Article Figures & Data
Figures
- Fig. 1.
LMP1-expressing cells exhibit a CD44high CD24low profile associated with stem cells. (A) KR4 is an EBV-positive type III lymphoblastoid cell line. KH1 and KH2 are type II cell lines derived from the fusion of KR4 and HeLa cells. Levels of LMP1 expression by these cell lines were revealed by Western blotting. (B) FACS analysis of the cell surface markers CD44 and CD24 in these cells. (C) (Left) CD44high CD24low cells and CD44low CD24high cells were separated by FACS. (Right) Phase-contrast images of tumor spheres seeded by CD44low CD24high (top) and CD44high CD24low (bottom) cells. (D) Quantification of tumor spheres formed by cells from the FACS-separated CD44high CD24low, CD44high CD24high, and CD44low CD24high populations of KH1 cells. The data are mean numbers of tumor spheres per 2,000 seeded cells ± standard deviations (P < 0.001).
- Fig. 2.
LMP1-expressing nasopharyngeal epithelial cells express the CD44high CD24low phenotype. (A) Morphological changes in newly established AdAH nasopharyngeal epithelial cell lines expressing retroviral vector pFB-Neo or pFB-LMP1 in standard growth medium. Phase-contrast images of representative clones are shown. Magnification ×100. (B) FACS analysis of the cell surface markers CD44 and CD24 in the cells described in the legend to panel A. (C) AdAH cells expressing pFB-Neo or pFB-LMP1 were analyzed by Western blotting for CD44, CD24, LMP1, and tubulin. (D) Quantification of tumor spheres formed by AdAH cells with or without LMP1 expression. The data are mean numbers of tumor spheres per 2,000 seeded cells ± standard deviations (P < 0.001).
- Fig. 3.
LMP1 induces rapid cell growth and high anchorage-independent growth. (A) Growth curves of AdAH cells expressing pFB-Neo or pFB-LMP1. The proliferation activities of LMP1 clones were significantly higher than those of the controls (P < 0.05). (B) CFSE intensity as measured by FACS at day 0 (red lines), day 2 (blue lines), and day 4 (green lines) in AdAH-pFB-Neo and AdAH-pFB-LMP1 cells. (C) Quantification of the anchorage-independent growth of AdAH cells expressing pFB-Neo or pFB-LMP1. The data are mean numbers of colonies per field ± standard deviations (P < 0.001).
- Fig. 4.
LMP1 silencing reduced the CD44high CD24low profile and cellular proliferation in an EBV-positive NPC cell line. (A) Analysis of LMP1 transcripts by RT-PCR in C666-1 cells transfected with either control siRNA or LMP1 siRNA. β-Actin was used as a loading control. (B) FACS analysis of the cell surface markers CD44 and CD24 in C666-1 cells transfected with either control siRNA or LMP1 siRNA. (C) Quantification of the anchorage-independent growth of C666-1 cells with or without LMP1. The data are mean numbers of colonies per field ± standard deviations (P < 0.001).
- Fig. 5.
LMP1 induces the epithelial-mesenchymal transition. (A) Expression levels of mRNAs encoding Twist, Snail, E-cadherin (E-Cad), N-cadherin (N-Cad), fibronectin (Fibro), and vimentin (Vim) in AdAH-pFB-LMP1 cells relative to those in AdAH-pFB-Neo cells as determined by real-time PCR. GAPDH mRNA was used to normalize the variability in template loading. The data are means ± standard deviations. (B) Western blot analysis of expression of Twist, Snail, E-cadherin, N-cadherin, fibronectin, and vimentin in AdAH cells expressing pFB-Neo or pFB-LMP1. Tubulin was used as a loading control. (C) Immunofluorescence images of mock-expressing (left) or LMP1-expressing (right) cells stained using antibodies against LMP1, Twist, or Snail. (D) Immunofluorescence images of mock-expressing (left) or LMP1-expressing (right) cells stained using antibodies against E-cadherin, vimentin, fibronectin, or N-cadherin.
- Fig. 6.
LMP1 induces several CSC surface markers and undifferentiated cytokeratins (CKs). (A) Nasopharyngeal epithelial cells stably expressing LMP1 express several CSC surface markers. AdAH-pFB-Neo and AdAH-pFB-LMP1 cells were analyzed for the expression of cell surface markers. (B) LMP1 expression leads to changes in cytokeratin profiles to an undifferentiated status. Shown are images of AdAH cells with or without LMP1 immunostained with antibodies against CK14, -18, and -19. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). (C) Western blot analysis of AdAH-pFB-Neo and AdAH-pFB-LMP1 cells. CK14, CK18, CK19, and LMP1 were analyzed. Τubulin was used as a loading control.
- Fig. 7.
LMP1 induces phenotypes of cancer stem/progenitor cells. (A) Expression levels of mRNAs encoding ABCG2, Nanog, nestin, Oct4, KLF4, and c-Myc in AdAH-pFB-LMP1 cells relative to those in AdAH-pFB-Neo cells as determined by real-time PCR. GAPDH mRNA was used to normalize variability in template loading. The data are means ± standard deviations. (B) Western blot analysis of AdAH-pFB-Neo and AdAH-pFB-LMP1 cells. ABCG2, Nanog, nestin, Oct4, KLF4, c-Myc, and LMP1 were analyzed. Τubulin was used as a loading control. (C) Flow cytometric profiles of SP cells in AdAH-pFB-LMP1 and AdAH-pFB-Neo cultures. SP cell profiles in the presence or absence of verapamil are shown. Percentages of SP cells are given.
Tables
- Table 3.
Hypothetical model: LMP1 induces cancer progenitor cells but not primitive cancer stem cells
Characteristic Phenotypea Cancer stem cells Cancer progenitor cells Cancer cells Cell surface marker(s) ABCG2− CD44+ ABCG2+ CD133+? integrin α2β1+ CD44+ ABCG2− Stem cell-like genesb High level Low level None Self-renewal capacity High Low None Proliferative potential High Low None Cell cycling Slow Rapid None Regulator Other EBV products, LMP2A? LMP1
