Limits...
HAb18G/CD147 cell-cell contacts confer resistance of a HEK293 subpopulation to anoikis in an E-cadherin-dependent manner.

Ma XK, Wang L, Li Y, Yang XM, Zhao P - BMC Cell Biol. (2010)

Bottom Line: When HAb18G/CD147 was downregulated, E-cadherin expression in HEK293ar cells was significantly suppressed; however, knockdown of E-cadherin by E-cadherin siRNA or blocking of E-cadherin binding activity with a specific antibody and EDTA had no significant effect on HAb18G/CD147 expression.Our results provide new evidence that HAb18G/CD147-mediated cell-cell contact confers anoikis resistance in an E-cadherin-dependent manner; and cell-cell contact mediated resistance to anoikis implicates PI3K pathway in a highly relevant cell model (HEK293ar).Our results also contribute to a better understanding of the biology of HEK293 cell spheroids, a major workhorse for producing human therapeutic agents and viral vaccines.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: Acquisition of resistance to "anoikis" facilitates the survival of cells under independent matrix-deficient conditions, such as cells in tumor progression and the production of suspension culture cells for biomedical engineering. There is evidence suggesting that CD147, an adhesion molecule associated with survival of cells in tumor metastasis and cell-cell contacts, plays an important role in resistance to anoikis. However, information regarding the functions of CD147 in mediating cell-cell contacts and anoikis-resistance remains limited and even self-contradictory.

Results: An anoikis-resistant clone (HEK293ar), derived from anoikis-sensitive parental Human Embryonic Kidney 293 cells, survived anoikis by the formation of cell-cell contacts. The expression of HAb18G/CD147 (a member of the CD147 family) was upregulated and the protein was located at cell-cell junctions. Upregulation of HAb18G/CD147 in suspended HEK293ar cells suppressed anoikis by mediating the formation of cell-cell adhesions. Anoikis resistance in HEK293ar cells also required E-cadherin-mediated cell-cell contacts. Knock-down of HAb18G/CD147 and E-cadherin inhibited cell-cell contacts formation and increased anoikis sensitivity respectively. When HAb18G/CD147 was downregulated, E-cadherin expression in HEK293ar cells was significantly suppressed; however, knockdown of E-cadherin by E-cadherin siRNA or blocking of E-cadherin binding activity with a specific antibody and EDTA had no significant effect on HAb18G/CD147 expression. Finally, pretreatment with LY294002, a phosphoinositide 3-kinase (PI3K/AKT) inhibitor, disrupted cell-cell contacts and decreased cell number, but this was not the case in cells treated with the extracellular signal-regulated kinase (ERK) inhibitor PD98059.

Conclusions: Our results provide new evidence that HAb18G/CD147-mediated cell-cell contact confers anoikis resistance in an E-cadherin-dependent manner; and cell-cell contact mediated resistance to anoikis implicates PI3K pathway in a highly relevant cell model (HEK293ar). Understanding of the role of HAb18G/CD147 cell-cell contacts in anoikis resistance may help in understanding the survival of cells in anchorage-independent growth, such as cells in tumor metastasis and suspension culture produced for biomedical engineering. Our results also contribute to a better understanding of the biology of HEK293 cell spheroids, a major workhorse for producing human therapeutic agents and viral vaccines.

No MeSH data available.


Related in: MedlinePlus

HAb18G/CD147 knockdown reduces the expression of E-cadherin. A. E-cadherin expression and the formation of cell-cell contacts with time after HAb18G/CD147 siRNA treatment (n = 4-6). Magnification: × 400. B. Western blotting to reveal E-cadherin in HAb18G/CD147-siRNA-treated HEK293ar cells in suspension culture (n = 4-6). α-Tubulin was used as a loading control. C. Comparison of the gray scale ratio of E-cadherin/α-tubulin in HAb18G/CD147-siRNA treatment (n = 4-6). ** p < 0.01, siRNA versus scrambled. Each value represents the mean ± SD of triplicate determinations. Results are the representative of three similar experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2864199&req=5

Figure 4: HAb18G/CD147 knockdown reduces the expression of E-cadherin. A. E-cadherin expression and the formation of cell-cell contacts with time after HAb18G/CD147 siRNA treatment (n = 4-6). Magnification: × 400. B. Western blotting to reveal E-cadherin in HAb18G/CD147-siRNA-treated HEK293ar cells in suspension culture (n = 4-6). α-Tubulin was used as a loading control. C. Comparison of the gray scale ratio of E-cadherin/α-tubulin in HAb18G/CD147-siRNA treatment (n = 4-6). ** p < 0.01, siRNA versus scrambled. Each value represents the mean ± SD of triplicate determinations. Results are the representative of three similar experiments.

