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Published OnlineFirst July 20, 2010; DOI: 10.1158/0008-5472.CAN-10-0688

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cular and Cellular Pathobiology

raction of TAp73 and Breast Cancer–Associated Gene 3ances the Sensitivity of Cervical Cancer Cells in Response

R

rradiation-Induced Apoptosis

as Ho-Yin Leung and Hextan Yuen-Sheung Ngan

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tification of proteins that are involved in the sensitivity of radiotherapy of cancers is important to en-the response to cancer treatment. Expression of TAp73 is associated with the sensitivity of radiotherapyical cancer patients, suggesting it plays an important role in controlling radiosensitivity. Here, by usingtwo-hybrid system, we identify breast cancer–associated gene 3 (BCA3) as the first and novel proteincting partner of TAp73. By coimmunoprecipitation and Western blot analysis, we confirm that TAp73with and stabilizes BCA3 in cervical cancer cell line HeLa. Immunofluorescence staining indicates thatis localized in the cytoplasm and nucleus. Interestingly, when coexpressed with TAp73, BCA3 interactslocalizes with TAp73 at the mitochondria. Mutagenesis reveals that the oligomerization domain ofis responsible for the interaction with BCA3. Furthermore, BCA3 augments the transactivation activity73 on bax promoter and protein expression. In addition, the expression of BCA3 also increases thevity of TAp73-transfected cells in response to γ-irradiation–induced apoptosis. Western blot analysisows that TAp73 and BCA3 induce activation of caspase-7 and caspase-9. In summary, these findingsted that BCA3 is a novel protein partner of TAp73, and they cooperate with each other to exert tumor-
sugges
suppressive functions and sensitize the response of cervical cancer cells to radiotherapy. Cancer Res; 70(16);

6486–96. ©2010 AACR.

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vical cancer is one of the major women malignancieswide and in Hong Kong. Radiotherapy remains thetay of treatment, especially in advanced cervical can-though there are extensive studies in the carcinogene-cervical cancer, the detail molecular mechanismslying the development and resistance to radiotherapylargely unclear. Thus, understanding of the signaling

ay associated with cell death and survival in responseiation may provide valuable information in developingtrategies for malignant therapeutics.candidate tumor suppressor gene p73 was identifiedghad and colleagues in 1997 (1). It encodes a proteinignificant similarity to p53. Unlike p53, somatic muta-f p73 gene is extremely rare. p73 mainly exists in two: the NH2-terminal transactivation domain containing(TAp73) and the dominant-negative isoforms lacking

ion domain (DNp73). TAp73 exhibits growth-or-suppressive, and proapoptotic functions,

promTAp73with TportanlizesTAp73and TIn con(the oOur d

n: Department of Obstetrics and Gynaecology, LKSe, The University of Hong Kong, Pokfulam, HKSAR

thor: Hextan Yuen-Sheung Ngan, Department of Ob-cology, 6/F Professorial Block, Queen Mary Hospital,. Phone: 852-22554260; Fax: 011-852-22550947;kucc.hku.hk.

5472.CAN-10-0688

ssociation for Cancer Research.

16) August 15, 2010

Research. on March 30, 202cancerres.aacrjournals.org ded from

as DNp73 promotes oncogenic activity and abolishesnctions of TAp73 (2). Functionally, p73 is able to inducecle arrest at the G1 phase and apoptosis (1, 3).ence from our previous finding revealed an associa-etween p73 expression and radiosensitivity of cervicalr, and suggested that p73 might play an important roletrolling cellular radiosensitivity (4, 5). However, theetween p73 expression and cervical cancer cell survivalpoptosis in response to radiation is still unclear.he present study, by using yeast two-hybrid screening,ve identified breast cancer–associated gene 3 (BCA3) asvel binding partner of p73. BCA3 plays an importantsubstrate localization, transcriptional regulation, ass actin cytoskeleton remodeling (6–8). We reportand BCA3 at the first time that they interact with eachand associate with the mitochondria. Because mito-rial control of apoptosis is one of the important path-that is involved in controlling cancer cell death, thispts us to investigate the functional significance of-BCA3 interaction. We confirm that BCA3 interactsAp73, and the oligomerization domain of TAp73 is im-t for their binding. We also indicate that TAp73 stabi-BCA3 in cervical cancer cells. BCA3 enhances the-mediated transcriptional activation of bax promoterAp73-dependent apoptosis in response to γ-irradiation.trast, BCA3 has marginal or no effect on TAp73-L371P
ligomerization domain–defective mutant) or DNp73.ata strongly suggest that BCA3 cooperates with TAp73
0. © 2010 American Association for Cancer

