Biology Reference
In-Depth Information
corresponding di- and triphosphate metabolites.
179
Since DNA repair requires cellular dNTPs, the depletion
of nucleotide pools can prevent the timely repair of
lesions formed from endogenous and exogenous agents,
including chemotherapeutic agents. This mechanism is
discussed more thoroughly using the pyrimidine
analog, gemcitabine, as an example (see section on
Self-Potentiating Activity of Gemcitabine below).
Another important cytotoxic effect of purine analogs
that is independent of direct inhibition of DNA
synthesis is through the induction of apoptosis caused
by the activation of caspases. A classic feature of
apoptosis is internucleosomal cleavage of genomic
DNA that occurs after caspase activation. One particular
caspase, caspase-3, is important by virtue of its ability to
be activated in the cytosol by dATP and cytochrome c.
180
Purine nucleoside analogs such as F-ara-ATP mimic
dATP and thus may function as surrogates to activate
dATP-dependent caspases to initiate apoptosis. Indeed,
treatment with F-ara-ATP causes the induction of
apoptosis both in cell culture systems and in primary
chronic lymphocytic leukemia (CLL) cells.
181
These
results are important as they provide a mechanism for
how nucleoside analogs such as F-ara-ATP cause cell
death in quiescent cells that are not actively undergoing
DNA synthesis.
F-ara-ATP by various human DNA polymerases has
been extensively studied.
170
e
175
Replicative polymerase
including pol
a
, pol
b
, pol
g
, and pol
3
, incorporate
F-ara-ATP after which DNA synthesis is inhib-
ited.
170
e
175
In vitro
studies reveal that the concentration
of F-ara-TP required to inhibit DNA synthesis by 50%
(IC
50
value) varies considerably across these human
DNA polymerases. For example, F-ara-ATP inhibits
pol
a
and pol
e
most potently, displaying
in vitro
IC
50
values of 1.6 and 1.3
m
M, respectively. Other polymer-
ases such as pol
b
and pol
g
are ~10-fold less sensitive
to F-ara-ATP and display IC
50
values of 24 and 44
m
M,
respectively. In all cases, the inhibitory effects of F-ara-
TP can be alleviated by increasing concentrations of
the natural nucleotide substrate, dATP, and validates
that F-ara-ATP competes with dATP for incorporation
opposite thymine in DNA. One incorporated into
DNA, F-ara-AMP is a poor substrate for subsequent
DNA elongation, making it an unusually effective chain
terminator. Quantitative analyses of DNA extracted
from cells incubated with [
3
H]F-ara-A reveal that F-
ara-AMP is present at terminal positions.
176
It is impor-
tant to note that the overall stability of F-ara-AMP at the
3
0
-terminus of DNA indicates that the analog is resistant
to the exonuclease to proofreading activities of pol
d
or
pol
3
that could remove the analog.
176
Another effect of F-ara-AMP on nucleic acid metabo-
lism is through the inhibition of DNA ligase I, an ATP-
dependent enzyme that plays an essential role in joining
DNA during DNA replication and repair.
177
DNA ligase
1 uses ATP to catalyze the formation of a phosphodiester
linkage from the 3
0
-hydroxyl and the 5
0
-phosphate of
adjacent deoxynucleotides. Human DNA ligase I is
inhibited by F-ara-ATP by two mutually exclusive mech-
anisms.
178
The most straightforward mechanism occurs
by F-ara-ATP competing with the binding of ATP to
prevent DNA ligation. The second mode is somewhat
more complicated and is dependent upon F-ara-AMP
incorporation into specific locations in DNA. Specifi-
cally, DNA ligase I cannot enzymatically join adjacent
pieces of DNA when F-ara-AMP is present as the 3
0
-
terminal nucleoside monophosphate. Collectively, the
ability of F-ara-AMP to inhibit DNA synthesis and
subsequent ligation causes inactivation of DNA
synthesis, and the inability of the cell
Clinical Activity of Fludarabine
as a Monotherapeutic Agent
Chronic lymphocytic leukemia (CLL), the most
common form of leukemia in the Western countries, is
a clonal disease characterized by proliferation and accu-
mulation of small CD5-positive B cells.
182
This form of
leukemia is most commonly diagnosed in the elderly,
exhibiting a median age of 65 years at diagnosis. Until
recently, the standard treatment of CLL patients
involved prednisone- or chlorambucil-containing
regimens.
183
While effective in generating modest
responses, these regimens suffer from low long-term
survival rates.
183
However, the development of purine
nucleoside analogs such as fludarabine has significantly
improved efficacy in treatment-na¨ve or pretreated
patients with CLL. Fludarabine is currently the most
effective and most extensively studied purine analog
used in hematological disorders such as CLL and indo-
lent B-cell malignancies.
184
Standard doses of fludara-
bine range between 25 and 30/mg/m
2
given over 30
minutes for five consecutive days. Plasma concentra-
tions of 3
m
M F-ara-A are achieved within 30 minutes,
and peak concentrations of F-ara-ATP are achieved
approximately 4 hours after the start of infusion.
185
Despite heterogeneity among individuals with respect
to rate
to complete
DNA replication induces apoptosis.
Other Cytotoxic Mechanisms of Fludarabine
In addition to directly inhibiting DNA synthesis,
nucleoside analogs can generate indirect effects on
nucleic acid metabolism. The predominant effect is by
depleting cellular nucleotide pools via the inhibition of
ribonucleotide reductase and various kinases involved
in the conversion of natural nucleoside to their
of F-ara-ATP accumulation, peak drug
