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Molecular Cancer Research 1:532-540 (2003)
© 2003 American Association for Cancer Research

Signaling and Regulation

Retinoic Acid Differentially Regulates Cancer Cell Proliferation via Dose-Dependent Modulation of the Mitogen-Activated Protein Kinase Pathway1

David L. Crowe1, Randie Kim1 and Roshantha A.S. Chandraratna2

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

Requests for reprints: David L. Crowe, Center for Craniofacial Molecular Biology, University of Southern California, 2250 Alcazar Street, Los Angeles, CA 90033. Phone: (323) 442-3170; Fax: (323) 442-2981.


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
The chemotherapeutic agent retinoic acid (RA) and its derivatives have been used to treat many tumor types. The antitumor effects of retinoids are in part due to their ability to inhibit proliferation of cancer cells. However, smokers receiving dietary vitamin A and ß carotene in chemoprevention studies had a higher incidence of lung cancer. These studies imply that lower doses of retinoids may have tumor-promoting activity. The effects of RA are mediated by a family of ligand-dependent transcription factors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXR). We examined the effects of low- and high-dose RA treatment on proliferation of human squamous cell carcinoma lines in vitro. Low concentrations of RA (20 nM) increased proliferation of SCC lines by epidermal growth factor (EGF) activation of the mitogen-activated protein kinase ERK1. These changes were accompanied by increased expression of S- and G2 phase cyclins and cyclin-dependent kinases (cdk), increased Rb phosphorylation, and increased E2F-1 DNA binding activity. In contrast, higher doses of RA (40 nM to 1 µM) inhibited ERK1 expression, caused accumulation of G1 phase cyclins and cdks, decreased Rb phosphorylation, and increased Rb/E2F-1 association. Overexpression of ERK1 or dominant negative ERK1 was sufficient to reproduce the effects of low- and high-dose RA, respectively. Treatment with receptor selective retinoids revealed that both RAR{alpha} and RAR{gamma} mediated the effects of RA on SCC lines. We concluded that low-dose RA induced proliferation by increased EGF signaling while higher concentrations inhibited cell division by decreasing ERK1 activation.


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
The vitamin A metabolite retinoic acid (RA) and its derivatives are effective chemotherapeutic agents in the treatment of a number of types of cancer, including tumors of lung, breast, head and neck, and blood (1–3). In animal models, retinoids have antitumor activity against cancer xenografts and induced regression of mammary carcinoma (4, 5). In vitro, RA and its derivatives inhibit proliferation of a variety of tumor types including lung, breast, leukemia, and rhabdomyosarcoma (6–10). However, smokers who took dietary doses of retinoids in chemoprevention studies had a higher incidence of lung cancer (11), suggesting that these compounds may also have tumor-promoting effects.

In the nucleus, the retinoic acid receptors (RAR{alpha}, -ß, -{gamma}) mediate the effects of RA on gene expression (for review, see 12). The RARs are members of a large family of ligand-dependent transcription factors that include steroid, thyroid hormone, and vitamin D receptors (for review, see 13). Numerous synthetic retinoids with receptor selective activities have been characterized (9, 14). RARs have functional domains for RA and DNA binding, dimerization with other factors, and transcriptional activation. The DNA binding domain contains two zinc finger motifs. RARs interact with cognate response elements in the promoters of many genes. RARs bind DNA as heterodimers with retinoid X receptors (RXRs) (15). RXRs bind 9-cis RA and possess homology to other members of the superfamily (for review, see 16).

The mitogen-activated protein kinase (MAPK) cascade transmits both growth-promoting and antiproliferative signals in many cell types (for review, see 17–20). The MAPK pathway transduces signals from a variety of growth factor receptors critical for passage through G1 phase of the cell cycle. Autophosphorylation of these receptors by their kinase domains activates ras which binds raf, the most upstream kinase in the cascade (21). Raf then phosphorylates MEK, the next kinase downstream in the cascade (21). MEK phsophorylates the extracellular signal-regulated kinase 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 both normal and tumor cells.

