Study of the G2/M cell cycle checkpoint in irradiated mammary epithelial cells overexpressing Cul-4A gene

PII S0360-3016(01)02739-0

BIOLOGY CONTRIBUTION

STUDY OF THE G2/M CELL CYCLE CHECKPOINT IN IRRADIATEDMAMMARY EPITHELIAL CELLS OVEREXPRESSING Cul-4A GENE

ANU GUPTA, PH.D.,* LI-XI YANG, M.D., PH.D.,† AND LING-CHUN CHEN, PH.D.*

*Geraldine Brush Cancer Research Institute, California Pacific Medical Center, San Francisco, CA;†Department of RadiationOncology, St. Mary’s Medical Center, California Pacific Medical Center Research Institute, San Francisco, CA

Purpose: Members of the cullin gene family are known to be involved in cell cycle control. One of the cullin genes,Cul-4A, is amplified and overexpressed in breast cancer cells. This study investigates the effect of Cul-4Aoverexpression upon G2/M cell cycle checkpoint after DNA damage induced by either ionizing or nonionizingradiation.Methods and Materials: The normal mammary epithelial cell line MCF10A was stably transfected withfull-length Cul-4A cDNA. Independent clones of MCF10A cells that overexpress Cul-4A proteins were selectedand treated with either 8 Gy of ionizing radiation or 7 J/M2 of UV radiation. The profile of cell cycle progressionand the accumulation of several cell cycle proteins were analyzed.Results: We found that overexpression of Cul-4A in MCF10A cells abrogated the G2/M cell cycle checkpoint inresponse to DNA damage induced by ionizing irradiation, but not to DNA damage induced by nonionizingradiation. Analysis of cell cycle proteins showed that after ionizing irradiation, p53 accumulated in themock-transfected MCF10A cells, but not in the Cul-4A transfectants.Conclusion: Our results suggest a role for Cul-4A in tumorigenesis and/or tumor progression, possibly throughdisruption of cell cycle control. © 2002 Elsevier Science Inc.

Cul-4A, Cell cycle, p53, Breast cancer.

INTRODUCTION

DNA amplification is a common mechanism used by cancercells to upregulate the activity of certain critical genes,making amplicons fertile areas for the discovery and char-acterization of genes contributing to cancer. We recentlyreported the cloning of a gene from an amplified region ofchromosome 13, human Cul-4A (Hs-Cul-4A), in breastcancer (1). Its involvement in breast carcinogenesis wassuggested by our finding that Hs-Cul-4A was amplified in16% of primary breast cancers and overexpressed in 47%(1).

Cul-4A is a member of the novel conserved gene family,the cullins (2). The normal function of Cul-4A is unknown.Recently, Cul-4A has been shown to be associated withROC (regulator of cullins) protein, which is an essentialsubunit of the SCF complex (Skp family/Cullin family/F-box protein) (3–5). SCF, which is known to be a ubiquitinligase, plays a role in regulation of the cell cycle by degra-dation of certain key proteins (6–12). Also a reasonablespeculation is that Cul-4A might have a ubiquitin ligase

activity, as some other members of the cullin family areknown to have (13, 14). For example, Cdc53, the cul-1homolog in yeast, is a subunit of the SCF complex, which inthis system mediates ubiquitin-dependent degradation of G1cyclins and S-phase cyclin-dependent kinase inhibitors (6,8). In mammalian cells, Cul-1 is also part of the SCFcomplex that mediates degradation of cyclin A, cyclin D,CDK inhibitors p21 and p27, E2F1,�-catenin, and��B�(11, 15–20). Another member of the cullin family, Cul-2,forms a stable complex with the von Hippel–Landau tumorsuppressor gene product (pVHL), and elongin B/C (21, 22).This complex is structurally similar to SCF and also con-tains ubiquitin ligase activityin vitro (23, 24). Still anothercullin, Cul-3, has been shown to be associated with cyclin Ein mammalian cells; overexpression of Cul-3 leads to in-creased ubiquitination of cyclin E (14).

