In view of the data available from randomized trials, gemcitabine has been established as a new standard for the treatment of pancreatic cancer. It was shown to improve clinical benefit response, time to progression, and survival when compared with agents such as 5-fluorouracil or metalloproteinase inhibitors. In one trial, the combination of cisplatin and gemcitabine significantly improved tumor response and time to progression as compared with gemcitabine alone, while a significant impact on survival yet needs to be shown. No significant, clinically meaningful survival benefit was observed when gemcitabine was combined with bolus or infusional 5-fluorouracil, capecitabine, metalloproteinase inhibitors, or the FTI tipifarnib. Numerous ongoing randomized trials are presently investigating gemcitabine-based combination regimens involving such agents as cisplatin, oxaliplatin, irinotecan, docetaxel, 5-fluorouracil, capecitabine, or pemetrexed.

Characterization of intracellular signaling pathways should lead to a better understanding of ovarian epithelial carcinogenesis and provide an opportunity to interfere with signal transduction targets involved in ovarian tumor cell growth, survival, and progression. Challenges toward such an effort are significant because many of these signals are part of cascades within an intricate and likely redundant intracellular signaling network (Fig.1). For instance, a given signal may activate a dual intracellular pathway (ie, MEK1-MAPK and PI3K/Akt required for fibronectin-dependent activation of matrix metalloproteinase 9). A single pathway also may transduce more than one biologic or oncogenic signal (ie, PI3K signaling in epithelial and endothelial cell growth and sprouting of neovessels). Despite these challenges, evidence for therapeutic targeting of signal transduction pathways is accumulating in human cancer. For instance, the EGF-specific tyrosine kinase inhibitor ZD 1839 (Iressa) may have a beneficial therapeutic effect on ovarian epithelial cancer. Therapy of this cancer may include inhibitors of PI kinase (quercetin), ezrin and PIP kinase (genistein). The G protein-coupled family of receptors, including LPA, also is an attractive target to drugs, although their frequent pleiotropic functions may be at times toxic and lack specificity. Because of the lack of notable toxicity, PI3K/Akt pathway inhibitors such as FTIs are a promising targeted therapy of ovarian epithelial cancer. Increasing insight into the oncogenic pathways involved in ovarian epithelial cancer also is helping clinicians to understand better the phenomenon of chemoresistance in this malignancy. Oncogenic activation of gamma-synuclein promotes cell survival and provides resistance to paclitaxel, but such a resistance is partially overcome by an MEK inhibitor that suppresses ERK activity. Ovarian epithelial cancer is a complex group of neoplasms with an overall poor prognosis. Comprehension of this cancer pathobiology suffers because of an incomplete understanding of precursor lesions and the absence of an orthotopic animal model until very recently. It can be predicted with confidence, however, that the discovery of potent inhibitors of signal transduction and the development of discovery tools, such as proteomics and metabolomics, may change the way by which clinicians may now address basic biomedical questions in this insidious and lethal disease.

Neurotrophin (NT)-3 is expressed in the neuronal target tissue of the developing rat cochlea and has been shown to promote the survival and neurite outgrowth of spiral ganglion (SG) neurons, suggesting a role for this protein during the innervation of the organ of Corti. In other neurons, NT-3 can mediate neuritogenesis and survival via a number of intracellular signal pathways. To date, the intracellular transduction pathways involved in the mediation of NT-3 effects have not been investigated in SG neurons. To determine whether the activities of NT-3 on SG neurons are dependent on the activation of mitogen-activated protein kinase kinases (MEK)/extracellular-signal-regulated kinases (ERK), Ras, and/or p38, SG explants from postnatal-day 4 rats were cultured with NT-3 and increasing concentrations of the MEK inhibitor U0126, the Ras farnesyl-transferase inhibitor (FTI)-277, and the p38 inhibitor SB203580. After fixation and immunocytochemical labeling, neurite growth was evaluated. A dose-dependent decrease of the effects of NT-3 on length and number of processes was observed in the U0126- and FTI-277-treated SG neurons. In contrast, SB203580 had no significant effect on NT-3-mediated stimulation of neurite growth, in terms of either number or length. The results suggest that NT-3 effects on SG neurons are mediated primarily by the Ras/MEK/ERK signaling pathway.

