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[Cancer Research 64, 1114-1121, February 1, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Cullin 3 Promotes Proteasomal Degradation of the Topoisomerase I-DNA Covalent Complex

Hua-Feng Zhang1, Akihiro Tomida1, Ritsuko Koshimizu1, Yasunari Ogiso1, Shuhong Lei1 and Takashi Tsuruo1,2

1 Institute of Molecular and Cellular Biosciences, The University of Tokyo, and 2 Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DNA topoisomerase I (TOP1)-DNA covalent complexes are the initial lesions produced by antitumor camptothecins (CPTs). The TOP1-directed drugs stimulate degradation of TOP1 via the ubiquitin-proteasome pathway. We found that proteasome inhibition prevents degradation of DNA-bound TOP1 and sustains high levels of covalent complexes, thus enhancing CPT-induced cell death. Consistent with this, increased degradation of TOP1-DNA covalent complexes was seen in acquired CPT-resistant cells. We found that the resistant cells showed elevated expressions of Cul3, a member of the cullin family of E3 ubiquitin ligases. The reduction in Cul3 expression by small interfering RNA decreased degradation of TOP1-DNA covalent complexes. Conversely, Cul3 overexpression by stable transfection promoted covalent complex degradation and reduced CPT-induced cell death without affecting basal TOP1 expression levels. These results indicate that Cul3, by promoting proteasomal degradation of TOP1-DNA covalent complexes, becomes an important regulator for cellular CPT sensitivity.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DNA topoisomerase I (TOP1) releases torsional stress of DNA through a single-strand breakage/rejoining reaction and plays critical roles in replication, transcription, and DNA damage repair in mammalian cells (1, 2, 3) . During the catalytic cycle, the active site tyrosine of TOP1 links covalently to a 3' phosphate of DNA at the break site (4) . The TOP1-DNA covalent complex, often referred to as the cleavable complex or cleavage complex, is the target of the antitumor drug camptothecin (CPT) and its clinically useful derivatives, such as irinotecan and topotecan (5, 6, 7) . The predominant cytotoxic mechanism of CPT is its stabilization of the cleavable complexes by inhibiting the religation step and ultimately generating double-strand DNA breaks as a result of a collision between moving replication forks and the CPT-stabilized cleavable complexes (8 , 9) . CPT shows an S-phase-specific cytotoxicity (9) .

Studies of a panel of colorectal cancer cell lines have revealed that the levels of CPT-induced cleavable complex, rather than cellular CPT accumulation or TOP1 mRNA and protein expression, correlate with cellular sensitivity to CPT (10) . However, downstream events from the cleavable complexes also have been reported to be critical for CPT cytotoxicity (9, 10, 11) . Recent studies have suggested that proteasome-dependent TOP1 degradation in response to CPT is an important downstream event leading to CPT resistance (12 , 13) . Among breast cancer cell lines, cells proficient in the CPT-induced TOP1 down-regulation show resistance to CPT, and the proteasome inhibitor carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132) increases CPT sensitivity (13) . The proteasomal degradation of TOP1 occurs in a transcription-dependent manner, suggesting its involvement in a collision between moving RNA polymerase complexes and the CPT-stabilized cleavable complexes (14 , 15) . CPT consistently and transiently inhibits transcription, and the recovery of transcription depends on proteasomal degradation of TOP1 and functional transcriptional-coupled repair (14) . Therefore, proteasomal TOP1 degradation may result in exposure of single-strand breaks for transcriptional-coupled repair (14) .

Ubiquitin conjugation to proteins destined for proteasomal degradation is carried out by the sequential action of three enzymes: E1, E2, and E3 (16) . E3 ubiquitin ligases determine the specificity of the substrate protein (17 , 18) , and cullins are the major components of a series of ubiquitin ligases (19 , 20) . The human cullin protein family consists of seven members: Cul1, Cul2, Cul3, Cul4A, Cul4B, Cul5, and Cul7 (21 , 22) . Cullins generally, if not always, function as scaffold proteins in the E3 ligases, and all cullin family members bind to the ring finger protein Rbx1/Roc1, an essential component for E3 ligase activity (23 , 24) . For example, the Rbx1-Skp1-Cul1-F-box complex is a well-characterized E3 ubiquitin ligase that plays an essential role in a wide variety of cellular activities (19) . Cul2 also forms an E3 ligase complex with the tumor suppressor VHL protein, elongin B, elongin C, and Rbx1 (25) . Unlike these cullins, Cul3-based E3 ligase complex remains to be determined. Although the physiologic function of Cul3 is largely unknown, it has been shown to play an important role in controlling cell proliferation (26) .