MAPK-driven progression through G1 phase of the cell cycle is mediated by the activity of cyclin-dependent kinases (cdk) and their cyclin regulatory subunits (27). Cdk4 and cdk6 phosphorylate members of the retinoblastoma (Rb) protein family which release E2F transcription factors, 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
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
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 µM RA. All five cell lines tested showed increased proliferation at 20 nM RA but decreasing growth rates at concentrations of 40 nM and higher. To determine whether this effect was dependent on RAR or RXR activity, we repeated the proliferation experiments using selective ligands. As shown in Fig. 1B, treatment with the RAR selective ligand AGN192890 essentially reproduced the dose-dependent effects of RA. Cellular proliferation was increased at the 20-nM concentration but decreased at 40 nM and higher in all five lines tested. Both the RAR{alpha} selective ligand AGN195183 and the RAR{gamma} selective compound AGN194433 showed effects on cell growth similar to AGN192890, but the RXR selective ligand AGN194204 failed to show any effects on proliferation of SCC lines (data not shown). These data indicate that RA acting via RARs differentially regulates the proliferation of SCC lines in a dose-dependent manner.



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  		FIGURE 1. RA differentially regulates proliferation of human SCC lines in a dose-dependent manner. A. SCC lines were plated in triplicate and grown for 4 days in all-trans-RA concentrations of 20 nM to 1 µM. Cells were trypsinized and counted with a hemacytometer. B. RA-dependent regulation of SCC proliferation is mediated by RARs. SCC lines were plated as described above and treated with 20 nM to 1 µM of the RAR selective retinoid AGN192890 or RXR selective compound AGN194204. Cells were trypsinized and counted with a hemacytometer. Data for the RAR selective retinoid are shown since the RXR selective compound had no effect on SCC proliferation. These experiments were performed three times with similar results. , vehicle; , 20 nM; , 40 nM; , 100 nM; , 1 µM. Error bars, SE.

 
To determine whether the decreased cell number observed after high-dose RA treatment of SCC lines was due to apoptosis, we examined internucleosomal DNA fragmentation by terminal deoxynucleotidyl transferase (Tdt)-mediated nick end labeling (TUNEL) assay. As shown in Fig. 2A, neither treatment with 20 nM nor 1 µM RA induced significant increases in the number of TUNEL-positive cells. By contrast, treatment of SCC25 cultures with the anti-Fas positive control antibody increased the fraction of TUNEL-positive cells from 1% to 50% in 16 h. These results indicate that RA did not induce apoptosis in these SCC lines. To determine if altered cell cycle progression could account for the effects of RA on SCC proliferation, we performed bromodeoxyuridine (BrdUrd) incorporation assays on cultures of all five cell lines treated with vehicle, 20 nM RA, or 1 µM RA. As shown in Fig. 2B, treatment with 20 nM RA increased the fraction of BrdUrd-positive cells from 15% to 25% on average, in agreement with the increased proliferation observed in these cell lines. In contrast, treatment with 1 µM RA decreased the fraction of BrdUrd-positive cells to 8% which correlated with the decreased cellular proliferation observed at this concentration. These results indicate that the effects of RA on proliferation of SCC lines is the result of altered cell cycle progression.



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  		FIGURE 2. RA treatment of SCC lines does not result in apoptosis but regulates the G1-to-S-phase transition of the cell cycle. A. SCC lines were plated in triplicate and treated with 20 nM to 1 µM all-trans-RA for 24 h. SCC25 cells were treated with anti-Fas antibody as the positive control for apoptosis. Cultures were subjected to TUNEL assay as described in "Materials and Methods." Numbers of TUNEL-positive cells were expressed as a percentage of total cells counted. , vehicle; , 20 nM RA; , 1 µM RA; , anti-Fas. B. SCC lines were plated in triplicate and treated with 20 nM to 1 µM all-trans-RA for 24 h. Cultures were subjected to BrdUrd labeling as described in "Materials and Methods." Numbers of BrdUrd-positive cells were expressed as a percentage of total cells counted. , vehicle; , 20 nM RA; , 1 µM RA. These experiments were performed three times with similar results. Error bars, SE.

 
To determine if altered cell cycle progression correlated with RA-mediated changes in cell cycle regulatory molecules, we examined expression of the DNA synthesis enzyme thymidine kinase (TK), the G1 phase markers cdk4 and cdk6, and the S-G2 phase proteins cyclin A, cyclin B, cdk1, and cdk2. As shown in Fig. 3, 20 nM RA treatment induced TK expression by 2-fold in SCC15 cells as determined by Northern blot, correlating with the increased S phase fraction observed in this treatment group. Similarly, induction of the S phase markers cyclin A and 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 µM 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 µM 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.



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  		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 µM RA for 16 h. Expression of the S phase marker TK was determined by Northern blot. Blots were stripped and hybridized to a ß-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.

 
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 µM 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.



