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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 7 (October 1), 1998:
pp. 2213-2219
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
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.
 |
ABSTRACT |
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
= 16) or resistant to chlorambucil (n = 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 ± 2.6 arbitrary units [a.u.] [range,
12.7 to 55.8 a.u.] versus 8.1 ± 2.8 a.u. [range, 0.9 to 44.5 a.u.], respectively (P < .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 = .875, P =.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.
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INTRODUCTION |
THE 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/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 DNA-bound 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 themselves 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 end-binding 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
cisplatin-resistant 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.
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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 = 16) or treated and resistant to nitrogen mustards (n = 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-2-yl]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% ± 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 ± 0.9 v 4.2% ± 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 × 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  70°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 ± 2.0 versus 19. 0 ± 2.8 mg/mL, P = .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% ± 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% ± 2.6%) and the resistant samples
(25.2% ± 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 protein complexes/[cpm in DNA complexes + cpm in free probe] × 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 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).
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).
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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 ± 2.6 a.u. v 8.1 ± 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).
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| Fig 1.
DNA-PK activity in CLL samples. Whole cell extracts (50 µg) 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 ( ) or mutated ( ) p53 peptide as indicated in Materials
and Methods. The panel represents the mean activity (±SE) for the two
groups of samples. *P value as determined by using an unpaired
Student's t-test.
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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 = 15) was found to be 3.3-fold more
resistant to CLB than the untreated one (n = 15) with a mean IC50 of 23.5 ± 4.7 µmol/L (range, 5.8 to 74 µmol/L)
versus 7.2 ± 2.4 µmol/L (range, 1.5 to 40 µmol/L),
respectively, P = .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 = .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 = .7), the lymphocyte count of the blood samples (P = .4), the
percentage of contaminating T cells in purified cell population (P = .98), or the protein concentration of the extracts
(P = .68). In addition, disease duration was not a significant
variable associated with the level of DNA-PK activity (P = .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 = 10) and those that were off therapy (n = 8) (26.1 ± 4.1 a.u. v 22.2 ± 2.9 a.u.,
respectively, P =.2). For the patients that were off therapy,
the DNA-PK activity did not correlate with the time since last
treatment was received (P = .7).
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| 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. ( ), Lymphocytes obtained from untreated patients;
(), 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.)
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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 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 25- to 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 contributes 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.
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| Fig 3.
Ku DEB activity in CLL extracts as determined by EMSA.
Protein extracts (0.5 µg) obtained from purified B-CLL cells were
incubated with 32P-labeled 25-bp DNA probe in the presence
of 1 µg 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.
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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% ± 3.5% v 22.8% ± 3.3%, P =.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 = .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).
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| 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. (),
Lymphocytes obtained from untreated patients; (), lymphocytes
obtained from treated-resistant patients.
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| |
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, 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 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 CD19+ 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.
| |
FOOTNOTES |
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: gchris1{at}po-box.mcgill.ca.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
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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Abstract
Introduction
Abstract