The vasculoprotective effects of hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors (statins) correlate with cholesterol lowering. HMG-CoA reductase inhibitors also disrupt cellular processes by the depletion of isoprenoids and dolichol. Insulin and insulin-like growth factor (IGF) signaling appear particularly prone to such disruption as intracellular receptor processing requires dolichol for correct N-glycosylation, whereas downstream signaling through Ras requires the appropriate prenylation (farnesol). We determined how HMG-CoA reductase inhibition affected the mitogenic effects of IGF-I and metabolic actions of insulin in 3T3-L1 cells and examined the respective roles of receptor glycosylation and Ras prenylation. IGF-I- and insulin-induced proliferation was significantly reduced by all statins tested, although cerivastatin (10 nm) had the greatest effect (p < 0.005). Although inhibitors of Ras prenylation induced similar results (10 microm FTI-277 89% +/- 7.4%, p < 0.01), the effect of HMG-CoA reductase inhibition could only be partially reversed by farnesyl pyrophosphate refeeding. Treatment with statins resulted in decreased membrane expression of receptors and accumulation of proreceptors, suggesting disruption of glycosylation-dependent cleavage. Glycosylation inhibitors inhibited IGF-I-induced proliferation (tunicamycin p < 0.005, castanospermine p < 0.01, deoxymannojirimycin p < 0.01). High concentrations of statin were necessary to impair insulin-mediated glucose uptake (300 nm = 33% +/- 12% p < 0.05), and this process was not effected by farnesyl transferase inhibition. Gycosylation inhibitors mimicked the effect of statin treatment (tunicamycin p < 0.001, castanospermine p < 0.05, deoxymannojirimycin p < 0.05), and there was insulin proreceptor accumulation. These data imply that HMG-CoA reductase inhibitors disrupt IGF-I signaling by combined effects on Ras prenylation and IGF receptor glycosylation, whereas insulin signaling is only affected by disrupted receptor glycosylation.

Treatment of prostate cancer (CaP) patients frequently involves androgen ablation, but resistance often develops and androgen-insensitive tumors emerge. The molecular basis for the development of refractory CaP that grows in an androgen-independent manner is poorly understood, but alterations in growth factor signaling pathways are likely to be involved. We examined the growth factor modulation of androgen-receptor element (ARE)-inducible luciferase reporter gene activity and consequent DNA synthesis as a measure of proliferative growth in androgen-dependent LNCaP or androgen-independent PC3 or DU145 CaP cells. The synthetic androgen R1881 stimulated ARE-inducible reporter gene activity and prostate-specific antigen expression in LNCaP cells and the MEK/ERK inhibitor U0126 or the anti-androgen bicalutamide (casodex) prevented both of these responses. Activated V12-Ha-Ras expression in LNCaP cells also stimulated ARE-inducible gene transcription, and U0126 or the farnesyltransferase inhibitor FTI-277 but not bicalutamide blocked this. ARE-inducible reporter gene activity was elevated already in PC3 cells, and ERK was constitutively activated in serum-starved LNCaP or DU145 cells. U0126 inhibited each of these responses and also inhibited DNA synthesis in all 3 CaP cell lines. These results demonstrate that chronic stimulation of the Ras-MEK-ERK signaling pathway can sustain ARE-inducible gene transcription and growth of CaP cells, and suggests that components of this pathway may offer targets for cancer therapy.

Hutchinson-Gilford progeria syndrome (HGPS) is caused by the accumulation of a farnesylated form of prelamin A (progerin). Previously, we showed that blocking protein farnesylation with a farnesyltransferase inhibitor (FTI) ameliorates the disease phenotypes in mouse model of HGPS (Lmna(HG/+)). However, the interpretation of the FTI treatment studies is open to question in light of recent studies showing that mice expressing a nonfarnesylated version of progerin (Lmna(nHG/+)) develop progeria-like disease phenotypes. The fact that Lmna(nHG/+) mice manifest disease raised the possibility that the beneficial effects of an FTI in Lmna(HG/+) mice were not due to the effects of the drug on the farnesylation of progerin, but may have been due to unanticipated secondary effects of the drug on other farnesylated proteins. To address this issue, we compared the ability of an FTI to improve progeria-like disease phenotypes in both Lmna(HG/+) and Lmna(nHG/+) mice. In Lmna(HG/+) mice, the FTI reduced disease phenotypes in a highly significant manner, but the drug had no effect in Lmna(nHG/+) mice. The failure of the FTI to ameliorate disease in Lmna(nHG/+) mice supports the idea that the beneficial effects of an FTI in Lmna(HG/+) mice are due to the effect of drug on the farnesylation of progerin.

