Emmanuel Conseiller

Restoration of transcriptional activity of p53 mutants in human tumour cells by intracellular expression of anti-p53 single chain Fv fragments

We report here the production and the properties of single chain Fv fragments (scFvs) derived from the anti-p53 monoclonal antibodies PAb421 and 11D3. 11D3 is a newly generated monoclonal antibody which exhibits properties very comparable to those of PAb421. The scFvs PAb421 and 11D3 are able to stably associate with p53 and to restore the DNA binding activity of some p53 mutants in vitro. When expressed in p53−/− human tumour cells, the scFv421 is essentially localized in the cytoplasm in the absence of p53, and in the nucleus when exogenous p53 is present. Thus, p53 is also able to stably associate with an anti-p53 scFv in cells. Cotransfection of p53−/− human tumour cells with expression vectors encoding the His273 p53 mutant and either scFv leads to restoration of the p53 mutant deficient transcriptional activity. These data demonstrate that, in human tumour cells, these scFvs are able to restore a function essential for the tumour suppressor activity of p53 and may represent a novel class of molecules for p53-based cancer therapy.

Definition of a p53 transactivation function-deficient mutant and characterization of two independent p53 transactivation subdomains.

The wild-type protein product of the p53 tumor suppressor gene can activate transcription of genes which are involved in mediating either growth arrest, e.g. WAF1 or apoptotis, e.g. BAX and PIG3. Additionally, p53 can repress a variety of promoters, which, in turn, may be responsible for the functional activities exhibited by p53. This study shows that the Q22, S23 double mutation, which is known to inactivate a p53 trans-activation subdomain located within the initial 40 residues of the protein, while abrogating transactivation from the WAF1 promoter, only attenuates apoptosis triggering, transactivation from other p53-responsive promoters and repression of promoters by p53. The Q53, S54 double mutation, which inactivates another p53 transactivation subdomain situated between amino acids 43 and 73 results in attenuation of all of the afore-mentioned p53 activities. In contrast to the Q22, S23 double mutation, this latter mutation set does not alter mdm-2-mediated inhibition and degradation of p53. Finally, mutation of all four residues results in complete abrogation of every p53 activity mentioned above.

Human fibulin‐4: analysis of its biosynthetic processing and mRNA expression in normal and tumour tissues

Here, we report the identification of a human orthologue of fibulin‐4, along with analysis of its biosynthetic processing and mRNA expression levels in normal and tumour tissues. Comparative sequence analysis of fibulin‐4 cDNAs revealed apparent polymorphisms in the signal sequence that could account for previously reported inefficient secretion in fibulin‐4 transfectants. In vitro translation of fibulin‐4 mRNA revealed the presence of full‐length and truncated polypeptides, the latter apparently generated from an alternative translation initiation site. Since this polypeptide failed to incorporate into endoplasmic reticulum membrane preparations, it was concluded that it lacked a signal sequence and thus could represent an intracellular form of fibulin‐4. Using fluorescence in situ hybridisation analysis, the human fibulin‐4 gene was localised to chromosome 11q13, this region being syntenic to portions of mouse chromosomes 7 and 19. Considering the fact that translocations, amplifications and other rearrangements of the 11q13 region are associated with a variety of human cancers, the expression of human fibulin‐4 was evaluated in a series of colon tumours. Reverse transcription‐polymerase chain reaction analysis of RNA from paired human colon tumour and adjacent normal tissue biopsies showed that a significant proportion of tumours had ∼2–7‐fold increases in the level of fibulin‐4 mRNA expression. Taken together, results reported here suggest that an intracellular form of fibulin‐4 protein may exist and that dysregulated expression of the fibulin‐4 gene is associated with human colon tumourigenesis.