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1.
PLoS One ; 8(2): e57099, 2013.
Article in English | MEDLINE | ID: mdl-23437320

ABSTRACT

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.


Subject(s)
Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/metabolism , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Animals , Biomarkers/metabolism , Cell Line, Tumor , Disease Models, Animal , Female , Humans , Mice , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Octamer Transcription Factor-3/metabolism , Ribosomal Protein S6 Kinases, 90-kDa/antagonists & inhibitors , Ribosomal Protein S6 Kinases, 90-kDa/metabolism , Spheroids, Cellular/drug effects , Transplantation, Heterologous , Tumor Burden/drug effects , Tumor Cells, Cultured , Tumor Stem Cell Assay
2.
PLoS One ; 7(8): e41401, 2012.
Article in English | MEDLINE | ID: mdl-22870217

ABSTRACT

Recently, there has been renewed interest in the role of tumor stem cells (TSCs) in tumorigenesis, chemoresistance, and relapse of malignant tumors including osteosarcoma. The potential exists to improve osteosarcoma treatment through characterization of TSCs and identification of therapeutic targets. Using transcriptome, proteome, immunophenotyping for cell-surface markers, and bioinformatic analyses, heterogeneous expression of previously reported TSC or osteosarcoma markers, such as CD133, nestin, POU5F1 (OCT3/4), NANOG, SOX2, and aldehyde dehydrogenase, among others, was observed in vitro. However, consistently significantly lower CD326, CD24, CD44, and higher ABCG2 expression in TSC-enriched as compared with un-enriched osteosarcoma cultures was observed. In addition, consistently higher CBX3 expression in TSC-enriched osteosarcoma cultures was identified. ABCA5 was identified as a putative biomarker of TSCs and/or osteosarcoma. Lastly, in a high-throughput screen we identified epigenetic (5-azacytidine), anti-microtubule (vincristine), and anti-telomerase (3,11-difluoro-6,8,13-trimethyl- 8H-quino [4,3,2-kl] acridinium methosulfate; RHPS4)-targeted therapeutic agents as candidates for TSC ablation in osteosarcoma.


Subject(s)
ATP-Binding Cassette Transporters/biosynthesis , Biomarkers, Tumor/biosynthesis , Chromosomal Proteins, Non-Histone/biosynthesis , Femoral Neoplasms/metabolism , Gene Expression Regulation, Neoplastic , Neoplasm Proteins/biosynthesis , Neoplastic Stem Cells/metabolism , Osteosarcoma/metabolism , Adolescent , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Azacitidine/pharmacology , Azacitidine/therapeutic use , Cell Line, Tumor , Femoral Neoplasms/drug therapy , Femoral Neoplasms/pathology , Humans , Male , Neoplastic Stem Cells/pathology , Osteosarcoma/drug therapy , Osteosarcoma/pathology , Vincristine/pharmacology , Vincristine/therapeutic use
3.
Biochemistry ; 50(33): 7218-27, 2011 Aug 23.
Article in English | MEDLINE | ID: mdl-21749055

ABSTRACT

Overexpression of S100A4, a member of the S100 family of Ca(2+)-binding proteins, is associated with a number of human pathologies, including fibrosis, inflammatory disorders, and metastatic disease. The identification of small molecules that disrupt S100A4/target interactions provides a mechanism for inhibiting S100A4-mediated cellular activities and their associated pathologies. Using an anisotropy assay that monitors the Ca(2+)-dependent binding of myosin-IIA to S100A4, NSC 95397 was identified as an inhibitor that disrupts the S100A4/myosin-IIA interaction and inhibits S100A4-mediated depolymerization of myosin-IIA filaments. Mass spectrometry demonstrated that NSC 95397 forms covalent adducts with Cys81 and Cys86, which are located in the canonical target binding cleft. Mutagenesis studies showed that covalent modification of just Cys81 is sufficient to inhibit S100A4 function with respect to myosin-IIA binding and depolymerization. Remarkably, substitution of Cys81 with serine or alanine significantly impaired the ability of S100A4 to promote myosin-IIA filament disassembly. As reversible covalent cysteine modifications have been observed for several S100 proteins, we propose that modification of Cys81 may provide an additional regulatory mechanism for mediating the binding of S100A4 to myosin-IIA.


Subject(s)
Cysteine/metabolism , Naphthoquinones/pharmacology , Nonmuscle Myosin Type IIA/metabolism , Recombinant Proteins/metabolism , S100 Proteins/metabolism , Chromatography, Liquid , Cysteine/genetics , Cytoskeleton , Humans , Nonmuscle Myosin Type IIA/antagonists & inhibitors , Nonmuscle Myosin Type IIA/genetics , Peptide Fragments/metabolism , Protein Binding , Recombinant Proteins/genetics , S100 Calcium-Binding Protein A4 , S100 Proteins/antagonists & inhibitors , S100 Proteins/genetics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , cdc25 Phosphatases/antagonists & inhibitors
4.
Stem Cells ; 28(4): 649-60, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20178109

ABSTRACT

Tumor stem cells or cancer initiating cells (CICs) are single tumor cells that can regenerate a tumor or a metastasis. The identification and isolation of CICs remain challenging, and a variety of putative CIC markers have been described. We hypothesized that cell lines of the NCI60 panel contain CICs and express putative CIC markers. We investigated expression of putative CIC surface markers (CD15, CD24, CD44, CD133, CD166, CD326, PgP) and the activity of aldehyde dehydrogenase in the NCI60 panel singly and in combination by six-color fluorescence-activated cell sorting analysis. All investigated markers were expressed in cell lines of the NCI60 panel. Expression levels of individual markers varied widely across the 60 cell lines, and neither single marker expression nor simple combinations nor co-expression patterns correlated with the colony-formation capacity of cell lines. Rather, marker expression patterns correlated with tumor types in multidimensional analysis. Whereas some expression patterns correlated with tumor entities such as basal breast cancer, other expression patterns occurred across different tumor types and largely related to expression of a more mesenchymal phenotype in individual breast, lung, renal, and melanoma cell lines. Our data for the first time demonstrate that tumor cell lines display CIC markers in a complex pattern that relates to the tumor type. The complexity and tumor type specificity of marker display creates challenges for the application of cell sorting and other approaches to isolation of putative tumor stem cell populations and suggests that therapeutic targeting strategies will need to take this into account.


Subject(s)
Biomarkers, Tumor/metabolism , Neoplastic Stem Cells/metabolism , Cell Line, Tumor , Cell Proliferation , Gene Expression Profiling , Humans , Neoplastic Stem Cells/cytology
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