To determine whether RhoA isoprenylation (geranylgeranylation) is required for agonist-induced actin cytoskeleton reorganization (measured by an increase in the filamentous F- to monomeric G-actin ratio), human airway smooth muscle cells were treated for 72 h with inhibitors of geranylgeranyltransferase I. Geranylgeranyltransferase inhibitor (GGTI)-2147 or -286 pretreatment completely blocked the increase in the F- to G-actin fluorescence ratio when cells were stimulated with lysophosphatidic acid (LPA), endothelin, or carbachol. In contrast, LPA or endothelin induced actin cytoskeletal reorganization in cells treated with farnesyltransferase inhibitor (FTI)-277 to inactivate Ras. Forskolin-induced adenylyl cyclase activity was inhibited by carbachol in GGTI-2147-pretreated cells, demonstrating that the effect of geranylgeranyltransferase I inhibition on stress fiber formation was not due to uncoupling of signaling between the heterotrimeric G(i) protein (the Ggamma subunit is isoprenylated) and distal effectors. These results demonstrate that selective GGTIs can inhibit agonist-induced actin reorganization.

Oncogenic ras has been shown to downregulate Fas receptor expression and increase Fas ligand expression and thus contribute to resistance to Fas-mediated cell death in several cell types. The effects of ras on Fas-mediated apoptosis have not been studied in melanoma. We studied the effects of activated N-ras by measuring Fas, Fas ligand, and FLIP expression as well as susceptibility to Fas-ligand-induced cell death in transfectants of WM35, a radial growth phase human melanoma cell line. Based on quantitative polymerase chain reaction and fluorescence-activated cell sorter analysis, we found that the ras transfectants expressed less Fas mRNA and surface Fas receptor. Cr51 release cytotoxicity assays demonstrated less susceptibility to Fas-mediated apoptosis in ras transfectants, correlating with the Fas mRNA and protein expression results. Ras inhibition with the specific inhibitor FTI-277 showed that downregulation of Fas in the ras transfectants could be reversed. This correlates with cytotoxicity experiments showing that ras inhibition increases susceptibility to Fas-mediated apoptosis. The control transfectants expressed FLIP but ras did not affect FLIP expression. The control and ras transfectants did not express Fas ligand as demonstrated by reverse transcriptase polymerase chain reaction and fluorescence-activated cell sorter analysis. Cytotoxicity assays further confirmed that these melanoma ras transfectants do not express functional Fas ligand. These results suggest that ras contributes to tumor progression by decreasing susceptibility to Fas-mediated cell death at least in part through downregulation of Fas receptor at the transcriptional level.

OBJECTIVE

To study the significance of breast size increment in pregnancy, and the impact of metformin during pregnancy on breastfeeding in women with polycystic ovary syndrome (PCOS).

METHODS

A follow-up study of a randomised controlled trial (the PregMet study).

METHODS

Eleven secondary care centres.

METHODS

Women with PCOS during pregnancy and postpartum.

METHODS

Women with PCOS were randomised to treatment with metformin or placebo from the first trimester to delivery. Questionnaires were sent to 240 participants 1 year postpartum: 186 responded.

METHODS

Pre-pregnancy and late-pregnancy brassiere size and breastfeeding patterns were registered, and androgen levels were measured in the mothers.

RESULTS

No difference in breast size increment and breastfeeding were found between the placebo and metformin groups. Breast size increment correlated positively with the duration of both exclusive and partial breastfeeding, whereas body mass index (BMI) correlated negatively with the duration of partial breastfeeding. Dehydroepiandrostenedione-sulphate (DHEAS), testosterone and free testosterone index (FTI) in pregnancy did not correlate with breast size increment or duration of breastfeeding. Women with no change in breast size were more obese, had higher blood pressure, serum triglycerides and fasting insulin levels, and had a shorter duration of breastfeeding compared with those with breast size increment.

CONCLUSIONS

Metformin and androgens had no impact on breastfeeding. Women with PCOS who had no breast size increment in pregnancy seem to be more metabolically disturbed and less able to breastfeed.

BACKGROUND

Some LMNA mutations responsible for lipodystrophies, and some HIV-protease inhibitors (PIs) induce accumulation of farnesylated prelamin A and premature senescence in some cell types. Patients with LMNA mutations or under PI-based therapy suffer from early atherosclerosis. The metalloprotease ZMPSTE24 is the key enzyme in prelamin A maturation.

