The inhibitors of protein prenylation have been proposed for chemotherapy of tumors. Lovastatin, a 3-hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA) reductase inhibitor, displays proapoptotic activity in tumor cells blocking the synthesis of isoprenoids compounds. To test whether HMG-CoA reductase inhibition can induce apoptosis in proliferating thyroid cells, we studied the effects of lovastatin in normal and neoplastic thyroid cells and in primary cultures from normal human thyroids. In an immortalized human thyroid cell line (TAD-2) and in neoplastic cells, lovastatin induced cell rounding within 24 h of treatment. After 48 h the cells were detached from the plate and underwent apoptosis, as evidenced by DNA fragmentation. Morphological changes and apoptosis did not occur in serum-starved quiescent TAD-2 cells or in primary cultures of normal thyrocytes. Mevalonate, the product of the HMG-CoA reductase enzymatic activity, and the protein synthesis inhibitor cycloheximide completely blocked the effects of lovastatin in a dose-dependent fashion. The geranylgeranyl transferase GGTI-298 inhibitor mimicked the effects of lovastatin on cell morphology and induced cell death, whereas the farnesyl transferase inhibitor FTI-277 was less effective to induce both cell rounding and apoptosis. Resistance to lovastatin-induced apoptosis by expression of the viral serpine CrmA and by the peptide inhibitor of caspases, Z-DEVD-fmk, demonstrated the involvement of CrmA-sensitive, caspase-3-like proteases. Inhibition of endogenous p53 activity did not affect the sensitivity of thyroid cells to lovastatin, demonstrating that this type of apoptosis is p53 independent. We conclude that lovastatin is a potent inducer of apoptosis in proliferating thyroid cells through inhibition of protein prenylation. This type of apoptosis requires protein synthesis, is CrmA sensitive and caspase-3-like protease dependent, and is independent from p53.

Endothelin-1 (ET-1) is a potent mitogen for many cells, especially when its levels are elevated under pathological conditions, as seen in tumor cell progression and astroglial activation in neuropathies. While ET-1 is known to cause astroglial proliferation, in the present study, multiple signaling pathways involved in ET-1-mediated astrocyte proliferation were characterized. Treatment with PD98059 and U0126 (MEK inhibitors) inhibited not only ET-1-induced cell proliferation but also ET-1-activated phosphorylation of extracellular signal-regulated protein kinase 1/2 (ERK1/2) in U373MG astrocytoma cells. Whereas the nonselective protein kinase C (PKC) inhibitor chelerythrine attenuated ET-1-induced cell proliferation, it was unable to block ET-1-induced ERK phosphorylation. However, ET-1 did not activate conventional or novel PKCs and did not elevate intracellular calcium. In addition, U73122 (a selective phospholipase C inhibitor), FTI-277 (an H-Ras inhibitor), as well as protein tyrosine kinase inhibitors also did not abolish ET-1-induced ERK1/2 phosphorylation. ET-1 treatment increased the activity of total Ras but not H-Ras. The phosphoinositide 3-kinase (PI3K) pathway appeared to be involved in signal transduction induced by ET-1, but it did not appear to participate in cross talk with the mitogen-activated protein kinase (MAPK) pathway. Activated ET receptors did not propagate signals either through protein tyrosine kinases or transactivation of EGF receptor tyrosine kinases, which typically trigger Ras-Raf-MAPK pathways. The results indicate that ET-1 stimulates cell proliferation by the activation of MAPK-, PKC-, and PI3K-dependent pathways that appear to function in a parallel manner. There is no apparent, direct "cross talk" between these pathways in U373MG cells, but rather, they might act on the independent but necessary components of the mitogenic effects of ET-1.

OBJECTIVE

Metabolic derangements, including insulin resistance and hyperlactatemia, are a major complication of major trauma (e.g., burn injury) and affect the prognosis of burn patients. Protein farnesylation, a posttranslational lipid modification of cysteine residues, has been emerging as a potential component of inflammatory response in sepsis. However, farnesylation has not yet been studied in major trauma. To study a role of farnesylation in burn-induced metabolic aberration, we examined the effects of farnesyltransferase (FTase) inhibitor, FTI-277, on burn-induced insulin resistance and metabolic alterations in mouse skeletal muscle.

METHODS

A full thickness burn (30% total body surface area) was produced under anesthesia in male C57BL/6 mice at 8 weeks of age. After the mice were treated with FTI-277 (5 mg/kg/day, IP) or vehicle for 3 days, muscle insulin signaling, metabolic alterations and inflammatory gene expression were evaluated.

RESULTS

Burn increased FTase expression and farnesylated proteins in mouse muscle compared with sham-burn at 3 days after burn. Simultaneously, insulin-stimulated phosphorylation of insulin receptor (IR), insulin receptor substrate (IRS)-1, Akt and GSK-3β was decreased. Protein expression of PTP-1B (a negative regulator of IR-IRS-1 signaling), PTEN (a negative regulator of Akt-mediated signaling), protein degradation and lactate release by muscle, and plasma lactate levels were increased by burn. Burn-induced impaired insulin signaling and metabolic dysfunction were associated with increased inflammatory gene expression. These burn-induced alterations were reversed or ameliorated by FTI-277.

