Effects of Acyclic Retinoid on Growth, Cell Cycle Control, Epidermal Growth Factor Receptor Signaling, and Gene Expression in Human Squamous Cell Carcinoma Cells

Chemicals.

ACR (NIK333; Nikken Chemicals Co., Ltd., Tokyo, Japan) was provided by Dr. Hisataka Moriwaki (Gifu University School of Medicine, Gifu, Japan; Ref. 6 ).

Cell Lines and Cell Culture.

The HCE7 human esophageal cancer cell line was provided by Dr. Curtis C. Harris (National Cancer Institute, Bethesda, MD; Ref. 18 ), and YCU-H891 and YCU-N861 human head and neck squamous cancer cell lines were provided by Dr. Mamoru Tsukuda (Yokohama City University, Yokohama, Japan; Ref. 23 ), and were maintained in RPMI-1640 containing 10% fetal bovine serum (RF-10; Invitrogen, San Diego, CA). Cells were treated with ACR and cultured in an incubator with humidified air at 37°C with 5% CO₂. As an untreated solvent control, cells were treated with DMSO (Sigma Chemical Co., St. Louis, MO) at a final concentration of <0.1%.

Cell Proliferation Assays.

Ten thousands cells of each cell lines were plated into six-well/35-mm diameter culture dishes and treated with the indicated concentrations of ACR for 48 h in RF-10. The numbers of cells were then determined using a Coulter Counter (Beckman Coulter Co., Fullerton, CA). Results were expressed as percentage of growth with 100% representing control cells treated with DMSO alone. To examine whether the growth-inhibitory effects of ACR were dependent on the treatment time, HCE7 cells were treated with 10 μm ACR for the indicated time (0, 3, 6, 12, 24, and 48 h), and this medium was then removed. The cells were more incubated in RF-10 until the indicated time (0, 24, 48, 72, and 96 h) and counted using a Coulter Counter (Beckman Coulter Co.).

Apoptosis Assays.

HCE7 cells were treated with DMSO or 10 μm ACR for the indicated time (0, 24, and 48 h), and the cell lysates were used for apoptosis assays. To quantify the induction of apoptosis, a DNA fragmentation assay was performed using the Cell Death Detection ELISA^PLUS kit (Roche Diagnostics Co., Indianapolis, IN), according to the manufacturer’s instructions. The level of DNA fragmentation found at 0 h was set to 1, and the increases were shown as fold activation.

Cell Cycle Assays.

HCE7 cells were seeded into 10-cm dishes (5 × 10⁵ cells/dish) in RF-10 and cultured overnight to allow for cell attachment. They were then treated with DMSO or 10 μm ACR for the indicated time (0, 24, and 48 h), harvested, fixed with 70% ethanol, centrifuged, resuspended in PBS containing 2 mg/ml RNase (Sigma), and stained with 400 μm of 0.1 mg/ml propidium iodide (Sigma). Samples were then analyzed for DNA histograms and cell cycle phase distribution by flow cytometer using a FACSCalibur instrument (Becton Dickinson, Franklin Lakes, NJ), and the data were analyzed by using the CELL Quest computer program (Becton Dickinson), as described previously (10) .

Protein Extraction and Western Blot Analysis.

HCE7 cells were treated with ACR for the indicated time (0, 3, 6, 12, 24, and 48 h) and concentrations (0, 1, 10, and 30 μm). Protein extracts were then prepared as described previously (10) . Cell lysates (40–80 μg/lane) were separated by SDS-PAGE using 7.5–15% polyacrylamide gels and transferred onto Immobilon-P transfer membranes (Millipore Co., Bedford, MA). The primary antibodies for cyclin D1, p16^INK4A, p21^CIP1, p27^KIP1, cyclin-dependent kinase (cdk) 4, cdk6, hyperphosphorylated form of retinoblastoma protein (ppRb), and actin were as described previously (10) . The RARα (C-20), RARβ (C-19), RARγ (C-19), RXRα (d-20), RXRβ (C-20), and RXRγ (Y-20) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antimouse IgG or antirabbit IgG antibodies (Amersham-Pharmacia Biotech, Buckinghamshire, United Kingdom) were used as the secondary antibodies. Each membrane was developed using an ECL-enhanced chemiluminescence system (Amersham-Pharmacia Biotech). The intensities of blots were quantified using NIH Image software version 1.62.

