[CANCER RESEARCH 64, 1114 1121, February 1, 2004]
Cullin 3 Promotes Proteasomal Degradation of the Topoisomerase I-DNA
Covalent Complex
Hua-Feng Zhang,1 Akihiro Tomida,1 Ritsuko Koshimizu,1 Yasunari Ogiso,1 Shuhong Lei,1 and Takashi Tsuruo1,2
1
Institute of Molecular and Cellular Biosciences, The University of Tokyo, and 2Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan
ABSTRACT
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 path-
way. We found that proteasome inhibition prevents degradation of DNA-
bound TOP1 and sustains high levels of covalent complexes, thus enhanc-
ing 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 cel-
lular CPT sensitivity.
INTRODUCTION
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 mam-
malian cells (13). 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 (57). The predominant cytotoxic mecha-
nism of CPT is its stabilization of the cleavable complexes by inhib-
iting 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 11). Recent studies have suggested that proteasome-
dependent TOP1 degradation in response to CPT is an important down-
stream 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
Received 9/10/03; revised 11/6/03; accepted 11/17/03.
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 from Zymed (South San Francisco, CA).
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@iam.u-tokyo.ac.jp.
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polymerase complexes and the CPT-stabilized cleavable complexes (14,
15). CPT consistently and transiently inhibits transcription, and the re-
covery 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 degra-
dation 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 physio-
logic 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). How-
ever, it remains to be determined whether the proteasomal TOP1
degradation pathway influences the S-phase specificity of CPT cyto-
toxicity and the levels of TOP1-DNA covalent complexes. In addi-
tion, little is known about the proteins that regulate proteasomal TOP1
degradation. In this report, we show that proteasome inhibition selec-
tively 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
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--t-butyl-L-glutamyl-L-alanyl-L-leucinal (PSI)
and MG132 were from the Peptide Institute Inc. (Osaka, Japan). These com-
pounds, 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 (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
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 g/ml of kanamycin. The human
 CUL3 AND TOP1 DEGRADATION

