AP-2gamma induces p21 expression, arrests cell cycle, and inhibits the tumor growth of human carcinoma cells

Abstract

Activating enhancer-binding protein 2gamma (AP-2gamma) is a member of the developmentally regulated AP-2 transcription factor family that regulates the expression of many downstream genes. Whereas the effects of AP-2alpha overexpression on cell growth are fairly well established, the cellular effects of AP-2gamma overexpression are less well studied. Our new findings show that AP-2gamma significantly upregulates p21 mRNA and proteins, inhibits cell growth, and decreases clonogenic survival. Cell cycle analysis revealed that forced AP-2gamma expression induced G1-phase arrest, decreased DNA synthesis, and decreased the fraction of cells in S phase. AP-2gamma expression also led to cyclin D1 repression, decreased Rb phosphorylation, and decreased E2F activity in breast carcinoma cells. AP-2gamma binding to the p21 promoter was observed in vivo, and the absence of growth inhibition in response to AP-2gamma expression in p21(-/-) cells demonstrated that p21 caused, at least in part, AP-2-induced cell cycle arrest. Finally, the tumor growth of human breast carcinoma cells in vivo was inhibited by the expression of AP-2gamma relative to empty vector-infected cells, suggesting that AP-2gamma acts as a tumor suppressor. In summary, expression of either AP-2gamma or AP-2alpha inhibited breast carcinoma cell growth; thus, these genes may be therapeutic targets for breast cancer.

Figure 1

Ad-AP-2α and Ad-AP-2γ adenovirus-infected MDA MB-231 cells showed increased AP-2 DNA-binding activity and decreased clonogenic survival. (A) MDA MB-231 cells were infected with increasing MOI of AP-2 adenoviruses, as indicated by ramps. Nuclear proteins were isolated 24 hours after infection. EMSAs were performed on nuclear extracts of both MDA MB-231 cells to establish AP-2 DNA-binding activity to an AP-2 consensus-binding site in vitro. Radiolabeled AP-2 consensus-binding sites were incubated with nuclear extracts under the following different conditions: lane 1: probe only; lane 2: untreated MDA MB-231 nuclear extract; lane 3: vector control Ad-Bgl II-treated cell extract; lanes 4 to 7: extracts from Ad-AP-2α-treated cells with increasing MOI from 50, 100, 200, and 200; lanes 8 to 11: extracts from Ad-AP-2γ-treated cells with increasing MOI from 50, 100, 200, and 200. To evaluate the specificity of this interaction, antibodies specific to AP-2α (lane 7) and AP-2γ (lane 11) were incubated with nuclear extracts from 200 MOI of Ad-AP-2-infected MDA MB-231 cells. (B) MDA MB-231 cells (2 x 10⁶) were plated in 100-mm dishes and infected with 100 MOI of Ad-Bgl II or Ad-AP-2. After 24-hour infection, cells were trypsinized and subcultured into 60-mm dishes at 500 cells/dish to determine clonogenic fractions. Surviving fractions were normalized to untreated cells. *P < .05 compared to untreated control cells and Ad-Bgl II-treated cells.

Figure 2

Not only AP-2αbut also AP-2γ overexpression arrested cell cycle and elevated p21 protein and mRNA levels in MDA MB-231 cells. (A) MDA MB-231 cells were plated and infected with 100 MOI of various adenoviruses, as indicated. After 24-hour incubation, cells were pulsed with BrdU and labeled with BrdU antibody, and flow cytometry was performed. The fractions of the cell population in different phases of the cell cycle were analyzed using CellQuest Software. (B) MDA MB-231 cells were infected with various MOI adenoviruses. Nuclear proteins and total RNA were isolated 24 hours after infection. Western blot analyses were carried out to determine p21 protein levels. β-Actin was used as the control for loading and transfer. (C and D) MDA MB-231 cells were treated as described in (B). Total mRNA was isolated 24 hours after infection. Quantitative RT-PCR was used to determine steady-state p21 mRNA levels. Results are expressed as mean ± SEM (n = 3). *P < .05 compared to untreated and vector control-treated mRNA samples. ^†P < .05 compared to vector control-treated cells.

