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[Cancer Research 60, 2025-2032, April 1, 2000]
© 2000 American Association for Cancer Research

Tumor Biology

S-phase Arrest and Apoptosis Induced in Normal Mammary Epithelial Cells by a Novel Retinoid1

Yuxiang Zhang, Arun K. Rishi, Marcia I. Dawson, Rainbow Tschang, Lulu Farhana, Madanamohan Boyanapalli, Uwe Reichert, Braham Shroot, Eric C. Van Buren and Joseph A. Fontana2

John D. Dingell VA Medical Center [Y. Z., A. K. R., R. T., L. F., M. B., J. A. F.] and Karmanos Cancer Institute, Detroit, Michigan 48201 [Y. Z., A. K. R., R. T., L. F., M. B., E. C. V. B., J. A. F.]; Molecular Medicine Research Institute, Mountain View, California 94043 [M. I. D]; and Center International de Rescherches Dermatologigues Galderma, F. 06902 Valbonne, France [U. R., B. S.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The addition of all-trans-retinoic acid has been found to mediate a G1 cell cycle phase arrest but not apoptosis in normal mammary epithelial cells. We have now found that addition of the novel retinoid 6-[3-(1-adamanty1)]-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437), which appears to function through a pathway independent of retinoic acid nuclear receptors, results in an S-phase arrest that is preceded by a 4-fold elevation in the levels of the cyclin-cyclin dependent kinase (cdk) inhibitor p21WAF1/CIP1. Failure to inhibit E2F-1 activation of genes through its phosphorylation by the cyclin cdk2 kinase has been shown to result in S-phase arrest and apoptosis in a number of cell types. Although exposure of the normal mammary cells to CD437 does not result in modulation of cyclin A or cdk2 levels, an increase in E2F-1 levels and a marked inhibition of cyclin A/cdk2 kinase activity are observed. Exposure to CD437 results in enhanced E2F-1 binding to its DNA consensus sequences and transcriptional activity during S phase. We hypothesize that this enhanced E2F-1 transcriptional activity results in S-phase arrest and subsequent apoptosis that has been observed in other systems.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Retinoids have been demonstrated to play important roles in the normal differentiation and development of a wide variety of vertebrate structures and tissues (1 , 2) . In addition, these compounds, through their antiproliferative activity, have been shown to inhibit the growth of a number of malignant and normal cell lines. Retinoids also function as chemopreventive agents through their ability to inhibit the transformation of preneoplastic diseases and have been shown to modify cellular activity predominately through their modulation of gene expression (3, 4, 5, 6) . This is accomplished through the binding of retinoids to specific nuclear transcription factors (RARs3 and RXRs), which, in turn, bind to specific consensus sequences (retinoic acid response elements and retinoid X response elements), resulting in the activation or inhibition of gene expression (7, 8, 9) . The specific genes involved in the antiproliferative activity of retinoids remain unclear, although a number of investigators have speculated that this is achieved through their anti-AP-1 activity (10, 11, 12) .

The growth of breast carcinoma cells has been shown to be inhibited by retinoids. This appears to be more dramatic in cells expressing high levels of RAR-{alpha}, in which significantly lower concentrations of retinoids are required for growth inhibition (13 , 14) . The ER in the presence of ligand has been shown to up-regulate RAR-{alpha}; whether the enhanced sensitivity of ER-positive cells to retinoid-mediated inhibition of cell growth is due to enhanced RAR-{alpha} expression is not clear (15, 16, 17, 18, 19) . The ability of retinoids to inhibit the growth of normal breast cells was investigated recently (19) . The addition of tRA resulted in G1 cell cycle arrest with accompanying inhibition of growth (20) . We have recently described a novel retinoid, 6-[3-(1-adamanty1)]-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437), which is a potent inducer of apoptosis in a number of malignant cell lines including both ER-positive and ER-negative breast carcinoma cells and appears to function through a RAR/RXR-independent pathway (21, 22, 23) . We have previously shown that the induction of apoptosis is preceded by the induction of the cyclin/cdk complex inhibitor p21WAF1/CIP1 and G1 cell cycle arrest. We have now examined the effect of CD437 on the growth of normal breast cells and found that in marked distinction to tRA, CD437 induced an S-phase cell cycle arrest in these cells, followed by the onset of apoptosis.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
CD437 was synthesized as described previously (24) dissolved in DMSO to a concentration of 5 mM, and stored at -80°C under a nitrogen atmosphere.

