Rebelión como herramienta de combate

0.8 - 0.6 - 0.4 - 0.2 - : cyclin E-dependent kinase activity : DNA synthesis

4 .8 Mutant p53 abolishes p2lWAFi/Cipi induction

The cell-cycle inhibitor p2lW AFi/cipi was discovered as a gene induced after activation of wild type p53 (el-Deiry et al., 1993). In NSARafER cells, the coexpression of a dominant negative p53 mutant understood to abolish wild-type p53 transcriptional activity towards specific targets was able to overcome the Raf-induced cell cycle arrest (see chapter 4.0, Figure IV.9). If the induction of p2lWAFi/Cipi jp response to activated Raf was p53- dependent, then NSARafER cells coexpressing mutant p53 should exhibit a clear decrease of p2lWAFi/Cipi protein levels compared to arrested NSARafER cells.

To examine p2lW AFi/cipi protein levels in Schwann cells coexpressing dominant negative p53, NSARafER mtpSS'*^^, NSARafER Puro and NSARafER SV40 LT cells were induced with 4-hydroxy tamoxifen. Protein lysates were prepared from induced and uninduced control cells 26 hours after induction, a time point at which the p2lW AFi/clpi protein level peaks (see Figure IV.1 0). Activated Raf in Schwann cells coexpressing p5 3R i7 5H or SV40 LT cells did, however, not induce p2lWAFi/Cipi (Figure IV. 12). In NSARafER Puro control cells the protein levels p2lW AFi/clpi were clearly induced 26 hours after 4-hydroxy tamoxifen induction as previously observed (Figure IV. 12). Thus, the induction of p2lWAFi/Cipi by activated ARaf occurred in a p53-dependent manner. This finding further established a correlation of p2lWAFi/Cipi induction with growth arrest and the decrease of p2lW AFi/cipi with cell cycle progression since Schwann cells coexpressing mutant p53 continued to progress through the cell cycle regardless of Raf activation (see Figure IV.10).

Figure IV.12: Raf induces p2lW AFi/cipi py a p53-dependent

mechanism

NSARafER Puro, NSARafER or NSARafER LT were grown for 30 hours in the presence or absence of 1 pM 4-hydroxy tamoxifen. Subsequently protein lysates were prepared. Cells were harvested after this time period and 50 pg of total cell protein were analysed on a SDS-PAGE. Western blotting analysis with an p2lWAFi/Cipi. specific antibody was carried out.

Figure IV.13: Cyciin D1 is induced in NSARafER pS3i75 ceiis in response to constitutiveiy active Raf

NSARafER Puro, NSARafER p5 3"i7 5 and NSARafER LT cells were grown as specified above in the presence (+) or absence (-) of 1 pM 4-hydroxy tamoxifen. Cells were harvested after this time period and 50 pg of total cell protein were analysed on a SDS-PAGE. Western blotting analysis with an cyclin D1 specific antibody was carried out.

p21

TM

y y

^ cyclin D1

TM

y

4 .9 Cyclin D1 induction by Raf does not contribute to G1 cell cycle arrest in Schwann ceils

The superinduction of cyclin D1 in response to Ras expression and transformation has been previously reported (Liu et al., 1995; Winston et al., 1996) and it has been suggested that superinduction of cyclin D1 can result in cell cycle arrest in some cell types. To test whether activated Raf can also superinduce cyclin D1 protein and whether this possible superinduction is correlated with cell cycle arrest in Schwann cells, cyclin D1 protein levels were examined in arrested and proliferating Schwann cells. NSARafER Puro, NSARafER p5 3i7 5 and NSARafER LT cells were grown in the presence or absence of 1 pM 4-hydroxy tamoxifen for 30 hours. Protein lysates were prepared and subsequently analysed by western blotting with a cyclin D1 specific antibody. Cyclin D1 protein levels are induced in response to activated Raf (Figure IV.13). However, the induction of cyclin D1 appears not to contribute to the G1 cell cycle arrest, since protein levels of cyclin D1 are similar in arrested NSARafER Puro and proliferating NSARafER p63‘*^5 and NSARafER LT cells (Figure IV.13).

4 .1 0 Raf induction and p53 transactivation activity

The induction of the cell-cycle inhibitor p2lW AFi/clpi ;g necessary and sufficient to arrest Schwann cells in the G1 phase of the cell-cycle (this thesis; Lloyd et al., 1997). This induction depends on p53 transactivation activity, since a dominant negative p5 3 mutant abrogates the p2lWAFi/Cipi induction (this thesis; Lloyd et al., 1997). However, as the evidence was indirect, I sought a more direct approach to investigate whether p53's transactivation activity was actually increased after Raf activation.

To investigate a possible change in p53 transactivation activity in response to activated Raf, transient transfection experiments introducing p53-responsive reporter genes were performed. These experiments were not successful. Schwann cells can be only transfected with very low efficiency and did not grow any further after transfection. It has been reported that in eukaryotic cells p53 is activated after transient transfections and can cause growth arrest which may also occur in Schwann cells. To overcome these difficulties, I decided to construct a retroviral vector containing a p53-responsive reporter gene and infect NSARafER cells.

4.10.1 Construction of retroviral vector containing a

p53 binding site reporter gene

To directly measure p53 transactivation activity in NSARafER cells after activation of Raf, I constructed a reporter gene with a p53 binding consensus sequence. Since Schwann cells have to be infected for a successful introduction of genes, the luciferase reporter gene was cloned into a modified version of retroviral vector pBPSTRI (Paulus et al.,1995). The DNA-sequences in pBPSTRI responsible for the tetracycline regulation of the pBPSTRI vector were substituted by a minimal thymidine kinase promoter and an adjacent luciferase gene. Oligomers containing a p53 consensus binding sequence (Chen et ai., 1993) were cloned in front of the minimal thymidine kinase promoter and luciferase reporter gene (Figure IV. 14) to measure luciferase gene expression in a p53-dependent way.

Figure IV.14: Retroviral P53-reporter construct pBPSTR WTp53-tk-luc2F

To test p53 transactivation activity directly, the retroviral p53-reporter construct pBPSTR WTp53-tk-luc2F was constructed as described in Materials and Methods. A p53 DNA binding site from the Ribosomal Gene Cluster was integrated in front of a tk promoter and luciferase reporter gene into the retrovirus pBSTR.

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