Vesnarinone, a differentiation inducing drug, directly activates p21waf1 gene promoter via Sp1 sites in a human salivary gland cancer cell line

We previously demonstrated that a differentiation inducing drug, vesnarinone induced the growth arrest and p21waf1 gene expression in a human salivary gland cancer cell line, TYS. In the present study, we investigated the mechanism of the induction of p21waf1 gene by vesnarinone in TYS cells. We constructed several reporter plasmids containing the p21waf1 promoter, and attempted to identify vesnarinone-responsive elements in the p21waf1 promoter. By the luciferase reporter assay, we identified the minimal vesnarinone-responsive element in the p21waf1 promoter at −124 to −61 relative to the transcription start site. Moreover, we demonstrated by electrophoretic mobility shift assay that Sp1 and Sp3 transcription factors bound to the vesnarinone-responsive element. Furthermore, we found that vesnarinone induced the histone hyperacetylation in TYS cells. These results suggest that vesnarinone directly activates p21waf1 promoter via the activation of Sp1 and Sp3 transcription factors and the histone hyperacetylation in TYS cells. British Journal of Cancer (2002) 87, 1042–1046. doi:10.1038/sj.bjc.6600592 www.bjcancer.com © 2002 Cancer Research UK

We have previously demonstrated that a differentiation inducing drug, vesnarinone inhibits the growth of a human salivary gland cancer cell line, TYS, and induces the expression of p21 waf1 , a potent inhibitor of cyclin dependent kinase (Sato et al, 1997a;Kawamata et al, 1998). Vesnarinone is currently used as a chemotherapeutic agent for head and neck cancer combined with radiation in several countries, such as Japan (Sato et al, 1997b,c), the United States and India. p21 waf1 is a gene functioning as a cell cycle blocker, and its expression is usually regulated at transcriptional level. p21 waf1 is known to inhibit cyclin dependent kinase activity in p53-mediated cell cycle arrest induced by DNA damage (El-Deiry et al, 1993). Further studies have indicated that p21 waf1 is also regulated by other transcription factors during cell differentiation and growth arrest (Dulic et al, 1994;Jiang et al, 1994). p21 waf1 promoter contains not only p53-binding sites but also several transcription factor responsive elements (Datto et al, 1995;Nakano et al, 1997). One of the responsive elements is for a transcription factor, Sp1. Sp1 responsive elements are located on the upstream of TATA box of p21 waf1 promoter. It is reported that several extracellular stimuli including butyrate (Nakano et al, 1997), transforming growth factor-b (Datto et al, 1995), phorbol esters (Biggs et al, 1996), okadaic acid (Biggs et al, 1996) and retinoic acid (Liu et al, 1996) activate the transcription of p21 waf1 gene through the Sp1 responsive elements.
Because TYS cells are reported to have a mutated p53 gene (Sato et al, 1997a), the expression of p21 waf1 gene and the growth arrest induced by vesnarinone may be conducted by the p53-independent pathway in TYS cells. In order to use vesnarinone more effectively on the patients with several malignancies, including head and neck cancer, the molecular mechanisms of the growth inhibitory effect of vesnarinone should be studied. In this experiment, we attempted to identify the vesnarinone-responsive elements in the p21 waf1 promoter, and clarify the molecular mechanisms of transcriptional activation of p21 waf1 gene by treatment with vesnarinone in a human salivary gland cancer cell line, TYS.

MATERIALS AND METHODS
Cell culture and reagents TYS cells (Yanagawa et al, 1986) were grown in Dulbecco's modified Eagle medium (DMEM; Life Technologies, Inc., Gaithersburg, MD, USA) supplemented with 10% foetal calf serum (FCS; Bio-Whittaker, Walkersville, MD), 100 mg ml 71 streptomycin, 100 U ml 71 penicillin (Life Technologies, Inc.), and 0.25 mg ml 71 amphotericin B (Life Technologies, Inc.) in a humidified atmosphere of 95% air and 5% CO 2 at 378C. Vesnarinone (Otsuka Pharmaceutical Company, Tokyo, Japan) was dissolved in dimethyl sulphoxide (DMSO; Sigma, St. Louis, MO, USA) at a concentration of 10 mg ml 71 as the first stock solution, and the first stock solution was diluted with the complete culture medium described above. Trichostatin A (TSA; Wako, Osaka, Japan) was dissolved in ethanol at a concentration of 1 mg ml 71 , and diluted with the complete culture medium at 10 mg ml 71 .

