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1.
Int J Cancer ; 134(11): 2572-82, 2014 Jun 01.
Article in English | MEDLINE | ID: mdl-24214898

ABSTRACT

Hypoxia and dysfunctional tumor vessels represent a prominent feature of pancreatic cancer, being, at least in part, responsible for chemotherapy resistance and immune suppression in these tumors. We tested whether the increase of oxygen delivery induced in vivo by myo-inositol trispyrophosphate (ITPP) can reverse hypoxia, control tumor growth and improve chemotherapy response. Tumor size, metastatic development (microcomputed tomography scan follow-up) and the survival of rats and nude or NOD.SCID mice, (bearing syngenic rat and MiaPaCa2- or patient-derived pancreatic tumors), were determined on ITPP and/or gemcitabine treatment. Partial oxygen pressure, expression of angiogenic factors and tumor histology were evaluated. Infiltration and oxidative status of immune cells, as well as chemotherapy penetration in tumors, were determined by fluorescence-activated cell sorting, fluorometry, nitric oxide release assays, Western blot and confocal microscopy. Weekly intravenous ITPP application resulted in the inhibition of metastasis development and restricted primary tumor growth, showing a superior effect on the rats' survival compared with gemcitabine. ITPP treatment restored tumor normoxia and caused a reduction in hypoxia inducible factor-1α levels, with subsequent VEGF and Lox downregulation, resulting in improved vessel structure and decreased desmoplasia. The latter effects translated into elevated immune cells influx and improved susceptibility to gemcitabine treatment. Growth of human pancreatic tumor xenografts was strongly inhibited by administration of ITPP. ITPP exploits a two-stage mechanism causing rapid, early and sustainable late stage normoxia. This is due to the angiogenic factor modulation and vascular normalization, leading to enhanced chemotherapy delivery and synergistic life prolongation, on combination with low doses of gemcitabine.


Subject(s)
Antimetabolites, Antineoplastic/therapeutic use , Deoxycytidine/analogs & derivatives , Hypoxia/drug therapy , Inositol Phosphates/therapeutic use , Liver Neoplasms/drug therapy , Pancreatic Neoplasms/drug therapy , Animals , Apoptosis/drug effects , Blotting, Western , Cell Proliferation/drug effects , Deoxycytidine/therapeutic use , Drug Synergism , Fluorescent Antibody Technique , Humans , Hypoxia/metabolism , Hypoxia/pathology , Hypoxia-Inducible Factor 1, alpha Subunit/antagonists & inhibitors , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Liver Neoplasms/metabolism , Liver Neoplasms/secondary , Male , Mice , Mice, Inbred NOD , Mice, SCID , Neovascularization, Pathologic , Oxygen/metabolism , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , RNA, Messenger/genetics , Rats , Rats, Inbred Lew , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, Cultured , Gemcitabine
2.
Br J Cancer ; 105(2): 288-95, 2011 Jul 12.
Article in English | MEDLINE | ID: mdl-21712827

ABSTRACT

BACKGROUND: The transcription factor HOXC8 regulates many genes involved in tumour progression. This study was to investigate the role of HOXC8 in pancreatic ductal adenocarcinoma (PDAC) growth and metastasis. METHODS: The Hoxc8 expression was determined in 15 PDAC cell lines and human specimens by RT-polymerase chain reaction and/or immunohistochemistry. The effects of HOXC8 silencing by RNA interference were investigated by functional tests. RESULTS: The Hoxc8 mRNA expression in PDAC cell lines was negatively related to their growth in vivo. Except for Suit2-007 cells, only those with low Hoxc8 mRNA expression grew in nude rats. Successful down-regulation of HOXC8 expression caused increased proliferation, migration (P ≤ 0.05) and colony formation (P ≤ 0.05) in Suit2-007, Panc-1 and MIA PaCa-2 PDAC cells, respectively. The Hoxc8 mRNA levels in diseased human pancreas tissues were significantly increased over normal in PDAC and autoimmune chronic pancreatitis specimens (P<0.01, respectively), but negatively related to tumour stage (P=0.09). In primary and metastatic tumour samples, immunohistochemical staining for HOXC8 was stronger in surrounding than in neoplastic tissues. Furthermore, grading of primary carcinomas was negatively associated with HOXC8 staining (P=0.03). Liver metastases showed the lowest HOXC8 expression of all neoplastic lesions. CONCLUSION: These data indicate that HOXC8 expression is inversely related to PDAC progression and metastasis.


Subject(s)
Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/pathology , Homeodomain Proteins/genetics , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Adolescent , Adult , Aged , Aged, 80 and over , Animals , Case-Control Studies , Cell Line, Tumor , Disease Progression , Female , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/physiology , Homeodomain Proteins/antagonists & inhibitors , Homeodomain Proteins/physiology , Humans , Male , Middle Aged , Neoplasm Metastasis , RNA, Small Interfering/pharmacology , Rats , Rats, Nude , Xenograft Model Antitumor Assays , Young Adult
3.
Cancer Immunol Immunother ; 59(9): 1389-400, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20514540

