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Cancer Res-2004-Zhang-1114-21-2 PDF
Cancer Res-2004-Zhang-1114-21-2 PDF
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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 4°C. After removing insoluble materials by centrifugation, the supernatants
were cultured at 37°C 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 737–755 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 manufacturer’s
(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 Dunnett’s 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 25°C. 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 4°C. 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
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
Fig. 1. Enhanced camptothecin (CPT)-induced cell death with stabilization of DNA cells treated with 0.1 g/ml of the drug (Fig. 2B). Although the
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
proteasome inhibitor MG132 increased the SN38-induced covalent
S-phase-synchronized cells (A–D) were treated for 4 h with the indicated concentrations complexes in both cell lines, the increments were more dramatic in
of CPT (A and B), SN38 (C), and topotecan (D) in the presence or absence of lactacystin St-4/CPT cells than in St-4 cells (Fig. 2B). Furthermore, when the
(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 cells were treated with 1 g/ml of SN38, the total expression level of
complex of TOP assay in S-phase-synchronized HT-29 cells that were treated with 100 TOP1 was decreased rapidly in St-4/CPT cells compared with St-4
ng/ml of CPT for the indicated periods (E) or 4 h (F) in the presence or absence of cells (Fig. 2C). The decrease in TOP1 protein was suppressed almost
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 completely by MG132. These results indicated that the proteasome-
M). Four g of DNA were slot-blotted and probed with anti-TOP1 antibody. G, dependent TOP1 degradation pathway was enhanced in the Cul3-
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
overexpressing St-4/CPT cells.
LCT, as in A. 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,
cells was almost the same with or without lactacystin (21.0 ⫾ 0.5 and designated Cul3 si262, si520, and si737. Immunoblot analysis of
21.5 ⫾ 0.2 ng of CPT/107 cells, respectively). We then determined the extracts from siRNA-transfected HT1080 cells revealed that the Cul3-
effect of proteasome inhibitors on CPT-induced TOP1-DNA cleav- directed siRNAs effectively reduced expression of Cul3 without af-
able complexes using the in vivo complex of TOP assay, which detects fecting that of Cul1 or Cul2 (Fig. 3A). The si262 also reduced Cul3
TOP1 that covalently bound to cellular DNA. In HT-29 cells syn- expression in Cul3-overexpressing St-4/CPT cells to the level in St-4
chronized in the S phase, the TOP1-DNA covalent complex rapidly cells (Fig. 3B). Although no alteration in total expression level of
reached a peak level, within 30 min after 100 ng/ml of CPT was TOP1 was observed in these siRNA-treated cells, si262 significantly
added, and then decreased gradually despite the presence of CPT (Fig. increased the amounts of SN38-induced TOP1-DNA covalent com-
1E). Lactacystin effectively prevented the decrease of the TOP1-DNA plex as compared with control siRNA (Fig. 3, C and D). Proteasome
covalent complex and kept it at high levels during the 4-h CPT inhibition increased the levels of the covalent complex in the Cul3-
treatment, although the peak level (0.5 h) was hardly increased by the and control siRNA-treated cells, and consequently, the differences in
proteasome inhibitor. Likewise, PSI and MG132 retained the CPT- covalent complex formation disappeared, indicating that loss of Cul3
induced TOP1-DNA covalent complex at high levels (Fig. 1F). In impaired proteasome-dependent degradation of the TOP1-DNA co-
those experiments, however, the TOP1-DNA covalent complex was valent complex.
below the detectable level without CPT. Similar results were obtained Enhanced TOP1 Degradation by Ectopic Expression of Cul3.
in A2780 cells (data not shown). To address whether overexpression of Cul3 promotes CPT-induced
We also determined the total cellular expression levels of TOP1 in TOP1 degradation, we established stable transfectants using an ex-
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CUL3 AND TOP1 DEGRADATION
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CUL3 AND TOP1 DEGRADATION
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CUL3 AND TOP1 DEGRADATION
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
Fig. 7. SN38 resistance by stable Cul3 overexpression. A–D, mock- and Cul3- of topologic stress in DNA. In that situation, the majority of TOP1
transfected cells (CLN1 and CLN2), as well as untransfected HT1080 cells (A), were proteins remain free from covalent complexes, and available TOP1
treated with 30 ng/ml of SN38 (A) or the indicated concentrations of SN38 (B), cisplatin (possibly sumoylated) may be recruited to dissolve the topologic
(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 stress in DNA. On recruitment to DNA, the TOP1 protein will be
3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay (B–D), re- trapped in the covalent complex by CPT and degraded as described
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
previously. The futile cycles of recruitment and degradation may lead
(MG132; 5 M) or the solvent was added 1 h before the SN38 addition (time 0). The cells eventually to overall reduction in TOP1 proteins. However, our pres-
additionally were cultured in drug-free medium up to the indicated time points. The dead ent results also showed that proteasome inhibition only keeps the
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 levels of TOP1-DNA covalent complex but does not change the peak
survival of the mock group with the CLN1 and CLN2 groups, respectively (A and B). levels (Fig. 1E), which appear to depend on CPT concentration. Thus,
ⴱP ⬍ 0.001. it may be that proteasome inhibition affects the recruiting and the
degradation process. Alternatively, the defined levels, depending on
transfected cells that were treated with SN38 alone. Although MG132 CPT doses, may be determined by other repair mechanism(s), possi-
also sensitized the mock-transfected cells to SN38, the sensitization bly by the tyrosyl-DNA phosphodiesterase Tdp1, which removes
effect was relatively transient as compared with that seen in CLN1. TOP1 from the covalent complex (38).
