Bulgar, A. D.

Donze, J. R.

Gerson, S. L.

Kirkland, E. B.

Li, Lan

Liu, Lili

Snell, M.

Xu, Yan

Yang, Shuming

English

EngagedScholarship @ Cleveland State University

Cleveland State University
EngagedScholarship@CSU
Chemistry Faculty Publications Chemistry Department
2010
Targeting Base Excision Repair Suggests a New
Therapeutic Strategy of Fludarabine for The
Treatment of Chronic Lymphocytic Leukemia
A. D. Bulgar
Cleveland State University
M. Snell
Case Western Reserve University
J. R. Donze
Case Western Reserve University
E. B. Kirkland
Case Western Reserve University
Lan Li
Cleveland State University
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Recommended Citation
Bulgar, A. D.; Snell, M.; Donze, J. R.; Kirkland, E. B.; Li, Lan; Yang, Shuming; Xu, Yan; Gerson, S. L.; and Liu, Lili, "Targeting Base
Excision Repair Suggests a New Therapeutic Strategy of Fludarabine for The Treatment of Chronic Lymphocytic Leukemia" (2010).
Chemistry Faculty Publications. 217.
https://engagedscholarship.csuohio.edu/scichem_facpub/217
Authors
A. D. Bulgar, M. Snell, J. R. Donze, E. B. Kirkland, Lan Li, Shuming Yang, Yan Xu, S. L. Gerson, and Lili Liu
This letter to the editor is available at EngagedScholarship@CSU: https://engagedscholarship.csuohio.edu/scichem_facpub/217
A.D. Bulgar, M. Snell, J.R. Donze, E.B. Kirkland, L. Li, S. Yang, Y. Xu, S.L. Gerson, L. Liu
1795
fragment (Figure 1b). As shown in Figure 1c, the production of
18 mer fragments, produced through UDG mediated excision
of F ara A, was proportionally increased with increasing
concentrations of HL60 cell extracts, demonstrating a direct
relationship between UDG activity and the levels of excised
F ara A. However, the UDG cleavage activity was abolished
when the cell extracts were pre incubated with uracil glycosy
lase inhibitor (Ugi), a bacteriophage encoded short polypeptide
that depletes UDG activity through the binding and formation of
a tight UDG:uracil glycosylase inhibitor complex (Figure 1d).
The results confirm that the cellular activity of UDG has an
important role in the removal of incorporated fludarabine
from DNA.
We next examined both UDG expression and activity in
primary CLL cells obtained from 18 patients. Interestingly, we
found that UDG was highly expressed in CLL cells. The levels of
both UDG protein (Figure 1e) and mRNA (Figure 1f) in CLL cells
were higher (6.7 and 4.3 fold, respectively) than that in normal
cells (lymphocytes and bone marrow cells). As expected, the
higher activity of UDG in CLL cells (Figure 1g) produced more
AP sites, which was proportionally increased with the concen
trations of fludarabine. In contrast, only a moderate increase in
Figure 1 UDG removes incorporated fludarabine from DNA. (a) Schematic diagram represents the preparation of oligonucleotide substrates
containing F ara A:Thymidine (F:T) mispairs and the cleavage reaction by DNA glycosylase/AP endonuclease (APE) when reacted with the
oligonucleotide substrates. (b) Oligonucleotide duplexes containing F:T were incubated with 5 DNA glycosylases (UDG, OGG (8-oxoguanine-
DNA glycosylase), TDG (thymine DNA glycosylase), AAG (Alkyl adenine glycosylase) and MutY (the Escherichia coli adenine glycosylase)) at
37 1C for 1 h, followed by incubation with APE at 37 1C for 30min. Reaction products were resolved by electrophoresis through denaturing 20%
polyacrylamide gels. UDG has activity to excise F Ade, but has no activity to cleave the normal substrates containing A:T. (c) Oligonucleotide
duplex containing F:T base pair was incubated with HL60 cell extracts (2.5 40mg) at 37 1C for 30min. (d) UDG activity in HL60 cell extract was
inhibited by uracil glycosylase inhibitor (Ugi was pre incubated with cell extracts at 37 1C for 30min). (e) CLL and normal bone marrow cell lysates
were subjected to western blot analysis with UDG specific antibodies. (f) Levels of UDG mRNA in CLL (n 18) versus normal lymphocytes and
bone marrow (normal donor (ND), n 14) samples were determined by quantitative reverse transcription PCR (nPo0.02). (g) Comparison of UDG
