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Molecular Biology and Genetics |
Departments of Oncology Research [P. C., M. L. B., P. W. F., M. R. M., B-B. S. Z.], Bioinformatics [M. R. H.], and Molecular Biology [Y. Z.], GlaxoSmithKline Pharmaceuticals, King of Prussia, Pennsylvania 19406, and Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111 [V. S., S. A. J., T. J. Y.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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How does Chfr delay cell entry into mitosis? Initiation of early mitotic events, including nuclear membrane breakdown and chromosome condensation, requires the activation of mitotic kinases such as cyclin B/cdc2 (8) . Because cyclin B/cdc2 activity remains high during the Chfr-dependent delay, its activity might not be the target of the Chfr-dependent pathway (7) . Alternatively, Chfr may be involved in controlling cyclin B/cdc2 substrates that regulate chromosome condensation and nuclear membrane breakdown. Chfr contains a FHA3 domain, which could bind phosphorylated peptides (9 , 10) ; thus, protein kinases might act upstream of Chfr and regulate its activity. Chfr also contains a RING-finger domain, which is found in several ubiquitin ligases (11) . In addition to ubiquitin ligase (E3), protein ubiquitination also involves the action of a ubiquitin-activating enzyme (E1) and a ubiquitin-conjugating enzyme (E2; Ref. 12 ). The spindle assembly checkpoint is associated with a ubiquitin-dependent pathway and is regulated by the ubiquitin ligase activity of the APC/C (13) . Recently, a subunit of APC/C, which contains a RING-finger domain, APC11, was shown to be the ubiquitin ligase in the APC/C (14 , 15) . Because Chfr has a RING-finger domain, it was of interest to see whether it had a role to play in the ubiquitination process as an E3 and whether this activity of Chfr mediates its function in the mitotic stress response.
We report here that Chfr has ubiquitin ligase activity, which may be required for its role in delaying chromosome condensation in cells stressed by tubulin poisons such as Taxol. Chfr expression increases cellular survival after Taxol treatment, consistent with a role for Chfr in surviving mitotic stress.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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R) was created by a restriction digestion approach. The pACcG2T vector containing full-length chfr was digested with BamHI and EcoRI to delete the following amino acids constituting the RING domain: CIICQDLLHDCVSLQPCMHTFCAACYSGWMERSSLCPTCRCPVE. The RING-fingerless domain fragment was then religated in frame. Both Chfr and ChfrR were also subcloned into mammalian expression vector pWSgfp (16)
for transfection experiments.
Chfr or ChfrR construct in pACcG2T vector was transfected into Sf-9 cells, and the virus from the transfection was harvested for amplification. The high titer viral stocks were additionally optimized for expression and scaleup. The harvested cells were lysed, and the protein was purified by sequential chromatography steps including glutathione Sepharose and Superdex G75 columns at 4°C.
Cell Lines.
Human SW480 was cultured in RPMI 1640 containing 10% FBS, human SAOS2 was maintained in McCoys 5A medium containing 15% FBS, HeLa, and DLD1 cell lines were maintained in DMEM with 10% FBS (complete medium). For creating stable cell lines, DLD1 cells were transfected with pWSgfp-Chfr or pWSgfp-ChfrR using the LipofectAMINE method (Life Technologies, Inc., Gaithersburg, MD). Clones were selected in G418 and confirmed both by expression of the GFP-Chfr protein using a fluorescent microscope and by Western blotting with anti-GFP and anti-Chfr antibody. The stable cell lines including DLD1/Chfr and DLD1/ChfrR were maintained in complete medium containing 400 µg/ml of G418. For synchronization, the cells were treated with 2 mM thymidine for 18 h, released for 6 h, then treated with 5 µg/ml of aphidicolin for 20 h. To induce mitotic stress, cells were treated with 300 nM Taxol for 18–20 h.
Antibodies.
Rabbit polyclonal antibodies against Chfr were generated against a NH2-terminal peptide of human Chfr: MERPEEGKQSPPPQPWGRLLRC-COOH and affinity purified. Antibodies against GFP and HA tag were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Western Blotting and Immunoprecipitation.
Nuclear lysates were prepared using an extraction kit from Pierce (Rockford, IL). The protein in the lysates was estimated and an equal amount of protein (150 µg) was immunoprecipitated with affinity-purified antibody at 4°C for 2 h followed by incubation with protein A-Sepharose for 1 h at 4°C. The samples were analyzed by SDS-PAGE. The gel was then transferred onto a nitrocellulose membrane. The membrane was first blocked in 3% milk followed by addition of primary antibody for 2 h at room temperature in milk and the secondary antibody for 1 h at room temperature. The blot was visualized by enhanced chemiluminescence (NEN, Boston, MA).
