RESUMO
OBJECTIVE: To delineate the activities of decorin and biglycan in the progression of post-traumatic osteoarthritis (PTOA). DESIGN: Three-month-old inducible biglycan (BgniKO) and decorin/biglycan compound (Dcn/BgniKO) knockout mice were subjected to the destabilization of the medial meniscus (DMM) surgery to induce PTOA. The OA phenotype was evaluated by assessing joint structure and sulfated glycosaminoglycan (sGAG) staining via histology, surface collagen fibril nanostructure and calcium content via scanning electron microscopy, tissue modulus via atomic force microscopy-nanoindentation, as well as subchondral bone structure and meniscus ossification via micro-computed tomography. Outcomes were compared with previous findings in the inducible decorin (DcniKO) knockout mice. RESULTS: In the DMM model, BgniKO mice developed similar degree of OA as the control (0.44 [-0.18 1.05] difference in modified Mankin score), different from the more severe OA phenotype observed in DcniKO mice (1.38 [0.91 1.85] difference). Dcn/BgniKO mice exhibited similar histological OA phenotype as DcniKO mice (1.51 [0.97 2.04] difference vs control), including aggravated loss of sGAGs, salient surface fibrillation and formation of osteophyte. Meanwhile, Dcn/BgniKO mice showed further cartilage thinning than DcniKO mice, resulting in the exposure of underlying calcified tissues and aberrantly high surface modulus. BgniKO and Dcn/BgniKO mice developed altered subchondral trabecular bone structure in both Sham and DMM groups, while DcniKO and control mice did not. CONCLUSION: In PTOA, decorin plays a more crucial role than biglycan in regulating cartilage degeneration, while biglycan is more important in regulating subchondral bone structure. The two have distinct activities and modest synergy in the pathogenesis of PTOA.
Assuntos
Biglicano/deficiência , Decorina/deficiência , Progressão da Doença , Osteoartrite/patologia , Animais , Biglicano/genética , Osso Esponjoso/patologia , Cartilagem Articular , Decorina/genética , Modelos Animais de Doenças , Meniscos Tibiais/patologia , Camundongos Knockout , Ossificação Heterotópica/patologia , Osteoartrite/genética , Osteófito/patologia , Lesões do Menisco Tibial/patologiaRESUMO
In an effort to develop a new therapy for prostate cancer (PCa) bone metastases, we have created Ad.dcn, a recombinant oncolytic adenovirus carrying the human decorin gene. Infection of PC-3 and DU-145, the human prostate tumor cells, with Ad.dcn or a non-replicating adenovirus Ad(E1-).dcn resulted in decorin expression; Ad.dcn produced high viral titers and cytotoxicity in human prostate tumor cells. Adenoviral-mediated decorin expression inhibited Met, the Wnt/ß-catenin signaling axis, vascular endothelial growth factor A, reduced mitochondrial DNA levels and inhibited tumor cell migration. To examine the antitumor response of Ad.dcn, PC-3-luc cells were inoculated in the left heart ventricle to establish bone metastases in nude mice. Ad.dcn, in conjunction with control replicating and non-replicating vectors were injected via tail vein. The real-time monitoring of mice, once a week, by bioluminescence imaging and X-ray radiography showed that Ad.dcn produced significant inhibition of skeletal metastases. Analyses of the mice at the terminal time point indicated a significant reduction in the tumor burden, osteoclast number, serum tartrate-resistant acid phosphatase 5b levels, osteocalcin levels, hypercalcemia, inhibition of cancer cachexia and an increase in the animal survival. Based on these studies, we believe that Ad.dcn can be developed as a potential new therapy for PCa bone metastasis.
Assuntos
Neoplasias Ósseas/secundário , Neoplasias Ósseas/terapia , Decorina/metabolismo , Terapia Viral Oncolítica , Vírus Oncolíticos/metabolismo , Neoplasias da Próstata/terapia , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Decorina/genética , Decorina/farmacologia , Modelos Animais de Doenças , Técnicas de Transferência de Genes , Humanos , Masculino , Camundongos Nus , Vírus Oncolíticos/genéticaRESUMO
Transformation of hematopoietic cells by the p210bcr/abl tyrosine kinase appears to require the expression of a functional MYC protein, suggesting that simultaneous targeting of BCR-ABL and c-myc might be a rational strategy for attempting treatment of Phil-adelphia leukemia. To test this hypothesis, severe combined immunodeficiency mice injected with Philadelphia leukemic cells were treated systemically with equal doses of bcr-abl or c-myc antisense oligodeoxynucleotides (ODNs) or with both ODNs in combination. Compared with the mice treated with individual agents, the disease process was much slower in the group treated with both ODNs, as revealed by flow cytometry, clonogenic assay, and reverse transcriptase-polymerase chain reaction analysis to detect leukemic cells in mouse tissue cell suspensions, and by enumeration of liver metastases. The retardation of the disease process was positively correlated with a markedly increased survival of leukemic mice treated with both ODNs. These data demonstrate the therapeutic potential of targeting multiple cooperating oncogenes.
