Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Apr;49(4):1277-86.
doi: 10.1002/hep.22743.

CD133+ liver cancer stem cells from methionine adenosyl transferase 1A-deficient mice demonstrate resistance to transforming growth factor (TGF)-beta-induced apoptosis

Wei Ding  1 , Marialena Mouzaki, Hanning You, Joshua C Laird, Jose Mato, Shelly C Lu, C Bart Rountree
Affiliations

CD133+ liver cancer stem cells from methionine adenosyl transferase 1A-deficient mice demonstrate resistance to transforming growth factor (TGF)-beta-induced apoptosis

Wei Ding et al. Hepatology. 2009 Apr.

Abstract

Methionine adenosyltransferase (MAT) is an essential enzyme required for S-adenosylmethionine biosynthesis. Hepatic MAT activity falls during chronic liver injury, and mice lacking Mat1a develop spontaneous hepatocellular carcinoma by 18 months. We have previously demonstrated that CD133(+)CD45(-) oval cells isolated from 16-month-old Mat1a(-/-) mice represent a liver cancer stem cell population. The transforming growth factor beta (TGF-beta) pathway constitutes a central signaling network in proliferation, apoptosis, and tumorigenesis. In this study, we tested the response of tumorigenic liver stem cells to TGF-beta. CD133(+)CD45(-) oval cells were isolated from premalignant 16-month-old Mat1a(-/-) mice by flow cytometry and expanded as five clone lines derived from a single cell. All clone lines demonstrated expression of both hepatocyte and cholangiocyte markers and maintained a small population (0.5% to 2%) of CD133(+) cells in vitro, and three of five clone lines produced tumors. Although TGF-beta1 inhibited cell growth equally in CD133(-) and CD133(+) cells from each clone line, the CD133(+) population demonstrated significant resistance to TGF-beta-induced apoptosis compared with CD133(-) cells. Furthermore, CD133(+) cells demonstrated a substantial increase in mitogen-activated protein kinase (MAPK) pathway activation, as demonstrated by phosphorylated extracellular signal-regulated kinase levels before and after TGF-beta stimulation. MAPK inhibition using mitogen-activated protein kinase kinase 1 (MEK1) inhibitor PD98059 led to a significant increase in TGF-beta-induced apoptosis in CD133(+) cells. Conversely, a constitutively active form of MEK1 blocked the apoptotic effects of TGF-beta in CD133(-) cells.

Conclusion: CD133(+) liver cancer stem cells exhibit relative resistance to TGF-beta-induced apoptosis. One mechanism of resistance to TGF-beta-induced apoptosis in CD133(+) cancer stem cells is an activated mitogen-activated protein kinase/extracellular signal-regulated kinase pathway.

PubMed Disclaimer

Conflict of interest statement

Potential conflict of interest: Nothing to report.

