Progeria is a rare genetic disorder characterized by premature aging that eventually leads to death and is noticed globally. Despite alarming conditions, this disease lacks effective medications; however, the farnesyltransferase inhibitors (FTIs) are a hope in the dark. Therefore, the objective of the present article is to identify new compounds from the databases employing pharmacophore based virtual screening. Utilizing nine training set compounds along with lonafarnib, a common feature pharmacophore was constructed consisting of four features. The validated Hypo1 was subsequently allowed to screen Maybridge, Chembridge, and Asinex databases to retrieve the novel lead candidates, which were then subjected to Lipinski's rule of 5 and ADMET for drug-like assessment. The obtained 3,372 compounds were forwarded to docking simulations and were manually examined for the key interactions with the crucial residues. Two compounds that have demonstrated a higher dock score than the reference compounds and showed interactions with the crucial residues were subjected to MD simulations and binding free energy calculations to assess the stability of docked conformation and to investigate the binding interactions in detail. Furthermore, this study suggests that the Hits may be more effective against progeria and further the DFT studies were executed to understand their orbital energies.

The optimal temporal sampling rate in electrocardiograph-gated myocardial SPECT is questionable: low rates, typically 8 frames per cardiac beat (8fr/cb), favor image quality, whereas high rates, typically 16 frames per cardiac beat (16fr/cb), favor the accuracy of left ventricular (LV) functional parameters. We examined whether Fourier temporal interpolation (FTI) from 8fr/cb to 16fr/cb can combine the advantages of low and high rates.

METHODS

In 34 patients imaged after stress injection of (99m)Tc-sestamibi, 4 sets of reconstructed gated slices were compared: a raw 16fr/cb acquisition (R16), a raw 8fr/cb acquisition (R8), a 16fr/cb set obtained by FTI of 8fr/cb projections (IP), and a 16fr/cb set obtained by FTI of 8fr/cb reconstructed slices (IS). LV ejection fraction (LVEF), end-diastolic volume (EDV), and end-systolic volume (ESV) obtained from the final LV volume curve were compared for the 4 datasets. Deviation of the whole LV volume curve was quantified for IP and IS with respect to R16. Image quality was evaluated by consensus reading of end-diastolic slices of the 4 sets. For R16, IP, and IS, cine display fluidity was quantified by a roughness index calculated from the LV volume curve.

RESULTS

No differences in EDVs or ESVs were found among R16, IP, and IS, whereas R8 gave smaller EDVs and larger ESVs. LVEF was lower with R8, IP, and IS than with R16: -3.9%, -1.2%, and -1.3%, respectively. The LV volume curve was closer to R16 with IP than with IS. Image quality was better with IP and IS than with R8 and better with R8 than with R16. Cine display fluidity was better with IP than with R16 and better with R16 than with IS.

CONCLUSIONS

FTI improved image quality not only over that provided by R16 but even over that provided by R8. The sole worsened LV functional parameter was LVEF, which was slightly underestimated with respect to that estimated by R16. Of the 2 FTI variants, IP was superior to IS for cine display fluidity and accuracy of the LV volume curve with respect to the data obtained with R16. Therefore, FTI to 16fr/cb performed before reconstruction on a pixel-by-pixel basis on 8fr/cb projections improves image quality and cine display fluidity over those of both R8 and R16 acquisitions at the sole cost of a 1% underestimation of LVEF.

Thirty-one (43%) of 68 patients with primary depression were found to have a blunted thyroid-stimulating hormone (TSH) response to thyrotropin-releasing hormone (TRH). Increased thyroid activity, as measured by the free thyroxine index (FTI), was present in 16 (24%) of the patients. Patients with blunted responses had a higher mean FTI level than those with normal responses. Patients with blunted responses were significantly more likely to exhibit the symptoms of depersonalization, derealization and agitation. There was no clear association between blunting and any particular diagnostic category of depression. Patients with blunted responses and high FTI values were more likely to report significant long-term environmental difficulties than patients with blunted responses and normal FTI values. It is suggested that there may be more than one mechanism responsible for blunting of the TSH response in depressed patients. In some patients blunting may be due to negative feedback from increased output of thyroid hormones, possibly released as part of a stress response. In other patients blunting may be due to a different mechanism, possibly involving pituitary gland dysfunction. These mechanisms would not necessarily be mutually exclusive in any one patient.