OBJECTIVE

We studied whether altered expression of ZMPSTE24 could contribute to vascular cell dysfunction in response to LMNA mutations or PI treatments.

METHODS

Protein expression of prelamin A and ZMPSTE24 were evaluated in patients' cells and in human cultured VSMCs. Oxidative stress, inflammation, senescence and transdifferentiation/calcification were evaluated in VSMCs.

RESULTS

Fibroblasts from LMNA-mutated lipodystrophic patients (mutations R482W, D47Y or R133L) and peripheral blood mononuclear cells from PI-treated-HIV-infected patients expressed increased prelamin A and decreased ZMPSTE24, which was also observed in VSMCs overexpressing mutant LMNA or treated with PIs. These alterations correlated with oxidative stress, inflammation, senescence and calcification (all p < 0.05). ZMPSTE24 silencing in native VSMCs recapitulated the mutant LMNA- and PI-induced accumulation of farnesylated prelamin A, oxidative stress, inflammation, senescence and calcification. A negative regulator of ZMPSTE24, miRNA-141-3p, was enhanced in LMNA-mutated or PI-treated VSMCs. The farnesylation inhibitors pravastatin and FTI-277, or the antioxidant N-acetyl cysteine, partly restored ZMPSTE24 expression, and concomitantly decreased oxidative stress, inflammation, senescence, and calcification of PI-treated VSCMs.

CONCLUSIONS

ZMPSTE24 downregulation is a major contributor in VSMC dysfunctions resulting from LMNA mutations or PI treatments that could translate in early atherosclerosis at the clinical level. These novel pathophysiological mechanisms could open new therapeutic perspectives for cardiovascular aging.

Lamin A (LaA) is a component of the nuclear lamina, an intermediate filament meshwork that underlies the inner nuclear membrane (INM) of the nuclear envelope (NE). Newly synthesized prelamin A (PreA) undergoes extensive processing involving C-terminal farnesylation followed by proteolysis yielding non-farnesylated mature lamin A. Different inhibitors of these processing events are currently used therapeutically. Hutchinson-Gilford Progeria Syndrome (HGPS) is most commonly caused by mutations leading to an accumulation of a farnesylated LaA isoform, prompting a clinical trial using farnesyltransferase inhibitors (FTI) to reduce this modification. At therapeutic levels, HIV protease inhibitors (PI) can unexpectedly inhibit the final processing step in PreA maturation. We have examined the dynamics of LaA processing and associated cellular effects during PI or FTI treatment and following inhibitor washout. While PI reversibility was rapid, with respect to both LaA maturation and associated cellular phenotype, recovery from FTI treatment was more gradual. FTI reversibility is influenced by both cell type and rate of proliferation. These results suggest a less static lamin network than has previously been observed.

BACKGROUND

Relative androgen deficiency in ageing males is assumed to have adverse health effects. This study assessed the effect of 12 months' standard dose, oral testosterone, on symptoms attributed to testosterone deficiency in older men with plasma testosterone levels in the low-normal range for young men.

METHODS

Testosterone undecanoate (TU, 80 mg bid) or placebo was administered for one year to 76 healthy men, 60 years or older, with a free testosterone index (FTI) of 0.3-0.5 and significant symptoms on a questionnaire designed to evaluate androgen deficiency (ADAM). The ADAM was completed at baseline, 6 and 12 months. Hormone and safety data were collected at baseline, 1, 3, 6 and 12 months.

RESULTS

After 12 months, plasma total testosterone was unchanged in both groups and sex hormone binding globulin decreased in the testosterone group (P = 0.01). FTI and calculated bioavailable testosterone (cBT) were greater in the testosterone group as compared with the placebo group (P = 0.021 and 0.025, respectively). There was no significant difference in total symptom score between testosterone and placebo groups after 12 months of oral TU. However, there were trends toward improvements in sadness/grumpiness (P = 0.063), reduced erection strength (P = 0.059) and decreased work performance symptoms (P = 0.077), particularly in men with baseline cBT levels below 3.1 nmol/l.

CONCLUSIONS

This study concludes that 80 mg bid oral TU does not improve overall ADAM questionnaire scores in older men with low-normal gonadal status. Oral TU may preserve mood and erectile function, as assessed by this questionnaire, particularly in men with the lowest testosterone levels.

