In a study using asynchronous exponentially growing cells, it was reported that DNA-PKcs cell lines containing site-directed mutation at the S2056/T2609 clusters as well as the C-terminal phosphatidylinositol 3-kinase (PI3K) kinase domain were radiosensitive (15
). The synchronized DNA-PKcs mutant cell lines irs-20 and V3 were very radiosensitive when irradiated in the G1
phase (nearly the same as Ku80-deficient xrs-5 cells) but displayed rapidly increasing cell survival (decreasing radiosensitivity) when they were irradiated throughout S phase, peaking at late S phase and then declining (13
). Because other DNA repair processes such as HRR also contribute to survival but operate virtually exclusively during S and G2
, it is important to study radiosensitivity with synchronous G1
-phase cells for proper interpretation of results. The present study was performed with cells incubated in isoleucine-deficient medium to synchronize the cell populations in G0
). The radiosensitivity of cells synchronized in G1
by isoleucine deprivation is similar to that of cells synchronized in G1
by allowing harvested mitotic cells to progress into G1
The experiments were carried out after all cell line designations were coded blindly as lines 1–12 and 14 without knowing the genotype of each cell line. Radiation effects on survival and chromosomal aberrations were examined in the site-directed mutant cell lines with mutations at the S2056 cluster, the T2609 cluster and the PI3K domain ( and ). shows cell killing after irradiation in G0/G1 as well as radiation-induced total chromosomal aberrations in late S/G2-phase cells among the various Chinese hamster cell lines compared to the DNA-PKcs site-directed mutant cell lines. We measured the D0 and D10 from complete survival curves for determining relative radiosensitivity as described in the last column and footnotes in . These survival curves are shown graphically in .
Gamma-Ray Sensitivity and Chromosomal Aberration Induced in DNA-PK Mutant Cell Lines
FIG. 2 Panel A: Survival curves of site-directed mutant cell lines with amino acid replacement in the S2056 and T2609 clusters. Colored areas indicate differential radiosensitivities: green, D0 = 1.0–1.4 Gy; yellow, D0 = 0.6–0.9 Gy; red, D0 = (more ...)
Both the L-2 and L-3 cell lines contain the mutated T2609 cluster, in which all six serine/threonine sites were replaced with alanine (A). When irradiated in the G0/G1 phase, these cell lines were very radiosensitive and fell in the same shaded area on the dose–survival plot as xrs-5 and V3 cells (L-7 empty vector) (). L-14 cells also containing a mutated T2609 cluster with three threonine residues were replaced with alanine (T2609A/T2638A/T2674A) and showed intermediate radiosensitivity, but they were more radiosensitive than L-6 cells, in which only a single amino acid was replaced (T2609A) ().
The single-site replacement of alanine in L-5 (S2056A) and L-6 (T2609A) mutant cell lines resulted in only mild hypersensitivity to radiation. However, L-4 (S2056A/T2609A) cells displayed synergistically increased radiosensitivity compared to cell lines with complete loss of the T2609 cluster (L-3) or complete loss of the S2056 cluster. The hyper-radiosensitivity of L-4 cells in G0
is distinctive from the mild radiosensitivity when exponentially growing cells were irradiated, whereas L-5 (S2056A) cells showed similar radiosensitivity to γ rays in G0
phase or in asynchronous conditions (18
It has been reported that the measurement of NHEJ direct end joining relative to alternative microhomology-directed end-joining activity increased dramatically in cells defective in NHEJ compared to the normal cell lines (34
). Although there was no significant difference in ionizing radiation-induced cell killing between S2056A (L-5) and S2056A/T2609A (L-4) cells when they were irradiated in asynchronous cultures, measurement of microhomology-directed end joining showed a significant increase (~80%) in S2056A/T2609A (L-4) cells as well as in V3 (L-7) cells, whereas S2056A (L-5) and T2609A (L-6) cells showed less induction (~60%) of microhomology-directed end joining (19
). The difference in microhomology-directed end joining suggests that NHEJ repair is severely compromised in L-4 (S2056A/T2609A) cells, reflecting the hyper-radiosensitivity of L-4 cells irradiated in G0
. The results also implied that the two sites (S2056 and T2609) may have some kind of interaction or synergistic effect on radiosensitivity. It is not clear why there are large differences in sensitivity for radiation-induced cell killing between L-4 and L-5/L-6 cells. Although the S2056 and T2609 clusters are 553 amino acids apart (), this does not exclude a closer location that depends on the three-dimensional structure of DNA-PKcs leading to synergistic interaction that could increase radiosensitivity in the L-4 cell line.
