Retinoic Acid Differentially Regulates Cancer Cell Proliferation Via Dose-Dependent Modulation of the Mitogen-Activated Protein Kinase Pathway

532 Vol. 1, 532–540, May 2003 Molecular Cancer Research

Retinoic Acid Differentially Regulates Cancer Cell Proliferation via Dose-Dependent Modulation of the Mitogen-Activated Protein Kinase Pathway

David L. Crowe,1 Randie Kim,1 and Roshantha A.S. Chandraratna2

1Center for Craniofacial Molecular Biology, University of Southern California, Los Angeles, CA and 2Division of Retinoid Research, Allergan, Irvine, CA

Abstract treatment of a number of types of cancer, including tumors The chemotherapeutic agent retinoic acid (RA) and its of lung, breast, head and neck, and blood (1–3). In animal derivatives have been used to treat many tumor types. models, retinoids have antitumor activity against cancer The antitumor effects of retinoids are in part due to their xenografts and induced regression of mammary carcinoma ability to inhibit proliferation of cancer cells. However, (4, 5). In vitro, RA and its derivatives inhibit proliferation of a smokers receiving dietary vitamin A and B carotene in variety of tumor types including lung, breast, leukemia, and chemoprevention studies had a higher incidence of lung rhabdomyosarcoma (6–10). However, smokers who took cancer. These studies imply that lower doses of dietary doses of retinoids in chemoprevention studies had a retinoids may have tumor-promoting activity. The effects higher incidence of lung cancer (11), suggesting that these of RA are mediated by a family of ligand-dependent compounds may also have tumor-promoting effects. transcription factors, the retinoic acid receptors (RARs) In the nucleus, the retinoic acid receptors (RARa,-h,-g) and the retinoid X receptors (RXR). We examined the mediate the effects of RA on gene expression (for review, effects of low- and high-dose RA treatment on prolifer- see 12). The RARs are members of a large family of ation of human squamous cell carcinoma lines in vitro. ligand-dependent transcription factors that include steroid, Low concentrations of RA (20 nM) increased proliferation thyroid hormone, and vitamin D receptors (for review, of SCC lines by epidermal growth factor (EGF) activation see 13). Numerous synthetic retinoids with receptor selective of the mitogen-activated protein kinase ERK1. These activities have been characterized (9, 14). RARs have changes were accompanied by increased expression of functional domains for RA and DNA binding, dimerization

S- and G2 phase cyclins and cyclin-dependent kinases with other factors, and transcriptional activation. The DNA (cdk), increased Rb phosphorylation, and increased binding domain contains two zinc finger motifs. RARs E2F-1 DNA binding activity. In contrast, higher doses of interact with cognate response elements in the promoters of RA (40 nM to 1 MM) inhibited ERK1 expression, caused many genes. RARs bind DNA as heterodimers with retinoid

accumulation of G1 phase cyclins and cdks, decreased X receptors (RXRs) (15). RXRs bind 9-cis RA and possess Rb phosphorylation, and increased Rb/E2F-1 associa- homology to other members of the superfamily (for review, tion. Overexpression of ERK1 or dominant negative see 16). ERK1 was sufficient to reproduce the effects of low- and The mitogen-activated protein kinase (MAPK) cascade high-dose RA, respectively. Treatment with receptor transmits both growth-promoting and antiproliferative signals selective retinoids revealed that both RARA and RAR; in many cell types (for review, see 17–20). The MAPK mediated the effects of RA on SCC lines. We concluded pathway transduces signals from a variety of growth factor that low-dose RA induced proliferation by increased receptors critical for passage through G1 phase of the cell EGF signaling while higher concentrations inhibited cell cycle. Autophosphorylation of these receptors by their kinase division by decreasing ERK1 activation. domains activates ras which binds raf, the most upstream kinase in the cascade (21). Raf then phosphorylates MEK, Introduction the next kinase downstream in the cascade (21). MEK The vitamin A metabolite retinoic acid (RA) and its phsophorylates the extracellular signal-regulated kinase derivatives are effective chemotherapeutic agents in the ERK1 (22). The current model for MAPK signaling relies on this three-kinase module (23). RA has been shown to activate or inhibit MAPK signaling in various cellular contexts (24–26). Regulation of this critical signaling pathway by RA may therefore modulate proliferation of Received 4/4/02; revised 4/3/03; accepted 4/4/03. both normal and tumor cells. The costs of publication of this article were defrayed in part by the payment of MAPK-driven progression through G1 phase of the cell page charges. This article must therefore be hereby marked advertisement in cycle is mediated by the activity of cyclin-dependent kinases accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Grant support: NIH grant DE10966. (cdk) and their cyclin regulatory subunits (27). Cdk4 and Requests for reprints: David L. Crowe, Center for Craniofacial Molecular cdk6 phosphorylate members of the retinoblastoma (Rb) Biology, University of Southern California, 2250 Alcazar Street, Los Angeles, CA 90033. Phone: (323) 442-3170; Fax: (323) 442-2981. protein family which release E2F transcription factors, Copyright D 2003 American Association for Cancer Research. promoting entry into S phase (28). Induction of cyclin

