The pharmacokinetics and regional tissue distribution of two IgG2b immunoglobulins were studied i... more The pharmacokinetics and regional tissue distribution of two IgG2b immunoglobulins were studied in athymic mice with D54MG human glioma xenografts. Monoclonal antibody (Mab) 81C6, an antiglioma antibody, had a plasma half-life of 2.7 +/- 0.3 (SE) days; 45.6, a control immunoglobulin, had a plasma half-life of 3.3 +/- 0.4 days. The immunoreactive fraction of 81C6 in plasma fell slowly from 0.37 to 0.23 over 9 days. The blood-to-tissue transfer constant (K1) of Mab was 0.11 +/- 0.05 ml/g/h in brain xenografts and 0.07 +/- 0.02 ml/g/h in s.c. xenografts. In contrast, K1 in muscle (0.005 +/- 0.002) and brain (0.0004 +/- 0.0001 ml/g/h) was much lower. The equilibration half-time of Mab in extracellular space was 1.1 +/- 0.2 h in the brain xenografts, 3.6 +/- 1.4 h in s.c. xenografts, and 8.1 and 24 h in muscle and brain, respectively. Distribution and binding of 81C6 was heterogeneous in the xenografts. A binding potential of 5-14 was found centrally and a binding potential of 0.8-1.0 was found peripherally in the brain xenografts. In the s.c. xenografts, the binding potential was higher peripherally than centrally. The exposure of D54MG xenograft tissue to Mab 81C6 was not significantly limited by the permeability of the blood vessels or blood flow due to the long plasma half-life of the immunoglobulin. A comparison of Mab and alpha-aminoisobutyric acid influx constants suggests that Mab entry into intracerebral xenografts occurs through large pores without significant sieving or steric restriction. Under such conditions the differences in influx constants between immunoglobulin and smaller immunoglobulin fragments will be proportional to the differences in their aqueous diffusion constants.
Posttranslational glycosaminoglycan attachment to decorin, a chondroitin/dermatan sulfate proteog... more Posttranslational glycosaminoglycan attachment to decorin, a chondroitin/dermatan sulfate proteoglycan, was studied by expression of a wild-type decorin cDNA and several mutagenized forms in two types of mammalian cells. Transfection of the wild-type cDNA resulted in the synthesis of an authentic chondroitin/dermatan sulfate proteoglycan similar to the decorin molecule synthesized by cultured human fibroblasts. Conversion of the serine residue that serves as the attachment site for the sole glycosaminoglycan chain in decorin to a threonine residue greatly reduced the efficiency of the glycosaminoglycan substitution. Less than 10% of the threonine-mutated core protein acquired a glycosaminoglycan chain, whereas most of the core protein was secreted without such substitution. Expression of cDNA in which an alanine residue had been introduced into the substituted serine position resulted in the secretion of core protein with no detectable glycosaminoglycan. Conversion to alanine of either one of the glycine residues that are adjacent to the substituted serine yielded the proteoglycan form of decorin. These results show that the xylosyltransferase responsible for the initiation of the glycosaminoglycan chain on the core protein can use a threonine residue for this substitution instead of a serine residue, but that such substitution is only partial, creating a "part-time" proteoglycan. Moreover, variations are possible in the sequence context of a glycosaminoglycan-substituted serine residue without loss of glycosaminoglycan substitution. The conformation of the substitution site may therefore be important for xylosyltransferase recognition.
