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Department of Radiological Health Sciences, Colorado State University, Fort Collins, Colorado 80523-1673 [R. O., J. S. B.]; Department of Radiation Oncology-Biology Division, University of Texas Medical Branch, Galveston, Texas 77555-0656 [R. O., K. S., Y. Y., R. L. U.]; and National Radiological Protection Board, Chilton, Didcot, Oxon OX11 0RQ, United Kingdom [A. S., R. C.]
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ABSTRACT |
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ray-induced DSBs as compared with
cells from all of the other commonly used strains and F1
hybrids of C57BL/6 and BALB/c mice. The BALB/c repair phenotype was
accompanied by a significantly reduced expression level of DNA-PKcs
protein as well as a lowered DNA-PK activity level as compared with the
other strains. In conjunction with published reports, these data
suggest that natural genetic variation in nonhomologous end joining
processes may have a significant impact on the in vivo
radiation response of mice. |
Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Differences in radiosensitivity and tumor susceptibility among various strains of mice are well recognized (5 , 6) . Among several commonly used inbred mouse strains, BALB/c has been consistently found to be unusually sensitive to the lethal effects of radiation (5 , 7 , 8) and to the development of various types of spontaneous as well as radiation-induced solid tumors (6) . Clonogenic assays of cell survival in jejunal crypt cells showed a greatly increased radiosensitivity in BALB/c mice compared with C57BL/6/BALB/c hybrids (9) . It also has been shown that the reduction in the lethal effects of radiation as a result of lowering the dose rate is substantially reduced in BALB/c as compared with C57Bl/6 mice (10) . This dose rate effect is commonly attributed to the operation of repair processes. Cellular studies on radiation-induced murine mammary cancer have demonstrated that strain-dependent differences in susceptibility result from differences in sensitivity to neoplastic initiation (11) . More recently, this laboratory has shown that mammary cells from BALB/c mice are more susceptible to radiation-induced genomic instability than those from C57BL/6 or F1 hybrids of these two strains (12 , 13) . These observations support the hypothesis that in certain genetic settings, such instability is associated with early events in radiation-induced tumorigenesis (12, 13, 14) .
Cells derived from highly radiosensitive SCID mice have a DNA DSB repair deficiency caused by a mutation in the DNA-PKcs gene (15, 16, 17) ; they also show minisatellite instability (18) and increased susceptibility to neoplastic transformation and genomic instability (19) . The defect for numerous independently isolated radiosensitive mutant cell lines has also been traced to involve DNA-PKcs (2) . However, depending on the mutation, the phenotypic defect has not always been as severe as that for SCID cells (e.g., irs 20 cells; Refs. 20 and 21 ). For this reason, we first chose to extend the phenotypic characterizations of these mouse strains showing differences in radiosensitivity to include the status of DNA DSB rejoining and the DNA-PK protein complex in kidney cells freshly isolated from BALB/c, C57BL/6, CXB6F1 (F1 hybrid of BALB/c and C57BL/6), A/J, C3H, and DBA strains. Our findings demonstrate that BALB/c carries a defect in DNA DSB rejoining that is quantitatively different from that of SCID mice. Although these observations do not, of course, establish a cause and effect relationship, the possible implications of these data for the in vivo radiation response of mice are discussed in the context of postirradiation lethality, apoptosis, and tumorigenesis.
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Materials and Methods |
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Cell Dissociation.
Female mice, 10–26 weeks of age, were anesthetized, and the kidneys
were extracted. Cell suspensions were prepared by mincing the kidneys
and incubating fragments in 199 medium containing collagenase (Type
III; 200 units/ml) at 37°C for 4–5 h with gentle agitation. The
resultant cells were extensively washed in 
-MEM medium containing
trace amount of fetal bovine serum and resuspended in -MEM medium
containing 10% fetal bovine serum, antibiotics, and fungizone for
kidney cell attachment and growth. After 2–3 days of incubation after
one medium change, cells were ready for use. Exponentially growing
cells without passage or with one passage were used.
Irradiation.
Irradiation was carried out using a 137Cs
irradiator with a dose rate of 6.70 Gy/min at 0°C. For a study of DNA
DSB rejoining kinetics over periods >2 h, cells were irradiated at
room temperature.
