CD44(high) /ALDH1(high) head and neck squamous cell carcinoma cells exhibit mesenchymal characteristics and GSK3ß-dependent cancer stem cell properties.

BACKGROUND: CD44 and aldehyde dehydrogenase 1 (ALDH1) have been shown to be useful markers for identification of cancer stem cells (CSCs). We previously reported that glycogen synthase kinase 3ß (GSK3ß) is involved in regulation of the self-renewal ability of head and neck squamous cell carcinoma (HNSCC) CSCs. The purpose of the present study was to clarify the role of GSK3ß in CD44(high) /ALDH1(high) HNSCC cells. METHODS: Cells with greater expression of CD44 and higher ALDH1 enzymatic activity were FACS sorted from the OM-1 HNSCC cell line. The self-renewal ability of CD44(high) /ALDH1(high) cells was then examined using a tumor sphere formation assay. mRNA expressions of the stem cell markers Sox2, Oct4, and Nanog, as well as GSK3ß were evaluated by real-time RT-PCR. RESULTS: CD44(high) /ALDH1(high) cells exhibited higher tumor sphere forming ability and increased expression of stem cell markers as compared with CD44(high) /ALDH1(low) cells. Interestingly, spindle-shaped cells positive for vimentin were found in the CD44(high) /ALDH1(high) but not the CD44(high) /ALDH1(low) cell population. In addition, the ALDH1 activity and sphere forming ability of CD44(high) /ALDH1(high) cells was significantly inhibited by GSK3ß knockdown. On the other hand, CD44(high) /ALDH1(low) cells exhibited high epidermal growth factor receptor (EGFR) expression and increased cell growth. CONCLUSIONS: Our results show that GSK3ß plays a major role in maintenance of stemness of CD44(high) /ALDH1(high) HNSCC cells. Additionally, they indicate a close relationship between CSC and mesenchymal characteristics in HNSCC.

In vitro and in vivo derived porcine embryos possess similar, but not identical, patterns of Oct4, Nanog, and Sox2 mRNA expression during cleavage development.

In vitro culture conditions stress the cleavage stage mammalian embryo and can contribute to reduced developmental potential of cultured embryos. One process that may be altered during embryo culture is the establishment and maintenance of pluripotency. Pluripotency is largely controlled by three genes: Oct4, Nanog, and Sox2. The objective of this study was to determine the expression pattern of Oct4, Nanog, and Sox2 in cleavage stage porcine embryos obtained in vivo or by in vitro fertilization and parthenogenetic activation. We used quantitative, real time PCR to assess the relative amount of each transcript in cleavage stage embryos. We found that Oct4 was transiently activated at the 2-cell stage (P-value <0.05) while Nanog and Sox2 were activated at the 4-cell stage (P-value <0.05) in in vitro embryos. Embryos derived in vivo showed a similar but not identical pattern of expression of Nanog mRNA been in highest abundance both at the 4 cell and the blastocyst stage. The activation observed at the 4-cell stage for Nanog and Sox2 was shown to be RNA polymerase II dependent (P-value <0.05). This study showed that Oct4, Nanog, and Sox2 possess similar, but not identical, patterns of expression between in vitro and in vivo derived porcine embryos. The difference between the amount of transcripts may reflect the reduced developmental potential observed in in vitro cultured embryos.