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Suppressive expression of CD274 increases tumorigenesis and cancer stem cell phenotypes in cholangiocarcinoma.

Tamai K, Nakamura M, Mizuma M, Mochizuki M, Yokoyama M, Endo H, Yamaguchi K, Nakagawa T, Shiina M, Unno M, Muramoto K, Sato I, Satoh K, Sugamura K, Tanaka N - Cancer Sci. (2014)

Bottom Line: Its malignant phenotypes may be assumed by cancer stem cells (CSC).Furthermore, the CD274(low) cells possess several CSC-related characteristics, such as high aldehyde dehydrogenase (ALDH) activity, reduced reactive oxygen species production and a dormant state in the cell cycle.Furthermore, depletion of CD274 expression by shRNA in RBE cells enhances their tumorigenicity and increases ALDH activity.

View Article: PubMed Central - PubMed

Affiliation: Division of Cancer Biology and Therapeutics, Miyagi Cancer Center Research Institute, Natori, Japan; Department of Cancer Science, Tohoku University Graduate School of Medicine, Sendai, Japan.

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Related in: MedlinePlus

(On the previous page) Evaluation of cancer stem cell (CSC)-related phenotypes in CD274low subpopulations. (a,b) CD274low and CD274high populations were sorted from the RBE and HuCCT1 cell lines as described in Supporting Information Fig. S1. Aldehyde dehydrogenase (ALDH) activity of the RBE (a) and HuCCT1 (b) subpopulations was measured using the Aldefluor assay followed by FACS analysis. A specific inhibitor of ALDH, diethylaminobenzaldehyde (DEAB), was used to control for background fluorescence. (c,d) Measurement of all-trans retinal metabolism in CD274high and CD274low subpopulations in RBE (c) and HuCCT1 (d) cells. (e,f) Quantification of the reactive oxygen species (ROS) production in RBE (e) and HuCCT1 (f) subpopulations. Each subpopulation was incubated with or without H2O2, stained with 2', 7'-Dichlorodihydrofluorescin diacetate (DCFH-DA) and subjected to FACS analysis. (g,h) Analysis of cell cycling in the RBE (g) and HuCCT1 (h) subpopulations. CD274low and CD274high cells were fixed with ethanol, stained with Ki-67 FITC and propidium iodide and subjected to FACS analysis. (i,j) Expression profile of stem cell-related genes in CD274high and CD274low cells. mRNA from CD274high and CD274low cells derived from the RBE (i) and HuCCT1 (j) cell lines were analyzed using real-time PCR.
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fig02: (On the previous page) Evaluation of cancer stem cell (CSC)-related phenotypes in CD274low subpopulations. (a,b) CD274low and CD274high populations were sorted from the RBE and HuCCT1 cell lines as described in Supporting Information Fig. S1. Aldehyde dehydrogenase (ALDH) activity of the RBE (a) and HuCCT1 (b) subpopulations was measured using the Aldefluor assay followed by FACS analysis. A specific inhibitor of ALDH, diethylaminobenzaldehyde (DEAB), was used to control for background fluorescence. (c,d) Measurement of all-trans retinal metabolism in CD274high and CD274low subpopulations in RBE (c) and HuCCT1 (d) cells. (e,f) Quantification of the reactive oxygen species (ROS) production in RBE (e) and HuCCT1 (f) subpopulations. Each subpopulation was incubated with or without H2O2, stained with 2', 7'-Dichlorodihydrofluorescin diacetate (DCFH-DA) and subjected to FACS analysis. (g,h) Analysis of cell cycling in the RBE (g) and HuCCT1 (h) subpopulations. CD274low and CD274high cells were fixed with ethanol, stained with Ki-67 FITC and propidium iodide and subjected to FACS analysis. (i,j) Expression profile of stem cell-related genes in CD274high and CD274low cells. mRNA from CD274high and CD274low cells derived from the RBE (i) and HuCCT1 (j) cell lines were analyzed using real-time PCR.

Mentions: We then investigated the CSC-related characteristics of the CD274low and CD274high populations derived from RBE and HuCCT1 cells. They were sorted by anti-CD274 antibody staining and the resulting populations were assessed for ALDH activity using the Aldefluor assay system followed by FACS analysis. The RBE CD274low population was 46.1% ALDH+, whereas the RBE CD274high population was 10.2% ALDH+ (Fig.2a). The HuCCT1 CD274low and CD274high populations showed 52.4% and 38.3% ALDH+, respectively. We then measured the retinal metabolism in vitro, because ALDH metabolizes all-trans retinals to all-trans retinoic acids. The level of retinoic acids produced by the CD274low population was significantly higher than that produced by the CD274high population (Fig.2c,d) in both RBE and HuCCT1 cells. These results are compatible with those of the Aldefluor assay. The CD274low and CD274high populations were further assessed for ROS levels. The ROS levels were significantly lower in the CD274low populations than in the CD274high populations derived from both RBE and HuCCT1 cells (Fig.2e,f). These results indicate that the CD274low populations of both cholangiocarcinoma cell lines predominantly exhibit CSC-related characteristics such as high ALDH1 activity and low ROS production.


