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RAPID COMMUNICATION
DNA-Dependent Protein Kinase Activity Correlates With Clinical and In Vitro
Sensitivity of Chronic Lymphocytic Leukemia Lymphocytes to Nitrogen Mustards
By Catherine Muller, Garyfallia Christodoulopoulos, Bernard Salles, and Lawrence Panasci
The objective of this study is to investigate the role of
DNA-dependent protein kinase (DNA-PK) in the chronic
lymphocytic leukemia (CLL) lymphocyte response to nitrogen mustard therapy. DNA-PK is a nuclear serine/threonine
kinase that functions in DNA double-strand break repair and
in the joining process in recombination mechanisms. In a
series of 34 patients with B-CLL, either untreated (n 5 16) or
resistant to chlorambucil (n 5 18), the kinase activity of the
complex, as determined by its capacity to phosphorylate a
peptide substrate in vitro, is increased in the resistant
samples as compared with the untreated ones (24.4 6 2.6
arbitrary units [a.u.] [range, 12.7 to 55.8 a.u.] versus 8.1 6 2.8
a.u. [range, 0.9 to 44.5 a.u.], respectively (P F .0001]),
independent of other clinical and biological factors. Linear
regression analysis shows an excellent correlation between
the level of DNA-PK activity and the inherent in vitro sensitivity of CLL lymphocytes to chlorambucil (r 5 .875, P 5.0001).
The regulation of DNA-PK activity was associated with
increased DNA-binding activity of its regulatory subunit, the
Ku heterodimer, in resistant samples. These results suggest
that this activity is a determinant in the cellular response to
chlorambucil and participates in the development of nitrogen mustard–resistant disease. The increase in DNA-PK
activity might contribute to the enhanced cross-link repair
that we previously postulated to be a primary mechanism of
resistance to nitrogen mustards in CLL.
r 1998 by The American Society of Hematology.
T
excision step of ICL repair is not rate-limiting in CLB-resistant
lymphocytes6,9,10 and suggest that the development of enhanced
repair of DNA cross-links in CLB-resistant CLL is likely to be
associated with an enhanced recombination pathway. In a
preliminary study, using a small sample of patients, we found an
increase in the DNA-dependent protein kinase activity in
resistant samples.11
DNA-dependent protein kinase (DNA-PK) is a recently
identified nuclear protein serine/threonine kinase comprising a
large catalytic subunit of 460 kD, DNA-PKcs, and a DNA
binding sub-unit, the Ku autoantigen (a dimer of the Ku 70 and
Ku 86 proteins). Ku binds to DNA double-strand ends and other
discontinuities in the DNA12-14 and recruits the catalytic subunit
of the complex.15 The active DNA-PK complex then acquires
the capacity, at least in vitro, to phosphorylate many DNAbound proteins in the vicinity.16 DNA-PK has been unequivocally identified as an important mammalian DNA repair complex involved in recombination processes.17 Mutations in either
DNA-PKcs or in the 86-kD subunit of Ku result in DNA
double-strand break (DSB) repair defects that manifest them-
HE NITROGEN MUSTARDS (NMs), mechlorethamine,
melphalan, chlorambucil, and cyclophosphamide, are
used in the treatment of a wide variety of cancers. Alkylation of
the DNA and, more importantly, the interstrand cross-linking of
DNA, is considered to be responsible for the toxicity of NMs.1-3
Although these drugs have been in use for over 30 years, factors
underlying in vivo–acquired resistance of human malignancies
to NMs are still poorly understood. In an effort to develop a
clinically applicable model, we have been studying the mechanisms of resistance to NMs in primary human tumor cells by
using B-cell chronic lymphocytic leukemia (B-CLL) as a
model. CLL is a disease characterized by the proliferation of
abnormal, developmentally regulated immature B cells that
accumulate in the peripheral blood of affected patients. The
nitrogen mustard, chlorambucil (CLB), is commonly used as
first-line therapy for CLL, with an initial response rate of 60%
to 80%, but the gradual development of resistance eventually
renders the drug ineffective.4 We have previously shown that
lymphocytes from treated-resistant patients have an enhanced
capacity to remove cross-links compared with those from
untreated patients.5 In addition, we recently observed that the
development of CLB resistance in CLL appears to be specifically associated with cross-resistance to other bifunctionnal
alkylating agents, which produces interstrand cross-links (ICL)
in DNA.6 Thus, enhanced ICL-specific repair appears to be one
of the primary mechanisms of NM resistance in CLL. DNA
interstrand cross-links and certain double-strand breaks need
information supplied by another chromosome or chromatid for
error-free repair. Based on the genetic and biochemical evidence from bacterial and yeast systems, it is thought that
cross-links are removed from the DNA of mammalian cells by
the combined actions of excision repair (NER) and recombination systems.7,8 It has been suggested that there may be at least
two recombinational mechanisms by which interstrand DNA
cross-links can be removed, which include the following: (1) an
intramolecular pathway operating throughout the cell cycle
requiring ERCC-1 and ERCC-4 plus possibly other proteins
from the nucleotide excision repair pathway; and (2) a second
intermolecular pathway operating between sister chromatids
after replication.8 However, our results suggest that the incision/
Blood, Vol 92, No 7 (October 1), 1998: pp 2213-2219
From the Institut de Pharmacologie et de Biologie Structurale (CNRS
UPR 9062), Toulouse, France; and the Lady Davis Institute for Medical
Research, The Sir Mortimer B. Davis-Jewish General Hospital, Montreal, Quebec, Canada.
