©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Effects of Tumor Necrosis Factor- on Antimitogenicity and Cell Cycle-related Proteins in MCF-7 Cells (*)

(Received for publication, May 4, 1995)

Doo-il Jeoung (1) Baiqing Tang (1) Martin Sonenberg (1) (2)(§)

From the  (1)Memorial Sloan-Kettering Cancer Center and the (2)Department of Medicine, Cornell University Medical College, New York, New York 10021

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Tumor necrosis factor-alpha (TNF-alpha) demonstrated antimitogenic activity in MCF-7 cells (estrogen receptor-positive human breast cancer cells) in a dose- and time-dependent manner (EC-50 of 2.5 ng/ml). This antimitogenic effect of TNF-alpha was accompanied by a decreased number of cells in S phase in a dose- and time-dependent manner. Based on growth arrest experiments using aphidicolin, it is apparent that TNF-alpha acted in early G(1) phase. It did not show antimitogenic effects once cells reentered the S phase based on [^3H]thymidine incorporation into DNA and cell cycle analysis. Specificity of TNF-alpha was established by using monoclonal anti-human TNF-alpha antibody. On the basis of Western immunoblot analysis of Rb, p53 and cell cycle inhibitory protein (Cip1) (p21) proteins, TNF-alpha decreased Rb protein expression in a dose- and time-dependent manner whereas it increased the expression level of tumor suppressor p53 protein. TNF-alpha also increased the expression level of Cip1 (p21) protein in a dose-dependent manner. This induction of Cip1 (p21) protein was preceded by the induction of p53 protein in MCF-7 cells. Cip1 (p21) protein associated with cyclin D was also increased. Tumor suppressor Rb protein expression was increased during G(1) to S phase progression. Cyclin D protein expression levels were not changed in response to TNF-alpha treatment, although serine/threonine kinase inhibitors such as H7 and the protein kinase C inhibitor staurosporine decreased cyclin D expression levels in MCF-7 cells. Based on experiments with staurosporine, it appears that TNF-alpha does not utilize a protein kinase C pathway in MCF-7 cells. Other cell cycle-related proteins such as Cdk2, Cdc2, and Cdk4 did not show any change in response to TNF-alpha. TNF-alpha did not affect complexes between cyclin D and Cdk2, Cdk4, and Rb proteins in MCF-7 cells. Taken together these results suggest that Rb, p53, and Cip1 (p21) proteins mediate TNF-alpha antimitogenic activity, and TNF-alpha induces growth arrest in the G(1) phase in MCF-7 cells.


INTRODUCTION

Tumor necrosis factor-alpha (TNF-alpha) (^1)is a macrophage-derived multi-functional cytokine that acts as a cytostatic or cytotoxic agent in many transformed and normal cells (1, 2, 3, 4, 5) . There have been reports of functional roles of TNF-alpha such as phosphorylation of epidermal growth factor receptor (6, 7) and cellular molecules, notably hsp27 and hsp70 protein(8, 9, 10, 11, 12, 13, 14, 15, 16) . Overexpression of hsp27 and hsp70 proteins protect cells against the cytotoxic effects of TNF-alpha(12, 17, 18) . It also induces phosphorylation of NF-KB (19) and the eukaryotic initiation factor EIF 4E(20) . The cellular responses to TNF-alpha are necrotic or apoptotic killing, DNA fragmentation, changes in arachidonic acid metabolism, lipid peroxidation, inhibition of mitochondrial electron transfer(21) , and increased production of prostaglandin E(22) . Although TNF-alpha is a well known antimitogenic agent, the mechanisms explaining these pleiotropic effects have not been well understood.

Rb protein is known to be cell cycle-regulated(23, 24, 25, 26, 27, 28) . This protein has tumor suppressing activity when the wild type Rb gene is transfected into transformed mammalian cells, which do not express functional Rb protein. In MCF-7 cells, as well as in osteocarcomas, Rb protein was shown to be abnormally expressed(29, 30) . The hypophosphorylated form of Rb protein acts as a functional tumor suppressor(30) . This hypophosphorylated Rb protein is associated with oncoproteins such as SV40 T antigen and adenovirus E1A protein, indicating their involvement in cellular transformation(31) . Phosphorylation of Rb protein is dependent on the growth state of cells (23, 26) , and antimitogenic agents or mitogenic agents exert their effects by changing the phosphorylation state of Rb protein(32) .

