Identification of proteins that are involved in the sensitivity of radiotherapy of cancers is important to enhance the response to cancer treatment. Expression of TAp73 is associated with the sensitivity of radiotherapy in cervical cancer patients, suggesting it plays an important role in controlling radiosensitivity. Here, by using yeast two-hybrid system, we identify breast cancer–associated gene 3 (BCA3) as the first and novel protein interacting partner of TAp73. By coimmunoprecipitation and Western blot analysis, we confirm that TAp73 binds with and stabilizes BCA3 in cervical cancer cell line HeLa. Immunofluorescence staining indicates that BCA3 is localized in the cytoplasm and nucleus. Interestingly, when coexpressed with TAp73, BCA3 interacts and colocalizes with TAp73 at the mitochondria. Mutagenesis reveals that the oligomerization domain of TAp73 is responsible for the interaction with BCA3. Furthermore, BCA3 augments the transactivation activity of TAp73 on bax promoter and protein expression. In addition, the expression of BCA3 also increases the sensitivity of TAp73-transfected cells in response to γ-irradiation–induced apoptosis. Western blot analysis also shows that TAp73 and BCA3 induce activation of caspase-7 and caspase-9. In summary, these findings suggested that BCA3 is a novel protein partner of TAp73, and they cooperate with each other to exert tumor-suppressive functions and sensitize the response of cervical cancer cells to radiotherapy. Cancer Res; 70(16); 6486–96. ©2010 AACR.

Cervical cancer is one of the major women malignancies worldwide and in Hong Kong. Radiotherapy remains the mainstay of treatment, especially in advanced cervical cancer. Although there are extensive studies in the carcinogenesis of cervical cancer, the detail molecular mechanisms underlying the development and resistance to radiotherapy remain largely unclear. Thus, understanding of the signaling pathway associated with cell death and survival in response to radiation may provide valuable information in developing new strategies for malignant therapeutics.

The candidate tumor suppressor gene p73 was identified by Kaghad and colleagues in 1997 (1). It encodes a protein with significant similarity to p53. Unlike p53, somatic mutation of p73 gene is extremely rare. p73 mainly exists in two forms: the NH2-terminal transactivation domain containing form (TAp73) and the dominant-negative isoforms lacking the transactivation domain (DNp73). TAp73 exhibits growth-inhibitory, tumor-suppressive, and proapoptotic functions, whereas DNp73 promotes oncogenic activity and abolishes the functions of TAp73 (2). Functionally, p73 is able to induce cell cycle arrest at the G1 phase and apoptosis (1, 3).

Evidence from our previous finding revealed an association between p73 expression and radiosensitivity of cervical cancer, and suggested that p73 might play an important role in controlling cellular radiosensitivity (4, 5). However, the link between p73 expression and cervical cancer cell survival and apoptosis in response to radiation is still unclear.

In the present study, by using yeast two-hybrid screening, we have identified breast cancer–associated gene 3 (BCA3) as the novel binding partner of p73. BCA3 plays an important role in substrate localization, transcriptional regulation, as well as actin cytoskeleton remodeling (68). We report TAp73 and BCA3 at the first time that they interact with each other and associate with the mitochondria. Because mitochondrial control of apoptosis is one of the important pathways that is involved in controlling cancer cell death, this prompts us to investigate the functional significance of TAp73-BCA3 interaction. We confirm that BCA3 interacts with TAp73, and the oligomerization domain of TAp73 is important for their binding. We also indicate that TAp73 stabilizes BCA3 in cervical cancer cells. BCA3 enhances the TAp73-mediated transcriptional activation of bax promoter and TAp73-dependent apoptosis in response to γ-irradiation. In contrast, BCA3 has marginal or no effect on TAp73-L371P (the oligomerization domain–defective mutant) or DNp73. Our data strongly suggest that BCA3 cooperates with TAp73 in mediating bax expression, activating caspase-7 and caspase-9, and apoptosis, which ultimately contributes to the sensitivity of cervical cancer cells to γ-irradiation. Further studies on the molecular mechanism between p73 and BCA3 may give insight into tumor suppressor function of p73 in cervical cancer.

Yeast two-hybrid screening

DNp73 was cloned into the GAL4 DNA binding domain in pGBKT7 vector (BD Biosciences Clontech). Library screening was performed by yeast mating. The AH109 strain, transformed with pGBKT7 DNp73, was mated with Y187-pretransformed human HeLa MATCHMAKER cDNA library (BD Biosciences Clontech). Mated cells were plated onto YPD selection media. Positive colonies were isolated, and the plasmid DNA in the yeast was prepared and transformed into Escherichia coli DH5α. Direct sequencing was performed to determine the identity of potential protein-interacting partners of DNp73.

