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Originally published In Press as doi:10.1074/jbc.M305624200 on October 9, 2003

J. Biol. Chem., Vol. 278, Issue 52, 52093-52101, December 26, 2003
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Cell Cycle Arrest and Apoptosis Induction by Activator Protein 2{alpha} (AP-2{alpha}) and the Role of p53 and p21WAF1/CIP1 in AP-2{alpha}-mediated Growth Inhibition*

Narendra Wajapeyee{ddagger} and Kumaravel Somasundaram§

From the Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India

Received for publication, May 29, 2003 , and in revised form, September 8, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Activator protein 2{alpha} (AP-2{alpha}) is a sequence-specific DNA-binding transcription factor implicated in differentiation and transformation. In this study, we have made a replication-deficient recombinant adenovirus that expresses functional AP-2{alpha} (Ad-AP2). Cells infected with Ad-AP2 expressed induced levels of AP-2{alpha} protein, which bound to DNA in a sequence-specific manner and activated the AP-2-specific reporter 3X-AP2. Expression of AP-2{alpha} from Ad-AP2 inhibited cellular DNA synthesis and induced apoptosis. Ad-AP2 infection resulted in efficient inhibition of growth of cancer cells of six different types. In addition, prior expression of AP-2{alpha} increased the chemosensitivity of H460, a lung carcinoma cell line, to adriamycin (2.5-fold) and cisplatin (5-fold). Furthermore, the growth inhibition by AP-2{alpha} was found to be less efficient in the absence of p53 or p21, which correlated with reduced apoptosis in p53 null cells and lack of DNA synthesis inhibition in p21WAF1/CIP1 null cells by AP-2{alpha}, respectively. These results suggest that AP-2{alpha} inhibits the growth of cells by inducing cell cycle arrest and apoptosis and that the use of AP-2{alpha} should be explored as a therapeutic strategy either alone or in combination with chemotherapy.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Sequence-specific DNA-binding transcription factors regulate gene expression in response to intracellular and extracellular stimuli, and they often play a central role in determining cell fate by controlling the fundamental mechanism of gene transcription. Activator protein 2 (AP-2)1 is a family of cell type-specific developmentally regulated transcription factors that have been implicated as critical regulators of gene expression during vertebrate development, embryogenesis, and transformation (1–5). There are four known members of the AP-2 gene family: AP-2{alpha}, AP-2{beta}, AP-2{gamma}, and AP-2{delta}, each encoded by a separate gene (6–12). AP-2 isoforms have a highly conserved dimerization/DNA-binding region, and they can homodimerize and heterodimerize through a unique C-terminal helix-span-helix motif and bind palindromic DNA recognition sequences consensus 5'-GCCN3GGC-3' through the basic domain that lies immediately N-terminal of the dimerization motif (6, 13–16). Several genes with a variety of functions like cell growth, cell morphology, and cell communication have been shown to possess sequences similar to the AP-2-binding sequence in their promoter regions or be regulated by AP-2 (4, 5, 9, 17–25).

Evidence from many laboratories suggests that AP-2{alpha} is a tumor suppressor gene. Reduced expression or loss of expression is found to be associated with breast cancer, colon carcinoma, and cutaneous malignant melanoma (26–28). Dominant negative mutant of AP-2{alpha} has been shown to increase invasiveness and tumorigenicity (29–31). Overexpression of AP-2{alpha} has been shown to induce p21WAF1/CIP1 and inhibit cellular DNA synthesis and colony formation (4). Significant correlation between AP-2{alpha} expression and p21WAF1/CIP1 has been observed in breast cancer, colorectal cancer, and malignant melanoma (26, 28, 32).

In this study, we have developed a replication-deficient recombinant adenovirus, which expresses functional AP-2{alpha}. AP-2{alpha} overexpression from Ad-AP2 resulted in cell cycle arrest in G1 phase, induction of apoptosis, and inhibition of growth of a variety of cancer cells. In addition, prior expression of AP-2{alpha} resulted in increased chemosensitivity of cancer cells to anticancer drugs. Moreover, the growth inhibition by AP-2{alpha} was found to be partially defective in p53 null or p21WAF1/CIP1 null cells. These results suggest that growth inhibition by AP-2{alpha} involves both cell cycle arrest and apoptosis induction, and the use of Ad-AP2 should be explored as a therapeutic strategy for cancer treatment.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids—pSG5/AP-2 and 3X-AP2, described before, were kindly provided by Dr. P. Kannan (Metro Health Medical Center, Case Western Reserve University). pAdTrack-CMV and pAdEasy-1, described previously (3), were kindly provided by Dr. Bert Vogelstein (Johns Hopkins University).

