Radiation induced expression of signaling molecules in mouse splenocytes

INDEX

Introduction

Ionization

Affect of ionizations on cells

Characteristics of DNA damage by radiation exposure

Biological effects differ by type of radiation
Direct effect
Indirect effect

Cellular sensitivity to radiation

Organ sensitivity to radiation

Effect of radiation dose

Biological Response to High Doses of Radiation

Signalling molecules
a) PKC
b) Protein kinase C-delta (PKCδ)
c) MAPKs (Mitogen-Activated Protein Kinases)
d) P44/42
e) p38

Cytoprotective pathways

Apoptosis pathways

Cellular response to radiation

Materials and Method

Results and Discussion

References

Introduction

The hazards of exposure to ionizing radiation were recognised shortly after Roentgen’s discovery of x-rays in 1895. Acute skin cancer, leukaemia and other biological damage were observed in the individuals working with x-ray generator. In the year, 1898 Becquerel performed the first recorded experiment in radiobiology, from this earlier study of radiobiology began. Since that time, a tremendous amount of research has been done attempting to interpret the reactions which take place from the moment that radiation enters a living cell until some permanent damage is produced. From beginning to end, these initial reactions are probably completed in a millionth of a second, making them very difficult to study. For this reason, it is still not known which of the many chemical or biochemical reactions brought about by ionizing radiation are responsible for initiating biological damage.

Ionizing radiation is energy transmitted by X-rays, gamma rays, beta particles (high-speed electrons), alpha particles (the nucleus of the helium atom), neutrons, protons, and other heavy ions such as the nuclei of argon, nitrogen, carbon, and other elements. X-rays and gamma rays are electromagnetic waves like light, but their energy is much higher than that of light (their wavelengths are much shorter). Ultraviolet (UV) light is a radiation of intermediate energy that can damage cells like sunburns, but UV light differs from the forms of electromagnetic radiation mentioned above in that it does not cause ionization (loss of an electron) in atoms or molecules, but rather excitation (change in energy level of an electron). The other forms of radiation particles are either negatively charged (electrons), positively charged (protons, alpha rays, and other heavy ions), or electrically neutral (neutrons).

Ionization

As an example of ionization, beta rays are fast electrons that lose energy as they pass through cells and interact with molecules. The transferred energy is high enough to disrupt chemical bonds, which results in radical formation (or ionization). When an electron passes through a cell, it releases its energy along its path (called a track) by interacting with the electrons of nearby molecules. The released energy is absorbed by atoms near the track, resulting in either excitation (a shift in the orbit of an electron to a higher energy level) or ionization (release of an electron from the atom), which makes the atoms very unstable. Such unstable atoms are called radicals and are chemically very reactive. Some radicals are so reactive that they exist only for as short a time as a microsecond. X-ray and gamma rays differ from beta particles in that they release high-speed electrons from atoms first. Positively charged particles transfer energy to molecules in cells by essentially the same mechanisms. Neutrons are somewhat different since they are electrically uncharged, and their main effect is to impact the nuclei of hydrogen atoms, namely protons. Since the masses of a neutron and a proton are similar, the impact results in an elastic scattering process like in billiards. The ejected protons behave as charged particles.

Effect of ionizations on cells

Radiation-induced ionizations may act directly on the cellular component molecules or indirectly on water molecules, causing water-derived radicals. Radicals react with nearby molecules in a very short time, resulting in breakage of chemical bonds or oxidation (addition of oxygen atoms) of the affected molecules. The major effect in cells is DNA breaks. Since DNA consists of a pair of complementary double strands, breaks of either a single strand or both strands can occur. However, the latter is believed to be much more important biologically. Most single-strand breaks can be repaired normally, due to double-stranded nature of the DNA molecule (the two strands complement each other, so that an intact strand can serve as a template for repair of its damaged, opposite strand). In the case of double-strand breaks, however, repair is more difficult and erroneous re-joining of broken ends may occur. These so-called misrepairs result in induction of mutations, chromosome aberrations, or cell death.