CONCLUSIONS

Our data demonstrate that burn increased FTase expression and protein farnesylation along with insulin resistance, metabolic alterations and inflammatory response in mouse skeletal muscle, all of which were prevented by FTI-277 treatment. These results indicate that increased protein farnesylation plays a pivotal role in burn-induced metabolic dysfunction and inflammatory response. Our study identifies FTase as a novel potential molecular target to reverse or ameliorate metabolic derangements in burn patients.

Myelodysplastic syndrome (MDS) is a heterogeneous hematopoietic stem cell disorder characterized by bone marrow dysplasia and peripheral cytopenias. Eighty percent of patients found to have MDS are older than 60 years and therefore not eligible for the only potentially curative therapy, bone marrow transplantation. Currently, there is no standard for treating MDS; therapies range from supportive care with transfusions or hematopoietic growth factors and low-intensity cytarabine therapy, to intensive anti-acute myeloid leukemia-type chemotherapy. Some of these treatments induce a limited hematologic response, but none consistently extends survival. Many are highly toxic. More than half of patients with MDS die within 3 to 4 years of infections, bleeding complications, or progression to acute leukemia. Agents in development for MDS include all-trans retinoic acid (ATRA), decitabine, and thalidomide. Farnesyltransferase inhibitors modulate many of the cancer-signaling pathways implicated in MDS initiation or progression and may therefore be well suited for treatment of these biologically diverse hematologic malignancies. Phase I and II clinical studies in our center show that the oral FTI ZARNESTRA (formerly R115777, Ortho Biotech Oncology, Raritan, NJ) has promising anti-MDS activity, suggesting that further investigation of this agent and of this class in MDS is warranted.

Synergistic drug combinations enable enhanced therapeutics. Their discovery typically involves the measurement and assessment of drug combination index (CI), which can be facilitated by the development and applications of in-silico CI predictive tools. In this work, we developed and tested the ability of a mathematical model of drug-targeted EGFR-ERK pathway in predicting CIs and in analyzing multiple synergistic drug combinations against observations. Our mathematical model was validated against the literature reported signaling, drug response dynamics, and EGFR-MEK drug combination effect. The predicted CIs and combination therapeutic effects of the EGFR-BRaf, BRaf-MEK, FTI-MEK, and FTI-BRaf inhibitor combinations showed consistent synergism. Our results suggest that existing pathway models may be potentially extended for developing drug-targeted pathway models to predict drug combination CI values, isobolograms, and drug-response surfaces as well as to analyze the dynamics of individual and combinations of drugs. With our model, the efficacy of potential drug combinations can be predicted. Our method complements the developed in-silico methods (e.g. the chemogenomic profile and the statistically-inferenced network models) by predicting drug combination effects from the perspectives of pathway dynamics using experimental or validated molecular kinetic constants, thereby facilitating the collective prediction of drug combination effects in diverse ranges of disease systems.

OBJECTIVE

Tipifarnib, a farnesyltransferase inhibitor, has antitumor activity in heavily pretreated metastatic breast cancer patients. Preclinical data suggest that FTIs could restore tamoxifen responsiveness in tamoxifen-resistant disease. Thus, combining FTIs and tamoxifen may be a promising clinical approach after relapse or progression on tamoxifen.

METHODS

Postmenopausal patients with measurable estrogen receptor- and/or progesterone receptor-expressing metastatic breast cancers were enrolled. Only patients with disease progression on tamoxifen were eligible, but there was no limitation regarding prior chemotherapy or hormone therapy regimens. Patients were immediately treated with 300 mg (n = 12) or 200 mg (n = 10) tipifarnib twice daily for 21 of 28-day cycles plus tamoxifen once daily. Serum was collected at baseline and after 8 weeks of treatment to enable proteomic comparison and identify possible predictive response markers.

RESULTS

Twenty patients were enrolled and evaluated for efficacy: one patient had an objective response (liver metastasis) and nine had stable disease after 6 months for a clinical benefit rate of 50%; median duration of benefit was 10.3 (range, 7.4-20.2) months. The proteomic analysis by SELDI-TOF and LTQ-FT-Orbitrap identified a known peptide of fibrinogen alpha, the intensity of which was significantly increased in patients with progression compared with patients who benefited from the combined treatment after 8 weeks.

CONCLUSIONS

Because the primary end point of efficacy (three objective responses) was not achieved, the study is negative. Nevertheless, the identified peptide could be of interest in discriminating, at 8 weeks of treatment, responders from nonresponders.