enzymatic activities of CLL and normal lymphocytes (nPo0.05). UDG activity was determined based on the cleaved products obtained after
oligonucleotide substrates were incubated with 5 mg cell extracts and quantified by using ImageQuant software (GE HealthCare, Piscataway,
NJ, USA). (h) The dose dependent relationship between the numbers of AP sites and the concentrations of fludarabine. CLL cells and normal
lymphocytes were cultured in complete RPMI 1640 containing 10% fetal calf serum, 2mM L glutamine, 100U/ml penicillin, and 100mg/ml
streptomycin (Invitrogen, Carlsbad, CA, USA). CLL and normal lymphocytes were treated with fludarabine alone (0 20mM) for 24 h. Genomic
DNA was extracted and AP sites were measured using aldehyde reactive probe reagent (nPo0.05). Results are representative of at least three
experiments. Ugi, uracil glycosylase inhibitor.
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AP site formation was detected in normal cells when treated
with high concentrations of fludarabine (Figure 1h). Owing to
the fact that AP site formation is the results of fludarabine
removed from DNA by UDG, the AP sites detected in CLL cells
indicate that fludarabine can actively incorporate into DNA.
Most importantly, these results support the findings that
subclonal cell populations of CLL are substantially replicating.7
In these cells, cellular BER mechanism presumably controls
the cytotoxic effect of fludarabine.
The identification of the role of UDG in the removal of
F ara A from DNA not only uncovers an important mechanism
responsible for the cellular resistance to fludarabine, but also
provides a rationale for fludarabine MX combinatorial thera
pies. MX has been shown to potentiate the cytotoxicity of
several alkylating therapeutic agents3,4 through the covalent
binding of an AP site. This biochemical reaction converts a
repairable AP site into a structurally modified AP site, which is
refractory to the lyase activity of AP endonuclease and poly
merase b, and consequently blocks the downstream effectors of
the BER pathway. The MX bound AP sites subsequently stall
DNA replication, induce severe metaphase chromosomal
aberrations (that is, chromosome fragmentation and sister
chromatin exchange events), and trigger apoptotic death.5,6
Consistent with previous results, the combination of fludar
abine and MX significantly induced DNA double strand breaks
quantified by the comet assay (Figures 2a and b). This result was
Figure 2 MX enhances cytotoxicity of fludarabine in leukemia cells. (a) Comet images assayed by neutral electrophoresis show that increased
DNA double stranded breaks detected in cells treated with combination of fludarabine and MX. HL60 cells were treated with fludarabine alone
(5mM) or fludarabine plus MX (3mM) for 4 h. (b) Tail length of the comet detected by neutral electrophoresis in HL60 cells after treatments with
fludarabine alone (0 20mM) and fludarabine plus MX (3mM, nPo0.05, compared with the treatment with fludarabine alone). (c) HL60 cells were
continuously treated with drugs (5 mM fludarabine alone and fludarabine plus 3mM MX) and collected at 24, 48 and 72h. Induction of expression
levels of gH2AX and topoisomerase IIa proteins was detected by western blotting. (d) The percentage of Annexin V positive cells induced by
fludarabine alone and fludarabine plus MX at 24, 48 and 72h (nPo0.02 compared with the treatment with fludarabine alone). (e) Induction of the
cleaved PARP and Bax proteins was detected by western blotting in cells after treatments with fludarabine alone and in combination with MX. No
changes in Bcl2 protein levels. (f). Induction of BER proteins was analyzed by western blotting in cells after drug treatment over 72 h. Samples
analyzed in experiments (c f) were cells collected from the same treatments: control (lane 1), fludarabine alone (24, 48 and 72h, lanes 2, 4, 6),
fludarabine plus MX (24, 48 and 72h, lanes 3,5,7), MX alone (72 h exposure, lane 8). Results are representative of at least three experiments.
