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Blood, Vol. 94 No. 6 (September 15), 1999:
pp. 1998-2006
ATM Is Upregulated During the Mitogenic Response in Peripheral
Blood Mononuclear Cells
By
Toshiyuki Fukao,
Hideo Kaneko,
Geoff Birrell,
Magtouf Gatei,
Hideaki Tashita,
Toko Yoshida,
Simone Cross,
Padmini Kedar,
Dianne Watters,
Kum Kum Khana,
Ihor Misko,
Naomi Kondo, and
Martin F. Lavin
From the Department of Pediatrics, Gifu University School of
Medicine, Gifu, Japan; the Queensland Cancer Fund Research Unit, the
Queensland Institute of Medical Research, PO Royal Brisbane Hospital,
Herston, Brisbane, Australia; and the Department of Surgery, the
University of Queensland, PO Royal Brisbane Hospital, Herston,
Brisbane, Australia.
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ABSTRACT |
Patients with the human genetic disorder ataxia-telangiectasia (A-T)
are characterized by immunodeficiency and a predisposition to develop
lymphoid malignancies. The gene mutated in A-T patients, ATM,
codes for a high molecular weight protein that is implicated in DNA
damage recognition and cell cycle control. The ATM protein does not
change in amount or cellular distribution throughout the cell cycle or
in response to DNA damaging agents. Because peripheral blood
mononuclear cells (PBMCs) are largely in a state of quiescence and can
be readily stimulated to enter a proliferative phase and because A-T
cells exhibit growth abnormalities and senescence, indicative of a
general intracellular defect in signalling, we chose PBMCs to examine
the relationship of ATM to the proliferative status of the cell. We
show here that ATM protein is present at low levels in freshly isolated
PBMCs and increases approximately 6-fold to 10-fold in response to a
mitogenic stimulus, reaching a maximum after 3 to 4 days. A similar,
but delayed response, was evident in the presence of serum only. This
increase in ATM protein was accompanied by an increase in ATM kinase
activity. While expression of ATM protein increased during
proliferation, ATM mRNA expression was unchanged in stimulated
and unstimulated cells and there was no evidence for increased ATM
protein stability in the phytohemagglutinin (PHA)-treated cells. In
keeping with the reduced levels of ATM in quiescent cells, the extent
of radiation-induction of the p53 pathway was significantly lower than
in mitogen-stimulated cells. Basal levels of p21 were elevated in
quiescent cells, and the response to radiation was negligible or
reduced compared with proliferating cells over a 2-hour period.
Overall, the data suggest that the increase in ATM protein in
proliferating cells is due to posttranscriptional regulation and points
to a role for ATM in more general signalling.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
ATAXIA-TELANGIECTASIA (A-T) is a
primary immunodeficiency disease characterized by defects in both
humoral- and cell-mediated immunity.1,2 Reduction in T-cell
helper activity in A-T patients does not appear to be sufficient to
account for the complete absence of IgA, which is observed in some
cases, suggesting that there exists either an intrinsic defect in the
maturation of IgA and IgE producing cells or a more general defect in
genetic recombination. Absence of, or abnormal development of the
thymus, is also a consistent feature of A-T.1 The thymic
abnormalities do not appear to be due to atrophy, but rather to a
defect in development.2 The
CD4+/CD8+ T-cell ratio in A-T patients is
reversed compared with normals due to a decrease in the total numbers
of CD4+ cells.3 Disturbances in recombination
are also observed in T cells as elevated recombination of T-cell
receptor genes4 and increased rates of spontaneous
intrachromosomal recombination with exogenously added DNA.5
However, signal and coding joint formation are both normal in V(D)J
recombination.6
One of the major hallmarks of A-T is predisposition to
malignancy.7 As many as one third of A-T patients develop
cancer with the majority of these being of the lymphoid
type.8 Most of the leukemias are T cell in origin; acute
lymphocytic leukemia in younger patients and T-cell prolymphocytic
leukemia (T-PLL) in older patients.9 In contrast to the
leukemias, the lymphomas, which represent up to 50% of
malignancies, are of both T-cell and B-cell
origin.10 A smaller proportion (15% to 20%) of tumors in
A-T patients are nonlymphoid.9,11 Nonrandom chromosomal translocations and inversions are frequently observed in A-T
lymphocytes, and some of these are associated with the activation and
expression of genes such as TCL1 and MTCP1, which are implicated in the
pathogenesis of T-PLL.12
The cloning of the gene mutated in A-T patients,
ATM,13 has provided greater insight into the nature
of the defect and the elevated prevalence of lymphoid tumors in A-T.
