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CD26 expression on T cell lines increases SDF-1-alpha-mediated invasion.

Havre PA, Abe M, Urasaki Y, Ohnuma K, Morimoto C, Dang NH - Br. J. Cancer (2009)

Bottom Line: This process was regulated in part by the PI-3K and MEK1 pathways, as indicated by increased phosphorylation of p44/42 MAP kinase and Akt in the presence of SDF-1-alpha and the effect of their respective inhibitors on MMP-9 secretion and in vitro invasion.In addition, CD26-associated enhancement of SDF-1-alpha-induced invasion was decreased when CD45 was inhibited.Our results indicate that the expression of CD26 in T cell lines leads to increased SDF-1-alpha-mediated invasion in an in vitro system and that this is controlled in part by the PI-3K and MEK1 pathways.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology/Oncology, University of Florida, Gainesville, FL 32610, USA.

ABSTRACT

Background: CD26 is a multifunctional membrane-bound glycoprotein that regulates tumour growth in addition to its other activities. Because disease aggressiveness is correlated with CD26 expression in several T-cell malignancies, we decided to investigate the invasiveness of cells expressing different levels of CD26.

Methods: To assess CD26 involvement in cell invasion, we performed in vitro invasion assays with human T cell lines expressing different levels of CD26. These included the parental CD26-positive T-lymphoblast cell line HSB-2 and clones infected with a retrovirus expressing siRNA vectors that either targeted CD26 or encoded a missense siRNA, and the parental CD26-negative T-leukaemia cell line Jurkat and clones expressing CD26. CD26 expression in these cell lines was evaluated by flow cytometry and western immunoblotting. CXCR4 expression, phosphorylation of signalling kinases, and MMP-9 secretion were also evaluated by western immunoblotting, whereas MMP-9 activity and the effect of kinase and CD45 inhibitors on activity were measured by zymography of conditioned media.

Results: The presence of CD26 enhanced stromal-cell-derived factor-1-alpha (SDF-1-alpha)-mediated invasion of T cell lines. This process was regulated in part by the PI-3K and MEK1 pathways, as indicated by increased phosphorylation of p44/42 MAP kinase and Akt in the presence of SDF-1-alpha and the effect of their respective inhibitors on MMP-9 secretion and in vitro invasion. In addition, CD26-associated enhancement of SDF-1-alpha-induced invasion was decreased when CD45 was inhibited.

Conclusions: Our results indicate that the expression of CD26 in T cell lines leads to increased SDF-1-alpha-mediated invasion in an in vitro system and that this is controlled in part by the PI-3K and MEK1 pathways. The data also suggest that CD26 enhancement of invasion may be mediated by CD45, however, more studies are required to confirm this involvement.

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MMP-9 is induced in HSB-2 cells but not in CD26-depleted clones. Cells were incubated with or without SDF-1-α (see Materials and methods). Cells were harvested at 24 h and the conditioned media was run on gels. (A) Equal volumes of conditioned media were loaded on an acrylamide gel containing 0.1% gelatin. Following staining, clear zones corresponding to digested gelatin could be detected. (B) Conditioned media was run on a 7.5% acrylamide gel and then probed with anti-MMP-9 antibody following transfer to nitrocellulose. (C) Inhibitors were added 30 min before the addition of SDF-1-α to both HSB-2 and H1-2 cells. The PI-3K inhibitor, LY294002, was added at a concentration of 12 μ, the MEK1 inhibitor, PD98059, was at 11 μ, and the CD45 inhibitor, N-(9,10-dioxo-9,10-dihydro-phenanthrene-2-yl)-2,2-dimethyl-propionamide, was at 1.5 μ. No SDF (clear bars), SDF and vehicle (solid bars), SDF and MEK1 inhibitor (dots, low density), SDF and CD45 inhibitor (dots, high density), SDF and PI-3K inhibitor (bricks). Gels were scanned and the clear zones quantified after inverting the image. Data depicted are representative of three experiments.
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fig5: MMP-9 is induced in HSB-2 cells but not in CD26-depleted clones. Cells were incubated with or without SDF-1-α (see Materials and methods). Cells were harvested at 24 h and the conditioned media was run on gels. (A) Equal volumes of conditioned media were loaded on an acrylamide gel containing 0.1% gelatin. Following staining, clear zones corresponding to digested gelatin could be detected. (B) Conditioned media was run on a 7.5% acrylamide gel and then probed with anti-MMP-9 antibody following transfer to nitrocellulose. (C) Inhibitors were added 30 min before the addition of SDF-1-α to both HSB-2 and H1-2 cells. The PI-3K inhibitor, LY294002, was added at a concentration of 12 μ, the MEK1 inhibitor, PD98059, was at 11 μ, and the CD45 inhibitor, N-(9,10-dioxo-9,10-dihydro-phenanthrene-2-yl)-2,2-dimethyl-propionamide, was at 1.5 μ. No SDF (clear bars), SDF and vehicle (solid bars), SDF and MEK1 inhibitor (dots, low density), SDF and CD45 inhibitor (dots, high density), SDF and PI-3K inhibitor (bricks). Gels were scanned and the clear zones quantified after inverting the image. Data depicted are representative of three experiments.