fibrosarcoma HT1080 cells were grown in RPMI 1640 supplemented with in the presence of protease inhibitor mixture, and nuclei were rocked for 1 h
10% heat-inactivated fetal bovine serum and 100 g/ml of kanamycin. They at 4C. After removing insoluble materials by centrifugation, the supernatants
were cultured at 37C in a humidified atmosphere containing 5% CO2. All were recovered as nuclear extracts. Protein concentrations were determined
experiments were performed using exponentially growing cells and were using a protein assay kit (Bio-Rad).
repeated at least twice. Immunoblot and Immunoprecipitation. For immunoblot analysis, equal
Synchronization and Treatments. For the synchronized culture, we amounts of proteins were resolved on an SDS-polyacrylamide gel and elec-
trapped cells in M phase of the cell cycle by treating them with 40 ng of troblotted onto a nitrocellulose membrane (Schleicher & Schuell; Ref. 32).
nocodazole/ml (Wako Pure Chemical Industries, Osaka, Japan) for 9 h, col- Membranes were probed with specific antibodies and appropriate secondary
lected cells by gentle pipetting, and reseeded them in fresh culture medium (28, antibodies, and the specific signals were detected using an enhanced chemi-
29). Populations of G1- and S-phase cells constituted the majority (typically luminescence detection system (Amersham Pharmacia Biotech, Tokyo, Japan).
60 70%) of the total cell population at 3 h and 9 h after the release, respec- The densitometric analysis was done as described previously.
tively (28, 29). For treatment of these cells, TOP1-directed drugs were added Immunoprecipitation of TOP1 was performed as described previously (32).
into the medium at 4 h (G1 phase) and 10 h (S phase) after the release. The Briefly, nuclear extracts were adjusted to 150 mM NaCl using the appropriate
proteasome inhibitor lactacystin was added 1 h after nocodazole was removed buffer without NaCl. Equal amounts of proteins were precleared and immu-
to avoid any effects on the release from M phase and cell cycle progression noprecipitated with the anti-TOP1 antibody and a Sepharose-immobilized
(29). MG132 and PSI were added 1 h before the addition of TOP1-directed protein L (Pierce, Rockford, IL). The immunocomplexes were washed five
drugs. For the colony formation assay, cells were treated for 4 h with TOP1- times with a washing buffer consisting of 50 mM Tris-HCl (pH 7.4), 150 mM
directed drugs, diluted appropriately with fresh medium, and cultured to form NaCl, 0.5% NP40, 5 mM MgCl2, 5 mM N-ethylmaleimide, and the protease
colonies for 7 8 days (29). The cell survival (mean SD in triplicate) was inhibitor mixture and were resuspended subsequently in 2 SDS sample
calculated by setting each of the appropriate control survivals as 1. buffer. After boiling for 5 min, the complexes were evaluated using immuno-
Cellular Accumulation of CPT. Intracellular CPT accumulation was de- blot analysis.
termined as described previously (30). Briefly, S-phase-synchronized HT-29 RNA Interference. Three Cul3-specific small interfering RNAs (siRNAs),
cells (1 107) were treated for 4 h with CPT (10 g/ml) in the presence or si262, si520, and si755, were designed based on the Cul3 cDNA sequence
absence of lactacystin at 7.5 M. The cells were washed twice, as quickly as (GenBank accession no. NM003590). These correspond to nucleotides 262
possible, with cold PBS [137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 1.5 280 for si262, 520 538 for si520, and 737755 for si755, when the transla-
mM KH2PO4 (pH 7.4)] containing 0.01 N HCl and were suspended in the same tional start codon is numbered as nucleotide 1. The control siRNA was a
buffer. After sonication and centrifugation, the supernatants were used, with double-stranded 21-mer unrelated to the Cul3 sequence. All of the double-
spectrofluorometry (excitation at 380 nm and emission at 430 nm), to deter- stranded RNAs were purchased from Qiagen-Xeragon (Tokyo, Japan). Tran-
mine CPT contents. sient transfection of siRNA was performed either with oligoamine reagent
MTT and Flow Cytometric Cytotoxicity Assays. For the 3-(4,5-dimeth- (Qiagen-Xeragon) for HT1080 cells or with lipofectamin/plus reagent (Invitro-
ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma Chemical Co., gen, San Diego, CA) together with pcMyc vector as a carrier for St-4/CPT
St. Louis, MO) assay, cells were cultured overnight in 96-well plates. Drug cells, according to the manufacturers protocols. Three days after transfection,
solutions were added directly to the culture medium, and the cells were the cells were used for experimentation.
cultured for an additional 24 h (for stable transfectants of Cul3) or 72 h (for Plasmids and Stable Transfection. The pcMyc vector was produced by
