From the Medicine Branch, Division of Clinical Sciences, NCI,
National Institutes of Health, Bethesda, Maryland 20892, the
Department of Pharmacology, Duke University Medical
Center, Durham, North Carolina 27710, and the
§ Laboratory of Chemoprevention, Division of Basic Sciences,
NCI, National Institutes of Health, Bethesda, Maryland 20892
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ABSTRACT |
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Progression through the cell cycle is
controlled by the induction of cyclins and the activation of cognate
cyclin-dependent kinases. The
3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor
lovastatin induces growth arrest and cell death in certain cancer cell
types. We have pursued the mechanism of growth arrest in PC-3-M cells,
a p53-null human prostate carcinoma cell line. Lovastatin treatment
increased protein and mRNA levels of the cyclin-dependent kinase inhibitor p21WAF1/CIP1,
increased binding of p21 with Cdk2, markedly inhibited cyclin E- and
Cdk2-associated phosphorylation of histone H1 or GST-retinoblastoma protein, enhanced binding of the retinoblastoma protein to the transcription factor E2F-1 in vivo, and induced the
activation of a p21 promoter reporter construct. By using p21 promoter
deletion constructs, the lovastatin-responsive element was mapped
to a region between 93 and
64 relative to the transcription start site. Promoter mutation analysis indicated that the
lovastatin-responsive site coincided with the previously identified
transforming growth factor-
-responsive element. These data indicate
that in human prostate carcinoma cells an inhibitor of the HMG-CoA
reductase pathway can circumvent the loss of wild-type p53 function and induce critical downstream regulatory events leading to transcriptional activation of p21.
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INTRODUCTION |
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Cell cycle progression is controlled by the orderly activation of cyclin-dependent kinases (Cdks)1 (1-4). Cdk activation is regulated at multiple levels including the induction and degradation of cyclin protein, Cdk phosphorylation by CAK, the cyclin-activating kinase, and the synthesis of Cdk-inhibitory proteins (5). Cdk inhibitors can negatively regulate cell cycle progression in response to a variety of antiproliferative signals including DNA damage, differentiation, contact inhibition, and senescence (6-8). p21WAF1/CIP1, the first Cdk inhibitor to be identified, is required for p53-induced growth arrest (9). The p21 protein negatively regulates cell cycle progression by inhibiting Cdk catalytic activity and by inhibiting proliferating cell nuclear antigen, which is required for DNA synthesis (10). By inhibiting the activity of G1 cyclin-Cdk complexes, p21 blocks phosphorylation of the retinoblastoma protein (pRB). This leaves pRB in its active, hypophosphorylated form, in which it is able to induce G1 arrest through negative regulation of the heterodimeric transcription factor E2F/DP (11-13).
We have found that human prostate carcinoma cells are very sensitive to
the induction of growth arrest and cell death by the HMG-CoA reductase
inhibitor lovastatin (14).2
Lovastatin blocks the rate-limiting step in the cholesterol-synthesis pathway, the formation of mevalonic acid from HMG-CoA (15-17). The
study presented here investigates the mechanism by which lovastatin induces cell cycle arrest in the androgen-independent prostate carcinoma line PC-3-M, which is lacking both p53 alleles (18). The data
show that lovastatin treatment caused a marked shift of pRB to its
underphosphorylated form and enhanced the in vivo binding of
pRB and E2F-1. In addition, lovastatin inhibited Cdk activity, and this
inhibition was associated with elevated protein and RNA expression of
the Cdk inhibitor p21, similar to the induction of p21 by lovastatin in
human breast cells (19). We investigated the mechanism of
lovastatin-induced p21 expression using p21 promoter-reporter assays.
Lovastatin treatment induced transcriptional activation of the p21
promoter as indicated by increased activity of a p21 promoter-luciferase reporter construct, and this transcriptional response did not require the p53 response element. All effects of
lovastatin were completely reversed by addition of mevalonic acid.
These data demonstrate a link between the mevalonate pathway and
activation of the promoter of the critical cell cycle-regulatory protein p21. To begin to understand the mechanism of lovastatin-induced transcription, promoter deletion and mutation studies were performed. This analysis demonstrated that lovastatin responsiveness is mediated by a promoter-proximal region that coincides with the recently identified transforming growth factor- (TGF-
)-responsive
element.
