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Biologic Basis for Radiation Oncology

Biologic Basis for Radiation Oncology

ABSTRACT: Improved understanding of the underlying biologic mechanisms that pertain to radiation oncology is providing an explanation for the cellular and tissue responses to ionizing radiation and is leading to the potential for novel therapeutic strategies. Among the areas of intensive investigation are: DNA recombination and repair, signal transduction, gene regulation, apoptosis, the cellular stress response, and the effect of the tumor microenvironment. These new biologic concepts, coupled with the superior technical capabilities now available for treatment delivery, are paving the way for new clinical approaches to improving both the quality and quantity of life for the cancer patient. [ONCOLOGY 10(3):399-415, 1996]

Introduction

The ongoing revolution in tumor, cellular, molecular, and structural
biology is having a profound impact on the approaches that will
be taken in cancer therapy. The current rate of generation of
new knowledge is extraordinarily rapid, and the steady elucidation
of the processes involved in tumor development and progression
is revealing a wide range of new possibilities for the prevention
and treatment of cancer. Advances in the understanding of the
principles underlying radiation biology have paralleled those
in other basic science fields. Furthermore, many concepts that
previously had been investigated primarily in radiation biology
laboratories, such as DNA repair, cell-cycle perturbation, cellular
stress response, and the tumor microenvironment, including tumor
and stromal interaction, are of major interest to a wide range
of investigators,

This paper will focus on emerging biologic concepts that are of
interest to the oncology practitioner. Conceptually, it will focus
on translational research--the bridge between the basic science
laboratory and the clinic. Given the limits of space and the rapid
growth of new knowledge, it is recognized that this paper provides
only a small sample of the ongoing developments in radiation oncology
and biology. These developments are taking place in parallel with
new technical advances, such as conformal radiation therapy, improved
brachytherapy techniques, new hyperthermia treatment capabilities,
and particle-based radiotherapy [1].

Current Research and the "Classical"
Models

Figure 1 is a schematic illustrating the current scope of radiation
biology research. The biotechnical revolution of the 1980s brought
new complexity and understanding to the field of radiation biology,
as compared with the previous "classical" models. These
classical models, however, are still of interest and importance
to the clinician and scientist.

Classical chemotherapy models have established the importance
of dose and the use of non-cross-resistant agents. Likewise, the
classical radiation models are of use in clinical investigation
and practice. These include:

  1. the understanding of the shape of the radiation cell-survival
    curves of tumors and normal tissues, which serves as the basis
    for altered fractionation (eg, hyperfractionation);
  2. in vitro assays of clonogenic tumor cell survival and tumor
    growth kinetics as possible predictors of treatment outcome;
  3. tumor hypoxia as a predictor of overall tumor response to
    therapy; and
  4. cell-cycle effects and DNA damage repair, which have led to
    the development of combined-modality regimens with radiation and
    chemotherapeutic agents.

Therefore, even though the mechanisms behind the models are not
fully understood, they serve as useful scientific and clinical
paradigms.

The processes illustrated in Figure 1 will serve as the basis
for the discussions that follow. The potential clinical importance
of each of these processes also will be reviewed.

DNA Repair and Recombination

DNA is felt to be the primary target and mediator of radiation-induced
cell killing [2,3]. Ionizing radiation deposits energy in a distribution
that results in closely spaced damage, termed "locally multiply
damaged sites." [2] Many varieties of DNA base damage and
cross-links are produced [2,3], which lead to both single- and
double-strand breaks. The accumulation of unrepaired double-strand
breaks correlates closely with the loss of cell survival [3].

Within a double-strand break, the etiology of the damage on each
of the strands of DNA may be different, including a deletion on
one strand, and a repair-induced break produced by an endonuclease
on the other [3]. Radiation-induced DNA lesions can undergo enzymatic
repair involving base excision repair [2,3] or recombination [4,5].
The specific enzymes involved are being identified, and include
enzymes that recognize, excise, and ligate damaged regions; those
with DNA helicase activity [3]; and others involved in DNA recombination
[4,5].

DNA Repair Deficiencies

DNA repair deficiencies are often identified from mutant cell
lines or from patients with clinical syndromes characterized by
sensitivity to x-ray-induced DNA damage, such as ataxia-telangiectasia
[6,7] and severe combined immunodeficiency (SCID) [4,5]. Patients
with defective DNA repair have an increased sensitivity to radiation-induced
damage, a phenomenon that can be observed in vitro using clonogenic
assays on irradiated fibroblasts derived from affected individuals
[8].

Another type of repair recently determined to be important in
genetic hypermutability and cancer progression is called mismatch
repair [9]. This surge in new knowledge of the various types of
DNA repair should lead to a better understanding of tumor progression,
and the cellular response to radiation.

Cell Survival

Of equal significance to the clinician and biologist is the shape
of the radiation survival curve for each cell type of interest;
with sensitive and resistant cells showing more or less cell killing,
respectively, for a given dose of radiation. The terms commonly
used to define cell survival are D0, the slope of the survival
curve, or SF 2 Gy, the surviving fraction of cells in culture
after a clinically relevant dose of 2 Gy.

