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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 promotes HEK293ar cells survival by mediating cell-cell contacts. A. Western blotting to reveal HAb18G/CD147 expression in HEK293ar and parental HEK293 cells in suspension culture. Two major forms of HAb18G/CD147 (43-66 and 35 kDa) were analyzed. α-Tubulin was used as a loading control (n = 4-6). B. Immunofluorescence of HAb18G/CD147 under laser scanning confocal microscope (Olympus FV1000, Tokyo, Japan; n = 4-10). The nuclei were counterstained with DAPI (4',6-diamidino-2-phenylindole). Magnification: ×1200. C. Western blotting to reveal HAb18G/CD147 expression in HEK293ar cells in HAb18G/CD147 RNA interference (n = 4-6). α-Tubulin was used as a loading control. D. Comparison of the gray scale ratio of HAb18G/CD147/α-tubulin in HAb18G/CD147 RNA interference (n = 4-6). ** p < 0.01, siRNA versus scrambled. E. Flow cytometric quantification of anoikis after siRNA treatment (n = 4-6). Anoikis was determined as described in Fig. 1A. F. Cell-cell contacts formation with time after treatment of targeted HAb18G/CD147-siRNA under the phase-contrast microscope (n = 4-6). Magnification: × 400. Each value represents the mean ± SD of at least triplicate determinations. Results are the representative of three similar experiments.
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Figure 2: HAb18G/CD147 promotes HEK293ar cells survival by mediating cell-cell contacts. A. Western blotting to reveal HAb18G/CD147 expression in HEK293ar and parental HEK293 cells in suspension culture. Two major forms of HAb18G/CD147 (43-66 and 35 kDa) were analyzed. α-Tubulin was used as a loading control (n = 4-6). B. Immunofluorescence of HAb18G/CD147 under laser scanning confocal microscope (Olympus FV1000, Tokyo, Japan; n = 4-10). The nuclei were counterstained with DAPI (4',6-diamidino-2-phenylindole). Magnification: ×1200. C. Western blotting to reveal HAb18G/CD147 expression in HEK293ar cells in HAb18G/CD147 RNA interference (n = 4-6). α-Tubulin was used as a loading control. D. Comparison of the gray scale ratio of HAb18G/CD147/α-tubulin in HAb18G/CD147 RNA interference (n = 4-6). ** p < 0.01, siRNA versus scrambled. E. Flow cytometric quantification of anoikis after siRNA treatment (n = 4-6). Anoikis was determined as described in Fig. 1A. F. Cell-cell contacts formation with time after treatment of targeted HAb18G/CD147-siRNA under the phase-contrast microscope (n = 4-6). Magnification: × 400. Each value represents the mean ± SD of at least triplicate determinations. Results are the representative of three similar experiments.

Mentions: We next explored whether HAb18G/CD147 was involved in HEK293ar cell spheroid survival by mediating cell-cell contacts. HAb18G/CD147 expression was compared between HEK293ar and HEK293 cells in suspension culture. HEK293ar cells expressed significantly more HAb18G/CD147 than the parental cells after 24 h (Fig. 2A). Positive immunofluorescence staining for CD147 was restricted to the cell-cell contacts of HEK293ar (Fig. 2B, merge, arrowhead). These results may imply a correlation between HAb18G/CD147expression and cell-cell contacts-directed anoikis resistance, as the level of HAb18G/CD147 expression was similar in both cell types in adhesion culture (data not shown). To obtain further insight into this correlation, HEK293ar cells were treated with HAb18G/CD147 siRNA in adhesion culture, and apoptosis and cell-cell contacts formation were determined in suspension culture. When HAb18G/CD147 expression was reduced by ~70% (Figs. 2C, D; **p < 0.01), HEK293ar cells underwent anoikis with a sub-G1 proportion of 37.9 ± 2.1% (Fig. 2E). Cell-cell contacts formation was totally inhibited after suspension, whereas the control cells formed multicellular aggregates after as little as 12 h in suspension (Fig. 2F). These results indicate that HAb18G/CD147 confers anoikis resistance of HEK293ar cells to anoikis by mediating cell-cell contacts formation.


