|
https://www.abbs.info ISSN 0582-9879 |
Restoration
of T Cell-specific V(D)J Recombination in DNA-PKcs-/- Mice by
Ionizing Radiation: The Effects on Survival, Development, and Tumorigenesis
LI
Xiao-Ling*, SHEN Shou-Rong, WANG Sa1, OUYANG Hong-Hai1,
LI Gloria C1
(
Cancer Research Institute,
Xiang Ya School of Medicine,
Central South University,
Changsha 410078,
China;
1Memorial
Sloan-kettering Cancer Center, New
York, NY 10021, USA )
Abstract
DNA-dependent protein kinase (DNA-PK) is a DNA-activated nuclear serine/threonine
protein kinase. DNA-PK consists of
a heterodimeric Ku subunit
(composed of a 70 and 86 kD subunit) which binds DNA ends and targets
the catalytic subunit DNA-PKcs to DNA strand breaks. DNA-PK plays a major role in the repair of double-strand
breaks (DSB) induced in DNA after exposure to ionizing radiation. To better understand the nature of DNA
repair defect associated with DNA-PKcs deficiency, we have established DNA-PKcs-/- mouse embryo
fibroblast cell lines and DNA-PKcs-/- null mice, and investigated the response of these
mutant cells and mice to DNA damage.
DNA-PKcs-/- cells are hypersensitive to g-irradiation, as evidenced by their low survival as assayed by colony
formation efficiencies. Consistent
with the radiation hypersensitive phenotype of the cell lines, DNA-PKcs-/- mice also
display an extreme radiosensitivity,
characterized by enhanced mortality after g-irradiation.
Treatment of newborn DNA-PKcs-/- mice with sublethal doses of
ionizing radiation restores T cell receptor (TCR)b
recombination and T cell maturation. However,
radiation does not restore B cell development. All these mice eventually developed thymic lymphoma. These observations suggest an
interrelationship between DSB repair,
V(D)J recombination and lymphomagenesis, and provide an in vivo model to elucidate the
critical pathways between the regulation of DNA DSB repair, V(D)J recombination, and the molecular mechanism of lymphoid
neoplasia.
Key
words DNA-PKcs; DNA DSB repair; V(D)J
recombination; apoptosis; tumorigenesis
Among
the various forms of DNA damage,
DNA double-strand breaks (DSB) are potentially the most lethal lesions
produced by ionizing radiation.
These DSBs also arise endogenously as intermediates of DNA rearrangement
during certain biological processes,
such as V(D)J recombination in early T- and B-lymphocyte
development, class switch
recombination occuring exclusively in mature B cells, and meiosis for reproduction. If unrepaired,
DSB are likely to result in lethality; if misrepaired or rejoined incorrectly, they can lead to mutations. All organisms, from yeast to human, have proficient machinery for DNA DSB
repair. In yeast, homologous recombination represents the
major mechanism for DSB repair,
whereas non-homologous end-joining plays a more important role in DSB
repair in mammalian cells. Many of
the yeast genes involved in homologous recombination were identified more than
a decade ago. However, genes involved in non-homologous
end-joining have been discovered only recently. The DNA-dependent protein kinase (DNA-PK) was among the
first few genes identified. Four
complementation groups of mammalian cell mutants have been described that are
specifically defective in DSB repair[1, 2]. Three of these groups were shown to
correspond to components of DNA-PK,
whereas the fourth mutant was deficient in the gene encoding XRCC4. Although, the biochemical properties to the XRCC4 gene are less clear[3], much more is known about the function
of DNA-PK. DNA-PK is a serine/threonine
kinase that consists of a 465 kD catalytic subunit (DNA-PKcs), and a DNA-targeting regulatory
component Ku, which itself is a
heterodimer of 70 kD and 80 kD polypeptides (Ku70 and Ku80). When assembled on a suitable DNA
molecule in vitro, DNA-PK
becomes activated and in turn phosphorylates many transcription factors
including Sp1, Oct1, c-fos, c-jun, the
tumor suppressor p53[4],
and the 34 kD subunit of replication protein A[5], Recent genetic and biochemical studies
strongly support the importance of DNA-PK in DNA DSB repair, in V(D)J recom-bination and in the
regulation of transcription[6].
To
better understand the nature of DNA repair defect associated with DNA-PKcs
deficiency, we have established
DNA-PKcs-/- mouse embryo fibroblast cell lines and DNA-PKcs-/-
null mice, and investigated the
response of these mutant cells and mice to DNA damage. DNA-PKcs-/- cells are
hypersensitive to g-irradiation, as evidenced by their low survival
shown by colony formation efficiencies.
