Neuroinflammation and oxidative stress underlie the pathogenesis of various neurodegenerative disorders. Here we demonstrate that sodium phenylbutyrate (NaPB), an FDA-approved therapy for reducing plasma ammonia and glutamine in urea cycle disorders, can suppress both proinflammatory molecules and reactive oxygen species (ROS) in activated glial cells. Interestingly, NaPB also decreased the level of cholesterol but involved only intermediates, not the end product of cholesterol biosynthesis pathway for these functions. While inhibitors of both geranylgeranyl transferase (GGTI) and farnesyl transferase (FTI) inhibited the activation of NF-κB, inhibitor of GGTI, but not FTI, suppressed the production of ROS. Accordingly, a dominant-negative mutant of p21(rac), but not p21(ras), attenuated the production of ROS from activated microglia. Inhibition of both p21(ras) and p21(rac) activation by NaPB in microglial cells suggests that NaPB exerts anti-inflammatory and antioxidative effects via inhibition of these small G proteins. Consistently, we found activation of both p21(ras) and p21(rac)in vivo in the substantia nigra of acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease. Oral administration of NaPB reduced nigral activation of p21(ras) and p21(rac), protected nigral reduced glutathione, attenuated nigral activation of NF-κB, inhibited nigral expression of proinflammatory molecules, and suppressed nigral activation of glial cells. These findings paralleled dopaminergic neuronal protection, normalized striatal neurotransmitters, and improved motor functions in MPTP-intoxicated mice. Consistently, FTI and GGTI also protected nigrostriata in MPTP-intoxicated mice. Furthermore, NaPB also halted the disease progression in a chronic MPTP mouse model. These results identify novel mode of action of NaPB and suggest that NaPB may be of therapeutic benefit for neurodegenerative disorders.

OBJECTIVE

A potential risk of testosterone replacement therapy is an increase in the incidence of prostate cancer, but it is unclear whether higher levels of serum testosterone are associated with a higher risk of prostate cancer. We prospectively evaluated serum androgen concentrations and prostate cancer risk.

METHODS

Included were 794 members of the Baltimore Longitudinal Study of Aging. We estimated the rate ratio (RR) of prostate cancer by entering serial measures of serum total testosterone, dehydroepiandrosterone sulfate, sex hormone binding globulin, calculated free testosterone, and free testosterone index (FTI) into a Cox proportional hazards regression model with simple updating.

RESULTS

Higher calculated free testosterone was associated with an increased age-adjusted risk of prostate cancer {RRs by quartile: 1.00, 1.52 [95% confidence interval (95% CI), 0.93-2.50], 1.16 (95% CI, 0.61-2.20), 2.59 (95% CI, 1.28-5.25); P(trend) = 0.03}, which persisted after excluding measures in men <45 years of age [RRs by quartile: 1.00, 1.33 (95% CI, 0.78-2.25), 1.26 (95% CI, 0.68-2.33), 1.89 (95% CI, 0.99-3.61); P(trend) = 0.03]. Compared to men with eugonadal FTI >> or = 0.153), men with hypogonadal FTI had a decreased risk of prostate cancer (RR, 0.51; 95% CI, 0.31-0.82).

CONCLUSIONS

Higher levels of calculated serum free testosterone are associated with an increased risk of prostate cancer. These findings suggest that men receiving testosterone therapy should be regularly monitored for prostate cancer and underscore the need for prospective trials of testosterone therapy incorporating incidence of prostate cancer as a primary safety end point.

The farnesyltransferase (FTase) inhibitor FTI-277 is highly effective at blocking oncogenic H-Ras but not K-Ras4B processing and signaling. While inhibition of processing and signaling of oncogenic K-Ras4B is more sensitive to the geranylgeranyltransferase I (GGTase I) inhibitor GGTI-286 than it is to FTI-277 in K-Ras4B-transformed NIH3T3 cells, the sensitivity of K-Ras as well as H- and N-Ras to the CAAX peptidomimetics in human tumor cell lines is not known. Here, we report that a panel of five human carcinoma cell lines from pancreatic, pulmonary, and bladder origins all express H-, N-, and K-Ras, and their respective prenylation sensitivities to the FTase and GGTase I inhibitors is variable. In all of the cell lines investigated, the prenylation of N-Ras was highly sensitive to FTI-277, and in two of the cell lines, N-Ras showed slight sensitivity to GGTI-298, an analog of GGTI-286. Although the prenylation of H-Ras was also sensitive to FTI-277, complete inhibition of H-Ras processing even at high concentrations of FTI-277 and/or GGTI-298 was never achieved. The prenylation of K-Ras, on the other hand, was highly resistant to FTI-277 and GGTI-298. Most significantly, treatment of human tumor cell lines with both inhibitors was required for inhibition of K-Ras prenylation. In one cell line, the human lung adenocarcinoma A-549, prenylation of K-Ras was highly resistant even when co-treated with both inhibitors. Furthermore, soft agar experiments demonstrated that in all the human tumor cell lines tested inhibition of K-Ras prenylation was not necessary for inhibition of anchorage-independent growth. In addition, although GGTI-298 had very little effect on soft agar growth, the combination of FTI-277 and GGTI-298 resulted in significant growth inhibition. Therefore, the results demonstrate that while FTI-277 inhibits N-Ras and H-Ras processing in the human tumor cell lines evaluated, inhibition of K-Ras processing requires both an FTase inhibitor as well as a GGTase I inhibitor, and that inhibition of human tumor growth in soft agar does not require inhibition of oncogenic K-Ras processing.

