Lovastatin-induced E2F-1 modulation and its effect on prostate cancer cell death
Chaehwa Park,
Inkyoung Lee and
Won Ki Kang,1
Department of Medicine and Cancer Center, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-Dong, Kangnam-Ku, Seoul, Korea
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Abstract
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Lovastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, induces growth arrest in a variety of cancer cell lines. Its mechanism of action, however, has not been completely elucidated. E2F-1 is thought to act as an oncogene and a tumour suppressor, with its action probably dependent upon the cellular context. We have shown in this study that transcriptional regulation and proteasomal degradation of E2F-1 are critical regulatory events in lovastatin-induced cell death. Accompanying this is a reduction in the E2F-1-regulated expression of cell cycle genes such as c-myc, cyclin D1, cyclin A and cyclin B1. Cell cycle analysis demonstrated that the accumulation of apoptotic cells was preceded by a progressive decrease in the S-phase cell population in response to lovastatin. Although expression of E2F-1 was reduced in three prostate cancer cell linesPC-3, LNCaP and DU-145the p21 and p27 protein levels were not increased in all the cell lines treated, suggesting that increase in p21 and p27 protein expression per se is not responsible for lovastatin-mediated down-regulation of E2F-1. The subsequent apoptotic death of these cells in the presence of lovastatin can be prevented by forced ectopic expression of E2F-1. Taken together, these facts imply that E2F-1 is the target of an HMG-CoA inhibitor and critical cell death mediator in prostate cancer cells.
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Introduction
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Human cancer cells are sensitive to the induction of growth arrest and cell death by a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, lovastatin (1). Lovastatin blocks the rate-limiting step in the cholesterol biosynthetic pathway that leads to the formation of mevalonic acid from HMG-CoA (24). Normally, E2F-1 controls cell growth both positively and negatively in a tissue-specific fashion, although the molecular mechanisms governing these events are poorly understood (5,6). E2F-1 uses both p53-dependent and p53-independent pathways to kill cells. E2F-1 induces the transcription of the p53 homologue, p73 (7), and activation of p73 provides a means by which E2F-1 induces death in the absence of p53 (8). Several groups have described the inhibition of p73 function by tumour-derived p53 mutants (9,10). Therefore, deregulated E2F-1 activity and the activation of p73 might act as an anti-tumourigenic safeguard mechanism. E2F-1 controls the transcription of a group of genes that are regulated at the G1S-phase transition and that encode proteins important for S-phase events (11,12). Therefore, E2F-1 modulation is critical in maintaining control of normal cell proliferation. Because it has previously been established that E2F-1 expression and activity are involved in proliferation (13,14), an analysis of E2F-1 modulation would appear to be central to elucidating the mechanisms that initiate growth arrest in a cell. According to a previously published papers, E2F-1 mRNA is down-regulated in response to growth inhibitors, with cell type-specific factors involved in the destabilization of E2F-1 mRNA. Keratinocyte growth arrest is characterized by a reduction in the activity and expression of E2F-1 (16). Therefore, there may be mechanisms for limiting the levels of free E2F-1, and the failure of these mechanisms could compromise cell survival (1719). E2F-1 gene expression is also controlled by a regulatory network involving oncogenes, such as cyclin D1, and potential tumour suppressor genes (20). These observations strongly suggest that E2F-1 may be a target of growth-inhibitory factors such as lovastatin. In this study, we undertook to clarify the involvement of E2F-1 in lovastatin-induced cell death.
