Abstract
Relationship between X-ray doses and mRNA levels in hematopoietic cells was examined. Induction of p21 and mdm2 mRNAs and repression of c-myc mRNA were used as indicator of before growth-arrest, and enhancement of puma and bax mRNAs as apoptosis. These mRNA levels were quantified, based on a precise method of real-time RT-PCR with improvements as follows; 1) control of the amount and quality of template RNA; 2) optimization of reverse transcription reaction, and 3) simultaneous PCR using reference plasmid as standard.
A murine macrophage cell line, RAW264.7 was used as the first model. The cells were irradiated at various doses of X-rays, and were harvested at several time-points to examine the levels of mRNA, the rate of apoptosis by TUNEL-staining and the degree of proliferation by BrdU-staining. Before the increase in the rate of apoptotic cells, puma mRNA reached to the peak level. Attenuation of the growth determined by BrdU-staining was simultaneously occurred with decrease in c-myc mRNA level, followed by increase in mRNA levels of p21 and mdm2 to the peak. These peak levels of mRNA of p21, mdm2 and puma increased x-ray dose-dependently within the range from 0.1 to 1.0 Gy.
As the second model, peripheral blood and bone marrow cells from whole-body X-irradiated C3H/He mice were used. The levels of x-ray responsive induction of mRNAs were varied by the circadian rhythm of mice. Only the increase of puma and bax mRNA levels in marrow cells were dose-dependent manner between 0.1 to 1.0 Gy with less contribution of circadian rhythm. The results show that the precise quantification of suitable mRNAs can be used to estimate radiation damage.