Assuntos
Proteínas de Fusão bcr-abl/genética , Genes myc , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Oligonucleotídeos Antissenso/uso terapêutico , Animais , Sequência de Bases , Primers do DNA/química , Expressão Gênica , Masculino , Camundongos , Camundongos SCID , Dados de Sequência Molecular , Metástase Neoplásica , Neprilisina/análise , Oncogenes , RNA Mensageiro/genética , RNA Neoplásico/genética , Células Tumorais CultivadasRESUMO
After 24 h of continuous labeling with radioactive precursors, a high molecular weight heparan sulfate proteoglycan (HS-PG) was isolated from both the medium and cell layer of human colon carcinoma cells (WiDr) in culture. The medium HS-PG eluted from a diethylaminoethyl anion exchange column with 0.45-0.50 M NaCl, had an average density of 1.46-1.49 g/ml on dissociative CsCl density-gradient ultracentrifugation, and eluted from Sepharose CL-2B with a Kav = 0.57. This proteoglycan had an estimated Mr of congruent to 8.5 X 10(5), with glycosaminoglycan chains of Mr = 3 X 10(4) which were all susceptible to HNO2 deaminative cleavage. Deglycosylation of the HS-PG with polyhydrogen fluoride resulted in a 3H-core protein with Mr congruent to 2.4 X 10(5). The cell layer contained a population of HS-PG with characteristics almost identical to that released into the medium but with a larger Mr = 9.5 X 10(5). Furthermore, an intracellular pool contained smaller heparan sulfate chains (Mr congruent to 1 X 10(4)) which were mostly devoid of protein core. In pulse chase experiments, only the large cell-associated HS-PG was released (approximately 58%) into the medium as intact proteoglycan and/or internalized and degraded (approximately 42%), with a t1/2 = 6 h. However, the small intracellular component was never released into the medium and was degraded at a much slower rate. When the cells were subjected to mild proteolytic treatment, only the large cell-associated HS-PG, but none of the small component, was displaced. Addition of exogenous heparin did not displace any HS-PG into the medium. Both light and electron microscopic immunocytochemistry revealed that the cell surface reacted with antibody against an HS-PG isolated from a basement membrane-producing tumor. Electron microscopic histochemistry using ruthenium red and/or cuprolinic blue revealed numerous 10-50-nm diam granules and 70-220-nm-long electron-dense filaments, respectively, on the surface of the tumor cells. The results indicate that colon carcinoma cells synthesize HS-PGs with distinct structural and metabolic characteristics: a large secretory pool with high turnover, which appears to be synthesized as an integral membrane component and localized primarily at the cell surface, and a small nonsecretory pool with low turnover localized predominantly within the cell interior. This culture system offers an opportunity to investigate in detail the mechanisms involved in the regulation of proteoglycan metabolism, and in the establishment of the neoplastic phenotype.
Assuntos
Proteoglicanas de Sulfatos de Condroitina/biossíntese , Neoplasias do Colo/metabolismo , Glicosaminoglicanos/biossíntese , Heparitina Sulfato/biossíntese , Proteoglicanas/biossíntese , Aminoácidos/análise , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Proteoglicanas de Sulfatos de Condroitina/isolamento & purificação , Neoplasias do Colo/ultraestrutura , Glicosaminoglicanos/isolamento & purificação , Proteoglicanas de Heparan Sulfato , Heparina , Heparitina Sulfato/isolamento & purificação , Humanos , Cinética , Microscopia Eletrônica , Peso Molecular , Proteoglicanas/isolamento & purificação , TripsinaRESUMO
Decorin is a member of the expanding group of widely distributed small leucine-rich proteoglycans that are expected to play important functions in tissue assembly. We report that mice harboring a targeted disruption of the decorin gene are viable but have fragile skin with markedly reduced tensile strength. Ultrastructural analysis revealed abnormal collagen morphology in skin and tendon, with coarser and irregular fiber outlines. Quantitative scanning transmission EM of individual collagen fibrils showed abrupt increases and decreases in mass along their axes. thereby accounting for the irregular outlines and size variability observed in cross-sections. The data indicate uncontrolled lateral fusion of collagen fibrils in the decorindeficient mice and provide an explanation for the reduced tensile strength of the skin. These findings demonstrate a fundamental role for decorin in regulating collagen fiber formation in vivo.
