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Carcinogenesis, Vol. 21, No. 7, 1303-1312, July 2000
© 2000 Oxford University Press

Cancer Biology

Phorbol ester-induced production of cytostatic factors by normal and oncogenic Ha-ras-transformed human breast cell lines

Meng Guo and John J. Reiners, Jr1

Institute of Chemical Toxicology, Wayne State University, 2727 Second Avenue, Room 4000, Detroit, MI 48201, USA


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on cell cycle progression were examined in the human breast cell line MCF10A-Neo and a derivative line which expresses a Ha-ras oncogene (MCF10A-NeoT cells). Exposure of MCF10A-Neo cultures to TPA induced a G1 arrest that lasted ~16–24 h (IC50 ~0.5 nM). TPA-treated cultures produced a cytostatic conditioned medium. Cytostatic activity was detectable within 1 h of TPA treatment, peaked 3–7 h later and disappeared between 16 and 24 h post-treatment. However, cytostatic conditioned medium could be quickly regenerated by re-feeding previously treated cultures with new medium. Removal of latent transforming growth factor ß (TGFß) from the culture medium, supplementing the culture medium with anti-TGFß or soluble TGFßII receptor, or pre-absorption of conditioned medium with anti-TGFß all reduced the cytostatic effects of TPA or conditioned medium on MCF10A-Neo proliferation by ~50%. Co-treatment with the serine protease inhibitors aprotinin or plasminogen activator inhibitor-1 also suppressed the cytostatic activity of TPA ~50%. Conditioned medium isolated from TPA-treated MCF10A-Neo cultures was transiently cytostatic to MCF10A-NeoT cells. The proliferation of MCF10A-NeoT cultures, in contrast to MCF10A-Neo cells, was suppressed at least 72 h following TPA exposure. Conditioned medium isolated from TPA-treated MCF10A-NeoT cultures also suppressed MCF10A-NeoT proliferation for ~72 h, but suppressed MCF10A-Neo proliferation for <24 h. These studies suggest that TPA quickly activates proteolytic processes in MCF10A-Neo cells leading to the activation of latent TGFß supplied by the serum in the culture medium. TPA also stimulates the production of an additional cytostatic factor(s) which signals via a mechanism not involving the TGFßII receptor. Lastly, expression of an activated Ha-ras oncogene alters both the types of cytostatic factors produced following TPA treatment and responsiveness to these factors.

Abbreviations: ER, estrogen receptor; PAI-1, plasminogen activator inhibitor-1; PBS, phosphate-buffered saline; rTGFßII, transforming growth factor ß type II receptor; TGFß, transforming growth factor ß; tPA, tissue plasminogen activator; TPA, 12-O-tetradecanoylphorbol-13-acetate; uPA, urokinase plasminogen activator.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) has diverse effects on the proliferation of cultured cells. It functions as a mitogen or co-mitogen when added to the PMK-R1 keratinocyte cell line (1), some subclones of NIH 3T3 fibroblasts (2), normal human melanocytes (3) and the B cell component of primary splenocyte cultures (4). Conversely, TPA also arrests the growth of a wide range of cell types (5–17). In some cases this arrest reflects the induction of terminal differentiation (5) or apoptosis (6). In other situations TPA is cytostatic as a consequence of its induction of a cell cycle block (7–9).

The human breast adenocarcinoma cell line MCF-7 undergoes a protracted G1 arrest following TPA exposure (8,9,16). This arrest is generally obvious within 48 but not 24 h of TPA treatment (12,16,17). Several studies have demonstrated that the cytostatic effects of TPA on MCF7 proliferation are accompanied by an accumulation of transforming growth factor ß (TGFß) mRNA and the production of a conditioned medium containing active TGFß (14,15,18). TGFß is cytostatic both in vivo and in culture to normal breast tissue and some neoplastic breast cell lines (15,19–22). Hence, the effects of TPA on MCF-7 proliferation are thought to reflect the consequences of autocrine production and activation of TGFß and subsequent TGFß-stimulated production of cyclin-dependent kinase inhibitors (14,15).

The MCF10A cell line originated as a spontaneously immortalized outgrowth of human fibrocystic breast tissue (23). By several criteria it appears to be neither transformed nor tumorigenic and it is commonly referred to as a normal human breast epithelial cell (23–25). MCF10A cells do not grow when transplanted into nude mice (23,24). However, a derivative (i.e. MCF10A-NeoT) of MCF10A cells engineered to express a transduced Ha-ras oncogene does form benign ductal aggregates upon xenografting (26). With time these lesions undergo neoplastic progression (26).

