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CELLULAR AND MOLECULAR
Department of Pharmacology (J.W.W., D.T.L., J.J.R., R.R.M.) and Institute of Environmental Health Sciences (M.D.K., J.J.R.) and Environmental Health Sciences Center in Molecular and Cellular Toxicology with Human Applications (J.J.R., R.R.M.), Wayne State University, Detroit, Michigan; Medicinal Chemistry and Molecular Pharmacology and Cancer Center, Purdue University, West Lafayette, Indiana (F.F., R.A.G., R.F.B.); and Barbara Ann Karmanos Cancer Institute, Programs in Proteases (J.J.R.) and Molecular Biology and Human Genetics (R.R.M.), Detroit, Michigan
Received December 24, 2007; accepted March 25, 2008.
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Abstract |
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). NF1 presents with an array of clinical manifestations arising during early development through adulthood, including increased pigmentation of the skin (café au lait macules), Lisch nodules of the iris, learning disabilities, and abnormal development of the skeletal system. NF1 is characterized by the development of benign peripheral nerve sheath tumors or neurofibromas. Approximately 10% of NF1 patients experience tumor transformation to the more aggressive malignant peripheral nerve sheath tumors (MPNSTs) (Ward and Gutmann, 2005). Progression toward MPNST is a leading cause of increased mortality for NF1 patients. Therapies are limited to excision of neurofibromas, radiation of plexiform neurofibromas, and the use of cytotoxic compounds. Although excision of tumors is the primary form of treatment, the tumors tend to return (Packer et al., 2002). A targeted molecular therapy designed against the background of NF1 may reveal more effective approaches for treatment of NF1 (Dilworth et al., 2006).
The molecular pathogenesis of NF1 was better understood following the discovery of the NF1 gene, which encodes the protein neurofibromin (Nf). Nf contains a Ras-GTPase-activating protein (GAP) domain (DeClue et al., 1991). This domain is responsible for decreasing Ras signaling by increasing the intrinsic rate of Ras-GTP to Ras-GDP (Eccleston et al., 1993). Germline mutations of the NF1 gene result in reduced Nf expression and a loss of Ras-GAP activity. The consequence of losing Ras-GAP activity is aberrant Ras signaling that can potentially lead to the development of NF1 (Basu et al., 1992; Feldkamp et al., 1999). Our laboratory and others have previously targeted downstream signaling partners of Ras by treating MPNST cell lines with MEK inhibitors (Tang et al., 1998; Chadee and Kyriakis, 2004; Mattingly et al., 2006; Roth et al., 2007). We have shown that PD184352 (Mattingly et al., 2006) (CI-1040) induced apoptosis in MPNST cell lines, confirming the dependence of the Ras-MAPK pathway in this disease.
Ras proteins are translated as inactive precursor molecules that must undergo a series of post-translational modifications before becoming fully functional (Gibbs et al., 2001). The first necessary step is the covalent addition of a prenyl group, either a 15C farnesyl or a 20C geranylgeranyl group, to the C-terminal "CaaX" box (Basso et al., 2006).
Reducing the prenylation of proteins to treat NF1 has been recognized as a potential therapeutic approach. For example, the farnesyl transferase inhibitor (FTI) BMS-186511 (Mazieres et al., 2003) reduces proliferation of MPNST cell line ST88-14 (Yan et al., 1995), and FTI L-739,749 reduces proliferation of Nf-deficient mouse Schwann cells (Kim et al., 1997). A phase I clinical trial using FTI tipifarnib to treat plexiform neurofibromas was tolerated well in children, yet no objective responses were achieved (Widemann et al., 2006). Although this study has advanced to an ongoing phase II trial (NCT00029354
[ClinicalTrials.gov]
), it is likely that further development of this treatment approach will be required.
Our laboratory is interested in using FTIs and lovastatin, an inhibitor of the hydroxymethylglutaryl (HMG)-CoA reductase, to reduce prenylation of proteins as a potential therapy for numerous diseases. We have previously reported that lovastatin, in combination with FTI 3-allylfarnesol, induces relocation of RhoB from the membrane fraction to the cytosolic fraction following treatment in A10 vascular smooth muscle cells. The translocation of RhoB from the membrane to the cytosol is the result of inhibiting RhoB prenylation (Mattingly et al., 2002). A prodrug analog of 3-allylfarnesol phosphate was also shown to inhibit RhoB prenylation in STS-26T MPNST cells when used in combination with lovastatin, resulting in reduced cell proliferation (Clark et al., 2007).
