Cancer Research Infection and Cancer: Biology, Therapeutics, and Prevention Translational Medicine Conference in Israel
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Farhana, L.
Right arrow Articles by Fontana, J. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Farhana, L.
Right arrow Articles by Fontana, J. A.
[Cancer Research 62, 3842-3849, July 1, 2002]
© 2002 American Association for Cancer Research

Tumor Biology

Cyclin B and E2F-1 Expression in Prostate Carcinoma Cells Treated with the Novel Retinoid CD437 Are Regulated by the Ubiquitin-mediated Pathway1

Lulu Farhana, Marcia Dawson, Arun K. Rishi, Yuxiang Zhang, Eric Van Buren, Charu Trivedi, Uwe Reichert, Guowei Fang, Marc W. Kirschner and Joseph A. Fontana2

John D. Dingell VA Medical Center and Karmanos Cancer Institute, and Department Internal Medicine, Wayne State University, Detroit, Michigan 48201 [L. F., A. K. R., Y. Z., C. T., E. V. B., J. A. F.]; Burnham Institute, La Jolla, California 92037 [M. D.]; Galderma R&D, Sophia Antipolis, France [U. R.]; and the Department Cell Biology, Harvard Medical School, Boston, Massachusetts 02015 [G. F., M. W. K.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
E2F-1 and cyclin B are important regulators of the cell cycle, and their expressionand degradation are tightly regulated. Proteolysis of both molecules is mediated by the ubiquitin degradation pathway involving the activation of specific E3 ubiquitin ligases. Treatment of prostate carcinoma cells with the novel retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437/AHPN) results in the enhanced expression of E2F-1 and rapid degradation of cyclin B in the absence of the modulation of mRNA levels; this is accompanied by the S phase arrest of the cells and subsequent apoptosis. The elevated level of E2F-1 is because of the enhanced stability of the molecule, as indicated by pulse-labeling studies, demonstrating a prolonged half-life. The enhanced E2F-1 stability is associated with the concomitant acetylation of E2F-1, the disassociation of E2F-1 from the E2F-1 E3 ligase p45SKP2, and decreased E2F-1 ubiquitination, suggesting CD437 inhibition of E-3 E2F-1 ligase activity. Exposure of the cells to CD437 also results in the enhanced association of the cyclin B E3 ligase APC with cyclin B and the rapid proteolysis of cyclin B. The CD437-enhanced proteolysis of cyclin B is blocked in the presence of the ubiquitin proteolysis inhibitor N-acetyl-leu-leu-norleu-al. Thus, CD437 modulates the expression of E2F-1 and cyclin B through the simultaneous stimulation and inhibition of the cyclin B and E2F-1 E3 ligases, respectively.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The ability of retinoids to modulate the growth of both normal and transformed cells has been well described (1) . Retinoid-mediated alterations of gene expression through binding to their nuclear receptor, i.e., RARs3 and RXRs, are felt to be responsible for the biological actions of these compounds (2) . The retinoid/retinoid nuclear receptor complexes bind in turn to specific motifs, RAR elements and RXR elements located in the regulatory regions of genes, and, thus, modify their expression (3) . Retinoid activation of these receptors in turn is known to play an important role in retinoid-mediated growth inhibition of different carcinoma cell types. Prostate carcinoma cells display resistance to the antiproliferative effects of retinoids (4 , 5) . The reason for this resistance is not clear, but it has been speculated that it may be secondary to the loss of RARß nuclear receptor expression (4) . We have described previously a novel retinoid CD437/AHPN, which induces S phase arrest and apoptosis in LNCaP and PC-3 prostate carcinoma cells (6) .

The induction of S phase cell cycle arrest and apoptosis has been found to be often associated with the inappropriate expression of the nuclear transcription factor E2F-1 (7, 8, 9) . The expression and biological activity of the E2F family of transcriptional activators are regulated tightly throughout the cell cycle (10) . E2F-1 is tightly bound by the unphosphorylated Rb protein during the early and midportions of the G1 phase of the cell cycle. Phosphorylation of Rb by cyclin D complexed to Cdk 4/cdk 2 or by cyclin E/cdk2 results in the release of E2F-1 and subsequent binding of E2F-1/DP-1 heterodimer to the E2F-1 consensus sequences located in the promoters of a number of genes (11, 12, 13, 14) . This allows for the activation of these genes and progression of cells through the G1 checkpoint and into the S phase of the cell cycle (9 , 15) . In addition, the E2F-1 promoter also contains an E2F-1 consensus sequence allowing for enhanced E2F-1 gene transcription and production (16) . Because DP-1 is always in excess, this allows for an even greater formation of DP-1/E2F-1 heterodimers and gene transcription (10) . Inactivation of the E2F-1/DP-1 heterodimer through phosphorylation of DP-1 by cyclin A/cdk 2 with loss of E2F-1/DP-1 binding to its consensus sequence is also necessary for normal progression through S phase (17 , 18) . Failure to inhibit the E2F-1/DP-1 heterodimer binding has been found to result in S phase cell cycle arrest with subsequent apoptosis (17, 18, 19) . A number of studies has now demonstrated that E2F-1 expression is regulated by the ubiquitin proteasome pathway involving a specific motif located in the COOH terminus of the E2F-1 molecule (20, 21, 22) .

Cyclin B1 also plays an important role in cell cycle progression. Cyclin B1 expression is minimal at the initiation of S phase and peaks at the G2-M border; this peak of cyclin B1 activity is required for cells to enter mitosis (23) . Regulation of cyclin B1 expression is complex and is found to occur at the transcriptional and post-transcriptional, as well as post-translational levels (23, 24, 25, 26, 27, 28) . The cyclin B1 promoter displays cell cycle regulation with minimal activity at G1 and maximal activity at the G2 phase of the cell cycle (23) . Activation of p53 can result in the inhibition of cyclin B1 promoter activity at the G2-M phase of the cell cycle resulting in G2-M arrest (29) . Cyclin B1 mRNA levels are also regulated through the presence of cis-stability motifs and the corresponding trans-factors (24) . Expression of these trans-factors appears to be cell cycle regulated such that the stability of cyclin B1 mRNA is minimal at G1 but peaks at G2-M (24) . In addition, cyclin B1 is degraded by the ubiquitin pathway through the activation of the APC (27 , 28) ; activation of the APC through specific phosphorylation of its components and the synthesis or activation of cyclin B1-directing components with the subsequent degradation of cyclin B appears to be necessary for the exit from mitosis (27) .

We have found that exposure of the prostate carcinoma cell lines LNCaP, DU145, and PC-3 to CD437 results in the rapid increase in E2F-1 levels accompanied by a concurrent decrease in cyclin B levels. CD437 modulation of the E2F-1 and cyclin B levels occurs through the ubiquitin pathway with the simultaneous inhibition of E2F-1 degradation and activation of cyclin B degradation.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and Antibodies.
AHPN/CD437 was synthesized as described previously (30) , dissolved in DMSO to a concentration 5 mM, and stored at -80°C. DMEM, DMEM-F12 medium, FBS, and dialyzed FBS were purchased from Life Technologies, Inc. (Grand Island, NY). Anticyclin B1, anti-E2F-1, anti-CDC20, anti-p45 SKP-2, and antiubiquitin antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); anti-Cdc27 antibody was obtained from Transduction Laboratories (Lexington, KY). The acetylated lysine antibody was obtained from Cell Signaling (Beverly, MA). The hCDH1 antibody has been described previously (31 , 32) . The proteasome inhibitor LLnL, horseradish peroxidase-conjugated secondary antibody, and histone H1 were purchased from Sigma (St. Louis, MO).