W e have previously reported the sequence of the integrin a9 subunit, a partner of the pl subunit... more W e have previously reported the sequence of the integrin a9 subunit, a partner of the pl subunit that is expressed in basal keratinocytes, hepatocytes, airway epithelial cells, and smooth and skeletal muscle. In the present study, we have stably expressed a9pl on the surface of the human embryonic kidney cell line 293 and the human colon carcinoma cell line SW480 and used these transfected cell lines to identify ligand(s) for this integrin. Transfected cells did not appear to utilize a9pl for attachment to the extracellular matrix proteins fibronectin, laminin, vitronectin, fibrinogen, thrombospondin, or type I or IV collagen. However, in contrast to mock transfectants, both 293 cells and SW480 cells expressing a9pl adhered to intact chicken tenascin. By utilizing a variety of recombinant fragments of tenascin, we were able to localize the binding site for a9p1 to the third type I11 repeat. This repeat contains the arginineglycine-aspartic acid (RGD) tripeptide that has been shown to serve as a binding site in tenascin for w-integrins. However, the RGD site does not appear to be the binding site for a9p1, as the attachment of a9 transfectants to this fragment was not inhibited by RGD peptide, nor by changing the RGD site to RAD or RAA. Integrins are transmembrane glycoproteins, consisting of ap heterodimers, that can serve as receptors for both extracellular matrix and cell surface ligands (1). In addition to mediating cell adhesion, integrins are known to participate in a variety of signaling pathways (2, 3) and to play important roles in fetal development, morphogenesis, cell migration, wound healing, and malignant transformation (4-7). There are at least 8 known integrin p subunits and 15 known a subunits that can combine to form at least 21 distinct receptors. We have described the sequence and tissue distribution of an integrin a subunit, a9, which forms a heterodimer with the p subunit, pl (8). By immunohistochemistry, a9 is expressed in squamous epithelia, in smooth muscle, in skeletal muscle, and in hepatocytes, but no ligand has previously been reported for a9pl. In the present study, we have stably transfected two different cell lines (the immortalized human embryonic kidney cell line 293 and the colon carcinoma cell line SW480) with human a9 Grants HJJA133259, HL47412, and HL25816 (to D. S.), CA53259 and
We have examined genomic sequences and mRNA species hybridizing to a cDNA clone of a yolk sac car... more We have examined genomic sequences and mRNA species hybridizing to a cDNA clone of a yolk sac carcinoma chondroitin sulfate proteoglycan designated PG19. Genomic blot hybridizations with cDNAs covering the majority of the PG19 mRNA sequence revealed 15 to 17 gene fragments. Similar analysis with probes representing either the propeptide or the combined core protein COOH-terminal domain and 3' untranslated sequences revealed single genomic fragments indicating that a single gene codes for the PGl9 proteoglycan. Genomic blot analysis with cDNA sequences coding for the serine-glycine repeat of the core protein identified the same gene fragments observed with the entire PG19 cDNA, indicating that this coding region is homologous with sequences present in multiple genes. The same probes were also used to examine mRNA expression. In addition to the PG19 mRNA, several PG19-related mRNAs could be seen. These PG19-related mRNAs had homology with the serine-glycine coding sequence of the PG19 cDNA. These mRNAs may be coding for proteoglycans. The mRNA coding for PG19 appeared to be uniquely expressed in parietal yolk sac and mast cell lineages. The PG19 mRNA existed in different forms in parietal yolk sac and mast cell lines due to cell-type-specific differences in the length of the 5' untranslated sequences. These results indicate that expression of the PG19 proteoglycan gene is regulated both in terms of cell-type-specific transcription and selection of a transcriptional start site.
In the present study, we evaluated the effects of A4.6.1 (100 μg twice weekly, ip) on growth and ... more In the present study, we evaluated the effects of A4.6.1 (100 μg twice weekly, ip) on growth and angiogenic activity of spheroids of the human prostatic cell line DU 145 (diameter 700 μm at implantation) implanted in dorsal skinfold chambers in nude mice (n = 11). An antibody of ...