ABSTRACT

Despite spontaneous or vaccination-induced immune responses, pancreatic cancer remains one of the most deadly immunotherapy-resistant malignancies. We sought to comprehend the spectrum of pancreatic tumor-associated antigens (pTAAs) and to assess the clinical relevance of their immunogenicity. An autologous SEREX-based screening of a cDNA library constructed from a pancreatic T3N0M0/GIII specimen belonging to a long-term survivor (36 months) revealed 18 immunogenic pTAA. RT-PCR analysis displayed broad distribution of the identified antigens among normal human tissues. PNLIPRP2 and MIA demonstrated the most distinct pancreatic cancer-specific patterns. ELISA-based screening of sera for corresponding autoantibodies revealed that although significantly increased, the immunogenicity of these molecules was not a common feature in pancreatic cancer. QRT-PCR and immunohistochemistry characterized PNLIPRP2 as a robust acinar cell-specific marker whose decreased expression mirrored the disappearance of parenchyma in the diseased organ, but was not related to the presence of PNLIPRP2 autoantibodies. Analyses of MIA-known to be preferentially expressed in malignant cells-surprisingly revealed an inverse correlation between intratumoral gene expression and the emergence of autoantibodies. MIA(high) patients were autoantibody-negative and had shorter median survival when compared with autoantibody-positive MIA(low) patients (12 vs. 34 months). The observed pTAA spectrum comprised molecules associated with acinar, stromal and malignant structures, thus presenting novel targets for tumor cell-specific therapies as well as for approaches based on the bystander effects. Applying the concept of cancer immunoediting to interpret relationships between gene expression, antitumor immune responses, and clinical outcome might better discriminate between past and ongoing immune responses, consequently enabling prognostic stratification of patients and individual adjustment of immunotherapy.


Subject(s)
Adenocarcinoma/diagnosis , Adenocarcinoma/immunology , Pancreatic Neoplasms/diagnosis , Pancreatic Neoplasms/immunology , Adenocarcinoma/blood , Adenocarcinoma/genetics , Adenocarcinoma/mortality , Aged , Antigens, Tumor-Associated, Carbohydrate/genetics , Antigens, Tumor-Associated, Carbohydrate/immunology , Antigens, Tumor-Associated, Carbohydrate/metabolism , Autoantibodies/blood , Cloning, Molecular , Enzyme-Linked Immunosorbent Assay , Female , Gene Library , Humans , Immunochemistry , Lipase/genetics , Lipase/immunology , Lipase/metabolism , Male , Middle Aged , Neoplasm Staging , Pancreas/metabolism , Pancreas/pathology , Pancreatic Neoplasms/blood , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/mortality , Prognosis
4.
Clin Exp Immunol ; 154(2): 216-23, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18778363

ABSTRACT

The chemokine receptor CXCR6 has been described on lymphoid cells and is thought to participate in the homing of activated T-cells to non-lymphoid tissue. We now provide evidence that the chemokine receptor CXCR6 is also expressed by activated polymorphonuclear neutrophils (PMN) in vivo: Examination of biopsies derived from patients with pancreatic carcinoma by confocal laser scan microscopy revealed a massive infiltration of PMN that expressed CXCR6, while PMN of the peripheral blood of these patients did not. To answer the question whether CXCR6 expression is a property of infiltrated and activated PMN, leucocytes were collected from patients with localized soft tissue infections in the course of the wound debridement. By cytofluorometry, the majority of these cells were identified as PMN. Up to 50% of these PMN were also positive for CXCR6. Again, PMN from the peripheral blood of these patients were nearly negative for CXCR6, as were PMN of healthy donors. In a series of in vitro experiments, up-regulation of CXCR6 on PMN of healthy donors by a variety of cytokines was tested. So far, a minor, although reproducible, effect of tumour necrosis factor (TNFalpha) was seen: brief exposure with low-dose TNFalpha induced expression of CXCR6 on the surface of PMN. Furthermore, we could show an increased migration of PMN induced by the axis CXCL16 and CXCR6. In summary, our data provide evidence that CXCR6 is not constitutively expressed on PMN, but is up-regulated under inflammatory conditions and mediates migration of CXCR6-positive PMN.


Subject(s)
Bacterial Infections/immunology , Neoplasm Proteins/metabolism , Neutrophils/immunology , Pancreatic Neoplasms/immunology , Receptors, Chemokine/metabolism , Receptors, Virus/metabolism , Acute Disease , Chemotaxis, Leukocyte/immunology , Humans , Ligands , Microscopy, Confocal/methods , Neutrophil Activation/immunology , Osteomyelitis/immunology , Prosthesis-Related Infections/immunology , Receptors, CXCR6 , Soft Tissue Infections/immunology , Tumor Cells, Cultured , Up-Regulation/immunology
5.
Br J Cancer ; 99(5): 760-7, 2008 Sep 02.
Article in English | MEDLINE | ID: mdl-18728667

ABSTRACT

Efficacy of chemotherapy for pancreatic cancer may be improved by tailoring it to individual chemosensitivity profiles. Identification of nonresponders before initiation of treatment may help to avoid side effects. In this study, primary pancreatic cancer cells were isolated from 18 patients undergoing pancreaticoduodenectomy for pancreatic cancer. Eight commonly used pancreatic cancer cell lines were used as controls. Ex vivo chemosensitivity for gemcitabine, 5-fluorouracil, mitomycin-C, cisplatinum, oxaliplatinum, paclitaxel and a combination of gemcitabine with oxaliplatinum or mitomycin-C was determined using a cellular ATP-based tumour chemosensitivity assay (ATP-TCA). Quantitative real-time-polymerase chain reaction was performed to determine RNA expression levels of genes implicated in chemoresistance. Chemosensitivity towards cytotoxic agents was highly variable in primary pancreatic cancer cells and pancreatic cancer cell lines. ATP-TCA results for gemcitabine correlated to the tissue expression of human equilibrative nucleoside transporter-1 (hENT1). Time to relapse in patients with gemcitabine-sensitive tumours was significantly higher than in patients with chemoresistant pancreatic cancers (P=0.01; 71 vs 269 days). Furthermore, time to relapse in gemcitabine-treated patients was related to hENT1 expression (P=0.0067). Thus, chemosensitivity testing using ATP-TCA in pancreatic cancer is feasible and correlated with time to relapse in gemcitabine-treated patients. This suggests that ATP-TCA testing could be used as a decision-making tool in the adjuvant treatment of pancreatic cancer.