Consistent with the radiation hypersensitive phenotype of the cell
lines, DNA-PKcs-/- mice
also display an extreme radiosensitivity,
characterized by enhanced mortality after g-irradiation. Treatment of newborn DNA-PKcs-/-
mice with sublethal doses of ionizing radiation restores T cell receptor (TCR)b recombination and T cell
maturation. However, radiation does not restore B cell
development. All these mice
eventually developed thymic lymphoma.
These observations suggest an interrelationship between DSB repair, V(D)J recombination and
lymphomagenesis, and provide an in
vivo model to elucidate the critical pathways between the regulation of DNA
DSB repair, V(D)J
recombination, and the molecular
mechanism of lymphoid neoplasia.
1Materials and Methods
1.1
Generation and screening of DNA-PKcs-/- mice
DNA-PKcs-/-
mice (C57BL/6-129/sv) were generated from DNA-PKcs+/- intercrosses
and screened using Southern blot or PCR analysis as described[7]. The breeding and maintenance were under
defined flora conditions.
1.2
Cell culture
Mouse
embryonic fibroblast (MEF) cells were established from 13 1/2-day-old DNA-PKcs+/-, DNA-PKcs-/- and wild type
mouse embryos following standard protocol and immortalized by SV40 transfection
via calcium phosphate precipitation.
All cells were maintained in DMEM supplemented with 10% fetal bovine
serum and antibiotic.
1.3
Irradiation
To
determine radiosensitivity in cell cultures, exponentially growing,
monolayers of embryonic fibroblast cells were exposed to graded dose of g-irradiation
(dose rate, 250 cGy/min), cells were then trypsinized, counted, serially diluted and plated on to 60 mm petri dishes, and incubated at 37 ℃
for colony formation. Ten to
twelve days later, cells were
fixed and stained, and colonies
containing greater than 50 cells were counted for clonogenic survival. Cell survival was always normalized to
the cloning efficiency of untreated controls. All experiments were performed at least three times and
yielded consistent results. To
determine the effect of nonlethal low dose of ionizing radiation on T- and
B-lymphocyte development, newborn
mice from litters of DNA-PKcs+/- intercrosses (DNA-PKcs+/+
, DNA-PKcs+/- and
DNA-PKcs-/-) were irradiated within 72 h after birth. Unirradiated sex-matched littermate
controls were used for comparison.
At different times after irradiation, animals were sacrificed, cells from thymus,
bone marrow and spleen were obtained for cellularity, flow cytometry and Southern blot
analyses. For in vivo
survival experiments, 2―3
month-old DNA-PKcs+/+ and DNA-PKcs-/- animals ( n=4―6
mice for each group) were irradiated at 300, 350, 400，
600 cGy, respectively. Mice survivals were monitored daily for
18 days.
1.4
Assay of apoptotic fraction
At
various time points after heating,
the medium containing the detached cells was collected, and the detached cells were recovered
by centrifugation. The attached
cells were trypsinized from the flasks and washed with phosphate-buffered
saline (PBS). The detached and the
attached cells were then pooled and used for the assay of the apoptotic
fraction. The pooled cells were
fixed with a 3∶1
mixture of methanol and acetic acid,
spread on slides, and
stained with 10 mg/L acridine orange in PBS[8, 9]. The morphology of cell nuclei was
viewed by phase-contrast and fluorescence microscopy. Apoptotic cells were recognized by their condensed or
fragmented chromatin[8, 9].
At least 200 cells were counted to calculate the fraction of apoptotic
cells. Each experiment was
performed at least three times to assure reproducible results. Data presented are from representative
individual experiments; analysis
of the pooled data yields the same conclusions.
1.5
Flow cytometry analysis of lymphocyte development
Thymus, bone marrow, and spleen were harvested at various times (e.g., 2, 4, 6―8
weeks) after irradiation (100 cGy) and single cell suspensions were
prepared. Thymocytes were stained
with a combination of phycoerythrin (PE)-conjugated anti-CD4, and fluorescent isothiocyanate
(FITC)-conjugated anti-CD8,
PE-conjugated anti-T cell receptor a b
chain (TCRab)
or FITC-conjugated anti-IL-2 receptor,
respectively. Cells from
bone marrow were stained with combination of FITC-conjugated anti-CD43 and
PE-conjugated anti-B220, while
cells from spleen were stained with combination of FITC-conjugated anti-IgM and
PE-conjugated anti-B220. In some
experiments, lymph nodes were
isolated from 8-week-old mice and lymphocytes were stained for CD4, CD8 and TCRab
markers. The intracellular
staining was done as described[10]. Briefly,
thymocytes were obtained from 1-week-old mice after irradiation (100
cGy) and fixed in 40 g/L paraformaldehyde for 20 min at 4 ℃, followed by pereabilization with 21 g/L
saponin. Cells were stained with
FITC-conjugated anti-CD4 versus PE-conjugated anti-TCRab
and analyzed by flow cytometry.