Loss of heterozygosity studies and mutation analysis of the ATM
gene have shown the presence of biallelic mutations, which lead to
premature truncations or alterations in the ATM gene product in
non-A-T patients with T-PLL, a leukemia seen frequently in
A-T.14-18 These observations suggest that ATM is a
tumor suppressor gene, the inactivation of which is important in the
development of T-PLL.
It seems unlikely that immunodeficiency is responsible for the pattern
of tumors that develop in A-T, but it is conspicuous that these tumours
arise primarily in T and B cells. To understand whether ATM has a
special role in lymphoid cells, we initially compared the amount of ATM
protein in quiescent and mitogen-stimulated peripheral blood
mononuclear cells (PBMCs). In response to phytohemagglutinin (PHA) or
serum, the level of ATM protein increased markedly in PBMCs, but mRNA
levels were comparable in the 2 populations. The extent of the
radiation-induced p53 response was reduced in quiescent cells, in
keeping with reduced or absent ATM. This is the first description of
alteration in ATM protein in response to a mitogenic stimulus.
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MATERIALS AND METHODS |
Preparation and culture of PBMCs.
PBMCs were isolated from heparinized blood samples from healthy
volunteers by Ficoll gradient centrifugation (Pharmacia Biotech, Piscataway, NJ). After 3 washes with phosphate-buffered
saline (PBS), an aliquot was stored at  80°C as freshly
isolated PBMCs. The rest of the PBMCs were resuspended at a density of
2 × 106 cells/mL in RPMI 1640 medium supplemented
with 10% fetal calf serum (FCS), 15 mmol/L HEPES, 2 mmol/L
L-glutamine, 100 µg/mL penicillin, and 100 µg/mL streptomycin and
incubated under an atmosphere of 5% CO2 at 37°C. For
preparation of PHA-stimulated lymphoblasts, PHA (PHA-P; Sigma, St
Louis, MO) was added at a concentration of 10 µg/mL and
stimulation was monitored by 3H-thymidine uptake for 4 hours in 0.2 mL of culture. For immunoblotting, cells were harvested by
centrifugation and after 3 washes with PBS, the cell pellets were
stored at 80°C until use. An Epstein-Barr virus
(EBV)-transformed A-T cell line, L3, was kindly provided by Dr Yosef Shiloh (Department of Human Genetics, Sackler School of
Medicine, Tel Aviv, Israel).
Immunoblotting.
Cells were harvested and lysed in a lysis buffer (50 mmol/L Tris-HC1 pH
7.6, 150 mmol/L NaCl, 0.2% Triton X-100, 0.3% NP40, proteinase
inhibitors added [0.1 mmol/L phenylmethylsulfonyl fluoride, leupeptin
5 mg/mL, aprotinin 1 mg/mL]). Total protein concentration of cell
extracts was determined by the Bradford microassay. Protein extracts
were solubilized in 0.2 vol of 5x concentrated sample buffer (0.25 mol/L Tris-HC1, pH 6.8, 0.4 mol/L dithiothreitol [DTT], 5%
sodium dodecyl sulfate (SDS), 0.5% bromophenol blue and 10%
glycerol) and separated on a 4.5% SDS-polyacrylamide gel for ATM
and a 12% gel for p53, p21, and  -actin. After electrophoresis, proteins were transferred to Hybond C or Hybond ECL
(Amersham, Arlington Heights, IL). The filters were
blotted in 5% skim milk overnight, probed with antibodies, and
visualized using the ECL method (Amersham). Densitometric scanning was
used to quantify protein.
Quantitative reverse transcriptase-polymerase chain reaction
(RT-PCR).