Assuntos
Colágeno/metabolismo , Proteoglicanas/metabolismo , Dermatopatias/metabolismo , Animais , Decorina , Proteínas da Matriz Extracelular , Feminino , Homozigoto , Masculino , Camundongos , Camundongos Knockout , Proteoglicanas/genética , Pele/metabolismo , Pele/patologia , Dermatopatias/genética , Dermatopatias/patologiaRESUMO
Thrombospondin (TSP) 2, and its close relative TSP1, are extracellular proteins whose functions are complex, poorly understood, and controversial. In an attempt to determine the function of TSP2, we disrupted the Thbs2 gene by homologous recombination in embryonic stem cells, and generated TSP2-null mice by blastocyst injection and appropriate breeding of mutant animals. Thbs2-/- mice were produced with the expected Mendelian frequency, appeared overtly normal, and were fertile. However, on closer examination, these mice displayed a wide variety of abnormalities. Collagen fiber patterns in skin were disordered, and abnormally large fibrils with irregular contours were observed by electron microscopy in both skin and tendon. As a functional correlate of these findings, the skin was fragile and had reduced tensile strength, and the tail was unusually flexible. Mutant skin fibroblasts were defective in attachment to a substratum. An increase in total density and in cortical thickness of long bones was documented by histology and quantitative computer tomography. Mutant mice also manifested an abnormal bleeding time, and histologic surveys of mouse tissues, stained with an antibody to von Willebrand factor, showed a significant increase in blood vessels. The basis for the unusual phenotype of the TSP2-null mouse could derive from the structural role that TSP2 might play in collagen fibrillogenesis in skin and tendon. However, it seems likely that some of the diverse manifestations of this genetic disorder result from the ability of TSP2 to modulate the cell surface properties of mesenchymal cells, and thus, to affect cell functions such as adhesion and migration.
Assuntos
Moléculas de Adesão Celular/fisiologia , Colágeno/fisiologia , Tecido Conjuntivo/anormalidades , Transtornos Hemorrágicos/complicações , Trombospondinas/deficiência , Animais , Densidade Óssea , Adesão Celular , Camundongos , Camundongos Knockout , Fenótipo , Cauda/anormalidades , Tendões/anormalidades , Trombospondinas/fisiologiaRESUMO
Leucine-rich repeats and immunoglobulin-like domains-1 (LRIG1) is a transmembrane protein with an ectodomain containing 15 leucine-rich repeats (LRRs) homologous to mammalian decorin and the Drosophila kekkon1 gene. In this study, we demonstrate that a soluble ectodomain of LRIG1, containing only the LRRs, inhibits ligand-independent epidermal growth factor receptor (EGFR) activation and causes growth inhibition of A431, HeLa and MDA-468 carcinoma cells. In contrast, cells that do not express detectable levels of EGFR fail to respond to soluble LRIG1. However, when a functional EGFR gene is introduced in these cells, they become growth-inhibited by soluble LRIG1 protein. Furthermore, we demonstrate the existence of high-affinity (K(d)=10 nM) binding sites on the A431 cells that can be competitively displaced (up to 75%) by molar excess of EGF. Even more powerful effects are obtained with a chimeric proteoglycan harboring the N-terminus of decorin, substituted with a single glycosaminoglycan chain, fused to the LRIG1 ectodomain. Both proteins also inhibit ligand-dependent activation of the EGFR and extracellular signal-regulated protein kinase 1/2 signaling in a rapid and dose-dependent manner. These results suggest a novel mechanism of action evoked by a soluble ectodomain of LRIG1 protein that could modulate EGFR signaling and its growth-promoting activity. Attenuation of EGFR activity without physical downregulation of the receptor could represent a novel therapeutic approach toward malignancies in which EGFR plays a primary role in tumor growth and survival.
Assuntos
Receptores ErbB/metabolismo , Regulação da Expressão Gênica/fisiologia , Glicoproteínas de Membrana/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Proliferação de Células , Cricetinae , Cricetulus , Decorina , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Genes Dominantes , Células HeLa , Humanos , Ligantes , Dados de Sequência Molecular , Ligação Proteica , Proteoglicanas/genética , Proteoglicanas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/patologiaRESUMO
Epidermolysis bullosa (EB) in a group of genodermatoses characterized by the fragility of skin. Previous studies on the dystrophic (scarring) forms of EB have suggested abnormalities in anchoring fibrils, morphologically recognizable attachment structures that provide stability to the association of the cutaneous basement membrane to the underlying dermis. Since type VII collagen is the major component of the anchoring fibrils, we examined the genetic linkage of dominant dystrophic EB (EBDD) and the type VII collagen gene (COL7A1) locus, which we have recently mapped to chromosome 3p, in three large kindreds with abnormal anchoring fibrils. Strong genetic linkage of EBDD and COL7A1 loci was demonstrated with the maximum logarithm of odds (LOD) score of 8.77 at theta = 0. This linkage was further confirmed with two additional markers in this region of the short arm of chromosome 3, and these analyses allowed further refinement of the map locus of COL7A1. Since there were no recombinants between the COL7A1 and EBDD loci, our findings suggest that type VII collagen is the candidate gene that may harbor the mutations responsible for the EB phenotype in these three families.
