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Experimental Therapeutics |
Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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JNK1 is also activated by DNA-damaging agents such as UV and ionizing radiation (17, 18, 19, 20) , whereas cell types that are resistant to the lethal effects of radiation do not exhibit JNK1 activation (18 , 19) . Similar activation of JNK1 has been demonstrated in cells responding to DNA adducts after treatment with the alkylating agents mitomycin C and cisplatin (21 , 22) and to DNA strand breaks associated with the topoisomerase inhibitors camptothecin and etoposide (10 , 21) . In addition, JNK1 and p38 are activated in response to inhibition of DNA replication caused by the nucleoside analogue ara-C (23, 24, 25) . Thus, diverse types of DNA damage can serve as initial signals for the activation of JNK1.
Nucleoside analogues are agents that induce selective DNA damage in that they act only upon incorporation into DNA and therefore are specific for S-phase cells (26) . F-ara-A, the nucleoside of the therapeutic agent fludarabine, specifically interferes with DNA replication (27 , 28) and repair (29 , 30) . The most extensively characterized action of fludarabine involves the misincorporation of its triphosphate F-ara-ATP into DNA, resulting in termination of DNA strand elongation (27) . Furthermore, F-ara-A-terminated DNA fragments resist excision repair by the 3'-5' exonuclease activity associated with DNA polymerases (31) , and ligation to adjacent DNA strands is inhibited (32) , thus creating a situation recognized by the cell as DNA damage.
Although it has been demonstrated that the c-abl tyrosine kinase functions upstream to JNK1 in the cellular response to ara-C and ionizing radiation (17 , 23) , the precise cascade of kinases involved in the DNA damage-induced activation of JNK1 is unclear. Furthermore, the consequences of this activation on nucleoside analogue-induced cell death remain unknown. Therefore, the objective of this investigation was to assess the importance of the JNK1 signaling pathway in F-ara-A-induced apoptosis. Specifically, we sought to determine: (a) whether the JNK1 signaling cascade was activated in response to distress stimuli initiated by incorporation of F-ara-ATP into DNA; and (b) whether the JNK1 signaling pathway facilitated F-ara-A-initiated DNA damage signals into a cell death response.
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MATERIALS AND METHODS |
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Quantitation of Intracellular F-ara-ATP and Its Incorporation
into DNA.
Quantitation of cellular F-ara-ATP and its incorporation into DNA was
done as described previously (27)
. Briefly, cells were
incubated with 3 or 10 µM F-ara-A for various times,
washed with cold PBS, and treated with 0.4 N perchloric
acid to extract nucleotides. The intracellular F-ara-ATP was
quantitated by HPLC analysis. To determine the incorporation of F-ara-A
into DNA, ML-1 cells were incubated with 3 and 10 µM
[3
H]F-ara-A for the indicated times and washed
twice with ice-cold PBS. Cells were then lysed in 10 mM
Tris-HCl (pH 7.4), 100 mM NaCl, 25 mM EDTA, and
0.5% SDS containing 2 mg/ml proteinase K for 12 h at 50°C,
extracted with phenol and chloroform, and precipitated in ethanol. The
precipitated nucleic acids were then dissolved in a final volume of 1
ml of TE buffer [10 mM Tris-HCl (pH 7.8), 1 mM
EDTA]. An aliquot of the total nucleic acid sample was treated with
RNase A (50 mg/ml) at 37°C for 2 h and then extracted with
phenol and chloroform. DNA was precipitated twice with three volumes of
ethanol, dissolved in water, and quantitated in terms of UV absorbance
at 260 nm. The [3
H]F-ara-AMP content in DNA was
measured by liquid scintillation counting. These data were used to
calculate the pmol of F-ara-AMP/mg of DNA. Furthermore, using the
equation 1 x 1012 pmol = 6.02 x 1011 molecules, we
calculated the number of molecules of F-ara-AMP incorporated into the
DNA under experimental conditions. The percentage of cells in S-phase
was determined by flow cytometry (
40%) and then used to calculate
the number of molecules of F-ara-AMP/S-phase cell in each experiment.
Pulsed-Field Gel Electrophoresis.
