Ethanol upregulates the expression of p21WAF1/CIP1 and ... - Nature

Oncogene (1997) 15, 1143 ± 1149  1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00

Ethanol upregulates the expression of p21WAF1/CIP1 and prolongs G1 transition via a p53-independent pathway in human epithelial cells Wentong Guo1,2, Marcel A Baluda2,3 and No-Hee Park1,2,4 1

Dental Research Institute, 2School of Dentistry, 3School of Medicine, 4Jonsson Comprehensive Cancer Center, University of California, Los Angeles, California 90095-1668, USA

The control of cell cycle progression is necessary for accuracy in the replication of DNA and the distribution of genetic information to daughter cells. Disturbances in progression of the cell cycle may result in the loss of genomic integrity, a `hallmark' of cancer cells. Extensive consumption of alcoholic beverages is a risk factor associated with the development of various human epidermoid cancer including oral and pharyngeal squamous cell carcinomas. However, eects of ethanol on cell cycle progression and on the expression of genes associated with the cell cycle have not been studied. We report here that exposure of human epithelial cells to ethanol, at concentrations (100 ± 200 mM) that do not cause cell death, (a) does not aect or only reduces slightly the cellular level of p53 protein, (b) upregulates the transcription of the WAF1/CIP1 gene, (c) inhibits the Cdk2 activity, and (d) reduces the rate of cellular proliferation by inducing a delay in G1 phase transition. The results also indicate that, at these non-cytotoxic concentrations, ethanol exhibits its eects through a p53independent mechanism. Keywords: ethanol; p53; p21WAF1/CIP1; cell cycle

Introduction Cell cycle progression is regulated by cyclin dependent kinases (cdks) which are activated by cyclins and inactivated by cdk inhibitors, such as p21WAF1/CIP1 (ElDiery et al., 1993; Harper et al., 1993; Xiong et al., 1993; Li et al., 1994; Macleod et al., 1995). The p21WAF1/CIP1 displays selective inactivation of G1/S cdk, that results in the accumulation of hypophosphorylated pRB protein which in turn causes cell cycle arrest in the G1 phase (Sherr and Roberts, 1995). Expression of the WAF1/CIP1 gene is upregulated by wild-type tumor suppressor protein p53, whose cellular level is elevated by genotoxic stress leading to either cell cycle arrest or cell death (Kastan et al., 1991; Xiong et al., 1993; ElDiery et al., 1993; Gujuluva et al., 1994). The p21WAF1/ CIP1 gene can also be upregulated via p53-independent pathways. For example, TGF-b (Datto et al., 1995), phorbol 12-myristate-13 acetate and octatic acid (Biggs et al., 1996) directly stimulate the WAF1/CIP1 gene promoter, resulting in G1 arrest. Paradoxically, whereas an increased level of p53 leads to programmed cell death Correspondence: No-Hee Park, UCLA Dental Research Institute, 73017 CHS, 10833 Le Conte Avenue, Los Angeles, CA 90095-1668, USA Received 17 March 1997; revised 16 May 1997; accepted 16 May 1997

(apoptosis), an elevated level of p21WAF1/CIP1 is frequently, albeit not always, associated with the survival of mammalian cells (Kobayashi et al., 1995; Gorospe et al., 1996). For example, in tumor cells overexpression of p21WAF1/CIP1 is correlated with resistance to chemotherapeutic agents (Zhang et al., 1995) and protects cells against apoptosis by arresting cell cycle progression in the G1 phase (Gorospe et al., 1996). On the other hand, overexpression of p21WAF1/CIP1 in cultured murine thymocytes, myelid leukemic cells and colon cancer cells is associated with apoptosis (Yonish-Rouach et al., 1991; Shaw et al., 1992; Lowe et al., 1993). Extensive consumption of alcohol is correlated with an increased risk of carcinogenesis (Mufti, 1992; Anderson et al., 1995; Deleyiannis, 1996). However, the molecular mechanism of ethanol action is poorly understood. It has been reported that ethanol aects a signal transduction pathway in lymphocytes by altering the cellular cAMP and calcium level (Rabin and Molino, 1983; Spinozzi et al., 1993; Diamond et al., 1994) and increasing the cytosolic and membraneassociated protein kinase C (PKC) activities (DePetrillo and Liou, 1993). Ethanol also inhibits cell mitosis by arresting cell cycle progression in G2/M phase in human lymphocytes (Ahluwalia et al., 1995) and induces apoptosis of murine thymocytes (Ewald and Shao, 1993; Shao and Ewald, 1995). However eects of ethanol on the proliferation, cell cycle progression, and the expression of genes controlling the cell cycle in human epithelial cells have not been investigated. In the present study, we have shown that ethanol delays the cell cycle progression through the G1 phase by upregulating the p21WAF1/CIP1 gene expression and inhibiting the Cdk2 kinase activity in human epithelial cells. We have further shown that these eects occur independently of the cellular p53 status.

