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The University of Texas M. D. Anderson Cancer Center, Science Park–Research Division, Smithville, Texas 78957, USA
2Correspondence: The University of Texas M. D. Anderson Cancer Center, Science Park, Research Division, P.O. Box 389, Smithville, TX 78957, USA. E-mail: pwong{at}sprd1.mdacc.tmc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: ATM protein kinase • differentiation • thiol • cellular redox activity • DNA turnover
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
4
). Mutations in the Atm gene result in multisystem
disorders. Cells, primarily germ line, lymphocytes, astrocytes, and
endothelial cells, which can rapidly alter and expose their DNA during
postnatal differentiation activity or after genetic injury, are most
vulnerable to loss of ATM. When ATM-dependent DNA metabolic activities
during postnatal development become faulty, profound changes in cell
fates ensue. In lymphoid cells, either apoptotic cell death or lymphoid
tumor formation is the end result (5)
.
ATM is activated primarily by breaks in DNA or chromatin that occur
during meiosis, mitosis, or V(D)J recombination, or with
oxidant-induced DNA breaks. On activation, ATM, along with its many
family members, can rapidly activate many proteins that inhibit or
modulate cell cycling at multiple checkpoints (2
, 3
, 6)
.
ATM can also rapidly modulate the thiol-dependent histone
acetylase-deacetylase system that controls chromatin and DNA exposure,
thereby controlling mRNA production (7)
. ATM may also be
involved in oxidative defense and can induce the major antioxidative
systems (i.e., catalase, glutathione peroxidase, superoxide dismutase,
and glutathione reductases) in the cytoplasm (8)
. Barlow
et al. (9)
found evidence of oxidative damage in brain,
testes, and thymus of young Atm-/- mice. In embryonic
neurons undergoing terminal differentiation and containing unrepaired
DNA lesions, ATM was shown to be required to initiate the
redox-sensitive p53/caspase 3 apoptotic pathways to neuronal death
(4
, 10)
. In our previous searches for means to
therapeutically restore the missing pleiotropic ATM functions, we noted
that the early death of primary lymphoid cells isolated from an AT
patient in serum-depleted media could be prevented by the permeant
thiol N-acetylcysteine (NAC) (11)
.
In this study, using primary murine thymocytes that are undergoing
progressive selection and DNA turnover, we noted that thiol redox
activity, as indicated by reduction of the tetrazolium MTS
[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium],
and DNA turnover, as indicated by incorporation of
[3H]thymidine into DNA, were both greatly
increased in Atm-/- thymocytes. This increased thymidine
incorporation and redox activity could be completely suppressed by the
permeant thiol NAC. It is possible, therefore, that ATM may suppress
DNA turnover by providing reduced thiols. This down-regulation of
developmental DNA turnover by ATM should result in suppression of
chromosomal instability and prevent the inevitable tumor development in
the Atm-/- lymphoid cells (2)
.
We also showed that in differentiated splenocytes, mitogens proportionally increased both the incorporation of [3H]thymidine and the rate of reduction of the tetrazolium MTS. These mitogen-induced activities in splenocytes were not affected by the presence or absence of ATM but were completely suppressed by the calcineurin-dependent inhibitor FK-506. FK-506, however, had no effect on the elevated DNA turnover activities in Atm-/- thymocytes. From these data, we conclude that the increased [3H]thymidine incorporation and reducing power indicate increased cell cycling in mitogenically stimulated splenocytes, whereas these two indicators represent increased FK506-independent DNA turnover activities in thymocytes.
Our findings further suggest that ATM’s abilities to stabilize chromatin, suppress DNA turnover and oncogenic activity, and prevent oxidative damage may all be modulated by its ability to regulate cellular thiol redox pathways.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. For each experiment, the
different genotypes used (wild type, heterozygous, and homozygous) were
restricted to littermates between 30 and 50 days old. Histopathological
examination indicated that none of the animals used in this study had
developed tumors.