We have found that TPA is cytostatic to MCF10A and MCF10A-Neo cells (a derivative of MCF10A stably transduced with an expression vector containing the Neo resistance gene). However, in contrast to what is observed in MCF-7 cells and several other human breast tumor lines, the cytostatic effects of TPA on the MCF10A lines develop very rapidly. In the current study we have investigated the basis for the rapid antiproliferative effect of TPA on MCF10A-Neo cells and compared them with the response of the MCF10A-NeoT cell line. Previous studies with other cell lines have shown that the Ha-ras oncogene influences both the production of and responsiveness to TGFß (19,27–31). Our studies demonstrate that TPA is cytostatic to MCF10A-Neo cells via a mechanism involving both TGFß and other cytokines that work independently of the TGFß signaling pathway. However, the source of the TGFß was unexpected and unusual since it did not originate from the cells. Instead, it was derived from the latent TGFß present in the serum used to supplement the medium. TPA rapidly stimulated proteolytic processes involved in activation of latent TGFß. Furthermore, we show that oncogenic p21 Ras expression influences both the types of cytostatic factors produced by MCF10A cells and susceptibility to these factors.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Chemicals
Aprotinin, recombinant-derived human TGFß1, TPA, control rabbit IgG and a neutralizing polyclonal pan rabbit antibody made to a mixture of TGFß1 + TGFß1.2 + TGFß2 + TGFß5 (IgG fraction, product no. T929) were obtained from Sigma (St Louis, MO). Normal goat IgG purified by protein G affinity chromatography, recombinant truncated human TGFß type II receptor (rTGFßII), polyclonal neutralizing goat antibody (IgG fraction) to native porcine TGFß2 (product no. AB-112-NA), affinity-purified neutralizing polyclonal goat antibody (IgG fraction) to chicken TGFß3 (product no. AF-243-NA) and an ELISA kit which recognizes processed human TGFß1 were purchased from R&D Systems (Minneapolis, MN). Recombinant-derived mutant human plasminogen activator inhibitor-1 (PAI-1) was from CalBiochem (San Diego, CA). Recombinant protein G–agarose, trypsin, penicillin/streptomycin solution and horse serum were obtained from Life Technologies (Grand Island, NY). [Methyl-3H]thymidine (6.7 Ci/mmol) was purchased from NEN Life Sciences (Boston, MA).

Cell lines and culture conditions
The MCF10A, MCF10A-Neo and MCF10A-NeoT cell lines were obtained from the Cell Lines Resource, Karmanos Cancer Institute (Detroit, MI). MCF10A-Neo and MCF10A-NeoT were derived by transfection of the MCF10A cell line with plasmid pHo6 or pHo6 containing a Ha-ras oncogene derived from the human T24 bladder carcinoma cell line and subsequently selected for G418 resistance. Derivation and characterization of these cell lines have been described in detail (23,24). The estrogen receptor (ER)-negative 139-2-8 cell line and the ER-positive 139B6 cell line were obtained from Dr S.Brooks (Wayne State University, Detroit, MI). These latter cell lines were derived by transfection of MCF10A cells with the pHß-Apr-neo vector or a vector containing the human ER sequence and subsequent selection in G418. Derivation and characterization of these two cell lines have been published (32).

Cells were cultured in Dulbecco's modified Eagle's medium/Ham's F-12 medium supplemented with 10% horse serum and a variety of growth factors as described by Basolo et al. (25) in a humidified atmosphere containing 5% CO2 at 37°C.

Culture treatments
Proliferation studies were performed with cultures plated at a density that ensured exponential growth for a minimum of 5 days. Cultures were generally treated 40–48 h after plating. Details of culture treatment are provided in the text. TPA was dissolved and diluted with DMSO. Organic solvent never exceeded 0.1% of total culture volume. Aprotinin and the truncated rTGFßII were diluted in sterile water. The various antibody preparations used in this study were reconstituted/diluted according to the manufacturer's instructions [sterile water, phosphate-buffered saline (PBS) or medium lacking serum and growth factors were primarily used]. Cells were harvested by exposure to a solution of 0.25% trypsin, 0.1 mM EDTA. They were subsequently counted and assessed for ability to exclude trypan blue.

Feeder cultures for conditioned medium studies were established in 100 mm dishes. Initial platings of 0.03x106, 0.2x106 and 0.8x106 cells yielded ~48 h later cell densities of 0.148 ± 0.019x106, 0.889 ± 0.067x106 and 4.175 ± 0.318x106 cells, respectively (n = 2–6 experiments). Two days after plating the feeder cultures were treated with nothing, DMSO or TPA. At various times after treatment the media were aspirated and the cultures were washed three times with PBS and subsequently re-fed with fresh growth medium. PBS and medium were pre-warmed to 37°C prior to use. Medium from feeder cultures was harvested at various times and centrifuged at 3000 g for 5 min to remove any cells or debris. The resulting supernatant fluid was used as conditioned medium. It was passaged to recipient cultures that had been plated 2 days earlier and washed with PBS three times immediately before receipt of the conditioned medium.

Protein G column chromatography of serum
Culture medium supplemented with horse serum was incubated with 10 µg/ml of a polyclonal rabbit pan TGFß IgG antibody or 10 µg/ml of control rabbit IgG for 2 h at 37°C. The media were then chromatographed on columns of protein G–agarose that had been washed and equilibrated in 0.15 M NaCl, 0.01 M NaH2PO4, pH 7.0. The eluant was used as a source of TGFß-depleted medium. Removal of latent/active TGFß was confirmed by ELISA.

ELISA determination of TGFß1
Serum and medium contents of latent and processed TGFß1 were determined according to the instructions of the manufacturer of the Human TGF-ß1 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN).

[3H]thymidine incorporation studies
Thymidine incorporation studies were performed with cultures grown in 35 mm culture dishes. Cultures were pulsed with [methyl-3H]thymidine (1 µCi/ml culture medium) at various times after treatment with solvent, TPA or conditioned medium for 1 h. At the end of the pulse the medium was aspirated and the cells were rinsed twice with cold PBS. The rinsed cultures were fixed by exposure to cold 5% trichloracetic acid for a minimum of 1 h. The procedure for the extraction of fixed cells has been described in detail (33). A second set of non-fixed dishes (three per treatment) were treated with trypsin/EDTA to estimate cell numbers. [3H]thymidine was detected by scintillation counting and expressed as d.p.m./103 cells.

