Cancer Research Audrey Hepburn Protein Translation and Cancer
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ammanamanchi, S.
Right arrow Articles by Brattain, M. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ammanamanchi, S.
Right arrow Articles by Brattain, M. G.
[Cancer Research 64, 2509-2515, April 1, 2004]
© 2004 American Association for Cancer Research

Regular Articles

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

Sudhakar Ammanamanchi1, Manoranjani P. M. Tillekeratne2, Tien C. Ko3 and Michael G. Brattain1

1 Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York; 2 Department of Pharmacology, Medical College of Ohio, Toledo, Ohio; and 3 Department of Surgery, The University of Texas Medical Branch, Galveston, Texas


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tumor progression due to loss of autocrine negative transforming growth factor-ß (TGF-ß) activity was reported in various cancers of epithelial origin. Estrogen receptor expressing (ER+) breast cancer cells are refractory to TGF-ß effects and exhibit malignant behavior due to loss or inadequate expression of TGF-ß receptor type II (RII). The exogenous TGF-ß effects on the modulation of cell cycle machinery were analyzed previously. However, very little is known regarding the endogenous control of cell cycle progression by autocrine TGF-ß. In this study, we have used a tetracycline regulatable RII cDNA expression vector to demonstrate that RII replacement reconstitutes autocrine negative TGF-ß activity in ER+ breast cancer cells as evidenced by the delayed entry into S phase by the RII transfectants. Reversal of the delayed entry into S phase by the RII transfectants in the presence of tetracycline in addition to the decreased steady state transcription from a promoter containing the TGF-ß responsive element (p3TP-Lux) by TGF-ß neutralizing antibody treatment of the RII transfected cells confirmed that autocrine-negative TGF-ß activity was induced in the transfectants. Histone H1 kinase assays indicated that the delayed entry of RII transfectants into phase was associated with markedly reduced cyclin-dependent kinase (CDK)2 kinase activity. This reduction in kinase activity was due to the induction of CDK inhibitors p21/waf1/cip1 and p27/kip, and their association with CDK2. Tetracycline treatment of RII transfectants led to the suppression of p21/waf1/cip1and p27/kip expression, thus, directly demonstrating induction of CDK inhibitors by autocrine TGF-ß leading to growth control of ER+ breast cancer cells.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Transforming growth factor-ßs (TGF-ß) belong to a large group of closely related multifunctional polypeptides, which regulate many cellular processes through cell surface receptors. These receptors are referred to as type I (RI), type II (RII), and type III (RIII). RI and RII are serine/threonine kinases, and an active receptor complex consists of two molecules each of RI and RII (1 , 2) . RIII, a proteoglycan, contains a short cytoplasmic tail with no signaling motif (1) . After TGF-ß binding and subsequent activation of RI by RII, RI phosphorylates smad2 and/or smad 3, which associate with smad 4 and translocate to nucleus where binding to the target DNA or other DNA-binding protein occurs. Smad 7 was reported to antagonize the TGF-ß signaling pathway by binding to RI and consequently preventing the activation of smad 2 and smad 3 (1) .

One of the biological effects of TGF-ß is to inhibit the epithelial cell proliferation by inducing cell cycle arrest (3, 4, 5) . Cell cycle progression involves sequential assembly, activation, and subsequent inactivation of a series of serine/threonine protein kinases that consist of a catalytic cyclin-dependent kinase (CDK) subunit and a regulatory cyclin subunit (6) . G1-S cyclins and their CDK partners regulate G0 to S progression whereas D-type cyclins (D1, D2, and D3) oversee the progression through the G1 restriction point. Cyclin E as well as cyclin A control S phase entry and progression (7, 8, 9) . The phosphorylation and dephosphorylation status of CDK kinase subunit and programmed degradation of the cyclin regulatory subunits modulate cyclin-CDK kinase activity (6) . It has also been shown that CDK inhibitory proteins bind to cyclin-CDK complexes and inhibit their activities (10) . CDK inhibitors p14/p15, p16, p18, and p19 specifically bind to cyclinD-CDK4/6 complexes and inhibit their activities. However, p21/waf1/cip1, p27/kip, and p57/kip have been shown to be potent inhibitors of a variety of cyclin-CDK kinases (10) .