Plasmids construction

The full-length coding region of BCA3 was amplified by PCR with gene-specific primers containing KpnI restriction site. The PCR products were digested with KpnI and were subsequently cloned into pcDNA3.1+ expression vector containing Myc or FLAG epitope and pEGFP expression vector.

Site-directed mutagenesis of TAp73

Site-directed mutagenesis was performed to generate an oligomerization-defective mutants of TAp73 (TAp73-L371P) and DNp73 (DNp73-L322P). These mutants were shown to be defective in forming homotetramer or heterotetramer through oligomerization (9, 10). Specific mutation was introduced by three-step PCR. The first PCR was performed using primer set T7 and TP73-L371P-R: 5′-GCT CTC TTT CGG CTT CAT CAG-3′, and the second PCR was performed using primer set TP73-L371P-F: 5′-CTG ATG AAG CCG AAA GAG AGC-3′ and BGH-R. The final PCR product amplified by T7 and BGH was then cloned into the pcDNA3.1+ vector through the HindIII site.

Cell lines

Cervical cancer cell lines C33A, HeLa, ME180, and SiHa were obtained from the American Type Cell Collection and maintained in MEM supplemented with 10% fetal bovine serum and 100 units of penicillin/streptomycin.

Coimmunoprecipitation

BCA3 was cotransfected with TAp73 into HeLa cells by Lipofectamine 2000 reagent (Invitrogen). Transfected cells were lysed with NET lysis buffer. Cell lysates were then incubated with anti-Myc antibody, and the immunoprecipitates were collected by protein G-Sepharose (Amersham Pharmacia Biotech). Total cell lysates and the immunoprecipitates were subjected to SDS gel electrophoresis and Western blotting. Anti-Myc and anti–green fluorescent protein (GFP) antibodies were used to detect Myc-tagged BCA3 and GFP-tagged TAp73, respectively.

Western blot analysis

Proteins were analyzed by Western blotting using antibodies including bax (Santa Cruz), caspase-7 and cleaved caspase-7, caspase-9 and cleaved caspase-9 (Cell signaling), and horseradish peroxidase–conjugated secondary antibody, and detected with enhanced chemiluminescence. β-Actin antibody (Sigma) was probed as a loading control.

Immunofluorescence staining

Cells were seeded on coverslips and transfected with Myc-tagged BCA3 plasmid or together with GFP-tagged p73 plasmids. Immunofluorescence staining was performed as describe previously (11). Nuclei were stained by 4′,6-diamidino-2-phenylindole. To stain mitochondria, Mitochondria marker Mitotracker Red (Molecular Probes) was added to the culture medium, and cells were incubated for 15 minutes before harvest.

Protein stability assay

To determine the stability of BCA3, Myc-tagged BCA3 was transiently transfected into HeLa cells. Twenty-four hours after transfection, cells were treated with cycloheximide (100 μg/mL). Cell lysates from different time points were collected and analyzed by Western blotting.

Luciferase assay

HeLa cells in a 24-well plate were transfected with different combinations of plasmids using Lipofectamine 2000. Plasmids used including pcDNA3.1+ TAp73, pcDNA3.1+ BCA3, the internal control pRL-SV40, and pGL3-bax-luc. Bax-luciferase reporter construct was kindly provided by Prof. L Tuosto (University 'La Sapienza', Via Dei Sardi 70, 00185 Rome, Italy). Twenty-four hours after transfection, cell lysates were collected and prepared for the Dual Luciferase Reporter Assay (Promega, Clontech). Transfection efficiency was normalized with the Renilla luciferase activity. Each transfection was done in triplicate, and three independent experiments were performed.

Small interfering RNA

Small interfering RNA (siRNA) for BCA3 and the nontargeting control (NTC) siRNA were purchased from Applied Biosystems. Transfection of siRNA and NTC siRNA (50 pmol in 2 mL medium) in six-well plates was done by Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.

Quantitative real-time PCR

Total RNA of cells were isolated by Trizol reagent according to the manufacturer's protocol (Invitrogen). cDNA was then synthesized from 1 μg of total RNA by High-Capacity RNA-to-cDNA Master Mix (Applied Biosystems). Quantitative real-time PCR was performed by using the Applied Biosystems Taqman system. Expression of TBP mRNA was used as an internal control.

Apoptotic assay

HeLa or SiHa cells in 96-well plate were transfected with different combinations of plasmids or siRNA. Twenty-four hours after transfection, cells were treated with 10 Gy γ-irradiation and were allowed to grow for 24 hours. Apoptotic assay (In situ cell death detection kit, Roche) was performed to study apoptotic response of the cells to γ-irradiation. The percentage of apoptotic cells was determined as the percentage of GFP-positive cells with TMR red positively stained cells. At least 200 GFP-positive cells were counted for each experiment.