Mitotic Index.
Overnight, 2 x 104 cells/well in 24-well plates were grown in 37°C, 5% CO2. Taxol (300 nM) dissolved in culture medium was applied to cells, and cells were incubated for 20 h. Medium was aspirated, and cells were washed and fixed in 3% paraformaldehyde for 5 min. Permeabilization was accomplished using 5% Triton X-100 for 5 min. DNA was stained with 100 ng/ml Hoechst dye for 5 min after which the cells were washed and fresh PBS applied. Condensed chromosome and total cell counts were done manually using a fluorescent microscope. Mitotic index was calculated based on the percentage of cells with condensed chromosomes.
In Vitro Ubiquitination Assay.
Ubiquitin ligase activity was determined as described previously (17)
. Reaction mixture contained the following in a volume of 10 µl: 40 mM Tris-HCl (pH 7.4), 1 mg/ml BSA, 1 mM DTT, 5 mM MgCl2, 10 mM phospho-creatine, 50 mg/ml creatine phosphokinase, 50 mM ubiquitin, 1 mM ubiquitin aldehyde, 1 pmol of E1, and 1.4 mg/ml wild-type Chfr or the Chfr mutants. The E2 enzymes were then added. After incubation at 30°C for 30 min, samples were subjected to electrophoresis on a 7.5% polyacrylamide-SDS gel. After transfer the membrane was probed with anti-Chfr and anti-ubiquitin antibodies. Purified recombinant UbcH5 was purchased from BostonBiochem (Boston, MA); purified recombinant UbcH4 was a generous gift of Dr. Hongtao Yu.
In Vivo Ubiquitination Assay.
HeLa cells (2 x 106) in Petri dishes were transiently cotransfected with 10 µg GFP-tagged Chfr or ChfrR and HA-tagged ubiquitin constructs. HA-tagged ubiquitin construct was a generous gift of Dr. Dale Haines. Total cell lysates were prepared and immunoprecipitated with anti-GFP antibody and immunoblotted with anti-Chfr antibody.
Transient Transfection for Immunofluorescence.
Dishes of DLD-1 cells (35 mm) containing coverslips were transfected with 2 µg of pWSgfp-Chfr or pWSgfp-ChfrR using Fugene6 according to manufacturer’s directions (Roche Molecular, Indianapolis, IN). Coverslips were harvested for staining 20–24 h after transfection. Cells were first fixed in 3.5% PBS-buffered paraformaldehyde for 7 min and then solubilized in KB + 0.5% Triton X-100 for 5 min, followed by a 5-min rinse in KB buffer [10 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.1% BSA]. Cells were incubated with CENP-F antibody for 30 min at 37°C in a humidified chamber, rinsed in KB, followed by incubation with Alexa-Fluor 594 goat antirabbit IgG (2 µg/ml; Molecular Probes, Eugene, OR) for 30 min at 37°C in a humidified chamber. Cells were subsequently incubated with 4',6-diamidino-2-phenylindole dihydrochloride (Molecular Probes; 0.25 µg/ml) for 15 min at room temperature, and mounted onto slides. Coverslips were scanned using a Nikon Microphot.
XTT Assay.
Inhibition of growth of various cell lines by Taxol in a 72-h incubation was assayed by a standard XTT protocol (18)
. Viability was calculated from the ratio of the absorbance at 450 nm for the test sample divided by the absorbance at 450 nm for the standard (untreated control) sample, measured on a microplate reader (Dynatech, Lorton, VA). Triplicate determinations were performed, with standard deviation less than 15%.
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RESULTS |
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Chfr Is a Ubiquitin Ligase Dependent on Its RING-Finger Domain.