Here, we describe our efforts to characterize the effectiveness of two novel FTase inhibitors, 1 (Clark et al., 2007) and 2, on human NF1 MPNST cell lines, NF90-8 and ST88-14. The prodrug structures are shown in Fig. 1. Prodrug 1 is highly lipophilic, so analog 2 was prepared in which a carboxylate side chain, which would be ionized at physiologic pH, replaced the N-methyl group on the prodrug moiety. The entire prodrug moiety is released from the inhibitor following activation inside the cell (Clark et al., 2007), so this modification will have no effect on inhibitor affinity. Our objective was to determine the efficacy of these compounds on Ras prenylation and cell proliferation when used in combination with lovastatin. The data show reduction of Ras prenylation in both cell lines with cell cycle G1 arrest and increased caspase activity following FTI/lovastatin combination treatment but lack of toxicity in normal rat Schwann cells.
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Materials and Methods |
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). Prodrug 2 was synthesized via an analogous route using amino ester 3 instead of methyl chlorobutylamine, followed by selective hydrolysis of the methyl ester (Fig. 1). Lovastatin (Sigma-Aldrich, St. Louis, MO) aliquots were prepared in dimethyl sulfoxide and stored at –80°C. JC-1 and Hoëchst 33342 (Invitrogen, Carlsbad, CA) were prepared in dimethyl sulfoxide and stored at 4°C. MitoTracker Orange CM-H2 TM Ros (Invitrogen) aliquots were prepared in dimethyl sulfoxide and stored at –20°C.
Cell Culture. NF90-8 and ST88-14 MPNST cell lines were generously donated by T. Glover (University of Michigan, Ann Arbor, MI). Cells were maintained as adherent cultures in RPMI 1640 (Invitrogen) with 5% fetal bovine serum (HyClone Laboratories, Logan, UT), 100 U/ml penicillin, and 100 µg/ml streptomycin (Invitrogen). Primary normal rat Schwann cells were isolated from the sciatic nerves of neonatal Sprague-Dawley Rats and grown on poly-D-lysine-coated coverslips as described previously (Skoff et al., 1998). These cells were grown in Eagle's medium with 10% calf serum before experimental manipulations. Normal, spontaneously immortal rat Schwann cell clones (iSCs) isolated from sciatic nerves were a generous gift from E.M. Shooter (Stanford University, Stanford, CA) and described previously (Bolin et al., 1992). These cells were maintained in minimal essential medium supplemented with 10% horse serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. All cells were maintained in a humidified incubator under 5% CO2. For all experiments, cells were plated 24 h before drug treatment. Immediately before drug treatment, the medium was replaced with fresh growth medium for the duration of the experiment.
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). Samples were then separated on polyacrylamide-SDS gels and electrophoretically transferred to nitrocellulose. Ras was detected with a 1:250 dilution of anti-pan Ras monoclonal antibody (BD Transduction Laboratories, San Jose, CA). Caspase-3 was detected with a 1:1000 dilution of anti-caspase-3 polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) as described previously (Menard et al., 2005). Retinoblastoma protein (pRb) phosphorylated Ser780 was detected with a 1:1000 dilution of anti-phospho-Rb Ser780 polyclonal antibody (Cell Signaling Technology Inc., Danvers, MA). The nitrocellulose membrane was stripped and reprobed for total pRb with a 1:1000 dilution of a mouse monoclonal antibody (Cell Signaling Technology Inc.). β-Tubulin was immunoblotted with a 1:2000 dilution of the E7 monoclonal antibody (Developmental Studies Hybridoma Bank, Iowa City, IA).
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Flow Cytometry. NF90-8 and ST88-14 cells were treated and collected for DNA analysis as described previously (Reiners et al., 1999). DNA content was analyzed using a FACScalibur instrument (BD Biosciences, San Jose, CA). A minimum of 104 cells/sample was analyzed to determine the percentage of apoptotic cells and cells in G1, S, and G2/M phase (MODFIT; Variety Software, Topsham, ME).
DEVDase Activity Assay. Lysates of NF90-8 and ST88-14 cultures were prepared and used in DEVDase assays as described previously (Caruso et al., 2004). Changes in fluorescence over time were converted into picomoles of product by comparison with a standard curve made with 7-amino-4-methylcoumarin. DEVDase-specific activities are reported as nanomoles of product per minute per milligram of protein. The bicinchoninic acid assay, using bovine serum albumin as a standard, was used to estimate protein concentrations.
