Inhibition of aberrant proliferation and induction of apoptosis in HER-2/neu oncogene transformed human mammary epithelial cells by N-(4-hydroxyphenyl)retinamide

Hiromitsu Jinno1,3, Melissa G. Steiner2, Rajendra G. Mehta4, Michael P. Osborne1,3 and Nitin T. Telang1,3,5

1 Division of Carcinogenesis and Prevention, Strang Cancer Research Laboratory, The Rockefeller University, 1230 York Avenue, New York, NY 10021,
2 Departments of Otolaryngology and
3 Surgery, Cornell University Medical College, 1300 York Avenue, New York, NY and
4 Department of Surgical Oncology, University of Illinois College of Medicine, 840 South Wood Street, Chicago, IL, USA


    Abstract
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 Abstract
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 Materials and methods
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Epithelial cells from non-cancerous mammary tissue in response to exposure to chemical carcinogens or transfection with oncogenes exhibit hyperproliferation and hyperplasia prior to the development of cancer. Aberrant proliferation may, therefore, represent a modifiable early occurring preneoplastic event that is susceptible to chemoprevention of carcinogenesis. The synthetic retinoid N-(4-hydroxyphenyl)retinamide (HPR), has exhibited preventive efficacy in several in vitro and in vivo breast cancer models, and represents a promising chemopreventive compound for clinical trials. Clinically relevant biochemical and cellular mechanisms responsible for the chemopreventive effects of HPR, however, are not fully understood. Experiments were performed on preneoplastic human mammary epithelial 184-B5/HER cells derived from reduction mammoplasty and initiated for tumorigenic transformation by overexpression of HER-2/neu oncogene, to examine whether HPR inhibits aberrant proliferation of these cells and to identify the possible mechanism(s) responsible for the inhibitory effects of HPR. Continuous 7-day treatment with HPR produced a dose-dependent, reversible growth inhibition. Long-term (21 day) treatment of 184-B5/HER cells with HPR inhibited anchorage-dependent colony formation by ~80% (P < 0.01) relative to that observed in the solvent control. A 24 h treatment with cytostatic 400 nM HPR produced a 25% increase (P = 0.01) in G0/G1 phase, and a 36% decrease (P = 0.01) in S phase of the cell cycle. HPR treatment also induced a 10-fold increase (P = 0.02) in the sub-G0 (apoptotic) peak that was down-regulated in the presence of the antioxidant N-acetyl-L-cysteine. Treatment with HPR resulted in a 30% reduction of cellular immunoreactivity to tyrosine kinase, whereas immunoreactivity to p185HER remained essentially unaltered. HPR exposure resulted in time-dependent increase in cellular metabolism of the retinoid as evidenced by increased formation of the inert metabolite N-(4-methoxyphenyl)retinamide (MPR) and progressive increase in apoptosis. Thus, HPR-induced inhibition of aberrant proliferation may be caused, in part, by its ability to inhibit HER-2/neu-mediated proliferative signal transduction, retard cell cycle progression and upregulate cellular apoptosis.

Abbreviations: AFU, arbitrary fluorescence unit; CFE, colony-forming efficiency; DMSO, dimethyl sulfoxide; FITC, fluorescein isothiocyanate; HPLC, high-performance liquid chromatography; HPR, N-(4-hydroxyphenyl)retinamide; Ig, immunoglobulin; MPR, N-(4-methoxyphenyl)retinamide; NAC, N-acetyl-L-cysteine; PBS, phosphate-buffered saline; PI, propidium iodide.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Breast cancer is one of the prevalent causes of female mortality in the USA. Recent estimates indicate a 29% incidence (178 700 of new breast cancer cases) and a 17% mortality (43 500 breast cancer-related deaths) of all female cancers in 1998 (1). Recent progress in early detection of the disease together with progress in surgical and adjuvant treatments contributes towards improved survival rate (27). Identification of specific and sensitive biomarkers for disease progression and mechanism-based evaluation for chemopreventive efficacy of natural as well as synthetic compounds may provide important leads for reduction of breast cancer incidence through effective primary and secondary prevention.

Our previous investigations have utilized in vitro cell culture models developed from non-cancerous mammary tissue to identify a panel of molecular, biochemical and cellular surrogate endpoint biomarkers that represent quantitative parameters to evaluate for induction and modulation of preneoplastic transformation (813). Recent experiments on human mammary epithelial cells have demonstrated that treatment with chemical carcinogen or transfection with oncogenes results in induction of aberrant cell cycle progression, alteration in cellular metabolism of 17-ß-estradiol and down-regulation of apoptosis in vitro prior to tumorigenesis in vivo. Several natural phytochemicals are effective in inhibiting the perturbation of these biomarkers (913).