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 promotes HEK293ar cells survival by mediating cell-cell contacts. A. Western blotting to reveal HAb18G/CD147 expression in HEK293ar and parental HEK293 cells in suspension culture. Two major forms of HAb18G/CD147 (43-66 and 35 kDa) were analyzed. α-Tubulin was used as a loading control (n = 4-6). B. Immunofluorescence of HAb18G/CD147 under laser scanning confocal microscope (Olympus FV1000, Tokyo, Japan; n = 4-10). The nuclei were counterstained with DAPI (4',6-diamidino-2-phenylindole). Magnification: ×1200. C. Western blotting to reveal HAb18G/CD147 expression in HEK293ar cells in HAb18G/CD147 RNA interference (n = 4-6). α-Tubulin was used as a loading control. D. Comparison of the gray scale ratio of HAb18G/CD147/α-tubulin in HAb18G/CD147 RNA interference (n = 4-6). ** p < 0.01, siRNA versus scrambled. E. Flow cytometric quantification of anoikis after siRNA treatment (n = 4-6). Anoikis was determined as described in Fig. 1A. F. Cell-cell contacts formation with time after treatment of targeted HAb18G/CD147-siRNA under the phase-contrast microscope (n = 4-6). Magnification: × 400. Each value represents the mean ± SD of at least 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 2: HAb18G/CD147 promotes HEK293ar cells survival by mediating cell-cell contacts. A. Western blotting to reveal HAb18G/CD147 expression in HEK293ar and parental HEK293 cells in suspension culture. Two major forms of HAb18G/CD147 (43-66 and 35 kDa) were analyzed. α-Tubulin was used as a loading control (n = 4-6). B. Immunofluorescence of HAb18G/CD147 under laser scanning confocal microscope (Olympus FV1000, Tokyo, Japan; n = 4-10). The nuclei were counterstained with DAPI (4',6-diamidino-2-phenylindole). Magnification: ×1200. C. Western blotting to reveal HAb18G/CD147 expression in HEK293ar cells in HAb18G/CD147 RNA interference (n = 4-6). α-Tubulin was used as a loading control. D. Comparison of the gray scale ratio of HAb18G/CD147/α-tubulin in HAb18G/CD147 RNA interference (n = 4-6). ** p < 0.01, siRNA versus scrambled. E. Flow cytometric quantification of anoikis after siRNA treatment (n = 4-6). Anoikis was determined as described in Fig. 1A. F. Cell-cell contacts formation with time after treatment of targeted HAb18G/CD147-siRNA under the phase-contrast microscope (n = 4-6). Magnification: × 400. Each value represents the mean ± SD of at least triplicate determinations. Results are the representative of three similar experiments.
Mentions: We next explored whether HAb18G/CD147 was involved in HEK293ar cell spheroid survival by mediating cell-cell contacts. HAb18G/CD147 expression was compared between HEK293ar and HEK293 cells in suspension culture. HEK293ar cells expressed significantly more HAb18G/CD147 than the parental cells after 24 h (Fig. 2A). Positive immunofluorescence staining for CD147 was restricted to the cell-cell contacts of HEK293ar (Fig. 2B, merge, arrowhead). These results may imply a correlation between HAb18G/CD147expression and cell-cell contacts-directed anoikis resistance, as the level of HAb18G/CD147 expression was similar in both cell types in adhesion culture (data not shown). To obtain further insight into this correlation, HEK293ar cells were treated with HAb18G/CD147 siRNA in adhesion culture, and apoptosis and cell-cell contacts formation were determined in suspension culture. When HAb18G/CD147 expression was reduced by ~70% (Figs. 2C, D; **p < 0.01), HEK293ar cells underwent anoikis with a sub-G1 proportion of 37.9 ± 2.1% (Fig. 2E). Cell-cell contacts formation was totally inhibited after suspension, whereas the control cells formed multicellular aggregates after as little as 12 h in suspension (Fig. 2F). These results indicate that HAb18G/CD147 confers anoikis resistance of HEK293ar cells to anoikis by mediating cell-cell contacts formation.

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