Thymocytes from age-matched wild type and DNA-PKcs-/- mice
were stained for comparison. All
antibodies were purchased from PharMingen.
1.6
Southern blot analysis of TCRαβ
rearrangement
Genomic
DNA was prepared from thymuses of irradiated newborn (IRNB) mice at the
indicated ages or from thymic lymphomas developed spontaneously in IRNB mice
and digested with PvuII overnight at 37 ℃. TCRab
rearrangement was analyzed by hybridizing with 5′-triphos phate-labeled TCR Cb
cDNA probe (kindly provided by Jayne S.
Danska, University of
Toronto, Ontario, Canada).
1.7
Histopathological analysis
Tissues
were fixed in 10% buffered formalin,
embedded in a paraffin blocks,
sectioned at 4―5
microns, and stained with
hematoxylin and eosin. Sections
were photographed at 100 ×
magnification as indicated.
2
Results
2.1
Radiation sensitivity of DNA-PKcs-/- mice and DNA-PKcs-/-
fibroblasts
The
radiation sensitivity of adult (2―3
months old) wild-type, DNA-PKcs+/-and
DNA-PKcs-/- mice was compared [Fig.1(A), left panel]. At
200 cGy, all wild-type and
DNA-PKcs-/- mice survived.
However, DNA-PKcs-/-
mice lost most of their hair within a month after irradiation. After an additional 1―2
months, their hair grew back, but it lacked the original agouti-color
pigmentation and appeared grey [Fig.1(A),
right panel]. At 400
cGy, all wild-type and
heterozygote mice survived this treatment. In contrast, all
irradiated DNA-PKcs-/- mice died in 16 days after 400 cGy g-ray
irradiation. At a higher dose of
600 cGy, all DNA-PKcs-/-
mice died in 9 days post-irradiation;
on the other hand, all
wild-type and heterozygote mice survived even after this treatment.
Survival
curves of the immortalized wild-type,
DNA-PKcs+/- and DNA-PKcs-/- cell lines were
detemined by measuring the colony-forming ability of irradiated cell
populations. Cells were plated
post-irradiation into 60 mm petri dishes and incubated at 37 ℃
for 10―12
days to allow for colony formation.
The experiment was repeated at least 3 times and shown by one
representative result. It is
clearly demonstrated in [Fig.1(B)] that DNA-PKcs-/- MEFs are more
sensitive to ionizing radiation than the wild-type or DNA-PKcs+/-
cells. DNA-PKcs-/-
cells showed significantly decreased ability to form colonies after ionizing
radiation as compared with DNA-PKcs+/- and wild-type controls. For example, after 400 cGy,
the survival is 20% for wild-type fibroblasts, and only 5% for DNA-PKcs-/- cells. The relative sensitivity between Ku80-/-
and DNA-PKcs-/- cells was also compared [Fig.1(B)]. Our data showed that Ku80-/-
MEFs are much more sensitive to ionizing radiation than the DNA-PKcs-/-
MEFs.
Fig.1 Radiation sensitivity of DNA-PKcs-/-
mice and DNA-PKcs-/- fibroblasts
(A) Survival of wild type and DNA-PKcs-/-
mice subjected to various doses of g-irradiation. Adult (2.5 months old) DNA-PKcs-/-
mice were exposed to 300 cGy (□), 350 cGy (●), 400 cGy(■)
or 600 cGy(▲)
and then monitored daily for survival. Note that irradiation resulted in the appearance of
gray hair in DNA-PKcs-/- mice 2 months after the treatment. In contrast, the color coat of wild-type mice was not affected
[Fig.1(A), right panel]. (B) Wild
type(○),
DNA-PKcs+/- (■),
DNA-PKcs-/- (▲),
and Ku80-/- (●)
embryo fibroblast cells,
immortalized by SV40 transfection,
were exposed to graded doses of g-irradiation.
2.2
Effect of DNA-PKcs on radiation-induced apoptosis
Monolayers
of DNA-PKcs-/- and DNA-PKcs+/+ cells were exposed to
graded doses of g-ray
irradiation, and at different
times after irradiation, the
medium containing the detached cells was collected and recovered by
centrifugation. The attached cells
were typsinized from the dishes and rinsed with phosphate-buffered saline
(PBS). The detached and the
attached cells were then pooled and used for the assay of the apoptotic
fraction. The time courses of the
development of apoptosis for the different cell lines irradiated with 10 Gy are
shown in Fig.2(left). The fraction
of apoptotic cells increased rapidly with time after irradiation. The time dependence of apoptosis in
these cells was similar.
