טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentEgozi Dana
SubjectTelomerase and Cell Cycle Regulatory Proteins in Human
Embryonic Stem Cells
DepartmentDepartment of Medicine
Supervisors Professor Karl Skorecki
Dr. Maty Zukerman
Full Thesis textFull thesis text - English Version


Abstract

Embryonic stem cells have a unique cell cycle structure with features more similar to malignant than normal somatic cells.  Some of these features were the focus of the present study.  Specifically, the first issue to focus on was the G1-S transition cell cycle regulator, Skp2, which is the ubiquitin ligase subunit that targets p27 for degradation, and thus is essential for the control of cell cycle progression and proliferation.  In undifferentiated human embryonic stem cells (hES cells), elevated Skp2 levels were observed together with low p27 levels.  This ratio was reversed during the process of differentiation, such that Skp2 levels decreased and p27 increased.  Within the embryoid bodies, various cell populations with diverse morphologies were observed, each expressing different levels of Skp2, either low or high, always reciprocally associated with p27 levels.  Interestingly, cells that maintained a high Skp2/p27 ratio displayed poorer differentiation than differentiated cells expressing a high p27/Skp2 ratio.  This suggests a role for Skp2 in the differentiation process that extends beyond its role in the proliferation process.  Changes in Skp2 expression are secondary to differences in the protein degradation rate and not in the transcription rate, as evidenced by the stability of RNA levels during the differentiation process, as well as the Skp2 protein stability that were observed only in undifferentiated cells.  During differentiation, however, Skp2 is degraded, allowing upregulation of p27 levels.  Proliferating somatic cells are characterized by elevated Skp2 levels in the S and G2/M phases of the cell cycle.  By contrast, Skp2 levels in undifferentiated hES cells are elevated in the G1 phase as well.  Taken together, these results suggest that Skp2 and p27 regulation, mediated by the ubiquitin-proteasome pathway, are different than the regulation in normal somatic cells, and has an important role in the regulation of hES proliferation and differentiation programs.

Elevated telomerase activity is another characteristic shared by hES and malignant cells.  With hES cell differentiation, telomerase activity decreases.  Clones that expressed the green fluorescence protein (GFP) driven by the hTERT promoter (either 3.3 kb or the core, which consist of 300 bp) were established.  The decrease of telomerase during differentiation was demonstrated to occur at the transcription level.  The core promoter was shown to be sufficient for the repression of telomerase activity in the differentiation process.  Scriptaid, a histone deacetylase inhibitor, caused re-expression of GFP in differentiated embryonic bodies; however, only 3-4% of the cells re expressed GFP, while the remaining cells were not affected by this treatment.