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Biological Basis of Radiation Sensitivity

Biological Basis of Radiation Sensitivity

ABSTRACT: Recent studies have elucidated some of the molecular and cellular mechanisms that determine the sensitivity or resistance to ionizing radiation. These findings ultimately may be useful in devising new strategies to improve the therapeutic ratio in cancer treatment. Despite the rapid advances in knowledge of cellular functions that affect radiosensitivity, we still cannot account for most of the clinically observed heterogeneity of normal tissue and tumor responses to radiotherapy, nor can we accurately predict which individual tumors will be controlled locally and which patients will develop more severe normal tissue damage after radiotherapy. However, several candidate genes for which deletion or loss of function mutations may be associated with altered cellular radiosensitivity (eg, ATM, p53, BRCA1, BRCA2, DNA-PK) have been identified. Some of the differences in normal tissue sensitivity to radiation may stem from mutations with milder effects, heterozygosity, or polymorphisms of these genes. Finally, molecular mechanisms linking genetic instability, radiosensitivity, and predisposition to cancer are being unraveled. [ONCOLOGY 14 (5):741-757, 2000]

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.

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