OBJECTIVE

Posttranslational farnesylation is required for Ras activation. Farnesyl transferase inhibitors (FTIs) selectively block protein farnesylation and reduce the growth of many Ras-transformed cells in vitro and in vivo. Activated Ras transforms rat intestinal epithelial (RIE-1) cells by a mechanism distinct from NIH 3T3 fibroblasts in that an epidermal growth factor receptor (EGFR) autocrine loop contributes significantly to the Ras-transformed RIE-1 phenotype.

METHODS

The ability of FTIs to block growth of Ras-transformed RIE-1 cells was evaluated, and these results were correlated with decreased EGFR ligand production.

RESULTS

FTI L744,832 caused a selective, dose-dependent, reversible blockade in proliferation of H-Ras-transformed RIE-1 cells, whereas control cell lines, K-Ras-transformed cells, and activated raf-transfected RIE cells were unaffected. The growth-inhibitory effects of L744,832 correlated with loss of farnesylated H-Ras protein and a marked reduction in transforming growth factor (TGF)-alpha and amphiregulin expression. Inhibition of proliferation of H-Ras RIE-1 cells by L744,832 was overcome by exogenous TGF-alpha, and enhanced growth inhibition was achieved by EGFR blockade in combination with L744,832. +

CONCLUSIONS

These data suggest that one mechanism by which FTIs inhibit growth of H-Ras-transformed epithelial cells is by reducing Ras-induced EGFR ligand production.

Src transformation of NIH3T3 mouse fibroblasts has been shown to be dependent on Ras function. Since we recently showed that the signaling pathways that mediate Ras transformation of RIE-1 rat intestinal epithelial cells are distinct from those that cause Ras transformation of fibroblasts, we utilized three approaches to determine if Src transformation of RIE-1 cells is dependent on Ras. First, although both Ras and Src cause upregulation of an epidermal growth factor (EGF) receptor-dependent autocrine growth loop, only Ras transformation required this activity. Second, whereas both Src and Ras caused upregulation of the p42 and p44 mitogen-activated protein kinases (MAPKs), only Ras transformation was blocked by the inhibition of MAPK activation by treatment with the PD 98059 MEK inhibitor. Third, treatment with the farnesyltransferase inhibitor FTI-277 blocked Ras, but not Src, transformation. Taken together, these observations suggest that Src transformation of RIE-1 cells is not dependent on Ras. Finally, we determined that Ras activation of Raf-independent pathways alone is sufficient to cause growth transformation of RIE-1 cells. Thus, both Ras and Src cause transformation of RIE-1 cells via pathways distinct from those required to cause transformation of NIH3T3 cells.

Pancreatic ductal adenocarcinoma is a highly lethal malignancy that is resistant to traditional cytotoxic therapy. High rates of activating codon 12 K-Ras mutations in this disease have generated considerable interest in the therapeutic application of novel farnesyl transferase inhibitors (FTIs). However, a comprehensive analysis of the effects of FTI treatment on pancreatic cancer cells has not been performed. Treatment of five different human pancreatic cancer cell lines with FTI L-744,832 resulted in inhibition of anchorage-dependent growth, with wide variation in sensitivity among different lines. Effective growth inhibition by L-744,832 correlated with accumulation of cells with a tetraploid (4N) DNA content and high levels of cyclin B1/cdc2 kinase activity, implying cell cycle arrest downstream from the DNA damage-inducible G2/M cell cycle checkpoint. In addition, sensitive cell lines underwent apoptosis as evidenced by changes in nuclear morphology and internucleosomal DNA fragmentation. L-744,832 at a concentration of 1 microM additively enhanced the cytotoxic effect of ionizing radiation, apparently by overriding G2/M checkpoint activation. The effects of FTI treatment on cell growth and cell cycle regulation were associated with changes in posttranslational processing of H-Ras and N-Ras, but not K-Ras. The results confirm the potential therapeutic efficacy of FTI treatment in pancreatic cancer, and suggest that farnesylated proteins other than K-Ras may act as important regulators of G2/M cell cycle kinetics.

Indices of thyroid function were measured in 108 infants born at 23-31 weeks gestation, after birth, at 24 and 72 h, and at 1, 3, 4, 5 and 6 weeks of age. This group was characterised by low serum thyroxine (T4), normal thyroid stimulating hormone (TSH), low-normal thyroid binding globulin (TBG), low free thyroxine index (FTI) and low triiodothyronine (T3). The incidence of hypothyroxinaemia defined as a serum T4 value of less than 65 nmol/l was 58% after birth, increasing to 84% at 1 week, after which there was progressive reduction to 36% by 6 weeks of age. Mean T4 values were inversely proportional to gestational age during this study period. Infants of 23-28 weeks gestation had significantly lower T4, TBG, FTI and T3 values compared to those of 29-31 weeks gestation. Infants who had hyaline membrane disease (HMD) had significantly lower T4 and FTI values compared to those without HMD for up to 3 weeks of age. Similar differences were found between deaths and survivors in the first week after birth. This study suggests that there is increasing delay in maturation of the hypothalamic-pituitary-thyroid axis control with increasing prematurity. In addition, the data suggest that infants who were extremely preterm or those with HMD had worse and more persistent abnormalities of thyroid function secondary to their illness and metabolic stress. The significance of our findings, in particular that of prolonged hypothyroxinaemia, is uncertain. The role of thyroid replacement therapy in these very preterm infants therefore need to be assessed with a randomised clinical trial.