An international meeting focused on farnesyl transferase inhibitors (FTIs) was held in Naples on 12 April 2002 and represented an excellent occasion to gather most of the clinicians who are involved in clinical trials with this class of new compounds. Oncogene mutations of the gene occur in approximately 30% of all human cancers and may have prognostic significance. Ras protein is normally synthesized as pro-Ras, which undergoes a number of post-translational modifications, among which farnesylation. Processed Ras proteins localize to the inner surface of the plasma membrane, and function as a molecular switch that cycles between an inactive and an active form. When in its active form, either because of the binding of an external ligand or because of its constitutive activation, Ras activates several downstream effectors, such as Raf-1, Rac, Rho and phospahtidylinositol-3 kinase, which mediate important cellular functions, such as proliferation, cytoskeletal organization and others. Interruption of the Ras signaling pathway can be basically achieved in three ways, i.e. inhibition of Ras protein expression through antisense oligonucleotides, prevention of Ras membrane localization and inhibition of Ras downstream effectors. SCH 66336 (lonafarnib; Sarasar), a tricyclic orally active FTI, has been the first of these compounds to undergo clinical development. The toxicity profile observed in all completed phase I/II trials has been fairly similar, since gastrointestinal tract toxicity (nausea, vomiting and diarrhea) and fatigue have generally qualified as dose-limiting toxicity (DLT). One objective response in a patient with pretreated non-small cell lung cancer (NSCLC) was observed. Based on preclinical evidence of synergism between lonafarnib and other anticancer agents, combination studies have been started. In particular, lonafarnib has been combined both with gemcitabine and with paclitaxel in phase I studies. Nausea, vomiting, diarrhea and myelosuppression represented DLTs in these studies, in which an encouraging clinical activity was observed, in particular in pancreatic carcinoma (lonafarnib plus gemcitabine) and in NSCLC (lonafarnib plus paclitaxel). R115777 (Zarnestra) is another novel orally active FT competitive inhibitor in clinical development. Single-agent phase I/II studies have shown that myelotoxicity and neurotoxicity are DLTs, intermittent schedule is probably better tolerated and antitumor activity is observed particularly in breast cancer. A number of combination studies with R115777 have been carried out; taken as a whole, they show that the drug can be easily combined with several anticancer agents and phase III trials exploring the potential benefit from incorporation of R115777 into active chemotherapy regimens are indicated. Two other FTIs are in an earlier stage of clinical development. BMS-214662 has the main advantage of being cytotoxic in nature, rather than cytostatic; in particular, potent antitumor activity in human tumor xenografts of different histologies has been reported. A major drawback for BMS-214662 is its severe gastrointestinal and liver toxicities, which prevent the achievement of adequate systemic exposures following the oral route. L-778,123 has been stopped in its clinical development due to its severe and unexpected toxicity, i.e. grade 4 thrombocytopenia and significant Q-T prolongation.

Farnesyl transferase inhibitors (FTIs) have displayed limited efficacy in clinical trials, possibly because of their relatively limited cytotoxic effects against most human cancer cells. Therefore, efforts to leverage the clinical utility of FTIs may benefit from learning how these agents elicit p53-independent apoptosis in mouse models of cancer. Knockout mouse studies have established that gain of the geranylgeranylated isoform of the small GTPase RhoB is essential for FTI to trigger apoptosis. Here we demonstrate that Cyclin B1 is a crucial target for suppression by RhoB in this death program. Steady-state levels of Cyclin B1 and its associated kinase Cdk1 were suppressed in a RhoB-dependent manner in cells fated to undergo FTI-induced apoptosis. These events were not derivative of cell cycle arrest, because they did not occur in cells fated to undergo FTI-induced growth inhibition. Mechanistic investigations indicated that RhoB mediated transcriptional suppression but also accumulation of Cyclin B1 in the cytosol at early times after FTI treatment, at a time before the subsequent reduction in steady-state protein levels. Enforcing Cyclin B1 expression attenuated apoptosis but not growth inhibition triggered by FTI. Moreover, enforcing Cyclin B1 abolished FTI antitumor activity in graft assays. These findings suggest that Cyclin B1 suppression is a critical step in the mechanism by which FTI triggers apoptosis and robust antitumor efficacy. Our findings suggest that Cyclin B1 suppression may predict favorable clinical responses to FTI, based on cytotoxic susceptibility, and they suggest a rational strategy to address FTI nonresponders by coinhibition of Cdk1 activity.