To detect high molecular weight DNA fragmentation, 1–2 x 106 cells were incubated as described
(26)
with 3 or 10 µM F-ara-A for the
indicated times and then washed once with PBS (pH 7.0). The cells were
embedded in 0.6% agarose plugs containing 75 mM NaCl, 5
mM EDTA, and 5 mM Tris-HCl (pH 7.8). The plugs
were allowed to solidify at 4°C for 20 min and then incubated in
lysis buffer containing 1% Sarkosyl, 50 mM EDTA, 50
mM Tris-HCl (pH 7.8), and 0.2 mg/ml proteinase K at 45°C
for 16 h. The plugs were analyzed by pulsed-field gel
electrophoresis (CHEF-DR II; Bio-Rad Laboratories, Richmond, CA) at 200
V with a switch time of 50 s for 16 h at 7°C in an
electrophoresis buffer containing 50 mM Tris-borate (pH
8.2) and 1 mM EDTA. After electrophoresis, the gel was
stained with ethidium bromide and photographed. Large DNA fragments
were quantitated by densitometric scanning of the negative films.
TUNEL Assay.
Cells were incubated with 3 or 10 µM F-ara-A for various
times, washed twice with cold PBS, fixed in 1% paraformaldehyde for 20
min on ice, and stored in 70% ethanol. The cells were analyzed for
apoptosis using the APO-DIRECT system. Briefly, cells were stained to
simultaneously assess DNA nicks by TUNEL assay and DNA content by
propidium iodide uptake according to the manufacturer’s instructions
and then analyzed by flow cytometry using an Epics Profile II flow
cytometer (Coulter Electronics, Miami, FL). The percentages of
TUNEL-positive and -negative cells were obtained by standard analysis
techniques using Elite software (Coulter Electronics). The cell cycle
profiles were obtained using Multicycle software (Phoenix Flow Systems,
San Diego, CA). The TUNEL-negative regions were further divided into
analysis regions to quantitate the percentage of cells in
G1, S, and G2-M phases of
the cell cycle.
Inhibition of JNK1 by Forskolin and Examination of Apoptotic Cell
Morphology.
Forskolin has been demonstrated previously to inhibit JNK1 in a time-
and dose-dependent fashion (33
, 34)
. ML-1 cells were
pretreated with 10 µM forskolin for 2 h and then
treated with 10 µM F-ara-A for an additional 3.5 h.
The cells were then centrifuged onto glass slides, fixed in methanol,
and stained with Wright-Giemsa stain. Using a Nikon HFX-II microscope,
cell morphology was examined, and the numbers of cells undergoing
apoptotic changes such as nuclear condensation, fragmentation of
nuclei, and rupture into debris were quantified.
Inhibition of JNK1 by Antisense Oligonucleotides and Examination
of Apoptotic Morphology.
JNK1 sense [SnJNK1 (sense oligonucleotide to JNK1 mRNA),
5-ATCATGAGCAGAAGCAAGCGTGAC-3] and antisense [AsnJNK1 (antisense
oligonucleotide to JNK1 mRNA), 5-GTCACGCTTGCTTCTGCTCATGAT-3]
oligonucleotides were synthesized under phosphorothioate-modified
conditions and purified by HPLC (Life Technologies, Inc., Gaithersburg,
MD). These sequences represent the amino acid codons 1 to 7 of JNK1
(10)
. The oligonucleotides were dissolved in 30
mM HEPES (pH 7.0) and added into culture media. Continued
exposure to either oligonucleotides proved nonspecifically toxic to
ML-1 cells; therefore, U937 myeloid leukemia cells were pretreated with
100 µM SnJNK1 or 100 µM AsnJNK1
oligonucleotides for 72 h and then exposed to 10 µM
F-ara-A for 6 h and centrifuged onto glass slides. Cells showing
apoptotic morphology were scored as described above.
Protein Kinase Assays.
Cells (1 x 107) were harvested,
and nuclear and cytosolic fractions were collected as described
(17
, 19)
. Nuclear JNK1 activities were immunoprecipitated
by anti-JNK1 antibodies. The precipitates were washed twice with lysis
buffer, twice with LiCl buffer [500 mM LiCl, 100
mM Tris-Cl (pH 7.6)] and twice with kinase buffer [20
mM 4-morpholinepropanesulfonic acid (pH 7.2), 2
mM EGTA, 10 µM MgCl2, 1
mM DTT, 0.1% Triton X-100, and 0.1 mM
Na3VO4]. The pellets were
mixed with 2 µg of GST-c-jun-1–79(1–79), 15 µM cold ATP,
and 5 µCi of [
-32P]ATP in 30 µl of
kinase buffer for 20 min at 30°C. The reaction was terminated with an
equal volume of Laemmli sample buffer, and the products were resolved
by 10% SDS-PAGE and autoradiography. The relative kinase activities
were normalized to the amounts of immunoprecipitated JNK1 assayed by
immunoblotting and visualized by chemiluminescence. Normalized JNK1
activity in control cultures was set at a value of 1.