Subject(s)
Antineoplastic Agents/therapeutic use , Deoxycytidine/analogs & derivatives , Gene Expression Profiling , Pancreatic Neoplasms/drug therapy , Adult , Aged , Cell Line, Tumor , Deoxycytidine/therapeutic use , Drug Screening Assays, Antitumor , Female , Humans , Male , Middle Aged , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Polymerase Chain Reaction , Gemcitabine
6.
J Pathol ; 215(2): 195-203, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18421760

ABSTRACT

In chronic pancreatitis (CP), both the progressive loss of acinar parenchyma and aggressive fibro-inflammatory reactions ultimately lead to irreversible organ destruction. Dying cells are normally removed by macrophages and elimination is associated with anti-inflammatory cytokine switch. We investigated whether defective clearance of damaged acini by macrophages such as compromised phagocytosis or altered cytokine reaction occurs in CP and thus represents a causative link between acinar loss and fibro-inflammation. In a checkerboard-like co-culture system, we assessed normal and CP macrophages for their phagocytic and cytokine responses to dying pancreatic acinar cells of normal or CP origin by FACS, confocal microscopy, QRT-PCR, and ELISA. In CP, phagocytosis of apoptotic acini by macrophages was not impaired; however, the associated cytokine responses were gradually perturbed. Most interestingly, only normal acini suppressed TGFbeta1 expression and accumulation specifically in normal macrophage cultures, while CP acini lost this ability. Both types of apoptotic acini induced pro-inflammatory cytokine bursts of varying strength in both types of macrophages; however, the most significant difference (more than 50-fold higher expression of IL-1beta, IL-6, and IL-8) was evident between CP/CP and normal/normal combinations, indicating that acinar and macrophage alterations synergistically lead to the ultimate CP-specific bias. In combination with in situ data comparing circulating inflammatory cells to pancreatic resident ones, our results indicate that cytokine expression in inflammatory cells undergoes spatiotemporal modulation, most likely through a successive interplay of acinar, stromal, and circulating factors. Thus, clearance of injured pancreatic acini by macrophages is associated with a unique cytokine reaction which may constitute a basis for progression of SAPE (sentinel acute pancreatitis event) to the irreversible fibro-inflammation in CP.


Subject(s)
Macrophages/physiology , Pancreas/immunology , Pancreatitis, Chronic/immunology , Apoptosis , Coculture Techniques , Enzyme-Linked Immunosorbent Assay/methods , Flow Cytometry , Humans , Interleukin-1/immunology , Interleukin-6/immunology , Interleukin-8/immunology , Macrophages/immunology , Microscopy, Confocal , Pancreas/pathology , Pancreatitis, Chronic/pathology , Phagocytosis , Polymerase Chain Reaction/methods , Tumor Necrosis Factor-alpha/immunology
7.
J Cell Mol Med ; 11(5): 1162-74, 2007.
Article in English | MEDLINE | ID: mdl-17979891

ABSTRACT

ADAM8 belongs to a family of transmembrane proteins implicated in cell-cell interactions, proteolysis of membrane proteins, and various aspects of carcinogenesis. In the present study, we aimed to evaluate the expression and function of ADAM8 in pancreatic cancer. ADAM8 mRNA levels were analysed by quantitative RT-PCR and correlated to patient survival. Immunohistochemistry was performed to localize ADAM8 in pancreatic tis-sues. Silencing of ADAM8 expression was carried out by transfection with specific siRNA oligonucleotides. Cell growth and invasion assays were used to assess the functional consequences of ADAM8 silencing. SELDI-TOF-MS was performed to detect the proteolytic activity of ADAM8 in pancreatic cancer cells. ADAM8 mRNA was significantly overexpressed in pancreatic ductal adenocarcinoma (PDAC) compared with normal pancreatic tissues (5.3-fold increase; P= 0.0008), and high ADAM8 mRNA and protein expression levels correlated with reduced survival time of PDAC patients (P= 0.048 and P= 0.065, respectively). Silencing of ADAM8 expression did not significantly influence pancreatic cancer cell growth but suppressed invasiveness. In addition, decreased proteolytic activity was measured in cell culture supernatants following silencing of ADAM8. In conclusion, ADAM8 is overexpressed in PDAC, influences cancer cell invasiveness and correlates with reduced survival, suggesting that ADAM8 might be a potential target in pancreatic cancer therapy.