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MATERIALS AND METHODS |
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Cells-- The human prostate carcinoma cell line PC-3-M was a gift of Dr. James Kozlowski, Department of Urology, Northwestern University Medical School (20). Cells were cultured as described previously (21).
Antibodies-- Polyclonal antibodies to cyclin E, Cdk2, Cdk4, and monoclonal anti-E2F-1 (clone 20) were obtained from Santa Cruz Biotechnology. Monoclonal anti-pRB antibody (clone G3-245) and monoclonal anti-p21 antibody were purchased from PharMingen.
Cell Cycle Analysis-- Cells were fixed in 50% ethanol and incubated with RNase A and the DNA intercalating dye propidium iodide, and cell cycle phase analysis was performed by flow cytometry using a Becton Dickinson FACStar flow cytometer and Becton Dickinson Cell Fit software.
Immunoprecipitation and Western Blot Analysis-- Immunoprecipitations were performed as described (22). Cell lysates were incubated with anti-Cdk2 polyclonal antibody, anti-E2F-1, or normal rabbit serum. The immunocomplex was collected on protein A-Sepharose (Sigma) and analyzed by SDS-polyacrylamide gel electrophoresis using enhanced chemiluminescence detection (Amersham Pharmacia Biotech). Western blot analysis was performed as described (23).
Northern Blot Analysis-- Total RNA was isolated from cells lysed in guanidinium thiocyanate solution, and Northern blot analysis was carried out as described previously (24) using 32P-labeled (ICN) cDNA probes and 30 µg of total RNA per lane. The human p21 DNA probe was a gift of Dr. David Givol.
Immune Complex Kinase Assay--
Immune complex kinase assays
were performed as described (22). Briefly, cell lysates were incubated
with primary antibody, and immune complexes were collected on protein
A-Sepharose beads and resuspended in kinase assay mixture containing
[-32P]ATP (ICN) and GST-RB or histone H1 (Life
Technologies, Inc.) as substrate. The GST-RB fusion protein containing
amino acids 379-928 of human pRB (25) was a gift of Dr. Frederic Kaye,
NCI, National Institutes of Health. The proteins were separated on 10%
SDS-polyacrylamide gels, and bands were detected by autoradiography.
p21 Promoter-Luciferase Constructs--
Drs. Bert Vogelstein and
Todd Waldman, The Johns Hopkins Oncology Center, provided the
full-length 2.4-kb human p21 promoter-chloramphenicol acetyltransferase
constructs as well as p21 promoter deletion constructs, in which 300 base pairs were progressively removed from the 3' terminus. Three
constructs were tested as follows: a 2.4-kb p21 promoter fragment
containing the p21 transcription initiation start site at its 3' end
and a p53 response element at the 5' end, a 2.1-kb fragment consisting
of the 2.4-kb fragment from which 300 base pairs, including the p53
response element, had been deleted from the 5' end, and a 0.3-kb
fragment consisting of the 300 base pairs most proximal to the
transcription start site (9). The original chloramphenicol
acetyltransferase constructs were digested with HindIII and
XhoI and subcloned into the
HindIII-XhoI sites of the luciferase reporter
vector pGL2-basic (Promega Corp.), to create p21 promoter-luciferase
constructs. The deletion constructs p21P 93-S (0 to 93), p21P Sma
1 (0 to
63) p21P Sma
2 (full-length p21 promoter from
which the region
64 to
110 was deleted) and p21P (0 to
100), and
the p21P 93-S mutants were created as described previously (26).
Luciferase Assay--
PC-3-M cells were co-transfected with the
p21 promoter-luciferase reporter constructs and with the
-galactosidase reporter vector pCMV
(Promega) using LipofectAMINE
(Life Technologies, Inc.), as recommended by the manufacturer.
Following transfection the cells were incubated for 6 h; the
medium was exchanged, and the cells were incubated for an additional
36 h in the presence or absence of 10 µM lovastatin.
The cells were then lysed, and luciferase activity in the lysates was
assayed using a Dynatech ML1000 luminometer (Dynatech Laboratories).