Simvastatin inhibits 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, which catalyses conversion of HMG-CoA to mevalonate, a rate-limiting step in cholesterol synthesis. We demonstrated that simvastatin at 1 microM markedly inhibited adipocyte differentiation measured by Oil Red O staining in preadipocyte cells (3T3-L1), while expression of leptin, a marker of adipocyte differentiation, was suppressed by 1 muM simvastatin for up to 12 days of culture. Next, to elucidate mechanisms underlying the reduction of leptin expression induced by simvastatin, differentiated 3T3-L1 adipocytes were treated with various inhibitors with mevalonate or its metabolite in the presence or absence of simvastatin. Simvastatin time- and dose-dependently suppressed leptin mRNA expression. Heterogeneous nuclear RNA related to leptin mRNA was inhibited by 10 muM simvastatin, while stability of the mRNA was not changed by treatment with simvastatin in transcription-arrested 3T3-L1 cells. Simvastatin inhibition of leptin gene transcription was not abrogated by pre-treatment with cycloheximide, an inhibitor of protein synthesis. Addition of mevalonate or geranylgeranyl pyrophosphate (GGPP), a mevalonate metabolite, abolished simvastatin-induced inhibition of leptin expression in 3T3-L1 cells. Suppression of expression was observed upon addition of GGTI-298, a geranylgeranyl transferase I inhibitor, but not FTI-277, a farnesyl transferase inhibitor. Expression was suppressed by treatment with hydroxyfasudil, a protein prenylation inhibitor. Treatment with phosphatidylinositol 3-kinase (PI3K) inhibitors, LY294002 and wortmannin, reduced leptin expression in 3T3-L1 cells. Simvastatin dose-dependently increased intra-cellular cyclic AMP (cAMP) concentrations in 3T3-L1 cells, with maximal stimulation obtained at 10 muM. Addition of GGPP abolished simvastatin-induced stimulation of cAMP accumulation and protein kinase A (PKA) activity. H89, an inhibitor of PKA, completely abolished simvastatin-induced suppression of leptin expression. These results suggested that simvastatin reduced geranylgeranylprotein prenylation followed by deactivation of PI3K, leading to cAMP accumulation and subsequent activation of PKA in differentiated 3T3-L1 adipocytes. Finally, PKA inhibited leptin gene transcription without new protein synthesis.

Neurofibromatosis type 1 (NF1) is a genetic disorder that is driven by the loss of neurofibromin (Nf) protein function. Nf contains a Ras-GTPase-activating protein domain, which directly regulates Ras signaling. Numerous clinical manifestations are associated with the loss of Nf and increased Ras activity. Ras proteins must be prenylated to traffic and functionally localize with target membranes. Hence, Ras is a potential therapeutic target for treating NF1. We have tested the efficacy of two novel farnesyl transferase inhibitors (FTIs), 1 and 2, alone or in combination with lovastatin, on two NF1 malignant peripheral nerve sheath tumor (MPNST) cell lines, NF90-8 and ST88-14. Single treatments of 1, 2, or lovastatin had no effect on Ras prenylation or MPNST cell proliferation. However, low micromolar combinations of 1 or 2 with lovastatin (FTI/lovastatin) reduced Ras prenylation in both MPNST cell lines. Furthermore, this FTI/lovastatin combination treatment reduced cell proliferation and induced an apoptotic response as shown by morphological analysis, procaspase-3/-7 activation, loss of mitochondrial membrane potential, and accumulation of cells with sub-G(1) DNA content. Little to no detectable toxicity was observed in normal rat Schwann cells following FTI/lovastatin combination treatment. These data support the hypothesis that combination FTI plus lovastatin therapy may be a potential treatment for NF1 MPNSTs.

We studied the effects of testosterone substitution on serum concentrations of lipids, lipoproteins, apoproteins and on the activity of hepatic lipase (HL) and lipoprotein lipase (LPL) in postheparin plasma and on the activity of LPL in adipose tissue (AT-LPL) in 13 male hypopituitary patients. The activities of LPL and HL in postheparin plasma were markedly increased by 1 week after a testosterone enanthate injection (P less than 0.001). The HL activity remained elevated (P less than 0.05) after 1 month's treatment, but the LPL activity declined to presubstitution levels. The prolonged substitution decreased serum apoproteins A-I and A-II (P less than 0.05). The changes of apo A-I and A-II correlated inversely with those of the free testosterone index (FTI) (r = -0.74, r = -0.67, P less than 0.05). Serum HDL-cholesterol level decreased slightly by 1 week and it correlated inversely with the increase in testosterone and the FTI (r = -0.67, r = -0.85, P less than 0.05). The results suggest that testosterone increases the activity of both lipolytic enzymes in postheparin plasma. The effect on HL appears to be more persistent than that on LPL. The data support a role for androgens in the regulation of serum lipoprotein and HDL-cholesterol levels.

