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Cell Growth & Differentiation Vol. 10, 677-683, October 1999
© 1999 American Association for Cancer Research


Articles

The Expression of p53 Tumor Suppressor Gene in Breast Cancer Cells Is Down-Regulated by Cytokine Oncostatin M1

Jingwen Liu2, Cong Li, Thomas E. Ahlborn, Michael J. Spence, Lou Meng and Linda M. Boxer

Veterans Affairs Palo Alto Health Care System, Palo Alto, California 94304 [J. L., C. L., T. E. A., L. M. B.]; Mountain States Medical Research Institute and Department of Veterans Affairs Medical Center, Boise, Idaho 83702 [M. J. S.]; Department of Pathology, University of Miami, Miami, Florida 33136 [L. M.]; and Department of Medicine, Stanford University School of Medicine, Palo Alto, California 94305-5112 [L. M. B.]

Abstract

Previously (J. Liu, et al., Cell Growth Differ., 8: 667–676, 1997), we showed that oncostatin M (OM), a cytokine produced by activated T cells and macrophages, inhibited the proliferation of breast cancer cells derived from solid tumors and malignant effusions. OM-treated cells showed reduced growth rates and differentiated phenotypes. Because the p53 tumor suppressor protein plays an important role in cellular proliferation, we examined p53 protein expression in three OM-responsive breast cancer cell lines, MCF-7, MDA-MB231, and H3922. Western blot analysis showed that p53 protein levels in all three of the cell lines were decreased by OM treatment. Reduction of p53 protein was detected after 1 day of OM treatment and reached maximal suppression of 10–20% of control after 3 days in H3922 and 40% of control after 4 days in MCF-7 cells. A comparison of p53 mRNA in OM-treated cells versus untreated control cells showed that exposure to OM reduced the steady-state levels of p53 mRNA transcripts to an extent similar to that of the p53 protein levels. This observation suggests that the effect of OM on p53 protein expression does not occur at the posttranslational level. Nuclear run-on assays verified that OM decreased the number of actively transcribed p53 mRNAs, which suggests a transcriptional regulatory mechanism. The effect of OM on p53 expression seems to be mediated through the extracellular signal-regulated kinase (ERK) pathway, inasmuch as the inhibition of ERK activation with a specific inhibitor (PD98059) to the ERK upstream kinase mitogen/extracellular-regulated protein kinase kinase abrogated the OM inhibitory activity on p53 expression in a dose-dependent manner. In addition to OM, we showed that the p53 protein expression in MCF-7 cells was also decreased by phorbol 12-myristate 13-acetate treatment (PMA). Because both OM and PMA induce MCF-7 cells to differentiate, our data suggest that p53 expression in breast cancer cells is down-regulated during the differentiation process.

Introduction

OM,3 a Mr 28,000 glycoprotein, is a cytokine derived from activated T lymphocytes and macrophages (1, 2, 3) . OM is a member of the IL-6 family cytokines, which includes IL-6, IL-11, LIF, ciliary neurotrophic factor, and cardiotrophin-1 (4, 5, 6, 7) .

As a pleiotrophic cytokine, OM elicits many different biological functions in different cell types, among which its ability to regulate cell growth and differentiation is most notable. OM stimulates the growth of normal fibroblasts (8 , 9) , normal rabbit vascular smooth muscle cells (10) , myeloma cells (11) , and AIDS-related Kaposi’s sarcoma cells (12) . OM also has been shown to inhibit the proliferation of a number of cell lines derived from human tumors including breast carcinoma, melanoma, and lung carcinoma (8 , 9 , 13, 14, 15, 16) . The inhibitory or stimulatory effects of OM on cell growth seem to depend on target cell type.

The growth regulatory activity of OM has been examined in a number of breast cancer cell lines, including MCF-7, MDA-MB231, and H3922 (8 , 13, 14, 15, 16) . The common responses of breast cancer cells to OM are reduced growth rates and the appearance of differentiative phenotypes. The growth-inhibitory effects of OM in these cell lines were accompanied by striking morphological changes (13 , 15) . Similar to the morphological changes seen in H3922 cells (13) , the appearance of cytoplasmic vacuoles and enlargement of cytoplasm were observed in MCF-7 and MDA-MB231 cells. In addition, OM-treated MDA-MB231 cells became spindle shaped, and the intercellular junctions were severely disrupted. In MCF-7 cells, a large amount of lipid droplets appeared after OM treatment. These phenotypic changes have been described as signs of differentiation of breast cancer cells (17) . The examination of several breast cancer cell lines that were treated with OM did not show a significant number of apoptotic cells, which suggests that OM does not induce apoptosis in breast cancer cells. The expression of the proto-oncogene c-myc is induced by OM within 1 h and is subsequently suppressed by OM after 24–48 h (13 , 14) . The molecular and cellular mechanisms underlying the growth-inhibitory activity of OM have not been elucidated.