Mentions: As HAb18G/CD147 and E-cadherin are both related to cell-cell contact-directed survival of HEK293ar cells in suspension, they may be functional connected. To test this hypothesis, we investigated the relationship between these two molecules by RNAi. Figs. 4A, B and 4C show that E-cadherin expression decreased dramatically in HAb18G/CD147 siRNA-treated HEK293ar cells according to immunofluorescence staining density (Figs. 4A, analyzed by Image Pro Plus 6.0 3-DS software, **p < 0.01, data not shown) and western blotting (Fig.4C, **p < 0.01). Interestingly, with time after HAb18G/CD147 siRNA treatment, cell-cell contacts and spheroids gradually disrupted (Fig. 4A). In contrast, blocking of E-cadherin binding by an anti-E-cadherin and inhibition of cell-cell contacts with EDTA showed no significant effect on HAb18G/CD147 expression at the single cell level according to immunofluorescence staining (Fig. 5A; fluorescence density was analyzed by Image Pro Plus 6.0 3-DS, **p < 0.01, data not shown), although the degree of cell aggregation was decreased in suspension culture (Fig. 5B, **p < 0.01). Furthermore, treatment of HEK293ar cells with E-cadherin siRNA under adhesion conditions did not alter the level of HAb18G/CD147 expression of HEK293ar in suspension (Figs. 5C, D, ** p < 0.01; Fig. 5E, p > 0.05). Together, these results indicate that the effect of HAb18G/CD147 on cell-cell adhesion and anoikis suppression is mediated by E-cadherin.


HAb18G/CD147 cell-cell contacts confer resistance of a HEK293 subpopulation to anoikis in an E-cadherin-dependent manner.

Ma XK, Wang L, Li Y, Yang XM, Zhao P - BMC Cell Biol. (2010)

HAb18G/CD147 knockdown reduces the expression of E-cadherin. A. E-cadherin expression and the formation of cell-cell contacts with time after HAb18G/CD147 siRNA treatment (n = 4-6). Magnification: × 400. B. Western blotting to reveal E-cadherin in HAb18G/CD147-siRNA-treated HEK293ar cells in suspension culture (n = 4-6). α-Tubulin was used as a loading control. C. Comparison of the gray scale ratio of E-cadherin/α-tubulin in HAb18G/CD147-siRNA treatment (n = 4-6). ** p < 0.01, siRNA versus scrambled. Each value represents the mean ± SD of triplicate determinations. Results are the representative of three similar experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2864199&req=5

Figure 4: HAb18G/CD147 knockdown reduces the expression of E-cadherin. A. E-cadherin expression and the formation of cell-cell contacts with time after HAb18G/CD147 siRNA treatment (n = 4-6). Magnification: × 400. B. Western blotting to reveal E-cadherin in HAb18G/CD147-siRNA-treated HEK293ar cells in suspension culture (n = 4-6). α-Tubulin was used as a loading control. C. Comparison of the gray scale ratio of E-cadherin/α-tubulin in HAb18G/CD147-siRNA treatment (n = 4-6). ** p < 0.01, siRNA versus scrambled. Each value represents the mean ± SD of triplicate determinations. Results are the representative of three similar experiments.
Mentions: As HAb18G/CD147 and E-cadherin are both related to cell-cell contact-directed survival of HEK293ar cells in suspension, they may be functional connected. To test this hypothesis, we investigated the relationship between these two molecules by RNAi. Figs. 4A, B and 4C show that E-cadherin expression decreased dramatically in HAb18G/CD147 siRNA-treated HEK293ar cells according to immunofluorescence staining density (Figs. 4A, analyzed by Image Pro Plus 6.0 3-DS software, **p < 0.01, data not shown) and western blotting (Fig.4C, **p < 0.01). Interestingly, with time after HAb18G/CD147 siRNA treatment, cell-cell contacts and spheroids gradually disrupted (Fig. 4A). In contrast, blocking of E-cadherin binding by an anti-E-cadherin and inhibition of cell-cell contacts with EDTA showed no significant effect on HAb18G/CD147 expression at the single cell level according to immunofluorescence staining (Fig. 5A; fluorescence density was analyzed by Image Pro Plus 6.0 3-DS, **p < 0.01, data not shown), although the degree of cell aggregation was decreased in suspension culture (Fig. 5B, **p < 0.01). Furthermore, treatment of HEK293ar cells with E-cadherin siRNA under adhesion conditions did not alter the level of HAb18G/CD147 expression of HEK293ar in suspension (Figs. 5C, D, ** p < 0.01; Fig. 5E, p > 0.05). Together, these results indicate that the effect of HAb18G/CD147 on cell-cell adhesion and anoikis suppression is mediated by E-cadherin.

