Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M400732200 on May 20, 2004

J. Biol. Chem., Vol. 279, Issue 32, 33079-33084, August 6, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/32/33079    most recent
M400732200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
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 arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Duncan, R. E.
Right arrow Articles by Archer, M. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Duncan, R. E.
Right arrow Articles by Archer, M. C.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Mevalonate Promotes the Growth of Tumors Derived from Human Cancer Cells in Vivo and Stimulates Proliferation in Vitro with Enhanced Cyclin-dependent Kinase-2 Activity*

Robin E. Duncan{ddagger}, Ahmed El-Sohemy{ddagger}, and Michael C. Archer{ddagger}§

From the Departments of {ddagger}Nutritional Sciences and §Medical Biophysics, University of Toronto, Toronto, Ontario M5S 3E2, Canada

Received for publication, January 1, 2004 , and in revised form, May 19, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Malignant cells are known to have elevated rates of mevalonate synthesis because of increased levels and catalytic efficiency of 3-hydroxy-3-methylglutaryl-CoA reductase. Whether this increased mevalonate synthesis occurs as a consequence of increased requirements for a mevalonate-derived metabolite in response to rapid growth or whether mevalonate promotes the growth of tumor cells is unknown. To address this question, we administered mevalonate via miniosmotic pumps to nude mice inoculated with MDA-MB-435 human cancer cells. After 13 weeks of growth, tumors in mevalonate-treated mice were significantly larger than tumors in saline-treated, control mice (1.52 ± 0.26 g versus 0.81 ± 0.27 g respectively, p < 0.05). The cancer cells treated in culture with mevalonate also demonstrated increased proliferation rates associated with accelerated entry of cells into S phase. These cells had enhanced total and cyclin A-immunoprecipitable cyclin-dependent kinase-2 (CDK-2) activity, increased activating phosphorylation of CDK-2, and decreased inhibitory binding of CDK-2 to p21Cip1. Our findings demonstrate that mevalonate promotes tumor growth and suggest that an increase in mevalonate synthesis in extrahepatic tissues following cholesterol-lowering therapy may explain the elevated risk of cancer shown in some studies.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
HMG-CoA1 reductase is the rate-limiting enzyme in cholesterol biosynthesis that catalyzes the formation of mevalonate (1). In addition to being a precursor of cholesterol, mevalonate is required for a number of cellular processes including DNA synthesis and proliferation (2). Mevalonate is also the precursor of non-sterol isoprenoids that have a variety of functions including prenylation of growth-regulating proteins and oncoproteins (3, 4). Elevated mevalonate synthesis has been reported in malignant breast (5), lung (6), leukemia and lymphoma (7), and hepatoma cells (8). Whether increased mevalonate synthesis occurs in malignant cells simply as a consequence of increased requirements for a mevalonate-derived metabolite in response to rapid growth or whether mevalonate promotes the growth of tumor cells is unknown.

HMG-CoA reductase is regulated through a multivalent feedback mechanism that is controlled, in part, by intracellular cholesterol levels (1). Cells meet their cholesterol requirements by de novo synthesis or by uptake from plasma of cholesterolrich low density lipoprotein. A fall in circulating levels of low density lipoprotein causes a decrease in its uptake, and the resultant drop in intracellular cholesterol levels leads to a compensatory stimulation of mevalonate synthesis through up-regulation of HMG-CoA reductase (1). We have shown that a diet rich in cholesterol that raised circulating cholesterol levels also significantly decreased mammary gland HMG-CoA reductase activity (5) and inhibited the development of chemically induced mammary tumors in rats (5, 9). We have also shown in mice that a diet rich in cholesterol protects against the development of chemically induced preneoplastic lesions of the colon (10). Conversely, oral administration of the bile acid-binding resin cholestyramine to rats during the promotion phase of mammary carcinogenesis has been shown to lower circulating cholesterol levels (11), increase cholesterol synthesis in the mammary gland (12), and increase the incidence of malignant mammary tumors (11). The action of circulating low density lipoprotein cholesterol as a feedback regulator of HMG-CoA reductase has been suggested to explain these observations (5, 9-12). In support of this notion, treatment of mice with cholestyramine has recently been shown to increase prenylation of the growth-regulating protein K-Ras in the lung (13). Because statins, like cholestyramine, reduce circulating low density lipoprotein, we reasoned that the increased risk of breast, gastrointestinal, and total cancer associated with the use of statins in some studies (14-17) may be related to an increase in mevalonate synthesis in extrahepatic cells.

Statins are the class of cholesterol-lowering drugs most widely used for the prevention of cardiovascular disease. They function to reduce serum cholesterol levels by competitively inhibiting HMG-CoA reductase activity in the liver (18, 19). Recent epidemiological studies have suggested there may be an increased risk of some types of cancer following the use of pravastatin (16, 17), although not all studies have found this association (20, 21). No increased risk, however, has been associated with the use of simvastatin (22) or lovastatin (23). The mechanism whereby some statins may increase cancer risk is unknown (16). Bile acid-binding resins are used less commonly in cholesterol reduction therapy than statins but are still prescribed in North America (24). As discussed above, data from rodent studies strongly suggest that increased mevalonate synthesis mediates the growth-promoting effects of cholestyramine on mammary tumorigenesis. The direct effect of mevalonate on tumor growth, however, has not been investigated.

Rats and mice that are commonly used in experimental cancer studies are generally unresponsive to the hypocholesterolemic effect of statins (25, 26). This precludes the use of these rodent models for direct investigation of the hypothesis that a statin-mediated increase in mevalonate synthesis in extrahepatic tissues could promote cancer development or tumor growth. Therefore, we investigated whether mevalonate supplied directly via miniosmotic pumps to human cancer cells growing in athymic nude mice promotes the growth of tumors derived from these cells. To provide mechanistic evidence supporting a role for elevated mevalonate in the promotion of tumor growth, we investigated the effect of mevalonate on the growth of MDA-MB-435 cells in culture. Furthermore, we investigated whether mediators of cell cycle progression play a role in the growth-promoting effects of increased concentrations of mevalonate.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—All reagents for electrophoresis and immunodetection were purchased from Bio-Rad. Mevalonic acid lactone as a racemic mixture and all other reagents and chemicals were obtained from Sigma.