RhoB expression is reduced in most invasive tumors, with loss of RhoB expression correlating significantly with tumor stage. Here, we demonstrate that upregulation of RhoB by the potent anticancer agent NSC126188 induces apoptosis of NUGC-3 human gastric carcinoma cells. The crucial role of RhoB in NSC126188-induced apoptosis is indicated by the rescue of NUGC-3 cells from apoptosis by knockdown of RhoB. In the presence of NSC126188, c-Jun N-terminal kinase (JNK) signaling was activated, and the JNK inhibitor SP600125 reduced RhoB expression and suppressed the apoptosis of NUGC-3 cells. Knockdowns of mitogen-activated protein kinase kinase (MKK) 4/7, JNK1/2 and c-Jun downregulated RhoB expression and rescued cells from apoptotic death in the presence of NSC126188. The JNK inhibitor SP600125 suppressed transcriptional activation of RhoB in the presence of NSC126188, as indicated by a reporter assay that used luciferase under the RhoB promoter. The ability of NSC126188 to increase luciferase activity through both the p300-binding site and the inverted CCAAT sequence (iCCAAT box) suggests that JNK signaling to upregulate RhoB expression is mediated through both the p300-binding site and the iCCAAT box. However, the JNK inhibitor SP600125 did not inhibit the upregulation of RhoB by farnesyltransferase inhibitor (FTI)-277. The p300-binding site did not affect activation of the RhoB promoter by FTI-277 in NUGC-3 cells, suggesting that the transcriptional activation of RhoB by NSC126188 occurs by a different mechanism than that reported for FTIs. Our data indicate that NSC126188 increases RhoB expression via JNK-mediated signaling through a p300-binding site and iCCAAT box resulting in apoptosis of NUGC-3 cells.

Ras proteins play fundamental roles in cell signal transduction pathways that regulate cell growth, differentiation, proliferation, and survival. ras mutations are among the most frequently encountered genetic abnormalities in human cancers and play a key role in tumorigenesis. The enzymatic attachment of a 15- or 20-carbon moiety to the Ras protein through farnesylation or geranylgeranylation, respectively, is a required step in the proper localization and activation of Ras. Inhibition of the catalytic enzymes, farnesyl transferase and geranylgeranyl transferase, is a novel, mechanism-based, targeted approach to cancer therapy development. Geranylgeranyl transferase inhibitors suppress tumor growth by accumulating cells in the G(1)/S cell cycle phase. One mechanism by which farnesyl transferase inhibitors suppress tumor growth is by inhibiting bipolar spindle formation, thereby blocking progression from prophase to metaphase. Although the exact molecular target responsible for the antitumor activity of farnesyl transferase inhibitors is unclear, at least in some tumor cells, inhibition of phosphoinositide-3-OH kinase/Akt-mediated cell survival pathways may play a critical role. Identifying the farnesylated proteins that are targeted by farnesyl transferase inhibitors and the tumor molecular signatures that dictate which set of patients will respond to farnesyl transferase inhibitors are critical end points for future mechanistic studies.

The aim of this study was to assess cardiac deformation patterns in myocarditis applying feature tracking imaging (FTI) to cardiovascular magnetic resonance (CMR) images. Thirty-six patients (31 males) with acute myocarditis and 36 age- and gender-matched healthy volunteers were studied. CMR examinations were performed in a 1.5 T MR-scanner including late gadolinium enhancement (LGE). FTI was applied to standard cine images of long and short axis views. Global peak circumferential, longitudinal and radial systolic strains as well as long axis strain (LAS) were measured. Patients showed significantly impaired global peak circumferential (-24.4 ± 4.2 % vs. -28.8 ± 3.8 %, p < 0.0001), longitudinal (-17.6 ± 4.4 % vs. -23.8 ± 3.1 %, p < 0.0001) and radial (26.1 ± 5.4 % vs. 37.9 ± 7.6 %, p < 0.0001) systolic strains. Even patients with a preserved ejection fraction (pEF, ≥55 %) had significantly reduced longitudinal (-20.0 ± 4.8 % vs. -23.8 ± 3.1 %, p < 0.01) and radial (27.7 ± 5.5 % vs. 37.9 ± 7.6 %, p < 0.0001) strains. The extent of LGE in patients did not correlate to their respective strains. Regarding the differentiation between patients and controls, the addition of global peak systolic strains to ejection fraction led to a significant improvement of the logistic regression model (χ(2) 48.7 vs. 71.5; p < 0.001) resulting in a high AUC of 0.98. Applying previously published reference values, 75 % or 31 % of patients with pEF showed at least one strain value or a LAS, which fell below the limit of 1 or respectively 2 standard deviations from the reference mean value. Cardiac strains measured by CMR-FTI are significantly impaired in patients with acute myocarditis even in those with pEF. Therefore, strain assessment may improve the diagnostic accuracy of CMR for myocarditis.