Recently we have shown that in fibroblasts (NIH 3T3 and Rat-1 cells) inhibition of protein geranylgeranylation leads to a G0/G1 arrest, whereas inhibition of protein farnesylation does not affect cell cycle distribution. Here we demonstrate that in human tumor cells the geranylgeranyltransferase-I (GGTase-I) inhibitor GGTI-298 blocked cells in G0/G1, whereas the farnesyltransferase (FTase) inhibitor FTI-277 showed a differential effect depending on the cell line. FTI-277 accumulated Calu-1 and A-549 lung carcinoma and Colo 357 pancreatic carcinoma cells in G2/M, T-24 bladder carcinoma, and HT-1080 fibrosarcoma cells in G0/G1, but had no effect on cell cycle distribution of pancreatic (Panc-1), breast (SKBr 3 and MDAMB-231), and head and neck (A-253) carcinoma cells. Furthermore, treatment of Calu-1, Panc-1, Colo 357, T-24, A-253, SKBr 3, and MDAMB-231 cells with GGTI-298, but not FTI-277, induced the protein expression levels of the cyclin-dependent kinase inhibitor p21WAF. HT-1080 and A-549 cells had a high basal level of p21WAF, and GGTI-298 did not further increase these levels. Furthermore, GGTI-298 also induces the accumulation of large amounts of p21WAF mRNA in Calu-1 cells, a cell line that lacks the tumor suppressor gene p53. There was little effect of GGTI-298 on the cellular levels of another cyclin- dependent kinase inhibitor p27KIP as well as cyclin E and cyclin D1. These results demonstrate that GGTase-I inhibitors arrest cells in G0/G1 and induce accumulation of p21WAF in a p53-independent manner and that FTase inhibitors can interfere with cell cycle events by a mechanism that involves neither p21WAF nor p27KIP. The results also point to the potential of GGTase-I inhibitors as agents capable of restoring growth arrest in cells lacking functional p53.

OBJECTIVE

Atherosclerosis is a chronic immuno-inflammatory disease involving the recruitment of monocytes and T lymphocytes to the vascular wall of arteries. Chemokines and their receptors, known to induce leukocyte migration, have recently been implicated in atherogenesis. Recent in vitro and in vivo studies have suggested that statins (3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors) have anti-inflammatory properties beyond their lipid-lowering effects. Thus, the aim of the present study was to investigate whether simvastatin reduces the expression of chemokines and chemokine receptors in two major cell types implicated in atherogenesis and to test isoprenoid intermediates involved in their regulation.

RESULTS

We performed in vitro experiments on human vascular endothelial cells and human primary macrophages. First, we have shown by ELISA that 1 microM simvastatin significantly reduced MCP-1 in endothelial cells (ECs) and macrophages stimulated with TNF-alpha or IFN-gamma, respectively. Messenger RNA analysis revealed that expression of the chemokines MCP-1, MIP-1alpha and MIP-1beta, as well as the chemokine receptors CCR1, CCR2, CCR4 and CCR5, was decreased by simvastatin, both in ECs and macrophages. Furthermore, the statin effects were reversed by mevalonate and mimicked by the geranylgeranyl transferase inhibitor (GGTI), whereas the farnesyl transeferase inhibitor (FTI) had no effect. These results suggests that statins act via inhibition of the geranylgeranylation of proteins.

CONCLUSIONS

Our results demonstrate that statins reduce chemokine and chemokine receptor expressions in human ECs and macrophages via inhibition of the geranylgeranylpyrophosphate pathway. Thus, our data provide further evidence that statins have anti-inflammatory properties beyond their lipid-lowering effects. These findings highlight specific novel therapeutic targets for cardiovascular diseases to reduce inflammation mediated by chemokines and their receptors.