Assuntos
Colágeno/genética , Epidermólise Bolhosa Distrófica/genética , Ligação Genética , Mapeamento Cromossômico , Genes Dominantes , Marcadores Genéticos , Humanos , Mutação , Polimorfismo de Fragmento de RestriçãoRESUMO
Decorin belongs to a family of secreted, small, leucine-rich proteoglycans that affect matrix assembly and cellular growth. Ectopic expression of decorin proteoglycan or protein core as a mutated form lacking any glycosaminoglycan side chains induced growth suppression in neoplastic cells of various histogenetic origins, including tumor cells derived from gastrointestinal, genital, skeletal, cutaneous, or bone marrow tissues. Exogenously added recombinant decorin also suppressed overall growth of the parental cell lines. In all stably-transfected clones, growth retardation was specifically associated with induction of the potent cyclin-dependent kinase inhibitor p21, but not p27, and subsequent translocation of p21 protein into the nuclei of decorin-expressing cells. This led to a greater proportion of the cells arrested in G1 phase of the cell cycle. These changes were independent of functional p53 or retinoblastoma protein. De novo expression of decorin in HCT116 human colon carcinoma cells harboring a disrupted p21 gene failed to induce growth suppression, in contrast to the wild-type cells in which p21 and growth arrest could be induced. These findings indicate that ectopic production of decorin protein core can retard the growth of a variety of tumor cells and that endogenous p21 is a required downstream effector of this biological axis.
Assuntos
Proteínas de Ciclo Celular , Divisão Celular/fisiologia , Ciclinas/biossíntese , Inibidores Enzimáticos/metabolismo , Proteoglicanas/biossíntese , Proteoglicanas/farmacologia , Proteínas Supressoras de Tumor , Animais , Células CHO , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Cricetinae , Inibidor de Quinase Dependente de Ciclina p21 , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/antagonistas & inibidores , Citomegalovirus , Decorina , Proteínas da Matriz Extracelular , Vetores Genéticos , Células HeLa , Humanos , Cinética , Pulmão , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/farmacologia , Pele , Transfecção , Células Tumorais CultivadasRESUMO
Distinct genetic abnormalities (loss-of-function mutations of APC and p53 and oncogenic activation of Ki-ras) are associated with specific stages of the sporadic, most common types of colorectal tumors. However, the inability to maintain primary colon epithelial cells in culture has hindered the analysis of the pathogenetic role of these abnormalities in colorectal tumorigenesis. We have now established primary cultures of epithelial cells from the colon crypts of p53-deficient mice; these cells are nontumorigenic as indicated by their failure to form colonies in soft agar and to grow as tumors in immunodeficient SCID mice and in immunocompetent syngeneic hosts. Upon ectopic expression of an activated Ki-ras gene, p53-deficient colon epithelial cells form colonies in soft agar and highly invasive subcutaneous tumors in both immunodeficient and immunocompetent mice. Ectopic expression of wild-type p53, but not of a DNA-binding-deficient mutant, markedly suppressed the colony-forming ability of the Ki-ras-transformed p53-deficient epithelial cells. Together, these findings establish a functional synergism in colorectal tumorigenesis dependent on the effects of an oncogenic Ki-ras in a p53-deficient background. This model of tumorigenic conversion of colon epithelial cells might be useful to identify genetic changes associated with disease progression and to evaluate the therapeutic response to conventional and novel anticancer drugs.