Assays for the state of phosphorylation of EGFR, extracellular signal-regulated kinase (ERK), and Stat3 were performed using specific antibodies as described previously (23) . To determine cellular levels of TGF-α, EGFR, ERK, Stat3, and the phosphorylated forms of these molecules, HCE7 cells were incubated in RPMI 1640 minus serum for 24 h and then treated with 10 μm ACR for the indicated time course. Proteins were then extracted and Western blot analysis was performed as described above. TGF-α (Ab-1) antibody was purchased from Oncogene Research Products (San Diego, CA).

RNA Extraction and Reverse Transcription-PCR (RT-PCR) Analysis.

Total RNA was isolated from frozen HCE7 cells using TRIzol reagent as recommended by the manufacturer (Invitrogen). cDNA was amplified from 1 μg of total RNA using SuperScript one-step RT-PCR with the platinum Taq system (Invitrogen). We chose the 22-, 25-, 28-, and 28-cycle rounds of PCR to analyze the expression levels of cyclin D1, p21^CIP1, RARβ, and TGF-α mRNA, respectively, because a semiquantitative assessment indicated that under these conditions the reaction had not reached a plateau and was still in the log phase. Cyclin D1 and actin specific primer sets were used as described elsewhere (10) . The sequences for RARβ-, p21^CIP1-, and TGF-α-specific primers were as follows: RABF1 (5′-AAA AAG ACC AAC AGC CTA CG-3′) and RABR1 (5′-AGC CCT TAC ATC CCT CAC AG-3′), CIF4 (5′-CTG GGG ATG TCC GTC AGA AC-3′) and CIR4 (5′-AAT CTG TCA TGC TGG TCT GC-3′), TAF2 (5′-CAG GCC TTG GAG AAC AGC AC-3′) and TAR2 (5′-CTG GGC TCT TCA GAC CAC TG-3′), respectively. The primers were designed using published sequences (24, 25, 26) . At the same time, amplified PCR products obtained with actin-specific primers served as internal controls. Each amplification cycle consisted of 0.5 min at 94°C for denaturation, 0.5 min at 55°C for primer annealing, and 1 min at 72°C for extension (10) . After PCR amplification, the PCR fragments were stained with ethidium bromide and analyzed by agarose gel electrophoresis.

Cyclin D1, c-fos, and Activator Protein (AP-1) Reporter Assays.

Reporter assays were performed as described previously (27) . The cyclin D1 promoter luciferase reporter plasmid –1745CD1LUC was kindly provided by Dr. Richard G. Pestell (Georgetown University, Washington, DC; Ref. 28 ). The c-fos promoter luciferase reporter plasmid pFos-wt-luc was described previously (27) . The pAP-1-luciferase plasmid was kindly provided by Dr. Jacalyn H. Pierce (National Cancer Institute, Bethesda, MD; Ref. 29 ). The pcDNA3 plasmid DNA (Invitrogen) was used as a control vector and used as needed to achieve the same total amount of plasmid DNA/transfection. Cells (2 × 10⁵) were transfected using Lipofectin reagent (Invitrogen) in six-well/35-mm diameter plates. One μg of DNA of the indicated luciferase reporter plasmid was transfected into HCE7 cells in opti-MEM I medium (Life Technologies, Inc.). After 24 h, the medium was changed to RF-10 containing the indicated concentrations of ACR (1, 10, or 30 μm) or DMSO. Cells were then incubated for 24 or 48 h, and luciferase activities were determined using a luciferase assay system (Program Co., Madison, WI). In all of these reporter assays the cells were also cotransfected with a cytomegalovirus-β-galactosidase (βgal) reporter. Luciferase activities were normalized with respect to the β-gal activities, to correct for differences in transfection efficiency. Luciferase activity obtained with cells treated with 0.1% DMSO was used as the 100% value at each time point.

