Radim Havelek - Academia.edu (original) (raw)

Papers by Radim Havelek

International Journal of Radiation Biology, 2013

The objective of the study was to investigate differences in the radiosensitivity of rat peripher... more The objective of the study was to investigate differences in the radiosensitivity of rat peripheral blood lymphocyte subsets identified by expression of surface clusters of differentiation markers (CD3, CD4, CD8, CD45RA, CD161) after whole-body in vivo gamma-ray irradiation and to assess their individual histone H2AX phosphorylation as an early cell response to irradiation. The relative representations of CD45RA B-lymphocytes, CD161 natural killer cells (NK cells), CD3CD4 T-lymphocyte subset and CD3CD8 T-lymphocyte subset in the rat peripheral blood were studied 24-72 hours after irradiation in a dose range of 0-5 Gy. Their intracellular H2AX phosphorylation (γ-H2AX) after 4 Gy and 9 Gy whole-body in vivo irradiation was assessed by multicolour flow cytometry. We determined the linear dose response of radioresistant CD161 NK cells (24 h), both radiosensitive T-lymphocyte subsets (24 h) and CD45RA B-lymphocytes (72 h) after in vivo irradiation. CD45RA B-lymphocytes showed the highest radiosensitivity and we observed pronounced H2AX phosphorylation which remained expressed in these cells for over 4 h after irradiation. The combination of the surface immunophenotyping together with intracellular detection of γ-H2AX offers the possibility to assess the absorbed dose of ionizing irradiation with high sensitivity post irradiation and could be successfully applied to biodosimetry.

Acta Veterinaria Brno, 2011

The aim of our study was to determine whether phosphorylation of histone H2AX can be used as an i... more The aim of our study was to determine whether phosphorylation of histone H2AX can be used as an indicator of received dose of gamma radiation after whole-body irradiation of rats.

Medical oncology (Northwood, London, England), 2015

The aim of the present study is to evaluate the role of ATM (KU55933) and DNA-PK (NU7441) inhibit... more The aim of the present study is to evaluate the role of ATM (KU55933) and DNA-PK (NU7441) inhibitors in the repair of double-strand breaks and downstream signaling of DNA damage introduced by ionizing radiation. The irradiation of MCF-7 cells alone increased the proportion of cells in the G1 phase in comparison with mock-treated cells. After ATM inhibitor pretreatment, the cells were more accumulated in the G2 phase, whereas DNA-PK inhibitor application increased the percentage of cells in the G1 phase. ATM and DNA-PK inhibitor application alone increased the sensitivity of MCF-7 cells to ionizing radiation; however, combining both inhibitors together resulted in a further enhancement of cell death. Unexpectedly, combining both inhibitors decreased the percentage of senescent cells and increased G2 cell cycle arrest 3 days after treatment. After irradiation, the p21 protein was increased and Chk1 and Chk2 were activated. These proteins were not increased in cells pretreated with the...

Biochemical and Biophysical Research Communications, 2014

Present-day oncology sees at least two-thirds of cancer patients receiving radiation therapy as a... more 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.

Chemicke Listy

This article summarizes basic information about the Amaryllidaceae alkaloids with cytotoxic and a... more This article summarizes basic information about the Amaryllidaceae alkaloids with cytotoxic and apoptosis-inducing potential in diverse cancer cell lines and tumour xenografts. The review focuses on molecular mechanisms leading to cell death mediated by Amaryllidaceae alkaloids. Since some of them possess high cytotoxic efficiency in cancer cell lines study of Amaryllidaceae alkaloids could generate chemical structures for potential drugs. The biological activities that could be useful especially in topical cancer treatment are indicated. We have tested cyto¬toxic and antiproliferative activities of lycorine, galanthine and tazzetine in breast adenocarcinoma cells SK-BR-3 using label-free and real-time monitoring of the xCELLigence cell viability system.

Stem Cells and Development, 2010

Adult human dental pulp contains stem cells (DPSCs) that are capable of differentiation into oste... more 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.