Increasing levels of E2 in gonadotropin-treated women stimulated hepatic synthesis of SHBG and TBG, and consequently increases in T4 concentration. Nevertheless, unchanged FTI and TSH suggested that a euthyroid state was maintained. The temporal patterns for the rise in serum concentrations of TBG and SHBG during gonadotropin therapy suggest that the synthesis of these proteins by the liver has different sensitivities to E2.

We have previously reported that angiotensin II (ANG II) stimulated Src tyrosine kinase via a pertussis toxin-sensitive type 2 receptor, which, in turn, activates MAPK, resulting in an increase in nitric oxide synthase (NOS) expression in pulmonary artery endothelial cells (PAECs). The present study was designed to investigate the pathway by which ANG II activates Src leading to an increase in ERK1/ERK2 phosphorylation and an increase in NOS protein in PAECs. Transfection of PAECs with G alpha(i3) dominant negative (DN) cDNA blocked the ANG II-dependent activation of Src, ERK1/ERK2 phosphorylation, and increase in NOS expression. ANG II stimulated an increase in tyrosine phosphorylation of sequence homology of collagen (Shc; 15 min) that was prevented when PAECs were pretreated with 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo-[3,4-d]pyrimidine (PP2), a Src inhibitor. ANG II induced a Src-dependent association between Shc and growth factor receptor-bound protein 2 (Grb2) and between Grb2 and son of sevenless (Sos), both of which were maximal at 15 min. The ANG II-dependent increase in Ras GTP binding was prevented when PAECs were pretreated with the AT(2) antagonist PD-123319 or with PP2 or were transfected with Src DN cDNA. ANG II-dependent activation of MAPK and the increase in endothelial NOS (eNOS) were prevented when PAECs were transfected with Ras DN cDNA or treated with FTI-277, a farnesyl transferase inhibitor. ANG II induction of Raf-1 phosphorylation was prevented when PAECs were pretreated with PD-123319 and PP2. Raf kinase inhibitor 1 prevented the ANG II-dependent increase in eNOS expression. Collectively, these data suggest that G alpha(i3), Shc, Grb2, Ras, and Raf-1 link Src to activation of MAPK and to the AT(2)-dependent increase in eNOS expression in PAECs.

We have shown that systolic pressure-volume area (PVA), which is equivalent to the total mechanical energy generated by ventricular contraction, correlates linearly with myocardial oxygen consumption, VO2, in canine left ventricle. Systolic force-time integral, FTI, also correlates with VO2. In this study, stroke volume was increased from 0 (isovolumic) in isolated cross-circulated canine left ventricle in a stable contractile state while keeping PVA constant with a servo pump. Ventricular total force was calculated from ventricular pressure and volume by the force-equilibrium equation and was integrated from the end of diastole to the end of systole as identified with the time of Emax to yield FTI. Under the conditions of constant PVA, FTI significantly (p less than 0.001) decreased by 10 +/- 7% and 25 +/- 8% with increases in stroke volume from 0 to 8 +/- 4 ml and 17 +/- 3 ml and in ejection fraction from 0 to 0.24 +/- 0.10 and 0.61 +/- 0.05, respectively, while VO2 and Emax remained constant. Therefore, we conclude that in a stable contractile state, it is possible to keep PVA constant even when stroke volume and ejection fraction are varied and that under such conditions, FTI no longer predicts VO2. PVA remains a reliable predictor of VO2 regardless of ventricular loading conditions.

Autonomously functioning thyroid nodules (AFTNs) are presumably independent of TSH for growth and function and appear "hot" on scintiscan because they selectively concentrate radionuclide to a greater extent than the remaining thyroid gland, which is controlled by the normal TH-TSH feedback mechanism. Such autonomously functioning tissue may occur in "patchy" areas, as a solitary nodule, or as multiple nodules (classic Plummer's disease), with the mass of hyperfunctioning tissue and the related secretion of thyroid hormones determining whether the patient is euthyroid or hyperthyroid. Important diagnostic tests include a 99mTc thyroid scan, T4 RIA, T3 uptake, FTI, TSH RIA, and occasionally T3 RIA ("T3 thyrotoxicosis"). Solitary autonomous nodules in adult patients characteristically progress slowly over many years, with toxicity rarely developing in nodules less than 2.5 cm in diameter and occurring primarily in nodules 3 cm or larger and in older patients. The decision to treat a solitary nodule depends upon the size and degree of function of the nodule and the patient's age. Surgery and radioactive iodine are effective therapies. Hyperfunctioning thyroid nodules in children and adolescents (under age 18) have a more rapidly progressive course than those in adults and should be treated by thyroid lobectomy at the time of diagnosis. Subtotal thyroidectomy is the preferred treatment for most patients with toxic multinodular goiter, because it achieves prompt control of the hyperthyroidism and removes the goiter. Radioiodine therapy and long-term antithyroid drug therapy are alternative forms of treatment for patients who are poor surgical risks or who develop recurrent hyperthyroidism following thyroid surgery.

