are uncertain of the cause for this discrepancy although this may be due to technical differences between laboratories, differences in cell lines or differences in the duplex sequence used for gene knockdown. CD133 shows plasticity of expression in CRC cell lines We used FACS to sort SW480 into pure CD133+ and CD1332 populations and, by mRNA quantification, showed that this was due to transcriptional repression rather than changes in glycosyl- ation. The mechanistic basis of inhibiting CD133 expression is uncertain. Epigenetic silencing due to hypermethylation of CD133 promoter has been reported as a means of controlling CD133 expression [31]. Alternatively other mechanisms such as mRNA degradation may be involved. Prolonged culture of pure CD133+/ CD1332 populations resulted in both populations reverting to a bimodal pattern although in neither case did the pattern revert to that of the original cell line. Thus, after 3 months, the cultured pure CD133+ population developed a CD133+/CD1332 split of 70%/30% respectively. These data are consistent with other studies showing that tumours derived from CD133+ populations ultimately become bimodal. However, it is uncertain why the CD133+ population did not revert to 40% as seen in the original SW480 cell line. The cultured pure CD1332 population developed a stable CD133+ population of around 17%. The study by O’Brien found that CD1332 sorted populations could induce tumours containing a CD133+ population and attributed this to contamination of the CD1332 cells by CD133+ cells. We think that contamination is unlikely to be an explanation in our case as our time-course studies References 1. Dalerba P, Cho RW, Clarke MF (2007) Cancer Stem Cells: Models and Concepts. Annual Review of Medicine 58: 267–284. 2. Reya T, Morrison SJ, Clarke MF, Weissman IL (2001) Stem cells, cancer, and cancer stem cells. Nature 414: 105–111. 3. Alison MR, Islam S (2009) Attributes of adult stem cells. The Journal of Pathology 217: 144–160. 4. Michor F, Hughes TP, Iwasa Y, Branford S, Shah NP, et al. (2005) Dynamics of chronic myeloid leukaemia. Nature 435: 1267–1270. 5. Singh SK, Clarke ID, Terasaki M, Bonn VE, Hawkins C, et al. (2003) Identification of a Cancer Stem Cell in Human Brain Tumors. Cancer Res 63: 5821–5828. 6. Yuan X, Curtin J, Xiong Y, Liu G, Waschsmann-Hogiu S, et al. (2004) Isolation of cancer stem cells from adult glioblastoma multiforme. Oncogene 23: 9392–9400. 7. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF (2003) Prospective identification of tumorigenic breast cancer cells. Proceedings of the National Academy of Sciences of the United States of America 100: 3983– 3988. 8. O’Brien CA, Pollett A, Gallinger S, Dick JE (2007) A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature 445: 106–110. 9. Ricci-Vitiani L, Lombardi DG, Pilozzi E, Biffoni M, Todaro M, et al. (2007) Identification and expansion of human colon-cancer-initiating cells. Nature 445: 111–115. 10. Tetsuhiro C, Kaoru K, Yun-Wen Z, Osamu Y, Hiromitsu S, et al. (2006) Side population purified from hepatocellular carcinoma cells harbors cancer stem cell-like properties. pp 240–251. 11. Li C, Heidt DG, Dalerba P, Burant CF, Zhang L, et al. (2007) Identification of Pancreatic Cancer Stem Cells. pp 1030–1037. 12. Addla SK, Brown MD, Hart CA, Ramani VAC, Clarke NW (2008) Characterization of the Hoechst 33342 side population from normal and malignant human renal epithelial cells. pp F680–687. 13. Wright M, Calcagno A, Salcido C, Carlson M, Ambudkar S, et al. (2008) Brca1 breast tumors contain distinct CD44+/CD24- and CD133+ cells with cancer stem cell characteristics. pp R10. 14. Yin AH, Miraglia S, Zanjani ED, Almeida-Porada G, Ogawa M, et al. (1997) AC133, a Novel Marker for Human Hematopoietic Stem and Progenitor Cells. pp 5002–5012. 