To observe the changes in cell cycle distribution induced by lovastatin treatment, we grew cells in the absence or presence of 10 µM lovastatin for 24 h. The proportion of cells in S-phase was approximately 24% among untreated PC-3 cells (Figure 1A
). When the cells were cultured with lovastatin, the S-phase fraction decreased gradually to 3% after 24 h of culture. We used this culture system to investigate the effects of lovastatin on E2F-1, unless otherwise indicated. The lovastatin-induced decrease in the number of S-phase cells, with a concomitant increase in the G1 and G2/M parts of the cell cycle, led us to investigate the expression levels of cell cycle-associated genes. Cyclins D1 and A were markedly reduced at both the mRNA and protein levels, while cyclin B mRNA was significantly reduced after 24 h of lovastatin treatment (Figure 1B
). Furthermore, expression of c-myc, an E2F-1-modulated gene, was also dramatically reduced. To further examine the mechanism of S-phase reduction, we performed western blot analysis to ascertain the effects of lovastatin on levels of E2F-1. Both E2F-1 transcripts (Figure 2A
) and the E2F-1 protein (Figure 2B
) were significantly reduced at 24 h after the addition of lovastatin. The reduction of E2F-1 expression by lovastatin was blocked by incubating the cells with 3 mM mevalonate, indicating that the down-regulation of E2F-1 is mediated by a component of the mevalonate pathway. Down-regulation of E2F-1 was also observed in two other widely used prostate carcinoma cell lines, LNCaP and DU-145 (Figure 2C
). We also investigated the effects of lovastatin on p21 and p27 expression in other prostate cancer cells. Although E2F-1 was reduced in all cell lines, neither p21 nor p27 protein levels changed in DU-145 cells (Figure 2C
).

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Fig. 1. Effects of lovastatin on the cell cycle. The human prostate carcinoma cell line PC-3 was obtained from the American Type Culture Collection (Rockville, MD). Lovastatin was generously provided by Merck (Darmstadt, Gemany). Mevalonate was purchased from Sigma (St Louis, MO). Exponentially growing PC-3 cells were treated with 10 µM lovastatin for 24 h. Cells were fixed with 70% ethanol and incubated with RNase A and the DNA intercalating dye propidium iodide. (A) Flow cytometric cell cycle analysis was performed comparing untreated control cells with lovastatin-treated cells. The results of a representative experiment from three independent experiments are presented, and the means and standard deviations of the results are given in the table. (B) To examine the effects of lovastatin on the cell cycle-associated genes c-myc, and cyclins A, D1 and B1, total RNA was prepared for northern blot analysis and total cell lysates were prepared for western blot analysis.
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Fig. 2. Effects of lovastatin on E2F-1 in relation to p21WAF1/CIP1. (A) After incubation with 10 µM lovastatin for the times indicated, total RNA was prepared from PC-3 cells and E2F-1 mRNA was detected by northern blot analysis. (B) Cell lysates were subjected to western blot analysis using an anti-E2F-1 antibody followed by enhanced chemiluminescence detection. (C) The effects of lovastatin on E2F-1 expression were examined in two other prostate cancer cell lines, LNCaP and DU-145. (D) p21WAF1/CIP1-deficient Rat-1 cells were incubated with increasing concentrations of lovastatin and the corresponding E2F-1 levels were assayed by western analysis. (E) Cyclin D1, an E2F-1-regulated protein, was monitored in the presence and absence of 10 µM lovastatin in PC-3 and Rat-1 cells.
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Previously, others have demonstrated that lovastatin induces cell death by the induction of p21WAF1/CIP1, which accompanies G1 arrest (21). Because p21WAF1/CIP1 suppresses the promoter activity of E2F-1 (22), and lovastatin induces activation of p21WAF1/CIP1 (21), p21WAF1/CIP1 was investigated as a mediator of lovastatin-induced E2F-1 regulation. To explore this idea, we tested the ability of lovastatin to deregulate E2F-1 in Rat-1 cells, which do not express p21WAF1/CIP1 (23). Rat-1 cells were sensitive to the E2F-1-inhibitory properties of lovastatin (Figure 2D
), and cyclin D1 was down-regulated, as demonstrated in PC-3 cells. Our results, however, clearly indicate that lovastatin modulates E2F-1 by a p21WAF1/CIP1-independent mechanism in Rat-1 cells (Figure 2E
).