Phytomedicine, 2014

Plants from the Amaryllidaceae family have been shown to be a promising source of biologically ac... more 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.

Medicinal Chemistry, 2012

Modern chemotherapy is interested in developing new agents with high efficiency of treatment in l... more 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.

International Journal of Radiation Biology, 2013

This study evaluates early changes in human mesenchymal stem cells (MSC) isolated from dental pul... more 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. MSC were irradiated with 2 and 20 Gy by (60)Co. 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. 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. 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.

International Journal of Radiation Biology, 2013

The objective of the study was to investigate differences in the radiosensitivity of rat peripher... more The objective of the study was to investigate differences in the radiosensitivity of rat peripheral blood lymphocyte subsets identified by expression of surface clusters of differentiation markers (CD3, CD4, CD8, CD45RA, CD161) after whole-body in vivo gamma-ray irradiation and to assess their individual histone H2AX phosphorylation as an early cell response to irradiation. The relative representations of CD45RA B-lymphocytes, CD161 natural killer cells (NK cells), CD3CD4 T-lymphocyte subset and CD3CD8 T-lymphocyte subset in the rat peripheral blood were studied 24-72 hours after irradiation in a dose range of 0-5 Gy. Their intracellular H2AX phosphorylation (γ-H2AX) after 4 Gy and 9 Gy whole-body in vivo irradiation was assessed by multicolour flow cytometry. We determined the linear dose response of radioresistant CD161 NK cells (24 h), both radiosensitive T-lymphocyte subsets (24 h) and CD45RA B-lymphocytes (72 h) after in vivo irradiation. CD45RA B-lymphocytes showed the highest radiosensitivity and we observed pronounced H2AX phosphorylation which remained expressed in these cells for over 4 h after irradiation. The combination of the surface immunophenotyping together with intracellular detection of γ-H2AX offers the possibility to assess the absorbed dose of ionizing irradiation with high sensitivity post irradiation and could be successfully applied to biodosimetry.

International Journal of Radiation Biology, 2013

The objective of the study was to investigate differences in the radiosensitivity of rat peripher... more The objective of the study was to investigate differences in the radiosensitivity of rat peripheral blood lymphocyte subsets identified by expression of surface clusters of differentiation markers (CD3, CD4, CD8, CD45RA, CD161) after whole-body in vivo gamma-ray irradiation and to assess their individual histone H2AX phosphorylation as an early cell response to irradiation. The relative representations of CD45RA B-lymphocytes, CD161 natural killer cells (NK cells), CD3CD4 T-lymphocyte subset and CD3CD8 T-lymphocyte subset in the rat peripheral blood were studied 24-72 hours after irradiation in a dose range of 0-5 Gy. Their intracellular H2AX phosphorylation (γ-H2AX) after 4 Gy and 9 Gy whole-body in vivo irradiation was assessed by multicolour flow cytometry. We determined the linear dose response of radioresistant CD161 NK cells (24 h), both radiosensitive T-lymphocyte subsets (24 h) and CD45RA B-lymphocytes (72 h) after in vivo irradiation. CD45RA B-lymphocytes showed the highest radiosensitivity and we observed pronounced H2AX phosphorylation which remained expressed in these cells for over 4 h after irradiation. The combination of the surface immunophenotyping together with intracellular detection of γ-H2AX offers the possibility to assess the absorbed dose of ionizing irradiation with high sensitivity post irradiation and could be successfully applied to biodosimetry.

Acta Veterinaria Brno, 2011

The aim of our study was to determine whether phosphorylation of histone H2AX can be used as an i... more The aim of our study was to determine whether phosphorylation of histone H2AX can be used as an indicator of received dose of gamma radiation after whole-body irradiation of rats.