Subject(s)
ADAM Proteins/genetics , Carcinoma, Pancreatic Ductal/pathology , Membrane Proteins/genetics , Pancreatic Neoplasms/pathology , ADAM Proteins/chemistry , ADAM Proteins/metabolism , Adult , Carcinoma, Pancreatic Ductal/genetics , Cell Hypoxia , Cell Line, Tumor , Cell Proliferation , Gene Expression Regulation, Neoplastic , Humans , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Middle Aged , Neoplasm Invasiveness , Pancreatic Neoplasms/genetics , Protein Processing, Post-Translational , Protein Structure, Tertiary , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Survival Analysis
8.
J Clin Pathol ; 59(1): 21-7, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16394277

ABSTRACT

BACKGROUND: Recurrent inflammation in chronic pancreatitis (CP) is not well understood. AIMS: To investigate whether decorin, an extracellular matrix (ECM) proteoglycan with macrophage modulating activity, is a pathogenic factor allowing diseased pancreatic stroma to sustain inflammation by affecting the cytokine profile of accumulating inflammatory cells. METHODS: Decorin was examined in 18 donors and 32 patients with CP by quantitative reverse transcription polymerase chain reaction (QRT-PCR), western blotting, and immunohistochemistry of pancreatic specimens. QRT-PCR was used to assess cytokine expression in donor peripheral blood mononuclear cells (PBMC), exposed or not to decorin in vitro, and to compare it with the cytokine profile of circulating and resident mononuclear cells (MNC) of patients with CP. RESULTS: In CP, desmoplasia is associated with overexpression of decorin in the growing ECM and enlarged pancreatic nerves. In culture, exposure of MNC to decorin stimulated expression of the MNC recruiting chemokine MCP-1. In biopsies, MNC infiltrates in decorin rich CP tissue showed a 300-fold upregulation of MCP-1 compared with decorin free peripheral blood, whereas no difference was found in basal MCP-1 expression in PBMC of patients versus donors. This effect was specific for MCP1-other inflammatory cytokines, such as interleukin 1beta and tumour necrosis factor alpha, were not affected. CONCLUSION: Decorin is a molecular marker of desmoplasia in CP, and excessive decorin may allow fibrotic masses to nourish and protract inflammation by deregulating the process of MNC accumulation and activation. These data provide a molecular basis for surgical resection of diseased tissue as a treatment option in CP.


Subject(s)
Pancreatitis, Chronic/metabolism , Proteoglycans/physiology , Adolescent , Adult , Aged , Blotting, Western , Cells, Cultured , Chemokine CCL2/blood , Decorin , Extracellular Matrix/metabolism , Extracellular Matrix Proteins , Female , Gene Expression Regulation, Neoplastic , Humans , Immunoenzyme Techniques , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Male , Middle Aged , Pancreas/innervation , Proteoglycans/metabolism , Proteoglycans/pharmacology , RNA, Messenger/genetics , Recurrence , Reverse Transcriptase Polymerase Chain Reaction/methods , Up-Regulation
9.
Gut ; 55(4): 519-28, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16174661

ABSTRACT

BACKGROUND: Success of chemotherapy and alleviation of pain are frequently less than optimal in pancreatic cancer patients, leading to increasing interest in new pharmacological substances, such as vanilloids. Our study addressed the question of whether vanilloids influence pancreatic cancer cell growth, and if vanilloids could be used for pain treatment via the vanilloid 1 receptor (VR1) in pancreatic cancer patients. METHODS: In vitro, the effect of resiniferatoxin (vanilloid analogue) on apoptosis and cell growth in pancreatic cancer cells--either alone, combined with 5-fluorouracil (5-FU), or combined with gemcitabine--was determined by annexin V staining, FACS analysis, and MTT assay, respectively. VR1 expression was evaluated on RNA and protein level by quantitative polymerase chain reaction and immunohistochemistry in human pancreatic cancer and chronic pancreatitis. Patient characteristics--especially pain levels--were registered in a prospective database and correlated with VR1 expression. RESULTS: Resiniferatoxin induced apoptosis by targeting mitochondrial respiration and decreased cell growth in pancreatic cancer cells without showing synergistic effects with 5-FU or gemcitabine. Expression of VR1 was significantly upregulated in human pancreatic cancer and chronic pancreatitis. VR1 expression was related to the intensity of pain reported by cancer patients but not to the intensity of pain reported by patients with chronic pancreatitis. CONCLUSIONS: Resiniferatoxin induced apoptosis in pancreatic cancer cells indicates that vanilloids may be useful in the treatment of human pancreatic cancer. Furthermore, vanilloid might be a novel and effective treatment option for neurogenic pain in patients with pancreatic cancer.


Subject(s)
Antineoplastic Agents/therapeutic use , Diterpenes/therapeutic use , Pain/prevention & control , Pancreatic Neoplasms/drug therapy , Adult , Aged , Antimetabolites, Antineoplastic/therapeutic use , Apoptosis/drug effects , Cell Division/drug effects , Cell Line, Tumor , Chronic Disease , Deoxycytidine/analogs & derivatives , Deoxycytidine/therapeutic use , Drug Synergism , Drug Therapy, Combination , Female , Fluorouracil/therapeutic use , Humans , Immunohistochemistry/methods , Male , Middle Aged , Mitochondria/drug effects , Oxidative Stress , Pancreas/chemistry , Pancreatic Neoplasms/complications , Pancreatitis/drug therapy , Prospective Studies , TRPV Cation Channels/analysis , Gemcitabine
10.
Cancer Biol Ther ; 4(7): 740-6, 2005 Jul.
Article in English | MEDLINE | ID: mdl-15970685