We have previously developed and tested a muscle model that predicts the effect of stimulation frequency on muscle force responses. The aim of this study was to enhance our isometric mathematical model to predict muscle forces in response to stimulation trains with a wide range of frequencies and intensities for the quadriceps femoris muscles of individuals with spinal cord injuries. Isometric forces were obtained experimentally from 10 individuals with spinal cord injuries (time after injury, 1.5-8 years) and then compared to forces predicted by the model. Our model predicted accurately the force-time integrals (FTI) and peak forces (PF) for stimulation trains of a wide range of frequencies (12.5-80 HZ) and intensities (150-600-mus pulse duration), and two different stimulation patterns (constant-frequency trains and doublet-frequency trains). The accurate predictions of our model indicate that our model, which now incorporates the effects of stimulation frequency, intensity, and pattern on muscle forces, can be used to design optimal customized stimulation strategies for spinal cord-injured patients.

Background: Mortality in patients with stages 4 and 5 chronic kidney disease (CKD) is higher than in the general population. Body composition predicts mortality. Our objective was to evaluate the effect of body composition on mortality in patients with stages 4 and 5 non-dialysis CKD. Methods: We performed a prospective study of 356 patients with stages 4 and 5 non-dialysis CKD. At baseline, we recorded general characteristics, history of cardiovascular events, body composition, serum inflammatory markers, nutrition and cardiac biomarkers. Body composition was analysed using bioimpedance spectroscopy. We recorded the lean tissue index (LTI), fat tissue index (FTI) and overhydration (OH). During a median (range) follow-up of 22 (3-49) months, we recorded mortality, cardiovascular events and progress to renal replacement therapy. Results: At baseline, mean (± standard deviation) age was 67 ± 13 years (men 64%; diabetes 36%). Mean body mass index was 28.2 ± 12.8 kg/m2, the FTI was 12.3 ± 5.6 kg/m2, the LTI was 15.7 ± 3.4 kg/m2 and median (interquartile range) OH was 0.6 (-0.4 to 1.5) L. Sixty-four (18%) patients died during follow-up. The univariate Cox analysis showed an association between mortality and age, low LTI, high Charlson comorbidity index, previous cardiovascular events, OH, low albumin and prealbumin levels, and high C-reactive protein levels. Kaplan-Meier analysis revealed higher survival in patients with a higher LTI (log-rank, 9.47; P = 0.002). The multivariate Cox analysis confirmed an association between mortality and low LTI (P = 0.031), previous cardiovascular events (P = 0.003) and high Charlson comorbidity index (P = 0.01). We did not find any association between body composition and cardiovascular events or renal replacement therapy. Conclusions: A low LTI is an independent factor for mortality in patients with stages 4 and 5 CKD.

Treatment of older patients with acute myeloid leukemia (AML) represents a significant challenge. There is little evidence that outcomes are improving for patients subjected to conventional intensive treatment. In addition, there is a large population of patients who are not considered fit for a more intensive treatment approach, and treatment options are limited for these patients. As molecular mechanisms and characteristics of the disease become clearer, they represent potential targets for therapy. One such mechanism is the process of farnesylation, which is required for the activation of RAS proteins and potentially other deranged pathways. The first farnesyltransferase inhibitor (FTI) to undergo assessment in AML is tipifarnib, which has achieved a complete remission in 4% of patients who have relapsed and in 15% of untreated elderly patients. Responses were not restricted to patients with RAS protein mutations and the treatment was well tolerated in general. Several randomized comparative and combination trials have been set up that will establish the position of tipifarnib in the treatment of AML.