Cultures and Cell Cycle Analysis.
The human prostate carcinoma cell lines DU145, LNCaP, and PC-3 were maintained in DMEM-F12 medium supplemented with 5% FBS, at 37°C in a humidified incubator with 5% CO2. For flow cytometry analysis, DU145 cells were treated with 1 µM CD437 for 24 and 48 h, and the cells were pulse labeled with BrdUrd, a thymidine analogue, for 2 h using a BrdUrd flow kit (PharMingen Laboratories, San Diego, CA). BrdUrd incorporation was detected with a FITC-conjugated anti-BrdUrd antibody, and DNA content was labeled with 7-AAD.

Flow cytometry was performed on a FACScan flow cytometer (BDIS, San Jose, CA) equipped with an argon ion laser tuned to 15 mW at 488 nm for fluorescence excitation and light scattering, controlled by a Power Macintosh G3 (Apple Computer, Cupertino, CA) running CELLQuest software (BDIS). FITC fluorescence was collected in the FL1 detector using a 530/30-nm bandpass filter, and 7-AAD fluorescence was reflected with a 560 short pass filter to be collected by the FL3 detector using a 650-nm long pass filter. Electronic compensation for the spectral overlap of the fluorochromes was not used. The Doublet Discrimination Module identified cell aggregates (33) . Typically, 20,000 events of list mode data were saved and analyzed with CELLQuest software run on a Macintosh G3 computer. Routine quality control of the FACScan was performed using FACSComp software with CaliBRITE beads (BDIS) and CELLQuest software with DNA Quality Control particles (BDIS). Cells in S phase were identified by BrdUrd expression using Cell Quest when possible or else computed from 7-AAD histograms using ModFit LT (Verify Software House, Topsham, ME).

Western Blot and Immunoprecipitation.
Cells were lysed with lysis buffer [50 mM Tris (pH 7.5), 100 mM NaCl, 1 mM EDTA, 0.5% NP40, 0.5% Triton X-100, 2.5 mM sodium orthovanadate, 10 µl/ml protease inhibitor cocktail (Sigma), and 1 mM phenylmethylsulfonyl fluoride], and the protein concentration was determined using the Bio-Rad assay system (Bio-Rad Laboratories, Hercules, CA). Proteins (75 µg) were fractionated using 12% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with 5% nonfat dried milk in either PBS or Tris-buffered saline buffer containing 0.1% Tween 20 and then incubated with the appropriate primary antibody. Horseradish peroxidase-conjugated antirabbit or antimouse IgG was used as the secondary antibody, and the protein bands were developed using the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Piscataway, NJ).

In immunoprecipitation studies, 500-1000 µg of protein were incubated with 1 µg of appropriate antibody and 20 µl of Protein G Sepharose 4 Fast Flow (Amersham Pharmacia Biotech) overnight, centrifuged, and washed three times with TT buffer [50 mM Tris-HCI (pH 7.5), 150 mM NaC1, and 0.5% Tween 20] and twice with lysis buffer. Coimmunoprecipitation studies were performed using antibody and the procedure of Fang et al. (32) . Proteins were eluted with Laemmli sample buffer and fractionated using 12% SDS-PAGE, and Western blots were performed.

Cyclin B Kinase Assay.
Cells were lysed with lysis buffer containing 5 µg/ml aprotinin, 5 µg/ml pepstatin A, and 5 µg/ml leupeptin. Proteins (500 µg) were immunoprecipitated with anticyclin B1 polyclonal antibody, and the immunoprecipitates were washed three times with TT buffer and two times with kinase buffer [20 mM HEPES (pH 7.5), 20 mM ß-glycerol phosphate, 10 mM p-nitrophenyl phosphate, 10 mM MgC12, 1 mM DTT, and 0.5 mM sodium orthovanadate]. The washed protein beads were suspended in kinase buffer containing 20 µM unlabeled ATP and histone H1 (3 µg) and incubated with 5 µCi of [{gamma}-32P] ATP (3000 Ci/mmol; NEN, Boston, MA) for 30 min at 30°C. The reactions were stopped by the addition of 1 x Laemmli sample buffer and fractionated on 12% SDS-PAGE. The gels were fixed, and the labeled bands were visualized by autoradiography.

Pulse Chase Experiment.
DU145 cells were grown in DMEM medium supplemented with 5% FBS and then incubated in DMEM medium without methionine and cysteine and supplemented with 5% dialyzed FBS for 1 h. Cells were labeled with L-[35S] methionine (100 µCi) and L-[35S] cysteine (1000 Ci/mmol; Amersham Pharmacia Biotech) for 2 h and then incubated in DMEM, supplemented with L-methionine and L-cysteine both at 5 mg/ml, 10% FBS, and in the presence and absence of 1 µM CD437. Cells were trypsinized and lysed with lysis buffer at 0, 2, 4, and 6 h after the addition of unlabelled L-methionine and L-cysteine. Protein extracts (1000 µg) were immunoprecipitated with anti-E2F-1 antibody and resolved on 12% SDS-PAGE. The gel was then fixed and exposed to film, and the bands were quantified using laser densitometry.

Gel Mobility Shift Assay.
Nuclear protein extract from DU145 was prepared as described by Ausbel et al. (34) . The gel mobility shift assays and the labeling of the E2F-1 consensus sequence probe were performed as described by Zhang et al. (35) . Supershifts were performed using E2F-1 antibody obtained from Santa Cruz Biotechnology, Inc.

Northern Blots.
RNA was extracted, and Northern blots were performed as we have described previously (36) .


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CD437 Induces S Phase Arrest and Apoptosis in DU145 Cells.
We have found previously that exposure of PC-3 and LNCaP cells to CD437 results in S phase cell cycle arrest followed by apoptosis (6) . We determined whether similar results would be found with the DU145 prostate carcinoma cell line. Exposure of DU145 cells to 1 µM CD437 results in the progressive increase in cells accumulating in S phase of the cell cycle (Fig. 1, A–D)Citation ; these cells arrest in S phase after 24- (85% in S phase) and 48-h (83% in S phase) exposure to CD437 as opposed to exposure to vehicle only, which results in only 32 and 35% in S phase at 24 and 48 h. Although these cells accumulate in S phase, this is accompanied by a progressive decrease in BrdUrd labeling. This decrease in BrdUrd labeling is secondary to the rapid onset of apoptosis in these cells with 20 and 60% undergoing apoptosis by 24 and 48 h, respectively (Fig. 1, E and F)Citation . There is a progressive shift in the cells arrested in S phase to the G0-G1 phase (Fig. 1DCitation compared with Fig. 1CCitation ); this represents the progressive accumulation of apoptotic cells. The S phase arrest noted in the prostate carcinoma cell lines was in distinct contrast to breast carcinoma cells in which exposure to CD437 resulted in a G1 phase arrest (6 , 36) .



