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1.
Immunotherapy ; 14(18): 1467-1480, 2022 12.
Article in English | MEDLINE | ID: mdl-36597724

ABSTRACT

Aims: To determine whether antigen-independent targeting of the TOP1 inhibitor exatecan to tumor with a pH-sensitive peptide (CBX-12) produces superior synergy with immunotherapy compared with unconjugated exatecan. Materials & methods: In vitro and ex vivo functional assays were performed via FACS and ELISA assays. In vivo efficacy was evaluated in the syngeneic CT26 model. Results: CBX-12 combined with anti-PD-1 or anti-CTLA4 results in delayed tumor growth and complete response, with cured animals displaying long-term antitumor immunity. CBX-12 stimulates expression of MHC 1 and PD-L1 and is an inducer of immunogenic cell death, producing long-term immune recognition of tumor cells and resultant antitumor immunity. Conclusion: The authors' data provide the rationale for exploring immunotherapy combinations with CBX-12 in clinical trials.


Although combinations of chemotherapy and immunotherapy have shown great promise for cancer treatment, they have also demonstrated significant safety concerns that require dose reductions. Targeting chemotherapy to the tumor can avoid these safety issues, thereby enhancing efficacy of combination therapies. CBX-12 is a novel peptide-drug agent targeting the TOP1-inhibiting drug exatecan to tumor via pH-sensitive peptide. Unlike tumor targeting via antibody, CBX-12 universally targets all solid tumors. CBX-12 avoids the immune cell toxicity of nontumor-targeted exatecan and safely synergizes with immunotherapies. CBX-12 treatment causes tumor cells to express and secrete molecules that result in activation of immune components to recognize and eliminate tumor cells. These data support the upcoming clinical trials of CBX-12 in combination with immunotherapy.


Subject(s)
Neoplasms , Animals , Neoplasms/drug therapy , Camptothecin/therapeutic use , Immunotherapy/methods , Immunity , Cell Line, Tumor , Tumor Microenvironment
3.
NAR Cancer ; 3(2): zcab021, 2021 Jun.
Article in English | MEDLINE | ID: mdl-34316708

ABSTRACT

Topoisomerase inhibitors are potent DNA damaging agents which are widely used in oncology, and they demonstrate robust synergistic tumor cell killing in combination with DNA repair inhibitors, including poly(ADP)-ribose polymerase (PARP) inhibitors. However, their use has been severely limited by the inability to achieve a favorable therapeutic index due to severe systemic toxicities. Antibody-drug conjugates address this issue via antigen-dependent targeting and delivery of their payloads, but this approach requires specific antigens and yet still suffers from off-target toxicities. There is a high unmet need for a more universal tumor targeting technology to broaden the application of cytotoxic payloads. Acidification of the extracellular milieu arises from metabolic adaptions associated with the Warburg effect in cancer. Here we report the development of a pH-sensitive peptide-drug conjugate to deliver the topoisomerase inhibitor, exatecan, selectively to tumors in an antigen-independent manner. Using this approach, we demonstrate potent in vivo cytotoxicity, complete suppression of tumor growth across multiple human tumor models, and synergistic interactions with a PARP inhibitor. These data highlight the identification of a peptide-topoisomerase inhibitor conjugate for cancer therapy that provides a high therapeutic index, and is applicable to all types of human solid tumors in an antigen-independent manner.

