Profiling of Secreted Proteins from Human Ovarian Cancer Cell Lines by Surface-Enhanced Laser Desorption Ionization Time-of-Flight Mass Spectrometry

Surface‐enhanced laser desorption ionization (SELDI) time‐of‐flight mass spectrometry (TOF MS) was used to profile six human ovarian cancer cell lines. Non‐confluent cell cultures were exchanged into serum free medium and allowed to grow for either 24 or 48 hours, at which time the medium was collected and analyzed using a Ciphergen SELDI‐TOF MS and a QSTAR Pulsar QqTOF mass spectrometer fitted with a SELDI source. The spectra showed the presence of several low molecular weight species, as well as differences and similarities in the proteins detected in the media of the six ovarian tumor cell lines. The same species were detected at 24 and 48 hours and reproducible changes in their relative abundances were observed

A small molecule (pluripotin) as a tool for studying cancer stem cell biology: proof of concept.

BACKGROUND: Cancer stem cells (CSC) are thought to be responsible for tumor maintenance and heterogeneity. Bona fide CSC purified from tumor biopsies are limited in supply and this hampers study of CSC biology. Furthermore, purified stem-like CSC subpopulations from existing tumor lines are unstable in culture. Finding a means to overcome these technical challenges would be a useful goal. In a first effort towards this, we examined whether a chemical probe that promotes survival of murine embryonic stem cells without added exogenous factors can alter functional characteristics in extant tumor lines in a fashion consistent with a CSC phenotype. METHODOLOGY/PRINCIPAL FINDINGS: The seven tumor lines of the NCI60 colon subpanel were exposed to SC-1 (pluripotin), a dual kinase and GTPase inhibitor that promotes self-renewal, and then examined for tumorigenicity under limiting dilution conditions and clonogenic activity in soft agar. A statistically significant increase in tumor formation following SC-1 treatment was observed (p<0.04). Cloning efficiencies and expression of putative CSC surface antigens (CD133 and CD44) were also increased. SC-1 treatment led to sphere formation in some colon tumor lines. Finally, SC-1 inhibited in vitro kinase activity of RSK2, and another RSK2 inhibitor increased colony formation implicating a role for this kinase in eliciting a CSC phenotype. CONCLUSIONS/SIGNIFICANCE: These findings validate a proof of concept study exposure of extant tumor lines to a small molecule may provide a tractable in vitro model for understanding CSC biology

Detailed DNA methylation profiles of the E-cadherin promoter in the NCI-60 cancer cells

E-cadherin (E-cad) is a transmembrane adhesion glycoprotein, the expression of which is often reduced in invasive or metastatic tumors. To assess E-cad\u27s distribution among different types of cancer cells, we used bisulfite-sequencing for detailed, base-by-base measurement of CpG methylation in E-cad\u27s promoter region in the NCI-60 cell lines. The mean methylation levels of the cell lines were distributed bimodally, with values pushed toward either the high or low end of the methylation scale. The 38 epithelial cell lines showed substantially lower (28%) mean methylation levels compared with the nonepithelial cell lines (58%). The CpG site at -143 with respect to the transcriptional start was commonly methylated at intermediate levels, even in cell lines with low overall DNA methylation. We also profiled the NCI-60 cell lines using Affymetrix U133 microarrays and found E-cad expression to be correlated with E-cad methylation at highly statistically significant levels. Above a threshold of approximately 20% to 30% mean methylation, the expression of E-cad was effectively silenced. Overall, this study provides a type of detailed analysis of methylation that can also be applied to other cancer-related genes. As has been shown in recent years, DNA methylation status can serve as a biomarker for use in choosing therapy