The role of tenascin in mediating tumor cell migration was studied using two cell migration model... more The role of tenascin in mediating tumor cell migration was studied using two cell migration models. In migration/invasion Transwell assays U251.3 glioma cells rapidly migrated through the 8 µm pore size membranes onto tenascin-and fibronectin-coated surfaces. In this assay the number of cells migrating onto tenascin was 52.2±9.6% greater than on fibronectin within 4 hours. To assess cell migration rates and cell morphology, U251.3 migration was examined in a two-dimension spheroid outgrowth assay. The radial distance migrated by U251.3 cells from tumor spheroids was found to be 53.8±4.9% greater on tenascin than on fibronectin. Cells migrating on tenascin display a very motile appearance, while cells migrating on fibronectin spread and maintain close intercellular contacts. Cell migration in the presence of integrin blocking antibodies demonstrated that migration on tenascin and fibronectin is mediated by distinct integrins, α2β1 and αvβ5/αvβ3, respectively. Since tenascin is coexpressed in malignant tumor matrices with fibronectin, we assessed the effects of tenascin on U251.3 cell migration mediated by fibronectin. Tenascin was found to provide a positive effect on fibronectin-mediated migration by altering cell morphology and enhancing cell motility. These effects of tenascin on fibronectin-mediated cell migration were inhibited by blocking β1 and α2β1 integrins. The results suggest that tenascin may play a significant role in promoting tumor cell migration and invasiveness by modulating cell responses to normal matrix components.
Adherence of eosinophils to vascular endothelium and their accumulation at sites of allergen chal... more Adherence of eosinophils to vascular endothelium and their accumulation at sites of allergen challenge are hallmarks of allergic inflammation. However, the molecular mechanisms mediating eosinophil adhesion under conditions of blood flow are not well understood. The present studies were performed to identify the receptors on human eosinophils involved in initiating adhesion to activated endothelium at physiologic shear rates in vivo. We have compared the relative contribution of L-selectin, VLA-4 (CD49d), and CD18 integrins in mediating eosinophil adhesion to microvascular endothelial cells in the rabbit mesentery by using intravital video microscopy. Eosinophils were found to roll in venules, but not arterioles, and this rolling could be stimulated by activation of endothelium with IL-1. In contrast to neutrophil rolling, which is predominantly L-selectin-dependent, eosinophil rolling was mediated by L-selectin, and also VLA-4. mAbs to L-selectin and VLA-4 alpha, but not CD18, sign...
Human umbilical vein endothelial cells were found to attach and partially spread on human tenasci... more Human umbilical vein endothelial cells were found to attach and partially spread on human tenascin. The attachment of endothelial cells to tenascin results in elongated cells with interconnecting processes and is distinct from the flattened appearance of endothelial cells on fibronectin, collagen, vitronectin or laminin substrata, suggesting a role for tenascin in modulating cell adhesion and motility. Endothelial attachment to tenascin was partially inhibitable by the SRRGDMS peptide derived from human tenascin and completely inhibitable by anti-integrin antibodies to α2β1 and αvβ3. Endothelial cell attachment to tenascin could be inhibited up to 80% with anti-x2 and anti-β1 monoclonal antibodies P1E6 and P4C10, respectively, and this was associated with a complete loss in cell spreading. In contrast, pretreatment of endothelial cells with the anti-(XVβ3 monoclonal antibody LM609, resulted in a 35% inhibition in cell attachment but did not alter cell spreading. In combination the a...
Knowledge of the stromal microenvironment is crucial for understanding the hematopoietic system. ... more Knowledge of the stromal microenvironment is crucial for understanding the hematopoietic system. We took advantage of an assay that permits analysis of primary stroma-initiating cells (SICs) on the clonal level, and further characterized SICs and the factors that regulate SICs. Stroma formation in this assay is dependent on a high-molecular-weight factor secreted by the stromal cell line AC3.U. Here we show that this factor is identical to macrophage colony-stimulating factor (M-CSF), and that purified M-CSF is sufficient for induction of stroma formation. M-CSF, isolated from the line AC3.U, as well as from L929 cells and COS cells transfected with an expression vector encoding M-CSF, migrated in two peaks as 160- and 650-kD species after gel filtration. These molecular-weight species encompassed all stroma-inducing activity, and both stimulated macrophage colony formation. Affinity chromatography and blocking studies with antibodies specific for M-CSF and c-fms confirmed M-CSF as ...
