Endogenous Control of Cell Cycle Progression by Autocrine Transforming Growth Factor β in Breast Cancer Cells

Cell Culture.

The breast cancer cell lines were obtained from American Type Culture Collection. The BT20 strain in our laboratory has a constitutively active mutated estrogen receptor. Hence, we refer to it as ER⁺. Cells were grown in McCoy’s 5A medium supplemented with 10% fetal bovine serum (Sigma, St. Louis, MO), amino acids, antibiotics, pyruvate, and vitamins (Life Technologies, Inc., Gaithersburg, MD), and were maintained at 37°C in a humidified atmosphere of 5% CO₂. The tetracycline regulatable RII cDNA expression vector was stably transfected along with a Neomycin vector into BT20 and ZR75 limiting dilution clones as described previously (12) . The control Neo clones are referred to as BT20 Neo and ZR75 Neo, whereas RII expressing clones are referred to as BT20 SRII 15 and ZR75 SRII 7.

RNA Analysis.

Total RNA from the breast cancer cells was extracted by guanidine thiocyanate homogenization and ultracentrifugation through a cesium gradient as described previously (16) . RII, fibronectin (FN), and actin probes were described previously (12) . RNase protection assays were performed as described previously (16) . Briefly, radioactive riboprobes were allowed to hybridize overnight with the RII and FN mRNA in 40 μg of total RNA. After RNase A and T1 treatment, the protected double-stranded RNA fragments were analyzed by urea-PAGE and visualized by autoradiography. Actin was used to normalize sample loading.

Receptor Cross-Linking.

Simian TGF-β1 was purified as described (16) and iodinated by the chloramine T method (16) . Cells were seeded at a density of 6 × 10⁴/well in a six-well plate. After the cells reached ∼80% confluency, receptor binding studies were carried out using 200 pm of ¹²⁵I TGF-β1 as described previously (16) . Labeled cells were solubilized in 200 μl of 1% Triton X-100 with 1 mm phenylmethylsulfonylfluoride. Equal amounts of cell lysate protein were separated by 4–10% SDS-PAGE under reducing conditions and exposed for autoradiography.

DNA Synthesis Activity.

Cells were seeded at a density of 5 × 10⁴/well in a six-well plate, and DNA synthetic activity was measured every 24 h for 7 days using [³H]thymidine as described previously (12) .

TGF-β Autocrine Activity.

One × 10³ cells/well were plated in a 96-well plate on day 0, and either control IgG or TGF-β neutralizing antibody (10 μg/ml; R&D Systems) was added on days 3 and 4. DNA synthetic activity was measured using [³H]thymidine as described (12) .

Luciferase Assay.

The TGF-β responsive promoter-reporter construct (p3TP-Lux) was used for transient transfections, and luciferase assays were performed as described previously (16) . BT20 Neo, ZR75 Neo, and their RII transfectants were transfected with 30 μg of p3TP-Lux and 10 μg of β-galactosidase plasmid by electroporation with a Bio-Rad gene pulser at 250 V and 960 μF. The electroporated cells were plated into six-well tissue culture plates. After the attachment of cells, control IgG (10 μg/ml) or TGF-β neutralizing antibody (10 μg/ml) was added. Cells were harvested with 200 μl of lysis buffer (Luciferase assay system; Promega). Luciferase activity was measured in the first 10 s after substrate addition using a luminometer (Berthold Lumat LB 9501) and expressed as relative units after normalized with β-galactosidase activity.

Flow Cytometry.

Cells were seeded at a density of 5 × 10⁴/well in a six-well plate either in the presence or absence of tetracycline (0.1 μg/ml) on day 0 and harvested on days 3–5. Cells were trypsinized, washed, and stained for DNA by resuspending them in stain solution I [50 μg/ml propidium iodide (Sigma), 3% polyethylene glycol, 0.1% Triton X-100, and 4 mm sodium citrate] with 40 μg/ml of RNase A, followed by incubation for 30 min at 37°C. Equal volumes of stain solution II (50 μg/ml propidium iodide, 3% polyethylene glycol, 0.1% Triton X-100, and 400 mm sodium chloride) was added to the samples and stored at 4°C for at least 1 h before being analyzed on a FACScan flow cytometer (Becton Dickinson). Cell cycle compartments were analyzed using a Modfit LT program (Verity Software House Inc.).

Histone H1 Kinase Assay.

