Biological Basis of Radiation Sensitivity

Introduction

Clinical and experimental studies of the acute and late effects of radiation and chemotherapy at the levels of tissues, organs, and gross pathology have enhanced our knowledge of how normal tissues and tumors respond to these agents. These studies have led to the optimization of radiation treatment schedules and to more precise modes of radiation delivery.

Improvements in radiation delivery include hyperfractionated radiotherapy (ie, smaller dose fractions delivered two or three times per day); computerized treatment planning and the use of multiple noncoplanar radiation portals (three-dimensional conformal radiotherapy) to reduce the volume of normal tissue irradiated; intensity-modulated radiation therapy, a technique designed to reduce the radiation dose to surrounding normal tissue for a given dose to the tumor volume; stereotactic radiosurgery and radiotherapy, allowing the delivery of single-dose or fractionated radiotherapy to precisely defined target volumes in the brain; improved combinations of radiotherapy and chemotherapy; and precisely targeted particle-beam therapy (eg, proton-beam treatment).[56-59] Given the state of the art, further refinements of this type are likely to lead to incremental rather than quantum improvements in cancer treatment.

If significant improvements in treatment outcome are to be achieved, they are more likely to result from the clinical application of the relative explosion in knowledge of the molecular mechanisms of the cell response to stress, including genotoxic agents, such as ionizing radiation and many chemotherapeutic drugs. The major sources of this knowledge have included: (1) identification of a series of yeast genes, and their mammalian counterparts, for which mutation or loss result in radiosensitivity; (2) studies of genetic mechanisms and cellular consequences of tumor-suppressor gene mutations (eg, p53, ATM, BRCA1/2, DNA-PK); (3) the discovery of the role of growth factor/cytokine signal transduction and oncogene activation in modulating radiosensitivity; and (4) elucidation of the molecular mechanisms of apoptosis, a form of programmed cell death.

The first part of this two-part review, published in last month’s issue, summarized the clinical and tissue kinetic factors that govern the sensitivity of normal tissues to ionizing radiation. This second part will characterize recent insights into the cellular and molecular pathways that determine the sensitivity of normal and tumor cells to ionizing radiation.

Factors Regulating Apoptosis

Mutations of tumor-suppressor genes, some of which are found in the human counterparts of yeast radiation response genes, and oncogene activation occur predominantly in malignant tumors,[60-64] while growth factor/cytokine signal transduction and apoptosis occur in normal development, tissue response to injury, as well as benign and malignant tumors.[61-64] Apoptosis, the major form of programmed cell death in animal cells, is a genetically programmed cascade of events activated in response to cell stress.[66-68] Apoptosis is characterized by nuclear DNA fragmentation, chromatin condensation, and a characteristic cytoplasmic and nuclear morphology, before the cell is eliminated by phagocytosis.

Apoptosis is an important mechanism by which radiation and chemotherapy kill cells.[69-71] As will become evident from the discussion below, tumor-suppressor mutations, oncogene activation, and growth factor signaling all modulate the induction of apoptosis by ionizing radiation. Thus, knowledge of the factors that regulate apoptosis is of paramount importance in understanding the molecular basis of the tumor and normal tissue response to radiation.

A genetic framework for understanding apoptosis in mammalian cells has been established by the study of the cell death (CED) genes of the nematode Caenorhabditis (C) elegans.[67,68] Three of the major CED gene products (and their human homologs) are CED-9 (Bcl-2), CED-4 (Apaf-1), and CED-3 (caspase-3). CED-9/Bcl-2, a pore-forming protein of the outer mitochondrial membrane, is thought to block apoptosis by binding to CED-4/Apaf-1 and/or preventing the release of cytochrome c from mitochondria. The apoptosis-activating factors (Apafs 1-3) are recently identified factors that mediate deoxyadenosine triphosphate (dATP)/cytochrome c–dependent cleavage of procaspases into active enzymes (see below).

The apoptotic cascade (illustrated in Figure 1) results in the activation of an enzyme (flipase) that causes the translocation of phosphatidylserine from the inner to the outer cell membrane. The phosphatidylserine on the cell surface is recognized by annexins on the surface of macrophages, resulting in the elimination of apoptotic cells by phagocytosis, so that they do not generate an inflammatory response.