Luciferase activity was normalized to
-galactosidase activity which
was assayed using the
-galactosidase Enzyme Assay System
(Promega).
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RESULTS |
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Lovastatin Induces G1 Arrest and Cell Death-- 24 h after 10 µM lovastatin addition there was an increase in the percent of PC-3-M cells in G1. By 48 h following lovastatin addition greater than 90% of the PC-3-M cells were arrested in G1 (Table I). Cell viability was consistently greater than 75% in cells treated for 48 h, after which there was a marked loss of viability, culminating in cell death (data not shown).
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Loss of Phosphorylated pRB in Cells Exposed to Lovastatin-- In cells treated with lovastatin pRB appeared as a single band on Western blot analysis, migrating in the position characteristic of unphosphorylated or hypophosphorylated pRB (27) (Fig. 1A). The position of pRB after lovastatin coincided with the position of pRB detected with an antibody specific for the un- or hypophosphorylated form (Pharmingen clone G99-549, data not shown).
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Lovastatin Enhances Association of pRB and E2F-1-- Association of pRB with the transcription factor E2F-1 was almost undetectable by co-immunoprecipitation analysis of the untreated log phase cells (Fig. 1B). However, 36 h after addition of lovastatin there was a strong increase in the association of pRB and E2F-1.
Levels of Cdk2, Cdk4, and Cyclin E Protein Are Unchanged following Lovastatin Treatment-- The G1/S transition is regulated by D-type cyclins which bind to and activate Cdk4 and Cdk6 and cyclin E, which activates Cdk2. Because lovastatin arrests PC-3-M cells in G1, we tested whether lovastatin decreased protein levels of Cdk4, Cdk2, or cyclin E, and as demonstrated in Fig. 2A, there was little detectectable change in the level of these proteins, even after 48 h of exposure to lovastatin. It should be noted, however, that the actual effects of lovastatin on these proteins may be larger than is apparent in the Western blot because of the nonlinearity of the chemiluminescence detection system.
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Lovastatin Inhibits Cdk2 and Cyclin E-associated Kinase Activity-- Cdk2- and cyclin E-associated kinase activity were reduced to undetectable levels after 36 h treatment with lovastatin (Fig. 2B). These results indicated that the inhibition of Cdk2- and cyclin E-associated kinase activity might involve binding of a Cdk inhibitor protein to the Cdk2-cyclin E complex.
Lovastatin Induces p21 Protein and Association of p21 with Cdk2-- Western blot analysis showed that lovastatin induced a marked increase in the level of the Cdk inhibitor p21 (Fig. 3A). We next asked whether the p21 protein induced by lovastatin treatment was associated with Cdk2 in vivo. As shown in Fig. 3B, p21 was detected in anti-Cdk2 immunoprecipitates from lovastatin-treated cells and not in immunoprecipitates from untreated cells. Thus lovastatin induced the association of p21 with Cdk2, suggesting that lovastatin-induced p21 blocks Cdk2 kinase activity by direct binding to the cyclin-Cdk2 complex.
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Lovastatin Induces p21 mRNA-- To examine the mechanism by which lovastatin induces p21 we performed Northern blot analysis of the effect of lovastatin on the level of p21 mRNA. The 2.4-kb p21 transcript was increased in PC-3-M cells as early as 12 h after lovastatin treatment (Fig. 4). The kinetics of induction were consistent with the time course of inhibition of Cdk activity. The increase in p21 mRNA induced by lovastatin was blocked by actinomycin D (data not shown), suggesting that lovastatin may be regulating p21 mRNA levels through an effect on p21 transcription.
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Transcriptional Activation of the p21 Promoter by Lovastatin-- To test for transcriptional regulation of p21 by lovastatin p21 promoter-luciferase reporter constructs were transfected into PC-3-M cells, and luciferase activity was measured in the presence and absence of lovastatin. The full-length 2.4-kb promoter construct containing a p53 response element was activated 3-4-fold by lovastatin (Fig. 5A). Activation was not seen with a control vector consisting of the pGL2-basic luciferase reporter vector alone, and activation of the p21 promoter by lovastatin was completely blocked by incubation of the cells with 5 mM mevalonic acid, demonstrating that the ability of lovastatin to activate the p21 promoter is mediated by a component of the HMG-CoA reductase pathway (data not shown).