Immunoblot Assays.
Cell lysates (30 µg protein) were resolved by 10% SDS-PAGE and then
electrophoretically transferred onto Immobilon P nitrocellulose
membranes (Millipore). After blocking with 4% nonfat dry milk in PBS-T
(PBS with 0.05% Tween 20) for 1 h, the membranes were probed
variously with antibodies specific to JNK1, actin, phospho-c-jun or
PARP for 1 h. The membranes were washed in TBS-T and incubated
with horseradish peroxidase-conjugated goat antirabbit for 1 h
before visualization using an enhanced chemiluminescence detection
system (Amersham International). The relative expression of JNK1 was
quantitated using a densitometer and normalized to the value obtained
for actin within the same samples.
Analysis of DNA Fragmentation.
Cells (5 x 106) were harvested,
washed, and incubated in 50 µl of 50 mM Tris-HCl (pH
8.0), 10 mM EDTA, 0.5% SDS, and 0.5 mg/ml proteinase K
(Sigma) for 6 h at 50°C. The samples were then incubated with 50
µl of 10 mM EDTA (pH 8.0) containing 2% (w/v)
low-melting-point agarose and 40% sucrose for 10 min at 70°C. The
DNA was separated in 2% agarose gels. After treatment with RNase, the
gels were visualized by UV illumination.
Statistical Analyses.
Quantitative data were averaged and expressed as the mean ± SE from at least three separate experiments. Differences among
groups were statistically analyzed first by one-way ANOVA and then by
Bonferroni’s post hoc test. Comparisons between two
experimental groups were based on the two-tailed t test.
P < 0.01 was considered significant.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Thus, the phosphorylation of F-ara-A to its triphosphate
occurred in a time- as well as dose-dependent manner in ML-1 cells. In
parallel, DNA was extracted from similarly treated cells to quantitate
the amounts of F-ara-AMP incorporated into DNA and to calculate the
number of analogue molecules per S-phase cell. The incorporation of
F-ara-AMP into DNA was time and concentration dependent during the
first 2 h (Fig. 1B)
. Subsequently, similar levels were
achieved in each culture, suggesting that F-ara-AMP incorporation was
self-limiting.
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and
approached 50% of the total population by 4 h. In contrast, it
took 4 h of exposure to 3 µM F-ara-A for
TUNEL-positive cells to appear. The relative constancy of the
G1 and G2 populations over
this relatively brief time course (data not shown) is consistent with
the conclusion that S-phase cells were specifically affected by F-ara-A
treatment (Fig. 1C)
. Incorporation of between 3 and
6 x 105 molecules of F-ara-AMP
into DNA appeared to be essential for triggering apoptosis. Thus,
F-ara-A-induced damage to the DNA of actively replicating cells
appeared to initiate distress signals that finally resulted in the
apoptotic death specifically in the S-phase population.
Activation of JNK1 in F-ara-A-induced Apoptosis.
The effect of F-ara-A on the kinase activity associated with JNK1 was
quantitated to determine the action of the analogue on this signaling
pathway. There was a visible change in JNK1 phosphorylation status
(Fig. 2A
, lower blot) in cells treated with 10 or 30
µM F-ara-A that was accompanied by robust
activation of its kinase activity (Fig. 2, A
, upper
blot and B). In contrast, cells incubated with 1 or 3
µM F-ara-A showed no discernable change in
electrophoretic mobility and correspondingly exhibited much less JNK1
activation at 2 h. Examination of the time course of JNK1
activation demonstrated that in cells treated with 10
µM F-ara-A (Fig. 3A
, upper blot) JNK1 activity was initiated by
1.5 h, was nearly maximal by 2 h, and was sustained
thereafter. In contrast, 3 µM F-ara-A elicited
initial JNK1 activation at 2.5 h, which continued to increase over
time. Again, there was a visible change in the JNK1 phosphorylation
status that coincided with activation of the protein kinase (Fig. 3A
, middle blot). This indicated that the
temporal activation of JNK1 was commensurate with the amounts of
analogue incorporated into DNA at that time. Once activated, JNK1
phosphorylates c-jun, among other targets. c-jun is phosphorylated on
multiple sites, notably on Ser-63 and Ser-73, which increases its
transactivating potential and DNA binding activity (36)
.
Therefore, we assessed the levels of endogenous c-jun that was
phosphorylated in response to activation of JNK1. Exponentially growing
ML-1 cells demonstrated a low basal level of c-jun phosphorylation.