We compared the extent and origin of muscle fatigue induced by short-pulse-low-frequency [conventional (CONV)] and wide-pulse-high-frequency (WPHF) neuromuscular electrical stimulation. We expected CONV contractions to mainly originate from depolarization of axonal terminal branches (spatially determined muscle fiber recruitment) and WPHF contractions to be partly produced via a central pathway (motor unit recruitment according to size principle). Greater neuromuscular fatigue was, therefore, expected following CONV compared with WPHF. Fourteen healthy subjects underwent 20 WPHF (1 ms-100 Hz) and CONV (50 μs-25 Hz) evoked isometric triceps surae contractions (work/rest periods 20:40 s) at an initial target of 10% of maximal voluntary contraction (MVC) force. Force-time integral of the 20 evoked contractions (FTI) was used as main index of muscle fatigue; MVC force loss was also quantified. Central and peripheral fatigue were assessed by voluntary activation level and paired stimulation amplitudes, respectively. FTI in WPHF was significantly lower than in CONV (21,717 ± 11,541 vs. 37,958 ± 9,898 N·s P<0,001). The reductions in MVC force (WPHF: -7.0 ± 2.7%; CONV: -6.2 ± 2.5%; P < 0.01) and paired stimulation amplitude (WPHF: -8.0 ± 4.0%; CONV: -7.4 ± 6.1%; P < 0.001) were similar between conditions, whereas no change was observed for voluntary activation level (P>> 0.05). Overall, our results showed a different motor unit recruitment pattern between the two neuromuscular electrical stimulation modalities with a lower FTI indicating greater muscle fatigue for WPHF, possibly limiting the presumed benefits for rehabilitation programs.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) preferentially induces apoptosis in transformed or malignant cells, thus exhibiting potential as a tumor-selective apoptosis-inducing cytokine for cancer treatment. Many studies have shown that the apoptosis-inducing activity of TRAIL can be enhanced by various cancer therapeutic agents. R115777 (tipifarnib) is the first farnesyltransferase inhibitor (FTI) that showed clinical activity in myeloid malignancies. In general, R115777, like other FTIs, exerts relatively weak effects on the induction of apoptosis in cancer cells with undefined mechanism(s). In the current study, we studied its effects on the growth of human lung cancer cells, including induction of apoptosis, and examined potential underlying mechanisms for these effects. We showed that R115777 induced apoptosis in human lung cancer cells, in addition to inducing G(1) or G(2)-M arrest. Moreover, we found that R115777 up-regulated the expression of death receptor 5 (DR5), an important death receptor for TRAIL, and exhibited an augmented effect on the induction of apoptosis when combined with recombinant TRAIL. Blockage of DR5 induction by small interfering RNA (siRNA) abrogated the ability of R115777 to enhance TRAIL-induced apoptosis, indicating that R115777 augments TRAIL-induced apoptosis through up-regulation of DR5 expression. Thus, our findings show the efficacy of R115777 in human lung cancer cells and suggest that R115777 may be used clinically in combination with TRAIL for treatment of human lung cancer.

Acquired resistance to endocrine therapy in breast cancer is associated with an increase in peptide growth factor signaling that results in cross-talk activation of the estrogen receptor (ER). Small molecule signal transduction inhibitors (STIs) can target components of these intracellular pathways, and may prove effective in anticancer therapy. However, early phase II clinical trials with various STIs as monotherapy in advanced breast cancer have shown only a modest level of efficacy for these intracellular inhibitors. Preclinical data suggest that combinations of tamoxifen with STIs may provide significantly greater growth inhibition than either therapy alone, and, furthermore, may delay the emergence of endocrine resistance. There are now several trials assessing the efficacy of combinations of small molecule tyrosine kinase inhibitors (TKIs), such as gefitinib and lapatinib, with either tamoxifen or aromatase inhibitors both in the second-line, endocrine-resistant and first-line, hormone-sensitive setting. Similar trials continue with both farnesyltransferase inhibitors (FTIs) and mTOR antagonists, where there are strong preclinical data to suggest additive or synergistic effects for either of these agents in combination with endocrine therapies. Biomarker studies in the presurgical setting are also being utilized as an alternative approach to investigate whether combined endocrine/STI therapy is an effective clinical strategy. This article reviews some of the preclinical evidence supporting this strategy, together with the current status of clinical trials in this area.

OBJECTIVE

To study the prevalence and severity of bone mineral loss and its relationship to sex hormone levels in men on long-term neuroleptic therapy.

METHODS

Sixteen men with schizophrenia who were from 19 to 62 years old and had taken neuroleptic medication for 1 to 30 years had bone mineral density (BMD) of the lumbar spine and proximal femur measured by dual-energy x-ray absorptiometry. Results were compared with those from 16 age-matched control subjects. Serum testosterone, sex hormone-binding globulin, free testosterone index (FTI), prolactin, and pituitary gonadotropins were assayed and compared with age-matched Red Cross blood donor controls (N = 23 to 90 for the various assays).

RESULTS

At the lumbar spine and at two of the three sites in the proximal femur (trochanter and Ward's triangle but not femoral neck), the BMD was lower in patients than in controls. A statistically significant increase in prolactin and sex hormone-binding globulin and a significantly decreased luteinizing hormone and FTI were found in the treated versus the control group. In the patient group, a significant inverse relationship existed between age and BMD at all sites. Lumbar spine density was related directly to FTI (r = 0.607; P=0.05) and inversely to duration of treatment (r = -0.767; P<0.001), although both correlations were accounted for mainly by age associations in multiple stepwise linear regression. Prolactin values did not correlate with either BMD or FTI.

CONCLUSIONS

BMD was significantly lower with long-term neuroleptic use. In the lumbar spine, these changes may be related to the associated findings of decreased free testosterone and hyperprolactinemia, although the significance of other factors such as an independent drug effect, the psychiatric disease itself, and lifestyle cannot be excluded.

