Gamma Radiation Induces p53 Mediated Cell Cycle Arrest in Bone Marrow Cells | Apoptosis | Calcium In Biology

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Gamma Radiation Induces p53 Mediated Cell Cycle Arrest in Bone Marrow Cells
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  Chapter Number 1 Gamma Radiation Induces p53-Mediated 2 Cell Cycle Arrest in Bone Marrow Cells 3 Andrea A. F. S. Moraes et al. *   4 Federal University of São Paulo – Unifesp, 5 Santa Cruz State University – Uesc 6 Brazil 7 1. Introduction 8 The hematopoietic system is organized in a hierarchical manner in which rare hematopoietic 9 stem cells initiate the hierarchy and have the ability to self-renew, proliferate and 10 differentiate into different lineages of peripheral blood cells as well as to intermediate 11 hematopoietic progenitor cells. Most hematopoietic stem cells are quiescent under steady- 12 state conditions and function as a stock population to protect the hematopoietic system from 13 exhaustion due to various stressful conditions. In contrast, hematopoietic progenitor cells 14 are rapidly proliferating cells with limited self-renewal ability. The proliferation and 15 differentiation of hematopoietic progenitor cells fulfills the requirements of normal 16 hematopoiesis allowing the hematopoietic system to react promptly and effectively to meet 17 the demand for increasing the output of mature cells during hematopoietic crisis such as 18 loss of blood, hemolysis, infection, the depletion of HPCs by chemotherapy and/or 19 radiotherapy (Reya, 2003; Weissman et al. , 2001; Walkley et al. , 2005). 20 Reactive oxygen species (ROS) are produced in organisms due to radiation, 21 biotransformation of dietary chemicals, some diet components, transient metal ions, 22 inflammatory reactions and during normal cellular metabolism. 23 The effect of gamma radiation ionization affects the main components of biological material 24 such as carbon, hydrogen, oxygen, nitrogen. Radiobiology is described as the action of 25 ionizing radiation on living things. On the molecular and cellular levels direct ionizing 26 radiation can affect molecules in cells, especially DNA, lipids and proteins, promoting 27 breakage and / or modifications or indirectly acting on water molecules and generating 28 excitation ionization products of water radiolysis which include free radicals present and 29 reactive oxygen species (ROS). Both direct and indirect effects of radiation on cells and 30 tissues have biological effects in the short or long term. Such effects can be mitigated or 31 eliminated by the antioxidant system and cell repair system that work against oxidizable 32 stress and / or cellular stress (Figure 1). 33 * Lucimar P. França, Vanina M. Tucci-Viegas, Fernanda Lasakosvitsch, Silvana Gaiba, Fernanda L. A. Azevedo, Amanda P. Nogueira, Helena R. C. Segreto, Alice T. Ferreira and Jerônimo P. França Federal University of São Paulo – Unifesp, Santa Cruz State University – Uesc Brazil     Flow Cytometry Book 1 2 RadiationDirect effectIndirect effect PROTEINSPHOSPHOLIPIDSDNASTRUCTURES OTHERSExcitation IonizationH O 2 H O 2 H O 3 H + OHOH +DissociationOH , H , e H , H O , OH, H O Products of water radiolysis 222aq3+ H O + e 2+- + H O 2 nH O 2aq e + ii iii   1 Fig. 1. Direct and indirect effect of radiation produced by free radicals of water radiolysis. 2 Modified scheme of Stark, 1991 3 The pro-oxidant/antioxidant balance leads to a disturbance which, in turn, results in a 4 condition of oxidative stress subsequently oxidizing cell components, activating cytoplasmic 5 and/or nuclear signal transduction pathways, modulating gene and protein expression and 6 changing both DNA and RNA polymerases activities. Normal cellular metabolism seems to 7 be a primary source for endogenous ROS (such as the participation of oxidatively damaged 8 DNA and repair in aging or cancer development). Oxidative damage to cellular DNA often 9 causes mutagenesis as well as programmed cell death. While mutagenesis might result in 10 carcinogenesis, programmed cell death often causes degenerative disorders (Nakabeppu et 11 al. , 2007). Hydroxyl radicals generated from ionizing radiation attack DNA resulting in 12 single strand breaks and oxidative damage to sugar and base residues. Hydroxyl radicals 13 cause ionization of DNA bases as well as of other cellular components. Unsaturated fatty 14 acids play an important role, since lipid peroxidation yields a plethora of stable derivates, 15 which add to nucleic acids forming exocyclic DNA adducts of high miscoding potential, as 16 well as DNA-DNA and DNA-protein cross-links (Bartsch et al. , 2004). 