RET proto-oncogene rearrangements (RET/PTC) are considered tumor-initiating events in the development of papillary thyroid carcinoma (PTC). Nevertheless, the role of RET/PTC in determining the aggressiveness or the progression of the tumors is still controversial. Recently, several genetic-clinical correlation studies have shown a better outcome for thyroid neoplasia harboring RET/PTC, implying a "protective" role of RET rearrangements towards thyroid cancer dedifferentiation and aggressiveness. In particular, RET/PTC is generally not found in poorly differentiated and anaplastic thyroid carcinomas. Aim of this work was to determine if RET/PTC expression could impair the aggressive phenotype of undifferentiated human thyroid cancer cells in vitro. For this purpose the WRO cell line, derived from a poorly differentiated human thyroid cancer, was stably transfected with a plasmid containing the RET/PTC1 cDNA under the control of a zinc-inducible metallothionein promoter. Three cell clones (WRO-PTC-1, -2, and -11) were selected by Western blot analysis on the basis of inducible and high expression of RET/PTC1 after treatment with ZnSO4 for 48h. As controls, WRO cells transfected with the empty vector (WRO-NEO) and untransfected parental cells (WRO) were used. Cell proliferation and viability as well as occurrence of apoptotic cell death were evaluated in the different cell lines grown in the presence or absence of ZnSO4 for up to 12 days. ZnSO4-induced activation of RET/PTC1 determined a dramatic reduction of cell growth (from 5 to 20 fold decrease at day 6) in the WRO-PTC-1, -2, and -11 clones compared to control cells (WRO-NEO and WRO). The lower proliferation rate of WRO/PTC clones persisted up to day 12. This effect was associated with a significant decrease in cell viability (WRO-PTC clones, from 56% to 70%; WRO-NEO, 85%; WRO, 92% at day 6). FACS analysis after staining with propidium iodide showed an increase in hypodiploid cell population (indicative of apoptotic cell death) in RET/PTC1 expressing clones compared to control cells (WRO-PTC clones, from 24 to 38%; WRO-NEO, 9%; WRO, 2% at day 2). TUNEL assay confirmed the occurrence of significant apoptosis in the ZnSO4-treated WRO/PTC clones compared to the controls. In conclusion, these data show that "de novo" activation of RET/PTC1 tyrosine-kinase function in poorly differentiated cells strongly impairs their proliferation rate, mainly through the induction of a proliferation block and apoptotic cell death. These results may contribute to explain the rare occurrence of RET/PTC expression in poorly differentiate and anaplastic thyroid carcinomas.
EXPRESSION OF THE RET/PTC1 ONCOGENE INHIBITS PROLIFERATION AND INDUCES APOPTOSIS OF POORLY DIFFERENTIATED HUMAN THYROID CANCER CELLS.
F Moretti;A Farsetti;
2001
Abstract
RET proto-oncogene rearrangements (RET/PTC) are considered tumor-initiating events in the development of papillary thyroid carcinoma (PTC). Nevertheless, the role of RET/PTC in determining the aggressiveness or the progression of the tumors is still controversial. Recently, several genetic-clinical correlation studies have shown a better outcome for thyroid neoplasia harboring RET/PTC, implying a "protective" role of RET rearrangements towards thyroid cancer dedifferentiation and aggressiveness. In particular, RET/PTC is generally not found in poorly differentiated and anaplastic thyroid carcinomas. Aim of this work was to determine if RET/PTC expression could impair the aggressive phenotype of undifferentiated human thyroid cancer cells in vitro. For this purpose the WRO cell line, derived from a poorly differentiated human thyroid cancer, was stably transfected with a plasmid containing the RET/PTC1 cDNA under the control of a zinc-inducible metallothionein promoter. Three cell clones (WRO-PTC-1, -2, and -11) were selected by Western blot analysis on the basis of inducible and high expression of RET/PTC1 after treatment with ZnSO4 for 48h. As controls, WRO cells transfected with the empty vector (WRO-NEO) and untransfected parental cells (WRO) were used. Cell proliferation and viability as well as occurrence of apoptotic cell death were evaluated in the different cell lines grown in the presence or absence of ZnSO4 for up to 12 days. ZnSO4-induced activation of RET/PTC1 determined a dramatic reduction of cell growth (from 5 to 20 fold decrease at day 6) in the WRO-PTC-1, -2, and -11 clones compared to control cells (WRO-NEO and WRO). The lower proliferation rate of WRO/PTC clones persisted up to day 12. This effect was associated with a significant decrease in cell viability (WRO-PTC clones, from 56% to 70%; WRO-NEO, 85%; WRO, 92% at day 6). FACS analysis after staining with propidium iodide showed an increase in hypodiploid cell population (indicative of apoptotic cell death) in RET/PTC1 expressing clones compared to control cells (WRO-PTC clones, from 24 to 38%; WRO-NEO, 9%; WRO, 2% at day 2). TUNEL assay confirmed the occurrence of significant apoptosis in the ZnSO4-treated WRO/PTC clones compared to the controls. In conclusion, these data show that "de novo" activation of RET/PTC1 tyrosine-kinase function in poorly differentiated cells strongly impairs their proliferation rate, mainly through the induction of a proliferation block and apoptotic cell death. These results may contribute to explain the rare occurrence of RET/PTC expression in poorly differentiate and anaplastic thyroid carcinomas.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
