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Lowdose Radiation Program Workshop IV
Abstract
Title:
Delayed genomic instability in human lymphoblasts exposed
to 137Cs y-rays radiation
Authors: Jeffrey L. Schwartzb, Robert
Jordanb, Marek Lenarczyka and Howard L. Libera
Institutions: aDepartment of Environmental
and Radiological Health Sciences,
Colorado State University, Ft. Collins, CO, 80523 bDepartment
of Radiation Oncology, University of Washington, Seattle, WA,
98195
The objective of this project is to explore mechanisms of
ionizing radiation-induced
delayed genomic instability. Our initial hypothesis was that
instability develops after
radiation-induced DNA damage [the signal] alters the normal
p53-dependent controls that signal for elimination of cells
with short telomeres [the target]. The overall strategy for
our research project is to create a series of isogenic human
cell lines that differ in key elements of cell cycle checkpoints,
apoptosis, or DNA repair in response to radiation induced
damage, and evaluate their sensitivity to radiation-induced
instability.
The dose and TP53 dependence for the induction of instability
at the levels of
chromosome aberrations (dicentrics) and point mutations (thymidine
kinase gene) were examined in two human lymphoblastoid cell
lines: TK6, a TP53-normal cell line, and NH32, a TP53-knockout
created from TK6. Series of clones were isolated after either
no exposure, or exposure to 5 or 10 cGy, or up to 200 cGy
of 137Cs gamma rays; genomic instability was determined approximately
35 generations later.
Studies at the chromosomal level
- Spontaneous and radiation-induced instability
was compared in TK6 and NH32 cells. Unexposed TK6 cells had
low levels of
chromosomal instability (0.002 ± 0.001 dicentrics/cell).
Exposure of TK6 cells to the
lowest dose of 5 cGy gamma rays increased chromosome instability
levels nearly 10-fold to 0.019 ± 0.008 dicentrics/cell.
There was no further increase in instability levels beyond
5 cGy. In contrast to TK6, unexposed cultures of NH32 had
much higher levels of chromosome instability of 0.041 ±
0.009, but showed little if any effect of radiation on chromosome
instability levels. These results suggest that radiation exposure
alters the normal p53-dependent cell cycle checkpoint controls
that recognize alterations in telomere structure and activate
apoptosis (Rad. Res, 159:730-736,
2003).
To further examine the role of apoptosis in suppressing chromosome
instability in
TK6 cells, spontaneous and radiation-induced instability was
quantified in a TK6
derivative that over expresses bcl-2 and is more resistant
to apoptosis induction than is TK6. Bcl-2 over expression
was associated with higher spontaneous levels of
chromosome instability as compared to TK6 cells, and gamma
ray exposure of TK6-
BCL2 had little if any effect on chromosome instability levels.
Thus the TK6-BCL2 cell line behaved just like NH32 in its
instability characteristics. We also examined telomere lengths
in TK6, NH32 and TK6-BCL2 clones. While telomere lengths were
shorter in NH32 and TK6-BCL2 clones, there was no obvious
relationship between telomere length and dicentric frequency
when all three lines were compared. Furthermore,
there was no evidence for a radiation-induced change in telomere
length in TK6 cells even though dicentric frequencies were
increased ten-fold. The results suggest that the targets for
radiation-induced instability are the genetic elements that
recognize chromosome alterations and activate apoptosis (manuscript
in preparation).
Studies at the level of gene mutation
- In the experiments with mutagenesis we have analyzed large
sets of more than 140 TK6-derived clones, and more than 130
NH32- derived clones collected after exposure to 0, 10 and
200 cGy of gamma-rays for evidence of radiation induced genetic
instability at the thymidine kinase locus. Based on the preliminary
analysis it appears likely that there is no effect of either
dose of gamma-ray exposure on genomic instability.
During the next few months we plan to collect clones of the
TK6 and NH32 cell line
treated during perturbation of the NHEJ system, using siRNA
knockdown for DNA-PKcs (DE-FG03-02ER63365)