Recent studies have suggested that the proteasomal degradation is a repair mechanism for TOP1-mediated DNA damage (12 , 27) . However, it remains to be determined whether the proteasomal TOP1 degradation pathway influences the S-phase specificity of CPT cytotoxicity and the levels of TOP1-DNA covalent complexes. In addition, little is known about the proteins that regulate proteasomal TOP1 degradation. In this report, we show that proteasome inhibition selectively sensitizes S-phase cells to CPT and prevents reduction in TOP1 that covalently bound to DNA. Furthermore, we demonstrate that expression level of Cul3 is an important determinant for efficient TOP1 degradation by the ubiquitin-proteasome system and for CPT resistance.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and Antibodies.
CPT and SN38 were provided by Yakult Co., Ltd. (Tokyo, Japan). Topotecan was a gift from GlaxoSmithKline Co. (King of Prussia, PA); cisplatin and etoposide were from Bristol-Myers Squibb Co. (Tokyo, Japan); and lactacystin was purchased from Kyowa Medex (Tokyo, Japan). Carbobenzoxy-L-isoleucyl-{gamma}-t-butyl-L-glutamyl-L-alanyl-L-leucinal (PSI) and MG132 were from the Peptide Institute Inc. (Osaka, Japan). These compounds, except topotecan (in distilled water), were dissolved in DMSO and added to culture media with the solvents <0.1%. Antibodies against TOP1 (clone C-21.1), ubiquitin (clone 1B3), TOP2{alpha} (clone KF4), and Myc (clone 9E10) were obtained from PharMingen (San Diego, CA), MBL (Nagoya, Japan), Sigma-Genosys (Cambridge, United Kingdom), and Roche Molecular Biochemicals (Indianapolis, IN), respectively. Antibodies against Cul1 and Cul2 were from NeoMarkers (Fremont, CA). Antibodies against Cul3, SUMO-1, and tubulin were from Zymed (South San Francisco, CA).

Cell Culture.
The human colon carcinoma HT29 cells, gastric carcinoma St-4 cells, lung cancer A549 cells, their CPT-resistant variants (HT29/CPT, St-4/CPT, and A549/CPT, respectively), and ovarian cancer A2780 cells were maintained in RPMI 1640 (Nissui, Tokyo, Japan) supplemented with 5% heat-inactivated fetal bovine serum and 100 µg/ml of kanamycin. The human fibrosarcoma HT1080 cells were grown in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum and 100 µg/ml of kanamycin. They were cultured at 37°C in a humidified atmosphere containing 5% CO2. All experiments were performed using exponentially growing cells and were repeated at least twice.

Synchronization and Treatments.
For the synchronized culture, we trapped cells in M phase of the cell cycle by treating them with 40 ng of nocodazole/ml (Wako Pure Chemical Industries, Osaka, Japan) for 9 h, collected cells by gentle pipetting, and reseeded them in fresh culture medium (28 , 29) . Populations of G1- and S-phase cells constituted the majority (typically 60–70%) of the total cell population at 3 h and 9 h after the release, respectively (28 , 29) . For treatment of these cells, TOP1-directed drugs were added into the medium at 4 h (G1 phase) and 10 h (S phase) after the release. The proteasome inhibitor lactacystin was added 1 h after nocodazole was removed to avoid any effects on the release from M phase and cell cycle progression (29) . MG132 and PSI were added 1 h before the addition of TOP1-directed drugs. For the colony formation assay, cells were treated for 4 h with TOP1-directed drugs, diluted appropriately with fresh medium, and cultured to form colonies for 7–8 days (29) . The cell survival (mean ± SD in triplicate) was calculated by setting each of the appropriate control survivals as 1.

Cellular Accumulation of CPT.
Intracellular CPT accumulation was determined as described previously (30) . Briefly, S-phase-synchronized HT-29 cells (1 x 107) were treated for 4 h with CPT (10 µg/ml) in the presence or absence of lactacystin at 7.5 µM. The cells were washed twice, as quickly as possible, with cold PBS [137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 1.5 mM KH2PO4 (pH 7.4)] containing 0.01 N HCl and were suspended in the same buffer. After sonication and centrifugation, the supernatants were used, with spectrofluorometry (excitation at 380 nm and emission at 430 nm), to determine CPT contents.

MTT and Flow Cytometric Cytotoxicity Assays.
For the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma Chemical Co., St. Louis, MO) assay, cells were cultured overnight in 96-well plates. Drug solutions were added directly to the culture medium, and the cells were cultured for an additional 24 h (for stable transfectants of Cul3) or 72 h (for St-4 and St-4/CPT cells). MTT (Sigma Chemical Co.) was added subsequently to the culture medium, and the absorbance of each well was determined as described previously (31) . Relative cell survival (mean ± SD in sextuplicate) was calculated by setting each of the appropriate control absorbance as 1. For the flow cytometric assay, cells were cultured overnight in six-well plates, treated with drugs as indicated, and stained with 7-amino-actinomycin D (PharMingen, San Diego, CA). Dead cell populations (mean ± SD in three independent experiments) were determined using a Beckman Coulter flow cytometer with Cytomics FC500 RXP software (Fullerton, CA). Statistical significance of cell survival and dead cell populations was evaluated using a one-way ANOVA with Dunnett’s test.

