Project 2: Mechanisms of Bystander Mutagenesis

Project Leader: Tom K. Hei

Overiew
Project 2 addresses the mechanisms of the bystander effect using either primary human fibroblasts or a well defined mutagenic assay based on the human hamster hybrid A_L cells and asks a number of mechanistic questions including the roles of peroxynitrite anions and mitochondrial functions in the bystander effect. The effects of COX-2 enzyme and cytoplasmic irradiation in mediating the bystander effect will be critically evaluated in both human and hamster cell lines. A final goal of this project is to determine whether the signaling molecules that induce bystander response can induce genomic instability in mammalian cells as well.

Research Aims
The central hypothesis of this Program Project is that the bystander effect involves multiple pathways and that an initial event in the hit cells and a subsequent downstream signaling step involving the arachidonic acid cascade in the bystander cells play an important role in mediating the process. In this Project, 4 specific goals are proposed to address the mechanism of the bystander response including roles of reactive nitrogen species, mitochondrial functions, COX-2 enzyme, contributions from cytoplasmic irradiation, and the relationship in mechanism between bystander versus genomic instability.

Research Highlights
In our quest to identify the signaling pathways involved in radiation induced bystander effect, we first focused on the genes that are differentially expressed among the bystander versus control cells. Since the microbeam can only irradiate one cell at a time and a large number of cells are needed for gene array analyses, we employed a novel double mylar dish approach to define the bystander response (diagram below).

Using a signal transduction pathway specific SuperArray, we compared the differentially expressed genes among the non-irradiated control normal human lung fibroblasts (NHLF) cells and the bystander cells. Transcription level of one gene, cyclooxygenase-2 (COX-2), was found to be consistently up-regulated by more than three-fold, while the RNA level of insulin growth factor binding protein-3 (IGFBP3) was found to be consistently lower by more than seven-fold in multiple analyses of multiple bystander samples. Addition of the COX-2 inhibitor NS-398 (50 µM) suppressed COX-2 activity in NHLF and finally, after 24 hours, reduced the COX-2 protein level in bystander cells to a non-detectable level. This corresponded to a significant reduction in the bystander mutagenesis at the hypoxanthine guanine phosphoribosyl-transferase (HPRT) locus in the NHLF cells. These results indicated that expression of COX-2 is associated with the bystander effect.

Insulin growth factor and other cytokines activate mitogen activated protein kinase (MAPK) signaling cascade; and activation of extracellular signal-related kinase (ERK) by phosphorylation is a critical upstream event preceding COX-2 expression. As shown below, there was a strong up-regulation of phospho-ERK and MAPKp38 levels in both a-irradiated and bystander NHLF four hours after treatment. To further confirm the activation of ERK in bystander cells, we used PD 98059 (50 µM), a specific inhibitor of MEK-ERK, which had been added to cell cultures immediately after irradiation for a period of four hours. In the presence of PD 98059, the phosphorylated form of ERK and its activation as well as the induction of COX-2 levels were all suppressed in both alpha particle irradiated and bystander cells.

Role of Nuclear Factor kappa B in bystander response
Since NFκB is an important transcription factor for many signaling genes including COX-2, it is likely that NFκB participates in the bystander response. There is clear evidence that alpha particle irradiation upregulates NFκB binding activity in both directly irradiated and bystander cells, while Bay 11-7082, a pharmacological inhibitor of IKK/NFκB, efficiently suppresses this up-regulation and also reduces levels below the basal amount. This inhibitor of NFκB activity also efficiently down-regulates COX-2 and iNOS-expression levels in both directly irradiated and bystander fibroblasts. Earlier studies using confluent human skin fibroblasts exposed to low fluences of alpha particles show a rapid up-regulation of NFκB, JNK and ERK in the exposed population and suggested activation of these stress inducible signaling pathways in bystander cells. Furthermore, addition of the antioxidant superoxide dismutase (SOD) was found to suppress the induction. Since induction of NFκB binding activity can be found in both directly irradiated and bystander cells, its role in the bystander response in this study is equivocal.

Role of mitochondria in the bystander response
The observations that extracellularly applied antioxidant enzymes such as superoxide dismutase and catalase can inhibit the medium-mediated bystander response suggest a role for reactive radical species in the bystander process. Since mitochondria are the main source of energy production as well as generators of free radicals in cells, especially in pathological and stressful conditions, they are the prime target for the source of these radical species. There is recent evidence that point mutations in the mitochondrial genome as well as an increase in mitochondrial mass are induced among either directly irradiated human papillomavirus-immortalized human keratinocytes exposed to a 5Gy dose of gamma-rays or by exposure to bystander factor(s) obtained from such cells. Using human fibroblasts that are devoid of mitochondrial DNA and, consequently, reduced mitochondrial DNA functions, there is evidence that mitochondria play an important role in the regulation of radiation-induced bystander effects.

ρºcells show a higher bystander response than wild type ρ⁺ cells
To explore the role of mitochondria in the radiation-induced bystander effect, a microbeam was used to lethally irradiate either ρº or ρ⁺ cells with 20 alpha particles each in a mixed, confluent culture, and the bystander response was determined in the non-irradiated fraction. ρº cells, when compared with ρ⁺ cells, showed a higher bystander HPRT^- mutagenic response in confluent monolayer when 10% of the same population was lethally irradiated. However, using mixed cultures of ρº and ρ⁺ cells and targeting only one population of cells with a lethal dose of alpha particles, a decreased bystander mutagenesis was uniformly found with both cell types indicating that mitochondrial deficient cells cannot effectively communicate the bystander signals to wild type cells; or alternatively, signals from one cell type can modulate expression of the bystander response in another cell type.

