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Oncogenic Ras signalling in many primary cell types results in a cell cycle arrest associated with the induction of CDKIs, which is mediated by the Raf/MAPK pathway. However, the mechanism responsible for the induction of these molecules appears to differ between cell types. For example, in primary MEFs oncogenic Ras expression results in the induction of p21^‘‘’\ which is thought to be due to p i 9^^*" m ediated activation of p53, and increased levels o f and (Serrano et aL, 1997); Palmero et a l, 1998; Malumbres et al.,

2000). In contrast in primary HDFs, the oncogenic R as/R af induced arrest is associated with the induction o f p l6^^^"^^ and p21^'^hn a p i 9"^^^ independent manner (Serrano et at.,

1997; Zhu, 1998; Wei et at., 2001).

In primary Schwann cells activation of R af results in a p53 dependent cell cycle arrest that is mediated, at least in part, by the induction of p21^'^^ (Lloyd et at., 1997). I have shown that R af activation also induces pl5^^’^'^’’ expression, which may also be involved in mediating the cell cycle arrest in these cells. This is similar to the situation observed with prim ary M EFs, which arrest in a dependent manner in response to oncogenic Ras (Malumbres et at., 2000). In contrast, I observed that p i 9^*^"“ levels decrease in response to R af activation in prim ary Schwann cells, w hilst p i8°^^'*^ levels remain unchanged.

Therefore neither p i 9^^"^ nor p i8^*^"*^ are involved in mediating the Raf induced cell cycle

arrest. This is not especially unexpected as pl9°^*^'^‘’ and p i8^*^"^ have been implicated in

mediating cell cycle arrests during development and terminal differentiation of a number of cell types, including myoblasts and adipocytes, rather than inducing an arrest following oncogenic stress (Zindy et aL, 1997a; Zindy et aL, 1997b; Phelps et aL, 1998).

Surprisingly I observed that p i l e v e l s also decrease in response to R af activation and therefore this CDKI is not involved in mediating the cell cycle arrest in primary Schwann cells. This is in contrast to a num ber of prim ary cell types where plb^^'*^ expression is induced in response to oncogenic Ras/Raf signalling (Serrano et aL, 1997; Zhu, 1998). M oreover in HDFs, p l6^^"^^ induction is associated with a cell cycle arrest in p53 or

null cells and mutation of in HDFs derived from a familial melanoma patient

appears to abrogate the Ras induced arrest, suggesting that p i i s an important mediator of the oncogenic Ras/Raf induced cell cycle arrest in this cell type (Zhu, 1998; Wei et aL,

2001). p i l e v e l s can be regulated by a number of transcription factors (Ohtani et aL,

2001; Jacobs et aL, 1999), however, the reduction in plb*^^'*^ protein levels following Raf activation was not due to reduced mRNA levels, suggesting that an as yet unidentified post- transcriptional mechanism is responsible for this effect. The reduction in plb^*^'^ protein levels and the induction of cyclin D1 may form part of the mechanism by which Ras/Raf signalling prom otes cell cycle progression in prim ary Schwann cells under appropriate environmental conditions.

The importance of p21^’’’^ in mediating the R af induced arrest in primary Schwann cells has been clearly demonstrated using p21^'^^antisense RNA, which partially abrogates the cell cycle arrest (Lloyd et aL, 1997). The mechanism of p21^'^^ induction by oncogenic Ras/Raf in these cells is unclear, however, I found that p21^'^^ induction is associated with elevated p2 icipi levels, which is consistent with increased transcription. Since the R af induced

arrest in prim ary Schwann cells is p53 dependent (Lloyd et aL, 1997), an increase in p53 transcriptional activity may be responsible for the increased p21^'^^ levels. H ow ever, although p21*^’P^ is a transcriptional target of p53 in response to stimuli such as DNA damage and oncogenic stress, p21^'^'can also be induced independently of p53 in response to oncogenic R as/R af by the transcription factors E2F1 and S p l/2 in other cell types (Serrano et aL, 1997; W oods et aL, 1997; Kivinen et aL, 1999; C artel et aL, 2000). Moreover, in other circumstances p21^'^^induction can be mediated by transcription factors such as IRF-1 that require the presence of basal levels of p53 (Tanaka et aL, 1996). A lternatively the elevated levels of p21‘^’’’^ RNA could indicate an increase in RNA stability.

Since the increase in p21^'^^ RNA is relatively small compared to the fold induction of protein, it is possible that other mechanisms are also involved in increasing p2 1^'^*protein

levels. It has been demonstrated that p21^'^^ levels can be regulated post-transcriptionally. F or exam ple C /E B P a (C C A A T/ enhancer -b in d in g protein a ) induces elevated p2 1 ^'P^protein levels via a combination of increased p2 1^'^'RNA and post-translational

stabilisation of the p21^'^^protein (Timchenko et aL, 1996), whilst conditional expression of oncogenic Ras in NIH3T3 cells resulted in increased protein synthesis in the absence of enhanced transcription or protein stability, suggesting that increased translation was occurring (Kivinen et aL, 1999). However, I found that R af activation in primary Schwann cells does not increase p2 1^'^‘protein stability.

