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[Cancer Research 64, 4155-4161, June 15, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Involvement of Interferon Regulatory Factor 1 and S100C/A11 in Growth Inhibition by Transforming Growth Factor ß1 in Human Hepatocellular Carcinoma Cells

Masahiro Miyazaki1, Masakiyo Sakaguchi1, Ichiro Akiyama1, Yoshihiko Sakaguchi2, Seishi Nagamori3 and Nam-ho Huh1

Departments of 1 Cell Biology and 2 Bacteriology, Okayama University Graduate School of Medicine and Dentistry, Okayama, and 3 Department of General Medicine, Kyorin University School of Medicine, Tokyo, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Growth inhibition by transforming growth factor (TGF)-ß1 has been attributed to the induction of cyclin-dependent kinase inhibitors, among which p21/Waf1 plays a major role in many biological contexts. In the present study, two new intracellular mediators for the induction of p21/Waf1 by TGF-ß1 were identified in a human hepatocellular carcinoma cell line (JHH-5) expressing mutant-type p53. After addition of TGF-ß1 to JHH-5 cells, a marked increase of the p21/Waf1 expression preceded the inhibition of DNA synthesis. Expression of IFN regulatory factor (IRF)-1, a known transacting factor for p21/Waf1 promoter, was elevated just before or in parallel with the increase of p21/Waf1. Transduction of antisense IRF-1 inhibited the increase in p21/Waf1 in JHH-5 cells treated with TGF-ß1 and partially released the cells from the growth arrest by TGF-ß1. Expression of S100C/A11, a member of the Ca2+-binding S100 protein family, also markedly increased after addition of TGF-ß1. S100C/A11 protein was translocated to and accumulated in nuclei of TGF-ß1-treated JHH-5 cells, where p21/Waf1 was concomitantly accumulated. When a recombinant S100C/A11 protein was introduced into nuclei of JHH-5 cells, DNA synthesis was markedly inhibited in a dose-dependent manner in the absence of TGF-ß1. Prior transfection of p21/Waf1-targeted small interfering RNA efficiently blocked decrease of DNA synthesis in JHH-5 cells caused by TAT-S100C/A11 or TGF-ß1 and markedly inhibited expression of p21/Waf1 protein in the cells. These results indicate that IRF-1 and S100C/A11 mediate growth inhibition by TGF-ß1 via induction of p21/Waf1.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Transforming growth factor (TGF)-ß is a pleiotropic cytokine and elicits a broad range of cellular responses, including the regulation of cell growth, differentiation, matrix production, and apoptosis. Among these, growth inhibition by TGF-ß for epithelial cells, endothelial cells, and hematopoietic cells has been of central interest because it may be instrumental in preventing malignant conversion of cells in the body (1) . Indeed, tumor cells of diverse tissue origins lose their sensitivity to TGF-ß-induced growth inhibition during the steps of malignant transformation (2 , 3) .

TGF-ß exerts its various effects via two transmembrane serine/threonine kinases known as type I and type II receptors. During the latter half of the 1990s, understanding of the intracellular pathways by which TGF-ß signals are mediated has been advanced by studies of the Smad family of signal transducers (4) . The ligand-activated type II receptor associates with, phosphorylates, and activates the type I receptor, which in turn phosphorylates pathway-specific Smad 2 and Smad 3. These activated Smads then associate with Smad 4 and translocate to the nucleus, where they regulate transcription by associating with nuclear transcription factors and/or by binding directly to DNA. The inhibitory Smad 6 and Smad 7 target the first step in the intracellular transduction pathway, namely, phosphorylation of the pathway-specific Smads by the type I receptor (4) .

The effectors of TGF-ß-induced growth inhibition are cyclin-dependent kinase inhibitors (CKIs), among which p21/Waf1 plays a major role in many biological contexts (5) . Sp1, a ubiquitous transcription factor, participates in the regulation of the p21/Waf1 gene by TGF-ß (6) . The TGF-ß signal transducers Smad 3 and Smad 4 functionally cooperate with Sp1 to activate the p21/Waf1 promoter (7) . There is a GC-rich Sp1-binding element in the proximal region of the p21/Waf1 promoter for transcriptional activation (8) . Smads themselves do not bind to the proximal Sp1-binding element but exert a transcription-enhancing effect by binding to Sp1 and increasing the affinity of Sp1 to the element (8) . Jun family proteins are also known to mediate TGF-ß signaling to activate p21/Waf1 through interaction with Sp1 (9) . Thus, Sp1 is a key molecule and absolutely necessary for the transcriptional activation of p21/Waf1 by TGF-ß (6, 7, 8, 9) .