In Vivo Complex of TOP Bioassay.
We performed the in vivo complex of TOP assay as described previously (32) . Immediately after CPT treatment, cells were lysed with 1% sarkosyl in 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA. The cell lysates were layered gently on top of a CsCl solution (1.5 g/ml density) and centrifuged at 438,000 x g for 5 h at 25°C. The cellular DNA pellet was dissolved in 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA, and the concentrations were measured by a spectrophotometer. Fixed amounts of DNA were blotted on a nitrocellulose membrane (Schleicher and Schuell, Dassel, Germany) using a slot-blot device (Bio-Rad, Hercules, CA). Blots were probed subsequently with anti-TOP1 antibody. Band intensities were quantified using NIH image 1.62 software (Bethesda, MD).

Preparation of Total Cellular and Nuclear Extracts.
Total cellular and nuclear extracts were prepared as described previously (32) . For preparation of total cell extracts, cells were lysed with ice-cold, high-salt lysis buffer [50 mM Tris-HCl (pH 7.4), 800 mM NaCl, 0.5% NP40, 5 mM MgCl2, and 5 mM N-ethylmaleimide] in the presence of the protease inhibitor mixture for mammalian cells (Sigma Chemical Co.). After removing insoluble materials by centrifugation, the supernatants were recovered as total cell extracts. For preparation of nuclear extracts, cells were suspended in ice-cold nucleus buffer [150 mM NaCl, 1 mM KH2PO4 (pH 6.4), 5 mM MgCl2, 1 mM EDTA, 1 mM PMSF, 0.2 mM DTT, 5 mM N-ethylmaleimide, and 0.3% Triton-X100] with gentle rocking for 10 min at 4°C. After centrifugation, the isolated nuclei were resuspended in ice-cold nucleus extraction buffer [500 mM NaCl, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 10% glycerol, and 5 mM N-ethylmaleimide] in the presence of protease inhibitor mixture, and nuclei were rocked for 1 h at 4°C. After removing insoluble materials by centrifugation, the supernatants were recovered as nuclear extracts. Protein concentrations were determined using a protein assay kit (Bio-Rad).

Immunoblot and Immunoprecipitation.
For immunoblot analysis, equal amounts of proteins were resolved on an SDS-polyacrylamide gel and electroblotted onto a nitrocellulose membrane (Schleicher & Schuell; Ref. 32 ). Membranes were probed with specific antibodies and appropriate secondary antibodies, and the specific signals were detected using an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Tokyo, Japan). The densitometric analysis was done as described previously.

Immunoprecipitation of TOP1 was performed as described previously (32) . Briefly, nuclear extracts were adjusted to 150 mM NaCl using the appropriate buffer without NaCl. Equal amounts of proteins were precleared and immunoprecipitated with the anti-TOP1 antibody and a Sepharose-immobilized protein L (Pierce, Rockford, IL). The immunocomplexes were washed five times with a washing buffer consisting of 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% NP40, 5 mM MgCl2, 5 mM N-ethylmaleimide, and the protease inhibitor mixture and were resuspended subsequently in 2x SDS sample buffer. After boiling for 5 min, the complexes were evaluated using immunoblot analysis.

RNA Interference.
Three Cul3-specific small interfering RNAs (siRNAs), si262, si520, and si755, were designed based on the Cul3 cDNA sequence (GenBank accession no. NM003590). These correspond to nucleotides 262–280 for si262, 520–538 for si520, and 737–755 for si755, when the translational start codon is numbered as nucleotide 1. The control siRNA was a double-stranded 21-mer unrelated to the Cul3 sequence. All of the double-stranded RNAs were purchased from Qiagen-Xeragon (Tokyo, Japan). Transient transfection of siRNA was performed either with oligoamine reagent (Qiagen-Xeragon) for HT1080 cells or with lipofectamin/plus reagent (Invitrogen, San Diego, CA) together with pcMyc vector as a carrier for St-4/CPT cells, according to the manufacturers’ protocols. Three days after transfection, the cells were used for experimentation.

Plasmids and Stable Transfection.
The pcMyc vector was produced by ligating oligo-DNA encoding N-terminal Myc epitope to the HindIII site of pcDNA3 (Invitrogen). Human Cul3 cDNA was generated by PCR and cloned in frame into the pcMyc vector at the KpnI site. The constructs were confirmed by DNA sequencing. Transfections of plasmids containing no insert (mock) and Myc-tagged Cul3 were performed using the FuGENE 6 Transfection Reagent (Roche Molecular Biochemicals) according to the manufacturer’s protocol. After 24 h of drug-free incubation, the cells were cultured for 1 week in culture medium containing 300 µg/ml of G418 (Invitrogen). G418-resistant cells were cloned subsequently and maintained in culture medium containing 300 µg/ml of G418. The clones were cultured in G418-free medium for experiments to avoid any effects from G418.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Enhanced CPT-Induced Cell Death with Stabilization of TOP1-DNA Complex by Proteasome Inhibition.
We examined the cytotoxic effects of CPT in combination with a proteasome inhibitor, lactacystin, using a synchronization culture system. In the experiments, lactacystin was used in concentrations at which it decreased the colony-forming ability of cells to 70–85%. Consistent with the fact that CPT shows an S-phase-specific cytotoxicity, a 4-h treatment with CPT reduced profoundly the colony-forming ability of HT-29 cells during S phase but only marginally during G1 phase (Fig. 1A)Citation . In the presence of lactacystin (7.5 µM), the cell-killing effect was enhanced strongly in the S-phase cells but not in the G1-phase cells, suggesting that lactacystin potentiated the CPT-mediated cytotoxicity. Similar potentiation by lactacystin (5 µM) was observed in S-phase-synchronized A2780 cells (Fig. 1B)Citation . Lactacystin also enhanced the cytotoxicity of SN38 (the active metabolite of irinotecan; Ref. 33 ) and topotecan during S phase in HT-29 cells (Fig. 1, C and D)Citation . Other proteasome inhibitors, such as PSI and MG132, also enhanced CPT cytotoxicity during S phase (data not shown).