We have examined genomic sequences and mRNA species hybridizing to a cDNA clone of a yolk sac car... more We have examined genomic sequences and mRNA species hybridizing to a cDNA clone of a yolk sac carcinoma chondroitin sulfate proteoglycan designated PG19. Genomic blot hybridizations with cDNAs covering the majority of the PG19 mRNA sequence revealed 15 to 17 gene fragments. Similar analysis with probes representing either the propeptide or the combined core protein COOH-terminal domain and 3' untranslated sequences revealed single genomic fragments indicating that a single gene codes for the PG19 proteoglycan. Genomic blot analysis with cDNA sequences coding for the serine-glycine repeat of the core protein identified the same gene fragments observed with the entire PG19 cDNA, indicating that this coding region is homologous with sequences present in multiple genes. The same probes were also used to examine mRNA expression. In addition to the PG19 mRNA, several PG19-related mRNAs could be seen. These PG19-related mRNAs had homology with the serine-glycine coding sequence of the ...
Stromal cells play an important role in regulating early hematopoiesis. How stromal cells exert t... more Stromal cells play an important role in regulating early hematopoiesis. How stromal cells exert their different functions and the factors that regulate stromal cells themselves remain to be elucidated definitively, however. We describe here a limiting dilution assay for primary stroma colonies from murine marrow. This system permits a critical analysis of stromal cell function and regulation on the clonal level. We report that stroma formation was dependent on an activity secreted by the long-term cultured stromal line AC-3.U. Differential ultrafiltration of AC-3.U supernatant (SN) suggests that this potentially novel activity is represented by molecules with apparent molecular weights (m.w.) of > 100 < 300 kD and > 300 kD. In contrast to the AC-3.U activity, hydrocortisone (HC) acts as a negative regulator of stroma colony formation. We used the stroma colony assay to analyze potential stromal cell heterogeneity. We found that most, if not all, primary stromal colonies sup...
The yolk sac carcinoma cell line L2 secretes a chondroitinldermatan sulfate proteoglycan that has... more The yolk sac carcinoma cell line L2 secretes a chondroitinldermatan sulfate proteoglycan that has an M , 10,000 core protein and carries an average of 14 glycosaminoglycan chains. The amino acid sequence of the mature core protein has been determined from cloned cDNA (
Proteines hybrides renfermant des proteines represseurs et des sites de liaison de recepteurs sub... more Proteines hybrides renfermant des proteines represseurs et des sites de liaison de recepteurs substitues, des sequences amino acides et ADN codant les proteines hybrides. Methode d'elaboration de ces proteines hybrides.
Stable transfection of U251.3 glioma cells with cDNA encoding MT-MMP-1 resulted in increased cell... more Stable transfection of U251.3 glioma cells with cDNA encoding MT-MMP-1 resulted in increased cell surface expression of MT-MMP-1 and TIMP-2, constitutive activation of MMP-2 proenzyme and increased collagen degradation. In tumor spheroid outgrowth assays, cell migration of MT-MMP-1 transfectants relative to control was enhanced on collagen and decreased on vitronectin and fibronectin. These effects were reversed by TIMP-2 and were not associated with any substantial changes in cell adhesion. Binding of U251.3 cells to the C-terminal domain of MMP-2 was specifically inhibited by anti-(alpha)vss3 integrin blocking antibody indicating that MMP-2 interacts with (alpha)vss3 through the enzyme's C-terminal portion at or near the integrin's matrix adhesion sites. We propose that these mechanisms could govern directed matrix degradation in the tumor cells' microenvironment by sequestration of active MMP-2 on the cell surface. Our data suggest that activation of MMP-2 and its pro...