4.
Acta Diabetol ; 56(3): 365-371, 2019 Mar.
Article in English | MEDLINE | ID: mdl-30539233

ABSTRACT

AIMS: Bone morphogenetic proteins (BMPs) are involved in the development and homeostasis of multiple organs and tissues. There has been a significant focus on understanding the role of BMPs in pancreatic ß-cell dysfunction associated with type 2 diabetes (T2D). Our objective was to investigate the relationship between BMP6 and glucose homeostasis. METHODS: Ob/ob mice were treated with BMP6 for 6 days and analyzed for insulin release, body weight, lipid parameters and glucose tolerance. Quantitative real-time PCR, chromatin immunoprecipitation and glucose output assays were used to assess BMP6 effect on gluconeogenesis in rat hepatoma H4IIE cells. Specificity of BMP6 receptors was characterized by the utilization of various receptor Fc fusion proteins in luciferase reporter gene and glucose output assays in INS1 and H4IIE cells. RESULTS: Treatment of ob/ob mice with BMP6 for 6 days resulted in a reduction of circulating glucose and lipid levels, followed by a significantly elevated plasma insulin level in a dose-dependent manner. In addition, BMP6 improved the glucose excursion during an oral glucose tolerance test, lowering the total glycemic response by 21%. In rat H4IIE hepatoma cells, BMP6 inhibited gluconeogenesis and glucose output via downregulation the PepCK expression. Moreover, BMP6 inhibited glucose production regardless of the presence of cAMP, antagonizing its glycogenolytic effect. BMP6 acted on pancreatic and liver cells utilizing Alk3, Alk6 and ActRIIA serine/threonine kinase receptors. CONCLUSIONS: Collectively, we demonstrate that BMP6 improves glycaemia in T2D mice and regulates glucose metabolism in hepatocytes representing an exciting prospect for future treatments of diabetes.


Subject(s)
Bone Morphogenetic Protein 6/pharmacology , Diabetes Mellitus, Experimental/metabolism , Glucose/metabolism , Homeostasis/drug effects , Animals , Blood Glucose/genetics , Blood Glucose/metabolism , Bone Morphogenetic Protein 6/physiology , CHO Cells , Cells, Cultured , Cricetinae , Cricetulus , Diabetes Mellitus, Experimental/pathology , Diabetes Mellitus, Type 2/metabolism , Diabetes Mellitus, Type 2/pathology , Homeostasis/genetics , Insulin/metabolism , Male , Mice , Mice, Obese , Rats , Recombinant Proteins/pharmacology
5.
PLoS One ; 11(2): e0150102, 2016.
Article in English | MEDLINE | ID: mdl-26907598

ABSTRACT

Reduced peripheral serotonin (5HT) in mice lacking tryptophan hydroxylase (TPH1), the rate limiting enzyme for 5HT synthesis, was reported to be anabolic to the skeleton. However, in other studies TPH1 deletion either had no bone effect or an age dependent inhibition of osteoclastic bone resorption. The role of 5HT in bone therefore remains poorly understood. To address this issue, we used selective breeding to create rat sublines with constitutively high (high-5HT) and low (low-5HT) platelet 5HT level (PSL) and platelet 5HT uptake (PSU). High-5HT rats had decreased bone volume due to increased bone turnover characterized by increased bone formation and mineral apposition rate, increased osteoclast number and serum C-telopeptide level. Daily oral administration of the TPH1 inhibitor (LX1032) for 6 weeks reduced PSL and increased the trabecular bone volume and trabecular number of the spine and femur in high-5HT rats. High-5HT animals also developed a type 2 diabetes (T2D) phenotype with increased: plasma insulin, glucose, hemoglobin A1c, body weight, visceral fat, ß-cell pancreatic islets size, serum cholesterol, and decreased muscle strength. Serum calcium accretion mediated by parathyroid hormone slightly increased, whereas treatment with 1,25(OH)2D3 decreased PSL. Insulin reduction was paralleled by a drop in PSL in high-5HT rats. In vitro, insulin and 5HT synergistically up-regulated osteoblast differentiation isolated from high-5HT rats, whereas TPH1 inhibition decreased the number of bone marrow-derived osteoclasts. These results suggest that constitutively elevated PSL is associated with bone loss and T2D via a homeostatic interplay between the peripheral 5HT, bone and insulin.


Subject(s)
Bone Diseases/blood , Diabetes Mellitus, Type 2/complications , Serotonin/blood , Animals , Bone Diseases/metabolism , Bone Diseases/pathology , Bone Diseases/physiopathology , Bone Remodeling , Female , Male , Mice , Organ Size , Osteoblasts/pathology , Osteoclasts/pathology , Phenotype , Rats
6.
J Am Soc Nephrol ; 22(4): 681-92, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21415150

