Limits...
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.

Show MeSH

Related in: MedlinePlus

SDF-1-α-mediated invasion is higher in HSB-2 cells than in CD26-depleted clones. (A) Cell-surface expression of CD26 on HSB-2 cell lines. (a) HSB-2 parental (b) H1-2 (c) 2E5 (d) 2F8 (e) 2G9. Left: cells incubated with PE-labeled isotypic control IgG. Right: cells incubated with PE-labeled anti-CD26 antibody. (B) Expression of CD26 in whole-cell extracts. Equal amounts of protein were run in each lane of a 7.5% gel, transferred to nitrocellulose, and probed with anti-CD26 antibody. Lanes contained HSB-2 parental cells, H1-2, and four clones depleted of CD26: 2E5, 2F8, 2F10, and 2G9. Note that CD26 runs as a dimer (see Materials and Methods). The multiple bands are due to differences in glycosylation. (C) Invasion assay (details in Materials and Methods). Assays were carried out for 24 h, at which time the cells were counted in all wells. Percentage invasion is the number of cells which passed through the transwell divided by the total cell number. Error bars are shown for the standard error of the mean.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2743358&req=5

fig1: SDF-1-α-mediated invasion is higher in HSB-2 cells than in CD26-depleted clones. (A) Cell-surface expression of CD26 on HSB-2 cell lines. (a) HSB-2 parental (b) H1-2 (c) 2E5 (d) 2F8 (e) 2G9. Left: cells incubated with PE-labeled isotypic control IgG. Right: cells incubated with PE-labeled anti-CD26 antibody. (B) Expression of CD26 in whole-cell extracts. Equal amounts of protein were run in each lane of a 7.5% gel, transferred to nitrocellulose, and probed with anti-CD26 antibody. Lanes contained HSB-2 parental cells, H1-2, and four clones depleted of CD26: 2E5, 2F8, 2F10, and 2G9. Note that CD26 runs as a dimer (see Materials and Methods). The multiple bands are due to differences in glycosylation. (C) Invasion assay (details in Materials and Methods). Assays were carried out for 24 h, at which time the cells were counted in all wells. Percentage invasion is the number of cells which passed through the transwell divided by the total cell number. Error bars are shown for the standard error of the mean.

Mentions: HSB-2 cell lines were evaluated for CD26 surface expression by flow cytometry as described in Materials and methods. HSB-2 cells and cells infected with missense siRNA, H1-2, expressed CD26 (Figure 1A). In contrast, CD26 expression was undetectable for the three clones 2E5, 2F8, and 2G9. To confirm CD26 expression, we ran whole-cell lysates on SDS gels, transferred to nitrocellulose, and probed with an anti-CD26 antibody. Both HSB-2 and H1-2 expressed CD26, but four CD26-depleted clones had a much lower level of CD26 than either the parental HSB-2 or H1-2 cells (Figure 1B). Note that because extracts were heated at 37°C instead of 100°C before loading, the detected size of CD26 was approximately 220 kDa instead of 110 kDa (Ghersi et al, 2002).


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)

SDF-1-α-mediated invasion is higher in HSB-2 cells than in CD26-depleted clones. (A) Cell-surface expression of CD26 on HSB-2 cell lines. (a) HSB-2 parental (b) H1-2 (c) 2E5 (d) 2F8 (e) 2G9. Left: cells incubated with PE-labeled isotypic control IgG. Right: cells incubated with PE-labeled anti-CD26 antibody. (B) Expression of CD26 in whole-cell extracts. Equal amounts of protein were run in each lane of a 7.5% gel, transferred to nitrocellulose, and probed with anti-CD26 antibody. Lanes contained HSB-2 parental cells, H1-2, and four clones depleted of CD26: 2E5, 2F8, 2F10, and 2G9. Note that CD26 runs as a dimer (see Materials and Methods). The multiple bands are due to differences in glycosylation. (C) Invasion assay (details in Materials and Methods). Assays were carried out for 24 h, at which time the cells were counted in all wells. Percentage invasion is the number of cells which passed through the transwell divided by the total cell number. Error bars are shown for the standard error of the mean.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2743358&req=5

fig1: SDF-1-α-mediated invasion is higher in HSB-2 cells than in CD26-depleted clones. (A) Cell-surface expression of CD26 on HSB-2 cell lines. (a) HSB-2 parental (b) H1-2 (c) 2E5 (d) 2F8 (e) 2G9. Left: cells incubated with PE-labeled isotypic control IgG. Right: cells incubated with PE-labeled anti-CD26 antibody. (B) Expression of CD26 in whole-cell extracts. Equal amounts of protein were run in each lane of a 7.5% gel, transferred to nitrocellulose, and probed with anti-CD26 antibody. Lanes contained HSB-2 parental cells, H1-2, and four clones depleted of CD26: 2E5, 2F8, 2F10, and 2G9. Note that CD26 runs as a dimer (see Materials and Methods). The multiple bands are due to differences in glycosylation. (C) Invasion assay (details in Materials and Methods). Assays were carried out for 24 h, at which time the cells were counted in all wells. Percentage invasion is the number of cells which passed through the transwell divided by the total cell number. Error bars are shown for the standard error of the mean.
Mentions: HSB-2 cell lines were evaluated for CD26 surface expression by flow cytometry as described in Materials and methods. HSB-2 cells and cells infected with missense siRNA, H1-2, expressed CD26 (Figure 1A). In contrast, CD26 expression was undetectable for the three clones 2E5, 2F8, and 2G9. To confirm CD26 expression, we ran whole-cell lysates on SDS gels, transferred to nitrocellulose, and probed with an anti-CD26 antibody. Both HSB-2 and H1-2 expressed CD26, but four CD26-depleted clones had a much lower level of CD26 than either the parental HSB-2 or H1-2 cells (Figure 1B). Note that because extracts were heated at 37°C instead of 100°C before loading, the detected size of CD26 was approximately 220 kDa instead of 110 kDa (Ghersi et al, 2002).

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