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DNA Topoisomerase I-Targeting Drugs as Radiation Sensitizers

DNA Topoisomerase I-Targeting Drugs as Radiation Sensitizers

ABSTRACT: Combination chemoradiation, alone or as an adjuvant to surgery, has been shown to improve treatment outcomes in a number of human malignancies, but may be limited by normal tissue toxicities. A primary challenge in radiation oncology is the development of drugs that can selectively enhance the cytotoxicity of ionizing radiation against tumor cells. Mammalian DNA topoisomerase I is the major cytotoxic target of a number of newly developed anticancer drugs that have shown efficacy against solid tumors, including colon cancer, ovarian cancer, lung cancer, cancer of the head and neck, and pediatric cancers. Topoisomerase I-targeting drugs exert their cytotoxic effect by producing enzyme-mediated DNA damage, rather than by directly inhibiting enzyme catalytic activity. DNA topoisomerase I recently has been established as a biochemical mediator of radiosensitization in cultured mammalian cells by camptothecin derivatives. Interestingly, this sensitization appears to be schedule-dependent, cell cycle phase-specific, cell line-dependent, and not strictly dependent on drug cytotoxicity. Clinical chemoradiation trials using camptothecin derivatives are currently ongoing. Future studies aimed at better understanding the underlying mechanisms of molecular radiosensitization with topoisomerase I-targeting drugs are pivotal to the clinical application of these agents, as well as in guiding the development of more effective radiosensitizers. [ONCOLOGY 13(Suppl 5):39-46,1999]


Introduction

Combined-modality therapy with chemotherapy and
radiation therapy has gained increasing importance in the treatment
of human solid tumors.[1,2] In addition to controlling potential or
overt metastatic disease, a number of chemotherapeutic drugs also
enhance the cytotoxic effect of ionizing radiation [3,4] and help
locally control the primary disease. However, the efficacy and use of
currently available chemoradiation regimens are limited by their
toxicities to normal tissue. The development of more effective
chemoradiation regimens using radiosensitizers, which can
preferentially enhance the cytotoxicity of ionizing radiation in
cancer cells, remains a major challenge in radiation oncology.

DNA topoisomerase I is a novel cellular target of a number of
antineoplastic compounds,[5] including the camptothecin
derivatives[6-9] and the newly identified DNA minor groove-binding
ligands (MGBLs) such as Hoechst 33342.[10,11] Drug interference with
topoisomerase I-mediated cleavage rejoining of DNA strands is thought
to be the common mechanism of action of these drugs.[6-11] Instead of
direct inhibition of catalytic activity of topoisomerase I,
topoisomerase I drugs produce their cytotoxic effects by converting
an essential DNA topology-modifying activity into a DNA-breaking
poison, which damages DNA through interactions with cellular
processes such as DNA replication.[5,12-15] The presence of
up-regulated levels of topoisomerase I in tumor cells compared to
normal cells suggests a therapeutic advantage of topoisomerase I-targeting
drugs selective against slow-growing as well as rapidly
proliferating tumors.[16-18] Recent advances in the molecular
mechanisms of cytotoxic action and radiosensitization of DNA
topoisomerase I-targeting drugs offer a unique opportunity for
developing more effective chemoradiation therapy against human cancers.

DNA Topoisomerase I is an Essential Nuclear Enzyme

Only one type-I human DNA topoisomerase has been identified as a
molecular target of anticancer drugs. The human topoisomerase I,
encoded by a single-copy TOP1 gene on chromosome 20, is a monomeric
100 kDa protein. It relaxes both positively and negatively
supercoiled DNA and requires no energy cofactor for its
activity.[19-21] The activities of topoisomerase I are important for
many aspects of DNA metabolism, including the initiation and
elongation of RNA transcription, DNA replication, and the regulation
of DNA supercoiling, which is essential for maintaining the stability
of the genome.[19-21]

During a typical catalytic cycle, topoisomerase I cleaves the DNA
backbone, allowing the passage of DNA strands, and then reseals the
DNA backbone in two successive transesterification reactions. As
illustrated in Figure 1, a key
covalent topoisomerase I-DNA intermediate is formed between the
tyrosine-723 residue of the enzyme and a 3'-phosphate at the break
site during the transient DNA cleavage stage. The 5'-end of the
broken DNA appears to be held by noncovalent interactions within the
enzyme.[22,23] All the known topoisomerase I-targeting drugs damage
DNA by trapping this key covalent reaction intermediate, called the
“topoisomerase I cleavable complex.”[5]

Cytotoxic Mechanism of DNA Topoisomerase
I-Targeting Drugs

Camptothecin and its derivatives (Figure
2
) are the best characterized human topoisomerase I-targeting
drugs. Camptothecin was originally isolated from the tree Camptotheca
acuminata
. Its broad spectrum antitumor activity in animal
models, especially against colon tumors, led to clinical trials in
the early 1970s. However, trials were terminated due to observations
of excessive toxicity with the ring-open form of camptothecin
(camptothecin, sodium salt, NSC-100880). It is now clear that the
ring-open form of camptothecin is inactive against its molecular
target, topoisomerase I.[24]

Among camptothecin derivatives, topotecan is positively charged and
irinotecan (CPT-11[Camptosar]) is a prodrug that generates its active
metabolite SN-38 intracellularly via carboxyl esteration. Both
topotecan and irinotecan demonstrated efficacy in clinical
trials,[24,25] and in 1997 were approved by the US Food and Drug
Administration (FDA) for the treatment of recurrent colon cancer and
ovarian cancer, respectively. S-phase-specific
cytotoxicity,[12,13,15] selective cytotoxicity against tumorigenic
over nontumorigenic cells,[26,27] and the ability to overcome
MDR1-mediated drug resistance,[28,29] are features of camptothecin
derivatives that may contribute to their excellent anticancer activity.