Cells were lysed in NP40 lysis buffer [50 mm Tris-HCl (pH 7.4), 150 mm NaF, 1 mm NaVo3, 1 mm phenylmethylsulfonylfluoride, 1 mm DTT, 25 μg/ml aprotinin, 25 μg/ml trypsin inhibitor, and 25 μg/ml leupeptin] at 4°C. The supernatants were cleared by centrifugation. Fresh total cell lysates (100 μg) were immunoprecipitated for 2 h at 4°C with rabbit polyclonal antibody against CDK2 (M2; Santa Cruz Biotechnology), followed by incubation with immobilized protein-A agarose beads (Life Technologies, Inc.) for another 2 h at 4°C with rotation. The beads were then washed three times with kinase buffer [20 mm Tris-HCl (pH 7.5) and 4 mm MgCl₂]. Phosphorylation of Histone H1 was measured by incubating the beads with 10 μl of reaction mixture containing 10 μCi [³²P]-χ-ATP (3000Ci/mm; NEN) and 2.4 μg Histone H1 (Sigma) in kinase buffer for 30 min at 37°C. The reaction was stopped by placing the samples on ice. The samples were then boiled in 2× sample buffer [100 mm Tris-HCl (pH 6.8), 2% SDS, 20% glycerol, 0.04% bromphenol blue, and 4% β-mercaptoethanol] for 5 min, and resolved by 10% SDS-PAGE. The gel was dried and subjected to autoradiography.

CDK4 Kinase Assay.

Total cell lysates were immunoprecipitated for 2 h at 4°C with rabbit polyclonal antibody against CDK4 (C-22; Santa Cruz Biotechnology), followed by incubation with immobilized protein-A agarose beads (Life Technologies, Inc.) for another 2 h at 4°C with rotation. After washing the beads, phosphorylation of the glutathione S-transferase-retinoblastoma substrate was measured by incubating the beads with 10 μl of reaction containing 10 μCi [³²P]χ-ATP and analyzed by Western blot as described above for the Histone H1 kinase assay.

Immunological Analysis.

Cells were lysed in NP40 lysis buffer. The supernatants were cleared by centrifugation. Equal amounts of cell lysates were boiled in 2× sample buffer, resolved by 12% SDS-PAGE, and transferred to nitrocellulose membranes (Amersham) for Western blot analysis. The blots were probed with various primary antibodies at a concentration of 1 μg/ml [anti-p21/waf1/cip1, anti-CDK2, anti-cyclin A, anti-p27/kip (C-19; Santa Cruz Biotechnology), and anti-cyclin E (HE12l Santa Cruz Biotechnology)], followed by incubation with 0.2 μg/ml horseradish peroxidase-conjugated antirabbit IgG or antimouse IgG (Santa Cruz Biotechnology). The proteins were then detected by the enhanced chemiluminescence system (Amersham). Wherever indicated, the cells were grown in the presence of 0.1 μg/ml of tetracycline, harvested, and analyzed for p21/waf1/cip1 and p27/kip expression.

Immunoprecipitation followed by Western blot analysis was performed to detect protein levels in the complex. Equal amounts of total cell lysates were immunoprecipitated overnight at 4°C with anti-CDK2 (M2-G; Santa Cruz Biotechnology) and then incubated with immobilized protein G agarose (Life Technologies, Inc.) for 1 h. The beads were washed three times with lysis buffer and boiled in 2× sample buffer for 5 min. The eluted samples were subjected to 12% SDS-PAGE followed by Western blot analysis.

Small Interfering RNA (siRNA) Transient Transfection and DNA Synthesis Activity.

SRII 7 cells at 60% confluency were transiently transfected with 25 nm p21 and p27 Si RNA (Cellogenetics, Inc) using GeneEraser reagent (Stratagene). Seventy-two h post-transfection, Western analysis using p21, p27, and actin antibodies, and DNA synthesis activity using [³H]thymidine was performed.

Soft Agarose Assay.

Soft agarose assays were performed as described previously (12) to compare clonogenic potential of control and RII-transfected cells in semi-solid medium. Briefly, cells were suspended (3 × 10³ cells/well) in 1 ml of 0.4% Sea plaque agarose (Sigma) in McCoy’s 5A medium containing 10% fetal bovine serum and plated on top of 1 ml of 0.8% agarose in the same medium in triplicate in six-well tissue culture plates. Plates were incubated for 2–3 weeks at 37°C with 5% CO₂ in a humidified incubator. Cell colonies were visualized by staining with 0.5 ml of p-iodonitrophenyl tetrazolium violet staining (Sigma).

Expression of RII in ER⁺ BT20 and ZR75 Cells.