Results Reversible eect of ethanol upon cellular replication RKO cells expressing wild-type p53 were cultured in the presence or absence of 200 mM ethanol. In McCoy's modi®ed culture medium supplemented with 10% fetal bovine serum, the number of cells doubled approximately every 17 h. In the continuous presence of 200 mM of ethanol added to the culture medium, the cells were able to proliferate, but at a reduced rate (the cells doubled approximately every 32 h) (Figure 1). If the cells were exposed to 200 mM ethanol for only 24 h, 24 h after plating then cultured in ethanol-free medium, the cells resumed their normal proliferation rate after ethanol removal (Figure 1).

Ethanol prolongs the G1 phase via p21WAF1/CIP1 W Guo et al

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To eliminate the possibility that 24 h exposure to ethanol had killed 47% of the cells (from extrapolation of curves to ordinate in Figure 1) and con®rm that the ethanol eect upon cellular proliferation was reversible, we determined the colony forming ability of ethanolexposed RKO cells in two dierent types of experiments in which the cells were treated with ethanol either before or after plating. In the ®rst type of experiment, approximately 1000 RKO cells from cultures either exposed to ethanol for 24 h or unexposed were plated and the colonies formed after 8 days of cultures were counted. Similar number of colonies developed with either the ethanol-exposed or unexposed cells (512+26 vs 518+92, respectively). In the second type of experiment, approximately 1000 cells were plated and cultured overnight. The cultures were divided into two groups: Group 1 was exposed to ethanol for 24 h and subsequently cultured for 6 additional days in ethanol-free medium; and group 2 was cultured in ethanol-free medium for 7 days. Again, there was no signi®cant dierence in the number of colonies formed with either ethanol treated or control cells (292+12 vs 276+12, respectively) albeit the ethanol treated colonies tended to be smaller in size. These data indicate that 200 mM of ethanol is not cytotoxic and its anti-proliferative eect is reversible. Ethanol prolongs the G1 phase of the cells cycle To determine reason for the reduced rate of cellular proliferation in the presence of ethanol, we investigated

Figure 1 Eect of ethanol on the proliferation of RKO cells. 46104 RKO cells were plated and cultured for 24 h. After that time, the cultures were divided into three groups: group 1: control (no ethanol) (open circle); group 2: cells exposed to ethanol (200 mM) for 24 h and cultured for ®ve additional days in ethanol-free medium (closed circle); and group 3: cells exposed to ethanol (200 mM) for 6 days (triangle). For each time point two culture ¯asks were used to count the cells as described in Materials and methods