Submitted February 3, 1998; accepted July 2, 1998.
C.M. and G.C. contributed equally to this investigation.
Supported by the Cancer Research Society, Montreal, Quebec,
Canada. L.P. is a recipient of the Gertrude and Stanley Vineberg
Clinical Scientist Award. C.M. is a recipient of a postdoctoral fellowship from the ‘‘Comité leucémie de la Fondation de France.’’
Address reprint requests to Lawrence Panasci, MD, Lady Davis
Institute for Medical Research, The Sir Mortimer B. Davis-Jewish
General Hospital, 3755 Côte Sainte Catherine, Montreal, Quebec,
Canada, H3T 1E2; e-mail: [email protected].
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.
r 1998 by The American Society of Hematology.
0006-4971/98/9207-0053$3.00/0
2213
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2214
MULLER ET AL
selves as x-ray sensitivity and impaired V(D)J recombination.18,19 In addition, previous reports showed that mutant cells
deficient either in DNA-PKcs or in the Ku DNA-end binding
activity also exhibit significant hypersensitivity to NMs (melphalan and mechlorethamine) and cisplatin.20,21 Furthermore, the
hypersensitivity of the rodent xrs6 cell line (which lacks the
86-kD subunit of Ku) to NMs appears to be related to the
DNA-PK defect because enhanced resistance to NMs was
regained along with bleomycin resistance and Ku DNA endbinding activity in a revertant cell line, xrs6rev.20 Similar
restoration of NMs sensitivity was observed in the xrs6/Ku80
cell line stably transfected with the human Ku 86 gene (C.M.,
unpublished results). In addition, we have found that cisplatinresistant L1210 murine cell lines exhibited cross-resistance to
ionizing radiation and to NMs, associated with an overexpression of the Ku 86 sub-unit (P. Frit, Y. Canitrot, J.M. Barret, P.
Calsou, and B. Salles, submitted for publication). Although its
precise mechanism of action remains unknown, these results
provide evidence in favor of a role for the DNA-PK complex in
regulating cell sensitivity to NMs.
The aim of the present study is to determine whether
regulation of DNA-PK activity is involved in the in vivo
development of CLB resistance in CLL. To test this hypothesis,
we examined the activity of DNA-PK in lymphocytes obtained
from 34 CLL patients either untreated or resistant to NM
therapy. Our results clearly show that changes in DNA-PK
activity correlate with CLB resistance, suggesting that DNA-PK
plays an important role in regulating tumor treatment response
to cross-linking agents as demonstrated both at the clinical and
in vitro levels.
MATERIALS AND METHODS
Patients. Thirty-four patients with a diagnosis of B-CLL according
to cytologic and immunologic criteria who were followed at the Jewish
General Hospital of Montreal between October 1993 and May 1997
were enrolled in the study after informed consent.22 Their main clinical
features are summarized in Table 1. Patients were classified into two
groups as previously described.23 Patients were either untreated (n 5 16)
or treated and resistant to nitrogen mustards (n 5 18). All the patients
with resistant CLL had received 4 to 6 mg of CLB daily for 6 months or
more. All the patients with resistant disease had previously responded to
CLB with the exception of two patients who showed de novo resistance.
