Introduction

Top Summary Introduction Materials and Methods Results Discussion References

Camptothecin (CPT) is isolated from extracts of the Camptotheca accumminata tree native to China (Wall and Wani, 1996). A number of CPT analogs, such as 9-aminocamptothecin, topotecan (TPT), and irinotecan, have shown impressive antitumor activity against a variety of tumors in clinical trials (Rothenberg, 1997). DNA topoisomerase I (top I) is the target of CPT. top I is an essential nuclear enzyme which can change the topological state of DNA by breaking one strand of a DNA double helix, allowing free rotation of the cleaved strand, and then rejoining the phosphodiester backbone of DNA (Porter and Champoux, 1989). top I relaxes both positively and negatively supercoiled DNA and plays important roles in a variety of cellular processes involving DNA such as DNA replication, RNA transcription, and DNA repair (Downes and Johnson, 1988; Roca, 1995; Wang, 1996).

The mechanism of top I involves a transient covalent bond between top I and the 3' end of the cleaved DNA strand. CPT stabilizes this "cleavable complex" which results in the accumulation of top I-DNA adducts known as protein-linked DNA breaks (PLDBs) (Hsiang et al., 1985). PLDB formation is reversible once CPT is removed. However, if a DNA synthesis fork collides with the PLDB, irreversible DNA damage occurs, leading to cell death (Pommier et al., 1994; Liu et al., 1996).

Preventing the collision of DNA synthesis forks with PLDBs, either by inhibiting DNA synthesis or by reducing the amount of top I available to form PLDBs, can protect cells against CPT-mediated cytotoxicity. The DNA synthesis inhibitor aphidicolin (APH) could protect cells from CPT cytotoxicity (Hsiang et al., 1989; Kaufmann, 1998). Several CPT-resistant cells have been selected that have reduced top I activity resulting from mutant top I genes, reduced top I expression, or postranslational modification of top I such as phosphorylation or poly-ADP ribosylation (Pommier et al., 1996).

Prolonged treatment of cells with CPT also leads to top I down-regulation, resulting in a reduction in PLDBs. D'Arpa and colleagues (Desai et al., 1997) have reported that top I protein is ubiquitinated and then degraded by the proteasome in response to CPT treatment. We confirm these results and report that CPT-induced top I down-regulation occurs in the nucleus of cells from the human epidermoid carcinoma cell line (KB cells) and is independent of cell death. We also examine the effects of several other top I poisons on top I expression and find that 6-N-formylamino-12,13-dihydro-1,11-dihydroxy-13-(-D-glucopyranosyl)-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione (NB506) and a novel topoisomerase inhibitor, camptothecin-(para)-4-amino-4'-O-demethyl Epipodophyllotoxin (W1), do not induce top I down-regulation.

	Materials and Methods

Top Summary Introduction Materials and Methods Results Discussion References

Cells

KB cells, derived from an epidermoid carcinoma in the mouth of an adult male Caucasian, were purchased from the American Type Culture Collection (Rockville, MD). The cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂. The growth medium used was RPMI 1640 supplemented with 5% fetal bovine serum, and 100 µg/ml kanamycin. CPT-resistant cells, KBCPT100 (Beidler et al., 1996), were maintained in RPMI 1640 medium supplemented with 100 nM CPT, 10% fetal bovine serum, and 100 µg/ml kanamycin. Before each experiment, KBCPT100 cells were cultured in CPT-free medium for 3 days.

Drugs and Antibodies

Mouse monoclonal antibody C21, an antitop I-specific antibody, was developed in this laboratory (Zhou et al., 1989). TPT was obtained from the National Cancer Institute (Bethesda, MD). NB506 was kindly provided by Dr. T. Yoshinari and Dr. S. Nishimura from the Banyu Tsukuba Research Institute, Tsukuba, Japan. W1 and camptothecin-(ortho)-4-amino-4'-O-demethyl Epipodophyllotoxin (W2) were kindly provided by Dr. K. H. Lee from the University of North Carolina. Lactacystin was kindly provided by Dr. E. J. Cory from Harvard University. trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E64) and i-BuNH-Eps-Leu-Pro-OH (IBU) were kindly provided by Dr. Barbara J. Gour from McGill University, Montreal, Canada. APH was purchased from Sigma Chemical Co. (St. Louis, MO). MG132, calpain inhibitor I, 3, 4-dichloroisocoumarin (DCI), and tosyl-phenylalanine chloromethyl ketone (TPCK), were purchased from Boehringer Mannheim Corporation (Indianapolis, IN). Interleukin-1-converting enzyme inhibitor I, Ac-Tyr-Val-Ala-Asp-chloromethyl ketone (YVAD-CMK), and carbobenzoxyl-L-leucyl-L-leucyl-L-leucinal (MG132) was purchased from Calbiochem-Novabiochem Corporation (La Jolla, CA).