Assuntos
Neoplasias do Colo/genética , Genes p53 , Genes ras , Animais , Sequência de Bases , Transformação Celular Neoplásica/genética , Colo/metabolismo , Colo/patologia , Neoplasias do Colo/etiologia , Neoplasias do Colo/patologia , Neoplasias Colorretais/etiologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Primers do DNA/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Knockout , Camundongos SCID , Microscopia Eletrônica , Retroviridae/genética , Transfecção , Ensaio Tumoral de Célula-TroncoRESUMO
A hallmark of systemic sclerosis (SSc) is the development of tissue fibrosis. Excessive production of several connective tissue components normally present in the dermis, including type I, III, V, and VI collagens as well as fibronectin and proteoglycans, is a consistent finding in the skin of SSc patients. Type VII collagen is a major constituent of anchoring fibrils, present in the skin at the dermal-epidermal basement membrane zone. TGF-beta has been shown to upregulate the expression of the type VII collagen gene. In this study, we assessed the expression of type VII collagen and TGF-beta in the skin of patients with SSc. Indirect immunofluorescence showed an abundance of type VII collagen in the patients' skin, including the dermis. Ultrastructural analysis of SSc skin revealed an abundance of fibrillar material, possibly representing type VII collagen. The increased expression of type VII collagen epitopes was accompanied by the elevated expression of immunodetectable TGF-beta 1 and TGF-beta 2. Dermal fibroblasts cultured from the affected individuals showed a statistically significant (P < 0.02) increase in the expression of type VII collagen at the mRNA level, as detected by reverse transcription-PCR with a mutated cDNA as an internal standard, and increased deposition of the protein as assessed by indirect immunofluorescence. Thus, type VII collagen is abundantly present in SSc patients' dermis, a location not characteristic of its normal distribution, and its aberrant expression may relate to the presence of TGF-beta in the same topographic distribution. The presence of type VII collagen in the dermis may contribute to the tightly bound and indurated appearance of the affected skin in SSc patients.
Assuntos
Colágeno/biossíntese , Escleroderma Sistêmico/metabolismo , Pele/metabolismo , Fator de Crescimento Transformador beta/fisiologia , Adulto , Idoso , Sequência de Bases , Células Cultivadas , Colágeno/genética , Epitopos , Feminino , Imunofluorescência , Humanos , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Dados de Sequência Molecular , RNA Mensageiro/análise , Pele/ultraestrutura , Fator de Crescimento Transformador beta/análise , Fator de Crescimento Transformador beta/imunologiaRESUMO
Decorin, a small leucine-rich proteoglycan, is capable of suppressing the growth of various tumor cell lines when expressed ectopically. In this report, we investigated the biochemical mechanism by which decorin inhibits cell cycle progression. In A431 squamous carcinoma cells, decorin proteoglycan or protein core induced a marked growth suppression, when either exogenously added or endogenously produced by a transgene. Decorin caused rapid phosphorylation of the EGF receptor and a concurrent activation of mitogen-activated protein (MAP) kinase signal pathway. This led to a protracted induction of endogenous p21, a potent inhibitor of cyclin-dependent kinases, and ultimate cell cycle arrest. Biglycan, a related proteoglycan, had no effect. Moreover, decorin activated the EGF receptor/MAP kinase/ p21 axis in cell lines of various histogenetic backgrounds. These results provide the first evidence that EGF and decorin converge functionally to regulate the cell cycle through activation of a common pathway which ultimately leads to growth suppression.
Assuntos
Carcinoma de Células Escamosas/patologia , Receptores ErbB/fisiologia , Proteoglicanas/fisiologia , Tirfostinas , Células 3T3 , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/biossíntese , Decorina , Ativação Enzimática , Proteínas da Matriz Extracelular , Humanos , Camundongos , Nitrilas/farmacologia , Fosforilação , Quinazolinas/farmacologia , Células Tumorais CultivadasRESUMO
Perlecan, a ubiquitous heparan sulfate proteoglycan, possesses angiogenic and growth-promoting attributes primarily by acting as a coreceptor for basic fibroblast growth factor (FGF-2). In this report we blocked perlecan expression by using either constitutive CMV-driven or doxycycline- inducible antisense constructs. Growth of colon carcinoma cells was markedly attenuated upon obliteration of perlecan gene expression and these effects correlated with reduced responsiveness to and affinity for mitogenic keratinocyte growth factor (FGF-7). Exogenous perlecan effectively reconstituted the activity of FGF-7 in the perlecan-deficient cells. Moreover, soluble FGF-7 specifically bound immobilized perlecan in a heparan sulfate-independent manner. In both tumor xenografts induced by human colon carcinoma cells and tumor allografts induced by highly invasive mouse melanoma cells, perlecan suppression caused substantial inhibition of tumor growth and neovascularization. Thus, perlecan is a potent inducer of tumor growth and angiogenesis in vivo and therapeutic interventions targeting this key modulator of tumor progression may improve cancer treatment.