Interestingly, there is no
significant difference neither in the kinetics of induction nor in the
magnitude of radiation-induced apoptosis between DNA-PKcs+/+ and
DNA-PKcs-/- MEFs. In
contrast, Ku80-/- MEFs
are much more susceptible to radiation-induced apoptosis than the wild type and
DNA-PKcs-/- cells.
Fig.2(right) shows the dose response curves of radiation-induced
apoptosis for DNA-PKcs-/- cells and wild type controls determined 48
h after irradiaiton. For
comparison, the dose response
curves of Ku80-/- and p53-/- cells were also shown. Our data clearly demonstrated that
cells deficient in DNA-PKcs apoptose after g-irradiation
in a time- and dose-dependent manner
silmilar to the wild-type control cells. On the other hand,
the Ku80-/- cells are more susceptible, while p53-/- cells are more
resistant to radiation-induced apoptosis.
Fig.2 The time courses of the development of apoptosis for the
different cell lines irradiated with 10 Gy(left); The dose response curves of radiation-induced
apoptosis for DNA-PKcs-/- cells and wild-type controls determined 48
h after irradiaiton (right)
2.3
Effect of sublethal doses of ionizing radiation on T cell and B cell
development
Recent studies have shown that ionizing
radiation can effect the
differentiation of double negative (DN) thymocytes to double positive (DP)
thymocytes and lead to significant increase in thymus cellularity in both
severe combined immune deficiency (SCID) and recombinase activation gene (RAG)-/-
mice[10―13]. Here, we investigated the kinetics and mechanisms of radiation-induced
DP thymocyte development in DNA-PKcs-/- mice. Animals were given a single non-lethal
dose of radiation (100 cGy) within 72 h of birth. Fig.3 shows that radiation induced restoration of DP
thymocyte development in DNA-PKcs-/- mice. Two weeks after irradiation, irradiated newborn DNA-PKcs-/- (IRNB-DNA-PKcs-/-)
mice had an average of 59% (ranged from 22% to 85%) DP thymocytes
[Fig.3(A)]. Immature transitional
CD4–CD8+ were seen at 8 weeks (mean: 5%, range 2% to 8%).
In addition, thymus
cellularity in 2-, 4-, and 8-week-old IRNB-DNA-PKcs-/- mice was
about 2, 10, 32 times greater than that in
age-matched, non-irradiated
DNA-PKcs-/- controls.
In wild-type mice, the DN
to DP transition is accompanied by loss of IL-2Ra
and a reduction in cell size reflecting the exit of DP thymocytes from the cell
cycle[14]. These
phenotypic transitions were also observed in IRNB-DNA-PKcs-/- mice
by 1―2 weeks, concomittently with the appearance of DP cells
[Fig.3(A)]. On the other
hand, unlike wild-type DP
thymocytes, radiation-induced DP
cells in DNA-PKcs-/- mice did not express significantly detectable
surface TCRb[Fig.3(A)]. Several weeks after the
radiation-induced appearance of DP cells in DNA-PKcs-/- mice, mature CD4+ or CD8+
single positive cells were consistantly found in the thymus and lymph nodes
[Fig.3(A) and Fig.3(B)],
suggesting that some rescued DP thymocytes can undergo further
maturation. In contrast to DP
thymocytes, matured thymocytes and
peripheral T cells in IRNB-DNA-PKcs-/- mice always expressed a
surface TCRβ
[Fig.3(B)], but at slightly lower
density than that of peripheral T cells from wild-type controls (data not
shown).
In
contrast to the rescued development of DP-thymocytes in DNA-PKcs-/-
mice, g-irradiation
failed to simultaneously restore B cell development. B cells from DNA-PKcs-/- bone marrow remained at
the immature CD43+ B220+ pro-B cell stage [Fig.3(C), top] and spleenocytes lacked surface
expression of IgM [Fig.3(C),
bottom]. In addition, the frequency of early B cell
progenitors (B220+ sIgM–) in bone marrow and spleen was
similar in the irradiated and control non-irradiated DNA-PKcs-/-
animals [Fig.3(C)]. Thus, irradiation did not rescue IgM rearrangement or
promote the transition of CD43+
pro-B cells to CD43–
pre-B cells in DNA-PKcs-/- mice [Fig.3(C)]. Together, the data confirm that low dose irradiation fails to restore
B cell development in DNA-PKcs-/- mice.
Fig.3 Analysis of T- and B-lymphocyte
development in irradiated newborn (IRNB)-DNA-PKcs-/- mice
(A) T cell development as a function of
time after irradiation. Thymocytes
(Thy) were isolated at different times after irradiation (100 cGy) of newborn
(1- to 3-day-old) DNA-PKcs+/- mice and analyzed for expression of
CD4, CD8, TCRb or IL-2R by multiparameter flow
cytometry. (B) Rescued CD4+ CD8+
thymocytes undergo further matruation and present in lymph node (LN). Cells were isolated from 8-week-old-DNA-PKcs-/-
mice after irradiation (100 cGy) and stained with CD4, CD8 or TCRb markers. (C). Lack of B
cell development in IRNB-DNA-PKcs-/- mice. Bone marrow (BM) and spleen (Spl) cells were obtained 6
weeks after irradiation (100 cGy).
