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
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