Fork Collision Model

Our current understanding of the cytotoxic mechanism of camptothecin
is demonstrated by the fork collision model (Figure
3
), which was proposed based on studies both in cultured cells
and in cell-free extracts.[5] Upon binding of topoisomerase I to DNA,
two different reaction intermediates, the “noncleavable
complex” and the “cleavable complex,” are formed. In a
relaxation reaction in the absence of drugs, the cleavable complex
and the noncleavable complex are at equilibrium.

By inhibiting the rejoining step, topoisomerase I drugs perturb this
equilibrium by trapping a reversible topoisomerase I-camptothecin-DNA
ternary reaction intermediate, the topoisomerase I cleavable complex.
The perturbed equilibrium can be rapidly reversed by removing drug
molecules from the medium. Studies using cell synchronization
techniques and specific inhibitors of DNA polymerases indicate the
involvement of active DNA synthesis in the induction of the highly
S-phase-specific camptothecin cytotoxicity.[30,31] It is currently
hypothesized that the collision between the replication machinery and
the drug-trapped topoisomerase I cleavable complex leads to eventual G2-phase
cell-cycle arrest and cell death. A similar cytotoxic mechanism has
been proposed for some newly identified topoisomerase I-targeting
drugs, including the MGBLs Hoechst 33342 and Hoechst 33258.

Radiosensitization Mechanism of
Topoisomerase I-Targeting Drugs

Understanding the mechanism of interaction between topoisomerase
I-targeting drugs and ionizing radiation is a prerequisite toward
successful use of their combination in cancer treatment.
Controversial early studies using cultured cells and human xenografts
suggested that camptothecin derivatives modulate the cytotoxic
effects of ionizing radiation.[32-39] To answer key questions, such
as whether camptothecin derivatives are radiosensitizers and whether
DNA topoisomerase I is involved in such radiosensitization, we
conducted clonogenic survival assays using cultured mammalian
cells.[40] We found that drug incubation with camptothecin
derivatives radiosensitized log-phased human MCF-7 breast cancer
cells in a schedule-dependent manner (Table 1).[40] The
radiosensitization effect was observed when the cells were exposed to
drug treatment before or concurrent with radiation treatment, but not
after radiation treatment (Table 1).
The implication based on this observation is that camptothecin
derivatives should be administered before or concurrently with
radiation during chemoradiation clinical trials to optimize the
radiosensitization effect.

Stereo-Specific Interaction

Camptothecin derivatives interact with DNA topoisomerase I in a
stereo-specific fashion.[30] For example, assayed by the ability to
induce topoisomerase I-mediated DNA cleavage, the 20(S)-stereoisomer
of 10,11-methylenedioxycamptothecin is 10,000-fold more active than
its 20(R)-isomer (see Figure 4A).
This pair of camptothecin derivatives was used to investigate the
role of DNA topoisomerase I in mediating radiosensitization. As shown in
Figure 4B
, only the 20(S)-10,11-methylenedioxycamptothecin
radiosensitized human breast cancer MCF-7 cells. The prerequisite
role of such an intact stereo-specific interaction in the induction
of radiosensitization was further supported by the observation that
the mutant topoisomerase I-containing DC3F cells were relatively
resistant to radiosensitization.[40-42]

DNA Repair Inhibition

Some investigators have suggested DNA repair inhibition as a
mechanism of radiosensitization by camptothecin derivatives.[37] If
this theory is correct, a larger radiosensitizing effect should be
observed in cells that are growth inhibited (G0/G1
cells) to maximize their repair function for potentially lethal
damages.[37] We found that camptothecin only minimally enhanced the
cytotoxic effect of radiation in plateau phase cells, which were
arrested by growing to confluency, as well as in synchronized
G1-phase cells obtained by mitotic shake-off technique.[40] This
finding may indicate a possible therapeutic advantage of camptothecin
derivatives to radiosensitize actively proliferating cancer cells selectively.

The molecular mechanism of radiosensitization of DNA topoisomerase
I-targeting drugs remains to be defined. Based on available
information, a plausible mechanism of radiosensitization of
topoisomerase I drugs is proposed (Figure
5
). It is possible that induction of radiosensitization is
originally initiated by the topoisomerase I-trapped cleavable
complex. This drug-stabilized cleavable complex, with a concealed
single-strand DNA break, may be viewed as a “potentially
sublethal” DNA damage. Interaction with as yet undefined
cellular processes such as DNA replication, RNA transcription, and
DNA repair may transform such “potentially sublethal” DNA
damage into “sublethal” DNA damage. It is plausible that
such “sublethal” DNA damage could then be converted into
“lethal” DNA damage with the addition of radiation-induced
DNA damage.

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