DNA DSB Assay.
DNA DSBs were measured by clamped homogenous electric field gel
electrophoresis (22)
. Cells were incubated with 0.01
µCi/ml [14C]thymidine
and 5 µM cold thymidine for at least 2 days before each
experiment. Cells were irradiated in plastic dishes, and DNA repair was
allowed to proceed at 37°C in air:CO2 (95:5).
At each repair point, cells were trypsinized and washed, and the
resultant cell pellet was immediately embedded in 0.5% agarose (Incert
agarose; FMC). These agarose samples were cut and immersed in an
ice-cold lysis solution containing 0.5 M EDTA, 0.01
M Tris, 2% Sarcosyl, and 0.2 mg/ml proteinase K (pH 8.0)
for 1 h, followed by an overnight incubation at 50°C. For the
initial (0-h repair) point, cells were embedded in agarose and
irradiated in ice-cold medium, followed by an immediate lysis at 0°C
for 1 h and 50°C overnight. After the overnight lysis, samples
were washed for 1 h in 0.1 M EDTA, 0.01 M
Tris at pH 8.0 and treated with 0.1 mg/ml RNase A for 1 h at
37°C.
Electrophoresis was carried out in 0.5 x TBE buffer (45 mM Tris, 45 mM boric acid, and 1.5 mM EDTA, pH 8.2) in a clamped homogenous electric field gel box (Bio-Rad) in 0.8% agarose gel (Bio-Rad) at 14°C. The applied voltage was 200 V with 60 s pulse time for the first 9 h, followed by a 120-s pulse time for the last 15 h. After electrophoresis, gels were stained and cut to separate the plug from the lane for each sample. The 14C activity of each piece was measured in a scintillation counter, and the FAR was calculated as the dpm of a lane divided by the total dpm (lane + plug) per sample. The FAR value for nonirradiated control cells was 1–3%.
Western Blotting.
For analysis of Ku protein, cells were lysed in buffer containing 25
mM Tris (pH 7.4), 50 mM NaCl, 0.5% sodium
deoxycholate, 2% NP40, 0.2% SDS, 1 mM
phenylmethylsulfonyl fluoride with 50 µg/ml aprotinin and 50
µM leupeptin freshly added. Electrophoresis was performed
on 10 µg of protein per sample on 10% SDS-polyacrylamide gels. For
DNA-PKcs, cells were lysed in buffer containing 50 mM Tris,
150 mM NaCl, 2 mM EDTA, 2 mM EGTA,
25 mM NaF, and 25 mM ß-glycerolphosphate (pH
7.5), 0.2% Triton X-100, 0.3% NP40, 0.1 mM sodium
vanadate, 0.1 mM phenylmethylsulfonyl fluoride, 5 µg/ml
leupeptin and 5 µg/ml aprotinin freshly added
(22
, 23)
. Forty µg of protein were applied per sample
for 6% SDS-PAGE.
The separated gels were transferred to nitrocellulose membranes for 24 h at 4°C. The membrane was washed with 1x TBST [25 mM Tris (pH 8.0), 125 mM NaCl, and 0.025% Tween 20] and incubated in blocking buffer containing 25 mM Tris (pH 8.0), 125 mM NaCl, 0.025% Tween 20, and 5% dry milk for 1 h at room temperature. The primary antibodies used were Ku 70 and Ku 80 polyclonal goat antimouse antibodies (Santa Cruz Biotechnology) for Ku (1:200 dilution) and mouse monoclonal DNA-PK Ab-4 for DNA-PKcs (1:500 dilution; Lab Vision, Fremont, CA) and applied for 1 h at room temperature. The membranes were washed and incubated with secondary antibodies labeled with horseradish peroxidase, diluted (1:2000) in blocking solution for 1 h. The secondary antibodies used were donkey antigoat IgG for Ku (Santa Cruz Biotechnology) and antimouse F(ab')2 for DNA-PKcs (Amersham). Subsequently, the membranes were washed and incubated in ECL Western blotting detection reagent (Amersham RPN 2106) for 1 min. The blot was exposed to X-ray film for 5 min. All of the membranes used for the blot were stained with Ponceau S solution (Sigma) to ascertain that an equal amount of protein was loaded to each lane.