RARE CAT-ELISA Assays.

The RARE-CAT reporter plasmid and retinoid receptor expression vectors (RARα, RARβ, RARγ, and RXRα) were kindly provided by Dr. David A. Talmage (Columbia University, New York, NY; Ref. 30 ). Transient transfection assays using a RARE promoter reporter were performed as described above. Two μg of a RARE-CAT plasmid and 1 μg of each expression vector were cotransfected into HCE7 cells in six-well dishes in opti-MEM I medium (Invitrogen). After 24 h, the medium was changed to RF-10 containing 10 μm ACR or DMSO. The cells were then incubated for 12 h, and RARE-CAT activity was measured by using an ELISA kit (CAT ELISA; Roche Diagnostics Co.) according to the manufacturer’s instructions. The level of RARE-CAT activity found in control DMSO-treated cells was set to a value of 1, and changes observed in the treated cultures were expressed as fold activation.

Reversibility Studies.

HCE7 cells were treated with 10 μm ACR for 12 h, and this medium was then removed. The cells were then treated with or without 10 μm ACR for an additional 36 h (total 48 h), and extracts were prepared for Western blot analysis or RT-PCR analysis as described above.

Statistical Analysis.

Statistical analyses of the DNA fragmentation assays, cyclin D1, c-fos, and AP-1 reporter assays, and RARE-CAT ELISA assays were analyzed by either Student’s or Welch’s t test. The results were considered statistically significant if the P < 0.05.

ACR Inhibits the Growth of Human SCC Cells.

We found that ACR inhibited growth of the HCE7, YCU-N861, and YCU-H891 cell lines, with IC₅₀ values of ∼10, 25, and 40 μm, respectively, when the cells were grown in RF-10 (Fig. 1A)⇓ . Because the HCE7 cell line was the most sensitive to the growth-inhibitory effect of ACR, additional studies were then done with these cells. When we examined the time-dependent growth-inhibitory effects of ACR, we found that treatment for only 12 h was sufficient to cause irreversible growth inhibition of HCE7 cells (Fig. 1B)⇓ .

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Fig. 1.

Inhibition of cell growth, induction of apoptosis, and progression of cell cycle by acyclic retinoid (ACR) in human squamous cell carcinoma (SCC) cell lines. A, dose-dependent effects of ACR in human SCC cell lines. The human esophageal SCC cell line HCE7, and the human head and neck SCC cell lines YCU-H891 and YCU-N861, were treated with the indicated concentrations of ACR for 48 h, and the total numbers of cells were then determined using a Coulter counter. B, time-dependent growth-inhibitory effects of ACR in the HCE7 cell line. HCE7 cells were treated with 10 μm ACR for the indicated times and then the medium was replaced with fresh medium lacking ACR. The number of cells were then counted at the indicated times. Values are means of triplicate assays. C, induction of apoptosis by ACR in the HCE7 cell line. HCE7 cells were treated with 10 μm ACR or DMSO for the indicated times, and cell lysates were examined for apoptosis using the DNA fragmentation assay. Each ∗ indicates a significant difference (P < 0.05) between the control untreated cells and the ACR-treated cells. D, exponential cultures of HCE7 cells were treated with DMSO or 10 μm ACR, and the cells were then analyzed by DNA flow cytometry at the indicated times. The distribution of cells in the sub-G₁, G₀-G₁, S, and G₂-M phases of the cell cycle were calculated and plotted. Bars, SD.

ACR Induces Apoptosis in HCE7 Cells.