Mentions: Because previous studies have reported a link between MMP-9 production and invasion (Kim et al, 2001; Redondo-Munoz et al, 2006), we examined potential MMP-9 induction following a 24 h treatment with SDF-1-α. Conditioned media was analysed by zymography and by western blots. For zymography, clear zones represent digested gelatin present in the gel. MMP-9 was induced in the CD26-expressing parental HSB-2 cells and H1-2 when SDF-1-α was present. In contrast, MMP-9 was not induced in the CD26-depleted clone 2E5, but moderately induced in clones 2F8 and 2G9 as shown by the clear zones (Figure 5A). This pattern parallels the one obtained for invasion where 2E5 showed no increase in invasion in the presence of SDF-1-α, whereas the other two clones, 2F8 and 2G9, showed a moderate increase in invasion (Figure 1A). Western blotting of the conditioned media further confirmed that MMP-9 secretion increased in HSB-2 and H1-2 cells, but did not increase for 2E5 and moderately increased for 2F8 and 2G9 (Figure 5B).


CD26 expression on T cell lines increases SDF-1-alpha-mediated invasion.

Havre PA, Abe M, Urasaki Y, Ohnuma K, Morimoto C, Dang NH - Br. J. Cancer (2009)

MMP-9 is induced in HSB-2 cells but not in CD26-depleted clones. Cells were incubated with or without SDF-1-α (see Materials and methods). Cells were harvested at 24 h and the conditioned media was run on gels. (A) Equal volumes of conditioned media were loaded on an acrylamide gel containing 0.1% gelatin. Following staining, clear zones corresponding to digested gelatin could be detected. (B) Conditioned media was run on a 7.5% acrylamide gel and then probed with anti-MMP-9 antibody following transfer to nitrocellulose. (C) Inhibitors were added 30 min before the addition of SDF-1-α to both HSB-2 and H1-2 cells. The PI-3K inhibitor, LY294002, was added at a concentration of 12 μ, the MEK1 inhibitor, PD98059, was at 11 μ, and the CD45 inhibitor, N-(9,10-dioxo-9,10-dihydro-phenanthrene-2-yl)-2,2-dimethyl-propionamide, was at 1.5 μ. No SDF (clear bars), SDF and vehicle (solid bars), SDF and MEK1 inhibitor (dots, low density), SDF and CD45 inhibitor (dots, high density), SDF and PI-3K inhibitor (bricks). Gels were scanned and the clear zones quantified after inverting the image. Data depicted are representative of three experiments.
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Related In: Results  -  Collection