Neuromuscular electrical stimulation can improve motor function in those affected by paralysis, but its use is limited by a high rate of muscular fatigue. Variable stimulation patterns have been examined in young adults with and without spinal cord injury, but much less investigation has been devoted to studying the effects of variable stimulation patterns administered to older adults or those paralyzed by stroke. Significant changes occur in the neuromuscular system with age that may affect the response to variable stimulation patterns. We administered three, 3-min intermittent stimulation patterns to the median nerves of 10 individuals with hemiplegia from stroke and 10 age-matched able-bodied adults: (1) constant 20 HZ, (2) a pattern that began at 20 HZ and progressively increased to 40 HZ in the latter half of the task, and (3) a 20-HZ pattern that switched to a 20-HZ doublet pattern after 90 s. In the able-bodied group the doublet pattern produced significantly higher force time integrals (FTI) (1409.72 +/- 3.15 N s) than the 20-40-HZ pattern (1067.46 +/- 1.15 N s) or the 20-HZ pattern (831 +/- 1.87 N s). In the poststroke individuals the doublet pattern also produced the highest FTI (724.04 +/- 2.02 N s), and there was no significant difference between the 20-40-HZ (636.42 +/- 1.65 N s) and 20-HZ (583.64 +/- 3.02 N s) patterns. These results indicate that protocols that incorporate doublets in the later stages of fatigue are effective in older adults and in older adults with paralysis from stroke.

Farnesyltransferase inhibitors (FTIs) were designed to block the action of Ras oncoproteins which depend on posttranslational modification by adding a farnesyl isoprenoid membrane anchor. However, off-target actions are believed to account for most of their antitumor activity. We recently reported the induction of autophagy in cancer cells in a dose-dependent manner by FTIs. We observed similar results of autophagy in a panel of tumor cell lines for the three FTIs tested. Therefore, the induction of autophagy is very likely a pharmacological class effect of inhibition of farnesyltransferase. In this addendum, we discuss the possible mechanisms underlying the induction of autophagy by FTIs, including reactive oxygen species-, DNA damage- and Ras-mediated pathways as alternatives to Rheb-mediated regulation of mTOR and autophagy.

Our randomized trial examined the effectiveness of preventive interventions in increasing positive cognitive attributions and reducing negative cognitive attributions in children of depressed parents. In addition, it tested the role of attribution changes in mediating the intervention effects on children's depressive and emotional symptoms. The participants were 109 Finnish families with at least one parent in treatment for affective disorder, for a total of 145 children, 8-16 years of age. Families were randomized into two groups: the "family talk intervention" (FTI, a whole-family approach enhancing communication and child resilience, Beardslee et al., 1997) group, and an active control, the "let's talk about the children" (LTC, a parent-only psycho-educational approach, Solantaus, Paavonen, Toikka, & Punamäki, 2010) group. Children reported their cognitive attributions (CASQ-R, Children's Attributional Style Questionnaire-Revised (Thompson, Kaslow, Weiss, & Nolen-Hoeksema, 1998)), depressive (CDI/BDI, Child Depression Inventory (Kovacs, 1981)/Beck Depression Inventory (Beck, Steer, & Garbin, 1988)) and emotional (SDQ, Strengths and Difficulties Questionnaire (Goodman, 1997)) symptoms, and mothers reported their children's emotional symptoms (SDQ at baseline (T1) and 10-month (T2) and 18-month (T3)) follow-ups. Contrary to our hypothesis, no beneficial attribution changes were found in the FTI group across the follow-ups. Instead, positive cognitive appraisals increased in the LTC group, especially from T2 to T3. The increase of positive attribution further served as a mediator for changes in children's emotional and depressive symptoms. The findings suggest that a short preventive intervention can enhance beneficial cognitive processes in high-risk families in routine adult psychiatric care.

OBJECTIVE

To evaluate the malignancy rate of focal thyroid incidentaloma (fTI) in a population of patients undergoing a 18F-fluorodeoxyglucose positron emission tomography-computed tomography (FDG PET-CT) for a non-thyroid purpose.

METHODS

We conducted a prospective cohort study from January 2013 to November 2014. All consecutive patients referred for a FDG PET-CT were prospectively screened. Patients with known neoplastic thyroid disease were excluded from the analysis. All patients presenting one or more fTI and who accepted to benefit from a complementary thyroid ultrasonography (US) were included and managed according to the French endocrine society consensus. Prevalence of fTI in our population and malignancy rate was assessed.

RESULTS

During the inclusion period, 10,171 patients were referred for a FDG PET-CT in our center. Fifty-three patients presenting a known thyroid disease were excluded. Among the remaining 10,118 patients, 127 (1.3%) with 131 fTI were individualized. US could not be performed in 37 patients. The remaining 90 patients (92 fTI) were explored by US ± fine-needle aspiration biopsy (FNAB). US results demonstrated a nodule aspect in 80 cases of which 60 benefited from FNAB. Nineteen of 92 fTI underwent surgery with 10 malignant lesions among the 60 patients performing both US and FNAB.