As described above, the involvement of Smad, Jun family proteins, and Sp1 in the induction of p21/Waf1 by TGF-ß has been well studied, but there may still be other intracellular mediators for CKI induction by TGF-ß. In human gingival fibroblasts, p38 mitogen-activated protein kinase was shown to mediate TGF-ß-induced transcriptional activation of collagen type 3 gene (10) . Down-regulation of phospholipase C or protein kinase C activity resulted in blocking of activation of a TGF-ß-responsive element (11) . The Smad pathway itself is modulated by a number of proteins including SARA (Smad anchor for receptor activation), which functions during the initiation of signaling, and Smurf1, which can negatively regulate Smad signaling (12) .

In the present study, we examined two possible candidate proteins, IFN regulatory factor (IRF)-1 and S100C/A11, for being involved in the TGF-ß-signaling, which emerged as such through our previous studies. IRF-1 was originally found as a regulator of the IFN system (13) , but later revealed to function as a transacting factor involved in cell growth regulation and apoptosis (14 , 15) . IRF-1 transcriptionally activates p21/Waf1 in cooperation with p53 in mouse embryonic fibroblasts (16) . We showed that p21/Waf1 was up- and down-regulated by TGF-ß1 in a human cholagiocarcinoma cell line and normal human fibroblasts, respectively, and that the transcriptional state of p21/Waf1 gene paralleled with IRF-1 mRNA levels (17) . S100C/A11 (calgizzarin) is a member of the Ca2+-binding S100 protein family with an EF-hand domain (18) . Recently, we found that S100C/A11 mediates growth inhibition of normal human fibroblasts at confluence (19) and that of normal human keratinocytes induced by high Ca2+ (20) . On exposure of the cells to the growth inhibitory conditions, cytoplasmic S100C/A11 protein was specifically phosphorylated, bound to nucleolin, and transferred to nuclei. In the nuclei, S100C/A11 liberated Sp1/3 from nucleolin, and the resulting free Sp1/3 transcriptionally activated p21/Waf1. Based on these observations, we examined in the present study whether IRF-1 and S100C/A11 are involved in the TGF-ß-signaling to induce p21/Waf1 in a human hepatoma cell line, JHH-5 (21) .


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and Culture.
Human hepatocellular carcinoma (HCC) cell lines HLE and HuH-7 and JHH-4, JHH-5, and JHH-6 were established and maintained at the Department of Cell Biology, Okayama University Graduate School and at the Department of General Medicine, Kyorin University School of Medicine, respectively (21 , 22, 23, 24, 25) . The human HCC cell line PLC/PRF/5 was obtained from American Type Culture Collection (Rockville, MD). A human embryonic lung fibroblast cell strain TIG-7 (26) and an immortalized human keratinocyte line HaCat (27) were kind gifts from Dr. Kaji (University of Shizuoka, Japan) and Dr. Fusenig (German Cancer Research Center, Heidelberg, Germany), respectively. These cells were seeded at a density of 2.5 x 104 cells/cm2 in DMEM (Nissui Paharmaceutical, Tokyo, Japan) containing 10% fetal bovine serum (Intergen, Purchase, NY) and 100 µg/ml kanamycin. The culture medium was renewed 1 d after seeding and thereafter as indicated.

TGF-ß1 Treatment.
TGF-ß1 from porcine platelets (R&D, Minneapolis, MN) was added at concentrations of 0.01–5 ng/ml to the human HCC cell cultures 24 h after cell seeding, unless otherwise indicated. After addition of TGF-ß1, no medium change was carried out.

Measurement of [3H]Thymidine Incorporation.
For measurement of DNA synthesis, cells on 24-well plates were labeled for 2–24 h with 1 µCi/ml/well [methyl-3H]thymidine ([3H]TdR; 71 Ci/mmol; ART-178B; American Radiolabeled Chemicals Inc., St. Louis, MO) before cell harvest. After radiolabeling, the cells were washed in situ twice each with ice-cold PBS, 5% trichloroacetic acid, and 95% ethanol. The cells were then lysed with 200 µl of 0.3 N NaOH. Aliquots of the cell lysates were neutralized with HCl, and the radioactivity was measured in a liquid scintillation counter.

Fluorescence-Activated Cell Sorter Analysis of Cell Cycle.
JHH-5 cells were harvested by trypsin digestion 1 and 4 days after seeding, pelleted and washed with PBS, and resuspended in 0.2% Triton X-100 in PBS, followed by gentle pipetting and filtration through Cell-Strainer (Becton Dickinson Labware, Franklin Lakes, NJ). Propidium iodide and RNase A were added to the cell suspensions at concentrations of 37.5 and 50 µg/ml, respectively. Samples were then subjected to fluorescence-activated cell sorting.

