Assembly of mTORC3 Involves Binding of ETV7 to Two Separate Sequences in the mTOR Kinase Domain.

mTOR plays a crucial role in cell growth by controlling ribosome biogenesis, metabolism, autophagy, mRNA translation, and cytoskeleton organization. It is a serine/threonine kinase that is part of two distinct extensively described protein complexes, mTORC1 and mTORC2. We have identified a rapamycin-resistant mTOR complex, called mTORC3, which is different from the canonical mTORC1 and mTORC2 complexes in that it does not contain the Raptor, Rictor, or mLST8 mTORC1/2 components.

Haploinsufficiency of the lysosomal sialidase NEU1 results in a model of pleomorphic rhabdomyosarcoma in mice.

Rhabdomyosarcoma, the most common pediatric sarcoma, has no effective treatment for the pleomorphic subtype. Still, what triggers transformation into this aggressive phenotype remains poorly understood. Here we used Ptch1/ETV7 mice with enhanced incidence of rhabdomyosarcoma to generate a model of pleomorphic rhabdomyosarcoma driven by haploinsufficiency of the lysosomal sialidase neuraminidase 1.

Phosphorylation of TSC2 by PKC-δ reveals a novel signaling pathway that couples protein synthesis to mTORC1 activity.

Downstream of insulin-like growth factor receptor, the TSC1/2/ TCB1D7 (tuberous sclerosis complex) and mTOR (mechanistic target of rapamycin) pathways are implicated in many human diseases, including cancer and diabetes. Targeting this pathway is currently an important approach for palliating or eradicating cancer. Downstream of mTOR, translational machinery targeting holds great promise for anticancer drug development.

ETV7 is an essential component of a rapamycin-insensitive mTOR complex in cancer.

The mechanistic target of rapamycin (mTOR) serine/threonine kinase, a critical regulator of cell proliferation, is frequently deregulated in human cancer. Although rapamycin inhibits the two canonical mTOR complexes, mTORC1 and mTORC2, it often shows minimal benefit as an anticancer drug. This is caused by rapamycin resistance of many different tumors, and we show that a third mTOR complex, mTORC3, contributes to this resistance.

Modeling of the human alveolar rhabdomyosarcoma Pax3-Foxo1 chromosome translocation in mouse myoblasts using CRISPR-Cas9 nuclease.

Many recurrent chromosome translocations in cancer result in the generation of fusion genes that are directly implicated in the tumorigenic process. Precise modeling of the effects of cancer fusion genes in mice has been inaccurate, as constructs of fusion genes often completely or partially lack the correct regulatory sequences. The reciprocal t(2;13)(q36.

Etanercept enhances preservation of osteochondral allograft viability.

Background: Osteochondral allografts are an increasingly popular treatment for the repair of articular cartilage lesions. Current tissue bank protocols require bacteriological testing that takes from 21 to 28 days to process. During this time, tumor necrosis factor-alpha (TNF-α, a proapoptotic cytokine) is upregulated, resulting in loss of chondrocyte viability.

Enhanced flexor tendon healing through controlled delivery of PDGF-BB.

A fibrin/heparin-based delivery system was used to provide controlled delivery of platelet derived growth factor BB (PDGF-BB) in an animal model of intrasynovial flexor tendon repair. We hypothesized that PDGF-BB, administered in this manner, would stimulate cell proliferation and matrix remodeling, leading to improvements in the sutured tendon's functional and structural properties. Fifty-six flexor digitorum profundus tendons were injured and repaired in 28 dogs.

Variation of mesenchymal cells in polylactic acid scaffold in an osteochondral repair model.

Objective: To achieve osteochondral regeneration utilizing transplantation of cartilage-lineage cells and adequate scaffolds, it is essential to characterize the behavior of transplanted cells in the repair process. The objectives of this study were to elucidate the survival of mesenchymal cells (MCs). In a polylactic acid (PLA) scaffold and assess the possibility of MC/PLA constructs for osteochondral repair.

Controlled-release kinetics and biologic activity of platelet-derived growth factor-BB for use in flexor tendon repair.

Purpose: Surgically repaired intrasynovial tendons are at greatest risk of failure in the first 3 weeks after surgery. Attempts to improve the strength of repair by modifying rehabilitation parameters have not always been successful. Manipulation of the biological environment of the sutured tendon holds great promise for accelerating the repair process.

Effects of biglycan deficiency on myocardial infarct structure and mechanics.