We transfected a tetracycline-regulatable RII cDNA expression plasmid (12) and a control Neo vector into BT20 Cl.9 and ZR75 Cl.16, typical limiting dilution clones of the cell lines BT20 and ZR75. The transfectants were initially screened for increased expression of RII mRNA by RNase protection assay and one high RII expressing positive clone each of BT20 (SRII 15) and ZR75 (SRII 7) was selected for biological characterization (Fig. 1A) ⇓ .

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Fig. 1.

Transforming growth factor-β receptor type II (RII) mRNA, and cell-surface receptor expression in BT20 and ZR75 transfectants. A, typical limiting dilution clones of BT20 and ZR75 were stably transfected with a tetracycline regulatable RII expression vector and selected in geneticin. The stable clones (designated SRII 15 and SRII 7) were compared with the Neo transfection controls for RII mRNA levels by RNase protection assay. Transfected RII mRNA levels are shown. Actin mRNA levels was used for normalization of sample loading. B, receptor cross-linking assays were used to verify cell surface expression of RII. Confluent monolayer cultures of BT20 NEO, ZR75 Neo, and their RII transfectants were incubated with 200 pm ¹²⁵I transforming growth factor-β1, cross-linked with 0.3 mm disulfosuccinimidyl suberate and separated by 4–10% gradient SDS-PAGE under reducing conditions. Wherever indicated, the cells were treated with 0.1 μg/ml of tetracycline to repress the RII expression and thereby demonstrated the specificity of binding.

Because RI and RII are interdependent for TGF-β binding and signaling, we carried out receptor binding studies with ¹²⁵I TGF-β to determine whether RII expression permitted TGF-β binding to RI. The data indicate that expression of RII resulted in the cell surface binding of TGF-β to RII as well as RI (Fig. 1B) ⇓ . Increased binding for RIII was also noted. This phenomenon has also been observed after RII transfection in our previous studies (12 , 13) . Binding specificity was demonstrated by adding 0.1 μg/ml of tetracycline, which led to the repression of RII expression and the resultant loss of ¹²⁵I TGF-β binding to the receptors.

Restoration of Autocrine-Negative TGF-β Activity.

DNA synthesis activity for RII clones (BT20 SRII 15 and ZR75 SRII 7) and their Neo counterparts were analyzed to determine whether RII expression led to alteration of growth parameters in tissue culture (Fig. 2, A and B) ⇓ . Compared with their Neo counterparts, RII transfectants exhibited a decrease in the DNA synthesis. To determine whether the decrease in DNA synthesis of the RII transfectants is due to reconstitution of autocrine-negative TGF-β activity, the RII and Neo clones were treated with 10 μg/ml of either TGF-β neutralizing antibody or control IgG (Fig. 2, C and D) ⇓ . RII transfectants displayed 60–65% stimulation in DNA synthesis confirming that RII replacement restored growth control through reactivation of autocrine-negative TGF-β activity.

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Fig. 2.

DNA synthesis activity and the effect of transforming growth factor β1 neutralizing antibody on DNA synthesis in BT20, ZR75 Neo, and receptor type II transfectants. A and B, cells were plated at 5 × 10⁴ cells/well in a six-well plate and DNA synthetic activity was measured using [³H]thymidine every 24 h for 7 days and plotted as cpm/cells. C and D, BT20, ZR75 Neo, and receptor type II transfectants were plated at a density of 1 × 10³ cells/well in a 96-well plate and were treated with 10 μg/ml of control IgG or transforming growth factor β neutralizing antibody on days 3 and 4. DNA synthetic activity was measured using [³H]thymidine; bars, ±SD.

To confirm the restoration of autocrine TGF-β activity after RII replacement, we compared the activity of a TGF-β-responsive promoter in control cells with that in RII transfectants. The p3TP-Lux promoter contains the TGF-β response elements from the plasminogen activator gene inserted upstream of the luciferase reporter gene and has been extensively used as a marker for TGF-β responsiveness (2) . Therefore, it would be expected that autocrine TGF-β activity would result in enhanced steady state expression of the p3TP-Lux construct in RII transfectants relative to Neo control cells. Thus, the TGF-β-responsive promoter-luciferase construct was transiently transfected into Neo control and RII transfectants. The activity of the promoter is reflected by luciferase activity. Fig. 3A ⇓ shows that BT20 as well as ZR75 RII transfectants expressed significantly higher levels of luciferase activity than Neo cells. If increased luciferase activity of p3TP-Lux construct was due to autocrine TGF-β activity, TGF-β neutralizing antibody treatment would reduce expression of the reporter construct. As shown in Fig. 3 ⇓ A, TGF-β neutralizing antibody treatment resulted in a substantial decrease in luciferase reporter activity in RII transfectants, whereas it had no effect on Neo control cells.

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Fig. 3.