In some cases, patients also received cyclophosphamide either alone (50
to 100 mg daily for 3 months or more) or in combination (CHOP
[cyclophosphamide, doxorubicin, vincristine, prednisone] and CVP
[cyclophosphamide, vincristine, prednisone] protocols).
Isolation of CLL lymphocytes and cell culture. Lymphocytes were
isolated from the peripheral blood of CLL patients by sedimentation
centrifugation on Ficoll Hypaque (Pharmacia, Uppsala, Sweden) as
previously described.6 Isolated lymphocytes were washed twice with
phosphate-buffered saline, pelleted by centrifugation at 500g, and either
resuspended in culture medium for MTT (3-[4,5-dimethylthiazol-2yl]2,5-diphenyl-tetrazolium bromide) cytotoxic assay (see below) or
stored as a pellet in liquid nitrogen before use for whole cell extract
preparation (see below). The percentage of contaminating T cells was
determined using fluorescence-activated cell sorting analysis with CD3
antibody. The mean T-cell contamination in our population was 4.4% 6
0.9% (SE), with a range of 1% to 8%. The percentage of contaminating
T cells was not statistically different between the untreated and resistant
samples (4.7 6 0.9 v 4.2% 6 0.9%, respectively; not significant [NS]).
The MO59J and MO59K cell lines24 were provided by Dr J.A. Turner
(Cross Cancer Institute, Edmonton, Canada). Cells were grown in
Dulbecco’s modified Eagle’s medium (DMEM; GIBCO-BRL, Grand
Island, NY) supplemented with 10% fetal calf serum, 2 mmol/L
glutamine, 125 U/mL penicillin, and 125 µg/mL streptomycin.
MTT cytotoxic assay. After the purification procedure, the B-CLL
lymphocytes were immediately resuspended in culture medium (RPMI
1640, 10% fetal calf serum, 20 mmol/L HEPES, 10 µg/mL gentamycin)
and the samples were screened for their sensitivity to CLB using the
MTT assay, as previously described.6
Cell extracts. Whole cell extracts were prepared as previously
described with slight modifications.25 Briefly, cell pellets were quickly
thawed and resuspended in extraction buffer (1 3 108 cells per 100 µL)
containing 50 mmol/L NaF, 20 mmol/L HEPES (pH 7.8), 450 mmol/L
NaCl, 25% glycerol, 0.2 mmol/L EDTA, 0.5 mmol/L dithiothreitol in
the presence of proteinase inhibitors (0.5 mmol/L phenylmethylsulfonyl
fluoride, 0.5 µg/mL apoprotin, 0.5 µg/mL leupeptin and 1.5 µg/mL
pepstatin), then frozen on dry ice and thawed at 30°C three times. After
centrifugation for 30 minutes at 4°C, supernatants were stored at
270°C. Protein concentrations were determined using the Bio-Rad
protein assay (Bio-Rad, Hercules, CA). The protein concentration
ranged from 7.4 to 39.0 mg/mL and was not significantly different
between the untreated and treated-resistant samples (13.4 6 2.0 versus
19. 0 6 2.8 mg/mL, P 5 .11).
DNA-PK activity. The DNA-PK ‘‘pulldown’’ kinase assay was
performed as previously described.25,26 For each sample, three kinase
assays were conducted in parallel: 1 in the absence of peptide, 1 in the
presence of a wild-type peptide (EPPLSQEAFADLLKK) that is a good
substrate for DNA-PK, and 1 in the presence of a mutated peptide
(EPPLSEQAFADLLKK) that is an ineffective DNA-PK substrate.
Values plotted are mean values of at least two experiments and were
derived as previously described27 by subtracting the value for no
peptide from the values for wild-type and mutated peptide and then
dividing these two resulting figures by the no peptide value. The results
are expressed as arbitrary units (a.u.). Consistent with previous work,24
we found that, whereas the radioresistant human malignant glioma
M059K cell line contains DNA-PK activity, the radiosensitive M059J
cell line does not (data not shown). In preliminary experiments, we
verified that no significant differences in DNA-PK activity were found
between extracts from fresh and frozen samples (data not shown).
