Regulation of Apoptosis and Differentiation by p53 in Human ...

CHAPTER 4: RNAi to explore role of p53 in hESC
However, there are a few upregulated genes that are known to have a pro-apoptotic role. NRGN
(neurogranin), also known as RC3, a calcium/calmodulin binding protein can induce induction of
apoptosis due to cytokine IL-2 deprivation in lymphoid cells (Devireddy and Green, 2003). In this
model, levels of intracellular Ca 2+ levels play a role in T-cell apoptosis. Bin1 (bridging integrator 1)
is a tumour suppressor that mediates apoptosis by interacting with c-myc (Sakamuro et al., 1996),
is involved in the negative regulation of progression through cell cycle and can inhibit
transformation by mutant p53 (Elliott et al., 1999). It has also been reported that Bin1 participates
in a caspase-independent cell death program (Elliott et al., 2000). These genes can be part of
simultaneous or alternative mechanisms to suppress the appearance of genetic abnormalities in
the hESC population.
The biological processes of the genes downregulated in p53 shRNA transduced cell lines are
genes include transport, regulation of transcription, nucleic acid metabolic process, cell cycle and
protein amino acid phosphorylation. Some of the downregulated genes can also contribute to
tumorigenesis. NME1 (non-metastatic cells 1) which is involved in negative regulation of
progression through cell cycle, was identified because of its reduced mRNA transcript levels in
highly metastatic cells (Driouch et al., 1998). CST6 (cystatin E/M) is a secreted inhibitor of
lysosomal cysteine proteases and its loss of expression is likely associated with the progression
of a primary tumour to a metastatic phenotype (Ai et al., 2006). TRIM22 (tripartite motif-containing
22) is a p53 target gene that confers reduced clonogenic growth and differentiation of leukemic U-
937 cells (Obad et al., 2004). This expression of this gene has also been shown to increase in
response to DNA-damaging treatments in B-lymphoblastoid cell line TK6 (Amundson et al., 2005)
and in response to cisplatin in TGCTs (Kerley-Hamilton et al., 2005). TMEM43 (transmembrane
protein 43) is another gene regulated by p53 identified by a genome-wide analysis under hypoxic
conditions (Hammond et al., 2006). Although the cell cycle inhibitor p21 (CDKN1A) protein was
not detected (section 3.9 of Chapter 2), levels of this p53 downstream target inhibitor of cell cycle
were found to be reduced in p53 shRNA transduced cell lines. On the other hand, Bax which was
found to be downregulated by western blotting analysis did not display differential expression on
the microarray analysis. In order to validate these results Q-PCR should be performed.
p53 had a small but reproducible effect in inhibiting spontaneous differentiation. Preliminary
studies to address the role of p53 in differentiation of hESC revealed a slight resistance of p53
shRNA transduced cell lines to BMP4-induced differentiation but the opposite was observed in
relation to Activin A treatment, based on expression of Nanog and Oct4. Because these
morphogens operate through different pathways, p53 may participate differently in the
differentiation process induced by them. In GFP shRNA transduced cells p53 levels decreased
upon differentiation with BMP4. Perhaps more hESC differentiated progeny show regulation of
p53 similar to somatic cells, where this tumor suppressor gene is kept at low levels. A very
interesting study performed by Dazard and colleagues (Dazard et al., 2000) explored the
p53/MDM2 regulatory loop during different transitions of the human epidermal differentiation
program. They showed that maximum expression of p53 was found in proliferating keratinocytes
and that a transient induction of MDM2 and a downregulation of p53 characterized the transition
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