΔEGF products were able to inhibit the binding of G3 to the cell surface. G3ΔEGF expression inhibited secretion of endogenous versican in astrocytoma cells and also inhibited the secretion of mini-versican in COS-7 cells co-transfected with the mini-versican and G3ΔEGF constructs. The effect seems to depend on the expression efficiency of G3ΔEGF, and it occurred via the carbohydrate recognition domain.

Epidermal growth factor induces cell cycle arrest and apoptosis of squamous carcinoma cells through reduction of cell adhesion

Most squamous epithelial cells are strictly anchorage‐dependent cell types. We observed that epidermal growth factor (EGF) promoted the growth of A431 squamous carcinoma cells in suspension cultures but suppressed cell growth and induced apoptosis in monolayer cultures, suggesting that loss of adhesion is responsible for the effects observed in monolayer culture, before cell death. Consistent with this finding, we demonstrated that EGF reduced cell attachment, cell‐cell interaction, and cell spreading. Treatment with EGF increased cell adhesion‐regulated expression of p21 but suppressed expressions of cyclin A, D1, cdk2, and retinoblastoma protein (pRb), leading to cell cycle arrest and adhesion‐regulated programmed cell death. To test directly whether promoting cell adhesion could reduce the effects of EGF, we grew cultures on plates coated with type II collagen. On these plates, cell adhesion was enhanced and EGF treatment had little effect on cell adhesion and apoptosis when cells were attached to the collagen. The collagen effects were dose dependent, and cell cycle and cell cycle‐associated proteins were altered accordingly. Finally, when cultures were plated on bacterial Petri dishes, which completely disrupted cell attachment to substratum, the level of apoptosis was greatly higher and cell cycle was arrested as compared with monolayer cultures. Taken together, our results strongly suggest that the EGF‐induced cell cycle arrest and apoptosis in monolayer cultures was the result of a decline in cell adhesion. J. Cell. Biochem. 77:569–583, 2000.

The roles of matrix molecules in mediating chondrocyte aggregation, attachment, and spreading

The most abundant macromolecules in cartilage are hyaluronan, collagen, aggrecan, and link protein, which are believed to play roles in maintaining a unique three‐dimensional network for a functional joint. This study was designed to investigate the roles of the major extracellular molecules in mediating chondrocyte‐matrix interactions. We employed specific approaches to remove components individually or in combination: hyaluronan was digested with hyaluronidase; type II collagen was digested with collagenase; aggrecan expression was inhibited with antisense and β‐xyloside approaches; and link protein expression was inhibited with antisense oligonucleotides. Digestion of hyaluronan induced chondrocyte attachment to tissue culture plates, collagen‐coated plates, and fibroblast‐like chondrocyte cultures, and induced chondrocyte aggregation. Treated chondrocytes exhibited a fibroblast‐like morphology, and the effects of hyaluronidase were dose‐dependent. Conversely, the effect of collagenase on chondrocyte adhesion and aggregation was far less pronounced. Treatment with Arg‐Gly‐Asp peptide inhibited chondrocyte‐collagen interaction. Chondrocyte attachment was enhanced by antisense oligonucleotides complementary to aggrecan and link protein and by β‐xyloside treatment. Nevertheless, hyaluronan seems to predominate over the other molecules in mediating chondrocyte‐matrix interactions. J. Cell. Biochem. 79:322–333, 2000.

Analysis of the regulatory motifs in eukaryotic initiation factor 4E-binding protein 1

Mammalian target of rapamycin complex 1 (mTORC1) phosphorylates proteins such as eukaryotic initiation factor 4E‐binding protein 1 (4E‐BP1) and the S6 kinases. These substrates contain short sequences, termed TOR signalling (TOS) motifs, which interact with the mTORC1 component raptor. Phosphorylation of 4E‐BP1 requires an additional feature, termed the RAIP motif (Arg–Ala–Ile–Pro). We have analysed the interaction of 4E‐BP1 with raptor and the amino acid residues required for functional RAIP and TOS motifs, as assessed by raptor binding and the phosphorylation of 4E‐BP1 in human cells. Binding of 4E‐BP1 to raptor strongly depends on an intact TOS motif, but the RAIP motif and additional C‐terminal features of 4E‐BP1 also contribute to this interaction. Mutational analysis of 4E‐BP1 reveals that isoleucine is a key feature of the RAIP motif, that proline is also very important and that there is greater tolerance for substitution of the first two residues. Within the TOS motif, the first position (phenylalanine in the known motifs) is most critical, whereas a wider range of residues function in other positions (although an uncharged aliphatic residue is preferred at position three). These data provide important information on the structural requirements for efficient signalling downstream of mTORC1.