ABSTRACT

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies, with an overall 5-year survival rate of less than 5%. Invasive tumor growth and early metastasis are two important reasons for this dismal prognosis. Osteopontin (OPN) is a secretory protein with a variety of functions, for example in cell adhesion and migration, inflammatory reaction and apoptosis. In this study the functional role of OPN in human pancreatic cancer and its potential use as a disease marker were analyzed. By real time quantitative PCR, there was a 2.2-fold and 1.6-fold increase of OPN mRNA in pancreatic cancers (n = 23) and chronic pancreatitis samples (n = 22), respectively, compared to normal pancreatic tissues (n = 20). Immunohistochemical analysis demonstrated OPN staining in 60% of the primary pancreatic tumors and in 72% of the lymph node and liver metastases. ELISA analysis of serum samples obtained from pancreatic cancer patients (n = 70), chronic pancreatitis patients (n = 12), and healthy donors (n = 20) showed a 1.6-fold increase in OPN serum levels in patients with tumors and a 1.9-fold increase in patients with chronic pancreatitis. Recombinant human OPN significantly increased the invasiveness of pancreatic cancer cells, without having any impact on cell proliferation. In addition, down regulation of OPN by specific siRNA molecules decreased pancreatic cancer cell invasion. In conclusion, OPN serum levels in pancreatic cancer and chronic pancreatitis patients are not significantly different, thereby restricting its role as a prognostic or follow-up marker. Our results do suggest, however, that blockade of OPN might be useful as a therapeutic approach to inhibit invasion and metastasis of pancreatic cancer cells.


Subject(s)
Pancreatic Neoplasms/pathology , Pancreatitis, Chronic/metabolism , Sialoglycoproteins/physiology , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Biomarkers, Tumor/metabolism , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/pathology , Case-Control Studies , Humans , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Liver Neoplasms/secondary , Lymphatic Metastasis/pathology , Neoplasm Invasiveness/pathology , Neoplasm Staging , Osteopontin , Pancreas/metabolism , Pancreatic Neoplasms/genetics , Pancreatitis, Chronic/genetics , Prognosis , RNA, Messenger/metabolism , RNA, Small Interfering/pharmacology , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sialoglycoproteins/antagonists & inhibitors , Tumor Cells, Cultured
11.
J Pharmacol Exp Ther ; 298(3): 1172-8, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11504817

ABSTRACT

Exaggerated or inappropriate signaling by the platelet-derived growth factor receptor (PDGFR) tyrosine kinase has been implicated in a wide variety of diseases. Thus, a series of piperazinyl quinazoline compounds were identified as potent antagonists of the PDGFR by screening chemical libraries. An optimized analog, CT52923, was shown to be an ATP-competitive inhibitor that exhibited remarkable specificity when tested against other kinases, including all members of the closely related PDGFR family. The PDGFRs and stem cell factor receptor were inhibited with an IC(50) of 100 to 200 nM, while 45- to >200-fold higher concentrations of CT52923 were required to inhibit fms-like tyrosine kinase-3 and colony-stimulating factor-1 receptor, respectively. Other receptor tyrosine kinases, cytoplasmic tyrosine kinases, serine/threonine kinases, or members of the mitogen-activated protein kinase pathway were not significantly inhibited at 100- to 1000-fold higher concentrations. In addition, this compound also demonstrated specificity for inhibition of cellular responses. Platelet-derived growth factor-induced smooth muscle cell migration or fibroblast proliferation was found to be blocked by CT52923 with an IC(50) of 64 and 280 nM, respectively, whereas 50- to 100-fold higher concentrations were required to inhibit these responses when induced with fibroblast growth factor. To investigate the effect of CT52923 on PDGFR signaling, in vivo studies demonstrated that CT52923 could significantly inhibit neointima formation following carotid artery injury by oral administration in the rat. Therefore, PDGFR antagonism by CT52923 could be a viable strategy for the prevention of clinical restenosis or the treatment of other human diseases involving PDGFR signaling.


Subject(s)
Carotid Artery Injuries/pathology , Neovascularization, Pathologic/prevention & control , Receptors, Platelet-Derived Growth Factor/antagonists & inhibitors , Angioplasty, Balloon , Animals , CHO Cells , Cell Division/drug effects , Cell Movement/drug effects , Cricetinae , Cytoplasm/drug effects , Cytoplasm/enzymology , DNA, Complementary/biosynthesis , DNA, Complementary/genetics , Humans , Neovascularization, Pathologic/pathology , Phosphorylation , Receptor, Platelet-Derived Growth Factor beta/antagonists & inhibitors , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Signal Transduction/drug effects , Thymidine/metabolism
12.
J Antibiot (Tokyo) ; 54(5): 405-14, 2001 May.
Article in English | MEDLINE | ID: mdl-11480883

ABSTRACT

RP-1776, a novel cyclic peptide, was isolated from the culture broth of Streptomyces sp. KY11784. RP-1776 selectively inhibited the binding of PDGF BB to the extracellular domain of the PDGF beta-receptor with an IC50 value of 11 +/- 6 microM. Detailed binding experiments suggested that RP-1776 directly interacts with PDGF BB. RP-1776 inhibited the phosphorylation of the PDGF beta-receptor induced by PDGF BB. These results suggested that RP-1776 antagonizes the signaling of PDGF BB probably through the inhibition of PDGF BB binding to the PDGF beta-receptor.