Transient transfection and luciferase assay
TYS cells (5610 5 cells dish 71 ) were seeded in 35 mm culture dish (Falcon; Becton Dickinson Labware, Lincoln Park, NJ, USA) in DMEM supplemented with 10% FCS. Twenty-four hours later, the cells were transfected with 5 mg of reporter plasmid DNA by using Superfect reagent (QIAGEN, Hilden, Germany). Fifteen hours after transfection, vesnarinone (50 mg ml 71 ) was added, and 20 h later, cell lysates were collected. Luciferase activities were measured by Promega luciferase assay Kit (Promega). The luciferase activities were normalised by the amount of protein. Each experiment was repeated at least three times.

Histone acetylation in TYS cells by vesnarinone treatment
TYS cells were seeded in 100 mm culture dishes. Twenty-four hours later, vesnarinone (50 mg ml 71 ) or TSA (10 mg ml 71 ) was added to the medium. Sixteen hours later, the cells were collected and the nuclear extracts were prepared as follows; cells were suspended in 400 ml of hypotonic buffer (20 mM HEPES-KOH (pH 7.9) containing 1 mM EDTA, 1 mM DTT, 20 mM NaF, 1 mM sodium orthovanadate, 0.5 mM PMSF, 0.2% NP-40, 1 mg ml 71 leupeptin, 10 units ml 71 aprotinin, and 1 mg ml 71 pepstatin A). Samples were centrifuged at 15 000 r.p.m. and the pellets were resuspended in 200 ml of hypertonic buffer (20 mM HEPES-KOH (pH 7.9) containing 1 mM EDTA, 1 mM DTT, 20 mM NaF, 1 mM sodium orthovanadate, 0.5 mM PMSF, 0.2% NP-40, 420 mM NaCl, 20% glycerol, 1 mg ml 71 leupeptin, 10 units ml 71 aprotinin, and 1 mg ml 71 pepstatin A). Samples were incubated on ice for 20 min and were centrifuged at 15 000 r.p.m. for 15 min. The supernatants were used as nuclear extracts. The protein concentrations of samples were determined with a Bio-Rad protein assay. Samples were electrophoresed on SDS-polyacrylamide gel. Proteins from gels were transferred to nitrocellulose (Bio-Rad) and were detected with an anti-acetylated Histone H3 antibody (Upstate Biotechnology, Lake Placid, NY, USA) and an Amersham ECL kit (Amersham Pharmacia Biotech.).

RESULTS
Effect of vesnarinone on the activation of p21 waf1 promoter Several reporter plasmids ( Figure 1) were transiently transfected in TYS cells, and luciferase activity was examined. Vesnarinone apparently enhanced the luciferase activity from the pGL3-WWP reporter plasmid in TYS cells when compared with untreated control or DMSO treatment (Figure 3). Vesnarinone also enhanced the luciferase activity from the pGL3-WWP-0.2 plasmid, which contained a 215 bp promoter fragment lacking two p53 binding sites ( Figure  3). Surprisingly, vesnarinone also enhanced the luciferase activity from the pWP124 containing only a 124 bp promoter fragment. However, vesnarinone did not activate a 60 bp promoter fragment of p21 waf1 in pWPdel-SmaI reporter plasmid (Figure 3).

Electrophoretic mobility-shift assay
According to the results from the luciferase assay, the vesnarinoneresponsive element exists within 77 bp region relative to the TATA element. This 77 bp region harbours four independent and two overlapping nearly consensus binding sites for transcription factor Sp1. They are tentatively termed Sp1-1, Sp1-2, Sp1-3, Sp1-4, Sp1-5 and Sp1-6 from the upstream (Figure 2). To determine if Sp1 or other proteins can interact with the vesnarinone-responsive element, electrophoretic mobility-shift assay was performed using the oligonucleotides containing the Sp1-binding sites. The Sp1-A contains Sp1-1 and Sp1-2 sites, and the Sp1-B contains Sp1-4, Sp1-5 and Sp1-6 sites ( Figure 2). After treatment with vesnarinone for 30 min, we detected the shifted band when using the Sp1-A as a probe ( Figure 4A). However, when we used the Sp1-B, we could not detect any shifted bands after vesnarinone treatment ( Figure  4A). As shown in Figure 4B, the mobility shift was detectable at 30 min after treatment with 50 mg ml 71 vesnarinone, and the intensity of the shifted band increased at 45 min after treatment. Moreover, this band completely disappeared by adding excess unlabelled Sp1-A oligonucleotide (data not shown).

