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Characterization of side population cells from spontaneously transformed mouse lung carcinoma cell lines

ABSTRACT: Background The side population (SP) phenotype, a subset of cells that extrude the nucleic acid dye Hoechst 33342, has been reported to be enriched for stem cells in several human normal tissues, cancers and cell lines, and thus may be useful for the identification and isolation of cancer stem cells. Methods We demonstrated the presence of SP fractions in all analyzed tumor cell lines ranging between 7- 20% of cells. To identify gene expression patterns that contribute to SP phenotype, microarray analysis of SP and non-SP cells was performed. We additionally confirmed regulation of some genes by qRT-PCR. Results Surprisingly, only a subset of few genes in SP cells showed altered gene expression. A total of 11 genes in A2C12, 103 genes in cRAF_cMYC and 101 genes in beta5 SP cells were regulated. Most regulated genes are involved in transcription / transcriptional regulation and transport. In addition, we found no enrichment of previously described stem cell marker like CD24a, CD90 or CD133 and also the ABC transporter ABCG2 was only slightly increased in side population fraction of two cell lines. But despite the few differences between SP and non-SP cells, the beta5 tumor cells were highly tumorigenic due to their capacity to form an original murine tumor when injected in NOD/SCID mice. Conclusion These findings stand in contrast to other observations but indicate that there are further factors responsible for the SP phenotype and that SP cells alone are not suitable as a universal stem cell marker.

ORGANISM(S): Mus musculus

PROVIDER: GSE22275 | GEO | 2016/12/31

SECONDARY ACCESSION(S): PRJNA128715

REPOSITORIES: GEO

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Project description:Side population (SP) cells isolated from limbal and conjunctival epithelia derive from cells that are slow cycling in vivo, a known feature of tissue stem cells. The purpose of this study was to define the molecular signature of the conjunctival side population cells by global differential gene expression to identify markers and signaling pathways associated with this cell phenotype. Four overnight cultures of freshly isolated human conjunctival epithelial cells stained with Hoechst 3342 were cytometrically sorted into SP and non-SP cohorts. RNA was isolated and processed for microarray analysis using a commercial oligonucleotide array representing more than 55,000 transcripts derived from about 30,000 different genes. Selected microarray results were validated at the gene and protein levels by quantitative PCR, and immunological methods. Data mining methods were used to identify cellular processes relevant for stem cell function. Comparative analysis of transcripts expression based on expression levels and present/absent software calls across 4 replicate experiments identified 16993 expressed conjunctival epithelial transcripts including 10,266 unique known genes. Of those genes, 1254 and 363 were over expressed (> 2-fold ) or under expressed (< 0.5-fold), respectively, in the SP. The overexpressed set included genes coding for non-epithelial genes (e.g., CD62E/E-selectin and CD93), genes that have been associated with stem cell function in other cellular systems, including several homeodomain genes and genes whose over- or under-expression may underpin the stem cell slow cycling phenotype ( e.g., dual specificity phosphatases and cyclin kinases). Experiment Overall Design: Whole human conjunctivae from unidentifiable cadaver donors, aged 55 to 65, were obtained from the National Disease Research Interchange (NDRI, Philadelphia, PA) within 48 hours of collection. No donor details apart from age, sex, and cause of death were released. The use of human tissue in the study was in accordance with the provisions of the Declaration of Helsinki and sanctioned by the Institutional Review Board. Fresh rabbit tissue was obtained from local abattoirs within one hour of sacrifice. Unless stated otherwise all reagents were procured from Sigma (St. Louis, Mo). Experiment Overall Design: Eight microarray experiments using side population (Hoechst 33342-transporting cells; SP) or non side population (Hoechst 33342-stained cells; nSP) cells from four cadavers.

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Characterization of side population cells from spontaneously transformed mouse lung carcinoma cell lines

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