TGF-ß resistance due to loss of either RI or RII has been reported in various cell types (11, 12, 13, 14, 15) . Estrogen receptor expressing (ER+) breast cancer cells are refractory to TGF-ß effects due to the absence or inadequate expression of RII (16) . RII expression restored response to exogenous TGF-ß leading to reduction in the malignancy of ER+ MCF-7L cells (12) . Exogenous TGF-ß has been shown to induce the cell cycle arrest either by down-regulating the levels/activities of G1-S cyclins, CDKs (17, 18, 19, 20, 21) , or by stimulating the expression of CDK inhibitory proteins p15, p21/waf1/cip1 (10 , 22) , and p27/kip (10 , 23) . Smad protein family was shown to cooperate with Sp1 in the TGF-ß-induced p21/WAF1/CIP1 expression in hepatic cells (24) . The involvement of the mitogen-activated protein/extracellular signal-regulated kinase pathway has been reported in the TGF-ß-mediated p21/WAF1/CIP1 induction in HaCaT cells (25) . Autocrine TGF-ß activity has been reported to induce radiation-mediated p21/WAF1/CIP1 expression in the p53-mutant pancreatic cancer cells (26) . A post-translational mechanism leading to p21/WAF1/CIP1 stabilization caused TGF-ß-mediated cell cycle arrest in human colon carcinoma cells (27) . Previous work from our laboratory demonstrated that in colon carcinoma cells suppression of autocrine TGF-ß activity by constitutively repressing endogenous TGF-ß expression led to a more progressed phenotype (28 , 29) . However, TGF-ß antisense transfected cells retained their functional receptor complexes and, hence, the ability to respond to exogenous TGF-ß. These results indicated that autocrine TGF-ß, rather than response to exogenous TGF-ß, is an important deterrent to malignant progression. Although there are reports analyzing the effects of exogenous TGF-ß on the modulation of cell cycle machinery, very little is known regarding the endogenous control of cell cycle progression by autocrine TGF-ß. Hence, identifying the mechanism(s) by which autocrine TGF-ß functions is important for our understanding of the carcinogenic process and malignant progression.

We have used a tetracycline regulatable RII expression vector to demonstrate that RII replacement reconstitutes autocrine negative TGF-ß activity in the ER+ BT20, ZR75 breast cancer cells. In addition, our data show that autocrine TGF-ß induces CDK inhibitors p21/waf1/cip1 and p27/kip in the RII transfectants, which associate with CDK2 and contribute to the reduction in the CDK2 kinase activity. However, autocrine TGF-ß did not affect the CDK4 expression levels or its kinase activities. The specificity of induction of CDK inhibitors by autocrine TGF-ß was demonstrated by repressing the expression of RII in the presence of tetracycline, which resulted in the abrogation of autocrine TGF-ß activity and the resultant suppression of p21/waf1/cip1 and p27/kip induction. These results indicate that p21/waf1/cip1 and p27/kip are involved in the autocrine TGF-ß-mediated regulation of the cell cycle.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
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-ß1 was purified as described (16) and iodinated by the chloramine T method (16) . Cells were seeded at a density of 6 x 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-ß1 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 x 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-ß Autocrine Activity.
One x 103 cells/well were plated in a 96-well plate on day 0, and either control IgG or TGF-ß 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-ß 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 ß-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-ß 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 ß-galactosidase activity.

Flow Cytometry.
Cells were seeded at a density of 5 x 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]-{chi}-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 2x sample buffer [100 mM Tris-HCl (pH 6.8), 2% SDS, 20% glycerol, 0.04% bromphenol blue, and 4% ß-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]{chi}-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 2x 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 2x 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 x 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).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
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)Citation .



View larger version (41K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Transforming growth factor-ß 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-ß1, 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-ß binding and signaling, we carried out receptor binding studies with 125I TGF-ß to determine whether RII expression permitted TGF-ß binding to RI. The data indicate that expression of RII resulted in the cell surface binding of TGF-ß to RII as well as RI (Fig. 1B)Citation . 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-ß binding to the receptors.

Restoration of Autocrine-Negative TGF-ß 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)Citation . 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-ß activity, the RII and Neo clones were treated with 10 µg/ml of either TGF-ß neutralizing antibody or control IgG (Fig. 2, C and D)Citation . RII transfectants displayed 60–65% stimulation in DNA synthesis confirming that RII replacement restored growth control through reactivation of autocrine-negative TGF-ß activity.



View larger version (24K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. DNA synthesis activity and the effect of transforming growth factor ß1 neutralizing antibody on DNA synthesis in BT20, ZR75 Neo, and receptor type II transfectants. A and B, cells were plated at 5 x 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 x 103 cells/well in a 96-well plate and were treated with 10 µg/ml of control IgG or transforming growth factor ß neutralizing antibody on days 3 and 4. DNA synthetic activity was measured using [3H]thymidine; bars, ±SD.