Northern Blot Analysis of Gene Expression.
At various times after addition of TGF-ß1, total RNA was isolated from JHH-5 cells by the guanidinium thiocyanate-phenol method and stored at –80°C until Northern blot analysis, which was carried out essentially as reported previously (28) .

cDNA Probes.
The following human cDNA probes were used: p53, obtained from Dr. H. Saya (Department of Tumor Biology and Pathology, Graduate School of Medicine, Kyoto University, Japan); p21/Sdi1/Waf1, obtained from Dr. A. Noda (Department of Radiation Biophysics and Genetics, Kobe University School of Medicine, Japan); and cyclins A, D1, and E, obtained from Dr. M. Ohtsubo (Division of Molecular Genetics, Institute of Life Science, Kurume University, Japan). The human S100C/A11 cDNA used was described previously (29) .

Other human cDNA fragments were prepared by reverse transcription-PCR. Total RNA was isolated from TIG-7 human embryonic lung fibroblasts (26) by the guanidinium thiocyanate-phenol method, and 1 µg of RNA was used for cDNA synthesis. The resulting products were amplified under the following conditions: initial incubation at 94°C for 4 min followed by 30 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s; and then a final step at 72°C for 5 min. The primers and expected length of products were as follows: [E2F-1], 774 bp; sense strand, 5'-ccaggaggtcacttctgagg-3'; antisense (AS) strand, 5'-ggccgaaagtgcagttagag-3'; [p27/Kip1], 748 bp; sense strand, 5'-ggcctcagaagacgtcaaac-3'; AS strand, 5'-tttttgccccaaactacctg-3'; [IRF-1], 552 bp; sense strand, 5'-cctgatgaccacagcagcta-3'; AS strand, 5'-caggtcctgcttgcctagag-3'; and [glyceraldehyde-3-phosphate dehydrogenase (GAPDH)], 694 bp; sense strand, 5'-cagcctcaagatcatcagca-3'; AS strand, 5'-tgaggaggggagattcagtg-3'.

GAPDH was used as an internal control. The amplified products were purified using a QIAquick Gel Extraction kit (Qiagen Science, German Town, MD) after electrophoresis on 1% agarose gel, and their sequences were confirmed by direct sequencing.

p53 Sequencing Analysis.
Total RNA was isolated from JHH-5 cells at semiconfluence by the guanidine-thiocyanate-phenol method, and 1 µg of RNA was used for cDNA synthesis. The resulting products were used for amplification of p53 cDNA (1,182 bp) identical to the coding region of the gene with the primers (sense strand, 5'-atggaggagccgcagtcagatccta-3'; AS strand, 5'-tcagtctgagtcaggcccttctgtc-3') under the following conditions: initial incubation at 94°C for 4 min followed by 30 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min; and then a final step at 72°C for 5 min. The PCR products were purified using a QIAquick Gel Extraction kit (Qiagen Science) after electrophoresis on 1% agarose gel, subcloned into a pGEM-T easy vector (Promega, Madison, WI), and then directly sequenced.

Gel Shift Assay.
A gel mobility-shift assay was performed as described previously (30) . We used a double-stranded oligonucleotide for p53 binding as a probe (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The consensus sequence is 5'-TACAGAACATGTCTAAGCATGCTGGGG-3'. The 32Plabeled probe (1 x 104 cpm) was mixed with crude nuclear extracts of TIG-7 or JHH-5 cells, incubated for 20 min on ice, and electrophoresed in a 5% polyacrylamide gel under nondenaturing conditions. For super-shift experiments, 1 µg of a mouse monoclonal antibody recognizing human wild-type p53 (OP-33; Oncogene Research Products, San Diego, CA) was added to the reaction mixture.

Western Blot Analysis.
Cells were washed twice with ice-cold PBS, and their protein extracts were subjected to Western blot analysis as described previously (19) . The primary antibodies used were mouse monoclonal antibodies against p53 of human origin, which recognizes both wild-type and mutant-type p53 (Bp53-12; Santa Cruz Biotechnology), and human IRF-1 (BD Biosciences, San Jose, CA), rabbit polyclonal antibody against human p21/Waf1 (Santa Cruz Biotechnology), and mouse monoclonal antibody against human tubulin (Sigma Chemical Co., St. Louis, MO), and the second antibodies were horseradish peroxidase-conjugated goat antimouse IgG and sheep antirabbit IgG (MBL, Nagoya, Japan).