The kinase activity of DNA-PKcs is essential for NHEJ repair because kinase-dead DNA-PKcs mutant cell lines (L-8, -9, -10 and -11) with site-directed mutations within the conserved PI3K domain significantly lowered DNA-PKcs kinase activity and increased radiosensitivity, even though they contained different amino acid substitutions or truncation (15
). All four kinase-dead mutant cell lines were very sensitive when the cells were irradiated in G0
phase ( and ) and displayed five to ten times greater radiosensitivity in G0
phase than in asynchronous cell populations with 2 Gy (15
In addition to investigating radiosensitivity of each mutant cell lines in G0/G1, we found that aneuploidy occurred in several DNA-PKcs mutant cell lines, as shown in . The L-2 and L-3 cell lines, in which all six phosphorylation residues of the T2609 cluster were substituted to alanine, were both very radiosensitive and aneuploid. Aneuploidy was also found in L-4 (S2056A/T2609A) cells but not in the single-site mutated L-5 (S2056A) and L-6 (T2609A) cell lines. On the other hand, two L-12 cell lines (L-12-10, L-12-15) in which all five putative phosphorylation residues of the S2056 cluster were substituted to alanine showed only mild radiosensitivity in G0/G1 phase ( and ) but displayed severe aneuploidy (). All four kinase-dead mutant cell lines with different types of mutations at the PI3K domain were very radiosensitive ( and ), but only the L-9 cell line was aneuploid (). While there was no direct cause-and-effect relationship seen between aneuploidy and radiosensitivity, it cannot be ruled out that certain DNA-PKcs mutations can lead to a propensity to develop aneuploidy.
Total spontaneous chromosomal aberrations as well as chromosomal aberrations induced by 0.5 Gy γ rays in G2
were analyzed in wild-type (CHO and AA8) and NHEJ mutated cells (Ku 70/80 deficient xrs-5 and DNA-PKcs site-directed mutant cell lines). There were higher frequencies of spontaneous chromosomal aberrations in NHEJ mutated cells than in wild-type CHO and AA8 cells (). This may suggest that these mutant cell lines developed genetic instability and/or aneuploidy during the process of establishing stable mutant clones ( and ). An assay of radiation-induced G2
chromosomal aberrations was performed by irradiating exponentially growing cells followed by 4 h of Colcemid treatment starting 30 min after irradiation. Mitotic cells collected under this protocol would have been in late S/G2
phase at the time of irradiation (48
). The G2
-phase chromosomal assay has been applied to number of radiosensitivity and cancer predisposition syndromes (35
). Chinese hamster cells deficient in either NHEJ or HRR showed similar increases in radiation-induced chromosomal aberrations in late S/G2
). After 0.5 Gy of γ rays, total chromosomal aberrations in late S/G2
-phase cells were also significantly elevated for all NHEJ mutant cell lines compared with the wild-type CHO and AA8, except the L-5 and L-6 cell lines (). These two cell lines showed near normal radiosensitivities and near diploid chromosome numbers ( and ). It is notable that L-12 cells, which contain the mutated S2056 cluster, in which all five serine sites were replaced with alanine, displayed a high frequency of spontaneous and radiation-induced chromosomal aberrations, although L-12 cells showed only mild radiosensitivity in G0
phase ( and ).
The relationship between DNA-PKcs activity and DSB repair underlying the NHEJ mechanism has been widely studied. Results from the current study provide further insight into the contributions of different modifications of DNA-PKcs activity (phosphorylations and kinase activity) to radiosensitivity phenotypes in G0
-phase cells. Mice deficient in DNA-PKcs and NHEJ components have been characterized by increased sensitivity to agents causing DNA damage as well as to chromosomal instability, immunodeficiency and predisposition to thymic lymphomas (43
). Studies of clinical samples have also correlated DNA-PKcs activity with cancer risk and prognosis. A reduction of DNA-PKcs expression or DNA-PK kinase activity in peripheral blood lymphocytes (PBL) has been found in bronchial epithelial cells (a progenitor cell for lung cancer) (44
) and was associated with a risk for breast and uterine cervix cancer as well as an increased frequency of chromosomal aberrations (45
). Negative expression of DNA-PKcs has also been correlated with tumor progression and poor patient survival in gastric cancer (46
). Recently, several point mutations of DNA-PKcs have been identified in breast tumor samples; one such mutation, Thr2609Pro, occurred specifically at the T2609 phosphorylation cluster (47
). Taken together, these results suggest a tumor suppressor role of DNA-PKcs in the development of cancer.
In summary, this study focused on radiosensitivity and chromosomal instability in site-directed DNA-PKcs mutant cell lines after γ irradiation in the G0/G1 phase. Very clear differences in radiosensitivity were observed among cell lines mutated in the T2609 cluster, the S2056 cluster and the PI3K kinase domain. Additionally, an interesting synergism between defects in phosphorylation sites that occur together in both the S2056 and T2609 clusters was found to be critical for conferring maximum hypersensitivity to radiation-induced cell killing, comparable to DNA-PKcs null mutants.