Anna Aprile

p53 mutants can often transactivate promoters containing a p21 but not Bax or PIG3 responsive elements.

The human p53 protein acts mainly as a stress inducible transcription factor transactivating several genes involved in cell cycle arrest (e.g. p21) or apoptosis (e.g. Bax, PIG3). Roughly half of all human tumours contains p53 missense mutations. Virtually all tumour-derived p53 mutants are unable to activate Bax transcription but some retain the ability to activate p21 transcription. Identification of these mutants may have valuable clinical implications. We have determined the transactivation ability of 77 p53 mutants using reporter yeast strains containing a p53-regulated ADE2 gene whose promoter is regulated by p53 responsive elements derived from the regulatory region of the p21, Bax and PIG3 genes. We also assessed the influence of temperature on transactivation. Our results indicate that a significant proportion of mutants [16/77 (21%); 10/64 (16%) considering only tumour-derived mutants] are transcriptionally active, especially with the p21 promoter. Discriminant mutants preferentially affect less conserved (P<0.04, Fishers exact test), more rarely mutated (P<0.006, Fishers exact test) amino acids. Temperature sensitivity is frequently observed, but is more common among discriminant than non-discriminant mutants (P<0.003, Fishers exact test). Finally, we extended the analysis to a group of mutants isolated in BRCA-associated tumours that surprisingly were indistinguishable from wild type in standard transcription, growth suppression and apoptosis assays in human cells, but showed gain of function in transformation assays. The incidence of transcriptionally active mutations among this group was significantly higher than in the panel of mutants studied previously (P<0.001, Fishers exact test). Since it is not possible to predict the behaviour of a mutant from first principles, we propose that the yeast assay be used to compile a functional p53 database and fill the gap between the biophysical, pharmacological and clinical fields.

Characterization of the p53 mutants ability to inhibit p73β transactivation using a yeast-based functional assay

p53 is the most frequently altered tumor suppressor gene in a wide spectrum of human tumors. The large majority of p53 mutations observed in tumors are missense mutations. The p73 gene, encoding a protein with significant sequence similarity to p53, expresses multiple transcription-competent spliced variants, or transcription-incompetent forms (i.e. ΔNp73). It was clearly shown that p73 transactivation from a p53-responsive promoter is inhibited by some tumor-derived p53 mutants in eucaryotic cells. In this study, we adapted a yeast-based p53 functional assay for the analysis of the influences of different p53 mutants on the activity of one of the p73 isoforms, namely p73β. We determined the ability of a panel of 61 p53 mutants to inhibit p73β activity following the net transcription of the ADE2 color (red/white) reporter gene driven by a p53-responsive promoter. By analysing a large number of mutants, we could conclude that interference: (a) is a quite frequent phenomenon (more than 70% of p53 mutants analysed are interfering); (b) is not confined to p53 mutations located in particular topological regions of the DNA binding domain; (c) does not appear to be dependent on the kind of side chains introduced at a specific position; (d) appears to significantly correlate with evolutionary conservation of the mutated p53 codon, frequency of occurrence of the mutation in tumors. The influence of a common R/P polymorphism at codon 72 on the ability of p53 mutants to interfere with p73β was also studied. Two sets of polymorphic variants (R and P) for 14 mutants were constructed and analysed. In all cases, the R/P 72 polymorphism was phenotypically irrelevant. In conclusion, our results suggest that the interpretation of the biological effects of p53 mutants should take into consideration the possibility that p53 mutants show loss or gain of function also through the interference with p53 family members.

Relationship between DNA Methylation and Mutational Patterns Induced by a Sequence Selective Minor Groove Methylating Agent