As with Rb protein, cyclins are known to be cell cycle-related (33, 34, 35, 36, 37, 38, 39) . Levels of cyclin-associated histone H1 kinase activities are also cell cycle-regulated(35, 37, 39, 40) . Some of these cyclins are known to have oncogenic activities associated with Rb activity, and a shortened G(1) phase(41) . These cyclins are associated with other cell cycle-regulated proteins such as Cdc2 and Cdk2 in many mammalian cells(42) . Complexes between Rb and these cyclins are assumed to regulate Rb protein functions in many mammalian cells(33, 43, 44, 45, 46) .

Cdc2 and Cdk2 proteins are known to phosphorylate Rb protein both in vitro and in vivo(47, 48) . This phosphorylation of Rb protein is believed to be achieved by physical association of these proteins and cyclins.

Tumor suppressor cell cycle inhibitory protein (Cip1) (p21) was found to be a potent inhibitor of cyclin-dependent kinases(50) . It is known to be associated with various cyclin-Cdk complexes. The main role of Cip1 (p21) protein is to inhibit cyclin-dependent kinase activities. Cip1 (p21) protein is known to be induced in mammalian cells undergoing G(1) arrest or apoptosis(51) . G(1) arrest is linked to p53 gene induction, and it is believed that induction of p53 in turn induces expression of Cip1 (p21) protein. The fact that oncogenes such as SV40 T antigen inhibits induction of Cip1 (p21) protein suggests that Cip1 (p21) protein might act as an anti-oncogene.

We investigated the antimitogenic effects of TNF-alpha in MCF-7 cells as determined by expression levels of cell cycle-related proteins such as Rb, Cip1 (p21), p53, and cyclins to extend our understanding of the mechanisms of TNF-alpha action in MCF-7 cells. We found by cell cycle analysis that TNF-alpha acted during the G(1) phase of the cell cycle. This was associated with a decrease in the expression of Rb and increased expressions of Cip1 (p21) and p53. There was no effect on cyclin D1, Cdk2, or Cdk4 proteins.


MATERIALS AND METHODS

Cell Culture and Reagents

MCF-7 (estrogen receptor-positive human breast cancer) cells were cultured in a humidified air atmosphere (5% CO(2)) and Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS. Human recombinant TNF-alpha was purchased from Sigma. Monoclonal antibodies to human cyclin A, cyclin B, Rb protein, and TNF-alpha were purchased from Upstate Biotechnology, Inc. (UBI, Lake Placid, NY). Polyclonal antibodies to human Cdc2, Cdk2, Cdk4, and cyclin D were also purchased from UBI. Polyclonal rabbit anti-human Cip1 (p21) Ab was purchased from Santa Cruz Biotech (Santa Cruz, CA). Monoclonal anti-human Rb antibody was purchased from Pharmingen (San Diego, CA). Aphidicolin and propidium iodide were purchased from Sigma and RNase A (Bovine) was purchased from Boehringer Mannheim. ECL (enhanced chemiluminescence) kit was purchased from Amersham Corp. [^3H]Thymidine was purchased from DuPont NEN. H7 and H8 were purchased from Sigma. Staurosporine was purchased from UBI.

[^3H]Thymidine Incorporation Assay

MCF-7 cells (2 10^4/well) were incubated with TNF-alpha for various time intervals. [^3H]Thymidine (20 µCi/ml) was added to each well for 1 h, washed with 1 HBSS (Hanks' buffered salt solution) three times, and cell lysates were prepared with 0.5% (w/v) SDS solution. To the latter was added 20% trichloroacetic acid (v/v) solution and incubation continued for 30 min on ice. Trichloroacetic acid-precipitated samples were washed with 20% trichloroacetic acid (v/v), 10% trichloroacetic acid (v/v), and 90% ethanol (v/v) successively, and filters were dried. Radioactivity was determined in a scintillation counter. [^3H]Thymidine incorporation data were normalized with cell counts and protein concentration of each sample.

Cell Cycle Analysis

MCF-7 cells treated with various concentrations of TNF-alpha were trypsinized. Cell pellets were resuspended in 1 ml of HBSS buffer, and were fixed by HBSS buffer containing 70% (v/v) ethanol for 1 h at 4 °C. Cells were centrifuged at 1,000 rpm for 10 min. Cell pellets were resuspended in HBSS buffer containing 50 µg/ml RNase A and 50 µg/ml propidium iodide. Incubation continued for 1 h at room temperature. Cells were filtered through nylon mesh (41 µm) and DNA content was measured in an Epics II flow cytometer. Propidium iodide staining does not distinguish G(0) cells from G(1).