Flow cytometry

For cell cycle analysis, cells were transfected with control vector, TAp73 or TAp73, and BCA3. Twenty-four hours after transfection, cells were treated with 10 Gy γ-irradiation and allowed to grow for 24 hours. Cells were then fixed with 80% cold ethanol and then washed with cold PBS twice. Fixed cells were stained with propidium iodide, and DNA profile was analyzed by flow cytometry.

Clonogenic assay

Cells were seeded in each well of a six-well plate in triplicate. Twenty-four hours after transfection, cells were treated with 0, 2, 6, or 10 Gy γ-irradiation and allowed to grow for 2 weeks. Cells were fixed and then stained with crystal violet, and colonies with >50 cells were counted. The surviving fraction (SF) was calculated as the ratio of the number of colonies formed/the number of cells plated × the plating efficiency (1215). X-Y log scatter plot was applied for plotting the curve, and the value of D0 and SF2 were obtained from the graph. D0 is defined as the dose required to reduce cell fraction to 0.37, whereas SF2 is the surviving fraction of growing cells that were irradiated at the clinically relevant dose of 2 Gy.

Identification of BCA3 as a protein binding partner of p73

To get more insight into the role of p73 in cervical cancer development, we attempted to identify novel protein-interacting partners of p73 protein. We performed yeast two-hybrid screening of HeLa cDNA library using the full-length form of DNp73 as bait. In total, 2 × 106 transformants were screened and positive clones were confirmed by β-galactosidase activity. DNA sequencing analysis showed that two of the positive clones (clone 6 and 16) were full-length clones with complete 5′-end and displayed identical nucleotide sequence to the 3′-end of chromosome 11 open reading frame 17 (C11orf17) or BCA3, transcript variant 2 (GI: 116174749).

In vivo interaction of p73 and BCA3

The physical interactions of BCA3 and TAp73 or DNp73 were confirmed by coimmunoprecipitation (Fig. 1A). TAp73 was able to coimmunoprecipitate with BCA3 in HeLa cells. Under the same condition, EGFP-empty vector control failed to coimmunoprecipitate BCA3. In contrast, we also found that DNp73 weakly associated with BCA3. These results suggested that both TAp73 and DNp73 can interact with BCA3 in vivo. The association between TAp73 and BCA3 is stronger than DNp73 and BCA3.

Figure 1.

A, interaction between GFP-p73 and Myc-BCA3 was shown in HeLa by coimmunoprecipitation. B, localization of BCA3 in cervical cancer cell line SiHa. DAPI, 4′,6-diamidino-2-phenylindole. C, colocalization of TAp73-BCA3 and DNp73-BCA3 at the perinuclear region in SiHa cells were shown in yellow color by immunofluorescence staining. Scale bar, 10 μm. D, subcellular localization of BCA3 in cervical cancer cell line. HeLa cells plated on coverslips were cotransfected with GFP-TAp73 and Myc-BCA3 expression plasmids. Mitochondria were stained by MitoTracker Red. The merged image shows mitochondria colocalization of TAp73 and BCA3. Scale bar = 10 μm.

Figure 1.

A, interaction between GFP-p73 and Myc-BCA3 was shown in HeLa by coimmunoprecipitation. B, localization of BCA3 in cervical cancer cell line SiHa. DAPI, 4′,6-diamidino-2-phenylindole. C, colocalization of TAp73-BCA3 and DNp73-BCA3 at the perinuclear region in SiHa cells were shown in yellow color by immunofluorescence staining. Scale bar, 10 μm. D, subcellular localization of BCA3 in cervical cancer cell line. HeLa cells plated on coverslips were cotransfected with GFP-TAp73 and Myc-BCA3 expression plasmids. Mitochondria were stained by MitoTracker Red. The merged image shows mitochondria colocalization of TAp73 and BCA3. Scale bar = 10 μm.

Close modal

Colocalization and interaction of p73 with BCA3 in vivo

Because the commercially available anti-BCA3 antibody failed to detect the endogenous BCA3 protein, we detected the Myc-tagged BCA3 in HeLa and SiHa cells. Previous studies have shown that both TAp73 and BCA3 are primarily localized in the nucleus (3, 16). Our immunofluorescence staining showed that BCA3 localized in the nucleus and cytoplasm (Fig. 1B). Transient expression of BCA3 together with TAp73 or DNp73 in SiHa cells displayed colocalization of BCA3 with both p73 isoforms with speckle staining pattern at the perinuclear region (Fig. 1C). Such speckle staining pattern suggests colocalization of the two proteins at mitochondria. To further confirm the observation, we applied MitoTracker Red as a marker of mitochondria. We observed that TAp73 and BCA3 were in fact colocalized at the mitochondria (Fig. 1D). Thus, these results indicated that both TAp73 and BCA3 translocated to mitochondria upon coexpression.