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  		FIGURE 4. RA differentially regulates Rb phosphorylation and association with E2F-1 in a dose-dependent manner. SCC lines were treated with 0.1% DMSO vehicle, 20 nM RA, or 1 µM RA for 16 h. Rb protein was immunoprecipitated (IP Rb) from cellular lysates as described in "Materials and Methods." Blots were probed with anti-phosphoRb antibody (anti-ppRb) to determine the level of Rb phosphorylation in response to RA. Blots were stripped and incubated with anti-E2F-1 antibody to determine association of this transcription factor with Rb. Blots were stripped and incubated with anti-Rb antibody to ensure equal amounts of immunoprecipitated protein in each lane. These experiments were performed three times with similar results. Representative blots are shown.

 
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, 20 nM RA treatment produced a reproducible increase in E2F DNA binding activity. In contrast, treatment with 1 µM RA resulted in a 3-fold decrease in E2F DNA binding activity. The presence of E2F-1 in the binding complex was confirmed by the presence of a supershifted band after incubation with an anti-E2F-1 antibody. Binding of E2F-1 to the probe was effectively competed by unlabeled oligonucleotide but not by one containing a mutated binding site. Increased E2F-1 DNA binding activity correlated with 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 SCC25 cells. Treatment with 1 µM RA inhibited promoter activity by 50% compared to control levels. Transfection of an E2F-1 expression vector as the positive control for transactivation produced an 8-fold increase in reporter gene activity. These results indicate that RA can regulate the DNA binding and transactivation properties of E2F-1 in a dose-dependent manner, consistent with its effects on proliferation of SCC lines.



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  		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 µM 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 µM RA for 16 h. Luciferase activity was normalized to ß-galactosidase levels for each sample. These experiments were performed three times with similar results. , vehicle; , 20 nM RA; , 1 µM RA; , E2F1. Error bars, SE.

 
Given that EGF signaling via the MAPK pathway is an important upstream regulator of cellular proliferation in human SCC lines (20, 21, 30), we wondered whether RA could regulate EGF expression and ERK1 activation and thus provide a potential mechanism for the effects of the retinoid on cell cycle regulatory proteins. Treatment of SCC4 and SCC9 cells with 20 nM RA stimulated EGF secretion (Fig. 6A) and induced activated ERK1 expression by nearly 2-fold (Fig. 6B). In contrast, treatment with 1 µM RA inhibited EGF production by 90% and ERK1 activation by 2-fold. This inhibition was accompanied by a decrease in total ERK1 expression. Treatment with both low- and high-dose RA inhibited EGFR expression in SCC4 and SCC9 cells (see "Discussion"); however, levels of ERK2, ERK3/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.



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  		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 µM RA for 16 h. A. EGF secretion was determined by enzyme-linked immunosorbent assay (ELISA) as described in "Materials and Methods." , con; , 20 nM; , 1 µM. B. ERK1 activation was determined by Western blot using antihuman antibody to activated ERK1. Blots were stripped and incubated with total ERK1 antibody to verify the amount of protein in each lane. Expression of additional kinases (EGFR, ERK2, ERK3, JNK1, and p38) was also determined by Western blot. These experiments were performed three times with similar results. Representative blots are shown.

 
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 wild-type ERK1 construct showed high levels of protein expression, kinase activation, and Elk1 phosphorylation (Fig. 7B). These levels were only marginally affected by 1 µM RA treatment, likely due to the constitutive expression of the ERK1 construct. Conversely, clones expressing the dominant negative ERK1 construct showed a 3-fold decrease in activation compared to ERK1 cells, with a corresponding reduction in Elk1 phosphorylation. RA treatment of dominant negative ERK1 clones decreased Elk1 phosphorylation by 5-fold due to inhibition of the residual activity of endogenous ERK1. Elk1 phosphorylation in RA-treated dominant negative ERK1 clones was 3-fold less than in similarly treated control cells. Since the ERK1 antibody recognized both wild-type and dominant negative forms of the protein, total ERK1 protein levels were higher in overexpressing clones than in control cells. ERK1 activation in control clones was half that of ERK1 overexpressing cells but higher than in dominant negative ERK1 cells as expected. RA treatment of control clones markedly inhibited ERK1 activation and Elk1 phosphorylation. Expression of the dominant negative ERK1 construct resulted in a decrease in BrdUrd-positive cells from 15% to 8% (Fig. 7C). Wild-type ERK1 overexpression caused an increase in the number of BrdU-positive cells to 20%. Interestingly, while treatment with 1 µM RA produced marked decreases in the number of 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.



