Activity of a Novel Camptothecin Analogue, Homocamptothecin, in Camptothecin-resistant Cell Lines with Topoisomerase I Alterations

CPT²derivatives are among the most promising anticancer drugs recently introduced in the clinic. Topotecan (Hycamtin; SmithKline Beecham) has been approved for the treatment of cisplatin-refractory ovarian carcinoma and for second-line therapy in small cell lung cancer. Irinotecan (CPT-11, Camptosar; Pharmacia) has been approved in the United States for the treatment of colorectal cancer. Current clinical trials indicate that CPT derivatives will be useful in a variety of other human malignancies. DNA top1 inhibitors such as CPT and its derivatives convert top1 into a cellular poison by inhibiting the religation step of the DNA nicking-closing reaction and thereby trapping top1 in a covalent complex with DNA. The cytotoxic lesions probably result from stable top1 covalent complexes associated with double-strand breaks that are generated after collision of DNA and RNA polymerases with the top1 cleavage complexes (for review, see Ref.1). hCPT is a novel CPT analogue that differs from CPT by the presence of an additional methylene group in the E-ring. Thus, hCPT has a seven-member β-hydroxylactone E-ring (Fig. 1; Ref. 2). This modification enhances the stability of the lactone ring, which, in the case of CPT and its derivatives, opens to form an inactive carboxylate derivative at physiological pH (3, 4). Thus, hCPT has a slow and irreversible hydrolysis of the E-ring instead of the fast equilibrium observed in the case of CPT. This enhanced stability may account for the superior activity of hCPT in vivo and in the presence of purified top1 (2, 5). In addition, this E-ring modification decreases the drug binding to human serum albumin (3).

The different mechanisms of resistance to top1 inhibitors can be grouped into three categories (1): (a) the precleavage complex mechanisms related to drug metabolism and uptake;(b) top1 alterations that result in reduced levels of cleavage complexes; (c) the postcleavage complex mechanisms related to the multiple pathways leading to cell death, including apoptosis, cell cycle regulation and checkpoints, and DNA repair (for review, see Ref. 1). In this report, we examine the activity of hCPT in CPT-resistant cell lines with top1 alterations to evaluate the role of top1 in the cytotoxicity of the drug and the effect of known top1 point mutations on the activity of hCPT against top1.

Human prostate carcinoma DU-145, leukemia CEM cells and their CPT-resistant subclones, DU-145/RC1 (1) and CEM/C2(6, 7), were cultured in RPMI 1640 (Life Technologies,Inc., Gaithersburg, MD) containing 10% FCS in a 5%CO₂ incubator at 37°C. The CPT-resistant DU-145 subline DU-145/RC1 was established by Dr. Panayotis Pantazis and colleagues (Brown University, Providence, RI). Murine leukemia P388 and its CPT-resistant subclone, P388/CPT45, were provided by Michael R. Mattern and Randall K. Johnson (SmithKline Beecham) and cultured in RPMI 1640 containing 20% FCS and 10 μmβ-mercaptoethanol (8, 9). The Chinese hamster lung fibroblast DC3F cell line and its CPT-resistant subline, DC3F/C10(10), were grown in MEM with Earle’s salt, supplemented with 10% FCS, 0.1 mm nonessential amino acids, and 1 mm sodium pyruvate (ABI, Columbia, MD). CPT was obtained from the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, National Cancer Institute (Rockville, MD). hCPT was kindly provided by Drs. Dennis Bigg and Olivier Lavergne (Institut Henri Beaufour, Les Ulis, France). Other drugs were purchased from Sigma (St. Louis, MO). [α-³²P]dGTP was purchased from New England Nuclear (Boston, MA). Human top1 was purified from Sf9 cells by using a baculovirus construct(11).

The MTT assay was used to determine drug sensitivity. Cells(3,000–15,000) were seeded as a suspension (100 μl/well) in 96-well microtiter plates. The cells were incubated at 37°C in the continuous presence of drug for 3 or 5 days. Cell viability was assayed by adding of 50 μg of MTT dye (in PBS). After a 4-h incubation period, during which activated cells reduced the yellow MTT salt to purple formazan,the stain was eluted into the medium by the addition of 100 μl of 2-propanol (containing 0.04 N HCl) or DMSO. Optical densities were measured at 550 nm. Determinations for all experiments were made in triplicate, and the results were expressed as means and SDs.