A-cdk2 and cyclin B-cdk1 complexes has been shown to regulate progression through S and G2 phases of the cell cycle before mitosis (27). RA has been shown to regulate cyclin and cdk expression in human bronchial epithelial cells (29) and synthetic receptor selective retinoids exhibit differential effects on the proliferation of lung cancer cell lines (9). These studies suggest that RA and its derivatives are able to exert pleiotropic effects on the proliferation of cancer cell lines in vitro. Given that RA can inhibit cancer cell proliferation in vivo and in vitro and in light of clinical studies suggesting tumor- promoting effects of dietary retinoids (5, 11), we proposed to elucidate potential molecular mechanisms for these seemingly contradictory findings. We examined the role of retinoid dose-dependent and receptor selective effects on cell cycle progression in human squamous cell carcinoma lines. We determined that RA and its synthetic derivatives can regulate cell cycle progression by differentially modulating MAPK activity.

Results To determine if RA exerted dose-dependent effects on the growth of human SCC lines, we used all-trans-RA and selective ligands in proliferation assays in vitro. As shown in Fig. 1A, treatment with 20 nM RA resulted in a 30% increase in cell number in all five lines compared to vehicle-treated controls. There were no differences in cell number between vehicle-treated cultures and those exposed to 10 nM RA (data not shown). In contrast, cultures treated with 40–100 nM RA showed a 20–30% decrease in proliferation of all lines compared to vehicle-treated cells. The highest degree of growth inhibition (50%) was seen in cultures treated with 1 AM RA. All five cell lines tested showed increased proliferation at FIGURE 1. RA differentially regulates proliferation of human SCC 20 nM RA but decreasing growth rates at concentrations of lines in a dose-dependent manner. A. SCC lines were plated in triplicate 40 nM and higher. To determine whether this effect was and grown for 4 days in all-trans-RA concentrations of 20 nM to 1 AM. dependent on RAR or RXR activity, we repeated the Cells were trypsinized and counted with a hemacytometer. B. RA- proliferation experiments using selective ligands. As shown dependent regulation of SCC proliferation is mediated by RARs. SCC lines were plated as described above and treated with 20 nM to 1 AM of in Fig. 1B, treatment with the RAR selective ligand the RAR selective retinoid AGN192890 or RXR selective compound AGN192890 essentially reproduced the dose-dependent effects AGN194204. Cells were trypsinized and counted with a hemacytometer. Data for the RAR selective retinoid are shown since the RXR selective of RA. Cellular proliferation was increased at the 20-nM compound had no effect on SCC proliferation. These experiments were concentration but decreased at 40 nM and higher in all five performed three times with similar results. , vehicle; , 20 nM;, lines tested. Both the RARa selective ligand AGN195183 and 40 nM; , 100 nM;,1AM. Error bars, SE. the RARg selective compound AGN194433 showed effects on cell growth similar to AGN192890, but the RXR selective ligand AGN194204 failed to show any effects on proliferation that RA did not induce apoptosis in these SCC lines. To of SCC lines (data not shown). These data indicate that RA determine if altered cell cycle progression could account for acting via RARs differentially regulates the proliferation of the effects of RA on SCC proliferation, we performed SCC lines in a dose-dependent manner. bromodeoxyuridine (BrdUrd) incorporation assays on cultures To determine whether the decreased cell number observed of all five cell lines treated with vehicle, 20 nM RA, or 1 AM after high-dose RA treatment of SCC lines was due to RA. As shown in Fig. 2B, treatment with 20 nM RA apoptosis, we examined internucleosomal DNA fragmentation increased the fraction of BrdUrd-positive cells from 15% to by terminal deoxynucleotidyl transferase (Tdt)-mediated nick 25% on average, in agreement with the increased proliferation end labeling (TUNEL) assay. As shown in Fig. 2A, neither observed in these cell lines. In contrast, treatment with 1 AM treatment with 20 nM nor 1 AM RA induced significant RA decreased the fraction of BrdUrd-positive cells to 8% increases in the number of TUNEL-positive cells. By which correlated with the decreased cellular proliferation contrast, treatment of SCC25 cultures with the anti-Fas observed at this concentration. These results indicate that the positive control antibody increased the fraction of TUNEL- effects of RA on proliferation of SCC lines is the result of positive cells from 1% to 50% in 16 h. These results indicate altered cell cycle progression.