OBJECTIVE

To evaluate a simple and economical technique to improve xylitol production using concentrated xylose solutions prepared from rice straw hemicellulosic hydrolysate.

RESULTS

Experiments were carried out with rice straw hemicellulosic hydrolysate containing 90 g l-1 xylose, with and without the addition of nutrients, using the yeast Candida guilliermondii previously grown on the hydrolysate (adapted cells) or on semi-defined medium (unadapted cells). By this method, the yield of xylitol increased from 17 g l-1 to 50 g l-1, and xylose consumption increased from 52% to 83%, after 120 h of fermentation. The xylitol production rates were very close to that (0.42 g l-1 h-1) attained in a medium simulating hydrolysate sugars.

CONCLUSIONS

Yeast strain adaptation to the hydrolysate showed to be a suitable method to alleviate the inhibitory effects of the toxic compounds. Adapted cells of Candida guilliermondii can efficiently produce xylitol from hydrolysate with high xylose concentrations.

CONCLUSIONS

Yeast adaptation helps the bioconversion process in hydrolysate made from lignocellulosic materials. This low-cost technique provides an alternative to the detoxification methods used for removal of inhibitory compounds. In addition, the use of adapted inocula makes it possible to schedule a series of batch cultures so that the whole plant can be operated almost continuously with a concomitant reduction in the overall operation time.

A long-standing goal in cancer research is to identify cellular functions that have selective roles in regulating neoplastic pathophysiology. Farnesyl-transferase inhibitors (FTIs) are a novel class of cancer chemotherapeutics which have little effect on normal cell physiology but which inhibit or reverse malignant cell phenotypes. FTIs were originally developed as a strategy to inhibit oncogenic Ras, the activity of which depends upon posttranslational farnesylation. However, recent work indicates the antineoplastic effects of FTIs are not linked to Ras inhibition but instead to alteration of RhoB, a small GTPase of the Rho family of cytoskeletal regulators that controls trafficking of cell surface receptors. Rho proteins integrate signals from integrins and cytokine receptors with cell shape via the actin cytoskeleton. A connection between FTIs and Rho alteration is interesting given that histological differences have long been used to define clinical cancer. RhoB is dispensable for normal cell growth and differentiation in mice. Thus, research into the antineoplastic effects of FTIs has led to the identification of a function(s) that is unnecessary for normal cell physiology but crucial for controlling malignant phenotypes.

The purpose of this work is to study mechanisms underlying anti-tumor effects of farnesyltransferase inhibitors (FTIs) in non-small cell lung cancer (NSCLC). We demonstrate that mRNA and protein levels of Ras homologue enriched in brain (Rheb) are highly expressed both in NSCLC tissues and in NSCLC cell lines. Rheb expression levels correlate with phosphorylation of its downstream target S6 and the sensitivity of NSCLC cells to FTIs (R115777 and SCH66336)-induced growth inhibition and apoptosis. FTIs effectively and preferentially inhibited Rheb downstream signaling in NSCLC cells. Moreover, inhibition of Rheb functions by FTIs or dominant-negative Rheb mutants enhance the effects of cisplatin on NSCLC cells. Rheb-CSVL, a FTIs-resistant mutant, reduces the effects of FTIs on NSCLC cells. Our results suggest that Rheb is a critical target for FTIs therapy in NSCLC.

The ras family of proto-oncogenes are upstream mediators of several essential cellular signal transduction pathways involved in cell proliferation and survival. Point mutations of ras oncogenes result in constitutively active Ras and have been shown to be oncogenic. However, ras activation can occur in the absence of ras mutations secondary to upstream receptor activation. The first important step in Ras activation is farnesylation by farnesyl transferase, and inhibitors of this enzyme have been demonstrated to inhibit Ras signaling, and have anti-tumor effects. However, it is now clear that farnesyl transferase inhibitors (FTIs) have activity independent of Ras, most likely due to effects on prenylated proteins downstream of Ras, which explains their activity in several malignancies, including breast cancer, where ras mutations are rare. Several FTIs are in clinical development for the treatment of solid tumors. Preclinical evidence suggests that FTIs can inhibit breast cancers in vitro and in vivo, and a phase II trial of the FTI, R115777, in patients with advanced breast cancer produced encouraging results. Based on prior successful outcomes with agents targeting the estrogen and epidermal growth factor receptor pathways in breast cancer, the FTIs, used alone or more likely with other agents, may be the next exciting targeted therapy in breast cancer.

