Breast epithelium is capable of completely and functionally
regenerating upon transplantation. This impressive renewal
capacity has been ascribed to the function of a multipotent
mammary gland stem/progenitor cell population that resides
and persists throughout the
Elucidating the role of stem/progenitor cells in preneoplasia
may be crucial to understanding the etiology of breast cancer,
and may lead to better chemopreventative strategies. Mutations
or epigenetic changes either in long-term stem cells or their
immediate progeny, the transit amplifying multipotent
progenitors, have been suggested to be the foundation of
malignancy (Clarke and Fuller, 2006). However, functional
studies of progenitor cells in a preneoplastic model have not
In normal development, the Wnt pathway has been shown
to be important in stem cell survival and maintenance in early
embryogenesis (Wang and Wynshaw-Boris, 2004),
maintaining the stem cell pool in the adult skin (Silva-Vargas
et al., 2005) and intestinal epithelium (Pinto and Clevers,
2005b; Pinto et al., 2003), and in regulating hematopoietic stem
cells in their niche environment (Rattis et al., 2004).
Dysregulation of the Wnt/?-catenin pathways in stem cell
regulation has been proposed to be one of the signaling
pathways responsible for carcinogenesis in the hematopoietic
system, the intestine and the epidermis (Reya and Clevers,
2005). Studies in chronic myelogenous leukemia indicate that
the elevated levels of nuclear ?-catenin exist in a minor
population of progenitor cells, resulting in their enhanced
capacity for self-renewal and increased leukemic potential
(Jamieson et al., 2004). Further evidence of dysregulation of
stem/progenitor cell self-renewal and maintenance by the
Wnt/?-catenin pathway have been demonstrated in lung
cancer, colorectal cancer and gastrointestinal cancer (Brabletz
et al., 2005; He et al., 2005; Mishra et al., 2005; Reya and
Clevers, 2005; Yardy and Brewster, 2005).
Stem cell antigen 1 (Sca1), a marker of hematopoietic stem
cells, is one of the putative markers used to isolate and enrich
for mammary gland progenitors. Previous studies by our
laboratory have demonstrated that primary mammary gland
progenitor cells isolated by using Sca1 lack differentiation
markers, are enriched in label-retaining, relatively quiescent
cells, and have enhanced outgrowth activity compared to the
Sca1–cells (Welm et al., 2003).
The COMMA-D ?-geo (CD?geo) cell line is an
immortalized cell line isolated from the parent COMMA-D
(CD), preneoplastic, mouse mammary cell line by both
transfection and selection using a dominant-selectable gene
transfer vector and by limiting dilution (Deugnier et al., 2006).
Recent studies by Deugnier et al. have demonstrated that the
CD?geo cells contain a permanent population of mouse
mammary epithelial progenitor cells with basal characteristics,
The COMMA-D? ?-geo cell line has been shown to contain
a permanent subpopulation of progenitor cells that are
enriched in outgrowth potential. Using the COMMA-D? ?-
geo cell line as a model, we sought to study the
radioresistance of mammary progenitor cells. Using the
putative progenitor cell marker stem cell antigen 1 (Sca1),
we were able to isolate a discrete subpopulation of Sca1+
multipotent cells from the immortalized COMMA-D? ?-geo
murine mammary cell line. At a clinically relevant dose, the
Sca1+cells were resistant to radiation (2 Gy). Sca1+cells
contained fewer ? ?-H2AX+DNA damage foci following
irradiation, displayed higher levels of endogenous ? ?-
catenin, and selectively upregulated survivin after
radiation. Expression of active ? ?-catenin enhanced self-
renewal preferentially in the Sca1+
suppressing ? ?-catenin with a dominant negative, ? ?-
engrailed, decreased self-renewal of the Sca1+cells.
Understanding the radioresistance of progenitor cells may
be an important factor in improving the treatment of
cancer. The COMMA-D? ?-geo cell line may provide a useful
model to study the signaling pathways that control
mammary progenitor cell regulation.
Key words: ?-catenin, Mammary gland, Stem, Progenitor, Cell line,
COMMA-D, ?-geo, Sca1
Wnt/? ?-catenin mediates radiation resistance of Sca1+
progenitors in an immortalized mammary gland cell
Mercy S. Chen1,*, Wendy A. Woodward2,*, Fariba Behbod1, Sirisha Peddibhotla1, Maria P. Alfaro1,
Thomas A. Buchholz2and Jeffrey M. Rosen1,‡
1Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, M638a Houston, TX 77030-3498, USA
2Department of Radiation Oncology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, USA
*These authors contributed equally to this work
‡Author for correspondence (e-mail: email@example.com)
Accepted 15 November 2006
Journal of Cell Science 120, 468-477 Published by The Company of Biologists 2007
Journal of Cell Science
Radiation resistance of mammary progenitor cells
which express putative stem cell markers such as stem cell
antigen 1 (Sca1), CD24 and CD49f, among others. These
investigators demonstrated that Sca1+cells have at least
fourfold enrichment in outgrowth potential compared with
Sca1–cells. In addition, they have observed that Sca1+cells
were able to form 3D acini on Matrigel, whereas Sca1–cells
grew poorly. Staining for cell-type-specific markers indicated
that the 3D-acini contained keratin 5+(K5+) basal cells, K8+
luminal and ?-SMA+myoepithelial cells within the acinus
(Deugnier et al., 2006).