These findings, showing that cullins function in ubiq-uitin-dependent degradation of cell cycle proteins, suggestthe general importance of these genes in cell cycle control.In the nematodeCaenorhabditis elegans, cul-1 functions asa negative cell cycle regulator. Cells with a null mutation of

Reprint requests to: Dr. Ling-Chun Chen, Geraldine BrushCancer Research Institute, California Pacific Medical Center, 2330Clay Street, San Francisco, CA 94115. Tel: (415) 561-1745; Fax:(415) 561-1390; E-mail: [email protected]

This work was supported by PO1 CA44768 from the NationalCancer Institute, DAMD17-99-1-9018 from the U.S. Army Med-ical Research Acquisition Activity, and The Susan G. Komen

Breast Cancer Foundation.Acknowledgments—We thank Ms. Judy Cheung for her technicalassistance, Dr. Terry Donlon for his contribution on irradiation ofcells, and Drs. Simone Nicholson and Andrew P. Smith for criticalevaluation of the manuscript.

Received Jul 10, 2001, and in revised form Oct 18, 2001.Accepted for publication Oct 22, 2001.

Int. J. Radiation Oncology Biol. Phys., Vol. 52, No. 3, pp. 822–830, 2002Copyright © 2002 Elsevier Science Inc.Printed in the USA. All rights reserved

0360-3016/02/$–see front matter

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cul-1 were unable to exit the normal cell cycle, resulting inexcessive cell division and abnormally small cells (gener-alized hyperplasia in all larval tissues) (2). In contrast, C.elegans cul-2 has been shown to be a positive cell cycleregulator. In the absence of cul-2, C. elegans germ cellsundergo G1 phase arrest (13). In addition, cul-2 is alsorequired for mitotic chromosome condensation (13). There-fore, we hypothesized that a possible link between Cul-4Aoverexpression and breast cancer could be through disrup-tion of the cell cycle. In the present study, we have testedthis hypothesis by overexpressing Cul-4A in the mammarycell line MCF10A, then exposing the cells to ionizing radi-ation (IR) or nonionizing radiation to induce DNA damage.Though there was no effect of Cul-4A overexpression onthe progression of these cells’ cell cycles under normalconditions, the cells exhibited a loss of the G2/M checkpointwhen exposed to ionizing radiation.

METHODS AND MATERIALS

Stable transfection of MCF10A cells with GFP-Cul-4Aand cell culture

MCF10A cells were maintained in 50% Dulbecco’s mod-ified Eagle’s medium H16 and 50% Ham’s nutrient mixtureF12 with the addition of supplements as described (25).Full-length cul-4A cDNA derived from heart tissue wascloned in PCR2.1 (Invitrogen, Carlabad, CA) using primers5� GGA TCC GGT CTT CTC AGC CGG GAC GCCAGC3� (sense) and 5� GGA TCC TCA GGC CAC GTAGTG GTA CTG ATT 3� (antisense) (1). The cul-4A cDNAwas subcloned in pEGFP-C1 vector (Clontech, Palo Alto,CA). The authenticity and correct reading frame of theGFP-Cul-4A fusion cDNA were confirmed by sequenceanalysis. The MCF10A cells were transfected with eitherthe empty vector or the GFP-Cul-4A construct using Lipo-fectin (GIBCO). The stable transfectants were selected byG418 at 300 �g/mL. Stable transfectants that expressedgreen fluorescent protein (GFP) were subcloned for analysisusing cloning cylinders.

Colony formation in soft agarSingle-cell suspensions were plated in complete medium

containing 0.3% agarose at 3 � 103 cells per 16-mm culturewell; the wells were precoated with 0.6% agarose. Plateswere incubated at 37°C and fed twice weekly. Colony-forming efficiency was evaluated after 2 weeks by countingcolonies of greater than 50 cells.