Subject(s)
Anti-Bacterial Agents/isolation & purification , Anti-Bacterial Agents/pharmacology , Peptides, Cyclic/isolation & purification , Peptides, Cyclic/pharmacology , Platelet-Derived Growth Factor/antagonists & inhibitors , Receptors, Platelet-Derived Growth Factor/metabolism , Streptomyces/chemistry , Animals , Anti-Bacterial Agents/chemistry , Becaplermin , CHO Cells , Cricetinae , Depsipeptides , Fermentation , Inhibitory Concentration 50 , Magnetic Resonance Spectroscopy , Microbial Sensitivity Tests , Peptides, Cyclic/chemistry , Phosphorylation/drug effects , Platelet-Derived Growth Factor/metabolism , Protein Structure, Tertiary , Proto-Oncogene Proteins c-sis , Soil Microbiology , Streptomyces/metabolism
13.
Nat Cell Biol ; 3(5): 517-21, 2001 May.
Article in English | MEDLINE | ID: mdl-11331882

ABSTRACT

Platelet-derived growth factor (PDGF) has been directly implicated in developmental and physiological processes, as well as in human cancer, fibrotic diseases and arteriosclerosis. The PDGF family currently consists of at least three gene products, PDGF-A, PDGF-B and PDGF-C, which selectively signal through two PDGF receptors (PDGFRs) to regulate diverse cellular functions. After two decades of searching, PDGF-A and B were the only ligands identified for PDGFRs. Recently, however, database mining has resulted in the discovery of a third member of the PDGF family, PDGF-C, a functional analogue of PDGF-A that requires proteolytic activation. PDGF-A and PDGF-C selectively activate PDGFR-alpha, whereas PDGF-B activates both PDGFR-alpha and PDGFR-beta. Here we identify and characterize a new member of the PDGF family, PDGF D, which also requires proteolytic activation. Recombinant, purified PDGF-D induces DNA synthesis and growth in cells expressing PDGFRs. In cells expressing individual PDGFRs, PDGF-D binds to and activates PDGFR-beta but not PDGFR-alpha. However, in cells expressing both PDGFRs, PDGF-D activates both receptors. This indicates that PDGFR-alpha activation may result from PDGFR-alpha/beta heterodimerization.


Subject(s)
Lymphokines , Platelet-Derived Growth Factor/chemistry , Platelet-Derived Growth Factor/physiology , 3T3 Cells , Amino Acid Sequence , Animals , Base Sequence , Binding, Competitive , Bromodeoxyuridine/metabolism , DNA, Complementary/metabolism , Dimerization , Dose-Response Relationship, Drug , Enzyme Activation , Enzyme-Linked Immunosorbent Assay , Fibroblasts/metabolism , Humans , Mice , Molecular Sequence Data , Phosphorylation , Polymerase Chain Reaction , Receptors, Platelet-Derived Growth Factor/metabolism , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Signal Transduction , Tissue Distribution , Tumor Cells, Cultured , Tyrosine/metabolism
14.
J Exp Med ; 190(3): 411-21, 1999 Aug 02.
Article in English | MEDLINE | ID: mdl-10430629

ABSTRACT

Mice with a null mutation of the gene encoding interferon consensus sequence-binding protein (ICSBP) develop a disease with marked expansion of granulocytes and macrophages that frequently progresses to a fatal blast crisis, thus resembling human chronic myelogenous leukemia (CML). One important feature of CML is decreased responsiveness of myeloid cells to apoptotic stimuli. Here we show that myeloid cells from mice deficient in ICSBP exhibit reduced spontaneous apoptosis and a significant decrease in sensitivity to apoptosis induced by DNA damage. In contrast, apoptosis in thymocytes from ICSBP-deficient mice is unaffected. We also show that overexpression of ICSBP in the human U937 monocytic cell line enhances the rate of spontaneous apoptosis and the sensitivity to apoptosis induced by etoposide, lipopolysaccharide plus ATP, or rapamycin. Programmed cell death induced by etoposide was specifically blocked by peptides inhibitory for the caspase-1 or caspase-3 subfamilies of caspases. Studies of proapoptotic genes showed that cells overexpressing ICSBP have enhanced expression of caspase-3 precursor protein. In addition, analyses of antiapoptotic genes showed that overexpression of ICSBP results in decreased expression of Bcl-X(L). These data suggest that ICSBP modulates survival of myeloid cells by regulating expression of apoptosis-related genes.


Subject(s)
Apoptosis/immunology , Consensus Sequence/immunology , Hematopoietic Stem Cells/pathology , Interferons/pharmacology , Repressor Proteins/physiology , Amino Acid Chloromethyl Ketones/pharmacology , Animals , Apoptosis/genetics , Bone Marrow Cells , Caspases/biosynthesis , Caspases/genetics , Caspases/metabolism , Cells, Cultured , Cysteine Proteinase Inhibitors/pharmacology , Etoposide/antagonists & inhibitors , Etoposide/pharmacology , Gene Expression Regulation/drug effects , Gene Expression Regulation/immunology , Hematopoietic Stem Cells/enzymology , Hematopoietic Stem Cells/immunology , Hematopoietic Stem Cells/metabolism , Humans , Interferon Regulatory Factors , Lymph Nodes , Mice , Mice, Knockout , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/biosynthesis , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Repressor Proteins/biosynthesis , Repressor Proteins/genetics , Spleen , U937 Cells , bcl-X Protein
15.
J Virol ; 73(8): 7093-5, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10400816