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DISCUSSION |
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The hormone refractory prostate cancer cell line studied here represents the most advanced and least treatable form of adult carcinoma (29, 30). Progressive malignant tranformation in epithelial tumors has been associated with a variety of genetic changes (31). Recent data suggest that mutations in cell cycle genes are the most common genetic change in tumor cells (32). Although inactivation or deletion of the RB gene itself is limited to a subset of human tumors (33), it has been suggested that continued molecular characterization may reveal that virtually all tumors harbor a mutation in one of the genes responsible for regulating G1 progression (32) and that the final common target of the genetic changes in G1 regulatory proteins is the functional inactivation of pRB (13).
Passage from G1 to S phase is regulated by D-type cyclins and their cognate kinases Cdk4 and Cdk6, which act by phosphorylating and inactivating pRB prior to the restriction point at which cells commit to DNA synthesis (34), and by cyclin E-Cdk2, which acts later in G1 (35). Genetic changes associated with G1 cyclins and their Cdks in human cancers include overexpression of cyclin D1, overexpression or mutation of Cdk4, loss of expression of the cyclin D-directed Cdk inhibitor p16, mutation or deletion of p53 and RB, and overexpression of cyclin E (36-39). The data presented here demonstrate that in the human androgen-independent prostate carcinoma cell line PC-3-M, the mevalonate pathway inhibitor lovastatin induces G1 arrest, blocks pRB phosphorylation, and increases in vivo binding of pRB to E2F-1, consistent with restoration of the growth-regulatory activity of pRB. Furthermore, lovastatin blocks cyclin E-Cdk2 kinase activity, in association with enhanced expression of the Cdk inhibitor p21.
Lovastatin has been shown previously to increase the protein level of the Cdk inhibitor p27KIP1 in HeLa cells, HL60 cells, and normal fibroblasts (40, 41). There was no effect on p27KIP1 in the PC-3-M cells (data not shown), and thus G1 arrest appears to be independent of p27KIP1 in the prostate cancer cells studied.
Inhibition of the mevalonate pathway has many ramifications including loss of the protein isoprenylation modifications farnesylation and geranylgeranylation, loss of sterol synthesis, and potential perturbation of N-linked glycosylation through inhibition of dolichol synthesis (42). To begin to map the target of lovastatin action within the mevalonate pathway, we incubated PC-3-M cells with the squalene synthase inhibitor zaragozic acid (43). We found that zaragozic acid treatment had no effect on p21 levels or cell cycle distribution (data not shown). Because lovastatin effects on the cell cycle were completely reversible by mevalonate and not mimicked by zaragozic acid, the critical point in the mevalonate pathway appears to be between HMG-CoA reductase and squalene synthase. One possible mechanism of lovastatin action is through Ras inactivation due to inhibition of Ras isoprenylation. Evidence suggesting that Ras may not be the mediator of the lovastatin response was presented by De Clue et al. (44) who showed that lovastatin arrests the growth of Ras-transformed cells even when the transforming Ras protein utilizes a myristate-dependent rather than farnesyl-dependent membrane-targeting moiety, and thus is not dependent on the mevalonate pathway. These data do not, however, eliminate Ras inactivation as the mechanism of lovastatin-induced G1 arrest, and further experimental approaches, including studies with specific farnesyltransferase inhibitors, will be required.
Although lovastatin itself may not be the optimal HMG-CoA reductase inhibitor for cancer treatment, it has been shown to induce growth arrest and cell death in tumor cells in vitro and in athymic mice in vivo (44-47). A phase I study of lovastatin was completed recently at the National Cancer Institute, in which it was demonstrated that doses as high as 25 times the standard anti-hypercholesterolemic dose were well tolerated (48). More precise identification of the target of lovastatin action would aid in developing new anticancer drugs that act through perturbation of the HMG-CoA reductase pathway.