BACKGROUND

Assessment of left (LV) ventricular function is one of the most important tasks of cardiovascular magnetic resonance (CMR). Impairment of LV deformation is a strong predictor of cardiovascular outcome in various cardiac diseases like ischemic heart disease or cardiomyopathies. The aim of the study was to provide reference values for myocardial deformation derived from the CMR feature tracking imaging (FTI) algorithm in a reference population of healthy volunteers.

METHODS

FTI was applied to standard short axis and 2-, 3- and 4-chamber views of vector-ECG gated CMR cine SSFP sequences of 150 strictly selected healthy volunteers (75 male/female) of three age tertiles (mean age 45.8 yrs). Global peak and mean radial, circumferential and longitudinal endo- and myocardial systolic strain values as well as early diastolic strain rates were measured using FTI within a standard protocol on a 1.5T whole body MR scanner.

RESULTS

Global peak systolic values were 36.3 ± 8.7% for radial, -27.2 ± 4.0% for endocardial circumferential, -21.3 ± 3.3% for myocardial circumferential, -23.4 ± 3.4% for endocardial longitudinal and -21.6 ± 3.2% for myocardial longitudinal strain. Global peak values were -2.1 ± 0.5 s(-1) for radial, 2.1 ± 0.6 s(-1) for circumferential endocardial, 1.7 ± 0.5 s(-1) for circumferential myocardial, 1.8 (1.5-2.2) s(-1) for longitudinal endocardial, 1.6 (1.4-2.0) s(-1) for longitudinal myocardial early diastolic strain rates. Men showed a higher radial strain than women whereas the circumferential and longitudinal strains were lower resulting in less negative values. Circumferential and longitudinal strain rates were significantly higher in female subjects. Radial strain increased significantly with age whereas the diastolic function measured by the radial, circumferential and longitudinal strain rates showed a decrease. The coefficients of variation determined in ten further subjects, who underwent two CMR examinations within 12 days, were -4.8% for circumferential and -4.5% for longitudinal endocardial mean strains.

CONCLUSIONS

Myocardial deformation analysis using FTI is a novel technique and robust when applied to standard cine CMR images providing the possibility of a reliable, objective quantification of global LV deformation. Since strain values and strain rates differed partly between genders as well as between age groups, the application of specific reference values as provided by this study is recommendable.

OBJECTIVE

A challenge of pulmonary vein isolation (PVI) in catheter ablation for paroxysmal atrial fibrillation (PAF) is electrical reconnection of the PV. EFFICAS I showed correlation between contact force (CF) parameters and PV durable isolation but no prospective evaluation was made. EFFICAS II was a multicentre study to prospectively assess the impact of CF guidance for an effective reduction of PVI gaps.

RESULTS

Pulmonary vein isolation using a radiofrequency (RF) ablation catheter with an integrated force sensor (TactiCath™) was performed in patients with PAF. Operators were provided EFFICAS I-based CF guidelines [target 20 g, range 10-30 g, minimum 400 g s force-time integral (FTI)]. Conduction gaps were assessed by remapping of PVs after 3 months, and gap rate was compared with EFFICAS I outcome. At follow up, 24 patients had 85% of PVs remaining isolated, compared with 72% in EFFICAS I (P = 0.037) in which CF guidelines were not used. The remaining 15% of gaps correlated to the number of catheter moves at creating the PVI line, quantified as Continuity Index. For PV lines with contiguous lesions and low catheter moves, durable isolation was 81% in EFFICAS I and 98% in EFFICAS II (P = 0.005). At index procedure, the number of lesions was reduced by 15% in EFFICAS II vs. EFFICAS I.

CONCLUSIONS

The use of CF with the above guidelines and contiguous deployment of RF lesions in EFFICAS II study resulted in more durable PVI in catheter ablation of PAF.

Farnesyltransferase inhibitors (FTIs) represent a novel class of anticancer drugs that exhibit a remarkable ability to inhibit malignant transformation without toxicity to normal cells. However, the mechanism by which FTIs inhibit tumor growth is not well understood. Here, we demonstrate that FTI-277 inhibits phosphatidylinositol 3-OH kinase (PI 3-kinase)/AKT2-mediated growth factor- and adhesion-dependent survival pathways and induces apoptosis in human cancer cells that overexpress AKT2. Furthermore, overexpression of AKT2, but not oncogenic H-Ras, sensitizes NIH 3T3 cells to FTI-277, and a high serum level prevents FTI-277-induced apoptosis in H-Ras- but not AKT2-transformed NIH 3T3 cells. A constitutively active form of AKT2 rescues human cancer cells from FTI-277-induced apoptosis. FTI-277 inhibits insulin-like growth factor 1-induced PI 3-kinase and AKT2 activation and subsequent phosphorylation of the proapoptotic protein BAD. Integrin-dependent activation of AKT2 is also blocked by FTI-277. Thus, a mechanism for FTI inhibition of human tumor growth is by inducing apoptosis through inhibition of PI 3-kinase/AKT2-mediated cell survival and adhesion pathway.