View larger version (62K):
[in this window]
[in a new window]
  		Fig. 1. Induction of S phase arrest and apoptosis in DU145 cell lines by CD437. DU 145 cells were seeded in DMEM medium supplemented with 5% FBS and 25 µg of gentamicin/ml and at a cell concentration of 1 x 106 cells/ml. The cells were incubated for 24 h, after which, CD437 was added to a final concentration of 1 µM. Cells were harvested at 24 and 48 h after exposure to CD437, and BrdUrd labeling of cells, flow cytometric analysis, and apoptosis were assessed, as described in "Materials and Methods." A, vehicle-treated cells stained with 7-AAD only; B, vehicle-treated cells stained with 7-AAD and anti-BrdUrd-FITC; C, cells treated for 24 h with 1 µM CD437 and stained with 7-AAD and anti-BrdUrd-FITC; D, cells treated for 48 h with 1 µM CD437 and stained with 7-AAD and anti-BrdUrd-FITC; E, apoptosis in DU145 cells exposed to vehicle; F, apoptosis in DU145 cells exposed to 1 µM CD437 for 24 h.

 
Exposure to CD437 Results in Elevated E2F-1 Levels.
Inappropriate elevation of E2F-1 levels has been demonstrated to induce S phase arrest and apoptosis in several cell lines (17, 18, 19) . Therefore, we examined E2F-1 levels in DU145, LNCaP, and PC-3 after exposure to CD437. Elevation of E2F-1 levels was noted within 6 h of CD437 addition to the cells and persisted for >48 h (Fig. 2A)Citation . Exposure to CD437 resulted in a 4- and 6-fold, 2- and 3-fold, and 2- and 2.5-fold increase in E2F-1 levels in DU145, LNCaP, and PC-3 cells, respectively, at 6 and 24 h. E2F-1 mRNA levels did not increase after the addition of CD437 in any of the cell lines (Fig. 2B)Citation , suggesting that the CD437-mediated increase in E2F-1 levels may occur through enhanced stability of the protein. Pulse chase experiments were performed in which E2F-1 was labeled with [35S] cysteine and [35S] methionine, and E2F-1 degradation was assessed (Fig. 3, A and B)Citation . Exposure to CD437 results in a significant increase in the half-life of E2F-1, confirming increased stability of E2F-1 in the presence of CD437 (Fig. 3, A and B)Citation .



View larger version (43K):
[in this window]
[in a new window]
  		Fig. 2. CD437-mediated increase in E2F-1 expression. Cells were seeded as described in the legend to Fig. 1Citation . CD437 was added to a final concentration of 1 µM, and the cells were harvested at various times. Western (A) and Northern (B) blots were performed as described in "Materials and Methods." Equal loading in the Western blots was assessed by actin levels.

 


View larger version (18K):
[in this window]
[in a new window]
  		Fig. 3. Exposure of prostate carcinoma cells to CD437 results in enhanced E2F-1 stability. DU 145 cells were grown as described in the legend to Fig. 1Citation . Cells were washed, grown in L-methionine- and L-cysteine-depleted medium supplemented with 5% dialyzed FBS, labeled with L-[35S] methionine and L-[35S] cysteine, and then grown in DMEM supplemented with 5 mg/ml L-cysteine and L-methionine and 5% FBS, as described in "Materials and Methods" in the presence and absence of 1 µM CD437. Cells were harvested, and immunoprecipitations using E2F-1 antibody were performed as described in "Materials and Methods." The immunoprecipitates were fractionated on SDS-PAGE. In A, the dried gels were exposed to X-ray film; in B, autoradiographs were quantified using a laser densitometer. The E2F-1 levels are described in arbitrary units relative to the control levels, which were assigned an arbitrary level of 1. The results are presented as the mean of three determinations. The error bars represent the standard errors of the mean.

 
Role of p45SKP2 in CD437-mediated E2F-1 Stability.
Degradation of E2F-1 through the ubiquitin proteasome pathway plays an important role in the regulation of E2F-1 levels (20, 21, 22) . It has been demonstrated recently that the disruption of the interaction between E2F-1 and the ubiquitin-protein ligase SCFSKP2 (p45SKP2) results in marked reduction in the ubiquitination of E2F-1 and the subsequent stabilization of E2F-1 levels (37) . To determine whether the addition of CD437 to the prostate carcinoma cells results in a decrease in the association between E2F-1 and p45SKP2, cells were grown in the absence and presence of CD437, p45SKP2 immunoprecipitated, and the associated E2F-1 levels were determined by Western blot (Fig. 4)Citation . Exposure to CD437 resulted in a significant decrease in the E2F-1-associated p45SKP2 (Fig. 4)Citation . Interestingly, a marked decrease in the p45SKP2 E2F-1-associated levels was noted at 6 h, followed by a mild increase at 24 and 48 h; the p45SKP2 E2F-1-associated levels noted at 24 and 48 h were still significantly below those noted in the vehicle-treated cells. The decreased E2F-1-bound p45SKP2 was not because of a decrease in p45SKP2 levels because no decrease in total p45SKP2 was noted after exposure to CD437 (Fig. 4)Citation . The specificity of the interaction between E2F-1 and p45SKP2 was indicated by the fact that no E2F-1 is detected if a non-p45SKP2 antibody is used to perform the immunoprecipitations.



View larger version (14K):
[in this window]
[in a new window]
  		Fig. 4. Exposure of prostate carcinoma cells to CD437 results in decreased association between E2F-1 and p45SKP2. Prostate carcinoma cells were exposed to 1 µM CD437 for various times. Cells were harvested, and immunoprecipitations using p45SKP2 antibody were performed as described in "Materials and Methods." The immunoprecipitates were fractionated using SDS-PAGE, and Western blots were performed with E2F-1 antibody, as described in "Materials and Methods."

 
CD437 Enhances E2F-1 Acetylation.
CD437 treatment of prostate carcinoma cells followed by 12% SDS-PAGE suggested the presence of two E2F-1 bands (Fig. 3)Citation . To further demonstrate the presence of these two bands and assess whether CD437 modulated their expression, DU145 cells were treated and exposed to CD437 in the presence and absence of cycloheximide, and the fractionation of proteins was performed using 10% SDS-PAGE, allowing for separation of the bands. Exposure to CD437 resulted in the increase in total E2F-1 and a faster migrating E2F-1 species (Fig. 5ACitation , Lane 2). Exposure to CD437 and cycloheximide decreased the expression of the slower migrating E2F-1 band, but there was no modulation of the faster migrating band level. This result suggested that incubation with CD437 resulted in the post-translational modification of E2F-1.



View larger version (27K):
[in this window]
[in a new window]
  		Fig. 5. CD437 increased acetylated E2F-1 levels. DU 145 cells (1 x 106 cells/ml) were exposed to vehicle or 1 µM CD437 in the presence and absence of cycloheximide (20 µg/ml). In A, cells were harvested at 6 h, and Western blots were performed using 10% SDS-PAGE, as described in "Materials and Methods." Lane 1, cells treated with vehicle alone; Lane 2, cells treated with CD437; Lane 3, cells treated with cycloheximide; Lane 4, cells treated with cycloheximide and CD437. The results depicted are representative of two independent experiments. Equal loading was assessed using actin levels. B, CD437-enhanced acetylation of E2F-1. Prostate carcinoma cells were exposed to 1 µM CD437 for 24 h. The cells were harvested and lysed, and immunoprecipitation with E2F-1 antibody was performed as described in "Materials and Methods." Western blots were performed using antiacetylated lysine antibody as described in "Materials and Methods."