�-irradiation and cell cycle analysisBoth the control MCF10A cells and the Cul-4A trans-

fected cells were seeded at 2 � 105 cells per 25 cm2 flask.Forty-eight hours later, the cells were treated with ionizingradiation at a dose rate of 1.17 Gy/min using a VarianClonac 2100C Accelerator. At various time points afterirradiation, cells were removed from the flasks, washed withphosphate-buffered saline (PBS), and fixed in ice-cold 70%ethanol for 1 h at 4°C. After being washed with PBS, cells

were treated with PBS containing RNaseA at 1 mg/mL andpropidium iodide at 50 �g/mL, as previously described(26). DNA content was analyzed on a fluorescence-acti-vated cell analyzer (FACScan, Becton Dickinson). The per-centage of cells in G1, S, and G2 phases was determinedusing the MODFIT program (Verity Software, Topahsm,ME).

Western blot analysisCells were rinsed in cold PBS and lysed in lysis buffer

(1% Triton X-100, 1% sodium deoxycholate, 0.1% sodiumdodecyl sulfate (SDS), 150 mM NaCl, and 10 mM Tris-HCl, pH 7.4) containing 1� concentration of CompleteMini Protease Inhibitor Cocktail (Boehringer Mannheim,Indianapolis, IN). The lysates were incubated for 30 min onice and clarified by centrifugation at 14,000 rpm for 5 min.The protein content was measured using the bicinchoninicacid (BCA) method (Pierce, Rockford, IL). Thirty micro-grams of protein from each cell line was separated on 10%NuPAGE gels with 2-(N-morpholino)ethane sulfate (MES)-SDS buffer (Invitrogen, Carlabad, CA) at 200 V for 60 minand transferred to nitrocellulose membranes at 25 V for 1 h.To detect the GFP-Cul-4A fusion protein, the blot wasprobed with the polyclonal antibody against Cul-4A ob-tained by immunizing New Zealand white specific pathogenfree (SPF) female rabbits with the purified Cul-4A-HisAfusion protein at Caltag Laboratories (Healdsburg, CA) orthe rabbit anti-GFP antibody (Clontech, Palo Alto, CA).The blot was then probed with horseradish peroxidase(HRP)-labeled secondary antibody (1:3000, DAKO,Carpinteria, CA) and developed using enhanced chemilu-minescence (Amersham) according to the manufacturer’sinstructions. For cell cycle protein analysis, the Westernblot was first probed with antibody against p53 (Santa Cruz,CA, Bp53-12). The blot was then stripped by submerging instripping buffer (100 mM �-ME, 2% SDS 62.5 mM Tris-HCl pH 6.7) and incubating at 60°C for 30 min. The blotwas subsequently probed with antibodies against cyclin B1(Santa Cruz, CA, GNS1) and p21WAF1 (Santa Cruz, CA,C19). Antibody against actin (Chemicon, MAB1501) wasused to normalize the loaded amounts. The films werescanned with Adobe Photoshop and quantitated by ImageQuant. The relative amounts of p53 and p21WAF1 in eachsample were expressed as density ratios to actin.

RESULTS

Overexpression of Cul-4A leads to morphologic changesand anchorage-independent growth of MCF10A cells

To study the effect of Cul-4A overexpression on breastcancer tumorigenesis, we transfected MCF10A with theCul-4A expression vector. This vector allows for constitu-tive expression of the full-length Cul-4A with N-terminal–tagged GFP. Stable transfectants that expressed abundantgreen fluorescent protein were selected by cloning cylindersand examined for full-length GFP-Cul-4A expression byNorthern blot and Western blot analysis. Two stably trans-

823Cul-4A and G2/M cell cycle checkpoint ● A. GUPTA et al.

fected cell lines, Cul-4A #1 and Cul-4A #2, which ex-pressed the 3.3-kb full-length GFP-Cul-4A mRNA (Fig.1A, lanes 3 and 4) and the predicted 105 kD GFP-Cul-4Afusion protein (Fig. 1B and 1C, lanes 2 and 3), were selectedfor further analysis. Expression of Cul-4A fusion protein isslightly higher in Cul-4A #2 clone than in Cul-4A #1 clone.The endogenous 80-kD Cul-4A protein was also expressedin these cells (Fig. 1B). The spontaneously immortalized,nontumorigenic mammary epithelial cell line MCF10A ex-presses a low level of endogenous Cul-4A (Fig. 1, lane 1).Both the parental MCF10A cells and cells transfected withempty pEGFP vector (Fig. 1A, lanes 1 and 2) were used ascontrolled cells for the following experiments.

