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¹ Grupo de Biología Molecular del Cáncer, Departamento de Biologia Molecular, Unidad de Biomedicina-CSIC, Universidad de Cantabria, Santander, Spain; ² Servicio de Hematología, Hospital Universitario Marqués de Valdecilla, Santander, Spain; ³ Unidad de Genética Molecular, Hospital Universitario Marqués de Valdecilla, Santander, Spain; and ⁴ Laboratori di Oncogenesi Molecolare, Istituto Regina Elena, Rome, Italy

Requests for reprints: Javier León, Departamento de Biologia Molecular, Facultad de Medicina, 39011 Santander, Spain. Phone: 34-942-201952; Fax: 34-942-201945. E-mail: leonj{at}unican.es

Amifostine is used as a cytoprotective agent in cancer treatments. Amifostine protects from apoptosis in some models and has been used as hematopoiesis stimulator in myeloid malignancies. As the apoptosis induced by many antitumoral agents is mediated by p53, we studied the effect of amifostine on p53-mediated apoptosis. We used human myeloid leukemia K562 and NB4 cells expressing the temperature-conditional p53-Val¹³⁵ mutant. Both cell lines undergo apoptosis at 32°C due to the presence of p53 in wild-type conformation. We found that amifostine dramatically reduced apoptosis by p53 in both cell lines, as assessed by cell morphology, annexin V binding, fraction of sub-G₁ cells, and DNA laddering. To explore the mechanism responsible for this apoptosis protection, we tested the effect of amifostine on p53 transcriptional activity. We found that amifostine reduced p53-mediated transactivation of target promoters in NB4 and K562. Macroarray analysis confirmed that several p53 target genes as p21^Waf1, mdm2, gadd45, pig8, and pig3 were down-regulated at the mRNA level by amifostine in NB4 and K562. Also, c-myc was up-regulated by amifostine in K562 in the presence of p53, consistently with the impairment of p53-mediated apoptosis exerted by c-Myc in these cells. We conclude that amifostine impairs p53-dependent apoptosis of myeloid leukemia cells by reducing the activation of apoptosis-related genes. Our results open the possibility that amifostine could reduce the effectiveness of antitumoral treatments when it is dependent on active p53.

Grant support: Grants PM98-109 and SAF02-4193 from Dirección General de Investigación Científica y Técnica, Spanish Government and grant FIS01-1129 from Spanish Ministerio de Sanidad y Consumo.

Received 5/14/03; revised 6/20/03; accepted 6/26/03.