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Lowdose Radiation Program Workshop IV
Abstract
Title:
12.5 keV Xray Microbeam Bystander Studies With Human Mammary
Epithelial Cells and Fibroblasts
Authors: E. A. Blakely1, R. I. Schwarz1,
A. C. Thompson2, K. A. Bjornstad1, P.
Y. Chang1,3 C.J. Rosen1, and D. Sudar1
Institutions: Divisions of 1Life
Sciences and 2Advanced Light Source, Lawrence Berkeley
National Laboratory, Berkeley, CA 94720-8174 USA and 3SRI
International, Menlo Park, CA 94025 USA.
We are using a novel X-ray Microprobe Beamline at the Advanced
Light Source
(ALS) at LBNL to investigate bystander effects of low doses
in wellcharacterized
human mammary epithelial cells (HMEC) and human skin fibroblasts
(HSF). The ALS facility is capable of producing a beam of
12.5 keV X-rays with a focussed spot size of __m_ and a wide
range of doses and dose-rates. Unlike normal X-ray sources,
this beam has a very small background of either low- or high-energy
X-rays. In initial studies, cultures grown in microwell slide
chambers have been irradiated with precise stripes of dose
up to 100_m wide. We are using fluorescence microscopy on
a high-precision-controlled microscope stage to evaluate several
classes of radiation-induced signals, how these signals are
communicated across cell compartments, and how radiation changes
cell signaling both acutely and chronically. We are investigating
the radiation induction of p21Cip1 (CDKN1a), and phosphorylation
of H2AX and p53 serine-15 as endpoints. Our preliminary results
indicate that there is a dose- and celltype- dependent expression
of p53 serine-15P within 10 minutes after exposure to a ____m
wide stripe of dose. Immunohistochemistry of p53-serine-15P-positive
cells traversed by the beam illuminates the path of the X-ray
microbeam, with epithelial cells responding more rapidly and
with greater intensity than fibroblasts. The intensity of
the immunofluorescence scales with the dose. Using stripes
of dose we are able to evaluate the spatial dependence of
intercellular bystander effects. The number and fluorescence
intensity of p53- serine-15P-positive cells in the unirradiated
cell populations at perpendicular distances away from the
dose stripe are being scored as a measure of the bystander
effect, and compared to appropriate controls. We will report
on cellular responses to doses of 400 cGy down to 10 cGy examined
in a time course from 10 min to 6 hours after exposure.
This
work was supported by the U. S. DOE's Low Dose Radiation Research
Program under Contract No. DE-AC03-76SF00098.
