Jirina Vavrova

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.

Phosphoproteomics: searching for a needle in a haystack.

Most of the cellular processes are regulated by reversible phosphorylation of proteins, which in turn plays a critical role in the regulation of gene expression, cell division, signal transduction, metabolism, differentiation, and apoptosis. Mass spectrometry of phosphopeptides obtained from tryptic protein digests has become a powerful tool for characterization of phosphoproteins involved in these processes. However, there is a general need to significantly enrich the phosphopeptide content to compensate their low abundance, insufficient ionization, and suppression effects of non-phosphorylated peptides. This paper aims to give a comprehensive overview on the methods involved in recent phosphoproteomics. It presents a description of contemporary enrichment techniques with references to particular studies and compares different approaches to characterization of phosphoproteome by mass spectrometry.

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.

Ataxia-telangiectasia Mutated Kinase (ATM) as a Central Regulator of Radiation-induced DNA Damage Response

Ataxia-telangiectasia mutated kinase (ATM) is a DNA damage-inducible protein kinase, which phosphorylates plethora of substrates participating in DNA damage response. ATM significance for the cell faith is undeniable, since it regulates DNA repair, cell-cycle progress, and apoptosis. Here we describe its main signalling targets and discuss its importance in DNA repair as well as novel findings linked to this key regulatory enzyme in the terms of ionizing radiation-induced DNA damage.

The effect of Amaryllidaceae alkaloids haemanthamine and haemanthidine on cell cycle progression and apoptosis in p53-negative human leukemic Jurkat cells

Plants from the Amaryllidaceae family have been shown to be a promising source of biologically active natural compounds of which some selected are currently in pre-clinical development. Regardless of interesting pioneer works, little is known about Amaryllidaceae alkaloids that have shown promising anti-cancer activities. The crinane group of the Amaryllidaceae, including haemanthamine and haemanthidine, was amongst the first of these compounds to exhibit an interesting cytotoxic potential against cancer cell lines. However, the mechanism of cytotoxic and anti-proliferative activity is not yet entirely clear. The primary objectives of the current study were to investigate the effects of haemanthamine and haemanthidine on the induction of apoptosis and the cell cycle regulatory pathway in p53-null Jurkat cells. Results indicate that haemanthamine and haemanthidine treatment decreases cell viability and mitochondrial membrane potential, leads to a decline in the percentage of cells in the S phase of the cell cycle, induces apoptosis detected by Annexin V staining and increases caspase activity. Dose dependent apoptosis was cross verified by fluorescence and bright field microscopy through Annexin V/propidium iodine staining and morphological changes which characteristically attend programmed cell death. The apoptotic effect of haemanthamine and haemanthidine on leukemia cells is more pronounced than that of gamma radiation. Contrary to gamma radiation, Jurkat cells do not completely halt the cell cycle 24h upon haemanthamine and haemanthidine exposure. Both Amaryllidaceae alkaloids accumulate cells preferentially at G1 and G2 stages of the cell cycle with increased p16 expression and Chk1 Ser345 phosphorylation. Concerning the pro-apoptotic effect, haemanthidine was more active than haemanthamine in the Jurkat leukemia cell line.

Local Environmental Factors Determine Hematopoietic Differentiation of Neural Stem Cells

Stem cells exhibit unique properties and hold high therapeutic promise, but factors influencing their differentiation after transplantation need to be recognized and defined for this promise to be fully met. Here, we demonstrate that endogenous colony-forming unit spleen (CFU-S) colonies are not generated in lethally irradiated mice transplanted with neural stem cells obtained from brain tissue of syngeneic donors. We investigated the proportion of transplanted neural stem cells that contributed to hematopoietic reconstitution and compared the distribution of transplanted cells in nonsplenectomized to that of splenectomized mice following sublethal whole-body irradiation. We also used clonogenic assays, colony assays, and histochemical analyses to explore conditions under which transplanted, beta-galactosidase-tagged neural stem cells underwent hematopoietic differentiation. Our results suggest that neural stem cells do undergo extramedullary hematopoiesis, even while no endogenous hematopoietic colonies develop in the spleen. Furthermore, we found that neural stem cells effectively colonized the bone marrow of splectomized recipients. We conclude that the hematopoietic differentiation of neural stem cells is highly dependent on the extramedullary environment. We also conclude that the bone marrow does not provide an environment supportive of hematopoietic differentiation by neural stem cells.

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.

Early changes in L-arginine-nitric oxide metabolic pathways in response to the whole-body gamma irradiation of rats

Abstract Purpose: Nitric oxide (NO), a reactive radical, is formed in higher amounts from L-arginine by inducible NO synthase (iNOS) during early response to ionizing radiation presumably as a part of signal transduction pathways. This study investigated the changes in L-arginine-NO metabolic pathways within a 24-hour period after whole-body gamma irradiation of rats with the range of low to supra-lethal doses. Materials and methods: Young adult female Wistar rats received either 0–50 Gy whole-body irradiation or an intraperitoneal injection of bacterial lipopolysaccharide (LPS, 10 mg/kg). Exhaled NO was monitored using chemiluminiscence, nitrite + nitrate (NOx) and L-arginine were assayed by high-performance liquid chromatography, and expression of iNOS was determined using Western blot. Results: Irradiation resulted in a dose-dependent increase of plasma NOx to maximum levels which were 4-fold higher compared to controls (p < 0.001). The NOx levels increased less in the bronchoalveolar lavage fluid (BAL) (1.7-fold, p < 0.001) and liver homogenate (2.5-fold, p < 0.05), respectively, and were dose-independent. Exhaled NO, lung NOx, plasma and BAL L-arginine, and the expression of iNOS in lung and liver tissues of irradiated rats and controls were similar. LPS caused a considerable increase (p < 0.001) in exhaled NO (61-fold), NOx levels (plasma 34-fold, BAL 6-fold, lung 5-fold, liver 4-fold), and in iNOS expression, respectively. Conclusion: In contrast to the LPS treatment of rats, the radiation-induced changes in L-arginine-NO metabolic pathways are modest, particularly in the airways and lungs. Noninvasive measurement of exhaled NO within a 24-h period following the exposure of rats to ionizing radiation has no value for biodosimetry.

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.