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  		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 described in "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 µM 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 µM 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 µM 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
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
We report here that proliferation of human SCC lines is regulated by RA dose-dependent mechanisms via EGF and the MAPK pathway. Low concentrations of RA increased EGF production, ERK1 activity, and the proliferation of SCC lines which was characterized by concomitant changes in cell cycle regulatory protein expression, Rb phosphorylation, and E2F-1 activation. Conversely, high concentrations of RA decreased EGF secretion, ERK1 activation, and cellular proliferation of SCC lines. We have shown previously that inhibiting ERK1 activation in these cells potently inhibits their proliferation (31). Interestingly, 20 nM RA treatment inhibited EGFR expression while inducing ERK1 activation and cellular proliferation. In a previous study, we determined that the EGFR overexpression commonly observed in SCC lines not only resulted in growth factor-independent proliferation but also limited EGF responsiveness due to limiting amounts of the downstream adaptor protein Grb2 (32). Pretreatment of SCC lines with RA restored EGF responsiveness to these cells by inhibiting EGFR expression and restoring stoichiometric balance between the receptor and the adaptor protein. Grb2 overexpression increased proliferation and restored EGF responsiveness in these cell lines. These results have important implications for retinoid refractoriness in SCC chemotherapy. Taken together, these two studies describe an important mechanism by which RA differentially regulates cellular proliferation via the EGF/MAPK signaling pathway.

A previous study using normal epidermal keratinocytes from RAR null mutant mice revealed that RAR{gamma} mediated the effects of RA on cellular proliferation (33). However, our present study using synthetic retinoids did not reveal similar receptor selectivity. Recently, other RAR{gamma} selective retinoids were shown to mediate growth inhibition in SCC lines (34). Whether additional properties of RAR{gamma} also mediate growth inhibition of SCC lines remains to be determined.

RA has been shown to differentially regulate MAPK activity in a variety of studies. During myeloid differentiation of HL-60 cells, RA was shown to activate ERK2 but not JNK or p38 kinases (24, 26). However, we did not observe changes in expression of these MAPKs following RA treatment of SCC lines. These results suggest that MAPKs other than ERK1 may not participate in RA-mediated inhibition of SCC proliferation. The mechanism by which ERK1 but not other MAPKs is regulated by RA treatment in SCC lines will be important in future studies. Blockade of MAPK activation inhibited the ability of RA to induce differentiation of HL-60 cells (24, 26). However, in human bronchial epithelial cells and lung cancer lines, RA was shown to inhibit both ERK- and JNK-dependent signaling pathways (26, 35). These effects were abrogated by the use of RAR antagonists. Recently, decreased ERK activity in aged human skin was shown to be reversed by RA treatment (36). We show here that RA can exert opposite effects on ERK1 activation in a dose-dependent manner. Taken together, these studies demonstrate that regulation of MAPK activity by RA is dependent on dose, receptor selectivity, and cellular context.

Another important result of this study is the induction of RA resistance by ERK1 overexpression. Resistance to the growth inhibitory effects of RA would be expected to provide a significant advantage to tumorigenic clones. ERK1 phosphorylates members of the ets transcription factor family such as Elk1 (21). Elk1 is part of the ternary complex which binds to and activates the serum response element in the c-fos promoter, thereby inducing its expression. Increased fos expression may lead to induction of AP-1 activity; binding sites for these factors are found in the promoters of many genes regulating cellular proliferation (37). Constitutive ERK1 overexpression may inhibit the anti-AP-1 effects of RA by activating c-fos, resulting in accelerated growth and an RA-resistant phenotype. These clones will provide a useful model system for investigating other aspects of the RA-resistant phenotype of cancer cells.

In summary, RA can differentially regulate MAPK activity and cancer cell proliferation by dose-dependent mechanisms. Recently, Rb protein has been shown to associate with histone deacetylase to repress transcription (38, 39). In future experiments, we will examine the role of RA in recruitment of histone deacetylase to cell cycle regulatory complexes and how these proteins regulate proliferation and retinoid resistance in human cancer cells.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Cell Culture and Stable Transfection
The human SCC lines used in this study were purchased from the American Type Culture Collection, Rockville, MD and have been described previously (40, 41). Cells were cultured in DMEM, 10% charcoal-stripped fetal bovine serum, 40 µg/ml gentamicin at 37°C in a humidified atmosphere of 5% CO2. SCC71 cells were transfected with 5 µg expression vectors for ERK1, a dominant negative kinase-deficient ERK1 (10), or neomycin resistance plasmid alone using LipofectAMINE reagent according to manufacturer's recommendations (Life Technologies, Inc., Carlsbad, CA). Cells were selected in 400 µg/ml G418 for 14 days. Resistant clones were picked for expansion and characterization.