CONCLUSIONS

The prevalence of fTI discovered on FDG PET-CT in our population was 1.3% with 10 malignant lesions among the 60 patients performing both US and FNAB.

BACKGROUND

Farnesyltransferase (FT), an essential enzyme at the downstream of mevalonate pathway, was reported to be upregulated in hypertrophic cardiomyocytes of spontaneously hypertensive rats (SHRs) compared with myocardium of Wistar-Kyoto rats (WKYs). This upregulation was accompanied with cardiac remodeling. This study was designed to determine whether FT inhibition can alter cardiac remodeling in SHRs.

METHODS

Twelve-week-old SHRs were randomized to receive infusion of either NS or FTI-276 (307 μg/kg/d i.v. each n=10). WKY rats served as normal controls (n=6). Echocardiography was performed before and after intervention. SHR hearts were perfused ex vivo for the evaluation of cardiac performance, collagen deposition and biochemical changes (activation of Ras, extracellular-signal regulated kinases/ERK1/2, procollagen type І/Ш, TGF-β1, connective tissue growth factor/CTGF, and bone morphogenetic protein-7/BMP-7 expression).

RESULTS

FTI-276 intervention decreased interventricular septum wall thickness at end- diastole (IVSd) and relative wall thickness (RWT) of SHRs (P<0.05). Three week intervention with FTI-276 attenuated hydroxyproline content (P<0.05), collagen deposition (P<0.01), Ras activation, ERK1/2 phosphorylation (P<0.01) and mRNA expression of procollagen type I, TGF-β1 and CTGF and elevated mRNA expression of BMP-7 (P<0.05) in left ventricle of SHRs.

CONCLUSIONS

The present study indicated that FT inhibition could attenuate myocardial fibrosis and partly improve cardiac remodeling in SHRs. The beneficial effects might be mediated through suppression of the activation of Ras and ERK1/2 phosphorylation pathway. The enhanced mRNA expression of BMP-7 with inhibition of TGF-β1 and CTGF mRNA expression might be an important mechanism.

Farnesyltransferase inhibitors (FTIs) are currently under investigation for leukemia treatment. We evaluated the FTI manumycin A (manumycin) in two myeloid leukemia cell lines (U937 and HL-60). Manumycin induced nitric oxide production and apoptosis of the leukemia cells. Nitric oxide or other reactive oxygen species may induce oxidative DNA damage, and the number of apurinic sites increased after manumycin treatment, which was reversed by concurrent treatment with N-acetyl-L-cysteine. Since repair of DNA damage is important to cell survival, we hypothesized that methoxyamine, an inhibitor of base-excision repair, would enhance the antineoplastic effect of manumycin. The combination of manumycin and methoxyamine resulted in enhanced apoptosis by six criteria increased annexin V binding, release of mitochondrial cytochrome c into the cytosol, activation of caspase-9, activation of caspase-3, specific cleavage of poly-adenosyl ribose polymerase, and increase in the sub-G1 cell cycle fraction. The drug combination enhanced inhibition on the soft agar clonogenic assay and on the formazan dye cell viability assay. The effects of manumycin or manumycin plus methoxyamine on apoptosis were blocked by N-acetyl-L-cysteine, and partially by nitric oxide synthase inhibitors or scavenger of peroxide. We conclude that methoxyamine enhances manumycin-induced apoptosis in myeloid leukemia cells.

BACKGROUND

Farnesyltransferase inhibitors (FTIs) are anticancer agents with a spectrum of activity in Ras-dependent and independent tumor cellular and xenograph models. How inhibition of protein farnesylation by FTIs results in reduced cancer cell proliferation is poorly understood due to the multiplicity of potential FTase targets. The low toxicity and oral availability of FTIs led to their introduction into clinical trials for the treatment of breast cancer, hematopoietic malignancy, advanced solid tumor and pancreatic cancer treatment, and Hutchinson-Gilford Progeria Syndrome. Although their efficacy in combinatorial therapies with conventional anticancer treatment for myeloid malignancy and solid tumors is promising, the overall results of clinical tests are far below expectations. Further exploitation of FTIs in the clinic will strongly rely on understanding how these drugs affect global cellular activity.

METHODS

Using FTase inhibitor I and genome-wide chemical profiling of the yeast barcoded deletion strain collection, we identified genes whose inactivation increases the antiproliferative action of this FTI peptidomimetic. The main findings were validated in a panel of cancer cell lines using FTI-277 in proliferation and biochemical assays paralleled by multiparametric image-based analyses.

RESULTS

ABC transporter Pdr10 or p-21 activated kinase (PAK) gene deletion increases the antiproliferative action of FTase inhibitor I in yeast cells. Consistent with this, enhanced inhibition of cell proliferation by combining group I PAK inhibition, using IPA3, with FTI-277 was observed in melanoma (A375MM), lung (A549) and colon (HT29), but not in epithelial (HeLa) or breast (MCF7), cancer cell lines. Both HeLa and A375MM cells show changes in the nuclear localization of group 1 PAKs in response to FTI-277, but up-regulation of PAK protein levels is observed only in HeLa cells.