Tumor Cell Lines and Culture Conditions—Human cancer cell lines 293, SW480, H460, U2OS, HeLa, HT1080, and HCT116 were described previously (34, 35). HCT116 p53-/- (36) and HCT116 p21-/- (37) were kindly provided by Dr. Bert Vogelstein (Johns Hopkins University). Transfections into 293 cells were carried out by using Lipofectamine (Invitrogen). For normalizing the variation between samples during transfection, 1 µg of pCMV-LacZ (Invitrogen) was added, and the {beta}-galatosidase levels were measured by orthonitrophenyl-beta-D-galactoside liquid assay as described previously (38).

Adenovirus Reagents and Infections—The following adenoviruses were used in this study. Ad-LacZ and Ad-p53 lacks both E1A and E1B but carry {beta}-galactosidase and p53, respectively (39). Ad-AP2 lacks both E1A and E1B but carries human AP-2{alpha}. It was constructed using a procedure described previously (33, 40). Briefly, AP-2{alpha} was cloned as a XbaI/XhoI fragment into pAdTrack-CMV. pAdTrack-CMV-AP-2 was linearized with PmeI and co-transformed along with pAdEasy-1 into Escherichia coli BJ5183 selecting for kanamycin resistance. Clones carrying appropriate recombinant plasmid were identified, plasmid DNA extracted, linearized with PacI, and transfected into 293 cells. The recombinant virus plaques were identified as localized areas of clearing consisting of round cells, which fluoresce green under fluorescent microscopy. Adenoviral infections were carried out as described (41).

CAT Assay, Western Analysis, and Immunohistochemical Analysis— CAT assay was performed as described previously (38). Western analysis and immunohistochemical staining were performed as described before (34) with mouse anti-human p21WAF1/CIP1 monoclonal (Ab-1; Oncogene), mouse anti-human p53 monoclonal (Ab-2; Oncogene), mouse anti-human Rb monoclonal (Ab5; Oncogene), mouse anti-human PARP monoclonal (Ab-2; Oncogene), rabbit anti-human AP-2 polyclonal (sc-184; Santa Cruz), and goat anti-human actin polyclonal (sc-1616; Santa Cruz). The cells were harvested or fixed at 24 h or appropriate time point as described after virus infection and subjected to analysis.

Electrophoretic Mobility Shift Assay—Electrophoretic mobility shift assay was essentially carried as described before (14). An AP-2-binding site (5'-GATCGAACTGACCGCCCGCGGCCCGT-3') was used as probe. The following reaction conditions were used: 10 mM Tris, pH 7.9, 4.5% Ficoll 400, 60 mM KCl, 4 mM MgCl2, 0.1 mM EDTA, 50 µg/ml bovine serum albumin, 0.2% Nonidet P-40, and 40 ng/ml poly(dI-dC). The sequence of the nonspecific competitor was as follows: 5'-GATCCGAATTCGGTACC-3'. Nuclear extracts were prepared after 24 h of virus infection of HT1080 cells as described before (42).

DNA Synthesis Inhibition—Bromodeoxyuridine (BrdU) incorporation and [3H]thymidine incorporation were done as described earlier (4, 34). BrdU (20 µM) or [3H]thymidine (5 µCi/ml) was added after 20 h of virus infection. The experiment was terminated 4 h after addition of these compounds, and DNA synthesis was measured either by using anti-BrdU antibody (Ab-3; Oncogene) or measuring the radioactivity in a scintillation counter.

Cell Cycle Analysis—H460 cells were infected with Ad-lacZ, Ad-p53, or Ad-AP2 virus at 20 MOI. At indicated time points, the cells were washed with PBS twice and harvested by trypsinization. The cells were washed again with PBS and fixed with cold 70% ethanol for 1 h. The cells were washed with PBS once and then incubated with 4 µg of ribonuclease A (Roche Applied Science) for 30 min at room temperature. Propidium iodide was added to the cell suspension at a final concentration of 20 µg/ml and incubated for 30 min. The cells were then analyzed by flow cytometry using FACScan (Becton Dickinson). The results were quantified by using the software Cell Quest (Becton Dickinson). For two-color FACS, 20 µM BrdU was added to cells during the last 4 h of their time, at which point they are harvested. The cells were washed with PBS twice and harvested by trypsinization. The cells were washed again with PBS and fixed with cold 70% ethanol for 1 h. Subsequently, the cells were centrifuged, and the pellet was resuspended in 2 N HCL and incubated at room temperature for 30 min. The cells were washed thrice with PBS and incubated with 1 µg of anti-BrdU antibody (Ab-3; Oncogene) for 30 min. The cells were washed again twice with PBS and incubated with 2 µg of fluorescein isothiocyanate-conjugated goat antimouse antibody (Oncogene) for 30 min at room temperature. The cells were washed with PBS once and then incubated with 4 µg of ribonuclease A (Roche Applied Science) for 30 min at room temperature. Propidium iodide was added to the cell suspension at a final concentration of 20 µg/ml and incubated for 30 min. The cells were then analyzed by flow cytometry using FACScan (Becton Dickinson). The results were quantified by using the software WinList (Verity Software House Inc).