Cell cycle analyses
Cells were plated in 100 mm culture dishes at densities that ensured exponential growth at the time of harvest. The harvesting and processing protocols used for propidium iodide detection of DNA by flow cytometry have been described in detail (34). Cells were analyzed with a Becton Dickinson FACScalibur instrument and the percentages of cells in the G1, S and G2/M stages of the cell cycle were determined with a DNA histogram-fitting program (MODFIT; Verity Software, Topsham, ME). A minimum of 104 events/sample were collected for subsequent analyses.

Statistical analyses
Data were analyzed by the Tukey HSD test. The Statistica 5.0 software package (StaSoft Inc., Tulsa, OK) was used to perform these calculations. Differences were considered statistically significant if P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Cytostatic activity of TPA
The proliferation of MCF10A-Neo cultures was suppressed by concentrations of TPA >= 1 nM (Figure 1AGo). This suppresion represented a cytostatic effect since culture viability was not affected by TPA (M.Guo and J.J.Reiners, unpublished observation). The cytostatic effects of TPA were transient. A semi-log plot of cell number versus culture time shows that the suppressive effects of TPA were lost within the first 24 h of treatment (Figure 1BGo). Analyses of [3H]thymidine incorporation indicated a maximum suppression of DNA synthesis ~16 h post-TPA treatment (Figure 1CGo). Thereafter, the cells recovered to their original DNA synthetic capacities.



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  		Fig. 1. Effects of TPA on the proliferation of MCF10A-Neo cells. MCF10A-Neo cells were plated at low density and allowed to grow for 24 h before being treated with DMSO or various concentrations of TPA. Cultures were harvested at various times after treatment for analyses of cell numbers (A and B), [3H]thymidine incorporation into DNA (C) and cellular DNA contents (D–F). Data in (A)–(C) represent means ± SD of three culture dishes. [3H]Thymidine incorporation data reported in (C) were first calculated as d.p.m./cell prior to being normalized to the DMSO response. Treatments and cell cycle designations are provided in the figure. *Significantly less than DMSO control, P <= 0.02.

 
Flow cytometric analyses of cellular DNA content were used to determine if the cytostatic effects of TPA reflected an arrest at a specific cell cycle checkpoint (Figure 1D–FGo). Cells having G0/G1 DNA content accumulated during the first 16 h of TPA treatment (Figure 1DGo). The accumulation of G0/G1 cells was accompanied by losses of both S and G2/M phase cells (Figure 1DGo). The arrested population re-entered the cell cycle 16–24 h post-TPA treatment (Figure 1EGo). By 48 h TPA-treated cells had a cell cycle distribution similar to that seen in solvent-exposed cultures (Figure 1FGo).

TPA-generated extracellular cytostatic factors
MCF10A-Neo cultures exposed to TPA produced a conditioned medium which, when added to non-treated MCF10A-Neo cultures, suppressed proliferation of the recipient cultures (Figure 2Go). This anti-proliferative effect represented a cytostatic activity since the viability of recipient cells was not affected by conditioned medium (M.Guo and J.J.Reiners, unpublished observation). Production of cytostatic conditioned medium occurred rapidly. In the study presented in Figure 2Go donor cultures were treated with TPA for 1 h prior to being washed and re-fed with fresh medium. Conditioned medium harvested within 30 min of re-feeding suppressed the proliferation of recipient cells. The level of suppression depended upon both the number of cells treated with TPA and the time elapsed between TPA treatment and the harvesting of conditioned medium. Specifically, the cytostatic activity of conditioned medium harvested 30 min after re-feeding correlated positively with the number of cells used for conditioning. However, the influence of cell density became insignificant if conditioned medium was harvested 8 h after re-feeding. Presumably, the process responsible for the cytostatic effect was saturable. An optimal concentration of cytostatic factor(s) could be reached by either having a large number of feeder cells or, in the case of less dense cultures, by allowing additional time for accumulation of factor(s) in the medium.



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  		Fig. 2. Effects of feeder density and duration of TPA conditioning on the production of cytostatic factor(s). MCF10A-Neo feeder cultures were plated at various densities such that they contained ~0.15, 0.9 or 4.2x106 cells/dish at the time of treatment (see Materials and methods). Approximately 48 h after plating the feeder cultures were treated with nothing, DMSO or 10 nM TPA for 1 h. Feeders were subsequently washed and re-fed with fresh medium. Conditioned medium was passed to recipient cultures 0.5–8 h later. Analyses of recipient culture cell numbers were made ~24 h later. Data represent means ± SD of three dishes. Treatments are noted in the figure. Recipient cultures were plated 2 days prior to exposure to conditioned medium. The numbers of cells on the recipient cultures at the time of treatment (T0) were determined for each of the three time points. All 4 and 8 h TPA groups and the 0.9 and 4.2x106 density, 30 min TPA groups are significantly less than the corresponding non-treated, DMSO-treated or 4.2x106 re-fed groups, P <= 0.04.