The spindle assembly checkpoint involves a ubiquitindependent pathway, but it is unclear whether the Chfr-dependent mitotic stress response is mediated by ubiquitination. Chfr contains a RING finger at its COOH terminus. As demonstrated previously (reviewed in Ref. 11
), most RING finger-containing proteins have the potential for ubiquitin protein ligase activity, so we examined whether Chfr has intrinsic ubiquitin protein ligase activity dependent on its RING-finger domain. For these experiments, GST fusion proteins with full-length Chfr and RING-finger domain deletion mutant (ChfrR) were expressed and purified from baculovirus. GST-Chfr and GST-ChfrR were assayed for self-ubiquitination in the presence of purified recombinant E1, E2, and ubiquitin. Four different human E2 subfamily members, Ubc10, Ubc2, Ubc4, and Ubc5, were tested in the assay. As a control, a blank reaction without Chfr was included. As shown in Fig. 1A
, in the presence of two different E2s namely Ubc4 or Ubc5, high molecular Chfr conjugates were formed (Fig. 1A
, Lane 4 and 5). On the other hand, no Chfr conjugates were found with two other E2s, Ubc10 and Ubc2, or in the control reaction (Fig. 1A
, Lane 1–3). To confirm that the Chfr conjugates are polyubiquitinated, the same reaction mixtures were probed with antiubiquitin antibody (Fig. 1B)
. These results show that Chfr is a ubiquitin ligase capable of catalyzing its own ubiquitination, and it prefers the Ubc4/5 family as its E2. In addition, because no conjugates were formed in the reactions where ChfrR was used (Fig. 1, A
and B, Lanes 6–10), Chfr requires an intact RING-finger domain for its ubiquitin ligase activity.
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R (pSWgfp-ChfrR) construct. Negative controls were the GFP vector (pSWgfp) with ubiquitin construct, GFP-Chfr, or GFP-ChfrR alone. Lysates from transfected cells were immunoprecipitated with anti-GFP antibody and blotted with anti-Chfr antibody. GFP-Chfr was conjugated with the HA-ubiquitin to produce a ladder of ubiquitinated products (Fig. 2
, Lane 4). No conjugates were detected when HA-ubiquitin was omitted from the transfection (Fig. 2
, Lane 3). In lysates prepared from GFP-ChfrR and HA-ubiquitin cotransfections, no significant high molecular weight laddering was seen (Fig. 2
, Lane 2). Lysates from GFP-Chfr and GFP-ChfrR transfections without HA-ubiquitin or the HA-ubiquitin transfection alone did not show any dark staining of ubiquitinated products (Fig. 2
, Lanes 1, 3, and 5). These results suggest that Chfr exhibits RING domain-dependent auto-ubiquitination activity in vivo.
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R), or vector alone (DLD1/Vector). The cells were treated with 300 nM Taxol for 20 h, fixed in paraformaldehyde, permeabilized, stained with Hoechst dye, and observed microscopically. The cells that looked clearly rounded (mitotic) also had condensed chromosomes, whereas the cells that were attached firmly had diffused nuclei. After Taxol treatment, various cells showed increased mitotic indices as a result of spindle checkpoint arrest at metaphase. As expected, DLD1 cells with vector alone had high mitotic index (52%) after Taxol treatment, whereas DLD1 expressing wild-type Chfr, SW480, and SAOS-2 cells have somewhat lower mitotic indices (Fig. 3)
consistent with a cell cycle delay or arrest before mitosis. On the other hand, DLD1 cells stably expressing ChfrR had a high mitotic index similar to the parental chfr-deficient DLD1 cells (Fig. 3)
. These results indicate that in the presence of Chfr the stressed cells are delayed in mitotic progression and that the RING-finger domain is required for this function.
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R) were included in the experiments to determine the role of the RING-finger domain in this mitotic stress response function of Chfr. Cells were treated with increasing concentrations of Taxol, and their viabilities were assayed at 72 h. As shown in Fig. 4A
, our results indicate that at as low as 10 nM Taxol, the DLD1/Chfr and SW480 cells were >60% viable compared with the cells expressing the RING-finger deletion mutant (ChfrR) or the vector alone (DLD1/vector), or the DLD1 cells, which were at about 20–30% viability (Fig. 4A)
. Three different DLD1/Chfr clones gave very similar results (data not shown). These findings confirm that the sensitivity of DLD1 cells to Taxol is correlated with their defects in Chfr-dependent mitotic delay, an activity also requiring RING domain. Also to confirm that Chfr has a role to play specifically in response to mitotic stress, we repeated the same XTT experiment with cells treated with increasing concentrations of Topotecan, an anticancer drug that transforms topoisomerase I-DNA-transient intermediates into lethal DNA lesions and has been suggested to activate a DNA-damage checkpoint (19, 20, 21)
. The presence or absence of Chfr made no difference to the response of cells to DNA damage, indicating that Chfr has a role limited to mitotic stress response pathway (Fig. 4B)
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R cells were synchronized at the G1-S boundary by a double block with thymidine and aphidicolin, and released for various times. As indicated in Fig. 5B
, FACS analysis was performed to ascertain the phase of the cell cycle at various times. Because Chfr is a nuclear protein and the antibody used only recognizes Chfr specifically in the nuclear fraction, nuclear extracts were used to study protein expression. The Western blot results indicate that cells in G1 express low levels of Chfr (Fig. 5A
, Lanes 2 and 3) as compared with asynchronous cells (Fig. 5A
, Lane 1) and that the protein levels are markedly increased during late S phase (Fig. 5A
, Lanes 4-6). Because Chfr is believed to function in prophase, this expression pattern is consistent with its role in the mitotic stress response. Because Chfr has autoubiquitin ligase activity, we tested whether the RING-finger domain had any role to play in this cell cycle-dependent expression pattern and whether ubiquitin ligase activity was required for this process. The same experiment was repeated with DLD1 cells stably expressing ChfrR cells; in the absence of the RING-finger domain, Chfr expression remained unchanged throughout the cell cycle (Fig. 5C)
. Consistent with the observation that the Chfr level is low in G1 and high before mitosis in DLD1 cells stably expressing Chfr, thymidine-treated SW480 cells with endogenous wild-type Chfr have a lower Chfr level, whereas Taxol-treated cells have a higher Chfr level in comparison with that of untreated controls (data not shown).