 
Recent studies have demonstrated that modification of E2F-1 either through its phosphorylation or acetylation markedly modulates the stability of E2F-1 (38 , 39) . Although phosphorylation of E2F-1 enhances its degradation, acetylation of E2F-1 results in its enhanced stability, as well as faster migration on SDS-PAGE (38 , 39) . Therefore, we examined whether exposure to CD437 resulted in the acetylation of E2F-1. Immunoprecipitation of E2F-1 from CD437-treated cells, followed by the assessment for acetylated lysine using antiacetyl lysine antibody and Western blots, confirmed the presence of increased acetylated lysine in the E2F-1 in the CD437-treated cells (Fig. 5B)Citation . Incubation of the cells with CD437 resulted in a 3-, 3-, and 2-fold increase in acetylated levels of E2F-1 in DU145, LNCaP, and PC-3 cells, respectively, at 24 h; this was accompanied by an increase in total E2F-1. These increased levels of acetylated E2F-1 correlated with the increased E2F-1 levels after CD437 exposure (Fig. 2)Citation .

Ubiquitination of E2F-1.
To further document that CD437 inhibited ubiquitin-mediated proteolysis of E2F-1, we examined ubiquitinated E2F-1 levels in the cells after exposure to CD437, the proteasome inhibitor LLnL, and the combination of these two agents. Treatment with CD437 resulted in a significant decrease in ubiquitin E2F-1 levels in both DU145 and LNCaP cells both in the presence and absence of LLnL (Fig. 6)Citation . This decrease in ubiquitin E2F-1 levels most likely is because of CD437-mediated inhibition of the association between E2F-1 and p45SKP2, which functions as the E3 ligase in the ubiquitination of E2F-1 (37) . The addition of the proteasome inhibitor LLnL markedly enhanced the E2F-1 ubiquitin levels but in combination with CD437 did not inhibit CD437-induced decrease in these levels (Fig. 6)Citation .



View larger version (66K):
[in this window]
[in a new window]
  		Fig. 6. CD437 inhibition of E2F-1 ubiquitination. DU145 and LNCaP cells were exposed to CD437 (1 µM), LLnL (50 µM), or the combination for various times. The cells were then harvested, ubiquitinated proteins were immunoprecipitated using antibody to ubiquitin, and Western blots were performed using E2F-1 antibody.

 
CD437-mediated Increase in E2F-1 Levels Results in Enhanced E2F-1 Binding to the E2F-1 Consensus Sequence.
We have found that CD437-mediated increase in E2F-1 levels is associated with S phase arrest and apoptosis in normal mammary epithelial cells (35) . Previous studies have demonstrated that enhanced binding of E2F-1 to its consensus sequence is associated with E2F-1-mediated apoptosis (19) . Therefore, we determined whether the CD437-mediated increase in E2F-1 levels is associated with enhanced E2F-1 binding to its consensus sequence. The appearance of an additional E2F-1 complex binding to the E2F-1 consensus sequence after exposure to CD437 is demonstrated in Fig. 7Citation (see arrow). This binding indeed represented E2F-1 because it was only competed by an excess of unlabelled E2F-1 consensus sequence and not by a 1000-fold excess of unlabeled nonspecific probe (double-stranded p21WAF1/CIP1 3'untranslated region of 108 bp; Fig. 7ACitation ) and supershift of the binding activity in the presence of E2F-1 antibodies (arrow, Fig. 7BCitation ).



View larger version (119K):
[in this window]
[in a new window]
  		Fig. 7. CD437-mediated increase in E2F-1 levels is associated with increased binding of E2F-1 to its consensus sequence. Nuclear extracts were prepared from DU145 cells after exposure to vehicle or 1 µM CD437 for 24 h; labeling of probe and gel shifts were performed as described in "Materials and Methods." A, Lane 1, probe alone; Lane 2, probe and nuclear protein extracts from untreated cells; Lane 3, probe nuclear protein extracts from untreated cells and 1000-fold excess of cold, unlabeled probe fragment DNA; Lane 4, probe nuclear protein extract from untreated cells and 1000-fold excess of cold, unlabeled, and double-stranded p21WAF1/CIP1 3'-untranslated region of 108 bp; Lane 5, probe nuclear protein extracts from CD437-treated cells; Lane 6, probe nuclear protein extracts from CD437-treated cells and 1000-fold excess of cold, unlabeled probe fragment DNA; Lane 7, probe nuclear protein extracts from CD437-treated cells and 1000-fold excess of cold, unlabeled, and double-stranded p21WAF1/CIP1 3'-untranslated region of 108 bp. B, Lane 1, probe alone; Lane 2, probe nuclear protein extracts from untreated cells; Lane 3, probe nuclear protein extracts from untreated cells and E2F-1 antibody; Lane 4, probe nuclear protein extracts from CD437-treated cells; Lane 5, probe nuclear protein extracts from CD437-treated cells and E2F-1 antibody. The results are representative of two independent experiments. Arrow, the appearance of the new E2F-1 complex after CD437 exposure.

 
CD437 Enhances Cyclin B Proteolysis and Cyclin B Ubiquitin Levels.
Although CD437 treatment of the prostate carcinoma cell lines resulted in an elevation of E2F-1, a marked decrease in cyclin B levels (Fig. 8A)Citation and cyclin B kinase activity (Fig. 8B)Citation in the three prostate carcinoma cell lines was also noted. Cyclin B mRNA levels remain unchanged. This result suggested that whereas CD437 inhibited the ubiquitin pathway-mediated proteolysis of E2F-1, it may enhance degradation of cyclin B through the same pathway. To document that the ubiquitin pathway is involved in the CD437-mediated lowering of cyclin B levels, the prostate carcinoma cells were grown in the absence or presence of the ubiquitin proteolysis inhibitor LLnL and in the absence and presence of CD437. If the CD437-induced decrease in cyclin B levels is modulated through the ubiquitin pathway, then inhibition of this pathway should block CD437-mediated decrease in cyclin B levels. Incubation with the proteolysis inhibitor LLnL inhibited the CD437-mediated decrease in cyclin B levels, demonstrating that CD437 enhanced ubiquitin pathway-mediated proteolysis of cyclin B (Fig. 9)Citation . Activation of the E-3 ubiquitin-protein ligase APC has been shown to play an essential role in cyclin B degradation (27 , 40) . To document the potential APC involvement in the CD437-mediated decrease in cyclin B levels, prostate carcinoma cells were exposed to CD437, cyclin B immunoprecipitates were fractionated on SDS-PAGE, and Western blots performed with anti-Cdc 27 antibody; Cdc27 is an essential component of APC (38) . An increased association of Cdc 27 with cyclin B was noted in the prostate carcinoma cells after exposure of the cells to CD437 (Fig. 10A)Citation . Targeting of activated APC to cyclin B requires the activation of hCDH1 and its binding to APC (31 , 41, 42, 43, 44, 45, 46) . Therefore, we investigated whether exposure to CD437 results in enhanced binding of hCDH1 to APC; increased binding of hCDH1 to Cdc27 was indeed found after exposure of the cells to CD437, whereas no modulation of cellular hCDH1 levels was detected (Fig. 10B)Citation . The specificity of the interaction between hCDH1 and Cdc 27 was indicated by the lack of a Cdc 27 band if a non-hCDH1 antibody was used for the immunoprecipitations. If CD437 exposure results in the increased association of cyclin B with APC ligase, then enhanced cyclin B ubiquitin levels should be noted after the exposure of the cells to CD437. Increased cyclin B ubiquitin levels were indeed noted after exposure of DU145, LNCaP, and PC3 cells to CD437 (Fig. 10C)Citation .