 
In order to better characterize the kinetics of cytostatic factor production we treated semi-confluent (~70%) cultures with TPA for varying lengths of time before washing and re-feeding. Conditioned medium was harvested 30 min after re-feeding and transferred to recipient cultures. The data presented in Figure 3Go show that a 30 min, but not a 10 min, exposure to TPA is sufficient for the production of conditioned medium having maximal cytostatic activity. Hence, the data presented in Figures 2 and 3GoGo collectively suggest that MCF10A-Neo cultures produce a conditioned medium having cytostatic activity within 1 h of TPA exposure (30 min TPA exposure + 30 min conditioning).



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  		Fig. 3. Duration of TPA exposure required for cytostatic factor(s) production. MCF10A-Neo feeder cultures were allowed to grow for 48 h (~4.2x106 cells/100 mm dish) before being treated with 10 nM TPA or DMSO for 10–90 min. At various times after treatment feeder cultures were washed and re-fed with fresh medium. The resulting conditioned medium was transferred to recipient cultures after 0.5 h. Analyses of recipient culture cell numbers were made ~24 h later. Data represent means ± SD of three dishes. Symbols represent conditioned medium isolated from cultures treated with nothing ({blacklozenge}), DMSO ({blacksquare}) or TPA ({blacktriangleup}). *Significantly less than corresponding controls, P <= 0.05.

 
Conditioned medium harvested from TPA-treated MCF10A-Neo cultures temporarily suppressed the proliferation of MCF10A-Neo recipient cultures (Figure 4Go). Indeed, the extent to which TPA-conditioned medium suppressed the proliferation of MCF10A-Neo cells was similar to that obtained following direct addition of TPA (compare Figures 1A and 4GoGo).



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  		Fig. 4. Responsiveness of MCF10A-Neo cultures to repeated exposure to TPA-conditioned medium. MCF10A-Neo feeder cultures (~4.2x106 cells/100 mm dish) were treated with DMSO or 10 nM TPA for 1 h. Feeder cultures were subsequently washed and re-fed with fresh medium. Four hours later the conditioned media were transferred onto recipient cultures (time 0). Approximately 24 h later some of the recipient cultures were washed and re-fed with a second similarly derived preparation of TPA-conditioned medium. Data represent means ± SD of three culture dishes. Recipient cultures were treated as follows: DMSO-conditioned medium ({blacksquare}); DMSO-conditioned medium followed by TPA-conditioned medium ({square}); TPA-conditioned medium ({blacktriangleup}); TPA-conditioned medium followed by TPA-conditioned medium ({triangleup}). The arrow indicates time of exposure to the second preparation of conditioned medium. *Significantly less than DMSO-conditioned medium control, P <= 0.002. **Significantly less than single exposure DMSO- and TPA-conditioned medium groups, P < 0.0003.

 
The observation that conditioned medium temporarily arrested the proliferation of MCF10A-Neo cultures suggested that either (i) the recipient cells become refractory to the cytostatic activity of the conditioned medium or (ii) the cytostatic factors are unstable and transiently made following TPA exposure. To test the former possibility we exposed cultures recovering from the cytostatic effects of TPA-conditioned medium to a fresh preparation of TPA-conditioned medium (Figure 4Go). Re-exposure suppressed proliferation. However, the proliferation of control cultures appeared to be more affected by the conditioned medium (Figure 4Go). Results similar to those reported in Figure 4Go were obtained in a second experiment (M.Guo, unpublished data). Hence, TPA-treated MCF10A-Neo cells appear to become partially refractive to the cytostatic factors present in the conditioned medium.

In order to characterize the time span of cytostatic factor production by TPA-treated cultures, we examined the cytostatic activity of conditioned media harvested at various times after re-feeding TPA-treated cultures. Based upon analyses of [3H]thymidine incorporation (Figure 5AGo) or cell counts (Figure 5BGo), it appears that TPA-treated MCF10A-Neo cultures produce a cytostatic conditioned medium for ~16 h. Conditioned medium harvested 24 h after TPA treatment had no cytostatic activity.



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  		Fig. 5. Kinetics of cytostatic medium production by MCF10A-Neo cultures. Two-day-old MCF10A-Neo feeder cultures (~4.2x106 cells/100 mm dish) were treated with DMSO or 10 nM TPA for 1 h. Feeder cultures were subsequently washed and re-fed with fresh medium. Conditioned media were subsequently harvested at varied times afterwards (1–48 h) and added to recipient cultures. (A) Recipient cultures were exposed to conditioned medium for 4 h prior to addition of [3H]thymidine. Three cultures per treatment group were harvested 1 h later for analyses of [3H]thymidine incorporation, estimation of cell numbers and calculation of d.p.m./cell. The d.p.m./cell values calculated at each time point for cultures treated with DMSO-conditioned medium were set as 100%. Data in (A) represent means ± SD of the TPA response, relative to the DMSO response, at each time point. (B) Recipient cultures were harvested ~24 h after addition of conditioned medium for cell counts. The increase in cell numbers occurring in the DMSO-conditioned medium treatment group during the 24 h was set as 100%. Data in (B) represent means ± SD of the TPA response, relative to the DMSO response, at each time point. Each treatment group at each time point employed three culture dishes. *Significantly less than corresponding DMSO control, P <= 0.03.