Immunocytochemistry.
To visualize p53 and actin or S100C/A11 and p21/Waf1 simultaneously, cells fixed with 4% paraformaldehyde were treated with mouse monoclonal antibody against human p53 (Bp53-12; Santa Cruz Biotechnology) and Oregon Green 488 phalloidin against human actin (Molecular Probes, Inc., Eugene, OR) or rabbit antihuman S100C/A11 antibody prepared in our laboratory (19) and mouse antihuman p21/Waf1 monoclonal antibody (Santa Cruz Biotechnology) overnight at 4°C. Then the cells were treated at 37°C for 1 h with the second antibodies, i.e., Cy3-conjugated goat antimouse IgG antibody (Sigma) for p53, tetramethylrhodamine isothiocyanate-conjugated goat antirabbit IgG antibody (Sigma) for S100C/A11, and FITC-conjugated goat antimouse IgG antibody (Sigma) for p21/Waf1, as reported previously (19) .

Construction of AS IRF-1 Expression Vector pTRE2/AS-IRF-1.
The human IRF-1 cDNA was amplified by PCR with the plasmid pHuIRF-3-1 containing wild-type human IRF-1 cDNA in a CDM8 vector (31) as a template. The primers used for IRF-1 were 5'-aacatgcccatcactcggatgcg-3' (sense) and 5'-gacacgctgtagactcagcccaa-3' (AS). The PCR product was purified using a QIAquick Gel Extraction kit (Qiagen Science) after electrophoresis on 1% agarose gel and subcloned into a pGEM-T easy vector (Promega). The plasmid DNA was amplified in Escherichia coli DH5{alpha} cells, and the inserted IRF-1 cDNA was excised with NotI restriction enzyme and purified. Then the IRF-1 cDNA was cloned in the 3'-5' (AS) orientation into the multicloning site of pTRE2/hyg mammalian expression vector encoding the tetracycline response element (Clontech Laboratories, Inc., Palo Alto, CA).

Transfection.
JHH-5 cells were doubly transfected with the regulator plasmid pTet-On (Clontech Laboratories, Inc.) and the expression plasmid pTRE2/AS-IRF-1. The pTet-On plasmid expresses the reverse tet-controlled transactivator, and the reverse tet-controlled transactivator binds to the tetracycline response element and activates transcription of target gene in the presence of tetracycline or its derivative doxycycline. The cells were grown to 50–60% confluence in 60-mm dishes and were transfected with the regulator plasmid pTet-On (10 µg) in a serum-free medium Opti-MEM (Life Technologies, Inc.) using LipofectAMINE (Life Technologies, Inc.). After an 18-h incubation, 2 volumes of 20% FBS-containing DMEM were added, and the cultures were incubated for 2 days. The cells were subcultured at a split ratio of 1:3 and further cultured for 24 h in 10% FBS-containing DMEM. For selection of transfected cells, the cultures were then treated with G418 at 200 µg/ml for 7 days, and the G418-resistant cells were isolated and named JHH-5/Tet-On-4 cells.

Under similar conditions to those described above, JHH-5/Tet-On-4 cells were transfected with the plasmid DNA pTRE2/AS-IRF-1 (50 µg), and the transfected cells were selected by treatment with hygromycin at 80 µg/ml and named JHH-5/AS-IRF-1 cells.

Introduction of Proteins into Cells.
Recombinant human S100C/A11 protein with a relatively low molecular mass of 11 kDa was introduced into cells by the aid of the protein transduction domain of TAT protein (YGRKKRRQRRR) from human immunodeficiency virus (32) . TAT-flanked glutathione S-transferase (GST) protein served as a control.

Small Interfering RNA (siRNA).
An optimal target site of p21/Waf1 mRNA for siRNA was determined as 5'-CUUCGACUUUGUCACCGAG-3' by a program provided by Qiagen (Chiba, Japan), and the synthetic siRNA was purchased from the same company. The siRNA was transfected to JHH-5 cells 1 d after seeding using LipofectAMINE 2000 (Invitrogen, Carlsbad, CA).

Quantification of Band Intensity.
The signal intensities of Northern and Western blots were quantified using Image Quant version 3.3 (Molecular Dynamics).

Statistical Analysis.
Data are presented as means ± SD. Statistical analyses were performed by the ANOVA method, and P < 0.05 was considered statistically significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
TGF-ß1 causes growth inhibition in many cell types via induction of CKIs, such as p21/Waf1 (5) , p27/Kip1 (33 , 34) , and p15/Ink4B (35) . Among these, p21/Waf1 is known to play a major role and to be induced by either a p53-dependent (36) or a p53-independent mechanism (37) . When we determined the status of the p53 gene in human HCC cell lines by a yeast functional assay, six of 11 cell lines expressed mutant-type p53 (38) . To circumvent the influence of p53, we examined the effect of TGF-ß1 on growth of these six HCC cell lines with mutant-type p53. TGF-ß1 dose-dependently inhibited the [3H]TdR incorporation into DNA in four cell lines (JHH-5, HuH-7, JHH-6, and PLC/PRF/5) but not in HLE and JHH-4 (data not shown). We chose JHH-5 for additional mechanistic study, because this cell line was most sensitive to TGF-ß1.

p53 mRNA was detected in JHH-5 cells, and the level slightly increased after addition of TGF-ß1 (data not shown). The p53 protein was abundantly present in JHH-5 cells and located in the nuclei (Fig. 1, A and B)Citation . By reverse transcription-PCR and direct sequencing, however, we found a deletion of 12 nucleotides (from codons 190 to 193) in a region of the p53 gene encoding the specific DNA-binding domain in JHH-5 cells (Fig. 1C)Citation . The p53 protein in JHH-5 cells did not bind to the consensus sequence for p53 binding as shown in Fig. 1DCitation .



