The history of radiotherapy began in 1895, when Röntgen discovered X rays, and in the following year, radiation was used for medical treatment. In the early days, the development of radiotherapy was based extensively on empiricism.
Radiotherapists worked closely with radiation biologists in attempting to describe and understand the phenomena produced by ionizing radiation in the clinic and in biological systems [6.1]. During the ensuing 120 years, radiotherapy has been improved significantly and, in addition to radiation biology, medical physics has played an important role in the design and development of equipment, quality assurance and dosimetry.
Over recent decades, advances have been made in the field of molecular biology. Currently available techniques enable us to elucidate the molecular mechanisms of cellular response to ionizing irradiation, and it is anticipated that the role and contributions of radiation biology in radiotherapy will remain relevant.
This chapter describes the clinically important biological points, including knowledge from current molecular biology.
6.2. MECHANISM OF CELL KILL BY IONIZING RADIATION 6.2.1. Types of ionizing radiation
Ionization is the process of removing one or more electrons from atoms by incident radiation, leaving behind electrically charged particles (an electron and a positively charged ion) which may subsequently produce significant biological effects in the irradiated material [6.2]. Ionizing radiation may be divided into directly ionizing and indirectly ionizing radiation according to its biological effects. Most of the particulate types of radiation (protons, neutrons, carbon ions)
radiation, namely X and γ rays, is indirectly ionizing because the rays do not produce chemical and biological damage themselves, but produce secondary electrons (charged particles) after energy absorption in matter.
6.2.2. Interaction of ionizing radiation with biological matter
Biological effects of radiation arise when ionizing radiation interacts with an organism or tissue, leaving some energy behind. The process by which electromagnetic photons are absorbed in matter depends on their energy and the atomic number of the absorbing material. Photons passing through matter transfer their energy through the following three main processes: photoelectric absorption; Compton scattering; and pair production. The photoelectric effect is the dominant energy transfer mechanism for X and γ ray photons having energies below 50 keV, but it is much less important for higher energies. The principal absorption mechanism for X and γ rays in the intermediate energy range from 100 keV to 10 MeV (therapeutic radiation range) is Compton scattering.
6.2.3. Radiation chemistry: Direct and indirect effects
The physical interaction of ionizing radiation with matter leads to loss of radiation energy, ionization and free radicals. These radicals react rapidly (10–10 s) with neighbouring molecules and produce secondary DNA or lipid radicals. Free radicals are fragments of molecules having unpaired electrons. They have high reactivity with cellular molecules and, therefore, a short life. Free radicals are generated in great number by ionizing radiation due to the process of energy absorption and breakage of chemical bonds in molecules. These radicals play a major role in radiation effects on biological tissues and organisms.
The absorption of energy depends on the abundance of material in the radiation path. When ionizing radiation energy is deposited in a macromolecule associated with observable biological effects such as DNA, it is called a direct effect of radiation. Alternatively, photons may be absorbed in the water of an organism causing excitation and ionization in the water molecules. Water is the predominant molecule in living organisms (about 80% of the mass of a living cell is water). Therefore, a major proportion of radiation energy deposited will be absorbed in cellular water. A complex series of chemical changes occur in water after exposure to ionizing radiation. This process is called water radiolysis.
About two thirds of the biological damage by low linear energy transfer (LET) radiation or sparsely ionizing radiation such as X rays or electrons is due to indirect action. Several lines of evidence indicate that the biological effects of radiation are mainly derived from damage to chromosomal DNA, a critical target of ionizing radiation in the human body [6.3–6.5]. Cancer cells whose DNA is
damaged beyond repair stop dividing or die. When the damaged cells die, they are broken down and eliminated by the body’s natural processes.
6.2.4. DNA damage and repair
Ionizing radiation induces several types of DNA damage, such as base damage, single strand breaks (SSBs), double strand breaks (DSBs) and cross-links [6.3, 6.4]. Since cells have repair pathways corresponding to each type of radiation induced DNA damage, they are able to recover from the radiation induced damage. Persistent or unrepaired DSBs may determine the anti-tumour effects of ionizing radiation by inducing apoptosis, necrosis, mitotic catastrophe or permanent growth arrest. About 40 DSBs/cell are generated by irradiation with 1 Gy. In theory, if only one DSB remains in an important gene, the cell might be sterilized or even die. Therefore, the efficiency of DSB repair capacity of a cell is a very important factor in radiotherapy. Eukaryotic cells repair DNA DSBs mainly by either non-homologous end joining (NHEJ) or homologous recombination (HR).