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iating bax expression, activating caspase-7 and caspase-apoptosis, which ultimately contributes to the sensi-f cervical cancer cells to γ-irradiation. Further studiesmolecular mechanism between p73 and BCA3 may
sight into tumor suppressor function of p73 in cervical Weste
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two-hybrid screening73 was cloned into the GAL4 DNA binding domainBKT7 vector (BD Biosciences Clontech). Librarying was performed by yeast mating. The AH109transformed with pGBKT7 DNp73, was mated withretransformed human HeLa MATCHMAKER cDNA(BD Biosciences Clontech). Mated cells were platedPD selection media. Positive colonies were isolated,e plasmid DNA in the yeast was prepared and trans-d into Escherichia coli DH5α. Direct sequencing wasmed to determine the identity of potential protein-cting partners of DNp73.

ids constructionfull-length coding region of BCA3 was amplified byith gene-specific primers containing KpnI restrictionhe PCR products were digested with KpnI and wereuently cloned into pcDNA3.1+ expression vector con-g Myc or FLAG epitope and pEGFP expression vector.

irected mutagenesis of TAp73-directed mutagenesis was performed to generate anerization-defective mutants of TAp73 (TAp73-L371P)Np73 (DNp73-L322P). These mutants were shown tofective in forming homotetramer or heterotetramerh oligomerization (9, 10). Specific mutation was intro-by three-step PCR. The first PCR was performed usingr set T7 and TP73-L371P-R: 5′-GCT CTC TTT CGG CTTAG-3′, and the second PCR was performed using prim-TP73-L371P-F: 5′-CTG ATG AAG CCG AAA GAG AGC-BGH-R. The final PCR product amplified by T7 andas then cloned into the pcDNA3.1+ vector throughndIII site.

nesvical cancer cell lines C33A, HeLa, ME180, and SiHabtained from the American Type Cell Collection andained in MEM supplemented with 10% fetal bovineand 100 units of penicillin/streptomycin.

munoprecipitation3 was cotransfected with TAp73 into HeLa cells byctamine 2000 reagent (Invitrogen). Transfected cellsysed with NET lysis buffer. Cell lysates were then incu-with anti-Myc antibody, and the immunoprecipitatesollected by protein G-Sepharose (Amersham Pharma-
otech). Total cell lysates and the immunoprecipitatessubjected to SDS gel electrophoresis and Western
temsas an

acrjournals.org

Research. on March 30, 202cancerres.aacrjournals.org Downloaded from

ng. Anti-Myc and anti–green fluorescent proteinantibodies were used to detect Myc-tagged BCA3FP-tagged TAp73, respectively.

rn blot analysisteins were analyzed by Western blotting using anti-s including bax (Santa Cruz), caspase-7 and cleavede-7, caspase-9 and cleaved caspase-9 (Cell signaling),orseradish peroxidase–conjugated secondary antibody,etected with enhanced chemiluminescence. β-Actindy (Sigma) was probed as a loading control.

nofluorescence stainings were seeded on coverslips and transfected with Myc-d BCA3 plasmid or together with GFP-tagged p73ids. Immunofluorescence staining was performed asbe previously (11). Nuclei were stained by 4′,6-diamidino-ylindole. To stain mitochondria, Mitochondria markeracker Red (Molecular Probes) was added to the culturem, and cells were incubated for 15 minutes beforet.

in stability assaydetermine the stability of BCA3, Myc-tagged BCA3 wasently transfected into HeLa cells. Twenty-four hourstransfection, cells were treated with cycloheximideg/mL). Cell lysates from different time points were col-and analyzed by Western blotting.

rase assaya cells in a 24-well plate were transfected with differ-mbinations of plasmids using Lipofectamine 2000.ids used including pcDNA3.1+ TAp73, pcDNA3.1+, the internal control pRL-SV40, and pGL3-bax-luc.ciferase reporter construct was kindly provided byL Tuosto (University 'La Sapienza', Via Dei Sardi 70,Rome, Italy). Twenty-four hours after transfection, cellwere collected and prepared for the Dual Luciferase

ter Assay (Promega, Clontech). Transfection efficiencyormalized with the Renilla luciferase activity. Eachection was done in triplicate, and three independentments were performed.

interfering RNAll interfering RNA (siRNA) for BCA3 and the nontarget-ntrol (NTC) siRNAwere purchased from Applied Biosys-Transfection of siRNA and NTC siRNA (50 pmol in 2 mLm) in six-well plates was done by Lipofectamine 2000ogen) according to the manufacturer's instructions.

titative real-time PCRal RNA of cells were isolated by Trizol reagent accord-the manufacturer's protocol (Invitrogen). cDNA wasynthesized from 1 μg of total RNA by High-Capacityo-cDNA Master Mix (Applied Biosystems). Quantitativeme PCR was performed by using the Applied Biosys-
Taqman system. Expression of TBP mRNA was usedinternal control.
Cancer Res; 70(16) August 15, 2010 6487