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



View larger version (35K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. A, effect of transforming growth factor-ß (TGF-ß) 1 neutralizing antibody on the transcription of a TGF-ß responsive promoter. BT20, ZR75 Neo, and their receptor type II transfectants were transiently transfected with 30 µg of p3TP-Lux and 10 µg of ß-galactosidase plasmid. Control IgG and TGF-ß1 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-ß activity. Previous work from our laboratory showed that autocrine TGF-ß controlled steady state levels of extracellular matrix protein, Fibronectin (13) . Consequently, induction of autocrine TGF-ß should be associated with increased extracellular matrix production. Therefore, we determined FN mRNA levels in the RII transfectants. As shown in Fig. 3BCitation , RII transfected cells showed a 4–5-fold increase in FN mRNA levels compared with Neo control cells. Tetracycline treatment reversed the autocrine TGF-ß-mediated enhanced FN mRNA expression in the ZR75 RII-transfected cells.

The data presented above demonstrated that RII replacement reconstituted autocrine-negative TGF-ß 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-ß modulates the cell cycle machinery.

Flow Cytometry.
Flow cytometry was performed to determine whether autocrine-negative TGF-ß 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)Citation . Tetracycline treatment reversed the autocrine TGF-ß effects on the cell cycle distribution in the RII-transfected cells.


View this table:
[in this window]
[in a new window]

  		Table 1 Flow cytometric analysis in ZR75 Neo and SRII 7 cells

Cells were plated at 5 x 104 cells/well in 6-well plates either in the presence or absence of tetracycline, and harvested on days 3–5. Their cell cycle distribution was analyzed by flow cytometric analysis of DNA content as described in "Materials and Methods."

 
Autocrine TGF-ß 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)Citation . The CDK4-associated kinase activities were found to be similar between Neo and RII transfectants indicating that autocrine TGF-ß did not affect CDK4 kinase activity.



View larger version (26K):
[in this window]
[in a new window]
[Download PPT slide]
  		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 x 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 x 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-ß 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)Citation . 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)Citation , cyclin A, and cyclin E protein levels (Fig. 4C)Citation .

Autocrine TGF-ß 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)Citation . To confirm the induction of CDK inhibitors by autocrine TGF-ß, RII transfectants were treated with 0.1 µg/ml of tetracycline to repress RII expression and, thus, abrogate autocrine TGF-ß activity. The cells were then analyzed for p21/waf1/cip1 and p27/kip proteins by Western blots. As shown in Fig. 4DCitation , abrogation of autocrine TGF-ß 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)Citation . 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-ß effects.
To additionally confirm that autocrine TGF-ß-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)Citation . 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-ß-mediated growth control.



View larger version (28K):
[in this window]
[in a new window]
[Download PPT slide]
  		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-ß 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. 6Citation , 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)Citation .



View larger version (52K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 6. Anchorage-independent growth of BT20, ZR75 Neo, and receptor type II transfectants. Exponentially growing cells (3 x 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.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Loss or reduced expression of TGF-ß receptors has been implicated in TGF-ß resistance leading to tumor formation and progression (11, 12, 13, 14, 15, 16) . Studies have shown that re-expression of RII restored response to exogenous TGF-ß and reversed the malignant behavior of various cell lines (12 , 13) . Previous work from our laboratory indicated that in colon carcinoma cells, suppression of autocrine TGF-ß activity by constitutively repressing endogenous TGF-ß expression led to a more progressed phenotype (28 , 29) . TGF-ß antisense-transfected cells retained their full TGF-ß receptor expression and, hence, the ability to respond to exogenous TGF-ß. These results indicated that autocrine negative TGF-ß rather than response to exogenous TGF-ß is an important deterrent to malignant progression. Although there are reports analyzing the effects of exogenous TGF-ß on the modulation of cell cycle machinery, very little is known regarding the endogenous control of cell cycle progression by autocrine TGF-ß. Hence, identifying the mechanism(s) by which autocrine TGF-ß functions is important for our understanding of the carcinogenic process and malignant progression.