Retinoids, natural and synthetic derivatives of vitamin A, play an important role in the control of mammary cell proliferation and differentiation (1419). These agents also exhibit anticancer activity in breast and other organ sites (27, 20). The synthetic retinoid N-(4-hydroxyphenyl)retinamide (HPR) has been shown to be an effective chemopreventive agent in several in vivo models of organ-site carcinogenesis (1419). Because of its mild toxicity (7,17,21), HPR represents a promising agent for human clinical trials. The mechanism responsible for its chemopreventive and therapeutic effect, however, remains to be elucidated. Several recent studies suggest that HPR exhibits its growth inhibitory effect by inducing apoptosis (2225) and that generation of reactive oxygen species may be involved in these effects of HPR (24,26).

Recent studies have shown that aberrant hyperproliferation induced by chemicals or oncogenes is an early-occurring event detectable in vitro prior to tumor formation in vivo during the multistep process of mammary carcinogenesis (913). Targeted overexpression of such positive growth regulators as ras, myc or HER-2/neu oncogenes induces aberrant proliferation and confers tumorigenic transformation in non-cancerous mammary epithelial cells (9,10,27,28). In this multistep carcinogenic process, cellular proliferation and apoptosis represent two regulatory events that are involved in the maintenance of cellular homeostasis. Promotion of cellular proliferation and down-regulation of apoptosis results in aberrant proliferation leading to the expression of tumor cell phenotype (1113,29,30).

Experiments in the present study were designed to (i) examine whether HPR inhibits aberrant proliferation in HER/neu oncogene expressing preneoplastic human mammary epithelial cells, and (ii) identify possible mechanisms responsible for the preventive efficacy of this synthetic retinoid.


    Materials and methods
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 Materials and methods
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Cell line
The 184-B5/HER cell line was developed by introducing the HER-2/neu oncogene into an immortalized non-tumorigenic human mammary epithelial 184-B5 cell line (28). The stable transfectants were grown in chemically defined, serum-free medium, prepared by mixing equal amounts of minimum essential medium (Life Technologies, Grand Island, NY) and keratinocyte basal medium, i.e. modified MCDB153 (Clonetics, San Diego, CA), supplemented with 10 ng/ml epidermal growth factor, 0.5 µg/ml hydrocortisone (Sigma, St Louis, MO), 0.24 IU/ml insulin (Eli Lilly, Indianapolis, IN), 10 µg/ml transferrin (Collaborative Biomedical Products, Bedford, MA) and 5 µg/ml gentamycin (Life Technologies). Routinely, the stock cultures of 184-B5/HER cells were maintained in the presence of Geneticin (200 µg/ml) (Sigma) to eliminate the expression of spontaneous revertants, and were subcultured at 1:10 split when they reached ~70% confluency. Cells were maintained at 37°C in a humidified atmosphere of 95% air:5% CO2 (1113,28,31).

Chemicals
HPR (Sigma) was dissolved in dimethyl sulfoxide (DMSO) (Sigma) at the concentration of 10 mM and stored in aliquots at –20°C. All procedures involving HPR were performed under yellow lights to minimize photoisomerization.

Antibodies
Anti-HER-2/neu antibody, FITC-conjugated anti-Bcl-2 antibody, FITC-conjugated anti-p53 antibody, non-specific rabbit antibody, FITC-conjugated swine anti-rabbit antibody and FITC-conjugated non-specific murine antibody were obtained from Dako (Carpinteria, CA). Anti-tyrosine kinase antibody was from ICN (Costa Mesa, CA) and Apoptag-Fluorescein Kit was from Oncor, (Gaithersburg, MD). All primary antibodies recognized the specified human protein. The antibodies were used at dilutions recommended by the commercial vendors.