Because the p53 tumor suppressor protein plays important roles in cellular proliferation and transformation (18) , we investigated the possibility that OM-induced growth inhibition and induction of differentiation are associated with alterations in p53 protein expression. In this study, we show that the p53 protein and the p53 mRNA in breast cancer cells were down-regulated by OM. Nuclear run-on studies and an analysis of p53 promoter activity both suggested that OM suppressed the transcription of the p53 gene. Interestingly, the effect of OM on p53 expression seemed to be mediated at least in part through the MAP kinase, also known as the ERK pathway, because the inhibition of ERK activation partially abolished the inhibitory effect of OM on p53 expression.

Results

To investigate whether OM regulates p53 expression, we initially examined the effect of OM on H3922 breast cancer cells. H3922 is a breast cancer cell line derived from an infiltrating ductal carcinoma. We characterized this cell line with regards to OM receptor, LIF receptor, EGF receptor, and estrogen receptor expression status (13) . H3922 cells express highly abundant OM-specific receptors and respond to OM with a strong growth inhibition. Treatment of these cells with OM for 3 days reduced DNA synthesis to 20% of control at concentrations of 10–20 ng/ml (13 , 14) . H3922 cells were exposed to OM at different concentrations for 3 days, and then untreated and OM-treated cells were lysed. Total cell lysate was harvested, and 50 µg of soluble protein from each sample were loaded on a 10% SDS gel and separated by electrophoresis, transferred to PVDF membrane, and blotted with anti-p53 monoclonal antibody. Fig. 1Citation shows that the p53 protein level was decreased in OM-treated cells in an OM dose-dependent manner. A maximal effect of 80% suppression was observed at 10 ng/ml and higher. In contrast to p53, the level of ß-actin was not altered by OM.



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Fig. 1. Dose-dependent suppression of p53 protein expression by OM. Total cell lysate was isolated from H3922 cells that were untreated or treated with 0.1, 1, 10, or 100 ng/ml OM for 3 days. Untreated control cells and OM-treated cells were lysed simultaneously at the end of treatment. Fifty µg of total cell lysate per sample were analyzed for p53 protein expression by Western blot. Anti-ß-actin monoclonal antibody was used to normalize the amount of cellular protein being used. The normalized p53 protein levels expressed as % of control were determined by densitometric analysis of the immunoblot. The normalized p53 protein levels (% of control) are: control, 100; OM 0.1 ng/ml, 83; OM 1 ng/ml, 68; OM 10 ng/ml, 9; OM 100 ng/ml, 15. The figure shown is representative of two separate experiments.

 
We next examined the time course of OM regulation on p53 protein expression. H3922 cells were untreated or treated with 50 ng/ml OM for different period of times. The results showed that the levels of p53 protein in untreated control cells remained unchanged during the experiment but were markedly decreased by OM treatment. The amount of p53 in OM-treated cells decreased to 50% of control after 9 h, lowered to 40% after 24 h, and further declined to the level of 10% of control after 48 h (Fig. 2A)Citation . To establish that the effect of OM on down-regulation of p53 expression is not the result of growth arrest, H3922 cells were cultured in serum-free medium for 48 h, which led to growth arrest as assayed by [3H]thymidine incorporation (data not shown). Then cell proliferation was initiated by replacing the culture medium with IMDM containing 10% FBS in the absence or the presence of OM for 24 h. Fig. 2BCitation shows that the p53 protein level in serum-starved cells decreased 30% (Lane 2) as compared with that in cells cultured in regular growth medium (Lane 1). Adding serum back to the growth arrested cells stimulated p53 expression (compare Lane 3 with Lane 2) in the absence of OM. However, OM produced a similar inhibitory effect on p53 protein expression as seen in Fig. 2ACitation . These experiments suggest that OM has a direct effect on p53 expression independent of the growth status of the cells. However, these studies cannot rule out completely the possibility that the growth arrest induced by OM contributes partially to the decreased expression of p53.