the eect of ethanol on cell cycle progression. As shown in Figure 2a, in the absence of ethanol RKO cells showed a cell cycle distribution of 35% in G0/G1, 52% in S and 13% in G2/M phases. After a 16 h exposure to 200 mM ethanol, there was an increase in the percentage of cells in G0/G1 i.e. 72% with 16% in S and 12% in G2/M phases. Since enhanced cellular level of wild-type p53 results in G1 arrest and RKO cells express wild-type p53 (Kastan et al., 1991), the ethanol-induced eect could be via a p53 pathway. If such is the case, ethanol should not induce a cell cycle delay in cells which do not express p53. To investigate this possibility, we compared the eect of ethanol on the cell cycle progression in secondary NHOK expressing wild-type p53 and in the SCC-9 oral cancer cell line which does not express p53 (Min et al., 1994). As found with the RKO cells, a 16 h ethanol exposure induced an increase in the percentage of cells in G1 in both the NHOK and SCC-9 cells (Figure 2b and c). These data indicate that ethanol delays G1 phase transition regardless of the presence or absence of p53. The kinetics of cell cycle progression (Figure 3) demonstrated that after 16 h the percentage (70%) of RKO cells in the G1 phase remained at a constant, signi®cantly increased level as long as ethanol was present in the culture medium. Ethanol eect upon the cellular level of p53, GADD45 and p21WAF1/CIP1 G1 arrest is associated with elevated cellular GADD45 (Smith et al., 1994) and p21WAF1/CIP1 levels whose expression can be upregulated by wild-type p53 or by a p53-independent pathway. We analysed levels of p53,

Figure 2 Ethanol arrests the cell cycle progression of NHOK, RKO and SCC-9 cells. The cells were cultured in the absence or presence of 200 mM of ethanol for 16 h then analysed for cell cycle arrest as described in Materials and methods. (a) Percentage of RKO cell cultures in G1/G0, S, and G2/M phases when cultured in the absence or presence of 200 mM in culture medium. (b) Percentage of secondary NHOK cultures in G1/G0, S, and G2/ M phases when cultured in the absence or presence of 200 mM in culture medium. (c) Percentage of SCC-9 cell cultures in G1/G0, S, and G2/M phases when cultured in the absence or presence of 200 mM in culture medium


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Figure 3 Kinetics of cell cycle progression in RKO cells exposed to 200 mM ethanol for 0, 16, 24, 36 and 48 h. Cell cycle analysis was performed as described in Materials and methods and the percentage of cells in G1/G0 (open circle), S (closed circle) and G2/M (triangle) phases determined for each time point

GADD45, and p21WAF1/CIP1 in RKO cells to con®rm that the ethanol eects upon G1 occurs in a p53dependent manner. As shown in Figure 4a, ethanol exposure of RKO cells for longer than 4 h slightly reduced the level of wild-type p53 and did not alter the GADD45 level, but increased the p21WAF1/CIP1 level by 3 ± 10-fold. As shown in Figure 4b, a 15 h ethanol exposure of NHOK and RKO cells which express wild-type p53 enhanced the p21WAF1/CIP1 protein level without increasing the p53 level. A similar ethanol exposure of SCC-4 cells which express mutant p53 and SCC-9 cells which do not express any p53 also increased the p21WAF1/CIP1 protein level without aecting p53 expression. These ®ndings indicate that ethanol increases the cellular concentration of p21WAF1/CIP1 independently of p53. We also examined the level of the WAF1/CIP1 mRNA in SCC-4 and RKO cells to determine whether the enhanced p21WAF1/CIP1 level was due to increased expression of the gene. Ethanol increases the WAF1/ CIP1 mRNA level by 2.5-fold in SCC-4 and RKO cells (Figure 5a and b), but there was no increase in the level of p53 mRNA (Figure 5c). Ethanol upregulates the WAF1 promoter Since ethanol increased the level of WAF1/CIP1 mRNA in cells, we investigated the direct eect of ethanol upon the WAF1/CIP1 gene promoter using the plasmid WWP-Luc which contains a luciferase reporter gene under the control of a 2.4 kb WAF1/CIP1 promoter. As shown in Figure 6, ethanol exposure for 24 h resulted in a 2.4-fold increase of luciferase activity in RKO cells which were transiently transfected with the plasmids WWP-Luc as compared to the unexposed cells. To further demonstrate that ethanol enhances transcription of the WAF1/CIP1 gene in a p53independent manner, we constructed deletion mutants

