This research was sponsored by the National Science Foundation Grant NSF PHY-931478

Bloch, C.

Klein, S.B.

Ramesh, M.A.

Zolan, M.E.

English

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PRELIMINARY ANALYSIS OF PROTON RADIATION DAMAGE RESPONSE IN 
DEFECTIVE, GAMMA-RADIATION-SENSITIVE CELLS 
S.B. Klein 
Institute for Ce llular and Molecular Biology, 
Indiana University, Bloomington, Indiana, 4 74 05 
@. Bloch 
Indiana University Cyclotron Facility, Bloomington, Indiana, 4 74 08 
M.A. Ramesh and M.E. Zolan 
Biology Department, Indiana University, Bloomington, Indiana 4 74 05 
Proton radiation emanating from solar events poses a serious problem for prolonged 
earth-orbiting space projects. A comprehensive understanding of the differences between 
cellular responses to gamma radiation and proton radiation, and possible differences in 
the mechanisms of repair, is required to develop radiation protection or resiliency. We 
have isolated and characterized 37 mutants of the mushroom Coprinus cinereus that are 
sensitive to gamma radiation. These mutants define at least 10 complementation groups 
for survival of gamma damage. Four of these genes are also required for normal progression 
through meiosis. It is the objective of the current investigation to determine the extent of 
overlap of the subsets of mutants exhibiting defects in proton sensitivity, gamma sensitivity, 
and meiotic defect. 
To establish the validity of mutant variation, the proton sensitivity characteristics of 
the congenic wild type C. cinereus (Java 6 strain) were examined. Mitotically derived, 
haploid spores termed oidia, were obtained by mechanical scraping of hyphae. The cells 
were counted and spread on growth medium at a cell number previously determined to 
produce sufficient viable colonies for scoring following irradiation. Identical samples were 
plated and irradiated by either of two methods within 24 hours. This time frame had been 
determined previously to be well within accept able viability limits.' The number of viable 
colonies arising from the surviving oidia were scored for 7 days following irradiation and 
the results were plotted as per cent survival of the un-irradiated control sample vs. dose 
(Fig. 1 )  At 1.67 kradstmin, little difference was noted between the survival of gamma 
irradiated oidia and proton irradiated oidia when they were exposed to equal total doses of 
radiation. Small differences in the shapes of the shoulders for the two curves may indicate 
subtle differences in repair processes. These experiments will be repeated at lower dose 
rates that will emphasize these differences, although maintenance of viability will be more 
difficult. 
Because dose rate is of major importance to a number of cellular functions that de- 
termine the survival of irradiated cells, the response of proton-irradiated oidia exposed at 
a rate of 1.67 krads/min was compared to oidia irradiated at 0.17 kradslmin (see Fig. 2). 
Once radiation starts to have an effect, the survival percentage declines exponentially with 
slope Do (the increased dose required to produce a 37% decrease in survival). The amount 
of radiation required (Do) was drastically increased and the quasi-threshold dose (D,, the 
place where the exponential crosses 100%) was reduced for the lower dose rate. This sur- 
Figure 1. Dose response curves for 100 
gamma- and proton-irradiated - 
E C. cinereus. Duplicate samples of $ 
r, 10 oidia were consecutively irradiated 0) 
a 
under identical conditions by either 
I( gamma-rays or protons at a dose rate Q 
* 
.L( 
1 
of 1.67 kradslmin. The linear por- * 
& tions of the curves on this semi-log a 
C/3 plot have been extended to emphasize 0.1 
the differences in Do and D,. -- gamma 
-A- proton  
0.01 1 I I I 
0 20 40 60 80 
Dose, krads 
4 Figure 2. Dose rate dependence 
exhibited in wild type C. cinereus. 2 
0) Identical samples of oidia were pro- rr 
CI ton irradiated at 1.67 and - 
a 0.167 kradslmin. The linear portion 
-2 
* of the curves has been extended to L 
1 
emphasize differences. Background w 
neutron radiation effects were evalu- 
ated at the higher dose rate. 
.- A- 1.67 krad/min 
----+---- 
0.01 Neutron background 1 I I 
Proton dose, krads' 
viva1 dependence upon proton delivery rate has also been demonstrated in a number of 
mammalian systems. The influence of the relatively high neutron background dose was 
also examined in this experiment by placing samples in proximity to the proton beam, but 
distant from its direct influence. The experiment indicated no detectable difference be- 
tween neutron-irradiated and control samples. Future work will examine the relationship 
between gamma- and proton-radiation dose rate as well as the effect on mutant damage 
repair. 
Figure 3. Screen for mutants differentially sensitive to gamma and proton radiation. 
Small chunks of mutant haphae were explanted and irradiated with low dose rate, high LET 
protons or gamma radiation from a 60Co source. The un-irradiated mutants (A) grew at 
the same rate as the congenic wild type, Rad+, strain (photographed at +3 days). Mutants 
exposed to 40 krads of either gamma (B) or proton (C) radiation grew considerably slower 
than the unirradiated samples at +5 days). The Rad+ control is located 
in the upper right corner of each photograph . 
Of the 20,000 mut angenized cultures screened for gamma radiation sensitivity, 37 re- 
tained sensitivity through at least one outcrossing to a wild type Of these, 16 
mutants were backcrossed to the same wild type strain five additional times. Backcrossing 
reduced the extraneous gene mutations, increasing the probability that a single mutated 
gene was responsible for gamma radiation sensitivity. These 16 mutants were individu- 
ally cultured; a small square "chunk" was excised, and they were transplanted to fresh 
growth medium. The chunks were irradiated at 20, 40, and 60 krads with either gamma 
radiation or protons. Three mutants indicated differential sensitivity to the two types 
of radiation. This difference in response indicated specific mutations that contributed to 
gamma-radiation sensitivity but less so to proton sensitivity. This implied that the genes 
responsible for the mutations were more important for gamma-radiation repair. The most 
dramatic difference was demonstrated by the mutant rad9-1 (Fig. 3). The rad9 gene has 
been isolated and sequenced, and found to encode a new metabolism protein whose func- 
tion is likely conserved in ev~lut ion .~  Because the initial screen was performed for gamma 
radiation sensitivity, genes specific for proton radiation repair would not be disclosed in 
these preliminary experiments. Primary screens for proton sensitivity are planned for the 
future. 
1. G. Valentine, Y.J. Wallace, F.R. Turner, M.E. Zolan, Molec. Gen. Genet. in press 
(1995). 
2. M.E. Zolan, C.J. Tremel, P.J. Pukkila, Genetics 120, 379 (1988). 
3. L. Seitz, K. Tang, W.J. Cummings, M.E. Zolan, in preparation. 


Preliminary Analysis of Proton Radiation Damage Response in Defective Gamma-Radiation-Sensitive Cells

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Preliminary Analysis of Proton Radiation Damage Response in Defective Gamma-Radiation-Sensitive Cells

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