MTT Assay—MTT assay was carried out as described previously (43, 44). 1.5 x 103 cells/well were plated in a 96-well plate. After 24 h of plating, the cells were infected with Ad-AP-2 at the indicated MOI. MTT (20 µl of 5 mg/ml) was added 48 h after infection. MTT is a tetrazolium salt that is converted by living cells into blue formazan crystals. The medium was removed from the wells 3 h after MTT addition, and 200 µl of Me2SO was added to dissolve the formazan crystals, and then the absorbance was measured at 550 nm in an enzyme-linked immunosorbent assay reader.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of Functional AP-2{alpha} in Ad-AP2-infected Cells— To detect the expression of AP-2{alpha} protein in Ad-AP2 infected cells, AP-2{alpha} protein level was monitored after infecting different cancer cells with Ad-AP2 or the control virus Ad-LacZ at a MOI of 20. At 24 h after infection, the cells were fixed and stained for AP-2{alpha} protein. Ad-AP2 infection resulted in intense nuclear staining in comparison with Ad-LacZ infection in SW80 (Fig. 1a, compare panel D with panel A), H460 (Fig. 1a, compare panel E with panel B), and HT1080 (Fig. 1a, compare panel F with panel C) cells. We also monitored the induced expression of AP-2{alpha} protein upon Ad-AP2 infection by Western blotting. Upon Ad-AP2 infection, SW480 (Fig. 1b, compare lane 2 with lane 1), H460 (Fig. 1c), and HT1080 (Fig. 1d) cells showed several fold higher amounts of AP-2{alpha} protein in comparison with Ad-LacZ infection. AP-2{alpha} expression in Ad-AP2-infected cells is also time-dependent with maximum expression seen between 24 and 36 h after infection (Fig. 1d).



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  		FIG. 1.
Expression of AP-2{alpha} in Ad-AP2-infected cells. a, SW480, H460, or HT1080 cells were infected with either Ad-LacZ or Ad-AP2 at a MOI of 20. The cells were fixed after 24 h and analyzed by immunohistochemical staining for AP-2. b–d, SW480 (b), H460 (c), and HT1080 (d) cells were infected with Ad-LacZ or Ad-AP2 at a MOI of 20. Total cell extract were made after 24 h (b and c) and at the indicated time points (d) and analyzed by Western blotting for AP-2 protein.

 
Next we checked whether AP-2{alpha} expressed from Ad-AP2 is functional by monitoring the sequence-specific DNA binding and activation of AP-2-specific reporter by AP-2{alpha} protein. The nuclear extract made from Ad-AP2-infected but not Ad-LacZ-infected HT1080 cells showed a specific DNA-protein complex (Fig. 2a, lane 3), which is a specific AP-2-DNA complex because it was competed by unlabeled AP-2-binding oligonucleotide (Fig. 2a, lanes 4–6) but not by a nonspecific oligonucleotide (Fig. 2a, lanes 7–9). To measure the function of AP-2{alpha} expressed from Ad-AP2, we used an AP-2-specific reporter 3X-AP2-CAT (45). 3X-AP2-CAT reporter contains three copies of AP-2-binding sequences cloned in tandem upstream of thymidine kinase minimal promoter. As a positive control, we used a construct pSG5/AP-2, which expresses AP-2{alpha} from SV40 promoter (3). As expected, AP-2{alpha} expressed from pSG5-activated 3X-AP2-CAT very efficiently (Fig. 2b, compare lane 3 with lane 2). Similarly, AP-2{alpha} expressed from Ad-AP2 also activated 3X-AP2-CAT (Fig. 2b, compare lane 5 with lane 4) to comparable levels. AP-2{alpha} has been shown to activate p21WAF1/CIP1 (4). In our experiments, AP-2{alpha} expressed from Ad-AP2 also activated p21WAF1/CIP1in a time-dependent manner (Fig. 4). Thus, it is evident that infection of cells with Ad-AP2 results in increased levels of functionally active AP-2{alpha}.



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  		FIG. 2.
Analysis of DNA binding activity and transcriptional activation by AP-2{alpha} expressed from Ad-AP2. a, electrophoretic mobility shift assay was performed using nuclear extract (Nuc.Ext.) made from either Ad-AP2- or Ad-LacZ-infected cells as described under "Materials and Methods." Competitions were performed by adding increasing amounts (1.0, 2.5, and 5.0 pmol) of specific (lanes 4–6) or nonspecific (lanes 7–9) competitor as indicated. b, HT1080 cancer cells were transfected with 5 µg of 3X-AP2 and where indicated by +, control vector pSG5 (5 µg) or pSG5 expressing AP-2{alpha} (5 µg). The total amount of DNA/transfection was kept constant at 10 µg. After 6 h of transfection, the cells were infected with Ad-AP2 (lane 4) or Ad-LacZ (lane 5) at a MOI of 20. Transfections were carried out using Lipofectin transfection reagent. The lysates were prepared and analyzed for CAT reporter activity 24 h post-transfection as described under "Materials and Methods." All of the samples were done in duplicate.