Seven day growth inhibition assay
To evaluate a cytostatic dose–response, 184-B5/HER cells were plated into T-25 flasks at an initial seeding density of 1x105 cells/flask. After a 24 h attachment period, the cultures were treated with HPR (100–500 nM) continuously for 7 days with replenishment of the fresh medium every 48 h. The solvent controls received a treatment of 0.1% DMSO, comparable with that present in the highest dose of HPR tested. The number of surviving cells was determined by hemocytometer cell counts of triplicate samples per treatment group. This dose–response assay identified IC10 and IC90 concentrations of HPR for further study. The cytostatic nature of HPR exposure at IC90 concentration (400 nM HPR) was further confirmed by the reversibility of growth inhibition after HPR withdrawal and determination of the cell number 7 days post-treatment. In addition, parallel cultures were continuously exposed either to 0.1% DMSO or to 400 nM HPR for the entire 14-day duration of the experiment. These cells represented the controls.

Anchorage-dependent growth
The anchorage-dependent colony forming assay was used to evaluate the long-term growth inhibitory effect of HPR (12). Briefly, 184-B5/HER cells were seeded at a density of 300 cells/well in six-well plates, and after a 24 h attachment period the cultures were continuously treated by 100 nM HPR (IC10) or the solvent for 21 days. The medium containing HPR or the solvent was replaced every 48 h during the 21 day period. Macroscopic colonies formed were fixed in 100% methanol and stained in 1:20 diluted Giemsa stain (Sigma) and counted. The data were expressed as the colony-forming efficiency (CFE), which was calculated as: CFE (%) = no. of colonies/initial seeding densityx100.

Cell cycle analysis and detection of apoptosis
Analysis of cell cycle progression and detection of apoptosis was performed using our recently optimized methods (1113) and a modification of the method described by Nicoletti et al. (32). Briefly, 184-B5/HER cells were plated into T-75 flasks at an initial seeding density of 5x105 cells/flask. After a 48 h incubation, the cells were treated with 400 nM HPR or the solvent 0.1% DMSO for the subsequent 24 h. Trypsinized cells were then fixed in 80% ethanol for 1 h at –20°C, washed twice with phosphate-buffered saline (PBS) and then incubated with 150 µl RNase A (final concentration: 500 U/ml; Worthington Biochemical, Freehold, NJ) at 37°for 20 min. The cells were stained with 150 µl propidium iodide (PI) solution (final concentration: 50 mg/ml in PBS; Calbiochem, La Jolla, CA) in the dark at 4°C overnight. Stained cells were analyzed on an EPICS 752 flow cytometer (Coulter, Hialeah, FL) equipped with 488 nm excitation and 630 nm long pass filter for the collection of PI fluorescence. DNA analysis was performed using MPLUS software (Phoenix Flow Systems, San Diego, CA). The data were expressed as percentage distribution of cells in sub-G0/G1, G0/G1, S and G2/M phases of the cell cycle.

To confirm the effect of HPR on the onset of apoptosis, cellular epifluorescence in the cultures stained with fluorescein isothiocyanate (FITC)-labeled Apoptag was determined by the quantitative flow cytometric assay. The protocol used was essentially similar to that optimized for determining the expression of cell cycle regulatory gene products (see below). The data were expressed as arbitrary fluorescence units after correction for non-specific immunofluorescence from FITC-labeled immunoglobulin (Ig)G.

Determination of cell cycle regulatory gene product expression
To investigate whether growth inhibitory effect of HPR was associated with inhibition of HER-2/neu-mediated signal transduction, and whether induction of apoptosis by HPR was associated with modulation of p53 and/or Bcl-2 expression, cellular immunoreactivity to p185HER, tyrosine kinase, Bcl-2 and p53 gene products was determined by flow cytometry using antibody against each protein. Briefly, 184-B5/HER cells were treated with 400 nM HPR for 24 h. The cells were then trypsinized and fixed in 0.25% paraformaldehyde (Polysciences, Warrington, PA) in PBS for 30 min. After washing with PBS twice, cells were incubated with 0.1% Triton X-100 (Sigma) at room temperature for 3 min. Permeabilized cells were then washed with PBS twice and incubated with 1:100 dilution of FITC-conjugated antibodies at 4°C for 30 min in the dark. The negative control represented cells incubated with FITC-conjugated non-specific murine IgG. Following two washes with PBS that contained 2% fetal calf serum, the cells were resuspended in PBS and analyzed on a flow cytometer using 488 nm excitation and a 520 nm band pass filter for the collection of FITC fluorescence. For HER-2/neu immunoreactivity, trypsinized cells were fixed in 80% ethanol at –20°C overnight. After washing with PBS twice, cells were incubated with rabbit anti-HER-2/neu antibody at 4°C for 30 min. The negative control was incubated with non-specific rabbit antibody. Cells were washed with PBS containing 2% bovine serum albumin and incubated with FITC-conjugated anti-rabbit antibody at 4°C for 30 min. Following two more PBS washes, the cells were analyzed on the Coulter flow cytometer as above. The data were expressed as arbitrary fluorescence units per 104 fluorescence events after correcting for the non-specific fluorescence of the negative control.