ABSTRACT

Bone morphogenetic proteins (BMPs) participate in organ regeneration through autocrine and paracrine actions, but the existence and effects of these proteins in the systemic circulation is unknown. Using liquid chromatography-mass spectrometry, we identified BMP6, GDF15, and the BMP1-3 isoform of the Bmp1 gene in plasma samples from healthy volunteers and patients with CKD. We isolated the endogenous BMP1-3 protein and demonstrated that it circulates as an active enzyme, evidenced by its ability to cleave dentin matrix protein-1 in vitro. In rats with CKD, administration of recombinant BMP1-3 increased renal fibrosis and reduced survival. In contrast, administration of a BMP1-3-neutralizing antibody reduced renal fibrosis, preserved renal function, and increased survival. In addition, treating with the neutralizing antibody was associated with low plasma levels of TGFß1 and connective tissue growth factor. In HEK293 cells and remnant kidneys, BMP1-3 increased the transcription of collagen type I, TGFß1, ß-catenin, and BMP7 via a BMP- and Wnt-independent mechanism that involved signaling through an integrin ß1 subunit. The profibrotic effect of BMP1-3 may, in part, be a result of the accompanied decrease in decorin (DCN) expression. Taken together, inhibition of circulating BMP1-3 reduces renal fibrosis, suggesting that this pathway may be a therapeutic target for CKD.


Subject(s)
Bone Morphogenetic Protein 1/blood , Bone Morphogenetic Protein 2/blood , Bone Morphogenetic Protein 3/blood , Kidney Diseases/pathology , Kidney/pathology , Adult , Aged , Animals , Bone Morphogenetic Protein 7/metabolism , Cells, Cultured , Chronic Disease , Collagen Type I/metabolism , Disease Models, Animal , Female , Fibrosis , HEK293 Cells , Humans , Kidney/metabolism , Kidney Diseases/metabolism , Male , Middle Aged , Protein Isoforms/metabolism , Rats , Rats, Sprague-Dawley , Transforming Growth Factor beta1/metabolism , beta Catenin/metabolism
7.
J Pharmacol Exp Ther ; 333(1): 2-13, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20089807

ABSTRACT

PF-04840082 is a humanized prototype anti-Dickkopf-1 (Dkk-1) immunoglobulin isotype G(2) (IgG(2)) antibody for the treatment of osteoporosis. In vitro, PF-04840082 binds to human, monkey, rat, and mouse Dkk-1 with high affinity. After administration of PF-04840082 to rat and monkey, free Dkk-1 concentrations decreased rapidly and returned to baseline in a dose-dependent manner. In rat and monkey, PF-04840082 exhibited nonlinear pharmacokinetics (PK) and a target-mediated drug disposition (TMDD) model was used to characterize PF-04840082 versus Dkk-1 concentration response relationship. PK/pharmacodynamic (PK/PD) modeling enabled estimation of antibody non-target-mediated elimination, Dkk-1 turnover, complex formation, and complex elimination. The TMDD model was translated to human to predict efficacious dose and minimum anticipated biological effect level (MABEL) by incorporating information on typical IgG(2) human PK, antibody-target association/dissociation rates, Dkk-1 expression, and turnover rates. The PK/PD approach to MABEL was compared with the standard "no adverse effect level" (NOAEL) approach to calculating clinical starting doses and a pharmacological equilibrium method. The NOAEL method gave estimates of dose that were too high to ensure safety of clinical trials. The pharmacological equilibrium approach calculated receptor occupancy (RO) based on equilibrium dissociation constant alone and did not take into account rate of turnover of the target or antibody-target complex kinetics and, as a result, it likely produced a substantial overprediction of RO at a given dose. It was concluded that the calculation of MABEL according to the TMDD model was the most appropriate means for ensuring safety and efficacy in clinical studies.


Subject(s)
Antibodies, Monoclonal/pharmacology , Intercellular Signaling Peptides and Proteins/immunology , Models, Biological , Osteoporosis/blood , Adult , Aged , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/pharmacokinetics , Bone Diseases, Metabolic/blood , Female , Humans , Intercellular Signaling Peptides and Proteins/blood , Macaca fascicularis , Male , Mice , Middle Aged , Osteoporosis/drug therapy , Osteoporosis, Postmenopausal/blood , Rats , Rats, Sprague-Dawley
8.
ACS Med Chem Lett ; 1(5): 219-23, 2010 Aug 12.
Article in English | MEDLINE | ID: mdl-24900198

ABSTRACT

As part of a strategy to deliver short-acting calcium-sensing receptor (CaSR) antagonists, the metabolically labile thiomethyl functionality was incorporated into the zwitterionic amino alcohol derivative 3 with the hope of increasing human clearance through oxidative metabolism, while delivering a pharmacologically inactive sulfoxide metabolite. The effort led to the identification of thioanisoles 22 and 23 as potent and orally active CaSR antagonists with a rapid onset of action and short pharmacokinetic half-lives, which led to a rapid and transient stimulation of parathyroid hormone in a dose-dependent fashion following oral administration to rats. On the basis of the balance between target pharmacology, safety, and human disposition profiles, 22 and 23 were advanced as clinical candidates for the treatment of osteoporosis.