The binding of PRAS40 to 14-3-3 proteins is not required for activation of mTORC1 signalling by phorbol esters/ERK

PRAS40 binds to the mTORC1 (mammalian target of rapamycin complex 1) and is released in response to insulin. It has been suggested that this effect is due to 14-3-3 binding and leads to activation of mTORC1 signalling. In a similar manner to insulin, phorbol esters also activate mTORC1 signalling, in this case via PKC (protein kinase C) and ERK (extracellular-signal-regulated kinase). However, phorbol esters do not induce phosphorylation of PRAS40 at Thr(246), binding of 14-3-3 proteins to PRAS40 or its release from mTORC1. Mutation of Thr(246) to a serine residue permits phorbol esters to induce phosphorylation and binding to 14-3-3 proteins. Such phosphorylation is apparently mediated by RSKs (ribosomal S6 kinases), which lie downstream of ERK. However, although the PRAS40(T246S) mutant binds to 14-3-3 better than wild-type PRAS40, each inhibits mTORC1 signalling to a similar extent. Our results show that activation of mTORC1 signalling by phorbol esters does not require PRAS40 to be phosphorylated at Thr(246), bind to 14-3-3 or be released from mTORC1. It is conceivable that phorbol esters activate mTORC1 by a distinct mechanism not involving PRAS40. Indeed, our results suggest that PRAS40 may not actually be involved in controlling mTORC1, but rather be a downstream target of mTORC1 that is regulated in response only to specific stimuli, such as insulin.

Steryl glucoside concentration declines with Cycas micronesica seed age

Neurotoxins contained in the seeds of Cycas micronesica K.D. Hill have been implicated in the Guam neurological disease cluster, amyotrophic lateral sclerosis-parkinsonism dementia complex (ALS-PDC). Some of these neurotoxins remain in the washed cycad seed flour that was historically an important part of the Chamorro diet. Of these, variant steryl glucosides have been identified by us as a possible etiological factor in the disease. In vitro and in vivo animal studies have strongly supported a role for these molecules in some forms of neurodegeneration. As part of a series of studies, we have now determined the concentrations of several steryl glucosides and their sterol precursors as affected by the age of C. micronesica seeds. The concentration of these molecules declined with seed age from 2.0 to 30.5 months. Following log-transformation of both axes, the decline was linear. Similarly, concentration of all but one of the molecules declined with age when samples were restricted to gametophyte tissue. Factors suspected of influencing phenotypic plasticity must be addressed when interpreting plant physiology data. Our results confirm for the first time that tissue age must be documented and reported in cycad seed biochemistry studies to remove ambiguities from results. Past studies in this important area of research have failed to account for the potential impact of seed age, rendering previous outcomes and interpretations of cycad neurotoxins in their impact on ALS-PDC ambiguous.

Published guidelines versus real-life practice in the diagnosis and treatment of essential thrombocythemia.

intensity [RIC]), primary disease, and status at transplant. Thrombocytopenia was distinguished as persistent or transient in the presence of a or <30 day duration of platelet count reduction, respectively. Clinical conditions related or associated with the development of thrombocytopenia (i.e. cGVHD, infectious complications, relapse, microangiopathy, or other) were evaluated. Quantitative variables were tested for normal distribution using the Shapiro-Wilk test. Comparisons between groups were performed with t test or U-Mann Whitney test, depending on Shapiro-Wilk test results. g tests were used to analyze categorical values; when assumptions for g test were not verified, Fisher’s exact test was used. Survival curves were estimated using the Kaplan-Meier method. Overall survival (OS) was analyzed using log-rank tests and Cox proportional hazard models, after the proportional hazards assumption had been verified.

TTI-621 (SIRPαFc), a CD47-blocking cancer immunotherapeutic, triggers phagocytosis of lymphoma cells by multiple polarized macrophage subsets

Tumor-associated macrophages (TAMs) are heterogeneous and can adopt a spectrum of activation states between pro-inflammatory and pro-tumorigenic in response to the microenvironment. We have previously shown that TTI-621, a soluble SIRPαFc fusion protein that blocks the CD47 “do-not-eat” signal, promotes tumor cell phagocytosis by IFN-γ-primed macrophages. To assess the impact of CD47 blockade on diverse types of macrophages that are found within the tumor microenvironment, six different polarized human macrophage subsets (M(-), M(IFN-γ), M(IFN-γ+LPS), M(IL-4), M(HAGG+IL-1β), M(IL-10 + TGFβ)) with distinct cell surface markers and cytokine profiles were generated. Blockade of CD47 using TTI-621 significantly increased phagocytosis of lymphoma cells by all macrophage subsets, with M(IFN-γ), M(IFN-γ+LPS) and M(IL-10 + TGFβ) macrophages having the highest phagocytic response. TTI-621-mediated phagocytosis involves macrophage expression of both the low- and high-affinity Fcγ receptors II (CD32) and I (CD64), respectively. Moreover, macrophages with lower phagocytic capabilities (M(-), M(IL-4), M(HAGG+IL-1β)) could readily be re-polarized into highly phagocytic macrophages using various cytokines or TLR agonists. In line with the in vitro study, we further demonstrate that TTI-621 can trigger phagocytosis of tumor cells by diverse subsets of isolated mouse TAMs ex vivo. These data suggest that TTI-621 may be efficacious in triggering the destruction of cancer cells by a diverse population of TAMs found in vivo and support possible combination approaches to augment the activity of CD47 blockade.