ABSTRACT

The unique Gag polyprotein of the replication-defective virus responsible for murine AIDS (MAIDS) induces B-cell activation, proliferation, and differentiation, including immunoglobulin class switch-recombination to immunoglobulin E (IgE). Secretion of IgE normally requires the serial induction of interleukin 4 (IL-4), engagement of the IL-4 receptor, activation of signal transducer and activator of transcription (STAT) 6, and induction of Iepsilon germline transcripts as a prelude to switching. Remarkably, expression of IgE is equivalent in normal and IL-4-deficient mice with MAIDS (Morawetz et al., J. Exp. Med. 184:1651-1661, 1996). To understand this anomaly, we studied mice with a null mutation of STAT6. Lymphoproliferation and immunodeficiency, the hallmarks of MAIDS, developed with comparable kinetics and degree in normal and mutant mice. In addition, serum IgE levels were indistinguishable in mice of either genotype. We conclude that B cells from mice with MAIDS activate unique IL-4- and STAT6-independent signaling pathways for B-cell activation and differentiation.


Subject(s)
Immunoglobulin E/biosynthesis , Leukemia Virus, Murine/immunology , Murine Acquired Immunodeficiency Syndrome/immunology , Trans-Activators/physiology , Animals , Interleukin-4/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , STAT6 Transcription Factor , Trans-Activators/genetics
16.
J Antibiot (Tokyo) ; 52(3): 235-44, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10348038

ABSTRACT

Nine azaphilones designated RP-1551-1, -2, -3, -4, -5, -6, -7, -M1, and -M2 were isolated from the culture broth of Penicillium sp. SPC-21609 as inhibitors of PDGF binding to its receptor. RP-1551s inhibit the binding of PDGF AA to the extracellular domain of PDGF alpha-receptor with IC50 values ranging from 0.1 to 2 microM without affecting PDGF BB binding to the extracellular domain of PDGF beta-receptor. PDGF binding was not restored after the PDGF alpha-receptor extracellular domain was washed in an attempt to remove the RP-1551-1 bound to the receptor. This result suggests that RP-1551-1 may irreversibly interact with the PDGF alpha-receptor. Since many azaphilone compounds possess high reactivity with an amino group, RP-1551-1 may prevent PDGF AA binding by reacting with amino groups on the alpha-receptor extracellular domain.


Subject(s)
Anti-Bacterial Agents/pharmacology , Benzopyrans/pharmacology , Penicillium/metabolism , Platelet-Derived Growth Factor/metabolism , Receptors, Platelet-Derived Growth Factor/metabolism , Anti-Bacterial Agents/biosynthesis , Anti-Bacterial Agents/isolation & purification , Benzopyrans/isolation & purification , Culture Media , Extracellular Space/metabolism , Fermentation , Humans , Protein Binding , Receptor, Platelet-Derived Growth Factor alpha , Receptor, Platelet-Derived Growth Factor beta , Recombinant Proteins/metabolism , Serum Albumin, Bovine/metabolism
17.
Arterioscler Thromb Vasc Biol ; 19(4): 900-9, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10195916

ABSTRACT

Restenosis remains a significant clinical problem associated with mechanical interventional procedures for arterial revascularization or repair, including coronary angioplasty and stenting. Studies with rodents have established that platelet-derived growth factor (PDGF), a potent chemotactic and mitogenic agent for vascular smooth muscle cells, is a key mediator of lesion formation after vascular injury. To further explore this hypothesis in a more clinically relevant model, neutralizing monoclonal antibodies (mAbs) were used to examine the effect of selective inhibition of alpha or beta PDGF receptor (PDGFR) on neointima formation in nonhuman primates. Carotid arteries were injured by surgical endarterectomy and femoral arteries by balloon catheter dilatation. Immunostaining revealed that both injuries induced cell proliferation and the upregulation of beta PDGFR but not alpha PDGFR. By 7 days after injury, beta PDGFR staining was limited to the luminal region of the media, the small areas of neointima, and the adventitia. Nearly all bromodeoxyuridine-positive cells were found in these regions as well. After 30 days, a concentric neointima that stained strongly for beta PDGFR had formed in the carotid and femoral arteries. Treatment of baboons with anti-beta PDGFR mAb 2A1E2 for 6 days after injury reduced the carotid artery and femoral artery lesion sizes by 37% (P<0.05) and 48% (P<0.005), respectively, when measured at 30 days. Under the same conditions, treatment with anti-alpha PDGFR mAb 2H7C5 had no effect. These findings suggest that PDGF mediates neointima formation through the beta PDGFR, and that antagonism of this pathway may be a promising therapeutic strategy for reducing clinical restenosis.