Cell Line—The human cancer cell line MDA-MB-435 was generously provided by Dr. J. E. Price (Department of Cell Biology, M. D. Anderson Cancer Center, Houston, TX) (27). Cells were routinely cultured in 75-cm2 flasks at 37 °C with 5% CO2, Iscove's modified Dulbecco's medium with 1% penicillin/streptomycin (Sigma), and 5% fetal bovine serum (Invitrogen).

Tumorigenesis Experiment—Forty female athymic nude mice (CD1-Nu/Nu) were purchased from Charles River Laboratories (Wilmington, MA) at 7 weeks of age and acclimatized for 1 week prior to study commencement. Mice were housed in microisolator cages with sterile bedding and were handled in a unidirectional laminar airflow hood. They were maintained throughout the study on a standard AIN-93G diet (Dyets, Bethlehem, PA) that was sterilized by exposure to 60Co (Steris Isomedix, Whitby, Ontario, Canada) (28). The tumor cell inoculation procedure has been described in detail elsewhere (27). We used the MDA-MB-435 human cell line, which is highly tumorigenic when xenografted into nude mice (27). Briefly, cells were grown to 70-90% confluence, and the medium was changed 24 h prior to harvesting by trypsinization. The cells were resuspended in complete medium and kept on ice prior to use (28). Under ketamine/xylazine anesthesia, mice received a small incision (1-2 mm) to expose the right thoracic mammary fat pad. Fifty µl of inoculum containing 106 cells were injected, and the wound was closed with tissue glue. Only those mice that developed a palpable, growing tumor were included in final analyses (28). One week later, under ketamine/xylazine anesthesia, mice were subcutaneously implanted with 200-µl miniosmotic pumps (flow rate 0.25 µl/h, 28-day pumping life) (Alzet Corp., Palo Alto, CA) in the intrascapular area and then randomized to two groups. Pumps delivered either mevalonolactone (1.5 µmol/h) or isotonic saline (control). We have shown previously that subcutaneously implanted miniosmotic pumps are an effective means of delivering mevalonate to the mammary glands of mice (29). The wound was closed with skin clips. At 28-day intervals, mice were anesthetized, and spent pumps were surgically removed and replaced with fresh pumps. Mice were weighed weekly. Tumors were measured with Vernier calipers by a single, blinded observer during the weeks when the wound clips were removed and wound healing was complete (i.e. weeks 2, 3, and 4 after surgery). The cross-sectional tumor area was calculated as described by Rose et al. (28). Mice were cared for throughout the study in accordance with recommendations of the Canadian Council on Animal Care and the University of Toronto Animal Care Policies and Guidelines. The tumorigenesis protocol used in the present study was reviewed and received ethical approval by the University of Toronto Animal Care Committee.

Cell Proliferation Assay—After 24 h of growth, cells were washed with phosphate-buffered saline, and growth was continued for 60 h in serum-free medium containing 0, 0.5, 1, or 5 mM mevalonate. For proliferation assays, cells were seeded in 96-well plates at an average density of 103 cells/well. Cell proliferation rates were measured according to the manufacturer's instructions by quantifying the incorporation of bromodeoxyuridine into DNA using a cell proliferation enzyme-linked immunosorbent assay kit (Roche Applied Science).

Flow Cytometry—Cells were harvested by trypsinization followed by repeated washing with ice-cold phosphate-buffered saline (minus Ca2+/Mg2+). The cells were then treated with 1 ml of 70% EtOH for 30 min to permeabilize membranes. Nuclei were stained with 500 µl of solution containing 20 µg/ml propidium iodide, 0.38 mM sodium citrate, and 100 µl of RNase. DNA content was determined using a FACStar Plus flow cytometer, and the proportion of cells in each cell cycle phase was calculated from DNA histograms using the FACStation software package (BD Biosciences).

Western Blotting and Immunoprecipitation—Cells were harvested and disrupted in lysis buffer (5 mM Tris-HCl, 5 mM EDTA, 10 mM EGTA, 50 mM NaF, 150 mM NaCl, 0.1% Nonidet P-40, 50 µg/ml phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 2 µg/ml leupeptin) using a glass minihomogenizer. Supernatants from cell lysates were precleared by centrifugation for 10 min at 12,000 x g, and then 2-75 µg of total cellular protein was solubilized in Laemmli buffer, heated for 5 min at 95 °C, electrophoresed in SDS-10, 12, or 15% polyacrylamide gels, and transferred to polyvinylidene difluoride membranes. After blocking overnight, membranes were probed first with antibodies to p21Cip1, p27Kip1, cyclin-dependent kinase-2 (CDK-2), CDK-4, cyclin D1, cyclin E, or cyclin A (Upstate Biotechnology, Lake Placid, NY) and then with horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies (Santa Cruz Biotechnologies, Santa Cruz, CA), and signals were detected by enhanced chemiluminescence (Amersham Biosciences). Expression patterns of {beta}-actin and Coomassie Blue staining of total proteins were used to ensure equal loading.

For immunoprecipitation, 500 µg of protein from cell lysates was diluted in lysis buffer to 1 mg/ml. Samples were precleared by gentle rocking for 1 h at 4 °C with 30 µl of protein-agarose A beads (Sigma) followed by immunoprecipitation with anti-CDK-2 antibody (4 µg) overnight. Immunocomplexes were captured by rocking with 100 µl of protein-agarose A beads at 4 °C for 2 h. Washed immunoprecipitates were resuspended in 100 µl of 2x Laemmli buffer with 2-mercaptoethanol and boiled. Aliquots of supernatant were subjected to Western blotting using SDS-12% polyacrylamide gels as described above and probed with antibodies for p21Cip1 and p27Kip1.