Processing of Cell Lysates

Total Cell Lysates. Cells (5 × 10⁵) were plated in a T-25 flask in 5 ml of growth medium overnight. The next day, a drug was added and the cells were grown for the indicated time. At the end of the incubation, cells were washed twice with PBS, incubated in drug-free medium for 30 min, and then treated with pancreatin to dislodge the cells from the flask. The cells were centrifuged and washed with PBS. The resulting cell pellet was resuspended in 1× Laemmli loading buffer (63 mM Tris, pH 6.8, 2% SDS, 5% 2-mercaptoethanol, 10% glycerol) and boiled for 5 min. The cell lysates were either used immediately or stored at 70°C. All samples were boiled again for another 5 min immediately before SDS-polyacrylamide gel electrophoresis (PAGE) gel analysis. Samples were normalized with an equal cell number per lane.

High Molecular Weight top I Analysis. The method of Desai (Desai et al., 1997) was followed. After drug treatment, cells were quickly harvested by pancreatin treatment and cell pellets were chilled on ice for 5 min. Ice-cold high-salt solution (50 mM Tris, pH 7.4, 0.8 M NaCl, 0.5% Nonidet P-40, 5 mM MgCl₂, 5 µg/ml leupeptin, 5 µg/ml pepstatin, 5 µg/ml aprotinin, 5 mM N-ethylmaleimide) was added to the cell pellet. The lysate was then added to an equal volume of 10 mM cysteine in water. Finally, one-third volume of 3 × SDS-PAGE sample buffer was added to the lysate. Samples were normalized to equal cell number before loading on the SDS-PAGE gel.

Cell Fractionation. KB cells were treated with 25 µM CPT for 10 min, 30 min, 1 h, 2 h, and 4 h. At each time point, cells were quickly harvested with pancreatin, washed twice with cold PBS, resuspended in buffer N (50 mM Tris, pH 7.4, 0.5% NP40, 5 mM MgCl₂, 5 µg/ml leupeptin, 5 µg/ml pepstatin, 5 µg/ml aprotinin), and incubated on ice for 30 min. The cytosolic fraction was obtained by centrifugation for 5 min at 5000 rpm in a microcentrifuge and then collecting the supernatant. Unfractionated cells and the nuclei were extracted with ice-cold high-salt buffer. Samples from an equal number of cells were loaded in each lane for SDS-PAGE analysis.

Western Blotting

Proteins were separated by either a 7% acrylamide gel or a 4 to 12% gradient gel. After electrophoresis, proteins were transferred onto a nitrocellulose blotting membrane, BioTrace NT (Pall Gelman Science, Ann Arbor MI) with a Bio-Rad mini trans-blot electrophoretic transfer cell (Bio-Rad Laboratories, Hercules CA). The resulting filter was blocked with 5% nonfat dried milk in PBST (0.15% Tween 20 in PBS) overnight with shaking at 4°C. The blot was incubated with the primary antibody, either antitop I antibody (C21, 1/1000) or antiactin in a blocking buffer for 2 h at room temperature. The blot was then washed at room temperature three times for 15 min and incubated with a secondary antibody (1/1000) in blocking solution for 1 h at room temperature. The secondary antibody was either antimouse IgM (µ chain specific, peroxidase conjugated, 1/1000; Sigma, St. Louis, MO) or antimouse polyvalent Ig (peroxidase conjugated, 1/1000, Sigma). After another three 15-min washes at room temperature with PBST, enhanced chemiluminescence (DuPont, Wilmington, DE) was used to detect the peroxidase conjugate by exposure to X-ray film.

PLDB Analysis

PLDBs were quantified by a potassium/SDS coprecipitation assay (Beidler and Cheng, 1995). PLDB formation reached a plateau level (defined as 100%) when cells were treated with 10 µM CPT.