 
MCF10A-Neo activation of a latent cytostatic factor found in culture medium
As a control for the conditioned medium studies we examined the effects of re-feeding treated cultures with fresh medium. The addition of fresh medium to DMSO-treated cultures had little effect on the kinetics of cell proliferation (Figure 6Go). However, the addition of fresh medium to recovering TPA-treated cultures transiently suppressed cell proliferation (Figure 6Go). This latter result, coupled with the data reported in Figure 5Go, suggested that culture medium may contain a latent cytostatic factor that is activated by TPA-treated MCF10A-Neo cells.



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  		Fig. 6. Activation of a latent cytostatic activity in culture medium by TPA-treated MCF10A-Neo cultures. Two-day-old MCF10A-Neo cultures were treated with DMSO or 10 nM TPA. Approximately 24 h later half the cultures were washed and re-fed with fresh culture medium. Data represent means ± SD of three dishes. DMSO alone ({blacksquare}); TPA alone ({blacktriangleup}); DMSO + fresh medium ({square}); TPA + fresh medium ({triangleup}). The arrow indicates time of medium change. *Significantly less than TPA group, P = 0.025. **Significantly less than DMSO control or DMSO + fresh medium group, P <= 0.0003.

 
TGFß is a component of the conditioned medium
The cytokine TGFß is cytostatic towards breast cells and is produced by some breast cell lines following exposure to TPA (14,15,19–22). A recombinant preparation of human TGFß1 partially suppressed MCF10A-Neo proliferation (Figure 7AGo). The suppression was temporary in nature and equivalent to ~50% of the cytostatic activity obtained with TPA (Figure 7AGo). The cytostatic activity of the TGFß1 preparation could be blocked by the addition of a neutralizing pan TGFß antiserum to culture medium just prior to TPA addition (Figure 7BGo).



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  		Fig. 7. Effects of TGFß1 on the proliferation of MCF10A-Neo cells. (A) MCF10A-Neo cultures were plated at low density and allowed to grow for 48 h before being treated with DMSO, 10 nM TPA or varying amounts of TGFß1. (B) MCF10A-Neo cultures were treated with DMSO, TGFß1, neutralizing pan antibody to TGFß or control Ig. Antibody and Ig preparations were added to cultures immediately prior to TGFß1 addition. Cultures were harvested at various times after treatment for cell counting. Treatments are given in the Figure. Data represent means ± SD of three culture dishes. *Significantly less than DMSO control and 0.1 ng/ml TGFß group, P <= 0.004. **Significantly less than all DMSO treatment groups and TGFß + 6 or 24 ng/ml TGFß Ab groups, P <= 0.0007.

 
Supplementation of MCF10A-Neo cultures with neutralizing pan TGFß antisera suppressed the cytostatic activity of TPA by ~50% (Figure 8AGo). Incubation of TPA-conditioned medium with the same neutralizing pan antibody also suppressed the cytostatic activity of the conditioned medium by ~50% (Figure 8BGo). As a complement to this latter study we pretreated TPA-conditioned medium with the pan antibody and used protein G–agarose to remove Ig–TGFß complexes prior to transfer to non-treated MCF10A-Neo cultures (Table IGo). This approach indicated that a TGFß species constituted ~64% of the cytostatic activity of conditioned medium. Control immunoglobulin had no effect on the cytostatic activity of either TPA or TPA-conditioned medium (Figure 8Go and Table IGo).



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  		Fig. 8. Suppression of the cytostatic effects of TPA (A) and TPA-conditioned medium (B) with a pan TGFß antibody. (A) MCF10A-Neo cultures were plated at low density and allowed to grow for 24 h prior to sequential treatment with a neutralizing pan TGFß antibody or control Ig, and DMSO or 10 nM TPA. (B) Two-day-old MCF10A-Neo feeder cultures (~4.2x106 cells) were treated with DMSO or 10 nM TPA for 1 h. Feeders were then washed and re-fed with fresh medium. Conditioned media were harvested 4 h later. Varying amounts of neutralizing TGFß antibody or control Ig were added to the conditioned media. After a 2 h incubation period the conditioned media were added to recipient cultures. Analyses of recipient culture cell numbers were made 24–48 h later. Data in both panels represent means ± SD of three culture dishes. Treatments are identified in the figure. *Significantly less than all DMSO treatment groups, P < 0.0003. **Significantly less than all DMSO treatment groups and all TPA + TGFß Ab treatment groups, P < 0.0007.

 

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  		Table I. Cytostatic effects of TPA-conditioned medium preabsorbed with a pan antibody to TGFßa
 
The pan TGFß antibody used in the studies presented in Figures 7 and 8GoGo and Table IGo recognizes TGFß1, TGFß2 and TGFß4. Antibodies made specifically to human TGFß2 and TGFß3 were also tested in neutralization and absorption protocols for their abilities to inhibit the cytostatic activity of TPA and TPA-conditioned medium. These antibodies had no protective effect in any protocol (M.Guo, unpublished data).

The cytostatic effects of TGFß family members are dependent upon their interaction with the rTGFßII component of the TGFß receptor complex. TGFß binding to its cellular receptor can be suppressed by inclusion of a soluble truncated form of rTGFßII. The truncated soluble receptor inhibits by virtue of its competition with the cellular receptor for ligand. Addition of soluble, truncated rTGFßII to MCF10A-Neo cultures immediately prior to the addition of TPA partially suppressed (~50%) the cytostatic effects of TPA (Table IIGo).