WAF1 Figure 4 Ethanol eect upon the cellular levels of p53, GADD45 and p21WAF1/CIP1 proteins. (a) Ethanol eect upon wild-type p53, GADD45, and p21WAF1/CIP1 in RKO cells exposed to 200 mM ethanol for 0, 2, 4, 6, 8 or 10 h. At each time point, the cellular levels of p53, GADD45, and p21WAF1/CIP1 were determined by Western blot analysis. (b) Ethanol eect upon p53 and p21WAF1/ CIP1 in cells expressing wild-type p53 (NHOK and RKO cells), in cells expressing mutant p53 (SCC-4 cells) or no p53 (SCC-9 cells). The level of p53 and p21WAF1/CIP1 in NHOK, SCC-4, SCC-9 and RKO cultured in the absence (7) or presence (+) of 200 mM of ethanol for 15 h were determined using Western blot analysis

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Figure 5 Ethanol eect upon WAF1/CIP1 and p53 mRNA levels. (a) Northern blot analysis of WAF1/CIP1 messages in RKO cells in the presence of 0, 50, 100 and 200 mM ethanol for 15 h. (b) Northern blot analysis of WAF1/CIP1 messages in the absence or presence of 200 mM ethanol for 15 h in SCC-4 cells. (c) Northern blot analysis of p53 messages in the absence or presence of 100 or 200 mM ethanol in RKO cells. After autoradiography, the probes were stripped o the ®lter for rehybridization with a b-actin probe


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of the WWP-Luc plasmid which did not contain the p53 binding site of the WAF1/CIP1 promoter (Figure 6). Although the basal level of luciferase activity was notably diminished by the deletion, ethanol exposure increased the luciferase activity by 3.6-fold in RKO cells which were transfected with the p21D1.26-Luc compared to that in control cells. To de®ne the region of the WAF1/CIP1 promoter necessary for induction by ethanol, we created three additional deletion mutants of the WWP-Luc construct, i.e., p21M0.85-Luc, p21P0.22-Luc, and p21H0Luc (Figure 6b). Ethanol exposure of cells transfected with either p21M0.85 or p21P0.22-Luc enhanced the luciferase activity by 4.8- and 2.2-fold, respectively, compared to unexposed controls. Cells transfected with p21H0-Luc, a construct with complete deletion of the WAF1/CIP1 promoter, demonstrated only background luciferase activity in the presence or absence of ethanol. These data indicate that approximately 200 bp of the WAF1/CIP1 promoter upstream from the transcription initiation site of the WAF1/CIP1 gene is necessary for induction by ethanol. Ethanol inhibits Cdk2 activity via p21WAF1/CIP1 p21WAF1/CIP1 inhibits cdk activity by forming a complex with cyclin/cdk, and in doing so induces G1 arrest. To determine that ethanol-induced G1 prolongation is via the increase of p21WAF1/CIP1 and not by direct inhibition of the kinase activity, we examined the eect of ethanol upon the levels of Cdk2 and Cdk2 bound p21WAF1/CIP1. Cells were exposed to 0, 50, 100 and 200 mM ethanol

for 24 h and lysed for Western analysis. The level of Cdk2 was determined by Western blot using Cdk2 antibody. To determine the level of Cdk2 bound p21WAF1/CIP1, aliquots of the cell lysates were immunoprecipitated with anti-Cdk2 antibody followed by Western blot using p21WAF1/CIP1 antibody. As shown in Figure 7, ethanol did not alter the cellular level of Cdk2, but signi®cantly enhanced the level of p21WAF1/ CIP1 which formed complexes with Cdk2. The cell lysate aliquots immunoprecipitated with Cdk2 antibody were also used to determine the Cdk2 activity using histone H1 as substrate. Exposure of cells to 50, 100 or 200 mM ethanol reduced the Cdk activity by 1.2-, 1.5and twofold, respectively (Figure 7). These ®ndings indicate that ethanol decreases Cdk-2 activity by increasing the p21WAF1/CIP1 level. Discussion Our data show that ethanol enhances the expression of p21WAF1/CIP1 and delays cell cycle progression in G1 phase via a p53-independent pathway. This conclusion is based on the following observations: (1) ethanol reduces the rate of cellular proliferation in a reversible manner and prolongs the cell cycle in G1 phase regardless of the status of cellular p53; (2) ethanol does not alter the p53 mRNA level, but appears to slightly lower the cellular p53 protein level in some cells; (3) ethanol does not aect the expression of GADD45, a protein regulated by wild-type p53; and (4) ethanol upregulates the WAF1/CIP1 promoter in