Lymphocyte isolation and [3H]thymidine
incorporation into DNA
Thymocytes and splenocytes were isolated from the thymus and
spleen as described previously (12)
. (In comparative
studies, thymocytes and splenocytes from the same mouse were used.) The
isolated cells were washed and incubated in a plastic tissue culture
dish at 37°C for 1–2 h. The nonadherent cells were separated from
the adherent cells and resuspended in Dulbecco’s modified Eagle’s
medium supplemented with 10% heat-inactivated FCS (Life Technologies,
Grand Island, NY), 3 mM L-glutamine, 100 U/ml penicillin,
and 100 µg/ml streptomycin. The resulting thymocyte and splenocyte
suspensions were plated in 96-well plates, 200 µl/well, at 5 x
105 and 2.5 x 105
viable cells/well, respectively. Cell viability was determined by the
trypan blue exclusion assay. For spontaneous DNA synthesis assays, the
cells were pulsed with 0.5 µCi of
[3H]thymidine (Amersham Pharmacia Biotech,
Piscataway, NJ) per well at 37°C in a humidified 5%
CO2 atmosphere. Eight hours later, the cells were
harvested and [3H]thymidine incorporation was
measured with a scintillation counter (Packard). Results were expressed
as mean counts per minute ± SD in triplicate
cultures. For mitogen-stimulated proliferation assays, the cells were
cultured with different mitogens for 48 h. For the last 8 h,
the cells were pulsed with 0.5 µCi of
[3H]thymidine per well, and then they were
harvested and measured as just described.
Bioreduction of tetrazolium salt
To assay the cellular redox activity, we used the
thiol-sensitive tetrazolium salt assay. This assay is based on the
bioreduction of MTS to a colored formazan as described before
(13)
. MTS, unlike the more commonly used
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),
contains phenazine methosulfate, which is readily reduced by thiols and
forms a more soluble formazan on bioreduction. This has the advantages
the error-prone solubilization step that is required for the MTT assay
is eliminated (13)
and that MTS is readily reduced at pH
7.4 by thiols (unpublished data). The MTS assay is briefly described
here. Freshly isolated thymocytes and splenocytes were plated in
96-well plates at 5 x 105 viable cells/well
as described above. The cells were pulsed with 20 µl of a freshly
prepared solution of MTS plus phenazine methosulfate. After incubation
for 2–4 h at 37°C, the absorbance at 490 nm was recorded by using a
Spectra Count plate reader (Packard).
Cell cycle analysis of thymocytes
Freshly isolated thymocytes were fixed in 70% ethanol and
stained with propidium iodide containing RNase A. DNA content was
analyzed by flow cytometry on a Coulter Epics Elite flow cytometer.
Cell cycle analysis was performed with Multicycle software (Phoenix
Flow Systems, San Diego).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Atm-/- mice are
severely wasted despite their apparently normal food intake and fecal
output. Weight and cell loss in these mice is severe in the spleen and
thymus (Table 1
). To understand why, we examined the effect of ATM on primary
lymphocytes freshly isolated from the thymus and spleen of
Atm-/-, Atm+/-, and Atm+/+ mice. We
carried out our studies immediately after obtaining these cells from
the mice to reflect the in vivo situation as closely as possible. We
observed that thymidine incorporation was at first markedly elevated
(8- to 10-fold higher) in freshly isolated Atm-/-
thymocytes compared with that in Atm+/+ or
Atm+/- thymocytes (Fig. 1A
). The thymidine incorporation rates in Atm-/-
splenocytes, although very low, were also elevated compared with those
in Atm+/- or Atm+/+ splenocytes. In contrast,
thymidine incorporation was only slightly affected by ATM deficiency in
mitogenically activated mature splenocytes (Fig. 1B
).
Together, these findings indicate that ATM is activated and active only
in undeveloped thymocytes but not in cycling splenocytes. The data
further suggest that the lymphoid atrophy seen in Atm-/-
mice may be the result of unrepaired DNA breaks incurred during V(D)J
or other recombination activities. With unchecked DNA turnover activity
in Atm-/- lymphocytes, double-strand DNA breaks (DSBs) and
abnormal chromosomes accumulate, resulting in apoptotic cell loss,
organ atrophy, or tumors.