 



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  		FIG. 4.
Western analysis of Ad-AP2 virus-infected cells. H460 cells were infected with Ad-LacZ or Ad-AP2 at 20 MOI. After the indicated time points of virus infection, the total cell extracts were prepared and subjected to Western analysis for PARP, Rb, p21WAF1/CIP1, AP-2{alpha}, and actin proteins as described under "Materials and Methods."

 
Ad-AP2 Infection Induces Growth Arrest and Apoptosis—To see the effect of AP-2{alpha} expressed from Ad-AP-2 on cell cycle, we monitored the extent of cellular DNA synthesis and cell cycle profile in Ad-AP2-infected cells. The cellular DNA synthesis was measured by measuring BrdU incorporation and [3H]thymidine incorporation. Infection of H460 cells with Ad-AP2 resulted in a drastic reduction in percentage of cells incorporating BrdU in comparison with Ad-LacZ cells (Fig. 3a, compare panel F with panel D). The results obtained in Fig. 3a were quantified, and the percentage of BrdU incorporation under different conditions is shown in Fig. 3b. Similarly, infection of H460 cells with Ad-AP2 resulted in reduction of [3H]thymidine incorporation (Fig. 3c), which is indicative of DNA synthesis inhibition. The inhibition of DNA synthesis is also dose-dependent because the inhibition is more severe as the MOI of virus is increased (Fig. 3c). The phosphorylation status of Rb correlated with the extent of cellular DNA synthesis (Fig. 4). The hypophosphorylated Rb is seen in Ad-AP2-infected cells in comparison with Ad-LacZ-infected cells (Fig. 4) in a time-dependent manner.



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  		FIG. 3.
DNA synthesis inhibition and induction of apoptosis in Ad-AP2-infected cells. a, H460 cells were infected with Ad-LacZ or Ad-AP2 at a MOI of 20. The cells were allowed to incorporate BrdU for last 4 h. After 24 h of infection, BrdU-positive cells were identified by staining using anti-BrdU antibody and Texas Red-conjugated anti-mouse secondary antibody (panels B, D, and F). Panels A, C, and E of a are DAPI staining of cells of the same region as panels B, D, and F of a, respectively. BrdU-positive cells were calculated as percentages of BrdU incorporation from a and shown in b. The percentage of BrdU incorporation was calculated by dividing the number of cells showing BrdU incorporation by total number of cells counted. c, H460 cells were infected with Ad-LacZ (black bar) or Ad-AP2 (gray bar) virus at a MOI of 5, 10, or 20. The cells were allowed to incorporate [3H]thymidine during the last 4 h of the experiment. After 24 h of virus infection, [3H]thymidine incorporated into cellular DNA was measured as described under "Materials and Methods." d, FACS analysis of Ad-AP2-infected cells. H460 cells were infected with Ad-LacZ, Ad-p53, or Ad-AP2 at 20 MOI. The cells were harvested at 0, 24, 36, 48, and 72 h after infection and subjected to flow cytometry analysis as mentioned under "Materials and Methods." The proportion of cells having a 2 N amount of cells, which corresponds to the G1 phase of cell cycle, are quantified, and the values are given on the right side. The percentage of cells carrying less than a 2 N amount of DNA of the total cells analyzed, which represent apoptotic cells (A), is also measured by using the same software and indicated in each panel.

 
Fluorescence-activated cell sorting (FACS) was also carried out to analyze the cell cycle profile and induction of apoptosis in Ad-AP2-infected cells. The tumor suppressor p53 expressing adenovirus (Ad-p53) was used as a positive control. H460 cells were infected with Ad-LacZ, Ad-p53, or Ad-AP2 viruses, and the cells were collected at different time points after infection and subjected to FACS (Fig. 3d). The proportion of cells containing a 2 N amount of DNA, which represent G1 phase cells, increased significantly in Ad-p53- and Ad-AP2-infected cells in comparison with Ad-LacZ-infected cells as early as 24 h after infection (55.54–71.52% and 55.54–64.02%, respectively). Based on the above results from BrdU incorporation, [3H]thymidine incorporation, and FACS studies with supporting evidence like induction of p21WAF1/CIP1 and appearance of hypophosphorylated Rb in Ad-AP2-infected cells, we conclude that AP-2{alpha} expression leads to inhibition of cellular DNA synthesis resulting in arrest of cells in G1 phase of cell cycle.