Cells were stained with antibodies against the pan-B cell marker B220, surface IgM and the early B cell marker
CD43. In all experiments, age-matched wild-type mice were used as
controls.
To
elucidate the mechanism of resuced DP thymocyte development, we examined the patterns of TCRb
rearrangement in thymocytes obtained from control non-irradiated DNA-PKcs-/-
and IRNB-DNA-PKcs-/- mice.
Southern blot analysis of thymus DNA showed that diverse TCRb
rearrangements were induced concomittantly with the appearance of DP thymocytes
in 1- to 2-week old IRNB-DNA-PKcs-/- mice (Fig.4, lanes 3 to 7), whereas rearrangement of this locus was
not detected in the control non-irradiated DNA-PKcs-/- mice
(Fig.4, lanes 1 and 2). In thymus DNA from wild-type C57BL/6
mice, highly diverse
rearrangements involving multiple V,
D, and J loci gave rise to
a characteristic smearing of the Cb
germ line bands (data not shown)[10]. Some of the thymus DNA from 1- to 4-week-old IRNB-DNA-PKcs-/-
mice (Fig.4, lanes 3, 5, and 8) also showed this smearing, which suggests the polyclonal nature of the
rearrangements. However, by 6―8
weeks after irradiation,
predominant, oligoclonal
TCRb
rearrangements were visible in all samples (Fig.4, lanes 11,
12, and 13).
Fig.4 Rearrangement of the TCRb
locus occurs rapidly after irradiation of newborn DNA-PKcs-/- mice.
Genomic DNA was prepared from DNA-PKcs-/- mice and from individual
thymuses of IRNB-DNA-PKcs-/- mice of the indicated ages. Southern blot was performed by [32P]-dCTP-labeled
TCR Cb
cDNA probe
The
frequency of radiation-induced rescue of productive, in-frame TCRb
rearrangement was determined by flow cytometric analysis of intracellular TCRb
protein in IRNB-DNA-PKcs-/- thymocytes. Cells were first permeablized with 70% ethanol, and analyzed by two-color
immunofluorescence for CD4 and TCRb
expression. In all mice analyzed 1
week after irradiation, 13% to 37% of the CD4+
thymocytes co-expressed cytoplasmic TCRb
(TCRb+)(Fig.5). As expected, no CD4+ or TCRb+ cells were detected in control DNA-PKcs-/-
animals, whereas most wild-type
thymocytes co-expressed both CD4 and cytoplasmic TCRb.
Fig.5 Analysis of intracellular TCRb
chain expression in IRNB-DNA-PKcs-/- mice
Thymocytes were obtained from
IRNB-DNA-PKcs-/- mice 1 week after irradiation (100 cGy) and
analyzed for intracellular expression of CD4 and TCRb
by mutiparameter flow cytometry.
Age-matched wild type and DNA-PKcs-/- mice were set up as
controls.
2.4
Susceptibility of newborn DNA-PKcs-/- mice to
radiation-induced thymic lymphomas
We
have shown in the previous paragraph that treatment of newborn DNA-PKcs-/-
mice with g-irradiation
restored T cell receptor (TCR)b
recombination and T cell maturation.
However, most of these mice
(11 out of 15 animals) developed thymic lymphoma between 3 and 7 months of age. Tumors of B lymphoid or non-lymphoid
origin were not detected among the tumor-bearing animals examined. Histologically, the primary tumors consisted of
mononuclear, a typical cells with
cleaved nuclei, prominent nucleoli
and many mitotic figures [Fig.6(A)].
Immunohistochemical analysis revealed that the tumors were CD3+, confirming the diagnosis of T cell
lymphoma. In most cases, these tumors were involved in other
organs, such as lung, heart, liver and spleen.
Southern
blot analysis of three IRNB-DNA-PKcs-/- thymic lymphomas that arose
12 to 20 weeks after irradiation indicated that these tumors arose from
oligoclonal expansion of preneoplastic cells, since only two or three TCRb
rearrangements were seen in each tumor [Fig.6(B)].
Fig.6 Development of thymic tumors developed
in IRNB-DNA-PKcs-/- mice
(A) Histological analyiss of thymic
tumors: (a) A thymus form DNA-PKcs-/-
mouse; (b) A spontaneous thymic
tumor developed 4 months after irradiateion; (c) positive immunohistochemical surface staining against T
cell surface marker CD3. (B)
Southern blot analyses were performed on DNA extracted from tissue fragments of
each primary tumor and hybridized with TCR Cb
cDNA probles.