Assuntos
DNA Antissenso/uso terapêutico , Fatores de Crescimento de Fibroblastos , Proteoglicanas de Heparan Sulfato , Heparitina Sulfato/genética , Neoplasias Experimentais/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Proteoglicanas/genética , Animais , Carcinoma/tratamento farmacológico , Neoplasias do Colo/tratamento farmacológico , Fator 10 de Crescimento de Fibroblastos , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 7 de Crescimento de Fibroblastos , Expressão Gênica , Substâncias de Crescimento/metabolismo , Heparitina Sulfato/biossíntese , Heparitina Sulfato/metabolismo , Humanos , Melanoma Experimental/tratamento farmacológico , Camundongos , Transplante de Neoplasias , Ligação Proteica , Proteoglicanas/biossínteseRESUMO
Microbial adhesion to the host tissue represents an early, critical step in the pathogenesis of most infectious diseases. BORRELIA: burgdorferi, the causative agent of Lyme disease (LD), expresses two surface-exposed decorin-binding adhesins, DbpA and DbpB. A decorin-deficient (Dcn(-/-)) mouse was recently developed and found to have a relatively mild phenotype. We have now examined the process of experimental LD in Dcn(-/-) mice using both needle inoculation and tick transmission of spirochetes. When exposed to low doses of the infective agent, Dcn(-/-) mice had fewer Borrelia-positive cultures from most tissues analyzed than did Dcn(+/+) or Dcn(+/-) mice. When the infection dose was increased, similar differences were not observed in most tissues but were seen in bacterial colonization of joints and the extent of Borreila-induced arthritis. Quantitative PCR demonstrated that joints harvested from Dcn(-/-) mice had diminished Borrelia numbers compared with issues harvested from Dcn(+/+) controls. Histological examination also revealed a low incidence and severity of arthritis in Dcn(-/-) mice. Conversely, no differences in the numbers of Borreila-positive skin cultures were observed among the different genotypes regardless of the infection dose. These differences, which were observed regardless of genetic background of the mice (BALB/c or C3H/HeN) or method of infection, demonstrate the importance of decorin in the pathogenesis of LD.
Assuntos
Grupo Borrelia Burgdorferi/patogenicidade , Proteoglicanas/fisiologia , Animais , Grupo Borrelia Burgdorferi/imunologia , Decorina , Modelos Animais de Doenças , Proteínas da Matriz Extracelular , Feminino , Imunidade Inata , Ixodes , Doença de Lyme/imunologia , Doença de Lyme/microbiologia , Doença de Lyme/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Knockout , Proteoglicanas/genética , Proteoglicanas/imunologiaRESUMO
Cell-surface heparan sulfate proteoglycans have been shown to participate in lipoprotein catabolism, but the roles of specific proteoglycan classes have not been examined previously. Here, we studied the involvement of the syndecan proteoglycan family. First, transfection of CHO cells with expression vectors for several syndecan core proteins produced parallel increases in the cell association and degradation of lipoproteins enriched in lipoprotein lipase, a heparan-binding protein. Second, a chimeric construct, FcR-Synd1, that consists of the ectodomain of the IgG Fc receptor Ia linked to the highly conserved transmembrane and cytoplasmic domains of syndecan-1 directly mediated efficient internalization, in a process triggered by ligand clustering. Third, internalization of lipase-enriched lipoproteins via syndecan-1 and of clustered IgGs via the chimera showed identical kinetics (t1/2 = 1 h) and identical dose-response sensitivities to cytochalasin B, which disrupts microfilaments, and to genistein, which inhibits tyrosine kinases. In contrast, internalization of the receptor-associated protein, which proceeds via coated pits, showed a t1/2 < 15 min, limited sensitivity to cytochalasin B, and complete insensitivity to genistein. Thus, syndecan proteoglycans can directly mediate ligand catabolism through a pathway with characteristics distinct from coated pits, and might act as receptors for atherogenic lipoproteins and other ligands in vivo.
Assuntos
Lipoproteínas LDL/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteoglicanas/metabolismo , Animais , Células CHO , Cloroquina/farmacologia , Cricetinae , Citocalasina B/farmacologia , Relação Dose-Resposta a Droga , Genisteína/farmacologia , Heparina/farmacologia , Humanos , Lipase Lipoproteica/metabolismo , Lipoproteínas LDL/farmacocinética , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Glicoproteínas de Membrana/genética , Proteoglicanas/genética , Ratos , Receptores de IgG/genética , Receptores de IgG/metabolismo , Receptores Imunológicos/fisiologia , Receptores de LDL/fisiologia , Proteínas Recombinantes de Fusão/farmacologia , Sindecana-1 , Sindecanas , Trombospondinas/farmacologia , TransfecçãoRESUMO
Heparan sulfate proteoglycans (HSPG) play a critical role in the formation of distinct fibroblast growth factor (FGF)-HS complexes, augmenting high-affinity binding and receptor activation. Perlecan, a secreted HSPG abundant in proliferating cells, is capable of inducing FGF-receptor interactions in vitro and angiogenesis in vivo. Stable and specific reduction of perlecan levels in mouse NIH 3T3 fibroblasts and human metastatic melanoma cells has been achieved by expression of antisense cDNA corresponding to the N-terminal and HS attachment domains of perlecan. Long-term perlecan downregulation is evidenced by reduced levels of perlecan mRNA and core protein as indicated by Northern blot analysis, immunoblots, and immunohistochemistry, using DNA probes and antibodies specific to mouse or human perlecan. The response of antisense perlecan-expressing cells to increasing concentrations of basic FGF (bFGF) is dramatically reduced in comparison to that in wild-type or vector-transfected cells, as measured by thymidine incorporation and rate of proliferation. Furthermore, receptor binding and affinity labeling of antisense perlecan-transfected cells with 125I-bFGF is markedly inhibited, indicating that eliminating perlecan expression results in reduced high-affinity bFGF binding. Both the binding and mitogenic response of antisense-perlecan-expressing clones to bFGF can be rescued by exogenous heparin or perlecan. These results support the notion that perlecan is a major accessory receptor for bFGF in mouse fibroblasts and human melanomas and point to the possible use of perlecan antisense constructs as specific modulators of bFGF-mediated responses.