Smoking is related to decreased bone mass and increased risk of osteoporotic fractures. However, the harmful effects of smoking on bone have not been well characterized. The purpose of this study was to assess the repercussions of smoking on bone mass in premenopausal women, and the relationship between these effects and parameters of mineral metabolism and hormone profile. We measured bone mineral density (BMD) in 101 premenopausal women (47 smokers, 54 nonsmokers) with dual-energy X-ray absorptiometry (DeXA) of the proximal femur and lumbar spine. In a subgroup of the sample (16 smokers, 15 nonsmokers) we measured biochemical indicators of mineral metabolism and hormone profile. BMD in the femoral neck, Ward's triangle, and the intertrochanter region was significantly lower in smokers (P < 0.05) than in nonsmokers. Concentrations of sex hormone-binding globulin were higher, and free testosterone index (FTI) was lower (P < 0.05) in smokers. We found no significant differences between the groups in parameters of mineral metabolism. Concentrations of dehydroepiandrosterone sulfate and free testosterone index were directly correlated with values of BMD in different sites. Our findings show that smoking by premenopausal women is associated with decreased BMD and characteristic changes in the hormone profile.

BACKGROUND

Assessment of longitudinal function with cardiovascular magnetic resonance (CMR) is limited to measurement of systolic excursion of the mitral annulus (MAPSE) or elaborate strain imaging modalities. The aim of this study was to develop a fast assessable parameter for the measurement of long axis strain (LAS) with CMR.

METHODS

40 healthy volunteers and 125 patients with different forms of cardiomyopathy were retrospectively analyzed. Four different approaches for the assessment of LAS with CMR measuring the distance between the LV apex and a line connecting the origins of the mitral valve leaflets in enddiastole and endsystole were evaluated. Values for LAS were calculated according to the strain formula.

RESULTS

LAS derived from the distance of the epicardial apical border to the midpoint of the line connecting the mitral valve insertion points (LAS-epi/mid) proved to be the most reliable parameter for the assessment of LAS among the different approaches. LAS-epi/mid displayed the highest sensitivity (81.6 %) and specificity (97.5 %), furthermore showing the best correlation with feature tracking (FTI) derived transmural longitudinal strain (r = 0.85). Moreover, LAS-epi/mid was non-inferior to FTI in discriminating controls from patients (Area under the curve (AUC) = 0.95 vs. 0.94, p = NS). The time required for analysis of LAS-epi/mid was significantly shorter than for FTI (67 ± 8 s vs. 180 ± 14 s, p < 0.0001). Additionally, LAS-epi/mid performed significantly better than MAPSE (Delta AUC = 0.09; p < 0.005) and the ejection fraction (Delta AUC = 0.11; p = 0.0002). Reference values were derived from 234 selected healthy volunteers. Mean value for LAS-epi/mid was -17.1 ± 2.3 %. Mean values for men were significantly lower compared to women (-16.5 ± 2.2 vs. -17.9 ± 2.1 %; p < 0.0001), while LAS decreased with age.

CONCLUSIONS

LAS-epi/mid is a novel and fast assessable parameter for the analysis of global longitudinal function with non-inferiority compared to transmural longitudinal strain.

The importance of the post-translational lipid modifications farnesylation and geranylgeranylation in protein localization and function coupled with the critical role of prenylated proteins in malignant transformation has prompted interest in their biology and the development of farnesyl transferase and geranylgeranyl transferase inhibitors (FTIs and GGTIs) as chemical probes and anticancer agents. The ability to measure protein prenylation before and after FTI and GGTI treatment is important to understanding and interpreting the effects of these agents on signal transduction pathways and cellular phenotypes, as well as to the use of prenylation as a biomarker. Here we describe protocols to measure the degree of protein prenylation by farnesyl transferase or geranylgeranyl transferase in vitro, in cultured cells and in tumors from animals and humans. The assays use [(3)H]farnesyl diphosphate and [(3)H]geranylgeranyl diphosphate, electrophoretic mobility shift, membrane association using subcellular fractionation or immunofluorescence of intact cells, [(3)H]mevalonic acid labeling, followed by immunoprecipitation and SDS-PAGE, and in vitro transcription, translation and prenylation in reticulocyte lysates. These protocols require from 1 d (enzyme assays) to up to 3 months (autoradiography of [(3)H]-labeled proteins).