Exposure to 3 µM F-ara-A resulted in a 2-fold
increase in the levels of p-Ser-63-c-jun after 2 h, whereas cells
exposed to 10 µM F-ara-A demonstrated this
increase starting at 1.5 h (Fig. 3A
, lower blot).
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1 h (Fig. 3B)
.
Activation of JNK1 Is a Specific Signaling Response to
Incorporation of F-ara-AMP into DNA.
To determine whether nucleotide analogue incorporation is required for
the initiation of signaling to apoptosis, exponentially growing ML-1
cells were pretreated for 24 h with the DNA synthesis inhibitor
aphidicolin prior to incubation with 10 µM F-ara-A for
2.5 h. Exposure of cells to aphidicolin inhibited DNA synthesis by
>98% (data not shown). This allowed the analogue triphosphate to be
formed but prevented the nucleotide analogue from being incorporated
into the DNA. Consequently, the aphidicolin-pretreated cells did not
activate JNK1 (Fig. 4A
, upper blot); in contrast, ML-1 cells treated
with 10 µM F-ara-A alone showed a 50-fold
increase in the levels of activated JNK1 (Fig. 4A
,
upper blot). Blocking F-ara-A incorporation into DNA with
aphidicolin also inhibited the induction of apoptosis (Fig. 4B)
; in contrast, cells treated with 10
µM F-ara-A alone for 2.5 h exhibited
significant levels of high molecular weight DNA fragmentation (Fig. 4B)
. Thus, JNK1 activation is a specific signaling response
induced by incorporation of F-ara-A into DNA.
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In our first approach, ML-1 cells were pretreated with 10
µM forskolin for 2 h before incubation
with 10 µM F-ara-A for an additional 2 h.
Consequently, cells treated with forskolin alone and cells pretreated
with forskolin before treatment with F-ara-A did not activate JNK1
(Fig. 5A)
. In contrast, cells exposed to F-ara-A alone showed a
35-fold increase in the levels of JNK1 activity (Fig. 5A)
.
Blocking F-ara-A-induced activation of JNK1 with forskolin also had a
profound effect on apoptosis, as demonstrated by several parameters. As
DNA fragmentation assays demonstrated, cells treated with forskolin
prior to treatment with F-ara-A and cells treated with forskolin alone
did not undergo DNA fragmentation (Fig. 5B)
, whereas cells
treated with 10 µM F-ara-A exhibited
internucleosomal sized DNA fragments (Fig. 5B)
.
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. In contrast, cells treated with F-ara-A alone exhibited
the cleaved Mr 85,000 form of PARP
(Fig. 5C)
.
On microscopic examination of the cells for apoptotic morphology such
as nuclear condensation, fragmentation of nuclei, and presence of
cellular debris, 25 ± 2% of the F-ara-A-treated cells
showed signs of apoptosis (Fig. 5D)
versus only
5 ± 0% of the cells pretreated with forskolin and then
subsequently treated with F-ara-A (P < 0.01;
Fig. 5D
).
The second approach aimed at depleting JNK1 protein levels by targeting
its mRNA with antisense oligonucleotides. U937 myeloid leukemia cells
were pretreated for 72 h with 100 µM sense
(SnJNK1; as a nonspecific control) or antisense oligonucleotides
(AsnJNK1) directed specifically against JNK1 mRNA. As immunoblot assays
revealed, the total level of JNK1 protein in the cells treated with
AsnJNK1 decreased by 80% when compared with the levels in lysates from
either control cells or those treated with SnJNK1 (Fig. 6A
, upper gel). The levels of actin in these cells
were also compared to confirm the specificity of the antisense
oligonucleotide against JNK1 (Fig. 6A
, lower
gel). Furthermore, immunoprecipitates from cells treated with
either F-ara-A alone or with SnJNK1 and then with F-ara-A showed a
12–14-fold increase in the kinase activity of JNK1. In contrast, there
was no demonstrable JNK1 activity in immunoprecipitates from cells
pretreated with AsnJNK1 and then treated with 10
µM F-ara-A (data not shown). The consequences
of inhibiting JNK1 activation on the morphological appearance of
F-ara-A-induced apoptosis were then examined. Changes in morphology
typical of cells undergoing apoptosis were seen in 21 ± 4% of the cultures treated with F-ara-A alone and in 14 ± 3% of the cells pretreated with SnJNK1 before being exposed to
F-ara-A. In contrast, pretreatment of cells with AsnJNK1 reduced the
incidence of F-ara-A-induced apoptosis to only 2 ± 2%
(Fig. 6B)
. Thus, pretreatment with AsnJNK1 inhibited
F-ara-A-induced apoptosis by 88% when compared with treatment with
F-ara-A alone (P < 0.01) and by 85% when
compared with pretreatment with SnJNK1 and then treatment with F-ara-A
(P < 0.01). Thus, the data presented in
Figs. 5
and 6
indicate that inhibition of JNK1 activation caused cells
to become resistant to the cell death process induced by the
incorporation of F-ara-A into DNA.