Characteristics of DNA damage by radiation exposure

Deletion of DNA segments is the predominant form of radiation damage in cells that survive irradiation. It may be caused by (1) misrepair of two separate double-strand breaks in a DNA molecule with joining of the two outer ends and loss of the fragment between the breaks or (2) the process of cleaning (enzyme digestion of nucleotides, the component molecules of DNA) of the broken ends before re-joining to repair one double-strand break.

Biological effects differ by type of radiation

Radiations differ not only by their constituents (electrons, protons, neutrons) but also by their energy. Radiations that cause dense ionization along their track (such as neutrons) are called high-linear-energy-transfer (high-LET) radiation. Low-LET radiations produce ionizations only sparsely along their track hence, almost homogeneously within a cell. Radiation dose is the amount of energy per unit of biological material (i.e. number of ionizations per cell). Thus, high-LET radiations are more destructive to biological material than low-LET radiations--such as X rays and gamma rays, at the same dose, the low-LET radiations induce the same number of radicals more sparsely within a cell, whereas the high-LET radiations such as neutrons and alpha particles transfer most of their energy to a small region of the cell. The localized DNA damage caused by dense ionizations from high-LET radiations is more difficult to repair than the diffuse DNA damage caused by the sparse ionizations from low-LET radiations (Radiation effects research foundation).

Direct effect

If radiation interacts with the atoms of the DNA molecule, or some other cellular component critical to the survival of the cell, it is referred to as a direct effect. Such an interaction may affect the ability of the cell to reproduce and thus, survive. If enough atoms are affected such that the chromosomes do not replicate properly, or if there is significant alteration in the information carried by the DNA molecule, then the cell may be destroyed by “direct” interference with its life-sustaining system.

Indirect effect

If a cell is exposed to radiation, the probability of the radiation interacting with the DNA molecule is very small since these critical components make up such a small part of the cell. However, each cell, just as is the case for the human body, is mostly water. Therefore, there is a much higher probability of radiation interacting with the water that makes up most of the cell’s volume. When radiation interacts with water, it may break the bonds that hold the water molecule together, producing fragments such as hydrogen (H) and hydroxyls (OH). These fragments may recombine or may interact with other fragments or ions to form compounds, such as water, which would not harm the cell. However, they could combine to form toxic substances, such as hydrogen peroxide (H2O2), which can contribute to the destruction of the cell.

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Cellular sensitivity to radiation

Not all living cells are equally sensitive to radiation. Those cells which are actively reproducing are more sensitive than those which are not. This is because dividing cells require correct DNA information in order for the cell’s offspring to survive. A direct interaction of radiation with an active cell could result in the death or mutation of the cell, whereas a direct interaction with the DNA of a dormant cell would have less of an effect. As a result, living cells can be classified according to their rate of reproduction, which also indicates their relative sensitivity to radiation. This means that different cell systems have different sensitivities. Lymphocytes (white blood cells) and cells which produce blood are constantly regenerating, and are, therefore, the most sensitive. Reproductive and gastrointestinal cells are not regenerating as quickly and are less sensitive. The nerve and muscle cells are the slowest to regenerate and are the least sensitive cells. Cells, like the human body, have a tremendous ability to repair damage. As a result, not all radiation effects are irreversible. In many instances, the cells are able to completely repair any damage and function normally. If the damage is severe enough, the affected cell dies. In some instances, the cell is damaged but is still able to reproduce. The daughter cells, however, may be lacking in some critical life-sustaining component, and they die. The other possible result of radiation exposure is that the cell is affected in such a way that it does not die but is simply mutated. The mutated cell reproduces and thus perpetuates the mutation. This could be the beginning of a malignant tumor.

Organ sensitivity to radiation

The sensitivity of the various organs of the human body correlate with the relative sensitivity of the cells from which they are composed. For example, since the blood forming cells were one of the most sensitive cells due to their rapid regeneration rate, the blood forming organs are one of the most sensitive organs to radiation. Muscle and nerve cells were relatively insensitive to radiation, and therefore, so are the muscles and the brain.