Histone H1 Kinase Assay—Immunoprecipitates were prepared as described above, but in this step we used 4 µg of antibodies to CDK-2, cyclin A, or cyclin E and assayed for histone H1 kinase activity, essentially as described by Naderi and Blomhoff (30). Immunocomplexes were washed twice with lysis buffer and then three times with kinase reaction buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 2 mM EGTA, 1 mM dithiothreitol, 1 mM NaF, 0.1 mM NaVO4, 10 mM {beta}-glycerophosphate). Immunoprecipitates were then incubated for 30 min at 37 °C with 25 µl of kinase assay buffer containing 25 µM cold ATP, 5 µg of histone H1 (Upstate Biotechnology), and 10 µCi of [{gamma}-32P]ATP (PerkinElmer Life Sciences). Reactions were stopped by adding 30 µl of Laemmli buffer with 2-mercaptoethanol followed immediately by boiling for 5 min. The products were then electrophoresed on an SDS-13% polyacrylamide gel. Radioactive bands corresponding to histone H1 were visualized and quantified using a Packard Instant Imager (Canberra Packard Canada, Mississauga, Ontario, Canada).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Mevalonate Promotes the Growth of Tumors in Nude Mice—The primary purpose of this study was to determine the effect of elevated mevalonate levels on the in vivo growth of tumors derived from human cancer cells. To this end, 40 female nude mice were inoculated with MDA-MB-435 cells and then randomized and implanted with miniosmotic pumps that provided a continuous supply of either mevalonate or saline (control). Thirteen mice in each group developed growing, palpable tumors, and only these mice were included in tumor measurements (28). Accelerated tumor growth in the mevalonatetreated animals was measurable after 7 weeks (Fig. 1). The increasing divergence in tumor size resulted in significantly larger average tumor cross-sectional areas at 13 weeks in mevalonate-treated animals compared with saline-treated controls (215.0 ± 24.8 mm2 versus 133.1 ± 28.1 mm2, p < 0.05) (Fig. 1). Tumors in the mevalonate-treated group also weighed more at necropsy than tumors in the saline control group (1.52 ± 0.26 g versus 0.81 ± 0.27 g, p < 0.05).



View larger version (15K):
[in this window]
[in a new window]
  		FIG. 1.
Mevalonate promotes the growth of human tumors in athymic nude mice. Mice were inoculated with 106 MDA-MB-435 human cancer cells into the right thoracic mammary fat pad. One week after inoculation, mice were implanted with miniosmotic pumps containing either mevalonate (1 g/ml) or isotonic saline. Tumor growth was followed by a single, blinded observer who weekly measured tumor diameters and then calculated cross-sectional areas. The solid line indicates tumors in mevalonate-treated mice, and the dotted line shows the saline control group. *, p < 0.05 at week 13 (n = 13) as determined by Student's t test.

 
Mevalonate Promotes Proliferation in Vitro by Accelerating Entry of Cells into S Phase—We determined the effect of mevalonate treatment on cell proliferation by assaying the incorporation of bromodeoxyuridine into the nuclei of replicating cells. As shown in Fig. 2A, mevalonate added to the culture medium for 60 h increased the rate of MDA-MB-435 cell proliferation. This effect was significant at 1 mM mevalonate, a concentration that elicited a 57 ± 14% increase compared with control. Treatment of cells with 5 mM mevalonate did not produce a further increase in growth.



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 2.
Mevalonate promotes the growth of MDA-MB-435 human cancer cells in culture by accelerating entry into S phase. Cells were grown for 60 h in serum-free medium in the presence of increasing levels of mevalonate as indicated. A, cell proliferation rates following treatment with mevalonate were assessed by enzyme-linked immunosorbent assay measurement of bromodeoxyuridine incorporation into cells that were actively synthesizing DNA (means ± S.E., n = 8). B, analysis of the DNA content of cells treated with mevalonate by flow cytometry was used to determine the percentage of cells in G0/G1 (black bars), S (open bars), and G2/M (gray bars) phases (means ± S.E., n = 3). *, p < 0.01, and **, p < 0.001, versus control as tested by analysis of variance with Dunnett's post-hoc test.

 
Analysis of DNA histograms following fluorescence-activated cell sorter analysis showed that mevalonate treatment at all concentrations significantly decreased the proportion of cells in G0/G1 phase compared with untreated controls (Fig. 2B). This effect was largely accounted for by an approximate doubling of the proportion of cells in S phase (Fig. 2B). Mevalonate treatment caused no significant changes in the proportion of cells in G2/M (Fig. 2B).

Mevalonate Alters the Expression of G1 Regulatory Proteins—To determine whether alterations in cell cycle distribution detected by fluorescence-activated cell sorter analysis were associated with changes in expression of G1 regulatory proteins, we performed Western blot analyses of extracts of cells grown in the presence of increasing concentrations of mevalonate (Fig. 3). Cyclin E expression was similar in all groups, but expression of cyclin A increased by 20-25% in mevalonatetreated cells compared with controls. Total CDK-2 expression (active 33 kDa phosphorylated form plus inactive 34 kDa unphosphorylated form) did not change with mevalonate treatment. When the active and inactive forms of CDK-2 were resolved on a 15% gel, however, 20-25% greater levels of the activated form were present in extracts from cells treated with mevalonate. The expression of p21Cip1 decreased by ~35% in cells treated with 0.5 mM mevalonate and by >55% in cells treated with 1 or 5 mM mevalonate. Mevalonate treatment also decreased p27Kip1 expression by ~50% at all concentrations examined compared with controls.