Deregulation of Ras/ERK signaling in myeloid leukemias makes this pathway an interesting target for drug development. Myeloid leukemia cell lines were screened for idarubicin-induced apoptosis, cell-cycle progression, cell-cycle-dependent MAP kinase kinase (MEK-1/2) activation, and Top2 expression. Cell-cycle-dependent activation of MEK/ERK signaling was blocked using farnesyltransferase inhibitor (FTI) BMS-214,662 and dual prenyltransferase inhibitor (DPI) L-778,123 to disrupt Ras signaling. Idarubicin caused a G2/M cell-cycle arrest characterized by elevated diphosphorylated MEK-1/2 and Top2α expression levels. The FTI/DPIs elicited distinct effects on Ras signaling, protein prenylation, cell cycling and apoptosis. Combining these FTI/DPIs with idarubicin synergistically inhibited proliferation of leukemia cell lines, but the L-778,123+idarubicin combination exhibited synergistic growth inhibition over a greater range of drug concentrations. Interestingly, combined FTI/DPI treatment synergistically inhibited cell proliferation, induced apoptosis and nearly completely blocked protein prenylation. Inhibition of K-Ras expression by RNA interference or blockade of its post-translational prenylation led to increased BMS-214,662-induced apoptosis. Our results suggest that nearly complete inhibition of protein prenylation using an FTI + DPI combination is the most effective method to induce apoptosis and to block anthracycline-induced activation of ERK signaling.

BACKGROUND

Protein-energy wasting (PEW) and heightened inflammation are prevalent in patients undergoing continuous ambulatory peritoneal dialysis (CAPD) and is a strong risk factor for morbidity and mortality in these patients. Evaluation of PEW, prevalence of inflammation as well as interrelationship between various nutritional indices and inflammation has not been studied in much detail in patients undergoing CAPD.

OBJECTIVE

This study was conducted to evaluate the interrelationship between PEW and inflammation in patients undergoing CAPD.

METHODS

Sixty-three patients undergoing CAPD (M = 28, F = 35) were assessed with regard to their nutritional status and inflammation after a minimum of 3 months CAPD initiation. Nutritional status was assessed by dietary diary, anthropometry, subjective global assessment, and multifrequency bioelectrical impedance analysis (BIA). In addition, their serum albumin, prealbumin, transferrin, and cholesterol level were measured. Also, inflammation in these patients was assessed by High-Sensitivity C-Reactive Protein (hs-CRP>> 3 mg/L) and Interleukin-6 (IL-6>> 2 µg/mL). Later on, diagnosis of malnutrition was made based on different methods. Correlation between inflammation and various nutritional assessment indices were analyzed statistically.

RESULTS

Mean (SD) age of the patients was 57.6 (11.6) years. The average (SD) calorie and protein intake per day were 25.5 (4.6) kcal and 0.81 (0.2) mg, respectively. The mean and standard deviation of anthropometry variables of body mass index (BMI), mid-arm circumference (MAC), tricipital skin-fold thickness (TST), mid-arm muscle circumference (MAMC), and corrected mid-arm muscle area (cMAMA) were 23.7 ± 5 kg/m(2), 26.3 ± 4.5 cm, 1.624 ± 0.4 cm, 25.6 ± 4.5 cm, and 45.7 ± 19.7 cm(2), respectively. The mean values of serum protein, albumin, prealbumin, transferrin, cholesterol, triglyceride, hs-CRP, and IL-6 were 5.9 g/dL, 3.0 g/dL, 21.11 mg/dL, 130.6 mg/dL, 155.9 mg/dL, 136.1 mg/dL, 8.8 ± 7.6 mg/L, and 8.4 ± 12.2 µg/dL, respectively. Based on subjective global assessment (SGA); 11.63 (17.4%), 34.63 (54%), and 18.65 (28.6%) patients undergoing CAPD had normal, moderate, and severe malnutrition status, respectively. According to serum albumin level; 13.63 (21%), 39.63 (62%), and 11.63 (17%) patients undergoing CAPD had normal, moderate, and severe malnutrition status, respectively. Finally, based on BMI; 33.63 (52%), 23.63 (37%), and 7.63 (11%) patients undergoing CAPD had normal, moderate, and severe malnutrition status, respectively. About 76.1% and 9.5% of patients undergoing CAPD were malnourished based on lean tissue index (LTI) and fat tissue index (FTI), respectively. Based on hs-CRP and IL-6 findings, 70% (44/63) and 71.8% (45/63) of patients undergoing CAPD had high inflammation, respectively. High sensitive C-reactive protein correlated negatively (significantly) with serum albumin, prealbumin, and transferrin. Interleukin -6 correlated negatively (significantly) with MAC; MAMA; serum albumin, cholesterol, and transferrin. There was significant positive correlation between hs-CRP and IL-6. There is statistically significant difference in total protein intake (g/d), protein intake (g/kg/d), serum protein (g/dL), albumin (g/dL), transferrin (mg/dL), and cholesterol (mg/dL) between patients with and without inflammation.