Stabilization of BCA3 in cervical cancer cells by TAp73 but not by DNp73

From the result of coimmunoprecipitation, the binding of BCA3 with TAp73 was stronger than that with DNp73. In addition, the protein expression level of BCA3 was increased in TAp73-transfected cells. We therefore sought to determine whether TAp73 can modulate the protein stability of BCA3. We cotransfected various amount of TAp73 or DNp73 together with constant amount of BCA3 (2 μg) into HeLa cells. In TAp73-transfected cells, the BCA3 protein expression was enhanced with increased amount of TAp73 protein (Fig. 2A). In contrast, the expression of BCA3 did not change with increased amount of DNp73. Moreover, we investigated the effect of BCA3 on the stability of TAp73 or DNp73 by transfection with various amount of BCA3 and constant amount of TAp73 or DNp73 (2 μg). However, no observable differences on the protein expression of either TAp73 or DNp73 was detected (Fig. 2B). The result indicated that BCA3 protein was only stabilized by TAp73.

Figure 2.

A, TAp73 stabilized BCA3 in a dose-dependent manner. HeLa cells were transfected with various amounts of GFP-TAp73 or GFP-DNp73 together with constant amount of Myc-BCA3 expression plasmids. The protein expression level of BCA3 affected by TAp73 or DNp73 was evaluated with anti-Myc antibody. B, HeLa cells were transfected with various amounts of BCA3 together with constant amount of TAp73 or DNp73. The expression level of TAp73 or DNp73 affected by BCA3 was evaluated with anti-GFP antibody. C, BCA3 was transfected alone or cotransfected with TAp73. After transfection, cells were treated with 100 μg/mL cycloheximide and harvested at time points ranging from 0 to 360 min. The expression of TAp73 and BCA3 were detected by anti-GFP and anti-Myc antibodies. D, BCA3 band intensity was measured and normalized to t = 0 (100%). The natural logarithm (Ln) of the band intensity (% of the initial) was plotted against time. BCA3 protein half-life (t1/2) was calculated to the time point corresponding to the Ln (50% of the initial protein intensity).

Figure 2.

A, TAp73 stabilized BCA3 in a dose-dependent manner. HeLa cells were transfected with various amounts of GFP-TAp73 or GFP-DNp73 together with constant amount of Myc-BCA3 expression plasmids. The protein expression level of BCA3 affected by TAp73 or DNp73 was evaluated with anti-Myc antibody. B, HeLa cells were transfected with various amounts of BCA3 together with constant amount of TAp73 or DNp73. The expression level of TAp73 or DNp73 affected by BCA3 was evaluated with anti-GFP antibody. C, BCA3 was transfected alone or cotransfected with TAp73. After transfection, cells were treated with 100 μg/mL cycloheximide and harvested at time points ranging from 0 to 360 min. The expression of TAp73 and BCA3 were detected by anti-GFP and anti-Myc antibodies. D, BCA3 band intensity was measured and normalized to t = 0 (100%). The natural logarithm (Ln) of the band intensity (% of the initial) was plotted against time. BCA3 protein half-life (t1/2) was calculated to the time point corresponding to the Ln (50% of the initial protein intensity).

Close modal

Stability of BCA3 in cervical cancer cells

To determine the stability of BCA3 protein in cervical cancer cells, Myc-tagged BCA3 was transiently transfected into HeLa cells. The degradation rate of BCA3 was examined after exposure to cycloheximide (protein biosynthesis inhibitor) with different time durations. We found that BCA3 is an unstable protein that could be degraded rapidly within 120 minutes after cyclohexamide treatment (Fig. 2C, left). Nonetheless, stabilization of BCA3 by TAp73 was observed in TAp73 and BCA3-cotransfected HeLa cells. We found that TAp73 stabilized BCA3 and extended BCA3 protein at a detectable level to 240 minutes (Fig. 2C, right). The corresponding calculation of the protein half-life was shown in Fig. 2D. The half-life of BCA3 protein in HeLa cells was 38 minutes. However, when cotransfected with TAp73, the half-life of BCA3 protein was prolonged to 82 minutes.

Attenuation of TAp73-BCA3 interaction by oligomerization domain–defective mutants

TAp73 exerts its functions when forming homotetramers or heterotetramers (17, 18). To investigate whether BCA3 interacts only with the functional state of TAp73, we generated oligomerization domain–defective mutants of TAp73 and DNp73. The schematic diagram of their domain structures were shown in Fig. 3A. Coimmunoprecipitation was performed to evaluate the binding activity of TAp73-L371P and DNp73-L322P mutants with BCA3. As expected, the interaction of BCA3 with TAp73 was strong. Although TAp73-L371P and DNp73-L322P were also able to interact with BCA3, the extent of interaction by these mutants was largely reduced (Fig. 3B).