In addition to cell cycle arrest, FACS also reveals the induction of apoptosis in Ad-AP2-infected cells (Fig. 3d). Cells with less than 2 N content of DNA, which represent apoptotic cells, appeared in very high proportion by 36 h in Ad-p53-infected cells (25.6%) and by 48 h in Ad-AP2 infected cells (18.7%) in comparison with Ad-LacZ infected cells (Fig. 3d). The proportion of cells undergoing apoptosis increased even further in both Ad-p53-infected (41.8% by 48 h) and Ad-AP2-infected (49.2% by 72 h) cells. Induction of apoptosis was also confirmed by PARP cleavage assay. PARP, an enzyme involved in DNA repair mechanisms is cleaved by the caspase-3. Therefore, the cleavage of PARP from the native 115 to 89 kDa is considered as a hallmark of apoptosis. Ad-AP2 infection but not Ad-LacZ infection resulted in the appearance of an 89-kDa cleaved product of PARP by as early as 24 h after infection (Fig. 4; see also Fig. 8). Taken together, above data suggest that infection of cells with Ad-AP2 results in induction of growth arrest and apoptosis.



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  		FIG. 8.
Western analysis of Ad-AP2 virus-infected HCT116 WT and HCT116 p53-/- cells. HCT116 p53 WT (a) or HCT116 p53-/- (b) cells were infected with Ad-LacZ or Ad-AP2 at 20 MOI. After the indicated time points of virus infection, the total cell extracts were prepared and subjected to Western analysis for PARP, p21WAF1/CIP1, AP-2{alpha}, p53, and actin proteins as described under "Materials and Methods."

 
Ad-AP2 Inhibits the Growth and Increases Chemosensitivity of Cancer Cells—To assay the cytotoxic potential of the AP-2{alpha}, the ability of Ad-AP2 to inhibit the growth of six different cancer cell lines, which differ in p53 status, was analyzed. The following cell lines were used: H460 (a human lung carcinoma cell line), HT1080 (a fibrosarcoma cell line), HCT116 (a colon carcinoma cell line), and U2OS (an osteosarcoma cell line), all of which harbor wild type p53. SW480, a human colon carcinoma cell line, harbors mutant p53. In HeLa cells, which is a cervical cancer-derived cell line, p53 is degraded because of the presence of human papilloma virus oncogene E6. The cells were infected with varying MOI of Ad-AP2, and the cytotoxicity was measured by MTT assay (Fig. 5, a–f). Ad-AP2 inhibited the growth of all cell lines tested very efficiently regardless of their p53 status in comparison with Ad-LacZ (Fig. 5, a–f). Although the amount of Ad-AP2 virus required to inhibit the growth of different cell lines varied, the p53 status was not found to be the requirement for AP-2{alpha}-mediated growth inhibition. For example, HT1080 (WT p53) and HeLa (degraded p53) cells are the cells most sensitive to Ad-AP2 (Fig. 5, b and e). Similarly, HCT116 (WT p53) and SW480 (mutant p53) cells are equally sensitive (Fig. 5, c and f). These results suggest that Ad-AP2 is a potent inhibitor of cancer cell growth.



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  		FIG. 5.
Growth inhibitory activity of AP-2{alpha} a–f, Ad-AP2 inhibits the growth of cancer cells. H460 (a), HT180 (b), HCT116 (c), U2OS (d), HeLa (e), and SW480 (f) cells were infected with indicated MOI of either Ad-LacZ (filled triangle) or Ad-AP2 (filled square) virus. After 48 h of virus infection, the proportion of live cells was quantified by MTT assay as described under "Materials and Methods." The absorbance of control cells not infected the virus was considered as 100%. g, expression of AP-2{alpha} increases the chemosensitivity of cancer cells to chemotherapeutic drugs. H460 cells were treated with varying amount of adriamycin or cisplatin alone (empty circle), infected with Ad-LacZ (empty square), or Ad-AP2 (empty triangle) at a MOI of 10. After 48 h of virus infection, the proportion of live cells was quantified by MTT assay as described under "Materials and Methods." The absorbance of control cells not infected with the virus was considered to be 100%. The IC50 values are calculated and given below.

 
To determine whether AP-2{alpha} could enhance the drug cytotoxicity, we examined the sensitivity of Ad-AP2-infected cells to commonly used anticancer drugs adriamycin and cisplatin. Infection of H460 cells with Ad-AP2 resulted in several fold increase in sensitivity to adriamycin and cisplatin. The IC50 for adriamycin decreased by 2.5-fold in Ad-AP2-infected cells in comparison with Ad-LacZ-infected cells (Fig. 5g; from 0.100 to 0.038 µg/ml). Similarly, the IC50 for cisplatin also decreased by 5-fold (Fig. 5g; from 0.500 to 0.100 µg/ml). These results suggest that prior overexpression of AP-2{alpha} increases the sensitivity of cells to adriamycin and cisplatin.