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Leung and Ngan

Cance6488


totic assaya or SiHa cells in 96-well plate were transfected with dif-combinations of plasmids or siRNA. Twenty-four hoursransfection, cells were treated with 10 Gy γ-irradiationere allowed to grow for 24 hours. Apoptotic assay (In situeath detection kit, Roche) was performed to studytic response of the cells to γ-irradiation. The percent-apoptotic cells was determined as the percentage ofositive cells with TMR red positively stained cells. At00 GFP-positive cells were counted for each experiment.

cytometrycell cycle analysis, cells were transfected with control, TAp73 or TAp73, and BCA3. Twenty-four hours afterection, cells were treated with 10 Gy γ-irradiation andd to grow for 24 hours. Cells were then fixed with 80%thanol and then washed with cold PBS twice. Fixedere stained with propidium iodide, and DNA profilealyzed by flow cytometry.

genic assays were seeded in each well of a six-well plate in tripli-wenty-four hours after transfection, cells were treated, 2, 6, or 10 Gy γ-irradiation and allowed to grow for 2. Cells were fixed and then stained with crystal violet,lonies with >50 cells were counted. The surviving frac-F) was calculated as the ratio of the number of coloniesd/the number of cells plated × the plating efficiency). X-Y log scatter plot was applied for plotting the curve,e value of D0 and SF2 were obtained from the graph. D0

ned as the dose required to reduce cell fraction to 0.37,as SF2 is the surviving fraction of growing cells that wereted at the clinically relevant dose of 2 Gy.

lts

ification of BCA3 as a protein bindinger of p73get more insight into the role of p73 in cervical can-velopment, we attempted to identify novel protein-cting partners of p73 protein. We performed yeastybrid screening of HeLa cDNA library using the full-form of DNp73 as bait. In total, 2 × 106 transfor-were screened and positive clones were confirmedgalactosidase activity. DNA sequencing analysisd that two of the positive clones (clone 6 and 16)ull-length clones with complete 5′-end and displayedal nucleotide sequence to the 3′-end of chromosomeen reading frame 17 (C11orf17) or BCA3, transcriptt 2 (GI: 116174749).

o interaction of p73 and BCA3physical interactions of BCA3 and TAp73 or DNp73onfirmed by coimmunoprecipitation (Fig. 1A). TAp73ble to coimmunoprecipitate with BCA3 in HeLa cells.the same condition, EGFP-empty vector control failed
mmunoprecipitate BCA3. In contrast, we also foundNp73 weakly associated with BCA3. These results sug-
38 mihalf-li

r Res; 70(16) August 15, 2010


that both TAp73 and DNp73 can interact with BCA3. The association between TAp73 and BCA3 is strongerNp73 and BCA3.

alization and interaction of p73 with BCA3 in vivoause the commercially available anti-BCA3 antibodyto detect the endogenous BCA3 protein, we detectedyc-tagged BCA3 in HeLa and SiHa cells. Previous studieshown that both TAp73 and BCA3 are primarily localizednucleus (3, 16). Our immunofluorescence stainingd that BCA3 localized in the nucleus and cytoplasmB). Transient expression of BCA3 together with TAp73p73 in SiHa cells displayed colocalization of BCA3 with73 isoforms with speckle staining pattern at the peri-r region (Fig. 1C). Such speckle staining pattern sug-colocalization of the two proteins at mitochondria. Tor confirm the observation, we applied MitoTrackers a marker of mitochondria. We observed thatand BCA3 were in fact colocalized at the mitochon-ig. 1D). Thus, these results indicated that both TAp73CA3 translocated to mitochondria upon coexpression.

ization of BCA3 in cervical cancer cells by TAp73ot by DNp73m the result of coimmunoprecipitation, the binding ofwith TAp73 was stronger than that with DNp73. In ad-, the protein expression level of BCA3 was increased in-transfected cells. We therefore sought to determineer TAp73 can modulate the protein stability of BCA3.transfected various amount of TAp73 or DNp73 to-with constant amount of BCA3 (2 μg) into HeLa cells.

p73-transfected cells, the BCA3 protein expression wasced with increased amount of TAp73 protein (Fig. 2A).trast, the expression of BCA3 did not change withsed amount of DNp73. Moreover, we investigated theof BCA3 on the stability of TAp73 or DNp73 by trans-with various amount of BCA3 and constant amount

p73 or DNp73 (2 μg). However, no observable differ-on the protein expression of either TAp73 or DNp73etected (Fig. 2B). The result indicated that BCA3 pro-as only stabilized by TAp73.

