|dc.description.abstract||The transcription factor Interferon Regulatory Factor-1 (IRF-1) has been demonstrated
to suppress tumour growth through the regulation of many anti-oncogenic genes. Pro- and
anti-apoptotic factors, cell cycle control genes, DNA damage response genes and prometastatic
factors are all under the control of IRF-1, which effects both transcriptional
activation and repression. In addition to these cell autonomous tumour suppressor activities,
IRF-1 is also a key regulator of the immune system and, as such, mediates immune
surveillance of tumours. Numerous studies have confirmed that loss or mis-regulation of
IRF-1 is a key factor in several different types of cancer.
Despite strong evidence for the crucial role of IRF-1 in cancer, and frequent assertions
that this protein warrants further investigation as a drug target, very little is known about
its regulation. Furthermore, since recent studies have linked upregulation of IRF-1 to the
development of autoimmune diseases, it is particularly important that drugs be able to
decouple autoimmune and anti-cancer functions of IRF-1 to avoid harmful side effects.
This thesis describes how phosphorylation of IRF-1 in its regulatory C-terminal Mf1
domain modulates transactivatory and tumour suppressor activity. Phosphospecific antibodies
were developed as tools to study the C-terminal phosphorylation. Using these,
it was shown that treatment of cells with Interferon-γ(IFN-γ) not only causes accumulation
of IRF-1 protein, but also results in phosphorylation of IRF-1 at two sites in the
C-terminal Mf1 domain.
Phosphomimetic mutants demonstrated that these phosphorylations enhanced the transactivatory
activity of IRF-1 at various promoters, but did not affect repressor activity. Gel
shift assays revealed that dual phosphorylation of IRF-1 (IRF-1 D/D) promoted DNAbinding
and suggested this was through increased interaction with the cofactor/histone
acetylase p300 which induces a conformational change in IRF-1, favouring DNA-binding.
Acetylation by p300 appears to be important although it is not yet clear whether this directly
or indirectly affects IRF-1 activity.
Since the tumour suppressor activity of IRF-1 is of particular interest, the effect of
phosphorylation was examined in clonogenic and invasion assays. IRF-1 D/D more efficiently
suppressed colony formation in both anchorage dependent and independent assays,
and may improve inhibition of invasion in Transwell assays. Thus, cell treatment
with the therapeutic agent IFN-γ nduces phosphorylation of IRF-1, resulting in enhanced
DNA binding of IRF-1 through improved p300 binding. In cells the outcome is more effective
tumour suppression and inhibition of metastasis.||en