cdk2 (4-fold) was detected by Western blot as was increased expression of the G2 phase proteins cyclin B and cdk1 (3-fold). No change in cdk4 and cdk6 expression was observed at this RA concentration. These results indicate that increased proliferation of SCC lines in response to 20 nM RA correlates with induction of S and G2 phase cell cycle regulatory proteins. In contrast, treatment with 1 AM RA inhibited TK expression by 50% compared to vehicle- treated control cells, consistent with the decreased S phase fraction determined by BrdUrd incorporation. This correlated with induction of the G1 phase markers cdk4 and cdk6 (3-fold). In agreement with these results, decreased expression of the S and G2 phase markers cyclin A and B was observed after treatment with 1 AM RA. No changes in cdk1 and cdk2 expression were observed. These data indicate that dose-dependent alteration of cell cycle progression by RA is accompanied at the molecular level by changes in regulatory protein expression. Rb phosphorylation and release of E2F transcription factors regulate progression from G1 to S phase of the cell cycle (27). To determine if altered cdk expression correlated with changes in Rb phosphorylation, we immunoprecipitated Rb from SCC lines treated with vehicle or increasing RA concentrations. As shown in Fig. 4, treatment with 20 nM RA produced a 6-fold increase in Rb phosphorylation on serine residues. This was accompanied by decreased association with E2F-1, consistent with the increase in proliferation observed at this RA concentration. In contrast, treatment with 1 AM RA decreased Rb phosphorylation consistent with increased association with E2F-1. These results indicate that Rb phosphorylation and E2F-1 release is differentially regulated by RA in a dose-dependent manner in SCC lines. To determine if decreased E2F-1 association with Rb resulted in increased binding of this transcription factor to DNA, we performed electrophoretic mobility shift analysis on vehicle- and RA-treated SCC25 cells. As shown in Fig. 5A, FIGURE 2. RA treatment of SCC lines does not result in apoptosis but 20 nM RA treatment produced a reproducible increase in E2F regulates the G1-to-S-phase transition of the cell cycle. A. SCC lines were DNA binding activity. In contrast, treatment with 1 AM RA plated in triplicate and treated with 20 nM to 1 AM all-trans-RA for 24 h. resulted in a 3-fold decrease in E2F DNA binding activity. SCC25 cells were treated with anti-Fas antibody as the positive control for apoptosis. Cultures were subjected to TUNEL assay as described in The presence of E2F-1 in the binding complex was ‘‘Materials and Methods.’’ Numbers of TUNEL-positive cells were confirmed by the presence of a supershifted band after expressed as a percentage of total cells counted. , vehicle; , 20 nM incubation with an anti-E2F-1 antibody. Binding of E2F-1 to RA; , 1 AM RA; , anti-Fas. B. SCC lines were plated in triplicate and treated with 20 nM to 1 AM all-trans-RA for 24 h. Cultures were subjected to the probe was effectively competed by unlabeled oligonu- BrdUrd labeling as described in ‘‘Materials and Methods.’’ Numbers of cleotide but not by one containing a mutated binding site. BrdUrd-positive cells were expressed as a percentage of total cells Increased E2F-1 DNA binding activity correlated with counted. , vehicle; , 20 nM RA; , 1 AM RA. These experiments were performed three times with similar results. Error bars, SE. transactivation of a promoter containing E2F binding sites (Fig. 5B). Twenty nanomolars RA treatment produced 2- to 3-fold induction of reporter gene activity in SCC15 and To determine if altered cell cycle progression correlated SCC25 cells. Treatment with 1 AM RA inhibited promoter with RA-mediated changes in cell cycle regulatory mole- activity by 50% compared to control levels. Transfection of cules, we examined expression of the DNA synthesis an E2F-1 expression vector as the positive control for enzyme thymidine kinase (TK), the G1 phase markers transactivation produced an 8-fold increase in reporter gene cdk4 and cdk6, and the S-G2 phase proteins cyclin A, cyclin activity. These results indicate that RA can regulate the DNA B, cdk1, and cdk2. As shown in Fig. 3, 20 nM RA binding and transactivation properties of E2F-1 in a dose- treatment induced TK expression by 2-fold in SCC15 cells dependent manner, consistent with its effects on proliferation as determined by Northern blot, correlating with the of SCC lines. increased S phase fraction observed in this treatment group. Given that EGF signaling via the MAPK pathway is an Similarly, induction of the S phase markers cyclin A and important upstream regulator of cellular proliferation in