Previous studies have suggested that exposure to polychlorinated biphenyls (PCBs) may alter thyroid function, but data on effects of PCB exposure on other endogenous hormones has been lacking. The current study is ancillary to a larger investigation of the effects of Great Lakes fish consumption on PCBs and reproductive function. In the current study we examine associations of PCBs, 1,1-bis (4-chlorophenyl)-2,2-dichloroethene (DDE), and fish consumption with thyroid and steroid hormones in 178 men and PCBs, DDE, and fish consumption with thyroid hormones in 51 women from the original study. Serum PCB level and consumption of Great Lakes fish are associated with significantly lower levels of thyroxine (T(4)) and free thyroxine index (FTI) in women and with significantly lower levels of T(4) in men. Fish consumption, but not PCB level, is significantly and inversely associated with triiodothyronine (T(3)) in men. Results for thyroid-stimulating hormone (TSH) are inconsistent. Among men, there are significant inverse associations of both PCB and fish consumption with sex hormone-binding globulin (SHBG)-bound testosterone, but no association with SHBG or free testosterone. There are no significant overall associations of PCB, DDE, or fish consumption with estrone sulfate, follicle-stimulating hormone, luteinizing hormone, or dehydroepiandrosterone sulfate. The results of this study are consistent with previous studies showing effects of fish consumption and PCB exposure on thyroid hormones and suggest that PCBs may also decrease steroid binding to SHBG. Elucidation of specific mechanisms must await future investigations.

Hutchinson-Gilford progeria syndrome (HGPS) is a progeroid syndrome characterized by multiple aging-like disease phenotypes. We recently reported that a protein farnesyltransferase inhibitor (FTI) improved several disease phenotypes in mice with a HGPS mutation (Lmna(HG/+)). Here, we investigated the impact of an FTI on the survival of Lmna(HG/+) mice. The FTI significantly improved the survival of both male and female Lmna(HG/+) mice. Treatment with the FTI also improved body weight curves and reduced the number of spontaneous rib fractures. This study provides further evidence for a beneficial effect of an FTI in HGPS.

Comparison of the malaria parasite and mammalian protein prenyltransferases and their cellular substrates is important for establishing this enzyme as a target for developing antimalarial agents. Nineteen heptapeptides differing only in their carboxyl-terminal amino acid were tested as alternative substrates of partially purified Plasmodium falciparum protein farnesyltransferase. Only NRSCAIM and NRSCAIQ serve as substrates, with NRSCAIM being the best. Peptidomimetics, FTI-276 and GGTI-287, inhibit the transferase with IC(50) values of 1 and 32 nm, respectively. Incubation of P. falciparum-infected erythrocytes with [(3)H]farnesol labels 50- and 22-28-kDa proteins, whereas [(3)H]geranylgeraniol labels only 22-28-kDa proteins. The 50-kDa protein is shown to be farnesylated, whereas the 22-28-kDa proteins are geranylgeranylated, irrespective of the labeling prenol. Protein labeling is inhibited more than 50% by either 5 microm FTI-277 or GGTI-298. The same concentration of inhibitors also inhibits parasite growth from the ring stage by 50%, decreases expression of prenylated proteins as measured with prenyl-specific antibody, and inhibits parasite differentiation beyond the trophozoite stage. Furthermore, differentiation specific prenylation of P. falciparum proteins is demonstrated. Protein labeling is detected predominantly during the trophozoite to schizont and schizont to ring transitions. These results demonstrate unique properties of protein prenylation in P. falciparum: a limited specificity of the farnesyltransferase for peptide substrates compared with mammalian enzymes, the ability to use farnesol to label both farnesyl and geranylgeranyl moieties on proteins, differentiation specific protein prenylation, and the ability of peptidomimetic prenyltransferase inhibitors to block parasite differentiation.