Cell Growth.
The normal human mammary epithelial cell lines AG11132A and AG11134A were obtained from the National Institute of Aging, Aging Cell Culture Repository (Coriell Institute for Medical Research, Camden, NJ). All studies in these cell lines, which have been shown to possess a limited life span, were performed between passages 15 and 18. MCF-7 and MDA-MB-231 human breast carcinoma cells were obtained from Dr. Mark Lippman (Lombardi Cancer Center, Washington, D.C.) All cells were grown in Mammary Epithelial Cell Basal Medium (Clonetics, San Diego, CA) supplemented with 4 µl/ml bovine pituitary extract (Clonetics), 5 µg/ml insulin, 10 ng/ml epidermal growth factor, 0.5 mg/ml hydrocortisone, 10 µM isoproterenol, and 10 mM HEPES (pH 7.4). Cells were cultured at 37°C in a humidified incubator with 5% CO2:95% air.

Western Blots.
Western blots were performed essentially as described previously (25) . Logarithmically growing cells were treated with CD437 for various times, and cells were harvested and lysed in Laemmli lysis buffer [500 mmol/liter Tris-HCl (pH 6.8), 2 mmol/liter EDTA, 10% glycerol, 10% SDS, and 5% ß-mercaptoethanol]. Protein lysates (50 µg/lane) were electrophoresed on 12% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The filters were blocked with 5% nonfat dried milk in 1x PBS/0.5% Tween 20 and then incubated with the appropriate antibodies. Horseradish peroxidase-conjugated rabbit antimouse IgG (Bio-Rad Laboratories, Hercules, CA) was used as the secondary antibody, and the bands were developed using the Amersham enhanced chemiluminescence (Amersham, Arlington Heights, IL) nonradioactive method following the manufacturer’s instructions. Cyclin A antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA), PARP antibody was bought from PharMingen (San Diego, CA), and p21WAF1/CIP1, cdk2, and E2F-1 antibodies were obtained from Transduction Laboratories (Lexington, KY).

Apoptosis Quantification.
Apoptotic cells were stained with acridine orange as described by Whitacre et al. (26) . After exposure to CD437, cells were harvested, washed with PBS, and resuspended to a cell concentration of 1 x 106 cells/ml. Fifty µl of cell suspension were stained with 5 µl of an acridine orange solution (100 mg/ml in PBS) in the dark.

Northern Blots.
RNA was isolated using the single-step method of Chomczynski and Sacchi (27) . The full-length p21WAF1/CIP1 cDNA probe was kindly provided by Drs K. Kinzler and B. Vogelstein (John Hopkins University, Baltimore, MD). Electrophoresis, Northern transfer, washing conditions, and hybridizations were performed as described previously (28) . Equivalent loading was confirmed by 28S/18S RNA levels.

BrdUrd Labeling.
Cells were exposed to 50 or 100 nM CD437 or vehicle alone for 48 h and then pulse-labeled with BrdUrd using an in situ proliferation kit (Boehringer Mannheim, Indianapolis, Indiana) as directed by the manufacturer. Flow cytometric analysis was performed on a FACScan (BDIS, San Jose, CA), which uses an argon ion laser tuned to 15 mW at 488 nm for fluorescence excitation and light scattering. FITC fluorescence was collected in the FL1 detector using a 530/30 nm band pass filter, and propidium iodide fluorescence was collected in the FL2 detector using a 585/42 nm band pass filter. The Doublet Discrimination Module was used to remove aggregates. Data were acquired with LYSYS II software on a CONSORT 32 workstation (BDIS). Typically, 20,000 events were saved as list mode data and analyzed with CELLQUest software (BDIS) using a Power Macintosh 7500/100 computer (Apple Computer, Cupertino, CA). Routine quality control of the FACScan was performed using AutoCOMP software and CaliBRITE beads; in addition, CellFIT software and DNA Quality Control Particles were also used (BDIS).