	Results

Top Summary Introduction Materials and Methods Results Discussion References

Degradation of top I Is CPT Dosage- and Incubation Time-Dependent but Cell Death-Independent. It has been previously reported that top I protein expression is down-regulated in response to CPT treatment in the KB cell line (Beidler and Cheng, 1995) and in other cell lines (Desai et al., 1997). However, due to the cytotoxic effect of CPT on these cells, it is possible that the reduction in top I protein is related to cell death rather than being directly caused by CPT exposure. We compared the effect of CPT on top I expression in CPT-sensitive versus CPT-resistant cell lines in an attempt to uncouple top I down-regulation from CPT-induced apoptosis. The CPT-resistant KBCPT100 cell line was derived from human KB cells by selecting for growth in media supplemented with CPT. KBCPT100 cells are resistant to a variety of top I poisons but not to other classes of drugs, including topoisomerase II (top II) poisons. The mechanism of CPT resistance is unknown because KBCPT100 cells have normal top I expression and activity (Beidler et al., 1996). The LC₅₀ value of CPT, defined as the concentration that produced a 50% reduction in colony-forming ability after one generation time exposure, was 12 nm for kB cells and was 3500 nm, a 300-fold higher concentration, for CPT-resistant variant.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   CPT dose- and incubation time-dependent but protein synthesis-independent of top I down-regulation. 1A, KB and KBCPT100 cells were treated with different concentrations of CPT for 8 h. 1B, KB and KBCPT100 cells were treated with 1 µM CPT for 0, 2, 4, or 8 h. 1C, KB or KBCPT100 cells were incubated with 1 µM CPT and 4.5 µM cycloheximide (CHX) for 8 h. Cells were then washed and allowed to grow in a drug-free medium for another 30 min (enough time for reversal of cleavable complex). Cells were then harvested with pancreatin and the cell pellet was lysed directly in a cell lysis buffer. An equal number of cells were loaded in each lane. Western blots of the cell lysates were probed for top I with C-21 antibody.

View larger version (39K): [in this window] [in a new window]   Fig. 2.   Reversibility of down-regulated top I after CPT removal. KB cells were pretreated with 1 µM CPT and 7.5 µM APH for 12 h (KBCPT100 cells were treated with 1 µM CPT) and then cells were washed and grown in a drug-free medium for the times indicated. At each time point, 0, 4, 8, 24, 48, 72, and 96 h after drug removal, cells were harvested. Cell lysates were prepared as described in Materials and Methods. Equal amounts of protein (50 µg of protein of cell lysate for KB cells and 65 µg of protein of cell lysate for KBCPT100 cells) were loaded for each time point. Western blots were probed with C-21 and anti-Actin monoclonal antibody.

Degradation of top I Can Be Blocked by Selected Protease/Proteasome Inhibitors. We next investigated the possibility that proteolysis is responsible for the observed decline in top I protein levels in CPT-treated cells by using specific protease inhibitors. Eight different protease inhibitors were tested for their ability to block CPT-induced down-regulation (Fig. 3A). Two proteasome inhibitors, lactacystin and MG132 (Rock et al., 1994; Fenteany et al., 1995; Fenteany and Schreiber, 1998), blocked top I down-regulation. The inhibition of top I degradation by MG132 was dose-dependent (Fig. 3B). As a control, MG132 or lactacystin alone had no effect on top I expression (Fig. 3A, top gel). This result suggests the involvement of the ubiquitin/proteasome pathway in the degradation of top I and is consistent with a recent report that described ubiquitination of top I upon CPT treatment (Desai et al., 1997). Calpain inhibitor I, a Ca²⁺-dependent cysteine protease inhibitor, can also block top I degradation. However, two other cysteine protease inhibitors 1-trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane and IBU (Sreedharan et al., 1996), did not block the top I degradation. This result suggested that the ability of calpain inhibitor I to block top I degradation is not due to inhibition of cysteine proteases in general, but probably due to inhibition of a very specific cysteine protease. The down-regulation of top I was not affected by an interleukin-1-converting enzyme inhibitor I inhibitor, YVAD-CMK. Two serine protease inhibitors, DCI and TPCK, were evaluated. DCI could only partially block degradation and TPCK had no effect.

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Prevention of CPT-induced top I down-regulation by selected protease inhibitors. 3A, KBCPT100 cells were incubated with protease inhibitors (100 µM lactacystin, 20 µM MG132, 93 µM DCI, 200 µM TPCK, 50 µM calpain inhibitor I, 10 µM YVAD-CMK, 20 µM E-64, and 20 µM IBU) alone or with 1 µM CPT for 8 h. 3B, KBCPT100 cells were incubated with 1 µM CPT and increasing concentration of MG132 (0, 0.5, 1, 2, 5, 10, and 20 µM) for 8 h. Cells were washed free of drugs and the incubations were continued in drug-free medium for another 30 min. Cells were harvested and cell pellets were lysed directly in lysis buffer. Equal numbers of cells were loaded for each lane. Western blots were probed with C-21 antibody.