Detection of N-(4-methoxyphenyl)retinamide (MPR) in 184-B5/HER cells
Evaluation of MPR formation in the cells treated by HPR was performed using high-performance liquid chromatography (HPLC) (33,34). 184-B5/HER cells were treated with 200 nM HPR for 24, 48 and 72 h. Harvested cell pellets were extracted twice with 2 ml of hexane. The extracts were evaporated to dryness using a Speed-Vac (Savant Instruments, Framingdale, NY) and reconstituted in 125 ml of methanol. For metabolite analysis, 100 µl of methanol extract was injected in a reverse phase Partisil ODS column (10 µm) and eluted on a mobile phase of 70–100% methanol gradient in 30 min delivered with a flow rate of 1.3 ml/min. Detection was performed at 350 nm. These HPLC conditions eluted HPR at 27 min and MPR at 29 min. The identity of the parent compound and the metabolite was confirmed by comparing the elution profiles of authentic standards. The data were expressed as nanograms MPR formed per 106 cells.

Statistical analysis
The HPR dose–response experiment to identify IC10 and IC90 concentrations was performed with three independent determinations per treatment group. The experiment, designed to identify the cytostatic concentration of HPR by monitoring the reversibility of growth inhibition, was performed with three independent determinations per treatment group per time point. The long-term effect of HPR on anchorage-dependent colony formation was performed in duplicate (cumulative n = 12 per treatment group). The experiments for cell cycle progression and cellular apoptosis were replicated four times, whereas that for HPR metabolism was replicated three times. The data generated from replicate experiments were analyzed for the statistical significance of the differences between various treatment groups using the Student's t-test and probability values P < 0.05 were considered statistically significant.


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Seven day growth inhibition by HPR
The effect of HPR on HER-2/neu oncogene-induced aberrant proliferation in 184-B5/HER cells was examined after continuous exposure to HPR doses that ranged from 100 to 500 nM. This experiment was designed to identify IC10 and IC90 concentrations of HPR and to evaluate maximum cytostatic dose. HPR at IC10 was tested for long-term inhibitory activity, whereas HPR at IC90 was used to identify possible mechanisms responsible for its biological activity. HPR treatment for 7 days showed a dose-dependent growth inhibition (Figure 1aGo). A 7 day treatment with 100 nM HPR resulted in no detectable growth inhibition. The long-term inhibitory effects of this low dose of HPR were therefore examined using the 21 day anchorage-dependent colony formation assay. A 7 day treatment with 400 nM HPR resulted in ~95% inhibition of growth in 184-B5/HER cells relative to that observed in the DMSO-treated solvent controls. The high dose of HPR was therefore used for 24 h duration in experiments involving cell cycle progression and cellular apoptosis assays. The cytostatic effect of 400 nM HPR was evaluated by monitoring the reversibility of growth inhibition upon withdrawal of the agent. A 7 day continuous exposure to 0.1% DMSO or to 400 nM HPR exhibited a surviving cell population of 5.6 ± 0.1x106 cells per 25 cm2 and 0.6 ± 0.2x106 cells per 25 cm2, respectively. Thus, HPR treatment induced ~89.3% inhibition of growth relative to that in DMSO-treated cultures. At the 14 day time-point, cells continuously exposed to 0.1% DMSO had reached confluency and exhibited contact inhibition of growth. These cultures exhibited a surviving population of 5.7 ± 0.1x106 cells per 25 cm2. A 14 day continuous exposure to 400 nM HPR yielded a surviving population of 0.3 ± 0.1x106 cells per 25 cm2, thereby indicating a 94.7% growth inhibition relative to that observed in the solvent controls. In contrast, HPR-treated cells, upon withdrawal of the retinoid, exhibited a surviving population of 5.4 ± 0.5x106 cells per 25 cm2 at the 14 day time-point, indicating a 17-fold increase within 7 days of HPR withdrawal (Figure 1bGo). The data generated from this experiment demonstrated that 400 nM HPR is cytostatic in the present model, and that continuous exposure is required for its persistent growth inhibitory effect.



