In vivo evaluation of the role of DNp73alpha protein in regulating the p53-dependent apoptotic pathway after treatment with cytotoxic drugs.
The amino terminus truncated p73 isoform, DeltaNp73alpha, shows dominant negative behavior toward TAp73 and wild-type p53, and has oncogenic potential. By contrast, we recently showed that in HCT116 clones forced expression of DeltaNp73alpha did not increase in vitro cellular resistance to anticancer agents. The purpose of this study was to characterize in vivo models and to investigate the functional interaction between the DeltaNp73alpha isoform and the p53 pathway. Human colon carcinoma HCT116 clones expressing inducible DeltaNp73alpha (HCT116/DN3, HCT116/DN14) and HCT116/8a (transfected with the mock empty vector), transplanted in immunodeficient nude mice, were used to study the antitumor activity of cis-diammine-dichloro-platinum (cDDP) (4 mg/kg, i.v., q7d x 3) and Doxorubicin (DX) (7.5 mg/kg, i.v., q7d x 3), with or without tetracycline-induced DeltaNp73alpha overexpression. DeltaNp73alpha expression was confirmed by RT-PCR, immunoblotting and immunohistochemical analysis. DeltaNp73alpha subcellular localization after DX treatment was checked by an immunofluorescence assay. Western blot was used to analyze p53, p21, Bax, Bcl-2 and p53AIP1 expression. DeltaNp73alpha overexpression did not modify the antitumor activity of either DX or cDDP in xenograft models. DX reduced DeltaNp73alpha protein expression, without affecting its nuclear localization. p53, p21, Bax and p53AIP1 protein expression increased and Bcl-2 decreased in HCT116 clone derived tumors 24 hr after DX exposure, independently of the presence of DeltaNp73alpha. Overexpression of DeltaNp73alpha does not affect tumor growth in vivo, does not increase the resistance of established tumors to anticancer agents and does not antagonize p53 apoptotic functions.
Sabatino
Maria Antonietta
MA
Laboratory of Molecular Pharmacology, Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy.
Previdi
Sara
S
Broggini
Massimo
M
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology* / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D015153:Blotting, Western; D002945:Cisplatin / Q000494:pharmacology / Q000627:therapeutic use; D003110:Colonic Neoplasms / Q000235:genetics / Q000473:pathology / Q000517:prevention & control; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D004317:Doxorubicin / Q000494:pharmacology / Q000627:therapeutic use; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D045325:HCT116 Cells; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D018613:Microscopy, Confocal; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000502:physiology*; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D023041:Xenograft Model Antitumor Assays
D000970:Antineoplastic Agents; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D004317:Doxorubicin; D002945:Cisplatin