Cyclin A-dependent Kinase Assay.
Cells were collected and lysed at 4°C in lysis buffer containing 10 mM Tris (pH 7.5), 130 mM NaC1, 1% Triton X-100, 10 mM NaF, 10 mM sodium phosphate, 10 mM Na PPi, 16 µg/ml benzamide HCl, 10 µg/ml phenanthrolene, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride. Clarified extracts containing 500 µg of protein were incubated (4°C, 2 h) with 10 µl of agarose-conjugated anti-cyclin A antibody (sc-751 AC; Santa Cruz Biotechnology) in a total volume of 1 ml. The immunocomplexes were washed three times in lysis buffer and then suspended in 20 µl of kinase buffer containing 50 mM HEPES (pH7.0), 0.1 mM EDTA, 0.01% Brij 35, 0.1 mg/ml BSA, 0.1% ß-mercaptoethanol, 0.15 M NaCl, and 10 µl of ATP mix [930 µl of protein kinase assay buffer, 6 µl of 50 mM ATP (pH 7.0), 20 µl of 2 M MgCl2, and 44 µl of [32P]ATP (10 mCi/ml)]. Histone (2 µg) was added to the immunoprecipitates and incubated for 20 min at 30°C. Reactions were terminated by adding SDS-PAGE sample buffer, and phosphorylated proteins were resolved by SDS-PAGE.

Transient Transfection Assay.
Transient transfection assays were performed essentially as we have described previously (29) . The reporter construct containing the E2F-1 promoter linked to a luciferase gene was a kind gift of Dr. David Livingston (Dana-Farber Cancer Center, Boston, MA). In brief, cells were plated in a 100-mm dish at a density of 3 x 106 cells/dish and then cotransfected with pCMVß (Clonetech, Palo Alto, CA), which carries the Escherichia coli Lac Z gene under the control of the cytomegalovirus promoter and encodes for ß-galactosidase and the E2F-1 reporter construct. After a 6-h incubation with the plasmid DNAs, the cells were washed and incubated for an additional 48 h in the presence and absence of 50 nM CD437. The cells were harvested, and ß-galactosidase and luciferase were measured as described previously (29) .

Gel Mobility Shift Assays.
The double-stranded oligonucleotide containing the consensus binding site for E2F-1 transcription factor was obtained from Santa Cruz Biotechnology. The double-stranded oligonucleotide probe was phosphorylated using T4 polynucleotide kinase and ATP as per the standard methods. The double-stranded, phosphorylated oligonucleotide containing the E2F motif was then ligated into MluI-digested, end-filled pGL2-Promoter plasmid vector. A recombinant plasmid containing three E2F motifs ligated as concatamers was obtained. The sequence of the E2F inserts was confirmed by sequencing, followed by PAGE purification of a ~100-bp-long XhoI-XbaI fragment containing three concatamers of E2F motif. The ~100-bp subfragment was then labeled by end-filling using [32P]dCTP and Klenow fragment of DNA polymerase essentially as described previously (29) . DNA-protein binding was measured using gel electrophoretic mobility shift assays essentially as described previously (29) . Briefly, 30 µg of the nuclear protein extracts obtained from untreated and CD437-treated normal human mammary epithelial cell line AG11132A and 5 µg of the nuclear protein extracts from untreated, wild-type MCF-7 human breast carcinoma cells were incubated on ice for 15 min with 2 µg of polydeoxyinosinic-deoxycytidylic acid, 5 µg of BSA, and 1x binding buffer [10 mM HEPES (pH 7.5), 1 mM ß-mercaptoethanol, 10% glycerol (v/v), and 50 mM KCl]. After the addition of 32P-labeled probe DNA (50 pg of probe/reaction), the mixture was incubated for another 15 min on ice. Excess unlabeled specific and nonspecific competitor DNAs were added 15 min before the addition of labeled probe. The specific competitor DNA consisted of the above-mentioned 100-bp XhoI-XbaI DNA fragment containing three E2F consensus sequences, whereas nonspecific competitor DNA consisted of 110 bp of double-stranded GADD45 cDNA fragment (positions 580–690; Ref. 29 ). The binding reactions were electrophoresed onto a 5% nondenaturing polyacrylamide gel (acrylamide:bisacrylamide, 30:0.8) containing 5% glycerol at 150 V in 1 mM EDTA, 3.3 mM sodium acetate, and 6.7 mM Tris (pH 7.5). The gels were dried at 60°C, and DNA-protein binding was visualized by autoradiography.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CD437 Inhibition of MDA-MB-231, AG11132A, and AG11134A Proliferation.
The normal human mammary epithelial cell lines AG11132A and AG11134A express RAR-{alpha}, -ß, and -{gamma} and display a limited life span (20) . To compare the growth-regulatory properties of CD437 in the malignant MDA-MB-231 cells and in the AG11132A and AG11134A cell lines, the three cell lines were grown in defined media (see "Materials and Methods") because the addition of fetal bovine serum to the normal human mammary cell lines results in their death (data not shown). As demonstrated in Fig. 1ACitation , the addition of various concentrations of CD437 to the three cell lines resulted in marked inhibition of growth, with maximum growth inhibition noted with 50 nM CD437. The addition of 50 nM CD437 to the cells resulted in complete inhibition of growth over a 3-day period (Fig. 1B)Citation . The concentration of CD437 required to cause inhibition of growth in MDA-MB-231 cells was significantly lower than previously reported when the cells were grown in 5% fetal bovine serum (21) ; we and others (30) have demonstrated that the increased biological effect noted with retinoids in the absence of serum is due to decreased serum binding. Increasing serum concentrations necessitates increased CD437 concentrations for a biological effect because 98% of CD437 is serum-bound (data not shown).