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m) was assayed by two independent methods. In one series of experiments, JC-1 was added directly to the medium at a final concentration of 5 µg/ml and incubated for 10 min at 37°C. Attached cells were trypsinized and combined with growth media containing detached cells. The cells were collected at 500g for 5 min, washed twice in PBS, and suspended in PBS containing 1% fetal bovine serum for analysis by excitation at 530 nm and emission at 590 nm. Cells with active mitochondria accumulate red-emitting "JC-1 aggregates." In cells having depolarized mitochondria, JC-1 is present as monomers, which exhibit green, not red, fluorescence emission.
In a second series of experiments, MitoTracker Orange CM-H2 TM Ros (excitation, 554 nm; emission, 576 nm) was added directly to the medium of treated cultures at a final concentration of 50 nM and incubated for 10 min at 37°C. Hoëchst 33342 (excitation, 350 nm; emission, 461 nm) was added directly to the medium at a final concentration of 500 nM to reveal nuclear morphology and coincubated with the MitoTracker for 5 min at 37°C. Cultures were rinsed once with PBS and subsequently rinsed three times with fresh growth medium. Images were immediately acquired with an Axiovert 200M fluorescence microscope (Carl Zeiss Inc., Thornwood, NY). Cells with reduced m are unable to sequester and oxidize MitoTracker Orange and will exhibit reduced fluorescence emission.
Statistical Analysis. Two-tailed paired Student's t test was employed to determine the statistical significance of a G1 cell cycle arrest between FTI/lovastatin combination treatment cultures and the DMSO control cultures. The significance level for this analysis was set at p < 0.05.
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Results |
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). N-Ras can be alternatively prenylated with a geranylgeranyl pyrophosphate in the presence of FTIs (Whyte et al., 1997). Therefore, we tested whether our novel FTIs (1 and 2) could reduce Ras prenylation in MPNST cell lines NF90-8 and ST88-14 (Fig. 2). Inhibition of prenylation is indicated by the slower mobility band or up-shift by Western analysis. Single 1 µM treatments of 1, 2, or lovastatin had little to no detectable effect on Ras prenylation in either cell line. However, a reduction of Ras prenylation was observed in the NF90-8 cells following combination treatment of 1 µM 1 or 2 with 1 µM lovastatin. Ras prenylation was also reduced in ST88-14 FTI/lovastatin combination-treated cells compared with the single treatments of 1, 2, or lovastatin. The observed reduction of Ras prenylation in ST88-14 cells was moderate compared with that occurring in NF90-8 FTI-/lovastatin-treated cells.
Ras isoforms are known to regulate cell processes such as survival, growth, and proliferation. Since our FTI/lovastatin combination treatment reduced Ras prenylation in NF90-8 and ST88-14 cells (Fig. 2), we next determined the effect of this treatment on cell proliferation. The results from proliferation experiments with NF90-8 cells are shown in Fig. 3, A and B. Single treatments of 500 nM 1, 2, or lovastatin alone had no effect on cell proliferation compared with cultures that were treated with DMSO. However, combination treatments of 500 nM 1 plus 500 nM lovastatin were cytostatic after 24 h of treatment and reduced total cell number below initial plating after 72 h of treatment (Fig. 3A). A similar cytostatic response was observed following treatment with 500 nM 2 plus 500 nM lovastatin (Fig. 3B). Inhibition of proliferation also occurred in the presence of lower concentrations of lovastatin (33 and 100 nM) in combination with 500 nM 1 or 2. Synergism of the FTI compounds with 33 nM lovastatin is notable because this dose of lovastatin is pharmacologically achievable in humans treated with anticholesterol doses of statins (Thibault et al., 1996). Proliferation data for ST88-14 cells treated with nanomolar combinations of FTI/lovastatin are shown in Fig. 3, C and D. Single treatment with DMSO, 1, 2, or lovastatin had no effect on cell proliferation. However, when 500 nM 1 or 2 were used in combination with 500 nM lovastatin, we observed a reduction of ST88-14 proliferation. Unlike the NF90-8 cells, lower doses of lovastatin in combination with 500 nM 1 or 2 had less effect on cell proliferation.