CONCLUSIONS

Protein-energy wasting (80% - 85%) by various methods and inflammation (70%) was very prevalent among patients undergoing CAPD. Inflammatory markers show significant negative correlation with anthropometry and serological markers. Inflammatory markers are suggested to be included in the regular assessment of patients undergoing CAPD, for the better management of protein-energy wasting.

Farnesyltransferase inhibitors (FTIs) were developed to prevent Ras processing and thus to be effective agents for the treatment of cancers harbouring mutated ras. In the present study, HepG2 cells underwent internucleosomal DNA fragmentation after treatment with farnesyltransferase inhibitor manumycin (20 microM) for 12 h. Flow cytometric analysis showed that HepG2 cells were accumulated in the G2/M phase of the cell cycle and the number of apoptotic sub-G1 fraction of cells was increased after treatment with manumycin in a time-dependent manner. During the induction of apoptosis, expression of p53 and p21WAF1 was upregulated, phosphorylation of IkappaB-alpha was blocked, caspase substrates poly(ADP-ribose) polymerase (PARP) and lamin B were cleaved, and Bcl-2 and Bax protein expression remained unchanged. These results indicated that manumycin induced apoptosis in HepG2 cells. The induction of apoptosis by manumycin involved the upregulation of p53 and p21WAF1, the activation of caspases, and the inhibition of nuclear factor-kappaB (NF-kappaB) pathway. However, Bcl-2 and Bax are not associated with manumycin-mediated apoptosis.

Hutchinson-Gilford progeria syndrome (HGPS) is characterized by accelerated senescence due to a de novo mutation in the LMNA gene. The mutation produces an abnormal lamin A protein called progerin that lacks the splice site necessary to remove a farnesylated domain. Subsequently, progerin accumulates in the nuclear envelope, disrupting nuclear architecture, chromatin organization, and gene expression. These alterations are often associated with rapid telomere erosion and cellular aging. Here, we further characterize the cellular and molecular abnormalities in HGPS cells and report a significant reversal of some of these abnormalities by introduction of in vitro transcribed and purified human telomerase (hTERT) mRNA. There is intra-individual heterogeneity of expression of telomere-associated proteins DNA PKcs/Ku70/Ku80, with low-expressing cells having shorter telomeres. In addition, the loss of the heterochromatin marker H3K9me3 in progeria is associated with accelerated telomere erosion. In HGPS cell lines characterized by short telomeres, transient transfections with hTERT mRNA increase telomere length, increase expression of telomere-associated proteins, increase proliferative capacity and cellular lifespan, and reverse manifestations of cellular senescence as assessed by β-galactosidase expression and secretion of inflammatory cytokines. Unexpectedly, mRNA hTERT also improves nuclear morphology. In combination with the farnesyltransferase inhibitor (FTI) lonafarnib, hTERT mRNA promotes HGPS cell proliferation. Our findings demonstrate transient expression of human telomerase in combination with FTIs could represent an improved therapeutic approach for HGPS.

In hippocampal neurons cultured from brains of newborn rats, the glutamate receptor agonist N-methyl-D-aspartate induced the clustering of neuronal perikarya and the fasciculation of neurites. In addition, N-methyl-D-aspartate activated the small GTPase Rac1. Other stimuli of Rac activity, such as the Rho kinase inhibitors Y-27632, H-1152, and H89, as well as the cytotoxic necrotizing factor-1 from Escherichia coli, also caused neuronal clustering and neurite bundling. In neurons transiently transfected with dominant negative Rac1N17 neither N-methyl-D-aspartate nor Y-27632 induced clustering and fasciculation. In addition, the PI3-kinase inhibitors wortmannin and LY-294002 prevented these effects, as did a dominant negative form of p110PI3-Kgamma. Time-lapse microscopy showed that lethal toxin from Clostridium sordellii, which inhibits Rac, and wortmannin blocked the neuronal migration induced by Y-27632. In contrast, only lethal toxin reversed the clustering and fasciculation induced by pre-treatment with Y-27632. This effect of the toxin may be due to inactivation of Ras, since FTI-277, which prevents the farnesylation of Ras and thereby inactivates the GTPase, also dissolved the preformed clusters. We suggest that active Rac and a PI3-kinase synergistically induce neuronal migration, whereas a Ras isoform is responsible for the lasting attachment of neurons necessary for clustering and neurite fasciculation.