Ras is a small guanosine triphosphate-binding protein that plays an important role in signal transduction pathways that influence cellular proliferation, apoptosis, cytoskeletal organization, and other important biological processes. Prenylation of Ras proteins by the enzyme farnesyltransferase renders the protein hydrophobic, causing localization to the inner surface of the cell membrane, where it exerts its biological effects. Ras mutations that result in constitutive activation of the Ras pathway are common in certain human cancers, and transfection of cell lines with mutant Ras renders them tumorigenic. Farnesyltransferase inhibitors (FTIs) were initially developed to inhibit growth of cancers harboring Ras mutations, but preclinical data suggests that they also have antiproliferative effects in cell lines with wild-type Ras. Preclinical data suggest that FTIs have antiproliferative and antitumor effects in breast cancer cell lines, but the precise target(s) remain to be defined. One phase II trial has demonstrated that one orally administered FTI has significant antitumor activity in metastatic breast cancer. In addition, preclinical evidence suggests that FTIs may augment the activity of cytotoxic agents and hormonal therapy. Clinical trials are currently underway evaluating whether these agents have a useful role in the management of advanced breast cancer.

The RAS gene product is normally a membrane-localized G protein (N-Ras, K-Ras and H-Ras) of 21 kDa classically described as a molecular off/on switch. It is inactive when bound to guanosine diphosphate and active when bound to GTP. When mutated, the gene produces an abnormal protein resistant to GTP hydrolysis by GTPase, resulting in a constitutively active GTP-bound protein that stimulates a critical network of signal transduction pathways that lead to cellular proliferation, survival and differentiation. At least three downstream effector pathways have been described, including Raf/MEK/ERK, PI3K/AKT and RalGDS, but they are not completely understood. Ras pathways are also important downstream effectors of several receptor tyrosine kinases localized in the cell membrane, most notably the BCR-ABL fusion protein seen in patients with Philadelphia chromosome positive chronic myelogenous leukemia. An important consideration in designing strategies to block Ras stimulatory effect is that Ras proteins are synthesized in the cytosol, but require post-translational modifications and attachment to anchor proteins or membrane binding sites in the cell membrane to be biologically active. Farnesyl transferase inhibitors (FTIs) are probably the best-studied class of Ras inhibitors in hematologic malignancies. They block the enzyme farnesyl-transferase (FTase), which is essential for post-translational modification. However, it has been observed that the Ras proteins also can be geranylgeranylated in the presence of FTIs, thus allowing membrane localization and activation, which limits their effectiveness. It is now hypothesized that their mechanism of action may be through FTase inhibition involving other signal transduction pathways. S-trans, trans-farnesylthiosalicylic acid, which was first designed as a prenylated protein methyltransferase inhibitor, has shown in vitro activity against all activated Ras proteins by dislodging them from their membrane-anchoring sites. Here, Ras biology, its signaling pathways and its implications as a therapeutic target in hematologic malignancies are reviewed.

BACKGROUND

Volume status, lean and fat tissue are gaining interest as prognostic predictors in patients on dialysis. Comparative data in peritoneal dialysis (PD) versus haemodialysis (HD) patients are lacking.

METHODS

In a cohort of PD (EuroBCM) and HD (Euclid database) patients, matched for country, gender, age and dialysis vintage, body composition was assessed by bioimpedance spectroscopy (BCM, Fresenius Medical Care). Time-averaged volume overload (TAVO) was defined as the mean of pre- and post-dialysis volume overload (VO), and relative (%) (TA)VO as (TA)VO/ECV.

RESULTS

Four hundred and ninety-one matched pairs (55.2% males, median age 60.0 years) were included. The body mass index (BMI, PD = 26.5 ± 4.7 versus HD = 25.9 ± 4.6 kg/m(2), P = 0.18 in males and 27.4 ± 5.8 versus 27.5 ± 6.6 kg/m(2), P = 0.75 in females) and fat tissue index (males: 11.5 ± 5.3 versus 11.4 ± 5.4 kg/m(2), P = 0.90, females: 14.8 ± 6.7 versus 15.4 ± 7.2 kg/m(2), P = 0.30) were not different in PD versus HD patients, whereas the lean tissue index (LTI) was higher in PD versus HD patients (males: 14.5 ± 3.4 versus 13.7 ± 3.1 kg/m(2), P = 0.001, females: 12.6 ± 3.3 versus 11.5 ± 2.6 kg/m(2), P < 0.0001). VO/extracellular water (ECW) was not different between PD versus just before the HD treatment (males: 10.8 ± 12.1 versus 9.2 ± 10.2%, P = 0.09; females: 6.5 ± 10.8 versus 7.7 ± 9.4%, P = 0.19). The relative TAVO was higher in PD versus HD (10.8 ± 12.1% versus 3.2 ± 11.2%, and 6.5 ± 10.8% versus 1.2 ± 10.9%, both P < 0.0001).