3
Discussion
To
investigate the role of DNA-PKcs in mammalian DNA DSB repair, we have determined the effects of
ionizing radiation on DNA-PKcs-/- cells, and whole animals.
Our results show that DNA-PKcs-/- mice and cells are severely
affected by doses of radiation that have no noticeable effect on wild type controls. At non-lethal doses of around 100
cGy, similar to the response of
newborn SCID mice, radiation has
no significant effect on the growth or survival of DNA-PKcs-/-
mice. This phenotype is different
from Ku80-/- mice which are particularly susceptible to whole-body
radiation early in development[15]. For newborn Ku80-/- mice, doses of ＞50
cGy aggravate their growth deficiency and result in high frequency of
mortality, suggesting that early
in development DNA-PKcs-/- mice are less radiosensitive than Ku80-/-
mice. On the other hand, the radiation response of adult
DNA-PKcs-/- mice appears to be similar to adult Ku80-/-
and SCID mice. Doses ＞400
cGy produce severe toxic effects on the gastrointestinal tract, and 100% of animals die within two weeks
post-irradiation (Fig.1). Lower
doses (＜300
cGy) of ionizing radiation result in abnormalites in the hair pigmentation of
adult DNA-PKcs-/- mice,
similar to that observed in Ku80-/- mice, and probably due to the
hypersensitivity of DNA-PKcs-/- melanocytes to ionizing radiation.
In
newborn mice, sublethal doses of
ionizing radiation were found to promote DNA-PKcs-/- thymocyte
maturation to the CD4+ CD8+ DP stage. Furthermore, this T cell-specific differentiation resulted in an increase
in thymus cellularity as well as high frequencies of thymic lymphoma
development. On the other
hand, low doses of radiation did
not induce maturation and proliferation of B cells, nor induce tumors of B cell origin. These results are strikingly similar to
that observed for irradiated newborn SCID mice[10], but distinctly different from Ku80-/-
mice[15]. In newborn
Ku80-/- mice, low doses
of radiation promote Ku80-/- thymocytes to the CD4+ CD8+
DP stage; however, this T cell-specific differentiation did
not increase thymocyte cellularity,
nor result in thymic lymphomas.
Cellular DNA damage induced by radiation can cause apoptosis, cell cycle arrest and transactivation
of genes[8, 16―19]. Although the signaling pathway by which
irradiation induces development and expansion of DP thymocytes is not well
understood, it is possible that
radiation could stimulate other DNA repair activites that can functionally
complement the DNA-PKcs gene during T cell maturation. It is likely that in the absence of DNA-PKcs
or Ku80, irradiation does not
supply all the necessary signals to fully effect this developmental
transition. The lack of
significant expansion of Ku80-/- DP thymocytes, but not in DNA-PKcs-/-
mice, may be because of the
sensitivity of DP cells to apoptosis[20], which is further enhanced in the absence of Ku80 but not so
in the absence of DNA-PKcs.
Consistent with this idea,
we found that Ku80-/- MEFs and Ku80-/- pre-B cells
were much more susceptible to spontaneous and g-ray-induced
apoptosis than wild-type controls[15]. On the other hand,
DNA-PKcs-/- MEFs had very similar response to g-ray-induced
apoptosis when compared to their wild-type controls.
Severe
combined immune deficiency (SCID) mice are hypersensitive to radiation, deficient in DNA double-strand break
repair and imparitied in V(D)J recombination. It has been shown that the SCID defect correlates with
nonsense mutations at the extreme 3′
end of the DNA-PKcs gene[21―24]. The identity of the gene has been
verified recently, because
knockout mice carrying genetic inactivated DNA-PKcs gene via homologous
recombination were generated in various laboratories[7, 25，26]. DNA-PKcs null mice exhibit no growth
retardation nor a high frequency of T cell lymphoma development. In contrast to Ku70-/- and
Ku80-/- phenotype,
DNA-PKcs mice are blocked for V(D)J coding but not for signal end joint
formation. In the present
study, we further demonstrated
that DNA-PKcs-/- cells,
mice are hypersensitive to g-irradiation. Treatment of newborn DNA-PKcs-/-
mice with sublethal doses of ionizing radiation restores TCRb
recombination and T cell maturation,
but not B cell development.
All these animals eventually developed thymic lymphoma.