Quantitative PCR was performed to determine the level of ATM mRNA
expression in quiescent and PHA-stimulated PBMCs as described previously.19 Nucleotides 5122-5665 were amplified for
normal ATM yielding a fragment of 544-bp. Competitor DNA was a purified 402-bp fragment with exon 38 skipping amplified from ATJGifu 1 (GAT1).20 An equivalent point is reached when target ATM
cDNA (544 bp) is amplified to the same extent as competitor DNA
allowing for quantification of mRNA. A total of 5 µg of RNA was
reverse transcribed in a total volume of 20 µL with 30 pmol of an
ATM-specific antisense primer
(5'-6059CCATACAAACTATCTGGCTCC-3') by Superscript II reverse
transcriptase (GIBCO-BRL, Gaithersburg, MD,). The
efficiency of cDNA synthesis was approximately the same in all cases.
Amplification was performed with an amount of cDNA corresponding to 0.1 µg of RNA containing 1 µL of a series of 2-fold dilutions of the
competitor DNA from 1/2 × 10 attomoles to 1/256 × 10 attomoles. Cycle conditions were 35 cycles of 94°C for 1 minute,
54°C for 1 minute, and 72°C for 2 minutes.
Determination of protein half-life using cycloheximide.
Cells were resuspended at a density of 2 × 106
cells/mL in RPMI 1640 medium supplemented with 10% FCS, 15 mmol/L
HEPES, 2 mmol/L L-glutamine, 100 µg/mL penicillin, 100 µg/mL
streptomycin, and 25 µmol/L cycloheximide and incubated under an
atmosphere of 5% CO 2 at 37°C for 0, 4, 8, 16, and 32 hours. In the case of PHA-stimulated lymphoblasts, after 3 days PHA
stimulation, the experiment was started and PHA was also included in
the above cycloheximide-containing medium.
ATM kinase assays.
PBMCs were isolated, the cells were counted and divided into 2 flasks
(5 × 107/mL) with RPMI and 10% serum and PHA was
added to 1 flask. Both flasks were incubated at 37°C for 72 hours
and the cells lysed by sonication in TGN
buffer.21 After centrifugation, 700 µg of extract was
precleared with protein A and protein G sepharose beads, ATM was
immunoprecipitated with Ab-3 antibody (Oncogene Research, Cambridge,
MA) with protein A/B sepharose beads. Immunoprecipitates were washed twice with TGN buffer, once with 100 mmol/L Tris-HCl, pH
7.5, plus 0.5 mol/L LiC1 and twice with kinase buffer (10 mmol/L HEPES,
pH 7.5, 50 mmol/L glycerophosphate, 50 mmol/L NaCl, 10 mmol/L
MgCl2, 10 mmol/L MnCl2, 5 µmol/L adenosine
triphosphate (ATP), and 1 mmol/L DTT). Kinase reactions
were performed by resuspending washed beads in 25 µL of kinase buffer
containing 1 µg of GSTp53 (1-40) and 0.5 µCi [ 32p]
ATP, and incubated at 30°C for 30 minutes. Proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE), dried, and assayed by fluorography. Protein loading was
determined by running a parallel sample of immunoprecipitated lysate
and blotting with anti-ATM antibody, ATM5BA).
Immunofluorescence analysis.
Cells were attached to slides by centrifugation at 250xg for 5 minutes, fixed with 4% formaldehyde in PBS for 30 minutes, and
permeabilized with PBS containing 0.3% Triton X-100 for 5 minutes.
After treatment with 0.1 mol/L glycine in PBS for 1 hour, preparations
were incubated with ATM-4BA antibody raised in a rabbit (1:100) in PBS
containing 2% FCS at 4°C overnight. After 3 washes with PBS
containing 0.05% Tween-20 for 5 minutes, the preparations were
incubated with fluorescein-linked F(ab')2 fragment donkey
antirabbit IgG (Jackson Immunoresearch, West Grove, PA) at 1:50 dilution in PBS containing 2% FCS for 3 hours. After 3 washes
with PBS containing 0.05% Tween-20, the cover glass was mounted with
10 µg/mL 4', 6'-diamidino-2-phenylindole (DAPI) (Sigma) in antifade solution. The cells were visualized by
fluorescence microscopy (×250).
Enrichment for monocytes in resting PBMCs.
Heparinized blood (10 mL) was centrifuged on a Ficoll-Hypaque density
gradient and the PBMCs were collected and washed twice with RPMI 1640 medium. The cell pellet was resuspended in 10 mL of medium ( 106 cells/mL), and cells were allowed to adhere to a
plastic petri dish for 1 hour at 37°C in a metabolic incubator.