We subsequently investigated the effects of FTI/lovastatin treatment on NF90-8 and ST88-14 cell cycle progression. Figure 4 shows fluorescence-activated cell sorting analysis of NF90-8 cells treated with 500 nM 1, or with 500 nM lovastatin, or a combination of the two drugs for 48 h. Single treatments with these compounds yielded cell cycle profiles comparable with the DMSO control, which was also similar to untreated controls (Table 1; Supplemental Fig. 1). The combination treatment results in an increased percentage of cells in G1 and a significant percentage of cells undergoing apoptosis, compared with the control cultures. These data coincide with the proliferation data in Fig. 3A, where we observed a cytostatic effect of 500 nM 1 plus 500 nM lovastatin at 24 h and a cytotoxic effect at 48 h. Similar results were observed with 500 nM 2 in combination with 500 nM lovastatin (Table 1; Supplemental Fig. 1). As a control for these experiments, we also tested compound 4 (Fig. 1), an analog of compound 1 that is inactive as an FTI and has no effect on the proliferation of the spontaneous MPNST cell line, STS-26T [Compound 5d in Clark et al. (2007)]. The cell cycle distribution of NF90-8 and ST88-14 cells was not affected by treatment with 1 µM 4 alone or in combination with 1 µM lovastatin (data not shown). Cell cycle progression of ST88-14 cells was also analyzed at 24 and 48 h following treatment with either 500 nM 1 or 500 nM 2 with or without 500 nM lovastatin (Table 2; Supplemental Fig. 2). We observed a moderate G1 cell cycle arrest at 24 h that was maintained at 48 h following FTI/lovastatin combination treatment. Although we observed an increased percentage of apoptotic cells at 48 h, the effect was more modest than that observed in NF90-8 cultures.
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One mechanism that could underlie a G1 arrest would be if pRb phosphorylation was reduced. Therefore, we investigated the phosphorylation pattern of pRb in the NF90-8 and ST88-14 cell lines (Fig. 5). Single treatments of 1 µM 1, 2, or lovastatin did not reduce hyperphosphorylation of pRb. However, combining 1 µM 1 or 2 with 1 µM lovastatin significantly reduced the phosphorylated pRb signal. Total pRb expression was also reduced in 1 or 2 plus lovastatin-treated cultures. As a further control for these experiments, we also tested the inactive control compound 4. No effect on pRb phosphorylation was observed in either cell line following treatment with 1 µM 4 alone or in combination with 1 µM lovastatin (Fig. 5).
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The morphological characteristics of FTI/lovastatin-treated MPNSTs, coupled with increased numbers of cells with sub-G1 DNA contents, suggested the occurrence of apoptosis. To monitor for apoptosis, we used N-acetyl-Asp-Glu-Val-Asp-amino-4-methylcoumarin to assay for the activity of caspases-3 and -7 (Fig. 7) (Caruso et al., 2004). Treatment with 500 nM 1, 2, or lovastatin resulted in no detectable activation of DEVDase. However, high amounts of active DEVDase were observed in NF90-8 cells (Fig. 7, C and D), and moderate amounts were observed in ST88-14 (Fig. 7, A and B) cells, following treatment with 500 nM 1 or 2 and 500 nM lovastatin. In addition, 100 nM 1 with 500 nM lovastatin also activated DEVDase in NF90-8 cells. NF90-8 cells treated with 500 nM 1 plus 500 nM lovastatin also exhibited a time-dependent increase of cleaved procaspase-3 (Fig. 7E). The kinetics of procaspase-3 cleavage correlated with the time-dependent increases in DEVDase (Fig. 7C).
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Changes in m often precede/accompany the activation of procaspases and are generally indicative of an activation of the intrinsic apoptotic pathway (Kroemer et al., 1997). We employed two methods to monitor m. The first method involved live cell microscopy using MitoTracker Orange (Fig. 8A). NF90-8 cells treated with DMSO, 500 nM 1, or 500 nM lovastatin exhibited tubular mitochondrial staining at 24 and 48 h with little to no detectable chromatin condensation. However, following 24 h of combination treatment with 500 nM 1 and 500 nM lovastatin, the cells exhibited less red fluorescence. Within 48 h of treatment, both the cells and nuclei had shrunken, and there were dramatic reductions in red fluorescence (i.e., reduced m).