Fibroblasts derived from Hutchinson-Gilford progeria syndrome (HGPS) patients and dermal cells derived from healthy old humans in culture display age-dependent progressive changes in nuclear architecture due to accumulation of farnesylated lamin A. Treating human HGPS cells or mice expressing farnesylated lamin A with farnesyl transferase inhibitors (FTIs) reverses nuclear phenotypes and extends lifespan. Aging adult Caenorhabditis elegans show changes in nuclear architecture resembling those seen in HGPS fibroblasts, as well as a decline in motility, phenotypes which are also inhibited by the FTI gliotoxin. However, it was not clear whether these effects were due to loss of farnesylation or to side effects of this drug. Here, we used a different FTI, manumycin or downregulated polyprenyl synthetase with RNAi to test the roles of farnesylation in C. elegans aging. Our results show that the age-dependent changes in nuclear morphology depend on farnesylation. We also demonstrate that inhibition of farnesylation does not affect motility or lifespan, suggesting that the effects of blocking protein prenylation on nuclear morphology could be separated from their effects on motility and lifespan. These results provide further understanding of the role of lamin and farnesylation in the normal aging process and in HGPS.

Specific mutations in human LMNA or loss of ZMPSTE26 activity cause abnormal processing of lamin A and early aging diseases, including Hutchinson Gilford progeria syndrome (HGPS). HGPS fibroblasts in culture undergo age-dependent progressive changes in nuclear architecture. Treating these cells with farnesyl transferase inhibitors (FTIs) reverse these nuclear phenotypes and also extend lifespan of mice HGPS models. Dermal cells derived from healthy old humans also accumulate the abnormally processed lamin A. However, the effect of FTIs on normal aging cells was not tested. Aging adult C. elegans cells show changes in nuclear architecture similar to HGPS fibroblasts and down regulating lamin expression in adult C. elegans reduces their lifespan. Here, we show that nuclei of adult C. elegans, in which lamin is down-regulated, have similar phenotypes to normal aging nuclei, but at an earlier age. We further show that treating adult C. elegans with the FTI gliotoxin reverses nuclear phenotypes and improves motility of aging worms. However, the average lifespan of the gliotoxin-treated animals was similar to that of untreated animals. These results suggest that lamins are involved in the process of normal aging in C. elegans.

OBJECTIVE

To evaluate, in a prospective fashion, the clinical interchangeability between two brands of levothyroxine, Synthroid (Boots Pharmaceuticals, Inc., Lincolnshire, Illinois) and Levoxine (Daniels Pharmaceuticals, Inc., St. Petersburg, Florida), by using clinical scores of hyperthyroidism and hypothyroidism, free thyroxine index (FTI), sensitive thyroid-stimulating hormone (TSH), and thyrotropin-releasing hormone (TRH) stimulation testing.

METHODS

Twenty-three of the 31 patients with long-standing primary hypothyroidism (6 men, 25 women; age range 30 to 71 years, mean 47.2 +/- 2.2 SEM) were switched from Synthroid to Levoxine (group 1) and the remaining patients from Levoxine to Synthroid (group 2). After switching, each patient continued to receive the same dosage as previously. Clinical scores of hypothyroidism and hyperthyroidism (Billewicz and Crooks scoring systems, respectively), basal FTI, and TRH stimulation test were obtained before and 4 months after the switching. Comparison of the variables before and after switching was performed separately in each subgroup and in the entire group.

RESULTS

There was no statistically significant difference in the hypothyroid clinical scores (-40.1 +/- 1.2 versus -39.7 +/- 1.2), the hyperthyroid clinical scores (-19.6 +/- 0.9 versus -19.2 +/- 1.0), FTI (9.6 +/- 0.3 versus 9.6 +/- 0.3), basal TSH levels (1.4 +/- 0.2 versus 1.4 +/- 0.2 mIU/L), or the magnitude of TSH response to TRH (mean delta TSH 9.4 +/- 1.5 versus 9.2 +/- 1.4 mIU/L), whether the patients were receiving Synthroid or Levoxine.