9.
Bioorg Med Chem Lett ; 19(16): 4555-9, 2009 Aug 15.
Article in English | MEDLINE | ID: mdl-19625189

ABSTRACT

Synthesis and structure-activity relationship (SAR) studies on 5-trifluoromethylpyrido[4,3-d]pyrimidin-4(3H)-ones, a novel class of calcium receptor antagonists is described with particular emphasis on optimization of the pharmacokinetic/pharmacodynamic parameters required for a short duration of action compound. Orally-active compounds were identified which displayed the desired animal pharmacology (rapid and transient stimulation of parathyroid hormone) essential for bone anabolic effects.


Subject(s)
Anabolic Agents/chemistry , Pyrimidinones/chemistry , Receptors, Calcium-Sensing/antagonists & inhibitors , Administration, Oral , Anabolic Agents/administration & dosage , Anabolic Agents/pharmacokinetics , Animals , Male , Parathyroid Hormone/metabolism , Pyrimidinones/administration & dosage , Pyrimidinones/pharmacokinetics , Rats , Rats, Sprague-Dawley , Receptors, Calcium-Sensing/metabolism , Structure-Activity Relationship
11.
Bioorg Med Chem Lett ; 19(7): 2075-8, 2009 Apr 01.
Article in English | MEDLINE | ID: mdl-19250823

ABSTRACT

Sulfonamides, exemplified by 3a, were identified as highly selective EP(2) agonists. Lead optimization led to the identification of CP-533536, 7f, a potent and selective EP(2) agonist. CP-533536 demonstrated the ability to heal fractures when administered locally as a single dose in rat models of fracture healing.


Subject(s)
Osteogenesis/drug effects , Pyridines/chemistry , Receptors, Prostaglandin E/agonists , Animals , Male , Pyridines/administration & dosage , Pyridines/pharmacokinetics , Rats , Rats, Sprague-Dawley , Receptors, Prostaglandin E/metabolism , Receptors, Prostaglandin E, EP2 Subtype , Structure-Activity Relationship , Sulfonamides/chemical synthesis , Sulfonamides/chemistry , Sulfonamides/pharmacology
12.
Proc Natl Acad Sci U S A ; 104(25): 10619-24, 2007 Jun 19.
Article in English | MEDLINE | ID: mdl-17537919

ABSTRACT

Bone is accrued and maintained primarily through the coupled actions of bone-forming osteoblasts and bone-resorbing osteoclasts. Cumulative in vitro studies indicated that proline-rich tyrosine kinase 2 (PYK2) is a positive mediator of osteoclast function and activity. However, our investigation of PYK2-/- mice did not reveal evidence supporting an essential function for PYK2 in osteoclasts either in vivo or in culture. We find that PYK2-/- mice have high bone mass resulting from an unexpected increase in bone formation. Consistent with the in vivo findings, mouse bone marrow cultures show that PYK2 deficiency enhances differentiation and activity of osteoprogenitor cells, as does expressing a PYK2-specific short hairpin RNA or dominantly interfering proteins in human mesenchymal stem cells. Furthermore, the daily administration of a small-molecule PYK2 inhibitor increases bone formation and protects against bone loss in ovariectomized rats, an established preclinical model of postmenopausal osteoporosis. In summary, we find that PYK2 regulates the differentiation of early osteoprogenitor cells across species and that inhibitors of the PYK2 have potential as a bone anabolic approach for the treatment of osteoporosis.