FIGURE 3. Differential regulation of SCC proliferation by RA results in induction of phase-specific cell cycle regulatory proteins. SCC lines were treated with vehicle, 20 nM RA, or 1 AM RA for 16 h. Expression of the S phase marker TK was determined by Northern blot. Blots were stripped and hybridized to a h-actin probe to ensure equal amounts of mRNA in each lane. Expression of S and G2 phase markers (cyclin A, cyclin B, cdk1, and cdk2) and G1 phase markers cdk4 and cdk6 was determined by Western blot. These experiments were performed three times with similar results. Representative blots are shown.

human SCC lines (20, 21, 30), we wondered whether RA reduction in Elk1 phosphorylation. RA treatment of domi- could regulate EGF expression and ERK1 activation and thus nant negative ERK1 clones decreased Elk1 phosphorylation provide a potential mechanism for the effects of the retinoid by 5-fold due to inhibition of the residual activity of on cell cycle regulatory proteins. Treatment of SCC4 and endogenous ERK1. Elk1 phosphorylation in RA-treated SCC9 cells with 20 nM RA stimulated EGF secretion dominant negative ERK1 clones was 3-fold less than in (Fig. 6A) and induced activated ERK1 expression by nearly similarly treated control cells. Since the ERK1 antibody 2-fold (Fig. 6B). In contrast, treatment with 1 AM RA recognized both wild-type and dominant negative forms of inhibited EGF production by 90% and ERK1 activation by the protein, total ERK1 protein levels were higher in 2-fold. This inhibition was accompanied by a decrease in overexpressing clones than in control cells. ERK1 activation total ERK1 expression. Treatment with both low- and high- in control clones was half that of ERK1 overexpressing cells dose RA inhibited EGFR expression in SCC4 and SCC9 but higher than in dominant negative ERK1 cells as cells (see ‘‘Discussion’’); however, levels of ERK2, ERK3/ expected. RA treatment of control clones markedly inhibited p97MAPK, JNK1, and p38 proteins were unaffected by retinoid treatment. These results indicated that low-dose RA increased proliferation of SCC lines by induction of EGF and ERK1 while high doses of the retinoid inhibited EGF expression and ERK1 activation. To determine if altered ERK1 expression was sufficient to reproduce the effects of RA on SCC proliferation, we stably transfected ERK1 or dominant negative ERK1 constructs into SCC71 cells. Expression and activity of transfected ERK1 and dominant negative ERK1 proteins is shown in Fig. 7A. Clones expressing dominant negative ERK1 showed markedly decreased levels of total activity compared to vector-transfected controls (Fig. 7A). In contrast, clones overexpressing wild-type ERK1 demonstrated increased activity of this kinase. Clones stably transfected with the FIGURE 4. RA differentially regulates Rb phosphorylation and association with E2F-1 in a dose-dependent manner. SCC lines were treated with wild-type ERK1 construct showed high levels of protein 0.1% DMSO vehicle, 20 nM RA, or 1 AM RA for 16 h. Rb protein was expression, kinase activation, and Elk1 phosphorylation (Fig. immunoprecipitated (IP Rb) from cellular lysates as described in ‘‘Materials and Methods.’’ Blots were probed with anti-phosphoRb antibody (anti- 7B). These levels were only marginally affected by 1 AM RA ppRb) to determine the level of Rb phosphorylation in response to RA. treatment, likely due to the constitutive expression of the Blots were stripped and incubated with anti-E2F-1 antibody to determine ERK1 construct. Conversely, clones expressing the dominant association of this transcription factor with Rb. Blots were stripped and incubated with anti-Rb antibody to ensure equal amounts of immunopre- negative ERK1 construct showed a 3-fold decrease in cipitated protein in each lane. These experiments were performed three activation compared to ERK1 cells, with a corresponding times with similar results. Representative blots are shown.