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  		Fig. 1. Enhanced camptothecin (CPT)-induced cell death with stabilization of DNA topoisomerase I (TOP1)-DNA complex by proteasome inhibition. A–D, colony formation assay in synchronized HT29 (A, C, and D) and A2780 cells (B). The G1- (A) or S-phase-synchronized cells (A–D) were treated for 4 h with the indicated concentrations of CPT (A and B), SN38 (C), and topotecan (D) in the presence or absence of lactacystin (LCT) for HT-29 (7.5 µM) and A2780 (5 µM) cells. The data represent the mean value of cell survival, and the bars indicate the SD of triplicate determination. E and F, in vivo complex of TOP assay in S-phase-synchronized HT-29 cells that were treated with 100 ng/ml of CPT for the indicated periods (E) or 4 h (F) in the presence or absence of proteasome inhibitors LCT (7.5 µM), carbobenzoxy-L-isoleucyl-{gamma}-t-butyl-L-glutamyl-L-alanyl-L-leucinal (PSI; 5 µM), and carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132; 5 µM). Four µg of DNA were slot-blotted and probed with anti-TOP1 antibody. G, immunoblot analysis for TOP1 and TOP2{alpha} expression. S-phase-synchronized HT-29 and A2780 cells were exposed to solvent (DMSO) or 10 µg/ml of CPT for 4 h with or without LCT, as in A.

 
The intracellular CPT accumulation after a 4-h exposure of HT-29 cells was almost the same with or without lactacystin (21.0 ± 0.5 and 21.5 ± 0.2 ng of CPT/107 cells, respectively). We then determined the effect of proteasome inhibitors on CPT-induced TOP1-DNA cleavable complexes using the in vivo complex of TOP assay, which detects TOP1 that covalently bound to cellular DNA. In HT-29 cells synchronized in the S phase, the TOP1-DNA covalent complex rapidly reached a peak level, within 30 min after 100 ng/ml of CPT was added, and then decreased gradually despite the presence of CPT (Fig. 1E)Citation . Lactacystin effectively prevented the decrease of the TOP1-DNA covalent complex and kept it at high levels during the 4-h CPT treatment, although the peak level (0.5 h) was hardly increased by the proteasome inhibitor. Likewise, PSI and MG132 retained the CPT-induced TOP1-DNA covalent complex at high levels (Fig. 1F)Citation . In those experiments, however, the TOP1-DNA covalent complex was below the detectable level without CPT. Similar results were obtained in A2780 cells (data not shown).

We also determined the total cellular expression levels of TOP1 in HT-29 and A2780 cells. The TOP1, but not TOP2{alpha}, protein levels were decreased by a 4-h treatment with CPT, and the TOP1 decrease was suppressed almost completely when lactacystin was added (Fig. 1G)Citation . It should be noted that the overall TOP1 reduction in HT-29 cells was detected only when a relatively high concentration of CPT (10 µg/ml) was used as compared with the aforementioned in vivo complex of TOP assay. The high dose of CPT also induced a much higher level of TOP1-DNA covalent complex than did 100 ng/ml of the drug (data not shown). These results suggested that the CPT-induced degradation of TOP1 occurred preferentially for the enzyme that bound covalently to DNA and that high levels of the covalent complex formation were required to decrease the total TOP1 protein expression.

Overexpression of Cul3 and Enhanced TOP1 Degradation in CPT-Resistant Cells.
While searching for proteins that could mediate the proteasome-dependent degradation of TOP1, we found that Cul3 commonly was overexpressed in three CPT-resistant cell lines, HT-29/CPT, St-4/CPT, and A549/CPT, as compared with their parental lines (Fig. 2A)Citation . The elevated levels of Cul3 appeared to be selective because the expression levels of Cul1 and Cul2 were nearly equal between the parent and the CPT-resistant cells (Fig. 2A)Citation . As we reported previously (34) , the expression levels of TOP1 were reduced in HT-29/CPT and St-4/CPT but not in A549/CPT (Fig. 2A)Citation , suggesting that the Cul3 overexpression had little effect on the basal expression levels of TOP1.