View larger version (26K):
[in this window]
[in a new window]
  		Fig. 8. The addition of CD437 to prostate carcinoma cells results in decreased cyclin B levels (A) and decreased cyclin B kinase activity (B). Prostate carcinoma cells were exposed to 1 µM CD437 for various periods of times. Cells were harvested, and Western blot for cyclin B levels (A) and cyclin B kinase activities (B) were determined as described in "Materials and Methods."

 


View larger version (29K):
[in this window]
[in a new window]
  		Fig. 9. LLnL blocks CD437-mediated decrease in cyclin B levels. Prostate carcinoma cells were grown in the presence of vehicle alone, 1 µM CD437, 1 µM CD437 in the presence of various concentrations of LLnL, and various concentrations of LLnL alone. The cells were harvested after 24 h, and cyclin B levels were assessed by Western blot. DU145 cells: Lane 1, control; Lane 2, 1 µM CD437; Lane 3, 2.5 µM LLnL; Lane 4, 2.5 µM LLnL and 1 µM CD437; Lane 5, 5 µM LLnL; Lane 6, 5 µM LLnL and 1 µM CD437. PC-3 cells: Lane 1, control; Lane 2, 1 µM CD437; Lane 3, 2.5 µM LLnL; Lane 4, 2.5 µM LLnL and 1 µM CD437; Lane 5, 10 µM LLnL; Lane 6, 10 µM LLnL and 1 µM CD437.

 


View larger version (55K):
[in this window]
[in a new window]
  		Fig. 10. Exposure to CD437 results in the increased association between cyclin B and APC and hCDH1 and elevated cyclin B ubiquitin levels. Prostate carcinoma cells were exposed to 1 µM CD437 for various periods of time. In A, cells were harvested, and immunoprecipitations were performed with cyclin B antibody as described in "Materials and Methods." The immunoprecipitates were fractionated on SDS-PAGE gels, and Western blots were performed with Cdc 27 antibody as described in "Materials and Methods." In B, immunoprecipitations were performed with hCDH1 antibody as described in "Materials and Methods." The immunoprecipitates were fractionated using SDS-PAGE, and Western blots were performed with Cdc 27 antibody as described in "Materials and Methods." In C, DU145, LNCaP, and PC-3 were exposed to 1 µM CD437 for various times, ubiquitinated proteins were immunoprecipitated using antibody to ubiquitin, and Western blots were performed using cyclin B antibody as described in "Materials and Methods."

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
E2F-1 and cyclin B are important regulatory molecules, which play essential roles in the progression of cells along the cell cycle. The ubiquitin pathway-mediated degradation of these proteins is tightly regulated. In this study, we have demonstrated that exposure of the prostate carcinoma cell lines DU145, LNCaP, and PC-3 to the novel retinoid CD437 results in the enhanced stability of E2F-1 with the concomitant instability of cyclin B through CD437 modulation of the ubiquitin-mediated pathway; these modulations of E2F-1 and cyclin B levels are associated with the onset of S phase cell cycle arrest and subsequent apoptosis of these cells.

The activation of the cdc 2/cyclin B complex serves as the trigger for entry into mitosis (47) . Cyclin B while synthesized in the cytoplasm shuttles between the nucleus and the cytoplasm; phosphorylation of its nuclear translocation signal results in its rapid accumulation in the nucleus at the time of entry into mitosis (48 , 49) . Full activation of the cdc 2/cyclin B complex, which occurs both in the cytoplasm and nucleus, requires the dephosphorylation of cdc2 at Thr 14 and Tyr 15 by the cdc 25 family of protein phosphatases (47) . Cyclin B is degraded near the completion of mitosis through the activation of APC and ubiquitination of a nine amino acid sequence termed the destruction box (27) . The ordered activation of the ubiquitin protein ligase APC by CDC 20 in metaphase and by hCDH1 in telophase is complex and requires the phosphorylation and dephosphorylation of a number of APC subunits (42) . We have found that exposure of prostate carcinoma cell lines to CD437 results in the enhanced association of APC with cyclin B and degradation of cyclin B in early S phase of the cell cycle; the fact that CD437 activates cyclin B proteolysis through the ubiquitin-mediated pathway is supported by the observation that CD437-mediated degradation of cyclin B is prevented by the proteasome inhibitor LLnL. The mechanism(s) involved by which CD437 activates the APC remains to be defined. However, CD437 increases the association of hCDH1 with the APC; whether exposure to CD437 also results in the enhanced phosphorylation of specific APC subunits resulting in its activation is under investigation.

Although incubation of the prostate carcinoma cell lines with CD437 results in APC activation and subsequent cyclin B degradation, the addition of CD437 results in a rapid increase in E2F-1 levels through increased stability of the E2F-1 molecule. CD437-enhanced E2F-1 stability was documented by the demonstration that: (a) protein synthesis was not required for the CD437-mediated increase in E2F-1 levels; and (b) pulse chase labeling studies revealed a significantly longer half-life for E2F-1 in the CD437-treated cells.

Recent studies have demonstrated that E2F-1 is rapidly degraded in the S-G2 phase of the cell cycle through its interaction with the F-box-containing protein p45SKP2 (37) ; p45SKP2 is an essential component of the ubiquitin protein ligase SCFSKP2 (37) .

Expression of p45SKP2 is cell cycle regulated with enhanced expression in S phase, where it binds to and directs ubiquitination of E2F-1 (37) ; disruption of the association between p45SKP2 and E2F-1 results in the reduction of ubiquitination of E2F-1 (37) . The F box protein p45SKP2 forms complexes with p19SKP1 and CUL-1 and so directs ubiquitination of E2F-1 (37) . Recent data would suggest that p45SKP2 may not be the only mediator involved in E2F-1 ubiquitination. A SKP1, SKP2-independent mechanism has been described recently (50) . The ring finger protein ROC 1, an essential subunit of the CUL 1/CDC 53 ubiquitin ligase, has been found to play a major role in E2F-1 ubiquitination in the absence of SKP1 and SKP2 (50) . However, we found that there is a tight association between E2F-1 and p45SKP2 in the prostate carcinoma cells and that the increased levels of E2F-1 after the addition of CD437 are associated with decreased levels of p45SKP2 bound to E2F-1; this would suggest that p45SKP2 appears to be involved in the ubiquitination of E2F-1. Additional evidence is provided by the fact that the decreased levels of p45SKP2 bound to E2F-1 after exposure to CD437 are associated with decreased ubiquitination of E2F-1. If CD437 is inhibiting the E3 ligase, then decreased ubiquitination of E2F-1 would be the expected result, and the proteasome inhibitor LLnL should not prevent this, because the ligase functions at a step before the proteasome destruction of E2F-1.

The mechanism by which the association between E2F-1 and p45SKP2 is disrupted is not defined, but it has been speculated that acetylation of E2F-1 at critical lysine residues, which are NH2 terminal to the DNA-binding domain, may inhibit the ubiquitination of E2F-1 and its proteolysis (39) . We have found that exposure to CD437 results in enhanced acetylation of E2F-1, and, therefore, we hypothesize that acetylation of these residues interrupts the association between E2F-1 and p45SKP2.