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Flow cytometric analysis of m with JC-1 was employed to quantify mitochondrial membrane potential (Fig. 8B). NF90-8 cells were treated with drugs and then incubated with JC-1, a compound that forms red fluorescent aggregates in cells having m. After a loss of m, JC-1 does not aggregate and fluoresce red. As observed with MitoTracker Orange, treatment with DMSO, 500 nM lovastatin, or 500 nM 1 did not affect m in NF90-8 cells. However, after FTI/lovastatin combination treatment, a progressive loss of m occurred from 24 to 72 h. It is noteworthy that a significant loss of m occurred at 24 h, a time that precedes the activation of DEVDase (Fig. 7C) and procaspase-3 cleavage (Fig. 7E).
We have demonstrated that treatment of human MPNST cell lines NF90-8 and ST88-14 with 500 nM 1 or 2 in combination with 500 nM lovastatin greatly reduced cell proliferation and induced an apoptotic response. We tested the effects of this FTI/lovastatin treatment on normal primary Schwann cells isolated from the sciatic nerve of rat pups. Normal primary rat Schwann cells were treated for 72 h, and differential interference contrast images were recorded (Fig. 9A). Once again, single treatments of DMSO, 500 nM 1 or 2, or 500 nM lovastatin had no detectable toxicity. However, in a stark contrast to the effect on MPNST cells, combination treatments of 500 nM 1 or 2 with 500 nM lovastatin had no observable toxicity on normal primary Schwann cells. To further examine whether the FTI/lovastatin treatment would be toxic, we tested our compounds on an iSC (Bolin et al., 1992). Single treatments of 500 nM 1, 2, or lovastatin had no effect on proliferation (Fig. 9B). Combination treatments of 500 nM 1 or 2 with 500 nM lovastatin also did not significantly reduce proliferation of iSC cells.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). The loss of Nf results in aberrant Ras signaling in cells derived from the neural crest. Ras must localize and attach to membranes where the protein functions and signals downstream. This membrane attachment requires numerous post-translational modifications. The initial step is the covalent addition of a 15C farnesyl or a 20C geranylgeranyl group to the cysteine located on the C-terminal CaaX box (Gibbs et al., 2001). This modification is critical for proper Ras function and has been investigated as a potential target for treating NF1 MPNST cells.
We used novel farnesyl transferase inhibitors, 1 and 2, to reduce protein prenylation in MPNST cell lines NF90-8 and ST88-14. FTI 1 and 2 are farnesyl diphosphate-based FTase inhibitors. To augment the inhibition of FTase, we cotreated the MPNST cell lines with lovastatin. Lovastatin is an inhibitor of HMG-CoA reductase, the rate-limiting enzyme in cholesterol synthesis (Wong et al., 2002). In addition to blocking cholesterol synthesis, HMG-CoA reductase is responsible for the initial steps of farnesyl diphosphate and geranylgeranyl diphosphate synthesis (Morgan et al., 2003). Other labs have shown that micromolar doses of lovastatin reduce Ras prenylation and increase the recovery of the protein in cytosolic fractions (Mendola and Backer, 1990; Sebti et al., 1991). In addition, lovastatin reverted the learning disabilities of Nf+/– mice (Li et al., 2005). Recently, a clinical trial was opened to evaluate the safety of lovastatin in adults with NF1 (NCT00352599
[ClinicalTrials.gov]
). In the current study, we used pharmacologically achievable (Thibault et al., 1996) nanomolar doses of lovastatin, which alone had no effect on NF90-8 and ST88-14 MPNST cell line proliferation but showed synergy when used in combination with novel FTIs, 1 and 2.
Our laboratory previously described N-Ras as the predominant active Ras isoform expressed in human-derived MPNST cell lines NF90-8 and ST88-14 (Mattingly et al., 2006). N-Ras is a member of a group of proteins that can undergo two types of prenylation, either farnesylation or geranylgeranylation. This characteristic could allow an escape mechanism in which N-Ras could be alternatively prenylated with a geranylgeranyl pyrophosphate to maintain proper N-Ras localization and function in the presence of FTIs (Whyte et al., 1997). Indeed, NF1–/– hematopoietic cells confer a myeloproliferative disorder that is resistant to FTI L-744,832 (Kohl et al., 1995) treatment. This resistance occurs despite block of H-Ras prenylation and is proposed to be due to lack of efficacy against N-Ras and for K-Ras (Mahgoub et al., 1999). Since N-Ras is commonly overexpressed or mutated in cancer, designing a therapy that can reduce farnesylation and geranylgeranylation of N-Ras may be a logical approach.