Western Blot Analysis

Cell lysates were prepared from MCF-7 cells treated with TNF-alpha by extraction buffer (20 mM Hepes (pH 7.2), 1% Triton X-100 (v/v), 10% glycerol (v/v), 2 mM sodium fluoride, 1 mM sodium orthovanadate, 50 µg/ml leupeptin, and 0.5 mM phenylmethylsulfonyl fluoride). Cell lysates were cleared by centrifugation at 15,000 rpm for 30 min in a microcentrifuge and were loaded for SDS-PAGE. Samples were transferred to nitrocellulose filters by electroblot transfer for 2 h at 200 mA. The nitrocellulose filter was incubated with blocking buffer (1 TBS (Tris-buffered saline), Fraction V 3% BSA (w/v), 0.2% (v/v) Tween 20) for 1 h at room temperature and washed with 1 TBS buffer for 20 min. The nitrocellulose filter was incubated with primary antibody and dissolved in blocking buffer overnight at 4 °C (anti-human Rb Ab was used at a concentration of 1 µg/ml). On the following day the nitrocellulose filter was washed with 1 TBS for 20 min and incubated with either anti-mouse or anti-rabbit horseradish peroxidase conjugated with IgG for 1 h at room temperature. Secondary antibody was at a concentration of 1:1000 dilution. After washing with 1 TBS for 20 min at room temperature, detection of proteins of interest was carried out by the ECL method.

For immunoprecipitation, confluent MCF-7 cells (10^6/100-mm dish) were lysed and cell lysates were immunoprecipitated with polyclonal anti-human cyclin D Ab conjugated with protein A-Sepharose. Reaction was carried out at 4 °C for 4 h on a rotary shaker. Immune complexes were washed three times with lysis buffer, and 2 sample buffer was added to the beads. Boiled samples were loaded for SDS-PAGE. Western immunoblot analyses were followed according to standard procedures.


RESULTS

Antimitogenicity of TNF-alpha in MCF-7 Cells

To determine whether TNF-alpha has antimitogenic effects on MCF-7 cells, various concentrations of TNF-alpha were added to MCF-7 cells for 24 h at 37 °C and [^3H]thymidine incorporation into DNA was carried out. The antimitogenic effect of TNF-alpha was dose-related as shown in Fig. 1. TNF-alpha did not show any effect at low concentrations but showed clear antimitogenic effects at concentrations exceeding 0.5 ng/ml. TNF-alpha did not show mitogenic activity at any concentration. The antimitogenic effect was also seen in MCF-7 cells grown under SFM conditions, suggesting that the antimitogenic effect is due not to serum factors but to direct antimitogenic effects of TNF-alpha on MCF-7 cells. To determine the specificity of TNF-alpha, a blocking experiment using monoclonal anti-human TNF-alpha Ab was carried out. Briefly, TNF-alpha (10 ng/ml) was preincubated with various concentrations of monoclonal anti-human TNF-alpha Ab at 37 °C for 1 h before addition to each well. The next day [^3H]thymidine incorporation into DNA was carried out. As shown in Table 1, the antimitogenic effect of TNF-alpha was efficiently blocked by an excess concentration of monoclonal anti-human TNF-alpha Ab while antibody itself did not show any effect on MCF-7 cell growth. Table 2shows that this inhibition of TNF-alpha action is consistent with the cell cycle analysis of MCF-7 cells treated with TNF-alpha (10 ng/ml) alone or in combination with anti-TNF-alpha Ab (500 ng/ml).


Figure 1: Dose response of TNF-alpha in MCF-7 cells. Exponentially growing MCF-7 cells (10^5 cells/well) were treated with various concentrations of TNF-alpha at 37 °C. [^3H]thymidine incorporation into DNA was carried out at 24 h.