Biglycan, a small leucine-rich proteoglycan, has been shown to interact with extracellular matrix (ECM) collagen and may influence fibrillogenesis. We hypothesized that biglycan contributes to post-myocardial infarction (MI) scar development and that the absence of biglycan would result in altered scar structure and mechanics. Anterior MI was induced in biglycan hemizygous null and wild-type mice by permanent ligation of the left coronary artery.

mTORC1 signaling can regulate growth factor activation of p44/42 mitogen-activated protein kinases through protein phosphatase 2A.

The mTORC1 complex (mammalian target of rapamycin (mTOR)-raptor) is modulated by mitogen-activated protein (p44/42 MAP) kinases (p44/42) through phosphorylation and inactivation of the tuberous sclerosis complex. However, a role for mTORC1 signaling in modulating activation of p44/42 has not been reported. We show that in two cancer cell lines regulation of the p44/42 MAPKs is mTORC1-dependent.

PDGF-BB released in tendon repair using a novel delivery system promotes cell proliferation and collagen remodeling.

The purpose of this study was to promote fibroblast proliferation and collagen remodeling in flexor tendon repair through sustained delivery of platelet derived growth factor (PDGF-BB). The release kinetics of PDGF-BB from a novel fibrin matrix delivery system was initially evaluated in vitro. After the in vivo degradation rate of the fibrin matrix was determined using fluorescently tagged fibrin, PDGF-BB was delivered to the site of flexor tendon repair in vivo in a canine model.

Differential regulation of vascular endothelial growth factor by Akt and mammalian target of rapamycin inhibitors in cell lines derived from childhood solid tumors.

Levels of vascular endothelial growth factor (VEGF) are regulated, in part, through activation of the phosphatidylinositol 3'-kinase/Akt pathway. Using pharmacologic inhibitors, we have examined the relative contributions of Akt and mammalian target of rapamycin (mTOR) signaling to VEGF production in neuroblastoma and rhabdomyosarcoma cells growing under normoxic (21% O(2)) or hypoxic (1% O(2)) conditions. Exogenous VEGF stimulated both Akt and extracellular signal-regulated kinase 1/2 phosphorylation in six of seven rhabdomyosarcoma cell lines but in only one of seven neuroblastoma cells, suggesting autocrine stimulation predominantly in rhabdomyosarcoma cell lines.

Role of apoptotic and matrix-degrading genes in articular cartilage and meniscus of mature and aged rabbits during development of osteoarthritis.

Objective: To determine expression patterns of apoptotic and matrix-degrading genes during aging and development of osteoarthritis (OA), using a rabbit model of induced OA.

Methods: Six mature and 6 aged rabbits underwent anterior cruciate ligament transection and were killed 4 and 8 weeks after surgery, respectively, to create early-grade and advanced-grade OA. RNA from articular cartilage and menisci was examined for expression of the genes caspase 8, Fas, Fas ligand, p53, aggrecanase, matrix metalloproteinase 1 (MMP-1), and MMP-3.

Prolonged storage of osteochondral allografts: does the addition of fetal bovine serum improve chondrocyte viability?

Osteochondral plugs were harvested from eight fresh human femoral condyles within 96 hours of donor death. The plugs were either stored in a serum-free media containing glucose, salts, and amino acids or 10% fetal bovine serum at 4 degrees C. After 28 days of storage, the osteochondral plugs were analyzed for chondrocyte viability and viable cell density using confocal microscopy, proteoglycan synthesis by (35)SO4 incorporation, and glycosaminoglycan content.

Early healing of flexor tendon insertion site injuries: Tunnel repair is mechanically and histologically inferior to surface repair in a canine model.

Orthopedic injuries often require surgical reattachment of tendon to bone. Tendon ends can be sutured to bone by direct apposition to the bone surface or by placement within a bone tunnel. Our objective was to compare early healing of a traditional surface versus a novel tunnel method for repair of the flexor digitorum profundus (FDP) tendon insertion site in a canine model.

Characterization of pro-apoptotic and matrix-degradative gene expression following induction of osteoarthritis in mature and aged rabbits.

Objective: The genetic and molecular changes leading to the distinctive alterations of aged cartilage and its propensity for developing osteoarthritis (OA) are unknown. We hypothesized that pro-apoptotic and matrix-degradative gene expression in a rabbit model of induced OA using mature and aged animals might elucidate this relationship.