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Inhibition of both recombination-associated and mitogen-induced
thymidine incorporation by dexamethasone and inhibition of
mitogen-induced DNA synthesis by FK-506
The cause of the increased thymidine incorporation in
Atm-/- thymocytes is unclear. Because the protein
phosphatase calcineurin is known to be required for mitogen-induced
proliferation of lymphocytes, FK-506 (a specific inhibitor of
calcineurin and mitogen-induced cell cycling) was used to determine
whether the increased thymidine incorporation in Atm-/-
thymocytes was dependent on mitogen-induced signaling. As shown in Fig. 1C
, FK-506 had no effect on the increased thymidine
incorporation in Atm-/- thymocytes, thus indicating that
the increased thymidine incorporation is independent of calcineurin and
proliferation and therefore is not due to DNA synthesis but is due to
other DNA turnover activities. As expected, FK-506 completely
suppressed DNA synthesis in mitogenically activated mature
Atm-/- and Atm+/+ splenocytes (Fig. 1D
). These results indicate that only
calcineurin-independent DNA turnover pathways are increased in
Atm-/- thymocytes.
The glucocorticoid dexamethasone was shown to suppress both thymidine
incorporation in Atm-/- thymocytes (Fig. 1C
)
and mitogen-induced DNA synthesis in splenocytes (Fig. 1D
).
Thus, unlike ATM kinase, glucocorticoid inhibits both the DNA turnover
activity in thymocytes and the mitogen-induced DNA synthesis in
splenocytes.
Elevated thymidine incorporation in Atm-/-
thymocytes is quickly followed by cell death
The spontaneous thymidine incorporation by Atm-/-
thymocytes was short-lived. As shown in Fig. 2
, after a 24-h incubation in 10% serum, recombination activity ceased
in thymocytes, and by 48 h many of the cells were trypan blue
positive. However, splenocytes survived for days and remained
quiescent, as indicated by their ability to respond normally to
mitogens phorbol myristate acetate (PMA) and concanavalin A (ConA)
(data not shown).
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Effect of ATM on cell cycle distribution in primary thymocytes
More than 90% of primary thymocytes are in the
G1 phase, and loss of ATM results in only a
slight increase in the number of cells in the S phase (Fig. 3
). These observations further suggest that most of the primary
thymocytes are not cycling but that ATM can activate the S phase
checkpoint in the few thymocytes that are cycling in vitro. In rapidly
proliferating transformed cell lines, ATM is known to activate multiple
cell cycle checkpoints after DNA damage (14)
.
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Regulation of cellular thiol redox activity by ATM
Using bioreduction of tetrazolium salt (MTS) assays, we noted that
both thymocytes undergoing recombination and mature quiescent
splenocytes from Atm-/- mice reduced MTS at much faster
rates than did thymocytes or splenocytes from Atm+/+ or
Atm+/- mice (Fig. 4A
). The increased reduction of tetrazolium in
Atm-/- thymocytes was markedly inhibited by dexamethasone
but not by FK-506 (Fig. 4B
). FK-506, however, completely
inhibited the mitogen-induced reduction of tetrazolium salt in
mitogen-activated splenocytes (data not shown). Because MTS reduction
is an indicator of thiol redox activity, these observations suggest
that a primary function of ATM kinase may be to regulate thiol redox
activity and thereby suppress the DNA turnover activity in
Atm-/- thymocytes. Alternatively, ATM could directly
suppress recombination activities.
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Mitogens proportionally increase the rate of thymidine
incorporation and MTS reduction
Because thymocytes are relatively short-lived and do not respond
to mitogens after 48 h of culture, splenocytes were used to
explore the relationship between proliferation and MTS reduction. We
observed that with addition of ConA, a specific mitogen for T cells, or
PMA, a mitogen for T or B cells, plus the calcium ionophore ionomycin
to splenocytes, MTS reduction and DNA synthesis were both greatly and
proportionally increased (Fig. 5
).