Both Cul-4A #1 and Cul-4A #2 cells displayed flat ap-pearances and increased cell size as compared with thecontrol MCF10A cells (Fig. 2A, control; Fig. 2B, Cul-4A#2). The expression of the 105-kD GFP-Cul-4A fusionprotein in these clones was detected mainly in the nucleus,suggesting that it is a nuclear protein (Fig. 2B). In addition,approximately 10–30% of the exponentially growing Cul-4A–transfected cells were multinucleated (2–4 nuclei, Fig.2B), whereas less than 0.2% of the mock-transfected cellswere multinucleated. However, no significant differenceswere observed in either the cell cycle progression (seebelow) or the doubling time between the exponentiallygrowing Cul-4A transfectants and the control cells (data notshown). Interestingly, however, both Cul-4A–transfectedclones exhibited anchorage-independent growth in soft agar(Fig. 2C).

Cul-4A overexpression abrogates G2/M checkpoint afterionizing radiation–induced DNA damage

To study the role of Cul-4A in cell cycle control, thedistribution of cell cycle profiles of the exponentiallygrowing cells was analyzed by flow cytometry. As shownin Fig. 3, exponentially growing cells overexpressingCul-4A showed cell cycle profiles similar to those of

transfected control cells (Fig. 3, 0 h). The MCF10A cellline has been shown to contain a wild-type p53 gene andto exhibit G2 cell cycle arrest after IR treatment, similarto what has been reported in normal human mammaryepithelial cells (27, 28). Because the presence of multinu-cleated cells in the Cul-4A–overexpressing cultures maybe associated with a disorganization of the mitosis, wehypothesized that in these cells the G2/M cell cyclecheckpoint may have been disrupted. To test this, thesecells were exposed to ionizing irradiation at a dose of 8Gy to induce DNA damage. In the representative exper-iments, approximately 60 –70% of either the untrans-fected MCF10A cells or the pEGFP vector–transfectedMCF10A cells accumulated at G2/M after irradiation(Fig. 3, the pEGFP-transfected cells). In contrast, neitherof the Cul-4A–transfected cell lines arrested at G2/Mphase after ionizing irradiation, as indicated by lack ofaccumulation of cells at G2/M phase (Fig. 3, shown hereCul-4A #1 only). The lack of accumulation of Cul-4A #1and Cul-4A #2 cells in the G2/M phase is not because ofthe lack of cells in cycling after IR. We have monitoredthe cell growth after IR and found that the cell numbersof both Cul-4A #1 and Cul-4A #2 increased from (3.5 �0.4) � 105 to (6.2 � 0.5) � 105 48 h after IR. Therefore,IR did not prevent cells from cycling within this timeframe.

We next examined the expression of various cell cycleregulatory proteins, including p53, cyclin B1, andp21WAF. Cell lysates were prepared 17 h after ionizingradiation and analyzed by Western blots with specificantibodies. Accumulation of p53 and p21WAF was ob-served in the control cells (Fig. 4). In contrast, neither ofthe Cul-4A–transfected cell lines showed detectable ac-cumulation of p53 after ionizing radiation. In addition,the Cul-4A–transfected cell lines showed less accumula-tion of p21WAF than the control cells (12–16-fold in-crease in control cells vs. 2–3-fold in the Cul-4A–trans-

Fig. 1. Expression of Cul-4A in parental and transfected MCF10A cells: (A) Northern blot analysis: 10 �g of total RNAfrom (lane 1) parental MCF10A cells, (lane 2) MCF10A cells transfected with the empty vector, and (lanes 3 and 4) twoMCF10A cell lines stably transfected with GFP-Cul-4A were analyzed by Northern blot and probed with the full-lengthCul-4A cDNA. The blot was stripped and reprobed with �-actin as a loading control. (B and C) Western blot analyses:30 �g of cell lysates from MCF10A cells transfected with (lane 1) the empty vector and (lane 2 and 3) two MCF10Acell lines stably transfected with GFP-Cul-4A were analyzed on 10% NuPAGE gel and probed with polyclonal antibody(B) against Cul-4A or (C) with anti-GFP antibody.