Figure 6 Eect of ethanol upon the transcription of luciferase gene in reporter plasmid constructs with various WAF1/CIP1 promoter deletions. (a) Partial restriction map of the 2.4 kb WAF1/CIP1 promoter in the WWP-Luc plasmid. (b) Luciferase activities of various WAF1/CIP1 promoter deletion constructs in RKO cells. The RKO cells were transiently transfected with various constructs and cultured in the absence (7) or presence (+) of 200 mM ethanol for 20 h. The cells were then lysed and prepared to determine the luciferase activities. The right column shows the fold increase in luciferase activity in the presence of ethanol


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human epithelial cells, it could permit malignant conversion after carcinogen damage by by-passing the p53 pathway that leads to apoptosis. Several studies support this assumption (Kobayashi et al., 1995; Zhang et al., 1995; Goropse et al., 1996) e.g. resistance to apoptosis is an important factor in the development of malignant lymphomas (Vaux et al., 1988).

Materials and methods Cell culture

b — Cdk2 c — Histone H1

Figure 7 Ethanol eect upon the cellular level of p21WAF1/CIP1 bound to Cdk2 in RKO cells. (a) Ethanol eect upon p21WAF1/ CIP1 associated with Cdk2 in RKO cells cultured in the presence of 0, 50, 100 and 200 mM of ethanol for 15 h. The cells were immunoprecipitated with anti-Cdk2 antibody followed by Western analysis using p21WAF1/CIP1 antibody. (b) Western analysis of Cdk2 with Cdk2 antibody in RKO cells exposed to 0, 50, 100 or 200 mM ethanol for 15 h. (c) Level of histone H1 kinase in RKO cells exposed to 0, 50, 100 or 200 mM ethanol for 15 h

the deletion mutants of the WWP-Luc plasmid which do not contain the p53 binding site of the WAF1/CIP1 promoter. The ethanol responsive element is located within approximately 200 base pairs of the WAF1/CIP1 promoter upstream from the transcriptional start site. In this region there are four putative SP1 binding sites and a TGF-b responsive element (Datto et al., 1995). A preliminary study shows that ethanol does not enhance the expression of TGF-b1 in RKO cells (data not shown), suggesting that the overexpression of p21WAF1/ CIP1 by ethanol is not via the induction of TGF-b1. It has been reported that ethanol can also increase the transcription of several stress responsive genes such as GRP78, GRP90 and Hsc70 in neuronal cells (Miles et al., 1991; Wilke et al., 1994; Hsieh et al., 1996). The GRP78 gene is up-regulated by ethanol through the SP1 site. Therefore, it would be of interest to investigate how ethanol aects the transcriptional activity mediated by the SP1 sites. Epidemiological studies have suggested that the drinking of alcoholic beverages is correlated with the development of cancer in humans, particularly oral and pharyngeal carcinomas (Blot et al., 1994; Maier et al., 1994; Elmore and Horwitz, 1995; Ko et al., 1995; Longnecker, 1995; Deleyiannis et al., 1996). The present study suggests that ethanol could enhance the carcinogenicity of DNA damaging agents by conferring a survival advantage to cells (preneoplastic) which have survived DNA damage that was not completely repaired. It remains to be determined whether ethanol aects DNA repair in mammalian cells. Ethanol may prevent apoptosis, the most potent natural defense mechanism against cancer (Morgenbesser et al., 1994; Symonds et al., 1994), by upregulating the expression of p21WAF1/CIP1. Inasmuch as ethanol in non-cytotoxic concentrations does not seem to aect the p53 level in