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. Under the same conditions, nuclear lysates were tested
for their ability to phosphorylate GST-jun and showed no quenching of
JNK1 activation (Fig. 7B)
. Taken together with the finding
that cells compromised in their ability to activate JNK1 did not
exhibit PARP cleavage or DNA fragmentation (see Fig. 5, C and B
), events that usually occur downstream of caspase
activation, these results indicated that the JNK1 signaling cascade was
upstream to that of the caspases in the apoptotic program.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, and JNK1 activation
followed 30 min later (Fig. 3A)
. Treatment of cells with 3
µM F-ara-A produced a proportionally slower
accumulation of F-ara-ATP (Fig. 1A)
and incorporation into
DNA (Fig. 1B)
. However, once incorporation had reached
levels similar to those in cells treated with 10
µM analogue (3–6 x 105 molecules/S phase cell), JNK1 was also
activated within 30 min (Fig. 3A)
. Thus, there appears to be
a temporal linkage between this level of analogue incorporation and the
initiation of JNK1 signaling. This in turn suggests that a certain
amount of drug has to be incorporated into DNA before JNK1 can be
activated. Whether incorporation of the drug into specific replicons
has a role in initiating the cascade of DNA damage signals that result
in the activation of JNK1 remains to be determined. The time between
F-ara-A addition and JNK1 activation reflects the time required to
accumulate F-ara-ATP, its incorporation into DNA, and the recognition
of DNA damage. Although the nature of the DNA damage is likely to
involve termination of nascent DNA chains by the nucleotide analogue
(27)
, further work will be required to identify the sensor
molecules involved in the initial damage recognition process.
JNK1 is classically activated by cellular stresses that depend upon
membrane-initiated signaling cascades (4
, 13
, 14
, 38)
.
However, because F-ara-A-induced apoptotic stimuli are generated within
the nucleus (26)
, it was important to establish whether
JNK1 activation by F-ara-A was a specific signaling response to
F-ara-AMP incorporation into DNA or a generic cellular stress response.
Consistent with previous reports using various nucleoside analogues in
other cell lines (26)
, our experiments with F-ara-A
demonstrated that incorporation of drug into DNA was the critical event
required to initiate apoptosis. Inhibition of DNA synthesis by
aphidicolin, an agent that is not incorporated into DNA and does not
affect F-ara-ATP accumulation, did not activate JNK1 or induce high
molecular weight DNA fragmentation. When incorporation of F-ara-A into
DNA was inhibited by pretreating ML-1 leukemia cells with aphidicolin,
subsequent treatment with F-ara-A failed to either activate JNK1 or
induce high molecular weight DNA fragmentation (Fig. 4)
. Thus,
incorporation of F-ara-AMP into DNA was essential for JNK1 activation.
Two lines of evidence support the conclusion that JNK1 activation is required as a facilitator during F-ara-A-induced apoptosis:
(a) we used forskolin to inhibit JNK1 in ML-1 cells.
Subsequent treatment of these cells with F-ara-A failed to activate
JNK1 (Fig. 5A)
or to initiate apoptosis as measured by DNA
fragmentation (Fig. 5B)
, by cleavage of the downstream
caspase-3 target PARP (Fig. 5C)
, and by scoring for
apoptotic morphology (Fig. 5D)
. These results support the
conclusion that, after F-ara-A-induced DNA damage, a functional JNK1
signaling pathway appears to be necessary to translate distress signals
into cell death. These results differ from a previous report in which
cells transfected with a dominant-negative mutant of c-jun, one of
several downstream targets of JNK1, did not acquire resistance to
ara-C-mediated toxicity (39)
.