CONCLUSIONS

Our data support the view that group I PAKs are part of a pro-survival pathway activated by FTI treatment, and group I PAK inactivation potentiates the anti-proliferative action of FTIs in yeast as well as in cancer cells. These findings open new perspectives for the use of FTIs in combinatorial strategies with PAK inhibitors in melanoma, lung and colon malignancy.

BACKGROUND

In anorexia nervosa (AN) patients osteoporosis occurs within a framework of multiple hormonal abnormalities as a result of bone turnover uncoupling, with decreased bone formation and increased bone resorption. The aim of study was to evaluate the hormonal and nutritional relationships with both of these bone remodeling compartments and their eventual modifications with age.

METHODS

In a cohort of 115 AN patients (mean BMI:14.6 kg/m2) that included 60 mature adolescents (age: 15.5-20 years) and 55 adult women (age: 20-37 years) and in 28 age-matched controls (12 mature adolescents and 16 adults) we assessed: bone markers [serum osteocalcin, skeletal alkaline phosphatase (sALP), C-telopeptide of type I collagen (sCTX) and tartrate-resistant acid phosphatase type 5b (TRAP 5b)], nutritional markers [ body mass index (BMI, fat and lean mass), hormones (free tri-iodothyronine (T3), free T4, thyroid stimulating hormone (TSH), luteinizing hormone (LH), follicle stimulating hormone (FSH), 17 beta estradiol, free testosterone index (FTI), dehydroepiandrosterone (DHEAS), insulin-like growth factor 1 (IGF-1), growth hormone (GH) and cortisol], plasma methoxyamines (metanephrine and normetanephrine) and calcium metabolism parameters [parathyroid hormone (PTH), Ca, vitamin D3].

RESULTS

Osteocalcin reached similar low levels in both AN age subgroups. sCTX levels were found to be elevated in all AN subjects and higher in mature adolescents than in adult AN (11,567+/-895 vs. 8976+/-805 pmol/l, p<0.05). sALP was significantly lower only in mature adolescent AN patients, while there were no significant differences in the levels of TRAP 5b between AN patients and age-matched control groups. Osteocalcin correlated with sCTX in the control subjects (r=0.65) but not in the AN patients, suggesting the independent regulation of these markers in AN patients. Osteocalcin levels strongly correlated with freeT3, IGF-I, 17 beta estradiol and cortisol, while sCTX correlated with IGF-I, GH and cortisol in both age subgroups of the AN patients. Other hormones or nutritional parameters displayed age-related correlations with bone markers, leading to different stepwise regression models for each age interval. In mature adolescent AN patients, up to 54% of the osteocalcin variance was due to BMI, cortisol and 17 beta estradiol, while 54% of the sCTX variance was determined by GH. In adult subjects, freeT3 and IGF-I accounted for 64% of osteocalcin variance, while 65% of the sCTX variance was due to GH, FTI and methoxyamines.

CONCLUSIONS

We suggest a more complex mechanism of AN bone uncoupling that includes not only "classical" influence elements like cortisol, IGF-I, GH or 17 beta estradiol but also freeT3, catecholamines and a "direct" hormone-independent impact of denutrition. Continuous changes of these influences with age should be considered within the therapeutic approach to AN bone loss.

The aggressiveness of triple-negative breast cancer (TNBC), which lacks estrogen receptor, progesterone receptor and epidermal growth factor receptor 2 (HER2), represents a major challenge in breast cancer. Migratory and self-renewal capabilities are integral components of invasion, metastasis and recurrence of TNBC. Elevated hypoxia-inducible factor-1α (HIF-1α) expression is associated with aggressiveness of cancer. Nonetheless, how HIF-1α expression is regulated and how HIF-1α induces aggressive phenotype are not completely understood in TNBC. The cytotoxic effects of farnesyltransferase (FTase) inhibitors (FTIs) have been studied in cancer and leukemia cells. In contrast, the effect of FTIs on HIF-1α expression has not yet been studied. Here, we show that clinically relevant low-dose FTI, tipifarnib (300 nM), decreased HIF-1α expression, migration and tumorsphere formation in human MDA-MB-231 TNBC cells under a normoxic condition. In contrast, the low-dose FTIs did not inhibit cell growth and activity of the Ras pathway in MDA-MB 231 cells. Tipifarnib-induced decrease in HIF-1α expression was associated with amelioration of the Warburg effect, hypermetabolic state, increases in Snail expression and ATP release, and suppressed E-cadherin expression, major contributors to invasion, metastasis and recurrence of TBNC. These data suggest that FTIs may be capable of ameliorating the aggressive phenotype of TNBC by suppressing the HIF-1α-Snail pathway. J. Cell. Physiol. 232: 192-201, 2017. © 2016 Wiley Periodicals, Inc.