To determine whether antimitogenicity of TNF-alpha is cell cycle-related, FACS (fluorescence-activated cell sorter) analysis was carried out. As shown in Fig. 2A, TNF-alpha changed the percentage of cells in S phase. The decrease in the fraction of cells in S phase was dose-dependent, as evidenced by larger changes in response to higher concentrations of TNF-alpha. Changes in cell cycle phases were also time-dependent (data not shown). These results suggest that TNF-alpha might prevent cells from entering S phase. Based on these results, it is likely that TNF-alpha acts at the G(1)/S phase boundary. To prove this point, MCF-7 cells were growth-arrested at the G(1)/S boundary by aphidicolin treatment (5 µg/ml) for 24 h, washed with 1 HBSS buffer, and refed with 10% FCS/DMEM. At each interval after release from aphidicolin growth arrest, TNF-alpha (10 ng/ml) was added. MCF-7 cells were harvested for cell cycle analysis, and cell lysates for [^3H]thymidine incorporation into DNA. As shown in Fig. 2B, TNF-alpha added at 0, 3, 6, and 12 h after release from aphidicolin treatment showed antimitogenic effects, but not at 20 h. This result suggests that TNF-alpha acts in the G(1) phase but not after cells reenter the S phase. This suggests that TNF-alpha does not offset the mitogenic stimulation of serum. The antimitogenic effect of TNF-alpha was more apparent in a synchronized population of MCF-7 cells. To determine whether the blocking effect of TNF-alpha by antibody is also accompanied by changes in cell cycle phases, growth arrest experiments using aphidicolin were carried out as before. As shown in Table 1, monoclonal anti-human TNF-alpha Ab itself did not affect cellular growth, suggesting that endogenous TNF-alpha did not play a role in MCF-7 cell growth. TNF-alpha added at 0, 3, 6, and 10 h but not 20 h showed antimitogenic effects. When TNF-alpha was added along with monoclonal TNF-alpha Ab, the latter efficiently inhibited the antimitogenic effect of TNF-alpha.


Figure 2: A, temporal profile of TNF-alpha action in MCF-7 cells. MCF-7 cells (10^6 cells/100-mm dish) were treated with various concentrations of TNF-alpha for 16 h. Cell cycle analysis was carried out according to standard procedures. Columns a-f refer to observations made at different concentrations of TNF-alpha of 0, 1, 5, 10, 50, and 100 ng/ml. B, temporal profile of TNF-alpha action in MCF-7 cells. MCF-7 cells were growth-arrested by aphidicolin treatment (5 µg/ml) for 24 h, washed with 1 HBSS buffer extensively, and refed with 10% FCS/DMEM. TNF-alpha (10 ng/ml) was added at the indicated times. FACS analysis was carried out according to standard procedures.



Effects of TNF-alpha on Cell Cycle-related Proteins

Based on the previous results of the antimitogenic effects of TNF-alpha, it was necessary to determine whether TNF-alpha affects expression levels of cell cycle-regulated proteins. For this purpose, Western blot analysis using monoclonal anti-human Rb Ab was carried out. MCF-7 cells were treated with aphidicolin for 16 h, washed with 1 HBSS buffer, and incubated with 10% FCS/DMEM. TNF-alpha (10 ng/ml) was added at each time point, and cell lysates were prepared according to standard procedures. As shown in Fig. 3, TNF-alpha added at 0, 3, 6, 12, and 20 h after release showed decreased expression levels of Rb proteins. This result suggests that the decreased DNA synthesis seen in Fig. 1is consistent with decreased expression levels of Rb protein in response to TNF-alpha. This result also suggests that Rb protein is important for G(1)/S transition in the cell cycle. Based on these results, it is plausible that TNF-alpha acts, in part, by decreasing Rb tumor suppressor protein in MCF-7 cells.


Figure 3: TNF-alpha acts at G(1) phase to decrease Rb expression. Normally growing MCF-7 cells (10^6/100-mm dish) were growth-arrested by incubation with aphidicolin (5 µg/ml) for 16 h, washed with 1 HBSS buffer three times, and refed with 10% FCS/DMEM. TNF-alpha was subsequently added at the indicated times after aphidicolin release and harvesting at 24 h. Thus cells were exposed to TNF-alpha for 24, 21, 18, 12, and 4 h, respectively. Western blot using monoclonal anti-human Rb Ab (1 µg/ml) was carried out according to standard procedures.