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Fig. 1. Dose–response of HPR on 184-B5/HER cells. (a) Dose-dependent inhibition of growth is induced by continuous 7 day exposure to HPR. (b) Reversal of growth inhibition observed after withdrawal of HPR.

 
Inhibition of anchorage-dependent growth by HPR
The long-term effect of 100 nM HPR was evaluated by an anchorage-dependent colony-forming assay of 21 days' duration (Figure 2Go). As evidenced by a duplicate experiment (cumulative n = 12), HPR at 100 nM concentration decreased the colony-forming efficiency from 11.3 to 2.3% (P < 0.01), exhibiting a 79.6% inhibition of anchorage-dependent growth. These data are indicative of persistent down-regulation of growth in the presence of HPR.



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Fig. 2. Long-term inhibitory effect of low-dose (100 nM) HPR showing inhibition of anchorage-dependent colony formation. Each datum point is expressed as mean of colony-forming efficiency ± SEM (n = 12).

 
Effect of HPR on cell cycle progression
To examine whether HPR induces alteration in cell cycle progression, flow cytometric analysis was performed on 184-B5/HER cells treated with the maximum cytostatic dose of HPR (400 nM) for 24 h. As determined from four replicate experiments, treatment with HPR resulted in 25% increase (P = 0.01) in the percentage of cells in G0/G1 phase and 36% decrease (P = 0.01) in the percentage of cells in S phase, relative to solvent controls (Figure 3a and bGo). These results suggest that growth inhibitory effect of HPR may mainly be caused by accumulation of cells in the G0/G1 phase and inhibition of the S phase of the cell cycle.



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Fig. 3. Effect of HPR on cell cycle progression of 184-B5/HER cells. (a) Representative DNA histograms show that treatment with HPR resulted in accumulation of cells in G0/G1 phase, inhibition of S phase and induction of apoptosis relative to that observed in DMSO treated solvent control. (b) Cumulative data on the effect of HPR on cell cycle progression obtained from quadruplicate experiments. Data presented are the means ± SEM.

 
Induction of apoptosis by HPR
To determine whether the growth inhibitory effect of HPR is correlated with induction of apoptosis, the flow cytometric analysis for the detection of sub-G0/G1 (apoptotic) cell population was performed (Figures 3a and 4GoGo). After a 24 h treatment with cytostatic 400 nM dose of HPR, the cells exhibited a hypodiploid sub-G0/G1 peak, which was a manifestation of endonuclease-mediated cleavage of DNA at specific linker intervals, and is considered a marker for an apoptotic population (2226,32). Quadruplicate determinations showed significant induction of apoptosis by HPR (P = 0.02), as determined by the sub-G0/G1 analysis of PI-stained cell suspension.



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Fig. 4. Regulation of HPR-induced apoptosis by N-acetyl-L-cysteine. Each datum point is expressed as mean ± SEM of four separate experiments.

 
Because of the modest induction of apoptosis by HPR in this model, it was important to confirm this phenomenon. In an independent experiment, the onset of apoptosis was compared by sub-G0/G1 analysis and by FITC–Apoptag quantitative fluorescence assay in three replicate experiments. Cultures of 184-B5/HER cells at 100 and 25% confluency represented the positive and negative controls, respectively. The positive control exhibited 18.1 ± 2.1% sub-G0/G1 population and an arbitrary fluorescence unit (AFU) value of 16.0 ± 1.4 by the FITC–Apoptag assay. In contrast, the negative control exhibited 0.8 ± 0.2% sub-G0/G1 population and an AFU value of 0.5 ± 0.1. These data demonstrate a good correlation in detection of confluency-mediated spontaneous apoptosis by two independent assays. Treatment of cultures with 400 nM HPR resulted in a time-dependent increase in apoptotic cell population as determined by the two independent assays. Thus, sub-G0/G1 analysis exhibited 4.8 ± 0.37, 5.7 ± 0.3 and 7.3 ± 0.4% apoptic populations at 24, 48 and 72 h time points, respectively. This extent of apoptosis corresponded with the AFU values of 3.0 ± 0.1, 3.6 ± 0.1 and 4.6 ± 0.2, respectively, at identical time points. It is noteworthy that the onset of apoptosis in cultures treated with 0.1% DMSO exhibited a trend towards time-dependent increase. In these cultures, the maximum extent of apoptosis at the 72 h time point was only 1.3 ± 0.2%, which corresponds to the AFU value of 0.4 ± 0.1. This extent of apoptosis was at least 4.6-fold lower than that induced by HPR at the identical time point.