824 I. J. Radiation Oncology ● Biology ● Physics Volume 52, Number 3, 2002

fected cells in separate experiments). In the Cul-4A–transfected cells, moreover, an additional cyclin B1species with a smaller molecular weight was detected.The smaller cyclin B1 protein may represent the partiallydegraded cyclin B1 passing through the ubiquitin-prote-olysis pathway, as previously reported (29).

Overexpression of Cul-4A has no significant effect on cellcycle checkpoint after nonionizing radiation–inducedDNA damage

Because p53 is also involved in the response to DNAdamage induced by nonionizing radiation, we next exam-

ined the effect of UV radiation in the Cul-4A–overexpress-ing cells. Both the control MCF10A cells and the Cul-4A–transfected MCF10A cells were exposed to 7 J/M2 of UVradiation. The cell cycle progression was analyzed by flowcytometry. Both the control MCF10A cells and the Cul-4A–transfected cells underwent S-phase arrest at 8 h and 16 hafter UV radiation (Fig. 5). By 24 h after UV treatment, boththe control and Cul-4A–transfected cells recovered (data notshown). This observation is consistent with the previousreport that normal human mammary epithelial cells werearrested at S phase after UV radiation (27). Concordant withthe observed cell cycle arrest, p53 proteins were accumu-lated in both the control and the Cul-4A–transfectedMCF10A cells after UV treatment (Fig. 6). However, theS-phase arrest in the Cul-4A transfectants seems not asdramatic as in the control cells. It remains possible that theCul-4A–transfected cells may recover from the S-phasearrest sooner than the control cells. Further experimentsneed to be done to determine this effect.

Cul-4A–transfected MCF10A cells are more sensitive toionizing radiation

Because overexpression of Cul-4A disrupts the G2/Mblock after ionizing radiation, we next determined its effecton cell survival after radiation. Control MCF10A cells andCul-4A #1 and Cul-4A #2 cells were treated with 1, 2, 5, or8 Gy of ionizing radiation or with 7 J/M2 of UV radiation,respectively. Forty-eight hours post irradiation, cells weretransferred from small T25 flasks to larger T75 flasks.Ninety-six hours post irradiation, numbers of viable cells ineach flask were determined. Viable cell numbers in Cul-4A#1 and Cul-4A #2 were similar to those of the controlMCF10A cells after UV irradiation (data not shown). How-ever, Both Cul-4A #1 and Cul-4A #2 were more sensitive toIR. This effect was more significant when cells were irra-diated with low doses. As illustrated in Fig. 7, when cellswere treated with 1 Gy radiation, 100% of the controlMCF10A cells were viable; however, only 50% and 35% ofthe Cul-4A #1 and Cul-4A #2, respectively, were viable.Viabilities declined for the Cul-4A–transfected cells as theradiation doses increased. These results strongly suggestthat the Cul-4A–transfected cells bypass the G2/M arrestwithout repair of their radiation-induced DNA damage.These cells might carry radiation-induced chromosomal ab-errations and are presumably doomed to die. We concludedthat Cul-4A #1 and Cul-4A #2 were more sensitive toionizing radiation than the control cells were.

DISCUSSION

We have previously shown that Cul-4A is both amplifiedand overexpressed in a substantial number of cancer celllines and primary breast cancers (1). To obtain more directevidence for its role in mammary tumorigenesis, we exam-ined in this study the effect of its overexpression in theepithelial cell line MCF10A. To study the cellular localiza-tion of Cul-4A, we chose to use a GFP-Cul-4A fusion

Fig. 2. Multinucleation and formation of soft agar colonies byCul-4A–transfected MCF10A cells. (A) GFP empty vector–trans-fected MCF10A cells (200�). (B) GFP-Cul-4A–transfected cells,Cul-4A #2 (200�); the arrows indicate multinucleated cells. (C)Formation of soft agar colonies by control and GFP-Cul-4A–transfected MCF10A cells. The results represent the average oftwo separate experiments performed in triplicate. The bars repre-sent the standard deviation.