Assuntos
DNA Antissenso/farmacologia , Fator 2 de Crescimento de Fibroblastos/antagonistas & inibidores , Proteoglicanas de Heparan Sulfato , Heparitina Sulfato/genética , Proteoglicanas/genética , Células 3T3 , Animais , Divisão Celular/efeitos dos fármacos , DNA Antissenso/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Expressão Gênica , Heparina/farmacologia , Heparitina Sulfato/metabolismo , Humanos , Camundongos , Proteoglicanas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
BACKGROUND: Philadelphia cells are human chronic myelogenous leukemia (CML) cells that contain the BCR/ABL oncogene (a fusion of the BCR and ABL genes). Selective eradication of these cells in vitro can be achieved by combined treatment with antisense phosphorothioate oligodeoxynucleotides ([S]ODNs) specifically targeted to this oncogene (bcr/abl [S]ODNs) and a suboptimal (for use as a single agent) dose of mafosfamide (the in vitro active form of cyclophosphamide). PURPOSE: We evaluated the ability of bcr/abl antisense [S]ODNs, alone or subsequent to treatment with a single injection of cyclophosphamide, to suppress the leukemic process induced in severe combined immunodeficient (SCID) mice by Philadelphia cells (i.e., primary CML-blast crisis [CML-BC] cells). In addition, we studied potential mechanisms that might explain the efficacy of the bcr/abl antisense [S]ODN-mafosfamide combination against Philadelphia cells in vitro. METHODS: The effects of treating leukemic mice with cyclophosphamide (25 mg/kg body weight; 25% of the dose required to eradicate evidence of leukemia in SCID mice) and/or bcr/abl antisense [S]ODNs were assessed by analysis of survival, by examination of bone marrow for the presence of leukemia cells (using a colony formation assay or using coupled reverse transcription and the polymerase chain reaction to screen for bcr/abl messenger RNA), and by examination of a variety of tissues for the presence of infiltrating leukemia cells. The induction of apoptosis (a cell death program) in vitro in primary CML-BC cells following treatment with bcr/abl antisense [S]ODNs plus or minus prior treatment with mafosfamide was monitored by use of a commercial assay. Relative cellular uptake of [S]ODNs by CML-BC cells treated in vitro with or without prior treatment with mafosfamide was determined by use of confocal microscopy and flow cytometry (for fluorescent [S]ODNs) or by use of blotting techniques that employed radioactively labeled probes (for extracted, unlabeled [S]ODNs). Levels of specific proteins in treated and untreated cells were determined by use of western blotting methods. Reported P values are two-sided. RESULTS: The disease process in leukemic mice was retarded substantially by combination treatment with cyclophosphamide and specific bcr/abl antisense [S]ODNs (P < .001, relative to treatment with specific antisense [S]ODNs alone, cyclophosphamide alone, or cyclophosphamide plus nonspecific [i.e., control] antisense [S]ODNs); 50% of the mice treated with cyclophosphamide and specific antisense [S]ODNs appeared to be cured of leukemia. The combination treatment was associated with increased induction of apoptosis. In addition, cellular uptake of bcr/abl antisense [S]ODNs appeared to be increased twofold to sixfold by prior treatment with mafosfamide. This increased uptake of [S]ODNs was associated with enhanced suppression of p210bcr/abl protein levels. CONCLUSIONS AND IMPLICATIONS: Combination therapy with antisense [S]ODNs targeted to specific oncogenes and less toxic doses of anticancer drugs may represent a rational strategy to purpose for the treatment of human leukemias.