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Fig. 2. Time course of regulation of p53 protein expression by OM. A, H3922 cells were untreated or treated with 50 ng/ml OM for 9 h, 1, 2, 3, and 4 days respectively. At the indicated times, cells—untreated or treated with OM—were lysed simultaneously. Twenty µg of protein of total cell lysate per sample was analyzed for p53 and ß-actin protein expression by Western blot. The figure shown is representative of two separate experiments. B, cells were seeded in regular growth medium overnight and then switched to serum-free medium for 48 h to induce growth arrest. Cell proliferation was initiated by adding serum (10% FBS) back to the culture medium with or without OM for 24 h. At the end of the experiment, all of the cells were harvested simultaneously and analyzed for p53 and ß-actin expression by Western blot. Lane 1, cells cultured in regular growth medium for 48 h; Lane 2, cells cultured in serum-free medium for 48 h; Lane 3, after serum starvation, cells were incubated in regular growth medium for 24 h in the absence of OM; Lane 4, after serum starvation, cells were incubated in regular growth medium for 24 h in the presence of OM.

 
To determine whether the inhibitory effect of OM on p53 is limited to the H3922 cell line or is a common response of breast cancer cells to OM, the effect of OM on p53 protein expression was examined in two other OM-inhibited breast cancer cell lines, MCF-7 and MDA-MB231. As shown in Fig. 3ACitation , the p53 protein level in MCF-7 cells was decreased to 84% of control after 1 day of OM treatment, reaching a lowest level of 33% of control after 6 days. The parallel experiment of counting the cell number showed that treating MCF-7 cells with OM for 6 days reduced the number of viable cells to 42.3% of control. Comparing the treated cells with untreated cells, we did not observe a significant increase in the number of dying cells in OM-treated MCF-7 cells. Similar to MCF-7 and H3922 cells, the p53 protein expression in MDA-MB231 cells was also down-regulated by OM. In comparison with the control cells, the level of p53 protein in OM-treated cells decreased 30% after 1 day and 56% after 2 days (Fig. 3B)Citation . These data demonstrate that decreased p53 protein expression is associated with the growth inhibition exerted by OM on breast cancer cells.



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Fig. 3. Examination of regulation of p53 protein expression by OM in MCF-7, MDA-MB231, and H3477 cells. Cells cultured in 2% FBS IMDM were treated with 50 ng/ml OM for the indicated times, and the total cell lysate was isolated and analyzed for p53 protein by Western blot. A, total cell lysate of samples from MCF-7; B, total cell lysate of samples from MDA-MB231; C, total cell lysate of samples from H3477. The relative p53 protein levels were determined by densitometric analysis of the immunoblot and normalized to the signal of ß-actin.

 
To ensure that the observed effect of OM on p53 protein expression is a receptor-mediated event, we examined the regulation of OM on p53 in H3477 cells. H3477 cells have been shown not to express the OM-specific receptor, and OM does not inhibit the growth of these cells (13 , 19) . As we expected, the p53 protein expression in H3477 breast cancer cells was not suppressed by OM (Fig. 3C)Citation , which indicated that the suppression of p53 protein expression by OM is a receptor-mediated event.

Previously (17 , 20) , it had been reported that PMA induces MCF-7 cells to differentiate. This differentiation process was accompanied by the accumulations of lipid droplets and intracellular vacuoles. To investigate the possibility that PMA down-regulates p53 expression in MCF-7 cells, MCF-7 cells were treated with 100 ng/ml PMA for different time periods, and p53 protein expression was examined by Western blot. Fig. 4Citation showed that the incubation of MCF-7 cells with PMA for 1 day lowered the steady level of p53 protein to 42% of that seen in control cells, and the p53 protein level remained at that low level at longer time points. Compared with the kinetics of OM action on p53 protein expression, the inhibitory effect of PMA seemed to be faster than OM in MCF-7 cells. Similarly, the PMA-induced morphological changes of MCF-7 cells were observed after 1 day of treatment, but the OM-induced morphological changes were not apparent until after 3–4 days (data not shown). Nevertheless, the common effects of OM and PMA on cell differentiation and p53 expression suggest that p53 expression in breast cancer cells is down-regulated during the process of cell differentiation.