TGF-beta, a well known antimitogenic agent in many other mammalian cells, did not manifest any antimitogenic activity or induce decreased Rb protein levels in MCF-7 cells (data not shown). Therefore, it is likely that TNF-alpha activity is not mediated by TGF-beta, as was the case with interferon-alpha in human Burkitt lymphoma Daudi cells(32) . G(1) cyclins such as cyclin D did not show any change in expression levels in response to TNF alpha in MCF-7 cells. Cdk4 protein did not respond to TNF-alpha under these conditions. To determine whether cyclin D protein reflects the growth state of the MCF-7 cells, normally proliferating MCF-7 cells were treated with TNF-alpha (10 and 25 ng/ml), Bt(2)cAMP (1 mM), or staurosporine (1 nM) for 24 h and Western immunoblot analysis using polyclonal anti-human cyclin D Ab (1 µg/ml) was employed. As seen in Fig. 4, there was no significant reduction in the expression level of cyclin D in response to TNF-alpha. However, in response to H7 as well as staurosporine, cyclin D expression levels were significantly reduced. H7 and staurosporine were found to be strong antimitogenic agents in MCF-7 cells (data not shown). Cdk4 protein levels remained constant regardless of the treatments.


Figure 4: Growth-regulated expression of cyclin D1 in MCF-7 cells. Normally proliferating MCF-7 cells (10^6/100-mm dish) were treated with various concentrations of TNF-alpha (10, 25 ng/ml), Bt(2)cAMP (1 mM), and staurosporine (1 nM) for 24 h, and cell lysates were prepared for Western immunoblot analysis. Polyclonal anti-human cyclin D Ab was used at a concentration of 1 µg/ml.



Fig. 5shows that TNF-alpha-mediated regulation of Rb protein expression is specific in nature, as preincubation of TNF-alpha with monoclonal anti-human TNF-alpha Ab prevented TNF-alpha antimitogenicity in MCF-7 cells (lanes3, 5, 7, 9, and 11). This result suggests that endogenous TNF-alpha does not regulate Rb protein expression. This figure also shows that TNF-alpha did not have an effect on Rb protein expression once cells enter S phase (lane13), suggesting an important role of Rb protein in G(1)/S transition in the cell cycle of MCF-7 cells.


Figure 5: Specificity of TNF-alpha activity in MCF-7 cells. Normally proliferating MCF-7 (10^6/100-mm dish) cells were treated with aphidicolin (5 µg/ml) for 16 h, washed with 1 HBSS buffer, and refed with 10% FCS/DMEM. TNF-alpha was added at the indicated times. For the blocking experiment, TNF-alpha (10 ng/ml) was preincubated with monoclonal anti-human TNF-alpha Ab (500 ng/ml) for 1 h at 37 °C before adding to cells. Western immunoblot analysis using monoclonal anti-human Rb Ab (1 µg/ml) was carried out according to standard procedures. Detection of Rb protein was done by the ECL method. Columns1-13 correspond to the experimental conditions indicated below the numbers.



To determine which cellular molecules are important for cell cycle progression, cell synchronization studies were carried out. Normally proliferating MCF-7 cells (10^6/100-mm dish) were treated with aphidicolin (5 µg/ml) for 12 h, washed with 1 HBSS buffer extensively and refed with 10% FCS/DMEM. At each time after release from growth arrest by aphidicolin, cells and cell lysates were harvested for Western blot and FACS analysis. As shown in Fig. 6, tumor suppressor Rb protein showed induction at late G(1) phase as expected. This induction of Rb protein remained through S phase and then declined to normal levels. Cyclin D protein was induced in late G(1) phase and remained at high expression levels through the S phase. Cdk4 protein was similarly induced in the G(1) phase and remained elevated during S phase and subsequent cycles. Well known mitotic cyclin A showed expression patterns similar to that of Rb protein.


Figure 6: Kinetics of induction of cell cycle-regulated proteins in MCF-7 cells. Normally proliferating MCF-7 cells (10^6/100-mm dish) were treated with aphidicolin (5 µg/ml) for 10 h, washed with 1 HBSS buffer extensively, and refed with 10% FCS/DMEM. At each time after release from growth arrest by aphidicolin, cell lysates and cells were harvested for Western blot and FACS analysis. Monoclonal anti-human p53 Ab was used at a concentration of 0.5 µg/ml.



Fig. 7A shows that activation of protein kinase A by Bt(2)cAMP or inhibition of protein kinase C by staurosporine decreased Rb protein expression in MCF-7 cells. These results suggest that Rb expression might be regulated by phosphorylation, and Rb protein expression reflects the growth state of the MCF-7 cells.


