BETHESDA, MarylandAn understanding of how camptothecins
intrude between topoisomerase I and DNA is helping to refine the anticancer
activity of these drugs, and studies of the pathway from initial camptothecin
binding to final cell death may help unravel the mechanisms behind drug
resistance. Yves Pommier, MD, PhD, chief of the National Cancer Institute’s
Laboratory of Molecular Pharmacology reviewed recent developments in these
areas at the Vanderbilt University Symposium.
DNA topoisomerase inhibitors are among the most commonly used
anticancer and antibacterial drugs, Dr. Pommier noted. Camptothecins are
topoisomerase I inhibitors. "Topoisomerases can also be inhibited by DNA
damage and by chemotherapeutic alterations of DNA, such as DNA strand breaks,
abasic sites, mismatches, and base damage," he said.
Where Camptothecins Bind
Dr. Pommier explained that camptothecins all work in basically
the same wayby interfering with DNA religation through production of a
cleavable topoisomerase I-camptothecin complex. The basic camptothecin
structure presents many potential sites for creation of new derivatives, and
several of these are under active clinical study.
Understanding where camptothecins bind in the topoisomerase
I-DNA complex is important for understanding and perhaps refining their
mechanism of action, Dr. Pommier noted. This question has been explored through
analysis of topoisomerase I mutations that lead to camptothecin resistance.
"These mutations are clustered at the enzyme-DNA
interface," Dr. Pommier said. These studies have enabled researchers to
construct an image of exactly how camptothecin fits into the topoisomerase
I-DNA complex, which in turn has suggested new targets for new drug
Once this docking occurs, reversible topoisomerase I cleavage
complexes are converted to permanent DNA damage by replication fork collisions.
These can occur either during replication or during transcription and involve
ubiquitination (binding to ubiquitin) and degradation of the DNA (Figure 1).
The time course of the resulting apoptosis varies depending on
tumor type. Recent studies of camptothecin effects in different tumor cell
lines point to caspase dependence and requirement of protein synthesis as
important factors in delaying cell death in some treated cancers.
"Protein synthesis and caspase activity are required for
camptothecin-induced DNA fragmentation in HT29 colorectal cancer cells,"
Dr. Pommier said. Delayed apoptosis in these cells is due to delayed activation
of caspase-3 and to degradation of poly (ADP-ribose) polymerase induced by
Fas and Fas-ligand (Fas-L) also come into play. Dr. Pommier
said that after treatment with camptothecin, there is an accumulation of FasL
and of Fas just before onset of apoptosis in colorectal cancer cell lines.
Camptothecins also affect expression of p53, which mediates production of other
proteins important in the apoptotic response, such as BAX, Bcl-X2, and p21.
"Fas-dependent cell killing induced by camptothecins is
independent of Fas ligand," Dr. Pommier said. "We now believe that
camptothecin-induced apoptosis, at least in HT29 colorectal cancer cells,
involves the following pathway: Camptothecin produces DNA damage mediated by
topoisomerase I. This leads to alterations in macromolecular synthesis of BAX,
Fas, and perhaps Fas ligand. The upregulation of BAX and Fas activate caspase.
The result is apoptosis."