St-4 and St-4/CPT cells). MTT (Sigma Chemical Co.) was added subsequently ligating oligo-DNA encoding N-terminal Myc epitope to the HindIII site of
to the culture medium, and the absorbance of each well was determined as pcDNA3 (Invitrogen). Human Cul3 cDNA was generated by PCR and cloned
described previously (31). Relative cell survival (mean SD in sextuplicate) in frame into the pcMyc vector at the KpnI site. The constructs were confirmed
was calculated by setting each of the appropriate control absorbance as 1. For by DNA sequencing. Transfections of plasmids containing no insert (mock)
the flow cytometric assay, cells were cultured overnight in six-well plates, and Myc-tagged Cul3 were performed using the FuGENE 6 Transfection
treated with drugs as indicated, and stained with 7-amino-actinomycin D Reagent (Roche Molecular Biochemicals) according to the manufacturers
(PharMingen, San Diego, CA). Dead cell populations (mean SD in three protocol. After 24 h of drug-free incubation, the cells were cultured for 1 week
independent experiments) were determined using a Beckman Coulter flow in culture medium containing 300 g/ml of G418 (Invitrogen). G418-resistant
cytometer with Cytomics FC500 RXP software (Fullerton, CA). Statistical cells were cloned subsequently and maintained in culture medium containing
significance of cell survival and dead cell populations was evaluated using a 300 g/ml of G418. The clones were cultured in G418-free medium for
one-way ANOVA with Dunnetts test. experiments to avoid any effects from G418.
In Vivo Complex of TOP Bioassay. We performed the in vivo complex of
TOP assay as described previously (32). Immediately after CPT treatment,
RESULTS
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 Enhanced CPT-Induced Cell Death with Stabilization of TOP1-
density) and centrifuged at 438,000 g for 5 h at 25C. The cellular DNA DNA Complex by Proteasome Inhibition. We examined the cyto-
pellet was dissolved in 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA, and the
toxic effects of CPT in combination with a proteasome inhibitor,
concentrations were measured by a spectrophotometer. Fixed amounts of DNA
lactacystin, using a synchronization culture system. In the experi-
were blotted on a nitrocellulose membrane (Schleicher and Schuell, Dassel,
Germany) using a slot-blot device (Bio-Rad, Hercules, CA). Blots were probed ments, lactacystin was used in concentrations at which it decreased the
subsequently with anti-TOP1 antibody. Band intensities were quantified using colony-forming ability of cells to 70 85%. Consistent with the fact
NIH image 1.62 software (Bethesda, MD). that CPT shows an S-phase-specific cytotoxicity, a 4-h treatment with
Preparation of Total Cellular and Nuclear Extracts. Total cellular and CPT reduced profoundly the colony-forming ability of HT-29 cells
nuclear extracts were prepared as described previously (32). For preparation of during S phase but only marginally during G1 phase (Fig. 1A). In the
total cell extracts, cells were lysed with ice-cold, high-salt lysis buffer [50 mM presence of lactacystin (7.5 M), the cell-killing effect was enhanced
Tris-HCl (pH 7.4), 800 mM NaCl, 0.5% NP40, 5 mM MgCl2, and 5 mM strongly in the S-phase cells but not in the G1-phase cells, suggesting
N-ethylmaleimide] in the presence of the protease inhibitor mixture for mam- that lactacystin potentiated the CPT-mediated cytotoxicity. Similar
malian cells (Sigma Chemical Co.). After removing insoluble materials by
potentiation by lactacystin (5 M) was observed in S-phase-synchro-
centrifugation, the supernatants were recovered as total cell extracts. For
nized A2780 cells (Fig. 1B). Lactacystin also enhanced the cytotox-
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 icity of SN38 (the active metabolite of irinotecan; Ref. 33) and
PMSF, 0.2 mM DTT, 5 mM N-ethylmaleimide, and 0.3% Triton-X100] with topotecan during S phase in HT-29 cells (Fig. 1, C and D). Other
gentle rocking for 10 min at 4C. After centrifugation, the isolated nuclei were proteasome inhibitors, such as PSI and MG132, also enhanced CPT
resuspended in ice-cold nucleus extraction buffer [500 mM NaCl, 50 mM cytotoxicity during S phase (data not shown).
Tris-HCl (pH 7.5), 1 mM EDTA, 10% glycerol, and 5 mM N-ethylmaleimide] The intracellular CPT accumulation after a 4-h exposure of HT-29
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 CUL3 AND TOP1 DEGRADATION
Fig. 1. Enhanced camptothecin (CPT)-induced cell death with stabilization of DNA
topoisomerase I (TOP1)-DNA complex by proteasome inhibition. AD, colony formation
assay in synchronized HT29 (A, C, and D) and A2780 cells (B). The G1- (A) or
S-phase-synchronized cells (AD) 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 M) and A2780 (5 M) 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 M), carbobenzoxy-L-isoleucyl--t-butyl-L-glutamyl-L-