 DNA Damage–Activated Signaling Pathways

Progress in understanding how DNA damage is signaled to the cellular response machinery has been greatly facilitated by the identification of a group of yeast genes whose mutation results in enhanced radiosensitivity. These genes encode protein kinases and kinase targets, the activation of which by DNA damage ultimately results in cell-cycle arrest and the transcription of DNA repair genes.

Roles of ATM and Structurally Related Proteins

In one pathway, activation of yeast phosphatidylinositol (PI)-related kinases MEC1 and TEL1 by damaged DNA results in phosphorylation of the Rad9 protein, its association with the protein kinase Rad53, and the downstream events of cell-cycle arrest and induction of DNA repair genes (Figure 2).[72-74] The human homolog of MEC1 is ATM, the product of the ataxia-telangiectasia susceptibility gene (ataxia-telangiectasia mutated, or ATM), located on human chromosome 11q22-23.[75] This gene encodes a 350-kD protein containing a PI3 kinase–like domain. PI3 kinase is a substrate involved in signal transduction from growth factor receptors; mammalian and yeast proteins that share the PI3K domain are involved in meiotic recombination and DNA damage responses, as well as cell-cycle control.[76]

ATM also appears to relay information from DNA damaged by ionizing radiation to the p53 tumor-suppressor gene (Figure 3).[77] Thus, cells lacking ATM are defective in the induction of p53 by radiation.

Finally, a pathway linking DNA damage to mitotic arrest through the human homolog of the yeast protein CHK1 has recently been elucidated.[78,79] Like the pathway for ATM signaling to p53, the CHK1 pathway involves 14-3-3, a group of proteins that participate in growth factor receptor signaling and in regulating apoptosis.[80] The human 14-3-3 signaling proteins are the homologs of two yeast proteins implicated in regulation of the response to radiation, Rad24 and Rad25.

The mechanisms by which ATM regulates radiosensitivity and chromosome stability are not well established. However, studies using ATM deletion mutants suggest that the C-terminal PI3K domain is required for maintaining S-phase arrest, chromosomal stability, and normal radiosensitivity following ionizing radiation.[81] These functions of ATM also require protein interactions mediated through the leucine zipper region of the protein.

A recent study further suggests that cleavage of ATM by the protease caspase-3, which is activated during apoptosis, blocks its protein kinase activity but not its DNA-binding activity. The kinase-inactivated ATM then acts as a dominant inhibitor of DNA damage signaling and repair.[82] These findings suggest that signal transduction in the nucleus involving the ATM protein may play a major role in limiting damage from ionizing radiation. The mechanism may involve delaying cells with severe chromosomal damage from exiting the S-phase before the damage is repaired.

Most ATM mutations result in inactivation or loss of the protein.[83] It is interesting to speculate whether there exist more subtle alterations of the ATM gene, polymorphisms, or defective ATM-binding factors that account for some of the individual heterogeneity in the response to radiotherapy (see part 1).

Two other proteins structurally related to ATM are the ATM-related (ATR) protein and DNA-dependent protein kinase (DNA-PK). Cells defective in ATM or ATR are hypersensitive to ionizing but not ultraviolet radiation.[84,85] DNA-PK, a nuclear serine/threonine kinase that binds to and is activated by double-strand DNA breaks, is a multiprotein complex consisting of a 470-kD catalytic subunit and a dimeric regulatory subunit (Ku-70 and Ku-80) with DNA-binding activity. DNA-PK has been implicated in genomic surveillance, detection and signaling of DNA damage, and cell-cycle control.

The radiation-sensitive rodent cell line CHO xrs-6 is defective in its ability to repair double-stranded DNA breaks induced by ionizing radiation, and the human Ku-80 (XRCC5) gene product corrects this defect.[86] This protein binds to the broken DNA, leading to the binding and activation of the DNA-PK catalytic subunit. A mutation in DNA-PK catalytic subunit is responsible for the murine severe combined immunodeficiency defect, characterized by a defect in V(D)J recombination during B- and T-cell development, the inability to repair double-stranded DNA breaks, and increased radiosensitivity.[87] These findings implicate DNA-PK in genetic recombination.