In Vitro Protein Kinase Assay.
DNA-PK kinase activity was measured using a "pull-down" assay
(24)
with modification. Briefly, whole-cell extracts were
prepared from primary mouse cells as described (25)
. One
hundred µg of protein lysate were mixed with 40 µl of 1:1 slurry of
DNA-cellulose and incubated on ice for 15 min, followed by a brief
centrifugation. The supernatant was re-extracted with 20 µl of
DNA-cellulose. The pellets containing DNA-cellulose bound DNA-PKcs were
combined, washed, and resuspended in distilled water. The samples were
then incubated with biotinylated peptide substrate supplied in the
SignaTECT DNA-PK kinase assay kit (Promega) in the presence of
[-32P]ATP for 5 min at 30°C. A fraction of
reactant was spotted onto the SAM2 membrane
(supplied in the kit). The membrane was washed, air-dried according to
the manufacturer’s instructions, and then subjected to liquid
scintillation counting. The enzymatic activity of DNA-PK was calculated
and expressed as in pmol ATM
min-1·mg-1 of protein.
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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. The percentages of remaining DSB damage (normalized FAR values) are
shown as a function of postirradiation times. In these experiments,
cells were incubated at 37°C after exposure to radiation (50 Gy),
and at each time point, samples were prepared and lysed immediately. A
significant difference in repair kinetics was observed in cells from
SCID and BALB/c strains. Cells from C57BL/6, A/J, C3H, DBA, and
F1 cells rejoined DNA DSBs very efficiently. As
expected, cells from SCID mice showed severe deficiency of DSB
rejoining (26)
, whereas BALB/c cells gave an intermediate
response.
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Data on Ku 70, Ku 80, and DNA-PKcs protein expression obtained by
Western blotting in extracts from BALB/c and C57BL/6 mice are shown in
Fig. 2A
. The expression levels of Ku proteins are similar in all of
the nonirradiated and irradiated BALB/c and C57BL/6 samples, and the
expression does not seem to be affected significantly by radiation. By
contrast, there is a large difference in the expression level of
DNA-PKcs between BALB/c and C57BL/6 cells. Greatly reduced expression
of the original size DNA-PKcs (Mr
460,000) as well as an excess of degraded cross-reacting protein (a
band seen around Mr 202,000) has been
observed in all of the BALB/c samples. Radiation does not seem to have
a measurable effect on the expression of this cross-reacting material
1 h after irradiation. Cleavage of DNA-PKcs molecules into
Mr 
240,000 fragments has been
reported with exposure to various cytotoxic agents in Burkitt lymphoma
cells (23)
, and the possibility of a novel protein of this
size has also been suggested (16)
. Fig. 2B
is a
Western blot for DNA-PKcs using cell extracts from all of the strains
used in the DNA DSB repair experiment (Fig. 1
). As expected, no
DNA-PKcs expression was observed in SCID, and BALB/c showed a weak
expression, whereas all of the other strains showed similar substantial
DNA-PKcs levels with F1 being slightly lower than
C57BL/6 and the other four strains. This result is consistent with that
of DNA DSB rejoining kinetics shown in Fig. 1
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summarizes the results of the DNA-PK activity assay for all of the
mouse strains and indicates a substantially lower activity level for
BALB/c and the lowest level for SCID mice, as compared with all of the
other strains. Although the contribution of kinase activity level to
DNA DSB repair is not yet established, the functional assay data of
Fig. 3
are consistent with those on protein expression (Fig. 2
) and DNA
DSB rejoining results (Fig. 1
).
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Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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The degree of severity of the DNA DSB repair defect in highly
radiosensitive SCID mice is substantially greater than that of BALB/c
mice (Fig. 1
), implying that BALB/c radiosensitivity is likely to be
less pronounced. Importantly, however, the in vivo
radiosensitivity of BALB/c may be distinguished from that of other
inbred mouse strains. The interstrain whole body X-irradiation studies
of Grahn and Hamilton (7)
, Sandler and Gowen
(8)
, and Roederick (5)
spanning almost 30
strains consistently showed BALB/c to be maximally susceptible to the
lethal effects of radiation, largely a consequence of cell killing in
critical organs. In these studies, C57BL/6 mice fell into the
radioresistant category (e.g., Fig. 1
in Ref.