Role of p53 in AP-2{alpha}-mediated Growth Inhibition—There have been conflicting reports about the role of p53 in AP-2{alpha}-mediated growth inhibition. AP-2{alpha} has been shown to interact with tumor suppressor p53 and induce cell cycle arrest in a p53-dependent manner (46). In contrast to this observation, AP-2{alpha} has also been shown to inhibit DNA synthesis in SW480 human carcinoma cell line, which harbors mutant p53, leading to suppression of colony formation (4). Our results in this study indicate that Ad-AP2 inhibits growth of cancer cells efficiently independent of their p53 status (Fig. 5, a--f). Although these results suggest the existence of a p53-independent mechanism of growth inhibition by AP-2{alpha}, it does not rule out a cooperation between p53 and AP-2{alpha}. To determine the role of p53 in AP2{alpha}-mediated growth suppression, we studied the cytotoxicity profile of Ad-AP2 in the HCT116 p53-/- cell line (a somatic knock-out for p53 derived from HCT116 cell line) and compared it with that of HCT116 WT. Ad-AP2 inhibited the growth of HCT116 p53-/- cells less efficiently than that of HCT116 WT cells (Fig. 6a), suggesting that the growth inhibition by AP-2{alpha} is partially p53-dependent. To study mechanistically the contribution by p53 in AP-2{alpha}-mediated growth suppression, we carried out FACS to analyze the cell cycle profile and apoptosis induction in Ad-AP2-infected HCT116 WT and HCT116 p53-/- cells. The proportion of cells actively replicating DNA, which represent S phase cells, decreased drastically in both HCT116 WT cells (from 22.57 to 0.10%) and HCT116 p53-/- cells (from 25.22 to 0.01%) by 24 h after Ad-AP2 infection. Ad-AP2 infection also resulted in induction of p21WAF1/CIP1 in both HCT116 WT and HCT116 p53-/- cells (see Fig. 8). However, the p21WAF1/CIP1 induction by AP-2{alpha} was much stronger in HCT116 WT cells in comparison with HCT116 p53-/- cells (Fig. 8), which suggests that p53 may also cooperate with AP-2{alpha} in activating p21WAF1/CIP1 in good correlation with an earlier report (46). On the other hand, the fact that p21WAF1/CIP1 is induced by AP-2{alpha} in HCT116 p53-/- (Fig. 8) suggests that AP-2{alpha} can also activate p21WAF1/CIP1 independent of p53, which is sufficient enough to inhibit cellular DNA synthesis efficiently (Fig. 7b).



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  		FIG. 6.
Effect of p53 on growth inhibition by AP-2{alpha} a, HCT116 WT and HCT116 p53-/- cells were infected with indicated MOI of either Ad-LacZ or Ad-AP2 virus. After 48 h of virus infection, the proportion of live cells was quantified by MTT assay as described under "Materials and Methods." The absorbance of control cells not infected the virus was considered as 100%. b, HCT116 WT and HCT116 p53-/- cells were treated with varying amount of adriamycin alone or adriamycin treatment plus infection with Ad-LacZ or Ad-AP2 at a MOI of 10. After 48 h of virus infection, the proportion of live cells was quantified by MTT assay as described under "Materials and Methods." The absorbance of control cells not infected with the virus was considered as 100%. The IC50 values are calculated and given below.

 



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  		FIG. 7.
FACS analysis of Ad-AP2-infected cells. HCT116 p53 WT (a) or HCT116 p53-/- (b) cells were infected with Ad-LacZ, Ad-p53, or Ad-AP2 at 20 MOI. The cells were allowed to incorporate BrdU for last 4 h of time points at which they were collected. The cells were harvested at 0, 24, and 48 h after infection and subjected to flow cytometry analysis as mentioned under "Materials and Methods." S indicates the cells undergoing DNA synthesis (S phase) and A indicates cells containing less than the 2 N amount of DNA (apoptotic cells). The quantified values are given at the right top corner.

 
Cells with less than 2 N content of DNA, which represent apoptotic cells, appeared in similar proportion by 24 h in Ad-AP2-infected HCT 116 WT (11.4%; Fig. 7a) as well as HCT116 p53-/- (13.5%; Fig. 7b) cells. However, there was a substantial difference in the level of apoptosis induced by AP-2{alpha} in HCT116 WT versus HCT116 p53-/- cells (70 and 53%, respectively; Fig. 7) by 48 h. This result suggests that AP-2{alpha} induces apoptosis both by p53-dependent and -independent pathways. The above result was confirmed by PARP cleavage assay as well. Although the 89-kDa cleaved product of PARP is seen by 24 h in both HCT116 WT as well as HCT116 p53-/- cells, the proportion of cleaved PARP product was found to be 55% in HCT116 WT cells and 12% in HCT116 p53-/- cells by 48 h of virus infection (Fig. 8). A marked difference in the PARP cleavage between these two cell lines suggests that AP-2{alpha} may induce apoptosis by both p53-dependent and -independent mechanisms. Thus, from these experiments, we conclude that although p53 may cooperate with AP-2{alpha} in inducing p21WAF1/CIP1, AP-2{alpha} can also activate p21WAF1/CIP1 independent of p53. The levels of p21WAF1/CIP1 induced by AP-2{alpha} in the absence p53 may be sufficient to inhibit DNA synthesis. In addition, AP-2{alpha} can induce apoptosis both by p53-dependent and -independent mechanisms.

