Jana Cmielova

Gamma radiation induces senescence in human adult mesenchymal stem cells from bone marrow and periodontal ligaments.

Purpose: Mesenchymal stem cells isolated from bone marrow (BM-MSC) and periodontal ligament (PLSC) are cells with high proliferative potential and ability to self-renewal. Characterization of these cells under genotoxic stress conditions contributes to the assessment of their prospective usage. The aim of our study was to evaluate changes in BM-MSC and PLSC caused by ionizing radiation. Methods: Human BM-MSC and PLSC were irradiated with the doses up to 20 Gy by Co60 and observed 13 days; viability, proliferation, apoptosis and senescence induction, and changes in expression and phosphorylation status of related proteins were studied. Results: Irradiation with the doses up to 20 Gy significantly reduces proliferation, but has no significant effect on cell viability. The activation of tumor suppressor protein 53 (p53) and its phosphorylations on serines 15 and 392 were detected from the first day after irradiation by 20 Gy and remained elevated to day 13. Expression of cyclin-dependent kinases inhibitor 1A (p21Cip1/Waf1) increased. The cell cycle was arrested in G2 phase. Instead of apoptosis we have detected hallmarks of stress-induced premature senescence: increase in cyclin-dependent kinases inhibitor 2A (p16INK4a) and increased activity of senescence-associated β-galactosidase. Conclusion: Mesenchymal stem cells isolated from bone marrow and periodontal ligament respond to ionizing radiation by induction of stress-induced premature senescence without apparent differences in their radiation response.

Irradiation of Adult Human Dental Pulp Stem Cells Provokes Activation of p53, Cell Cycle Arrest, and Senescence but Not Apoptosis

Adult human dental pulp contains stem cells (DPSCs) that are capable of differentiation into osteoblasts, odontoblasts, adipocytes, and neuronal-like cells. Because these cells have potential use in tissue regeneration, herein we characterized the response of DPSC lines to ionizing radiation (IR). These DPSC lines have been developed from the extracted molars of healthy donors. DPSCs were cultivated in a unique media supplemented with epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). Since tissue homeostasis depends on a precise balance among cell proliferation, senescence, and cell death, we explored the effects of IR (2-20 Gy) on the proliferative activity of DPSCs and the molecular pathways involved. Even the highest dose used (20 Gy) did not induce DPSC apoptosis. After irradiation with doses of 6 and 20 Gy, DPSCs accumulated in the G2 phase of the cell cycle. DPSCs responded to IR (20 Gy) with senescence detected as SA-β-galactosidase positivity, beginning on the third day after irradiation. Twenty-four hours after irradiation, p53 and its serine 15 and 392 phosphorylated forms were detected. At this time, p21 (WAF1) was induced. Increases in protein p16 were observed from the third day following irradiation and continued till the end of the examination (Day 13). We conclude that DPSCs respond to IR-induced damage by permanent cell cycle arrest in the G2 phase and by stress-induced premature senescence.

Differences in Vanadocene Dichloride and Cisplatin Effect on MOLT-4 Leukemia and Human Peripheral Blood Mononuclear Cells

Modern chemotherapy is interested in developing new agents with high efficiency of treatment in low-dose medication strategies, lower side toxicity and stronger specificity to the tumor cells. Vanadocene dichloride (VDC) belongs to the group of the most promising metallocene antitumor agents; however, its mechanism of action and cytotoxicity profile are not fully understood. In this paper we assess cytotoxic effects of VDC in comparison to cisplatin using opposite prototype of cells; human peripheral blood mononuclear (PBMCs) cells and human acute lymphoblastic leukemia cell line (MOLT-4). Our findings showed cytotoxic effect of VDC on leukemia cells, but unfortunately on human peripheral blood mononuclear cells as well. VDC induces apoptosis in leukemia cells; the induction is, however, lower than that of cisplatin, and in contrary to cisplatin, VDC does not induce p53 up-regulation. Cytotoxic effect of VDC on leukemia cells is less pronounced than that of cisplatin and more pronounced in PBMCs than in MOLT-4 cells.