17 Damage of cells by ionizing radiation includes mainly modifications of DNA molecules, 18 such as single and double-strand breaks. While single-strand breaks are quickly repaired in 19 a process that requires poly-(ADP-ribose)-polymerase (PARP), double-strand breaks represent 20 potentially lethal damage and their repair is complicated. Imperfect DNA repair causes 21 mutations and contributes to genome instability. This is mostly manifested as chromosome 22 aberrations, interchromosomal and intrachromosomal rearrangements (dicentric aberrations, 23 translocations, or inversions). Detection of chromosomal aberrations in peripheral 24 lymphocytes is an important indicator of obtained dose of radiation (Kozubek, 2000). 25 When the DNA is damaged an interconnected network of signaling is activated, resulting in 26 damage repair, temporary or permanent cell cycle arrest or cell death. Cell cycle arrest 27 allows time for DNA damage repair. If the repair is unsuccessful, the cells are removed by 28   Gamma Radiation Induces p53-Mediated Cell Cycle Arrest in Bone Marrow Cells 3 apoptosis, necrosis or their proliferation is permanently suppressed by initiation of stress- 1 induced premature senescence (SIPS). Cmielová et al. , 2011) reported that it is possible that 2 the major mechanism of response of these tissues to irradiation is not apoptosis, but 3 induction of SIPS. Both pathways often work together to induce replicative and premature 4 senescence. In general, activation of p53 and upregulation of p21 in cells undergoing 5 senescence are transient (Toussaint et al. , 2000; Robles & Adami, 1998). 6 Increased p53 activity and p21 expression usually occur during the onset of senescence and 7 then subside when the expression of p16 starts to rise. Before p16 upregulation, inactivation 8 of p53 can prevent senescence induction in some cells. However, once p16 is highly 9 expressed cell cycle arrest becomes irreversible simply by downregulation of p53 (Campisi 10 et al. , 2005; Beausejour et al. , 2004; Narita et al. , 2004). This suggests that while both p53 and 11 p21 play an important role in the initiation of senescence, only p16 is required for the 12 maintenance of senescence. In agreement with this suggestion, we found that IR-induced 13 activation of p53 and upregulation of p21 occurred prior to the increased expression of p16 14 and p19 in murine BM HSCs (Meng et al. , 2003; Neben et al. , 1993; Wang et al. , 2006) 15 Recent studies showed that a majority of murine BM hematopoietic cells including HSCs 16 died by apoptosis after exposure to a moderate dose of IR in vitro . However, a subset of 17 these cells survived IR damage up to 35 days in a long-term BM-cell culture although 18 having lost their clonogenic function. These surviving cells exhibited an increased AS β  gal 19 activity, a biomarker for senescent cells, and expressed elevated levels of the proteins 20 (p16Ink4a and p19Arf) encoded by the Ink4a-Arf locus, whose expression has been 21 implicated in the establishment and maintenance of senescence by direct inhibition of 22 various cyclin-dependent kinases (CDKs), (Meng et al. , 2003; Dimri et al. , 1995; Lowe et al. , 23 2003; Sharpless et al. , 1999). 