The rate of reproduction of the cells forming an organ system is not the only criterion determining overall sensitivity. The relative importance of the organ system to the well being of the body is also important. One example of a very sensitive cell system is a malignant tumor. The outer layer of cells reproduces rapidly, and also has a good supply of blood and oxygen. Cells are most sensitive when they are reproducing, and the presence of oxygen increases sensitivity to radiation. Anoxic cells (cells with insufficient oxygen) tend to be inactive, such as the cells located in the interior of a tumor. As the tumor is exposed to radiation, the outer layer of rapidly dividing cells is destroyed, causing it to “shrink” in size. If the tumor is given a massive dose to destroy it completely, the patient might die as well. Instead, the tumor is given a small dose each day, which gives the healthy tissue a chance to recover from any damage while gradually shrinking the highly sensitive tumor. Another cell system that is composed of rapidly dividing cells with a good blood supply and lots of oxygen is the developing embryo. Therefore, the sensitivity of the developing embryo to radiation exposure is similar to that of the tumor, however, the consequences are dramatically different. Whole body sensitivity depends upon the most sensitive organs which, in turn, depend upon the most sensitive cells. As noted previously, the most sensitive organs are the blood forming organs and the gastrointestinal system. The biological effects on the whole body from exposure to radiation will depend upon several factors. Some of these are listed above. For example, a person, already susceptible to infection, who receives a large dose of radiation, may be affected by the radiation more than a healthy person.

Effect of radiation dose

Biological effects of radiation are typically divided into two categories. The first category consists of exposure to high doses of radiation over short periods of time producing acute or short term effects. The second category represents exposure to low doses of radiation over an extended period of time producing chronic or long term effects. High doses tend to kill cells and damage tissue and organs, this in turn may cause a rapid whole body response often called the Acute Radiation Syndrome (ARS). While low doses tend to damage or change over long periods of time don’t cause an immediate problem to any body organ (Biological Effects of Radiation).

Biological Response to High Doses of Radiation (Biological Effects of Radiation)

< 5 rad - No immediate observable effects

- 5 rad to 50 rad - Slight blood changes may be detected by medical evaluations.
- 50 rad to 150 rad - Slight blood changes will be noted and symptoms of nausea,fatigue, vomiting, etc. likely.
- 150 rad to1,100 rad - Death due to destruction of blood forming organs.
- 1,100 rad to -Death due to destruction of nervous system and GI

2,000 rad

Effect of low dose

There are three general categories of effects resulting from exposure to low doses of radiation. These are:

Genetic - The effect is suffered by the offspring of the individual exposed.

Somatic - The effect is primarily suffered by the individual exposed. Since cancer is the primary result, it is sometimes called the carcinogenic effect.

In-Utero - Some mistakenly consider this to be a genetic consequence of radiation exposure, because the effect, suffered by a developing embryo/ fetus, is seen after birth. However, this is actually a special case of the somatic effect, since the embryo/fetus is the one exposed to the radiation.

Signalling molecules

a) PKC: Twelve serine/threonine protein kinases constitute the Protein Kinase C (PKC) family which transduces a myriad of signals which regulates multiple physiological functions (Hug and Sarre, 1993). Three distinct subfamilies of PKC isoforms are identified according to their dependency on three combinations of activators:

Conventional PKCs (α, ßI, ßII, γ) require phosphatidylserine (PS), diacylglycerol (DAG), and Ca 2+.Novel PKCs (δ, ε, η, θ) need PS and DAG but not Ca 2+.A typical PKCs (z, i/λ, μ) are insensitive to both DAG and Ca 2+ (Hug and Sarre, 1993; Newton, 1997). The primary structure of PKC reveals the presence of four domains conserved across PKC isoforms (C1-C4) and five variable domains that are divergent (V1-V5). Two functional domains have been described in PKC: an amino terminal regulatory domain and a carboxyl terminal catalytic domain (Hug and Sarre, 1993). The regulatory domain (V1-V3) contains the so-called pseudo substrate site which is thought to interact with the catalytic domain to retain PKC in an inactive conformation. The regulatory domain also contains sites for the interaction of PKC with PS, DAG/phorbol ester, and Ca 2+. The Ca 2+ dependency is mediated by the C2 region (which is indeed absent in novel PKCs), while phorbol-ester binding requires the presence of two cysteine-rich zinc-finger regions within the C1 domain. Atypical PKCs lack one of the two cysteine-rich zinc-finger regions and therefore do not bind (and cannot be activated by) phorbol esters (Hug and Sarre, 1993). The catalytic domain (V3-V5) contains ATP binding site and is thought to interact with the substrates (Hug and Sarre, 1993). Fatty acids, D3- phosphorylated inositol lipids, phosphatidic acid (PA), and ceramide are additional molecules that are considered to be capable of activating PKC (Newton, 1997; Liu and Heckman, 1998).