The inappropriate elevation of E2F-1 levels has been associated with S phase cell cycle arrest and apoptosis in a number of cell types (17, 18, 19) . Initial results suggested that this effect of E2F-1 was p53 dependent, but later, investigations have documented that the E2F-1-mediated induction of S phase arrest and apoptosis can occur through a p53-independent pathway (7 , 51, 52, 53, 54) . We have found that CD437 induces increased E2F-1 levels and subsequent S phase arrest and apoptosis in both p53 wild-type and mutant prostate carcinoma cells, as well as in DU145 cells, which possess biallellic deletions of Rb, confirming that modulation of Rb is not involved (55) . Recent studies have also documented that only E2F-1 of the E2F family is capable of inducing apoptosis in cells, although E2F-2 and E2F-3 are capable of enhancing entry into and progression along S phase (56) . In addition, the heterodimer of E2F-1/DP-1 is a much more potent inducer of apoptosis than E2F-1 alone (57) . Binding of the heterodimer to the E2F-1 consensus sequence appears to be required for E2F-1-mediated apoptosis (18 , 19 , 58) . We have found that CD437-mediated elevation of E2F-1 levels is associated with a marked increase in E2F-1 bound to its consensus sequence. It has been demonstrated recently that acetylation of E2F-1 results in increased binding of E2F-1 to it consensus sequence and subsequent transactivation (59) ; thus, the enhanced binding of E2F-1 to its consensus sequence after exposure to CD437 may be the result of an increase in acetylated E2F-1 levels.

CD437 is a novel retinoid that, although binding to RAR{gamma}, has been shown by a number of investigators to mediate apoptosis through a pathway not involving the RARs or RXRs (60, 61, 62) ; thus, CD437 may exert its effect in the prostate carcinoma cells through a unique pathway not involving these retinoid nuclear receptors. The contention that the RARs are not involved in CD437 modulation of E2F-1 and cyclin B levels and its induction of apoptosis is supported by the observation that trans-retinoic acid, which is a much better activator of the RARs than CD437, neither inhibits the growth of nor induces apoptosis in these cell lines, even at concentrations of 10 µM (5 , 63) . Perhaps more importantly, exposure of these cells to CD437 results in their death; whether CD437 or its analogs will prove to demonstrate similar activity in vivo against prostate carcinoma is now under investigation.

In this study, we have made the unique observation that exposure to the novel retinoid CD437 results in inhibition of E2F-1 proteolysis but stimulation of cyclin B proteolysis, both through the ubiquitin-mediated pathway. The exact mechanisms by which CD437 exerts these paradoxical effects on the E3 ligases involved are now being examined.


   ACKNOWLEDGMENTS
 
We thank Bill Browning for the preparation of the figures and Donna Bennett for excellent secretarial assistance.


   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 by grants from Medical Research Services of the Department of Veteran Affairs (to A. K. R. and J. A. F.), NIH (PO1 CA51993; to M. D. and J. A. F.), and Detroit Medical Center and Allied Diseases (DMCIOAD) institutional grant (to A. R. K.). Flow cytometry studies were supported by NIH Grant P30 CA22453-20 to the Flow Cytometry Core Facility of the Karmanos Cancer Institute. Back

2 To whom requests for reprints should be addressed, at VA Medical Center, Oncology 11M-HO, 4646 John R Street, Detroit, MI 48201. Phone: (313) 576-3659; Fax: (313) 576-1122; E-mail: Joseph.Fontana{at}med.va.gov Back

3 The abbreviations used are: RAR, retinoic acid receptor; RXR, retinoid X receptor; 7-AAD, 7-aminoactinomycin D; FBS, fetal bovine serum; CD437/AHPN, 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid; BrdUrd, 5-bromo-2'-deoxyuridine; Rb, retinoblastoma; cdk, cyclin dependent kinase; BDIS, Becton Dickinson Immunocytometry Systems; APC, anaphase-promoting complex; LLnL, N-acetyl-leu-leu-norleu-al. Back