View larger version (49K):
[in this window]
[in a new window]
  		FIG. 3.
Mevalonate alters the expression and binding of G1 cell cycle regulatory proteins in MDA-MB-435 cells. Western blot analysis was performed on extracts from cells grown in the presence of increasing levels of mevalonate with antibodies specific for cyclin E, cyclin A, CDK-2, and the CDKIs p21Cip1 and p27Kip1. The CDK-2 antibody also detected phosphorylated CDK-2 (CDK-2-P). Relative binding of p21Cip1 to CDK-2 was determined by immunoprecipitation (IP) of the kinase followed by Western blotting with an antibody specific for p21Cip1. In all cases representative blots of samples from three independent experiments are shown.

 
Mevalonate Inhibits p21Cip1 Binding to CDK-2—Rao et. al. (31) have reported that relative binding of CDK inhibitors (CDKI) may be more important than expression levels in determining their net inhibitory effect on CDK enzyme activity. To determine whether decreases in expression of CDKIs in mevalonate-treated cells are paralleled by changes in binding to CDK-2, we immunoprecipitated CDK-2 from cellular extracts and performed Western blot analyses of associated p21Cip1 and p27Kip1 proteins. Quantification by density scanning of band intensities indicated a decline of 25-35% in the relative levels of p21Cip1 complexed to immunoprecipitated CDK-2 (Fig. 3). We were unable to visualize and, therefore, to quantify a band for p27Kip1 associated with CDK-2. This was likely because of both low expression levels of p27Kip1 and low binding of this CDKI to CDK-2.

Mevalonate Promotes Histone H1 Kinase Activity—The histone H1 kinase activities of immunoprecipitates of CDK-2 and cyclins A and E were assayed to determine whether mevalonate increased CDK-2 activity and whether this was the result of differences in activation of the enzyme by one or both of its regulatory cyclin subunits (Fig. 4). Visualization of radiolabeled histone H1 bands showed clearly that mevalonate treatment increased CDK-2 kinase activity (by ~3-fold at 1 and 5 mM mevalonate). This increase was found to be caused by ~5- and ~10-fold increases in cyclin A-immunoprecipitable kinase activity in cells treated with 1 and 5 mM mevalonate, respectively. Cyclin E-immunoprecipitable kinase activity increased modestly (~1.5-fold) with 1 mM mevalonate treatment but did not change in cells treated with mevalonate at any other concentration.



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 4.
Mevalonate increases CDK-2 activity. Immunoprecipitates of CDK-2 (A), cyclin E (B), and cyclin A (C) were prepared from extracts of cells grown in increasing concentrations of mevalonate, as indicated, and assayed for their ability to phosphorylate histone H1 using 32P-labeled ATP. Shown are representative blots with histograms illustrating the means from density scanning of duplicate blots.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we have demonstrated that mevalonate administered to nude mice via miniosmotic pumps promotes the growth of tumors derived from MDA-MB-435 human cancer cells. This cell line was derived from a pleural effusion in a patient with metastatic ductal adenocarcinoma of the breast (32). Until recently, this cell line was widely accepted as having originated from the breast tumor. More recent evidence, however, suggests that the cell line may have been derived from an occult melanoma (32, 33). Despite being of uncertain origin, the MDA-MB-435 cell line is a highly malignant model of human cancer. To the best of our knowledge, the present study is the first to demonstrate that mevalonate directly promotes tumor growth in vivo. In the saline-treated control group, tumors grew in accordance with growth kinetics previously reported for this cell line in nude mice (27). In the mevalonate-treated group, accelerated tumor growth was measurable after week 7, and significantly larger tumors were present by the end of the study. This growth pattern could be explained by a small but persistent increase in the proliferative rate of tumor cells exposed to mevalonate over the course of the experiment. During the early weeks of tumor development, a small difference in proliferation would result in an indiscernable increase in tumor size compared with controls. Over a more extended time period, however, an increasing divergence in tumor size between the two groups became apparent. Our findings are in accordance with those of others who report similar patterns of growth in studies of promoters of MDA-MB-435 cell-derived tumors in nude mice (34, 35). To investigate whether increased proliferation may, indeed, mediate the growth-promoting properties of mevalonate, we next studied the effects of mevalonate on the same cancer cells in vitro.

Concentrations of mevalonate used in cell culture experiments were designed to amplify physiologically relevant effects over a much shorter time period than required for tumor growth. Mevalonate at both 1 and 5 mM increased cell proliferation rates by more than 50% over a period of 60 h. Furthermore, mevalonate increased the proportion of cells in S phase by promoting passage through the G1 restriction point, as evident in the smaller proportion of cells in G0/G1. The proportion of cells in G2/M did not differ significantly between treatment groups and control. Similar effects of mevalonate on cell cycle distribution and proliferation have been observed previously in breast cancer cells (36) and lymphocytes (37), albeit at substantially higher concentrations of mevalonate (up to 23 mM). As might be expected, these effects are the converse of cell cycle events that follow mevalonate depletion in cultured cells treated with statins (2). It has been known for some time that a critical level of mevalonate or an as yet unidentified non-sterol metabolite of mevalonate is required for initiation of DNA synthesis (2). Inadequate intracellular levels of mevalonate prevent entry of cells into S phase, resulting in a characteristic G1 phase growth arrest (2, 38).

