Columbia University Medical Center
Center for Radiological Research

NIH Program Project on
Radiation Bystander Effects: Mechanism
PO1-CA 49062-23

Project 1: Role of Rad9 in Bystander Effects

Project Leader: Howard B. Lieberman

Overiew

The overall cellular response to ionizing radiation exposure is not limited to cells directly irradiated, but includes neighboring "bystanders". Essentially every endpoint used routinely to test for changes in cells directly exposed to radiation, such as mutation, apoptosis, micronuclei formation or cell cycle checkpoints, has been studied in bystanders. This phenomenon has caused a paradigm shift in thinking about biological effects and associated health risks of radiation exposure; the impacted cell population is larger than once believed. The mechanism regulating bystander effects is not well understood. This Project focuses on the function of Rad9, a gene known to play important multiple roles in the cellular response to direct ionizing radiation exposure, in regulating radiation-induced bystander effects.

Research Aims

This Project concerns the hypothesis that Rad9 plays an important role in radiation-induced bystander effects via mechanisms involving at least Cox-2, p21, Connexin 32 (Cxn32) and TCTP. There are five specific experimental goals that will address this hypothesis. We will determine whether Rad9 is important for nuclear versus cytoplasmic bystander signaling, identify the functional domains of the protein involved, establish whether the relationships between Rad9 and Cox-2, p21, Connexin 32, TCTP or other proteins are critical, assess whether Rad9 plays a role in bystander mutagenesis, and define in more depth the significance of junctional communication on the regulation and function of Rad9 with respect to the bystander response. Moreover, we have most recently focused on the use of prostate cancer model systems to test the hypothesis that RAD9 regulates signals induced by ionizing radiation in cancer stem and non-stem cells to elicit a bystander response in prostate smooth muscle cells, which could be at risk during radiotherapy to treat this disease.

Research Highlights

Fig.1. Double ring Mylar dishes used for bystander studies. Left: Outer ring with a base made of Mylar that is 6m thick, and contained within is the inner ring bearing several strips of 30m Mylar as a base. Outer and inner rings fit together; Right: The inner ring with strips of Mylar.

The goals of this project, which focus on defining the significance of Rad9 in mediating bystander effects, are based on several research findings. A Rad9 null mutation sensitizes mouse ES cells to 125 KeV/µm 3He alpha particle-induced bystander effects, specifically at least micronuclei formation and apoptosis. For these experiments, we used a specially made double Mylar two-ring dish to mediate direct and bystander radiation exposure of cells (Fig. 1). The outer ring has a base made of Mylar that is 6 µm thick, and the inner ring contains several strips of 30 µm Mylar as a base. The outer and inner rings fit together. After sterilizing in 70% ethanol and air-drying, both Mylar layers were coated with fibronectin solution. ES cells (5x105) were plated onto the double Mylar culture rings. Two days later, cells were approximately 90%-100% confluent, allowing direct cell-cell communication. Confluent ES cells were irradiated with 3He alpha particles (LET: 125keV/µm), or served as unirradiated controls. Dishes were irradiated from underneath using the track segment mode of a 4 MeV Van de Graaff accelerator located at the Radiological Research Accelerator Facility of Columbia University. Since alpha particles can penetrate the 6 µm but not the 30 µm Mylar, only cells growing on the lower, thinner surface were irradiated. However, the unirradiated cells on the 30 µm strips were in close physical proximity to the irradiated cells, and were the “bystander” population. After irradiation, the medium was immediately changed to remove dead floating cells and the dishes were incubated for 24 hrs. Irradiated and unirradiated cells were removed from the 6 µm and 30 µm Mylar strips separately using trypsin. Part of the trypsinized culture was replated onto a 2-well chamber slide coated with fibronectin for detecting micronuclei. Cells were also seeded onto a 6-well plate to monitor apoptosis.

