行政院國家科學委員會補助專題研究計畫成果報告:犬視網膜細胞凋亡調控基因(SFRP基因族)之選殖，及在犬正常組織及乳腺腫瘤細胞之表現及細胞凋亡變化之分析(2/2)

The secreted apoptosis related protein (also
named secreted frizzled related protein, sFRP)
family is implicated to have dual roles of
modulation of Wnt-Frizzled signal transduction
pathway and regulation of apoptosis. The
Wnt-Frizzled signaling plays an important role in
normal development and oncogenesis, particularly
in mammary neoplasia. Therefore, members of the
sFRP gene family are good candidates to
investigate their roles in apoptosis regulation and
mammary tumors of canine species.
The project is comprised of three major parts:
at the first stage, primary canine mammary gland
tumor (MGT) cell lines were established from
freshly excised MGT specimens. An aliquot of
tumor tissues was rinsed by sterile PBS and kept
in DMEM medium supplemented with 10% fetal
calf serum for primary cultures. We have
successfully established more native primary MGT
cell lines from surgically excised MGT specimens.
At the second stage, the primary MGT cell
lines were analyzed for mRNA and protein
expression of sFRPs. RNA was extracted from
MGT cells. extraction. Expression analysis
included mRNA in situ hybridization,
immunohistochemistry, RT-PCR, and northern
blotting hybridization. Expression revealed the
sFRP2 was abundantly expressed in canine MGT
cell lines, but not expressed in normal canine MG
cells nor human breast cancer cell line MCF7.
At the third stage, primary MGT cell lines
have been prepared for gene transfection. sFRP
genes were transfected into MGT cells using a
mammalian expression vector by lipofection and
UV-induced apoptosis (25 J/m2) was performed.
The apoptosis activity was investigated in
sFRP-transfected MGT and MG cells,
untransfected MGT and MG cells, and human
breast cancer cell line MCF7, respectively. Results
showed the sFRP2 transfected MGT cells
possessed marked anti-apoptotic activity,
compared to cells from untransfected MGT cells,
and normal MG cells.
The results of the project should offer
important and novel scientific information to
understand the roles of sFRP gene family in
apoptosis control of canine normal and 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 of
canine model of mammary tumors.