Poster Presentation 6th Annual Meeting for Australasian Society for Stem Cell Research 2013

TDGF-1 (CRIPTO-1) effect on cellular de-differentiation of human glioma cells (#102)

FAISAL ALOWAIDI 1 2 3 4 , SAEED HASHIMI , Naif AlQurashi , Adrian Meedenyia , Saso Ivanovski 2 , Brenton Cavanch 1 , Maria Nguyen 1 , Bernadette Bellette 1 , Alan Mackay-Sim 1 , Stephen Wood 1
  1. Eskitis Institute for Cell and Molecular Therapies, Griffith University, Brisbane, QLD, Australia
  2. School of dentistry and oral health, Griffith health institute, Griffith University, Gold Coast, QLD, Australia
  3. School of Medical science, Griffith University, Gold Coast, Australia
  4. College of Medicine and University Hospitals, King Saud University, Saudi Arabia, Riyadh, Saudi Arabia

Objective of the study:

The current study investigates the role of cripto-1 overexpression in modulating the de-differentiation of human glioblastoma cells.

Methods:

Cripto-1 was stably over expressed in U87 cells, a human glioblastoma cell line. Cells were harvested for functional analysis. RNA and Protein were extracted from cripto-1 clone and the control clone. Then, RNA was used for QPCR gene expression of specific signature of genes known to be over expressed during the undifferentiated phenotype. Western blot and proteome profiler were used to study the protein expression for the genes OCT-A, NANOG, SOX-2 & CD44. FACS analysis was used to assess the different population between the different clones using CD44 and EdU incorporation. Cells from both of the clones were also subjected for a method to test their ability to grow under starvation conditions using MTT reagents. Propodium Iodide (PI) was also utilised to assess the differences between tested clones in regard to their cell cycle checkpoints. Tumour sphere and limited formation assays were used to assess the ability of cells to self renew and grow in spheres under ultra low attachment conditions.

Results:

RNA expression analysis showed significant increase in OCT4, NANOG, SOX2, CD44, & Id1 gene expressions in Cripto-1 clones compared to control clones. Furthermore, MTT analysis indicates that cripto-1 over expressing clones are significantly faster in growth under starvation conditions compared to the cells from the control clones. Cell cycle analysis showed elevated levels of cells in all check points of the cell cycle with a significant increase in percentage of cells in S phase of cripto-1 clone compared to the control clone. Moreover, cripto-1 clone cells were higher in expressing OCT4-A, NANOG, SOX2 & CD44 compared to control clones at the protein level. Finally, Number of spheres produced by the cripto-1 clones were significantly higher than the ones produced by the control clones .

Conclusion:

It can be concluded that cripto-1 over-expression increases the self renewal capacity of glioma cells.