alanyl-L-leucinal (PSI; 5 M), and carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132; 5
M). Four g of DNA were slot-blotted and probed with anti-TOP1 antibody. G,
immunoblot analysis for TOP1 and TOP2 expression. S-phase-synchronized HT-29 and
A2780 cells were exposed to solvent (DMSO) or 10 g/ml of CPT for 4 h with or without
LCT, as in A.
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 cleav-
able complexes using the in vivo complex of TOP assay, which detects
TOP1 that covalently bound to cellular DNA. In HT-29 cells syn-
chronized 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). 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). 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
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HT-29 and A2780 cells. The TOP1, but not TOP2, 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). It should be noted that the overall TOP1 reduction in HT-29 cells
was detected only when a relatively high concentration of CPT (10
g/ml) was used as compared with the aforementioned in vivo com-
plex 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 deg-
radation 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 expres-
sion.
Overexpression of Cul3 and Enhanced TOP1 Degradation in
CPT-Resistant Cells. While searching for proteins that could medi-
ate 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 pa-
rental lines (Fig. 2A). 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). 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), suggest-
ing that the Cul3 overexpression had little effect on the basal expres-
sion levels of TOP1.
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 g/ml examined, than those in parental St-4
cells treated with 0.1 g/ml of the drug (Fig. 2B). 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). Furthermore, when the
cells were treated with 1 g/ml of SN38, the total expression level of
TOP1 was decreased rapidly in St-4/CPT cells compared with St-4
cells (Fig. 2C). 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 af-
fecting that of Cul1 or Cul2 (Fig. 3A). The si262 also reduced Cul3
expression in Cul3-overexpressing St-4/CPT cells to the level in St-4
cells (Fig. 3B). 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 com-
plex as compared with control siRNA (Fig. 3, C and D). 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 co-
valent complex.
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 ex-
 CUL3 AND TOP1 DEGRADATION
Fig. 2. Overexpression of Cul3 and enhanced DNA topoisomerase I (TOP1) degrada-
tion 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 M) or the solvent (DMSO) for 1 h, and the
indicated doses of SN38 then were added to culture media for 4 h. Four g 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 g/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 g/ml) with () or without () MG132 (5 M) pretreatment. Top and
middle panels show results of the same samples after long and short exposures, respec-
tively.
pression 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 immu-
noblot analysis using an anti-Myc epitope antibody (Fig. 4A). The
total expression levels of Cul3 in CLN1 and CLN2 were approxi-
mately four and three times, respectively, higher than those in control
mock-transfected and untransfected HT1080 cells (Fig. 4A). However,
the expression levels of TOP1, as well as Cul1 and Cul2, in the clones
were similar to those in the control cells (Fig. 4A; data not shown).
The Cul3-transfected clones proliferated well, and their growth rates
were almost the same as those of control cells (Fig. 4B).
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). 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
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levels, and consequently, the levels became essentially the same
between the cell lines (Fig. 5B). Thus, degradation of TOP1-DNA
covalent complexes was accelerated in CLN1 cells. The accelerated
degradation also was observed in CLN2 (Fig. 5C). 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 g/ml), it occurred more rapidly in CLN1
than in mock-transfected cells (Fig. 5D).
To address whether Cul3 overexpression influences ubiquitylation
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 M). The
SN38 concentrations we used were 100 ng/ml for HT1080 cells (C) and 3 g/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 g of DNA were slot-blotted
and probed with anti-TOP1 antibody.
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 (1 104/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.
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 CUL3 AND TOP1 DEGRADATION

