<p> Proper gene expression programs cellular activities, while aberrant manipulation of
transcription factors often leads to devastating consequences, such as cancer or cell death.
The transcription factor family activator protein-1 (AP-1) plays an important role in many
cellular activities including cell transformation, proliferation and survival (Shaulian and
Karin 2002). However, little has been done to obtain a global view of the role of
individual AP-1 members and how they cooperate in many cellular activities. We have
discovered that blocking the AP-1 pathway by a c-Jun dominant negative mutant,
TAM67, induced cell death in RSV-transformed primary chicken embryo fibroblasts
(CEF), suggesting that AP-1 activity is vital for cell survival upon v-Src transformation.
In addition, accumulation of cytoplasmic vesicles was observed in the cytoplasm of a
proportion of RSV-transformed CEF expressing TAM67. Oil-red staining of these
vesicles indicated the presence of lipid droplets in these cells, suggesting that the
inhibition of AP-1 promotes the adipogenic conversion of v-Src transformed CEF. To
understand the role of individual members of the AP-1 family, a retroviral-based shRNA
expressing system was designed to stably downregulate individual AP-1 members. This
retroviral-based RNAi system provided sustained gene downregulation of AP-1 family
members. Reduction of the c-Jun protein level by shRNA induced senescence in normal
CEF, while it modestly downregulated AP-1 activity in RSV -transformed CEF indicating
that c-Jun is not the main component of the AP-1 complex in RSV-transformed CEF.
Inhibition of JunD expression induced apoptosis and was deleterious to both normal and
RSV-transformed CEF, suggesting that JunD is crucial for the survival of CEF. Transient express10n reporter-assays also showed that loss-of-function of JunD by shRNA
dramatically repressed AP-1 activity. Hence JunD is the main component of the AP-1
complex that regulates the survival of CEF. Furthermore, we determined that loss of
JunD expression resulted in an elevated level of tumour suppressor p53. Co-inhibition of
p53 and JunD restored the transforming ability of v-Src transformed CEF, as indicated by
foci formation in soft agar assays. Hence, repression of p53 induction was able to bypass
the death signal released as a result of AP-1 inhibition in v-Src transformed CEF. Downregulation of Fra-2 (Fos-related antigen 2) level by shRNA did not affect the proliferation
of normal CEF. However, RSV -transformed CEFs expressing fra -2 shRNA were
transformation-defective with the presence of multiple vesicles in cytoplasm. Oil-red
staining of these vesicles indicated the presence of lipid droplets, which resembles the
effect of T AM67 in RSV -transformed CEF indicating that Fra-2 blocks differentiation.
These findings help us to understand the role of individual members of the AP-1
transcription factor family in normal and RSV -transformed CEF. Importantly, global
gene profiling of v-Src transformed CEF expressing shRNA for individual AP-1
members will improve our knowledge of the transformation process. Functional
characterization of the cascade will rely on the use of retroviral-based shRNA expressing
system as described above. </p> / Thesis / Doctor of Philosophy (PhD)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/19514 |
| Date | 05 1900 |
| Creators | Wang, Lizhen |
| Contributors | Bédard, P.A., Biology |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
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