ity of BCA3 in cervical cancer cellsdetermine the stability of BCA3 protein in cervical can-lls, Myc-tagged BCA3 was transiently transfected intocells. The degradation rate of BCA3 was examined af-posure to cycloheximide (protein biosynthesis inhibi-ith different time durations. We found that BCA3 isstable protein that could be degraded rapidly withininutes after cyclohexamide treatment (Fig. 2C, left).heless, stabilization of BCA3 by TAp73 was observed73 and BCA3-cotransfected HeLa cells. We found thatstabilized BCA3 and extended BCA3 protein at a

table level to 240 minutes (Fig. 2C, right). Theponding calculation of the protein half-life was shown. 2D. The half-life of BCA3 protein in HeLa cells was
nutes. However, when cotransfected with TAp73, thefe of BCA3 protein was prolonged to 82 minutes.
Cancer Research



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uation of TAp73-BCA3 interaction byerization domain–defective mutants73 exerts its functions when forming homotetramers ortetramers (17, 18). To investigate whether BCA3 inter-ly with the functional state of TAp73, we generated olig-zation domain–defective mutants of TAp73 and DNp73.hematic diagramof their domain structures were shown3A. Coimmunoprecipitation was performed to evaluateinding activity of TAp73-L371P and DNp73-L322P
ts with BCA3. As expected, the interaction of BCA3 withwas strong. Although TAp73-L371P and DNp73-L322P
remaintrast t

acrjournals.org


lso able to interact with BCA3, the extent of interactionse mutants was largely reduced (Fig. 3B).

-L371P failed to colocalize with BCA3 at thehondriaddition to coimmunoprecipitation, the binding activityen TAp73-L371P and BCA3 was also studied by immuno-scence staining. When coexpressed with BCA3, TAp73-was detected mainly in the nucleus, whereas BCA3

1. A, interaction between3 and Myc-BCA3own in HeLa bynoprecipitation.lization of BCA3 in cervicalcell line SiHa. DAPI,midino-2-phenylindole.calization of TAp73-BCA3p73-BCA3 at thelear region in SiHa cellsown in yellow color byfluorescence staining.ar, 10 μm. D, subcellulartion of BCA3 in cervicalcell line. HeLa cells platederslips were cotransfectedP-TAp73 and Myc-BCA3ion plasmids. Mitochondriaained by MitoTracker Red.rged image showsndria colocalization ofand BCA3. Scale bar =

ed its localization in the nucleus and cytoplasm. In con-o TAp73 and BCA3 (Fig. 3C), neither of TAp73-L371P or




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Leung and Ngan

Cance6490


localized at the mitochondria (Fig. 3D). This result sug-that oligomerization of TAp73 protein is necessary formation and relocalization of TAp73-BCA3 complex froms to mitochondria. Our observations in coimmunopreci-n and immunofluorescence staining further support theinteraction between TAp73 and BCA3. These data indi-that oligomerization of TAp73 is responsible for BCA3g and targeting TAp73-BCA3 complex to mitochondria.

-L371P failed to stabilize BCA3next determined whether TAp73-L371P mutant couldze BCA3. We cotransfected various amount of TAp73 or-L371P together with BCA3 into HeLa cells. Consistente previous result, BCA3 was stabilized by TAp73 proteinse-dependent manner (Fig. 3E). However, there was noable increase of BCA3 protein in TAp73-L371P–trans-cells. We also performed quantitative real-time PCR toine whether expression of TAp73 would affect BCA3 ex-n at transcriptional level; however, no obvious increase3mRNAexpressionwas found inTAp73-transfected cellsF). These results suggested that oligomerization of TAp73ted the formation of TAp73-BCA3 complex, which en-d the stability of BCA3 protein in cervical cancer cells.

enhanced the transactivation activity of TAp73 onromoter and protein expression
well known that TAp73 is able to activate the tran- (P < 0
and intensity (% of the initial) was plotted against time. BCA3 protein half-life (t1/2al protein intensity).

r Res; 70(16) August 15, 2010


of BCA3 on the transactivation activity of TAp73x promoter. As shown in Fig. 4A, TAp73 markedlysed the transcriptional activity of bax promoter by, whereas BCA3 alone had no effect on the bax promoterred with empty vector control. Notably, cotransfection73 and BCA3 increased the transcriptional activity ofon bax promoter by 17.6-fold. In contrast, DNp73 didow any significant effect on bax promoter with or with-e expression of BCA3. In addition, the protein expres-f bax was also upregulated in TAp73 and BCA3sfected cells (Fig. 4B). These results suggested thatenhanced the transactivation activity of TAp73 onx promoter, which ultimately increased bax proteinsion.