Prenylation is a post-translational modification critical for the proper function of multiple physiologically important proteins, including small G-proteins, such as Ras. Methods allowing rapid and selective detection of protein farnesylation and geranylgeranylation are fundamental for the understanding of prenylated protein function and for monitoring efficacy of drugs such as farnesyltransferase inhibitors (FTIs). Although the natural substrates for prenyltransferases are farnesyl pyrophosphate and geranylgeranyl pyrophosphate, farnesyltransferase has been shown to incorporate isoprenoid analogues into protein substrates. In this study, protein prenyltransferase targets were labeled using anilinogeraniol, the alcohol precursor to the unnatural farnesyl pyrophosphate analogue 8-anilinogeranyl diphosphate in a tagging-via-substrate approach. Antibodies specific for the anilinogeranyl moiety were used to detect the anilinogeranyl-modified proteins. Coupling this highly effective labeling/detection method with two-dimensional electrophoresis and subsequent Western blotting allowed simple, rapid analysis of the complex farnesylated proteome. For example, this method elucidated the differential effects induced by two chemically distinct FTIs, BMS-214,662 and L-778,123. Although both FTIs strongly inhibited farnesylation of many proteins such as Lamins, NAP1L1, N-Ras, and H-Ras, only the dual prenylation inhibitor L-778,123 blocked prenylation of Pex19, RhoB, K-Ras, Cdc42, and Rap1. This snapshot approach has significant advantages over traditional techniques, including radiolabeling, anti-farnesyl antibodies, or mass spectroscopy, and enables dynamic analysis of the farnesylated proteome.

Farnesyltransferase inhibitors (FTI) have been developed as anticancer drugs and are currently being evaluated in clinical trials. In this study, we have examined the effects of FTIs on Tsc-null cells to gain insight into their effects on farnesylated Rheb GTPase. This protein is involved in the activation of mTOR/S6K signaling and is down-regulated by the Tsc1/Tsc2 complex. Both Tsc1(-/-) and Tsc2(-/-) mouse embryonic fibroblasts exhibit constitutive activation of S6K and grow in the absence of serum. Two different FTI compounds, the clinical compound BMS-214662 and the newly described BMS-225975, inhibit the constitutive activation of mTOR/S6K signaling and block serum-free growth of the Tsc-null mouse embryonic fibroblasts. We have also found that Tsc-null mouse embryonic fibroblasts grow under anchorage-independent conditions and that both FTI compounds inhibit this soft agar growth. These FTI effects are similar to those observed with rapamycin. Another interesting phenotype of Tsc-null mouse embryonic fibroblasts is that they are round and contain actin filaments predominantly at the cell periphery. The addition of FTIs, but not rapamycin, led to the reappearance of intracellular actin filaments and reduction of peripheral actin filaments. The ability of FTI to rearrange actin filaments seems to be largely mediated by the inhibition of Rheb protein, as induction of intracellular actin filaments by FTI was much less efficient in Tsc2-null cells expressing Rheb (M184L), a geranylgeranylated mutant Rheb that can bypass farnesylation. These results reveal that FTIs inhibit Rheb, causing two different effects in Tsc-deficient cells, one on growth and the other on actin filament distribution.

CML (chronic myeloid leukaemia) is a myeloproliferative disease that originates in an HSC (haemopoietic stem cell) as a result of the t(9;22) translocation, giving rise to the Ph (Philadelphia chromosome) and bcr-abl oncoprotein. The disease starts in CP (chronic phase), but as a result of genomic instability, it progresses over time to accelerated phase and then to BC (blast crisis), becoming increasingly resistant to therapy. bcr-abl is a constitutively active tyrosine kinase that has been targeted by TKIs (tyrosine kinase inhibitors), including IM (imatinib mesylate), nilotinib and dasatinib. We have developed various flow cytometry techniques to enable us to isolate candidate CML stem cells from CP patients at diagnosis that efflux Hoechst dye, express CD34, lack CD38 and are cytokine-non-responsive in culture over periods of up to 12 days in growth factors. These stem cells have been shown to regenerate bcr-abl-positive haemopoiesis in immunocompromised mice upon transplantation. We previously demonstrated that IM was antiproliferative for CML stem cells but did not induce apoptosis. Clinical experience now confirms that IM may not target CML stem cells in vivo with few patients achieving complete molecular remission and relapse occurring rapidly upon drug withdrawal. Our recent efforts have focused on understanding why CML stem cells are resistant to IM and on trying to find novel ways to induce apoptosis of this population. We have shown that CML stem cells express very high levels of functional wild-type bcr-abl; no kinase domain mutations have been detected in the stem cell population. Dasatinib, a more potent multitargeted TKI than IM, inhibits bcr-abl activity more efficiently than IM but still does not induce apoptosis of the stem cell population. Most recently, we have tested a number of novel drug combinations and found that FTIs (farnesyl transferase inhibitors) have activity against CML. BMS-214662 is the most effective of these and induces apoptosis of phenotypically and functionally defined CML stem cells in vitro, as a single agent and in combination with IM or dasatinib. The effect against CML stem cells is selective with little effect on normal stem cells. The drug is also effective against BC CML stem cells and equally effective against wild-type and mutant bcr-abl, including the most resistant mutant T315I. In association with apoptosis, there is activation of caspase 8 and caspase 3, inhibition of the MAPK pathway, IAP-1 (inhibitor of apoptosis protein-1), NF-kappaB (nuclear factor kappaB) and iNOS (inducible nitric oxide synthase). Furthermore, BMS-214662 synergizes with MEK1/2 [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase 1/2] inhibitors, suggesting a second mechanism other that RAS inhibition for induction of apoptosis. Our intentions are now to explore the activity of BMS-214662 in other cancer stem cell disorders and to move this preclinical work to a clinical trial combining dasatinib with BMS-214662 in CML.