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  		Table II. Cytostatic effects of TPA on MCF10A-Neo cells cultured in the presence of soluble rTGFßIIa
 
Serum as the source of latent TGFß
Serum can contain significant levels of latent TGFß1. ELISA analyses of the culture media used for growing MCF10A-Neo cells (supplemented with 10% horse serum) indicated that it contained ~300 pg/ml latent TGFß1 (J.J.Reiners, unpublished data). Thus, does this source of latent TGFß contribute to the cytostatic effects of TPA? We examined this possibility by first immunodepleting the culture medium of latent TGFß and then assessing whether depleted medium could support the cytostatic activity of TPA. Table IIIGo demonstates that this approach eliminated ~50% of the cytostatic activity of TPA, a value similar to that obtained by treating cultures directly with neutralizing pan TGFß antibody (see Figure 8AGo).


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  		Table III. Cytostatic effects of TPA on MCF10A-Neo cells cultured in medium depleted of TGFßa
 
Suppression of TPA effects by protease inhibitors
Latent TGFß must be activated in order to be cytostatic to target cells (35–37). This activation is performed by a limited number of proteases, including plasmin. However, plasmin is derived from plasminogen (35,36). Both urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA) are serine proteases capable of converting plasminogen to plasmin. Co-treatment of MCF10A-Neo cultures with TPA and either the serine protease inhibitor aprotinin or PAI-1 (a specific protein inhibitor of uPA and tPA) partially suppressed the cytostatic activity of TPA (Table IVGo). The two protease inhibitors afforded approximately the same amount of protection. Co-treating cultures with PAI-1 and soluble, truncated rTGFßII prior to TPA addition suppressed the cytostatic actions of TPA (Table IVGo). However, combined PAI-1 and rTGFßII treatment offered no more protection than singular treatment with either agent (Table IVGo).


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  		Table IV. Cytostatic effects of TPA on MCF10A-Neo cells cultured in the presence of aprotinin, plasminogenin activator inhibitor or soluble rTGFßIIa
 
Cytostatic activity of TPA towards MCF10A-NeoT cells
The MCF10A-NeoT cell line was derived from MCF10A cells and expresses oncogenic p21 Ras (24). TPA suppressed the proliferation of MCF10A-NeoT (Figure 9AGo). However, unlike the results obtained with MCF10A-Neo cultures, the antiproliferative effects of TPA on MCF10A-NeoT proliferation were maintained 72 h after exposure. This suppression represented a cytostatic effect since the viability of MCF10A-NeoT cultures was not affected by TPA exposure (M.Guo, unpublished data). Furthermore, TPA-treated MCF10A-NeoT cells produced a conditioned medium that duplicated the sustained cytostatic effects of straight TPA treatment (Figure 9BGo).



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  		Fig. 9. Cytostatic effects of TPA on the growth of MCF10A-NeoT cells. (A) Two-day-old cultures of MCF10A-NeoT cells were treated with DMSO ({blacksquare}) or 10 nM TPA ({blacktriangleup}). (B) Two-day-old feeder cultures of MCF10A-NeoT cells were exposed to 10 nM TPA or DMSO for 1 h prior to washing and re-feeding with fresh medium. Conditioned media were harvested 4 h later and transferred to recipient MCF10A-NeoT cultures. A control group received fresh medium. Analyses of recipient culture cell numbers were made 24–72 h later. Fresh medium ({lozenge}); DMSO-conditioned medium ({blacksquare}); TPA-conditioned medium ({blacktriangleup}). All data represent means ± SD of three dishes. *Significantly less than values calculated for groups treated with DMSO, DMSO-conditioned medium or fresh medium, P < 0.002.

 
MCF10A-NeoT cells in the absence of any treatment produced a conditioned medium that inhibited the growth of MCF10A-Neo cultures (Figure 10AGo). In this study the medium was conditioned for 26 h prior to being transferred to MCF10A-Neo cultures. Preincubation of this conditioned medium with a neutralizing pan antibody to TGFß strongly inhibited the cytostatic activity of the medium towards MCF10A-Neo cultures (Figure 10BGo). The suppressive effects of MCF10A-NeoT culture medium on MCF10A-Neo growth were dependent upon the length of conditioning time. Specifically, MCF10-NeoT culture medium that had been conditioned for only 4 h, as opposed to 26 h, was not cytostatic towards MCF10A-Neo cultures (Figure 11AGo). These latter conditions were then used to examine whether conditioned medium obtained from TPA-treated MCF10A-NeoT cells exhibited sustained cytostatic activity towards MCF10A-Neo cells. Such medium was cytostatic to MCF10A-Neo cells (Figure 11AGo). However, it did not cause the sustained antiproliferative effect noted with MCF10A-NeoT cultures (compare Figures 11A and 9BGoGo). This differential response of MCF10A-Neo and MCF10A-NeoT cells to conditioned medium derived from TPA-treated MCF10A-NeoT cells raised the question of how MCF10A-NeoT cells would respond to conditioned medium isolated from TPA-treated MCF10A-Neo cultures. Conditioned medium derived from TPA-treated MCF10A-Neo cells transiently suppressed the proliferation of MCF10A-NeoT cells (Figure 11BGo).



