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IFN has diverse functions that include anti-viral, anti-proliferative, pro-apoptotic and immune modulation effects. The anti-proliferative effects of IFN are primarily translated through p21 and p27, which are produced from IFN dependent genes with IFN stimulation resulting in increased transcription of both. These molecules are cyclin dependent kinase (CDK) inhibitors. CDK is required during the cell cycle with inhibition causing arrest in the G1-S phase. IFN also downregulates c-myc which is responsible for activating cyclin:CDK complexes and inducing transcription of genes required for S phase [301] .

p27, also known as Kip 1, is expressed in most cells and, along with p21, is a member of the Cip/Kip family of cell cycle inhibitors [302]. The established role of p27 is to bind and inhibit cyclin-cyclin dependent kinase (CDK) complexes, thereby inhibiting cell cycle progression between G1 and S phase [303]. p27 binds to cyclin D and CDK4 in the cytoplasm which promotes translocation of the complex to the nucleus. In non-cycling cells, p27 binds to cyclinD-CDK4 complexes as well as cyclinE-CDK2. The p27-cyclinE- CDK2 complex results in inhibition of CDK2 resulting in cell cycle arrest. In proliferating cells levels of cyclinD-CDK4 increase and preferentially bind p27 resulting in degradation of p27, this produces release of cyclinE-CDK2 from p27. The liberation of CDK2 from p27 results in loss of inhibition, and subsequent stimulation of cell cycle progression [302], (Figure 3.3). This anti-proliferative effect gives p27 a significant role as a tumour suppressor gene. p27 is rarely mutated and is predominantly regulated at a post- transcriptional level by degradation in the ubiquitin-proteosome pathway [304]. As previously alluded to, some of the anti-proliferative effects of IFN are through p27, hence its inclusion in the current study.

Cell surface Cyclin D Nuclear translocation CDK 4 Cyclin D CDK 4 p27 Cyclin D CDK 4 p27 Cyclin E CDK 2 p27 Cyclin D CDK 4 p27 Cyclin E CDK 2 Nucleus Cytoplasm Cell-cycle progression Cell-cycle arrest

Figure 3.3 Cip/Kip protein (p27) regulates cyclin-CDK complexes in the cytoplasm. p27 acts as a bridge between cyclin D and cdk4 to promote their association. Following binding, p27 enhances the nuclear translocation of the complex. Once in the nucleus, nascent cyclin-D- CDK complexes titrate p27 from cdk2, thereby inducing cell cycle progression. Upon cell cycle arrest, the levels of p27 increase, saturate D type proteins and then bind to cyclin-E- cdk2 to block catalytic activity of the kinase.

With such a well documented tumour suppressor role, it is intriguing that such controversy exists as to the prognostic significance of p27, including its sub-cellular location. Low p27 expression has been associated with poor prognosis in a range of malignancies including breast, colorectal, ovary, prostate, bladder and pancreatic tumours [306-309]. Other studies contrast these findings with a loss of p27 producing a favourable prognostic outcome in pancreatic [310], colon, oesophageal and endometrial cancers [311]. Therefore the prognostic influence of p27 as part of the IFN pathway is clearly of interest, especially in view of the controversial findings of previous studies.

The pro-apoptotic effects of IFN are mediated through a number of pathways including Fas and TNF . IRFs are a family of transcription factors induced by type I and II IFN pathways and as such are associated with a variety of immune, cell cycle regulation and apoptotic effects. Interferon regulatory factor 1 (IRF1), a tumour suppressor gene, is thought to play a central role in producing the apoptotic effects of IFN [312]. This is primarily through the activation of caspase 1 [181] and discussed later. Levels of IRF1

Exposure of cells with high levels of functioning IFN receptor to IFN produces a rapid activation of STAT1, thereby inducing high levels of IRF1 which in turn trigger the apoptotic pathway. However, if cells have low levels of IFN receptor, then exposure to IFN produces lower levels of phosphorylated STAT1 and in consequence low levels of IRF1. These lower levels of IRF1 are not sufficient to trigger apoptosis [291]. This is illustrated in experiments in which over expression of IFN receptor in cells usually expressing low levels, changes the response to IFN exposure from an anti-apoptotic/ proliferative effect to a pro-apoptotic effect [291]. This may explain the differing responses of cell types to IFN. Another member of the IRF family, IRF2, acts as a suppressor of IRF1, by binding to the same gene promoter elements [313]. These are both produced at low levels in the non-stimulated cells; however IRF2 is more stable than IRF1 and so accumulates in the nucleus and represses the actions of IRF1 [314]. During IFN signalling the subsequent up-regulation of IRF1 allows competition with IRF2 and stimulates transcription of many IFN inducible genes [315].

Caspase 1 is a cysteine protease belonging to a family of caspases which represent the common final pathway for a number of apoptotic mechanisms. Caspases tend to act as the principle executors in these pathways and produce an amplification cascade between individual caspases. Caspases are expressed as inactive pro-enzymes prior to proteolytic activation via a number of pro-apoptotic stimuli [316]. Caspase 1 can be triggered by a number of mechanisms but is associated with producing IFN mediated apoptosis through IRF1 [181]. Hence, tumour cell evasion from immunosurveillance through defects in the IFN pathway may be through inhibition of the pro-apoptotic effects of IFN induced caspases. Therefore reduced expression of caspase 1 through a defective IFN-IRF1- caspase 1 pathway may result in a poorer prognosis.