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Evaluation of Cancer Stem Cell Markers CD133, CD44, CD24: Association with AKT Isoforms and Radiation Resistance in Colon Cancer Cells

Sahlberg, Sara Häggblad, Spiegelberg, Diana, Glimelius, Bengt, Stenerlöw, Bo, Nestor, Marika·2014·PLoS ONE
DOI 10.1371/journal.pone.0094621PMC PMC3997403
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Figure 1 Expression of CD133, CD44, CD24 and EGFR in three colon cancer cell-lines. A) HT-29, HCT116 and DLD-1. The expression patterns in the dotplots are from one representative flow cytometer experiment. The grid demonstrates the margin between high and low expression of the protein defined by isotype controls. B) The expression of CD24 positive cells in flow cytometry depend on the anti-CD24 antibody. The table shows the percent of CD24 positive cells using three different CD24 antibodies.
Figure 1 Expression of CD133, CD44, CD24 and EGFR in three colon cancer cell-lines. A) HT-29, HCT116 and DLD-1. The expression patterns in the dotplots are from one representative flow cytometer experiment. The grid demonstrates the margin between high and low expression of the protein defined by isotype controls. B) The expression of CD24 positive cells in flow cytometry depend on the anti-CD24 antibody. The table shows the percent of CD24 positive cells using three different CD24 antibodies.CC BY · PubMed Central
Figure 2 Radiation sensitivity of CD133/CD44 sorted cells. Clonogenic assay of A) HT-29, B) HCT116 and C) DLD-1 cells. The top and bottom 10 percent of CD133 and CD44 expressing cells were sorted by flow cytometry (CD133high/CD44high or CD133low/CD44low) and the radiation sensitivity was analyzed using clonogenic assays. The controls of both fractions were normalized and set to 100% survival. The error bars represent the standard error of the mean from at least two separate experiments with triplicate samples. A higher resistance to radiation in the CD133high/CD44high population compared to the CD133low/CD44low population was observed for all cell-lines. These differences were statistically significant at all radiation doses (2, 4 and 6 Gy) for DLD-1 cells (Figure 2C), at 4 and 6 Gy for HCT116 cells, and at 4 Gy for HT-29 cells (Figure 2A and B) with a P-value of <0.05 (one-way ANOVA). The percentage of cells positive for EGFR, CD24 with BD bioscience antibody or CD24 with Miltenyi biotech/MACS were analyzed with flow cytometry.
Figure 2 Radiation sensitivity of CD133/CD44 sorted cells. Clonogenic assay of A) HT-29, B) HCT116 and C) DLD-1 cells. The top and bottom 10 percent of CD133 and CD44 expressing cells were sorted by flow cytometry (CD133high/CD44high or CD133low/CD44low) and the radiation sensitivity was analyzed using clonogenic assays. The controls of both fractions were normalized and set to 100% survival. The error bars represent the standard error of the mean from at least two separate experiments with triplicate samples. A higher resistance to radiation in the CD133high/CD44high population compared to the CD133low/CD44low population was observed for all cell-lines. These differences were statistically significant at all radiation doses (2, 4 and 6 Gy) for DLD-1 cells (Figure 2C), at 4 and 6 Gy for HCT116 cells, and at 4 Gy for HT-29 cells (Figure 2A and B) with a P-value of <0.05 (one-way ANOVA). The percentage of cells positive for EGFR, CD24 with BD bioscience antibody or CD24 with Miltenyi biotech/MACS were analyzed with flow cytometry.CC BY · PubMed Central
Figure 3 AKT expression in CD133/CD44 sorted cells. A) DLD-1 cells were sorted by flow cytometry and different populations with CD44positive/CD133negative (Q1), CD44positive/CD133positive (Q2), CD44negativeCD133negative (Q3), were collected. B) The sorted cells were further analyzed with western blot for total AKT, AKT1 or AKT2 and betaactin expression.