ction of TAp73 and BCA3 sensitized cervicalr cells to γ-irradiation–induced apoptosisause TAp73 is recognized as an important mediator induction of apoptosis triggered by ionizing radiation, wenvestigated the effect of BCA3 on TAp73-mediated ap-is in response to γ-irradiation. HeLa and SiHa cellssed as a model, which were shown to be radioresistantprevious study (5) and showed relatively low BCA3 ex-on compared with the radiosensitive cell line C33AC). We observed a significant number of apoptotic cells73-transfected HeLa cells in response to γ-irradiation

.0001). More importantly, the number of apoptotic cells
on of p53-responsive genes (19). We investigated the was significantly increased in TAp73 and BCA3-cotransfected
2. A, TAp73 stabilized BCA3 in a dose-dependent manner. HeLa cells were transfected with various amounts of GFP-TAp73 or GFP-DNp73r with constant amount of Myc-BCA3 expression plasmids. The protein expression level of BCA3 affected by TAp73 or DNp73 was evaluatedti-Myc antibody. B, HeLa cells were transfected with various amounts of BCA3 together with constant amount of TAp73 or DNp73. The expressionTAp73 or DNp73 affected by BCA3 was evaluated with anti-GFP antibody. C, BCA3 was transfected alone or cotransfected with TAp73. Aftertion, cells were treated with 100 μg/mL cycloheximide and harvested at time points ranging from 0 to 360 min. The expression of TAp73 and BCA3tected by anti-GFP and anti-Myc antibodies. D, BCA3 band intensity was measured and normalized to t = 0 (100%). The natural logarithm (Ln)

) was calculated to the time point corresponding to the Ln (50% of

Cancer Research



Figurestructutheir cosequenTAp73,BCA3 wSubcelcotransGFP-TA(D), andimmunoantibodMitoTramitochoBCA3.BCA3 iwere trTAp73constanexpressDNp73F, HeLavector,BCA3.measurinternal control reference gene TBP.


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3. A, schematic diagram of the domainre of TAp73/DNp73 with wild-type andrresponding L371P/L322P mutantsces. B, the binding activity of DNp73,and their corresponding mutants withas examined by coimmunoprecipitation.lular localization of BCA3 in HeLafected with expression plasmids forp73 (C) or GFP-TAp73-L371P mutantMyc-BCA3 were detected byfluorescence staining using anti-Mycy. Mitochondria were stained bycker Red. The merged image showsndria colocalization of TAp73 andScale bar, 10 μm. E, TAp73 stabilizedn a dose-dependent manner. HeLa cellsansfected with various amounts ofor TAp73-L371P together with at amount (2 μg) of BCA3. The proteinion level of BCA3 affected by TAp73 orwas evaluated with anti-Myc antibody.cells were transfected with controlTAp73, or TAp73-L371P together withReal-time PCR was performed toe BCA3 mRNA expression with the


Research. on March 30, 2020. © 2010 American Association for Cancer.org


cells (in theFurtheptosisfectedcytomwas dcomptransf

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Leung and Ngan

Cance6492


P < 0.0001; Fig. 5A). In contrast, no significant increaseapoptotic cells was found in DNp73-transfected cells.rmore, we also evaluated DNA fragmentation (apo-) in control vector–, TAp73-, and TAp73/BCA3- trans-SiHa cells after γ-irradiation treatment by flow

etric analysis. Higher percentage of apoptotic cellsetected in TAp73/BCA3 cotransfected cells (12.1%)ared with control vector (1.3%) and TAp73 (8.6%)ectants (Fig. 5B).

-BCA3 enhanced the cleavage of caspase-7aspase-9onfirm that the induction of apoptosis by TAp73-BCA3ted to the mitochondrial pathway, the protein expres-of procaspase-7, procaspase-9, and their correspondingd forms (activated form) were studied. In nontrans-HeLa cells, very low expression of cleaved caspase-7spase-9 were found with or without γ-irradiation treat-The result suggested that no activation of mitochondrialotic pathway occurred in response to γ-irradiation inIn contrast, higher expression of cleaved caspase-7 ande-9 were found in TAp73-transfected, BCA3-transfected,p73/BCA3-cotransfected cells after γ-irradiation treat-

(Fig. 5C). The result further indicated that expression ofand BCA3 enhanced the sensitivity to irradiation and
d apoptosis through activation of the mitochondrialay.
nogenSiHa c

r Res; 70(16) August 15, 2010


ing of BCA3 reduced TAp73-induced apoptosis innse to γ-irradiationfurther confirm the role of BCA3 and TAp73 in γ-tion–induced mitochondrial pathway of apoptosis, wed siRNA to suppress the endogenous BCA3 expressiona cells. Transfection of siRNA profoundly reduced the ex-n of BCA3 by 60% in SiHa cells relative to NTC siRNA–ected cells (Fig. 6A). After siRNA transfection, cellshen transfected with GFP control vector or TAp73. Ap-s induction was determined in transfected cells afteriation treatment. We found that ectopic expression ofin NTC siRNA–transfected cells could induce apoptosisonse to γ-irradiation. However, significant reduction ofotic cells were observed in BCA3 siRNA–transfectedven with the expression of TAp73 (P = 0.0054; Fig. 6B).