The unifying model of bystander effect
The mechanism of the radiation-induced bystander effect, whether involving cell-cell contact or mediated by soluble factors, is not clear and is likely to be complex, involving multiple pathways. It is, however, clear that p53 gene function is not necessary for the effect since cells without normal p53 function (such as CHO cells) show a large bystander response in either bystander pathway. It is likely that multiple signaling cascades involving both an initiating event and downstream signaling steps are necessary to mediate the bystander process. Previous studies have shown that COX-2 is critically linked to the radiation-induced bystander effect in normal human fibroblasts. There is evidence that NO can induce expression of COX-2 in mouse skin and human cultured airway epithelial cells, and that the NFκB pathway is involved in the process. The recent findings that Bay 11-7082, a specific IKK/NFκB inhibitor, can eliminate bystander mutagenesis in both wild type and ρº cells, highlight the important role of this transcription factor in the bystander phenomenon.

A unifying model of the signaling pathways involved in radiation-induced bystander effects. Expression/secretion of the inflammatory cytokines strongly increased after exposure to ionizing radiation or oxidants. Secreted or membrane-associated forms of cytokines such as TNFα activate IKK-mediated phosphorylation of IκB, which releases NF-κB, that enters the nucleus and acts as a transcription factor for COX-2 and iNOS. TNFα also activates MAPK pathways (ERK, JNK and p38) that, via the AP-1 transcription factor, additionally up-regulates expression of COX-2 (Zhou et al 2005) and iNOS, which stimulate production of nitric oxide. Mitochondrial damage facilities the production of hydrogen peroxide that migrates freely across plasma membranes and is subjected to antioxidant removal. Activation of COX-2 provides a continuous supply of reactive radicals and cytokines for the propagation of bystander signals either through gap junctions or medium.

In vivo non-targeted mutagenic response
Recently, we have used the gpt delta transgenic mouse system, established in the laboratory of Dr. Takehiko Nohmi, to examine in vivo non-targeted effects of ionizing radiation. An area 1cm x 1cm square in the lower extremity of eight weeks old animals was exposed to a 5Gy dose of X-rays while the rest of the body was shielded with lead. At specified time points post-irradiation animals were anesthetized and 0.1 ml of blood from each animal was drawn from the orbital sinus to determine cytokine (TGFβ, TNFα, IL-6) levels by ELISA assay. Anesthetized animals were euthanized by decapitation and tissues (lung and liver) samples were obtained. The scattering dose was measured by insertion of mini-dosimeters into the lungs of the animals when irradiated and, for a 5 Gy dose, the scattered dose was estimated to be ~ 6 cGy. The following figure illustrates the positioning of the animal during irradiation.

Using western blotting, there is evidence that out of field (bystander) lung tissues of partially irradiated gpt delta mice show an induction of COX-2 that peaks at 24 hours post-irradiation in both male and female animals.

Induction of COX-2 protein expression in the out of field lung tissues of gpt delta mice irradiated with a single 5 Gy dose of X-rays in the lower abdominal extremity of animals at various time point post-irradiation. (Preliminary data from Chai and Hei)

To illustrate that the bystander induction of COX-2 in the lung tissue is not due to exposure of animals to the scatter dose of X-rays, experiments were repeated in female mice using a whole body irradiation with a 6 cGy dose of X-rays. While exposure of animals to either a 5 Gy whole body exposure or to a 5 Gy out of field (partial body) irradiation induced COX-2 induction, there was no induction with a 6 cGy dose of whole body exposure (data not shown). These results provide clear evidence that the bystander induction in animal was NOT induced as a result of the scattering dose generated as a result of the secondary photons.

Induction of bystander genotoxicity in lung tissues of gpt delta mice: To determine if bystander effects can be demonstrated in vivo, we collected the lung tissues of partial body irradiated gpt delta mice and quantify the incidence of Spi^– mutations. As shown in the following figure, there was a three fold increase in the mutant fraction in the non-targeted out of field lung tissues 24 hour post-irradiation. Corresponding lung tissues from animals receiving a whole body 6 cGy dose (equivalent to the scattering dose) resulted in no mutation induction (data not shown).

Induction of bystander Spi– mutagenesis in the lung tissues of gpt delta mice. Preliminary data from three independent data set of three animals each. Bar + SD of means.

Genomic instability in bystander mammalian cells
Using the human hamster hybrid (A_L) cells, which contain a complete set of CHO chromosomes and a single copy of human chromosome 11, the induction of genomic instability among the progeny of bystander cells were examined using the m-BAND assay. Approximately 20% of A_L cells were randomly selected by the image analysis system of the Columbia University microbeam and a precise number of alpha particles were delivered to the nucleus or the cytoplasm of the cells. After irradiation, individual cells were cloned and expanded in cultures. The presence of chromosomal re-arrangement on the human chromosome 11, which include translocation, duplication, paracentric or pericentric inversion, insertion, interstitial or terminal deletions together with whole chromosome 11 deletion or duplication were determined. There is evidence that the fraction of induced abnormal cells involving human chromosome 11 increases at 10 days post-irradiation (~15 population doublings) in the progeny of bystander cells (14.5%), in progeny of nuclear-irradiated cells (14.5%) and in progeny of cytoplasmic-targeted cells (9.7%) relative to the corresponding controls at 6.8%. These data indicate that genomic instability manifest following ionizing radiation exposure is not dependent on direct damage to the cell nucleus. Please refer to Project 3 for more details.