Of
significant interest there are several salient features of our study. First, the striking similarity between DNA-PKcs-/- and
SCID mice in terms of lymphocyte development and V(D)J recombination[7,
25], radiation
hypersenstivity as well as the restoration of T cell promotion and maturation
in irradiated newborn DNA-PKcs-/- mice suggest that the “leaky”
phenotype frequently observed in the lymphocyte development of SCID mice may
not be due to the “leakiness”
of DNA-PKcs expression. Our data
support the hypothesis that there may exist an alternate pathway in these
processes involving DNA double-strand breaks. Second, our
studies on Ku80-/-[15, 27] and DNA-PKcs-/- mice[28], clearly show that Ku has function in
V(D)J recombination and normal development that are distinct from that in terms
of a DNA-PK holoenzyme: loss of
Ku80 expression results in growth retardation defects and deficiency in
jointing coding ends as well as signal ends in mice[27, 29], whereas, DNA-PKcs-/- mice are normal in size and only
deficient in coding end joining[25, 26]. Third, our data
suggest that Ku may have DNA-PKcs-independent function in DNA damage
repair. This is evidenced by the
much more sensitive survival response of Ku80-/- MEFs than the
DNA-PKcs-/- MEFs [Fig.1(B)].
Furthermore, the much less
severe phenotype of DNA-PKcs-/- mice compared to the Ku-deficient
mice in terms of growth characteristics and response of newborn mice to
ionizing radiation indicate a role for Ku in cell proliferation control and
apoptosis in a DNA-PKcs-independent manner. It will be a challenge to reveal why different mutations in
different components of DNA-PK complex result in discrete phenotype, and to uncover the alternate pathway
which functionally complements DNA-PK deficiency.
References
1 Weaver DT. What to do at an end:
DNA double strand break repair. Trends
Genet, 1995, 11(10):
388―392
2 Jeggo PA , Taccioli GE,
Jackson SP. Menage a trois: Double strand break repair, V(D)J recombination and DNA-PK. Bioessays, 1995, 17(11): 949―957
3 Carlomagno F, Burnet NG,
Turesson I, Nyman J, Peacock JH, Dunning AM,
Ponder BA et al. Comparison of DNA repair protein
expression and activities between
human fibroblast cell lines with different radiosensitivities. Int J Cancer, 2000, 85(6): 845―849
4 Anderson CW. DNA damage and the DNA-activated protein kinase. Trends Biochem Sci, 1993, 18(11): 433―437
5 Pan ZQ, Amin AA, Gibbs
E, Niu H, Hurwitz J. Phosphorylation of the p34 subunit of human
single-stranded-DNA-binding protein in cyclin A-activated G1 extracts is
catalyzed by cdk-cyclin A complex and DNA-dependent protein kinase. Proc Natl Acad Sci USA,
1994, 91(18): 8343―8347
6 Hennequin C, Giocanti N,
Averbeck D, Favaudon V. DNA-dependent protein kinase
(DNA-PA), a key enzyme in the
re-ligation of double-stranded DNA breaks. Cancer Radiother, 1999, 3(4): 289―295
7 Kurimasa A, Ouyang H, Dong
LJ, Wang S, Li X, Cordon-Cardo C,
Chen DJ et al.
Catalytic subunit of DNA-dependent protein kinase: Impact on lymphocyte development and
tumorigenesis. Proc Natl Acad
Sci USA, 1999, 96(4): 1403―1408
8 Clarke AR, Purdie CA,
Harrison DJ, Morris RG, Bird CC, Hooper ML,
Wyllie AH. Thymocyte
apoptosis induced by p53-dependent and independent pathways. Nature, 1993, 362(6423): 849―852
9 Chen CH, Zhang J, Ling
CC. Transfected c-myc and c-Ha-ras
modulate radiation-induced apoptosis in rat embryo cells. Radiat Res,
1994, 139(3): 307―315
10 Danska JS, Pflumio F,
Williams CJ, Huner O, Dick JE, Guidos CJ. Rescue of T cell-specific V(D)J recombination
in SCID mice by DNA-damaging agents.
Science, 1994, 266(5184): 450―455
11 Murphy WJ, Durum SK, Anver
MR, Ferris DK, McVicar DW, O’Shea,
JJ, Ruscetti SK et al. Induction of T cell differentiation and
lymphomagenesis in the thymus of mice with severe combined immune deficiency
(SCID). J Immunol, 1994, 153(3): 1004―1014
12 Zuniga-Pflucker JC, Jiang D, Schwartzberg PL,
Lenardo MJ. Sublethal
gamma-radiation induces differentiation of CD4–/CD8– into
CD4+/CD8+ thymocytes without T cell receptor beta
rearrangement in recombinase activation gene 2-/- mice. J Exp Med, 1994 , 180(4): 1517―1521
13 Guidos CJ, Williams CJ, Wu
GE, Paige CJ, Danska JS. Development of CD4+CD8+ thymocytes in
RAG-deficient mice through a T cell receptor beta chain-independent pathway. J Exp Med, 1995, 181(3):
1187―1195
14 Pearse M, Wu L, Egerton
M, Wilson A, Shortman K, Scollay R. A
murine early thymocyte developmental sequence is marked by transient expression
of the interleukin 2 receptor.