Cells not adhering to the dish (mostly lymphocytes) were aspirated
gently and the fluid phase was completely removed. The dish was then
placed at 4°C for 30 minutes to detach the resting adherent cells
(mostly monocytes) and then flushed with medium +10% FCS, prewarmed to 37°C.22
Fluorescence-activated cell sorting (FACS) labelling.
Mouse antihuman monoclonal antibodies (Becton Dickinson, Sydney,
Australia) against CD16 fluorescein isothiocyanate (FITC) (Leu 11a, clone NKP15), CD33 PE (Leu M9, clone P67.6) and CD3 Per-CP
(Leu 4/clone SK7) were used to phenotype human cell subsets (Becton
Dickinson, Sourcebook). Cells were then permeabilized and fixed
(Pharmigen Cytofix Kit, Cytoperm Plus, Becton Dickinson) and incubated
either with preimmune (control) serum or the ATM-specific antibody,
ATM-4BA. Cells were kept at 4°C during the entire procedure. Both
control and ATM-4BA-treated cells were washed and then indirectly labelled with the donkey antirabbit Ig FITC (Silenus, Melbourne, Australia), washed again, and then subjected to FACS analysis.
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RESULTS |
Effect of PHA and serum on ATM protein level in PBMCs.
Exposure of PBMCs to mitogen leads to a dramatic increase in the number
of proliferating cells. To determine whether ATM levels changed in
response to a mitogenic signal, we performed immunoblotting with
quiescent freshly isolated PBMCs and cells exposed to PHA or serum.
When 30 µg of protein was loaded and Western blotting performed with
ATM-3BA, an antibody specific for ATM, no protein was detected in
extracts from 2 of 3 freshly isolated PBMC samples and only a low level
was present in the third sample (Fig 1). However, after stimulation with PHA(P) for 3 days, there was a dramatic
increase in ATM protein in all 3 samples (Fig 1). ATM protein also
increased in the presence of serum(S), but this was considerably less
marked than with PHA. Data using extracts from control (C3ABR) and A-T
lymphoblastoid cells (L3) are included. L3 had no ATM protein as
expected.23
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| Fig 1.
Immunoblotting of ATM in extracts from lymphocytes of
three individuals (1-3). F, extracts from freshly isolated lymphocytes;
S, extracts from lymphocytes incubated in the presence of 10% FCS for
3 days; and P, extracts from PHA-stimulated lymphocytes (3 days). A
total of 30 µg of protein extract was added to each lane of a 4.5%
SDS-PAGE gel and immunoblotting performed with ATM-3BA. Protein loading
was monitored by Ponceau S staining. Extracts were also included for an
A-T lymphoblastoid cell line (L3) (25 µg) and 4, 10, and 25 µg
loading for a control lymphoblastoid cell line (C3ABR).
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Time course of induction of ATM.
Because ATM increased markedly in PHA-stimulated PBMCs by 3 days, we
examined the time course of induction by immunoblotting over 5 days.
The results in Fig 2 provide data for 2 individuals. ATM protein was only detectable at low levels 1 day after
PHA addition, but increased markedly over a period of 2 to 5 days. PBMC
samples from other individuals showed that ATM levels were maximal at 3 to 4 days posttreatment (results not shown). When cells were exposed to
serum, there was a delay in the appearance of ATM compared with
PHA-treated cultures, but ATM increased by days 4 and 5 (Fig 2). This
delay was also evident in other samples (results not shown). For
comparison of the amount of ATM protein, we loaded 4, 10, and 25 µg
of protein extract from EBV-transformed lymphoblastoid (proliferating)
cells (C3ABR). The peak values obtained with PHA and serum are of the
same order as the 25-µg loading for the lymphoblastoid cell extract
(Figs 1 and 2).
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| Fig 2.
Effect of incubation time on the amount of ATM in PBMCs.
Lymphocytes were freshly prepared (F) and incubated for 1 to 5 days in
the presence of serum or PHA. A total of 30 µg of extract was used
for all lymphocyte samples and 10 and 25 µg of extract were used for
C3ABR, a proliferating EBV-transformed cell line. Immunoblotting was
performed using ATM-3BA antibody and protein loading was determined
with Ponceau S staining.