The G1 arrest that results when mevalonate is depleted by statins in cultured cells is associated with changes in several regulators of cell cycle progression but most consistently with an inhibition of CDK-2 (31, 39, 40). CDK-2 controls initiation of DNA synthesis and replication of the chromosomes and is a major regulator of transition through the G1/S restriction point (for reviews see Refs. 41-43). Overexpression of the CDK-2 activator cyclin E in fibroblasts prevents cell cycle arrest following mevalonate depletion by lovastatin (39), suggesting that CDK-2 is an important regulator of the effects of mevalonate on G1/S phase progression. In the present study, mevalonate increased total CDK-2 activity as determined by its ability to phosphorylate histone H1. Enhanced CDK-2 activity was also evident in immunoprecipitates of cyclin A, indicating that mevalonate increased kinase binding to this activating subunit. Mevalonate treatment resulted in little change in histone H1 kinase activity associated with cyclin E, suggesting that increased activation of CDK-2 by cyclin A was primarily responsible for the increased total kinase activity evident in cellular extracts. Elevated mevalonate also decreased expression of the CDKIs p21Cip1 and p27Kip1 and reduced the inhibitory binding of p21Cip1 to CDK-2 (31). Low expression levels of p27Kip1 precluded its detection in immunoprecipitates of CDK-2. Both p21Cip1 and p27Kip1 are regulators of the G1 restriction point and of CDK-2 activity (44, 45). Finally, we have shown that although total expression levels of CDK-2 did not change with mevalonate treatment, there were increased levels of the 33-kDa active form of CDK-2 that had been phosphorylated on its threonine 160 residue. Taken together, our results indicate that mevalonate increases CDK-2 activity through effects at multiple levels of regulation that are effectively the converse of events observed in mevalonate-depleted cells (31, 39, 40, 46). Increased CDK-2 activity may explain, at least in part, the growth-promoting effects of mevalonate on the cancer cells.

Our in vitro experiments strongly suggest that mevalonate promoted the growth of tumors in vivo, at least in part, through enhanced proliferation. It is possible, however, that mevalonate may also have altered the biology of either the host mouse or the tumor to enhance growth indirectly. Because mice used in this study were athymic, immunomodulation is an unlikely mediator of the effects we observed. Mevalonate may have promoted growth through effects on tumor vasculature. Neovascularization is critical to the expansion of tumors beyond ~1 mm in diameter (47). We have shown, however, that mevalonate at concentrations as high as 1 mM has no effect on the proliferation of human umbilical vein endothelial cells or their ability to form tubules in Matrigel.2 Furthermore, medium conditioned with mevalonate-pretreated MDA-MB-435 cells also had no effect on the proliferation of human umbilical vein endothelial cells in culture and, indeed, inhibited the formation of tubules by these cells in Matrigel.2 These observations suggest that mevalonate does not promote the growth of tumors by enhancing their ability to neovascularize and may even inhibit this process.

Our finding that mevalonate promotes the growth of human tumors in nude mice suggests that the increased risk of cancer associated with cholesterol reduction therapy in some studies may be related to an increase in mevalonate synthesis in extrahepatic cells. Bile acid-binding resins that promote mammary carcinogenesis in rats are unabsorbed and have been shown to increase HMG-CoA reductase activity in both the liver (48) and mammary gland (12). Because of the absence of a specific transport protein on most extrahepatic cells (19), hydrophilic pravastatin has been shown to inhibit HMG-CoA reductase only in the liver (19) and ileum (49). Pravastatin, therefore, like cholestyramine, will be unable to mitigate the increase in mevalonate synthesis in most extrahepatic tissues that may accompany the fall in circulating cholesterol levels caused by its hepatic effects. This mechanism may explain the increased risk of breast (16) and gastrointestinal cancer (17) and the increased overall risk of cancer (17) that follows the use of this statin in particular. Although the effect of pravastatin on the growth of experimental mammary tumors or gastrointestinal tumors in rodents has not been reported, at physiological concentrations, pravastatin does not inhibit the proliferation of human breast cancer cells (50) or gastric carcinoma cells (51) in culture. Interestingly, pravastatin does inhibit the growth of colon tumors in rats (52) and mice (53), in keeping with its ability to inhibit HMG-CoA reductase in intestinal cells (49, 54). A reduced risk of colorectal cancer has also been reported in one human trial of pravastatin (16).

In contrast to pravastatin, the lipophilic statins, including lovastatin, simvastatin, atorvastatin, and fluvastatin, inhibit HMG-CoA reductase activity equally well in cultured human hepatocytes and cells of extrahepatic origin (55). Although oral administration leads to efficient metabolism of these compounds by the liver, active metabolites can be found in the circulation (19). Moreover, orally administered lovastatin and simvastatin have both been demonstrated to inhibit cholesterol synthesis in a number of extrahepatic tissues in mice (49). Thus, as demonstrated previously in mononuclear leukocytes (56) and in contrast to pravastatin, the lipophilic statins would be able to mitigate the increase in reductase that accompanies cholesterol reduction therapy through direct interaction with extrahepatic cells. This may explain why the use of simvastatin has been associated with a reduction in overall risk of cancer (22) and why the use of lovastatin has been shown to significantly lower the risk of melanoma (23). Furthermore, studies in rodents show a protective effect of lipophilic statins on the growth of several diverse tumor types (57-65), likely by decreasing mevalonate synthesis.

In summary, we report that mevalonate promotes the growth of tumors derived from human cancer cells grown in nude mice. As confirmation of these observations, the cells treated with mevalonate in culture demonstrated increased proliferation with accelerated entry into S phase. Furthermore, these cells had enhanced total CDK-2 activity, enhanced CDK-2 activity in immunoprecipitates of cyclin A, increased activating phosphorylation of CDK-2, and decreased inhibitory binding of p21Cip1 to CDK-2. Our findings demonstrate that mevalonate promotes tumor growth, and this may explain the elevated risk of cancer associated with cholesterol-lowering therapy in some studies.

Note Added in Proof—A paper published after our manuscript was submitted indicates that the MDA-MB-435 cancer cells utilized in the present study most likely originated from breast epithelium but have undergone dedifferentiation during tumor progression to a melanocyte phenotype as a result of genetic instability (Sellappan, S., Grijalva, R., Zhou, X., Yang, W., Eli, M. B., Mills, G. B., and Yu, D. (2004) Cancer Res. 64, 3479-3485).