Figures

Fig. 1
Fig. 1
Single-cell isolation for clonal expansion. (A) Schematic representation of single-cell FACS isolation of CD133+CD45− cells from 16-month-old Mat1a−/− mice. On day 1, single cells were plated into individual wells of a 96-well plate. After 21 days, 10/96 wells showed colonies with growth over 50% of the well bottom. Five of these colonies were selected for expansion (CD133+ clone lines 1–5). (B) RT-PCR. In each clone line 1–5, coexpression of Albumin and CK-19 indicated bipotential stem cells, with strong expression of oval cell–associated transcription factors hepatocyte nuclear factor 1α and 3β (HNF1α and HNF3β) and ABCG2 transmembrane pump. Strong expression of growth factor receptors c-Met and epithelial growth factor receptor (EGFr) were also detected by RT-PCR. (C) Western blot. High levels of prominin-1 (CD133) and αFP protein were detected in each clone line (+ control = positive control for Western blot).
Fig. 2
Fig. 2
CD133+ Cancer stem cells. (A) All five CSC clone lines demonstrated anchorage-independent growth in soft agar at 21 days (4× and 20× objective), and (B) three of the five lines formed subcutaneous tumors in nude mice (2 × 106 cells injected, passage 5). Hematoxylin-eosin staining demonstrates tumor histology with mixed hepatoma-like populations of liver epithelial tumors (4× and 40× objectives).
Fig. 3
Fig. 3
CSC clone lines demonstrate growth inhibition in response to TGF-β stimulation. (A) Compared with serum-free baseline conditions, 24-hour TGF-β stimulation results in a significant decrease in cell cycling using bromodeoxyuridine 1-hour pulse (n = 3 lines, each in triplicate, + standard deviation). *P < 0.05. (B) TGF-β stimulation results in similar decrease in cell cycling in all three CSC cell lines. (C) No difference in cell cycle inhibition in response to TGF-β stimulation between C133+ and CD133− cells isolated from each CSC clone line using [3H]-thymidine incorporation. (B, C) Twenty-four-hour TGF-β stimulation, 2-hour [3H]-thymidine pulse, percent inhibition after TGF-β stimulation compared with serum-free controls, each line in triplicate, + standard deviation (P > 0.05 for each line CD133+ versus CD133−).
Fig. 4
Fig. 4
Real-time PCR analysis of CD133+ and CD133− cells in response to TGF-β stimulation. Real-time PCR analysis of TGF-β down-stream targets, p15 and p21, and cell cycle regulators, c-myc and cyclin D1, (A) 4 hours and (B) 12 hours after TGF-β stimulation (5 ng/mL). No significant difference between CD133+ and CD133− cells was detected (2 × 104 cells/cm2 plated, data presented as fold change in expression, n = 3 lines each in triplicate + standard error).
Fig. 5
Fig. 5
CD133+ cells demonstrate resistance to TGF-β–induced apoptosis. (A) Timing of TGF-β–induced apoptosis in CD133+ and CD133− cells with activated caspase-3 levels increasing from 4 to 16 hours using intracellular active caspase-3 FACS analysis (2 × 104 cells/cm2 plated from CSC clone line 1, each time point in triplicate + standard deviation). *P < 0.05. (B) CD133+ cells demonstrate less apoptosis compared with CD133− cells from the same culture plates from each of the CSC clone lines 1–3. Apoptosis determined using annexin V/PI staining and FACS analysis (2 × 104 cells/cm2 plated, each line in triplicate, 50,000 events counted/replicate + standard error). *P < 0.05.
Fig. 6
Fig. 6
Elevated p-Erk in CD133+ cells. (A) Western blot analysis demonstrates elevated p-Erk in CD133+ cells compared with CD133− cells in each of the CSC clone lines 1–3. pan-Erk was used as a loading control. (B) Densitometry analysis of all three CSC lines revealed a significant increase in p-Erk levels in CD133+ cells (2 × 104 cells/cm2 plated, n = 3 lines + standard error). *P < 0.05. (C) Western blot analysis demonstrated that TGF-β stimulation results in a decrease in p-Erk levels, with a smaller decrease in CD133+ cells.
Fig. 7
Fig. 7
Mek1 inhibition reverses survival advantage of CD133+ cells by decreasing p-Erk levels. The MEK1 inhibitor PD98059 was used to block Erk activation. (A) Erk phosphorylation decreased with increasing dose of PD98059. pan-Erk was used as a loading control. (B) Graph of densitometry comparing p-Erk with pan-Erk in CSC cell lines 1 and 3. (C) PD98059 reverses the survival advantages of CD133+ cells. TGF-β stimulation with PD98059 pretreatment resulted in a significant increase in apoptosis in both CD133+ and CD133− cells. Apoptosis measured using annexin V/PI staining and FACS analysis (2 × 104 cells/cm2 plated, n = 3, 50,000 events/replicate + standard error). #1,#2,#3P < 0.05.
Fig. 8
Fig. 8
Forced increase in p-Erk provides a survival advantage in CD133− cells. Adenovirus vectors with β-Gal or CA-MEK1 were used at different MOI. (A) Western blot of p-Erk and pan-Erk demonstrated a marked increase in p-Erk levels in cells infected with CA-MEK1 adenovirus compared with β-Gal adenovirus, with increased levels at higher MOI. (B) Graph of densitometry analysis of p-Erk/pan-Erk ratio demonstrates a marked increase in the p-Erk/pan-Erk ratio with increasing MOI of CA-MEK1 adenovirus. (C) CA-MEK1 confers a survival advantage in CD133− cells after TGF-β stimulation (2 × 104 cells/cm2 plated annexin V/PI FACS analysis, n = 3, 50,000 events/replicate + standard error). #1,#2,#3P < 0.05.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. El-Serag HB. Hepatocellular carcinoma: an epidemiologic view. J Clin Gastroenterol. 2002;35:S72–S78. - PubMed
    1. Llovet JM, Fuster J, Bruix J. Prognosis of hepatocellular carcinoma. Hepatogastroenterology. 2002;49:7–11. - PubMed
    1. Ricci-Vitiani L, Lombardi DG, Pilozzi E, Biffoni M, Todaro M, Peschle C, et al. Identification and expansion of human colon-cancer-initiating cells. Nature. 2007;445:111–115. - PubMed
    1. Gou S, Liu T, Wang C, Yin T, Li K, Yang M, et al. Establishment of clonal colony-forming assay for propagation of pancreatic cancer cells with stem cell properties. Pancreas. 2007;34:429–435. - PubMed
    1. Singh SK, Clarke ID, Terasaki M, Bonn VE, Hawkins C, Squire J, et al. Identification of a cancer stem cell in human brain tumors. Cancer Res. 2003;63:5821–5828. - PubMed

Publication types

MeSH terms