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  		Fig. 1. CD437 inhibition of AG11132A, AG11134A, and MDA-MB-231 cell proliferation. The cell lines were seeded in 24-well plates in medium as described in "Materials and Methods." The cells were incubated overnight, and then (A) various concentrations of CD437 or (B) 50 nM CD437 were added, and the cells were incubated for 3 days. The error bars represent the SDs derived from three independent experiments.

 
CD437 Induction of Cell Cycle Arrest.
Exposure of all three cell lines results in the inhibition of cell proliferation (Fig. 1)Citation . We have previously noted that CD437 induced G1 arrest in MDA-MB-231 cells grown in serum (21) . We therefore examined whether CD437 induced a cell cycle arrest in AG11132A and AG11134A cells grown under serum-free conditions. CD437 induced a S-phase cell cycle phase arrest in AG11132A cells (Fig. 2)Citation after exposure to either 50 or 100 nM CD437, with approximately 90% of the cells found to be in S phase, as demonstrated by the marked increase in BrdUrd incorporation. There was a progressive increase in the percentage of S-phase cells over time when AG11132A cells were exposed to CD437 (Fig. 3)Citation .



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  		Fig. 2. CD437 induction of S-phase cell cycle arrest. AG11132A cells were incubated in medium in the presence and absence of 50 or 100 nM CD437 for 48 h, and the cells were incubated with BrdUrd as described in "Materials and Methods." The results of two independent experiments are shown.

 


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  		Fig. 3. CD437-mediated S-phase arrest in AG11132A cells. Cells were seeded in medium and exposed to CD437 (50 nM). At the end of incubation period, the cells were harvested, and flow cytometric analysis for DNA content was performed as described in "Materials and Methods." The results are representative of two independent experiments.

 
CD437-induced p21WAF1/CIP1 mRNA and Protein Levels.
Enhanced p21WAF1/CIP1 expression has been associated with a G1 cell cycle arrest (Ref. 31 and the references therein). CD437 induced p21WAF1/CIP1 mRNA and protein expression and G1 cell cycle arrest in a number of breast carcinoma cell lines (21) . However, we found that CD437 induced an S-phase cell cycle arrest in AG11132A and AG11134A cells. We therefore investigated whether CD437 induced p21WAF1/CIP1 mRNA and protein expression in these cell lines. A 4-fold increase in p21WAF1/CIP1 mRNA expression was noted in the AG11132A and AG11134A cell lines after exposure to 50 nM CD437 at 24 h (Fig. 4, A and B)Citation . Exposure to CD437 also resulted in a corresponding increase in p21WAF1/CIP1 protein expression at 24 h (Fig. 5)Citation , with a 6-fold and 3-fold increase in p21WAF1/CIP1 expression noted in AG11132A and AG11134A, respectively, as determined by laser densitometry. This increase in p21WAF1/CIP1 mRNA and protein expression was similar to that noted previously when other breast carcinoma cell lines were exposed to CD437 (21) .