Alteration in immunoreactivity to cell cycle regulatory gene products by HPR
The effect of HPR on cellular immunoreactivity to p185HER, tyrosine kinase, Bcl-2 and p53 gene products were evaluated using the flow cytometry-based quantitative immunofluorescence assay (Table IGo). Whereas HPR treatment did not significantly alter the immunoreactivity to p185HER, it was found to inhibit tyrosine kinase expression by ~30% (P = 0.03). These data suggest that HPR may specifically down-regulate oncogene-mediated signal transduction via inhibition of tyrosine kinase activity. Lack of modulation in the expression of Bcl-2 and p53 gene products raises the possibility that induction of apoptosis by HPR does not involve the participation of these gene products, which are frequently associated with regulation of cellular apoptosis.


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Table I. Alteration in immunoreactivity to cell cycle regulatory gene products by N-(4-hydroxyphenyl)retinamide in 184-B5/HER cells
 
Regulation of HPR-induced apoptosis
To examine whether induction of apoptosis by HPR is caused by the metabolic generation of reactive oxygen species from the retinoid, the extent of modulation of apoptosis was determined in the presence of a potent antioxidant N-acetyl-L-cysteine (NAC). The data presented in Figure 4Go clearly demonstrated that 0.1 mM NAC was effective in inhibiting HPR-induced apoptosis. It is also noteworthy that NAC did not exhibit any effect on G0/G1, S and G2/M phases of the cell cycle, thus providing evidence for its specificity towards down-regulation of apoptosis.

Cellular metabolism of HPR
Unlike all-trans-retinoic acid that functions as an agonist for specific retinoid receptors (17,18,51), the molecular targets responsible for HPR action are not equivocally identified. Cellular cyp450 dependent metabolism of HPR, relative extent of formation of metabolites differing in their biological activity, and alteration in insulin like growth factor-I system (3,17,33, 34,58) have therefore been used as surrogate biomarkers for responsiveness to HPR. In the present study, the extent of conversion of HPR to its biologically inert metabolite MPR represented a marker for evaluating the responsiveness of 184-B5/HER cells to HPR (Table IIGo). Treatment of 184-B5/HER cells with HPR at IC20 (200 nM) dose levels resulted in a time-dependent increase in intracellular levels of MPR up to the 72 h time-point. This increase in HPR metabolism also corresponded with progressive increase in sub-G0 (apoptotic) cell population. The representative HPLC profiles from control cultures and HPR-treated cultures are presented in Figure 5a and bGo. In contrast to the elution profile from control cultures, the elution profile obtained from HPR-treated cultures exhibited two distinct peaks, non-metabolized HPR eluting at 27.54 min and MPR eluting at 29.03 min.


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Table II. Kinetics of metabolism of N-(4-hydroxyphenyl)retinamide and induction of apoptosis in 184-B5/HER cells
 


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Fig. 5. Metabolism of HPR by 184-B5/HER cells. (a) HPLC elution profile of the extract from 184-B5/HER cells treated with the solvent. (b) Elution profile of the extract from cultures treated with 200 nM HPR for 72 h. Note the presence of HPR and MPR.

 

    Discussion
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 Abstract
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 Materials and methods
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The experimental evidence for chemopreventive and chemotherapeutic efficacy of natural and synthetic retinoid derivatives, and the possible leads for clinical application of these agents, have been largely derived from in vivo studies on animal models for organ site carcinogenesis. These laboratory studies have provided strong evidence that retinoids, functioning as antiproliferative and differentiation-inducing agents, affect the promotional stage of carcinogenesis to inhibit chemical carcinogen-induced tumor development (1419). Data generated using in vitro models developed from mammary explant and cell culture systems have suggested that retinoids inhibit preneoplastic transformation via multiple mechanisms, which are dependent on the specific target tissue and the type of retinoid (33,3538). The laboratory data generated from in vivo and in vitro models are dependent on extrapolation for their clinical relevance. On the other hand, human tissue derived in vitro models that facilitate evaluation of preventive efficacy of retinoids against preneoplastic and/or neoplastic transformation offer a clinically relevant mechanistic approach. Such an approach should substantially decrease the need for extrapolation. The endocrine responsive breast tissue exhibits biochemical and phenotypic changes during the carcinogenic process, which results in altered responsiveness to estrogen and antiestrogens. In clinical breast cancer, amplification/overexpression of HER-2/neu oncogene is significantly correlated with down-regulation of estrogen receptor. These aggressive, receptor-negative tumors are frequently resistant to antiestrogen therapy (5761). Differences in HER-2/neu expression between estrogen-receptor-positive and -negative subgroups may provide leads for selection of patients for adjuvant tamoxifen and/or HPR therapy (3,4,6,60,61). A direct interactive influence of multidrug therapeutic intervention is evaluable, under stringently controlled conditions by the use of the present human tissue derived pre-clinical model.