In addition, we compared the phosphorylation of the wild-type and
mutated peptides from extracts that underwent the ‘‘pulldown’’ assay,
where protein binds selectively to double-stranded DNA-cellulose
beads, to extracts that were not subjected to the ‘‘pulldown’’ assay but
that were incubated in the presence of 10 µg/mL of salmon sperm DNA.
This was done with three untreated and three resistant samples. The
activity of the extracts that were not subjected to the ‘‘pulldown’’ assay
was 28.8% 6 2.9% of the activity determined with the pulldown assay.
In particular, there was no significant difference in the percentage of
activity between the untreated samples (32.4% 6 2.6%) and the
resistant samples (25.2% 6 1.3%).
Band shift assay. The double-stranded 25-mer DNA was prepared
as previously described.28 Briefly, radiolabeled DNA (4 ng, 100,000
cpm) was incubated with extracts (0.25 to 0.75 µg) in 20 µL of binding
buffer (10 mmol/L Tris-HCl pH 8.0, 1 mmol/L EDTA, 10% glycerol, 50
mmol/L KCl) at room temperature for 10 minutes. The samples were
electrophoresed on a 12% polyacrylamide gel at 4°C, for 2 hours at 100
V. The gel was dried on Whatman paper (Whatman, Maidstone, UK)
and exposed to x-ray film. Excised bands that corresponded either to
free probe or to DNA-protein complexes were quantified by scintillation counting. The Ku DNA-end binding activity was expressed as the
percentage of radioactivity that corresponded to the DNA-protein
complexes (cpm in DNA 2 protein complexes/[cpm in DNA 2
complexes 1 cpm in free probe] 3 100). To ensure quantitative
measurements of Ku DEB activity, we assessed in preliminary experiments that the results obtained in these experimental conditions are in
the linear range between 0.25 and 0.75 µg of protein extracts for the
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DNA-PK ACTIVITY AND NM SENSITIVITY IN CLL
2215
Table 1. Clinical Characteristics of the Patients
Patient
No.
Sex/
Age*
Lymphocyte
Count/µL
Stage†
80,700
28,800
83,100
76,200
31,000
49,900
48,300
58,500
40,200
53,500
29,800
136,400
49,900
35,600
42,000
110,400
B
B
B
B
C
A
A
B
A
A
A
B
A
A
B
B
Treatment
Received‡
Time Since
Last Treatment
DNA-PK
Activity§
Untreated patients
U1
U2
U3
U4
U5
U6
U7
U8
U9
U10
U11
U12
U13
U14
U15
U16
F/51
F/59
F/84
F/64
F/65
M/81
M/59
M/85
M/50
M/92
F/71
M/66
F/69
M/74
F/52
F/52
6.9
5.3
9.8
3.4
2.2
2.2
1.7
1.9
22.8
14.9
44.5
0.9
3.4
2.9
5.8
1.2
Treated patients with resistance to NMs
T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
T11
T12
T13
T14
T15
T16
T17
T18
M/53
F/78
F/59
F/87
F/82
F/72
F/56
F/60
M/63
M/86
F/76
F/70
M/54
M/76
M/69
M/77
M/66
M/52
57,500
233,500
74,800
77,800
32,700
128,100
102,100
158,500
104,700
58,100
127,300
61,900
127,300
167,800
398,500
88,400
31,300
56,700
C
C
C
C
C
C
B
C
C
B
C
B
C
B
C
B
B
B
CLB, CYC, CHOP
Fluda
CLB, CYC, Fluda
CLB
CLB, CYC
CLB
CLB, CYC
CLB, CYC
CLB, CYC
CLB
CLB
CLB
CLB
CLB, CVP, Fluda
CLB, CVP, Fluda
CLB
CLB, CYC, Fluda
CLB, CHOP CLB
12 mo
On treatment
6 mo
On treatment
On treatment
On treatment
On treatment
On treatment
3 mo
On treatment
On treatment
12 mo
1 mo
12 mo
3 mo
5 mo
On treatment
On treatment
19.0
13.0
13.4
16.9
12.7
32.5
28.3
26.3
16.2
28.8
55.8
35.6
32.3
20.7
19.4
25.6
34.9
14.9
Response to therapy to date for the untreated patients is as follows: U1, U2, U3, U6, U7, U8, U10, U11, U13, and U16 have not received any
treatment. U4 was treated for 81 months and had an excellent response to CLB (normalization of lymphocyte count, lymphadenopathy:
pretreatment node size of 3 to 4 cm v posttreatment node size of #0.5 cm). U5 and U12 were treated for 81 and 36 months, respectively, and had a
complete response to CLB (normalization of lymphocyte count, disappearance of lymphadenopathy, and no other evidence of disease). U9 had no
response to chlorambucil as previously defined24 (lymphocyte count .35,000 and no decrease in lymphadenopathy after 6 months of
chlorambucil treatment). U14 was treated with CLB for 3 months. He had an excellent response as evidenced by a normalization of his lymphocyte
count, disappearance of lymphadenopathy and splenomegaly, but his platelet count did not return to normal (95,000/µL). U15 did not tolerate CLB
treatment because of nausea and vomiting. She took 30% to 40% of the first two courses (CLB 20 mg and 12 mg every day 35 every 4 weeks). She
took only 1 day of the third course of CLB 4 mg daily. Despite this she showed a partial response as evidenced by a decrease in the size of a large
cervical lymph node mass (9 3 9 cm to ,3 3 3 cm).