Received 9/20/01. Accepted 4/22/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Hansen L. A., Sigman C. C., Andreola F., Ross S. A., Kelloff C. J., De Luca L. M. Retinoids in chemoprevention and differentiation therapy. Carcinogenesis (Lond.), 21: 1271-1279, 2000.[Abstract/Free Full Text]
  2. Niles R. M. Recent advances in the use of Vitamin A (Retinoids) in the prevention and treatment of cancer. Nutrition, 16: 1084-1090, 2000.[Medline]
  3. Dawson M. I., Zhang X., Hobbs P. D., Jong L. An update of biological aspects and clinical applications Livrea M. A. eds. . Vitamin A and Retinoids, 161-196, Birkhaeuser, Verlag Basel, Switzerland 2000.
  4. Campbell M. J., Park S., Uskohovic M. R., Dawson M. I., Koeffler H. P. Expression of retinoic acid receptor-ß sensitizes prostate cancer cells to growth inhibition mediated by combination of retinoids and a 19. nor hexafluoride vitamin D3 analog. Endocrinology, 139: 1972-1980, 1990.[Abstract/Free Full Text]
  5. Sun S-Y., Yue P., Lotan R. Implication of multiple mechanisms in apoptosis induced by the synthetic retinoid CD437 in human prostate carcinoma cells. Oncogene, 19: 4513-4522, 2000.[Medline]
  6. Liang J. Y., Fontana J. A., Rao J. N., Ordonez J. V., Dawson M. I., Shroot B., Wilber J. F., Feng P. A synthetic retinoid CD437 induces S-phase arrest and apoptosis in the human prostate cancer cells. LNCaP and PC-3. Prostate, 38: 228-236, 1999.[Medline]
  7. Qin X-Q., Livingston D. M., Kaelin W. G., Jr., Adams P. D. Deregulated transcription factor E2F-1 expression leads to S phase entry and p53-mediated apoptosis. Proc. Natl. Acad. Sci. USA, 91: 10918-10922, 1994.[Abstract/Free Full Text]
  8. Almasan A., Yin Y., Kelley R. E., Lee E. Y-H. P., Bradley A., Li W., Bertino J. R., Wahl G. M. Deficiency of retinoblastoma proteins leads to inappropriate S phase entry, activation of E2F-responsive genes and apoptosis. Proc. Natl. Acad. Sci. USA, 92: 5436-5440, 1995.[Abstract/Free Full Text]
  9. Kowalik T. F., Degregori J., Leone G., Jakoi L., Nevins J. R. E2F-1 specific induction of apoptosis and p53 accumulation which is blocked by MDM-2. Cell Growth Differ., 9: 113-118, 1998.[Abstract]
  10. Nevins J. R. Toward an understanding of the functional complexity of the E2F-1 and retinoblastoma families. Cell Growth Differ., 9: 585-593, 1998.[Medline]
  11. Lees J. A, Buchkovich K. J., Marshak D. R., Anderson C. W., Harlow E. The retinoblastoma protein is phosphorylated on multiple sites by human Cdc2. EMBO J, 10: 4279-4290, 1991.[Medline]
  12. Ewen M. E., Sluss H. K, Sherr C. J., Matsushime H., Kato J., Livingston D. M. Functional interactions of the retinoblastoma protein with mammalian D-type cyclins. Cell, 73: 487-497, 1993.[Medline]
  13. Hatakeyama M., Brill J. A., Fink G. R., Weinberg R. A. Collaboration of G1 cyclins in the functional inactivation of the retinoblastoma protein. Genes Dev., 8: 1759-1771, 1994.[Abstract/Free Full Text]
  14. Lundberg A. S., Weinberg R. A. Functional inactivation of the retinoblastoma protein requires sequential modification by at least two distinct cyclin-cdk complexes. Mol. Cell. Biol., 18: 753-761, 1998.[Abstract/Free Full Text]
  15. Helin K. Regulation of cell proliferation by the E2F transcription factors. Curr. Opin. Genet. Dev., 8: 28-35, 1998.[Medline]
  16. Johnson D. G., Ohtani K., Nevins J. R. Autoregulatory control of E2F-1 expression in response to positive and negative regulators of cell cycle progression. Genes Dev., 8: 1514-1525, 1994.[Abstract/Free Full Text]
  17. Xu M., Sheppard K. A., Peng C. Y., Yee A. S., Piwnica. Worms H. Cyclin A/CDK2 binds directly to E2F-1 and inhibits the DNA-binding activity of E2F-1/DP-1 by phosphorylation. Mol. Cell. Biol., 14: 8420-8431, 1994.[Abstract/Free Full Text]
  18. Krek W., Xu G., Livingston D. M. Cyclin A-kinase regulation of E2F-1 DNA binding function underlines suppression of an S phase checkpoint. Cell, 83: 1149-1158, 1995.[Medline]
  19. Hsieh J-K., Fredersdorf S., Kouzarides T., Martin K., Lu X. E2F-1-induced apoptosis requires DNA binding but not transactivation and is inhibited by the retinoblastoma protein through direct interaction. Genes Dev., 11: 1840-1852, 1997.[Abstract/Free Full Text]
  20. Hofmann F., Martelli F., Livingston D. M., Wang Z. The retinoblastoma gene product protects E2F-1 from degradation by the ubiquitin proteosoma pathway. Genes Dev., 10: 2949-2959, 1996.[Abstract/Free Full Text]
  21. Hateboer G., Kerkhoven R. M., Shvarts A., Bernards R., Beijersbergen R. L. Degradation of E2F by the ubiquitin-proteasome pathway: regulation by retinoblastoma family proteins and adenovirus transforming proteins. Genes Dev., 10: 2960-2979, 1996.[Abstract/Free Full Text]
  22. Campanero M. R., Flemington E. K. Regulation of E2F through ubiquitin-proteosome dependent degradation: stabilization by the RB tumor suppressor protein. Proc. Natl. Acad. Sci. USA, 94: 2221-2226, 1997.[Abstract/Free Full Text]
  23. Hwang A., Gillies McKenna W., Muschel R. J. Cell cycle-dependent usage of transcriptional start sites: a novel mechanism for regulation of cyclin B1. J. Biol. Chem., 273: 31505-31509, 1998.[Abstract/Free Full Text]
  24. Maity A., Gillies McKenna W., Muschel R. J. Evidence for post-transcriptional regulation of cyclin B1 mRNA in the cell cycle and following irradiation in Hela cells. EMBO J., 14: 603-609, 1995.[Medline]
  25. Farina A., Gaetano C., Crescenzi M., Puccini F., Manni I., Sacchi A., Piaggo G. The inhibition of cyclin B1 gene transcription in quiescent NIH3T3 cells is mediated by an E-box. Oncogene, 13: 1287-1296, 1996.[Medline]
  26. Farina A., Manni I., Fonte Maggi G., Tiainen M., Cenciarelli C., Bellorini M., Mantovani R., Sacchi A., Piaggio G. Down-regulation of cyclin B1 gene transcription in terminally differentiated skeletal muscle cells is associated with a loss of functional CCAAT-binding NF-Y complex. Oncogene, 18: 2818-2827, 1999.[Medline]
  27. Hershko A. Mechanisms and degradation of cyclin B. Philos. Trans. R. Soc. Lond. Biol. Sci., 354: 1571-1576, 1999.[Medline]
  28. Vorlavfer E., Peters J-M. Regulation of the cyclin B degradation by an inhibitor of mitotic proteolysis. Mol. Biol. Cell, 9: 1817-1831, 1998.[Abstract/Free Full Text]
  29. Park M., Chae H-D., Yun J., Jung M., Kim Y-S., Kim S-H., Han M. H., Shin D. Y. Constitutive activation of cyclin B1-associated cdc 2 kinase overrides p53-mediated G2-M arrest. Cancer Res., 60: 542-545, 2000.[Abstract/Free Full Text]
  30. Charpenter B., Bernardon J. M., Eustache J., Millois C., Martin B., Michel S., Shroot B. Synthesis, structure-affinity relationships and biological activities of ligands binding to retinoic acid receptor subtypes. J. Med. Chem., 38: 4993-5001, 1995.[Medline]
  31. Fang G., Yu H., Kirschner M. W. Direct binding of CDC 20 protein family members activates the anaphase-promoting complex in mitosis and G1. Mol. Cell, 2: 163-171, 1998.[Medline]
  32. Fang G., Yu H., Kirschner M. W. The checkpoint protein MAD2 and the mitotic regulator CDC 20 form a ternary complex with the anaphase-promoting complex to control anaphase initiation. Genes Dev., 12: 1871-1883, 1998.[Abstract/Free Full Text]
  33. Sharpless T., Traganos F., Darzynkiewicz Z., Melamed M. R. Flow cytofluorimetry: discrimination between single cell and cell aggregates by direct size mesurements. Acta Cytol., 19: 577-581, 1975.[Medline]