K-ras alterations have been reported in 20-30% of non-small cell lung cancer (NSCLC) and represent a suitable target for the development of novel anticancer agents, such as Farnesyl transferase inhibitors (FTi), a new class of agents inhibiting the post-translational modification of the K-ras proteins. The effectiveness of FTi SCH66336 in inhibiting cell proliferation and deranging cell cycle of NSCLC cell lines as well as its interaction with chemotherapy or radiation have been evaluated. The activity of FTi SCH66336, alone or in combination with paclitaxel, gemcitabine, and radiotherapy, was examined in 3 cell lines, A-549, LX-1 and CaLu-6, by colorimetric MTT assay. Cell cycle perturbation and apoptosis were also assessed by cytofluorimetric analysis. The activity of SCH 66336 was found to be concentration- and time-dependent. The effect of SCH 66336, as demonstrated by cell growth recovery experiments, resulted cytostatic and it was superimposable in both cell lines bearing 2 different K-ras mutations (A-549 and LX-1) and in K-ras wild-type Ca-Lu-6. In all cell lines the combination of SCH 66336 and paclitaxel resulted in a synergism of action when SCH 66336 followed paclitaxel treatment, whereas, antagonism was found when SCH 66336 preceded paclitaxel treatment. No significant synergism or addition with SCH 66336 followed by radiation treatment was noted. Different cell cycle phase blocks at various drug concentrations were observed. In conclusion, SCH 66336 displays concentration-dependent cytostatic antitumour activity and schedule-dependent synergy with 2 commonly used anticancer agents in NSCLC cell lines. Further clinical testing of these combinations is warranted.

During an electrically elicited isometric contraction, the metabolic cost of attaining is greater than of maintaining force. Thus fatigue produced during such stimulation may not simply be a function of the force-time integral (FTI), as previously suggested. The goal of the present study was to evaluate fatigue produced in human medial gastrocnemius by intermittent, isometric electrical stimulation with trains of different frequencies (20, 40, or 80 Hz) and durations (300, 600, or 1,200 ms) that produced different peak forces and FTIs. Each subject (n = 10) participated in a total of six sessions. During each session, subjects received a pre- and postfatigue testing protocol and a different, 150-train fatiguing protocol. Each fatiguing protocol used only a single frequency and duration. The fatigue produced by the different protocols was correlated to the initial peak force of the fatiguing protocols (r2= 0.74-0.85) but not to the initial or total FTI. All of the protocols tested produced a proportionately greater impairment of force in response to low- vs. high-frequency stimulation (i.e., low-frequency fatigue). There was no effect of protocol on low-frequency fatigue, suggesting that all the protocols produced comparable levels of impairment in excitation-contraction coupling. These results suggest that, for brief stimulated contractions, peak force is a better predictor of fatigue than FTI, possibly because of the different metabolic demands of attaining and maintaining force.

BACKGROUND

Low contact force and force-time integral (FTI) during catheter ablation are associated with ineffective lesion formation, whereas excessively high contact force and FTI may increase the risk of complications. We sought to evaluate the optimal FTI for pulmonary vein (PV) isolation based on atrial wall thickness under the ablation line.

RESULTS

Contact force parameters and FTI during anatomical ipsilateral PV isolation for atrial fibrillation and atrial wall thickness were assessed retrospectively in 59 consecutive patients for their first PV isolation procedure. The PV antrum was divided into 8 segments, and the wall thickness of each segment under the ablation line was determined using multidetector computed tomography. The FTI for each ablation point was divided by the wall thickness of the PV antrum segment where each point was located to obtain FTI/wall thickness. In total, 5335 radiofrequency applications were delivered, and 85 gaps in PV isolation ablation lines and 15 dormant conductions induced by adenosine were detected. The gaps or dormant conductions were significantly associated with low contact force, radiofrequency duration, FTI, and FTI/wall thickness. Among them, FTI/wall thickness had the best prediction value for gaps or dormant conductions by receiver operating characteristic curve analysis. FTI/wall thickness of <76.4 gram-seconds per millimeter (gs/mm) predicted gaps or dormant conductions with sensitivity (88.0%) and specificity (83.6%), and FTI/wall thickness of <101.1 gs/mm was highly predictive (sensitivity 97.0%; specificity 69.6%).

