Abstract #5094: Potential role of ASPP1 protein level for p53 dependent apoptosis in the germ cell tumor cell line NTERA
Authors: Christiane Markwardt, Ioanna Skorta, Thomas Mürdter, Heiko van der Kuip, Walter Aulitzky
Journal: Cancer Research (2009)

Purpose: Cisplatin is widely used for treatment of various malignant tumor entities. However, only a few, such as germ cell tumors, can actually be cured by such treatment. These entities harbor a unique phenotype which is characterized by hypersensitivity to Cisplatin. The well accepted hypothesis is that this hypersensitivity is mediated by a lower DNA repair capacity. We asked whether this low DNA repair capacity is causative for the hypersensitive phenotype or if an early induction of apoptosis may precede cell cycle arrest and DNA repair. Experimental procedure: Cisplatin-induced apoptosis was analyzed by Annexin-V staining. Removal of Cisplatin adducts from DNA was measured by atom absorption spectrometry and used as equivalent for DNA repair capacity. To compare cells with different Cisplatin sensitivity within the same cellular system we established an inducible model system basing on the testicular germ cell line NTERA. This cell line is characterized by an unusual high sensitivity to Cisplatin. Short term treatment of these cells with all-trans retinoic acid (ATRA) renders them about 10fold less sensitive to Cisplatin. Using this model system, we investigated proteins involved in DNA damage recognition, regulation of cell cycle arrest, DNA repair, and apoptosis. Results: As expected, NTERA responded to Cisplatin treatment by rapid induction of cell death and showed no attempts to arrest cell cycle or repair DNA damage. Pre-treatment with both the caspase inhibitor z-VAD as well as the p53 inhibitor Pifithrin completely abolished induction of Cisplatin-induced cell death suggesting a mechanism dependent on both caspase and p53 activity. Importantly, preventing apoptosis by z-VAD enabled NTERA to partially remove Cisplatin adducts from DNA indicating that the rapid induction of cell death upon Cisplatin precedes DNA repair in these cells. To investigate the mechanisms behind this rapid cell death we analyzed expression and activity of DNA damage regulators in NTERA cells and NTERA cells pre-incubated with ATRA. We found significant differences neither in DNA damage recognition proteins nor in p53 induction and Ser15 phosphorylation or proteins involved in DNA repair. Interestingly however, ASPP1 expression was markedly reduced in cells pre-treated with ATRA. In addition, the diminished sensitivity to Cisplatin in cells pre-treated with ATRA was mirrored by a reduced induction of BAX and NOXA. Conclusion: We found that NTERA cells still have the capability to repair DNA-Cisplatin adducts. However, induction of cell death precedes the onset of cell cycle arrest and DNA repair. Our results show that this very rapid cell death depends on caspase and p53 activity. This may at least partially depend on high ASPP1 protein levels, a transcriptional co-factor functioning as apoptosis stimulating protein of p53.   Citation Information:  In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 5094.