Abstract
PurposeTo determine the impact of exogenous transforming growth factor beta 1 (TGF-β1) on side population (SP) cells isolated from normal, papillary thyroid cancer and anaplastic thyroid cancer cell lines and from human thyroid tissues.MethodsAll cell populations were stained with Hoechst 33342 and analysed using dual wavelength flow cytometry to identify SP cells. This SP assay was used to assess the impact of TGF-β1 treatment and withdrawal of treatment on SP percentages. Semi-quantitative and quantitative PCR were used for molecular analysis of cells pre and post TGF-β1 treatment.ResultsAll cell lines expressed mRNA for both TGFB1 and its receptors, as well as showing variable expression of CDH1 and CDH2, with expressing of CDH1 being highest and CDH2 being lowest in the normal cell line. Exposure to exogenous TGF-β1 resulted in a reduction in mRNA expression of ABCG2 compared to controls which was significant between control and treated cancer cell lines. SP cells were isolated from primary human thyroid tissues, with numbers being significantly higher in papillary thyroid cancers. Exposure to TGF-β1 decreased the SP percentage in both thyroid cancer cell lines and completely abrogated these cells in the primary papillary thyroid cancer cultures. On withdrawal of TGF-β1 the SP phenotype was restored in the cancer cell lines and SP percentages increased to above that of untreated cells.ConclusionsTGF-β1 exposure transiently regulates thyroid cancer SP cells, leading to a reduction in SP percentages, while withdrawal of TGF-β1 results in restoration of the SP phenotype.
Highlights
We have previously reported on the presence of side population (SP) cells in the normal thyroid cell line N-thy ori-3-1, papillary thyroid cancer (PTC) cell line BCPAP and the ATC cell line SW1736 and reported that these cancer SP cells are responsive to hypoxia, with an increase in cell number upon hypoxia exposure [14]
We examined 4 different thyroid tissue samples for mRNA expression of ABCB1 and ABCG2 and all expressed both transporters, expression was higher in all cases for ABCG2 (Fig. 4H) SP data for each individual tissue analysed and patient information is provided (Table 2)
We have shown that we can isolate SP cells from primary cell cultures derived from normal thyroid tissue (NT), benign diseased thyroid tissue (BT) and PTC
Summary
TGF-β signalling and/or hypoxia have been reported to drive epithelial to mesenchymal transition (EMT) in many cancers contributing to cancer progression [2, 3]), including thyroid cancers were EMT makers have been found to be overexpressed in more aggressive and metastatic thyroid tumours in vivo and in vitro [4,5,6,7,8]. TGF-β1 has been reported to increase the expression of HMGA1 at both the gene and protein level in the thyroid cancer cell line SW579. While HMGA1 has been reported to be involved in the progression of several types of cancers including thyroid, this study supports a role for TGF-β1 in its induction [9].
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