Figure 3.

A, schematic diagram of the domain structure of TAp73/DNp73 with wild-type and their corresponding L371P/L322P mutants sequences. B, the binding activity of DNp73, TAp73, and their corresponding mutants with BCA3 was examined by coimmunoprecipitation. Subcellular localization of BCA3 in HeLa cotransfected with expression plasmids for GFP-TAp73 (C) or GFP-TAp73-L371P mutant (D), and Myc-BCA3 were detected by immunofluorescence staining using anti-Myc antibody. Mitochondria were stained by MitoTracker Red. The merged image shows mitochondria colocalization of TAp73 and BCA3. Scale bar, 10 μm. E, TAp73 stabilized BCA3 in a dose-dependent manner. HeLa cells were transfected with various amounts of TAp73 or TAp73-L371P together with a constant amount (2 μg) of BCA3. The protein expression level of BCA3 affected by TAp73 or DNp73 was evaluated with anti-Myc antibody. F, HeLa cells were transfected with control vector, TAp73, or TAp73-L371P together with BCA3. Real-time PCR was performed to measure BCA3 mRNA expression with the internal control reference gene TBP.

Figure 3.

A, schematic diagram of the domain structure of TAp73/DNp73 with wild-type and their corresponding L371P/L322P mutants sequences. B, the binding activity of DNp73, TAp73, and their corresponding mutants with BCA3 was examined by coimmunoprecipitation. Subcellular localization of BCA3 in HeLa cotransfected with expression plasmids for GFP-TAp73 (C) or GFP-TAp73-L371P mutant (D), and Myc-BCA3 were detected by immunofluorescence staining using anti-Myc antibody. Mitochondria were stained by MitoTracker Red. The merged image shows mitochondria colocalization of TAp73 and BCA3. Scale bar, 10 μm. E, TAp73 stabilized BCA3 in a dose-dependent manner. HeLa cells were transfected with various amounts of TAp73 or TAp73-L371P together with a constant amount (2 μg) of BCA3. The protein expression level of BCA3 affected by TAp73 or DNp73 was evaluated with anti-Myc antibody. F, HeLa cells were transfected with control vector, TAp73, or TAp73-L371P together with BCA3. Real-time PCR was performed to measure BCA3 mRNA expression with the internal control reference gene TBP.

Close modal

TAp73-L371P failed to colocalize with BCA3 at the mitochondria

In addition to coimmunoprecipitation, the binding activity between TAp73-L371P and BCA3 was also studied by immunofluorescence staining. When coexpressed with BCA3, TAp73-L371P was detected mainly in the nucleus, whereas BCA3 remained its localization in the nucleus and cytoplasm. In contrast to TAp73 and BCA3 (Fig. 3C), neither of TAp73-L371P or BCA3 localized at the mitochondria (Fig. 3D). This result suggested that oligomerization of TAp73 protein is necessary for the formation and relocalization of TAp73-BCA3 complex from nucleus to mitochondria. Our observations in coimmunoprecipitation and immunofluorescence staining further support the in vivo interaction between TAp73 and BCA3. These data indicated that oligomerization of TAp73 is responsible for BCA3 binding and targeting TAp73-BCA3 complex to mitochondria.

TAp73-L371P failed to stabilize BCA3

We next determined whether TAp73-L371P mutant could stabilize BCA3. We cotransfected various amount of TAp73 or TAp73-L371P together with BCA3 into HeLa cells. Consistent with the previous result, BCA3 was stabilized by TAp73 protein in a dose-dependent manner (Fig. 3E). However, there was no observable increase of BCA3 protein in TAp73-L371P–transfected cells. We also performed quantitative real-time PCR to determine whether expression of TAp73 would affect BCA3 expression at transcriptional level; however, no obvious increase of BCA3 mRNA expression was found in TAp73-transfected cells (Fig. 3F). These results suggested that oligomerization of TAp73 mediated the formation of TAp73-BCA3 complex, which enhanced the stability of BCA3 protein in cervical cancer cells.

BCA3 enhanced the transactivation activity of TAp73 on bax promoter and protein expression

It is well known that TAp73 is able to activate the transcription of p53-responsive genes (19). We investigated the effect of BCA3 on the transactivation activity of TAp73 on bax promoter. As shown in Fig. 4A, TAp73 markedly increased the transcriptional activity of bax promoter by 14-fold, whereas BCA3 alone had no effect on the bax promoter compared with empty vector control. Notably, cotransfection of TAp73 and BCA3 increased the transcriptional activity of TAp73 on bax promoter by 17.6-fold. In contrast, DNp73 did not show any significant effect on bax promoter with or without the expression of BCA3. In addition, the protein expression of bax was also upregulated in TAp73 and BCA3 cotransfected cells (Fig. 4B). These results suggested that BCA3 enhanced the transactivation activity of TAp73 on the bax promoter, which ultimately increased bax protein expression.