Subject(s)
Carotid Artery Injuries , Femoral Artery/injuries , Receptors, Platelet-Derived Growth Factor/physiology , Animals , Antibodies, Blocking/pharmacology , Antibodies, Monoclonal/pharmacology , Carotid Arteries/pathology , Catheterization , Cell Division/immunology , Endarterectomy , Femoral Artery/pathology , Male , Papio , Phosphorylation , Receptor, Platelet-Derived Growth Factor alpha , Receptor, Platelet-Derived Growth Factor beta , Receptors, Platelet-Derived Growth Factor/antagonists & inhibitors , Receptors, Platelet-Derived Growth Factor/biosynthesis , Receptors, Platelet-Derived Growth Factor/immunology , Tunica Intima/immunology , Tunica Intima/pathology
18.
Braz J Med Biol Res ; 31(1): 61-7, 1998 Jan.
Article in English | MEDLINE | ID: mdl-9686180

ABSTRACT

The contributions of cytokines to the development and progression of disease in a mouse model of retrovirus-induced immunodeficiency (MAIDS) are controversial. Some studies have indicated at etiologic role for type 2 cytokines, while others have emphasized the importance of type 1 cytokines. We have used mice deficient in expression of IL-4, IL-10, IL-4 and IL-10, IFN-gamma, or ICSBP-a transcriptional protein involved in IFN signaling-to examine their contributions to this disorder. Our results demonstrate that expression of type 2 cytokines is an epiphenomenon of infection and that IFN-gamma is a driving force in disease progression. In addition, exogenously administered IL-12 prevents many manifestations of disease while blocking retrovirus expression. Interruption of the IFN signaling pathways in ICSBP-/- mice blocks induction of MAIDS. Predictably, ICSBP-deficient mice exhibit impaired responses to challenge with several other viruses. This immunodeficiency is associated with impaired production of IFN-gamma and IL-12. Unexpectedly, however, the ICSBP-/- mice also develop a syndrome with many similarities to chronic myelogenous leukemia in humans. The chronic phase of this disease is followed by a fatal blast crisis characterized by clonal expansions of undifferentiated cells. ICSBP is thus an important determinant of hematopoietic growth and differentiation as well as a prominent signaling molecule for IFNs.


Subject(s)
Cytokines/physiology , Murine Acquired Immunodeficiency Syndrome/immunology , Receptors, Cytokine/physiology , Animals , Disease Models, Animal , Mice , Retroviridae Infections
19.
J Exp Med ; 186(9): 1535-46, 1997 Nov 03.
Article in English | MEDLINE | ID: mdl-9348311

ABSTRACT

Mice with a null mutation of the gene encoding interferon consensus sequence-binding protein (ICSBP) develop a chronic myelogenous leukemia-like syndrome and mount impaired responses to certain viral and bacterial infections. To gain a mechanistic understanding of the contributions of ICSBP to humoral and cellular immunity, we characterized the responses of control and ICSBP-/- mice to infection with influenza A (flu) and Leishmania major (L. major). Mice of both genotypes survived infections with flu, but differed markedly in the isotype distribution of antiflu antibodies. In sera of normal mice, immunoglobulin (Ig)G2a antibodies were dominant over IgG1 antibodies, a pattern indicative of a T helper cell type 1 (Th1)-driven response. In sera of ICSBP-/- mice, however, IgG1 antibodies dominated over IgG2a antibodies, a pattern indicative of a Th2-driven response. The dominance of IgG1 and IgE over IgG2a was detected in the sera of uninfected mice as well. A seeming Th2 bias of ICSBP-deficient mice was also uncovered in their inability to control infection with L. major, where resistance is known to be dependent on IL-12 and IFN-gamma as components of a Th1 response. Infected ICSBP-deficient mice developed fulminant, disseminated leishmaniasis as a result of failure to mount a Th1-mediated curative response, although T cells remained capable of secreting IFN-gamma and macrophages of producing nitric oxide. Compromised Th1 differentiation in ICSBP-/- mice could not be attributed to hyporesponsiveness of CD4(+) T cells to interleukin (IL)-12; however, the ability of uninfected and infected ICSBP-deficient mice to produce IL-12 was markedly impaired. This indicates that ICSBP is a deciding factor in Th responses governing humoral and cellular immunity through its role in regulating IL-12 expression.


Subject(s)
Carrier Proteins/immunology , Carrier Proteins/physiology , Interleukin-12/biosynthesis , Repressor Proteins , Transcription Factors/physiology , Animals , Cell Differentiation/immunology , Consensus Sequence/immunology , Disease Susceptibility , Influenza A virus/immunology , Interferon Regulatory Factors , Interferons/physiology , Interleukin-12/deficiency , Interleukin-12/metabolism , Leishmania major/immunology , Leishmaniasis, Cutaneous/genetics , Leishmaniasis, Cutaneous/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Orthomyxoviridae Infections/immunology , Th1 Cells/cytology , Th2 Cells/cytology
20.
Leukemia ; 11 Suppl 3: 167-9, 1997 Apr.
Article in English | MEDLINE | ID: mdl-9209332

ABSTRACT

The mechanisms responsible for development of profound immunodeficiency and extensive lymphoproliferation that characterize infection of different species with retroviruses are only partially understood. In mice, it has been shown the activities of an unusual Gag protein are necessary and sufficient to induce these abnormalities in a syndrome designated mouse AIDS (MAIDS). Current studies suggest that complex, antigen-driven interactions between T cells and B cells result in polyclonal activation of both types of lymphocytes, aberrant cytokine production and late lymphomas.


Subject(s)
Gene Products, gag/immunology , Leukemia Virus, Murine/immunology , Murine Acquired Immunodeficiency Syndrome/immunology , Murine Acquired Immunodeficiency Syndrome/virology , Viral Proteins/immunology , Animals , CD4-Positive T-Lymphocytes/immunology , Cytokines/biosynthesis , Cytokines/deficiency , Cytokines/genetics , Defective Viruses/immunology , Leukemia Virus, Murine/metabolism , Lymphocyte Activation , Mice , Mice, Knockout , Transcription, Genetic
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