CONCLUSIONS

Switching did not result in substantial clinical or laboratory changes in any individual patient. We conclude that the two brands of levothyroxine are clinically interchangeable.

Candida guilliermondii FTI 20037 was cultured in sugarcane bagasse hydrolysate supplemented with 2.0 g/L of (NH4)2SO4, 0.1 g/L of CaCl2 x 2H2O, and 20.0 g/L of rice bran at 35 degrees C; pH 4.0; agitation of 300 rpm; and aeration of 0.4, 0.6, or 0.8 vvm. The high xylitol production (20.0 g/L) and xylose reductase (XR) activity (658.8 U/mg of protein) occurred at an aeration of 0.4 vvm. Under this condition, the xylitol dehydrogenase (XD) activity was low. The apparent K(M) for XR and XD against substrates and cofactors were as follows: for XR, 6.4 x 10(-2)M (xylose) and 9.5 x 10(-3) mM (NADPH); for XD, 1.6 x 10(-1)M (xylitol) and 9.9 x 10(-2) mM (NAD+). Because XR requires about 10-fold less xylose and cofactor than XD for the condition in which the reaction rate is half of the Vmax, some interference on the overall xylitol production by the yeast could be expected.

BACKGROUND

Recurrent atrial flutter following cavotricuspid isthmus (CTI) ablation remains a significant problem. The prevalence of low contact force (CF) during CTI ablation using standard tools is unknown. Our aim was to characterize the prevalence of low CF applications when experienced operators performed CTI ablation using "traditional" markers of contact blinded to CF measurements.

RESULTS

Average CF (grams, g) and force-time integral (FTI) was analyzed in 458 lesions in 17 patients undergoing CTI ablation. The isthmus was divided into the annular, mid and caval segments for region-specific analysis. Despite "good" contact using traditional markers, there was significant variability in CF within each isthmus segment (e.g., annular CTI 1-57 g). A high proportion of lesions had a CF <10 g (40%). Lowest CF was the annular (median 9 g), followed by the mid (12 g) and the caval CTI (18 g, P < 0.001). Sites of acute CTI re-connection had a lower average CF and FTI than nonreconnected sites (P < 0.05). Each 1 g increase in CF was associated with a 16% reduction in risk of recovered CTI conduction (95% confidence interval: 4-27%, P = 0.01).

CONCLUSIONS

Use of surrogate markers of "good contact" during ablation by experienced operators in the absence of real-time CF sensing resulted in nearly half of all lesions being delivered with low CF with marked region-specific variability in CF. Low CF was implicated in longer time to achieve conduction block and increased risk of acute reconnection. These findings underscore the importance of real-time CF measurements for optimizing ablation of typical atrial flutter.

Protein farnesylation is required for the activation of multiple proteins involved in cell differentiation and function. In white adipose tissue protein, farnesylation has shown to be essential for the successful differentiation of preadipocytes into adipocytes. We hypothesize that protein farnesylation is required for PPARgamma2 expression and activation, and therefore for the differentiation of human mesenchymal stem cells (MSCs) into adipocytes. MSCs were plated and induced to differentiate into adipocytes for three weeks. Differentiating cells were treated with either an inhibitor of farnesylation (FTI-277) or vehicle alone. The effect of inhibition of farnesylation in differentiating adipocytes was determined by oil red O staining. Cell survival was quantified using MTS Formazan. Additionally, nuclear extracts were obtained and prelamin A, chaperon protein HDJ-2, PPARgamma, and SREBP-1 were determined by western blot. Finally, DNA binding PPARgamma activity was determined using an ELISA-based PPARgamma activation quantification method. Treatment with an inhibitor of farnesylation (FTI-277) arrests adipogenesis without affecting cell survival. This effect was concomitant with lower levels of PPARgamma expression and activity. Finally, accumulation of prelamin A induced an increased proportion of mature SREBP-1 which is known to affect PPARgamma activity. In summary, inhibition of protein farnesylation arrests the adipogenic differentiation of MSCs and affects PPARgamma expression and activity.