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  		Fig. 4. CD437 induction of p21WAF1/CIP1 mRNA in AG11132A and AG11134A cells. Cells were seeded in medium as described in "Materials and Methods," and after 24 h of incubation, CD437 (50 nM) was added, and the cells were harvested at various times. Northern blots were performed as indicated in "Materials and Methods." A, a representative Northern blot assay. B, quantitation of a representative experiment of two independent experiments. The values are expressed relative to respective controls, which were given an arbitrary value of 1.

 


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  		Fig. 5. CD437 induction of p21WAF1/CIP1 protein expression. Cells were seeded in medium and exposed to CD437 as described in the legend to Fig. 3Citation . Cells were harvested at various times after exposure to CD437, and Western blots were performed as described in "Materials and Methods." A representative Western blot is shown. Similar results were obtained from two independent experiments.

 
CD437 Induction of Apoptosis.
Exposure of breast carcinoma cells to CD437 results in the induction of apoptosis (21) . We therefore determined whether CD437 also induced apoptosis in the AG11132A and AG11134A cell lines; apoptosis was assessed using a number of criteria. As depicted in Fig. 6Citation , exposure to CD437 for 72 h resulted in fragmented nuclei and condensed chromatin with intact plasma membranes in both normal cell lines as well as in the MDA-MB-231 cell line. Less than 5% of the cells displayed these features of apoptosis at 48 h, but 35% and 75% apoptotic cells were noted at 72 and 96 h, respectively. To further confirm CD437-mediated apoptosis in the AG11132A and AG11134A cell lines, we determined whether exposure to CD437 resulted in activation of the caspase system; activation of the caspase system and cleavage of a number of substrates including PARP have been associated with apoptosis (32 , 33) . We quantitated the activation of caspase-3, -8, and -9 after exposure to CD437 using specific substrates for each caspase. Exposure to CD437 resulted in a progressive increase in caspase-3 activity with time. Data in Fig. 7Citation show an 8-fold increase in caspase-3 activity after 48 h of treatment with CD437. Activation of caspase-8 and -9 was not noted until 48 h of exposure to CD437, with a 3-fold increase in the activity of both. PARP cleavage was also assessed in these cells. AG11132A cells were exposed to 50 nM CD437, the adherent and floating cells were harvested separately, and PARP cleavage was assessed (Fig. 8)Citation . Cleavage of PARP from its normal Mr 115,000 to Mr 85,000 was noted in both the adherent and floating cells, with the floating cells that were already undergoing apoptosis demonstrating complete cleavage to the Mr 85,000 form (Fig. 8)Citation . PARP cleavage was not noted in the control cells.



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  		Fig. 6. CD437-mediated apoptosis in AG11132A, AG11134A, and MDA-MB-231 cells. Cells were exposed to 50 nM CD437 for 72 h; the cells were then harvested and stained with acridine orange as described in "Materials and Methods."

 


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  		Fig. 7. CD437 activation of caspase-3, -8, and -9. AG11132A and AG11134A cells were exposed to 50 nM CD437 for 48 h, and then the cells were harvested, and caspase-3, -8, and -9 activities were assessed as described in "Materials and Methods."

 


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  		Fig. 8. CD437-mediated PARP cleavage. AG11132A cells were grown as described in "Materials and Methods" and exposed to 50 nM CD437 for 72 h. Control (C) cells were treated with vehicle only. Adherent (AD) and floating (FL) cells were harvested separately, and Western blots were performed as described in "Materials and Methods."