*Sex: M, male; F, female; age in years.
†Staging is according to Binet et al.22
‡CYC, cyclosphosphamide; Fluda, fludarabine.
§DNA-PK activity is expressed in arbitrary units. For each sample, the results represent the mean of at least two experiments with variability
of ,15%.
control cell line MO59K cell line. For the CLL extracts, two different
protein concentrations were used (0.25 and 0.5 µg) for each experiment.
Western blot. Proteins were subjected to electrophoresis on an 8%
sodium dodecyl sulfate-polyacrylamide gel under reducing conditions.
The proteins were then electroblotted onto a nitrocellullose filter in a
Bio-Rad Trans-Blot chamber and detected as previously described.11
Antibodies used in Western blotting experiments were as follows. The
monoclonal antibodies (MoAbs) directed against human Ku 70 (MoAb
N3H10) and human Ku 86 (MoAb S10B1)29 were obtained from
Interchim (France), and used at a 1:1,000 dilution (secondary antibody
goat anti-mouse at a 1:2,500 dilution). The MoAb directed against the
catalytic subunit of DNA-PKcs antibody (MoAb 18-2, a gift from Dr T.
Carter, St John’s University, New York, NY)30 was used at a 1:2,500
dilution (secondary antibody goat anti-mouse at a 1:2,500 dilution).
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2216
MULLER ET AL
Statistical analysis. The P values for the group comparisons
(treated-resistant versus untreated) were obtained using an unpaired
t-test analysis. The r values for the correlation curves were obtained
using a simple linear regression analysis. All computations were
performed using StatView 512 V.12 (Calabasas, CA).
RESULTS
DNA-PK activity correlates with sensitivity of CLL lymphocytes to chlorambucil. In CLL extracts, the level of DNA-PK
activity varied considerably between the different samples
analyzed (see Table 1 for individual values). In the samples
obtained from resistant patients, the level of DNA-PK activity is
similar to that observed in a panel of established cell lines
extracts,11 whereas there is less activity in the majority of the
samples obtained from untreated patients. The mean DNA-PK
activity was found to be significantly higher in the resistant
samples compared with untreated ones (24.4 6 2.6 a.u. v 8.1 6
2.8 a.u., respectively, P , .0001) (Fig 1). No differences were
observed between the two groups regarding the incorporation
into the mutated peptide (Fig 1).
Previous clinical studies showed that 60% to 80% of the
patients with CLL will respond to CLB administered as
first-line therapy.4 Thus, it is expected that the vast majority of
the untreated patients included in our study will respond to this
drug if and when they are treated. Our laboratories have shown
previously, by using the MTT assay, that lymphocytes from
patients with B-CLL display in vitro sensitivity to nitrogen
mustards (chlorambucil and melphalan) that correlates with
their clinical status.6,11 Thus, to analyze the implication of
DNA-PK activity in the cellular response of B-CLL lymphocytes to nitrogen mustards, the relationship between this
activity and in vitro sensitivity to CLB was examined in 30 of
the B-CLL samples included in this study. As expected, the
treated-resistant population (n 5 15) was found to be 3.3-fold
more resistant to CLB than the untreated one (n 5 15) with a
Fig 1. DNA-PK activity in CLL samples. Whole cell extracts (50 mg)
derived from CLL lymphocytes obtained either from untreated or
treated-resistant patients were tested for DNA-PK activity using
standard DNA-PK microfractionation/peptide assays in the presence
of wild-type (j) or mutated (¢) p53 peptide as indicated in Materials
and Methods. The panel represents the mean activity (6SE) for the
two groups of samples. *P value as determined by using an unpaired
Student’s t-test.