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  		Fig. 1. Mutation of p53 in JHH-5 human HCC cell line. A, Western blot analysis for p53. TIG-7, normal human fibroblasts derived from embryonic lung; HaCat, an immortalized human keratinocyte line with mutant-type p53. Tubulin served as an internal control. B, immunocytochemical staining of JHH-5 cells for p53 (red) and actin (green). p53 is localized in nuclei of the cells. Bars = 20 µm. C, by reverse transcription-PCR and direct sequencing, a deletion of 12 nucleotides (codons 190–193) encoding PPQH was found in the p53 gene of JHH-5 cells. The deleted region corresponds to a central part of the p53-specific binding domain. D, a gel shift assay performed using the consensus sequence for p53 binding as a probe. Nuclear extracts (NE) from TIG-7, JHH-5, or Saos2 cells (Lane 1, 0 µg; Lanes 2 and 7, 1 µg; Lanes 3 and 8, 2 µg; Lanes 4–6 and 9–12, 5 µg) were mixed with the probe with or without an antibody against wild-type p53. Expected positions of shifted and super-shifted bands are indicated with {triangleup} and {blacktriangleup}, respectively. Saos2 cell line is known to have mutated p53.

 
Decrease in incorporation of [3H]TdR into DNA was appreciable at 0.1 ng/ml, and the extent of reduction became constant at concentrations higher than 1 ng/ml. We therefore treated the cells with 1 ng/ml TGF-ß1 in the following experiments. DNA synthesis of JHH-5 cells was unchanged for the first 12 h after addition of TGF-ß1, and it then declined remarkably over the next 12 h and had almost completely ceased at 36 h (Fig. 2A)Citation . Under the same culture conditions, population doubling time of untreated JHH-5 cells was estimated as 32.3 h. The growth fractions of JHH-5 cells did not remarkably change during the culture period, i.e., from the sparse culture at day 1 to the near-confluent culture at day 4, indicating that the cells were not synchronized and grew randomly (Fig. 2B)Citation .



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  		Fig. 2. Inhibition of [3H]TdR incorporation into DNA in JHH-5 cells by TGF-ß1. A, the cells were treated with TGF-ß1 at 1 ng/ml for 36 h. [3H]TdR (1 µCi/ml) was added to the cultures 2 h before cell harvest, and radioactivity in a cold trichloroacetic acid-insoluble fraction was measured. Each column represents the mean of three wells. Vertical bars indicate SD. B, cell cycle profile of JHH-5 cells was evaluated by propidium iodide staining and fluorescence-activated cell sorter analysis 1 and 4 days after seeding. Total number of cells per dish and percentages of cells in the G0-G1, S, and G2-M phases are indicated.

 
To understand the molecular mechanism of growth inhibition by TGF-ß1, we examined the expressions of the CKIs. After addition of TGF-ß1 to JHH-5 cell cultures, the expression level of p21/Waf1 mRNA increased by 3-fold in the first 3 h, continued to increase, and reached a maximal level at 24 h, showing a 24-fold increase (Fig. 3, A and B)Citation . The amount of p21/Waf1 protein also increased in proportion to the mRNA level (data not shown; see Fig. 4CCitation ). However, the mRNA level of another CKI, p27/Kip1, did not change after addition of TGF-ß1 (Fig. 3, A and B)Citation . Expressions of G1-S cyclins also showed remarkable changes after the addition of TGF-ß1. Cyclin D1 mRNA increased continuously after addition of TGF-ß1 and reached a maximal level at 24 h, showing a 17-fold increase (Fig. 3, A and B)Citation . In contrast, expression levels of cyclin E and cyclin A mRNAs had decreased to 37% and 6% of the respective control levels at 36 h after addition of TGF-ß1 (Fig. 3, A and B)Citation . The expressions of these two cyclins are known to be regulated by the transcription factor E2F-1 (39 , 40) . Consistent with this, the expression level of E2F-1 mRNA decreased after addition of TGF-ß1, showing a maximal reduction of 89% at 36 h (Fig. 3, A and B)Citation .



