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

Bloch, C.

Klein, S.B.

Zolan, M.E.

English

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RADIATION BIOLOGY 
PROTON IRRADIATION OF RADIATION-SENSITIVE 
MUTANTS OF COPRINUS CINEREUS 
S.B. Klein and C. Bloch 
Indiana University Cyclotron Facility, Bloomington, Indiana, 47408 
M.E. Zolan 
Biology Department, Indiana University, Bloomington, Indiana, 47405 
Proton radiation has important biological consequences in two specific cases: radiation 
therapy and high energy particle (particularly solar) radiation encountered by biological 
systems traveling outside of the Earth's magnetosphere. Thus there is a strong impetus 
to underst and the basic biology that has produced the empirically derived consequences 
of exposure to proton radiation. These consequences include cell death, neurological mal- 
function, accelerated aging, cataracts, genomic inst ability, mut agenesis, carcinogenesis, 
bone-mass loss, and shortened life span. Unfortunately, the classical methods of irradiat- 
ing mammalian cells in culture have limited application to the in vivo situation. We have 
selected the basidiomycete fungus (mushroom), Coprinus cinereus for a model system be- 
cause it is a genetically tractable, haploid organism that presents a synchronous meiotic 
(sexual) cycle. With this organism, it is possible to examine the nondifferentiated (stem) 
cells, differentiated cells (organs), meiotic (germ) cells, and haploid genetics (damage to 
single genes) not accessible in a mammalian tissue culture system. C. cinereus will be used 
to identify the genes involved in proton radiation sensitivity, the mechanisms responsible 
for cell death and genomic instability; the pathway overlaps between DNA repair, radia- 
tion sensitivity, and meiotic function; and the unique consequences of particle radiation 
compared to photon radiation. 
The proton radiation survival characteristics of vegetative spores (oidia) have been 
characterized for the wild-type fungus and four gamma radiation-sensitive mutants (see 
Fig. I).' These mutants each express different defective genes2 that are involved in the 
same radiation-sensitivity pathway as well as in m e i ~ s i s . ~  Small differences in radiation 
quality (protons vs. '37Cs radiation) and dose rate effects were noted. The data were 
fit by minimization of X 2  with the linear or quadratic equations traditionally used to 
describe i n  vitro radiation survival effects. The radiobiological effectiveness (RBE) of 
protons compared to 137Cs gamma radiation for cell killing was found to be between 1.27 
and 1.39. This is comparable to the values obtained for mammalian cell systems. The 
biphasic character of the survival curves for wild type C. cinereus was not maintained 
in the survival curves for the sets radl2, rad l l ,  and rad3; the survival followed a simple 
linear relationship. The standard interpret at ion of this phenomenon is that the mutant 
has lost the ability to repair specific types of DNA damage. Interestingly, the survival 
Figure 1. Survival of proton- 
irradiated vegetative spores. 
The oidia from wild type C. 
cinereus (filled circle), and four 
mutants: rad5-2 (hollow tri- 
angle), rad9- I (hollow circle), 
radll-1 (filled triangle), and 
(hollow square), were proton 
irradiated at increasing doses. 
The oidia were plated at clonal 
densities, and the number of 
colonies surviving following ir- 
radiation were scored. The 
data were fit by the equation, 
S = ~ e - ( " ~ + p ~ ~ ) ,  where S = 
survival, D = dose and a and /3 
are free parameters. 
0 10 20 30 40 50 60 70 
Dose (kRads) 
curves for rad9 maintained the biphasic character, did not indicate profound loss of repair 
function compared to wild type, and only exhibited a slightly increased sensitivity to proton 
radiation. 
Small explants of vegetative hyphae were also irradiated by 137Cs gamma radiation and 
protons. Unlike the oidia, which are held at the G2 cell cycle check point, the vegetative 
cells are cycling through mitosis, and are asynchronous. The explants were exposed to 
5 ,  10, 20, and 40 krads of 137Cs gamma radiation, or protons, and the sensitivity was 
assessed by the colony diameter at 2 days (see Fig. 2). Two mutants, radll and radl2, 
were extremely sensitive to both types of radiation, as had been indicated by the survival 
curves for irradiated oidia. In addition, the sets rad5 and rad9 exhibited sensitivity to 
both types of radiation, but unlike the oidia survival curves rad9 was drastically growth 
inhibited at 40 krads. Because the metabolism of the two vegetative cells differed, it was 
necessary to determine whether the difference between the survival-curve data and explant 
data was biological or artifactual. The clonal growth of irradiated oidia was reanalysed for 
growth rate (the number of colonies appearing each day). Figure 3 shows that although 
the mutant, rad9, eventually produced as many colonies as the wild type, the initiation of 
growth was delayed. The molecular character of the rad9 gene has been described4 and the 
functions of the rad9 protein in meiosis and radiation damage repair are currently under 
investigation. 
0 krad CONTROL 40 krads PROTON 
IRRADIATION 
Figure 2. Irradiation of mitotically-cycling vegetative explants. Control (A) panel shows 
the relative growth rates of unirradiated wild type and four mutant hyphae explants two 
days after transplantation. Proton irradiation at 40 krads (B) or at 10 krads (C) demon- 
strated the increased radiation sensitivity of the four meiotic mutant strains at four days 
post-irradiation. 
Figure 3. Growth-rate analysis of vegetative spores. Clonal survival was analyzed for 
growth rate by scoring the appearance of colonies each 24 hours. The results are presented 
as a percent of total colonies. The growth rate of the wild type (A), unirradiated (hollow 
circle) vs. irradiated at 60 krads (filled circle), and the growth rate of rad9-1 (B), unir- 
radiated (hollow circle), and irradiated at 40 (hollow triangle) and 60 (filled circle) krads 
are compared. 
100 - A 1 
80 - -  
0 1 2 3 4 5 6 7  
Days post-irradiation 
100 - 
90 
80 
70 
60 
50 - -  
40 - -  
30 - -  
- -  
B 
- -  
- -  
- -  
"/J& I 
0 
0 1 2 3 4 5 6 7  
Days post-irradiation 
1. G. Valentine, Y.J. Wallace, F.R. Turner and M.E. Zolan, Molec. Gen. Genet. 247, 
169 (1995). 
2. M.E. Zolan, J.R. Crittenden, N.K. Heyler and L.C. Seitz, Nucleic Acids Res. 20, 3993 
(1992). 
3. M.E. Zolan, N.Y. Stassen, M.A. Ramesh, B.C. Lu and G. Valentine, Can. J. Bot. 73, 
S226 (1995). 
4. L.C. Seitz, K. Tang, W.J. Cummings and M.E. Zolan, Genetics 142, 1105 (1996). 


Proton Irradiation of Radiation-Sensitive Mutants of Coprinus Cinereus

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Proton Irradiation of Radiation-Sensitive Mutants of Coprinus Cinereus

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