Similar results also were obtained using CLN2 and lactacystin (data The mechanisms behind Cul3-regulated TOP1 degradation remain
not shown). to be determined. According to the aforementioned model, it is
possible that Cul3 acts as the E3 ubiquitin-TOP1 ligase. We showed
DISCUSSION that after SN38 treatment, ubiquitylated TOP1 proteins were elevated
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Research.
CUL3 AND TOP1 DEGRADATION
more in the Cul3-overexpressing cells than in the mock-transfected 10. Goldwasser, F., Bae, I., Valenti, M., Torres, K., and Pommier, Y. Topoisomerase
I-related parameters and camptothecin activity in the colon carcinoma cell lines
cells (Fig. 6B). However, we have failed to detect binding between from the National Cancer Institute anticancer screen. Cancer Res., 55: 2116 –
Cul3 and TOP1 either in vitro or in vivo using immunologic tech- 2121, 1995.
niques. This may be because of technical problems; for example, it is 11. Murren, J. R., Beidler, D. R., and Cheng, Y. C. Camptothecin resistance related
to drug-induced down-regulation of topoisomerase I and to steps occurring after
difficult to extract DNA-linked TOP1 under mild experimental con- the formation of protein-linked DNA breaks. Ann. N. Y. Acad. Sci., 803: 74 –92,
ditions. Another difficulty is that cullin family proteins generally form 1996.
multiprotein complexes and function as a scaffold protein in the E3 12. Desai, S. D., Liu, L. F., Vazquez-Abad, D. and D’Arpa, P. Ubiquitin-dependent
destruction of topoisomerase I is stimulated by the antitumor drug camptothecin.
ligase complexes (19, 20). Thus, TOP1 recognition by Cul3 may J. Biol. Chem., 272: 24159 –24164, 1997.
require other protein(s), like F-box proteins in the Cul1-based Rbx1- 13. Desai, S. D., Li, T. K., Rodriguez-Bauman, A., Rubin, E. H., and Liu, L. F.
Skp1-Cul1-F-box E3 ligase. Alternatively, it also is possible that Cul3 Ubiquitin/26S proteasome-mediated degradation of topoisomerase I as a resistance
mechanism to camptothecin in tumor cells. Cancer Res., 61: 5926 –5932, 2001.
regulates an as-yet unidentified ubiquitin-TOP1 ligase(s) or the re- 14. Desai, S. D., Zhang, H., Rodriguez-Bauman, A., Yang, J. M., Wu, X., Gounder,
cently identified TOP1-interacting protein TOPRS, which is a poten- M. K., Rubin, E. H., and Liu, L. F. Transcription-dependent degradation of topoi-
tial E3 with a ring finger domain (39). Such multistep regulation of somerase I-DNA covalent complexes. Mol. Cell. Biol., 23: 2341–2350, 2003.
15. Wu, J., and Liu, L. F. Processing of topoisomerase I cleavable complexes into DNA
TOP1 ubiquitylation could be supported by the present observation damage by transcription. Nucleic Acids Res., 25: 4181– 4186, 1997.
that proteasome inhibition paradoxically reduced the amounts of 16. Hershko, A., Heller, H., Elias, S., and Ciechanover, A. Components of ubiquitin-
protein ligase system. Resolution, affinity purification, and role in protein breakdown.
ubiquitin-conjugated TOP1 proteins (Fig. 6B). Additional studies,
J. Biol. Chem., 258: 8206 – 8214, 1983.
especially identification of Cul3-containing E3 ligase complexes, will 17. Pray, T. R., Parlati, F., Huang, J., Wong, B. R., Payan, D. G., Bennett, M. K.,
be needed to establish the molecular-based roles of Cul3 in TOP1 Issakani, S. D., Molineaux, S., and Demo, S. D. Cell cycle regulatory E3 ubiquitin
ligases as anticancer targets. Drug Resist. Update, 5: 249 –258, 2002.
ubiquitylation. 18. Pickart, C. M. Mechanisms underlying ubiquitination. Annu. Rev. Biochem., 70:
Proteasome inhibitors have received much attention recently in the 503–533, 2001.
cancer chemotherapy field because of the potent antitumor activity of 19. Patton, E. E., Willems, A. R., Sa, D., Kuras, L., Thomas, D., Craig, K. L., and Tyers,
M. Cdc53 is a scaffold protein for multiple Cdc34/Skp1/F-box protein complexes that
this class of drugs (40 – 42). In addition, proteasome inhibitors have regulate cell division and methionine biosynthesis in yeast. Genes Dev., 12: 692–705,
been shown to enhance antitumor activities of various chemothera- 1998.
peutic agents, including TOP1-directed drugs (40). Combined use of 20. Yu, H., Peters, J. M., King, R. W., Page, A. M., Hieter, P., and Kirschner, M. W.