Farnesyltransferase (FTase) is a zinc enzyme that has been the subject of particular attention in anti-cancer research. This enzyme promotes the addition of a farnesyl group from farnesyl diphosphate (FPP) to a cysteine residue of a protein substrate containing a typical -CAAX motif at the carboxyl terminus. Initial interest in FTase inhibition was prompted by the finding that farnesylation was absolutely required for the oncogenic forms of ras proteins to transform cells, as ras proteins have been implicated in around 30% of all human cancers. This discovery led to frenetic search for FTase inhibitors (FTIs), with more than 400 patents registered in less than a decade. However, despite the very promising initial results, the outcome of Phase II and Phase III clinical trials was, is general, rather disappointing, with the most advanced FTIs failing to demonstrate anti-tumor activity in ras dependent cancers, presumably because K-ras, the most frequently mutated form of ras in human cancers, is able to bypass FTI blockade through cross-prenylation by the related enzyme geranylgeranyltransferase I (GGTase I). Surprisingly, several of these compounds were later shown to have anti-tumor activity against non-ras dependent cancers, launching the grounds for a new and exciting era in FTIs research and development, although the precise target for the FTIs activity of these compounds still remains unknown. This review reports the recent progress in the field, presenting a comprehensive summary of the most promising FTIs, in terms of their chemical structure and properties, taking into account the topology of the enzyme's active-site, and the most recent mechanistic results on the catalytic activity of FTase, both at the theoretical and mechanistic level. These features are presented in close linking with the available results on the biological activity of these inhibitors, and with the outcome of the most recent clinical trials.

The Pedar system is one of the most commonly used systems for in-shoe pressure measurement. Good repeatability is necessary to ensure the consistency of measurements on which clinical judgements are based. In addition, there is a need to establish a range of normal in-shoe pressure values, which will help to identify abnormalities. The aim of this study was to assess the repeatability of the Pedar system and determine the pressure values in normal subjects. Fifty-three subjects, 17 females (32%) and 36 males (68%), were recruited and measurements were performed twice with an average gap of 12 days (range 1-32 days) using only one brand of standardised running shoes (Donnay International). Peak pressure (PP), contact area (CA), contact time (CT), pressure-time integral (PTI), force-time integral (FTI) and instant of peak pressure (IPP) were calculated. The coefficient of repeatability (CR), expressed as a percentage of the mean, was no greater than 15.3% for all 122 parameters considered. The highest PP areas were under the great toe, with mean (S.D.) equal to 280.4 (83.0) kPa and heel 264.3 (44.1) kPa, followed by the first 248 (70.1) kPa, second 246.5 (48.3) kPa, and third 224.7 (50.4) kPa metatarsal heads. The CA was highest under the heel at 41.54 cm(2). The CT of the metatarsals was 77% to 87% of the total CT while that of the hallux was 75%. The PTI and FTI were highest under the heel. We concluded that the Pedar system was repeatable. The normal pressure values identified can therefore be used to provide a reference range in clinical practice using this specific type of footwear.

Recent results have shown that the ability of farnesyltransferase inhibitors (FTIs) to inhibit malignant cell transformation and Ras prenylation can be separated. We proposed previously that farnesylated Rho proteins are important targets for alternation by FTIs, based on studies of RhoB (the FTI-Rho hypothesis). Cells treated with FTIs exhibit a loss of farnesylated RhoB but a gain of geranylgeranylated RhoB (RhoB-GG), which is associated with loss of growth-promoting activity. In this study, we tested whether the gain of RhoB-GG elicited by FTI treatment was sufficient to mediate FTI-induced cell growth inhibition. In support of this hypothesis, when expressed in Ras-transformed cells RhoB-GG induced phenotypic reversion, cell growth inhibition, and activation of the cell cycle kinase inhibitor p21WAF1. RhoB-GG did not affect the phenotype or growth of normal cells. These effects were similar to FTI treatment insofar as they were all induced in transformed cells but not in normal cells. RhoB-GG did not promote anoikis of Ras-transformed cells, implying that this response to FTIs involves loss-of-function effects. Our findings corroborate the FTI-Rho hypothesis and demonstrate that gain-of-function effects on Rho are part of the drug mechanism. Gain of RhoB-GG may explain how FTIs inhibit the growth of human tumor cells that lack Ras mutations.

Farnesylation of the oncoprotein Ras is required for its cancer-causing activity. We have designed farnesyltransferase inhibitor (FTI)-276, a tetrapeptide mimetic of the carboxyl terminus of K-Ras4B, as a highly potent and selective inhibitor of Ras farnesylation in vitro and in vivo. FTI-276 blocked the growth in nude mice of a human lung carcinoma that expresses the two most prevalent genetic alterations in human cancers (K-Ras oncogenic mutation and deletion in the tumor suppressor gene p53). In contrast, FTI-276 did not inhibit tumor growth of a human lung carcinoma that harbors no Ras mutations. Furthermore, FTI-276 inhibited oncogenic signaling and tumor growth of NIH 3T3 cells transformed with the ras but not the raf oncogene. Inhibition of tumor growth in vivo was dose dependent and correlated with inhibition of Ras processing in tumors in vivo. The work described here identifies FTI-276 as a highly selective suppressor of Ras-dependent oncogenicity and suggests that a broad spectrum of human cancers with aberrant Ras function could benefit from farnesyltransferase inhibitor treatment.