Fig 2. Correlation of DNA-PK activity with CLB sensitivity as
determined in vitro. CLL lymphocytes were screened for CLB sensitivity in vitro by using the MTT assay as described in Materials and
Methods, and the IC50 for this drug was compared with DNA-PK
activity. Each point represents the result from an individual patient’s
sample. (h), Lymphocytes obtained from untreated patients; (j),
lymphocytes obtained from treated-resistant patients. (Four patients
were not included in this analysis bacause in vitro sensitivity to
melphalan was done instead of chlorambucil.)
mean IC50 of 23.5 6 4.7 µmol/L (range, 5.8 to 74 µmol/L)
versus 7.2 6 2.4 µmol/L (range, 1.5 to 40 µmol/L), respectively,
P 5 .002. However, there are variations in CLB sensitivity
within the group of untreated patients because some patients
might be de novo resistant to CLB. Linear regression analysis
shows a highly significant correlation between the level of
DNA-PK activity and in vitro CLB sensitivity (P 5 .0001;
Fig 2). Six of the patients in the untreated group have now been
treated with CLB. One patient who exhibited elevated DNA-PK
activity (U9) and who was treated with CLB is resistant to this
treatment as previously defined,23 while five other untreated
patients with low DNA-PK activity responded to chlorambucil
treatment (see legend of Table 1). The level of DNA-PK activity
did not correlate with the age of the patients (P 5 .7), the
lymphocyte count of the blood samples (P 5 .4), the percentage
of contaminating T cells in purified cell population (P 5 .98), or
the protein concentration of the extracts (P 5 .68). In addition,
disease duration was not a significant variable associated with
the level of DNA-PK activity (P 5 .2). Moreover, when the
group of previously treated patients was considered, the level of
DNA-PK activity was not different between the patients that
were on therapy (n 5 10) and those that were off therapy
(n 5 8) (26.1 6 4.1 a.u. v 22.2 6 2.9 a.u., respectively, P 5.2).
For the patients that were off therapy, the DNA-PK activity did
not correlate with the time since last treatment was received
(P 5 .7).
Regulation of DNA-PK activity in CLL extracts. We then
examined the mechanism(s) involved in the regulation of
DNA-PK activity in the CLL samples. First, we showed that the
DNA-PKcs protein was expressed at similar levels between the
different samples (Table 2). Second, the DNA-end binding
activity was determined by electrophoretic mobility shift assays
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DNA-PK ACTIVITY AND NM SENSITIVITY IN CLL
2217
Table 2. Western Blot Analysis
Samples
DNA-PKcs
Ku 86
Ku 70
Untreated
Treated-resistant
P value determined
by t-test
1.22 6 0.11
1.16 6 0.11
1.18 6 0.16
3.19 6 0.82
1.12 6 0.13
3.11 6 0.19
NS
P , .00001
P , .00001
Ku 86, Ku 70, and DNA-PKcs protein levels were determined in
B-CLL lymphocytes obtained from untreated and treated patients with
resistance to NMs. Data were normalized to a-tubulin in order to
account for loading differences and expressed relative to normalized
U1 sample extracts which was assigned the arbitrary value of 1 for the
Ku 86, Ku 70, and DNA-PKcs expression.
because the DNA end-binding (DEB) activity of the Ku
heterodimer is the early limiting step for the formation of an
active DNA-PK complex. In human cell lines, Ku represents the
major double-strand (ds) DEB protein and its activity can be
easily detected by using ds DNA fragments in an electrophoretic mobility shift assay (EMSA). It has been shown that a 25to 30-bp dsDNA fragment is the minimum length required for
the binding of a single Ku heterodimer.13 As previously
described,28 we used a 25-mer ds probe for EMSA analysis. As
shown in Fig 3, a unique DNA-protein complex that corresponded to Ku DEB activity (complex H) is detected in the
control cell line extracts. In some cases, when the CLL extracts
were considered, a second complex with faster electrophoretic
mobility is observed (see complex L, lanes 3 and 4). We have
previously shown that this complex corresponded to the expression in these cells of a variant form of the Ku 86 protein.11
Although the Ku 70/ Ku 86 variant heterodimer binds to DNA
ends, this altered form of the Ku heterodimer has a decreased
ability to recruit the catalytic subunit, DNA-PKcs, and contrib-
Fig 3. Ku DEB activity in CLL extracts as determined by EMSA.