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fig5: MMP-9 is induced in HSB-2 cells but not in CD26-depleted clones. Cells were incubated with or without SDF-1-α (see Materials and methods). Cells were harvested at 24 h and the conditioned media was run on gels. (A) Equal volumes of conditioned media were loaded on an acrylamide gel containing 0.1% gelatin. Following staining, clear zones corresponding to digested gelatin could be detected. (B) Conditioned media was run on a 7.5% acrylamide gel and then probed with anti-MMP-9 antibody following transfer to nitrocellulose. (C) Inhibitors were added 30 min before the addition of SDF-1-α to both HSB-2 and H1-2 cells. The PI-3K inhibitor, LY294002, was added at a concentration of 12 μ, the MEK1 inhibitor, PD98059, was at 11 μ, and the CD45 inhibitor, N-(9,10-dioxo-9,10-dihydro-phenanthrene-2-yl)-2,2-dimethyl-propionamide, was at 1.5 μ. No SDF (clear bars), SDF and vehicle (solid bars), SDF and MEK1 inhibitor (dots, low density), SDF and CD45 inhibitor (dots, high density), SDF and PI-3K inhibitor (bricks). Gels were scanned and the clear zones quantified after inverting the image. Data depicted are representative of three experiments.
Mentions: Because previous studies have reported a link between MMP-9 production and invasion (Kim et al, 2001; Redondo-Munoz et al, 2006), we examined potential MMP-9 induction following a 24 h treatment with SDF-1-α. Conditioned media was analysed by zymography and by western blots. For zymography, clear zones represent digested gelatin present in the gel. MMP-9 was induced in the CD26-expressing parental HSB-2 cells and H1-2 when SDF-1-α was present. In contrast, MMP-9 was not induced in the CD26-depleted clone 2E5, but moderately induced in clones 2F8 and 2G9 as shown by the clear zones (Figure 5A). This pattern parallels the one obtained for invasion where 2E5 showed no increase in invasion in the presence of SDF-1-α, whereas the other two clones, 2F8 and 2G9, showed a moderate increase in invasion (Figure 1A). Western blotting of the conditioned media further confirmed that MMP-9 secretion increased in HSB-2 and H1-2 cells, but did not increase for 2E5 and moderately increased for 2F8 and 2G9 (Figure 5B).

Bottom Line: This process was regulated in part by the PI-3K and MEK1 pathways, as indicated by increased phosphorylation of p44/42 MAP kinase and Akt in the presence of SDF-1-alpha and the effect of their respective inhibitors on MMP-9 secretion and in vitro invasion.In addition, CD26-associated enhancement of SDF-1-alpha-induced invasion was decreased when CD45 was inhibited.Our results indicate that the expression of CD26 in T cell lines leads to increased SDF-1-alpha-mediated invasion in an in vitro system and that this is controlled in part by the PI-3K and MEK1 pathways.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology/Oncology, University of Florida, Gainesville, FL 32610, USA.

ABSTRACT

Background: CD26 is a multifunctional membrane-bound glycoprotein that regulates tumour growth in addition to its other activities. Because disease aggressiveness is correlated with CD26 expression in several T-cell malignancies, we decided to investigate the invasiveness of cells expressing different levels of CD26.

Methods: To assess CD26 involvement in cell invasion, we performed in vitro invasion assays with human T cell lines expressing different levels of CD26. These included the parental CD26-positive T-lymphoblast cell line HSB-2 and clones infected with a retrovirus expressing siRNA vectors that either targeted CD26 or encoded a missense siRNA, and the parental CD26-negative T-leukaemia cell line Jurkat and clones expressing CD26. CD26 expression in these cell lines was evaluated by flow cytometry and western immunoblotting. CXCR4 expression, phosphorylation of signalling kinases, and MMP-9 secretion were also evaluated by western immunoblotting, whereas MMP-9 activity and the effect of kinase and CD45 inhibitors on activity were measured by zymography of conditioned media.

Results: The presence of CD26 enhanced stromal-cell-derived factor-1-alpha (SDF-1-alpha)-mediated invasion of T cell lines. This process was regulated in part by the PI-3K and MEK1 pathways, as indicated by increased phosphorylation of p44/42 MAP kinase and Akt in the presence of SDF-1-alpha and the effect of their respective inhibitors on MMP-9 secretion and in vitro invasion. In addition, CD26-associated enhancement of SDF-1-alpha-induced invasion was decreased when CD45 was inhibited.

Conclusions: Our results indicate that the expression of CD26 in T cell lines leads to increased SDF-1-alpha-mediated invasion in an in vitro system and that this is controlled in part by the PI-3K and MEK1 pathways. The data also suggest that CD26 enhancement of invasion may be mediated by CD45, however, more studies are required to confirm this involvement.

Show MeSH
Related in: MedlinePlus