 
S-phase Arrest and Apoptosis.
S-phase arrest and apoptosis have been associated with the inappropriate expression of E2F-1 or failure to inactivate the E2F-1/DP-1 complex through phosphorylation (34, 35, 36) . The cyclin A/cdk2 complex has been found to be responsible for the phosphorylation of the E2F-1/DP-1 complex and its subsequent inability to bind to E2F-1 consensus sequences (34 , 37) . We therefore examined whether modulation of E2F-1, cyclin A, or cdk2 levels occurred during CD437-mediated inhibition of growth and apoptosis of the normal breast cells. We did not observe any modulation of either cyclin A or cdk2 levels after exposure to CD437 (data not shown). However, we found a 3-fold increase in E2F-1 protein levels in AG11132A cells after a 24–48-h exposure to CD437 (Fig. 9)Citation . Inhibition of cyclin A/cdk2 kinase with subsequent failure to phosphorylate E2F-1/DP-1 due to inhibition of cyclin A-cdk2 kinase activity could also result in failure to inactivate E2F/DP1 (34 , 37) . We therefore examined cyclin A-dependent kinase activity in the AG11132A cells after exposure to CD437. Exposure to CD437 resulted in complete inhibition of cyclin A/cdk2 kinase activity (Fig. 10)Citation .



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  		Fig. 9. CD437 elevation of E2F-1 levels. Cells (AG11132A) were seeded in medium and exposed to 50 nM CD437 as described in the legend to Fig. 3Citation . Cells were harvested at either 24 or 48 h after exposure to either vehicle or CD437, and Western blots were performed as described in "Materials and Methods." A representative of two independent experiments is shown.

 


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  		Fig. 10. Cyclin A-dependent kinase activity in CD437-treated AG11132A cells. Cells were treated with 50 nM CD437 and harvested at 48 h. Cyclin A immunoprecipitation and the kinase assay were performed as described in "Materials and Methods."

 
Despite multiple attempts, we were not able to adequately immunoprecipitate E2F-1 to assess its phosphorylation by the cyclin A/cdk2 kinase. We therefore evaluated E2F-1-mediated transcriptional activity using an E2F-1 reporter construct in AG11132A cells after exposure to vehicle or 50 nM CD437. Exposure to CD437 resulted in a statistically significant (P < 0.001) 1.5-fold increase in E2F-1-mediated transcriptional activity in the CD437-exposed cells (Fig. 11)Citation . We further assessed whether inhibition of cyclin A/cdk2 activity resulted in decreased E2F-1 phosphorylation/inactivation and thus increased E2F-1 binding to its consensus sequence by performing gel shift assays using nuclear extracts from AG11132A cells exposed to vehicle or CD437. As shown in Fig. 12Citation , the level of E2F-1 bound to its consensus sequence was significantly increased in the CD437-treated cells.



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  		Fig. 11. CD437 enhanced E2F-1-mediated transcriptional activity. Transient transfections and exposure to CD437 are described in "Materials and Methods." The luciferase activity (expressed in light units) was normalized to ß-galactosidase activity (expressed as absorbance). The results are the mean of four independent experiments relative to the vehicle-treated cells, which was arbitrarily defined as 1:bar SE.

 