ER+ breast cancer cells acquire TGF-ß resistance due to inadequate expression of RII (16) . We have used a tetracycline regulatable expression vector to demonstrate that RII replacement is sufficient to reconstitute autocrine-negative TGF-ß activity. Restoration of autocrine TGF-ß activity reduced the DNA synthesis activity in RII transfectants in comparison with Neo control cells (Fig. 2, A and B)Citation . This autocrine TGF-ß-mediated reduction in DNA synthesis was reversed when the RII transfectants were treated with TGF-ß neutralizing antibodies (Fig. 2, C and D)Citation . Restoration of autocrine TGF-ß activity was additionally confirmed by the enhanced activity of a TGF-ß responsive promoter-reporter element (p3TP-Lux) and increased expression of fibronectin in the RII transfectants (Fig. 3, A and B)Citation . Thus, the above data demonstrated that RII replacement is vital and sufficient to restore autocrine-negative TGF-ß activity in the ER+ BT20 and ZR75 breast cancer cells.

Numerous studies have shown that exogenous TGF-ß inhibits cell proliferation by inducing cell cycle arrest (3, 4, 5) . This inhibition can be induced through a variety of mechanisms leading to modulation of the levels and/or activities of cyclins, CDKs, and CDK inhibitors. However, there are no reports characterizing the mechanistic basis for the inhibitory effects of autocrine TGF-ß. We used autocrine TGF-ß activity restored ZR75 breast cancer cell line (SRII 7) to analyze the mechanism of autocrine TGF-ß-mediated cell cycle arrest.

Autocrine TGF-ß-induced reduction in DNA synthesis of RII transfectants (SRII 7) was supported by the flow cytometric data (Table 1)Citation , which indicated there was a lower percentage of S phase and a higher percentage of G0-G1 phase cells in RII transfectants (SRII 7) than in Neo cells. These data suggest that autocrine TGF-ß affects the regulation of cell cycle, and the abrogation of autocrine TGF-ß activity due to tetracycline-mediated RII repression reverses these effects. Furthermore, this autocrine TGF-ß-mediated delayed S phase entry was associated with marked inhibition of CDK2 kinase activity (Fig. 4B)Citation suggesting that CDK2 is one of the downstream targets of the inhibitory effects of TGF-ß. However, the autocrine TGF-ß did not effect the CDK2 (Fig. 4B)Citation as well as cyclin A and cyclin E protein levels (Fig. 4C)Citation . The CDK4 kinase activity was similar between Neo and RII transfectants (Fig. 4A)Citation indicating that the down-regulation of CDK2-associated kinase activities in the RII transfectants was autocrine TGF-ß-mediated direct affect and not a consequence of the down-regulation of CDK4 kinase activity.

TGF-ß affects cell cycle progression by inducing the expression of and/or the activities of multiple CDK inhibitors. Specific inhibitors have their own specific CDK targets; however, they may also function collaboratively and/or concomitantly. In our study, autocrine TGF-ß activity led to the induction of CDK inhibitors p21/waf1/cip1 and p27/kip in the RII transfectants, whereas the expression was not detected in the Neo control cells (Fig. 4C)Citation . Tetracycline-mediated repression of RII and the concomitant abrogation of autocrine TGF-ß activity resulted in the suppression of CDK inhibitors p21/waf1/cip1 and p27/kip in the RII transfectants (Fig. 4D)Citation . Thus, our data indicated that p21/waf1/cip1 and p27/kip are the downstream effectors of autocrine TGF-ß-mediated growth regulation.

Western blot analysis followed by immunoprecipitation with CDK2 antibody revealed the association between CDK inhibitors (p21/waf1/cip1 and p27/kip) and CDK2 in the RII transfectants, whereas the complex was not detected in the Neo cells (Fig. 4E)Citation . This association may have contributed to the marked reduction in the CDK2 kinase activity in the RII transfectants (SRII 7). Si RNA-mediated endogenous knockdown of p21/p27 expression also confirmed the role of these inhibitors in the autocrine TGF-ß-mediated growth control of breast cancer cells.

Numerous mechanisms have been suggested for CDK inhibitors to inhibit CDK activity. First, inhibitors may inhibit or disrupt the cyclin-CDK complex formation (23) . Secondly, they may block the phosphorylation of CDKs by the CDK activating kinase (10 , 30) . Finally, inhibitors may associate with cyclin-CDK complexes and inhibit their catalytic activity (23) . In our study, CDK2-associated cyclin A and cyclin E levels are the same between Neo and RII transfectants (data not shown). This indicates that p21/waf1/cip1 and p27/kip neither prevents nor disrupts CDK2 complex formation.