Farnesyltransferase enzyme (FTase) is an interesting target for anticancer therapy that has been the subject of particular attention over the past decade. However, despite of the thrilling achievements in the development of farnesyltransferase inhibitors (FTIs) over the past few years, the farnesylation mechanism remains, to some degree, a mystery. This work describes the application of molecular dynamics simulations to the study of enzyme flexibility in the 4 key intermediate states formed during the FTase catalytic mechanism--FTase resting state, binary complex (FTase-FPP), ternary complex (FTase-FPP-Peptide), and product complex (FTase-Product)--thereby covering the main states in the mechanistic pathway of this mysterious enzyme, while relating, dissecting, and exploring, in minute detail, the set of structural and dynamical changes taking place with FPP binding, peptide coordination and product formation. This study reveals the existence of a series of variational patterns involving the mechanistic events taking place at the active site of the enzyme, increasing in magnitude away from the active-site, demonstrating that relatively small-scale events such as substrate binding or product formation cause minor changes at the neighboring residues and corresponding helices, but ultimately induce much more dramatic effects on the more external regions of the enzyme.

The expression of activated RAS oncogenes has been shown to increase radioresistance in a number of cell lines. The pathways by which RAS leads to radioresistance, however, are unknown. RAS activates several signal transduction pathways, with the RAF-MAP2K-MAP kinase pathway perhaps the best studied. MAP kinase has also been shown to be activated by radiation through this pathway. Given the important role of MAP kinase in multiple signaling events, we asked if radioresistance induced by RAS was mediated through the activation of MAPK. Cells of two human bladder carcinoma cell lines were used, one with a mutated oncogenic HRAS (T24) and other with a wild-type HRAS (RT4). The surviving fraction after exposure to 2 Gy of radiation (SF2) for the T24 cell lines was found to be 0.62, whereas that for RT4 cells was 0.40. Treatment with the farnesyl transferase inhibitor (FTI) L744,832, which inhibits RAS processing and activity, decreased the SF2 of T24 cells to 0.29, whereas the SF2 of RT4 cells remained unchanged after FTI treatment, thus demonstrating the importance of RAS activation to the radiosensitivity of cells with mutated RAS. MAP kinase activation was found to be constitutive and dependent on RAS in T24 cells, while it was inducible by radiation and was independent of RAS in RT4 cells. Treatment of both cell lines with the MAP2K inhibitor PD98059 inhibited MAPK activation; however, inhibiting MAPK activation had no effect on radiation survival of T24 or RT4 cells. These data indicate that MAPK activation does not contribute to RAS-induced radioresistance in this system.

Farnesyltransferase inhibitors (FTIs) are a novel class of cancer therapeutics that were developed to block the localization and thereby the activity of oncogenic Ras protein. Preclinical studies have established that FTIs are nontoxic yet capable of reversing malignant phenotypes. However, there is growing evidence that inhibition of Ras may not be crucial and that the antitransforming properties of FTIs are based at least in part upon alteration of Rho, a small GTPase which is involved in cell adhesion and cytoskeletal regulation. These recent developments are reviewed and their impact on the design of clinical trials is discussed. Copyright 1999 Harcourt Publishers Ltd.

A study of the prenyl transferase reactions was performed by fluorescence using rat brain cytosol fractions as an enzyme source. Four dansylated peptides corresponding to the C-terminal sequence of Ras isoforms were synthesised. The effects of different detergents on the farnesylation or geranylgeranylation of the four peptides were evaluated. Dose-dependent effects of dodecyl-maltoside, a non-ionic detergent, on the farnesyl transferase or geranylgeranyl transferase activities were observed with all peptide substrates. Additionally, the effect of temperature was investigated and these assays were applied to determine Michaelis-Menten constants (K(m)) of the substrates: dansyl-GCVLS (1.8 microM), dansyl-GCVVM (3.2 microM), dansyl-CVIM (3.4 microM) and dansyl-GCVLL (8.4 microM) and FPP (22.6 microM) for FTase activity. Using GGPP as co-substrate, GGTase activity was measured with K(m) values superior to 50 microM for all the three substrate dansyl-GCVLS, dansyl-GCVVM, or dansyl-CVIM, whereas values of 7.6 and 5.4 microM were calculated for the dansyl-GCVLL sequence and GGPP co-substrate, respectively. IC50 values of selective prenyl transferase inhibitors, B-581, FTI 276 and GGTI 287 have been measured to 34, 0.8 and 18 nM, respectively, using dansyl-GCVLS as substrate (FTase inhibition). When dansyl-GCVLL is used as substrate (GGTase inhibition) the IC50 values are 5100, 75 and 5 nM for B-581, FTI 276 and GGTI 287, respectively. Then, this developed method allowed to evaluate the selectivity of all the three inhibitors tested.