Figure 7: Growth-regulated expression of Rb protein in MCF-7 cells. A, confluent MCF-7 cells (10^6/100-mm dish) were treated with TNF-alpha, staurosporine (1 nM) or Bt(2)cAMP (1 mM) separately for 16 h. Monoclonal anti-human Rb Ab was used at a concentration of 1 µg/ml. B, confluent MCF-7 cells (10^6/100-mm dish) were treated with H7 and Western immunoblot analysis using monoclonal anti-human Rb Ab was carried out.



Fig. 7B shows that the serine/threonine kinase inhibitor H7 causes dephosphorylation of Rb protein in MCF-7 cells. This result suggests that Rb phosphorylation is achieved by serine/threonine kinases in MCF-7 cells. This is consistent with the previous results that showed phosphorylation of Rb protein at serine/threonine residues.

To determine whether TNF-alpha affects the expression level of another tumor suppressor Cip1 (p21) protein, MCF-7 cells were treated with TNF-alpha for 16 h and Western immunoblot analysis using polyclonal rabbit anti-human Cip1 (p21) Ab was carried out. As shown in Fig. 8, there was a significant increase in Cip1 (p21) protein expression level in response to TNF-alpha treatment. This is surprising in that Cip1 (p21) protein is known to be induced by DNA damaging agents such as UV and adriamycin, and requires functional p53 protein. This result suggests that Cip1 (p21) protein plays a major role in mediating antimitogenic effects of TNF-alpha in MCF-7 cells. In addition to TNF-alpha, antimitogenic agents such as H7 and staurosporine also induced Cip1 (p21) protein expression in MCF-7 cells (data not shown). MCF-7 cells have readily detectable amounts of p53 protein and are also known to have functional p53 protein(42) . To check whether TNF-alpha employs any specific kinase pathway, TNF-alpha was added to the MCF-7 cells with staurosporine or Bt(2)cAMP under SFM conditions and [^3H]thymidine incorporation assays were carried out according to standard procedures. Neither Bt(2)cAMP nor staurosporine was able to block the antimitogenic effect of TNF-alpha in MCF-7 cells (data not shown).


Figure 8: TNF-alpha induces Cip1 (p21) protein in MCF-7 cells. Confluent MCF-7 cells (10^6/100-mm dish) were treated with TNF-alpha (10 and 25 ng/ml each) for 16 h under SFM conditions. Polyclonal rabbit anti-human Cip1 (p21) Ab was used at a concentration of 0.5 µg/ml in Western immunoblot analysis.



Since Cip1 (p21) protein was induced in response to TNF-alpha treatment, it was of interest to determine whether Cip1 (p21) protein associated with cyclin D was also increased. To check this, 50 µg of cell lysates from MCF-7 cells treated with or without TNF-alpha (10 ng/ml) were immunoprecipitated with protein A-Sepharose conjugated with polyclonal anti-human cyclin D Ab and boiled samples were loaded onto 15% SDS-PAGE. As shown in Fig. 9, Cip1 (p21) protein interacts with cyclin D and the amount of Cip1 (p21) protein associated with cyclin D was increased in MCF-7 cells treated with TNF-alpha (10 and 25 ng/ml). Given the fact that cyclin D remained constant regardless of the treatment, it is possible that the decreased expression level of Rb protein in response to TNF-alpha is due to the induction of Cip1 (p21) protein. Cip1 (p21) protein is believed to be a part of cyclin D complex and an inhibitor of cyclin-associated kinase activity. To check this possibility, MCF-7 cells were treated with TNF-alpha (10 ng/ml) for 16 h and Western immunoblot analysis was carried out. Fig. 10A shows that TNF-alpha induced both p53 and Cip1 (p21) proteins in MCF-7 cells.


Figure 9: Effect of TNF-alpha on Cip1 protein associated with cyclin D1 in MCF-7 cells. MCF-7 cells were treated with TNF-alpha (10 and 25 ng/ml) for 16 h, and 50 µg of total cell lysates were immunoprecipitated with protein A-Sepharose conjugated with polyclonal anti-human cyclin D Ab. Immunoprecipitated samples were loaded onto 15% SDS-PAGE, and Western immunoblot analysis using polyclonal anti-human cyclin D (1 µg/ml) or anti-human Cip1 (p21) Ab (1 µg/ml) was carried out.




