AN Immunohistochemical, STUDY OF SURVIVIN EXPRESSION IN NORMAL AND IN TRANSFORMED CELLS
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Date
2006-04
Authors
ALIAS, ISKANDAR ZULKARNAIN
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Abstract
Survivin is a new member of the inhibitors of apoptosis protein (IAP) family, selectively
over-expressed in common human cancers but not in normal adult tissues. It is also
expressed in cancer cell lines. The study was performed generally to investigate the
basic and clinical roles of survivin in normal and transformed cells. Rabbits were
immunized with two synthetic oligopeptides, MGAPTLPP A WQP and
KEFEET AKKVRRAIEQLAAMD amino acids sequences of the survivin molecule.
Serum antibodies were purified by ammonium sulphate and caprilic acid and their
specificities were confirmed by immunoblo~ing and pre-absorption tests against
survivin positive tissues or synthetic survivin oligopeptides. These antibodies were used
to detect survivin in normal and transformed cells by immunohistochemistry in
formalin-fixed paraffin embedded tissue sections, evaluated by a standard scoring
system and chequerboard analysis. Normal cells were obtained frqm fetal and adult
tissues of mouse and rat whilst the transformed cells were obtained from the human
breast cancer cell line MCF-7 and the infiltrating ductal carcinoma (IDC) of the breast
patients. In the MCF-7 cell line experiment, the effects of chemotherapeutic drugs
namely doxorubicin, 5-fluorouracil, cyclophosphamide, and tamoxifen on the apoptosis
index measured by propidium iodide and acridine orange dyes. The relative cell viability
was measured by an MTT assay and survivin expression was measured by
immunocytochemistry. In IDC patients (n=382), survivin expression in tissues was
analyzed for its correlation with clinicalpathological factors, hormonal status, p53, bcl-2
and the survival rate. Patients and their tissue blocks were obtained from three general
hospitals in The East Coast of Malaysia. Autoantibodies to survivin were also
investigated in the sera of the same IDC patients population (n=57) and were compared
to the control population (n=44). For the immunohistochemistry assay, four rabbit
antiserum were produced and tested against survivin. The results of this study indicated
that the antigen retrieval buffer, pH 9 was superior than pH 6 and optimization
immunohistochemistry was obtained by chequerboard analysis. Furthermore, it was
found that survivin is expressed abundantly in normal growing fetal cells but not in
normal differentiated adult tissues of mouse and rat. In the MCF-7 cell line, the cell
viability was reduced in a dose-dependent pattern when incubated with the drugs. The
ICso estimation in MCF-7 cell line for doxorubicin was 6.0 Ilg/ml, cyclophosphamide
171.1 Ilg/ml, 5-fluorouracil 0.611lg/ml, and tamoxifen 0.71lg/ml, respectively. It was
found that most of the MCF-7 cells expressed survivin, predominantly in the cytoplasm.
The percentages of apoptotic celli; were increased with the increased concentrations of
the drugs. Among the IDe patients, the expression of survivin was 68.1 %, p53 29.6%,
and bcl-2 43.7%, respectively. There was a significant correlation (p<0.05) between
survivin expression and lymph node involvement, tumour sizes, p53, bcl-2 expression,
and survival rate among the IDC patients. Anti-survivin autoantibodies reactivities were
detected in 7% of the sera of IDC patients but not in normal sera. These autoantibodies
correlated with the positivity of survivin expression, and with advanced breast cancer. It
was concluded that survivin was abundantly and prominently expressed during fetal
development of rat and mouse. The polyclonal antibody SUR12A-CFI recognized rat
and mouse survivin. It was also concluded that survivin is frequently over-expressed in
IDC patients, and in most MCF-7 cells. Survivin expression has a predictive value in
predicting the aggressiveness of the tumour cells suggesting that survivin may be a
useful tool in assessing a prognosis.
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AN Immunohistochemical, STUDY OF SURVIVIN EXPRESSION , IN NORMAL AND IN TRANSFORMED CELLS