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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.

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Related in: MedlinePlus

Anoikis-resistant HEK293ar cells survives anoikis through cell-cell contacts. An anoikis-resistant HEK293 cell subpopulation (HEK293ar) was obtained by sequential cycles of adhesion and suspension culture. A. Anoikis in HEK293ar cells in suspension culture at different time points. Flow cytometry (histogram) shows that the apoptosis rate changed little during suspension culture of HEK293ar. The x and y-axes indicate the size of DNA and the number of cells counted, respectively. FL3-H, a standard term for flow cytometry, represents measurement of the fluorescence intensity of propidium iodide (PI) at a super-red wavelength (670 nm). The results are means ± S.D. (n = 4-6). B. The morphology of HEK293ar and parental HEK293 cells in suspension and adhesion culture revealed by microscopy (n = 4-10). Magnification: ×200. C. TEM of a multicellular HEK293ar spheroid. Chromatin masses of moderate electron density are dispersed in the nuclei and nucleoli are conspicuous (arrowed). The cells were observed in 4-6 independent sections. Bar, 2 μm. D. A junctional complex with thickened membranes (arrowed) in suspended HEK293ar cells viewed under SEM. Bar, 50 nm. The cells were viewed in 4-10 independent sections (at least 100 cells/section). E. EDTA (10 mmol/l) treatment disrupted the cell-cell contacts and resulted in massive apoptosis as determined by TUNEL assay. Arrowheads show that single cells detached from the aggregates give more intense TUNEL staining. Magnification: ×200. The apoptotic nuclei were counted in 4-10 independent sections (at least 500 nuclei/section).
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Figure 1: Anoikis-resistant HEK293ar cells survives anoikis through cell-cell contacts. An anoikis-resistant HEK293 cell subpopulation (HEK293ar) was obtained by sequential cycles of adhesion and suspension culture. A. Anoikis in HEK293ar cells in suspension culture at different time points. Flow cytometry (histogram) shows that the apoptosis rate changed little during suspension culture of HEK293ar. The x and y-axes indicate the size of DNA and the number of cells counted, respectively. FL3-H, a standard term for flow cytometry, represents measurement of the fluorescence intensity of propidium iodide (PI) at a super-red wavelength (670 nm). The results are means ± S.D. (n = 4-6). B. The morphology of HEK293ar and parental HEK293 cells in suspension and adhesion culture revealed by microscopy (n = 4-10). Magnification: ×200. C. TEM of a multicellular HEK293ar spheroid. Chromatin masses of moderate electron density are dispersed in the nuclei and nucleoli are conspicuous (arrowed). The cells were observed in 4-6 independent sections. Bar, 2 μm. D. A junctional complex with thickened membranes (arrowed) in suspended HEK293ar cells viewed under SEM. Bar, 50 nm. The cells were viewed in 4-10 independent sections (at least 100 cells/section). E. EDTA (10 mmol/l) treatment disrupted the cell-cell contacts and resulted in massive apoptosis as determined by TUNEL assay. Arrowheads show that single cells detached from the aggregates give more intense TUNEL staining. Magnification: ×200. The apoptotic nuclei were counted in 4-10 independent sections (at least 500 nuclei/section).

Mentions: To explore the role of cell-cell contacts in preventing anoikis, we generated a subpopulation (HEK293ar) with an anoikis-resistant phenotype derived from HEK293 cells using sequential cycles of adhesion and suspension culture [14]. Upon detachment, the apoptotic ratio in HEK293ar cells was lower than in the parental cells, as shown by quantifying the proportions in sub-G1 by flow cytometry (6.97 ± 3.1% versus 37.3 ± 2.3%, data not shown). When HEK293ar cells were cultured in suspension for 1, 10 and 30 days, the mean sub-G1 proportions were 8.7, 11.7, and 15.1%, respectively, with little change in the apoptotic ratio (Fig. 1A). Moreover, this subpopulation maintained the anoikis resistance phenotype in vitro for 6 months (data not shown). Additionally, after ~6-10 h suspension culture, the HEK293ar cells spontaneously and gradually formed cell-cell contacts and generated multi-cellular spheroids that could still grow as adhesion cultures, whereas the parental HEK293 cells did not (Fig. 1B). Ultrastructural examination demonstrated that the suspended spheroids comprised many single cells. In each spheroid, chromatin masses of moderate electron density were dispersed in the nuclei, and the nucleoli were conspicuous (Fig. 1C, arrowed). Cells within the multi-cellular spheroids adhered laterally to each other through diverse specialized intercellular junctions (Fig. 1D, arrowhead). Pretreatment with EDTA solution to disrupt calcium-dependent cell-cell contacts also disrupted the adhesions and caused massive DNA fragmentation in HEK293ar cells, as shown by TUNEL assay (Fig. 1E). Interestingly, single cells detached from the aggregates gave more intense TUNEL staining (Fig. 1E, arrowhead). In contrast, TUNEL assays and ultrastructural examination demonstrated that HEK293 cells clearly underwent anoikis (data not shown). It was previously reported that cell-cell contacts prevent anoikis in primary human colonic epithelial cells [15]. The present results also suggest that adjacent HEK293ar epithelial cells form cell-cell contacts, suppressing anoikis under matrix-deficient conditions.