ACR can induce apoptosis in human hepatoma cells (7, 8, 9) . To determine whether the growth inhibition we observed with HCE7 cells was associated with induction of apoptosis we carried out DNA fragmentation assays (Fig. 1C)⇓ . We found that when tested at 10 μm ACR caused a time-dependent induction of apoptosis; thus, the DNA fragmentation ratio increased by 2.3-fold after 24 h and by 2.9-fold after 48 h (Fig. 1C)⇓ . In contrast, no significant changes were observed in DMSO-treated cells during this time course (Fig. 1C)⇓ .

ACR Causes HCE7 Cells to Arrest in the G₀-G₁ Phase of the Cell Cycle.

To determine whether this growth inhibition was also associated with specific changes in cell cycle distribution, cell cycle analysis was performed using DNA flow cytometry (Fig. 1D)⇓ . Flow cytometric analysis indicated that when HCE7 cells were treated with 10 μm ACR, the percentage of cells in G₀-G₁ increased by 9.7% after 24 h and increased by 18.7% after 48 h, and this was associated with a concomitant decrease of cells in the S and G₂-M phases of cell cycle (Fig. 1D)⇓ . In contrast, no significant changes were observed in the DMSO-treated control cells during these time courses (Fig. 1D)⇓ .

ACR Induces p21^CIP1, Decreases Cyclin D1, and Inhibits Hyperphosphorylation of Retinoblastoma Protein.

Because we found that treatment of HCE7 cells with 10 μm ACR caused an increase of cells in the G₀-G₁ phase of the cell cycle during the subsequent 24–48 h (Fig. 1D)⇓ , we examined the effects of ACR on cellular levels of the G₁ cell cycle control proteins cyclin D1, cdk4, cdk6, and the cdk inhibitors p16^INK4a, p21^CIP1, and p27^KIP1, using Western blot analysis (Fig. 2A)⇓ . We also examined the cellular levels of cyclin D1 and p21^CIP1 mRNAs using semiquantitative RT-PCR analysis (Fig. 2B)⇓ . To examine time-dependent effects of ACR, HCE7 cells were treated with 10 μm ACR, and extracts were prepared at 0, 3, 6, 12, 24, and 48 h after the addition of the drug. We found that there was a marked increase in the levels of expression of the p21^CIP1 protein during the subsequent 6–12 h (Fig. 2A)⇓ . The expression levels of the cyclin D1 protein displayed a moderate decrease at 24 h and a marked decrease at 48 h (Fig. 2A)⇓ . These specific changes of cyclin D1 and p21^CIP1 proteins were correlated with the changes of their corresponding genes (Fig. 2B)⇓ . There were no significant changes in the expression levels of the p16^INK4a, p27^KIP1, cdk4, and cdk6 proteins (Fig. 2A)⇓ .

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Fig. 2.

Effects of acyclic retinoid (ACR) on the expression of cell cycle control molecules. HCE7 cells were treated with 10 μm ACR for the indicated times, and cell extracts were then examined by Western blot analysis using the respective antibodies (A) or examined by reverse transcription-PCR (RT-PCR) analysis using the cyclin D1 or p21^CIP1 specific primers (B), as described in “Materials and Methods.” C, to examine the time- and dose-dependent effects of ACR on levels of expression of both the cyclin D1 protein and mRNA, HCE7 cells were treated with ACR for the indicated times and concentrations, and cell extracts were then examined by Western blot analysis or RT-PCR analysis for levels of cyclin D1. An antibody to actin and actin specific PCR products served as controls. D, effects of ACR on transcriptional activity of the cyclin D1 promoter. The cyclin D1 promoter luciferase reporter plasmid-1745CD1LUC was transected into HCE7 cells, and the transfected cells were treated with ACR for the indicated times and concentrations. The extracts were then examined for luciferase activity. Each ∗ indicates a significant difference (P < 0.05) between control untreated cells and ACR-treated cells. Bars, SD.

The tumor suppressor retinoblastoma protein inhibits the G₁ to S transition, but when it becomes hyperphosphorylated (designated ppRb) it no longer exerts this inhibitory effect (31 , 32) . Therefore, we also examined whether treatment with ACR alters the cellular level of the ppRb. We found that the level of the ppRb displayed a marked decrease after 6–12 h, and was virtually undetectable at 24–48 h, after treatment with 10 μm ACR (Fig. 2A)⇓ .