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  		Fig. 10. Cytostatic effects of MCF10A-NeoT-conditioned medium on MCF10-Neo cells. (A) Two-day-old MCF10A-NeoT feeder cultures were washed and re-fed. Conditioned medium was isolated 26 h later and transferred to MCF10A-Neo cultures either undiluted ({blacktriangledown}) or diluted 1:2 ({triangleup}), 1:4 ({circ}) or 1:8 ({triangleup}). Control cultures received fresh medium ({blacksquare}). Analyses of recipient culture cell numbers were made ~24–47 h later. (B) Conditioned medium isolated from MCF10A-NeoT cultures was diluted 1:4 and added directly to recipient MCF10A-Neo cultures ({circ}) or incubated for 2 h with 12 ng/ml of a neutralizing pan antibody to TGFß before being added to recipient MCF10A-Neo cultures (•). Control cultures received fresh medium ({blacksquare}). Analyses of recipient culture cell numbers were made ~24–47 h later. Data represent means ± SD of three dishes. *Significantly less than corresponding cultures receiving fresh medium, P < 0.0004. **Significantly less than corresponding cultures receiving fresh medium or conditioned medium pretreated with TGFß antibody, P < 0.0003.

 


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  		Fig. 11. Cytostatic effects of TPA-conditioned medium on heterologous target cells. (A) Two-day-old MCF10A-NeoT feeder cultures were treated with DMSO or 10 nM TPA for 1 h prior to being washed and re-fed. Conditioned media were isolated 4 h later and transferred to MCF10A-Neo cultures. MCF10A-Neo cultures treated with nothing ({square}), DMSO-conditioned medium ({blacksquare}) or TPA-conditioned medium ({blacktriangleup}). (B) Two-day-old MCF10A-Neo feeder cultures were treated with DMSO or 10 nM TPA for 1 h prior to being washed and re-fed. Conditioned media were isolated 4 h later and transferred to MCF10A-NeoT cultures. MCF10A-NeoT cultures treated with DMSO-conditioned medium ({blacksquare}) or TPA-conditioned-medium ({blacktriangleup}). Data represent means ± SD of analyses of three dishes. *Significantly less than non-treated group or DMSO-conditioned medium group, P <= 0.04.

 
Influence of the ER on the antiproliferative activity of TPA
The MCF10A line, unlike MCF10A-NeoT cells, does not express the ER (38). ER status has been reported to influence the effects of TPA on human breast cell invasiveness, morphology and chemotaxis (39). In order to determine if ER status modulates the cytostatic effects of TPA, we examined the effects of TPA on the growth of clonal derivatives of MCF10A engineered to express the ER gene. 139B6 cells express a transduced human ER gene whereas 139-2-8 cells are stably transfected with just the expression vector (32). TPA transiently inhibited the proliferation of both cell lines to about the same extent (Figure 12Go). Hence, the differential responses of MCF10A-Neo and -NeoT cultures to TPA probably do not reflect differences in ER expression.



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  		Fig. 12. Influence of ER on anti-proliferative activity of TPA. Two-day-old cultures of ER-positive 139B6 cells (A) and ER-negative 139-2-8 cells (B) were exposed to DMSO ({blacksquare}) or 10 nM TPA ({blacktriangleup}) for varying lengths of time before being harvested for cell counts. Data represent means ± SD of analyses of three dishes. *Significantly less than corresponding DMSO treatment group, P <= 0.02.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
TPA was cytostatic to MCF10A-Neo cells. Several studies suggested that TGFß partially mediated some of this activity. First, supplementation of culture medium directly with a neutralizing pan antibody to TGFß suppressed the antiproliferative activity of TPA by ~50%. Second, immunodepletion of TGFß from TPA-conditioned medium prior to addition to target cells reduced the cytostatic activity of conditioned medium by ~50%. Third, supplementation of MCF10A-Neo culture medium with soluble, truncated rTGFßII prior to TPA addition suppressed the antiproliferative effects of TPA by ~50%. Hence, approximately half of the cytostatic effects of TPA or TPA-conditioned medium on MCF10A-Neo proliferation appear to be mediated by TGFß.

MCF-7 cultures, like MCF10A-Neo cells, undergo a G1 arrest following exposure to TPA (8). However, the development of cell cycle arrest occurs much slower in MCF-7 cells. Furthermore, the arrest is protracted, as opposed to transient (8,9,16), and accompanied by prolonged increases in TGFß mRNA and medium levels of activated TGFß (14,15,18). Although our studies implicate TGFß as a mediator of the cytostatic effects of TPA on MCF10A-Neo proliferation, the cells were not the source of TGFß. Instead, the TGFß was derived from latent TGFß supplied by the serum in the culture medium. This conclusion is based upon two observations. First, ELISA assays demonstrated that overall TGFß1 levels following TPA treatment never exceeded the amount of latent TGFß1 found in serum-supplemented medium. Second, the cytostatic effects of TPA were reduced ~50% (the amount that could be attributed to TGFß by a variety of criteria) if the culture medium was immunodepleted of latent TGFß prior to addition to the cultures.