Herein we report that CD ?-geo cells contain a minor
population of Sca1+cells that are able to self-renew, and
differentiate asymmetrically. In addition, we report that Sca1+
cells sustain less DNA damage as indicated by the formation
of ?-H2AX foci, contain higher levels of non-phosphorylated
or active ?-catenin, and are more resistant to radiation than
Sca1–cells. To date, this is the first report where functional
studies of progenitor cells have been carried out in a
preneoplastic model to explore mechanisms of therapeutic
resistance in the mammary gland.
COMMA-D ?-geo Sca1+cells are capable of self-
renewal and expansion
The CD?geo cell line, originally isolated from the midpregnant
mammary gland of BALB/c mice, has been shown to retain
stem and multipotent progenitor cell characteristics (Danielson
et al., 1984; Deugnier et al., 2006). Because CD?geo cells
retain stem/progenitor cell properties in vivo, we sought to
distinguish self-renewing cells from cells lacking this capacity.
The CD?geo cells are heterogeneous, containing Sca1+and
Sca1–populations. Routinely 13-20% Sca1+cells were
observed in culture (Fig. 1a). Clonogenic assays were used to
determine the replicative
subpopulations. Sca1+cells form 47-fold more colonies than
Sca1–cells (P<0.00002, Fig. 1b). Consistent with these data,
Deugnier et al. have shown that the Sca1–cells are also unable
to form attachments in Matrigel, or give rise to an organized
spheroid structure (Deugnier et al., 2006).
The hallmark of stem and multipotent progenitor cells is the
ability to asymmetrically self-renew thereby maintaining the
progenitor pool and giving rise to differentiated daughter cells.
To examine whether CD?geo cells were able to divide
asymmetrically, cells were first sorted into Sca1+and Sca1–
subpopulations. The original Sca1+cells were able to give rise
to both Sca1+(75±4.2%) and Sca1–cells (24±4.1%) (Fig. 1c).
By contrast, the Sca1–cells were less bipotent, giving rise to
predominantly Sca1–cells (94±1.5%) and very few Sca1+cells
(6±1.45%) (Fig. 1d).
competence of CD?geo
CD?geo cells can self-renew in suspension culture
To further explore the self-renewing potential of CD?geo
progenitor cells, we used the mammosphere in vitro self-
renewal assay described by Dontu et al. (Dontu et al., 2003),
which is based upon the hypothesis that progenitor cells are
able to survive in anchorage-independent conditions. By
retroviral tagging experiments, Dontu et al. showed previously
that mammospheres are clonally derived, and not a result of
aggregation (Dontu et al., 2003). In addition, mammospheres
are now known to comprise a heterogeneous population of
cells, with multipotent mammary stem cells within the core,
surrounded by progenitor cells in various stages of
differentiation. To determine whether CD?geo cells have
mammosphere-forming ability, we plated ten thousand cells on
low adherence plates in serum free medium supplemented with
EGF and bFGF as growth stimulants. Approximately 0.1-0.6%
of the CD?geo cells were able to form mammospheres. Next
we examined the expression patterns of putative stem cell
markers in the secondary mammospheres, such as CD49f,
TIE2, keratin 6 (K6), and epithelial-specific markers keratin 14
(K14) and keratin 18 (K18). In the secondary mammospheres
examined, CD49f was expressed mainly in the center of the
mammosphere (Fig. 2b). K14 staining was also localized in the
center of the mammosphere (Fig. 2d). Both TIE2 and K6 were
randomly distributed throughout the mammospheres (Fig.
2c,e) and luminal K18+cells were not detected (data not
shown). In previous studies, CD49f has been associated with
multi-potent mammary gland progenitors (Stingl et al., 2005),
TIE2 has been shown to identify quiescent hematopoietic stem
cells and is thought to function by maintaining these cells in
the bone marrow niche (Arai et al., 2004). K6 is expressed
within the body cells of the developing mammary terminal end
% of cells
% of cells
Number of Cells
Number of Colonies
Fig. 1. CD?geo Sca1+cells are capable of self-renewal and
expansion. (a) CD?geo cells were stained with FITC-conjugated
antibody against Sca1, and analyzed by flow cytometry. Two
populations can be distinguished based on Sca1 fluorescence: Sca1+
13.1%; and Sca1–67.4%. (b) Cells were sorted into Sca1+and Sca1–
populations directly into 96-well plates containing growth-factor-
reduced Matrigel at 500 cells/well, and clones were counted after 10
days. The Sca1+cells gave rise to 47±14 colonies in Matrigel,
whereas the Sca1–produced no colonies. Colonies were stained with
Crystal Violet and counted by two independent researchers.
(c) CD?geo Sca1+population give rise to 75±4.2% Sca1+and
24±4.1% Sca1–cells. (d) CD?geo Sca1–cells remained mostly Sca1–
after culture, and gave rise to 94±1.5% Sca1–cells and only 6±1.45%
Journal of Cell Science
Radiation resistance of mammary progenitor cells
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