CONCLUSIONS

The LTI was impaired, and this was more in males versus females, but was better preserved on PD versus HD, whereas fat tissue index (FTI) was increased, but not different between PD and HD. Volume overload was more present in PD versus HD when TAVO, but not when predialysis volume status, was used as a reference.

OBJECTIVE

To evaluate how different compartments of body composition can affect survival in hemodialysis (HD) patients.

METHODS

Multicenter longitudinal observational study of a cohort of patients in HD with 12 months of follow-up.

METHODS

Patients from 34 Nephrocare dialysis units in Portugal were included.

METHODS

A total of 697 patients on maintenance HD during 4 hours 3 days per week were enrolled.

METHODS

Dry weight, presence of diabetes, body mass index (BMI), lean tissue index (LTI), fat tissue index (FTI), body cell mass index (BCMI), albumin and hydration status were recorded at baseline. In all patients, the assessment of body composition was carried out using the Body Composition Monitor (BCM; Fresenius Medical Care a Deutschland GmbH, Germany).

METHODS

Survival during a 12-month period of follow-up.

RESULTS

Patient's mean (±standard deviation) age was 65.4 ± 14.3 years, and median (interquartile range) HD vintage was 41 (19-81) months. Patients who died during the study period, had higher age (P < .001), lower dry weight (P = .001), BMI (P < .001), albumin (P < .001), LTI (P = .015), and also lower BCMI (P = .046). Patients with diabetes (P = .045), BMI < 18.5 kg/m(2) (P < .001), albumin < 4.0 g/dL (P < .001), relative overhydration ≥ 15% (P = .001), low FTI (P = .019), and also those in the lowest tertile of BCMI (P = .022) displayed a significantly worse survival. In the Cox regression analysis, the overall mortality of patient was related to low FTI, relative overhydration, BMI < 18.5 kg/m(2), BCMI ≤ 5.2 kg/m(2), and albumin < 4.0 g/dL.

CONCLUSIONS

Several body composition parameters demonstrated to have an important role in predicting 1-year mortality in HD patients. Albumin, FTI, and BMI were useful predictors of mortality in these patients.

The aim of this study was to acquire static and dynamic foot geometry and loading in childhood, and to establish data for age groups of a population of 1-13 year old infants and children. A total of 10,382 children were recruited and 7788 children (48% males and 52% females) were finally included into the data analysis. For static foot geometry foot length and foot width were quantified in a standing position. Dynamic foot geometry and loading were assessed during walking on a walkway with self selected speed (Novel Emed X, 100Hz, 4 sensors/cm(2)). Contact area (CA), peak pressure (PP), force time integral (FTI) and the arch index were calculated for the total, fore-, mid- and hindfoot. Results show that most static and dynamic foot characteristics change continuously during growth and maturation. Static foot length and width increased with age from 13.1±0.8cm (length) and 5.7±0.4cm (width) in the youngest to 24.4±1.5cm (length) and 8.9±0.6cm (width) in the oldest. A mean walking velocity of 0.94±0.25m/s was observed. Arch-index ranged from 0.32±0.04 [a.u.] in the one-year old to 0.21±0.13 [a.u.] in the 5-year olds and remains constant afterwards. This study provides data for static and dynamic foot characteristics in children based on a cohort of 7788 subjects. Static and dynamic foot measures change differently during growth and maturation. Dynamic foot measurements provide additional information about the children's foot compared to static measures.

BACKGROUND

Recently, several indexes such as tension-time index (TTI), tension-time or force-time integral (FTI), rate-pressure product (RPP), pressure-work index (PWI), and systolic pressure-volume area (PVA) have been developed as predictors of myocardial oxygen consumption in experimental and clinical studies. However, it is still unclear whether these indexes are reliable predictors of myocardial oxygen consumption under various contractile states in human hearts.

RESULTS

We assessed the relation between TTI, FTI, RPP, PWI, and PVA and myocardial oxygen consumption per beat (VO2) in 13 patients with heart disease during volume loading. Left ventricular (LV) volume and pressure were measured simultaneously by the conductance catheter with the tipped micromanometer technique. VO2 was calculated from arterial coronary sinus oxygen content difference, and coronary sinus blood flow was measured by the thermodilution method. After z transformation of the correlation coefficients, mean z value for the VO2-PVA relation (1.83 +/- 0.60) was greater than those for the VO2-TTI relation (1.22 +/- 0.66; p < 0.005), VO2-FTI relation (1.18 +/- 0.61; p < 0.05), VO2-RPP relation (0.95 +/- 0.65; p < 0.05), and VO2-PWI relation (1.24 +/- 0.58; p < 0.05). During dobutamine infusion (5 micrograms.kg-1.min-1) in five of the 13 patients, VO2 also correlated best with PVA (z = 1.70 +/- 0.89) compared with TTI (z = 1.43 +/- 0.86), FTI (z = 1.48 +/- 0.95), RPP (z = 1.00 +/- 0.53), and PWI (z = 0.88 +/- 0.80). The contractile efficiency (38 +/- 14% to 38 +/- 20%), the reciprocal of the slope of the VO2-PVA relation, remained unchanged, whereas the VO2,PVA 0.8 (VO2 at PVA = 0.8 J per beat/100 g LV) increased from 1.48 +/- 1.16 to 2.06 +/- 1.13 J per beat/100 g LV (p < 0.05). These results show the parallel upward shift of the VO2-PVA relation during dobutamine infusion. Because increases in the VO2-intercept represent the VO2 for the increased excitation-contraction (E-C) coupling associated with the augmented contractile state, the parallelism of the VO2-PVA relation could discriminate between VO2 for mechanical work (PVA-dependent VO2) and VO2 for E-C coupling (PVA-independent VO2).