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Fig. 4. Down-regulation of p53 protein expression by PMA. MCF-7 cells were treated with 100 ng/ml PMA for the indicated times and the p53 protein was detected by Western blot. The normalized p53 protein levels against actin were expressed as % of control and were determined by densitometric analysis of the immunoblot.

 
Because the regulation of p53 protein expression has been shown to occur at levels of translation and transcription, the effect of OM on p53 mRNA expression was examined by Northern blot analysis. H3922 cells were treated with OM for different periods of times, and total RNA was isolated. Fig. 5Citation shows that the level of p53 mRNA, after normalization to GAPDH, was decreased to 60% of control after 9 h of OM treatment, was further lowered to 25% of control in cells treated with OM for 24 h, and the mRNA level remained constant at longer time points. These results indicate that the effects of OM on p53 mRNA and p53 protein expression are essentially correlated with regard to the kinetics and the degree of suppression.



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Fig. 5. Kinetics of p53 mRNA expression in H3922 cells treated with OM. H3922 cells cultured in IMDM containing 2% FBS were incubated with 50 ng/ml OM for different times as indicated. Untreated control cells and OM-treated cells were lysed simultaneously at the end of the treatment with the RNA isolation solution. Total RNA was isolated and 15 µg per sample was analyzed for p53 mRNA by Northern blot. The membrane was stripped and hybridized to a human GAPDH probe. The figure shown is a representative of three different experiments.

 
To further study the regulatory mechanism, nuclear run-on assays were conducted to measure the relative rate of transcription of p53. Nuclei were prepared from control H3922 cells and H3922 cells treated with OM for 24 h, and transcription was allowed to continue in the presence of [32P-]UTP for 30 min. The incorporation of 32P into p53-specific RNA was used as a measure of transcription rate. The transcription rate for GAPDH was also measured as an internal control. OM-treated cells contained only approximately 20% as many active p53 transcripts as observed in control cells (Fig. 6A)Citation . Data were normalized by the signals detected in GAPDH slots. The effect of OM on p53 transcription was further studied by analyzing p53 promoter activity. A human p53 promoter reporter construct, pGL3-p53LUC, and a human LDL receptor promoter reporter construct, pLDLR234LUC, were transiently transfected into H3922 cells, and the luciferase activities were measured. As shown in Fig. 6BCitation , OM treatment decreased p53 promoter activity to approximately 30% of control. In contrast, in the same experiment, the promoter activity of the LDL receptor was increased 2.5-fold, which is consistent with the stimulatory effect of OM on this promoter reported in other cell lines (21) . These results obtained from the studies of nuclear run-on and promoter activity suggest that transcriptional regulation is a major component of the observed OM-mediated suppression of p53 mRNA expression.



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Fig. 6. OM down-regulates p53 gene transcription. A, nuclear run-on analysis of p53 transcription: two slots were blotted onto each of two nylon membrane strips. One slot received 3 µg of the 2-kb fragment of the p53 cDNA. The second slot was loaded with 5 µg of the GAPDH plasmid. One nylon strip was hybridized to a 32P-radiolabeled nuclear run-on reaction prepared from 24-h OM-treated H3922 cells. The second was hybridized to a labeled nuclear run-on reaction prepared from untreated control cells. Equal amounts of radioactivity were used in each hybridization. Radioactive signals were detected by autoradiography and quantified by densitometric analysis. The figure shown is representative of two different experiments. B, analysis of human p53 promoter activity: H3922 cells were transfected with the pGL3-p53LUC and pLDLR234LUC. After transfection, cells were cultured in media minus or plus OM for 48 h. Assays of luciferase activity were conducted as described (21) . The data (mean ± SE) shown are representative of three separate experiments in which triplicate wells were assayed.