BrdU-positive cells (40–50% of vehicle-treated controls), RA failed to decrease the S phase fraction of ERK1-overexpressing clones. These results were reflected in proliferation assays using vehicle- and RA-treated control, ERK1, and dominant negative ERK1 clones (Fig. 7D). We concluded that alterations in ERK1 activity were sufficient to reproduce the effects of RA on cell proliferation, and that overexpression of this kinase induced an RA-resistant phenotype in the SCC71 line.

FIGURE 5. RA differentially regulates E2F-1 DNA binding activity and transactivation in a dose-dependent manner. A. Nuclear extracts from SCC25 cells treated with vehicle, 20 nM RA, or 1 AM RA were subjected to electrophoretic mobility shift analysis as described in ‘‘Materials and Methods.’’ Anti-E2F-1 antibody was included in some binding reactions to demonstrate the presence of the transcription factor in the shifted complexes (SS). To determine the specificity of E2F-1 binding to its binding site, 10- to 1000-fold molar excess of unlabeled competitor oligonucleotide (comp) was included in some binding reactions. No binding was detected using a labeled probe in which the E2F binding site had been mutated (mut. probe) nor did this oligonucleotide effectively compete for binding to the wild-type probe. The position of the free probe is shown. This experiment was performed three times with similar results. A representative gel is shown. B. SCC lines were transiently transfected with a heterologous promoter containing four tandem E2F binding sites cloned in the pGL3 luciferase reporter vector as described in ‘‘Materials and Methods.’’ A human E2F-1 expression vector or blank plasmid was cotransfected as controls for promoter activation. Cells were treated with 10 nM to 1 AM RA for 16 h. Luciferase activity was normalized to h-galactosidase levels for each sample. These experiments were performed three times with similar results. , vehicle; , 20 nM RA; , 1 AM RA; , E2F1. Error bars, SE. FIGURE 6. Retinoids differentially regulate epidermal growth factor (EGF) expression and ERK1 activity in a dose-dependent manner. SCC lines were treated with vehicle, 20 nM RA, or 1 AM RA for 16 h. A. EGF secretion was determined by enzyme-linked immunosorbent assay ERK1 activation and Elk1 phosphorylation. Expression of (ELISA) as described in ‘‘Materials and Methods.’’ , con; , the dominant negative ERK1 construct resulted in a decrease 20 nM;,1AM. B. ERK1 activation was determined by Western blot using antihuman antibody to activated ERK1. Blots were stripped and in BrdUrd-positive cells from 15% to 8% (Fig. 7C). Wild- incubated with total ERK1 antibody to verify the amount of protein in type ERK1 overexpression caused an increase in the number each lane. Expression of additional kinases (EGFR, ERK2, ERK3, JNK1, and p38) was also determined by Western blot. These of BrdU-positive cells to 20%. Interestingly, while treatment experiments were performed three times with similar results. Repre- with 1 AM RA produced marked decreases in the number of sentative blots are shown.

FIGURE 7. Overexpression of ERK1 or dominant negative ERK1 is sufficient to reproduce the dose-dependent effects of RA on proliferation of SCC lines. A. SCC71 cells were stably transfected with a human FLAG-tagged ERK1 construct, FLAG-tagged dominant negative ERK1 (dnERK) expression plasmid, or blank vector. Exogenous ERK1 proteins were immunoprecipitated with anti-FLAG antibody (IP FLAG) and subjected to in vitro kinase assay (ERK1 kinase). Total ERK1 protein also was immunoprecipitated with anti-ERK1 antibody (IP ERK1) and subjected to in vitro kinase assay (total ERK1 kinase). Blots were probed with antihuman ERK1 antibody to determine relative amounts of ERK1 protein in each lane. These experiments were repeated three times with similar results. Representative blots are shown. B. Lysates from vehicle- or RA-treated (+RA) SCC71 stable clones expressing ERK1, dominant negative ERK1, or blank vector were subjected to Western blotting with antibodies to activated ERK1, total ERK1, and phosphorylated Elk1 as describedin ‘‘Materials and Methods.’’ Representative blots are shown. C. Vector transfected control, ERK1, or dominant negative ERK1 (dnERK1)-expressing clones were plated in triplicate and treated with vehicle or 1 AM RA for 16 h. Cultures were subjected to BrdUrd incorporation analysis as described in ‘‘Materials and Methods.’’ BrdUrd-positive cells were expressed as a percentage of total cells counted. These experiments were performed three times with similar results. , vehicle; , 1 AM RA. Error bars, SE. D. Vector-transfected control (vec), ERK1 (erk), or dominant negative ERK1 (dn)-expressing clones were plated in triplicate and treated with vehicle or 1 AM RA (+RA) for up to 6 days. At 2-day intervals, cells were trypsinized and counted using a hemacytometer. These experiments were performed three times with similar results. , vec; , vec + RA; , erk; , erk + RA; , dn; , dn + RA.