Protein farnesyltransferase inhibitors (FTIs) inhibit Ras transformation and Ras-dependent tumor cell growth, but the biological mechanisms underlying these activities is unclear. In previous work, we presented support for the hypothesis that the anti-transforming effects of FTIs depend upon alterations in the function of RhoB, a member of the Rho family of proteins that regulate cytoskeletal actin, cell adhesion, and cell growth. A significant question that needed to be addressed was whether FTIs could directly alter the prenylation as well as the function of RhoB in cells. This issue is complex because farnesylated and geranylgeranylated forms of RhoB (RhoB-F and RhoB-GG) both exist in cells. Here, we show that RhoB farnesylation in vitro can be catalyzed by protein farnesyltransferase and that the peptidomimetic FTI L-739,749 inhibits the farnesylation of RhoB both in vitro and in intact cells. In drug-treated cells, the level of RhoB-GG increased in parallel with the decrease in RhoB-F. In addition to altering RhoB prenylation, L-739,749 suppressed RhoB-dependent cell growth. Taken together, the results suggest that the inhibitory effects of FTIs on RhoB function can be mediated by a relative loss of RhoB-F, a gain of RhoB-GG, or both. Our findings strengthen the causal link between RhoB inhibition and the anti-transforming effects of FTIs and indicate that differently prenylated forms of RhoB may have unique functions.

Farnesyl transferase (FT) inhibitors (FTI) are anticancer agents developed to target oncogenic Ras proteins by inhibiting Ras farnesylation. FTIs potently synergize with paclitaxel and other microtubule-stabilizing drugs; however, the mechanistic basis underlying this synergistic interaction remains elusive. Here we show that the FTI lonafarnib affects the microtubule cytoskeleton resulting in microtubule bundle formation, increased microtubule stabilization and acetylation, and suppression of microtubule dynamics. Notably, treatment with the combination of low doses of lonafarnib with paclitaxel markedly enhanced tubulin acetylation (a marker of microtubule stability) as compared with either drug alone. This synergistic effect correlated with FT inhibition and was accompanied by a synergistic increase in mitotic arrest and cell death. Mechanistically, we show that the combination of lonafarnib and paclitaxel inhibits the in vitro deacetylating activity of the only known tubulin deacetylase, histone deacetylase 6 (HDAC6). In addition, the lonafarnib/taxane combination is synergistic only in cells lines expressing the wild-type HDAC6, but not a catalytic-mutant HDAC6, revealing that functional HDAC6 is required for the synergy of lonafarnib with taxanes. Furthermore, tubacin, a specific HDAC6 inhibitor, synergistically enhanced tubulin acetylation in combination with paclitaxel, similar to the combination of lonafarnib and paclitaxel. Taken together, these data suggest a relationship between FT inhibition, HDAC6 function, and cell death, providing insight into the putative molecular basis of the lonafarnib/taxane synergistic antiproliferative combination.

This study compared the results of various testosterone assays in a cross-sectional study of 50 male subjects age 28 to 90 years. The purpose of the study was to determine the relationship of the various testosterone assays to one another. In addition, we determined week-to-week variability in testosterone and bioavailable testosterone in 16 subjects. The following assays were undertaken: total testosterone (T), free testosterone by equilibrium dialysis (FT(D)), bioavailable testosterone (BT), free testosterone by ultracentrifugation (FT(U)), and direct estimation of serum free T by an analog ligand radioimmunoassay (FT(A)). In addition, using total T and sex hormone-binding globulin (SHBG), we calculated the free androgen index (FAI = T/SHBG) and the free testosterone index (FTI) using the method of Vermeulen. In a second study, we measured the week-to-week variation in T and BT in a group of older males. Lastly, we demonstrated excellent stability of serum stored at -70 degrees C for up to 7 years for T and BT. Correlations of the various assays to increasing age were significant for all assays except total T (r = -.126). The best correlation was found with BT (r = -.744, P <.001). All measures were statistically significantly correlated with FT. The best correlations were FTI (r =.807, P <.007) and BT (r =.670, P <.001). If T was used to classify hypogonadism in comparison to BT, it resulted in misclassification in 42% of cases. In addition, we demonstrated a marked week-to-week variability in T and BT in older men with the T and BT being in the eugonadal range some weeks and hypogonadal on other occasions. This occurred in 8 of 16 men for T and 10 of 16 men for BT. Based on these data, we suggest that the FTI or BT are the most practical methods to determine hypogonadism. There is a need for increased awareness that marked week-to-week variability within a single individual can occur for measurements of both T and BT.