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  		Fig. 2. Overexpression of Cul3 and enhanced DNA topoisomerase I (TOP1) degradation in camptothecin (CPT)-resistant cells. A, immunoblot analysis for expression of Cul3, Cul1, Cul2, and TOP1 in exponentially growing HT29, HT29/CPT, St-4, St-4/CPT, A549, and A549/CPT cells. S, CPT-sensitive parent; R, CPT-resistant cells. B, in vivo complex of TOP assay. St-4 (S) and St-4/CPT (R) cells were pretreated with carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132; 5 µM) or the solvent (DMSO) for 1 h, and the indicated doses of SN38 then were added to culture media for 4 h. Four µg of DNA were slot-blotted and probed with anti-TOP1 antibody. Right, densitometric analysis of the image at left. Relative TOP1 levels binding to DNA were calculated by setting the level in St-4 cells treated with 0.1 µg/ml of SN38 alone as 1. C, immunoblot analysis for TOP1 and Cul3 expression at the indicated time points after St-4 and St-4/CPT cells were exposed to SN38 (1 µg/ml) with (+) or without (-) MG132 (5 µM) pretreatment. Top and middle panels show results of the same samples after long and short exposures, respectively.

 
To investigate whether the Cul3 overexpression was involved in the proteasome-dependent degradation of TOP1, we compared levels of TOP1-DNA covalent complex in St-4 and St-4/CPT cells. St-4/CPT cells were ~30 times more resistant to SN38 than the parent St-4 cells based on the comparison of IC50 values (~300 ng/ml for St-4/CPT) in a 72-h MTT assay. When St-4/CPT cells were treated for 4 h with SN38, the levels of TOP1-DNA complex were lower, at all of the doses of 0.1, 1, and 3 µg/ml examined, than those in parental St-4 cells treated with 0.1 µg/ml of the drug (Fig. 2B)Citation . Although the proteasome inhibitor MG132 increased the SN38-induced covalent complexes in both cell lines, the increments were more dramatic in St-4/CPT cells than in St-4 cells (Fig. 2B)Citation . Furthermore, when the cells were treated with 1 µg/ml of SN38, the total expression level of TOP1 was decreased rapidly in St-4/CPT cells compared with St-4 cells (Fig. 2C)Citation . The decrease in TOP1 protein was suppressed almost completely by MG132. These results indicated that the proteasome-dependent TOP1 degradation pathway was enhanced in the Cul3-overexpressing St-4/CPT cells.

Decreased TOP1 Degradation by Cul3 Knockdown.
To address whether Cul3 is required for CPT-induced TOP1 degradation, we attempted knockdown by siRNA. We used three different siRNAs, designated Cul3 si262, si520, and si737. Immunoblot analysis of extracts from siRNA-transfected HT1080 cells revealed that the Cul3-directed siRNAs effectively reduced expression of Cul3 without affecting that of Cul1 or Cul2 (Fig. 3A)Citation . The si262 also reduced Cul3 expression in Cul3-overexpressing St-4/CPT cells to the level in St-4 cells (Fig. 3B)Citation . Although no alteration in total expression level of TOP1 was observed in these siRNA-treated cells, si262 significantly increased the amounts of SN38-induced TOP1-DNA covalent complex as compared with control siRNA (Fig. 3, C and D)Citation . Proteasome inhibition increased the levels of the covalent complex in the Cul3- and control siRNA-treated cells, and consequently, the differences in covalent complex formation disappeared, indicating that loss of Cul3 impaired proteasome-dependent degradation of the TOP1-DNA covalent complex.



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  		Fig. 3. Decreased DNA topoisomerase I (TOP1) degradation by Cul3 knockdown. HT1080 (A and C) and St-4/camptothecin (CPT) cells (B and D) were transfected with control or Cul3-directed small interfering RNAs (siRNAs; si262, si520, and si737) as indicated. A and B, immunoblot analysis for expression of Cul3 and TOP1, as well as Cul1 and Cul2 (A), using total cell extracts of the siRNA-transfected cells. Tubulin expression was determined as a loading control (A and B), and Cul3 and TOP1 expression in untransfected St-4 cells (None) also was examined as a control (B, Lane 3). C and D, in vivo complex of TOP assay after 4-h treatments with SN38 or the solvent (DMSO) in the presence or absence of carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132; 5 µM). The SN38 concentrations we used were 100 ng/ml for HT1080 cells (C) and 3 µg/ml and 100 ng/ml for siRNA-transfected St-4/CPT and untransfected St-4 cells (None), respectively (D). MG132 was added 1 h before the SN38 addition. Four µg of DNA were slot-blotted and probed with anti-TOP1 antibody.

 
Enhanced TOP1 Degradation by Ectopic Expression of Cul3.
To address whether overexpression of Cul3 promotes CPT-induced TOP1 degradation, we established stable transfectants using an expression vector of Myc-tagged Cul3 with HT1080 cells. We isolated two independent clones designated CLN1 and CLN2. These clones expressed different levels of Myc-tagged Cul3, as shown by immunoblot analysis using an anti-Myc epitope antibody (Fig. 4A)Citation . The total expression levels of Cul3 in CLN1 and CLN2 were approximately four and three times, respectively, higher than those in control mock-transfected and untransfected HT1080 cells (Fig. 4A)Citation . However, the expression levels of TOP1, as well as Cul1 and Cul2, in the clones were similar to those in the control cells (Fig. 4ACitation ; data not shown). The Cul3-transfected clones proliferated well, and their growth rates were almost the same as those of control cells (Fig. 4B)Citation .



