Mechanisms by which p53 suppresses cell transformation

Through the manipulation of cells in culture, the role of p53 in suppressing tumourigenesis has been demonstrated as an ability to suppress the transformation of cells by a number of viral and activated cellular oncoproteins. The mechanism by which transformation is suppressed probably involves reduced cell growth and / or increased cell death as p53 is known to arrest growth (predominantly in the G1 phase of the cell cycle) and to induce an apoptotic response. However the relationship of these functions to the ability of p53 to suppress oncogenic transformation remains unclear. While p53 can suppress transformation by various oncoproteins, studies in this thesis have demonstrated that it is unable to suppress the transformation of cells by the SV40 large T antigen (LTag). In the presence of LTag, p53 also failed to inhibit cell growth. Interestingly, the function of LTag which enabled it to overcome both the growth and transformation suppressing effects of p53 was not the binding (and probable inactivation) of p53, but the binding and inactivation of another tumour suppressing e la te d op p^ote iaJ protein, pRt£ These observations indicate that*pRbKis required for the mechanism by which p53 suppresses cell growth and transformation. Although the adenovirus Ela proteins can also bind and inactivate pRb (and consequently overcome p53-dependent growth inhibition), their transformation-related activities were found to be suppressed by p53. Further experiments revealed that cells which express E la are susceptible to p53-dependent apoptosis. While p53 was also able to suppress transformation involving an activated form of ras (EJ-ras), further experiments showed that this could be attributed to a growth inhibitory rather than apoptotic effect of p53. It can therefore be proposed that p53 suppresses transformation either by causing a growth arrest (which is mediated by pRb) or by inducing apoptosis, and that the mechanism of suppression is determined by the activities of the transforming oncoproteins. Transformation by LTag would remain refractory to suppression due to the abolition of both cellular effects of p53. However since the studies in this thesis have shown that transformation by LTag does not require the binding (and inactivation) of p53, an additional anti-apoptotic function must be associated with this oncoprotein. It can also be proposed that the process of transformation usually requires cooperation between different oncoproteins (such as E la and EJ-ras) in order to overcome the activities of endogenous p53. While transformation by EJ-ras is normally prevented due to p53-dependent (pRb-mediated) growth inhibition, E la can overcome this effect. Furthermore, EJ-ras was shown to inhibit p53-dependent apoptosis which normally prevents transformation by Ela. However, this anti-apoptotic effect is probably limited to low levels of p53, as exogenous p53 can suppress transformation by Ela and EJ-ras. Through the use of various mutant forms of p53, studies presented in this thesis have shown that within the p53 protein, the regions which are important for growth inhibition are different to those which are important for apoptosis. Thus, inhibition of cell growth and induction of apoptotic cell death are genetically separable functions of p53. Since the studies also demonstrated that p53-dependent growth inhibition is neither sufficient nor necessary for p53-dependent apoptosis, one can suggest that these are alternative rather than sequential functions for suppressing tumourigenesis. This finding is consistent with the observations (as mentioned above) that growth inhibition and apoptosis occur in response to different types of oncoprotein activity. The activities of various mutant forms of p53 have also revealed that the growth inhibitory effect of p53 is neither sufficient nor necessary for the suppression of transformation by E la and EJ-ras. However, the ability of a mutant p53 protein to suppress this type of transformation was shown to correlate well with its ability to induce apoptosis. These observations are in agreement with the prediction (as explained above) that transformation by E la and EJ-ras would be suppressed by an apoptotic rather than growth inhibitory function of p53. Because the mechanisms involved in growth inhibition and apoptosis are ambiguous, the studies presented in this thesis have examined the relationships between the biochemical and cellular activities of p53. Thus, various mutant forms of p53 were used to demonstrate the regions of the protein which are important for activation and repression of gene transcription as well as inhibition of SV40 origin-dependent DNA replication (as this might reflect a role for p53 in regulating cellular DNA replication). Although there is considerable evidence that the transactivation of p21WAF1 is involved in p53-dependent growth arrest, studies in this thesis have shown that transcriptional activation is neither sufficient nor necessary for a growth inhibitory effect. It would therefore appear that p53 has more than one biochemical mechanisms for inhibiting cell growth and that only one of these pathways involves the transcriptional activation of p21WAF1. While transcriptional repression and inhibition of DNA replication were also found to be insufficient for growth inhibition, either of these biochemical activities may contribute to this cellular function of p53. As independent functions, transcriptional activation, transcriptional repression, and inhibition of DNA replication were also found to be insufficient for p53-dependent apoptosis. However, the mechanism by which p53 induces apoptosis may require any of these activities and the combined effects of transcriptional activation and transcriptional repression may even be sufficient for this type of response. In summary, the growth inhibitory and apoptotic activities of p53 have been shown to be separate and alternative functions which are important for suppressing cell transformation in response to different types of oncoproteins. The mechanisms by which these cellular functions occur appear to involve multiple biochemical activities of p53.