 
It has been proposed that the p53 gene is a target of the proto-oncogene c-myc. The c-Myc protein regulates the transcription of p53 through the c-Myc-responsive element present in the promoter region of the p53 gene (22) . We investigated the possibility that OM-mediated suppression of p53 transcription was due to an effect on c-Myc-mediated transcription. Northern blot analysis was used to examine the levels of c-myc mRNA and p53 mRNA in total RNAs isolated from H3922 cells untreated or treated with OM for different periods of time. Fig. 7Citation shows that transcription of the c-myc mRNA and the p53 mRNA were not concurrently regulated by OM. The c-myc mRNA was increased more than 3-fold by OM after 30 min and slowly decreased afterward. The suppression of c-myc transcription was not seen until 24 h after OM treatment. In contrast, the p53 mRNA level was steadily decreased during OM treatment. The biphasic effect of OM on c-myc mRNA was not seen. A similar result was obtained from MDA-MB231 cells. Furthermore, Western blot to examine the c-Myc protein level in untreated and OM-treated H3922 cells showed that the level of c-Myc protein did not decrease until after 2 days of OM treatment (data not shown). The decrease in p53 mRNA was maximal at 24 h in H3922 cells. These results suggest that either c-Myc is not involved in the regulation of p53 by OM or it is not the major transcriptional regulator. Other transcription factors could be involved in addition to c-Myc for the OM-induced down-regulation of p53.



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Fig. 7. OM regulates the mRNA expressions of p53 and c-myc with different kinetics. H3922 cells were incubated with 50 ng/ml OM for different times as indicated. Total RNA was isolated and 15 µg per sample were analyzed for p53 mRNA, c-myc mRNA, and GAPDH mRNA by Northern blot. The radioactive signals were detected and quantitated by a PhosphorImager. The figure shown (A) is representative of two different Northern blots. B, the normalized c-myc and p53 mRNA levels (% of control).

 
Two major signaling pathways can be activated by OM and its related cytokines IL-6 and LIF: (a) the Janus family tyrosine kinase/signal transducer and activator of transcription pathway (23) ; and (b) the Ras-MAP kinase pathway (21 , 24, 25, 26) . In OM-treated breast cancer cells, both STAT3 and MAP kinases ERK1 and ERK2 were activated (27) . To investigate whether the MAP kinase pathway is involved in the OM-mediated suppression of p53, H3922 cells were incubated with OM for 2 days in the presence of different concentrations of PD98059, which specifically blocks ERK activation by inhibiting the enzymatic activity of the ERK upstream kinase, MEK (28) . Incubation of cells with PD98059 alone, in up to a 30-µM concentration, did not change the levels of p53 protein (data not shown); however, the OM-mediated suppression of p53 protein expression was partially (maximal 50–60%) inhibited by PD98059 in a dose-dependent manner (Fig. 8)Citation . These results suggest that the suppression of p53 expression is a downstream event of the activation of MAP kinase pathway by OM in breast cancer cells. The fact that the OM-inhibitory activity could not be completely reversed by PD98059 at concentrations that effectively inhibited ERK activation (21 , 29) suggests that there are other signaling pathways, such as the STAT pathway, that may be involved.



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Fig. 8. The dose-dependent effects of MEK inhibitor PD98059 on OM regulation of p53 protein expression. H3922 cells were treated with OM for 2 days in the absence or the presence of the indicated concentrations of PD98059. Total lysate was harvested and analyzed for p53 protein by Western blot. The membrane was stripped and reprobed with anti-ß-actin monoclonal antibody. The normalized p53 protein levels expressed as % of control were determined by densitometric analysis of the immunoblot.

 
Discussion

Previous studies have established a differentiative role of OM in breast cancer cells (13, 14, 15, 16) . The general effects of OM on breast cancer cells include: (a) inhibition of cellular proliferation in monolayer culture and inhibition of colony formation in soft agar; (b) regulation of the cell cycle by increasing the proportion of cells in G0-G1 phase with a concomitant decrease in the number of cells in S phase; and (c) induction of a variety of morphological changes associated with the differentiated phenotype. These effects are believed to be mediated through the OM-specific receptor that consists of gp130 as a low-affinity ligand-binding subunit and OSMRß as the signal-transducing subunit (13 , 19) . In this study, we demonstrated that OM also down-regulates p53 expression in breast cancer cells.

The effect of OM on p53 protein expression was initially examined in four breast cancer cell lines, among which 3 cell lines—H3922, MCF-7, and MDA-MB231—were growth-inhibited by OM. The cell line H3477 does not respond to OM treatment because of its lack of expression of the signal-transducing subunit (OSMRß) of the OM-specific receptor. The incubation of cells with OM decreased the level of p53 protein in all three of the OM-responsive cell lines, with the most dramatic effect found in H3922 cells. The expression of p53 protein in H3477 cells was not inhibited at all by OM. The decrease in p53 protein was detected after 9 h of OM treatment and reached the lowest levels (10–20% of control in H3922 and 30–40% of control in MCF-7 and MDA-MB231) after 3–4 days. The extent of the suppression of p53 expression in different cell lines seems to correlate with the expression level of OSMRß, inasmuch as a previous study (19) using quantitative reverse transcription-PCR showed that H3922 cells express the highest mRNA level of OSMRß as compared with that detected in other cell lines.