nuclear extracts (Fig. 6D). Nevertheless, the MG132 cotreatment

decreased ubiquitin-conjugated TOP1 proteins (Fig. 6B). Thus,
MG132 may stabilize TOP1 not only by inhibiting proteasome activ-
ity 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). 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) and an MTT assay (Fig.
7B). 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). Con-
sistent with the higher level of Cul3 expression, CLN1 was more
resistant to SN38 than was CLN2 (compare Fig. 4A and Fig. 7A).
However, these Cul3 stable transfectants showed almost the same
sensitivity to cisplatin and etoposide as the mock transfectant (Fig. 7,
C and D). Thus, Cul3 overexpression selectively conferred resistance
to TOP1-directed drugs.
We also examined the effect of MG132 on the SN38 resistance in
CLN1 using the flow cytometric assay (Fig. 7E). 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-

Fig. 5. Enhanced DNA topoisomerase I (TOP1) degradation by stable Cul3 overex-

pression. AC, 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 M) or the solvent was added 1 h before the SN38 addition (B and C). Four
g 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 g/ml) with
() or without () MG132 (5 M) 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).

of TOP1, we immunoprecipitated TOP1 from nuclear extracts and

monitored the ubiquitylation status (Fig. 6). Comparable amounts of
TOP1 were immunoprecipitated when CLN1 and mock-transfected
cells were treated with or without 1 g/ml of SN38 for 30 min (Fig.
6A). Immunoblot analysis with antiubiquitin antibody revealed that
ubiquitylated TOP1 proteins increased in response to SN38 (Fig. 6B).
Consistent with the accelerated TOP1 degradation, ubiquitylated
TOP1 proteins were more abundant in Cul3-overexpressing CLN1
than in mock-transfected cells (Fig. 6B).
When the transfected cells were cotreated with SN38 and MG132,
total ubiquitylated proteins increased in the preimmunoprecipitated
1118
Fig. 6. Enhanced DNA topoisomerase I (TOP1) ubiquitylation by stable Cul3 over-
expression. Mock- and Cul3-transfected cells (CLN1) were treated with 1 g/ml of SN38
or the solvent for 30 min. Carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132; 5 M) 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. Im-
munoprecipitates were analyzed by immunoblot with anti-TOP1 (A), antiubiquitin (Ub;
B), or anti-SUMO-1 (SUMO) antibodies (C). The nuclear extracts (not immunoprecipi-
tated) also were probed with anti-Ub antibody (D).

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Research.
 CUL3 AND TOP1 DEGRADATION
Fig. 7. SN38 resistance by stable Cul3 overexpression. AD, 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 (BD), re-
spectively, as described in Materials and Methods. E, mock and CLN1 cells were treated
with 3 g/ml of SN38 or the solvent for 4 h. Carbobenzoxy-L-leucyl-L-leucyl-L-leucinal
(MG132; 5 M) 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 Dunnetts test, comparing the cell death or
survival of the mock group with the CLN1 and CLN2 groups, respectively (A and B).
P 0.001.
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
Proteasomal degradation of TOP1 has been implicated as an im-
portant step to repair CPT-induced DNA damage (12, 27). This is
supported additionally by our present findings that proteasome inhi-
bition sensitizes tumor cells to CPT cytotoxicity preferentially during
S phase of the cell cycle and suppresses degradation of TOP1-DNA
covalent complex (Fig. 1). 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. Ele-
vated levels of Cul3 were observed commonly in CPT-resistant cells
(Fig. 2A), and resistance to TOP1-directed drugs was reproduced by
ectopic expression of Cul3 (Fig. 7). The Cul3 overexpression pro-
moted proteasome-dependent TOP1 degradation and enhanced re-
moval of TOP1 from the covalent complexes (Fig. 5). Conversely,
Cul3 knockdown by siRNA reduced degradation of TOP1-DNA co-
valent complexes (Fig. 3). 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 iden-
tified as events immediately downstream from the TOP1-DNA cleav-
1119
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Research.
able complexes (12, 32, 35, 36). Both modifications can influence the
levels of CPT-induced TOP1-DNA covalent complex, but ubiquity-
lation and sumoylation appear to have opposite effects (Fig. 8). We
showed previously that sumoylation enhances the CPT-induced co-
valent 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 sumoy-
lated TOP1 proteins are linked covalently to DNA (32, 35). Con-
versely, 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).
The paradoxical responses may come from the complicated mech-
anisms of action of CPT and may provide an explanation why deg-
radation 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 pres-
ent results also showed that proteasome inhibition only keeps the
levels of TOP1-DNA covalent complex but does not change the peak
levels (Fig. 1E), 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), possi-
bly 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
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.
 CUL3 AND TOP1 DEGRADATION
more in the Cul3-overexpressing cells than in the mock-transfected
cells (Fig. 6B). However, we have failed to detect binding between
Cul3 and TOP1 either in vitro or in vivo using immunologic tech-
niques. This may be because of technical problems; for example, it is
difficult to extract DNA-linked TOP1 under mild experimental con-
ditions. 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 re-
cently identified TOP1-interacting protein TOPRS, which is a poten-
tial 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). 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 42). In addition, proteasome inhibitors have
been shown to enhance antitumor activities of various chemothera-
peutic 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-B
(44). In addition to this mechanism, it is likely that preventing deg-
radation 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-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 pro-
teasome inhibitors with TOP1 interactive agents in the clinic.
ACKNOWLEDGMENTS
We thank Drs. Mikihiko Naito and Naoya Fujita for helpful discussions.
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Cullin 3 Promotes Proteasomal Degradation of the
Topoisomerase I-DNA Covalent Complex
Hua-Feng Zhang, Akihiro Tomida, Ritsuko Koshimizu, et al.

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