Subject(s)
Focal Adhesion Kinase 2/physiology , Osteoblasts/physiology , Osteoclasts/physiology , Osteogenesis/physiology , Osteoporosis/therapy , Animals , Bone Marrow Cells/cytology , Bone Marrow Cells/physiology , Cell Differentiation , Cells, Cultured , Enzyme Inhibitors/therapeutic use , Female , Humans , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/physiology , Mice , Mice, Knockout , Ovariectomy , Rats , Rats, Sprague-Dawley
13.
J Bone Miner Res ; 21(4): 565-75, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16598377

ABSTRACT

UNLABELLED: CP432 is a newly discovered, nonprostanoid EP4 receptor selective prostaglandin E2 agonist. CP432 stimulates trabecular and cortical bone formation and restores bone mass and bone strength in aged ovariectomized rats with established osteopenia. INTRODUCTION: The purpose of this study was to determine whether a newly discovered, nonprostanoid EP4 receptor selective prostaglandin E2 (PGE2) agonist, CP432, could produce bone anabolic effects in aged, ovariectomized (OVX) rats with established osteopenia. MATERIALS AND METHODS: CP432 at 0.3, 1, or 3 mg/kg/day was given for 6 weeks by subcutaneous injection to 12-month-old rats that had been OVX for 8.5 months. The effects on bone mass, bone formation, bone resorption, and bone strength were determined. RESULTS: Total femoral BMD increased significantly in OVX rats treated with CP432 at all doses. CP432 completely restored trabecular bone volume of the third lumbar vertebral body accompanied with a dose-dependent decrease in osteoclast number and osteoclast surface and a dose-dependent increase in mineralizing surface, mineral apposition rate, and bone formation rate-tissue reference in OVX rats. CP432 at 1 and 3 mg/kg/day significantly increased total tissue area, cortical bone area, and periosteal and endocortical bone formation in the tibial shafts compared with both sham and OVX controls. CP432 at all doses significantly and dose-dependently increased ultimate strength in the fifth lumber vertebral body compared with both sham and OVX controls. At 1 and 3 mg/kg/day, CP432 significantly increased maximal load in a three-point bending test of femoral shaft compared with both sham and OVX controls. CONCLUSIONS: CP432 completely restored trabecular and cortical bone mass and strength in established osteopenic, aged OVX rats by stimulating bone formation and inhibiting bone resorption on trabecular and cortical surfaces.


Subject(s)
Aging/physiology , Bone Density/drug effects , Bone Diseases, Metabolic/drug therapy , Dinoprostone/agonists , Osteogenesis/drug effects , Receptors, Prostaglandin E/agonists , Receptors, Prostaglandin E/metabolism , Animals , Body Weight , Bone Density/physiology , Disease Models, Animal , Female , Femur/anatomy & histology , Lumbar Vertebrae/anatomy & histology , Molecular Structure , Organ Size/drug effects , Ovariectomy , Rats , Rats, Sprague-Dawley , Receptors, Prostaglandin E, EP4 Subtype , Substrate Specificity , Tibia/anatomy & histology
14.
Bioorg Med Chem Lett ; 16(7): 1799-802, 2006 Apr 01.
Article in English | MEDLINE | ID: mdl-16442794

ABSTRACT

Heptanoic acid lactams, exemplified by 2, were identified as highly selective EP4 agonists via high throughput screening. Lead optimization led to the identification of lactams with a 30-fold increase in EP4 potency in vitro. Compounds demonstrated robust bone anabolic effects when administered in vivo in rat models of osteoporosis.


Subject(s)
Bone Density/drug effects , Bone Development , Bone and Bones/drug effects , Organ Size/drug effects , Ovariectomy , Receptors, Prostaglandin E/agonists , Animals , Female , Osteoporosis , Rats , Rats, Sprague-Dawley , Receptors, Prostaglandin E, EP4 Subtype
15.
J Bone Miner Res ; 18(11): 2033-42, 2003 Nov.
Article in English | MEDLINE | ID: mdl-14606517