BACKGROUND

Farnesyltransferase inhibitors (FTIs) target multiple pathways implicated in the pathogenesis of solid and hematologic malignancies.

METHODS

Novel preclinical and clinical data on FTIs.

RESULTS

Results of clinical trials of FTIs are critically summarized: Phase I - II studies demonstrated that tipifarnib (the most extensively investigated FTI) had antileukemic activity. The rates of complete response (CR), partial response (PR) and/or CR with incomplete platelet recovery (CRp) in patients with MDS and refractory/poor-risk AML were 5 - 25% and 11 - 14%, respectively (hematological improvement, 17 - 35% and 8 - 9.5%, respectively). A Phase III study comparing tipifarnib with best supportive care, including hydroxyurea in patients with untreated AML ≥ 70 years old showed no survival benefit in the tipifarnib arm. A two-gene classifier (RASGRP1:APTX gene expression ratio) predicted response and survival, indicating that a two-gene expression assay may help select patients with AML who would benefit from tipifarnib.

CONCLUSIONS

Patient selection should become a priority for targeted agent drug development. Clinical trials selecting patients who would benefit from FTIs should be designed to define the role of FTIs in the treatment of hematological malignancies and solid tumors.

BCR/ABL, the oncoprotein responsible for chronic myeloid leukemia (CML), transforms hematopoietic cells through both Ras-dependent and -independent mechanisms. Farnesyl protein transferase inhibitors (FTIs) were designed to block mutant Ras signaling, but they also inhibit the growth of transformed cells with wild-type Ras, implying that other farnesylated targets contribute to FTI action. In the current study, the clinical candidate FTI SCH66336 was characterized for its ability to inhibit BCR/ABL transformation. When tested against BCR/ABL-BaF3 cells, a murine cell line that is leukemogenic in mice, SCH66336 potently inhibited soft agar colony formation, slowed proliferation, and sensitized cells to apoptotic stimuli. Quantification of activated guanosine triphosphate (GTP)-bound Ras protein and electrophoretic mobility shift assays for AP-1 DNA binding showed that Ras effector pathways are inhibited by SCH66336. However, SCH66336 was more inhibitory than dominant-negative Ras in assays of soft agar colony formation and cell proliferation, suggesting activity against targets other than Ras. Cell cycle analysis of BCR/ABL-BaF3 cells treated with SCH66336 revealed G2/M blockade, consistent with recent reports that centromeric proteins that regulate the G2/M checkpoint are critical farnesylated targets of FTI action. Mice injected intravenously with BCR/ABL-BaF3 cells developed acute leukemia and died within 4 weeks with massive splenomegaly, elevated white blood cell counts, and anemia. In contrast, nearly all mice treated with SCH66336 survived and have remained disease-free for more than a year. Furthermore, SCH66336 selectively inhibited the hematopoietic colony formation of primary human CML cells. As an oral, nontoxic compound with a mechanism of action distinct from that of ABL tyrosine kinase inhibition, FTI SCH66336 shows promise for the treatment of BCR/ABL-induced leukemia.

