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Nickoloff et al. Cancer Drug Resist 2021;4:244-63 I http://dx.doi.org/10.20517/cdr.2020.89 Page 248

tissue, thus the highest X-ray doses are near the skin at the entrance point. To concentrate X-ray doses
within tumors, beams are intensity modulated and delivered to patients from several angles, spreading
[3]
low doses to a large volume of normal tissue . Protons have a small mass and a single positive charge.
Proton interactions with tissue slow and eventually stop these particles at a defined depth (within a tumor),
[63]
termed the Bragg peak . This feature provides a clear benefit as normal tissue beyond the tumor receives
essentially no dose. Carbon ions with high mass and six positive charges are high LET radiation. Because
of their mass, carbon ions also stop at depth and eliminate exit dose, similar to protons. However, the high
mass and high charge of carbon ions produces dense ionization tracks, especially at the end of the track as
particles slow and stop [64,65] .

X-rays, protons, and carbon ions induce the same number of DSBs per unit dose (~40 DSBs/Gy). Exposures
to 1 Gy of X-rays or protons kills ~10%-20% of cells [66-68] . In contrast, the same dose of carbon ions kills
2-3-fold more cells, hence the relative biological effect (RBE) of carbon ions is ~2.5. Proton LET increases
somewhat in the distal region of the Bragg peak, and RBE correspondingly increases to perhaps as high
as 1.7 [65,69] . The high RBE of carbon ions reflects the fact that these ions efficiently induce clustered DSBs,
defined as two or more DSBs separated by < 200 bp [64,68,70] . Clustered DSBs are repaired inefficiently and are
hence more cytotoxic than isolated DSBs. Low LET X-rays and protons induce occasional clustered DSBs -
it is thought that these lesions primarily determine low LET radiation cytotoxicity, not the more prevalent
isolated DSBs [64,68,71-73] . The greater cytotoxicity (RBE) of carbon ions reflects their greater efficiency at
inducing clustered DSBs. NHEJ, the dominant DSB repair pathway, initiates with Ku70/Ku80 (Ku) binding
[51]
to DNA ends and recruitment of DNA-PKcs [Figure 2] . Ku appears to efficiently bind both large and
small DNA fragments, generated by isolated and clustered DSBs, respectively. However, short fragments
[74]
do not activate DNA-PKcs kinase , which has critical roles in NHEJ, HR, DDR signaling, and checkpoint
activation . Thus, short DNA fragments appear to be refractory to repair by NHEJ, and this may account
[75]
for both the greater cytotoxicity of clustered vs. isolated DSBs, and the shift from NHEJ toward HR in
cells exposed to high LET radiation [64,76-79] . A greater dependence on HR was also observed with protons
[80]
than X-rays , perhaps reflecting the higher proton LET in the Bragg peak. However, a more recent study
showed minimal differences when cells were treated with X-rays vs. protons, and inhibitors of NHEJ or
[81]
HR , suggesting additional factors determine repair pathway choices among cell types. That cells struggle
to repair clustered DSBs may reflect their rarity in nature and the lack of selective pressure to evolve repair
systems for this class of complex DNA lesion.

Low and high LET radiation are distinguished in two other ways. Low LET X-rays and protons induce ROS
most efficiently in well-oxygenated tissue. At low oxygen levels, the cytotoxic effects of X-rays and protons
[82]
is reduced ~3-fold, the so-called oxygen enhancement ratio (OER) . Importantly, high LET carbon ions
show far less reliance on oxygen (lower OER), owing to the greater ionization potential of these high
mass/high charge ions [82,83] . Radiosensitivity varies during the cell cycle. Low LET X-rays and protons
show highest cytotoxicity during G1 and M phases, and ~2-fold less cytotoxicity during S-phase, termed
[84]
S-phase radioresistance . Interestingly, high LET carbon ions show the opposite effect: ~2-3-fold S-phase
radiosensitivity relative to G1 cells (Kato, unpublished results). This suggests one mechanism by which
mixed high and low LET exposures might yield synergistic cell killing [85-87] .

The highly damaging effects of high LET radiation initially raised concerns about the safety of carbon ions
[88]
in radiotherapy , but serious side effects occur no more often than with X-rays or protons [89-91] . This safety
profile probably reflects the fact that high LET ions behave similarly to low LET X-rays and protons while
traveling through (normal) tissue at high speed, gaining their high LET properties only when slowing and
stopping at the end of their tracks (in tumors) [63,92] . Thus, carbon ion LET and RBE are relatively low in the
entrance region and increase dramatically in the Bragg peak, and the most damaging effects are confined to
the tumor volume [63,93,94] .