(b) The levels of JNK1 protein significantly decreased when
we treated U937 cells with antisense oligonucleotides directed against
JNK1 mRNA as opposed to sense JNK1 oligonucleotides (Fig. 6A)
. Although lysates from the cells treated with AsnJNK1
had a residual level of JNK1 protein, subsequent treatment of these
cells with F-ara-A caused no demonstrable JNK1 activation, as measured
by in vitro kinase assays (data not shown). Concordantly,
when the cells were challenged with F-ara-A after antisense treatment,
there was a significant inhibition of F-ara-A-induced apoptosis (Fig. 6B)
. In contrast, the percentage of apoptotic cells in
cultures treated with sense oligonucleotides prior to the addition of
F-ara-A did not differ significantly from cultures treated with F-ara-A
alone (P > 0.01). Furthermore, MCF-7 cells
coexpressing green fluorescent protein (GFP) as well as
dominant-inactive mutant of JNK1 exhibited resistance to
F-ara-A-induced apoptosis in comparison to cells expressing green
fluorescent protein and the vector
alone.4
Although the exact transcriptional targets of the JNK1 pathway in nucleoside-analogue induced apoptosis remain unknown, apoptosis induced by certain types of DNA damage in T-cell lines depends on the JNK1-mediated transcriptional up-regulation of the Fas ligand (40) . However, in our studies, ML-1 cells treated with anti-Fas antibody did not undergo cell death (data not shown), indicating that the Fas-mediated apoptotic pathway is not active in this cell line. In addition, the susceptibility of U937 leukemia cells to certain cytotoxic drugs was shown to be independent of the Fas receptor/ligand (3) . Therefore, up-regulation of Fas ligand expression is not a likely target of the JNK1 pathway during F-ara-A-induced apoptosis in these myeloid leukemia cell lines.
Caspases have roles in initiating as well as executing cellular
disassembly during apoptosis (41
, 42)
. For instance, they
regulate apoptosis by specifically cleaving a variety of substrates,
some of which help protect cells from apoptosis. Such substrates
include the antiapoptotic proteins bcl-2 or
bcl-XL (43)
, an inhibitor of caspase
activated DNA fragmentation factor ICAD (44)
and DNA-PK
(45)
. Caspases also directly contribute to apoptosis by
cleaving proteins required for the maintenance of subcellular integrity
(46)
. In our experiments with the pan-caspase inhibitor
FK009, cells treated with this agent did not exhibit high molecular
weight DNA fragmentation in response to F-ara-A (Fig. 7A)
.
This is consistent with earlier published reports that demonstrate that
DNA fragmentation factor (47
, 48)
, a protein species that
functions downstream to caspase-3, is responsible for the DNA
fragmentation seen during apoptosis. However, cells pretreated with
z-VAD-fmk continued to activate JNK1 in response to F-ara-A, although
DNA fragmentation was blocked (Fig. 7B)
. This suggests that
the JNK1 cascade functions upstream of the caspases. This conclusion is
supported by our finding that inhibition of JNK1 led to an inhibition
of PARP cleavage in ML-1 cells (Fig. 5C)
, indicating a lack
of activation of caspase-dependent protein degradation.
In summary, we have shown that: (a) F-ara-A incorporation into DNA is associated with the time- and dose-dependent activation of JNK1; (b) despite its effects on deoxynucleotide triphosphate pools and RNA synthesis, incorporation of F-ara-A into DNA is essential to activate JNK1 as well as trigger apoptosis; (c) the JNK1 signaling pathway acts upstream of the caspases in F-ara-A-induced apoptosis; and (d) activation of the JNK1 signaling pathway facilitates DNA damage-initiated distress stimuli into apoptotic cell death.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by Grant CA28596 from the
Department of Health and Human Services, Grant DHP-1 from the American
Cancer Society, and a research grant from Schering AG, Berlin,
Germany. 
2 To whom requests for reprints should be
addressed, at Department of Experimental Therapeutics, Box 71, The
University of Texas M. D. Anderson Cancer Center, 1515 Holcombe
Boulevard, Houston, Texas 77030. Phone: (713) 792-3335; Fax:
(713) 794-4316; E-mail: wplunket{at}mdanderson.org
3 The abbreviations used are: JNK1, c-Jun
NH2-terminal kinase 1; ara-C,
1-ß-D-arabinofuranosylcytosine; TUNEL, terminal
deoxynucleotidyl transferase-mediated dUTP nick end labeling; F-ara-A,
9-ß-D-arabinosyl-2-fluoroadenine; FK009, z-VAD-fmk; PARP,
poly(ADP-ribose) polymerase; HPLC, high-performance liquid
chromatography.
4 D. Sampath and W. Plunkett, unpublished
observations.
Received 11/10/99. Accepted 9/18/00.
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