One study investigated the mechanisms of apoptosis resistance of cells with stem-cell-like properties in both normal and malignant human epithelia, and whether altered cell cycle regulation played a role.
To address these questions, the following methodology was employed:
- Cells were isolated from fresh human head and neck carcinomas (n = 11), cell lines derived from head and neck, prostate and breast human carcinomas (n = 7), and from normal human oral mucosa (n = 5)
- All cells were exposed to various apoptosis-inducing stimuli (UV, tumour necrosis factor, cisplatin, etoposide, and neocarzinostatin)
- The subset of cells with stem-cell-like properties were identified by flow cytometry for CD44 and epithelial-specific antigen (ESA) expression, colony morphology, tumour sphere formation and rapid adherence assays
- Apoptosis, cell cycle and expression of various cell cycle checkpoint proteins were assessed by Western Blot and quantitative PCR (qPCR)
- Debromohymenialdisine (DBH) and small interfering RNA (siRNA) were used to investigate the role of G2-checkpoint regulators Chk1 and Chk2
- A subset of CD44high cells were found in both cancer biopsies and carcinoma cell lines that showed increased clonogenicity, a significantly lower rate of apoptosis, and a significantly higher proportion of cells in the G2-phase of the cell cycle. An inverse correlation between the Percentage of cells in G2-phase and the rate of apoptosis revealed an inverse correlation.
- It appeared that CD44high carcinoma cells spent longer time in G2, even in untreated controls as shown by pulse-chase with iododeoxyuridine (IdU). These cells expressed higher levels of G2 checkpoint proteins, and their release from G2 with BDH or Chk1 siRNA increased their rate of apoptosis.
- Interestingly, low passage cultures of normal keratinocytes were found to contain a subset of CD44high cells which also showed increased clonogenicity, and a similar pattern of G2-block associated with apoptotic resistance.
Conclusions
The results of this study suggested that not only malignant, but also normal human epithelial cells with stem-cell-like properties have extended G2 cell cycle phase which is associated with greater resistance to apoptosis, and that this property is not a consequence of neoplastic (cancerous) transformation (i.e. not unique to cancer cells). This knowledge provides an opportunity for novel therapeutic approaches targeting G2 checkpoint proteins to release these cells from the G2-block and make them more prone to apoptosis.Abbreviations
PCR = polymerase chain reaction; RNA = ribonucleic acid
Reference
Harper LJ, Costea DE, Gammon L, et al. Normal and malignant epithelial cells with stem-like properties have an extended G2 cell cycle phase that is associated with apoptotic resistance. BMC Cancer 2010, 10:166doi:10.1186/1471-2407-10-166.Free full-text article available at: http://www.biomedcentral.com/1471-2407/10/166