Figure 4.

A, HeLa cells were transfected with bax-luciferase reporter construct together with indicated plasmids. The results are expressed as fold induction over the basal activity of the vector control–transfected cells. B, HeLa cells were transfected with indicated plasmids. Equal amount of protein lysates were analyzed by Western blot with anti-bax antibody. β-Actin was probed as a loading control. C, expression of BCA3 mRNA in C33A, HeLa, ME180, and SiHa were evaluated by quantitative real-time PCR with the internal control reference gene TBP.

Figure 4.

A, HeLa cells were transfected with bax-luciferase reporter construct together with indicated plasmids. The results are expressed as fold induction over the basal activity of the vector control–transfected cells. B, HeLa cells were transfected with indicated plasmids. Equal amount of protein lysates were analyzed by Western blot with anti-bax antibody. β-Actin was probed as a loading control. C, expression of BCA3 mRNA in C33A, HeLa, ME180, and SiHa were evaluated by quantitative real-time PCR with the internal control reference gene TBP.

Close modal

Interaction of TAp73 and BCA3 sensitized cervical cancer cells to γ-irradiation–induced apoptosis

Because TAp73 is recognized as an important mediator in the induction of apoptosis triggered by ionizing radiation, we then investigated the effect of BCA3 on TAp73-mediated apoptosis in response to γ-irradiation. HeLa and SiHa cells were used as a model, which were shown to be radioresistant in our previous study (5) and showed relatively low BCA3 expression compared with the radiosensitive cell line C33A (Fig. 4C). We observed a significant number of apoptotic cells in TAp73-transfected HeLa cells in response to γ-irradiation (P < 0.0001). More importantly, the number of apoptotic cells was significantly increased in TAp73 and BCA3-cotransfected cells (P < 0.0001; Fig. 5A). In contrast, no significant increase in the apoptotic cells was found in DNp73-transfected cells. Furthermore, we also evaluated DNA fragmentation (apoptosis) in control vector–, TAp73-, and TAp73/BCA3- transfected SiHa cells after γ-irradiation treatment by flow cytometric analysis. Higher percentage of apoptotic cells was detected in TAp73/BCA3 cotransfected cells (12.1%) compared with control vector (1.3%) and TAp73 (8.6%) transfectants (Fig. 5B).

Figure 5.

A, HeLa cells were transiently transfected with indicated plasmids. Transfected cells were treated with 10 Gy γ-irradiation. After 24 h, cells were fixed and the apoptotic cells were detected by terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) labeling assay. B, cells undergo DNA fragmentation (apoptosis) after treated with 10 Gy γ-irradiation were evaluated by propidium iodide staining and analyzed by flow cytometry. Numbers in each panel, the percentage of apoptotic cells calculated by flow cytometric analysis. C, protein expression of caspase-7, cleaved caspase-7, caspase-9, and cleaved caspase-9 were evaluated in transfected cells with or without 10 Gy γ-irradiation treatment by Western blot analysis. β-Actin was probed as a loading control. The values below the blots represent the change in the protein expression of the bands normalized to the expression in control vector transfected cells.

Figure 5.

A, HeLa cells were transiently transfected with indicated plasmids. Transfected cells were treated with 10 Gy γ-irradiation. After 24 h, cells were fixed and the apoptotic cells were detected by terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) labeling assay. B, cells undergo DNA fragmentation (apoptosis) after treated with 10 Gy γ-irradiation were evaluated by propidium iodide staining and analyzed by flow cytometry. Numbers in each panel, the percentage of apoptotic cells calculated by flow cytometric analysis. C, protein expression of caspase-7, cleaved caspase-7, caspase-9, and cleaved caspase-9 were evaluated in transfected cells with or without 10 Gy γ-irradiation treatment by Western blot analysis. β-Actin was probed as a loading control. The values below the blots represent the change in the protein expression of the bands normalized to the expression in control vector transfected cells.

Close modal

TAp73-BCA3 enhanced the cleavage of caspase-7 and caspase-9

To confirm that the induction of apoptosis by TAp73-BCA3 is related to the mitochondrial pathway, the protein expressions of procaspase-7, procaspase-9, and their corresponding cleaved forms (activated form) were studied. In nontransfected HeLa cells, very low expression of cleaved caspase-7 and caspase-9 were found with or without γ-irradiation treatment. The result suggested that no activation of mitochondrial apoptotic pathway occurred in response to γ-irradiation in HeLa. In contrast, higher expression of cleaved caspase-7 and caspase-9 were found in TAp73-transfected, BCA3-transfected, and TAp73/BCA3-cotransfected cells after γ-irradiation treatment (Fig. 5C). The result further indicated that expression of TAp73 and BCA3 enhanced the sensitivity to irradiation and induced apoptosis through activation of the mitochondrial pathway.