Northern blot analysis to examine p53 mRNA expression in control and OM-treated cells demonstrated that OM down-regulated p53 mRNA expression to a degree similar to that observed in the levels of p53 protein in these cells. The decreased mRNA expression was due to a direct inhibition of transcription of the p53 gene by OM as demonstrated by nuclear run-on analysis. Collectively, these data establish that OM down-regulates the transcription of the p53 gene in breast cancer cells, with a resulting decrease in p53 protein level. The inhibitory effect of OM on p53 promoter activity suggests that an OM-responsive element(s) is present in the promoter region of the p53 gene. Interactions of this putative cis-acting element with an OM-inducible transcription factor may be responsible for the suppression of p53 transcription. The nature of this interaction has not been characterized in the present study; however, the fact that the MEK inhibitor PD98059 partially prevented the OM-inhibitory effect on p53 expression suggests that a substrate of ERK may be directly or indirectly involved in the OM-elicited signaling pathway that mediates this regulation.

It has been proposed that p53 is a target gene of c-Myc. The c-Myc protein was reported to transactivate the p53 promoter through the c-Myc-responsive element, E box (CATGTG) (22) . Because OM has been shown to regulate c-myc gene transcription in breast cancer cells and in M1 leukemia cells, one potential mechanism of OM suppression of p53 transcription would be suppression of c-myc transcription. However, the kinetics of the OM-induced down-regulation of p53 were different from the kinetics of the OM-induced down-regulation of the c-myc gene in these cells. The c-myc mRNA was transiently induced by OM within 0.5–8 h and was subsequently suppressed at later time points. The maximal suppression occurred after 2–3 days of OM treatment (Fig. 7Citation ; Refs. 13 , 14 ). In contrast, p53 mRNA was not induced by OM at any time point examined. Instead, it was gradually decreased during the period of OM treatment. A significant decrease in p53 mRNA level was observed after 8 h of OM treatment. This difference in kinetics between the expression of p53 mRNA and c-myc mRNA suggests that the effect of OM on p53 transcription is not a direct effect of OM on c-myc transcription alone. Additional studies to identify the cis-acting element in the p53 promoter that is responsible for the OM-mediated suppression of p53 transcription will clarify the relationship between p53 transcription and c-myc transcription in breast cancer cells.

The p53 tumor suppressor protein is involved in several central cellular processes that are critical for maintaining cellular homeostasis, including gene transcription, DNA repair, cell cycling, senescence, and apoptosis (18 , 30) . Compared with the vast information and knowledge available regarding the role of p53 protein in apoptosis, the function of p53 in cell differentiation is not well understood. This study is the first report to show that p53 expression is down-regulated in growth-inhibited and -differentiated breast cancer cells through a transcriptional mechanism. Additional studies to examine the functional role of p53 in the differentiation of breast cancer cells will be needed for a better understanding of the complexity of p53 functions that are essential for maintaining cell homeostasis.

Materials and Methods

Cells and Reagents.
The human breast cancer cell line H3922 was derived from an infiltrating ductal carcinoma, and the human breast cancer cell line H3477 was derived from primary solid tumor (13) . MDA-MB231 and MCF-7 cells were obtained from American Type Culture Collection (Manassas, VA). All of the cell lines were cultured in IMDM supplemented with 10% heat-inactivated FBS. Purified human recombinant OM was provided by Bristol-Myers Squibb (Princeton, NJ). PMA and anti-ß-actin monoclonal antibody were purchased from Sigma Chemical Co. (St. Louis, MO). The MEK inhibitor PD98059 was purchased from New England Biolabs (Beverly, MA). The antibodies against p53 and c-Myc were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Western Blot Analysis of p53 Protein.
Cells were cultured in 60-mm culture plates in 2% FBS IMDM with or without OM. Cells were rinsed with cold PBS and lysed with 0.25 ml of lysis buffer [50 mM Tris (pH 7.4), 1% NP40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM NaF, 5 µg/ml aprotinin, 1 µg/ml leupeptin, 1.25 µg/ml pepstatin, 1 mM Na3VO4, 10 µM okadaic acid, and 10 µM cypermethrin]. Concentration of soluble protein from total cell lysate was determined using BCA reagent with BSA as a standard (Pierce). Approximately 10–50 µg of protein of total cell lysate per sample was separated on 10% SDS PAGE, transferred to PVDF membrane, blotted with anti-p53 monoclonal antibody (DO-1, Santa Cruz Biotechnology) using an enhanced chemiluminescence (ECL) detection system (Amersham). Membranes were stripped and reblotted with anti-ß-actin monoclonal antibody to ensure that an equal amount of protein is loaded on gel. The signals were quantitated with a Bio-Rad Fluro-S MultiImager system. Densitometric analysis of autoradiographs in these studies included various exposure times to ensure linearity of signals.