24 The main function of mitochondria is ATP production, which occurs during mitochondrial 25 oxidative phosphorylation (ox-phos). In several cell types, mitochondria also act as a very 26 efficient Ca2+ buffer, taking up substantial amounts of cytosolic Ca2+ at the expense of 27 mitochondrial membrane potential ( ΔΨ m). The pathways of Ca2+ entry into mitochondrial 28 matrix are known as the mitochondrial calcium uniporter (MCU), the “rapid mode” 29 mechanism, and the mitochondrial ryanodine receptor. The main role of mitochondrial 30 Ca2+ is the stimulation of the ox-phos enzymes. In addition to ox-phos, mitochondria are 31 central players in cellular Ca2+ signaling by shaping and buffering cellular Ca2+ signals. As 32 a consequence of Ca2+ uptake, mitochondria can suffer Ca2+ overload, triggering the 33 opening of the permeability transition pore (PTP) which is associated with apoptosis via the 34 mitochondrial pathway or necrosis due to mitochondrial damage. PTPs have been shown to 35 be promoted by thiol oxidation and inhibited by antioxidants, supporting a role of ROS in 36 pore opening. In addition, it has been demonstrated that mitochondrial Ca2+ uptake can 37 lead to free radical production. From a thermodynamic point of view, however, it has been 38 noted that Ca2+ uptake occurring at the expense of membrane potential should result in a 39 decrease in ROS production (Crosstalk signaling between mitochondrial Ca2+ and ROS) 40 (Brookes et al. , 2004). Mitochondrial PTP is formed from a number of proteins within the 41 matrix, and mitochondrial inner and outer membranes (Ishas & Mazat, 1998; Brookes et al. , 42 2004). One of the processes through which mitochondria contribute to cell death is through 43 PTP opening (Bratton & Cohen, 2001). The PTP precise composition remains unclear, but it 44 is evident that this is a multi-subunit protein channel that spans the mitochondrial inner and 45   Flow Cytometry Book 1 4 outer membrane. Critical components appear to include the mitochondrial VDAC (voltage- 1 dependent anion channel), the ANT (adenine nucleotide translocase), and cyclophilin D 2 (Ishas & Mazat, 1998). It has also been shown that pre-treatment of isolated mitochondria 3 with pro-oxidants can lower the threshold at which the PTP opening occurs (Brookes & 4 Darley-Usmar, 2004). It seems that the opening of this Ca 2+ -dependent channel plays an 5 important role in controlling the commitment of the cell to death through apoptotic or 6 necrotic mechanisms (Kokoszka et al. , 2004; Baines et al. , 2005). 7 2. Objective 8 The purpose of this work is to evaluate ionizing radiation-induced apoptosis in mice bone 9 marrow cells and the role of p53, p21 and Ca++ in this process, cell cycle alterations and 10 indirect determination of reactive oxygen specimens (ROS). 11 3. Methods 12 3.1 Animals 13 Mice C57BL/10 (3 months) were provided by the Instituto Nacional de Farmacologia 14 (located at Rua Três de Maio 100, Vila Clementino, SP, Brazil). The animals were maintained 15 on standard mouse feed and water ad libitum . 16 3.2 Irradiation 17 Gamma irradiation was carried out with an Alcyon II 60 CO teletherapy unit with the mice at 18 a distance of 80 cm from the source. The dose rate was 1.35 Gy/min. Animals in batches of 19 ten were placed in a well–ventilated acrylic box with an individual cell for each mouse and 20 exposed whole – body to 7Gy (Segreto et al. , 1999). 21 3.3 Preparation procedure of bone marrow cells 22 The mice were killed by cervical dislocation 4 hours after gamma irradiation, and both 23 femurs were removed from each mouse. After cutting of the proximal and distal ends of the 24 femurs, the bone marrow cells were gently flushed out with 5ml suspension using 25 phosphate buffered saline (PBS) (Segreto et al. , 1999). 26 3.4 Intracellular reactive oxygen species 27 Intracellular peroxides were determined by incubating 2X10 6  cells/ml in medium (defined 28 above) with 5 nM 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) (Molecular Probes) 29 for 30 min at 37°C, then analyzed using a Becton Dickinson (BD) Bioscience Flow Cytometer 30 - model FACScalibur (San Jose, CA) equipped with an argon laser emitting at 488 nm 31 (Jagetia & Venkatesh, 2007). BD CellQuest Pro software was used for fast reliable 32 acquisition, analysis and presentation of information. 33 3.5 Measurement of intracellular Ca++ 34 Calcium was measured after incubation of the bone marrow cells with the fluorescence 35 indicator Fura- 2/AM in the form of acetoxymethyl ester (AM). The bone marrow cells at 36
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