Identification of a cullin homology region in a subunit of the anaphase-promoting
the first proteasome inhibitor, bortezomib (PS-341), and irinotecan complex. Science (Wash. DC), 279: 1219 –1222, 1998.
enhances antitumor activity without observable adverse effects in 21. Ou, C. Y., Lin, Y. F., Chen, Y. J., and Chien, C. T. Distinct protein degradation
mice, and that combination therapy now is in clinical trials (40, 43). mechanisms mediated by Cul1 and Cul3 controlling Ci stability in Drosophila eye
development. Genes Dev., 16: 2403–2414, 2002.
Previous studies showed that the enhanced antitumor activity involves 22. Dias, D. C., Dolios, G., Wang, R., and Pan, Z. Q. CUL7: A DOC domain-containing
inhibition of antiapoptotic transcriptional factor, nuclear factor-B cullin selectively binds Skp1. Fbx29 to form an SCF-like complex. Proc. Natl. Acad.
(44). In addition to this mechanism, it is likely that preventing deg- Sci. USA, 99: 16601–16606, 2002.
23. Kamura, T., Koepp, D. M., Conrad, M. N., Skowyra, D., Moreland, R. J., Iliopoulos,
radation of TOP1-DNA covalent complex may be associated with the O., Lane, W. S., Kaelin, W. G., Jr., Elledge, S. J., Conaway, R. C., Harper, J. W., and
effectiveness of the combined use, as shown by this and previous Conaway, J. W. Rbx1, a component of the VHL tumor suppressor complex and SCF
ubiquitin ligase. Science (Wash. DC), 284: 657– 661, 1999.
studies (13). Supporting this notion is the previous observation that 24. Ohta, T., Michel, J. J., Schottelius, A. J., and Xiong, Y. ROC1, a homolog of APC11,
nuclear factor-B activation in response to CPT depends on the represents a family of cullin partners with an associated ubiquitin ligase activity. Mol.
TOP1-DNA covalent complex (45). We additionally have shown, Cell., 3: 535–541, 1999.
25. Pause, A., Lee, S., Worrell, R. A., Chen, D. Y., Burgess, W. H., Linehan, W. M., and
herein, that elevated expression of Cul3 confers cellular resistance to Klausner, R. D. The von Hippel-Lindau tumor-suppressor gene product forms a stable
TOP1-directed drugs and promotes proteasomal degradation of complex with human CUL-2, a member of the Cdc53 family of proteins. Proc. Natl.
TOP1-DNA covalent complexes, both of which can be antagonized by Acad. Sci. USA, 94: 2156 –2161, 1997.
26. Du, M., Sansores-Garcia, L., Zu, Z., and Wu, K. K. Cloning and expression analysis
proteasome inhibition. Therefore, determination of Cul3 expression in of a novel salicylate suppressible gene, Hs-CUL-3, a member of cullin/Cdc53 family.
tumors may have an implication for effective combined use of pro- J. Biol. Chem., 273: 24289 –24292, 1998.
teasome inhibitors with TOP1 interactive agents in the clinic. 27. Beidler, D. R., and Cheng, Y. C. Camptothecin induction of a time- and concentra-
tion-dependent decrease of topoisomerase I and its implication in camptothecin
activity. Mol. Pharmacol., 47: 907–914, 1995.
28. Kim, H. D., Tomida, A., Ogiso, Y., and Tsuruo, T. Glucose-regulated stresses cause
ACKNOWLEDGMENTS degradation of DNA topoisomerase IIá by inducing nuclear proteasome during G1
cell cycle arrest in cancer cells. J. Cell. Physiol., 180: 97–104, 1999.
29. Ogiso, Y., Tomida, A., Lei, S., Omura, S., and Tsuruo, T. Proteasome inhibition
We thank Drs. Mikihiko Naito and Naoya Fujita for helpful discussions. circumvents solid tumor resistance to topoisomerase II-directed drugs. Cancer Res.,
60: 2429 –2434, 2000.
30. Kanzawa, F., Sugimoto, Y., Minato, K., Kasahara, K., Bungo, M., Nakagawa, K.,
Fujiwara, Y., Liu, L. F., and Saijo, N. Establishment of a camptothecin analogue
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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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