A, effect of transforming growth factor-β (TGF-β) 1 neutralizing antibody on the transcription of a TGF-β responsive promoter. BT20, ZR75 Neo, and their receptor type II transfectants were transiently transfected with 30 μg of p3TP-Lux and 10 μg of β-galactosidase plasmid. Control IgG and TGF-β1 neutralizing antibody were added at 10 μg/ml concentrations after the cells were attached to the plates. Cell extracts were prepared 48 h later. Luciferase activity was measured for the first 10 s after substrate addition in a luminometer. Each value is the mean of triplicate samples. B, fibronectin mRNA levels in BT20, ZR75 Neo, and receptor type II transfectants. Total RNA from control or tetracycline treated cells was isolated and hybridized with FN antisense probe (described in “Materials and Methods”). Actin mRNA levels was used to normalize loading of the samples; bars, ±SD.

Extracellular matrix protein induction is another parameter of TGF-β activity. Previous work from our laboratory showed that autocrine TGF-β controlled steady state levels of extracellular matrix protein, Fibronectin (13) . Consequently, induction of autocrine TGF-β should be associated with increased extracellular matrix production. Therefore, we determined FN mRNA levels in the RII transfectants. As shown in Fig. 3B ⇓ , RII transfected cells showed a 4–5-fold increase in FN mRNA levels compared with Neo control cells. Tetracycline treatment reversed the autocrine TGF-β-mediated enhanced FN mRNA expression in the ZR75 RII-transfected cells.

The data presented above demonstrated that RII replacement reconstituted autocrine-negative TGF-β activity leading to the restoration of growth control in the BT20 and ZR75 breast cancer cells. Consequently, additional studies were carried out using the ZR75 Neo and RII transfectant (SRII 7) to analyze the mechanism by which autocrine TGF-β modulates the cell cycle machinery.

Flow Cytometry.

Flow cytometry was performed to determine whether autocrine-negative TGF-β activity is delaying the RII transfected cells from entering S phase of the cell cycle as quickly as Neo cells. The analysis revealed there was a lower percentage of S phase and a higher percentage of G₀-G₁ phase cells in RII transfectants than in Neo cells (Table 1) ⇓ . Tetracycline treatment reversed the autocrine TGF-β effects on the cell cycle distribution in the RII-transfected cells.

Autocrine TGF-β Effect on CDK4 Kinase Activity.

One of the consequences of CDK4 activation is the phosphorylation of its substrate, retinoblastoma protein, which determines whether cell cycle progression will continue past the early G₁ phase of the cell cycle. We compared the CDK4 protein levels and its kinase activities by using glutathione S-transferase-retinoblastoma as a substrate in Neo and RII transfectants (Fig. 4A) ⇓ . The CDK4-associated kinase activities were found to be similar between Neo and RII transfectants indicating that autocrine TGF-β did not affect CDK4 kinase activity.

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Fig. 4.

A, comparison of cyclin-dependent kinase (CDK) 4 protein levels and kinase activity in ZR75 Neo and SRII 7 cells. Cells were seeded at 5 × 10⁴ cells/well in six-well plates and harvested on days 3 and 4. Fifty μg of total cell lysates were used for CDK4 Western and CDK4 kinase assay. C, CDK2 protein levels and kinase activity in ZR75 Neo and SRII 7. Cells were plated at 5 × 10⁴ cells/well in six-well plates, harvested in NP40 lysis buffer on days 3–5. Western analysis using antibody against CDK2 and CDK2 kinase activities were measured by immunoprecipitation of 100 μg of cell lysates with antibody against human CDK2, followed by histone H1 kinase assay as described in “Materials and Methods.” C, comparison of expression of cyclin A, cyclin E, p21/waf1/cip1 and p27/kip in ZR75 Neo and SRII 7 cells. Cells were plated and harvested as described in the legend to B. Fifty μg of total cell lysates were analyzed by Western blots using antibodies against cyclin A, cyclin E, p21/waf1/cip1, and p27/kip. D, suppression of CDK inhibitors, p21/waf1/cip1, and p27/kip in the RII transfected ZR75 cells (SRII 7). ZR75 SRII 7 cells were grown in the absence or presence of 0.1 μg/ml of tetracycline, harvested on days 3 and 4, and Western blot analysis using antibodies against p21/waf1/cip1, p27/kip, and actin was performed. E, association between CDK2 and CDK inhibitors in ZR75 Neo and SRII 7 cells. One hundred μg of cell lysates were subjected to the immunoprecipitation with goat anti-CDK2, followed by Western blot analysis with p21/waf1/cip1 and p27/kip antibodies.

Autocrine TGF-β Effect on CDK2 Kinase Activity.