Targeted overexpression of HER-2/neu oncogene confers tumorigenic transformation in non-cancerous human mammary epithelial cells in vitro as well as in transgenic mouse models in vivo (27,28,3941). At the clinical level, amplification and/or overexpression of HER-2/neu oncogene is detected in ~40–60% of in situ carcinomas and in ~12–15% of infiltrating breast carcinomas (4244). Detection of HER-2/neu oncogene in poorly differentiated, high-grade in situ carcinomas is associated with an increased risk of recurrence and of progression to metastatic disease (4547,57,59). HER-2/neu expressing preneoplastic human mammary epithelial cells, therefore, represent a clinically relevant and reliable experimental model to examine the antiproliferative activity of the synthetic retinoid HPR in an effort to understand possible mechanisms responsible for the preventive efficacy of this agent.

Initial dose–response experiment identified the concentrations of HPR that were capable of inhibiting HER-2/neu oncogene induced aberrant proliferation in 184-B5/HER cells by 10 and 90% relative to that observed in solvent-treated controls. The cytostatic nature of HPR exposure was evident by the lack of decrease in cell number after a 24 h exposure to IC90 levels and a reversal of growth inhibition after withdrawal of HPR. Prolonged exposure to low dose (IC10) HPR exhibited persistent inhibition of anchorage-dependent colony formation of 184-B5/HER cells. These observations taken together indicate that prolonged exposure to HPR is essential for its continued preventive activity and therefore, are consistent with previous reports where interruption of HPR treatment has been observed to result in regrowth of the tumor (1519) and clinical preventive interventions involve prolonged continuous administration of the retinoid (2,3,7,21).

To examine the short-term mechanistic effects of high-dose HPR, alterations in cell cycle progression were evaluated by flow cytometric analysis. The cell cycle analysis revealed that HPR-mediated growth inhibition of 184-B5/HER cells was caused by accumulation of cells in the G0/G1 phase, and inhibition of the S phase of the cell cycle. Relative to that in non-cancerous 184-B5 cells, preneoplastic 184-B5/HER cells exhibit increased proliferative activity and decreased apoptosis (11,12). This alteration is manifested as impaired cellular homeostatic growth control caused by an imbalance between positive regulation (proliferation) and negative regulation (apoptosis). Similar to that observed with HPR (48), our recent studies (1113,49) have shown that other mechanistically distinct classes of phytochemicals, such as glucosinolates, polyphenols and isoflavones, also exhibit down-regulation of aberrant cell cycle progression. These observations taken together provide evidence that cell cycle progression may represent a phenomenological marker to evaluate preventive efficacy.

This experiment, designed to identify molecular targets responsible for the observed inhibitory effect of HPR on cell cycle progression, measured the status of selected gene products including p185HER, tyrosine kinase, Bcl-2 and p53. The expression of these gene products is crucial for cell cycle progression and/or cellular apoptosis (29,30). The data generated from these experiments demonstrated that HPR treatment induced a down-regulation in cellular immunoreactivity to tyrosine kinase, whereas that to p185HER remained essentially unaltered. The cellular immunoreactivity to tyrosine-kinase-specific phosphotyrosine antibody is inducible by several members of the receptor tyrosine kinase family, which include HER-2/neu and EGF. This induction leads to phosphorylation of specific tyrosine residues during the process of proliferative signal transduction (27,28,50,62,63). In an independent unpublished study, we have observed that the absence of exogenous EGF substantially inhibits the growth of non-cancerous 184-B5 cells but not of HER-2/neu expressing preneoplastic 184-B5/HER cells. These cellular changes correspond with EGF-induced down-regulation of tyrosine phosphorylation of EGF receptor in 184-B5 but not in 184-B5/HER cells. Thus, 184-B5/HER cells, in part because of overexpression of HER-2/neu oncogene, appear to be less dependent on exogenous EGF.