   FOOTNOTES
 
* This work was supported by U. S. Army Medical Research Acquisition Activity Grant DAMD17-99-1-9409. 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. Back

Recipient of a Natural Sciences and Engineering Research Council of Canada Industrial Research Chair and supported by the member companies of the program in Food Safety, Nutrition and Regulatory Affairs of the University of Toronto. To whom correspondence should be addressed: Dept. of Nutritional Sciences, University of Toronto, FitzGerald Bldg., 150 College St., Toronto, Ontario M5S 3E2, Canada. Tel.: 416-978-8195; Fax: 416-971-2366; E-mail: m.archer{at}utoronto.ca.

1 The abbreviations used are: HMG, 3-hydroxy-3-methylglutaryl; CDK, cyclin-dependent kinase; CDKI, cyclin-dependent kinase inhibitor. Back

2 R. E. Duncan, A. El-Sohemy, and M. C. Archer, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank Suying Lu, Jim Chen, and Linda Wang for help with the nude mice experiments and Chris Lange for assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Goldstein, J. L., and Brown, M. S. (1990) Nature 343, 425-430[CrossRef][Medline] [Order article via Infotrieve]
  2. Quesney-Huneeus, V. R., Wiley, M. H., and Siperstein, M. D. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 5056-5060[Abstract/Free Full Text]
  3. Schmidt, R. A., Schneider, C. J., and Glomset, J. A. (1984) J. Biol. Chem. 259, 10175-10180[Abstract/Free Full Text]
  4. Casey, P. J., Solski, P. A., Der, C. J., and Buss, J. E. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 8323-8327[Abstract/Free Full Text]
  5. El-Sohemy, A., and Archer, M. C. (2000) Carcinogenesis 21, 827-831[Abstract/Free Full Text]
  6. Bennis, F., Favre, G., Le Gaillard, F., and Soula, G. (1993) Int. J. Cancer 55, 640-645[Medline] [Order article via Infotrieve]
  7. Harwood, H. J., Jr., Alvarez, I. M., Noyes, W. D., and Stacpoole, P. W. (1991) J. Lipid Res. 32, 1237-1252[Abstract]
  8. Kawata, S., Takaishi, K., Nagase, T., Ito, N., Matsuda, Y., Tamura, S., Matsuzawa, Y., and Tarui, S. (1990) Cancer Res. 50, 3270-3273[Abstract/Free Full Text]
  9. El-Sohemy, A., Bruce, W. R., and Archer, M. C. (1996) Carcinogenesis 17, 159-162[Abstract/Free Full Text]
  10. El-Sohemy, A., Kendall, C. W., Rao, A. V., Archer, M. C., and Bruce, W. R. (1996) Nutr. Cancer 25, 111-117[Medline] [Order article via Infotrieve]
  11. Melhem, M. F., Gabriel, H. F., Eskander, E. D., and Rao, K. N. (1987) Br. J. Cancer 56, 45-48[Medline] [Order article via Infotrieve]
  12. Rao, K. N., Melhem, M. F., Gabriel, H. F., Eskander, E. D., Kazanecki, M. E., and Amenta, J. S. (1988) J. Natl. Cancer Inst. 80, 1248-1253[Abstract/Free Full Text]
  13. Calvert, R. J., Tepper, S., Diwan, B. A., Anderson, L. M., and Kritchevsky, D. (2003) Biochem. Pharmacol. 66, 393-403[CrossRef][Medline] [Order article via Infotrieve]
  14. Beck, P., Wysowski, D. K., Downey, W., and Butler-Jones, D. (2003) J. Clin. Epidemiol. 56, 280-285[CrossRef][Medline] [Order article via Infotrieve]
  15. Coogan, P. F., Rosenberg, L., Palmer, J. R., Strom, B. L., Zauber, A. G., and Shapiro, S. (2002) Epidemiology 13, 262-267[CrossRef][Medline] [Order article via Infotrieve]
  16. Sacks, F. M., Pfeffer, M. A., Moye, L. A., Rouleau, J. L., Rutherford, J. D., Cole, T. G., Brown, L., Warnica, J. W., Arnold, J. M., Wun, C. C., Davis, B. R., and Braunwald, E. (1996) N. Engl. J. Med. 335, 1001-1009[Abstract/Free Full Text]
  17. Shepherd, J., Blauw, G. J., Murphy, M. B., Bollen, E. L. E. M., Buckley, B.M., Cobbe, S. M., Ford, I., Gaw, A., Hyland, M., Jukema, J. W., Kamper, A. M., Macfarlane, P. W., Meinders, A. E., Norrie, J., Packard, C. H., Perry, I. J., Stott, D. J., Sweeney, B. J., Twomey, C., and Westendorp, R. G. J. (on behalf of the PROSPER study group) (2002) Lancet 360, 1623-1630[CrossRef][Medline] [Order article via Infotrieve]
  18. Brown, M. S., and Goldstein, J. L. (1980) J. Lipid Res. 21, 505-517[Abstract]
  19. Hamelin, B. A., and Turgeon, J. (1998) Trends Pharmacol. Sci. 19, 26-37[CrossRef][Medline] [Order article via Infotrieve]
  20. Tonkin, A., and Simes, R. J. (1998) N. Engl. J. Med. 339, 1349-1357[Abstract/Free Full Text]
  21. Furberg, C. D., Wright, J. T., Davis, B. R., Cutler, J. A., Alderman, M., Black, H., Cushman, W., Grimm, R., Haywood, J., Leenen, F., Oparil, S., Probstfield, J., Whelton, P., Nwachuku, and C., Gordon, D. (2002) J. Am. Med. Assoc. 288, 2998-3007[Abstract/Free Full Text]
  22. Pedersen, T. R., Wilhelmsen, L., Faergeman, O., Strandberg, T. E., Thorgeirsson, G., Troedsson, L., Kristianson, J., Berg, K., Cook, T. J., Haghfelt, T., Kjekshus, J., Miettinen, T., Olsson, A. G., Pyorala, K., and Wedel, H. (2000) Am. J. Cardiol. 86, 257-262[CrossRef][Medline] [Order article via Infotrieve]
  23. Downs, J. R., Clearfield, D. O., Weis, S., Whitney, E., Shapiro, D. R., Beere, P. A., Langendorfer, A., Stein, E. A., Kruyer, W., and Gotto, A. M. (1998) J. Am. Med. Assoc. 279, 1615-1622[Abstract/Free Full Text]
  24. Avorn, J., Monette, J., Lacour, A., Bohn, R. L., Monane, M., Mogun, H., and LeLorier, J. (1998) J. Am. Med. Assoc. 279, 1458-1462[Abstract/Free Full Text]
  25. Endo, A., Tsujita, Y., Kuroda, M., and Tanzawa, K. (1979) Biochim. Biophys. Acta 575, 266-276[Medline] [Order article via Infotrieve]
  26. Krause, B. R. (1998) Atherosclerosis 140, 15-24[CrossRef][Medline] [Order article via Infotrieve]
  27. Price, J. E., Polyzos, A., Zhang, R. D., and Daniels, L. M. (1990) Cancer Res. 50, 717-721[Abstract/Free Full Text]
  28. Rose, D. P., Connolly, J. M., and Meschter, C. L. (1991) J. Natl. Cancer Inst. 83, 1491-1495[Abstract/Free Full Text]
  29. Duncan, R. E., El-Sohemy, A., and Archer, M. (2004) J. Pharmacol. Toxicol. Methods, in press
  30. Naderi, S., and Blomhoff, H. K. (1999) Blood 94, 1348-1358[Abstract/Free Full Text]
  31. Rao, S., Lowe, M., Herliczek, T. W., and Keyomarsi, K. (1998) Oncogene 17, 2393-2402[CrossRef][Medline] [Order article via Infotrieve]