Our data show that Ras prenylation was maintained following single treatments of 1, 2, or lovastatin in both MPNST cell lines. It is possible that these agents singularly inhibited FTase and reduced farnesylation. However, N-Ras may have undergone a compensatory alternative geranylgeranylation. Combination treatment of 1 or 2 with lovastatin induced a near-complete inhibition of Ras prenylation in NF90-8 cells and a moderate inhibition in ST88-14 cells. The combination treatment may have provided a more effective inhibition of FTase, but it could also be that lovastatin may have reduced geranylgeranyl diphosphate pools in the cell and so impaired alternate prenylation of Ras (Morgan et al., 2005).
Combination treatment of 1 or 2 with lovastatin impaired Ras prenylation in both NF1 MPNST cell lines, but to different degrees. The degree to which Ras prenylation was suppressed correlated with the antiproliferative and proapoptotic effects of the combination treatment. Although inhibition of Ras prenylation correlated strongly with cellular response, Ras is not the exclusive target protein for FTIs (Lebowitz et al., 1997; Ashar et al., 2000; Clark et al., 2007). Approximately 0.5% of proteins in the cell must undergo prenylation to function properly. Known farnesylated proteins include the Ras proteins, RhoB, Rheb, CENP-E, CENP-F, and the nuclear lamins (Tamanoi et al., 2001). The large list of potential FTI targets increases the difficulty in identifying the true therapeutic target and which diseases would respond to an FTI-based therapy. Nevertheless, because Ras activation drives the NF1 phenotype, inhibition of Ras prenylation probably contributes to the efficacy of the combinatorial drug treatment.
Combination FTI/lovastatin induced G1 arrest, coinciding with reduced pRb phosphorylation at 48 h. Total pRb content was also reduced in combination-treated cultures. Because pRb is a substrate for caspase-3 and -7 (Fattman et al., 1997), and combination treatment resulted in the activation of procaspase-3 and -7, it is possible that pRb was cleaved by active caspases and degraded. This reduction of total pRb may also have contributed to reduced pRb phosphorylation and the observed G1 arrest. Induction of G1 arrest by prenylation inhibitors that include a lactone structure may be through a p21-dependent mechanism that includes inhibition of the proteasome (Efuet and Keyomarsi, 2006). The active compounds in the current study, however, do not contain a lactone moiety. They also induce a G1 arrest in STS-26T cells (Clark et al., 2007), which do not express p21 protein (Mattingly et al., 2006).
Theoretically, toxicity to normal cells could be a concern following FTI treatment due to the large number of proteins that are prenylated. FTIs in combination with lovastatin may increase the number of proteins with impaired prenylation. However, we found a lack of detectable cytotoxicity in normal or immortalized rat Schwann cells following combined treatment. It is conceivable that the target protein of our FTIs responsible for the observed effects in the MPNST cell lines is not necessary for normal Schwann cell survival or proliferation.
NF1 MPNST cell lines have increased active Ras compared with non-NF1 cell lines. This makes Ras a rational therapeutic target for this disease. Ongoing clinical trials include one regarding lovastatin tolerance in adults with NF1 (NCT00352599
[ClinicalTrials.gov]
) and the use of the peptide-competitive FTI tipifarnib on treating NF1 in children [(Widemann et al., 2006) and NCT00029354
[ClinicalTrials.gov]
]. The novel FTIs described in this study are extremely effective against NF1 MPNST cell lines with a lack of toxicity against normal Schwann cells. We propose that a combination of FTI/statin treatment may be more efficacious in treatment of NF1 MPNSTs.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: NF1, neurofibromatosis type 1; MPNST, malignant peripheral nerve sheath tumor; Nf, neurofibromin; GAP, GTPase-activating protein; FTI, farnesyl transferase inhibitor; HMG, hydroxymethylglutaryl; FTase, farnesyl transferase; JC-1, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide; iSC, spontaneously immortal rat Schwann cell clone; pRb, retinoblastoma protein; m, mitochondrial membrane potential; PBS, phosphate-buffered saline; DMSO, dimethyl sulfoxide.
The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. 
Address correspondence to: Dr. Raymond R. Mattingly, Department of Pharmacology, Wayne State University, 540 East Canfield Ave., Detroit, MI 48201. E-mail: r.mattingly{at}wayne.edu
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