Submitted on May 1, 2008
Revised on July 2, 2008
Accepted on July 2, 2008
Characterization of the Hoechst33342 side population from normal and malignant human renal epithelial cells
Sanjai K Addla1, Michael David Brown1*, Claire A Hart1, Vijay AC Ramani2, and Noel W Clarke2
1 Paterson Institute for Cancer Research
2 Christie Hospital NHS foundation Trust
* To whom correspondence should be addressed. E-mail: mbrown{at}picr.man.ac.uk.
The fundamental changes which predispose for Renal Cell Carcinoma (RCC) are poorly characterised. It is hypothesised that "cancer stem cells" may be influential in carcinogenesis and the epithelial side population (SP) is enriched for stem-like cells in other epithelial cancers. In this study, we have isolated and characterised the SP and non-SP (NSP) populations from normal (NK) and malignant (RCC) human kidney tissue. NK specimens were taken from patients undergoing non-renal cancer surgery and paired malignant and macroscopically normal tissue samples were taken from patients undergoing surgery for RCC. The Hoechst 33342 dye efflux technique was used to isolate epithelial SP and NSP from normal and malignant human renal tissue. Cellular subpopulations were phenotyped for lineage, cell cycle and putative stem cell markers, and functionally characterised using in vitro colony forming and proliferation assays. The SP constituted 3.8±0.4% and 5.9±0.9% of epithelial cells in NK and RCC respectively, of which 14.1±3.5% and 13.2±3.6% were shown to be in G0. SP cells demonstrated greater proliferative potential in colony forming efficiency, long-term culture and spheroids assays, and were shown to be maintained upon tissue culture passage. We have shown that the renal SP is enriched for quiescent cells, with a high proliferative capacity and stem-like properties. The population is however heterogeneous, confirming that the terms SP cell and stem cell cannot be used interchangeably.