When activated by biomechanical stress or neuro hormonal mediators, G-protein coupled receptors separate heterotrimeric G-proteins to G-protein alpha-q/11 subunits and heterodimeric G-protein beta/gamma subunits. G-proteins bind and activate Phospholipase C beta ( PLC-beta ), recruit PLC-beta to the membrane where it hydrolyses Phosphatidylinositol 4,5 bisphosphate (PtdIns (4,5)P2 ) and releases Inositol 1,4,5-triphosphate ( IP3 ) and DAG. IP3 binds to receptors (IP3R) in the endoplasmic reticulum, releasing calcium Ca 2+. The increase in cytosolic Ca 2+ activates the protein phosphatase calcineurin. Calcineurin dephosphorylates several residues in the amino-terminal region of the transcription factor NF-AT, allowing it to translocate to the nucleus and activate transcription of hypertrophic response genes.

PKC-alpha, PKC-delta, PKC-epsilon, PKC-zeta and PKC-mu phosphorylate and activate PKC-potentiated inhibitor protein of 17kDa (CPI-17). CPI-17 specifically inhibits myosin light chain phosphatase (MLCP), leading to MELC phosphorylation by MLCK. MLCK in turn is activated by calmodulin (Zemlickova et al., 2004). One of the PKC-regulated pathways leads to the inhibition of a subset of Histone deacetylases (HDAC7) that specifically regulate cellular hypertrophy. In this pathway, PKC-delta activates another protein kinase, PKC-mu, that in its turn phosphorylates the HDAC7 leading to its export from the nucleus and consequent inactivation (Li et al., 2004).

PKC-mu activates the transcription factor Nuclear factor kappaB (NF-kB). PKC-mu phosphorylates the IKK beta, leading to I-kB degradation and subsequent NF-kB translocation into the nucleus (Storz et al.,2003). Activation of PKC-mu in response to oxidative stress requires its sequential phosphorylation by two kinases, tyrosine kinase cABL and PKC-delta (Storz et al., 2004). PKC-mu activation leads to the transcriptional activation of NUR77 via Myocyte enhancer factor 2 (MEF2)-binding sites in its promoter (Parra et al., 2005). v-Src sarcoma viral oncogene homolog (c-Src) phosphorylates and activates PKC-iota (Wooten et al.,2001). Atypical PKC-zeta is activated by Ceramide. This results in activation of NF-kB and continued survival of the cell (Wang et al., 1999).

The two members of the atypical protein kinase C (aPKC) subfamily of isozymes ( PKC-zeta and PKC-iota ) are involved in control of the NF-kB activity through IKKbeta activation. A PKC-binding protein Sequestosome 1(p62) selectively interacts with receptor-interacting protein RIPK1 as an adaptor. Sequestosome 1(p62) bridges atypical PKCs and RIPK1. The latter activates IKK gamma, and atypical PKCs phosphorylate and activate IKKbeta. Thereby, the interactions of Sequestosome 1(p62) with RIPK1 and the atypical PKCs lead to the activation of NF-kB signalling pathway (Sanz et al., 1999). The PKC-theta isoform also induces NF-kB activation. PKC-theta directly targets IKK beta for phosphorylation and activation, possibly via homodimeric IKKbeta complexes (Altman et al., 2003). PKC-alpha, PKC-beta, PKC-gamma, PKC-epsilon, and PKC-eta phosphorylate and activate v-Raf-1 murine leukemia viral oncogene homolog 1 (c-Raf-1 ) leading to the stimulation of the Mitogen-activated protein kinase kinase 1 and 2 ( MEK1 and MEK2 )/ Mitogen-activated protein kinases 1 and 3 ( ERK1/2 ) cascade and activation of the transcription factor Elk-1 (Hamilton et al., 2001).