The NHEJ pathway directly rejoins the two broken DNA ends. After the induction of DSBs, the Ku70/80 heterodimer recognizes the DSB sites, and DNA-PKcs are subsequently recruited and activated [6.3, 6.4, 6.6]. The activated DNA-PKcs phosphorylate themselves and other proteins involved in repair or damage signalling. The DNA ends are then processed by nucleases, such as Artemis and WRN, and DNA polymerases, such as pol λ and pol µ. In the final step, the broken ends are ligated together by the XRCC4/DNA ligase IV/XLF complex. NHEJ does not require the homologous DNA sequence, so it is available regardless of the cell cycle stage. However, during the end processing, the sequence information at the ligated site is lost, so NHEJ is an error-prone mechanism of repair.
HR is a repair pathway that utilizes the undamaged homologous DNA sequence, usually from the sister chromatid, as the template [6.3, 6.4, 6.7]. The initial step of HR involves the creation of single stranded regions by the MRN complex (Mre11-Rad50-Nbs1) and CTBP1 (C-terminal-binding protein 1).
The single stranded DNA formed around the breaks is immediately coated with
‘replication protein A’ (RPA). Subsequently, HR proteins, including RAD51, RAD52 and BRCA1/2, are recruited to form a nucleoprotein filament. RAD51 is the central protein in HR, since it mediates the search for homologous DNA and the strand invasion. Afterwards, DNA synthesis is performed with DNA polymerases. HR is error free repair, because it utilizes DNA with the same
The radiation induced reorganization of damaged chromatin, such as post-translational modifications and histone exchange, has recently been shown to play important roles in DNA repair. The phosphorylation of H2AX (γH2AX), a histone variant, is one of the best characterized radiation induced modifications of histones, and occurs within 5–30 min after the induction of DSBs. γH2AX forms nuclear foci, called γH2AX foci, at damaged sites and serves as a scaffold for the recruited repair proteins (Fig. 6.1). The number of γH2AX foci has been shown to correspond to the number of DSBs. The kinetics of γH2AX focus formation is widely used to analyse the induction of DNA damage, the ability of DSB repair and the radiosensitivity of cells [6.3, 6.4, 6.8].
FIG.6.1. Radiation induced focus formation of γH2AX. Immunofluorescence staining of a human fibroblast cell line at 30 min after 8 Gy irradiation. γH2AX (γH2AX) and DNA (PI) are shown in green and red, respectively, in the merged (merge) and 3-D reconstructed (3-D) images. Scale bars = 10 μm.
6.2.5. Cell death
In the context of radiation biology and cancer therapy, ‘cell death’ is defined as “the permanent loss of reproductive capacity”, except for terminally differentiated non-proliferating cells, such as muscle and nerve cells [6.3].
Previously, cell death was separated into only two types in the field of radiation biology, ‘interphase death’ and ‘mitotic death’, based on the period after radiation. Interphase death is defined as the death of irradiated cells before they reach mitosis. On the other hand, mitotic death is defined as the death of irradiated cells after they execute one or more cell divisions.
However, recent progress in cell death research has shown that cells can die in many different ways after irradiation, such as by apoptosis, autophagy, necrosis, mitotic catastrophe and senescence-like growth arrest [6.3, 6.4, 6.9].
Apoptosis, a form of rapid cell death after irradiation, is characterized by chromatin condensation, nuclear fragmentation and compartmentalization by densely staining globules. Autophagy is a process in which cellular components are self-digested through the lysosome machinery. Autophagy was originally considered as an important mechanism for cellular maintenance, through the exchange of damaged and newly synthesized proteins. Recently, it has been shown that autophagy is also involved in cell death induced by radiation. Necrosis is a type of cell death characterized by an increase in cell volume, with the swelling of organelles such as mitochondria, plasma membrane rupture, and the subsequent loss of intracellular contents. Mitotic catastrophe is a mode of cell death occurring from the inappropriate completion of cell division due to unrepaired or misrepaired DNA damage, and can be accompanied by morphological alterations such as micronucleation and multinucleation. Senescence-like growth arrest is defined as the permanent arrest of cell division in G1 phase with active metabolism. Senescence has been considered a tumour suppressing mechanism that prevents excessive cell growth after the accumulation of genomic mutations by radiation. However, many details of the molecular mechanisms of radiation induced cell death remain to be clarified.