To examine the dose-dependent effect of ACR on cyclin D1 expression, HCE7 cells were treated with 0, 1, 10, or 30 μm ACR for 24 or 48 h (Fig. 2C)⇓ . At 24 h the 1 μm dose had no effect but by 48 h even this low dose of ACR caused a marked decrease in the cellular level of both the cyclin D1 protein and mRNA (Fig. 2C)⇓ .

ACR Inhibits Transcription from the Cyclin D1 Promoter.

In view of our finding that ACR caused a decrease in cellular levels of both the cyclin D1 protein and mRNA (Fig. 2, A–C)⇓ , we examined whether these changes were associated with inhibition of de novo transcription of the cyclin D1 gene by using transient transfection cyclin D1 promoter-luciferase reporter assays (Fig. 2D)⇓ . After transfection of the luciferase reporter plasmid –1745CD1LUC, HCE7 cells were treated with increasing concentrations of ACR or DMSO for 24 or 48 h, and luciferase activity was then determined in the cell extracts. We found that treatment with ACR caused a dose- and time-dependent decrease in activity of the cyclin D1 promoter, especially in the assays done after 48 h. Thus, at 48 h 1 μm ACR caused a 35% inhibition and 10 μm caused a 48% inhibition of this activity (Fig. 2D)⇓ . These results provide evidence that ACR causes a decrease in the cellular level of cyclin D1 mRNA and protein by inhibiting the transcriptional activity of the cyclin D1 promoter. The time course aspects of these effects are considered in the “Discussion” section.

ACR Causes an Increase in the Cellular Level of Both the RARβ Protein and mRNA.

Because retinoids are thought to exert most of their effects by regulating gene expression by binding to specific nuclear receptors (13) , we examined the effects of ACR on cellular levels of the retinoid receptors RARs α, β, and γ, and RXRs α, β, and γ proteins using Western blot analysis (Fig. 3A)⇓ . When we examined the time-dependent effect of 10 μm ACR, we found that there was a marked and rapid increase in the levels of expression of the RARβ protein at 3 h after the addition of the drug; the protein expression ratio increased by 2.2-fold after 3 h (Fig. 3A)⇓ . However, there were no significant changes in the expression levels of the other molecules (Fig. 3A)⇓ . When we used semiquantitative RT-PCR analysis to examine levels of RARβ mRNA we found an increase in the RARβ band intensity within 3 h after the addition of the drug, and this induction increased and persisted for up to 48 h (Fig. 3B)⇓ . In addition, treatment of HCE7 cells with only 1 μm ACR caused a marked increase in the level of expression of both the RARβ protein and mRNA at 24 h (Fig. 3C)⇓ . Thus, 1 μm ACR can induce cellular levels of RARβ to the same extent as 10 μm ACR (Fig. 3C)⇓ .

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Fig. 3.

Effects of acyclic retinoid (ACR) on the expression of nuclear retinoid receptors. HCE7 cells were treated with 10 μm ACR for the indicated times, and cell extracts were then examined by Western blot analysis using the respective antibodies (A) or examined by reverse transcription-PCR (RT-PCR) analysis using the RARβ specific primer (B), as described in “Materials and Methods.” The results obtained in A were quantitated by densitometry and are displayed in the bottom panel of A. C, HCE7 cells were treated with ACR for the indicated times and concentrations, and cell extracts were then examined for levels of RARβ protein and mRNA, using Western blot or RT-PCR analysis, respectively. An antibody to actin and actin specific products served as controls. D, Effects of ACR on RARE-CAT reporter activity when HCE7 cells were cotransfected with various retinoid receptor expression vectors. The RARE-CAT reporter plasmid and the indicated retinoid receptor expression vectors were cotransfected into HCE7 cells, and the cells were then treated with 10 μm ACR or DMSO for 12 h. RARE-CAT activities were measured by using an ELISA system, as described in “Materials and Methods.” Each ∗ represents a significant difference (P < 0.05) between control untreated cells and ACR-treated cells. Bars, SD.

