Antitumor Genotoxic Agents, Such as Fludarabine*, Doxorubincin or Cis-Platine, Induce Activation of STAT1 in a p53 Protein Dependent Manner.

Introduction:

Chemotherapeutic drug such as Fludarabine*; doxorubicin or cis-platine induce cell cycle arrest and apoptosis via activation of p53. Convergent studies suggest that p53 and STAT1 cooperate in the induction of apoptosis, and that STAT1 favors p53 activation. However, to our knowledge, the role of p53 in the activation of STAT1 is not documented. We present our results suggesting that (i) genotoxic agents are STAT1 inducers, (ii) STAT1 activation depends on the presence of p53 protein, and (iii) this phenomenon is modulated by the tyrosine kinase inhibitor STI571.

Materials and Methods:

To analyse the role of p53 in STAT1 activation, we have used different cellular models with different p53 status: PRI (p53wt), BL2 (p53wt), BL41 (p53 mutated on Arg248, resulting in the loss of p53 DNA binding activity (p53mut)), Jurkat, HL60 and MEF (the 3 latter being p53 null). The following cDNAs were used for functional studies: p53wt, p53mut, MDM2 and MTBP (MDM2 transforming protein). These cDNAs were cloned either in a pcDNA3 vector or a pRT-1 inducible vector (in the latter, the gene of interest is expressed from a bidirectional doxycycline regulatable promoter allowing simultaneous expression of truncated NGF receptor, used as a surrogate marker of inducibility).

Results:

Treatment of the different cell lines with the 3 genotoxic drugs Fludarabine*, doxorubicin or cis-platine induced STAT1 activation in p53wt BL2 or PRI cells and in p53mut BL41 cells, but not in Jurkat cells neither in HL60 or MEF cells. Induction of STAT1 was also obtained in presence of the RNA synthesis inhibitor Actinomycin D or in presence of secretion inhibitor Brefeldine A. Over-expression of p53wt or p53mut markedly increased STAT1 activation in PRI cells. This effect was reversed by over-expression of MTBP. Complementation of both HL60 and MEF cells with both p53wt and p53mut cDNA induced constitutive STAT1 activation, an effect that was increased by treatment with doxorubine in transfected HL60 cells. This effect was reversed by over-expression of MDM2 in HL60 cells. Finally, we found that treatment of cells with the inhibitor STI 571 of c-Abl tyrosine kinase, a kinase known to be associated with ATM during p53 activation, decreased STAT1 activation by genotoxic drugs.

Conclusion:

Our results show that genotoxic agents are inducers of STAT1, that p53 protein but not p53 transcriptional activity is responsible for this STAT1 activation, and suggest a possible involvement the cABL tyrosine kinase.