To further investigate the down-regulation mechanism of E2F-1, we transiently transfected PC-3 cells with a CMV-promoter-driven E2F-1 expression vector, and examined the post-translational regulation of over-expressed E2F-1 following lovastatin treatment. Recombinant E2F-1 protein was partially degraded by lovastatin (Figure 3A
), whereas ectopically expressed E2F-1 mRNA remained intact (Figure 3B
), indicating that regulation is only partially post-transcriptional. E2F-1 is presumed to have been degraded by ubiquitin-dependent proteolysis (21). Therefore, the effects of specific inhibitors of proteasome protease (24,25), MG132 and lactacystin on E2F-1 expression were examined in relation to lovastatin. Western blot analysis of cell lysates revealed that lactacystin prevented the degradation of E2F-1 by lovastatin (Figure 3C
). MG-132 also showed similar results (data not shown). In contrast, calpain inhibitor II had little effect on the level of E2F-1 expression and did not influence the down-regulation of E2F-1 by lovastatin (Figure 3D
). These indications of lovastatin-mediated proteasomal modulation support the idea that lovastatin can destabilize E2F-1 through a p21WAF1/CIP1-independent mechanism. Furthermore, E2F-1 modulation was not completely restored by lactacystin, which means that transcriptional modulation is another critical regulatory mechanism.

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Fig. 3. Post-transcriptional modulation of E2F-1. PC-3 cells were transfected with a pCMV-E2F-1 expression vector using fugene (Roche, Indianapolis, IN), as recommended by the manufacturer. Plasmid pCMV-E2F-1 contains full-length E2F-1 cDNA cloned into the EcoRI site of the expression vector pcDNA3.1 (Invitrogen, Carlsbad, CA). Twenty-four hours after transfection, cells were washed with PBS and incubated in culture medium alone or in medium containing lovastatin. After incubation with 10 µM lovastatin for 24 h, cell lysates were prepared and E2F-1 levels were assayed by western blot analysis with a monoclonal antibody for E2F-1 (Santa Cruz Biotechnology, Santa Cruz, CA). Ectopically-expressed E2F-1 protein exceeded normal levels (A) and was significantly modulated by lovastatin. However, CMV-promoter-driven E2F-1 mRNA was not modulated by lovastatin, as shown by northern blot analysis (B). To examine the effects of proteasome proteases, (C) lactacystin and (D) calpain inhibitor II were added at concentrations of 20 µM with or without lovastatin. Only the proteasome inhibitor lactacystin inhibited lovastatin-induced E2F-1 reduction.
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In an effort to understand the effects of E2F-1 on lovastatin-mediated cell death, we transfected an E2F-1 expression vector (pCMV-E2F-1) into proliferating PC-3 cells. Immunolocalization of E2F-1 subunits showed that the protein accumulates in the nucleus when expressed ectopically, whereas it is reduced in both the nucleus and cytoplasm after 24 h of treatment with 10 µM lovastatin. (Figure 4A
). We also investigated whether forced expression of E2F-1 could prevent lovastatin-mediated growth inhibition in PC-3 cells. Indeed, ectopically over-expressed E2F-1 conferred a survival advantage on lovastatin-treated cells (Figure 4B
). Furthermore, FACS analysis of E2F-1-transfected cells demonstrated that lovastatin-induced cell cycle arrest was relieved by E2F-1 expression (Figure 4B
).

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Fig. 4. Ectopic expression of E2F-1 mitigated lovastatin-induced cell death. PC-3 cells were transfected with the pCMV-E2F-1 expression vector or the control pcDNA3.1 vector using fugene (Roche), as recommended by the manufacturer. Twenty-four hours after transfection, cells were washed with PBS and incubated in culture medium alone or medium containing 10 µM lovastatin. After the times indicated, the cultures were processed by indirect immunofluorescence microscopy to determine the intracellular distribution of E2F-1. E2F-1 is indicated by a signal from the secondary FITC-conjugated rabbit anti-mouse antibody (Santa Cruz Biotechnology); the DAPI signal shows the location of the cell nucleus. The data clearly show that lovastatin induces both nuclear and cytoplasmic clearing of E2F-1 (A). To investigate the role of E2F-1 in lovastatin-treated cell survival, an E2F-1-transfected cell fraction among total live cells was monitored following lovastatin exposure for the indicated times. Numbers of E2F-1-expressing cells were estimated by immunocytochemical staining using FITC-conjugated secondary antibody and the total surviving cell number was assessed by trypan-blue staining (B). Data represent the means of three independent experiments and are expressed as fold survival (± SD) of E2F-1 transfected cells with respect to the value obtained in control cells that were not transfected with E2F-1. Fold survival = (percentage survival of E2F-1-transfected cells)/(percentage survival of control cells). FACS analysis of E2F-1-transfected cells demonstrated that the lovastatin-induced cell cycle arrest was counteracted by the expression of transfected E2F-1 (B). Our results clearly demonstrate that E2F-1 expression confers a survival advantage on cells undergoing lovastatin-induced cell cycle arrest.