The cytotoxic effect of α-tomatine in MCF-7 human adenocarcinoma breast cancer cells depends on its interaction with cholesterol in incubation media and does not involve apoptosis induction

In recent years, α-tomatine has been studied for its anticancer activity. In the present study, we focused on the cytotoxic effect of α-tomatine in the MCF-7 human breast adenocarcinoma cell line, its mechanism of action, biotransformation and stability in the culture medium. We observed an inhibition of cell proliferation and viability at concentrations of 6 and 9 μM but then a recovery of cells occurred. The recovery was not caused by the biotransformation of α-tomatine in MCF-7 cells, but by a substantial decrease in the concentration of α-tomatine in the culture medium due to its binding with cholesterol. Regarding the mechanism of action of α-tomatine, we observed no DNA damage, no changes in the levels of the proteins p53 and p21WAF1/Cip1, and no apoptosis (neither activated caspase-8 and -9, nor sub-G1 peak, or morphological signs). We found a loss of ATP in α-tomatine-treated cells. These results support the conclusion that α-tomatine does not induce apoptosis in the MCF-7 cell line.

The effect of ATM kinase inhibition on the initial response of human dental pulp and periodontal ligament mesenchymal stem cells to ionizing radiation

Abstract Purpose: This study evaluates early changes in human mesenchymal stem cells (MSC) isolated from dental pulp and periodontal ligament after γ-irradiation and the effect of ataxia-telangiectasia mutated (ATM) inhibition. Methods: MSC were irradiated with 2 and 20 Gy by 60Co. For ATM inhibition, specific inhibitor KU55933 was used. DNA damage was measured by Comet assay and γH2AX detection. Cell cycle distribution and proteins responding to DNA damage were analyzed 2–72 h after the irradiation. Results: The irradiation of MSC causes an increase in γH2AX; the phosphorylation was ATM-dependent. Irradiation activates ATM kinase, and the level of p53 protein is increased due to its phosphorylation on serine15. While this phosphorylation of p53 is ATM-dependent in MSC, the increase in p53 was not prevented by ATM inhibition. A similar trend was observed for Chk1 and Chk2. The increase in p21 is greater without ATM inhibition. ATM inhibition also does not fully abrogate the accumulation of irradiated MSC in the G2-phase of the cell-cycle. Conclusions: In irradiated MSC, double-strand breaks are tagged quickly by γH2AX in an ATM-dependent manner. Although phosphorylations of p53(ser15), Chk1(ser345) and Chk2(thr68) are ATM-dependent, the overall amount of these proteins increases when ATM is inhibited. In both types of MSC, ATM-independent mechanisms for cell-cycle arrest in the G2-phase are triggered.

Specific inhibition of Wee1 kinase and Rad51 recombinase: A strategy to enhance the sensitivity of leukemic T-cells to ionizing radiation-induced DNA double-strand breaks

Present-day oncology sees at least two-thirds of cancer patients receiving radiation therapy as a part of their anticancer treatment. The objectives of the current study were to investigate the effects of the small molecule inhibitors of Wee1 kinase II (681641) and Rad51 (RI-1) on cell cycle progression, DNA double-strand breaks repair and apoptosis following ionizing radiation exposure in human leukemic T-cells Jurkat and MOLT-4. Pre-treatment with the Wee1 681641 or Rad51 RI-1 inhibitor alone increased the sensitivity of Jurkat cells to irradiation, however combining both inhibitors together resulted in a further enhancement of apoptosis. Jurkat cells pre-treated with inhibitors were positive for γH2AX foci 24h upon irradiation. MOLT-4 cells were less affected by inhibitors application prior to ionizing radiation exposure. Pre-treatment with Rad51 RI-1 had no effect on apoptosis induction; however Wee1 681641 increased ionizing radiation-induced cell death in MOLT-4 cells.