Figure 10: A, induction of p53 correlates with Cip1 (p21) protein in MCF-7 cells. Confluent MCF-7 cells (10^6/100-mm dish) were treated with TNF-alpha for 16 h and Western immunoblot analysis was carried out. Monoclonal anti-human p53 and polyclonal anti-human Cip1 (p21) Ab were used at concentrations of 0.5 and 0.1 µg/ml, respectively. B, induction of p53 precedes that of Cip1 (p21) protein in MCF-7 cells. Confluent MCF-7 cells (10^6/100-mm dish) were treated with TNF-alpha (10 ng/ml) for various times and cell lysates were prepared at each time point. Western immunoblot analysis was carried out as in panelA.



Fig. 10B shows the kinetics of induction of p53 and Cip1 (p21) protein in MCF-7 cells. p53 protein showed earlier induction than that of Cip1 (p21) protein. This is consistent with the fact that p53 protein is a transcriptional activator and induction of Cip1 (p21) protein is closely related to p53 induction. These results suggest that induction of Cip1 (p21) protein by TNF-alpha is mediated through p53. To determine the cellular molecules that interact with cyclin D, and the possible effect of TNF-alpha on these interactions, MCF-7 cells treated with TNF-alpha (10 ng/ml) were immunoprecipitated with polyclonal anti-human cyclin D Ab conjugated with protein A-Sepharose. These immunoprecipitates were loaded, and Western immunoblot analysis was carried out. As shown in Fig. 11, TNF-alpha did not break complexes formed between cyclin D and Cdk2 or Cdk4. There was no complex formation between cyclin D and Rb protein in MCF-7 cells. These results suggest that the decrease in Rb protein in response to TNF-alpha might be due to the increase in Cip1 (p21) protein, which in turn inhibits cyclin D-associated kinase activity. This decreased kinase activity might play a role in mediating the antimitogenic effect of TNF-alpha. No effect on these complexes was noted with TNF-alpha (Fig. 12).


Figure 11: Molecular interaction between cyclin D1 and cellular molecules in MCF-7 cells. 50 µg of cell lysates prepared from MCF-7 cells treated with TNF-alpha (10 ng/ml) were immunoprecipitated with polyclonal anti-human cyclin D Ab conjugated with protein A-Sepharose. Immunoprecipitates were loaded for SDS-PAGE. Western immunoblot analysis was carried out. Monoclonal anti-human Rb, polyclonal anti-human Cdk2, and Cdk4 antibodies were used at concentrations of 1 µg/ml.




Figure 12: Effect of phosphatase on Rb phosphorylation. 50 µg of cell lysates were immunoprecipitated with monoclonal anti-human Rb Ab conjugated with protein A-Sepharose. Immunoprecipitated samples were treated with 100 units of calf intestine alkaline phosphatase for 30 min at 37 °C. Samples were boiled and were loaded for SDS-PAGE. Detection of Rb protein was done by Western immunoblot analysis using monoclonal anti-human Rb Ab (1 µg/ml).




DISCUSSION

We have found that TNF-alpha shows antimitogenic effects in a dose- and time-dependent manner in MCF-7 cells. TNF-alpha did not show cytotoxic effects seen in other transformed and normal mammalian cells(1, 2, 3, 4, 5) . TNF-alpha decreased the fraction of cells in the S phase in cell cycle analysis in a time- and dose-dependent manner. Based on cell synchronization studies, it seems apparent that TNF-alpha acted at the G(1) phase to prevent cells from entering S phase but did not show effects once cells entered S phase. This antimitogenic effect of TNF-alpha was efficiently blocked by excess concentrations of monoclonal anti-human TNF-alpha Ab in MCF-7 cells in a dose-dependent manner. This blocking effect was also confirmed by cell cycle analysis and Western immunoblot analysis of Rb protein. This blocking experiment also proves that TNF-alpha-mediated regulation of Rb protein expression is specific in nature because TNF-alpha preincubated with monoclonal anti-human TNF-alpha Ab did not decrease Rb protein expression in MCF-7 cells.

