Studies of Bystander Effects in 3-D Tissue Systems Using a Low-LET Microbeam

PDF Version Also Available for Download.

Description

It is now accepted that biological effects may occur in cells that were not themselves traversed by ionizing radiation but are close to those that were. Little is known about the mechanism underlying such a bystander effect, although cell-to-cell communication is thought to be important. Previous work demonstrated a significant bystander effect for clonogenic survival and oncogenic transformation in C3H 10T(1/2) cells. Additional studies were undertaken to assess the importance of the degree of cell-to-cell contact at the time of irradiation on the magnitude of this bystander effect by varying the cell density. When 10% of cells were exposed to ... continued below

Creation Information

Brenner, David J. July 17, 2009.

Context

This report is part of the collection entitled: Office of Scientific & Technical Information Technical Reports and was provided by UNT Libraries Government Documents Department to Digital Library, a digital repository hosted by the UNT Libraries. More information about this report can be viewed below.

Who

People and organizations associated with either the creation of this report or its content.

Provided By

UNT Libraries Government Documents Department

Serving as both a federal and a state depository library, the UNT Libraries Government Documents Department maintains millions of items in a variety of formats. The department is a member of the FDLP Content Partnerships Program and an Affiliated Archive of the National Archives.

Contact Us

What

Descriptive information to help identify this report. Follow the links below to find similar items on the Digital Library.

Description

It is now accepted that biological effects may occur in cells that were not themselves traversed by ionizing radiation but are close to those that were. Little is known about the mechanism underlying such a bystander effect, although cell-to-cell communication is thought to be important. Previous work demonstrated a significant bystander effect for clonogenic survival and oncogenic transformation in C3H 10T(1/2) cells. Additional studies were undertaken to assess the importance of the degree of cell-to-cell contact at the time of irradiation on the magnitude of this bystander effect by varying the cell density. When 10% of cells were exposed to a range of 2-12 alpha particles, a significantly greater number of cells were inactivated when cells were irradiated at high density than at low density. In addition, the oncogenic transformation frequency was significantly higher in high-density cultures. These results suggest that when a cell is hit by radiation, the transmission of the bystander signal through cell-to-cell contact is an important mediator of the effect, implicating the involvement of intracellular communication through gap junctions. Additional studies to address the relationship between the bystander effect and the adaptive response were undertaken. A novel apparatus, where targeted and non-targeted cells were grown in close proximity, was used to investigate these. It was further examined whether a bystander effect or an adaptive response could be induced by a factor(s) present in the supernatants of cells exposed to a high or low dose of X-rays, respectively. When non-hit cells were co-cultured for 24 h with cells irradiated with 5 Gy alpha-particles, a significant increase in both cell killing and oncogenic transformation frequency was observed. If these cells were treated with 2 cGy X-rays 5 h before co-culture with irradiated cells, approximately 95% of the bystander effect was cancelled out. A 2.5-fold decrease in the oncogenic transformation frequency was also observed. When cells were cultured in medium donated from cells exposed to 5 Gy X-rays, a significant bystander effect was observed for clonogenic survival. When cells were cultured for 5 h with supernatant from donor cells exposed to 2 cGy and were then irradiated with 4 Gy X-rays, they failed to show an increase in survival compared with cells directly irradiated with 4 Gy. However, a twofold reduction in the oncogenic transformation frequency was seen. An adaptive dose of X-rays cancelled out the majority of the bystander effect produced by alpha-particles. For oncogenic transformation, but not cell survival, radioadaption can occur in unirradiated cells via a transmissible factor(s). A pilot study was undertaken to observe the bystander effect in a realistic multicellular three-dimensional morphology. We found bystander responses in a three-dimensional, normal human-tissue system. Endpoints were induction of micronucleated and apoptotic cells. A charged-particle microbeam was used, allowing irradiation of cells in defined locations in the tissue yet guaranteeing that no cells located more than a few micrometers away receive any radiation exposure. Unirradiated cells up to 1 mm distant from irradiated cells showed a significant enhancement in effect over background, with an average increase in effect of 1.7-fold for micronuclei and 2.8-fold for apoptosis. The surprisingly long range of bystander signals in human tissue suggests that bystander responses may be important in extrapolating radiation risk estimates from epidemiologically accessible doses down to very low doses where nonhit bystander cells will predominate. Finally, it would be of great benefit to develop a reproducible tissue system suitable for critical radiobiological assays. We have developed a reliable protocol to harvest cells from tissue samples and to investigate the damage induced on a single cell basis. In order to result in a valid tool for bystander experiments, the method focuses on processing and analyzing radiation damage in individual cells as a function of their relative position in the tissue. We have investigated the micronucleus formation following partial irradiation with 3.5 MeV protons in an artificial human skin construct. Following the optimization of the Cytochalasin-B concentration and incubation time necessary to obtain a reproducible and suitable number of binucleated cells, the induction of micronuclei across the samples is assessed for 3 dose. The reproducible and low background frequency of micronuclei measured in this system allowed us to detect small increases following the irradiation exposure. The effect is statistically significant at doses as low as 0.1 Gy and it shows evidence of a spatial dependency as it decreases in the cells further away from the directly exposed area. This experimental protocol represents the initial steps in the development of an in vivo-like assay for complex radiation damage in human tissues.

Language

Item Type

Identifier

Unique identifying numbers for this report in the Digital Library or other systems.

  • Report No.: DOE/ER/63632
  • Grant Number: FG02-03ER63632
  • DOI: 10.2172/959346 | External Link
  • Office of Scientific & Technical Information Report Number: 959346
  • Archival Resource Key: ark:/67531/metadc926463

Collections

This report is part of the following collection of related materials.

Office of Scientific & Technical Information Technical Reports

Reports, articles and other documents harvested from the Office of Scientific and Technical Information.

Office of Scientific and Technical Information (OSTI) is the Department of Energy (DOE) office that collects, preserves, and disseminates DOE-sponsored research and development (R&D) results that are the outcomes of R&D projects or other funded activities at DOE labs and facilities nationwide and grantees at universities and other institutions.

What responsibilities do I have when using this report?

When

Dates and time periods associated with this report.

Creation Date

  • July 17, 2009

Added to The UNT Digital Library

  • Nov. 13, 2016, 7:26 p.m.

Description Last Updated

  • Dec. 13, 2016, 12:17 p.m.

Usage Statistics

When was this report last used?

Yesterday: 0
Past 30 days: 0
Total Uses: 4

Interact With This Report

Here are some suggestions for what to do next.

Start Reading

PDF Version Also Available for Download.

Citations, Rights, Re-Use

Brenner, David J. Studies of Bystander Effects in 3-D Tissue Systems Using a Low-LET Microbeam, report, July 17, 2009; United States. (digital.library.unt.edu/ark:/67531/metadc926463/: accessed April 27, 2018), University of North Texas Libraries, Digital Library, digital.library.unt.edu; crediting UNT Libraries Government Documents Department.