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  		Fig. 3. TGF-ß1 enhances mRNA levels of p21/Waf1 and cyclin D1 but suppresses those of cyclin E, cyclin A, and E2F-1 in JHH-5 human HCC cells. A, Northern blot analysis was carried out with RNA samples derived from JHH-5 cells treated with TGF-ß1 at 1 ng/ml for different times. GAPDH was used as an internal control to compare the amounts of loaded RNA between lanes. B, relative signal intensities of p21/Waf1, p27/Kip1, cyclin D1, cyclin E, cyclin A, and E2F-1 mRNAs to GAPDH mRNA. Signal intensity was determined by using Image Quant.

 


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  		Fig. 4. Induction of IRF-1 by TGF-ß1 and abrogation of TGF-ß1-induced growth inhibition by AS IRF-1 in JHH-5 cells. A, Northern blot analysis for IRF-1 mRNA was carried out using JHH-5 cells treated with TGF-ß1 at 1 ng/ml for different times. GAPDH was used as an internal control for loaded amounts of RNA. Relative signal intensity of IRF-1 mRNA to GAPDH mRNA was determined using Image Quant. Experiments starting from 1, 2, and 3 days after seeding gave essentially the same results. Hence only an image of the first blot (top panel) and means with SD of relative band intensity in three different blots (bottom panel) are shown. B, Western blot analysis for IRF-1 in JHH-5 cells treated with TGF-ß1 (1 ng/ml). Because experiments starting from 1, 2, and 3 days after seeding gave essentially the same results, only the third blot is shown. Tubulin served as an internal control. C, Western blot analysis of expression of p21/Waf1 was carried out at different times after the addition of TGF-ß1 (1 ng/ml). Protein samples were derived from untreated control cultures or TGF-ß1-treated cultures of JHH-5 cells and their AS IRF-1-transduced cells (JHH-5/AS-IRF-1). Representative results of p21/Waf1 expression are shown. Tubulin served as an internal control. D, JHH-5 and JHH-5/AS-IRF-1 cells were treated for 18 h with or without TGF-ß1 at 1 ng/ml after 24-h pretreatment with doxycycline (2 µg/ml). [3H]TdR (1 µCi/ml) was added 6 h before the termination of TGF-ß1 treatment, and then cells were harvested for measurement of radioactivities in cold trichloroacetic acid-insoluble fractions. Each point represents the mean of three wells. Vertical bars indicate SD.

 
The results described above indicate that p21/WAF1 is the principal effector of TGF-ß1-induced growth inhibition in JHH-5 cells. An emerging question is which protein(s) mediates the signaling. Apparently, p53 cannot be a candidate because p53 was demonstrated to be mutated and transcriptionally inactive in JHH-5 cells (Fig. 1D)Citation . IRF-1 is a tumor suppressor gene and was shown to inhibit cell growth via transcriptional induction of p21/Waf1 (16 , 41) . In JHH-5 cells, expression of IRF-1 mRNA increased 27-fold by 3 h and then gradually decreased to the basal level by 36 h after addition of TGF-ß1 (Fig. 4A)Citation . The IRF-1 protein also markedly increased in parallel with the increase of its mRNA (Fig. 4B)Citation , indicating possible involvement of this transcription factor in the induction of p21/Waf1 following TGF-ß1 treatment. To test this possibility, we constructed an AS IRF-1 expression vector (pTRE2/AS-IRF-1) for depleting endogenous IRF-1. We transfected JHH-5/Tet-On-4 cells with the plasmid pTRE2/AS-IRF-1 and established a stable subline, JHH-5/AS-IRF-1. As shown in Fig. 4, C and DCitation , transduction of AS IRF-1 suppressed the induction of p21/Waf1 protein and substantially restored DNA synthesis even in the presence of TGF-ß1. Difference in the reduction of [3H]TdR incorporation into DNA by TGF-ß1 between control and pTRE2/AS-IRF-1-treated JHH-5 cells was statistically significant (P < 0.05). Thus, the transduction of AS IRF-1 endowed JHH-5 cells with partial resistance to TGF-ß1, indicating that IRF-1 is one of key mediators of growth inhibition by TGF-ß1 in JHH-5 cells.