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Modulation of DNA turnover and cell cycling by the permeant thiol
NAC
The increased incorporation of thymidine (i.e., DNA turnover
activity) seen in Atm-/- thymocytes was completely
prevented by the permeant thiol NAC (Fig. 6A
). Impermeant thiols (e.g., glutathione) were ineffective
(data not shown). In contrast, DNA synthesis in mitogen-activated
mature splenocytes was stimulated by NAC (Fig. 6B
). These
findings indicate that NAC, like ATM, can suppress DNA turnover
activity in thymocytes. However, in splenocytes NAC facilitates
mitogen-activated DNA synthesis.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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recently showed that
multiple organs in 2-month-old Atm-/- mice, especially the
brain and testes, underwent oxidative damage, which preceded the onset
of lymphoid tumor development. Previously, we noted that blood
mononuclear cells from an AT patient also rapidly die in culture. This
death could be prevented by the permeant thiol NAC and by various
growth-promoting signals (11)
. In this study, using
freshly isolated thymocytes and splenocytes from Atm-/-,
Atm+/-, and Atm+/+ mice, we showed that in
premature undifferentiated Atm-/- thymocytes, which are
under genomic stress because of rapid recombinational events
postnatally, both the DNA turnover process, as indicated by
incorporation of thymidine into DNA, and the thiol redox activity, as
indicated by reduction of thiol-sensitive MTS, are greatly increased
compared with Atm+/+ thymocytes. The Atm-/-
thymocytes also die in culture at faster rates. In primary
differentiated Atm-/- splenocytes that are quiescent,
mitogen-independent incorporation of thymidine was much lower than that
observed in thymocytes.
We also observed that stimulation of the quiescent splenocytes by
appropriate mitogens rapidly and proportionally increased both
thymidine incorporation and redox activity. These mitogen-stimulated
activities in splenocytes were not affected by the presence or absence
of ATM but were completely inhibited by FK-506. However, these two
activities in thymocytes were not inhibited by FK-506. These
observations suggest that these activities indicate increased DNA
turnover activity in the immature thymocytes but increased cell cycling
and proliferation in the differentiated splenocytes. Because MTS is
readily reduced by thiols, including NAC, ß-mercaptoethanol, and
reduced glutathione (unpublished data), it appears that MTS reduction
is a measure of turnover of cellular sulhydryls (thiol redox potential)
in response to DNA turnover activities. To evaluate this possibility
further, we investigated the role of the permeant thiol NAC on both DNA
turnover and proliferative activities in thymocytes and splenocytes. We
show that NAC suppressed the DNA turnover activity in
Atm-/- thymocytes but up-regulated the proliferative
activity in both Atm-/- and Atm+/+ splenocytes.
Dexamethasone, however, inhibited both thymidine incorporation and
redox activities in both thymocytes and splenocytes with or without
Atm. These findings suggest that the thiol-sensitive
activities in Atm-/- thymocytes represent DNA and
telomeric turnover activities and other illegitimate repair activities
(15
, 16
, 16a)
. In contrast, thiol-stimulated activity in
Atm-/- or Atm+/+ splenocytes, which is
inhibited by FK-506, represents proliferative DNA synthesis.
Glucocorticoids, which are well-known activators of cell death pathways
in lymphocytes (17)
, inhibit both thymidine incorporation
and redox activity in cells undergoing either proliferative expansion
or postnatal recombination and differentiation. From these findings, we
conclude that ATM is essential for survival and tumor prevention in
lymphocytes during differentiation (5)
.