Nitrogen-containing bisphosphonate drugs inhibit bone resorption by inhibiting FPP synthase and thereby preventing the synthesis of isoprenoid lipids required for protein prenylation in bone-resorbing osteoclasts. NE10790 is a phosphonocarboxylate analogue of the potent bisphosphonate risedronate and is a weak anti-resorptive agent. Although NE10790 was a poor inhibitor of FPP synthase, it did inhibit prenylation in J774 macrophages and osteoclasts, but only of proteins of molecular mass approximately 22-26 kDa, the prenylation of which was not affected by peptidomimetic inhibitors of either farnesyl transferase (FTI-277) or geranylgeranyl transferase I (GGTI-298). These 22-26-kDa proteins were shown to be geranylgeranylated by labelling J774 cells with [(3)H]geranylgeraniol. Furthermore, NE10790 inhibited incorporation of [(14)C]mevalonic acid into Rab6, but not into H-Ras or Rap1, proteins that are modified by FTase and GGTase I, respectively. These data demonstrate that NE10790 selectively prevents Rab prenylation in intact cells. In accord, NE10790 inhibited the activity of recombinant Rab GGTase in vitro, but did not affect the activity of recombinant FTase or GGTase I. NE10790 therefore appears to be the first specific inhibitor of Rab GGTase to be identified. In contrast to risedronate, NE10790 inhibited bone resorption in vitro without markedly affecting osteoclast number or the F-actin "ring" structure in polarized osteoclasts. However, NE10790 did alter osteoclast morphology, causing the formation of large intracellular vacuoles and protrusion of the basolateral membrane into large, "domed" structures that lacked microvilli. The anti-resorptive activity of NE10790 is thus likely due to disruption of Rab-dependent intracellular membrane trafficking in osteoclasts.

The goal of this study was to identify the clinical and biological patterns of hypogonadism in a cohort of 1040 elderly men. Residual androgenic activity was estimated by total testosterone as well as the apparent free testosterone concentration (AFTC) and free testosterone index (FTI) calculated on the basis of concentrations of SHBG and total testosterone using appropriate formulae. The lower limit of the normal range defined by 2 SD below the mean in 150 healthy, nonobese, and nonsmoking men, aged 19-40 yr, was calculated for total testosterone (9.26 nmol/liter), AFTC (146 pmol/liter), and FTI (0.14 nmol/nmol). The prevalence of hypogonadism increased with ageing. Hypogonadal men were older and heavier (due to a higher fat body mass) and had lower concentrations of 17 beta-estradiol and androstenedione than men with normal androgenic activity. Men with decreased AFTC had a slightly lower bone mineral density (BMD) at certain sites. Men with decreased FTI had lower appendicular skeletal muscle mass and relative skeletal muscle index. For all three measures of androgenic activity, hypogonadal men had increased levels of the markers of bone resorption. In the multiple regression models including both 17 beta-estradiol and testosterone (total, AFTC, or FTI), 17 beta-estradiol was the only significant determinant of BMD. In the multiple regression models including 17 beta-estradiol and AFTC or FTI, only testosterone was a significant determinant of the variability in bone formation markers, whereas both 17 beta-estradiol and testosterone were significant determinants of the variability of the markers of bone resorption. Hypogonadism was associated with an increase in the risk of falls, an impairment of static and dynamic balance, as well as the inability to stand up from a chair and to perform the tandem walk. Decreased AFTC (<146 pmol/liter) discriminated best men with functional disabilities (odds ratio, 1.54-7.95; P < 0.05-0.0001). Hypogonadal elderly men had increased bone resorption that was not adequately matched by an increase in bone formation, lower muscle strength, impaired static and dynamic balance, a higher risk of falls, and, in men with low AFTC, a slightly lower BMD. Low AFTC seems to have the best discriminative power for densitometric, biochemical, and functional parameters, followed by FTI, whereas total testosterone was the least discriminative. In multiple regression models, 17 beta-estradiol was the strongest determinant of BMD, and AFTC and FTI were significant determinants of the variability in bone formation markers, whereas both 17 beta-estradiol and testosterone determined the variability in bone resorption markers.