RNA Isolation and Northern Blot Analysis.
Cells were lysed in Ultraspec RNA lysis solution (Biotecx Laboratory, Houston, TX), and total cellular RNA was isolated according to the vendor’s protocol. Approximately 15 µg of each total RNA sample were used in Northern blot analysis as described previously. The p53 mRNA was detected with a 2-kb 32P-labeled human p53 cDNA probe, the c-myc mRNA was detected with a 1.4-kb probe containing c-myc exons 2 and 3, and the GAPDH mRNA was detected with a plasmid containing a human GAPDH cDNA. Differences in hybridization signals of Northern blots were quantitated by a PhosphorImager.

Nuclear Run-On Analysis.
Reaction was performed with the method described previously (14) . Briefly, the nuclei were harvested from H3922 cells that were untreated or treated with 50 ng/ml OM for 24 h. Approximately 2 x 107 nuclei in 100 µl were mixed with 100 µl of 2x reaction buffer containing 250 µCi [32P]rUTP. Approximately 2.0 x 106 cpm of each nuclear run-on reaction was used as a probe to hybridize a Hybond N membrane (Amersham) slot blot. Each slot blot contained 5 µg of GAPDH plasmid and 3 µg of the 2-kb fragment of the p53 cDNA as described in the Northern blot analysis. Probing the GAPDH plasmid allowed normalization of the p53 signals measured by densitometry.

Transient Transfection Assays.
The p53 promoter luciferase-reporter construct p53Ex1aLUC was generously provided by Dr. Peter Gruss at the Max-Planck-Institute (Göttingen, Germany). For construction of pGL3-p53LUC, the 550-bp insert containing the human p53 promoter region and Exon 1 (31) was released from p53Ex1aLUC by KpnI and BglII digestion and subcloned into pGL3-basic digested with KpnI and BglII. The plasmid vector pLDLR234LUC has been described previously (21) . H3922 cells, seeded in 12-well tissue culture plates, were transiently transfected with plasmid DNAs pGL3-p53LUC and pLDLR234LUC by the method of calcium phosphate coprecipitation. Fifteen h after transfection, OM (50 ng/ml) was added. After a 48-h treatment, cells were lysed. Equal amounts of cell lysate from each well were used for measuring luciferase activity. The data (mean ± SE) shown are representative of three separate experiments in which triplicate wells were assayed (Fig. 6)Citation .

Acknowledgments

We thank Jessy Dorn for her technical assistance in Western blot analysis of p53.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was supported by the Department of Veterans Affairs (Office of Research and Development, Medical Research Service); by Grant 94 MM4548 from the United States Army Medical Research and Development Command; and by NIH Grant CA69322. Back

2 To whom requests for reprints should be addressed, at Veterans Affairs Palo Alto Health Care System, 3801 Miranda Avenue, Palo Alto, CA 94304. Phone: (650) 493-5000 ext. 64411; Fax: (650) 849-0251; E-mail: liu{at}icon.palo-alto.med.va.gov Back

3 The abbreviations used are: OM, oncostatin M; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL, interleukin; IMDM, Iscove’s modified Dulbecco’s medium; LIF, leukemia-inhibitory factor; MAP, mitogen-activated protein; MEK, mitogen/extracellular-regulated protein kinase kinase; OSMRß, oncostatin M-specific receptor ß subunit; PMA, phorbol 12-myristate 13-acetate; LDL, low-density lipoprotein. Back

Received for publication 3/ 1/99. Revision received 7/12/99. Accepted for publication 8/18/99.

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