Cell Death and Proliferation Assays
The TUNEL assay for in situ cell death detection in SCC lines has been described previously (42). Cells were treated with 0.1% DMSO vehicle, up to 1 µM all-trans-RA, the RAR and RXR selective retinoids AGN192890 and AGN194204, or the RAR{alpha} and RAR{gamma} selective compounds AGN195183 and AGN194433 (9). Cells were fixed in 4% paraformaldehyde (pH 7.4), and permeabilized with 0.1% Triton X-100, 0.1% sodium citrate for 2 min on ice. A mouse IgM antihuman Fas antibody (Molecular Biology Laboratories, Watertown, MA) that induces apoptosis in sensitive cell lines was used as the positive control. An isotype-matched control antibody was used as the negative control. After washing with PBS, cells were incubated with fluorescein-dUTP and terminal deoxynucleotidyl transferase for 60 min at 37°C according to manufacturer's recommendations (Roche Molecular Biochemicals, Indianapolis, IN). After washing three times in PBS, apoptotic cells were visualized by fluorescence microscopy. Proliferation was analyzed by plating 5 x 104 cells in triplicate cultures and treating with vehicle or RA for up to 6 days. Cells were trypsinized and counted with a hemacytometer at 2- or 4-day intervals. To determine G1 to S phase progression, BrdUrd incorporation analysis was performed. Vehicle- and RA-treated cells were incubated with 10 µM BrdUrd for 1 h, washed in PBS, and fixed in 70% ethanol containing 50 mM glycine (pH 2) for 20 min at -20°C. After washing in PBS, the cells were incubated with mouse anti-BrdUrd primary antibody for 30 min at 37°C according to manufacturer's recommendations (Roche Molecular Biochemicals). The cells were washed in PBS and incubated with antimouse IgG secondary antibody conjugated to fluorescein. Following extensive washing in PBS, BrdUrd-positive cells were visualized by fluorescence microscopy, counted, and expressed as a percentage of the total cells counted in 10 random high-power fields.

Northern Blot
Two micrograms mRNA from vehicle- or RA-treated SCC cells were electrophoresed in 1% agarose gels containing 2.2 M formaldehyde using 1x 4-morpholinepropanesulfonic acid running buffer. mRNA was capillary transferred to nylon membranes (Schleicher and Schuell, Keene, NH), hybridized to 6 x 106 cpm/ml random primed 32P-labeled TK cDNA probe (purchased from the American Type Culture Collection) for 16 h at 42°C in 50% formamide, 5x SSPE, 1x Denhardt's solution, and 0.2% SDS. Blots were washed once in 2x SSC, 0.1% SDS for 45 min at 50°C followed by one wash in 0.2x SSC, 0.1% SDS for 45 min at 50°C. Blots were washed a final time in 0.2x SSC, 0.1% SDS for 45 min at 65°C and exposed to Kodak XAR5 autoradiographic film for 16 h at -80°C.

Western Blot
Seventy-five micrograms total protein from vehicle- or RA-treated cells were separated by SDS-PAGE on 10% resolving gels under denaturing and reducing conditions. Separated proteins were electroblotted to polyvinylidene difluoride membranes according to manufacturer's recommendations (Bio-Rad, Hercules, CA). Blots were incubated with antihuman primary antibodies to cyclins, cdks, EGFR, ERK1, activated ERK1, ERK2, ERK3, JNK1, p38, and activated Elk1 (Transduction Laboratories, San Diego, CA and Santa Cruz Biotechnology, Santa Cruz, CA) for 16 h at 4°C. After washing in Tris-buffered saline containing 0.1% Tween 20 (TBST, pH 7.5), blots were incubated for 30 min at room temperature with anti-IgG secondary antibody conjugated to horseradish peroxidase. After extensive washing in TBST at room temperature, bands were visualized by the enhanced chemiluminescence method (Roche Molecular Biochemicals).