The method used is a modification of a method described previously(6). Briefly, log-phase cultures containing 10⁷ cells were washed twice at 4°C using nucleus buffer (150 mm NaCl, 1 mmKH₂PO₄, 5 mmMgCl₂, and 1 mm EGTA) and recovered by centrifugation at 200 × g for 10 min. Cell pellets were resuspended in nucleus buffer containing 0.03%Triton X-100. After incubation at 4°C for 10 min, nucleus pellets were recovered by centrifugation at 3,000 × g for 10 min. After washing with ice-cold nucleus buffer, pellets were recovered by centrifugation at 3,000 × g for 10 min. Salt extraction of the nuclear pellet was achieved by adjusting the final NaCl concentration to 0.35 m and by gentle mixing at 4°C for 30 min. After centrifugation at 12,000 × g for 30 min,supernatants containing salt-soluble material were collected as nuclear extract.

Top1-mediated cleavage sites were mapped in the 161-bp PvuII-HindIII fragment of pBluescript (pSK)plasmid (Stratagene, La Jolla, CA; Ref. 12). The fragment was 3′-end labeled by a fill-in reaction. Briefly, linearized pSK (200 ng) was incubated with [α -³²P]dGTP in 1×labeling buffer [0.5 mm dATP, dCTP, and dTTP in 50 mm Tris-HCl (pH 8.0), 100 mm MgCl, and 50 mm NaCl]in the presence of 0.5 unit of the Klenow fragment of DNA polymerase I. Labeled DNA was purified by phenol chloroform extraction followed by ethanol precipitation and resuspension in water. For cleavage assays,labeled DNA (approximately 50 fmol/reaction) was incubated with purified top1 or nuclear extract for 30 min at 25°C with or without drug in 1× reaction buffer [10 mm Tris-HCl (pH 7.5), 50 mm KCl, 5 mmMgCl₂, 0.1 mm EDTA, and 15μg/ml BSA]. Reactions were stopped by adding 0.5% SDS (final concentration), and DNA was ethanol precipitated and resuspended in loading buffer [80% formamide, 45 mm sodium hydroxide, 1 mm sodium EDTA, 0.1% xylene cyanol,and 0.1% bromphenol blue (pH 8.0)]. Reaction products were separated in 7% denaturing polyacrylamide gels (7 m urea)in 1× Tris-borate EDTA for 2 h at 40 V × cm at 50°C. Imaging and quantitations were performed by using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Purified top1 and labeled pSK fragment (see above) were reacted for 30 min with 1 μm CPT or hCPT. DNA cleavage was reversed by heating samples to 50°C or by adding 0.25 mNaCl (final concentration) and keeping the samples at 25°C for the indicated times. Reactions were stopped with 0.5% SDS (final concentration), and DNA was ethanol precipitated and resolved by 7% denaturing PAGE.

Because hCPT is a known top1 inhibitor, we evaluated whether CPT-resistant mammalian cells with well-characterized top1 alterations were cross-resistant to hCPT. For this purpose, we used three cell lines with top1 mutations and a top1-deficient cell line. The three point mutant cell lines were chosen because their resistance is linked to mutations in different domains of the top1 polypeptide (Fig. 2). The human prostate cell line DU-145/RC1 has a mutation in core subdomain I (G364H; Ref.1).³The Chinese hamster DC3F/C10 cell line has a mutation in core subdomain III (G503S; Ref. 10), and the human leukemia CEM/C2 cell line has a mutation in the COOH-terminal domain (N722S; Ref. 6, 7). The top1-deficient cells are murine leukemia P388/CPT45 cells (8, 9). In these cells, top1 is not detectable by immunoblotting (13).

Fig. 3 and Table 1 demonstrate that the four cell lines studied were highly resistant to both CPT and hCPT. P388/CPT45 cells were the most resistant to both drugs (>2000-fold for CPT and 4286-fold for hCPT), whereas the DC3F/C10 cells were the least resistant to both CPT and hCPT (84-fold for CPT and 23-fold for hCPT; Fig. 3 and Table 1). Calculated IC₅₀ values were smaller for hCPT than for CPT for both parental and resistant cell lines. Interestingly, the IC₅₀ values for hCPT were in the submicromolar range in the CPT-resistant prostate DU-145/RC1 cells and in the lung fibroblast DC3F/C10 cells.

We next evaluated whether hCPT can stabilize top1 cleavage complexes in the presence of CPT-resistant top1. Nuclear extracts from parental(WT) and top1 mutant cell lines were used for cleavage assays using a pBluescript DNA fragment that was 3′-end labeled with ³²P (Fig. 4). No detectable cleavage was observed for the N722S mutation (CEM/C2 cell line) in the presence of hCPT. For the two other top1 mutations,G503S (DC3F/C10) and R364H (DU-145/RC1), cleavage was markedly reduced and only detectable at the highest hCPT concentration (Fig. 4). The distribution of DNA cleavage sites induced by hCPT and CPT did not show detectable differences in the DNA segment examined. Together, these observations indicate cross-resistance of the three point mutant top1 enzymes for hCPT, with a lack of detectable cleavage in the N722S mutant.