Discussion inhibits their proliferation (31). Interestingly, 20 nM RA We report here that proliferation of human SCC lines is treatment inhibited EGFR expression while inducing ERK1 regulated by RA dose-dependent mechanisms via EGF and activation and cellular proliferation. In a previous study, we the MAPK pathway. Low concentrations of RA increased determined that the EGFR overexpression commonly EGF production, ERK1 activity, and the proliferation of observed in SCC lines not only resulted in growth factor- SCC lines which was characterized by concomitant changes independent proliferation but also limited EGF responsive- in cell cycle regulatory protein expression, Rb phosphoryl- ness due to limiting amounts of the downstream adaptor ation, and E2F-1 activation. Conversely, high concentrations protein Grb2 (32). Pretreatment of SCC lines with RA of RA decreased EGF secretion, ERK1 activation, and cel- restored EGF responsiveness to these cells by inhibiting EGFR lular proliferation of SCC lines. We have shown previously expression and restoring stoichiometric balance between the that inhibiting ERK1 activation in these cells potently receptor and the adaptor protein. Grb2 overexpression increased

proliferation and restored EGF responsiveness in these cell Materials and Methods lines. These results have important implications for retinoid Cell Culture and Stable Transfection refractoriness in SCC chemotherapy. Taken together, these two The human SCC lines used in this study were purchased studies describe an important mechanism by which RA dif- from the American Type Culture Collection, Rockville, MD ferentially regulates cellular proliferation via the EGF/MAPK and have been described previously (40, 41). Cells were signaling pathway. cultured in DMEM, 10% charcoal-stripped fetal bovine serum, A previous study using normal epidermal keratinocytes from 40 Ag/ml gentamicin at 37jC in a humidified atmosphere of 5% RAR null mutant mice revealed that RARg mediated the effects CO2. SCC71 cells were transfected with 5 Ag expression of RA on cellular proliferation (33). However, our present study vectors for ERK1, a dominant negative kinase-deficient ERK1 using synthetic retinoids did not reveal similar receptor (10), or neomycin resistance plasmid alone using Lipofect- selectivity. Recently, other RARg selective retinoids were AMINE reagent according to manufacturer’s recommendations shown to mediate growth inhibition in SCC lines (34). Whether (Life Technologies, Inc., Carlsbad, CA). Cells were selected in additional properties of RARg also mediate growth inhibition 400 Ag/ml G418 for 14 days. Resistant clones were picked for of SCC lines remains to be determined. expansion and characterization. RA has been shown to differentially regulate MAPK activity in a variety of studies. During myeloid differentiation Cell Death and Proliferation Assays of HL-60 cells, RA was shown to activate ERK2 but not JNK The TUNEL assay for in situ cell death detection in SCC lines or p38 kinases (24, 26). However, we did not observe changes has been described previously (42). Cells were treated with 0.1% in expression of these MAPKs following RA treatment of SCC DMSO vehicle, up to 1 AM all-trans-RA, the RAR and RXR lines. These results suggest that MAPKs other than ERK1 may selective retinoids AGN192890 and AGN194204, or the RARa not participate in RA-mediated inhibition of SCC proliferation. and RARg selective compounds AGN195183 and AGN194433 The mechanism by which ERK1 but not other MAPKs is (9). Cells were fixed in 4% paraformaldehyde (pH 7.4), and regulated by RA treatment in SCC lines will be important in permeabilized with 0.1% Triton X-100, 0.1% sodium citrate for future studies. Blockade of MAPK activation inhibited the 2 min on ice. A mouse IgM antihuman Fas antibody (Molecular ability of RA to induce differentiation of