Mutations in genes encoding nuclear envelope proteins, particularly LMNA encoding the A-type lamins, cause a broad range of diverse diseases, referred to as laminopathies. The astonishing variety of diseased phenotypes suggests that different mechanisms could be involved in the pathogenesis of laminopathies. In this review we will focus mainly on two of these pathogenic mechanisms: the nuclear damages affecting the chromatin organization, and the oxidative stress causing un-repairable DNA damages. Alteration in the nuclear profile and in chromatin organization, which are particularly impressive in systemic laminopathies whose cells undergo premature senescence, are mainly due to accumulation of unprocessed prelamin A. The toxic effect of these molecular species, which interfere with chromatin-associated proteins, transcription factors, and signaling pathways, could be reduced by drugs which reduce their farnesylation and/or stability. In particular, inhibitors of farnesyl transferase (FTIs), have been proved to be active in rescuing the altered cellular phenotype, and statins, also in association with other drugs, have been included into pilot clinical trials. The identification of a mechanism that accounts for accumulation of un-repairable DNA damage due to reactive oxygen species (ROS) generation in laminopathic cells, similar to that found in other muscular dystrophies (MDs) caused by altered expression of extracellular matrix (ECM) components, suggests that anti-oxidant therapeutic strategies might prove beneficial to laminopathic patients.

The discovery that the transforming activity of oncogenic Ras depends upon its post-translational farnesylation has led to the development of farnesyl transferase inhibitors (FTIs). FTIs inhibit the growth of ras-transformed cells in vitro and induce tumor regression in Ras-dependent tumors. Currently, FTIs are undergoing clinical trials in various solid or hematological malignancies. In this review, we will summarize our current knowledge on cellular effect and molecular mechanism of FTIs. We will then describe recent clinical trials and propose some clues for their interpretation. Based on pre-clinical findings, we will emphasize on the optimal use of FTIs in anti-cancer strategy and lastly, we will insist on the interest of combining FTIs with chemotherapy, radiotherapy or other targeted agents.

OBJECTIVE

To investigate whether myocardial deformation imaging, assessed by feature tracking cardiac magnetic resonance (FTI-CMR), would allow objective quantification of myocardial strain and estimation of functional recovery in patients with first time ST-elevation myocardial infarction (STEMI).

METHODS

Cardiac magnetic resonance (CMR) imaging was performed in 74 consecutive patients 2-4 days after successfully reperfused STEMI, using a 1.5T CMR scanner (Philips Achieva). Peak systolic circumferential and longitudinal strains were measured using the FTI applied to SSFP cine sequences and were compared to infarct size, determined by late gadolinium enhancement (LGE). Follow-up CMR at 6 months was performed in order to assess residual ejection fraction, which deemed as the reference standard for the estimation of functional recovery.

RESULTS

During the follow-up period 53 of 74 (72%) patients exhibited preserved residual ejection fraction ≥50%. A cut-off value of -19.3% for global circumferential strain identified patients with preserved ejection fraction ≥50% at follow-up with sensitivity of 76% and specificity of 85% (AUC=0.86, 95% CI=0.75-0.93, p<0.001), which was superior to that provided by longitudinal strain (ΔAUC=0.13, SE=0.05, z-statistic=2.5, p=0.01), and non-inferior to that provided by LGE (ΔAUC=0.07, p=NS). Multivariate analysis showed that global circumferential strain and LGE exhibited independent value for the prediction of preserved LV-function, surpassing that provided by age, diabetes and baseline ejection fraction (HR=1.4, 95% CI=1.0-1.9 and HR=1.4, 95% CI=1.1-1.7, respectively, p<0.05 for both).

CONCLUSIONS

Estimation of circumferential strain by FTI provides objective assessment of infarct size without the need for contrast agent administration and estimation of functional recovery with non-inferior accuracy compared to that provided by LGE.

OBJECTIVE

Catheter-tissue contact is critical for effective lesion creation in radiofrequency catheter ablation (RFCA). In a multicentre prospective study, we assessed the effect of direct contact force (CF) measurement on acute procedural parameters during RFCA of atrial fibrillation (AF).