In primary MEFs the oncogenic Ras induced cell cycle arrest is associated with increased p53 levels (Serrano et aL, 1997). In contrast, I showed that p53 levels do not increase in response to R af activation in prim ary Schwann cells. Since p53 activity is norm ally associated with increased stability this may suggest that p53 is not actively involved in transcription o f p21^'^' in response to R af activation in these cells. A similar situation exists in HDFs where activation o f R af results in the induction of p21^'"^^in a p53 dependent manner, but is not associated with a detectable increase in p53 levels (Zhu 1998). However, p53 stabilisation is not essential for activation o f p53 in NIH3T3 cells and M EFs (Korgaonkar et aL, 2002), suggesting that p53 activity could be increased in prim ary Schwann cells in the absence of elevated p53 levels. In HDFs and MEFs p53 activity can also be enhanced by interaction with PM L in response to oncogenic R as/R af and it is possible a similar interaction may occur in primary Schwann cells, however this interaction might also be expected to result in increased p53 levels (Ferbeyre, 2000) Pearson et aL,

2000).

M utant p53 proteins, which are used as dominant negative inhibitors of wild-type p53 activity, have also been observed to inhibit the functions of the p53 homologues p63 and p73, raising the possibility that the R af induced arrest in prim ary Schwann cells is dependent on the functions of either or both molecules instead of p53 ([Yang, ; Di Como et aL, 1999; Strano et aL, 2002). However, p63 and p73 are rarely mutated in human cancers, p73 null mice are not predisposed to tumorigenesis and p73 inactivation is not required for viral transformation of cells, suggesting that p63 and p73 may not be tumour suppressor proteins (Marin et aL, 1998; Yang et aL, 2002). However, p63 may be involved in the p53 response to UV, and p73 is stabilised by y-irradiation and can be induced by oncogenes in p53 deficient cells resulting in apoptosis or the transcription o f p53 targets

(Zaika et aL, 2001; Yang et a l, 2002). Thus the importance of p63 and p73 in response to p53 activating stimuli remains unclear. It will be interesting to examine w hether R af activation in primary Schwann cells is associated with increased levels and activity of p63 or p73.

P I9ARF been implicated in the regulation of p53 stability and activity in response to

oncogenic stress in primary MEFs (Palmero et ah, 1998; Damalas et aL, 2001). In contrast, in HDFs the oncogenic Ras induced increase in p53 levels appears to be p l9^^^ independent (Wei et aL, 2001). In primary Schwann cells, I observed that R af activation results in the induction of p i 9^^^ RNA, which is associated with increased p i 9 ^ ^ protein levels. Increased p i 9 ^ ^ RNA levels may reflect increased p i 9 ^ ^ transcription, which may be mediated by the transcription factors E2F (Bates et aL, 1998) or D M P l (Inoue et aL,

1999). Alternatively R af activation may result in the reduction in levels or activity of the transcriptional repressors bmi-1. Twist or TBX2 (Jacobs et aL, 1999; Maestro et aL, 1999; Jacobs et aL, 2000) or may induce increased p i 9^^^ RNA by another m echanism independently of increased transcription. In primary MEFs the oncogenic Ras/Raf induced arrest is p i 9^^^ dependent (Kamijo et aL, 1997). However, the importance of p i 9^^^ in mediating the Raf induced arrest in Schwann cells and whether Raf activation increases p53 transcriptional activity via pl9^^^ induction has yet to be determined. A model for the mechanism of the oncogenic Ras/Raf induced cell cycle arrest based on these results and the previous findings of Lloyd et aL, is detailed in Fig. 6.1 (Lloyd et aL, 1997).

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Cell cycle arrest

Fig. 6.1. Model of the mechanism of the oncogenic Ras/Raf induced cell cycle arrest in primary Schwann cells.

Activation of the Raf/MAPK pathway results in a cell cycle arrest associated with elevated levels of the CDKIs p21^'P’ and p i i n a p53 dependent manner. However, and levels decrease and so these factors are not involved in inducing the arrest. Regulation of the levels of and by Raf may be mediated by the MAPK pathway, but this has yet to be confirmed. Raf activation also induces pl9^^^expression, which may result in p53 activation in the absence of stabilisation of p53 protein levels. Transcriptionally active p53 may directly induce p 21C ipl expression or other transcription factors may be required instead of or as well as p53. The mechanism of Raf/M APK induction of has yet to be investigated in these cells. Dashed arrows indicate potential interactions. See text for more details.