CONCLUSIONS

FTI/wall thickness is a strong predictor of gap and dormant conduction formation in PV isolation. An FTI/wall thickness ≈100 gs/mm could be a suitable target for effective ablation.

OBJECTIVE

In patients with advanced kidney disease, metabolic and nutritional derangements induced by uremia interact and reinforce each other in a deleterious vicious circle. Literature addressing the effect of dialysis initiation on changes in body composition (BC) is limited and contradictory. The aim of this study was to evaluate changes in BC in a large international cohort of incident hemodialysis patients.

METHODS

A total of 8,227 incident adult end-stage renal disease patients with BC evaluation within the initial first 6 months of baseline, defined as 6 months after renal replacement therapy initiation, were considered. BC, including fat tissue index (FTI) and lean tissue index (LTI), were evaluated by Body Composition Monitor (BCM, Fresenius Medical Care, Bad Homburg, Germany). Exclusion criteria at baseline were lack of a BCM measurement before or after baseline, body mass index (BMI) < 18.5 kg/m(2), presence of metastatic solid tumors, treatment with a catheter, and prescription of less or more than 3 treatments per week. Maximum follow-up was 2 years. Descriptive analysis was performed comparing current values with the baseline in each interval (delta analysis). Linear mixed models considering the correlation structure of the repeated measurements were used to evaluate factors associated with different trends in FTI and LTI.

RESULTS

BMI increased about 0.6 kg/m(2) over 24 months from baseline. This was associated with increase in FTI of about 0.95 kg/m(2) and a decrease in LTI of about 0.4 kg/m(2). Female gender, diabetic status, and low baseline FTI were associated with a significant greater increase of FTI. Age>> 67 years, diabetes, male gender, high baseline LTI, and low baseline FTI were associated with a significant greater decrease of LTI.

CONCLUSIONS

With the transition to hemodialysis, end-stage renal disease patients presented with distinctive changes in BC. These were mainly associated with gender, older age, presence of diabetes, low baseline FTI, and high baseline LTI. BMI increases did not fully represent the changes in BC.

Accumulating evidence suggests that cancer cells with stem cell-like phenotypes drive disease progression and therapeutic resistance in glioblastoma (GBM). NOTCH regulates self-renewal and resistance to chemoradiotherapy in GBM stem cells. However, NOTCH-targeted γ-secretase inhibitors (GSIs) exhibited limited efficacy in GBM patients. We found that farnesyltransferase inhibitors (FTIs) significantly improved sensitivity to GSIs. This combination showed significant antineoplastic and radiosensitizing activities in GBM stem cells, whereas non-stem GBM cells were resistant. These combinatorial effects were mediated, at least partially, through inhibition of AKT and cell-cycle progression. Using subcutaneous and orthotopic GBM models, we showed that the combination of FTIs and GSIs, but not either agent alone, significantly reduced tumor growth. With concurrent radiation, this combination induced a durable response in a subset of orthotopic tumors. These findings collectively suggest that the combination of FTIs and GSIs is a promising therapeutic strategy for GBM through selectively targeting the cancer stem cell subpopulation.

Cytarabine is a conventionally used chemotherapeutic agent for treating acute myeloid leukemia (AML). However, chemoresistance, toxic side-effects and poor patient survival rates retard the efficacy of its performance. The current study deals with the chemosensitization of AML cells using heteronemin, a marine natural product towards cytarabine chemotherapy. Heteronemin could effectively sensitize HL-60 cells towards sub-toxic concentration of cytarabine resulting in synergistic toxicity as demonstrated by MTT assay and [3H] thymidine incorporation studies, while being safe towards healthy blood cells. Flow cytometry for Annexin-V/PI and immunoblotting for caspase cleavage proved that the combination induces enhancement in apoptosis. Heteronemin being a farnesyl transferase inhibitor (FTI) suppressed cytarabine-induced, farnesyl transferase-mediated activation of Ras, as assessed by Ras pull-down assay. Upon pre-treating cells with a commercial FTI, L-744,832, the synergism was completely lost in the combination, confirming the farnesyl transferase inhibitory activity of heteronemin as assessed by thymidine incorporation assay. Heteronemin effectively down-regulated cytarabine-induced activation of MAPK, AP-1, NF-κB and c-myc, the down-stream targets of Ras signaling, which again validated the role of Ras in regulating the synergism. Hence we believe that the efficacy of cytarabine chemotherapy can be improved to a significant extent by combining sub-toxic concentrations of cytarabine and heteronemin.