The pharmacokinetics and regional tissue distribution of two IgG2b immunoglobulins were studied i... more The pharmacokinetics and regional tissue distribution of two IgG2b immunoglobulins were studied in athymic mice with D54MG human glioma xenografts. Monoclonal antibody (Mab) 81C6, an antiglioma antibody, had a plasma half-life of 2.7 +/- 0.3 (SE) days; 45.6, a control immunoglobulin, had a plasma half-life of 3.3 +/- 0.4 days. The immunoreactive fraction of 81C6 in plasma fell slowly from 0.37 to 0.23 over 9 days. The blood-to-tissue transfer constant (K1) of Mab was 0.11 +/- 0.05 ml/g/h in brain xenografts and 0.07 +/- 0.02 ml/g/h in s.c. xenografts. In contrast, K1 in muscle (0.005 +/- 0.002) and brain (0.0004 +/- 0.0001 ml/g/h) was much lower. The equilibration half-time of Mab in extracellular space was 1.1 +/- 0.2 h in the brain xenografts, 3.6 +/- 1.4 h in s.c. xenografts, and 8.1 and 24 h in muscle and brain, respectively. Distribution and binding of 81C6 was heterogeneous in the xenografts. A binding potential of 5-14 was found centrally and a binding potential of 0.8-1.0 was found peripherally in the brain xenografts. In the s.c. xenografts, the binding potential was higher peripherally than centrally. The exposure of D54MG xenograft tissue to Mab 81C6 was not significantly limited by the permeability of the blood vessels or blood flow due to the long plasma half-life of the immunoglobulin. A comparison of Mab and alpha-aminoisobutyric acid influx constants suggests that Mab entry into intracerebral xenografts occurs through large pores without significant sieving or steric restriction. Under such conditions the differences in influx constants between immunoglobulin and smaller immunoglobulin fragments will be proportional to the differences in their aqueous diffusion constants.
Posttranslational glycosaminoglycan attachment to decorin, a chondroitin/dermatan sulfate proteog... more Posttranslational glycosaminoglycan attachment to decorin, a chondroitin/dermatan sulfate proteoglycan, was studied by expression of a wild-type decorin cDNA and several mutagenized forms in two types of mammalian cells. Transfection of the wild-type cDNA resulted in the synthesis of an authentic chondroitin/dermatan sulfate proteoglycan similar to the decorin molecule synthesized by cultured human fibroblasts. Conversion of the serine residue that serves as the attachment site for the sole glycosaminoglycan chain in decorin to a threonine residue greatly reduced the efficiency of the glycosaminoglycan substitution. Less than 10% of the threonine-mutated core protein acquired a glycosaminoglycan chain, whereas most of the core protein was secreted without such substitution. Expression of cDNA in which an alanine residue had been introduced into the substituted serine position resulted in the secretion of core protein with no detectable glycosaminoglycan. Conversion to alanine of either one of the glycine residues that are adjacent to the substituted serine yielded the proteoglycan form of decorin. These results show that the xylosyltransferase responsible for the initiation of the glycosaminoglycan chain on the core protein can use a threonine residue for this substitution instead of a serine residue, but that such substitution is only partial, creating a &quot;part-time&quot; proteoglycan. Moreover, variations are possible in the sequence context of a glycosaminoglycan-substituted serine residue without loss of glycosaminoglycan substitution. The conformation of the substitution site may therefore be important for xylosyltransferase recognition.