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A summary of all of the data from 15 individuals is presented in
Fig 3. To put the data into perspective, we
have plotted the individual values on a scale derived from the
detection of ATM in known amounts of protein from the proliferating
lymphoblastoid cell line, C3ABR. Eleven of 15 extracts from freshly
isolated PBMCs were below the limit of detection (4 µg of C3ABR
protein) under the exposure conditions for ECL used when 30 µg of
total protein was loaded. ATM in the other 4 PBMC samples was detected in the 4 to 6 µg range on this scale (Fig 3). Of 15 PHA-stimulated samples, 9 showed 25 µg or greater ATM equivalents and the other 6 had 10 to 20 µg equivalents at 3 days after addition of PHA (Fig 3).
As was evident from earlier experiments, the increase in ATM after
addition of serum only was less and more variable, ranging from below
the limit of detection to 25-µg equivalents.
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| Fig 3.
Summary of immunoblotting data for extracts from 15 different individuals. F, freshly isolated ( ); S, serum-stimulated
( ) and P, PHA-stimulated ( ) for 3 days. ATM protein in
lymphocytes was standardized to the amount of protein in C3ABR
lymphoblastoid cells. A vertical line at 4 µg means the detection
limit in this experiment.
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Effect on ATM kinase activity.
Previous data show that ATM interacts with p5324 and after
exposure of cells to ionizing radiation, ATM phosphorylates p53 on
serine 15 presumably to stabilize and activate p53.21,24,25 In these investigations, ATM kinase activity increased 2-fold to 4-fold
above the basal level of activity in response to radiation. Because we
showed a significant increase in ATM protein in response to PHA, we
determined whether this increase might be accompanied by an increase in
ATM kinase activity. When extracts from untreated (serum only) and
PHA-stimulated PBMCs were immunoprecipitated with anti-ATM antibody,
followed by incubation with GST-p53 (1-40) and -32P ATP
substrate, there was a substantial increase in ATM kinase activity, in
keeping with the ATM protein response to PHA
(Fig 4). This is reflected in the amount of
ATM protein as determined by immunoblotting in the same experiment.
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| Fig 4.
Use of ATM immunoprecipitates to determine the effect of
PHA on protein kinase activity, using GST-p53 (1-40) as a substrate for
ATM kinase. PHA-treated or untreated PBMCs were collected after 72 hours of incubation at 37°C before preparation of lysates. The same
amount of total protein was immunoprecipitated with anti-ATM antibody
(Ab-3, Oncogene Research). The beads were washed with
lysis buffer twice, once with 0.1 mol/L Tris-HCI, pH 7.5 containing 0.5 mol/L LiCl, and finally twice with kinase buffer. Reactions were
performed in 25 µL containing 1 µg of GST-p53 (amino acids 1-40), 5 µCi of ATP-32-P in kinase buffer for 30 minutes at
30°C and analyzed for incorporation on SDS-PAGE. and + refer to without and with PHA in PBMCs from two individuals.
Immunoblotting with ATM-5BA antibody was used to detect ATM in the
immunoprecipitates.
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Distribution and expression of ATM in PBMCs.
The lower levels of ATM protein in freshly isolated PBMCs could reflect
lower levels in all cell types in the population or reduced numbers of
cells expressing protein. Immunofluorescence analysis showed that the
intensity of ATM staining in freshly prepared PBMCs was markedly less
than that in PHA-stimulated cells (Fig 5A).
Nuclear staining by DAPI is comparable in the 2 cell types. There was
some indication that a subpopulation of unstimulated PBMCs had more
intense labelling (Fig 5A). Because these cells were larger in size
than lymphocytes and because their proportion was approximately the
normal percentage of monocytes in PBMCs, we compared ATM by FACS
analysis in an adherent population of cells enriched by monocytes, but
also containing lymphocytes. Labelling resting cells with anti-CD33
antibody allowed us to compare the amount of ATM protein in monocytes
with that in lymphocytes (Fig 5B). After labelling with ATM-4BA, the
mean fluorescence intensity (MFI) of the lymphocytes (24) increased
approximately 6-fold (144) and the monocytes (76) approximately 3-fold
(208), but they expressed the same amount of ATM protein, as monocytes in preimmune sera have a higher autofluorescence than lymphocytes (76 compared with 24), indicating that there are similar levels of ATM
protein in both cell types (Fig 5B). The higher intrinsic autofluorescence seen in monocytes by FACS analysis, when compared with
lymphocytes, could explain the apparent difference observed between
these cells when immunofluorescence was used (Fig 5A).
