The recovery from hydroxyurea-induced DNA replication stress is

The displacement of DEK from damaged chromatin is a rather early event since its mobility was increased directly after DNA damage induction by laser micro-irradiation.

Long-term effects of DNA damage on DEK’s localization have not been studied so far.

Since DNA replication stress impacts on histone recycling and thereby on replication-dependent chromatin assembly (Jasencakova et al, 2010), it was asked whether DEK´s localization may be altered in particular in the course of recovery from DNA damage arising from DNA replication stress. To this end, HeLa S3 cells were synchronized by

5 RESULTS 47

mitotic shake off (MSO) and replication stress was induced by HU 14 h after seeding of mitotic cells. At this time the majority of cells resided in S phase (see CYCA staining in Figure 5.19 A, and (Fox, 2004)). HU was removed after 24 hours. The localization of DEK was monitored via immunocytochemistry using a polyclonal DEK antibody (Figure 5.19).

In interphase, DEK is known to localize to the nucleus where it mainly binds to chromatin (Figure 5.15, and Chapter 4.1.3.1). Interestingly, during the recovery from DNA replication stress DEK became virtually undetectable via immunocytochemistry from two to seven days after removal of HU (Figure 5.19 A, see images 4-6). Additionally, it became evident that this effect was cell cycle-independent since DEK was absent from all cells, whether CYCA-positive or -negative. The presence of both types of cells at later time points indicates that cells were de-synchronized after such a long incubation time. Two days after removal of HU, low levels of DEK could be detected in the cytoplasm only (Figure 5.19 B, see image 4). DEK re-localized to the nucleus approximately five days after HU removal, but was still hardly detectable via immunocytochemistry (Figure 5.19 B, see image 5). To rule out that MSO, which poses additional stress to the cells, had any impact on DEK localization during the recovery from HU-induced replication stress an alternative method was applied. S phase cells were pulse-labeled with EdU for one hour and then treated with HU for 24 hours. After removal of the drug and recovery from replication stress for 38 h, EdU positive cells, i.e. those cells that were in S phase when replication stress was induced by HU, were devoid of DEK, confirming the previous observations (Figure 5.19 C). These EdU-positive cells showed a very weak DEK signal in the cytoplasm but no nuclear DEK (data not shown) similarly to the cells shown in image (4) in Figure 5.19 B. The punctuated DEK staining pattern suggested a compartmentalization of DEK protein in the cytoplasm.

To further investigate DEK expression during the recovery phase from DNA replication stress, cells were lysed in cold SDS lysis buffer and subjected to SDS-PAGE and Western blot analysis. Strikingly, no change in DEK expression level could be detected using the polyclonal DEK antibody (Figure 5.20 A). Only the monoclonal DEK antibody which preferentially binds to a phosphorylated form of DEK suggested that DEK levels or its posttranslational modification status may change over time. Since the signal for phosphorylated DEK is weak in unsynchronized cells, strongest in mitotic cells and decreases again 14 h after synchronous release from mitosis, it can be concluded that the phosphorylation state of DEK is strongly cell cycle-dependent. This is in accordance with proteome-wide phosphorylation studies across the cell cycle which showed strongest phosphorylation of DEK during mitosis and G1 phase (Daub et al, 2008; Kappes et al, 2004; Olsen et al, 2010).

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Figure 5.19 | DEK changes its localization during recovery from HU-induced DNA replication stress.

(A) Confocal immunofluorescence images of HeLa S3 cells labeled with DEK- (red) and CYCA- (green) specific antibodies. DNA was counterstained using Hoechst33342 (blue). Cells were synchronized by mitotic shake-off (MSO) and cultured for 14 h (1). They were then treated with 1 mM HU which was removed after 24 h (2). Cells were allowed to recover for one, two, five and seven days (3-6). Scale bar: 10 µm. (B) Quantitation of DEK-specific fluorescence intensity of cells as treated in (A). At least 100 nuclei were evaluated per condition. Images show contrast-enhanced confocal images of cells after two days (4) and five days (5) of recovery from HU-induced replication stress (merge of DEK and DNA-specific signals). Scale bar: 5 µm. (C) Confocal images of HeLa S3 cells pulse labeled with EdU for 1 h and either left untreated for 52 h (upper row) or treated with 1 mM HU for 24 h followed by a 38 h recovery period (lower row). DEK was detected using a specific antibody (rabbit polyclonal) and EdU was labeled with Alexa 488-azide. Scale bar: 10 µm.