In previous studies, we identified S100C/A11 as a key mediator of growth arrest induced in normal human fibroblasts by confluency (19) and in normal human keratinocytes by high Ca2+ (20) . In the growing cells, S100C/A11 was present mostly in cytoplasm. On exposure to growth-arresting signals, S100C/A11 was phosphorylated, translocated into nuclei, and eventually inhibited DNA synthesis through the induction of p21/Waf1. When JHH-5 cells were exposed to TGF-ß1, S100C/A11 was partially but promptly transferred to nuclei (Fig. 5A)Citation , this being a hallmark of S100C/A11-mediated growth inhibition (19 , 20) . S100C/A11 protein that was mainly present in cytoplasm in untreated JHH-5 cells was translocated to nuclei as early as 1 h after exposure to TGF-ß1 and progressively accumulated in nuclei thereafter. To examine whether S100C/A11 inhibits DNA synthesis of JHH-5 cells, we introduced recombinant S100C/A11 protein into JHH-5 cells by the aid of the protein transduction domain of TAT protein (YGRKKRRQRRR; Ref. 32 ). The TAT-flanked S100C/A11 protein and a negative control, TAT-GST protein, were efficiently transferred into cell nuclei (data not shown). DNA synthesis in the TAT-S100C/A11-transduced cells was markedly inhibited in a dose-dependent manner in the absence of TGF-ß1, whereas there was no change in DNA synthesis in TAT-GST-transduced cells (Fig. 5B)Citation . Interestingly, p21/Waf1 protein concomitantly accumulated in the nuclei, where S100C/A11 was present in abundance (Fig. 5C)Citation . We further examined whether the TAT-flanked S100C/A11 protein directly inhibited DNA synthesis in JHH-5 cells or manifested its growth inhibitory effect via induction of p21/Waf1 protein in the cells. For this purpose, we transfected JHH-5 cells with p21/Waf1-targeted siRNA to prevent induction of p21/Waf1. siRNA against p21/WAF1 markedly inhibited the induction of p21/Waf1 protein by TGF-ß1 or TAT-S100C/A11 (Fig. 5D)Citation . TGF-ß1 and TAT-S100C/A11 reduced DNA synthesis to a level of ~5% of the control level in the p21/Waf1-intact cells, but the level was recovered to 67% and 90% in the p21/Waf1-depleted cells treated with TGF-ß1 and S100C/A11, respectively (Fig. 5E)Citation . Transfection of the siRNA or introduction of TAT-GST in itself showed no significant effect on DNA synthesis. The mRNA level of S100C/A11 markedly increased from 6 to 24 h and reached a maximal 16-fold higher level at 36 h (Fig. 5F)Citation .



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  		Fig. 5. Involvement of S100C/A11 in TGF-ß1-mediated induction of p21/WAF1 and growth inhibition in JHH-5 cells. A, nuclear translocation of S100C/A11 in JHH-5 cells by 1 ng/ml TGF-ß1. Bars = 20 µm. B, reduction of [3H]TdR incorporation into DNA in JHH-5 cells by exogenous S100C/A11. Recombinant TAT-flanked S100C/A11 or GST (control) was added to cultures of JHH-5 cells 24 h before cell harvest. [3H]TdR (1 µCi/ml) was added 6 h before cell harvest. Each column represents the mean of three wells. Vertical bars indicate SD. C, immunocytochemistry for S100C/A11 (red) and p21/Waf1 (green) in JHH-5 cells treated with 1 ng/ml TGF-ß1. Bars = 20 µm. D and E, JHH-5 cells were transfected with p21/Waf1-targeted siRNA 36 h before the introduction of the TAT-flanked S100C/A11 protein and TAT-GST protein or treatment with TGF-ß1. Cells were harvested after cultivating for an additional 24 h. Western blot analysis was performed for p21/WAF1 (D), or incorporation of [3H]TdR into an insoluble fraction was monitored (E). For the latter experiment, [3H]TdR (1µCi/ml) was added to the cultures 6 h before the cell harvest. Each column represents the mean of three wells. Vertical bars indicate SD. F, Northern blot analysis for S100C/A11 carried out under the conditions similar to Fig. 3Citation .

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have demonstrated that the tumor suppressor IRF-1 (14 , 15) and Ca2+-binding S100C/A11 (18, 19, 20) mediate growth inhibition by TGF-ß1 through induction of a CKI, p21/Waf1, in JHH-5 human HCC cells. p53 is not involved in this signaling because JHH-5 cells have a mutant-type p53 protein deficient in binding to the target promoter sequence (Fig. 1)Citation . After addition of TGF-ß1 to JHH-5 cell cultures, p21/Waf1 mRNA level increased by 3-fold within about 3 h and reached a maximal level of 24-fold increase at 24 h, and thereafter, the enhanced level was maintained at least until 36 h. IRF-1 mRNA level also increased by 27-fold within 3 h and then declined to the basal level by 36 h. On the other hand, S100C/A11 protein was translocated to and progressively accumulated in nuclei from 1 h after the onset of treatment with TGF-ß1 (Fig. 5A)Citation . S100C/A11 mRNA level increased by 4-fold over a period of 12 h and continued to increase at least until 36 h, showing a 16-fold increase.