Spontaneous and 125 keV/µm 3He Alpha Particle-Induced Micronuclei Formation. A set of mouse ES cells with different Mrad9 genotypes was assessed for micronuclei (MN) formation. Irradiated or unirriadiated cells were removed from the Mylar surface after 24 hrs using trypsin, plated on a 2-well chamber slide containing supplemented Knockout-DMEM medium and incubated for an additional 24 hrs. Cells were then fixed for 1 hr to overnight with alcohol acid (95% ethanol/5% acetic acid). Following fixation, slides were washed three times in PBS. After air-drying, the slides were stained with DAPI (Vector Laboratories). Cover slips were mounted onto the slides and cells were visualized using fluorescent microscopy. About 500 to 1000 cell nuclei were scored per sample. Fig. 2 indicates that spontaneous micronuclei frequencies in most populations ranged from 5 to 7%. However, Mrad9-/- cells had two to three times that frequency, which was reduced by ectopic expression of either mouse Mrad9 or the human HRAD9. Alpha particles at doses of 0.5 to 10 Gy significantly increased the levels above background, with peak induction occurring at 5 Gy for all populations (Fig. 2A). Most data points for the Mrad9+/+ and Mrad9+/- populations, as well as the Mrad9-/- cells ectopically expressing Mrad9 or HRAD9 differed statistically from the results for the Mrad9-/- population (p≤0.05) but not each other. The only exception was when the 10 Gy points for the Mrad9-/- cells and the same population with ectopic expression of the wild type Mrad9 gene were compared (p=0.07), due to the large error bars for the latter point. A similar pattern was observed when apoptosis was used as the endpoint (not shown).

Bystander Micronuclei Formation is Enhanced in Cells Deleted for Mrad9.
The ability of 125 keV/µm alpha particles to induce micronuclei in cells neighboring those directly irradiated was examined (Fig. 2B). With the exception of Mrad9-/- cells, doses in the 0.5 to 10 Gy range induced a minimal bystander effect, and usually any increase above background levels was not statistically significant. The largest increase was detected in the Mrad9-/- cells where 5 and 10 Gy induced statistically significant increases in bystander micronuclei formation above background levels. The Mrad9-/- cells ectopically expressing Mrad9 or HRAD9 did not demonstrate these high levels of micronuclei. Therefore, the Mrad9 null mutation sensitizes cells to alpha particle induced bystander micronuclei formation.  When radiation-induced bystander apoptosis was assessed, a similar enhancement by Rad9 null was revealed (not shown).

Fig.2. Induction of micronuclei by 125 keV/m 3He alpha particles. Panel A: Micronuclei formation after direct exposure to 0, 0.5, 5 or 10 Gy of alpha particles. Panel B: Micronuclei formation after bystander exposure to doses indicated. Mouse ES cells used have the following genotypes: Mrad9+/+, Mrad9+/-, Mrad9-/-, Mrad9-/- ectopically expressing Mrad9, Mrad9-/- ectopically expressing HRAD9. Columns, average of three independent experiments, S.E.M.