Me-lex, a methyl sulfonate ester appended to a neutral N-methylpyrrolecarboxamide-based dipeptide, was synthesized to preferentially generateN 3-methyladenine (3-MeA) adducts which are expected to be cytotoxic rather than mutagenic DNA lesions. In the present study, the sequence specificity for DNA alkylation by Me-lex was determined in the p53 cDNA through the conversion of the adducted sites into single strand breaks and sequencing gel analysis. In order to establish the mutagenic and lethal properties of Me-lex lesions, a yeast expression vector harboring the human wild-type p53 cDNA was treated in vitro with Me-lex, and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. The results showed that: 1) more than 99% of the lesions induced by Me-lex are 3-MeA; 2) the co-addition of distamycin quantitatively inhibited methylation at all minor groove sites; 3) Me-lex selectively methylated A’s that are in, or immediately adjacent to, the lex equilibrium binding sites; 4) all but 6 of the 33 independent mutations were base pair substitutions, the majority of which (17/33; 52%) were AT-targeted; 5) AT → TA transversions were the predominant mutations observed (13/33; 39%); 6) 13 out of 33 (39%) independent mutations involved a single lex-binding site encompassing positions A600–602 and 9 occurred at position 602 which is a real Me-lex mutation hotspot (n = 9, p < 10−6, Poisson’s normal distribution). A hypothetical model for the interpretation of mutational events at this site is proposed. The present work is the first report on mutational properties of Me-lex. Our results suggest that 3-MeA is not only a cytotoxic but also a premutagenic lesion which exerts this unexpected property in a strict sequence-dependent manner.

Hydrodynamic and mass spectrometry analysis of nearly-intact human fibrinogen, chicken fibrinogen, and of a substantially monodisperse human fibrinogen fragment X

The shape and solution properties of fibrinogen are affected by the location of the C-terminal portion of the Aalpha chains, which is presently still controversial. We have measured the hydrodynamic properties of a human fibrinogen fraction with these appendages mostly intact, of chicken fibrinogen, where they lack 11 characteristic 13-amino acids repeats, and of human fragment X, a plasmin early degradation product in which they have been removed. The human fibrinogen/fragment X samples were extensively characterized by SDS-PAGE/Western blotting and mass spectrometry, allowing their composition to be precisely determined. The solution properties of all samples were then investigated by analytical ultracentrifugation and size-exclusion HPLC coupled with multi-angle light scattering and differential pressure viscometry detectors. The measured parameters suggest that the extra repeats have little influence on the overall fibrinogen conformation, while a significant change is brought about by the removal of the C-terminal portion of the Aalpha chains beyond residue Aalpha200.

Interaction retinol-chromatin: an analysis of DNA from vitamin A-treated V79 Chinese hamster cells

When V79 cells are incubated in the presence of radiolabeled retinol, a small but consistent amount of radioactivity remains associated with nuclear DNA. Chromatographic analysis of enzymatic hydrolysates of DNA shows that no irreversible changes, such as adducts, have taken place on DNA. We present evidence that this radioactive incorporation may occur via a metabolic conversion of retinol, leading mainly to the formation of radiolabeled thymidine, which is then incorporated into newly made DNA. Mutagenic effects by retinol, given at concentrations well above physiological levels, have been also excluded.

Characterization of drug release from fibrin gels loaded with different pharmaceutical and experimental doxorubicin formulations

BACKGROUND Local delivery of anticancer drugs represents a desirable type of treatment. Nevertheless, characteristics such as availability, biocompatibility, ease of operation, and efficacy sometimes represent difficult to overcome hurdles. Fibrin gels (FBGs) may be attractive biomaterials for local treatment when loaded with different chemotherapeutics or with polymer-anticancer-drug conjugates and nanoparticles. These components, linked together, might represent candidates to counteract local recurrences or reduce the volume of inoperable tumors. In the present study we analyzed the features of in vitro release of different formulations of doxorubicin (DOXO) from FBGs, and in vivo FBGs degradation. METHODS In vitro DOXO release from FBGs was studied as a function of thrombin and Ca2+ ion concentrations. DOXO was loaded in FBGs either alone or pre-incorporated in nanoparticles characterized by different physical features. The FBGs in vivo degradation was analyzed after sc or ip positioning. RESULTS Our results suggest that each of the factors involved in the FBGs preparation may have different effects on drug release. In particular, the fibrinogen (FG) concentration and, above all, the DOXO formulation were found to have the greatest impact. Not surprisingly, we have also found a longer permanence in vivo of FBGs prepared at the highest thrombin, Ca2+ ion, and FG concentrations. CONCLUSIONS The aim of this work was to study the effect of several conditions for preparing drug delivery systems based on FBGs loaded with different clinical or experimental formulations of DOXO. Our data identify some of these modalities that will be tested in vivo to evaluate their antitumor activity.