To date, the clinical syndromes associated with abnormal DNA repair
and the mutant cell lines are characterized by decreased repair
capacity and not by excessively proficient repair. This is of
importance in the search for the causes of clinical resistance
to radiation and in the development of predictive assays for use
in the clinic. In addition to the shape of the radiation cell-survival
curve, other possible determinants of clinical resistance include:
the number of clonogenic cells in the tumor, the ability to undergo
apoptosis, and microenvironmental factors. These factors and clinical
predictive assays are discussed below.

Radiation-Induced Genes

Given that radiation induces changes in cellular phenotype, such
as growth and cell-cycle arrest, it is logical to assume that
it would also lead to alterations in gene expression. The expression
of a gene can be altered in a number of ways, including: gene
induction (production of new mRNA), stabilization of preexisting
mRNA, or post-translational modification (stabilization) of a
protein.

A range of genes and gene products have been shown to be induced
following x-irradiation to a cell. These include inflammatory
response molecules, such as tumor necrosis factor (TNF) and interleukin-1
(IL-1); growth factors, such as epidermal growth factor (EGF);
cell-cycle-related genes (gadd genes); and cell-surface
receptor genes (integrins), to name a few examples [10-13].

Studies demonstrating gene induction have been carried out at
a wide range of radiation doses. High radiation doses are often
used to highlight an effect in the laboratory; however, these
doses are often in a range at which few, if any, cells remain
viable. Therefore, the gene induction observed in these experiments
may not necessarily be clinically relevant to surviving cells
within a tumor. The importance of defining the sequence of genes
induced by irradiation at clinically relevant conditions is obvious,
as it will lead not only to the understanding of how a cell responds
to x-rays, but also to novel therapeutic strategies to alter that
response.

Transcription Factors

One way to study the effects of x-rays on gene expression is to
study the control regions of the genes, looking for radiation
responsive elements (RRE). Genes contain regulatory domains that
are proximal or upstream to the transcription site, which is the
starting site for mRNA synthesis. Whether a gene is induced or
repressed following cellular radiation depends on which elements
in the control region of the gene are bound by specific regulatory
proteins.

Genes are activated when proteins called transcription factors
bind to their proper sequence in the control region of the gene.
Similarly, other bound factors can repress or inhibit gene expression.
Several transcription factors have been shown to be activated
by ionizing radiation, including: AP-1 factors (fos, jun), EGR-1,
and the c-rel-family (NF kappa beta). These transcription factors,
in turn, can act on numerous downstream target genes.

Activated transcription factors characteristically form oligomers
prior to binding to the promoter region of the gene. The AP-1
group of transcription factors form two component complexes termed
"dimers." Homodimers are from the same subgroup (jun-jun),
and heterodimers contain factors from two subgroups (fos-jun).
The specific oligomers that are induced may determine whether
or not a transcription factor will activate one gene or another.
Furthermore, genes contain many transcription-factor binding sites,
so that activation often requires multiple factors from a range
of transcription factor groups.

Genes may be activated in sequence. Early response genes, such
as the AP-1 regulated genes, produce a protein that is itself
a transcription factor for a secondary gene, which, in turn, may
produce a cytokine, structural protein, or enzyme. Since radiation
is just one of a whole range of stimuli to which a cell is exposed,
it is not likely that there will be a specific RRE on either the
primary or secondary genes; however, some combination of response
elements may be more readily induced by x-rays than are others--a
concept that has led to the development of genetic radiotherapy.

Genetic Radiotherapy

In genetic radiotherapy, a promoter with a radiation-activated
sequence is attached to a gene that can make a product that would
enhance the efficacy of radiation. One sequence of interest has
been the serum response element (SRE), which is in the promoter
region of the EGR1 gene, an "immediate early response
gene. [14]" This promoter region has been attached to the
TNF-alpha gene, so that when a cell containing the EGR1-TNF-alpha
construct is irradiated, TNF is produced. When xenograft murine
tumors containing the transfected EGR1-TNF-alpha construct
were irradiated with a single large dose of 40 Gy, an improved
cure rate was demonstrated [15].

The efficacy of genetic radiation therapy, like all forms of gene
therapy, will depend on sufficient gene product being made to
achieve the desired effect. If used as a radiation modifier, sufficient
gene product (eg, TNF) must be induced by a clinically relevant
radiation dose (~2 Gy), the timing of gene activation must be
in proper relationship to the x-ray treatment to provide sensitization,
and the gene construct would need some extended stability to last
throughout a course of ionizing radiation. Since most promoters
can be activated by many different stimuli, the release of the
radiation-induced gene product may not be tightly controllable.
This may not necessarily be a disadvantage, as other aspects of
the tumor microenvironment may help activate the RRE. Despite
the many hurdles between a laboratory concept and a clinically
useful therapy, these early studies are important steps in assessing
the ultimate utility of this approach.

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