Protein extracts (0.5 mg) obtained from purified B-CLL cells were
incubated with 32P-labeled 25-bp DNA probe in the presence of 1 mg of
closed circular plasmid DNA, as a nonspecific competitor. The electrophoretic mobility of the protein-DNA complexes were analyzed in a
12% polyacrylamide gel as described in Materials and Methods.
The positions of protein-DNA complexes consisting of DNA-end
binding activity are indicated (H, heterodimer that corresponds to
full-length Ku 70/full-length Ku 86 subunits; L, heterodimer that
corresponds to full-length Ku 70/variant Ku 86 subunits). U, untreated
CLL lymphocytes; T, treated-resistant CLL lymphocytes; C, MO59K
control cells.
utes to a negative regulation of the kinase activity of the
complex.28,31 The expression of this altered form of the Ku
heterodimer was detected in 4 of the 16 samples obtained from
untreated patients (U5 , U7, U8, and U12), as shown in Fig 3 for
samples U7 and U12 (lanes 3 and 4). In addition, we verified by
Western blot that the variant form of the Ku 86 protein is
expressed in these four samples (data not shown). In these
samples, the expression of the altered form of the heterodimer
was associated with low DNA-PK activity (mean, 1.67 a.u.; see
Table 1 for individual values) and high sensitivity to CLB
(mean IC50, 2.4 µmol/L). Interestingly, the altered form of the
Ku heterodimer was not detected in samples obtained from
treated-resistant patients or in the patients that exhibited de
novo high levels of DNA-PK activity.
The Ku DEB activity was quantitated in 32 samples of our
series (see Materials and Methods). As for DNA-PK activity,
Ku DEB activity was significantly higher in the treated-resistant
samples as compared to the untreated ones (57.6% 6 3.5% v
22.8% 6 3.3%, P 5.0001). These results suggest that an
increase in the level of Ku DEB activity contributes to the
observed increase in DNA-PK activity. Indeed, as shown in Fig
4, in the 32 samples tested, a linear correlation was observed
between the Ku DEB activity and the kinase activity of the
complex (P 5 .0001), supporting the concept that regulation of
the activity of the complex occurs primarily at the Ku level.
Furthermore, the Western analyses confirms an increase in
Ku70/Ku86 protein levels but not in DNA-PKcs (Table 2).
DISCUSSION
To the best of our knowledge, the implication of DNA-PK in
regulating the response of primary tumor specimens to treatment (radiotherapy or chemotherapeutic treatment with NMs)
has never been characterized, although this activity is likely to
play a role in regulating cell sensitivity to these cytotoxic
agents, as shown in mutant cell lines.20,21 Our present data show
that, in CLL samples, DNA-PK activity is increased in samples
that exhibited a phenotype of resistance to NMs determined
either both in vivo and in vitro or in vitro only for those
untreated patients that have not started therapy at the time of the
analysis. In addition, the excellent linear correlation between
DNA-PK activity and in vitro CLB toxicity strongly suggest
that DNA-PK is likely to contribute to this phenotype. In fact,
the higher DNA-PK activity observed is unlikely to reflect a
transient induction by the treatment itself because the increase
in DNA-PK activity with a corresponding decrease in CLB
sensitivity in vitro was observed even in patients that were not
actively receiving therapy at the time of the sampling (8 of the
18 resistant samples analyzed and 3 of the 16 previously
untreated patients). Moreover, the level of DNA-PK activity
was not different between the resistant patients that were on
therapy compared with those who had interrupted treatment. In
accordance, no changes in the levels of Ku polypeptides or
DNA-PK activity have been detected after treatment of established cell lines with genotoxic compounds.24,32 The increase of
DNA-PK activity in CLB-resistant samples is unlikely to be
caused by the presence of a subpopulation of actively dividing
cells. Previous studies have suggested that Ku expression may
be dependent on the proliferative status of the cells.33-35
However, despite dramatic changes in Ku mRNA expression,
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2218
Fig 4. Ku DEB activity according to the two groups of samples. (A)
The Ku DEB activity was determined by EMSA experiments and
bands that corresponded to the Ku-DNA complexes were excised and
counted by scintillation as described in Materials and Methods. For
each sample, the results represent the mean of at least two experiments with variability of less than 10%. (B) Correlation of Ku DEB
activity with the DNA-PK kinase activity as determined for 32
samples. Each point represents the result from an individual patient’s
sample. (h), Lymphocytes obtained from untreated patients; (j),
lymphocytes obtained from treated-resistant patients.
