## 4 Discussion
In addition, IDA-induced apoptosis was accompanied by increased levels of tumor suppressor p53 and its target protein p21 in MV4-11 cells. A large drop in viability and PARP fragmentation were induced by all drugs in the OCI-AML2 cell line derived from an AML patient with DNMT3A R635W mutation. Ara-C-induced p53 expression was not followed by an increase in p21, but the simultaneous drop in XIAP and Mcl1 expressions likely contributed to extensive apoptosis despite survivinupregulation. Conversely, p53-independent p21 upregulation occurred after IDA treatment together with survivvin, XIAP, and Mcl1 decrease. A similarly unusual p53 regulation in the OCI-AML2 cell line, which has no reported p53 mutations, was also previously observed after NSC348883 treatment [55]. Combination of Ara-C with IDA canceled the p21 overexpression and further enhanced a fraction of dead cells. YM-155 substantially decreased the survivvin, XIAP, and Mcl1 levels and, in combination with Ara-C, it also caused p21 deregulation, leading to extensive apoptosis. In the OCI-AML3 cell line (with NPM1 mutation type A and DNMT3A R882C), survivin deregulation correlates with a viability drop induced by IDA or YM-155. Moreover, Ara-C and IDA upregulate p53, and extensive p21 induction is observed in all samples. Simultaneously, XIAP and Mcl1 remain unchanged, suggesting that survivin regulation plays an important role in apoptosis in these cells. Whether it may have a consequence with aberrant localization of mutated NPM remains to be investigated. Survivin mRNA upregulation after IDA was observed in two p53-null cell lines, KG-1 and HL-60. Whereas, in HL-60, the protein level also increased, KG-1 cells showed a decrease in survivin after IDA treatment. Different regulation of survivin at the mRNA vs. protein level has also been documented in esophageal cancer cells [12]. Interestingly, the viability was only slightly decreased in KG-1 although XIAP and Mcl1 expressions were substantially lowered. On the contrary, a high fraction of dead cells occurred in IDA-treated HL-60 cells although no considerable changes were detected in XIAP, Mcl1, and p21 levels. Importantly, YM-155 caused a viability drop, together with PARP fragmentation and survivin, XIAP, and Mcl1 downregulation in both these p53-null cell lines, and its effect was potentiated by combination with Ara-C. YM-155, thus, reliably reverts Ara-C-induced survivin upregulation and triggers apoptosis in p53-null cell lines. In accordance with the results shown in the literature [53], the level of antiapoptotic protein Bcl2 was unchanged (data not shown). Moreover, we did not notice any significant change in expression of NF-kB, which was reported to participate in response to YM-155 treatment in several tumor types [8; 16]. Lastly, immunofluorescence (IF) staining confirmed that enhanced survivin expression detected by immunoblotlot reflects a higher occurrence of survivin in the nuclei of interphase cells. We did not detect any mitotic cells in Ara-C- and/or IDA-treated cells. On the other hand, mitosis was not affected by YM-155 treatment, and localization of survivin in mitotic cells was identical in control and YM-155 samples. Therefore, YM-155 probably does not compromise the proliferation of normal cells, which corresponds with its good tolerability. Interestingly, GFP-tagged survivin is often found in the cytoplasm of transfected cells [56; 57], whereas we found prevalently nuclear localization in the interphase cells by immunofluorescence staining of endogenous survivin. As our finding correlates with IF data provided by various survivin antibody suppliers (Abcam, Cell Signaling Technology, Santa Cruz BioTechnology et al. ), we suggest that, if the GFP tag is used for small proteins, attention must be paid to pitfalls eventually arising from aberrant localization of the resulting fusion protein.