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| Fig 5.
Determination of the amount of ATM in resting and
PHA-stimulated PBMCs. (A) Immunofluorescence labelling of the 2 cell
types with ATM-4BA antibody. DAPI was used to stain nuclei. (B)
Plastic-adherent cells (resting) isolated from human PBMC were enriched
for monocytes (R2, CD33+) and contained some T
lymphocytes (R1, CD33-) as shown by FACS analysis. Cells were labelled
either with ATM-4BA or with preimmune rabbit sera as a control. The MFI
for each population is shown in brackets.
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ATM mRNA expression and protein stabilization.
Because the amount of ATM protein was very low in quiescent PBMCs, we
predicted that this might also be reflected in ATM mRNA levels.
Accordingly, we used quantitative PCR to compare ATM mRNA in
quiescent and PHA-stimulated lymphocytes as described
previously.19 In the experiments, a cDNA fragment (402 bp)
corresponding to an A-T mutant (5319G to A) lacking exon 38 was used as
a competitor for normal ATM cDNA amplification (nucleotides
5122-5665, 544 bp) in a series of 2-fold dilutions. In 2 samples of
unstimulated PBMCs, an equivalent point was reached at 10/24 and 10/32
attomoles/0.1 µg of RNA and the point was 10/48 and 10/64 for
PHA-stimulated cells (Fig 6), demonstrating
that mRNA levels in quiescent and PHA-stimulated cells were not
appreciably different and could not account for the increased ATM
protein in stimulated cells. ATM mRNA levels were of the same
order (10/16 attomole/0.1 µg of RNA) in proliferating lymphoblastoid
cells (C3ABR) (Fig 6).
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| Fig 6.
Quantitative RT-PCR for the ATM transcript. A control
EBV-transformed lymphoblast cell line (C3ABR) and 2 sets of freshly
isolated PBMCs and PHA-stimulated PBMCs from healthy controls (1 and 2)
were examined. Each template contained the same amounts of cDNA
corresponding to 0.1 µg of total RNA and 1 of 2-fold dilutions of a
competitor DNA from 1/2 × 10 to 1/256 × 10 attomole (lanes
a through h). Equivalent points where target cDNA and competitor DNA
were almost equivalent are indicated by arrow heads. The positions of
target cDNA and competitor DNA (Comp.) are also indicated by arrows.
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Another explanation for the higher levels of ATM in PHA-treated cells
could be increased stabilization of ATM protein. To investigate this,
we used cycloheximide to inhibit de novo synthesis of ATM and thus
measure the stability of existing protein. In proliferating
lymphoblastoid cells (C3ABR), the t 1/2 for ATM was
approximately 16 hours as determined by densitometric analysis of the
data in Fig 7. The t1/2 value for
PHA-stimulated PBMCs was between 16 and 32 hours. While it was more
difficult to measure t1/2 for ATM in resting cells (140 µg loaded
compared with 30 µg for PHA and C3ABR), it is evident that an initial
low level of ATM persists over 32 hours suggesting that ATM turnover is not more rapid in these cells compared with PHA-stimulated cells. Thus,
although there is a marked increase in ATM protein when PBMCs are
stimulated to proliferate, the level of mRNA remains approximately the
same in quiescent and dividing cells, and the t1/2 of the protein does
not change.
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| Fig 7.
Stability of ATM protein in PBMCs and lymphoblastoid
cells. Cells were incubated in cycloheximide (25 µg/mL) for the times
indicated before preparation of extracts for separation on SDS-PAGE and
immunoblotting with ATM-3BA antibody. A total of 30 µg of protein was
loaded for PHA-stimulated cells and for lymphoblastoid cells C3ABR,
while 140 µg of protein was used for unstimulated PBMCs. Controls
were included as in Fig 1. Exposure times differed among C3ABR,
PHA-stimulated cells, and freshly isolated PBMCs.
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Comparison of p53/p21-WAF1 response in stimulated and unstimulated
PBMCs.