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  		Fig. 12. Gel mobility shift assays. Binding of nuclear proteins from CD437-treated and -untreated normal mammary epithelial AG11132A cells and MCF-7 human breast carcinoma cells to a 32P-labeled, 100-bp, double-stranded DNA subfragment containing three E2F consensus sequences. Binding reactions were as described in "Materials and Methods." Lane 1, probe only; Lane 2, probe and the nuclear protein extracts from untreated AG11132A cells; Lane 3, probe, nuclear protein extracts from untreated AG11132A cells, and 1000-fold excess of the cold unlabeled probe fragment DNA; Lane 4, probe, nuclear protein extracts from untreated AG11132A cells, and 1000-fold excess of the cold, unlabeled, double-stranded, 110 bp of GADD45 cDNA fragment (positions 580–690; Ref. 29 ); Lane 5, probe, nuclear protein extracts from CD437-treated AG11132A cells; Lane 6, probe, nuclear protein extracts from CD437-treated AG11132A cells, and 1000-fold excess of the cold, unlabeled probe fragment DNA; Lane 7, probe, nuclear protein extracts from CD437-treated AG11132A cells, and 1000-fold excess of the cold, unlabeled, double-stranded, 110 bp of the GADD45 cDNA fragment (positions 580–690; Ref. 29 ); Lane 8, probe only; Lane 9, probe and the nuclear protein extracts from untreated MCF-7 cells; Lane 10, probe, nuclear protein extracts from untreated MCF-7 cells, and 1000-fold excess of the cold, unlabeled, probe fragment DNA; Lane 11, probe, nuclear protein extracts from untreated MCF-7 cells, and 1000-fold excess of the cold, unlabeled, double-stranded, 110 bp of GADD45 cDNA fragment (positions 580–690; Ref. 29 ); The arrow denotes the location of the putative E2F protein-DNA complex.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
CD437 has been found to induce cell cycle arrest and apoptosis in a variety of malignant cells (21, 22, 23 , 38, 39, 40, 41, 42) . The ability of this novel retinoid to influence the growth of normal cells has not been investigated. In this report, we examined the ability of CD437 to affect the growth of normal mammary epithelial cells. We used the normal mammary epithelial cell strains AG11132A and AG11134A, which were derived from reduction mammoplasty specimens and display a limited life span. As demonstrated by us and other investigators (20) , these cells possess all three RARs. The addition of tRA to similar cell lines (AG11132 and AG11134) resulted in G1 cell cycle arrest but not apoptosis (20) . The observed G1 cell cycle arrest induced by tRA appeared to be independent of p53 and was not associated with the induction of G1 cyclin/cdk inhibitors, i.e., p21 or p27 (20 , 43) . These results are in marked distinction to those noted with CD437, in which an S-phase arrest was noted with an associated increase in p21WAF1/CIP1 expression and subsequent apoptosis. In addition, whereas only 50 nM CD437 was required for S-phase cell cycle arrest and apoptosis, 1 µM tRA was required for G1 cell cycle arrest in these cells. An obvious explanation for the different results obtained between tRA and CD437 is that these retinoids most likely posses different mechanism(s) of action. tRA functions through the classical retinoid pathways involving the RARs, whereas a number of investigators have now found using a variety of cells that CD437 can function through a RAR/RXR-independent pathway(s) (20, 21, 22) . The mechanism through which CD437 mediates its actions remains to be defined.

We had previously found that CD437 induced a G1 cell cycle arrest in a number of breast carcinoma cells (21) ; however, in the present study, we found that CD437 induces an S-phase cell cycle arrest in the two normal mammary epithelial cell lines. The ability of CD437 to induce a S-phase cell cycle arrest has been documented in several studies using lymphoma or prostate carcinoma cell lines (22 , 42) . Why CD437 induces S-phase arrest in normal mammary epithelium cells but G1 arrest in breast carcinoma cells is not clear. There appears to be no correlation between the presence of functional p53 or induction of p21WAF1/CIP1 and the specific cell cycle phase in which the cells are arrested because breast carcinoma cells with a functional p53 arrested in G1, and normal mammary epithelial cells with a functional p53 arrested in S phase; p21WAF1/CIP1 is induced in all of these cell lines (21) . Whether the normal mammary cells possess an S-phase checkpoint that is lost in the carcinoma cell lines is not clear. Interestingly, this has been found to be the case in keratinocytes; normal keratinocytes possess an adhesion-dependent S-phase checkpoint that appears to be absent in immortalized and tumorigenic cell lines (44) .