Because hCPT exhibited a greater antiproliferative activity than CPT in both parental and CPT-resistant cell lines, we examined the stability of the cleavage complexes observed in the presence of hCPT. Heat and salt were used to test the reversibility of the top1 cleavage complexes. By heating the reactions to 50°C, cleavage was reversed rapidly within 5 min for both CPT and hCPT (Fig. 5,A). Under these conditions, we detected no difference in the stability of top1 cleavage complexes between CPT and hCPT. Salt reversal with NaCl (final concentration, 0.25 m)showed that DNA cleavage in the presence of hCPT persisted for up to 30 min and reversed more slowly than that for CPT. At 5 min, the amount of cleavage observed in the presence of hCPT remained as much as that seen at 0 min (Fig. 5 B). These results demonstrate that hCPT stabilizes more efficiently the top1 cleavage complexes than CPT(14).

hCPT contains a seven-member β-hydroxylactone in place of the six-member α-hydroxylactone found in CPT and its derivatives. This modification results in a more stable lactone and reduces the conversion of the drug to its carboxylate derivative(2), which, in the case of CPTs, is inactive against top1(1) and is highly bound to human serum albumin(15). Decreased binding to human plasma protein has been observed consistently for hCPTs (3, 4). hCPTs are also more potent top1 inhibitors than the corresponding CPT derivatives both in the presence of purified top1 (2) and in cancer cells,including some multidrug-resistant cells (2, 3). Until now, little was known about the activity of hCPT in CPT-resistant cells.

The high levels of resistance (>4000-fold; Table 1) of the top1-deficient p388/CPT45 cells (8, 9, 13) to hCPT and the cross-resistance of the three cell lines with top1 point mutations demonstrate that top1 is the cellular target of hCPT. Although all of the CPT-resistant cells were cross-resistant to hCPT, hCPT was more efficient than CPT in both parental and CPT-resistant cell lines. hCPT appears to be relatively more potent than CPT in the diverse mammalian cell lines examined: human prostate carcinoma; human leukemia; murine leukemia; and Chinese hamster lung fibroblasts (see Fig. 3 and Table 1). It is noticeable that, in the parental human prostate DU-145 cells, hCPT was 15-fold more effective than CPT. Recently Philippart et al. (5) compared the antiproliferative activity of CPT, topotecan, and SN-38 against that of two new hCPTs, BN80245 and BN80915, in 48 colon cancer cell types. The two hCPT derivatives exerted greater antiproliferative activity than the three anti-top1 reference compounds, with the highest activity observed for BN80915. We find that the IC₅₀values for hCPT in two of the CPT-resistant cell lines, DC3F/C10 and DU-145/RC1, are 10-fold lower than that in the CEM/C2 cell line (0.45,0.67, and 7 μm, respectively). Thus, hCPT can be effective at submicromolar concentrations in two of the CPT-resistant cell lines (Table 1), which suggests that because of its enhanced potency, hCPT could potentially overcome resistance to CPT in some cancer cells.

As shown in Fig. 2, the top1 point mutations leading to CPT resistance cluster in different regions that are highly conserved among eukaryotic top1s. According to crystal structure data, the top1 residues that are mutated in CPT-resistant cells and belong to the core domain are in close proximity to the catalytic tyrosine (Y723), the other residues involved in CPT resistance in the COOH-terminal domain and the bound DNA (1, 16). It is interesting to note that,in the present study, the three mutations led to resistance to both CPT and hCPT in the top1-DNA cleavage assays. This cross-resistance suggests that the two drugs share a common binding site in the top1-DNA complex. In the case of CPTs, this binding site has been proposed to be at the enzyme-DNA interface, with the drug stacked along the base pairs flanking the DNA cleavage site (16, 17, 18). We also found that, in the DNA segment examined, there was no difference in DNA cleavage patterns between CPT and hCPT. It should be noted, however,that some differences have been reported in the sequence specificity of drug-induced DNA cleavages in other DNA fragments (14). It is also noticeable that the top1 cleavage complexes formed in the presence of hCPT are less reversible in high salt than those induced by CPT (see Fig. 5; Ref. 14). Greater stability of top1 cleavage complexes can contribute to the greater cytotoxicity of top1 inhibitors (1). Together, these observations indicate that hCPT and CPT probably bind to a common binding site in the top1-DNA complexes and exert their antiproliferative activity in a top1-dependent manner. Stability of the lactone ring and enhanced molecular interactions with the top1-DNA complexes probably account for the greater antiproliferative activity of hCPT in all cell lines examined, including the CPT-resistant cell lines.

We thank Drs. Dennis Bigg and Olivier Lavergne (Institut Henri Beaufour) for providing hCPT.