Upload
nelson-duane-walton
View
213
Download
0
Tags:
Embed Size (px)
Citation preview
Increased Radiation Resistance of Mouse Mammary Side Population Stem/Progenitor Cells: Role of Wnt/ ß -Catenin Signaling
Wendy A. Woodward MD-PhD, Mercy S. Chen BS, Thomas A. Buchholz MD, and Jeffrey Rosen PhD U.T. M.D. Anderson Cancer Center and Baylor College of Medicine
INTRODUCTION
Mammary Stem Cells:
•Long replicative potential
•Capacity to self-renew and proliferate
•Attractive candidate for cell origin of cancer
•Required in adult mammary gland –Fulfill demands of pregnancy-dependent epithelial turnover (tissue renewal)–Respond to damage (tissue repair)
Hypothesis:
•Breast cancers arise from cancer stem cells, which may be resistant to conventional therapy, and are therefore a critical determinant of recurrence
Rationale:•Understanding the pathways involved in stem cell survival may identify new targets for molecular therapeutics
METHODS Primary mouse mammary epithelial cells (MECs) and immortalized Comma D (CD) mammary epithelial cells were irradiated in 60 mm culture dishes using a 137cesium cell irradiator. MECs were isolated from 6-8 week old wild type Balb/c mice, from mice containing a floxed exon III ß-catenin allele, which generates stabilized ß-catenin upon excision in culture with an adenovirus-driven Cre recombinase, from transgenic Wnt-1mice with mammary hyperplasias, from wild type (WT) MECs in mice of the same background as Wnt-1 transgenic mice. MECs were maintained in stem cell promoting media after plating for 2 days and irradiated on day 4. On day 5 cells were trypsinized and stained for 60 min with Hoechst dye prior to analysis using flow cytometry. CD cells were transduced with control ß-galactosidase, amino-terminal, stabilized ß-catenin, or dominant-negative ß-engrailed retroviruses. CD cells were transplanted into the cleared fat pads of Balb/c mice 48h after transduction, and outgrowths were biopsied after 8 weeks.
RESULTS:•Radiation increases the proportion of stem-like progenitor cells in a mouse mammary cell lines, CommaD cells (CD), and in primary mammary epithelial cells(MECs)•Radiation leads to a greater increase in stem-like progenitor cells in CD cells and MECs expressing stabilized ß-catenin•Outgrowths from transplanted CD cells expressing stabilized ß-catenin fill more of the fat pad compared to outgrowths from CD cells expressing ß-galactosidase, but form disorganized and hyperplastic appearing outgrowths. Outgrowths from CD cells expressing ß-engrailed appear to lack myoepithelial cells
Fig. 2 Floxed exon III ß-catenin allele generates stabilized ß-catenin upon excision in culture with an adenovirus-driven Cre recombinase (A). Clinically relevant doses of radiation lead to a greater increase the percentage of side population cells (stem-like progenitors) in primary mouse mammary epithelial cells expressing stabilized ß-catenin than in control cells (B).
Fig. 3 Clinically relevant doses of radiation lead to a greater increase the percentage of side population cells (stem-like progenitors) in primary mouse mammary epithelial cells from transgenic Wnt-1 mice with mammary hyperplasias.
Fig. 1 Clinically relevant doses of radiation increase the percentage of side population cells (stem-like progenitors) in primary mouse mammary epithelial cell culture. Magnitude of increase varies by mouse strain.
CONCLUSIONS:• Irradiation of immortalized cells and primary mammary epithelial cells increases the percentage of stem-like cells, likely due to stem cell radioresistance relative to the differentiated cells
•Expression of ß-Catenin, a stem cell survival factor, further increases the percentage of radiation-induced stem-like cells and promotes mammary outgrowth from immortalized cells
Stable CD
0
0.5
1
1.5
2
2.5
3
0 2 4 6
Dose (Gy)
Norm
ali
zed
SP
fold
ch
an
ge
A.
B.
A.
B.
100 101 102 103 104
FL2
0
500
1000
1500
# C
ells
66.1
100 101 102 103 104
FL2
0
500
1000
1500
# C
ells
77.4
5’ LTR +
-cateninmut
IRES-GFP3’ LTR
Amp
pMSCV- -cateninmut - IRES - GFP
pMSCV- IRES - GFP
5’ LTR +
GFP
IRES-GFP3’ LTR
Amp
C.
Fig 4. CD cells were transduced with MSCVvector containing IRES-GFP or a stabilized ß -cateninand IRES-GFP(A). Cells were sorted by flow cytometryand positive cells were cultured to enrich for GFP+ cells (B). Clinically relevant doses of radiation lead to a greater increase the percentage of side population cells (stem-like progenitors) in primary mouse mammary epithelial cells expressing stabilized ß-catenin than in control cells (C).
Figure 5. 10,000 CD cells transduced with stabilized ß-galactoidase ß -catenin or ß -engrailed were transplanted into cleared mammary fat pads. ß -catenin outgrowths were larger and filled more of the fat pad at 8 weeks. ß -Galactosidase outgrowths were roughly normal appearing on H&E sections. ß -Catenin outgrowths contain normal branchingand ductal elements but were highly disorganized and contain abnormal areas of undifferentiated cells (inset). ß -engrailed outgrowths were small and characterized by marked dilation of of the ducts with irregular or absent myoepithelial cells(inset: immunohistochemistry of same section with smooth muscle actin antibody).
ß-Galactosidase ß -Catenin ß -Engrailed (Dominant negative)
Harada et al., EMBO J . 18:5931, 1999
Baylor cow parade M. D. Anderson Cancer Center
WT MEC
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
0 2 4 6
XRT Dose (Gy)
Fold
In
cre
ase
BalbCC57/Black
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 2 4 6
Dose (Gy)
%S
P AdCreAdLacZ
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 2
Dose (Gy)
%S
P WT FVBWNT