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  • br concentration dependent obstructions during


    concentration-dependent obstructions during DNA replication. Seemingly, treating AsPC-1 cells with 1 induces a different kind of
    cell cycle rearrangements as for MCF-7 cells (Fig. 4). In the AsPC-1 samples subjected to 1 at 30 μM, cells are hindered to enter the S phase, thus inducing a mild accumulation in G0/G1. At the higher con-centrations M3814 (nedisertib) (50 and 75 μM), cells are found gathered at the S-to-G2 restriction point, whereas treatment at 100 μM results in an arrest in the S phase. This time changes are accompanied by a concentration-de-pendent amplification of apoptotic response (Figs. 3 and 4). Cell cycle alterations seen in samples incubated with 50 and 75 μM indicate that cells passed the process of chromosomal replication but were restrained to enter the final phase of mitotic division probably due to necessary DNA repair. In cells treated with 100 μM, division is delayed during replication, which suggests that 1 induces at such concentration a da-mage that had to activate DNA repair mechanisms before accessing S-to-G2 restriction point.
    As stated before, CDDP did not achieve any activity against AsPC-1 cells. Considering its relevance as a reference compound, M3814 (nedisertib) assessment has been performed and described previously [116]. Here, CDDP applied in a concentration range from 1 to 50 μM on MCF-7 cells induces a significant accumulation in G2/M phase (Fig. 4). In none of those samples the incidence of neither apoptotic nor necrotic events are notably different compared to non-treated control (Fig. 3). Significant augmentation in frequency of Annexin V-stained cells is found in the samples subjected to CDDP at the highest concentrations (75 and 100 μM) with both of them shifting cells to stop in S-to-G2 restriction point. CDDP is known for its ability to trigger various mechanisms that affect homeostasis and lead to apoptosis, with liable formation of DNA lesions [120–124]. Initially, such type of results were reported as ex-clusively depended on p53 status and drug concentration [125–129]. According to those reports, in cells protected with mutated p53, arrest at the G2 phase was found as the dominant characteristic, while in-crease in CDDP concentration induced cell cycle block in the S phase. On the contrary, in cells with wild-type (wt) p53, arrest in the G1 phase was found at all concentrations of CDDP. Later on, it was disclosed that CDDP induces arrest in the S phase in a p53-independent manner [130]. Nevertheless, He et al. [131] recently revealed that such type of cell cycle arrest strongly depends on pathways activated by particular DNA damaging agents, including CDDP, rather than on the p53 status of treated cells. MCF-7 cells used here are reported to have wt p53 status [132], which is not the case of AsPC-1 cells [133]. Thus, distribution of cells within mitotic division in MCF-7 samples afterwards treatment with CDDP (Fig. 4) correspond to previously described data [131]. The divergence in cell cycle response of MCF-7 cells to 1 and CDDP treat-ments can be addressed to different types of interference those two drugs induce during DNA replication. On the other hand, alterations in
    (caption on next page)
    Fig. 3. Incidences of apoptotic and necrotic deaths in cells treated with 1 (A) and CDDP (B) determined after 24 h incubation by means of Annexin V/PI dual staining method. In Annexin V/PI dot plots cells are discriminated as viable (non-stained cells, lower left quadrant), cells in early phase of apoptosis (Annexin V single-stained cells, lower right quadrant), cells in advanced phases of cell death of apoptotic death (double-stained cells, upper right quadrant), and necrotic cells (PI single-stained cells, upper left quadrant). Results are represented as the mean ± SD percentages of two replicates from independent experiments.