ABSTRACT

UNLABELLED: CP-533,536, a newly discovered, non-prostanoid EP2 receptor-selective PGE2 agonist, stimulates local bone formation and enhances fracture healing in rat models. INTRODUCTION: There is a significant medical need for agents that can stimulate local bone formation and enhance fracture healing. We tested the effects of CP-533,536, a newly discovered, non-prostanoid EP2 receptor-selective prostaglandin E2 (PGE2) agonist, in stimulating local bone formation and enhancing fracture healing in rat models. MATERIALS AND METHODS: In the first model, a single injection of CP-533,536 at doses of 0.3, 1, or 3 mg/kg to the proximal tibial metaphysis of 6-week-old male rats was given on day 1, and the local bone anabolic effect was determined on day 7. We then tested the effects of this compound in inducing bone formation on rat periosteum of the femur. A single dose of 0.3 mg of CP-533,536 incorporated in a poly-(D,L-lactide-co-glycolide) (PLGH) matrix was injected onto the periosteum of the femur in 3-week-old male rats, and local bone formation was determined on day 14. Finally, the ability of CP-533,536 in PLGH matrix in enhancing fracture healing was tested using the rat femoral fracture model. CP-533,536 in PLGH matrix at doses of 0.05, 0.5, or 5 mg was delivered to the local fracture site on the same day of fracture, and its efficacy was evaluated on day 21. RESULTS AND CONCLUSIONS: A single injection of CP-533,536 at doses of 0.3, 1, or 3 mg/kg to the proximal tibial metaphysis dose-dependently stimulated local lamellar bone formation on trabecular, endocortical, and periosteal surfaces, and thus increased bone mineral content and bone strength at the injected site. Similarly, a single injection of 0.3 mg of CP-533,536 incorporated in PLGH matrix onto the periosteum of the femur induced significantly local bone formation. In the rat femoral fracture model, CP-533,536 in PLGH matrix at doses of 0.05, 0.5, and 5 mg dose-dependently increased callus size, density, and strength compared with PLGH matrix alone. These results show that CP-533,536 stimulates new bone formation on trabecular, endocortical, and periosteal surfaces and enhances fracture healing. These data reveal that EP2 receptor-selective agonists provide therapeutic potential for local bone augmentation, bone repair, and bone healing in humans.


Subject(s)
Bone and Bones/drug effects , Dinoprostone/agonists , Femoral Fractures/drug therapy , Fracture Healing/drug effects , Pyridines/pharmacology , Pyridines/therapeutic use , Receptors, Prostaglandin E/metabolism , Animals , Bone and Bones/physiology , Disease Models, Animal , Fluorescence , Injections , Male , Molecular Structure , Pyridines/administration & dosage , Rats , Rats, Sprague-Dawley , Receptors, Prostaglandin E, EP2 Subtype , Substrate Specificity
16.
Biochem Biophys Res Commun ; 305(3): 598-604, 2003 Jun 06.
Article in English | MEDLINE | ID: mdl-12763036

ABSTRACT

As a part of the study to identify genes associated with hormone-refractory stage of human prostate cancer, we have recently identified several genetic and epigenetic changes that seem to be associated with the progression of androgen-sensitive to androgen-independent prostate tumor cells. In the present study, we report a novel gene, macrophage inhibitory cytokine-1 (MIC-1) also known as prostate derived factor (PDF), that was highly expressed in androgen-independent LNCaP-C81 cells and its metastatic variant LNCaP-Ln3 compared to androgen-sensitive LNCaP-C33 cells. The MIC-1/PDF expression was dysregulated (very low to non-detectable) in the androgen-independent PC3 and DU145 cells. Interestingly, serum factors demonstrated a differential regulation of MIC-1/PDF in the androgen-sensitive and the androgen-independent cells of LNCaP cells. Immunohistochemical analysis on 15 prostatic adenocarcinomas showed a weak staining in the benign prostatic glandular area (intensity score 2.38+/-0.25; n=13), while the immunoreactivity was significantly stronger (p<0.05) in areas of adenocarcinoma (score 7.33+/-0.88; n=15). Altogether, these data suggest that the serum factors (including androgens and cytokines) might contribute to the regulation of the MIC-1/PDF gene that seems to be associated with the progression of prostate cancer.


Subject(s)
Adenocarcinoma/metabolism , Cytokines/metabolism , Membrane Proteins/metabolism , Prostatic Neoplasms/metabolism , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Bone Morphogenetic Proteins , Cell Culture Techniques , Cytokines/analysis , Cytokines/genetics , Disease Progression , Gene Expression Regulation, Neoplastic , Growth Differentiation Factor 15 , Humans , Immunohistochemistry , Male , Membrane Proteins/analysis , Membrane Proteins/genetics , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , RNA, Messenger/biosynthesis , Tumor Cells, Cultured
17.
Mol Endocrinol ; 17(7): 1356-67, 2003 Jul.
Article in English | MEDLINE | ID: mdl-12677010