It is well-established that the ionizing radiation signalling pathway
operating through p53 and p21/WAF1 is defective in A-T cells.21,26-29 Furthermore, reducing the
level of ATM with antisense cDNA constructs in control cells abrogates
this response.30 Because ATM is approximately 6-fold to
10-fold lower in freshly isolated PBMCs, it would be expected that the
p53 and p21/WAF1 responses to radiation would be reduced in these
cells. The results in Fig 8 show that this is the case
for p53. In response to radiation exposure, the extent of stabilization
of p53 was considerably greater in extracts from PHA-stimulated cells
than in those from unstimulated cells. There was a high basal level of
p21 in both samples of unstimulated PBMCs (Fig 8), which is in
agreement with levels in other quiescent or differentiated
cells.31,32 Radiation exposure failed to increase further
the level of p21 in 1 patient's (1) cells, which is in agreement with
the very weak p53 response. There was a small increase in a second
patient (2), which was somewhat exaggerated by protein loading.
Stimulation with PHA increased the p21 response to radiation exposure
in both patients lymphocytes (Fig 8).
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| Fig 8.
Effect of ionizing radiation (6 Gy) on p53 and p21/WAF1
levels in freshly isolated (F) and PHA-stimulated (P) lymphocytes from
2 individuals. Cells were irradiated followed by incubation at 37°C
in 5% CO2 for 2 hours before preparation of extracts. C3
represents extracts from unirradiated C3ABR cells. A total of 20 µg
of protein was loaded in each lane of a 12% SDS-PAGE gel. -actin
was used as a loading control.
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DISCUSSION |
The product of the ATM gene plays a pivotal role in sensing
damage in DNA and as a consequence in modulating cell cycle
checkpoints.33 The predominant presence of ATM in the
nucleus in proliferating cells supports this role.24,34-37
However, it is also clear that ATM is present outside the nucleus and
is associated with vesicular structures.36 These vesicles
have now been identified as clathrin-coated endosomes38 and
peroxisomes (Watters et al, unpublished). Furthermore, there is some
evidence that distribution of ATM is influenced by the differentiation
state of the cell.39,40
Neither the subcellular distribution of ATM nor the total amount of
cellular ATM protein is influenced by exposure to ionizing radiation or
radiomimetic compounds, agents to which ATM
responds.24,34,37 In addition, ATM protein levels are
relatively constant throughout the cell cycle in human
fibroblasts.34 The novel aspect of the present study is the
demonstration that the amount of ATM changes dramatically in quiescent
lymphocytes in response to a mitogenic signal. In nondividing PBMCs,
only a small amount of ATM protein was present compared with that in
PHA-stimulated cells as determined by immunoblotting, FACS analysis,
and immunofluorescence. The increased levels of ATM protein in
proliferating lymphocytes were not due to a more stable ATM protein or
to an increase in ATM mRNA in response to PHA. Failure to
observe parallel changes at the level of mRNA suggests that regulation
is at the posttranscriptional/translational rather than transcriptional
level. Indeed, extensive structural diversity at the 5' and
3' untranslated regions of ATM mRNA might lead to synthesis of specific transcripts, which could account for
control at this level,41 ensuring a prompt response to
physiological stimuli such as response to antigen or mitogen in the
present context. The results obtained here suggest that ATM is
upregulated in proliferating lymphocytes and plays a more general role
than signalling DNA damage. Evidence for a broader role for ATM in intracellular signalling is derived from observations that show a
greater demand for growth factors in A-T fibroblast
survival42,43 and the poor growth capacity of embryonic
fibroblasts from Atm / mice.44
Defective signalling through the T-cell and B-cell receptors, as
evidenced by a reduced or absent mobilization of Ca2+ and
defective activation of phosphatidylinositol 3 (PI3)-kinase and phospholipase C1
(PLC1) in A-T lymphoid cells, has also been
reported,29,45 as well as a defect in the transmission of
mitogen-mediated signalling due to a failure of exocytosis in
PHA-stimulated lymphocytes.46 Thus, upregulation of ATM in lymphocytes induced to proliferate, and a likely requirement for ATM in
these cells, could account for the failure of A-T cells to initiate an
appropriate response to specific stimuli during ontogeny of B and T
cells. Indeed, there is evidence for a defective PHA
response in some A-T patients.1,47 More recently,
accelerated cell death has been observed in PBMCs from an A-T patient