The ability to form colonies in soft agarose is reflective of malignant transformation. Anchorage-independent growth assays indicated that RII transfectants exhibited 50–60% reduction in cloning efficiency (Fig. 6)Citation . The reduction in cloning efficiency can be partly attributed to the slower growth rates caused by the autocrine TGF-ß-mediated induction of CDK inhibitors, p21 and p27. Another important function of TGF-ß is the regulation of interaction between cell and extracellular matrix through induction of extracellular matrix proteins such as fibronectin, collagen, and laminin, and so forth. Restoration of autocrine-negative TGF-ß activity in SRII 7 cells also enhanced fibronectin expression. This may be an additional factor that contributes to the reduced cloning efficiency. Consequently, our studies demonstrate that reconstitution of TGF-ß RII is necessary and sufficient to restore autocrine TGF-ß activity, induce CDK inhibitors p21/waf1/cip1 and p27/kip, and inhibit endogenous control of cell cycle progression through association between CDK inhibitors and CDK2. This is the first report analyzing the mechanism by which autocrine TGF-ß functions to inhibit the epithelial cell growth and the subsequent reduction in malignancy.


   FOOTNOTES
 
Grant support: NIH Grants CA 38173, CA 50457, and CA 72001.

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.

Requests for reprints: Michael G. Brattain, Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263. Phone: (716) 845-8224; Fax: (716) 845-4437; E-mail: Michael.brattain{at}roswellpark.org

