Klin Padiatr 2012; 224 - A15
DOI: 10.1055/s-0032-1320183

Actinomycin-D treatment of high-risk ependymomas re-establishes the apoptotic function of p53

TD Tzaridis 1, 2, H Witt 1, 2, T Milde 2, 4, S Bender 1, 2, E Pfaff 1, 2, DTW Jones 1, AE Kulozik 2, P Lichter 5, A Korshunov 3, O Witt 2, 4, SM Pfister 1, 2
  • 1Division of Pediatric Neurooncology, German Cancer Research Center (DKFZ), Heidelberg, Germany
  • 2Department of Pediatric Oncology, Hematology and Immunology, University Hospital Heidelberg, Germany
  • 3Department of Neuropathology, University Hospital Heidelberg, Germany
  • 4Clinical Cooperation Unit Pediatric Oncology, German Cancer Research Center (DKFZ), Heidelberg, Germany
  • 5Division of Molecular Genetics, German Cancer Research Center (DKFZ), Heidelberg, Germany

Aim of study:

Ependymomas are brain tumours morphologically resembling the lining epithelium of the ventricular system of the CNS and account for 12% of all pediatric intracranial malignancies. Despite the absence of TP53 mutations, immunohistochemical accumulation of non-functional p53 is frequently observed in ependymomas and correlates with poor clinical outcome. Homozygous CDKN2A deletion is another common aberration characterizing high-risk ependymomas. Since this deletion results in excessive MDM-2 activity due to absence of p14, we believe it to comprise one important molecular mechanism of loss of p53 function and aim to reverse it by inhibiting MDM-2 through Actinomycin-D. Our study includes the treatment of two ependymoma cell lines with Actinomycin-D, in order to evaluate the potential of the agent to reactivate p53.

Methods:

TP53 mutation status of 130 primary ependymomas and two ependymoma cell lines was assessed by Sanger sequencing of exons 2–11. p53 protein expression was studied by immunohistochemistry on 398 intracranial ependymomas. Cell viability after treatment was assessed by MTS assay. Proapoptotic effects of Actinomycin-D treatment were analysed by flow cytometry. Transcriptome analyses were performed using Agilent 44K microarrays and were followed by validation via Western blotting and quantitative real-time PCR.

Results:

TP53 sequencing of primary ependymomas confirmed the low rate of somatic mutations (3%). Nevertheless, p53 accumulation occurred much more frequently (22%) and was strongly associated with unfavourable prognosis. After treatment of the cells with different concentrations of Actinomycin-D we calculated the IC-50 of the agent (0.2–0.7nM), thereby emphasising its low-dose effects. Flow cytometric analysis of treated and untreated cells demonstrated low-dose Actinomycin-D to be more effective in apoptosis induction. Subsequently, we performed transcriptome analyses of high-dose (100nM), low-dose (5nM) and untreated cells. Among various differenzially expressed genes between low-dose and high-dose Actinomycin-D we revealed a significant proportion of p53-dependent candidates (i.e. TP53I3, PUMA). We further validated by Western blotting the Actinomycin-D mediated reestablishment of the p53-complex (p21, MDM-2).

Conclusion:

These results demonstrate that Actinomycin-D is capable of reactivating p53 and thereby inducing apoptosis in ependymoma cell lines. Therefore it comprises a promising novel therapeutic approach for high-risk ependymoma patients, which we are currently evaluating in vivo.