construct for transfection. We found that, similar to othercullin protein, Cul-4A is expressed as a nuclear protein (13).In addition, overexpression of Cul-4A in epithelial cell lineMCF10A resulted in (1) anchorage-independent growth, (2)formation of pleiomorphic and multinucleated cells, and (3)disruption of the G2/M cell cycle checkpoint after ionizingradiation. Our data also suggest that lack of accumulation ofp53 may contribute to the defect in cell cycle checkpoint inthese cells. We cannot completely rule out the possibilitythat GFP fusion altered Cul-4A function. However, studiesof other GFP fusion proteins have demonstrated that theGFP motif does not change the physiologic properties of theprotein and has no effect on the growth of cells stablytransfected with GFP (30–32). Furthermore, we found that

overexpression of untagged Cul-4A in another cell line,Rat-1 fibroblasts, also resulted in anchorage-independentgrowth (data not shown).

Both parental MCF10A cells, as well as the pEGFPvector–transfected cells, arrested at G2 after IR-inducedDNA damage. This observation is consistent with previousreports that the immortalized breast epithelial cell lineMCF10A arrested at G2 after IR-induced DNA damagebecause of the loss of G1 checkpoint (27, 33). However,when we overexpressed Cul-4A in MCF10A, these cellsfailed to undergo cell cycle arrest after ionizing radiation,and this failure was correlated with an inability to accumu-late p53 protein. It has been established that p53 plays acritical role in the cellular response to DNA damage by

Fig. 3. Flow cytometric analysis of control and Cul-4A–transfected MCF10A cells after IR. Exponentially growingcontrol and Cul-4A–transfected MCF10A cells were either untreated or treated with 8 Gy of ionizing irradiation. Thepopulation of cells present in G1, S, and G2/M was determined using a FACScan flow cytometer (Becton Dickinson).The percentage of cells in G1, S, and G2/M was calculated using MODIFIT software (Becton Dickinson) and indicatedon the top right of each cell cycle profile.


serving as a cell cycle checkpoint determinant (34, 35). Thep53 protein is stabilized, and its level is increased in cells inresponse to DNA damage (35). The increased p53 levelsafter DNA damage are thought to be responsible for the cellcycle arrest observed under this condition (35, 36). Cellcycle arrest allows cells to repair the damaged DNA beforeproceeding to the next stage in the cell cycle, to maintaingenomic stability (37, 38). Concomitantly, induction ofCDK inhibitor p21 was also less in Cul-4A–overexpressingcells when compared to the control cells, consistent withp21 being induced downstream of p53 (39).

In contrast, both control and Cul-4A–overexpressingcells showed cell cycle arrest at the S phase after UV-induced DNA damage, as previously reported (27). The p53protein was stabilized and accumulated in both control andCul-4A–overexpressing cells after UV-induced DNA dam-age. These results demonstrate that both mock transfectionand Cul-4A transfection do not affect the wild-type p53gene in the parental MCF10A cells (28). We have furtheranalyzed and confirmed that p53 gene in the transfectedcells is not mutated by immunohistochemical staining ofp53 protein and single-strand conformation polymorphismmutational analysis of exons 2–9 of the p53 gene (data notshown).

We speculate that one of the mechanisms by whichoverexpressing Cul-4A abrogates G2/M cell cycle check-

point is through increased ubiquitination and proteolysis ofp53 protein based upon the following reasons. Normally,the turnover of p53 in vivo is mediated by the ubiquitinproteolysis pathway after IR irradiation (40). Based on itshomology with other cullins and its interaction with ROC1,an essential subunit of ubiquitin ligase E3 (5), Cul-4A isbelieved to be a subunit of E3 ligase involved in the ubiq-uitin proteasome proteolysis pathway. It is also known thatUV and IR irradiation activate p53 through different signal-ing pathways and that p53 is differentially modified underthese conditions (41, 42). Differences in modification resultin the absence of ubiquitinated p53 after UV irradiation(43). Therefore, the lack of accumulation of p53 in Cul-4A–overexpressing cells after IR irradiation but not after UVirradiation suggests that overexpression of Cul-4A mayenhance p53 ubiquitination. Future experiments will beneeded to test the hypothesis that Cul-4A overexpression

Fig. 4. Expression of cell cycle proteins in control and Cul-4A–transfected MCF10A cells after IR. Cell were either untreated (�)or treated (�) with 8 Gy of ionizing radiation. The cell lysateswere prepared 17 h after irradiation. The expression of cyclin B1,p53, p21, and �-actin was analyzed by 10% SDS gel electrophore-sis and Western blot with specific antibodies.