RESULTS

A new open-irrigated tip catheter with CF sensing (SmartTouch™, Biosense Webster Inc.) was used. All the patients underwent the first ablation procedure for paroxysmal AF with antral pulmonary vein (PV) isolation, aiming at entry and exit conduction block in all PVs. Ninety-five patients were enroled in nine centres and successfully underwent ablation. Overall procedure time, fluoroscopy time, and ablation time were 138.0 ± 67.0, 14.3 ± 11.2, and 33.8 ± 19.4 min, respectively. The mean CF value during ablation was 12.2 ± 3.9 g. Force time integral (FTI) analysis showed that patients achieving a value below the median of 543.0gs required longer procedural (158.0 ± 74.0 vs. 117.0 ± 52.0 min, P = 0.004) and fluoroscopy (17.5 ± 13.0 vs. 11.0 ± 7.7 min, P = 0.007) times as compared with those in whom FTI was above this value. Patients in whom the mean CF during ablation was >20 g required shorter procedural time (92.0 ± 23.0 vs. 160.0 ± 67.0 min, P = 0.01) as compared with patients in whom this value was <10 g. Four groin haematomas were the only complications observed.

CONCLUSIONS

Contact force during RFCA for PV isolation affects procedural parameters, in particular procedural and fluoroscopy times, without increasing complications.

We have used specific inhibitors for farnesyltransferase (FTase) and geranylgeranyltransferase (GGTase) I as well as combinations of lovastatin with geranylgeraniol (GGOH) or farnesol (FOH) to investigate the role of protein prenylation in platelet-derived growth factor (PDGF)-induced PDGF receptor tyrosine phosphorylation. NIH-3T3 cells treated with the highly specific FTase inhibitor FTI-277 had no effect on PDGF receptor tyrosine phosphorylation or PDGF activation of mitogen-activated protein kinase (MAPK) at doses that completely inhibit FTase-dependent processing. In contrast, treatment of these cells with GGTase I inhibitor GGTI-298 strongly inhibited receptor tyrosine phosphorylation, and co-treatment with FTI-277 had no additional effect. Interestingly, the inhibitory effect of GGTI-298 on PDGF activation of MAPK was only partial. Furthermore, although lovastatin, which inhibits both protein geranylgeranylation and protein farnesylation, blocked PDGF receptor tyrosine phosphorylation, co-treatment with GGOH, but not FOH, reversed the lovastatin block. In addition, although lovastatin was observed to block MAPK activation by PDGF, co-treatment with GGOH, but not FOH, restored its activation. Further investigations indicated that inhibition of receptor tyrosine phosphorylation was not due to decreased expression of the receptor or to inhibition of GGTase II. Thus, these results demonstrate that PDGF receptor tyrosine phosphorylation requires protein geranylgeranylation but not protein farnesylation and that the tyrosine phosphorylation levels of the receptor are modulated by a protein that is a substrate for GGTase I.

CpG oligodeoxynucleotides (ODN) activate immune cells to produce immune mediators by Toll-like receptor 9 (TLR9)-mediated signal transduction, which activates mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-kappaB) through the MyD88/IRAK/TRAF6 kinases cascade. However, the precise mechanisms of CpG ODN activation of immune cells have not been fully elucidated. The small GTP-binding protein Ras mediates MAPK activation in response to a variety of stimuli. Up to now, it is not clear whether Ras plays a role in CpG ODN signaling. In the present study, we found that the dominant-negative version of Ras (RasN17) and specific Ras inhibitor, FTI-277, inhibited CpG ODN-induced nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) production by murine macrophage cell line RAW264.7. While overexpression of wild-type Ras enhanced CpG ODN-induced extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and NF-kappaB activation, overexpression of RasN17 inhibited CpG ODN-induced ERK, JNK, and NF-kappaB activation. RasN17 overexpression also inhibited CpG ODN-induced IRAK1/TRAF6 complex formation. Further studies revealed that CpG ODN activated Ras in a time- and dose-dependent manner, and Ras associated with TLR9 in a CpG ODN-dependent manner. Most interestingly, activation of Ras preceded the association of Ras with TLR9, giving rise to a possibility that Ras activation might not be dependent on the interaction between Ras and TLR9. Our data demonstrate for the first time that Ras can be activated by CpG ODN in macrophages, and Ras is involved in CpG ODN signaling as an early event by associating with TLR9 and promoting IRAK1/TRAF6 complex formation, and MAPK and NF-kappaB activation.