W e have previously reported the sequence of the integrin a9 subunit, a partner of the pl subunit... more W e have previously reported the sequence of the integrin a9 subunit, a partner of the pl subunit that is expressed in basal keratinocytes, hepatocytes, airway epithelial cells, and smooth and skeletal muscle. In the present study, we have stably expressed a9pl on the surface of the human embryonic kidney cell line 293 and the human colon carcinoma cell line SW480 and used these transfected cell lines to identify ligand(s) for this integrin. Transfected cells did not appear to utilize a9pl for attachment to the extracellular matrix proteins fibronectin, laminin, vitronectin, fibrinogen, thrombospondin, or type I or IV collagen. However, in contrast to mock transfectants, both 293 cells and SW480 cells expressing a9pl adhered to intact chicken tenascin. By utilizing a variety of recombinant fragments of tenascin, we were able to localize the binding site for a9p1 to the third type I11 repeat. This repeat contains the arginineglycine-aspartic acid (RGD) tripeptide that has been shown to serve as a binding site in tenascin for w-integrins. However, the RGD site does not appear to be the binding site for a9p1, as the attachment of a9 transfectants to this fragment was not inhibited by RGD peptide, nor by changing the RGD site to RAD or RAA. Integrins are transmembrane glycoproteins, consisting of ap heterodimers, that can serve as receptors for both extracellular matrix and cell surface ligands (1). In addition to mediating cell adhesion, integrins are known to participate in a variety of signaling pathways (2, 3) and to play important roles in fetal development, morphogenesis, cell migration, wound healing, and malignant transformation (4-7). There are at least 8 known integrin p subunits and 15 known a subunits that can combine to form at least 21 distinct receptors. We have described the sequence and tissue distribution of an integrin a subunit, a9, which forms a heterodimer with the p subunit, pl (8). By immunohistochemistry, a9 is expressed in squamous epithelia, in smooth muscle, in skeletal muscle, and in hepatocytes, but no ligand has previously been reported for a9pl. In the present study, we have stably transfected two different cell lines (the immortalized human embryonic kidney cell line 293 and the colon carcinoma cell line SW480) with human a9 Grants HJJA133259, HL47412, and HL25816 (to D. S.), CA53259 and
We have examined genomic sequences and mRNA species hybridizing to a cDNA clone of a yolk sac car... more We have examined genomic sequences and mRNA species hybridizing to a cDNA clone of a yolk sac carcinoma chondroitin sulfate proteoglycan designated PG19. Genomic blot hybridizations with cDNAs covering the majority of the PG19 mRNA sequence revealed 15 to 17 gene fragments. Similar analysis with probes representing either the propeptide or the combined core protein COOH-terminal domain and 3' untranslated sequences revealed single genomic fragments indicating that a single gene codes for the PGl9 proteoglycan. Genomic blot analysis with cDNA sequences coding for the serine-glycine repeat of the core protein identified the same gene fragments observed with the entire PG19 cDNA, indicating that this coding region is homologous with sequences present in multiple genes. The same probes were also used to examine mRNA expression. In addition to the PG19 mRNA, several PG19-related mRNAs could be seen. These PG19-related mRNAs had homology with the serine-glycine coding sequence of the PG19 cDNA. These mRNAs may be coding for proteoglycans. The mRNA coding for PG19 appeared to be uniquely expressed in parietal yolk sac and mast cell lineages. The PG19 mRNA existed in different forms in parietal yolk sac and mast cell lines due to cell-type-specific differences in the length of the 5' untranslated sequences. These results indicate that expression of the PG19 proteoglycan gene is regulated both in terms of cell-type-specific transcription and selection of a transcriptional start site.
In the present study, we evaluated the effects of A4.6.1 (100 μg twice weekly, ip) on growth and ... more In the present study, we evaluated the effects of A4.6.1 (100 μg twice weekly, ip) on growth and angiogenic activity of spheroids of the human prostatic cell line DU 145 (diameter 700 μm at implantation) implanted in dorsal skinfold chambers in nude mice (n = 11). An antibody of ...