ing of BCA3 enhanced the survival of cells afteration treatmentclonogenic survival of C33A, HeLa, ME180, and SiHa

nvestigated after exposure to 0, 2, or 6 Gy ofγ-irradiation.und that C33A was the most sensitive cell lines in re-e to γ-irradiation (Fig. 6C), which is consistent with theus literature (20). The SF2 and D0 values for C33A,ME180, and SiHa cells were summarized in Table 1.mine whether BCA3 expression was related to the clo-
ic survival, BCA3 siRNA was transfected into C33A andervical cancer cell lines. Cells were then treated with
uresfeorteicateressbastrols wicaterotesteriboddingA3 m180quath

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4. A, HeLa cells werected with bax-luciferaser construct together withd plasmids. The results areed as fold induction overal activity of the vector–transfected cells. B, HeLaere transfected withd plasmids. Equal amountin lysates were analyzed byn blot with anti-baxy. β-Actin was probed as acontrol. C, expression ofRNA in C33A, HeLa,

, and SiHa were evaluatedntitative real-time PCRe internal control reference

Cancer Research

tion for Cancer


differerate intransfand Sirradiaa D0 v

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nt dosage of γ-irradiation. We found that the survivalBCA3-silenced cells were higher than NTC siRNA–

ected cell and nontransfected cells in C33A (Fig. 6D)iHa (Fig. 6E). The SF2 value of exponentially growing
ted BCA3 siRNA–transfected C33A cells was 0.4 withalue of 210 cGy, whereas the SF2 value for NTC siRNA–
of BCcells.

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ected C33A cells was 0.27 with the D0 value of 150 cGy.sult indicated that cells transfected with BCA3 siRNAmore resistant to γ-irradiation compared with NTC–transfected cells in C33A. We also tested the effect

5. A, HeLa cells were transientlyted with indicated plasmids.cted cells were treated with 10 Gyation. After 24 h, cells were fixed and thetic cells were detected by terminalucleotidyl transferase–mediated dUTPd labeling (TUNEL) labeling assay.undergo DNA fragmentation (apoptosis)ated with 10 Gy γ-irradiation wereed by propidium iodide staining andd by flow cytometry. Numbers in eachhe percentage of apoptotic cellsted by flow cytometric analysis.ein expression of caspase-7, cleavede-7, caspase-9, and cleaved caspase-9aluated in transfected cells with or10 Gy γ-irradiation treatment byn blot analysis. β-Actin was probed as acontrol. The values below the blotsnt the change in the protein expressionands normalized to the expression in

A3 knockdown in response to γ-irradiation in SiHaThe SF2 for BCA3 siRNA–transfected SiHa cells was




Figureor BCAAfter 24cervicasiRNA

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6. A, real-time PCR was performed to measure BCA3 mRNA expression in BCA3-silenced cells. B, SiHa cells were transfected with NTC siRNA3 siRNA followed by transfection of pEGFP control vector or TAp73 expression vector. Transfected cells were then treated with 10 Gy γ-irradiation.h, cells were fixed and the apoptotic cells were detected by TUNEL labeling assay. C, clonogenic survival of C33A, HeLa, ME180, and SiHa

l cancer cells were determined after exposure to the indicated doses of γ-irradiation. C33A cells (D) and (E) SiHa cells were transfected with NTCor BCA3 siRNA, and clonogenic survival were determined.

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ith a D0 value of 510 cGy, suggesting that BCA3 siRNA–cted SiHa cellsmore resistant in response toγ-irradiationTC siRNA–tranfected SiHa cells (SF2, 0.63; D0: 343 cGy).

ssion

he present study, we have identified BCA3 as a novelcting partner of p73. We have shown for the first timeteraction of TAp73, but not DNp73, stabilizes BCA3 inal cancer cells. Such differences in the consequence ofnding of TAp73 and DNp73 with BCA3 might explainposing functions of p73 in carcinogenesis. A previoushas shown that TAp73 is found predominantly in thes (3). In addition, BCA3 is originally found to be local-the nucleus (16). Here, we show that the ectopic expres-f BCA3 alone is localized in the nucleus and cytoplasm ofells. Interestingly, when coexpressing TAp73 and BCA3a cells, both proteins redistribute and translocate to mi-dria. This phenomenon suggests that the interaction ofand BCA3 facilitates TAp73-BCA3 complex in targetingondria. A study reported that TAp73 can be cleaved byse-3 and caspase-8 during apoptosis. TAp73 and itsd products were localized to the mitochondria, which in-the release of cytochrome C (21). Furthermore, p53 wasto rapidly translocate to the mitochondria upon expo-genotoxic stress and triggered mechanisms, which ulti-induced apoptosis (22–25). Our data on the localization73-BCA3 complex at the mitochondria therefore impli-at TAp73 may cooperate with BCA3 at the mitochon-induce the release of cytochrome C and ultimately