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. Examination of cells transfected with the GFP-ChfrR showed that it was expressed in mitotic cells and G1 or S cells that were also CENP-F negative (Fig. 5E)
. Thus, the loss of GFP-Chfr in mitotic cells correlates with the Western blot expression pattern. This loss is likely dependent on ubiquitination activity, because the RING domain mutant is stable in mitosis and G1 phase. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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An important role of a checkpoint protein is to coordinate cell cycle events and regulate mechanisms that enforce proper order when the cell perceives stress. When cells are subjected to mitotic stress, Chfr delays entry into metaphase (7)
. In cells expressing Chfr, the mitotic index was lower than that of either cells lacking Chfr or those expressing the RING-finger deletion mutant (Fig. 3)
. In cells with intact Chfr, viability was much greater in the presence of Taxol than in cells in which Chfr was either absent or presented as a RING-finger deletion mutant (Fig. 4)
, whereas no such difference in viability was observed in cells treated with Topotecan. These results suggest that Chfr has a role to play specifically on mitotic stress and that the ubiquitin ligase function is required to enable Chfr to delay mitotic progression in mitotic stress conditions. However, the RING domain itself is not sufficient for the function of Chfr, because a FHA domain deletion with intact RING domain is also functionally defective (7)
. Chfr may ultimately resemble another RING domain protein c-Cbl, of which the RING domain alone has no affinity for platelet-derived growth factor ß-receptor but rather requires a SH2 domain to interact with its substrate (23)
. In the case of Chfr, substrate binding could depend on the FHA domain, a phospho-amino acid motif (9
, 10)
. Such a substrate for Chfr has not yet been identified. It is very likely that Chfr-mediated ubiquitin-dependent degradation of yet unidentified proteins contributes to its role in the mitotic stress pathway.
Many cancers show resistance to the current antimitotic drugs including Taxol and Vinca alkaloids. Thus far very little is known about the mechanisms which lead to the multidrug resistance-independent resistance in cancer cells. Whereas multidrug resistance accounts for much of the clinical resistance to taxanes and Vinca alkaloids, other mechanisms are likely important. The present results suggest an additional basis for drug resistance in cancer cells, because the loss of Chfr results in increased sensitivity to mitotic poisons. Chfr function was found to be absent, either because of mutation or loss of expression, in four of the eight human cancer cell lines examined (7) . Thus, it would seem useful to conduct a thorough examination of the status of Chfr in various tumors, as well as the role of Chfr in the sensitivity of human cancers to antimicrotubule agents. Abrogation of the Chfr-dependent pathway could make current cancer therapy more effective.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by a postdoctoral fellowship from a National Cancer Institute Grant CA-50771 (to P. C.), by a fellowship from Human Frontiers Science Program (to V. S.), a NIH core grant (CA06927), and an appropriation from the Commonwealth of Pennsylvania (to T. J. Y.). 
2 To whom requests for reprints should be addressed, at Department of Oncology Research, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA 19406. Phone: (610) 270-6638; Fax: (610) 270-5005; Email: Bin-Bing_S_Zhou{at}gsk.com
3 The abbreviations used are: FHA, fork head-associated; APC/C, anaphase promoting complex/cyclosome; GST, glutathione S-transferase; FACS, fluorescence-activated cell sorter; GFP, green fluorescence protein; ubiquitin, ubiquitin; FBS, fetal bovine serum; XTT, 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt; HA-ubiquitin, HA-tagged construct of ubiquitin.
Received 10/11/01. Accepted 1/18/02.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and after Taxol treatment (
) were calculated based on the percentage of cells with condensed chromosomes.
; S,