HL-60 cells (24, 26). Biology Laboratories, Watertown, MA) that induces apoptosis in However, in human bronchial epithelial cells and lung cancer sensitive cell lines was used as the positive control. An isotype- lines, RA was shown to inhibit both ERK- and JNK-dependent matched control antibody was used as the negative control. After signaling pathways (26, 35). These effects were abrogated by washing with PBS, cells were incubated with fluorescein-dUTP the use of RAR antagonists. Recently, decreased ERK activ- and terminal deoxynucleotidyl transferase for 60 min at 37jC ity in aged human skin was shown to be reversed by RA according to manufacturer’s recommendations (Roche Molec- treatment (36). We show here that RA can exert opposite effects ular Biochemicals, Indianapolis, IN). After washing three times on ERK1 activation in a dose-dependent manner. Taken in PBS, apoptotic cells were visualized by fluorescence micro- together, these studies demonstrate that regulation of MAPK scopy. Proliferation was analyzed by plating 5 Â 104 cells in activity by RA is dependent on dose, receptor selectivity, and triplicate cultures and treating with vehicle or RA for up to cellular context. 6 days. Cells were trypsinized and counted with a hemacytometer Another important result of this study is the induction of at 2- or 4-day intervals. To determine G to S phase progression, RA resistance by ERK1 overexpression. Resistance to the 1 BrdUrd incorporation analysis was performed. Vehicle- and RA- growth inhibitory effects of RA would be expected to provide treated cells were incubated with 10 AM BrdUrd for 1 h, washed a significant advantage to tumorigenic clones. ERK1 phos- in PBS, and fixed in 70% ethanol containing 50 mM glycine (pH phorylates members of the ets transcription factor family such 2) for 20 min at À20jC. After washing in PBS, the cells were as Elk1 (21). Elk1 is part of the ternary complex which binds incubated with mouse anti-BrdUrd primary antibody for 30 min to and activates the serum response element in the c-fos at 37jC according to manufacturer’s recommendations (Roche promoter, thereby inducing its expression. Increased fos Molecular Biochemicals). The cells were washed in PBS and expression may lead to induction of AP-1 activity; binding sites incubated with antimouse IgG secondary antibody conjugated to for these factors are found in the promoters of many genes fluorescein. Following extensive washing in PBS, BrdUrd- regulating cellular proliferation (37). Constitutive ERK1 over- positive cells were visualized by fluorescence microscopy, expression may inhibit the anti-AP-1 effects of RA by activating counted, and expressed as a percentage of the total cells counted c-fos, resulting in accelerated growth and an RA-resistant in 10 random high-power fields. phenotype. These clones will provide a useful model system for investigating other aspects of the RA-resistant phenotype of cancer cells. Northern Blot In summary, RA can differentially regulate MAPK activity Two micrograms mRNA from vehicle- or RA-treated SCC and cancer cell proliferation by dose-dependent mechanisms. cells were electrophoresed in 1% agarose gels containing 2.2 M Recently, Rb protein has been shown to associate with histone formaldehyde using 1Â 4-morpholinepropanesulfonic acid deacetylase to repress transcription (38, 39). In future experi- running buffer. mRNA was capillary transferred to nylon ments, we will examine the role of RA in recruitment of histone membranes (Schleicher and Schuell, Keene, NH), hybridized to deacetylase to cell cycle regulatory complexes and how these 6 Â 106 cpm/ml random primed 32P-labeled TK cDNA probe proteins regulate proliferation and retinoid resistance in human (purchased from the American Type Culture Collection) for 16 h cancer cells. at 42jC in 50% formamide, 5Â SSPE, 1Â Denhardt’s solution,