Silencing of BCA3 reduced TAp73-induced apoptosis in response to γ-irradiation

To further confirm the role of BCA3 and TAp73 in γ-irradiation–induced mitochondrial pathway of apoptosis, we applied siRNA to suppress the endogenous BCA3 expression in SiHa cells. Transfection of siRNA profoundly reduced the expression of BCA3 by 60% in SiHa cells relative to NTC siRNA–transfected cells (Fig. 6A). After siRNA transfection, cells were then transfected with GFP control vector or TAp73. Apoptosis induction was determined in transfected cells after γ-irradiation treatment. We found that ectopic expression of TAp73 in NTC siRNA–transfected cells could induce apoptosis in response to γ-irradiation. However, significant reduction of apoptotic cells were observed in BCA3 siRNA–transfected cells even with the expression of TAp73 (P = 0.0054; Fig. 6B).

Figure 6.

A, real-time PCR was performed to measure BCA3 mRNA expression in BCA3-silenced cells. B, SiHa cells were transfected with NTC siRNA or BCA3 siRNA followed by transfection of pEGFP control vector or TAp73 expression vector. Transfected cells were then treated with 10 Gy γ-irradiation. After 24 h, cells were fixed and the apoptotic cells were detected by TUNEL labeling assay. C, clonogenic survival of C33A, HeLa, ME180, and SiHa cervical cancer cells were determined after exposure to the indicated doses of γ-irradiation. C33A cells (D) and (E) SiHa cells were transfected with NTC siRNA or BCA3 siRNA, and clonogenic survival were determined.

Figure 6.

A, real-time PCR was performed to measure BCA3 mRNA expression in BCA3-silenced cells. B, SiHa cells were transfected with NTC siRNA or BCA3 siRNA followed by transfection of pEGFP control vector or TAp73 expression vector. Transfected cells were then treated with 10 Gy γ-irradiation. After 24 h, cells were fixed and the apoptotic cells were detected by TUNEL labeling assay. C, clonogenic survival of C33A, HeLa, ME180, and SiHa cervical cancer cells were determined after exposure to the indicated doses of γ-irradiation. C33A cells (D) and (E) SiHa cells were transfected with NTC siRNA or BCA3 siRNA, and clonogenic survival were determined.

Close modal

Silencing of BCA3 enhanced the survival of cells after irradiation treatment

The clonogenic survival of C33A, HeLa, ME180, and SiHa were investigated after exposure to 0, 2, or 6 Gy of γ-irradiation. We found that C33A was the most sensitive cell lines in response to γ-irradiation (Fig. 6C), which is consistent with the previous literature (20). The SF2 and D0 values for C33A, HeLa, ME180, and SiHa cells were summarized in Table 1. To examine whether BCA3 expression was related to the clonogenic survival, BCA3 siRNA was transfected into C33A and SiHa cervical cancer cell lines. Cells were then treated with different dosage of γ-irradiation. We found that the survival rate in BCA3-silenced cells were higher than NTC siRNA–transfected cell and nontransfected cells in C33A (Fig. 6D) and SiHa (Fig. 6E). The SF2 value of exponentially growing irradiated BCA3 siRNA–transfected C33A cells was 0.4 with a D0 value of 210 cGy, whereas the SF2 value for NTC siRNA–transfected C33A cells was 0.27 with the D0 value of 150 cGy. The result indicated that cells transfected with BCA3 siRNA were more resistant to γ-irradiation compared with NTC siRNA–transfected cells in C33A. We also tested the effect of BCA3 knockdown in response to γ-irradiation in SiHa cells. The SF2 for BCA3 siRNA–transfected SiHa cells was 0.89 with a D0 value of 510 cGy, suggesting that BCA3 siRNA–transfected SiHa cells more resistant in response to γ-irradiation than NTC siRNA–tranfected SiHa cells (SF2, 0.63; D0: 343 cGy).

Table 1.