Fig. 5. Flow cytometric analysis of control and Cul-4A–transfectedMCF10A cells after UV radiation. Exponentially growing controland Cul-4A–transfected MCF10A cells were either untreated (�)or treated (�) with 7 J/M2 of UV radiation. The population of cellspresent in G1, S, and G2/M was determined using a FACScan flowcytometer (Becton Dickinson). The percentage of cells in G1, S,and G2/M was calculated using MODIFIT software (Becton Dick-inson) and indicated on the top right of each cell cycle profile.


may increase ubiquitinated p53 proteins and decrease p53protein stability in cells after ionizing irradiation.

Cyclin B1 is the regulatory subunit of the cdc2 kinase andis essential for entry into mitosis. Cyclin B1 is accumulatedduring G2/M and is degraded at the end of mitosis (44).After the MCF10A cells were treated with IR and under-went G2/M cell cycle arrest, we observed a twofold increaseof cyclin B1 protein level. This increase was not observed inthe Cul-4A transfectants. In addition, cyclin B1 species oflower molecular weight was detected in the Cul-4A–trans-fected MCF10A cells after ionization. Presence of thesmaller cyclin B1 has been observed during cyclin B1ubiquitination and degradation (29). Therefore, it is alsopossible that overexpression of Cul-4A may contribute toincreased degradation of cyclin B1, which results in earlyexit of these cells from mitosis. In this case, the effect ofCul-4A may also be mediated through regulation of cyclinB1 degradation. On the other hand, the decrease of cyclinB1 may not be the cause, but a result, of mitotic exit of thecell.

Associations of cullins with neoplasia have been docu-mented previously. Mutations of Cul-1 in C. elegans causedhyperplasia in all tissues of the larvae (2). Human Cul-2 hasbeen shown to form a complex with tumor suppressorprotein pVHL (21). Depending on the cell types, deletion ofCul-3 in mouse embryo results in either increased entry intoS phase or a block to enter S phase (14). In addition,overexpression of all cullins except Cul-5 was observed ina variety of human cancer cell lines (45). Cul-4A has beenshown to associate with damaged DNA-binding protein(DDB), which is known to bind to UV-damaged DNA forrepair (46). It has been proposed that Cul-4A may beinvolved in ubiquitination of DDB or other proteins in thepathway of DDB function (46). Although there was noeffect of Cul-4A overexpression on cell cycle control afterUV irradiation, it is possible that damaged DNA repairfunction might be reduced, resulting in an increase of UV-induced mutagenesis in Cul-4A–overexpressing cells. Wehave shown here that overexpression of Cul-4A disruptsG2/M cell cycle checkpoint after IR irradiation. Abrogationof the G2/M cell cycle checkpoint in turn can promotegenomic instability and oncogenesis (37). Patients withdiseases associated with G2/M checkpoint control, such asataxia telangiectasia (47) and Bloom’s syndrome (48), dem-onstrate increased tumor susceptibility. Therefore, the abil-ity of Cul-4A overexpression to override the G2/M cellcycle checkpoint may be a relevant factor in the develop-ment of breast cancer and possibly other cancer types.

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Fig. 6. Expression of p53 in control and Cul-4A–transfectedMCF10A cells after UV. Cell were either untreated (�) or treated(�) with 7 J/M2 of UV radiation. The cell extracts were prepared8 h after UV radiation. The level of p53 protein was analyzed by10% SDS gel electrophoresis and Western blot.

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Study of the G2/M cell cycle checkpoint in irradiated mammary epithelial cells overexpressing Cul-4A gene