Pancreatic and colorectal carcinomas frequently express oncogenic/mutant K-Ras that contributes to both tumorigenesis and clinically observed resistance to radiation treatment. We have previously shown that farnesyltransferase inhibitors (FTI) radiosensitize many pancreatic and colorectal cancer cell lines that express oncogenic K-ras at doses that inhibit the prenylation and activation of H-Ras but not K-Ras. In the present study, we have examined the mechanism of FTI-mediated radiosensitization in cell lines that express oncogenic K-Ras and found that wild-type H-Ras is a contributor to radiation survival in tumor cells that express oncogenic K-Ras. In these experiments, inhibiting the expression of oncogenic K-Ras, wild-type H-Ras, or epidermal growth factor receptor (EGFR) led to similar levels of radiosensitization as treatment with the FTI tipifarnib. Treatment with the EGFR inhibitor gefitinib led to similar levels of radiosensitization, and the combinations of tipifarnib or gefitinib plus inhibition of K-Ras, H-Ras, or EGFR expression did not provide additional radiosensitization compared with tipifarnib or gefitinib alone. Finally, supplementing culture medium with the EGFR ligand transforming growth factor alpha was able to reverse the radiosensitizing effect of inhibiting K-ras expression. Taken together, these findings suggest that EGFR-activated H-Ras signaling is initiated by oncogenic K-Ras to promote radiation survival in pancreatic and colorectal cancers.

Muscle mass and strength losses during aging may be associated with declining levels of serum testosterone (T) in men. Few studies have shown a direct relationship between T and muscle mass and strength. Subjects were 262 men, aged 24-90 yr, from the Baltimore Longitudinal Study of Aging, who had T and sex hormone-binding globulin sex hormone-binding globulin (SHBG) measurements, from which the free T index (FTI) was calculated (T/SHBG) from serum samples collected longitudinally since 1963, total body fat mass and arm and leg fat-free mass (FFM) by dual-energy X-ray absorptiometry and arm and leg strength by dynanomometry. Mixed-effects models estimated T and FTI at the time of mass and strength measurements. Age, total body fat, arm and leg FFM, T, and FTI were significantly associated with concentric and eccentric strength. FTI, not T, was modestly, but directly, related to arm and leg strength after fat, arm and leg FFM, height, and age were accounted for and indirectly through body mass. FTI is a better predictor of arm and leg strength than T in aging men.

Despite the success of protein farnesyltransferase inhibitors (FTIs) in the treatment of certain malignancies, their mode of action is incompletely understood. Dissecting the molecular pathways affected by FTIs is important, particularly because this group of drugs is now being tested for the treatment of Hutchinson-Gilford progeria syndrome. In the current study, we show that FTI treatment causes a centrosome separation defect, leading to the formation of donut-shaped nuclei in nontransformed cell lines, tumor cell lines, and tissues of FTI-treated mice. Donut-shaped nuclei arise during chromatin decondensation in late mitosis; subsequently, cells with donut-shaped nuclei exhibit defects in karyokinesis, develop aneuploidy, and are often binucleated. Binucleated cells proliferate slowly. We identified lamin B1 and proteasome-mediated degradation of pericentrin as critical components in FTI-induced "donut formation" and binucleation. Reducing pericentrin expression or ectopic expression of nonfarnesylated lamin B1 was sufficient to elicit donut formation and binucleated cells, whereas blocking proteasomal degradation eliminated FTI-induced donut formation. Our studies have uncovered an important role of FTIs on centrosome separation and define pericentrin as a (indirect) target of FTIs affecting centrosome position and bipolar spindle formation, likely explaining some of the anticancer effects of these drugs.

Treatment of H-Ras transgenic mice with the geranylgeranyltransferase I (GGTase I) inhibitor GGTI-2154 results not only in halting the growth of aggressive breast tumors but actually in inducing the regression (54 +/- 3%) of all 19 tumors analyzed. The farnesyltransferase (FTase) inhibitor FTI-2148 induced an average of 87 +/- 3% regression in the 13 tumors analyzed. GGTase I, but not FTase, is inhibited in breast tumors after treatment with GGTI-2154, whereas in tumors from mice treated with FTI-2148, only FTase is inhibited. The processing of the geranylgeranylated proteins RhoA, Rap1, and R-Ras, but not the farnesylated proteins H-Ras and HDJ-2, is inhibited in tumors obtained from mice treated with GGTI-2154. GGTI-2154 and FTI-2148 suppress constitutively activated phospho-Erk1/2 and phospho-Akt, induce apoptosis, and induce differentiation toward ductolobular breast epithelium. The data demonstrate that geranylgeranylated proteins are critical in H-Ras oncogenesis in vivo and give strong support for GGTase I as a target for anticancer drug discovery.