Transduction of AS IRF-1 inhibited the increase in p21/Waf1 in JHH-5 cells treated with TGF-ß1 and restored their DNA synthesis to a level of about 2-fold regardless of the presence of TGF-ß1 (Fig. 4D)Citation . We previously reported that TGF-ß1 increased the expression level of IRF-1 mRNA by 4-fold over the period of about 4 h in a human cholangiocarcinoma cell line, HuCCT1, that expresses mutant-type p53 (17 , 38) . p21/Waf1 was up-regulated in TGF-ß1-treated cells, leading to inactivation of cyclin E-associated kinase(s) and inhibition of DNA synthesis. In contrast, TGF-ß1 suppressed the expressions of both p53 and IRF-1 in human embryonic lung fibroblasts, IMR-90, thereby down-regulating expression of p21/Waf1. The reduced p21/Waf1 level resulted in activation of cyclin E-associated kinase(s) and stimulation of DNA synthesis (17) . These results also indicate that IRF-1 is closely associated with regulation of human cell growth by TGF-ß1.

We previously screened proteins down-regulated in immortalized human fibroblasts by two-dimensional gel electrophoresis (19) . S100C/A11 was among the proteins markedly down-regulated in immortalized human fibroblasts compared with their normal counterparts. When normal cells reached confluence, S100C/A11 was phosphorylated and then moved to and accumulated in the nuclei. In immortalized cells, however, S100C/A11 was not phosphorylated and remained in the cytoplasm even when the cells were in a confluent state. Microinjection of an anti-S100C/A11 antibody into normal confluent quiescent cells induced DNA synthesis. Thus, nuclear S100C/A11 mediates the contact inhibition of cell growth (19) . Furthermore, when S100C/A11 was expressed in HeLa cells as a conjugated form with a nuclear localization signal (PKKKRKV), the protein translocated into the nuclei and remarkably inhibited DNA synthesis via induction of p21/Waf1. In the present study, we found that TGF-ß1 induced accumulation of S100C/A11 and p21/Waf1 in the nuclei of JHH-5 cells in association with inhibition of DNA synthesis. Furthermore, when S100C/A11 protein was introduced into nuclei of JHH-5 cells by the aid of the protein transduction domain of TAT protein (YGRKKRRQRRR) from human immunodeficiency virus (32) , DNA synthesis was inhibited in a dose-dependent manner. Contrarily, prior transfection of JHH-5 cells with p21/Waf1-targeted siRNA efficiently blocked decrease of DNA synthesis in the cells caused by TAT-S100C/A11 or TGF-ß1 and markedly inhibited expression of p21/Waf1 protein in the cells. These results indicate that nuclear S100C/A11 mediates growth inhibition by TGF-ß1 in JHH-5 cells via induction of p21/Waf1.

We recently found that S100C/A11 was specifically phosphorylated on threonine 10 and serine 94 in human keratinocytes exposed to high Ca2+ (20) . We also found that the phosphorylation facilitated the binding of S100C/A11 to nucleolin, resulting in nuclear translocation of S100C/A11. In nuclei, S100C/A11 liberated Sp1 and Sp3 from nucleolin and the resulting free Sp1 and Sp3 transcriptionally activated p21/Waf1. In addition, introduction of anti-S100C/A11 antibody into the cells largely abolished the growth inhibition induced by Ca2+ and the induction of p21/Waf1 (20) . These results are consistent with the reported finding that Sp1 and Sp3 activate the p21/Waf1 promoter (6) . Furthermore, Smad 3 and Smad 4 were found to cooperate with Sp1 for transcriptional activation of the p21/Waf1 promoter in a human hepatoblastoma cell line, HepG2, (7) and in an immortalized human keratinocyte line, HaCat, exposed to TGF-ß1 (8) . These findings together with the findings in the present study of accumulation of S100C/A11 and p21/Waf1 in the nuclei induced by TGF-ß1 suggest that S100C/A11 mediates the induction of p21/Waf1 by liberating Sp1 and Sp3 from nucleolin in the nuclei of JHH-5 cells treated with TGF-ß1.

In conclusion, TGF-ß1 inhibits growth of some human HCC cell lines expressing mutant-type p53. IRF-1 and S100C/A11 may mediate the growth inhibition by TGF-ß1 via induction of p21/Waf1.


   ACKNOWLEDGMENTS
 
We thank Drs. Toshiya Tsuji, Yusuke Inoue, Kazuo Fushimi, Hirosuke Kouchi, and Tadahiro Uemura for their helpful support and Emiko Nakashima for her technical assistance in the present study.


   FOOTNOTES
 
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: Masahiro Miyazaki, Department of Cell Biology, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. Phone: 81-86-235-7395; Fax: 81-86-235-7400; E-mail: hiromiya{at}md okayama-u.ac.jp.

Received 9/ 2/03. Revised 3/ 8/04. Accepted 4/ 6/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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