ate apoptosis. Notably, TAp73-L371P fails to colocalizeCA3 at the mitochondria, suggesting that the oligomer-domain of TAp73 plays an essential role in BCA3 bind-

dmitochondrial targeting. Because p73 and p53 interactmitochondria through hetero-oligomerization (21),-L371P, which fails to form homo-oligomers or het-igomers, could not localize at the mitochondria.rexpression of TAp73 markedly increases the proteinty of BCA3. It is noteworthy that DNp73, which doesntain a transactivation domain, is unable to stabilizein HeLa cells. Furthermore, TAp73-L371P also fails toze BCA3, which further suggest that the monomericf TAp73 lost its properties in interacting with and sta-g the BCA3 protein. Like p53, TAp73 has been shown
rt its function as a homotetrameric or heterotetramericn (18). These results imply that the oligomerization do-
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acrjournals.org


is necessary for the specific functions of TAp73 on thection, stabilization, and colocalization of BCA3.t, we would like to address the functional significance73-BCA3 interaction in cervical cancer. We show thatression of BCA3 and TAp73 augments the transactiva-f bax promoter luciferase reporter construct and in-s the expression of bax protein. Consequently, TAp73CA3 coexpression potentiates TAp73-mediated apopto-cervical cancer in response to γ-irradiation. Similaration was found in the interaction of p73 with the Yes-ated protein. The physical interaction of p73 and Yes-ated protein resulted in enhancing the p73-mediatedosis by increasing the transactivation activity of p73x promoter and bax protein expression (26, 27).ences from our previous studies showed that differen-pression of TAp73 and DNp73 is related to radiosen-in cervical cancer (4, 5, 28). The implication of BCA3ancing the TAp73-mediated apoptotic signal inducedrradiation was shown by transient transfection of BCA3er with TAp73 into radioresistant cervical cancer cell lineIn addition, silencing of the endogenous BCA3 by siRNAther radioresistant cervical cancer cell line SiHa reducesmber of apoptotic cells in TAp73-overexpressing cells af-rradiation treatment. Clonogenic assay also reveals thating of BCA3 enhances cell survival after γ-irradiationent in cervical cancer cells (C33A and SiHa). The resultsBCA3 knockdown further confirm the involvement ofin sensitizing the response to γ-irradiation. Taken to-, our data suggest that BCA3 interacts with TAp73 atitochondria. The relocalization of TAp73-BCA3 complexces the expression of proapoptotic protein bax and in-the cleavage of caspase-7 and caspase-9. The interaction73 and BCA3 sensitizes the response of cervical canceres to γ-irradiation through induction of apoptosis.ummary, our findings in this study suggest an interest-tion on how TAp73-BCA3 complex associates to mito-ria and contributes to the tumor suppressor functions ofCA3 can interact with and potentiate TAp73 function inse to γ-irradiation–induced apoptosis. This is the firstto show the possible mechanism on how p73 contri-to the enhanced radiosensitivity of cervical cancer cells.r studies on the functional importance of TAp73-BCA3

g better understanding on their roles in cervical cancer.

osure of Potential Conflicts of Interest

otential conflicts of interest were disclosed.

Support

Wong Check She Charitable Foundation and the Research Fund fromartment of Obstetrics and Gynaecology, the University of Hong Kong.costs of publication of this article were defrayed in part by the paymentcharges. This article must therefore be hereby marked advertisement innce with 18 U.S.C. Section 1734 solely to indicate this fact.