Latent TGFß is activated by proteolysis (35–37). The cell adhesion molecule thrombospondin (40,41) and the protease plasmin (35,36) are capable of the appropriate proteolytic processing. However, plasmin normally exists in an inactive form (i.e. plasminogen) and needs to be activated by proteolysis. Two of the principal activators of plasminogen are the serine proteases tPA and uPA. Both are inhibited by the serine protease inhibitors aprotinin and PAI-1. Supplementation of MCF10A-Neo culture medium with either aprotinin or PAI-1 inhibited the cytostatic effects of TPA by ~50% (see Table IVGo). These inhibitor studies suggest that TPA treatment leads to the activation of pre-existing tPA or uPA or the de novo synthesis and subsequent activation of these proteases. Preliminary studies using a substrate recognized by both tPA and uPA and neutralizing antibodies to tPA/uPA show elevated uPA activity within 0.5–1 h of TPA treatment (M.Guo and J.J.Reiners, unpublished observation). Activation of uPA is not inhibited by cycloheximide. Furthermore, supplementing culture media with uPA antibody suppresses the cytostatic activity of TPA by ~50% (M.Guo and J.J.Reiners, unpublished observation). Presumably, activation of latent TGFß in TPA-treated MCF10A-Neo cells is mediated by a protease cascade involving uPA and plasminogen. We are currently characterizing the mechanism by which TPA increases uPA activity in MCF10A-Neo cultures.

TGFß accounted for only a portion of the cytostatic activity of TPA and TPA-conditioned medium. Human breast epithelial and/or myoepithelial cells make a variety of non-TGFß cytostatic factors, including activin and inhibin (19,42–44). These latter two cytokines are members of the TGFß supergene family and utilize the same SMAD proteins (vertebrate homologs to Sma and Mad gene products) required for signaling by TGFß (45–47). However, the three cytokines utilize different receptors (45,46). Susceptibility to these cytokines is dependent upon expression of the appropriate membrane receptors and there appears to be considerable variation amongst breast cell lines in expression of these receptors (19,42). MCF10A cells express the activin A receptor (42). We have found that supplementing the culture medium with a neutralizing antibody to activin A has no effect on the cytostatic activity of TPA (M.Guo and J.J.Reiners, unpublished observation). Hence, it seems unlikely that activin A contributes to the cytostatic activity of TPA. The identity of the non-TGFß factor(s) responsible for the cytostatic effects of TPA is still unknown.

MCF10A-NeoT cells constitutively produced and grew in a conditioned medium that was cytostatic to MCF10A-Neo cells. Immuno-neutralization studies suggested that TGFß was responsible for this cytostatic activity (see Figure 10BGo). Oncogenic p21 Ras increases the production of latent TGFß and stimulates its activation in some cell types (27–29). It also down-regulates expression of rTGFßII (19,30,31). We do not know if MCF10A-NeoT cells express rTGFßII. However, absence of the receptor would explain the ability of MCF10A-NeoT cells to proliferate in their own TGFß-containing conditioned medium and their resistance to the cytostatic effects of exogenous preparations of activated TGFß1 (M.Guo and J.J.Reiners, unpublished observation).

MCF10A-Neo and MCF10A-NeoT cells generated several cytostatic factors following TPA exposure. Specifically, MCF10A-Neo cultures produced a TPA-conditioned medium that transiently arrested the growth of both MCF10A-Neo and -NeoT cells. Since MCF10A-NeoT cells were refractory to the growth inhibitory effects of TGFß, a cytostatic factor other than TGFß must have been responsible for this activity. TPA-treated MCF10A-NeoT cultures produced a conditioned medium that suppressed MCF10A-NeoT proliferation for at least 72 h. The same conditioned medium only transiently arrested (<24 h) the growth of MCF10A-Neo cultures. Hence, the two cell lines differ markedly in their responses to the cytostatic factor(s) specifically produced by MCF10A-NeoT cultures. These studies suggest that MCF10A-Neo and MCF10A-NeoT cultures collectively produce/activate a minimum of three cytostatic factors following TPA treatment, including: (i) TGFß; (ii) the non-TGFß factor present in MCF10A-Neo conditioned medium; (iii) the non-TGFß factor produced by MCF10A-NeoT cells capable of inducing a protracted cell cycle arrest. It should be pointed out that the source of the latter two cytostatic factors is not known. They may be synthesized, excreted and activated by the MCF10A cell lines following TPA treatment. Conversely, they may be serum/medium components that exist in a latent form until activated by processes initiated by TPA treatment. To the best of our knowledge these are the first data to show: (i) that human breast cells produce multiple cytostatic agents following exposure to TPA; (ii) that oncogenic p21 Ras can modulate responses to cytostatic agents other than TGFß.

The biological activity of TGFß requires proteolytic processing of the latent, inactive form of the protein (35–37). Although several proteins are directly capable of activating latent TGFß, the processes which regulate their rapid in vivo activation are poorly understood. The studies described herein suggest that the MCF10A-Neo cell line may be an excellent model for the study of the components involved in latent TGFß activation and the signaling pathways that regulate the activities of these components.


   Notes
 
1 To whom correspondence should be addressed Email: john.reiners.jr{at}wayne.edu Back


   Acknowledgments
 
The authors thank Drs Michael McCabe Jr and Hyeong-Reh Kim for their critical reading of this manuscript. This research was supported by National Institutes of Health grant CA34469 and a Graduate Research Assistant Award provided by the Office of the Vice President, Wayne State University. This project was assisted by the Cell Culture Facility Core and the Cell Imaging and Cytometry Facility Core, which are supported by National Institutes of Environmental Health Sciences grant P30 ES06639.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received November 17, 1999; revised March 22, 2000; accepted March 29, 2000.


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