CONCLUSIONS

The results of the present study indicate that PVA is a reliable and valuable predictor of myocardial oxygen consumption under different contractile states in human hearts. The VO2-PVA relation could provide useful information about mechanoenergetics in diseased human hearts.

A major contributing factor to the high mortality rate associated with acute myeloid leukemia and multiple myeloma is the development of resistance to chemotherapy. We have shown that the combination of tipifarnib, a nonpeptidomimetic farnesyltransferase inhibitor (FTI), with bortezomib, a proteosome inhibitor, promotes synergistic death and overcomes de novo drug resistance in acute myeloid leukemia cell lines. Experiments were undertaken to identify the molecular mechanisms by which tipifarnib produces cell death in acute myeloid leukemia and multiple myeloma cell lines (U937 and 8226, respectively). Tipifarnib, but not other FTIs tested [N-[4-[2(R)-amino-3-mercaptopropyl]amino-2-phenylbenzoyl]methionine methyl ester trifluoroacetate salt (FTI-277) and 2'-methyl-5-((((1-trityl-1H-imidazol-4-yl)methyl)amino)methyl)-[1,1'-biphenyl]-2-carboxylic acid (FTI-2153), promotes elevations in intracellular free-calcium concentrations ([Ca(2+)](i)) in both cell lines. These elevations in [Ca(2+)](i) were accompanied by highly dynamic plasmalemmal blebbing and frequently resulted in membrane lysis. The tipifarnib-induced elevations in [Ca(2+)](i) were not blocked by thapsigargin or ruthenium red, but were inhibited by application of Ca(2+)-free extracellular solution and by the Ca(2+) channel blockers Gd(3+) and La(3+). Conversely, 2-aminoethoxydiphenyl borate (2-APB) potentiated the tipifarnib-evoked [Ca(2+)](i) overload. Preventing Ca(2+) influx diminished tipifarnib-evoked cell death, whereas 2-APB potentiated this effect, demonstrating a link between tipifarnib-induced Ca(2+) influx and apoptosis. These data suggest that tipifarnib exerts its effects by acting on a membrane channel with pharmacological properties consistent with store-operated channels containing the Orai3 subunit. It is noteworthy that Orai3 transcripts were found to be expressed at lower levels in tipifarnib-resistant 8226/R5 cells. Our results indicate tipifarnib causes cell death via a novel mechanism involving activation of a plasma membrane Ca(2+) channel and intracellular Ca(2+) overload.

The mevalonate (MEV) cascade is responsible for cholesterol biosynthesis and the formation of the intermediate metabolites geranylgeranylpyrophosphate (GGPP) and farnesylpyrophosphate (FPP) used in the prenylation of proteins. Here we show that the MEV cascade inhibitor simvastatin induced significant cell death in a wide range of human tumor cell lines, including glioblastoma, astrocytoma, neuroblastoma, lung adenocarcinoma, and breast cancer. Simvastatin induced apoptotic cell death via the intrinsic apoptotic pathway. In all cancer cell types tested, simvastatin-induced cell death was not rescued by cholesterol, but was dependent on GGPP- and FPP-depletion. We confirmed that simvastatin caused the translocation of the small Rho GTPases RhoA, Cdc42, and Rac1/2/3 from cell membranes to the cytosol in U251 (glioblastoma), A549 (lung adenocarcinoma) and MDA-MB-231(breast cancer). Simvastatin-induced Rho-GTP loading significantly increased in U251 cells which were reversed with MEV, FPP, GGPP. In contrast, simvastatin did not change Rho-GTP loading in A549 and MDA-MB-231. Inhibition of geranylgeranyltransferase I by GGTi-298, but not farnesyltransferase by FTi-277, induced significant cell death in U251, A549, and MDA-MB-231. These results indicate that MEV cascade inhibition by simvastatin induced the intrinsic apoptosis pathway via inhibition of Rho family prenylation and depletion of GGPP, in a variety of different human cancer cell lines.