The role of tenascin in mediating tumor cell migration was studied using two cell migration model... more The role of tenascin in mediating tumor cell migration was studied using two cell migration models. In migration/invasion Transwell assays U251.3 glioma cells rapidly migrated through the 8 µm pore size membranes onto tenascin-and fibronectin-coated surfaces. In this assay the number of cells migrating onto tenascin was 52.2±9.6% greater than on fibronectin within 4 hours. To assess cell migration rates and cell morphology, U251.3 migration was examined in a two-dimension spheroid outgrowth assay. The radial distance migrated by U251.3 cells from tumor spheroids was found to be 53.8±4.9% greater on tenascin than on fibronectin. Cells migrating on tenascin display a very motile appearance, while cells migrating on fibronectin spread and maintain close intercellular contacts. Cell migration in the presence of integrin blocking antibodies demonstrated that migration on tenascin and fibronectin is mediated by distinct integrins, α2β1 and αvβ5/αvβ3, respectively. Since tenascin is coexpressed in malignant tumor matrices with fibronectin, we assessed the effects of tenascin on U251.3 cell migration mediated by fibronectin. Tenascin was found to provide a positive effect on fibronectin-mediated migration by altering cell morphology and enhancing cell motility. These effects of tenascin on fibronectin-mediated cell migration were inhibited by blocking β1 and α2β1 integrins. The results suggest that tenascin may play a significant role in promoting tumor cell migration and invasiveness by modulating cell responses to normal matrix components.
Adherence of eosinophils to vascular endothelium and their accumulation at sites of allergen chal... more Adherence of eosinophils to vascular endothelium and their accumulation at sites of allergen challenge are hallmarks of allergic inflammation. However, the molecular mechanisms mediating eosinophil adhesion under conditions of blood flow are not well understood. The present studies were performed to identify the receptors on human eosinophils involved in initiating adhesion to activated endothelium at physiologic shear rates in vivo. We have compared the relative contribution of L-selectin, VLA-4 (CD49d), and CD18 integrins in mediating eosinophil adhesion to microvascular endothelial cells in the rabbit mesentery by using intravital video microscopy. Eosinophils were found to roll in venules, but not arterioles, and this rolling could be stimulated by activation of endothelium with IL-1. In contrast to neutrophil rolling, which is predominantly L-selectin-dependent, eosinophil rolling was mediated by L-selectin, and also VLA-4. mAbs to L-selectin and VLA-4 alpha, but not CD18, sign...
Human umbilical vein endothelial cells were found to attach and partially spread on human tenasci... more Human umbilical vein endothelial cells were found to attach and partially spread on human tenascin. The attachment of endothelial cells to tenascin results in elongated cells with interconnecting processes and is distinct from the flattened appearance of endothelial cells on fibronectin, collagen, vitronectin or laminin substrata, suggesting a role for tenascin in modulating cell adhesion and motility. Endothelial attachment to tenascin was partially inhibitable by the SRRGDMS peptide derived from human tenascin and completely inhibitable by anti-integrin antibodies to α2β1 and αvβ3. Endothelial cell attachment to tenascin could be inhibited up to 80% with anti-x2 and anti-β1 monoclonal antibodies P1E6 and P4C10, respectively, and this was associated with a complete loss in cell spreading. In contrast, pretreatment of endothelial cells with the anti-(XVβ3 monoclonal antibody LM609, resulted in a 35% inhibition in cell attachment but did not alter cell spreading. In combination the a...
Knowledge of the stromal microenvironment is crucial for understanding the hematopoietic system. ... more Knowledge of the stromal microenvironment is crucial for understanding the hematopoietic system. We took advantage of an assay that permits analysis of primary stroma-initiating cells (SICs) on the clonal level, and further characterized SICs and the factors that regulate SICs. Stroma formation in this assay is dependent on a high-molecular-weight factor secreted by the stromal cell line AC3.U. Here we show that this factor is identical to macrophage colony-stimulating factor (M-CSF), and that purified M-CSF is sufficient for induction of stroma formation. M-CSF, isolated from the line AC3.U, as well as from L929 cells and COS cells transfected with an expression vector encoding M-CSF, migrated in two peaks as 160- and 650-kD species after gel filtration. These molecular-weight species encompassed all stroma-inducing activity, and both stimulated macrophage colony formation. Affinity chromatography and blocking studies with antibodies specific for M-CSF and c-fms confirmed M-CSF as ...