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)

Anoikis-resistant HEK293ar cells survives anoikis through cell-cell contacts. An anoikis-resistant HEK293 cell subpopulation (HEK293ar) was obtained by sequential cycles of adhesion and suspension culture. A. Anoikis in HEK293ar cells in suspension culture at different time points. Flow cytometry (histogram) shows that the apoptosis rate changed little during suspension culture of HEK293ar. The x and y-axes indicate the size of DNA and the number of cells counted, respectively. FL3-H, a standard term for flow cytometry, represents measurement of the fluorescence intensity of propidium iodide (PI) at a super-red wavelength (670 nm). The results are means ± S.D. (n = 4-6). B. The morphology of HEK293ar and parental HEK293 cells in suspension and adhesion culture revealed by microscopy (n = 4-10). Magnification: ×200. C. TEM of a multicellular HEK293ar spheroid. Chromatin masses of moderate electron density are dispersed in the nuclei and nucleoli are conspicuous (arrowed). The cells were observed in 4-6 independent sections. Bar, 2 μm. D. A junctional complex with thickened membranes (arrowed) in suspended HEK293ar cells viewed under SEM. Bar, 50 nm. The cells were viewed in 4-10 independent sections (at least 100 cells/section). E. EDTA (10 mmol/l) treatment disrupted the cell-cell contacts and resulted in massive apoptosis as determined by TUNEL assay. Arrowheads show that single cells detached from the aggregates give more intense TUNEL staining. Magnification: ×200. The apoptotic nuclei were counted in 4-10 independent sections (at least 500 nuclei/section).
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Related In: Results  -  Collection

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Figure 1: Anoikis-resistant HEK293ar cells survives anoikis through cell-cell contacts. An anoikis-resistant HEK293 cell subpopulation (HEK293ar) was obtained by sequential cycles of adhesion and suspension culture. A. Anoikis in HEK293ar cells in suspension culture at different time points. Flow cytometry (histogram) shows that the apoptosis rate changed little during suspension culture of HEK293ar. The x and y-axes indicate the size of DNA and the number of cells counted, respectively. FL3-H, a standard term for flow cytometry, represents measurement of the fluorescence intensity of propidium iodide (PI) at a super-red wavelength (670 nm). The results are means ± S.D. (n = 4-6). B. The morphology of HEK293ar and parental HEK293 cells in suspension and adhesion culture revealed by microscopy (n = 4-10). Magnification: ×200. C. TEM of a multicellular HEK293ar spheroid. Chromatin masses of moderate electron density are dispersed in the nuclei and nucleoli are conspicuous (arrowed). The cells were observed in 4-6 independent sections. Bar, 2 μm. D. A junctional complex with thickened membranes (arrowed) in suspended HEK293ar cells viewed under SEM. Bar, 50 nm. The cells were viewed in 4-10 independent sections (at least 100 cells/section). E. EDTA (10 mmol/l) treatment disrupted the cell-cell contacts and resulted in massive apoptosis as determined by TUNEL assay. Arrowheads show that single cells detached from the aggregates give more intense TUNEL staining. Magnification: ×200. The apoptotic nuclei were counted in 4-10 independent sections (at least 500 nuclei/section).
Mentions: To explore the role of cell-cell contacts in preventing anoikis, we generated a subpopulation (HEK293ar) with an anoikis-resistant phenotype derived from HEK293 cells using sequential cycles of adhesion and suspension culture [14]. Upon detachment, the apoptotic ratio in HEK293ar cells was lower than in the parental cells, as shown by quantifying the proportions in sub-G1 by flow cytometry (6.97 ± 3.1% versus 37.3 ± 2.3%, data not shown). When HEK293ar cells were cultured in suspension for 1, 10 and 30 days, the mean sub-G1 proportions were 8.7, 11.7, and 15.1%, respectively, with little change in the apoptotic ratio (Fig. 1A). Moreover, this subpopulation maintained the anoikis resistance phenotype in vitro for 6 months (data not shown). Additionally, after ~6-10 h suspension culture, the HEK293ar cells spontaneously and gradually formed cell-cell contacts and generated multi-cellular spheroids that could still grow as adhesion cultures, whereas the parental HEK293 cells did not (Fig. 1B). Ultrastructural examination demonstrated that the suspended spheroids comprised many single cells. In each spheroid, chromatin masses of moderate electron density were dispersed in the nuclei, and the nucleoli were conspicuous (Fig. 1C, arrowed). Cells within the multi-cellular spheroids adhered laterally to each other through diverse specialized intercellular junctions (Fig. 1D, arrowhead). Pretreatment with EDTA solution to disrupt calcium-dependent cell-cell contacts also disrupted the adhesions and caused massive DNA fragmentation in HEK293ar cells, as shown by TUNEL assay (Fig. 1E). Interestingly, single cells detached from the aggregates gave more intense TUNEL staining (Fig. 1E, arrowhead). In contrast, TUNEL assays and ultrastructural examination demonstrated that HEK293 cells clearly underwent anoikis (data not shown). It was previously reported that cell-cell contacts prevent anoikis in primary human colonic epithelial cells [15]. The present results also suggest that adjacent HEK293ar epithelial cells form cell-cell contacts, suppressing anoikis under matrix-deficient conditions.

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