View larger version (31K): [in this window] [in a new window]   Fig. 4.   High molecular weight top I ladder. KB or KBCPT100 cells were treated with 25 µM CPT for 10 min, 30 min, 1 h, 2 h, and 4 h. At each time point, cells were quickly treated with pancreatin and the cell pellet was collected by centrifugation. The cell pellet was extracted with high-salt (0.8 M NaCl) lysis buffer (see Materials and Methods for details) and an equal number of cells were loaded in each lane. Western blots were probed with C-21 antibody.

Intracellular Localization of top I after CPT Treatment. We investigated the localization of top I during CPT-induced down-regulation in KB cells by subcellular fractionation. KB cells were treated with 25 µM CPT for various times and then fractionated into cytosol and nuclei fractions. Top I was present only in the nuclear fraction at all time points (Fig. 5). The high molecular weight ladder of top I immunoreactivity was also observed only in the nuclear fraction. This result suggests that top I is ubiquitinated and subsequently degraded within the nuclei. Alternatively, degradation could occur rapidly after ubiquitinated top I has been translocated to the cytoplasm.

View larger version (48K): [in this window] [in a new window]   Fig. 5.   Subcellular localization of top I. KB cells were treated with 25 µM CPT for 10 min, 30 min, 1 h, 2 h, and 4 h. At each time point, cells were fractionated into cytosol or nuclei fractions (see Materials and Methods). The nuclei fraction was extracted with 0.8 M NaCl. Cytosol or nuclear fractions from an equal number of cells were loaded for SDS-PAGE analysis. Blots were probed with C-21 antibody.

The Effect of Other top I Poisons on top I Protein Down-Regulation. Although CPT has been extensively characterized in the laboratory, it has limited utility in clinical settings due to its high toxicity (Rothenberg, 1997). Therefore, we wished to determine whether other top I poisons could also induce top I down-regulation. For this study, we chose two well known top I poisons, TPT and NB506 (Fukasawa et al., 1998, Yoshinari et al., 1995), and two newly designed drugs, W1 and W2. The new drugs are conjugates of CPT and the top II inhibitor, 4-amino-4'-O-demethyl epipodophyllotoxin (VP-16). The CPT moiety in both drugs is conjugated through the seven position of the B ringa modification known to have only minor effects on top I inhibitory activity (Wang et al., 1997). The major difference between W1 and W2 is that the linkage between the CPT moiety and the aromatic ring of the epipodophyllotoxin analog is para for W1 and ortho for W2 (Bastow et al., 1997). W1 and W2 have similar cytotoxic effects in KB cells with an LC₅₀ of 44 nM for W1 and 75 nM for W2 (JY Chang, X Guo, HX Chen, ZL Jiang, HK Wang, KF Bastow, XK Zhu, J Guan, KH Lee, YC Cheng, submitted). The CPT-resistant KBCPT100 cells are partially cross-resistant to both WI and W2, whereas the VP-16-resistant KB/7D cell line does not suggest that the primary mode of cytotoxicity of these conjugates is via top I (JY Chang et al., submitted).

View larger version (37K): [in this window] [in a new window]   Fig. 6.   Different effects of top I poisons on top I protein down-regulation. KB cells were treated with CPT, TPT, W1, W2, or NB506 at the indicated concentrations for 4 h. Then cells were washed and allowed to grow in drug-free medium for another 30 min (enough time for reversal of cleavable complex). Then cells were harvested with pancreatin and the cell pellet was lysed directly with cell lysis buffer. An equal number of cells were loaded for each lane. Western blots of the cell lysate were probed for top I with C-21 and anti-actin monoclonal antibody.

View larger version (61K): [in this window] [in a new window]   Fig. 7.   Different effects of top I poisons on the high molecular weight top I ladder formation. KBCPT100 cells were treated with 0, 1, 5, or 25 µM CPT, TPT, NB506, W1, or W2 for 10 min. Cells were quickly harvested by pancreatin treatment and the cell pellet was collected by centrifugation. The cell pellet was extracted with high-salt (0.8 M NaCl) lysis buffer (see Materials and Methods) and an equal number of cells were loaded in each lane. Western blots were probed with C-21 antibody.

View larger version (19K): [in this window] [in a new window]   Fig. 8.   PLDB formation induced by five topoisomerase poisons. KB cells were treated with 1 µM and 10 µM concentrrations of each drug, and the steady-state levels of PLDBs were measured with an in vivo K-SDS coprecipitation assay (Beidler and Cheng, 1995). The quantity of PLDBs induced by 10 µM CPT was defined as 100%.

