行政院國家科學委員會專題研究計畫成果報告:細胞凋亡調控基因(sFRP2)在犬乳腺腫瘤之細胞凋亡調控角色及相關調節因子的研究(3/3)
Date Issued
2005
Date
2005
Author(s)
DOI
932313B002022
Abstract
Mammary neoplasms are important and
common tumors in both animals and humans and
the etiology is complex. The secreted frizzled
related proteins (sFRPs) are newly identified
proteins and implicated to have dual roles of
modulation of Wnt-Frizzled signal transduction
pathway and regulation of apoptosis. We have
recently found that sFRP2 was expressed
abundantly in human and canine mammary gland
tumors (MGT) tissues but was undetectable in
normal canine mammary gland. To systematically
investigate the functional roles and molecular
mechanisms of sFRP2 in canine MGT, several
strategies are to be carried out as described below.
The project is comprised of six major parts for a
period of 3 years: In the 1st year, new primary cell
cultures from native canine MGT tissues has been
established and purified for mammary epithelial
cells. We have successfully established and
analyzed more native primary MGT cell lines
from surgically excised MGT specimens. The
cells are characterized for their cell origins,
proliferation rate (by MTT assay), expression of
sFRP2 by RT-PCR, in situ hybridization, and
immunohistochemistry, and Western blotting.
Expression experiments revealed the sFRP2 was
abundantly expressed in canine MGT cell lines,
but not expressed in normal canine MG cells nor
other commercial non-MGT cell lines (previous
NSC project, published in the Breast Cancer
Research and Treatment). Canine sFRP2 is cloned
into a mammalian expression vector with GFP
reporter gene and CMV promoter. The
GFP-sFRP2 is stably transfected into primary
canine MGT and commercial MGT cell lines by
lipofection for further analysis from the next stage
of the project.
In the 2nd year, apoptosis regulation
mediated by SFRP2 was investigated by
overexpression of SFRP2 in MGT and MCF7
cells. DNA fragmentation and caspase 3 activity
analyses showed that the susceptibility of the cells
to UV-induced apoptosis decreased in the context
of SFRP2 overexpression. To analyze the
pathways through which SFRP2 transduces
anti-apoptosis signals, co-immunoprecipitation
and cell adhesion assays were carried out. SFRP2
was found secreted from cells and associated with
the fibronectin-integrin protein complex and
could promote cell adhesion. Moreover, by using
heparin to block the SFRP2-fibronectin
interaction or anti-integrin 51 antibody to
interrupt the fibronectin-integrin connection, the
anti-apoptosis activity of SFRP2 was decreased.
Taken together, these results suggest that SFRP2
exert its anti-apoptotic function in mammary
cancer cells through association with the
fibronectin-integrin signal transduction pathway,
not the Wnt signaling as previous thought. The
important data has been published. In the 3rd year,
analysis of the relation of sFRP2 transduced
anti-apoptosis with other signaling and
transcription factors, multiple-color
immunofluorescence staining, immunoprecipitation,
and immunoblotting were carried out.
SFRP2 was found co-localized in the extracellular
matrix of MGTs and the tyrosine phosphorylation
of FAK was enhanced. Moreover, JNK was
suppressed and NF-kB was activated in the cells
expressing SFRP2 after UV-induced apoptosis
analyzed by immunoblotting and electrophoretic
mobility shift assay (EMSA). Taken together,
these results suggest that SFRP2 exerts its
anti-apoptotic function in mammary cancer cells
with NF-κB activation or JNK suppression.
The results of this project should offer
important scientific basis and information to
understand the roles of sFRP-mediated signaling
in canine mammary tumor cells. It also provides a
basis for further analysis of functions of different
members of the sFRP gene family and elucidation
of the complex etiology and signaling pathways
of mammary tumors.
Subjects
secreted apoptosis related protein
secreted frizzled related protein
secreted frizzled related protein
apoptosis
signal
transduction
transduction
gene transfection
mammary
neoplasia
neoplasia
SDGs
Publisher
臺北市：國立臺灣大學獸醫學系暨研究所
Type
report
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