Supershift assay
To elucidate whether the retarded bands represent the binding of Sp1 or Sp3, supershift assay was performed by the nuclear extracts pre-incubated with anti-Sp1 or anti-Sp3 antibody ( Figure  4C). In the presence of anti-Sp1 or anti-Sp3 antibody, the intensity of the shifted band was markedly reduced. The shifted band completely disappeared in the presence of anti-Sp1 and anti-Sp3 antibody together. When pre-immune rabbit-IgG was added as a

Experimental Therapeutics
Transcriptional activation of p21 waf1 by vesnarinone F Omotehara et al negative control, the intensity of the band was slightly reduced. However, the effect of pre-immune IgG was much weaker than that of anti-Sp1 or anti-Sp3 antibody. Thus, the effect of preimmune IgG was probably due to non-specific interference of the IgG protein with the binding of Sp1 or Sp3 protein and DNA.

DISCUSSION
In this study, we examined the molecular mechanisms of the transcriptional regulation of p21 waf1 gene by a differentiation inducing drug, vesnarinone. We identified the minimal vesnarinone-responsive element in the p21 waf1 promoter at 7124 to 761 relative to the transcription start site, and demonstrated that vesnarinone enhanced the binding of the transcription factors Sp1 and Sp3 to the vesnarinone-responsive element. Furthermore, we found that vesnarinone induced the histone acetylation in TYS cells. Sp1 is a ubiquitously expressed nuclear protein that is initially identified as a protein that binds and stimulates transcription of simian virus 40 early promoter (Dynan and Tjian, 1983). Sp1 protein binds to the GC-rich sequences present in a variety of cellular and viral promoters and stimulates their transcriptional activity (Lania et al, 1997). Sp3 belongs to the same family of Sp1 related transcription factor, and it also binds to the GC-rich sequences (Sp1 binding sites) (Lania et al, 1997). In the p21 waf1 promoter, there are four independent Sp1 binding sites (Sp1-1 -Sp1-4) and two overlapping Sp1 binding sites (Sp1-5, Sp1-6) ( Figure 2). We identified the Sp1-1 and Sp1-2 site as main vesnarinone-responsive elements of the p21 waf1 promoter. Generally, eukaryotic transcription is regulated by more than one transcription factor, and these transcription factors form a complex in specific promoter elements via interaction with various cofactors (Struhl and Moqtaderi, 1998). Sp1 and Sp3 are likely to be transcription factors that have low specificity to the extra-cellular stimuli, but they would be indispensable factors in p53-indepen-dent pathway on the p21 waf1 gene transcriptional activity in our system.
Vesnarinone induced histone acetylation in TYS cells. Recent studies demonstrated that there were various kinds of histone acetyltransferase (HAT) and histone deacetylase (HDAC) in mammalian cells, and the level of histone acetylation was controlled by equilibrium of the activities of HAT and HDAC (Grunstein, 1997). The transcriptional coactivators, p300 and CREB binding protein (CBP) are known to possess the HAT activity, and interact with a wide range of DNA binding proteins, including Sp1, p53, the RelA (p65) nuclear factor kB subunit, E2F, MyoD, activator protein 1, several nuclear receptors, and many others (Yuan et al, 1996;Avantaggiati et al, 1997;Gu and Roeder, 1997;Lee et al, 1998;Ikeda et al, 2000). Although data was not shown, we confirmed the expressions of p300, CBP and HDAC1 proteins in the nucleus of TYS cells.
Several histone acetylation inducing drugs show the growthinhibitory effect or differentiation-inducing effect, and are used as a chemotherapeutic agent on several human malignancies (Chen et al, 1997;Dion et al, 1997;McCaffrey et al, 1997). The molecular targets for the differentiation inducing drugs (or histone acetylation inducing drug) may be different from those for DNAdamaging drugs. Moreover, activating pathway of the target molecules by differentiation inducing drugs may also be different from those by DNA-damaging drugs. Thus, the differentiation inducing drugs, such as vesnarinone may act synergistically on the induction of p21 waf1 gene with the DNA-damaging therapy, such as radiation and the administration of conventional chemotherapeutic drugs. These informations are useful for creating new strategy for differentiation-inducing-therapy. Figure 5 Histone acetylation in TYS cells induced by vesnarinone. Nuclear extracts were prepared from TYS cells after treatment with 50 mg ml 71 vesnarinone or 10 mg ml 71 TSA for 16 h. Protein samples were subjected to SDS -PAGE, transferred to nitrocellulose, and detected with an anti-acetylated Histone H3 antibody and Amersham ECL kit.