ABSTRACT

The P2X7 nucleotide receptor is an ATP-gated ion channel expressed widely in cells of hematopoietic origin. Our purpose was to explore the involvement of the P2X7 receptor in bone development and remodeling by characterizing the phenotype of mice genetically modified to disrupt the P2X7 receptor [knockout (KO)]. Femoral length did not differ between KO and wild-type (WT) littermates at 2 or 9 months of age, indicating that the P2X7 receptor does not regulate longitudinal bone growth. However, KO mice displayed significant reduction in total and cortical bone content and periosteal circumference in femurs, and reduced periosteal bone formation and increased trabecular bone resorption in tibias. Patch clamp recording confirmed expression of functional P2X7 receptors in osteoclasts from WT but not KO mice. Osteoclasts were present in vivo and formed in cultures of bone marrow from KO mice, indicating that this receptor is not essential for fusion of osteoclast precursors. Functional P2X7 receptors were also found in osteoblasts from WT but not KO mice, suggesting a direct role in bone formation. P2X7 receptor KO mice demonstrate a unique skeletal phenotype that involves deficient periosteal bone formation together with excessive trabecular bone resorption. Thus, the P2X7 receptor represents a novel therapeutic target for the management of skeletal disorders such as osteoporosis.


Subject(s)
Bone Development/genetics , Bone Resorption/genetics , Receptors, Purinergic P2/physiology , Animals , Bone Marrow Cells/cytology , Cells, Cultured , Female , Femur/anatomy & histology , Femur/growth & development , Femur/pathology , Gene Expression Regulation, Developmental , Male , Mice , Mice, Knockout , Osteoblasts/physiology , Osteoclasts/physiology , Patch-Clamp Techniques , Receptors, Purinergic P2X7 , Tomography/methods
18.
Oncogene ; 21(27): 4212-9, 2002 Jun 20.
Article in English | MEDLINE | ID: mdl-12082608

ABSTRACT

Human astrocytic brain tumors select for mutations in the p53 tumor suppressor gene early in malignant progression. p53 is activated upon various kinds of cellular stress leading to apoptosis or cell cycle arrest, but is also implicated in complex biological processes such as inhibition of angiogenesis and metastasis. In an effort to shed light on consequences mediated by p53 inactivation in gliomas, we established the Tet-On system for p53 in the LN-Z308 glioblastoma cell line. The macrophage inhibitory cytokine-1 (MIC-1) gene was identified as a most prominent p53 target gene upon gene expression profiling. Oxygen deprivation, an important cellular stress, revealed MIC-1 as an anoxia responsive gene in glioblastoma cell lines. MIC-1 up-regulation by anoxia is mediated through an alternative, p53 and hypoxia inducible factor 1 (HIF-1) independent pathway. Furthermore, ectopic expression of MIC-1 in LN-Z308 cell line completely abolished its inherent tumorigenicity in nude mice, while proliferation in vitro was not affected. In the present experimental model MIC-1 may exert its anti-tumorigenic properties via a paracrine mechanism mediated by host cells in vivo. Taken together, these data suggest that MIC-1 is an important downstream mediator of p53 function, while acting itself as an intercessor of cellular stress signaling and exerting anti-tumorigenic activities.


Subject(s)
Brain Neoplasms/pathology , Cytokines/biosynthesis , DNA-Binding Proteins/physiology , Gene Expression Regulation, Neoplastic , Glioblastoma/pathology , Neoplasm Proteins/biosynthesis , Nuclear Proteins/physiology , Oxygen/pharmacology , Transcription Factors , Tumor Suppressor Protein p53/physiology , Animals , Brain Neoplasms/genetics , Brain Neoplasms/metabolism , Cell Hypoxia/genetics , Cytokines/genetics , Dexamethasone/pharmacology , Doxycycline/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Genes, p53 , Glioblastoma/genetics , Glioblastoma/metabolism , Growth Differentiation Factor 15 , Humans , Hypoxia-Inducible Factor 1 , Hypoxia-Inducible Factor 1, alpha Subunit , Mice , Mice, Nude , Neoplasm Proteins/genetics , Neoplasm Proteins/physiology , Neoplasm Transplantation , Recombinant Fusion Proteins/physiology , Signal Transduction , Transplantation, Heterologous
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