Medical oncology (Northwood, London, England), 2015

The aim of the present study is to evaluate the role of ATM (KU55933) and DNA-PK (NU7441) inhibit... more The aim of the present study is to evaluate the role of ATM (KU55933) and DNA-PK (NU7441) inhibitors in the repair of double-strand breaks and downstream signaling of DNA damage introduced by ionizing radiation. The irradiation of MCF-7 cells alone increased the proportion of cells in the G1 phase in comparison with mock-treated cells. After ATM inhibitor pretreatment, the cells were more accumulated in the G2 phase, whereas DNA-PK inhibitor application increased the percentage of cells in the G1 phase. ATM and DNA-PK inhibitor application alone increased the sensitivity of MCF-7 cells to ionizing radiation; however, combining both inhibitors together resulted in a further enhancement of cell death. Unexpectedly, combining both inhibitors decreased the percentage of senescent cells and increased G2 cell cycle arrest 3 days after treatment. After irradiation, the p21 protein was increased and Chk1 and Chk2 were activated. These proteins were not increased in cells pretreated with the...

Biochemical and Biophysical Research Communications, 2014

Present-day oncology sees at least two-thirds of cancer patients receiving radiation therapy as a... more 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.

Chemicke Listy

This article summarizes basic information about the Amaryllidaceae alkaloids with cytotoxic and a... more This article summarizes basic information about the Amaryllidaceae alkaloids with cytotoxic and apoptosis-inducing potential in diverse cancer cell lines and tumour xenografts. The review focuses on molecular mechanisms leading to cell death mediated by Amaryllidaceae alkaloids. Since some of them possess high cytotoxic efficiency in cancer cell lines study of Amaryllidaceae alkaloids could generate chemical structures for potential drugs. The biological activities that could be useful especially in topical cancer treatment are indicated. We have tested cyto¬toxic and antiproliferative activities of lycorine, galanthine and tazzetine in breast adenocarcinoma cells SK-BR-3 using label-free and real-time monitoring of the xCELLigence cell viability system.

Stem Cells and Development, 2010

Adult human dental pulp contains stem cells (DPSCs) that are capable of differentiation into oste... more 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.

Phytomedicine, 2014

Plants from the Amaryllidaceae family have been shown to be a promising source of biologically ac... more 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.

Medicinal Chemistry, 2012

Modern chemotherapy is interested in developing new agents with high efficiency of treatment in l... more 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.

International Journal of Radiation Biology, 2013

This study evaluates early changes in human mesenchymal stem cells (MSC) isolated from dental pul... more 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. MSC were irradiated with 2 and 20 Gy by (60)Co. 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. 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. 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.

International Journal of Radiation Biology, 2013

The objective of the study was to investigate differences in the radiosensitivity of rat peripher... more The objective of the study was to investigate differences in the radiosensitivity of rat peripheral blood lymphocyte subsets identified by expression of surface clusters of differentiation markers (CD3, CD4, CD8, CD45RA, CD161) after whole-body in vivo gamma-ray irradiation and to assess their individual histone H2AX phosphorylation as an early cell response to irradiation. The relative representations of CD45RA B-lymphocytes, CD161 natural killer cells (NK cells), CD3CD4 T-lymphocyte subset and CD3CD8 T-lymphocyte subset in the rat peripheral blood were studied 24-72 hours after irradiation in a dose range of 0-5 Gy. Their intracellular H2AX phosphorylation (γ-H2AX) after 4 Gy and 9 Gy whole-body in vivo irradiation was assessed by multicolour flow cytometry. We determined the linear dose response of radioresistant CD161 NK cells (24 h), both radiosensitive T-lymphocyte subsets (24 h) and CD45RA B-lymphocytes (72 h) after in vivo irradiation. CD45RA B-lymphocytes showed the highest radiosensitivity and we observed pronounced H2AX phosphorylation which remained expressed in these cells for over 4 h after irradiation. The combination of the surface immunophenotyping together with intracellular detection of γ-H2AX offers the possibility to assess the absorbed dose of ionizing irradiation with high sensitivity post irradiation and could be successfully applied to biodosimetry.