Bottom Line: When HAb18G/CD147 was downregulated, E-cadherin expression in HEK293ar cells was significantly suppressed; however, knockdown of E-cadherin by E-cadherin siRNA or blocking of E-cadherin binding activity with a specific antibody and EDTA had no significant effect on HAb18G/CD147 expression.Our results provide new evidence that HAb18G/CD147-mediated cell-cell contact confers anoikis resistance in an E-cadherin-dependent manner; and cell-cell contact mediated resistance to anoikis implicates PI3K pathway in a highly relevant cell model (HEK293ar).Our results also contribute to a better understanding of the biology of HEK293 cell spheroids, a major workhorse for producing human therapeutic agents and viral vaccines.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: Acquisition of resistance to "anoikis" facilitates the survival of cells under independent matrix-deficient conditions, such as cells in tumor progression and the production of suspension culture cells for biomedical engineering. There is evidence suggesting that CD147, an adhesion molecule associated with survival of cells in tumor metastasis and cell-cell contacts, plays an important role in resistance to anoikis. However, information regarding the functions of CD147 in mediating cell-cell contacts and anoikis-resistance remains limited and even self-contradictory.

Results: An anoikis-resistant clone (HEK293ar), derived from anoikis-sensitive parental Human Embryonic Kidney 293 cells, survived anoikis by the formation of cell-cell contacts. The expression of HAb18G/CD147 (a member of the CD147 family) was upregulated and the protein was located at cell-cell junctions. Upregulation of HAb18G/CD147 in suspended HEK293ar cells suppressed anoikis by mediating the formation of cell-cell adhesions. Anoikis resistance in HEK293ar cells also required E-cadherin-mediated cell-cell contacts. Knock-down of HAb18G/CD147 and E-cadherin inhibited cell-cell contacts formation and increased anoikis sensitivity respectively. When HAb18G/CD147 was downregulated, E-cadherin expression in HEK293ar cells was significantly suppressed; however, knockdown of E-cadherin by E-cadherin siRNA or blocking of E-cadherin binding activity with a specific antibody and EDTA had no significant effect on HAb18G/CD147 expression. Finally, pretreatment with LY294002, a phosphoinositide 3-kinase (PI3K/AKT) inhibitor, disrupted cell-cell contacts and decreased cell number, but this was not the case in cells treated with the extracellular signal-regulated kinase (ERK) inhibitor PD98059.

Conclusions: Our results provide new evidence that HAb18G/CD147-mediated cell-cell contact confers anoikis resistance in an E-cadherin-dependent manner; and cell-cell contact mediated resistance to anoikis implicates PI3K pathway in a highly relevant cell model (HEK293ar). Understanding of the role of HAb18G/CD147 cell-cell contacts in anoikis resistance may help in understanding the survival of cells in anchorage-independent growth, such as cells in tumor metastasis and suspension culture produced for biomedical engineering. Our results also contribute to a better understanding of the biology of HEK293 cell spheroids, a major workhorse for producing human therapeutic agents and viral vaccines.

No MeSH data available.


Related in: MedlinePlus