Received 8/25/03. Revised 12/16/03. Accepted 1/23/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Shi Y, Massague J Mechanisms of TGF-ß signaling from cell membrane to the nucleus. Cell, 113: 685-700, 2003.[CrossRef][Medline]
  2. Wrana JL, Attisano L, Carcamo J, et al TGF-ß signals through a heteromeric protein kinase receptor complex. Cell, 71: 1003-14, 1992.[CrossRef][Medline]
  3. Lyons RM, Moses HL Transforming growth factors and the regulation of cell proliferation. Eur J Biochem, 187: 467-73, 1990.[Medline]
  4. Massague J The transforming growth factor-ß family. Ann Rev Cell Biol, 6: 597-641, 1990.[CrossRef]
  5. Sporn MB, Roberts A TGF-ß: recent progress and new challenges. J Cell Biol, 119: 1017-21, 1992.[Free Full Text]
  6. Morgan DO Cyclin-dependent kinases: engines, clocks and microprocessors. Ann Rev Cell Dev Biol, 13: 261-91, 1997.[CrossRef][Medline]
  7. Koff A, Cross F, Fisher A, et al Human cyclin E, a new cyclin that interacts with two members of the CDC2 gene family. Cell, 66: 1217-28, 1991.[CrossRef][Medline]
  8. Ohtsubo M, Roberts JM Cyclin-dependent regulation of G1 in mammalian fibroblasts. Science, 259: 1908-12, 1993.[Abstract/Free Full Text]
  9. Resnitzky D, Hengst L, Reed SI Cyclin A-associated kinase activity is rate limiting for entrance into S phase and is negatively regulated in G1 by p27kip1. Mol Cell Biol, 15: 4347-52, 1995.[Abstract]
  10. Vidal A, Koff A Cell-cycle inhibitors: three families united by a common cause. Gene, 247: 1-15, 2000.[CrossRef][Medline]
  11. Park K, Kim S-J, Bang YJ, et al Genetic changes in the TGF-ß type II receptor gene in human gastric cancer cells: correlation with sensitivity to growth inhibition by TGF-ß. Proc Natl Acad Sci USA, 91: 8772-6, 1994.[Abstract/Free Full Text]
  12. Sun L-Z, Wu G, Willson JKV, et al Expression of TGF-b type II receptor leads to reduced malignancy in human breast cancer MCF-7L cells. J Biol Chem, 269: 26449-55, 1994.[Abstract/Free Full Text]
  13. Wang J, Sun L-Z, Myeroff L, et al Demonstration that mutation of the type II TGF-ß receptor inactivates its tumor suppressor activity in RER+ colon carcinoma cells. J Biol Chem, 270: 22044-9, 1995.[Abstract/Free Full Text]
  14. Wang J, Han W, Zborowska E, et al Reduced expression of TGF-ß type I receptor contributes to the malignancy of human colon carcinoma cells. J Biol Chem, 271: 17366-71, 1996.[Abstract/Free Full Text]
  15. Kang SH, Bang YJ, Im YH, et al Transcriptional repression of TGF-ß type I receptor gene by DNA methylation results in the development of TGF-ß resistance in human gastric cancer. Oncogene, 18: 7280-6, 1999.[CrossRef][Medline]
  16. Ammanamanchi S, Kim SJ, Sun LZ, Brattain MG Induction of TGF-ß receptor type II expression in ER+ breast cancer cells through Sp1 activation by 5 aza-2'dc. J Biol Chem, 273: 16527-34, 1998.[Abstract/Free Full Text]
  17. Geng Y, Weinberg RA TGF-ß effects on expression of G1 cyclins and cyclin-dependent protein kinases. Proc Natl Acad Sci USA, 90: 10315-9, 1993.[Abstract/Free Full Text]
  18. Ewen M, Sluss HK, Whitehouse LL, Livingston DM TGF-ß inhibition of CDK4 synthesis is linked to cell cycle arrest. Cell, 74: 1009-20, 1993.[CrossRef][Medline]
  19. Feng XH, Filvaroff EH, Derynck R TGF-ß induced down-regulation of cyclin A expression requires a functional TGF-ß receptor complex. J Biol Chem, 270: 24237-45, 1995.[Abstract/Free Full Text]
  20. Ko TC, Sheng HM, Reisman D, Thompson AE, Beauchamp DR TGF-ß1 inhibits cyclin D1 expression in intestinal epithelial cells. Oncogene, 10: 177-84, 1995.[Medline]
  21. Koff A, Ohtsuki M, Polyak K, Roberts JM, Massague J Negative regulation of G1 in mammalian cells: inhibition of cyclin E-dependent kinase by TGF-ß. Science, 260: 536-9, 1993.[Abstract/Free Full Text]
  22. Datto MB, Li Y, Panus JF, Howe DJ, Xiong Y, Wang X-F TGF-ß induces the cyclin-dependent kinase inhibitor p21 through a p53-independent mechanism. Proc Natl Acad Sci USA, 92: 5545-9, 1995.[Abstract/Free Full Text]
  23. Peter M, Herskowitz I Joining the complex: cyclin-dependent kinase inhibitory proteins and the cell cycle. Cell, 79: 181-4, 1994.[CrossRef][Medline]
  24. Moustakas A, Kardassis D Regulation of the human p21/WAF1/CIP1 promoter in hepatic cells by functional interactions between Sp1 and Smad family members. Proc Natl Acad Sci USA, 95: 6733-8, 1998.[Abstract/Free Full Text]
  25. Hu PP, Shen X, Huang D, Liu Y, Counter C, Wang XF The MEK pathway is required for stimulation of p21/WAF1/CIP1 by TGF-ß. J Biol Chem, 274: 35381-7, 1999.[Abstract/Free Full Text]
  26. Ahmed MM, Alcock RA, Chendil D., et al Restoration of TGF-b signaling enhances radiosensitivity by altering the Bcl2/Bax ratio in the P53 mutant pancreatic cancer cell line MIA PaCa-2. J Biol Chem, 277: 2234-46, 2002.[Abstract/Free Full Text]
  27. Gong JG, Ammanamanchi S, Ko TC, Brattain MG TGF-ß1 increases the stability of p21/WAF1/CIP1 protein and inhibits CDK2 kinase activity in human colon carcinoma FET cells. Cancer Res, 63: 3340-6, 2003.[Abstract/Free Full Text]
  28. Wu SP, Theodorescu D, Kerbel R, et al TGF-ß1 is an autocrine negative growth regulator of human colon carcinoma FET cells in vivo as revealed by transfection of an antisense expression vector. J Cell Biol, 116: 186-97, 1992.
  29. Wu SP, Sun L-Z, Willson JKV, Fields K, Humphrey LE, Brattain MG Repression of autocrine TGF-ß1 and ß2 in quiescent CBS colon carcinoma cells leads to progression of tumorigenic properties. Cell Growth Diff, 4: 115-23, 1993.[Abstract]
  30. Apprelikova O, Xiong Y, Liu ET Both p16 and p21 families of CDK inhibitors block the phosphorylation of cyclin-dependent kinases by the CDK-activating kinase. J Biol Chem, 270: 18195-7, 1995.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 BloodHome page
E. Esplugues, J. Vega-Ramos, D. Cartoixa, B. N. Vazquez, I. Salaet, P. Engel, and P. Lauzurica
Induction of tumor NK-cell immunity by anti-CD69 antibody therapy
Blood, June 1, 2005; 105(11): 4399 - 4406.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ammanamanchi, S.
Right arrow Articles by Brattain, M. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ammanamanchi, S.
Right arrow Articles by Brattain, M. G.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online