ACR Stimulates the Activity of a RARE-CAT Construct When the Cells Are Cotransfected with a RARβ Expression Vector.

RARs modulate the expression of target genes by interacting with the RARE element located in the promoter regions of target genes (13) . In view of our finding that ACR caused a rapid and marked increase in the levels of expression of both the RARβ protein and mRNA (Fig. 3, A–C)⇓ , we examined whether ACR stimulates the transcriptional activity of a RARE-CAT reporter in transient transfection assays (Fig. 3D)⇓ . In these studies, we also examined the effects of cotransfection with expression plasmids that encode the retinoid receptors RARα, RARβ, RARγ, and RXRα to determine whether ACR preferentially activates specific retinoid receptors. After transfection, the cells were incubated with or without 10 μm ACR for 12 h, and extracts assayed for CAT activity using an ELISA assay as described in “Materials and Methods.” In studies in which the cells were not cotransfected with an exogenous retinoid receptor, 10 μm ACR caused an ∼1.1-fold stimulation of reporter activity when compared with the DMSO-treated control cells (Fig. 3D)⇓ . Among the retinoid receptors tested only RARβ caused a significant enhancement of ACR stimulation of reporter activity (Fig. 3D)⇓ . Therefore, it appears that ACR preferentially interacts with RARβ in stimulating the RARE transcription element. The results we obtained with RARβ in these reporter assays (Fig. 3D)⇓ coupled with our findings that ACR preferentially induces the expression of RARβ in HCE7 cells (Fig. 3, A–C)⇓ suggest that the biological effects of ACR in HCE7 cells are mediated mainly through RARβ.

ACR Causes a Decrease in the Cellular Level of TGF-α mRNA and Protein, and a Decrease in the Phosphorylated Forms of the EGFR, Stat3, and ERK Proteins.

Because the EGFR and its ligand TGF-α appear to play critical roles in the growth of human esophageal SCC (20, 21, 22) , we examined the effects of ACR on expression of TGF-α/EGFR pathway (Fig. 4, A and B)⇓ . Using semiquantitative RT-PCR analysis, we found that when HCE7 cells were treated with 10 μm ACR there was a marked decrease in the cellular level of TGF-α mRNA at 12 h and an additional decline at 24 and 48 h (Fig. 4A)⇓ . There was also a marked decrease in the cellular level of the TGF-α protein at 24 h after addition of the drug (Fig. 4B)⇓ . We then examined the time-dependent effects of 10 μm ACR on cellular levels of the EGFR protein and the downstream signaling proteins, ERK and Stat3, as well as cellular levels of the corresponding phospho-proteins. We found that although ACR did not have an appreciable effect on the levels of any of these proteins at 24 and 48 h, there was a marked decrease in the levels of the phosphorylated (i.e., activated) forms of the EGFR protein, the ERK protein, and the Stat3 protein (Fig. 4B)⇓ .

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Fig. 4.

Effects of acyclic retinoid (ACR) on activation of the transforming growth factor (TGF)α/epidermal growth factor receptor (EGFR) signaling pathway. HCE7 cells were treated with 10 μm ACR for the indicated times, and cell extracts were then examined by reverse transcription-PCR using TGF-α specific primer (A) or examined by Western blot analysis using the respective antibodies (B), as described in “Materials and Methods.” Actin-specific PCR products and an antibody for actin served as controls. Effects of ACR on transcriptional activity of the c-fos (C) and activator protein 1 (AP; D) promoters. Either c-fos (C) or AP-1 (D) promoter luciferase reporter plasmids were transfected into HCE7 cells, and cells were treated with ACR for the indicated times and concentrations. Cell extracts were then examined for luciferase activity. Each ∗ indicates a significant difference (P < 0.05) between control untreated cells and ACR-treated cells. Bars, SD.