Next we checked the requirement of p53 in the ability of AP-2{alpha} to enhance the chemosensitivity to anticancer drugs. The HCT116 p53-/- cell line was slightly resistant to adriamycin as evidenced from the increase in IC50 of adriamycin (from 0.258 to 0.290 µg/ml; Fig. 6b). Upon Ad-AP2 virus infection, the IC50 of adriamycin is decreased by 2.7 times (from 0.230 to 0.085 µg/ml) in HCT116 WT cells compared with Ad-LacZ infection. However, there was only 1.8-fold decrease (from 0.278 to 0.155 µg/ml) of adriamycin IC50 in HCT116 p53-/- cells, suggesting that p53 may have a role to play in the enhancement of chemosensitivity of cancer cells by AP-2{alpha} (Fig. 6b).

Role of p21WAF1/CIP1 in AP-2{alpha}-mediated Growth Inhibition—To find out the role of p21WAF1/CIP1 in AP-2{alpha}-mediated growth suppression, we studied the ability of Ad-AP2 to inhibit the growth of HCT116 p21-/- cells, a somatic knock-out of both p21WAF1/CIP1 alleles (37). Ad-AP2 inhibited the growth of HCT116 p21-/- cells less efficiently than HCT116 WT cells (Fig. 9a). Approximately, twice the amount of Ad-AP2 virus was required to inhibit the growth of HCT116 p21-/- in comparison with HCT116 WT cells. The inability of Ad-AP2 to inhibit the growth of HCT116 p21-/- cells as efficiently as that of HCT116 WT cells could be attributed to the absence of p21WAF1/CIP1. This result suggests that p21WAF1/CIP1 is required at least partially for AP-2{alpha} to inhibit the growth of cells. Next we checked the ability of AP-2{alpha} to inhibit the cellular DNA synthesis in HCT116 p21-/- cells. Ad-AP2 inhibited cellular DNA synthesis in HCT116 WT cells very efficiently (Fig. 9b). However, Ad-AP2 failed to inhibit the cellular DNA synthesis in HCT116 p21-/- cells (Fig. 9b). These results suggest that p21WAF1/CIP1 is required for AP-2{alpha} to inhibit cellular DNA synthesis, and the growth inhibition by AP-2{alpha} is partially defective in the absence of p21WAF1/CIP1.



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  		FIG. 9.
Effect of p21WAF1/CIP1 on growth inhibition by AP-2{alpha} a, HCT116 WT and HCT116 p21-/- cells were infected with indicated MOI of either Ad-LacZ or Ad-AP2 virus. After 48 h of virus infection, the proportion of live cells was quantified by MTT assay as described under "Materials and Methods." The absorbance of control cells not infected the virus was considered to be 100%. b, HCT116 WT and HCT116 p21-/- cells were infected with Ad-LacZ or Ad-AP2 at a MOI of 20. The cells were allowed to incorporate BrdU for during the last 4 h of the experiment. After 24 h of infection, BrdU-positive cells were identified by staining the cells with anti-BrdU antibody and Texas Red-conjugated anti-mouse secondary antibody. BrdU-positive cells were calculated as the percentage of BrdU incorporation and shown. The percentage of BrdU incorporation was calculated by dividing the number of cells showing BrdU incorporation by total number of cells counted.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have developed a recombinant adenovirus that expresses functional AP-2{alpha}. We show for the first time that overexpression of AP-2{alpha} (from a replication-deficient recombinant adenovirus) results in inhibition of cellular DNA synthesis, leading to arrest of cells in the G1 phase of the cell cycle and also induction of apoptosis. Studies from several laboratories suggest that the expression AP-2{alpha} is inhibitory to cell growth because it has been shown to act as a tumor suppressor in several cancer types. Enforced expression of AP-2 in metastatic melanoma cells inhibited their growth and metastatic potential in nude mice by directly affecting the transcription of MCAM and c-KIT (47, 48). Loss of AP-2 expression was found in tumor specimens in advanced primary and metastatic lesions. In addition, expression of dominant negative AP-2{alpha} in primary cutaneous melanoma augmented its growth in nude mice (31). Progressive loss of AP-2{alpha} has been demonstrated during tumor progression from normal mammary epithelium to invasive breast cancer (26). Similarly, studies involving colon carcinoma have reported a decrease in AP-2{alpha} expression levels in Dukes's stage adenocarcinomas (27).