Since TNF-alpha specifically acts at the G(1) phase to prevent cells from entering S phase, it was necessary to check whether TNF-alpha affects cell cycle-regulated proteins in MCF-7 cells. Tumor suppressor Rb protein has been known to be involved in cellular growth and differentiation and exists as hypo- and hyperphosphorylated forms, depending on the growth state of the cells(49) . Its role in G(1) to S progression in the cell cycle has been well documented. Cell cycle-regulated proteins such as Rb protein showed decreases in response to TNF-alpha treatment in a time- and dose-dependent manner, whereas p53 and Cip1 (p21) protein expression levels increased in response to TNF-alpha. This result suggests that TNF-alpha-mediated antimitogenicity of MCF-7 cells specifically involves regulation of Rb and p53 protein expression. We did not detect two Rb protein bands frequently seen in normal mammalian cells. We assume that the Rb protein band seen in MCF-7 cells is hyperphosphorylated Rb protein and TNF-alpha acts by decreasing this hyperphosphorylated form of Rb protein. Supporting this is the fact that serine/threonine kinase inhibitor H7 increased mobility of Rb protein in MCF-7 cells (Fig. 7B).

G(1) cyclin, like cyclin D protein, did not show decreased expression levels in response to TNF-alpha treatment under normal culture conditions using asynchronous cells. H7 and Bt(2)cAMP decreased cyclin D expression. The expression level of cyclin D is increased at mid- or late G(1) phase based on cell synchronization studies. These results suggest that cyclin D protein expression is governed by growth states of MCF-7 cells. These results also suggest that other cell cycle-related proteins mediate the antimitogenic effect of TNF-alpha in MCF-7 cells. Mitotic cyclin such as cyclin A and B did not show apparent changes in their expression levels in response to TNF-alpha treatment (data not shown). Other cell cycle-related proteins such as Cdc2 (data not shown), Cdk2 (data not shown), and Cdk4 (Fig. 3) did not show any change in their expression levels in response to TNF-alpha treatment.

It is noteworthy that Cip1 (p21) protein was significantly induced in response to TNF-alpha treatment in MCF-7 cells. Cip1 (p21) protein associated with cyclin D was also increased in response to TNF-alpha in MCF-7 cells (Fig. 9). These results provide valuable information concerning the mechanism of TNF-alpha antimitogenicity in MCF-7 cells. The induction of Cip1 (p21) protein was preceded by p53 protein induction in MCF-7 cells (Fig. 10). This is consistent with the fact that Cip1 (p21) protein induction needs functional p53 protein and induction of p53 protein in response to DNA damaging agents. MCF-7 cells are known to have functional p53 protein(42) . The induction of Cip1 (p21) protein may affect the expression level of Rb protein in MCF-7 cells treated with TNF-alpha. Since Rb protein showed a decrease in response to TNF-alpha treatment, it was necessary to determine whether this is due to dissociation of cyclin D-Rb complex or a decreased cyclin D-associated kinase activity. There was no complex formation between cyclin D and Rb protein in MCF-7 cells. Therefore, it is not likely that decreased Rb protein expression in MCF-7 cells is due to breakdown of a cyclin D-Rb complex in MCF-7 cells. It was of further interest to determine the effect of TNF-alpha on molecular interactions between cyclin D and Cdk proteins since complexes between cyclins and Cdks have been shown to play a major role in phosphorylation of Rb protein in other mammalian cells. We detected complexes of cyclin D with Cdk2 or Cdk4. However, TNF-alpha did not have any effect on these complexes. The role of cellular oncogenes is not clear at present. Studies of these cellular oncogenes will provide further understanding of the mechanisms of TNF-alpha-mediated antimitogenicity in MCF-7 cells. Thus, it would appear that TNF-alpha acts to increase p53 protein which in turn induces Cip1 (p21) to decrease Rb protein expression with resultant antimitogenicity.


FOOTNOTES

*
This work was supported in part by Grant DK 41931 from the National Institutes of Health (to M. S.) and Grant CA 09512 from the Clinical Scholars Biomedical Research Training Program (to B. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: 1275 York Ave., New York, NY 10021. Tel.: 212-639-7878; Fax: 212-717-3053.

^1
The abbreviations used are: TNF-alpha, tumor necrosis factor-alpha; Cip1, cell cycle inhibitory protein; DMEM, Dulbecco's modified Eagle's medium; FACS, fluorescence-activated cell sorter; FCS, fetal calf serum; H7, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine; H8, N-(2-methylaminoethyl)-5-isoquinolinesulfoneamide; HBSS, Hank's balanced salt solution; PAGE, polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; TGF-beta, transforming growth factor-beta; SFM, serum-free medium.


ACKNOWLEDGEMENTS

We are grateful to Wayne Douglas for technical assistance. We thank Sigrid Whaley for help in preparation of the manuscript.


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