the total level of Ku has been reported to be only slightly (about
twofold) increased in proliferative versus quiescent cells.33,35 In
CLL, even in advanced disease, cell-cycle analysis consistently
showed that most malignant cells are in G0/G1 phase and very
few are in S phase.36,37 Our results suggest that a higher
DNA-PK activity is a marker for the presence of resistant cells
to NMs in the tumor cell population, and this activity appears to
play a determining role in the resistance phenotype.
In our study we have shown that DNA-PK activity is
positively regulated in cells that exhibited a resistant phenotype
to CLB. Our results also provide the basis for a new approach to
understanding the mechanisms that contribute to the development of drug resistance in CLL. Indeed, it is noticeable from our
results that most leukemic extracts obtained from untreated
MULLER ET AL
patients exhibited very low levels of DNA-PK activity (see Fig
1 and Table 1). Thus, resistance in CLL may be simply a state in
which tumor cells lose an abnormal sensitivity to alkylating
agents, rather than a process in which they gain an abnormal
insensitivity. Our results show that in CLL, changes in DNA-PK
activity appear to be regulated through a variation in the Ku
heterodimer DEB activity, but also, in some samples, potentially through the expression of an altered form of the heterodimer. Interestingly, we recently showed that the normal
human peripheral blood CD191 B lymphocytes express solely
this altered form of the Ku heterodimer that is no longer able to
recruit the catalytic component of the complex when bound to
DNA.31 Thus, our present results extend our previous reports11,31 and show that in B-CLL lymphocytes its expression
may be persistent despite the acquisition of a malignant
phenotype in some, but not all, samples. Independent of the
expression of this altered form of the Ku heterodimer, our
results show that the kinase activity of the complex is regulated
through the activity of its regulatory sub-unit Ku, because the
level of the kinase activity of the complex observed in CLL
samples correlated with the level of Ku DEB activity (see
Fig 4). In addition, we verified by Western blot that the level of
expression of the catalytic subunit of the complex was not
increased in the resistant samples with elevated levels of kinase
activity. Taken together, these results show that regulation of Ku
expression and function plays a pivotal role during the acquisition of resistance to NMs therapy. This regulation contributes to
the increased activity of the kinase of the complex as Ku
DNA-binding represents the predominant mechanism for
DNA-PK activation.15,25
In conclusion, although uncertainties remain concerning its
precise mechanism of action, our results strongly suggest that
Ku/DNA-PK activity contributes to resistance to NM therapy in
B-CLL. Because its appears that Ku/DNA-PK is tightly regulated in normal lymphoid tissues, it would be of interest to
determine if this activity is involved in the response of other
tumor cell types to this class of drugs or if these results are
restricted to tumors of lymphoid origin. Finally, to the extent
that enhanced kinase activity does contribute to resistance, it
should be possible to improve the efficacy of nitrogen mustards
against currently resistant tumors by inhibiting this activity.
Accordingly, results obtained in our laboratory show that
wortmannin, a nonspecific inhibitor of DNA-PK activity, is able
to potentiate CLB toxicity in resistant samples.38 Thus, our
findings point to new possibilities to improve the effectiveness
of NM therapy.
ACKNOWLEDGMENT
The authors thank Dr P. Calsou for critical reading of the manuscript.
We also thank Franka Sicilia for excellent technical support in T-cell
typing.
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1998 92: 2213-2219
DNA-Dependent Protein Kinase Activity Correlates With Clinical and In
Vitro Sensitivity of Chronic Lymphocytic Leukemia Lymphocytes to
Nitrogen Mustards
Catherine Muller, Garyfallia Christodoulopoulos, Bernard Salles and Lawrence Panasci
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