In addition to p53, recent studies have demonstrated activation of the
p21 promoter by TGF- (49, 50), vitamin D3 (51), phorbol
ester, okadaic acid (52-54), STAT1 (55), and butyrate (28). Activation
by vitamin D3 and STAT1 occurs more than 500 base pairs
upstream of the lovastatin-responsive region (51, 55). In the U937
myeloid leukemia cell line, it has been demonstrated that
activation of p21 transcription by phorbol esters and okadaic acid is
mediated by the promoter region between
154 and +16 and that Sp1
plays a critical role in this response. In K562, SW480, and HepG2 cells
the activation of p21 transcription by phorbol ester was mapped to the
region between
95 and
121, and AP2 was shown to act as a positive
regulator of the phorbol ester response. The T
RE is located between
71 and
86 relative to the p21 transcription start site (49, 50). We
found that the lovastatin response region maps between
64 and
93.
Furthermore, mutation of the T
RE abrogated lovastatin-induced p21
promoter activation. Thus the lovastatin response site corresponds with
the T
RE. One possible mechanism for the induction of p21 by
lovastatin in PC-3-M cells would be induction of TGF-
by lovastatin
and activation of p21 transcription by TGF-
. It is likely, however,
that lovastatin effects on p21 are unrelated to TGF-
because PC-3-M
cells have an attenuated response to TGF-
, and direct addition of
TGF-
1 to PC-3-M cells did not activate the p21 promoter reporter
construct (data not shown). Recently it was reported that butyrate
activates the p21 promoter and that this response maps to the T
RE
(28). Sp1 is required for p21 promoter activation in response to both TGF-
and butyrate (28, 50). We have shown that mutation of an Sp1
binding site also blocks lovastatin activation of the p21 promoter.
Several studies have shown that Sp1 is a critical factor in regulating
transcriptional responses to pRB. Sp1 binds to the pRB-responsive
element, and Sp1 binding is required and sufficient for promoter
activation in response to pRB (56-58). Although the overall level of
pRB protein decreases in PC-3-M cells in response to lovastatin, that
decrease is predominantly or entirely in the hyperphosphorylated form
(Fig. 1A and data not shown), and there is a substantial
increase in active pRB as assessed by the markedly enhanced E2F-pRB
binding in the in vivo co-immunoprecipitation experiment
(Fig. 1B). These data suggest that both pRB and Sp1 may play
a role in lovastatin-induced activation of the p21 promoter.
The data presented here demonstrate that lovastatin induces p21 transcription in the absence of wild-type p53. The most frequently mutated tumor suppressor in human cancer is p53. Therefore, p53-independent induction of p21 presents an attractive target for anticancer drug development (59). The data presented here suggest that perturbation of the mevalonate pathway may offer a new approach to transcriptional control of this critical negative growth-regulatory gene.
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ACKNOWLEDGEMENTS |
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We thank Drs. Bert Vogelstein and Todd Waldman, The Johns Hopkins Oncology Center, for kindly providing the p21 promoter constructs; Dr. David Givol for kindly providing the human p21WAF1/CIP1 DNA probe; and Dr. Frederic Kaye, NCI, National Institutes of Health, for kindly providing the cDNA encoding the GST-RB fusion protein containing amino acids 379-928 of human pRB. We thank Alfred W. Alberts, Merck, for generously providing lovastatin. We are grateful to Drs. Konrad Huppi and Frederic Kaye for critical review of the manuscript.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Medicine Branch, NCI, Bldg. 10, Rm. 12N226, National Institutes of Health, Bethesda, MD 20892. Tel.: 301-496-1547; Fax: 301-402-0172; E-mail: trepel{at}helix.nih.gov.
1
The abbreviations used are: Cdk,
cyclin-dependent kinase; pRB, retinoblastoma protein; GST,
glutathione S-transferase; TGF-, transforming growth
factor-
; T
RE, transforming growth factor-
-responsive element;
kb, kilobase pair; HMG-CoA, 3-hydroxy-3-methylglutaryl-coenzyme A.
2 W.-K. Kang, M. J. Ha, S. J. Lee, P. Nguyen, F. Pirnia, M. M. Borner, E. Solary, Y. Pommier, C. E. Myers, and J. B. Trepel, manuscript in preparation.
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REFERENCES |
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