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We have shown in this study that transcriptional regulation and proteasomal degradation of E2F-1 may be critical regulatory events, and that p21WAF1/CIP1 induction and G1 arrest are not general mechanisms in lovastatin-mediated cell death. The data presented here demonstrate that, in the human prostate carcinoma cell line PC-3, the HMG-CoA inhibitor lovastatin inhibits cell growth by decreasing the proportion of cells in S-phase, with a concomitant induction of G1 and/or G2/M arrest. To answer the question why the S-phase cells were lost, we investigated the level of E2F-1, an essential factor required for cell cycle progression to S-phase. Lovastatin markedly inhibited E2F-1 expression at both the mRNA and protein levels, which may contribute to SG2 cell cycle delay and increased cell susceptibility to apoptosis (27). Lovastatin is an effective inducer of cell death, which can be prevented by the simultaneous addition of mevalonate to the culture medium (2729). Previous studies have demonstrated that lovastatin induces the p53-independent transcriptional regulation of p21WAF1/CIP1 (21), and that E2F-1 activity is inhibited by p21WAF1/CIP1 (30). For example, the discoveries that transforming growth factor ß induces p21WAF1/CIP1 (31) and that E2F-1 expression can overcome transforming growth factor ß-induced growth arrest (32) suggest that transforming growth factor ß may inhibit growth by suppressing E2F activity through p21WAF1/CIP1. Recent data on the effects of
-irradiation (33) similarly suggest that radiation may inhibit growth by suppressing E2F activity through p21WAF1/CIP1. However, we found that lovastatin caused a reduction in E2F-1 in p21WAF1/CIP1-deficient Rat-1 cells (23). Therefore, E2F-1 appears to be a target and ultimate effector of lovastatin-mediated growth arrest, in both p21WAF1/CIP1-dependent and -independent ways, for the inhibition of cell proliferation. Even though lovastatin treatment causes G1 arrest in a wide variety of tumour cells, irrespective of their p53 or pRb status, the levels of p21 and p27 did not increase in all cell lines treated, suggesting that an increase in p21 and p27 protein expression per se is not responsible for lovastatin-mediated G1 arrest (34).
Inhibition of HMG-CoA has many ramifications, including the loss of the protein-isoprenylation modifications such as farnesylation and geranylgeranylation, the loss of sterol synthesis and the inhibition of dolichol synthesis (35,36). Lovastatin has been shown to induce growth arrest and cell death in tumour cells in vitro and in vivo (3740). Recent studies have demonstrated that farnesyl transferase inhibitor can effectively inhibit tumour cell growth with minimal side effects. Identification of the cell death mechanism of lovastatin would assist in further developing new anticancer drugs that act through perturbation of the HMG-CoA reductase pathway.
The ability of lovastatin to suppress cancer cell proliferation and survival has been extensively described. However, the molecular mechanisms involved in this growth perturbation are still unclear. This study provides evidence that the mechanism by which lovastatin induces prostate cancer cell death involves down-regulation of E2F-1. Furthermore, it indicates that over-expression of E2F-1 suppresses lovastatin-induced cell death and promotes cell survival.
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Notes
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1 To whom correspondence should be addressedEmail: wkkang{at}smc.samsung.co.kr 
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Acknowledgments
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This work was supported in part by the Samsung Grant No. SBRI C-A0-046-1 and Grant No. 2000-0-208-001-3 from the Basic Research Program of the Korea Sicience & Engineering Foundation.
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Received January 12, 2001;
revised July 3, 2001;
accepted July 5, 2001.