Figure 3 AKT expression in CD133/CD44 sorted cells. A) DLD-1 cells were sorted by flow cytometry and different populations with CD44positive/CD133negative (Q1), CD44positive/CD133positive (Q2), CD44negativeCD133negative (Q3), were collected. B) The sorted cells were further analyzed with western blot for total AKT, AKT1 or AKT2 and betaactin expression.CC BY · PubMed Central
Figure 4 Flow cytometry analysis of the expression of CD133, CD24 and CD44 in the colon cancer cell-line DLD-1 with its isogenic knock-out cell-lines of AKT1, AKT2 and AKT1/2. A) In the parental cells, approximately 10% of the cells were CD133 positive cells. However, in the AKT1 and AKT1/2 knock-outs, the CD133 positive cells were reduced to 0.3 and 0.1% respectively. This was not seen in the AKT2 knock-out cell-line, where 33% of the cells were positive for CD133. B) The mean fluorescent intensity of CD44 normalized to the DLD-1 parental cell-line increased to 150% in AKT1 KO, 160% in AKT2 KO and 300% in AKT1/2 KO cell-line. The error bars represent the standard deviation (SD) from at least two experiments. C) The percent of CD24 positive cells analyzed with two different CD24 antibodies from BD Biosciences and Miltenyi/MACS in flow cytometry. The standard deviations are from repeated experiments. D) Cell-cycle distribution in DLD-1 parental, AKT1 KO, AKT2 KO and AKT1/2 KO cells.
Figure 4 Flow cytometry analysis of the expression of CD133, CD24 and CD44 in the colon cancer cell-line DLD-1 with its isogenic knock-out cell-lines of AKT1, AKT2 and AKT1/2. A) In the parental cells, approximately 10% of the cells were CD133 positive cells. However, in the AKT1 and AKT1/2 knock-outs, the CD133 positive cells were reduced to 0.3 and 0.1% respectively. This was not seen in the AKT2 knock-out cell-line, where 33% of the cells were positive for CD133. B) The mean fluorescent intensity of CD44 normalized to the DLD-1 parental cell-line increased to 150% in AKT1 KO, 160% in AKT2 KO and 300% in AKT1/2 KO cell-line. The error bars represent the standard deviation (SD) from at least two experiments. C) The percent of CD24 positive cells analyzed with two different CD24 antibodies from BD Biosciences and Miltenyi/MACS in flow cytometry. The standard deviations are from repeated experiments. D) Cell-cycle distribution in DLD-1 parental, AKT1 KO, AKT2 KO and AKT1/2 KO cells.CC BY · PubMed Central
Figure 5 Protein and gene expression in DLD-1 parental, AKT1 KO, AKT2 KO, and AKT1/2 KO cells. A) Protein expression of CD44, CD133, phospho-FOXO, total FOX03a, phospho-GSK3β and total GSK3β from western blot analysis. B) Gene expression, up-regulation (+), down-regulation (−) or not changed (NC), of CD44, CD24, CD133, FOX01, FOX03, FOX04, GSK3β and LYN in DLD-1 AKT1 KO, AKT2 KO or AKT1/2 KO cells in comparison with DLD-1 parental cells.
Figure 5 Protein and gene expression in DLD-1 parental, AKT1 KO, AKT2 KO, and AKT1/2 KO cells. A) Protein expression of CD44, CD133, phospho-FOXO, total FOX03a, phospho-GSK3β and total GSK3β from western blot analysis. B) Gene expression, up-regulation (+), down-regulation (−) or not changed (NC), of CD44, CD24, CD133, FOX01, FOX03, FOX04, GSK3β and LYN in DLD-1 AKT1 KO, AKT2 KO or AKT1/2 KO cells in comparison with DLD-1 parental cells.CC BY · PubMed Central

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Metadata

Authors
Sahlberg, S. H., Spiegelberg, D., Glimelius, B., Stenerlöw, B., & Nestor, M.
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2014-04-23
Volume
9
Issue
4
Pages
e94621
ISSN
1932-6203
DOI
10.1371/journal.pone.0094621
PMC
PMC3997403
Cited by
181
References
2
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (unspecified)

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