Rad9 Null in Mouse ES Cells Enhances IR-Induced Bystander Chromatid Aberration Frequency.  We showed above that Rad9 null in mouse ES cells, relative to the Mrad9+/+ control, can enhance IR-induced bystander responses.  All populations demonstrated α-particle-induced bystander apoptosis, but that effect was most prominent in the Rad9-/- cells. Minimal α-particle induction of micronuclei in WT bystander cells was observed, but for the Rad9-/- mutant, a significant increase above background was detected. Therefore, the Rad9 null mutation selectively sensitizes mouse ES cells to spontaneous and high-LET radiation-induced bystander apoptosis and micronucleus formation. We extended this work to assess the impact of Rad9 null on bystander induced genomic instability, an endpoint that expresses many generations beyond the time of initial radiation insult and what we examined previously. We studied formation of chromatid and chromosome aberrations up to 28 days post-IR exposure.  For chromatid aberrations (breaks and gaps on only one arm of a replicated chromosome) (Fig. 3), there were no significant differences in yields for unirradiated controls at all time points regardless of Rad9 status.  Interestingly, WT α-particle irradiated (1 Gy) and bystander cells did not have significantly higher yields of chromatid aberrations compared to unirradiated controls (Fig. 3A). In contrast, directly irradiated Rad9-/- ES cells had ~4-5 fold higher levels at early time points that returned to control levels by 28 days post-IR (Fig. 3B). Bystander Rad9 null cells also had significantly higher yields at 7 days post-IR. Ectopic expression of Mrad9 restored low WT chromatid aberration levels to Mrad9-/- cells (Fig. 3C).  Spontaneous chromosome aberrations (mainly acentric fragments and rings, a few dicentrics) were also scored and found to be higher in Rad9 knockout cells compared to WT or mutant ectopically expressing Mrad9 (not shown). At 7 days post-IR, all exposed cells had equivalent, higher than control levels of chromosome-type aberrations. Chromosome aberration frequencies in bystander WT and mutant +Mrad9 populations were elevated relative to controls, up to 28 days post-IR.  Of note, in bystander Mrad9-/- cells, unlike for the patterns of chromatid aberrations, chromosome aberration yields were for the most part similar to those of WT controls (not shown).  Overall, the data support the findings of a role for RAD9 in genomic instability and bystander responses, but now demonstrated over many generations post-IR exposure. In addition, differential expression of chromatid and chromosome aberrations as a function of Rad9 status indicates the protein plays different roles in formation of delayed chromosomal aberrations in directly irradiated vs bystander cells.  Using site-directed mutagenesis at known functional domains, the precise activity of multi-functional RAD9 mediating these phenotypes is under investigation.

Figure 3.

Fig.3. Fig. 3. Chromatid aberration frequencies in unirradiated (clear first bars), α-particle-irradiated (1 Gy; second filled bars) and bystander (1 Gy; third stippled bars) mouse ES cells bearing Mrad9+/+ (A), Mrad9-/- (B), Mrad9-/- +Mrad9 (C).

Human Prostate Cancer Cells Transmit an IR-Induced Bystander Signal to Noncancer Prostate Smooth Muscle Cells.  Several investigators demonstrated that prostate cancer cells such as DU145 can send an IR-induced bystander signal to themselves or noncancer fibroblasts Particularly at risk during radiotherapy are prostate smooth muscle cells in the vicinity of a tumor, as those cells can incur damage as well as develop into sarcomas.  We used the no cell-cell contact media transfer setup for these preliminary studies and show that in our system DU145 cells transmit a bystander signal to themselves and, as well, prostate smooth muscle cells, PrSMC and WMPY-1, when 1 Gy track segment 3He ions (125 keV/μm) is used.  Micronuclei (MN) formation as an endpoint is shown (Fig. 4), and similar results for apoptosis were obtained (not shown).  It is clear that bystander responses can occur using DU145.  We similarly tested DU145 knocked down for RAD9.  For 24 hrs post-treatment with α particles (Fig. 4), reduced RAD9 abundance sensitized all bystanders to MN formation, more so than directly irradiated DU145 with inherent high RAD9 levels. We are extending these findings to assess if these results are limited to DU145 or found in other prostate cancer cells (PC-3, LNCaP), stem vs. non-stem cells, the mechanisms involved, and whether such an effect can be found in intact mice receiving partial body irradiation.

Figure 4.

Fig.4. DU145 cells can transmit an IR-induced bystander signal for micronuclei (MN) formation to DU145, WPMY-1 or PrSMC cells, which is enhanced when RAD9 is knocked down.  Track segment irradiation using specialized double ring mylar dishes to study medium transfer bystander signaling. DU145 or DU145shRAD9 cells directly irradiated with 1Gy 3He ions (125 keV/μm), using track segment mode of the 4 MeV Van de Graaff accelerator at Columbia University.  DU145, DU145shRAD9, WPMY-1 or PrSMC were bystanders. Irradiated and unirradiated controls incubated at 37°C for 24hrs, then processed for MN scoring. For all studies, about 500 bi-nucleated cells scored for MN per sample.  MN percentage: (# binucleate cells with MN/500 binucleate cells) X 100.