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  		Fig. 4. Establishment of stable transfectants overexpressing Cul3. A, total cell extracts of untransfected HT1080, mock, and Cul3 transfectants (CLN1 and CLN2) were subjected to immunoblot analysis using antibodies against Myc epitope, DNA topoisomerase I (TOP1), Cul3, and tubulin (loading control). B, cells (1 x 104/well) were plated in six-well plates (day 0), and the cell numbers were counted every 24 h. The data represent mean ± SD in triplicate determinations.

 
We measured the levels of TOP1-DNA covalent complex in these cells. On treatment with 100 ng/ml of SN38, the covalent complex reached a peak level within 30 min, and these levels were nearly equal in mock-transfected and CLN1 cells (Fig. 5A)Citation . The levels of covalent complex subsequently decreased in both cell lines, but the decrease occurred more rapidly and profoundly in CLN1 than in the mock transfectant. MG132 prevented the reduction of covalent complex levels, and consequently, the levels became essentially the same between the cell lines (Fig. 5B)Citation . Thus, degradation of TOP1-DNA covalent complexes was accelerated in CLN1 cells. The accelerated degradation also was observed in CLN2 (Fig. 5C)Citation . We examined the overall degradation of TOP1 in CLN1 and mock-transfected cells. Although TOP1 degradation was proteasome dependent in both cell lines in response to SN38 (1 µg/ml), it occurred more rapidly in CLN1 than in mock-transfected cells (Fig. 5D)Citation .



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  		Fig. 5. Enhanced DNA topoisomerase I (TOP1) degradation by stable Cul3 overexpression. A–C, in vivo complex of TOP assay. Cul3-stable transfectants CLN1 (A and B) and CLN2 (C), as well as mock transfectant, were exposed to 100 ng/ml of SN38 for the indicated periods (A and B) or for 4 h (C). Carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132; 5 µM) or the solvent was added 1 h before the SN38 addition (B and C). Four µg of DNA were slot-blotted and probed with anti-TOP1 antibody. In A, the band intensities were determined by densitometry, and the relative TOP1 levels binding to DNA were calculated by setting the level in SN38-treated mock-transfected cells at 30 min as 1 (bottom graph). D, immunoblot analysis for TOP1 and Myc-tagged Cul3 expression at the indicated time points after mock and CLN1 cells were exposed to SN38 (1 µg/ml) with (+) or without (-) MG132 (5 µM) pretreatment. Tubulin expression was determined as a loading control. The band intensities were determined by densitometry, and relative TOP1 levels were calculated by setting each control level (0 min; Lanes 1 and 7) as 1 (bottom graph).

 
To address whether Cul3 overexpression influences ubiquitylation of TOP1, we immunoprecipitated TOP1 from nuclear extracts and monitored the ubiquitylation status (Fig. 6)Citation . Comparable amounts of TOP1 were immunoprecipitated when CLN1 and mock-transfected cells were treated with or without 1 µg/ml of SN38 for 30 min (Fig. 6A)Citation . Immunoblot analysis with antiubiquitin antibody revealed that ubiquitylated TOP1 proteins increased in response to SN38 (Fig. 6B)Citation . Consistent with the accelerated TOP1 degradation, ubiquitylated TOP1 proteins were more abundant in Cul3-overexpressing CLN1 than in mock-transfected cells (Fig. 6B)Citation .



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  		Fig. 6. Enhanced DNA topoisomerase I (TOP1) ubiquitylation by stable Cul3 overexpression. Mock- and Cul3-transfected cells (CLN1) were treated with 1 µg/ml of SN38 or the solvent for 30 min. Carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132; 5 µM) or the solvent was added 1 h before the SN38 addition. The nuclear extracts were prepared, and equal amounts of proteins were immunoprecipitated with anti-TOP1 antibody. Immunoprecipitates were analyzed by immunoblot with anti-TOP1 (A), antiubiquitin (Ub; B), or anti-SUMO-1 (SUMO) antibodies (C). The nuclear extracts (not immunoprecipitated) also were probed with anti-Ub antibody (D).

 
When the transfected cells were cotreated with SN38 and MG132, total ubiquitylated proteins increased in the preimmunoprecipitated nuclear extracts (Fig. 6D)Citation . Nevertheless, the MG132 cotreatment decreased ubiquitin-conjugated TOP1 proteins (Fig. 6B)Citation . Thus, MG132 may stabilize TOP1 not only by inhibiting proteasome activity but also by affecting the TOP1 ubiquitylation system activated in response to SN38. The MG132 cotreatment also led to a decrease in the mean apparent molecular weights of SUMO-1-conjugated TOP1 (Fig. 6C)Citation . We speculated that the small species were those without additional ubiquitin conjugations.