APE, AP endonuclease; Pol b, polymerase b.
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supported by the finding that gH2AX, a well know marker of
DNA double strand break, was highly induced in cells treated
with the combination of fludarabine and MX, when compared
with treatment with fludarabine alone (Figure 2c). In agreement
with previous work,4 increased levels of gH2AX occurred
concomitantly with the upregulation of topoisomerase II a (topo
II a; Figure 2c), suggesting that MX bound AP sites act as a topo
II a poison, inducing topo II a mediated DNA double strand
break.6 Apoptosis analysis performed in HL60 cells revealed
that treatment with fludarabine alone resulted in low levels of
apoptotic cell death. A possible explanation for this is that the
p53 deficiency in HL60 cells results in decreased p53 dependent
apoptosis (Figure 2d). In contrast, cotreatment of fludarabine
with MX significantly increased the percentage of apoptotic cells
as evidenced by the level of Annexin V positive labeled cells.
At 72 h, B70% of cells were more likely to undergo apoptosis
(nPo0.02). The measurements of proteolytic cleavage of PARP
by caspases, a hallmark of apoptotic cell death, demonstrated a
time dependent apoptotic death, which was associated with the
induction of the proapoptotic protein Bax (Figure 2e) Altogether,
the data indicate that MX enhanced DNA lesions (MX bound
AP sites and double strand breaks) contribute to the propaga
tion of p53 independent apoptotic death. As p53 is the most
commonly mutated tumor suppressor in human cancer, this
finding highlights the importance of combined therapy with MX.
Indeed, various investigators have shown that B cell Chronic
Lymphocytic Leukemia and Acute Myeloid Leukemia patients
with mutations in p53 display fludarabine resistance.
The involvement of BER in response to the DNA damage
induced by the treatment with fludarabine alone or in
combination with MX was evident from the induction of several
BER proteins, including UDG, polymerase b and FEN1
(Figure 2f). UDG protein was significantly upregulated after
drug treatments, particularly in cells treated with the combina
tion treatment of fludarabine and MX. On the basis of previous
observation that UDG has a higher affinity for the products at
the AP site than the actual substrate,8 it has been proposed that
subsequent to a base release, UDG remains bound to the AP site
to protect the cell until the AP site is transferred to the
downstream BER pathway enzymes, AP endonuclease and
polymerase b. Although the upregulation of UDG in response
to fludarabine and MX occurs at the transcriptional level (data
not shown), it is also possible that the persistence of MX bound
AP sites results in the accumulation of UDG at un repairable
MX bound AP sites. In agreement with previous report,3 the
elevated polymerase b, the limiting factor for short patch BER,
and FEN1, the protein responsible for long patch BER process,
suggest that MX AP sites impact both short and long patch BER.
Although the mechanisms for the regulation of BER proteins by
fludarabine treatment are not yet clear, these results suggest that
BER proteins have an adaptive response to DNA damage that is
exaggerated by the combination of fludarabine and MX.
MX was capable of potentiating the antitumor effect of
fludarabine, in vitro and in vivo. In agreement with the
induction of apoptosis, a remarkable inhibition of cell growth
was observed by the combined treatment of fludarabine and MX
Figure 3 MX augments the chemotherapeutic effect of fludarabine in vitro and in vivo. (a) Growth inhibition induced by fludarabine and the
combination with MX in HL60 cells. HL60 cells were seeded in complete growth medium in a 6 well plate (Falcon, Becton Dickinson, Franklin
Lakes, NJ, USA) at a density of 5 105 cells per well. Cells were treated with fludarabine (5mM) or fludarabine plus MX (3mM). Cell viability was
determined by trypan blue exclusion every 24 h for 5 days. (b) MX enhanced antitumor effect of fludarabine in xenografts (HL60). (The arrowhead