Progression of cells from the G1 phase of the cell cycle to the S phase is associated with E2F-1 activation of a number of genes whose expression is required for the G1 to S transition (45 , 46) . In addition, cyclin A/cdk2 inactivation of E2F-1 binding activity is intimately associated with orderly progression along the S phase and entrance into the G2-M phase of the cell cycle (35) . Overexpression of E2F-1 or inhibition of cyclin A/cdk2 phosphorylation of E2F-1 has been associated with S-phase delay and subsequent apoptosis (34 , 47 , 48) . We therefore examined the levels of E2F-1, cyclin A and cdk2 after exposure to CD437. CD437 did not modulate the expression of cyclin A or cdk2, but it did increase the level of E2F-1 in these cells. Several studies have now documented that E2F-1 levels are up-regulated as a response to DNA damage (49 , 50) ; whether this is the mechanism through which CD437 enhances E2F-1 expression remains to be delineated. Inactivation of E2F-1 with subsequent failure of the E2F-1/DP-1 complex to bind to its consensus sequences requires the phosphorylation of these factors by the cyclin A/cdk2 kinase (37 , 51) . Exposure of cells to CD437 was found to result in marked inhibition of cyclin A/cdk2 kinase activity. Neither cyclin E, cyclin D, nor cyclin B can substitute for cyclin A in the phosphorylation of the E2F-1/DP-1 complex with its subsequent inactivation (37) ; thus, the CD437-mediated inhibition of cyclin A/cdk2 kinase activity would result in continued and inappropriate activation of E2F-1 and subsequent S-phase arrest and apoptosis. We speculate that the marked elevations in p21WAF1/CIP1 after incubation with CD437 was responsible for the decrease in cyclin A/cdk2 kinase activity. It has been demonstrated previously by a number of investigators that p21WAF1/CIP1 can indeed inhibit cyclin A/cdk2 kinase activity. Li et al. (52) demonstrated that overexpression of p21WAF1/CIP1 in human sarcoma cells leads to inhibition of phosphorylation of cyclin A-associated kinase activity and inhibition of phosphorylation of E2F-1, with resultant enhanced binding of E2F-1 to the E2F-1 consensus sequence. In addition, the kinase activity of cyclin A/cdk2 associated with p21WAF1/CIP1 is significantly lower than that of cyclin A/cdk2 kinase free of p21WAF1/CIP1, and as cells progress through the S phase, p21WAF1/CIP1 levels fall, and free cyclin A/cdk2 levels increase, with subsequent increases in kinase activity (53) . The CD437-mediated increase in p21WAF1/CIP1 expression would continue to inhibit cyclin A/cdk2 activity, even in S phase. Two observations suggest that the CD437-mediated inhibition of cyclin A/cdk2 kinase results in abnormal activation of E2F-1: (a) CD432-treated cells display enhanced E2F-1-mediated transcriptional activity; and (b) enhanced binding of E2F-1 to its consensus sequence. We have previously shown that CD437 enhances p21WAF1/CIP1 expression through enhanced stability of the mRNA (54) .

As opposed to tRA, CD437 induced apoptosis in both normal mammary epithelial cell lines after the S-phase arrest. Apoptosis was documented using a number of criteria. AG11132A and AG11134A cell lines displayed fragmented nuclei and condensed chromatin with intact plasma membranes after exposure to CD437. In addition, this process was preceded by the activation of caspase-3,-8, and -9 and cleavage of PARP. We have previously found that caspase-3 is activated in CD437-mediated apoptosis in leukemia cell lines (23) . Caspase-8, also known as Flice, is directly activated by Fas and tumor necrosis factor {alpha} through the accessory molecule FADD, whereas caspase-9 and -3 are activated using a separate pathway (55 , 56) . We have found that CD437-mediated apoptosis is not inhibited in cells expressing either a dominant negative FADD or Flice that blocked tumor necrosis factor {alpha}- and Fas-mediated apoptosis. Thus, CD437-mediated apoptosis appears to predominately involve the caspase-9 and -3 pathway.


   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 Supported in part by a Merit Review Award by the Medical Research Services of the Department of Veterans Affairs (to J. A. F.) and NIH Grant P01CA51993 (to M. I. D. and J. A. F.). Flow cytometry studies were supported by NIH Grant P30 CA22453-20 to the Molecular and Cellular Imaging and Analytical Cytometry Core Facility of the Karmanos Cancer Institute. Back

2 To whom requests for reprints should be addressed, at John D. Dingell VA Medical Center, Oncology (11 M-HO), 4646 John R. Street, Detroit, MI 48201. Phone: (313) 576-3661; Fax: (313) 576-1122. Back

3 The abbreviations used are: RAR, retinoic acid receptor; RXR, retinoid X receptor; BrdUrd, 5-bromo-2'-deoxyuridine; cdk, cyclin dependent kinase; ER, estrogen receptor; PARP, poly(ADP-ribose) polymerase; tRA, all-trans-retinoic acid; BDIS, Becton Dickinson Immunocytometry Systems. Back

Received 9/16/99. Accepted 2/ 2/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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