CPT Resistance by Ectopic Expression of Cul3.
We determined the sensitivity of Cul3 transfectants to SN38 using a flow cytometric assay with 7-amino-actinomycin D (Fig. 7A)Citation and an MTT assay (Fig. 7B)Citation . In both assays, the Cul3 transfectants CLN1 and CLN2 showed resistance to SN38 as compared with mock-transfected cells. CLN1 and CLN2 also exhibited resistance to CPT (data not shown). Consistent with the higher level of Cul3 expression, CLN1 was more resistant to SN38 than was CLN2 (compare Fig. 4ACitation and Fig. 7ACitation ). However, these Cul3 stable transfectants showed almost the same sensitivity to cisplatin and etoposide as the mock transfectant (Fig. 7, C and D)Citation . Thus, Cul3 overexpression selectively conferred resistance to TOP1-directed drugs.



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  		Fig. 7. SN38 resistance by stable Cul3 overexpression. A–D, mock- and Cul3-transfected cells (CLN1 and CLN2), as well as untransfected HT1080 cells (A), were treated with 30 ng/ml of SN38 (A) or the indicated concentrations of SN38 (B), cisplatin (C) or etoposide (D) for 24 h. The dead cell population and relative cell survival were determined by the flow cytometric assay with 7-amino-actinomycin D (7-AAD; A) and the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay (B–D), respectively, as described in "Materials and Methods." E, mock and CLN1 cells were treated with 3 µg/ml of SN38 or the solvent for 4 h. Carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132; 5 µM) or the solvent was added 1 h before the SN38 addition (time 0). The cells additionally were cultured in drug-free medium up to the indicated time points. The dead cell populations were determined by the flow cytometric assay, as in A. Statistical analysis was performed using a one-way ANOVA with Dunnett’s test, comparing the cell death or survival of the mock group with the CLN1 and CLN2 groups, respectively (A and B). *P < 0.001.

 
We also examined the effect of MG132 on the SN38 resistance in CLN1 using the flow cytometric assay (Fig. 7E)Citation . MG132 at a subtoxic dose, at which the dead cell population was <5%, significantly reduced the resistance of CLN1, and consequently, the dead cell population of CLN1 increased to a level nearly equal to mock-transfected cells that were treated with SN38 alone. Although MG132 also sensitized the mock-transfected cells to SN38, the sensitization effect was relatively transient as compared with that seen in CLN1. Similar results also were obtained using CLN2 and lactacystin (data not shown).


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Proteasomal degradation of TOP1 has been implicated as an important step to repair CPT-induced DNA damage (12 , 27) . This is supported additionally by our present findings that proteasome inhibition sensitizes tumor cells to CPT cytotoxicity preferentially during S phase of the cell cycle and suppresses degradation of TOP1-DNA covalent complex (Fig. 1)Citation . This study also shows that the expression level of Cul3 is a determinant for the efficiency of proteasomal TOP1 degradation and the cellular sensitivity to TOP1-directed drugs. Elevated levels of Cul3 were observed commonly in CPT-resistant cells (Fig. 2A)Citation , and resistance to TOP1-directed drugs was reproduced by ectopic expression of Cul3 (Fig. 7)Citation . The Cul3 overexpression promoted proteasome-dependent TOP1 degradation and enhanced removal of TOP1 from the covalent complexes (Fig. 5)Citation . Conversely, Cul3 knockdown by siRNA reduced degradation of TOP1-DNA covalent complexes (Fig. 3)Citation . However, neither overexpression nor knockdown of Cul3 affected the basal TOP1 protein levels, indicating that the Cul3-regulated TOP1 degradation pathway is activated in response to the drugs.

Conjugations of ubiquitin and SUMO-1 to TOP1 have been identified as events immediately downstream from the TOP1-DNA cleavable complexes (12 , 32 , 35 , 36) . Both modifications can influence the levels of CPT-induced TOP1-DNA covalent complex, but ubiquitylation and sumoylation appear to have opposite effects (Fig. 8)Citation . We showed previously that sumoylation enhances the CPT-induced covalent complex formation and proposed that this modification can be a recruiting signal of available TOP1 to DNA (32) . This notion also is supported by previous studies showing that the majority of sumoylated TOP1 proteins are linked covalently to DNA (32 , 35) . Conversely, ubiquitylation can negatively regulate the covalent complex by targeting TOP1 for proteasome-dependent degradation. Because Cul3 is a member of the cullin family of E3 ubiquitin-protein ligases (21 , 37) , the present evidence suggests strongly that it promotes the latter process. Proteasome inhibition may suppress this process and prolong the life span of the covalent complex, thereby leading to eventual cell death possibly through increased frequency of collisions with the replication forks, according to the collision model (8 , 9) .