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ika AI, Slade N, Erster SH, et al. ΔNp73, a dominant-negative in-itor of wild-type p53 and TAp73, is up-regulated in human tumors.xp Med 2002;196:765–80.t CA, Marin MC, Kaelin WG, Jr. p73 is a simian [correction of hu-n] p53-related protein that can induce apoptosis. Nature 1997;:191–4.SS, Chan KY, Cheung AN, Liao XY, Leung TW, Ngan HY. Expres-n of δNp73 and TAp73α independently associated with radiosen-vities and prognoses in cervical squamous cell carcinoma. Clinncer Res 2006;12:3922–7.SS, Chan KY, Leung RC, Law HK, Leung TW, Ngan HY.

hancement of the radiosensitivity of cervical cancer cells by over-pressing p73α. Mol Cancer Ther 2006;5:1209–15.KP, Itokawa T, ZhuML, Syam S, Seth A, Insogna K. Breast cancer-ociated gene 3 (BCA3) is a novel Rac1-interacting protein. J Boneer Res 2007;22:628–37.o N, Asamitsu K, Hibi Y, Ueno T, Okamoto T. AKIP1 enhances-κB-dependent gene expression by promoting the nuclear reten-and phosphorylation of p65. J Biol Chem 2008;283:7834–43.

o F, Cheng J, Shi T, Yeh ET. Neddylation of a breast cancer-ociated protein recruits a class III histone deacetylase that re-sses NFκB-dependent transcription. Nat Cell Biol 2006;8:1171–7.ika A, Irwin M, Sansome C, Moll UM. Oncogenes induce and ac-te endogenous p73 protein. J Biol Chem 2001;276:11310–6.in M, Marin MC, Phillips AC, et al. Role for the p53 homologue p732F-1-induced apoptosis. Nature 2000;407:645–8.ng TH, Ching YP, Yam JW, et al. Deleted in liver cancer 2 (DLC2)presses cell transformation by means of inhibition of RhoA activ-Proc Natl Acad Sci U S A 2005;102:15207–12.endil D, Ranga RS, Meigooni D, Sathishkumar S, Ahmed MM.rcumin confers radiosensitizing effect in prostate cancer cell line-3. Oncogene 2004;23:1599–607.KW, Mutter RW, Cao C, et al. Autophagy for cancer therapy

ough inhibition of pro-apoptotic proteins and mammalian targetrapamycin signaling. J Biol Chem 2006;281:36883–90.med MM, Sells SF, Venkatasubbarao K, et al. Ionizing radiation-
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wth factor-β signaling enhances radiosensitivity by altering thel-2/Bax ratio in the p53 mutant pancreatic cancer cell line MIACa-2. J Biol Chem 2002;277:2234–46.stri M, Barraclough DM, Carmichael PT, Taylor SS. A-kinase-eracting protein localizes protein kinase A in the nucleus. Proctl Acad Sci U S A 2005;102:349–54.oumanne A, Harms KL, Chen X. Structural basis for gene activa-n by p53 family members. Cancer Biol Ther 2005;4:1178–85.vison TS, Vagner C, Kaghad M, Ayed A, Caput D, Arrowsmith CH.3 and p63 are homotetramers capable of weak heterotypic inter-tions with each other but not with p53. J Biol Chem 1999;274:709–14.mamura Y, Lee WL, Goh MX, Ito Y. Role of TAp73α in induction ofoptosis by transforming growth factor-β in gastric cancer cells.BS Lett 2008;582:2663–7.xena A, Yashar C, Taylor DD, Gercel-Taylor C. Cellular responsechemotherapy and radiation in cervical cancer. Am J Obstetnecol 2005;192:1399–403.yan AE, Sayan BS, Gogvadze V, et al. P73 and caspase-cleaved3 fragments localize to mitochondria and augment TRAIL-inducedoptosis. Oncogene 2008;27:4363–72.hara M, Erster S, Zaika A, et al. p53 has a direct apoptogenic rolethe mitochondria. Mol Cell 2003;11:577–90.nsome C, Zaika A, Marchenko ND, Moll UM. Hypoxia death stim-s induces translocation of p53 protein to mitochondria. Detectionimmunofluorescence on whole cells. FEBS Lett 2001;488:110–5.majerova A, Wolff S, Petrenko O, Moll UM. Viral and cellular on-genes induce rapid mitochondrial translocation of p53 in primaryithelial and endothelial cells early in apoptosis. FEBS Lett 2005;9:6079–83.rchenko ND, Wolff S, Erster S, Becker K, Moll UM. Monoubiqui-tion promotes mitochondrial p53 translocation. EMBO J 2007;26:3–34.ano S, Munarriz E, Rossi M, et al. Physical interaction with Yes-sociated protein enhances p73 transcriptional activity. J Biolem 2001;276:15164–73.ano S, Monti O, Pediconi N, et al. The transcriptional coactivators-associated protein drives p73 gene-target specificity in re-onse to DNA Damage. Mol Cell 2005;18:447–59.SS, Leung RC, Chan KY, et al. p73 expression is associated with
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Cancer Research



2010;70:6486-6496. Published OnlineFirst July 20, 2010.Cancer Res Thomas Ho-Yin Leung and Hextan Yuen-Sheung Ngan to Irradiation-Induced ApoptosisEnhances the Sensitivity of Cervical Cancer Cells in Response

Associated Gene 3−Breast CancerInteraction of TAp73 and

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