Assuntos
Antineoplásicos/uso terapêutico , Ciclofosfamida/análogos & derivados , Proteínas de Fusão bcr-abl/efeitos dos fármacos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Oligonucleotídeos Antissenso/uso terapêutico , Cromossomo Filadélfia , Tionucleotídeos/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Medula Óssea/efeitos dos fármacos , Células da Medula Óssea , Ciclofosfamida/uso terapêutico , Sondas de DNA , Citometria de Fluxo , Proteínas de Fusão bcr-abl/biossíntese , Proteínas de Fusão bcr-abl/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Camundongos , Camundongos SCID , Reação em Cadeia da Polimerase , Análise de Sobrevida , Resultado do Tratamento , Células Tumorais CultivadasRESUMO
Proteoglycans are among the most complex and sophisticated molecules of mammalian systems in terms of their protein and carbohydrate moieties. These macromolecules are in a continuous interplay with each other and the cell surface signal-transducing pathways, some of which are beginning to be elucidated. Because of their domain structure, catalytic potential, and diversity, these molecules appear to be designed for integrating numerous signaling events. For example, some proteoglycans interact with hyaluronan and lectins, thereby linking cell surfaces and distant matrix molecules. Some interact with collagen during the complex process of fibrillogenesis and regulate this biological process fundamental to animal life. Others interact with growth factors and serve as depot available during growth or tissue remodeling. In this review, we center on the most recent developments of proteoglycan biology, focusing primarily on genomic organization and transcriptional and posttranscriptional control. We discuss only those proteoglycans whose gene and promoter elements have been characterized and proved to be functional. When possible, we correlate the effects of growth factors and cytokines on proteoglycan gene expression with the topology of cis-acting elements in their genomic control regions. The analysis leads to a comprehensive critical appraisal of the principles that underlie the regulation of proteoglycan gene expression and to the delineation of common regulatory mechanisms.
Assuntos
Regulação da Expressão Gênica , Proteoglicanas/genética , Processamento Pós-Transcricional do RNA , Transcrição Gênica , Animais , Sequência de Bases , DNA , HumanosRESUMO
Previous studies have shown that the invasion of V2 carcinoma cells in the rabbit mesentery is associated with marked extracellular matrix synthesis leading eventually to an overall increase in mesenteric mass. The purpose of the present study was to investigate the structural and biochemical composition of the extracellular matrix in tumor-free parts of rabbit mesenteries at various stages after intraperitoneal implantation of V2 carcinoma cells. The overall thickness of the tumor-implanted mesenteries increased progressively and peaked at about Day 14, when it was about 8 times greater than the untreated or liver-implanted controls. This was mainly the result of an accumulation of extracellular matrix components. In particular, there was a marked increase in both collagen fibers and proteoglycan granules, as well as filaments, probably hyaluronic acid, as visualized by ruthenium hexammine trichloride. Stereological analysis showed a 6-fold increase in collagen fibers and a significant increase in the density and average diameter of proteoglycan granules. Biochemical analysis revealed a marked elevation in uronic acid content in the tumor-implanted mesenteries. Specifically, they contained 2.6 and 8.6 times the amount of hyaluronic acid and chondroitin sulfate, respectively, than did controls. Furthermore, the relative percentage of chondroitin sulfate was elevated markedly (26 versus 6% in controls). However, the content of heparan or dermatan sulfate did not vary significantly. Stereological analysis of the fibroblasts showed that their absolute number had doubled and that the cell volume of the individual fibroblast had increased markedly. This suggests that the fibroblasts were responsible for the excessive production of the extracellular matrix. These results support the concept that carcinoma cells can modulate their surrounding extracellular environment by stimulating the synthesis of connective tissue in the host mesenchymal cells.
Assuntos
Carcinoma/metabolismo , Matriz Extracelular/metabolismo , Proteoglicanas/análise , Animais , Movimento Celular , Colágeno/análise , Glicosaminoglicanos/análise , Ácido Hialurônico/análise , Mesentério/análise , Mesentério/patologia , CoelhosRESUMO
The Wnt-5A gene codes for a secreted cysteine-rich growth factor that mediates cell to cell signaling via a paracrine mechanism during development and ontogeny. We have recently determine the genomic organization and chromosomal mapping of the human Wnt-5A, and observed distinct patterns of expression in developing human embryos. In this report, we have performed a detailed expression analysis of 100 adult human tissues and tumors and 10 human cell lines. Our data show a widespread expression of Wnt-5A in adult tissues and cells, and aberrant mRNA levels in lungs, breast, and prostate carcinomas and in melanomas. The up-regulation of Wnt-5A in human malignancy was not due to either gene rearrangement or amplification. These findings document an abnormal expression of this growth factor in malignancy and implicate Wnt-5A in the genesis of human cancer.