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  		Fig. 8. A model for regulation of DNA topoisomerase I (TOP1)-DNA covalent complex. Camptothecin (CPT)-induced TOP1-DNA cleavable complexes trigger two downstream events: sumoylation (left) and ubiquitylation of TOP1 (right). In this model, the TOP1 sumoylation serves as a recruiting signal of TOP1 to DNA and enhances the cleavable complex formation. Meanwhile, the TOP1 ubiquitylation prompts proteasomal degradation of TOP1, thereby decreasing the cleavable complexes and leading to CPT resistance. Cul3 promotes the latter process possibly by acting as an E3 ubiquitin (Ub) ligase.

 
The paradoxical responses may come from the complicated mechanisms of action of CPT and may provide an explanation why degradation of TOP1 in the covalent complex results in the overall reduction in TOP1 proteins. Once cells are treated with CPT, TOP1 activity will be inhibited, and the resulting TOP1-DNA covalent complex will be targeted for Cul3-regulated proteasomal degradation. At the same time, inhibition of TOP1 activity will cause accumulation of topologic stress in DNA. In that situation, the majority of TOP1 proteins remain free from covalent complexes, and available TOP1 (possibly sumoylated) may be recruited to dissolve the topologic stress in DNA. On recruitment to DNA, the TOP1 protein will be trapped in the covalent complex by CPT and degraded as described previously. The futile cycles of recruitment and degradation may lead eventually to overall reduction in TOP1 proteins. However, our present results also showed that proteasome inhibition only keeps the levels of TOP1-DNA covalent complex but does not change the peak levels (Fig. 1E)Citation , which appear to depend on CPT concentration. Thus, it may be that proteasome inhibition affects the recruiting and the degradation process. Alternatively, the defined levels, depending on CPT doses, may be determined by other repair mechanism(s), possibly by the tyrosyl-DNA phosphodiesterase Tdp1, which removes TOP1 from the covalent complex (38) .

The mechanisms behind Cul3-regulated TOP1 degradation remain to be determined. According to the aforementioned model, it is possible that Cul3 acts as the E3 ubiquitin-TOP1 ligase. We showed that after SN38 treatment, ubiquitylated TOP1 proteins were elevated more in the Cul3-overexpressing cells than in the mock-transfected cells (Fig. 6B)Citation . However, we have failed to detect binding between Cul3 and TOP1 either in vitro or in vivo using immunologic techniques. This may be because of technical problems; for example, it is difficult to extract DNA-linked TOP1 under mild experimental conditions. Another difficulty is that cullin family proteins generally form multiprotein complexes and function as a scaffold protein in the E3 ligase complexes (19 , 20) . Thus, TOP1 recognition by Cul3 may require other protein(s), like F-box proteins in the Cul1-based Rbx1-Skp1-Cul1-F-box E3 ligase. Alternatively, it also is possible that Cul3 regulates an as-yet unidentified ubiquitin-TOP1 ligase(s) or the recently identified TOP1-interacting protein TOPRS, which is a potential E3 with a ring finger domain (39) . Such multistep regulation of TOP1 ubiquitylation could be supported by the present observation that proteasome inhibition paradoxically reduced the amounts of ubiquitin-conjugated TOP1 proteins (Fig. 6B)Citation . Additional studies, especially identification of Cul3-containing E3 ligase complexes, will be needed to establish the molecular-based roles of Cul3 in TOP1 ubiquitylation.

Proteasome inhibitors have received much attention recently in the cancer chemotherapy field because of the potent antitumor activity of this class of drugs (40, 41, 42) . In addition, proteasome inhibitors have been shown to enhance antitumor activities of various chemotherapeutic agents, including TOP1-directed drugs (40) . Combined use of the first proteasome inhibitor, bortezomib (PS-341), and irinotecan enhances antitumor activity without observable adverse effects in mice, and that combination therapy now is in clinical trials (40 , 43) . Previous studies showed that the enhanced antitumor activity involves inhibition of antiapoptotic transcriptional factor, nuclear factor-{kappa}B (44) . In addition to this mechanism, it is likely that preventing degradation of TOP1-DNA covalent complex may be associated with the effectiveness of the combined use, as shown by this and previous studies (13) . Supporting this notion is the previous observation that nuclear factor-{kappa}B activation in response to CPT depends on the TOP1-DNA covalent complex (45) . We additionally have shown, herein, that elevated expression of Cul3 confers cellular resistance to TOP1-directed drugs and promotes proteasomal degradation of TOP1-DNA covalent complexes, both of which can be antagonized by proteasome inhibition. Therefore, determination of Cul3 expression in tumors may have an implication for effective combined use of proteasome inhibitors with TOP1 interactive agents in the clinic.


   ACKNOWLEDGMENTS
 
We thank Drs. Mikihiko Naito and Naoya Fujita for helpful discussions.


   FOOTNOTES
 
Grant support: Grant-in-Aid for Scientific Research on Priority Areas Cancer from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Takashi Tsuruo, Laboratory of Cell Growth and Regulation, Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan. Phone: 81-3-5841-8488; Fax: 81-3-5841-8487; E-mail: ttsuruo{at}iam.u-tokyo.ac.jp

Received 9/10/03. Revised 11/ 6/03. Accepted 11/17/03.


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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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