Trafficking of autoreactive leukocytes across the blood-nerve barrier and into peripheral nerves is an early pathological hallmark of Guillain-Barré syndrome (GBS). Tumor necrosis factor-α (TNF-α), a proinflammatory cytokine, promotes transendothelial migration by upregulating endothelial expression of inflammatory mediators, including CCL2, a chemokine implicated in GBS. We sought to determine the mechanism by which TNF-α induces expression and secretion of CCL2 from peripheral nerve microvascular endoneurial endothelial cells (PNMECs). Expression of CCL2 mRNA and protein in quiescent PNMEC cultures was minimal. In contrast, cultures treated with TNF-α exhibited increased CCL2 mRNA and protein content, as well as protein secretion. Simvastatin significantly attenuated TNF-α-induced CCL2 secretion without affecting CCL2 mRNA or protein expression. Co-incubation with geranylgeranyl pyrophosphate, but not farnesyl pyrophosphate, prevented the effect of simvastatin. By comparison, inhibiting protein isoprenylation with GGTI-298, but not FTI-277, mimicked the effect of simvastatin and significantly attenuated transendothelial migration in vitro. Inhibition of the monomeric GTPase Cdc42, but not Rac1 or RhoA-C, attenuated TNF-α-mediated CCL2 secretion. TNF-α-mediated trafficking of autoreactive leukocytes into peripheral nerves during GBS may proceed by a mechanism that involves Cdc42-facilitated secretion of CCL2.

Farnesyl transferase inhibitors (FTIs) target signal-transduction pathways responsible for the proliferation and survival of hematologic malignancies, including acute myelogenous leukemias (AML). Lonafarnib has been shown to be a potent inhibitor of Pgp-mediated drug efflux. On the basis of these findings, we examined the Pgp-inhibitory properties of tipifarnib and assessed its activity when combined with anthracyclines. The effects of tipifarnib on cell proliferation, induction of apoptosis and inhibition of Pgp-mediated anthracycline efflux were analyzed in two human leukemia cell lines overexpressing Pgp (CCRF-CEM and KG1a). Measurement of residual daunorubicin (DNR)-mediated fluorescence after incubation with DNR and tipifarnib demonstrated that tipifarnib significantly inhibited DNR efflux in CCRF-CEM with an IC(50) value less than 0.5 microM. Proliferation and apoptosis assays after exposure to DNR in the presence or absence of tipifarnib demonstrated synergistic inhibition of cellular proliferation, and induction of apoptosis with the combination of tipifarnib and DNR. Similar data was obtained with an enantiomer of tipifarnib that possesses no FTI activity. Incubation with tipifarnib and DNR did not interfere with inhibition of the post-translational processing of HDJ-2. These data suggest that tipifarnib possesses Pgp-inhibitory activity in addition to its FTI activity. In high risk and refractory patients these properties may be exploited as a dual targeting mechanism in the therapy of AML.

OBJECTIVE

Preliminary studies have demonstrated that the Ras family and related guanosine triphosphate-dependent proteins are overactivated in malignant gliomas and that inhibition of the activation of such proteins, by blockade of their post-translational processing, reduces tumor cell growth in vitro. The current study evaluates the utility of this therapeutic strategy in vivo, using preclinical glioma model systems.

METHODS

We examined the efficacy against U-87 human malignant glioma cells, in both subcutaneous and intracranial nude mouse models, of selective peptidomimetic inhibitors of farnesyltransferase (FTI-276) and geranylgeranyltransferase (GGTI-297), which are involved in critical steps in the post-translational processing of Ras and related guanosine triphosphate-dependent proteins. For the subcutaneous model, 2 x 10(5) U-87 cells were implanted; after measurable tumors were detected on Day 7, animals were treated with either FTI-276, GGTI-297, or vehicle, administered by continuous infusion for 7 days. Differences in tumor volumes among the treatment groups were examined for significance using a Student's t test. For the intracranial model, 2 x 10(5) U-87 cells were implanted in the right frontal lobe and treatment was initiated on Day 7. In initial studies, animals received a 7-day course of either FTI-276, GGTI-297, or vehicle. In subsequent studies, a 28-day treatment period was used. Comparisons of survival times among treatment groups were performed using a rank-sum test.

RESULTS

Although the two agents exhibited comparable antiproliferative activities in previous in vitro studies, an obvious difference in efficacy was apparent in this study. Whereas the geranylgeranyltransferase inhibitor failed to improve survival rates, compared with those observed for control animals, in either the subcutaneous or intracranial model, the farnesyltransferase inhibitor produced objective regression of tumor growth in the subcutaneous model and significant prolongation of survival times in the intracranial model, without apparent toxicity. In the subcutaneous model, tumor volumes for the control, GGTI-297-treated, and FTI-276-treated animals on Day 28 after implantation were 621+/-420, 107+/-104, and 18.5+/-12.7 mm3, respectively (P < 0.05). In the 7-day-treated intracranial model, survival times for the control, GGTI-297-treated, and FTI-276-treated groups were 27.7+/-2.9, 29.8+/-2.1, and 43.6+/-2.7 days, respectively (P < 0.001). In the 28-day-treated intracranial model, survival times for the control, GGTI-297-treated, and FTI-276-treated groups were 29.2+/-3.7, 28.3+/-3.9, and 58.7+/-6.2 days, respectively, with five of six animals in the latter group surviving more than 55 days after tumor implantation (P < 0.001).