Higher molecular weight species of DEK could be detected with the polyclonal DEK antibody 48 h after HU removal using hot SDS lysis buffer, most likely because this buffer preserves PTMs, such as conjugation to ubiquitin and ubiquitin-like proteins (Figure 5.20 B). Interestingly, DEK contains a consensus sumoylation motif Ψ-K-X-E (K261), and sumoylation is known to impact on protein stability, protein-protein interaction, and protein localization (Geiss-Friedlander & Melchior, 2007). Based on these observations, it was investigated whether DEK is a target for sumoylation. To this end, immunoprecipitation of endogenous DEK was performed in HeLa Kyoto whole cell lysates using rabbit anti-DEK polyclonal antibody. The precipitated material was analyzed by PAGE and Western blot using DEK and SUMO1-specific antibodies. DEK and a higher molecular weight form of DEK could be detected in untreated cells as well as in cells treated with HU for four hours. The high molecular weight form of DEK was detected also with the SUMO1-specific antibody, indicating sumoylation of DEK (Figure 5.20 C).

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Figure 5.20 | Western blot analysis of DEK expression in response to HU-induced DNA replication stress.

(A+B) Cells were synchronized by mitotic shake-off (MSO) and cultured for 14 h. They were treated then with 1 mM HU which was removed after 24 h. Cells were allowed to recover as indicated. Whole cell lysates were analyzed by SDS-PAGE and Western blot. (A) Cold SDS-lysis of HeLaS3 cells. DEK and actin were detected using specific antibodies. (B) Hot SDS lysis of HeLa S3 and HeLa Kyoto cells. DEK and PCNA were detected using specific antibodies. (C) Western blot analysis of immunoprecipitated DEK from untreated HeLa Kyoto cells. DEK and SUMO1 were detected using specific antibodies.

In sum, DEK is heavily modified in response to DNA replication stress. This modification is a late and persistent consequence of damage which does not become evident until the successive cell generations. DEK’s release from chromatin and its re-localization to the cytoplasm are most probably a consequence of changes in its PTM pattern. Among others, DEK may be a target for sumoylation. It seems likely that the massive change in DEK´s localization from nuclear to cytoplasmic during recovery from replication stress leads to a global change in chromatin structure. To my knowledge, this is the first report of a response to DNA replication stress affecting global chromatin composition. This response becomes manifest during the recovery phase from replication stress, persists for days after the actual insult and affects daughter cell generations.

6 DISCUSSION

Several groups have independently reported about DEK’s implication in cancer development resulting in its classification as a bona fide oncogene which can serve as a tumor marker in multiple malignancies. The activities responsible for DEK´s tumor promoting function are still matter of investigation. So far, the negative impact of DEK on cellular processes that protect against cancer development is considered to be the main cause for DEK-driven tumor promotion: (1) DEK affects apoptotic cell death by destabilization of p53 leading to transcriptional repression of p53 target genes including the pro-apoptotic protein BCL-2 resulting in apoptosis inhibition, and (2) DEK inhibits senescence and delays differentiation by so far unknown mechanisms, and (3) DEK impacts on cell adhesion by leading to a decreased expression of E-cadherin which is important for cell-cell interactions rendering cells more motile. On the other hand, DEK was demonstrated to facilitate the repair of DNA damage. This latter activity is hard to reconcile with a tumor promoting function, since the accumulation of DNA damage and genomic instability are hallmarks of cancer development.

In hyperproliferating cancer cells, DNA replication stress is a well-established source for unusual DNA secondary structures as well as DNA strand breaks which both trigger a DNA damage response. In healthy cells, this would result in slow down or arrest of the cell cycle to allow for proper damage repair. A persistent damage signaling that exceeds a certain threshold would lead to the induction of apoptosis or senescence: the replication stress-induced DNA damage barrier to cancerogenesis. However, during cancer development cells that are capable of circumventing these cell-intrinsic regulatory mechanisms are positively selected for. Possible routes for bypassing the DNA damage barrier are inactivation of downstream effectors of the DNA damage response (e.g. p53 inactivation) or partial reversal of genomic instability by facilitating DNA repair thereby promoting proliferation. The latter effect of genomic re-stabilization in cells that already underwent transformation can be considered pro-oncogenic. Early reports already pointed towards a functional link between DEK and replication: DEK affects replication efficiency in vitro and preferentially binds to cruciform DNA structures. Therefore, this thesis aimed to investigate DEK’s function in the response to DNA damage, in particular to replication stress, and it was hypothesized that DEK affects this response in a way that promotes tumor cell proliferation.

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