indicates drug treatment daily, nPo0.05 compared with the treatment with fludarabine alone). (c) Cytotoxicity of fludarabine or fludarabine plus
MX in primary CLL cells detected by trypan blue exclusion. CLL cells were treated with 5 mM fludarabine and fludarabine plus 3mM MX. Cell
viability was expressed as percentage of untreated control. The results are representative of 10 primary CLL samples. (d) Comparison of the
cytotoxicities induced by fludarabine alone and the combination of fludarabine and methoxyamine in normal bone marrow cells assayed by
clonogenic formation.
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in both HL60 and CLL cells (Figures 3a and c). MX enhanced
fludarabine cytotoxicity by threefold (nPo0.05) when compared
with fludarabine treatment alone. In addition, MX potentiated
the therapeutic efficacy of fludarabine as evidenced in HL60
xenografts in nude mice. As shown in Figure 3b and a, moderate
sensitivity to fludarabine alone was observed in HL60 xeno
grafts; however, significant inhibition of tumor growth was seen
after the combined fludarabine plus MX treatment. At 17 days,
HL60 xenografts treated with PBS (control group) had a mean
tumor volume of 2500±106mm3 compared with a mean tumor
volume of 1456±76mm3 and 643±142mm3 (nPo0.05) in
mice treated with either fludarabine alone or with the combined
fludarabine plus MX treatment, respectively. These results
demonstrate that the disruption of the BER by MX increased
the magnitude of the response to fludarabine. MX alone did not
affect tumor growth, displaying a similar growth rate to that
observed in control group. At these doses, mice did not present
any evidence of toxicity as evaluated by measurements of body
weight and whole blood toxicity analysis. To further confirm
that MX enhanced cytotoxicity of fludarabine is associated with
the cellular levels of UDG and topo II a, normal bone marrow
cells were treated with fludarabine alone and in combination
with MX. Results showed that MX did not appear to increase the
cytotoxicity observed with fludarabine alone in normal bone
marrow cells, presumably, because these cells express lower
levels of both UDG and topo II a compared with tumor cells.7
These results strongly suggest that the induced killing effect of
the combination treatment of fludarabine and MX would be
selective toward tumor cells, which would be relatively
protective of normal bone marrow cells.
In summary, we demonstrate that (i) a new mechanism, the
BER pathway, is involved in processing incorporated fludar
abine, as well as mis incorporated uridine in DNA through the
enzymatic activity of UDG; (ii) MX binding of AP sites generated
directly and indirectly by fludarabine induces un repairable DNA
damage that can block the BER pathway; and (iii) MX potentiates
the therapeutic efficacy of fludarabine, allowing for the possibility
of a novel therapeutic strategy to combine inhibitors of BER with
fludarabine for clinical treatment. Targeting BER as a target based
therapeutic strategy can be extended to the combination of MX
with a number of other drugs that incorporate into DNA either by
acting as nucleotide analogs or through the manipulation of the
nucleotide pools.
Conflict of interest
SL Gerson & L Liu have conflict of interest. Tracon Pharmaceuticals
Inc licenses intellectual property of Methoxyamine (MX).
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Post-print standardized by MSL Academic Endeavors, the imprint of the Michael Schwartz Library at Cleveland State University, 2017.
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Targeting Base Excision Repair Suggests a New Therapeutic Strategy of Fludarabine for The Treatment of Chronic Lymphocytic Leukemia

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Targeting Base Excision Repair Suggests a New Therapeutic Strategy of Fludarabine for The Treatment of Chronic Lymphocytic Leukemia

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