Suppressive expression of CD274 increases tumorigenesis and cancer stem cell phenotypes in cholangiocarcinoma.

Tamai K, Nakamura M, Mizuma M, Mochizuki M, Yokoyama M, Endo H, Yamaguchi K, Nakagawa T, Shiina M, Unno M, Muramoto K, Sato I, Satoh K, Sugamura K, Tanaka N - Cancer Sci. (2014)

(On the previous page) Evaluation of cancer stem cell (CSC)-related phenotypes in CD274low subpopulations. (a,b) CD274low and CD274high populations were sorted from the RBE and HuCCT1 cell lines as described in Supporting Information Fig. S1. Aldehyde dehydrogenase (ALDH) activity of the RBE (a) and HuCCT1 (b) subpopulations was measured using the Aldefluor assay followed by FACS analysis. A specific inhibitor of ALDH, diethylaminobenzaldehyde (DEAB), was used to control for background fluorescence. (c,d) Measurement of all-trans retinal metabolism in CD274high and CD274low subpopulations in RBE (c) and HuCCT1 (d) cells. (e,f) Quantification of the reactive oxygen species (ROS) production in RBE (e) and HuCCT1 (f) subpopulations. Each subpopulation was incubated with or without H2O2, stained with 2', 7'-Dichlorodihydrofluorescin diacetate (DCFH-DA) and subjected to FACS analysis. (g,h) Analysis of cell cycling in the RBE (g) and HuCCT1 (h) subpopulations. CD274low and CD274high cells were fixed with ethanol, stained with Ki-67 FITC and propidium iodide and subjected to FACS analysis. (i,j) Expression profile of stem cell-related genes in CD274high and CD274low cells. mRNA from CD274high and CD274low cells derived from the RBE (i) and HuCCT1 (j) cell lines were analyzed using real-time PCR.
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Related In: Results  -  Collection

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fig02: (On the previous page) Evaluation of cancer stem cell (CSC)-related phenotypes in CD274low subpopulations. (a,b) CD274low and CD274high populations were sorted from the RBE and HuCCT1 cell lines as described in Supporting Information Fig. S1. Aldehyde dehydrogenase (ALDH) activity of the RBE (a) and HuCCT1 (b) subpopulations was measured using the Aldefluor assay followed by FACS analysis. A specific inhibitor of ALDH, diethylaminobenzaldehyde (DEAB), was used to control for background fluorescence. (c,d) Measurement of all-trans retinal metabolism in CD274high and CD274low subpopulations in RBE (c) and HuCCT1 (d) cells. (e,f) Quantification of the reactive oxygen species (ROS) production in RBE (e) and HuCCT1 (f) subpopulations. Each subpopulation was incubated with or without H2O2, stained with 2', 7'-Dichlorodihydrofluorescin diacetate (DCFH-DA) and subjected to FACS analysis. (g,h) Analysis of cell cycling in the RBE (g) and HuCCT1 (h) subpopulations. CD274low and CD274high cells were fixed with ethanol, stained with Ki-67 FITC and propidium iodide and subjected to FACS analysis. (i,j) Expression profile of stem cell-related genes in CD274high and CD274low cells. mRNA from CD274high and CD274low cells derived from the RBE (i) and HuCCT1 (j) cell lines were analyzed using real-time PCR.
Mentions: We then investigated the CSC-related characteristics of the CD274low and CD274high populations derived from RBE and HuCCT1 cells. They were sorted by anti-CD274 antibody staining and the resulting populations were assessed for ALDH activity using the Aldefluor assay system followed by FACS analysis. The RBE CD274low population was 46.1% ALDH+, whereas the RBE CD274high population was 10.2% ALDH+ (Fig.2a). The HuCCT1 CD274low and CD274high populations showed 52.4% and 38.3% ALDH+, respectively. We then measured the retinal metabolism in vitro, because ALDH metabolizes all-trans retinals to all-trans retinoic acids. The level of retinoic acids produced by the CD274low population was significantly higher than that produced by the CD274high population (Fig.2c,d) in both RBE and HuCCT1 cells. These results are compatible with those of the Aldefluor assay. The CD274low and CD274high populations were further assessed for ROS levels. The ROS levels were significantly lower in the CD274low populations than in the CD274high populations derived from both RBE and HuCCT1 cells (Fig.2e,f). These results indicate that the CD274low populations of both cholangiocarcinoma cell lines predominantly exhibit CSC-related characteristics such as high ALDH1 activity and low ROS production.

Bottom Line: Its malignant phenotypes may be assumed by cancer stem cells (CSC).Furthermore, the CD274(low) cells possess several CSC-related characteristics, such as high aldehyde dehydrogenase (ALDH) activity, reduced reactive oxygen species production and a dormant state in the cell cycle.Furthermore, depletion of CD274 expression by shRNA in RBE cells enhances their tumorigenicity and increases ALDH activity.

View Article: PubMed Central - PubMed

Affiliation: Division of Cancer Biology and Therapeutics, Miyagi Cancer Center Research Institute, Natori, Japan; Department of Cancer Science, Tohoku University Graduate School of Medicine, Sendai, Japan.

Show MeSH
Related in: MedlinePlus