Recent studies have suggested that statins, the inhibitors for 3-hydroxy-3-methyglutaryl (HMG)-CoA reductase in the mevalonate pathway, exhibit anti-inflammatory effects. However, the immune modulatory effects of statins on the differentiation of CD4(+) T cells and their underlying mechanisms are still largely unknown. To address these issues, we examined the effect of simvastatin and inhibitors for protein farnesylation and geranylgeranylation on the differentiation of IL-17-producing T cells (T(h)17 cells) and Foxp3(+) CD4(+) T cells. Simvastatin inhibited the differentiation of T(h)17 cells through the inhibition of HMG-CoA reductase activity but enhanced the differentiation of Foxp3(+) CD4(+) T cells. Geranylgeranyltransferase I inhibitor, GGTI-298, but not farnesyltransferase inhibitor, FTI-277, mimicked the effects of simvastatin, indicating that the inhibition of protein geranylgeranylation is responsible for the effects. Moreover, Foxp3(+) CD4(+) T cells developed in the presence of transforming growth factor-beta and GGTI-298 functioned as regulatory T cells (Tregs) in in vitro T cell proliferation assay as well as in an autoimmune colitis model. Finally, GGTI-298 induced SOCS3 expression and inhibited IL-6-induced signal transducers and activators of transcription3 phosphorylation in CD4(+) T cells. Taken together, these results indicate that protein geranylgeranylation enhances the differentiation of T(h)17 cells and inhibits the differentiation of Foxp3(+) Tregs partly via the inhibition of SOCS3 expression.

During isometric contractions, no true work is performed, so the force-time integral (FTI) is often used to approximate isometric work. However, the relationship between FTI and metabolic cost is not as linear. We tested the hypothesis that this nonlinearity was due to the cost of attaining a given force being greater than that of maintaining it. The ATP consumed per contraction in the human medial gastrocnemius muscle (n = 6) was determined by use of (31)P-NMR spectroscopy during eight different electrical stimulation protocols. Each protocol consisted of 8 trains of a single frequency (20 or 80 Hz) and duration (300, 600, 1,200, or 1,800 ms) performed under ischemic conditions. The cost of force generation was determined from the ATP turnover during the short-duration trains that did not attain a steady force level. Estimates of the cost of force maintenance at each frequency were determined by subtracting the ATP turnover during the shorter-duration trains from the turnover during the long-duration trains. The force generation phase of an isometric contraction was indeed more metabolically costly than the force maintenance phase during both 20- and 80-Hz stimulation. Thus the mean rate of ATP hydrolysis appeared to decline as contraction duration increased. Interestingly, the metabolic costs of maintaining force during 20-Hz and 80-Hz stimulation were comparable, although different levels of force were produced.

To identify possible intracellular mediators of hair cell (HC) death due to ototoxins, we treated basal-turn, neonatal, rat HCs in vitro with several intracellular signaling inhibitors, prior to and during gentamicin exposure. The general guanine nucleotide-binding protein (G-protein) inhibitor, GDP-betaS (1 mM), provided potent HC protection, suggesting involvement of G-proteins in the intracellular pathway linking gentamicin exposure to HC death. ADP-betaS had minimal effect, indicating that the protection is specific to guanosine diphosphate (GDP)-binding, rather than a general reaction to nucleotides. Azido-GTP(32) photolabeling and gel electrophoresis indicated activation of an approximately 21 kDa G-protein in HCs after exposure to gentamicin. Spectroscopic analysis of peptide fragments from this band matched its sequence with H-Ras. The Ras inhibitors B581 (50 microM) and FTI-277 (10 microM) provided potent protection against damage and reduced c-Jun activation in HC nuclei, suggesting that activation of Ras is functionally involved in damage to these cells due to gentamicin.

Destruction of tumor cells is a key function of lymphocytes, but the molecular processes driving it are unclear. Analysis of signal molecules indicated that mitogen-activated protein kinase (MAPK)/extracellular regulated kinase 2 critically controlled lytic function in human NK cells. We now have evidence to indicate that target ligation triggers a Ras-independent MAPK pathway that is required for lysis of the ligated tumor cell. Target engagement caused NK cells to rapidly activate MAPK within 5 min, and PD098059 effectively blocked both MAPK activation and tumoricidal function in NK cells. Target engagement also rapidly activated Ras, detected as active Ras-GTP bound to GST-Raf-RBD, a GST fusion protein linked to the Raf protein fragment containing the Ras-GTP binding domain. However, Ras inactivation by pharmacological disruption with the farnesyl transferase inhibitor, FTI-277, had no adverse effect on the ability of NK cells to lyse tumor cells or to express MAPK activation upon target conjugation. Notably, MAPK inactivation with PD098059, but not Ras inactivation with FTI-277, could interfere with perforin and granzyme B polarization within NK cells toward the contacted target cell. Using vaccinia delivery of N17 Ras into NK cells, we demonstrated that IL-2 activated a Ras-dependent MAPK pathway, while target ligation used a Ras-independent MAPK pathway to trigger lysis in NK cells.