  34. Ausbel F. M. Brent R. Kingston R. E. Moore D. D. Seidman J. G. Smith J. A. Struhl K. eds. . Current Protocols in Molecular Biology, 3: 12.01-12.03, John Wiley and Sons 1990.
  35. Zhang Y., Rishi A. K., Dawson M. I., Tschang R., Farhana L, Boyananpalli M., Reichert U., Shroot B., Van Buren E. C., Fontana J. A. S phase arrest and apoptosis induced in normal mammary cells by a novel retinoid. Cancer Res., 60: 2025-2032, 2000.[Abstract/Free Full Text]
  36. Shao Z. M., Dawson M. I., Li X-S., Rishi A. K., Sheikh M. S., Han Q-X., Ordonez J. V., Shroot B., Fontana J. A. p53 Independent G1/Go arrest and apoptosis induced by a novel retinoid in human breast cancer cells. Oncogene, 11: 493-504, 1995.[Medline]
  37. Marti A., Wirbelauer C., Schneffner M., Krek W. Interaction between ubiquitin-protein ligase SCFSKP2 and E2F-1 underlies the regulation of E2F-1 degradation. Nat. Cell Biol., 1: 14-19, 1999.[Medline]
  38. Vandel L., Kouzarides T. Residues phosphorylated by TFII H are required for E2F-1 degradation during S-phase. EMBO J., 18: 4280-4291, 1999.[Medline]
  39. Martinez-Balbas M. A., Bauer U-M., Nielsen S. J., Brehm A., Kouzaeides T. Regulation of E2F-1 activity by acetylation. EMBO J., 19: 662-671, 2000.[Medline]
  40. Page A. M., Heitler P. The anaphase-promoting complex: new subunits and regulators. Annu. Rev. Biochem., 68: 583-609, 1999.[Medline]
  41. Vistin R., Prinz S., Amon A. CDC 20 and CDH1: a family of substrate-specific activators of APC-dependent proteolysis. Science (Wash. DC), 278: 460-463, 1997.[Abstract/Free Full Text]
  42. Schwab M., Lutum A. S., Seufert W. Yeast Hct 1 is a regulator of CLb2 cyclin proteolysis. Cell, 90: 683-693, 1997.[Medline]
  43. Zachariae W., Schwab M., Nasmyth K., Seufert W. Control of cyclin ubiquitinization by CDK-regulated binding of Hct 1 to the anaphase promoting complex. Science (Wash. DC), 282: 1721-1724, 1998.[Abstract/Free Full Text]
  44. Kramer E. R., Scheuringer N., Podtelejnikov A. V., Mann M., Peters J. M. Mitotic regulation of the APC activator proteins CDC 20 and CDH 1. Mol. Biol. Cell, 11: 1555-1569, 2000.[Abstract/Free Full Text]
  45. Prinz S., Hwang E. S., Visintin R., Amon A. The regulation of Cdc 20 proteolysis reveals a role for APC components Cdc 23 and Cdc 27 during S phase and early mitosis. Curr. Biol., 18: 750-760, 1998.
  46. Yudkovsky Y., Shteinberg M., Listovsky T., Brandeis M., Hershko A. Phosphorylation of Cdc 20/fizzy negatively regulates the mammalian cyclosome/APC in the mitotic checkpoint. Biochem. Biophys. Res. Commun., 271: 299-304, 2000.[Medline]
  47. DeSouza C. P., Ellem K. A., Gabrielli B. G. Centrosomal and cytoplasmic Cdc2/cyclin B1 activation precedes nuclear mitotic events. Exp. Cell Res., 257: 11-21, 2000.[Medline]
  48. Hagting A., Jackman M., Simpson K., Pines J. Translocation of cyclin B1 to the nucleus at prophase requires a phosporylation dependent nuclear import signal. Curr. Biol., 9: 680-689, 1999.[Medline]
  49. Yang J., Song H., Walsh S., Bardes E. S., Kornbluth S. Combinatorial control of cyclin B1 nuclear trafficking through phosphorylation at multiple sites. J. Biol. Chem., 276: 3604-3609, 2001.[Abstract/Free Full Text]
  50. Ohta T., Xiong Y. Phosphorylation- and Skp1-independent in vitro ubiquitination of E2F-1 by multiple ROC-cullin ligases. Cancer Res., 61: 1347-1353, 2001.[Abstract/Free Full Text]
  51. Philips A. C., Bates S., Ryan K. M., Helin K., Vousden K. H. Apoptosis. Induction of DNA synthesis and apoptosis are separable functions of E2F-1. Genes Dev., 15: 1853-1863, 1997.
  52. Gomez-Manzano C., Fueyo J., Alameda F., Kyritsis A. P., Yung W. K. Gene therapy for gliomas: p53 and E2F-1 proteins and the target of apoptosis. Int. J. Mol. Med., 3: 81-85, 1999.[Medline]
  53. Hunt K. K., Deng J., Liu T. J., Wilson-Helner M., Swisher S. G., Clayman G., Hung M. C. Adenovirus-mediated overexpression of the transcription factor E2F-1 induces apoptosis in human breast and ovarian carcinoma cell lines and does not require p53. Cancer Res., 57: 4722-4726, 1997.[Abstract/Free Full Text]
  54. Holmberg C., Helin K., Sehested M., Karlstrom O. E2F-1-induced p53 independent apoptosis in transgenic mice. Oncogene, 17: 143-155, 1998.[Medline]
  55. Bookstein R., Shew J., Chen P., Scully P., Lee W. Suppression of tumorigenicity of human prostate carcinoma cells by replacing a mutated RB gene. Science (Wash. DC), 247: 712-715, 1990.[Abstract/Free Full Text]
  56. De Gregori J., Leone G., Miron A., Jakoi L., Nevins J. R. Distinct roles for E2F proteins in cell growth control and apoptosis. Proc. Natl. Acad. Sci. USA, 94: 7245-7250, 1997.[Abstract/Free Full Text]
  57. Shan B., Farmer A. A, Lee W. H. The molecular basis of E2F-1/DP-1-induced S-phase entry and apoptosis. Cell Growth Differ., 7: 689-697, 1996.[Abstract]
  58. Hofferer M., Wirbelauer C., Humor B., Krek W. Increased levels of E2F-1-dependent DNA binding activity after UV-or {gamma} irradiation. Nucleic Acids Res., 15: 491-495, 1999.[Abstract/Free Full Text]
  59. Merzio G., Wagener C., Gutierrez M. I., Cartwright P., Helin K., Giacca M. E2F family members are differentially regulated by reversible acetylation. J. Biol. Chem., 275: 10887-10892, 2000.[Abstract/Free Full Text]
  60. Hsu C. A., Rishi A. K., Li X-S., Gerald T. M., Dawson M. I., Schiffer C., Reichert U., Shroot B., Poirer G., Fontana J. A. Retinoid induced apoptosis in leukemia cells through a retinoic acid nuclear receptor independent pathway. Blood, 89: 4470-4479, 1997.[Abstract/Free Full Text]
  61. Sun S-Y., Yue P., Shroot B., Hong W. K., Lotan R. Induction of apoptosis in human non-small cell lung carcinoma cells by a novel synthetic retinoid CD437. J. Cell. Physiol., 173: 279-284, 1997.[Medline]
  62. Adachi H., Adams A., Hughes F. M., Zhang J., Cidlowski J. A., Jetten A. M. Induction of apoptosis by the novel retinoid AHPN in human T-cell lymphoma cells involves caspase-dependent and independent pathways. Cell Death Differ., 5: 973-983, 1994.
  63. Chao W-R., Hobbs P. D., Jong L., Zhang X-K., Zheng Y., Wu Q., Shroot B., Dawson M. I. Effects of receptor class- and subtype-selective retinoids and an apoptosis inducing retinoid on the adherent growth of the NIH-OVCAR-3 ovarian cancer cell line in culture. Cancer Lett., 115: 1-7, 1997.[Medline]



This article has been cited by other articles:


Home page
 Cancer Res.Home page
L. Farhana, M. I. Dawson, and J. A. Fontana
Apoptosis Induction by a Novel Retinoid-Related Molecule Requires Nuclear Factor-{kappa}B Activation
Cancer Res., June 1, 2005; 65(11): 4909 - 4917.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Y. Zhang and A. W. Hamburger
Heregulin Regulates the Ability of the ErbB3-binding Protein Ebp1 to Bind E2F Promoter Elements and Repress E2F-mediated Transcription
J. Biol. Chem., June 18, 2004; 279(25): 26126 - 26133.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. K. Rishi, L. Zhang, M. Boyanapalli, A. Wali, R. M. Mohammad, Y. Yu, J. A. Fontana, J. S. Hatfield, M. I. Dawson, A. P. N. Majumdar, et al.
Identification and Characterization of a Cell Cycle and Apoptosis Regulatory Protein-1 as a Novel Mediator of Apoptosis Signaling by Retinoid CD437
J. Biol. Chem., August 29, 2003; 278(35): 33422 - 33435.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Farhana, L.
Right arrow Articles by Fontana, J. A.