Primary normal human oral keratinocytes (NHOK) were cultured in Keratinocyte Basal Medium (KBM) supplemented with the growth factor bullet kit (Clonetics, San Diego, CA) as previously described (Park et al., 1991). Primary NHOK were subcultured once and utilized in the present experiment. A colon carcinoma RKO cell line obtained from Dr M Brattain (Medical College of Ohio, Toledo, OH), was cultured in McCoy's 5A modi®ed medium (GIBCO/BRL, Grand Island, NY) supplemented with 10% fetal bovine serum. The oral cancer cell lines SCC-4 and SCC-9 were cultured in DMEM/F12 medium (GIBCO/BRL) supplemented with 10% fetal bovine serum and 0.4 mg/ml of hydrocortisone. NHOK and RKO cells express wild-type p53 (Kuerbitz et al., 1992; Park et al., 1991), whereas the SCC-4 and SCC-9 cancer cells express mutant p53 and no p53, respectively (Min et al., 1994). Ethanol treatment Ethanol was added to 25 cm2 tissue culture ¯asks containing approximately 70% con¯uent cells. The cultures were then incubated for 4 ± 24 h at 378C. To avoid the evaporation of ethanol, the ¯ask cap was immediately tightened after the addition of ethanol. Unexposed control cells were cultured and handled in a manner similar to cells exposed to ethanol. The cells were harvested by trypsinization for analysis. Determination of cell proliferation rate To determine the eect of ethanol on the proliferation of cells, 46104 RKO cells were plated in culture ¯asks and used 24 h later. The cultures were divided into three groups: First group was cultured in the absence of ethanol (control); second group was cultured in the continuous presence of 200 mM ethanol for 6 days; and third group was exposed to 200 mM ethanol for 24 h (from day 1 to day 2) and subsequently incubated in ethanol-free medium for four additional days. The caps of culture ¯asks were tightly closed to avoid the evaporation of ethanol. Every 24 h, the cells were harvested with trypsinization and the number was counted with a hemocytometer. Two ¯asks from each experimental group were used for each time point. Colony formation assay Two types of experiments were carried out: (a) 70% con¯uent RKO cultures were exposed to 200 m M ethanol for 24 h. After trypsinization approximately 1000 single cells from either ethanol-exposed or unexposed cultures were then plated in triplicates. The colonies formed after 8 days of cultures were stained with Wright's stain and counted. (b) In second experiment, approximately 1000 unexposed cells were plated in each of six 25 cm2 ¯ask and cultured overnight. Three ¯asks were then exposed to 200 mM ethanol for 24 h and subsequently cultured for six additional days in ethanol-free medium. The other three

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¯asks were cultured in the absence of ethanol for 7 days. The colonies developed in these ¯asks were stained with Wright's stain and counted. Cell cycle analysis RKO cells were exposed to 200 mM ethanol for 0, 16, 24, 36 or 48 h. SCC-9 and NHOK cells were exposed to 200 mM ethanol for 15 h. At the end of ethanol exposure, cells were labeled with 10 mM bromodeoxyuridine (BrdU, Sigma Biochemicals, St. Louis, MO) for 2 h and harvested by trypsinization. The state of the cell cycle progression was analysed from the harvested cells using a FACScan ¯ow cytometer (Beckton-Dickinson, San Jose, CA) at 480 nm excitation as previously described (Gujuvala et al., 1994). Western analysis and immunoblotting Seventy percent con¯uent cells cultured in the absence or presence of ethanol were harvested and processed for Western analysis to determine the cellular level of p53, GADD45, p21WAF1/CIP1, and Cdk2 proteins using the Western-Light kit (Tropic Inc., Bedford, MA) as previously described (Baek et al., 1994). Monoclonal antibodies to p53, GADD45, p21WAF1/CIP1 and Cdk2 were obtained from Oncogene Sciences (Uniondale, NY). Northern analysis Poly (A)+ RNA was extracted from cells using the Fasttrack mRNA isolation kit (Invitrogen, San Diego, CA) and subjected to Northern analysis using WAF1/CIP1 cDNA (from Dr B Vogelstein, Johns Hopkins University, Baltimore, MD) and b-actin cDNA probes to determine the expression of these genes. The probes were labeled with 32 P-a-dCTP (ICN Biomedicals, Costa Mesa, CA) by multiprime labeling kit (Amersham Corp., Arlington Heights, IL). Creation of deletions in WAF1/CIP1 promoter/reporter plasmids The human WAF1/CIP1 gene promoter construct, WWPLuc, containing a 2.4 kbp genomic fragment of the WAF1/ CIP1 promoter was obtained from Dr B Vogelstein. To create deletion mutations starting from 5'-terminal end to DraI (71235) and MscI (7835) restriction sites, the WWP-Luc plasmid was digested with BamHI, and the cohesive end was ®lled with dNTP to create a blunt end. The linear DNA fragment was then digested with HindIII to separate the vector plasmid, a small fragment between the blunt end and the 5'-end Hind III site, and the WAF1/ CIP1 promoter. The fragment containing the WAF1/CIP1 promoter was digested with either DraI or MscI, the resulting 1265 bp DraI ± HindIII and 853 bp MscI ± HindIII fragments were gel puri®ed and ligated to the vector to create plasmid p21D1.26-Luc and p21M0.85-Luc, respectively. To generate plasmid p21P0.22-Luc which contains approximately 200 bp of the WAF1/CIP1 promoter upstream from the transcription initiation site of the WAF1/CIP1 gene, the WWP-Luc plasmid was digested with PstI which yielded 209 bp of the WAF1/ CIP1 promoter upstream from the transcription initiation site of the gene. The larger fragment was ligated and named p21P0.22-Luc. The p21H0-Luc deletion plasmid which does not contain the WAF1/CIP1 promoter was