CONCLUSIONS

These studies demonstrate that farnesyltransferase inhibition is effective in diminishing the growth of human glioma cells in vivo. Evaluation of this treatment approach in clinical trials is warranted.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare progeroid syndrome caused by mutations in the LMNA gene. Currently there is no treatment available for HGPS, but promising results from several studies using farnesyl transferase inhibitors (FTIs) on cells and animal models of HGPS have been published and a clinical trial using FTIs has been started in patients with HGPS. However, the published data from animal models treated with FTIs come from studies where the treatment was started before pronounced disease development. This study used an inducible transgenic animal model of HGPS with abnormalities of the skin and teeth. After phenotype development, the transgenic expression was turned off and a rapid improvement of the phenotype was noted, within 4 weeks of transgenic suppression. After 13 weeks, the skin was almost indistinguishable from wild-type skin. This study shows that in these tissues, expression of the progeria mutation does not cause irreversible damage and that reversal of disease phenotype is possible, which gives promise for a treatment for this disease.

Chronic biofilm formation by Pseudomonas aeruginosa in cystic fibrosis (CF) lungs is a major cause of morbidity and mortality for patients with CF. To gain insights into effectiveness of novel anti-infective therapies, the inhibitory effects of fosfomycin, tobramycin, and a 4:1 (wt/wt) fosfomycin/tobramycin combination (FTI) on Pseudomonas aeruginosa biofilms grown on cultured human CF-derived airway cells (CFBE41o-) were investigated. In preformed biofilms treated for 16 h with antibiotics, P. aeruginosa CFU per mL were reduced 4 log10 units by both FTI and tobramycin at 256 mg L(-1) , while fosfomycin alone had no effect. Importantly, the FTI treatment contained five times less tobramycin than the tobramycin-alone treatment. Inhibition of initial biofilm formation was achieved at 64 mg L(-1) FTI and 16 mg L(-1) tobramycin. Fosfomycin (1024 mg L(-1)) did not inhibit biofilm formation. Cytotoxicity was also determined by measuring lactate dehydrogenase (LDH). Intriguingly, sub-inhibitory concentrations of FTI (16 mg L(-1)) and tobramycin (4 mg L(-1)) and high concentrations of fosfomycin (1024 mg L(-1)) prevented bacterially mediated airway cell toxicity without a corresponding reduction in CFU. Overall, it was observed that FTI and tobramycin demonstrated comparable activity on biofilm formation and disruption. Decreased administration of tobramycin upon treatment with FTI might lead to a decrease in negative side effects of aminoglycosides.

Treatment with statins, inhibitors of HMG-CoA reductase, extends the survival of septic mice. However, the molecular mechanisms underlying the cholesterol-lowering, independent beneficial effects of statins in sepsis are poorly understood. The inhibition of protein isoprenylation, namely farnesylation and geranylgeranylation, has been proposed as a mediator of the pleiotropic protective effects of statins, although direct evidence is lacking. Major features of sepsis-induced immune suppression include T-cell dysfunction, which is characterized by apoptosis of splenic T cells, increased CD4(+)Foxp3(+) regulatory T cells (Tregs), and suppression of type 1 helper T-cell response [e.g., interferon-γ (IFN-γ) secretion] in mice. Here, we show that the induction of sepsis by cecal ligation and puncture (CLP) resulted in increases in farnesyltransferase activity and farnesylated proteins in the spleen relative to sham operation. Treatment with farnesyltransferase inhibitor N-[4-[2(R)-amino-3-mercaptopropyl]amino-2-phenylbenzoyl]methionine methyl ester trifluoroacetate salt (FTI-277) (25 mg/kg b.wt. i.p.) at 2 h after CLP blocked the increase in farnesylated proteins and improved survival and bacterial clearance of septic mice. FTI-277 reverted to or mitigated sepsis-induced apoptosis in spleen and thymus, increased splenic CD4(+)Foxp3(+) Tregs, and suppressed IFN-γ secretion and proliferation of splenocytes in response to anti-CD3+CD28 antibodies in mice. Moreover, FTI-277 promoted macrophage phagocytotic activity in septic mice. These results indicate that elevation in protein farnesylation plays a role in derangements in immune function and mortality of septic mice. These findings suggest that prevention of immune dysfunction might contribute to FTI-277-induced improvement in survival of septic mice. These data highlight protein farnesyltransferase as a novel potential molecular target to reduce the mortality of patients with sepsis.