Biologic Basis for Radiation Oncology

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.