Pamidronate (PAM) and zoledronic acid (ZOL) are aminobisphosphonates (BPs) able to affect the isoprenylation of intracellular small G proteins. We have investigated the antitumor activity of BPs and R115777 farnesyl transferase inhibitor (FTI) against epidermoid cancer cells. In human epidermoid head and neck KB and lung H1355 cancer cells, 48 h exposure to PAM and ZOL induced growth inhibition (IC(50) 25 and 10 microM, respectively) and apoptosis and abolished the proliferative and antiapoptotic stimuli induced by epidermal growth factor (EGF). In these experimental conditions, ZOL induced apoptosis through the activation of caspase 3 and a clear fragmentation of PARP was also demonstrated. A strong decrease of basal ras activity and an antagonism on its stimulation by EGF was recorded in the tumor cells exposed to BPs. These effects were paralleled by impaired activation of the survival enzymes extracellular signal regulated kinase 1 and 2 (Erk-1/2) and Akt that were not restored by EGF. Conversely, farnesol induced a recovery of ras activity and antagonized the proapoptotic effects induced by BPs. The combined treatment with BPs and R115777 resulted in a strong synergism both in growth inhibition and apoptosis in KB and H1355 cells. The synergistic activity between the drugs allowed BPs to produce tumor cell growth inhibition and apoptosis at in vivo achievable concentrations (0.1 micromolar for both drugs). Moreover, the combination was highly effective in the inhibition of ras, Erk and Akt activity, while farnesol again antagonized these effects. In conclusion, the combination of BPs and FTI leads to enhanced antitumor activity at clinically achievable drug concentrations that resides in the inhibition of farnesylation-dependent survival pathways and warrants further studies for clinical translation.

Farnesyltransferase inhibitor (FTI) induces apoptosis of transformed cells. This involves changes in mitochondria, including decrease of mitochondrial membrane potential and the release of cytochrome c. The released cytochrome c then induces events leading to the activation of caspase-3. In this study, we report that purine derivative cyclin-dependent kinase (Cdk) inhibitors, roscovitine and olomoucine, dramatically enhance this FTI-induced apoptosis of human cancer cell lines. We noticed the synergy between Cdk inhibitors and FTI through our screen to identify compounds that enhance FTI-induced apoptosis of promyelocytic leukemic cell line HL-60. The Cdk inhibitors by themselves do not induce apoptosis at the concentrations used. Roscovitine synergizes with FTI to release cytochrome c from mitochondria. In addition, we detected synergistic effects of FTI and roscovitine to inhibit hyperphosphorylation of retinoblastoma protein. Enhancement of FTI-induced apoptosis by roscovitine is not unique to HL-60 cells, since similar synergy was observed with a leukemic cell line CEM and a prostate cancer cell line LNCaP. In LNCaP cells, in addition to roscovitine and olomoucine, phophatidylinositol 3-kinase (PI 3-kinase) inhibitor, LY294002, was effective in enhancing FTI-induced apoptosis. However, the effects of roscovitine appear to be distinct from those of LY294002, since roscovitine did not affect Akt activity while LY294002 significantly decreased the activity of Akt. Our finding of the synergy between FTI and Cdk inhibitor is significant for understanding the mechanism of action of FTI as well as for clinical use of FTI.

SCH 66336 is a potent farnesyl transferase inhibitor (FTI) in clinical development. It efficiently prevents the membrane association of H-ras, but not K- or N-ras. Yet, in soft agar, it reverts the anchorage-independent growth of human tumor cell lines (hTCLs) harboring H-ras, K-ras, and N-ras mutations, implying that blocking farnesylation of proteins besides ras may be responsible for this effect. Experiments show that SCH 66336 altered the cell cycle distribution of sensitive human tumor cells in two distinct ways. Most sensitive hTCLs accumulated in the G(2)->>M phase after the FTI treatment, but those with an activated H-ras accumulated in G(1) phase, suggesting that the biological effects induced by FTIs in cells with an activated H-ras are distinct from other sensitive cells. A careful genotypic comparison of the hTCLs revealed that those cells with wild-type p53 are especially sensitive to the FTIs. In these cells p53 and its downstream target gene p21(Cip1) are induced after treatment with SCH 66336 for 24 h. These data suggest that cell cycle effects, either G(1) or G(2)->>M accumulation, and p53 status are important for mediating the effects of FTIs on tumor cells.