Summary of the SF2 and D0 in cervical cancer cell lines

Cell linesSF2D0
C33A 0.27 150 cGy 
HeLa 0.86 415 cGy 
ME180 0.55 258 cGy 
SiHa 0.67 348 cGy 
Cell linesSF2D0
C33A 0.27 150 cGy 
HeLa 0.86 415 cGy 
ME180 0.55 258 cGy 
SiHa 0.67 348 cGy 

In the present study, we have identified BCA3 as a novel interacting partner of p73. We have shown for the first time that interaction of TAp73, but not DNp73, stabilizes BCA3 in cervical cancer cells. Such differences in the consequence of the binding of TAp73 and DNp73 with BCA3 might explain the opposing functions of p73 in carcinogenesis. A previous study has shown that TAp73 is found predominantly in the nucleus (3). In addition, BCA3 is originally found to be localized in the nucleus (16). Here, we show that the ectopic expression of BCA3 alone is localized in the nucleus and cytoplasm of HeLa cells. Interestingly, when coexpressing TAp73 and BCA3 in HeLa cells, both proteins redistribute and translocate to mitochondria. This phenomenon suggests that the interaction of TAp73 and BCA3 facilitates TAp73-BCA3 complex in targeting mitochondria. A study reported that TAp73 can be cleaved by caspase-3 and caspase-8 during apoptosis. TAp73 and its cleaved products were localized to the mitochondria, which induced the release of cytochrome C (21). Furthermore, p53 was shown to rapidly translocate to the mitochondria upon exposure to genotoxic stress and triggered mechanisms, which ultimately induced apoptosis (2225). Our data on the localization of TAp73-BCA3 complex at the mitochondria therefore implicate that TAp73 may cooperate with BCA3 at the mitochondria to induce the release of cytochrome C and ultimately stimulate apoptosis. Notably, TAp73-L371P fails to colocalize with BCA3 at the mitochondria, suggesting that the oligomerization domain of TAp73 plays an essential role in BCA3 binding and mitochondrial targeting. Because p73 and p53 interact in/on mitochondria through hetero-oligomerization (21), TAp73-L371P, which fails to form homo-oligomers or hetero-oligomers, could not localize at the mitochondria.

Overexpression of TAp73 markedly increases the protein stability of BCA3. It is noteworthy that DNp73, which does not contain a transactivation domain, is unable to stabilize BCA3 in HeLa cells. Furthermore, TAp73-L371P also fails to stabilize BCA3, which further suggest that the monomeric form of TAp73 lost its properties in interacting with and stabilizing the BCA3 protein. Like p53, TAp73 has been shown to exert its function as a homotetrameric or heterotetrameric protein (18). These results imply that the oligomerization domain is necessary for the specific functions of TAp73 on the interaction, stabilization, and colocalization of BCA3.

Next, we would like to address the functional significance of TAp73-BCA3 interaction in cervical cancer. We show that coexpression of BCA3 and TAp73 augments the transactivation of bax promoter luciferase reporter construct and increases the expression of bax protein. Consequently, TAp73 and BCA3 coexpression potentiates TAp73-mediated apoptosis in cervical cancer in response to γ-irradiation. Similar observation was found in the interaction of p73 with the Yes-associated protein. The physical interaction of p73 and Yes-associated protein resulted in enhancing the p73-mediated apoptosis by increasing the transactivation activity of p73 on bax promoter and bax protein expression (26, 27).

Evidences from our previous studies showed that differential expression of TAp73 and DNp73 is related to radiosensitivity in cervical cancer (4, 5, 28). The implication of BCA3 in enhancing the TAp73-mediated apoptotic signal induced by γ-irradiation was shown by transient transfection of BCA3 together with TAp73 into radioresistant cervical cancer cell line HeLa. In addition, silencing of the endogenous BCA3 by siRNA in another radioresistant cervical cancer cell line SiHa reduces the number of apoptotic cells in TAp73-overexpressing cells after γ-irradiation treatment. Clonogenic assay also reveals that silencing of BCA3 enhances cell survival after γ-irradiation treatment in cervical cancer cells (C33A and SiHa). The results from BCA3 knockdown further confirm the involvement of BCA3 in sensitizing the response to γ-irradiation. Taken together, our data suggest that BCA3 interacts with TAp73 at the mitochondria. The relocalization of TAp73-BCA3 complex enhances the expression of proapoptotic protein bax and induces the cleavage of caspase-7 and caspase-9. The interaction of TAp73 and BCA3 sensitizes the response of cervical cancer cell lines to γ-irradiation through induction of apoptosis.

In summary, our findings in this study suggest an interesting notion on how TAp73-BCA3 complex associates to mitochondria and contributes to the tumor suppressor functions of p73. BCA3 can interact with and potentiate TAp73 function in response to γ-irradiation–induced apoptosis. This is the first report to show the possible mechanism on how p73 contributes to the enhanced radiosensitivity of cervical cancer cells. Further studies on the functional importance of TAp73-BCA3 interaction and the biological functions of BCA3 are crucial in getting better understanding on their roles in cervical cancer.

No potential conflicts of interest were disclosed.

Grant Support: The Wong Check She Charitable Foundation and the Research Fund from the Department of Obstetrics and Gynaecology, the University of Hong Kong.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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