Rizzo S.,Institute of Cancer Research |
Hersey J.M.,Institute of Cancer Research |
Mellor P.,Institute of Cancer Research |
Dai W.,Imperial College London |
And 11 more authors.
Molecular Cancer Therapeutics | Year: 2011
Platinum-based chemotherapy, with cytoreductive surgery, is the cornerstone of treatment of advanced ovarian cancer; however, acquired drug resistance is a major clinical obstacle. It has been proposed that subpopulations of tumor cells with stem cell - like properties, such as so-called side populations (SP) that overexpress ABC drug transporters, can sustain the growth of drug-resistant tumor cells, leading to tumor recurrence following chemotherapy. The histone methyltransferase EZH2 is a key component of the polycomb-repressive complex 2 required for maintenance of a stem cell state, and overexpression has been implicated in drug resistance and shorter survival of ovarian cancer patients. We observed higher percentage SP in ascites from patients that have relapsed following chemotherapy compared with chemonaive patients, consistent with selection for this subpopulation during platinum-based chemotherapy. Furthermore, ABCB1 (P-glycoprotein) and EZH2 are consistently overexpressed in SP compared with non-SP from patients' tumor cells. The siRNA knockdown of EZH2 leads to loss of SP in ovarian tumor models, reduced anchoragein-dependent growth, and reduced tumor growth in vivo. Together, these data support a key role for EZH2 in the maintenance of a drug-resistant, tumor-sustaining subpopulation of cells in ovarian cancers undergoing chemotherapy. As such, EZH2 is an important target for anticancer drug development. ©2010 AACR.
Chen S.W.,Bioprocessing Technology Institute Centros |
Oh S.K.W.,Bioprocessing Technology Institute Centros
Methods in Molecular Biology | Year: 2010
The following protocols provide a rapid approach for establishing good working conditions for transfecting siRNAs for specific gene knockdown. By first using microscopy to evaluate efficient transfection of an inexpensive, fluorescent oligonucleotide, the researcher can later proceed with more expensive Western blot or quantitative real-time PCR (qRT-PCR) methods. Thus, the main culprit of ineffective knockdown, poor transfection, can be eliminated before engaging in tedious and time-consuming approaches for troubleshooting siRNA knockdown experiments. © 2010 Springer Science+Business Media, LLC.