Activating mutations of Ras that frequently occur during malignant transformation, enhance growth-promoting signal transduction, allowing cells to bypass stringent control of cell cycle progression, thereby rendering them highly proliferative. Abundantly expressed c-Ha-ras protein in human cervical HeLa cells is farnesylated and attached to the plasma membrane, inducing enhanced signal transduction. Exposure of HeLa cells to cisplatin very efficiently inhibits cell proliferation and induces apoptosis. Unfortunately, high doses of cisplatin are strongly cytotoxic, therefore, an alternative therapeutic strategy allowing dose reduction of cisplatin by inhibition of farnesylation could increase the curative effects of cisplatin, thereby benefiting cancer patients. We used two inhibitors of farnesyl protein transferase (FPTase), FTI, and L-744,832, to sensitize HeLa cells to the action of cisplatin. The combined administration of cisplatin and inhibitors of FPTase increased the cytostatic potency of cisplatin. L-744,832 exhibited a stronger synergistic effect in combination with cisplatin than FTI. Moreover, the efficiency of the combined therapy strongly depended on the treatment regimen: The highest efficiency was achieved after combined treatment for 24 h and post-incubation with an inhibitor of FPTase for 48 h. Following this optimized treatment, apoptosis was induced in approximately 50% of HeLa cells treated with 1 microM cisplatin, representing approximately a threefold increase as compared to cisplatin monotherapy. Combined treatment of HeLa cells with cisplatin and inhibitors of FPTase significantly increases the efficacy of the therapy and allows to reduce the dose of cisplatin. Importantly, best therapeutic effects can be achieved by post-treatment with inhibitors of FPTase.

BACKGROUND

We have previously shown that FTI-277, a farnesyl transferase inhibitor (FTI), enhances the efficacy of tamoxifen (Tam) in inhibiting the proliferation of the estrogen dependent MCF-7 cell line. As the cellular response to Tam is the result of an inhibition of both estrogen receptor-dependent and -independent pathways, we have used the estrogen receptor selective anti-estrogen ICI182,780 and N-pyrrolidine(-phenylmethyl-phenoxy)-ethanamine-HCl (PBPE), a selective ligand of anti-estrogen binding site (AEBS), to dissect out the mechanism(s) associated with the observed additivity resulting from combination treatment with FTI-277 and Tam. Moreover, for these studies, FTI-277 has been replaced by R115,777, a FTI currently in phase III clinical trials.

METHODS

The quantitative sulphorhodamine B (SRB) colorimetric assay was used to determine the growth inhibitory effect of agents on MCF-7 cells. Dose response interactions between R115,777-Tam, R115,777-ICI182,780 and R115,777-PBPE were evaluated, at the IC50 point, using the isobologram method. Apoptotic cell death (DNA fragmentation, nucleus condensation and cytokeratin 18 cleavage) and inhibition of the mevalonate pathway (western blot) were also determined.

RESULTS

Combinations of the specific FTI R115,777 with either ICI182,780 or PBPE exhibit a synergistic effect on MCF-7 cell growth inhibition, while its combination with Tam is additive, as previously reported for FTI-277. Apoptosis is detected after treatment with combinations of R115,777 with either Tam or PBPE but not with ICI182,780, suggesting that each combination inhibits cell proliferation by different mechanisms. Even though the ER pathway has not yet been deciphered, it is shown here that the AEBS pathway is able to interfere with the mevalonate pathway at the level of protein farnesylation.

CONCLUSIONS

Overall, this work reveals that combinations of R115,777 with either selective ER ligands or a selective AEBS ligand are able to induce large increases in their anti-proliferative activities on MCF-7 cells. Moreover, these results suggest that it may be of definite interest to evaluate combinations of R115,777 with different anti-estrogens in the treatment of ER positive breast tumours. Based on these experimental data, such combinations may prove beneficial in different clinical scenarios or when used in specific sequences; studying the combination of R115,777 with ICI182,780 for early treatment and reserving combinations with either Tam or a selective AEBS ligand, such as BMS-217380-01, for more resistant disease.