Proc Natl Acad Sci USA, 1989, 86(5): 1614―1618
15 Nussenzweig A, Sokol K, Burgman P, Li
L, Li GC. Hypersensitivity of Ku80-deficient cell
lines and mice to DNA damage: the
effects of ionizing radiation on growth,
survival, and
development. Proc Natl
Acad Sci USA, 1997, 94(25): 13588―13593
16 Kastan MB, Onyekwere O,
Sidransky D, Vogelstein B, Craig RW. Participation of p53 protein in the cellular response to DNA
damage. Cancer Res, 1991, 51(23 Pt 1): 6304―6311
17 Kastan MB, Zhan Q,
el-Deiry WS, Carrier F, Jacks T, Walsh WV,
Plunkett BS et al. A mammalian cell cycle checkpoint
pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia. Cell, 1992, 71(4):
587―597
18 Kuerbitz SJ, Plunkett BS,
Walsh WV, Kastan MB. Wild-type p53 is a cell cycle
checkpoint determinant following irradiation. Proc Natl Acad Sci
USA, 1992, 89(16): 7491―7495
19 Lowe SW, Schmitt EM,
Smith SW, Osborne BA, Jacks T. p53 is required for radiation-induced apoptosis in
mouse thymocyte. Nature,
1993， 362(6423): 847-849
20 Ferrier P, Covey LR, Li
SC, Suh H, Malynn BA, Blackwell TK,
Morrow MA et al. Normal recombination substrate VH to
DJH rearrangements in pre-B cell lines from scid mice. J Exp Med, 1990, 171(6): 1909―1918
21 Blunt T, Gell D, Fox
M, Taccioli GE, Lehmann AR, Jackson SP,
Jeggo PA.
Identification of a nonsense mutation in the carboxyl-terminal region of
DNA-dependent protein kinase catalytic subunit in the scid mouse. Proc Natl Acad Sci USA, 1996, 93(19):
10285―10290
22 Danska JS, Holland DP,
Mariathasan S, Williams KM, Guidos CJ. Biochemical and genetic defects in the DNA-dependent protein
kinase in murine scid lymphocytes.
Mol Cell Biol,
1996, 16(10): 5507―5517
23 Araki R, Fujimori A,
Hamatani K, Mita K, Saito T, Mori M,
Fukumura R et al. Nonsense mutation at Tyr-4046 in the
DNA-dependent protein kinase catalytic subunit of severe combined immune
deficiency mice. Proc Natl Acad
Sci USA, 1997, 94(6): 2438―2443
24 Shin EK, Perryman LE,
Meek K. A
kinase-negative mutation of DNA-PK(CS) in equine SCID results in defective
coding and signal joint formation.
J Immunol,
1997, 158(8): 3565―3569
25 Gao Y, Chaudhuri J,
Zhu C, Davidson L, Weaver DT, Alt FW. A
targeted DNA-PKcs-null mutation reveals DNA-PK-independent functions for Ku in
V(D)J recombination. Immunity, 1998, 9(3): 367―376
26 Taccioli GE, Amatucci AG,
Beamish HJ, Gell D, Xiang XH, Torres Arzayus MI,
Priestley A et al.
Targeted disruption of the catalytic subunit of the DNA-PK gene in mice
confers severe combined immunodeficiency and radiosensitivity. Immunity, 1998, 9(3): 355―366
27 Nussenzweig A, Chen C, da Costa Soares V,
Sanchez M, Sokol K, Nussenzweig MC, Li GC. Requirement for Ku80 in growth and immunoglobulin V(D)J
recombination. Nature, 1996, 382(6591): 551―555
28 Wang S, Guo M, Ouyang
H, Li X , Cordon-Cardo C, Kurimasse A, Chen DJ et al.
The catalytic subunit of DNA-dependent protein kinase selectively
regulates p53-dependent apoptosis but not cell-cycle arrest. Proc Natl Acad Sci USA, 2000, 97(4): 1584―1588
29 Ouyang H, Nussenzweig A,
Kurimasa A, Soares VC, Li XL, Cordon-Cardo C,
Li WH et al. Ku70 is required for DNA repair
but not for T cell antigen receptor gene recombination in vivo. J Exp Med, 1997, 186(6): 921―929
Received:
October 8, 2001
Accepted: October 23, 2001
This
work was supported by grants CA-31397 from National Institute of Health (USA)
*Corresponding
author: Tel, 86-731-4805446; Fax, 86-731-4805383; e-mail, [email protected]