Even though the above observations suggest that AP-2{alpha} is a negative regulator of cell growth, the mechanism of AP-2{alpha}-mediated growth inhibition is not clearly understood. Particularly, the regulatory roles played by AP-2{alpha} on cell cycle and apoptosis is not known yet. AP-2 acts as a negative regulator of transactivation by Myc (18). The transcriptional activation function of Myc is required for its ability to induce cell cycle progression, cellular transformation, and apoptosis. Transactivation by Myc is under negative control by the transcription factor AP-2. AP-2{alpha} has also been shown to activate p21WAF1/CIP1 leading to inhibition of cellular DNA synthesis and stable colony formation in colon carcinoma cells (4). Consistent with this view, significant correlation between AP-2{alpha} expression and p21WAF1/CIP1 levels have been reported in breast cancer, colorectal carcinoma, and stage I cutaneous malignant melanoma (26, 28, 32). Thus, it is believed that AP-2{alpha} may negatively regulate cell cycle progression by inhibiting c-Myc-mediated transactivation and inducing p21WAF1/CIP1 levels.

Using a transgenic mouse model, Zhang et al. (49) showed that targeted overexpression of AP-2{alpha} in mammary gland results in inhibition of mammary gland growth and morphogenesis. AP-2{alpha} overexpression also resulted in increased expression of parathyroid hormone-related protein, reduced cellular proliferation, and increased cell death. Our study provides evidence suggesting that overexpression of AP-2{alpha} leads to cell cycle arrest. FACS data clearly demonstrate that overexpression of AP-2{alpha} results in the arrest cells in the G1 phase of cell cycle, which is aptly supported by induction of p21WAF1/CIP1 and appearance of hypophosphorylated Rb in AP-2{alpha}-overexpressed cells.

In the transgenic mouse model, overexpression of AP-2{alpha} in the mammary gland specifically results in an increase in the number of cells undergoing apoptosis (49). Our study involving FACS provides direct evidence that adenovirus directed overexpression of AP-2{alpha} results in the induction of apoptosis. Induction of apoptosis by AP-2{alpha} is supported by PARP cleavage assay, which shows that PARP is cleaved by 48 h after Ad-AP2 infection.

Our results also show that Ad-AP2 is a potent growth inhibitor of many cancer cells. It was shown that the tumor suppressor activity of AP-2{alpha} is mediated through a direct interaction with p53 (46). Another report shows that AP-2{alpha} overexpression in mutant p53 containing colon cancer cell line results in induction of p21WAF1/CIP1 and inhibition of DNA synthesis (4). Although our study demonstrates that Ad-AP2 could inhibit growth of different cancer cells regardless of their p53 status, the use of HCT116 p53-/- cell line suggests that p53 may also contribute to AP-2{alpha}-mediated growth suppression. We suggest that the mechanism by which AP-2{alpha} inhibits the growth of cells may involve both p53-dependent and -independent pathways. We also show that cdk inhibitor p21WAF1/CIP1 is required for AP-2{alpha} to inhibit cellular DNA synthesis, and it contributes partially to AP-2{alpha}-mediated growth inhibition.

Conventional chemo- and radiotherapy procedures often fail because tumors develop resistance. Many preclinical and clinical studies suggest that a combination of tumor suppressor gene therapy (e.g. p53 based gene therapy) and chemo- and/or radiotherapy procedures leads to better outcome (50). Our study shows that infection of cells with Ad-AP2 results in many fold increase in the chemosensitivity of cancer cells to commonly used chemotherapeutic drugs adriamycin and cisplatin (Fig. 5g). It appears that AP-2{alpha} expression sensitizes the cancer cells to growth inhibition by chemotherapeutic drugs.

Further work is underway to identify other cell cycle regulatory molecules involved in AP-2{alpha}-induced G1 arrest and also to study the mechanism of apoptosis induction by AP-2{alpha}. Overall, the results of this work suggest that AP-2{alpha} inhibits cancer cell growth by arresting the cells in the G1 phase of cell cycle and inducing apoptosis. The use of Ad-AP2 should be explored as a therapeutic strategy either alone or in combination with chemotherapy.


   FOOTNOTES
 
* This work was supported in part by Program Support to the Indian Institute of Science from the Department of Biotechnology of the Government of India. 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. Back

{ddagger} Supported by a fellowship from University Grants Commission of the Government of India. Back

§ To whom correspondence should be addressed. Tel.: 91-80-2932973; Fax: 91-80-3602697; E-mail: skumar{at}mcbl.iisc.ernet.in.

1 The abbreviations used are: AP-2, activator protein 2; Ad-AP2, adenovirus expressing functional AP-2{alpha}; CAT, chloramphenicol acetyltransferase; PARP, poly(ADP-ribose) polymerase; BrdU, bromodeoxyuridine; MOI, multiplicity of infection; PBS, phosphate-buffered saline; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; FACS, fluorescence-activated cell sorting; WT, wild type. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Omana Joy for assistance in FACS analyses. The FACScan facility funded by the Department of Biotechnology of the Government of India is acknowledged.



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