Transcription factor AP4, also known as activating enhancer binding protein 4, was
discovered in 1988 because of its ability to bind to the enhancer of the SV40 viral
genome and activate its late transcription in vitro. As a ubiquitously expressed
transcription factor, it exclusively forms homodimers and controls the expression of its
target genes through binding to the E-box (CAGCTG) on the promoter region. On the
basis of increasing number of AP4 target genes discovered, it seems that AP4
participates in many important physiological activities via direct activation or
suppression on its target genes and coordinates the physiological progress by
cooperating with other molecules in the cells. The identified targets of AP4, such as
insulin-like growth factor binding protein-2, caspase 9, p21, and Pin1, are involved in
cell proliferation, cell death, and development and aging. Recently, AP4 was found to be
expressed at a high level in many kinds of cancer tissues, including pancreatic cancer,
colorectal cancer, gastric cancer, and hepatocellular carcinoma (HCC), suggesting that it
may be involved in cancer progression. However, it has been rarely reported the
function of AP4 in normal cells, and the physiological functions of AP4 in cancer
therapy is also need to be further investigated.
In this thesis, an inducible double stable Tet-On 3G system was used to generate
human retinal pigment epithelial (RPE) cell clones that overexpress AP4 or DN-AP4
protein in the presence of the inducer doxycycline (DOX). With these inducible stable
cell clones, we could avoid apoptosis induced by the ectopic expression of AP4 during
transient transfection with transfecting reagents, and control the expression level of AP4
at a specific level after a specific time. Another AP4 molecule was cloned called DNAP4,
which lacks the residues 46-60 in the DNA binding domain, could be used to form
dimer with endogenous AP4 and inactivate it, thus resulting in a similar action to
knocking down of AP4.
Using the inducible RPE AP4/DN-AP4 cell clones, a series experiments on the
roles of AP4 on the cell proliferation in non-confluent and post-confluent states of RPE
cells were performed: (1) the alternations on proliferation rate and individual cell
volume; (2) the expression of cell cycle related or senescence-associated proteins; (3)
the control of AP4 on the expression of p53; (4) the potential association of vimentin
and AP4; (5) the roles of AP4 on c-MYC-induced cellular response.
My study showed that in actively proliferating RPE cells, overexpression of AP4
decreased individual cell volume and had no significant effect on cell proliferation, and
a substantial suppression of AP4 on cell cycle related proteins may be partly responsible
for the decrease in individual cell volume. On the other hand, high expression level of
DN-AP4 led to lower proliferation rate and the increase in p21 level, a cell cycle
inhibitor, was the major cause of the decrease in cell proliferation. High expression of
AP4 had no significant effects on p53 mRNA expression, but it upregulated the level of
p53 protein after the addition of MG132, which inhibits the proteasomal degradation of
p53 protein, indicating that AP4 may regulate p53 protein indirectly.
Western blot analysis and immunofluorescence microscopy showed that the levels
of endogenous AP4 gradually decreased in post-confluent RPE cells. Based on the
observations that high expression of AP4 is found in many cancer tissues and that most
of the cells in human body are cell-cell contact-inhibited, long-term cultured postconfluent
RPE cells was used as a model to investigate the roles of AP4 in the biology
of the cell. We found that long-term overexpression of AP4 in RPE cells after the cells
reached confluence (ARC) induced cell enlargement and flattening accompanied with a
cessation of cell proliferation, which were the characteristics of senescent cells. By
contrast, RPE cells overexpressing DN-AP4 had no difference in cell morphology and
proliferation compared to controls. Furthermore, the enlarged, flattened cells could be
stained β-galactosidase positively and exhibited the senescence-associated secretory
phenotype, as well as high γH2AX activity. Moreover, exhibition of strong signals of
p53 and ARF in these senescent cells indicated that AP4 may induce cellular senescence
in post-confluent RPE cells through the ARF-p53 pathway.
Further investigation of Western blot analysis of the subcellular fractionated
protein extracts from the cells at Day 24 ARC showed that the induced AP4 was
primarily located in the nucleus while DN-AP4 was found in the cytoplasm, revealing
that AP4 functions in the nucleus as a transcription factor in inducing cellular
senescence. Importantly, AP4 induced the upregulation of both p53 mRNA and protein
as determined by RT-PCR and Western blot analysis, respectively. Further functional
characterization of the AP4 binding sites on the promoters of the p53 gene by the ChIPqPCR
assay showed that AP4 bound to two of the three E-boxes on the promoter of the
p53 gene in long-term culture cells. Under low serum concentration, high expression
level of AP4 could not induce p53 protein and cellular senescence, indicating that serum
was necessary for AP4 activation, and giving further supports that AP4-induced cellular
senescence in post-confluent RPE cells was p53- and serum-dependent. Besides, the colocalization
of cytoplasmic AP4 protein and vimentin bundles in senescent cells
displayed by immunofluorescence microscopy suggested that cytoplasmic AP4 may
facilitate and stabilize vimentin bundling, which confer the flat and enlarged cell shapes,
thereby contributing to AP4-induced cellular senescence.
Additionally, several RPE cell clones expressing different levels of c-MYC-ER, a
fusion protein between c-MYC and estrogen receptor mutant, which can be translocated
into the nucleus in the presence of 4-hydroxythamoxifen (4-OHT), were generated.
Analysis on the cellular responses of post-confluent RPE cells expressing high,
moderate, and low levels of c-MYC-ER revealed that high level of c-MYC resulted in
apoptosis, moderate expression of c-MYC induced cell death as well as senescence, and
low expression of c-MYC led to cellular senescence. The translocation of c-MYC-ER
fusion protein into the nucleus by incubating with 4-OHT induced the expression of
AP4. In comparison, under high and moderate levels of c-MYC that were translocated
into the nucleus, further knockdown of AP4 protein using a lentiviral delivery system in
RPE c-MYC-ER cells accelerated cell death and promoted only cell death without
senescence, respectively. After low level of c-MYC-ER was translocated into the
nucleus, decrease in p53 protein was observed accompanied by the depletion of AP4. In
contrast, knockdown of AP4 had no influence on p53 expression when the expression
level of c-MYC-ER was moderate, suggesting that AP4 mediated the effect of c-MYC
on p53 expression when c-MYC was low.
Overall, the findings in this study suggest that AP4 potentially affects cell
proliferation and individual cell volume in actively growing RPE cells, stimulates
cellular senescence via the activation of p53 in post-confluent RPE cells, and mediates
c-MYC-induced cellular senescence.
| Date of Award | 2 Oct 2015 |
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| Original language | English |
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| Awarding Institution | - City University of Hong Kong
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| Supervisor | Sung Kay David CHIU (Supervisor) |
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