and large amounts of serum or added cytokines only partially protected these cells against cell death.48 These results agree with
the likely explanation for the immunodeficiency in A-T, ie,
developmental defects in both T and B cells.49
The immunofluorescence data suggest that ATM is predominantly present
in the nucleus in the PHA-stimulated cells and also in quiescent cells
by the pattern of immunofluorescence staining in a cell type where the
nucleus occupies most of the intracellular space. This is supported by
immunoblotting with freshly isolated lymphocytes where ATM was largely
present in the nucleus, but there was some evidence of microsomal
labelling.34 The detection of ATM in freshly isolated PBMCs
in that study does not conflict with our results. They used 2 × 108 PBMCs to fractionate for analysis of nuclear and
microsomal ATM and used 10% of the resultant nuclear fraction for
immunoblotting, at which levels a signal would be evident by
immunoblotting. Taken together, these results show that ATM is still
primarily a nuclear protein in quiescent PBMCs. There is also evidence
that a related member of the PI3-kinase family DNA-dependent protein
kinase, catalytic subunit (DNA-PKcs) is present at very low levels in nuclei from resting PBMCs.50 On stimulation of PBMCs with
PHA, the activity of DNA-PK increased markedly by 48 hours. However, unlike the ATM protein, which increases in cells in amount and activity
after PHA stimulation, the changes in DNA-PK were due to subcellular
translocation. In resting lymphocytes, full DNA-PK activity, compared
with that in stimulated cells, was detected in whole cell extracts as
opposed to that in nuclear extracts. Immunoblotting also showed
comparable amounts of DNA-PKcs protein in whole cell extracts from
resting and stimulated PBMCs. Thus, in proliferating lymphocytes, the
amount and activity of ATM increases, whereas the activity increase for
DNA-PK is due to translocation to the nucleus.50
The reduced extent of the p53 response in quiescent cells compared with
PHA-stimulated cells postirradiation is compatible with reduced or
absent ATM. As observed elsewhere, we could not detect p53 in
unstimulated PBMCs, but after exposure to PHA, there was an increase in
p53, which correlates with cellular DNA replication.51 Several reports have demonstrated a deficient p53 response in A-T cells
after exposure to ionizing radiation.27,28,52
Coimmunoprecipitation data show that ATM and p53 interact
directly,24 and it has been demonstrated recently that p53
is a substrate for ATM in this pathway.21,25,26 Stimulation
of PBMCs with PHA led to an increase in basal levels of p53 and a more
marked response to radiation exposure, reflecting the increase in ATM
in these cells. As is already established, ATM plays a key role in cell
survival postirradiation, suggesting that its absence or lower level in
quiescent lymphocytes might contribute to the increased
radiosensitivity of these cells compared with proliferating
lymphocytes.53,54 In addition, there is evidence that DNA
repair capacity increases in PHA-stimulated lymphocytes.47,55-57 These data may provide additional
insight into the reported differences in radiosensitivity between
resting and stimulated lymphocytes.
We have demonstrated here that ATM increases markedly as PBMCs enter a
proliferative stage. Thus, it might be expected that ATM will increase
in lymphoid cells as they respond to antigenic or other proliferative
signals during ontogeny. Equally important, in the absence of ATM,
failure to integrate and transmit these signalling events could result
in T-cell and B-cell developmental abnormalities characteristic of A-T
patients. Furthermore, lymphoid cells induced to proliferate in A-T
patients would retain the reduced capacity for DNA repair observed in
quiescent cells,47 share the radiosensitivity of quiescent
cells, and thus might be expected to be more vulnerable to cellular
damage leading to genome instability and cancer predisposition.
| |
FOOTNOTES |
Submitted September 10, 1998; accepted May 14, 1999.
Supported in part by a Grant in Aid for Science Research, Ministry of
Education, Science, Sports and Culture of Japan, the Ministry of Health
and Welfare's Primary Immunodeficiency Research Grant for Specific
Diseases, Japan, a Grant from "Science Promotion Foundation of Gifu
University School of Medicine", the Australian National Health and
Medical Research Council, and the A-T Children's Foundation, Boca
Raton, FL.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Toshiyuki Fukao, MD, PhD, Department of
Pediatrics, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu,
Gifu 500-8076, Japan; email: toshi-gif{at}umin.u-tokyo.ac.jp.
| |
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ABSTRACT
INTRODUCTION