Quantifying a bystander response following microbeam irradiation using single-cell RT-PCR analyses

Abstract

There is growing recognition that the effects of ionizing radiation may extend to more than those cells that directly suffer damage to DNA in the cell nucleus. Data from several investigators have indicated that cells neighboring those that are irradiated also demonstrate several responses seen in hit cells--the so-called bystander effect. The microbeam facility at the Center for Radiological Research is particularly well suited for the study of this bystander effect, since it has the ability to place known numbers of charged particles (protons or alpha-particles at LETs from 20 to 180 KeV/microm) at defined positions relative to individual cells. That is, some known fraction of cells in a population can be irradiated through the nucleus, or the cytoplasm or even adjacent to cells through the media. Therefore, using the microbeam it is possible to examine individual cell responses in both hit and nonhit cells in the same population. Alterations in the cyclin-dependent kinase inhibitor CDKN1a (p21/Cip1/WAF1) were quantified at the mRNA level in single normal human fibroblasts following precise delivery of 0 or 10 alpha-particles per cell at 90 KeV/microm to 50% of cells in a population. Semiquantitative RT-PCR of individual hit cells demonstrated increases in the levels of CDKN1A message that followed the kinetics previously described for irradiated populations. Furthermore, nonhit bystander cells also showed increased (though lesser) levels of CDKN1a message. Data presented here demonstrate the power of this approach, which combines the ability of the microbeam to irradiate specific cells in a population and the ability to quantify the response to the irradiation in individual targeted and bystander cells.