15. Miraglia S, Godfrey W, Yin AH, Atkins K, Warnke R, et al. (1997) A Novel Five-Transmembrane Hematopoietic Stem Cell Antigen: Isolation, Character- ization, and Molecular Cloning. pp 5013–5021. CD133 and Colorectal Cancer did not show any difference in rates of proliferation between CD133+ and CD1332 cells. Another possibility is that CD133 was re-induced in these cells. Since CD133 mRNA was detected in the CD1332 cells, it shows that a low level of transcriptional activity may still have been occurring in cells which were negative for protein expression. This would fit with data from animal studies demonstrating that transit amplifying cells (which lie beyond the stem cell compartment) can re-acquire stem cell properties in the small intestine under appropriate conditions [32]. If this is the case, then it would question the existence of CSCs since, by extension, non-stem cells within a tumour could theoretically acquire stem cell features. In summary we have shown that there is only one CD133 splice variant expressed in colorectal tissues and there is plasticity of its expression. There is marked variation in the size of the CD133+ population within CRC cell lines although, given that some cell lines have no CD133+ cells and CD1332 cells from bimodal populations can be clonogenic, it is unlikely that CD133 is a specific stem cell marker. However CD133 expression is associated with some attributable to stemness and the mechanistic basis of these warrants further investigation. Author Contributions Conceived and designed the experiments: MI. Performed the experiments: TME. Analyzed the data: TME LMP ARR RS. Contributed reagents/ materials/analysis tools: AM ARS IT. Wrote the paper: TME MI. Helped do the flow cytometry: LMP. Helped do the functional assay studies: SK. Helped do PCR and QRT-PCR experiments: RS. Helped do cell culture and western blot: DJ. 16. Olempska M, Eisenach PA, Ammerpohl O, Ungefroren H, Fandrich F, et al. (2007) Detection of tumor stem cell markers in pancreatic carcinoma cell lines. Hepatobiliary Pancreat Dis Int 6: 92–97. 17. Suetsugu A, Nagaki M, Aoki H, Motohashi T, Kunisada T, et al. (2006) Characterization of CD133+ hepatocellular carcinoma cells as cancer stem/ progenitor cells. Biochemical and Biophysical Research Communications 351: 820–824. 18. Dalerba P, Dylla SJ, Park IK, Liu R, Wang X, et al. (2007) Phenotypic characterization of human colorectal cancer stem cells. Proc Natl Acad Sci U S A 104: 10158–10163. 19. Shmelkov SV, Butler JM, Hooper AT, Hormigo A, Kushner J, et al. (2008) CD133 expression is not restricted to stem cells, and both CD133+ and CD133aˆJ‘‘ metastatic colon cancer cells initiate tumors. The Journal of Clinical Investigation 118: 2111–2120. 20. Abdulkader A, Rashmi S, Darryl J, Ahmed B, Simon C, et al. (2009) C-terminal Tensin-like (CTEN) is an oncogene which alters cell motility possibly through repression of E-cadherin in colorectal cancer. pp 57–65. 21. Oliver MH, Harrison NK, Bishop JE, Cole PJ, Laurent GJ (1989) A rapid and convenient assay for counting cells cultured in microwell plates: application for assessment of growth factors. pp 513–518. 22. Hadas D-S, Eyal S, Eliezer L, Diana K, Nadir A (2007) Suppression of gastric cancer cell growth by targeting the beta-catenin/T-cell factor pathway. pp 188–197. 23. Arango M, Chamorro C, Cohen-Haguenauer O, Rojas M, Restrepo LM (2005) Human skin keratinocytes modified by a Friend-derived retroviral vector: a functional approach. Dermatol Online J 11: 2. 24. Ieta K, Tanaka F, Haraguchi N, Kita Y, Sakashita H, et al. (2008) Biological and Genetic Characteristics of Tumor-Initiating Cells in Colon Cancer. Annals of Surgical Oncology 15: 638–648. 25. Yu Y, Flint A, Dvorin EL, Bischoff J (2002) AC133-2, a novel isoform of human AC133 stem cell antigen. J Biol Chem 277: 20711–20716. 26. Liu G, Yuan X, Zeng Z, Tunici P, Ng H, et al. (2006) Analysis of gene expression and chemoresistance of CD133+ cancer stem cells in glioblastoma. pp 67. 27. Corbeil D, Roper K, Hellwig A, Tavian M, Miraglia S, et al. (2000) The human AC133 hematopoietic stem cell antigen is also expressed in epithelial cells and targeted to plasma membrane protrusions. J Biol Chem 275: 5512–5520. 28. Giebel B, Corbeil D, Beckmann J, Hohn J, Freund D, et al. (2004) Segregation of lipid raft markers including CD133 in polarized human hematopoietic stem and progenitor cells. pp 2332–2338. PLoS ONE | www.plosone.org 10 May 2010 | Volume 5 | Issue 5 | e10714