Figure 3

Forced AP-2 expression significantly decreased Rb phosphorylation, cyclin D1 mRNA and protein levels, and E2F responsive promoter activity. (A) Cells were infected with vector control or Ad-AP-2, as indicated. Total mRNA was harvested 24 hours after infection. Quantitative RT-PCR was used to determine cyclin D1 mRNA level in MDA MB-231 cells. Results are expressed as mean ± SEM (n = 3). * P < .05 compared to untreated and vector control-treated mRNA sample. (B) Cells were infected with 100 MOI of vector control or Ad-AP-2. Total cell lysates were harvested 24 hours after infection. Western blot analyses were conducted to determine cyclin D1 protein levels in both MDA MB-231 and HCT116 wt cells. β-Actin was used as a control for loading and transfer. (C) Cells were treated as described above (B). Western blot analyses were conducted to determine pRb (S780) protein level in MDA MB-231 cells. (D) MDA MB-231 cells were infected with Ad-Bgl II, Ad-AP-2α, or Ad-AP-2γ at 100 MOI and transfected with 5 µg of 4x E2F firefly luciferase reporter and 1 µg of Cytomegalovirus renilla luciferase reporter, as described in the Materials and Methods section. Total proteins from adherent cells were collected, and firefly luciferase activities were measured 24 hours after infection. *P < .05 compared with control and vector control-treated cells.

Figure 4

Overexpression of AP-2α or AP-2γ failed to induce p21 protein G₁ arrest and to inhibit BrdU incorporation in HCT116 p21 (-/-) cells. HCT116 parent and p21 (-/-) cells were infected with 100 MOI of vector control or Ad-AP-2 for 24 hours. (A) Total proteins were isolated, and Western blot analysis was carried out to detect p21 protein level. β-Actin was used as control for loading and transfer. (B) Cells were pulsed with BrdU and labeled with BrdU antibody. PI staining was used to detect total DNA content. Flow cytometry was carried out. Quantitative BrdU FITC level was calculated for every cell group. *P < .05 compared to untreated control cells and vector control-treated cells. Fractions of cells are shown as histograms of HCT116 wt cells (C) and HCT116 p21 (-/-) cells (D). *P < .05 compared to untreated control cells. ^†P < .05 compared to vector control-treated cells.

Figure 5

Both AP-2α and AP-2γ bind to p21 proximal promoter in vivo. MDA MB-231 cells were infected with vector control, Ad-AP-2α, or Ad-AP-2γ at 100 MOI. Twenty-four hours after infection, AP-2α and AP-2γ antibodies were used to immunoprecipitate protein-bound DNA. An equivalent amount of genomic DNA without immunoprecipitation was used as input control. Standard PCR was carried out to determine the extent of AP-2 binding to the p21 proximal promoter region illustrated in Zeng et al. [33].

Figure 6

AP-2-expressing MDA MB-231 cells showed less tumorigenicity, and adenoviral AP-2 infection significantly delayed established tumor growth in vivo. (A) MDA MB-231 cells were infected with 100 MOI of vector control or Ad-AP-2 in vitro for 24 hours before injection. Under sterile conditions, 7- to 8-week-old nude mice were injected subcutaneously (day 0). Tumor sizes were measured and calculated according to the description in the Materials and Methods section. For statistical analysis, time of tumor initiation was defined as the first day on which tumor volume exceeded 10 mm³. (B) Under sterile conditions, 7- to 8-week-old nude mice were injected subcutaneously with MDA MB-231 cells. When the average diameter reached 3 to 4 mm, animals were grouped to achieve the same average tumor volume per group. Then 1 x 10⁹ pfu of indicated adenoviruses was injected into each tumor (day 0). Adenoviral injections (a total of three injections) were performed every 5 days. Animals were sacrificed when any tumor reached 1000 mm³ in either flank. *P < .05 compared to untreated and Ad-Bgl II-treated groups.

Figure 7

A proposed model for cell growth inhibition by AP-2α and AP-2γ. In the presence of overexpressed wt AP-2α or AP-2γ, p21 mRNA and proteins are present at high levels, whereas cyclin D1 expression is inhibited. The consequent pRb hypophosporylation leads to E2F inactivation, which facilitates cell cycle arrest. When AP-2α or AP-2γ expression is lost, such as during tumor formation, p21 expression is also lost and cyclin D1 becomes more actively expressed. The subsequent phosphorylation of pRb and activation of E2F promote cell proliferation and tumor formation.