  32. Ross, D. T., Scherf, U., Eisen, M. B., Perou, C. M., Rees, C., Spellman, P., Iyer, V., Jeffrey, S. S., Van de Rijn, M., Waltham, M., Pergamenschikov, A., Lee, J. C., Lashkari, D., Shalon, D., Myers, T. G., Weinstein, J. N., Botstein, D., and Brown, P. O. (2000) Nat. Genet. 24, 227-235[CrossRef][Medline] [Order article via Infotrieve]
  33. Ellison, G., Klinowska, T., Westwood, R. F., Docter, E., French, T., and Fox, J. C. (2002) Mol. Pathol. 55, 294-299[Abstract/Free Full Text]
  34. Connolly, J. M., Liu, X. H., and Rose, D. P. (1996) Nutr. Cancer 25, 231-240[Medline] [Order article via Infotrieve]
  35. Connolly, J. M., Liu, X. H., and Rose, D. P. (1997) Nutr. Cancer 29, 48-54[Medline] [Order article via Infotrieve]
  36. Wejde, J., Blegen, H., and Larsson, O. (1992) Anticancer Res. 12, 317-324[Medline] [Order article via Infotrieve]
  37. Yachnin, S. (1982) Oncodev. Biol. Med. 3, 111-123[Medline] [Order article via Infotrieve]
  38. Keyomarsi, K., Sandoval, L., Band, V., and Pardee, A. B. (1991) Cancer Res. 51, 3602-3609[Abstract/Free Full Text]
  39. Ghosh, P. M., Moyer, M. L., Mott, G. E., and Kreisberg, J. I. (1999) J. Cell. Biochem. 74, 532-543[CrossRef][Medline] [Order article via Infotrieve]
  40. Ukomadu, C., Dutta, A. (2003) J. Biol. Chem. 278, 4840-4846[Abstract/Free Full Text]
  41. Coverley, D., Laman, H., and Laskey, R. A. (2002) Nat. Cell Biol. 4, 523-528[CrossRef][Medline] [Order article via Infotrieve]
  42. Morgan, D. O. (1995) Nature 374, 131-134[CrossRef][Medline] [Order article via Infotrieve]
  43. Sherr, C. J. (1996) Science 274, 1672-1677[Abstract/Free Full Text]
  44. Coats, S., Flanagan, W. M., Nourse, J., and Roberts, J. M. (1996) Science 272, 877-880[Abstract]
  45. Harper, J. W., Adami, G. R., Wei, N., Keyomarsi, K., and Elledge, S. J. (1993) Cell 75, 805-816[CrossRef][Medline] [Order article via Infotrieve]
  46. Tanaka, T., Tatsuno, I., Noguchi, Y., Uchida, D., Oeda, T., Narumiya, S., Yasuda, T., Higashi, H., Kitagawa, M., Nakayama, K., Saito, Y., and Hirai, A. (1998) J. Biol. Chem. 273, 26772-26778[Abstract/Free Full Text]
  47. Folkman, J. (1990) J. Natl. Cancer Inst. 82, 4-6[Free Full Text]
  48. Shand, J. H., and West, D. W. (1995) Lipids 30, 917-926[CrossRef][Medline] [Order article via Infotrieve]
  49. Koga, T., Shimada, Y., Kuroda, M., Tsujita, Y., Hasegawa, K., and Yamazaki, M. (1990) Biochim. Biophys. Acta 1045, 115-120[Medline] [Order article via Infotrieve]
  50. Seeger, H., Wallwiener, D., and Mueck, A. O. (2003) Exp. Clin. Endocrinol. Diabetes 111, 47-48[CrossRef][Medline] [Order article via Infotrieve]
  51. Gebhardt, A., and Niendorf, A. (1995) J. Cancer Res. Clin. Oncol. 121, 343-349[CrossRef][Medline] [Order article via Infotrieve]
  52. Narisawa, T., Morotomi, M., Fukaura, Y., Hasebe, M., Ito, M., and Aizawa, R. (1996) Jpn. J. Cancer Res. 87, 798-804[CrossRef]
  53. Narisawa, T., Fukaura, Y., Terada, K., Umezawa, A., Tanida, N., Yakazawa, K., and Ishikawa, C. (1994) Carcinogenesis 15, 2045-2048[Abstract/Free Full Text]
  54. Reimann, F. M., Winkelmann, F., Fellermann, K., and Stange, E. F. (1996) Atherosclerosis 125, 63-70[CrossRef][Medline] [Order article via Infotrieve]
  55. van Vliet, A. K., van Thiel, G. C., Huisman, R. H., Moshage, H., Yap, S. H., and Cohen, L. H. (1995) Biochim. Biophys. Acta 1254, 105-111[Medline] [Order article via Infotrieve]
  56. Stone, B. G., Evans, C. D., Prigge, W. F., Duane, W. C., and Gebhard, R. L. (1989) J. Lipid Res. 30, 1943-1952[Abstract]
  57. Matar, P., Rozados, V. R., Roggero, E. A., and Scharovsky, O. G. (1998) Cancer Biother. Radiopharm. 13, 387-393[Medline] [Order article via Infotrieve]
  58. Clutterbuck, R. D., Millar, B. C., Powles, R. L., Newman, A., Catovsky, D., Jarman, M., and Millar, J. L. (1998) Br. J. Haematol. 102, 522-527[CrossRef][Medline] [Order article via Infotrieve]
  59. Kusama, T., Mukai, M., Iwasaki, T., Tatsuta, M., Matsumoto, Y., Akedo, H., Inoue, M., and Nakamura, H. (2002) Gastroenterology 122, 308-317[CrossRef][Medline] [Order article via Infotrieve]
  60. Feleszko, W., Zagozdzon, R., Golab, J., and Jakobisiak, M. (1998) Eur. J. Cancer 34, 406-411[CrossRef][Medline] [Order article via Infotrieve]
  61. Hawk, M. A., Cesen, K. T., Siglin, J. C., Stoner, G. D., and Ruch, R. J. (1996) Cancer Lett. 109, 217-222[CrossRef][Medline] [Order article via Infotrieve]
  62. Maltese, W. A., Defendini, R., Green, R. A., Sheridan, K. M., and Donley, D. K. (1985) J. Clin. Investig. 76, 1748-1754[Medline] [Order article via Infotrieve]
  63. Sebti, S. M., Tkalcevic, G. T., and Jani, J. P. (1991) Cancer Commun. 3, 141-147[Medline] [Order article via Infotrieve]
  64. Alonso, D. F., Farina, H. G., Skilton, G., Gabri, M. R., De Lorenzo, M. S., and Gomez, D. E. (1998) Breast Cancer Res. Treat. 50, 83-93[CrossRef][Medline] [Order article via Infotrieve]
  65. Kikuchi, T., Nagata, Y., and Abe, T. (1997) J. Neurooncol. 34, 233-239[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
O. Fromigue, Z. Hamidouche, and P. J. Marie
Blockade of the RhoA-JNK-c-Jun-MMP2 Cascade by Atorvastatin Reduces Osteosarcoma Cell Invasion
J. Biol. Chem., November 7, 2008; 283(45): 30549 - 30556.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
J. Montero, A. Morales, L. Llacuna, J. M. Lluis, O. Terrones, G. Basanez, B. Antonsson, J. Prieto, C. Garcia-Ruiz, A. Colell, et al.
Mitochondrial Cholesterol Contributes to Chemotherapy Resistance in Hepatocellular Carcinoma
Cancer Res., July 1, 2008; 68(13): 5246 - 5256.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. Villagra, N. Ulloa, X. Zhang, Z. Yuan, E. Sotomayor, and E. Seto
Histone Deacetylase 3 Down-regulates Cholesterol Synthesis through Repression of Lanosterol Synthase Gene Expression
J. Biol. Chem., December 7, 2007; 282(49): 35457 - 35470.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
R. M. Adam, N. K. Mukhopadhyay, J. Kim, D. Di Vizio, B. Cinar, K. Boucher, K. R. Solomon, and M. R. Freeman
Cholesterol Sensitivity of Endogenous and Myristoylated Akt
Cancer Res., July 1, 2007; 67(13): 6238 - 6246.
[Abstract] [Full Text] [PDF]