	Discussion

Top Summary Introduction Materials and Methods Results Discussion References

Our laboratory has previously shown that CPT can induce top I down-regulation at all stages of the cell cycle (Beidler and Cheng, 1995). Here, using CPT-resistant cells, our results (Fig. 1) demonstrate that the down-regulation of top I is independent of CPT-induced cell death and that top I protein levels recover after CPT removal. These observations have important implications for the design of future clinical trials of top I poisons. Prolonged treatment with CPT analogs may reduce the amount of top I protein in target tumor cells, thereby reducing the efficacy of the treatment. Our results suggest that expression of top I in target tumors will recover after the top I poison has been metabolized. Therefore, we suggest that high dosages, short exposure times, and multiple cycles of treatment should be considered for future clinical trials of top I poisons. Alternatively, it may be advantageous to use a CPT conjugate like W2 that traps top I-DNA cleavable complexes without inducing top I down-regulation.

Loss of top I activity in response to CPT results from a dose- and concentration-dependent loss of top I protein. We tested a panel of protease inhibitors to determine whether a protease is responsible for top I down-regulation. MG132, a proteasome inhibitor, inhibited CPT-induced top I down-regulation in a dose-dependent manner, suggesting involvement of the proteasome. We confirmed proteasome involvement by inhibiting top I down-regulation with the more specific proteasome inhibitor lactacystin. Short-lived proteins such as cyclins (Krek, 1998), p53 (Whitesell et al., 1997), and IkBa (Ghoda et al., 1997) are marked for degradation by the proteasome by covalent modification with a polyubiquitin chain consisting of multiple 8.6-kDa adducts. In contrast to other proteins known to be degraded by the proteasome, top I has a long half-life in normal cells and is not cell cycle-regulated. Nevertheless, long exposure of Western blots revealed induction of a high molecular weight ladder of top I immunoreactivity 10 min after addition of CPT. These high molecular weight adducts are likely to be ubiquitinated top I as previously reported for CHO cells by Desai et al (1997). The high molecular weight top I-immunoreactive bands were observed before top I degradation induced by TPT and W2 as well as CPT, but were not seen after 10 min of incubation with W1 or NB506. Thus, there is a strong correlation between the induction of high molecular weight top I immunoreactivity and subsequent top I degradation.

It was suggested by others that when cells are treated with TPT, top I is rapidly redistributed (Buckwalter et al., 1996; Danks et al., 1996). Danks et al. (1996) also observed increased cytoplasmic concentrations of top I protein. However, our result (Fig. 5) and that of Buckwalter(1996) do not support such an observation. We detected reduced top I protein in the nuclear fraction upon CPT treatment but did not see increased cytoplasmic top I protein. Proteasomes are found in the nucleus as well as in the cytoplasm of liver cells (Rivett et al., 1992). However, cell type-dependent differences in proteasome localization have also been observed (Palmer et al., 1994). It would be very interesting to see whether this top I redistribution phenomenon, which was observed in tissue cells, could be duplicated in patient samples, or whether different types of cancer cells differ in top I redistribution.

In addition to the proteasome-specific inhibitors lactacystin and MG132, we observed that DCI and calpain inhibitor I also inhibited top I degradation (Fig. 3). The effect of DCI was partial and may represent a low potency inhibitory effect on the proteasome (Nannmark et al., 1996). For calpain inhibitor I, there are several possible inhibitory mechanisms. Calpain inhibitor I may act upstream of the 26S proteasome pathway. Another possibility is that calpain inhibitor I may prevent a post-translational modification of top I that is necessary for subsequent polyubiquitination. The third possibility invokes a novel lactacystin- and MG132-sensitive proteolysis pathway (Glas et al., 1998).

We compared the ability of five drugs to induce the formation of PLDBs and top I degradation. Although PLDBs were induced by all five drugs, NB506 and W1, a conjugate of CPT and VP-16, did not induce top I down-regulation. Therefore, the formation of top I-DNA-cleavable complexes is not sufficient to induce top I degradation. The crystal structure of a top I-DNA complex was recently solved (Redinbo et al., 1998; Stewart et al., 1998). The authors suggested a model based on their structure for the binding of CPT to top I-DNA complexes. Our current hypothesis is that CPT and W1 have different effects on the conformation of the top I-DNA complex that regulate its ability to be recognized by the ubiquitin-conjugating enzymes and, subsequently, to be degraded. W1 may be more useful for long-term chemotherapy due to its failure to induce top I down-regulation.