CDK2-associated kinase activity has been shown to be a limiting factor for progression from G₁ into S phase (6) . Consequently, we compared the CDK2 kinase activity in Neo and RII transfectants. Histone H1 phosphorylation assays revealed that RII transfectants had markedly reduced CDK2-associated kinase activity relative to Neo cells (Fig. 4B) ⇓ . This indicated that the delayed entry into S phase of RII transfectants was associated with reduced CDK2 kinase activity. Western blot analysis indicated that the suppression of CDK2 kinase activity in RII transfectants was not due to reductions of CDK2 (Fig. 4B) ⇓ , cyclin A, and cyclin E protein levels (Fig. 4C) ⇓ .

Autocrine TGF-β Induces CDK Inhibitors p21/waf1/cip1 and p27/kip.

We examined the possible roles of CDK2 inhibitors, p21/waf1/cip1 and p27/kip in the suppression of CDK2 kinase activity in RII transfectants by Western blots. The RII transfectants showed significant induction of p21/waf1/cip1 as well as p27/kip, whereas the expression of these CDK inhibitors was not detected in the Neo cells (Fig. 4C) ⇓ . To confirm the induction of CDK inhibitors by autocrine TGF-β, RII transfectants were treated with 0.1 μg/ml of tetracycline to repress RII expression and, thus, abrogate autocrine TGF-β activity. The cells were then analyzed for p21/waf1/cip1 and p27/kip proteins by Western blots. As shown in Fig. 4D ⇓ , abrogation of autocrine TGF-β activity resulted in the loss of expression of CDK inhibitors p21/waf1/cip1 and p27/kip in the RII transfectants. However, there was no difference in the actin levels, indicating the selectivity of p21/waf1/cip1 and p27/kip modulation.

Western blot analysis after immunoprecipitation with CDK2 antibody indicated that CDK2 forms complexes with the CDK inhibitors p21/waf1/cip1 and p27/kip in the RII transfectants. No such complexes were detected in the Neo cells (Fig. 4E) ⇓ . These results suggested that the induction as well as association of CDK inhibitors p21/waf1/cip1 and p27/kip with CDK2 is responsible for the reduced CDK2 kinase activity in RII transfectants.

p21 and p27 Si RNA Abrogates Autocrine TGF-β effects.

To additionally confirm that autocrine TGF-β-mediated p21 and p27 induction in the RII transfected cells was contributing to the restoration of growth control, we have knocked down the endogenous p21 and p27 in the RII transfected cells using p21 and p27 Si RNA molecules, and analyzed the DNA synthesis activity in these cells (Fig. 5) ⇓ . p21 and p27 Si RNA expression eliminated the inhibitory effects of RII restoration in the ZR75 cells, thus confirming the role of these inhibitors in the autocrine TGF-β-mediated growth control.

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Fig. 5.

p21/p27 small interfering (Si) RNA effects on DNA synthesis in SRII 7 cells. p21 and p27 Si RNA was transiently expressed in receptor type II-transfected cells (SRII 7). A, Western analysis using p21, p27, and actin antibodies in ZR75 Neo, SRII 7, and SRII 7 Si RNA-transfected cells. B, SRII 7 cells at 60% confluency were transiently transfected with p21/p27 Si RNA and DNA synthesis activity using [³H]thymidine was measured 72 h post-transfection as described in “Materials and Methods;” bars, ±SD.

Anchorage-Independent Growth.

The ability to form colonies in soft agarose is reflective of malignant transformation. Therefore, to assess the effect of the restoration of autocrine TGF-β activity on the malignant properties of BT20 and ZR75 RII transfectants, we compared the ability of the Neo and RII-transfected clones to form colonies in soft agarose. As shown in Fig. 6 ⇓ , BT20 RII transfectant showed 50% and ZR75 RII transfectant showed 60% lower cloning efficiencies in semisolid medium compared with their Neo counterparts. The number of colonies were counted and represented in a graph format (Fig. 6E) ⇓ .

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Fig. 6.

Anchorage-independent growth of BT20, ZR75 Neo, and receptor type II transfectants. Exponentially growing cells (3 × 10³) were suspended in 1 ml of 0.4% sea plaque agarose in McCoy’s 5A serum-free medium and plated on top of a 1 ml underlayer of 0.8% agarose in the same medium in a six-well plate. Cell colonies were visualized by staining with 0.5 ml of p-iodonitrotetrazolium violet after 2 weeks of incubation. A, BT 20 Neo; B, SRII 15; C, ZR 75 Neo; and D, SRII 7. E, the number of colonies were counted and represented in a graph format; bars, ±SD.