Home page
 Toxicol PatholHome page
W. O. Ward, D. A. Delker, S. D. Hester, S.-F. Thai, D. C. Wolf, J. W. Allen, and S. Nesnow
Transcriptional Profiles in Liver from Mice Treated with Hepatotumorigenic and Nonhepatotumorigenic Triazole Conazole Fungicides: Propiconazole, Triadimefon, and Myclobutanil
Toxicol Pathol, December 1, 2006; 34(7): 863 - 878.
[Abstract] [Full Text] [PDF]

Home page
 JAMAHome page
R. E. Duncan, A. El-Sohemy, and M. C. Archer
Statins and the Risk of Cancer
JAMA, June 21, 2006; 295(23): 2720 - 2720.
[Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. E. Morris, L. M. Schang, and D. N. Brindley
Lipid Phosphate Phosphatase-2 Activity Regulates S-phase Entry of the Cell Cycle in Rat2 Fibroblasts
J. Biol. Chem., April 7, 2006; 281(14): 9297 - 9306.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
Y. C. Li, M. J. Park, S.-K. Ye, C.-W. Kim, and Y.-N. Kim
Elevated Levels of Cholesterol-Rich Lipid Rafts in Cancer Cells Are Correlated with Apoptosis Sensitivity Induced by Cholesterol-Depleting Agents
Am. J. Pathol., April 1, 2006; 168(4): 1107 - 1118.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Epidemiol. Biomarkers Prev.Home page
R. E. Duncan, A. El-Sohemy, and M. C. Archer
Statins and Cancer Development
Cancer Epidemiol. Biomarkers Prev., August 1, 2005; 14(8): 1897 - 1898.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/32/33079    most recent
M400732200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
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 arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Duncan, R. E.
Right arrow Articles by Archer, M. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Duncan, R. E.
Right arrow Articles by Archer, M. C.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement