Microarrays of 41 human tumor cell lines for the characterization of new molecular targets: expression patterns of cathepsin B and the transferrin receptor.
ABSTRACT In recent years the use of the microarray technology has allowed the identification of numerous cancer-related genes and proteins. Today anticancer drug discovery is mainly target driven, which requires the characterization of molecular targets in existing cell lines or xenograft models. However, this analysis is time consuming and labor intensive. In order to ease this bottleneck, we have established tissue microarrays of 41 human tumor cell lines on glass slides. The purpose of our study was to (a) establish a simple and efficient method for cell line microarray construction, (b) apply the resulting array to the profiling of cathepsin B and transferrin receptor by immunohistochemistry and (c) verify the results in separate Western blot analyses. Ten to twenty million (1-2 x 10(7)) cells were harvested without trypsinization and fixed with Bouin's solution containing 8% formalin. Cell pellets 2-5 mm in diameter were embedded in paraffin. Microarrays were assembled using a tissue arrayer. Pellet biopsies 0.6 cm in diameter were taken and arrayed in duplicate in a new recipient paraffin block. About 60 slides can be obtained from one block. Citrate buffer was used for antigen retrieval. Expression of cathepsin B was granular and located in the cytoplasm. High cathepsin B levels were detected in 2 melanomas (MEXF 514L and MEXF 276L) and in the renal cell line RXF 486L. Twenty-five cell lines showed only minimal positivity. Nine cell lines of leukemia and lymphoma, breast, ovarian, prostate and renal cancer origin were positive for the transferrin receptor, while 32 cell lines were negative. Western blotting confirmed the results obtained by immunohistochemistry. Using these cell line microarrays, cell lines overexpressing a target of interest can be selected for in vitro evaluation of specific inhibitors.
Article: Role of transferrin receptor and the ABC transporters ABCB6 and ABCB7 for resistance and differentiation of tumor cells towards artesunate.[show abstract] [hide abstract]
ABSTRACT: The anti-malarial artesunate also exerts profound anti-cancer activity. The susceptibility of tumor cells to artesunate can be enhanced by ferrous iron. The transferrin receptor (TfR) is involved in iron uptake by internalization of transferrin and is over-expressed in rapidly growing tumors. The ATP-binding cassette (ABC) transporters ABCB6 and ABCB7 are also involved in iron homeostasis. To investigate whether these proteins play a role for sensitivity towards artesunate, Oncotest's 36 cell line panel was treated with artesunate or artesunate plus iron(II) glycine sulfate (Ferrosanol). The majority of cell lines showed increased inhibition rates, for the combination of artesunate plus iron(II) glycine sulfate compared to artesunate alone. However, in 11 out of the 36 cell lines the combination treatment was not superior. Cell lines with high TfR expression significantly correlated with high degrees of modulation indicating that high TfR expressing tumor cells would be more efficiently inhibited by this combination treatment than low TfR expressing ones. Furthermore, we found a significant relationship between cellular response to artesunate and TfR expression in 55 cell lines of the National Cancer Institute (NCI), USA. A significant correlation was also found for ABCB6, but not for ABCB7 in the NCI panel. Artesunate treatment of human CCRF-CEM leukemia and MCF7 breast cancer cells induced ABCB6 expression but repressed ABCB7 expression. Finally, artesunate inhibited proliferation and differentiation of mouse erythroleukemia (MEL) cells. Down-regulation of ABCB6 by antisense oligonucleotides inhibited differentiation of MEL cells indicating that artesunate and ABCB6 may cooperate. In conclusion, our results indicate that ferrous iron improves the activity of artesunate in some but not all tumor cell lines. Several factors involved in iron homeostasis such as TfR and ABCB6 may contribute to this effect.PLoS ONE 02/2007; 2(8):e798. · 4.09 Impact Factor