The data demonstrating susceptibility of HER-2/neu-mediated signal transduction (tyrosine kinase activity) to HPR, loss of dependency to exogenous EGF and lack of down-regulation of EGF receptor tyrosine phosphorylation in 184-B5/HER cells, taken together, suggest that HPR-induced inhibition of aberrant cell cycle progression may be caused by a direct effect of this retinoid on HER-2/neu specific tyrosine kinase activity. In this context it is noteworthy that synthetic tyrosine kinase inhibitors exert differential modulatory effects on diverse tyrosine kinases and protein kinases (6264). Clearly, further studies are essential to distinguish specific effects of HPR on HER-2/neu expression from those on oncogene-mediated signal transduction.

The antiproliferative effects of HPR on preneoplastic and neoplastic epithelial cells involves multiple mechanisms, including retinoid metabolism (7,14,17,33), generation of reactive oxygen species that damage cellular DNA (24,26), mechanisms involving retinoid receptors (51,52) and induction of cellular apoptosis (2226,53). In the present study HPR treatment resulted in a time-dependent induction of apoptosis as evidenced by a progressive increase in the sub-G0/G1 (apoptotic) peak and in activity of FITC–Apoptag, but failed to modulate cellular immunoreactivity to Bcl-2 or p53. Since Bcl-2 expression protects cells from apoptosis, whereas that of wild-type p53 arrests the cell cycle progression and favors apoptotic cell death of irreparably damaged cells (29,30,54), the lack of modulation in those two apoptosis-associated gene products suggests that HPR-induced apoptosis is independent of Bcl-2 and/or p53 expression. Consistent with present data, HPR has been reported to induce apoptosis in cervical carcinoma cells without modulating the cellular levels of Bcl-2 and Bax proteins (24). The lack of p53 expression in response to HPR treatment suggests that, at cytostatic levels, this retinoid may not be generating a type and/or extent of DNA damage that provokes expression of wild-type p53 and the resultant apoptosis. In this context it is noteworthy that inhibition of aberrant cell cycle progression in 184-B5/HER cells with natural phytochemicals induces Bcl-2 dependent apoptosis (49). In contrast, induction of apoptosis by the same phytochemicals in 184-B5 cells initiated for carcinogenesis by a chemical carcinogen involves a p53-dependent mechanism (13).

Generation of DNA damaging free radicals during cellular oxidative metabolism of HPR has been reported to be responsible for the antiproliferative and apoptotic effects of this agent (2426). This aspect was examined by determining whether HPR-induced apoptosis is modulated in the presence of an antioxidant, and whether cellular metabolism of HPR is positively correlated with induction of apoptosis. The data generated by these experiments clearly demonstrated that co-treatment of cells with HPR + NAC resulted in substantial reduction of HPR-induced apoptosis, and treatment with HPR alone resulted in time-dependent increases in the formation of the biologically inert metabolite MPR and in the extent of cellular apoptosis. Previous studies on rodent and human mammary tissue have demonstrated that HPR is preferentially sequestered in the epithelial and adipocyte cells and is subject to metabolism by the transformation sensitive target tissue (7,33,34,37,55). Our current data on 184-B5/HER cells support these previous observations by demonstrating that human mammary epithelial cells are metabolically competent to convert HPR to MPR and that biological consequence of metabolic process is the induction of apoptosis in HPR responsive cells. These observations taken together are consistent with those reported for HPR in other cell culture systems (2426,56) and suggest that HPR in the present experimental system functions as a potent pro-oxidant whose biological activity is dependent on its cellular metabolism and resultant generation of reactive oxygen radicals.

In conclusion, the present study conducted on preneoplastic human mammary epithelial 184-B5/HER cells has demonstrated that chemopreventive efficacy of HPR in this pre-clinical model may in part be caused by the ability of the retinoid to inhibit HER-2/neu-mediated proliferative signal transduction, retard aberrant cell cycle progression and induce apoptosis through the generation of reactive oxygen species.


    Acknowledgments
 
The authors wish to thank Milan Zvanovec for his excellent technical assistance. This work was supported in part by the Department of Defense grant (DAMD 17-94-J-4208), National Institutes of Health grant (P01CA29502) to N.T. H.J. was supported by the Peter Shea Research Fellowship and Iris Cantor Breast Cancer Research Fund.


    Notes
 
5 To whom correspondence should be addressed Email: telangn{at}rockvax.rockefeller.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received March 12, 1998; revised July 31, 1998; accepted August 11, 1998.