constructed by digesting the plasmid with HindIII (Figure 6a). Luciferase assay Approximately 60% con¯uent RKO cells were transiently transfected with the WWP-Luc, p21D1.26-Luc, p21M0.85Luc, p21P0.22-Luc or p21H0-Luc plasmid using lipofectin as previously described (Park et al., 1991). Six hours after transfection, lipofectin was removed from the culture media by replacing the media with fresh culture media. After 24 h incubation, the culture was exposed to ethanol (200 mM) for 24 h. After harvesting, washing, and lysis, the luciferase activity was determined using a Luciferase Assay Kit (Promega, Medison, WI) by integrating total light emission over 30 s using a Beckman scintillation counter as recommended by the manufacturer. The luciferase activities were normalized based on protein concentrations. Each experiment (transfection) was repeated at least twice, and the presented luciferase activity of each group was an average of six assays. Immunoprecipitation and kinase assay Cells were suspended in kinase-lysis buer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 20 mM EDTA and 1% Triton X-100 and lysed by passing through a 22 G needle for 12 ± 15 times. The cell lysates were then centrifuged at 14 000 r.p.m. for 15 at 48C and the supernatants were aliquoted and stored at 7708C. About 200 ± 500 mg of protein in the cell lysate was incubated with 10 ml of anti-Cdk2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at 48C, and the immunocomplexes were precipitated down by addition of protein G Sepharose (Oncogene Sciences, Uniondale, NY). The immunocomplexes were then washed three times with RIPA buer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% Nonidet P40, 1% sodium deoxycholate, 0.1% SDS, 2 m M EDTA, 1 mM phenylmethylsulfonyl ¯uoride) and once with kinase assay buer. Kinase assay was then performed by incubating the immunocomplexes with 2 mg of histone H1 (Sigma Biochemicals, St. Louis, MO) in a buer containing 20 mM Tris-HCl, pH 7.5, 4 mM MgCl2, 25 mM cold ATP, 10 mCi of g-32P-ATP (ICN Biomedicals, Costa Mesa, CA) and 3 mg of ovalbumin (Sigma Biochemicals, St. Louis, MO) at 378C for 30 min. The reactions were stopped by adding 26Laemmli SDS gel sample buer and boiled for 5 min. The samples were then separated on 15% SDS ± PAGE and histone H1 was visualized by Coomassie blue staining. The gel was then destained and dried. The 32P labeled histone H1 bands were detected by exposing the dried gel to Kodak X-ray ®lm. The intensity of the radiolabeled bands was analysed and compared by densitometric scanning of the bands.

Acknowledgements This work was supported in part by grants from the DE10598 (UCLA/King-Drew Regional Research Center for Minority Oral Health) and DE11229 funded by the National Institute of Dental Research at the National Institutes of Health and from the STRC, Inc., No. 0509. We thank Dr B Vogelstein for providing the WWP-Luc plasmid and thank Dr R Chiu for his critical review of the manuscript.

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