Several PKC isotypes (PKC-alpha, PKC-beta, PKC-gamma, PKC-delta, and PKC-eta ) phosphorylate Glycogen synthase kinase 3 beta ( GSK3-beta ) and inactivate it (Fang et al., 2002). GSK3-beta phosphorylates conserved serines of NF-AT. This phosphorylation promotes the nuclear exit of NF-AT, thereby opposing Ca 2+ - calcineurin signalling (Dorn et al., 2005).

b) Protein kinase C-delta (PKCδ): A member of the lipid-regulated serine/threonine PKC family has been implicated in a wide range of important cellular processes. In the past decade, the critical role of PKCδ in regulation of both intrinsic and extrinsic apoptosis pathways has been widely explored. In most cases, over-expression or activation of PKCδ results in the induction of apoptosis. The phosphorylations and multiple cell organelle translocations of PKCδ initiate apoptosis by targeting multiple downstream effectors. During apoptosis, PKCδ is proteolytically cleaved by caspase-3 to generate a constitutively activated catalytic fragment, which amplifies apoptosis cascades in nucleus and mitochondria. However, PKCδ also exerts its anti-apoptotic and pro-survival roles in some cases. Therefore, the complicated role of PKCd in apoptosis appears to be stimulus and cell type dependent (Meng et al., 2012).

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Figure. 1 Diagramatic activation mechanisms of PKC δ. (Ref: Meng Zhao • Li Xia • Guo-Qiang Chen Protein Kinase Cd in Apoptosis: A Brief Overview Arch. Immunol. Ther. Exp. (2012) 60:361–372).

PKCδ binds to DAG and it is phosphorylated by Ser/Thr and Tyr kinases, which changes the conformation and exposes the substrate binding site of PKC δ to get activated. PKCδ is proteolytically cleaved by caspase-3, which releases the

catalytic fragment, a constitutively active form.

c) MAPKs (Mitogen-Activated Protein Kinases)

These are Serine-threonine protein Kinases that are activated in response to a variety of extracellular stimuli and mediate signal transduction from the cell surface to the nucleus. MAPKs are expressed in multiple cell types including cardiomyocytes, vascular endothelial cells, and vascular smooth muscle cells. Three major MAPKs include ERKs (Extracellular signal-Regulated Kinases), JNKs (c-Jun NH (2)-terminal protein Kinases), and p38 Kinases. Members of the JNK/SAPK (Stress-Activated Protein Kinase) family of MAPKs are strongly stimulated by numerous environmental stresses, but also more modestly stimulated by mitogens, inflammatory cytokines, oncogenes, and inducers of cell differentiation and morphogenesis. Ten mammalian JNK isoforms have been identified and are encoded by three distinct genes, JNK1, JNK2, and JNK3, the transcripts of which are alternatively spliced to yield four JNK1 isoforms, four JNK2 isoforms, and two JNK3 isoforms. JNK1 and JNK2 are the products of alternative splicing of a single gene and are expressed in many tissues, but JNK3 is specifically expressed in brain. Members of the JNK family play crucial roles in regulating responses to various stresses, and in neural development, inflammation, and apoptosis. JNK activation is much more complex than that of ERK1/ERK2 owing to inputs by a greater number of MAPKKKs (Mitogen-Activated Protein Kinase Kinase Kinases) at least 13, including MEKK1 (MAP/ERK Kinase-Kinase-1)-MEKK4 (MAP/ERK Kinase-Kinase-4), ASK (Apoptosis Signal-regulating Kinase) and MLKs (Mixed-Lineage Kinases), which are activated by upstream Rho-family GTPases. These activate JNK MAPKKs MEK4 (MAPK/ERK Kinase-4) and MEK7 (MAPK/ERK Kinase-7), which further activate JNKs. The JNK MAPK modules are regulated by a number of different scaffold proteins, including JIP1 (JNK Interacting Protein-1), JIP2 (JNK Interacting Protein-2), JIP3 (JNK Interacting Protein-3), JIP4 (JNK Interacting Protein-4), Beta-Arrestin-2, Filamin and CrkII. The scaffold proteins presumably target the MAPK modules to different sites in the cell and play roles in kinase activation and/or substrate selection (Himes et al., 2006 & Moulin et al., 2004).

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