We have examined genomic sequences and mRNA species hybridizing to a cDNA clone of a yolk sac car... more We have examined genomic sequences and mRNA species hybridizing to a cDNA clone of a yolk sac carcinoma chondroitin sulfate proteoglycan designated PG19. Genomic blot hybridizations with cDNAs covering the majority of the PG19 mRNA sequence revealed 15 to 17 gene fragments. Similar analysis with probes representing either the propeptide or the combined core protein COOH-terminal domain and 3' untranslated sequences revealed single genomic fragments indicating that a single gene codes for the PG19 proteoglycan. Genomic blot analysis with cDNA sequences coding for the serine-glycine repeat of the core protein identified the same gene fragments observed with the entire PG19 cDNA, indicating that this coding region is homologous with sequences present in multiple genes. The same probes were also used to examine mRNA expression. In addition to the PG19 mRNA, several PG19-related mRNAs could be seen. These PG19-related mRNAs had homology with the serine-glycine coding sequence of the ...
Stromal cells play an important role in regulating early hematopoiesis. How stromal cells exert t... more Stromal cells play an important role in regulating early hematopoiesis. How stromal cells exert their different functions and the factors that regulate stromal cells themselves remain to be elucidated definitively, however. We describe here a limiting dilution assay for primary stroma colonies from murine marrow. This system permits a critical analysis of stromal cell function and regulation on the clonal level. We report that stroma formation was dependent on an activity secreted by the long-term cultured stromal line AC-3.U. Differential ultrafiltration of AC-3.U supernatant (SN) suggests that this potentially novel activity is represented by molecules with apparent molecular weights (m.w.) of > 100 < 300 kD and > 300 kD. In contrast to the AC-3.U activity, hydrocortisone (HC) acts as a negative regulator of stroma colony formation. We used the stroma colony assay to analyze potential stromal cell heterogeneity. We found that most, if not all, primary stromal colonies sup...
The yolk sac carcinoma cell line L2 secretes a chondroitinldermatan sulfate proteoglycan that has... more The yolk sac carcinoma cell line L2 secretes a chondroitinldermatan sulfate proteoglycan that has an M , 10,000 core protein and carries an average of 14 glycosaminoglycan chains. The amino acid sequence of the mature core protein has been determined from cloned cDNA (
Proteines hybrides renfermant des proteines represseurs et des sites de liaison de recepteurs sub... more Proteines hybrides renfermant des proteines represseurs et des sites de liaison de recepteurs substitues, des sequences amino acides et ADN codant les proteines hybrides. Methode d'elaboration de ces proteines hybrides.
Stable transfection of U251.3 glioma cells with cDNA encoding MT-MMP-1 resulted in increased cell... more Stable transfection of U251.3 glioma cells with cDNA encoding MT-MMP-1 resulted in increased cell surface expression of MT-MMP-1 and TIMP-2, constitutive activation of MMP-2 proenzyme and increased collagen degradation. In tumor spheroid outgrowth assays, cell migration of MT-MMP-1 transfectants relative to control was enhanced on collagen and decreased on vitronectin and fibronectin. These effects were reversed by TIMP-2 and were not associated with any substantial changes in cell adhesion. Binding of U251.3 cells to the C-terminal domain of MMP-2 was specifically inhibited by anti-(alpha)vss3 integrin blocking antibody indicating that MMP-2 interacts with (alpha)vss3 through the enzyme's C-terminal portion at or near the integrin's matrix adhesion sites. We propose that these mechanisms could govern directed matrix degradation in the tumor cells' microenvironment by sequestration of active MMP-2 on the cell surface. Our data suggest that activation of MMP-2 and its pro...
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Papers by Mario Bourdon