Methods: Groups of six mature and aged rabbits underwent anterior cruciate ligament transection (ACLT) and were sacrificed 4 weeks after surgery to create an Outerbridge grade II OA.

Does subchondral bone affect the fate of osteochondral allografts during storage?

Background: Osteochondral allografts currently are hypothermically stored for a minimum of 14 days to a maximum of 28 days before surgical implantation, making storage conditions increasingly important. Previous studies have suggested that graft deterioration during storage may result from degradative factors and residual marrow elements in the subchondral bone.

Hypothesis: Allografts stored with large bone-to-cartilage ratios will be compromised after prolonged storage compared with grafts with minimal bone.

Kinetics of collagen crosslinking in adult bovine articular cartilage.

Objective: Determine the kinetics of collagen crosslinking in adult bovine articular cartilage explants using radiolabel pulse-chase studies.

Methods: Explant cultures of adult bovine articular cartilage were radiolabeled with [14C]lysine in medium including fetal bovine serum and ascorbate, and then maintained for chase periods up to 28 days. In some samples, beta-aminopropionitrile (BAPN) was included during chase to inhibit lysyl oxidase-mediated collagen crosslinking.

Analysis of stored osteochondral allografts at the time of surgical implantation.

Background: To date, the morphological, biochemical, and biomechanical characteristics of articular cartilage in osteochondral allografts that have been stored have not been fully described.

Hypothesis: Osteochondral allografts procured and stored commercially for a standard period as determined by tissue banking protocol will have compromised chondrocyte viability but preserved extracellular matrix quality.

Study Design: Controlled laboratory study.

Effect of several growth factors on canine flexor tendon fibroblast proliferation and collagen synthesis in vitro.

Purpose: Growth factor delivery may be useful to accelerate the rate of tendon healing. Before in vivo use, however, the effects of growth factors on tendon cells need to be well characterized. The purpose of this study was to evaluate the effects of 4 growth factors on intrasynovial tendon fibroblast proliferation and collagen production in vitro.

Characterization of mature vs aged rabbit articular cartilage: analysis of cell density, apoptosis-related gene expression and mechanisms controlling chondrocyte apoptosis.

Objective: The prevalence of osteoarthritis (OA) is increased in aged individuals and a direct correlation between chondrocyte apoptosis and cartilage degradation secondary to OA has been demonstrated. To address the question of whether age predisposes articular cartilage to apoptosis, the objective of the present study was to characterize and compare in aged and mature non-OA rabbit articular cartilage, cell density and expression levels of specific genes associated with apoptosis. Mechanistic studies on the inhibition of induced apoptosis were also carried out.

Gefitinib enhances the antitumor activity and oral bioavailability of irinotecan in mice.

As a single agent the ERBB1 inhibitor, gefitinib (Iressa; ZD1839) showed minimal activity against a panel of 10 pediatric tumor xenografts that do not express the ERBB1 receptor. However, combined with irinotecan (CPT-11), significantly greater than additive activity was observed in four of eight models (P < 0.05), and the combination showed enhanced activity against three additional tumor lines.

Inhibition of mammalian target of rapamycin activates apoptosis signal-regulating kinase 1 signaling by suppressing protein phosphatase 5 activity.

Under serum-free conditions, rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), induces a cellular stress response characterized by rapid and sustained activation of the apoptosis signal-regulating kinase 1 (ASK1) signaling pathway and selective apoptosis of cells lacking functional p53. Here we have investigated how mTOR regulates ASK1 signaling using p53-mutant rhabdomyosarcoma cells. In Rh30 cells, ASK1 was found to physically interact with protein phosphatase 5 (PP5), previously identified as a negative regulator of ASK1.

Imatinib mesylate is a potent inhibitor of the ABCG2 (BCRP) transporter and reverses resistance to topotecan and SN-38 in vitro.

Imatinib mesylate (Gleevec, STI571) is a kinase inhibitor selective for Bcr-Abl, activated c-Kit kinases, and platelet-derived growth factor receptor tyrosine kinase. Imatinib mesylate, similar to many other tyrosine kinase inhibitors (TKIs), such as members of the 4-anilinoquinazoline class, competes for ATP binding. Previously, 4-anilinoquinazoline TKIs have been shown to inhibit the function of the breast cancer resistance-associated drug transporter (ABCG2), reversing resistance to camptothecin derivatives topotecan and SN-38.