Immunoprecipitation and in Vitro Kinase Assay
Vehicle- and RA-treated cells were lysed in 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 1 mM DTT, 1% NP40, 10% glycerol, 1 mM NaF, 0.1 mM sodium orthovanadate, and protease inhibitors for 30 min at 4°C. Lysates were centrifuged at 10,000 x g for 10 min and antihuman primary antibody to Rb (Santa Cruz Biotechnology) was incubated with the supernatants for 1 h at 4°C. For in vitro kinase assays, anti-FLAG antibody was used to immunoprecipitate transfected ERK1 and dnERK1. Anti-ERK1 antibody was used to immunoprecipitate total ERK1 proteins from these clones. Antigen-antibody complexes were precipitated by incubation with protein A/G agarose (Santa Cruz Biotechnology) for 1 h at 4°C. Immunoprecipitated proteins were washed three times with 1 ml lysis buffer. For kinase assays, immunoprecipitates were washed an additional time in kinase buffer [50 mM HEPES (pH 7.5), 10 mM MgCl2, 1 mM DTT, 2.5 mM EGTA, 1 mM NaF, 0.1 mM sodium orthovanadate, 20 µM ATP]. Antigen-antibody complexes were incubated in 20 µl kinase buffer containing 1 µg myelin basic protein (Santa Cruz Biotechnology) and 10 µCi [{gamma}-32P]ATP (3000 Ci/mmol; DuPont, Boston, MA) for 30 min at 30°C. Immunoprecipitated protein complexes were separated by SDS-PAGE as described above. Kinase blots were exposed to Kodak XAR5 autoradiographic film for 4 h at -80°C before developing. Rb blots were incubated with anti-phosphoRb antibody and anti-E2F-1 antibody (Santa Cruz Biotechnology) as described above. Blots were stripped and incubated with anti-Rb or anti-ERK1 antibody to determine amounts of immunoprecipitated protein in each lane.

Electrophoretic Mobility Shift Assay
107 SCC25 nuclei were extracted in 20 mM HEPES (pH 7.9), 25% glycerol, 1.5 mM MgCl2, 1.2 M KCl, 0.2 mM EDTA, 0.2 mM PMSF, and 0.5 mM DTT for 30 min at 4°C. Following centrifugation at 10,000 x g for 30 min at 4°C, the supernatant was removed and dialyzed against 20 mM HEPES (pH 7.9), 20% glycerol, 0.1 M KCl, 0.2 mM EDTA, 0.2 mM PMSF, and 0.5 mM DTT for 1 h at 4°C. Fifteen micrograms of dialyzed nuclear extract were incubated in binding reactions containing 2 µg poly(dI·dc)-poly(dI·dc) and 10,000 cpm 32P end labeled double-stranded oligonucleotide containing a canonical E2F site (5'-TTTCGCGC-3'; Santa Cruz Biotechnology). For binding competition analysis, 10- to 1000-fold molar excess of unlabeled probe or mutated oligonucleotide (5'-TTTCGCat-3') was included in the reactions. To determine if E2F-1 was present in the shifted complexes, 1 µl antihuman E2F-1 antibody (Santa Cruz Biotechnology) or control IgG was included in the binding reactions. Reactions were incubated at room temperature for 15 min and subjected to native PAGE using 0.5x Tris-borate-EDTA running buffer. Gels were dried and exposed to Kodak XAR5 autoradiographic film for 16 h at -80°C.

Transient Transfection and Reporter Gene Assays
SCC lines were plated in triplicate into six-well tissue culture plates. Cells were transiently transfected with 5 µg of a heterologous promoter containing four tandem repeats of the E2F recognition sequence (5'-TTTCGCGC-3') cloned in the pGL3 luciferase reporter vector (Promega, Madison, WI) using LipofectAMINE according to manufacturer's recommendations (Life Technologies). One microgram ß-galactosidase expression vector (Vical) was used to normalize for transfection efficiency. Cells were treated with vehicle or up to 1 µM RA for 24 h following transfection. Reporter gene activity was determined using a commercially available kit (Dual-light, Tropix). Luciferase activity was normalized to ß-galactosidase levels for each sample.

Enzyme-Linked Immunosorbent Assay
Triplicate cultures of SCC25 cells were treated with vehicle, 20 nM RA, or 1 µM RA for 16 h. Conditioned medium was collected from each culture and subjected to ELISA for secreted EGF using a commercially available kit (Quantikine, R&D Systems, Minneapolis, MN).


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We thank Dr. Karen Vousden for the E2F1 expression vector and Dr. Melanie Cobb for ERK1 expression plasmids.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
1 NIH grant DE10966. Back

Received April 4, 2002; revised April 3, 2003; accepted April 4, 2003.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

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