Together, our observations and those of others suggest that ATM
functions both as a "gatekeeper" and as a "caretaker"
(15)
in lymphoid cells. As a gatekeeper, ATM, which is
activated by exposed DNA with DSBs that may be produced during meiosis
(18
, 19)
, during recombination (5)
, or with
exposure to various oxidants including ionizing radiation
(20)
, can activate redox pathways that control cell death,
recombination, and proliferation rates. In Atm-/-
thymocytes, DSBs, which normally occur with V(D)J or nonhomologous
recombination during postnatal differentiation, may persist, leading to
accumulation of unrepaired DNA, translocated DNA and telomeric
fragments (16a)
, and abnormal chromosomes. This in time
will lead to oncogenic transformations (5)
.
As a caretaker, ATM, which also is a redox thiol-sensitive protein
kinase (7)
, functions by activating multiple
redox-sensitive or phosphorylation-sensitive mechanisms responsible for
maintaining genomic, telomeric, and chromosomal integrity under
conditions of genomic or redox stress primarily during postnatal
selection. Because both redox activity and thymidine incorporation, as
well as cell survival, were shown to be maintained optimally both by
ATM and by external permeant thiols, we suggest that a major caretaker
function of ATM is to maintain production of thiol reductants, probably
via the hexose monophosphate shunt NADPH production or by inhibiting
glycolytic flux and H+ production by modulation
of the redox-sensitive glycolytic dehydrogenase glyceraldehyde
phosphate dehydrogenase (21)
. Because this ATM-dependent
redox activity can suppress both the potentially genotoxic and
oncogenic DNA turnover activity and the redox-sensitive histone
deacetylase-acetylase system (7)
, it appears that
activated ATM can operate both as a genomic gatekeeper and as a
caretaker via its redox activity.
ATM thus appears to function as a global protector of broken genomes.
Activation of ATM by DSBs has been shown to lead to: 1) up-regulation
of gene expression via phosphorylation of nuclear tyrosine kinase c-Abl
(22)
; 2) arrest of cell cycling and activation of
apoptosis via up-regulation of tumor suppressor p53 (6)
;
3) up-regulation of replication protein A (RPA), which is involved in
DNA replication, DNA repair, and recombination activities
(3)
; 4) suppression of V(D)J recombination and thymic
lymphoma (5)
; 5) abnormal chromosome aberrations and
T-cell tumors (23)
; 6) activation of checkpoint kinases
Chk2 and Chk1, which rapidly check cycling at
G1/S and G2/M, respectively
(24)
; 7) phosphorylation of nibrin, one of a complex of
factors involved in rapid repair of DSBs (25)
; 8)
inhibition of the histone deacylase HDAC1 (7)
: because
activated ATM binds HDAC1 through the redox-sensitive motif LXCXE, the
rapid modulation of histone-DNA interactions with the resultant altered
transcription and cell death are also probably redox sensitive; 9)
prevention of ionizing radiation-induced necrotic cell death in
proliferating lymphocytes: this necrotic pathway is probably mediated
by oxidation of membrane lipids in the presence of iron (26
, 27)
; and 10) prevention of postnatal oxidative damage in brain
cells, germ cells (9)
, human erythrocytes
(28)
, and circulating lymphocytes (11)
.
In conclusion, the data presented in this report suggests that
activated ATM, utilizing its multiple protein kinase activities, may
control cell fates by modulating the cellular thiol redox activities in
cells with genomic damage or in cells undergoing pre- or postnatal
development. One possibility is that ATM can divert the glycolytic flux
into hexose monophosphate shunt pathway. This would result in decreased
production of H+ (21)
but increased
production of NADPH, mitochondrial glutathione, and nuclear
thioredoxin. Release of apoptogenic factors and opening of the
redox-sensitive transition pores in mitochondria would also be
curtailed (29)
. The redox-sensitive pathways in the
nucleus that are responsible for recombination or repair pathways would
also be curtailed in favor of proliferative or quiescent transcription
pathways. Because our observation suggests that these redox functions
of activated ATM can be replaced by permeant thiols, bypass therapy
using permeant thiols may become possible for AT as well as for other
disorders characterized by chromosomal instability that leads to
neuroimmunodegeneration and cancer.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication September 11, 2000.
Revision received December 12, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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