行政院國家科學委員會補助專題研究計畫期中進度報告: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(2/3)
Date Issued
2004
Date
2004
Author(s)
DOI
922313B002133
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
3
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.
To elucidate the role of SFRP2 in the
tumorigenesis of MGTs, 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 α5β1 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 and accepted by the Journal of
Biological Chemistry (impact factor: 7.0).
The results of this stage of the project should
offer important scientific basis and information to
understand the roles of sFRP gene family in canine
neoplastic 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
neoplasi
neoplasi
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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