ACR Inhibits the Transcriptional Activity of the c-fos and AP-1 Promoters.

AP-1 is one of the important downstream effectors of ERK and Stat3 (33 , 34) . In view of our finding that ACR inhibited activation of the TGF-α/EGFR pathway (Fig. 4, A and B)⇓ , we examined the effects of ACR on the transcriptional activity of the c-fos and AP-1 promoters, using transient transfection luciferase reporter assays (Fig. 4, C and D)⇓ . After transfection with the c-fos or AP-1 luciferase reporter plasmid, HCE7 cells were treated with increasing concentrations of ACR for 24 or 48 h, and luciferase activities were determined in cell extracts. Treatment with ACR caused a dose- and time-dependent decrease in activity of both the c-fos (Fig. 4C)⇓ and AP-1 (Fig. 4D)⇓ luciferase reporters. These effects were greater at the 48 h than the 24 h time point and were greater with the AP-1 reporter than the c-fos reporter (Fig. 4, C and D)⇓ . Significant inhibitory effects were seen with only 1 μm ACR (Fig. 4, C and D)⇓ . At 48 h 10 μm ACR, the IC₅₀ concentration for growth inhibition caused about 50% inhibition of AP-1 activity (Fig. 4D)⇓ . Therefore, treatment of HCE7 cells with concentrations of ACR that are in the same range as those that inhibit growth also inhibit the transcriptional activities of both the c-fos and AP-1 promoters.

Treatment with ACR for 12 h Causes Sufficient Molecular Changes to Inhibit the Growth of HCE7 Cells.

Because we found that treatment of HCE7 cells with 10 μm ACR for 12 h caused an irreversible growth inhibition (Fig. 1B)⇓ , we examined whether treatment for only 12 h was sufficient to alter the expression of RARβ, cyclin D1, ppRb, and suppression of the TGF-α/EGFR pathway (Fig. 5)⇓ . HCE7 cells were treated with 10 μm ACR for 12 h. The medium was then removed and replaced with fresh medium containing or lacking 10 μm ACR, and the cells were then incubated for an additional 36 h (total 48 h), and extracts were prepared. We found that there was a marked increase in the levels of expression of both the RARβ protein and mRNA (Fig. 5A)⇓ , and a significant decrease in the levels of expression of both the cyclin D1 protein and mRNA, and in the ppRb protein (Fig. 5B)⇓ , in both the cells that were or were not exposed to ACR during the 12–48 h interval. Activation of the TGF-α/EGFR pathway was also suppressed to the same extent in both types of cell cultures (Fig. 5C)⇓ . These findings suggest that treatment of HCE7 cells with ACR for only 12 h is sufficient to induce a cascade of changes in gene expression that persists for at least an additional 36 h. Presumably this explains why treatment with ACR for only 12 h is also sufficient to cause irreversible inhibition of cell growth (Fig. 1B)⇓ .

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Fig. 5.

Time-dependent effects of acyclic retinoid (ACR) on the expression of retinoid acid receptor (RAR)β and cell cycle control molecules, and on activation of the transforming growth factor (TGF)α/epidermal growth factor receptor (EGFR) pathway. HCE7 cells were treated with 10 μm ACR for 12 h, and this medium was then removed. The cells were then treated with fresh medium containing 10 μm ACR (ACR 48 h) or fresh medium without ACR (ACR 12 h) for an additional 36 h (total 48 h), and extracts were prepared for Western blot analysis or reverse transcription-PCR analysis. The control cells were treated with final concentration of 0.1% DMSO for 48 h (0.1% DMSO). The respective antibodies or specific primers for RARβ (A), cyclin D1 and hyperphosphorylated form of protein retinoblastoma (ppRb; B), and TGFα, phosphorylated (p) -EGFR, phosphorylated extracellular signal-regulated kinase (p-ERK), and phosphorylated (p) -Stat3 (C) are described in “Materials and Methods.” Actin-specific PCR products and an antibody for actin served as controls.