7
). Further, Kallman (10)
showed that the
loss of effectiveness commonly observed with a reduction in dose rate
and generally attributed to repair processes is greatly reduced for the
lethal effect of radiation in BALB/c as compared with C57BL/6 mice.
Regarding radiosensitivity at the cellular level, Hanson et
al. (9)
showed a large difference in the
dose-survival response of jejunal crypt cells by clonogenic survival
measurements for BALB/c versus C57BL/6/BALB/c hybrid mice.
The former were more radiosensitive by nearly a factor of two
(9)
.
Almost all in vivo and cellular measures of radiosensitivity will be quantitative genetic traits influenced by the specific distribution of variant germ-line sensitivity/resistance alleles between different mouse strains. On this basis, the in vivo data on postirradiation lethal effects might be explained if BALB/c were to carry a major sensitivity allele with respect to cellular radiation response that distinguishes it from most other commonly studied mice. Some support for this contention is available from the genetic investigations of Mori et al. (30) on mouse loci that determine the in vivo apoptotic response of thymocytes to X-rays. In these studies, BALB/c was shown to carry a recessive chromosome 16-encoded variant gene (Rapop1) that represents a major determinant of susceptibility to apoptotic response. The fact that Rapop1 was mapped by Mori et al. (30) to the same mouse chromosome 16 genomic segment now assigned to the DNA-PKcs (Prkdc) gene provides further circumstantial evidence to suggest a direct genetic association between the recessive DNA DSB repair/DNA-PK protein expression phenotype of BALB/c and in vivo radiosensitivity.
The extent of the data available on interstrain variation in radiation tumorigenesis in mice does not allow specific genetic correlations to be suggested, but nevertheless, the quantitative tumorigenesis data of Storer et al. (6) on C57BL/6, RFM, C3H, and BALB/c mice are not inconsistent with the view that a tumor susceptibility allele is carried by BALB/c.
Relating DNA DSB repair deficiency and DNA-PKcs expression in mice to patterns of tumorigenesis is not straightforward. SCID mice are characterized by excess lymphomas rather than solid cancers (31) , as are DSB repair-deficient Ku 70-/- knock-outs (32) . Whether this lymphoma susceptibility reflects the severity of the repair defect and poor cell survival or, alternatively, the influence of genetic background remains to be resolved. In the case of BALB/c, which has an unremarkable lymphoma incidence (6) , the partial defect in DSB repair may, in principle, target different organs.
The data presented here provide initial evidence that a partial BALB/c
defect in DNA-PKcs protein expression may underlie the DNA DSB repair
phenotype. Instability of DNA-PKcs suggested by the data of Fig. 2
,
leading to reduced kinase activity (Fig. 3
), would be expected to lead
to a reduction in the efficiency of DNA DSB repair, particularly in
cells where DNA-PKcs tended to be expressed at low levels
(33)
. However, whether these cellular phenotypes and their
associations with in vivo radiation response are a
reflection of specific variation at the mouse Prkdc locus
will demand genetic mapping of controlling loci and sequence analysis
of DNA-PKcs in appropriate strains.
In conclusion, the studies reported here show that natural genetic variation between inbred mice can provide the means to explore the relationships between the expression of specific proteins, DNA repair deficiency, genomic instability, and in vivo radiation response.
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Acknowledgments |
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FOOTNOTES |
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1 This work was supported by Grants CA71438,
CA43322, and CA 73729/CA 73926 (jointly funded by the National Cancer
Institute and National Aeronautics and Space Administration), a
grant from the College of Veterinary Medicine and Biomedical Sciences,
Colorado State University, and Grant FIGH-CT1999-00035 (to R. C.) from
the European Union. 
2 To whom requests for reprints should be
addressed, at Department of Radiological Health Sciences, Colorado
State University, Fort Collins, CO 80523-1673. Phone: (970) 491-7024;
Fax: (970) 491-7742; E-mail: rokayasu{at}cvmbs.colostate.edu
3 The abbreviations used are: DSB, double strand
break; NHEJ, nonhomologous end joining; SCID, severe combined
immunodeficient; FAR, fraction of activity released.
Received 1/26/00. Accepted 6/29/00.
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