and 0.2% SDS. Blots were washed once in 2Â SSC, 0.1% SDS centrifugation at 10,000 Â g for 30 min at 4jC, the supernatant for 45 min at 50jC followed by one wash in 0.2Â SSC, 0.1% was removed and dialyzed against 20 mM HEPES (pH 7.9), 20% SDS for 45 min at 50jC. Blots were washed a final time in glycerol, 0.1 M KCl, 0.2 mM EDTA, 0.2 mM PMSF, and 0.5 mM 0.2Â SSC, 0.1% SDS for 45 min at 65jC and exposed to DTT for 1 h at 4jC. Fifteen micrograms of dialyzed nuclear Kodak XAR5 autoradiographic film for 16 h at À80jC. extract were incubated in binding reactions containing 2 Ag poly(dIÁdc)-poly(dIÁdc) and 10,000 cpm 32P end labeled double- Western Blot stranded oligonucleotide containing a canonical E2F site (5V- Seventy-five micrograms total protein from vehicle- or RA- TTTCGCGC-3V; Santa Cruz Biotechnology). For binding treated cells were separated by SDS-PAGE on 10% resolving competition analysis, 10- to 1000-fold molar excess of gels under denaturing and reducing conditions. Separated unlabeled probe or mutated oligonucleotide (5V-TTTCGCat- proteins were electroblotted to polyvinylidene difluoride 3V) was included in the reactions. To determine if E2F-1 was membranes according to manufacturer’s recommendations present in the shifted complexes, 1 Al antihuman E2F-1 antibody (Bio-Rad, Hercules, CA). Blots were incubated with antihuman (Santa Cruz Biotechnology) or control IgG was included in the primary antibodies to cyclins, cdks, EGFR, ERK1, activated binding reactions. Reactions were incubated at room temper- ERK1, ERK2, ERK3, JNK1, p38, and activated Elk1 (Trans- ature for 15 min and subjected to native PAGE using 0.5Â Tris- duction Laboratories, San Diego, CA and Santa Cruz borate-EDTA running buffer. Gels were dried and exposed to j Biotechnology, Santa Cruz, CA) for 16 h at 4jC. After washing Kodak XAR5 autoradiographic film for 16 h at À80 C. in Tris-buffered saline containing 0.1% Tween 20 (TBST, pH 7.5), blots were incubated for 30 min at room temperature with Transient Transfection and Reporter Gene Assays anti-IgG secondary antibody conjugated to horseradish perox- SCC lines were plated in triplicate into six-well tissue culture idase. After extensive washing in TBST at room temperature, plates. Cells were transiently transfected with 5 Agofa bands were visualized by the enhanced chemiluminescence heterologous promoter containing four tandem repeats of the method (Roche Molecular Biochemicals). E2F recognition sequence (5V-TTTCGCGC-3V)clonedinthe pGL3 luciferase reporter vector (Promega, Madison, WI) using LipofectAMINE according to manufacturer’s recommendations Immunoprecipitation and in Vitro Kinase Assay (Life Technologies). One microgram h-galactosidase expression Vehicle- and RA-treated cells were lysed in 50 mM HEPES vector (Vical) was used to normalize for transfection efficiency. (pH 7.5), 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 1 mM DTT, Cells were treated with vehicle or up to 1 AM RA for 24 h following 1% NP40, 10% glycerol, 1 mM NaF, 0.1 mM sodium transfection. Reporter gene activity was determined using a orthovanadate, and protease inhibitors for 30 min at 4jC. commercially available kit (Dual-light, Tropix). Luciferase Lysates were centrifuged at 10,000 Â g for 10 min and activity was normalized to h-galactosidase levels for each sample. antihuman primary antibody to Rb (Santa Cruz Biotechnology) was incubated with the supernatants for 1 h at 4jC. For in vitro kinase assays, anti-FLAG antibody was used to immunopreci- Enzyme-Linked Immunosorbent Assay pitate transfected ERK1 and dnERK1. Anti-ERK1 antibody was Triplicate cultures of SCC25 cells were treated with vehicle, used to immunoprecipitate total ERK1 proteins from these 20 nM RA, or 1 AM RA for 16 h. Conditioned medium was clones. Antigen-antibody complexes were precipitated by collected from each culture and subjected to ELISA for secreted incubation with protein A/G agarose (Santa Cruz Biotechnol- EGF using a commercially available kit (Quantikine, R&D ogy) for 1 h at 4jC. Immunoprecipitated proteins were washed Systems, Minneapolis, MN). three times with 1 ml lysis buffer. For kinase assays, immunoprecipitates were washed an additional time in kinase Acknowledgments buffer [50 mM HEPES (pH 7.5), 10 mM MgCl2,1mM DTT, We thank Dr. Karen Vousden for the E2F1 expression vector and Dr. Melanie Cobb for ERK1 expression plasmids. 2.5 mM EGTA, 1 mM NaF, 0.1 mM sodium orthovanadate, 20 AM ATP]. 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Downloaded from mcr.aacrjournals.org on October 1, 2021. © 2003 American Association for Cancer Research. Retinoic Acid Differentially Regulates Cancer Cell Proliferation via Dose-Dependent Modulation of the Mitogen-Activated Protein Kinase Pathway11NIH grant DE10966.

David L. Crowe, Randie Kim and Roshantha A.S. Chandraratna

Mol Cancer Res 2003;1:532-540.

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