Anti-CD20 monoclonal antibodies (mAbs) are successfully used in the management of non-Hodgkin lymphomas and chronic lymphocytic leukemia. We have reported previously that statins induce conformational changes in CD20 molecules and impair rituximab-mediated complement-dependent cytotoxicity. Here we investigated in more detail the influence of farnesyltransferase inhibitors (FTIs) on CD20 expression and antitumor activity of anti-CD20 mAbs. Among all FTIs studied, L-744,832 had the most significant influence on CD20 levels. It significantly increased rituximab-mediated complement-dependent cytotoxicity against primary tumor cells isolated from patients with non-Hodgkin lymphomas or chronic lymphocytic leukemia and increased CD20 expression in the majority of primary lymphoma/leukemia cells. Incubation of Raji cells with L-744,832 led to up-regulation of CD20 at mRNA and protein levels. Chromatin immunoprecipitation assay revealed that inhibition of farnesyltransferase activity was associated with increased binding of PU.1 and Oct-2 to the CD20 promoter sequences. These studies indicate that CD20 expression can be modulated by FTIs. The combination of FTIs with anti-CD20 mAbs is a promising therapeutic approach, and its efficacy should be examined in patients with B-cell tumors.

OBJECTIVE

In an in vitro model, the authors tested the hypotheses that: 1) lesion dimensions correlate with lesion size index (LSI); and 2) LSI could predict lesion dimensions better than power, contact force (CF), and force-time integral (FTI).

BACKGROUND

When performing radiofrequency (RF) catheter ablation for cardiac arrhythmias, reliable predictors of lesion quality are lacking. The LSI is a multiparametric index incorporating time, power, CF, and impedance recorded during ablation.

METHODS

RF lesions were created on porcine myocardial slabs by using an open-tip irrigated catheter capable of real-time monitoring of catheter-tissue CF. Initially, 3 power settings of 20, 25, and 30 W were used with a fixed CF of 10 g. A fixed power of 20 W was then set with a CF of 20 and 30 g, thereby yielding a total of 5 ablation groups. In each group, LSI values of 5, 6, 7, and 8 were targeted. Sixty RF lesions were created by using 20 ablation protocols (3 lesions for each protocol).

RESULTS

Lesion width and depth were not correlated with power or CF, but the results significantly correlated with FTI (p < 0.01) and LSI (p < 0.0001). Four steam pops occurred with power set at 30 W; no pops were noted with 20 or 25 W even when high LSI values were targeted.

CONCLUSIONS

In this in vitro model, FTI and LSI predicted RF lesion dimensions, whereas power and CF did not. The LSI predictive value was higher than that of FTI. Steam pops occurred only using high ablation power levels, regardless of the targeted LSI.

To evaluate the temporal coupling between spontaneous kisspeptin and luteinizing hormone (LH) pulsatile releases in polycystic ovary syndrome (PCOS) patients.

We examined 71 patients diagnosed with PCOS. A 2 h pulsatility study was performed to evaluate serum kisspeptin and LH pulse frequency and concentration, sampled every 10 min; baseline follicle-stimulating hormone (FSH), estradiol (E2), prolactin (PRL), cortisol, 17-hydroksy-progesterone (17OHP), testosterone (T), free testosterone index (FTI, and insulin levels were also measured. Detect and Specific Concordance (SC) algorithms were used to evaluate the temporal coupling associations between spontaneous episodic secretion of kisspeptin and LH.

All PCOS patients demonstrated LH and kisspeptin pulsatile secretions. When the SC index was calculated across the sample of PCOS patients (n = 71), no temporal coupling was observed between kisspeptin and LH pulses. When PCOS patients were subdivided according to their menstrual cyclicity, oligomenorrheic patients demonstrated elevated kisspeptin pulse frequency. Additionally, the SC index reveled a temporal coupling between kisspeptin and LH secretory peaks only in eumenorrheic patients (n = 30, intermenstrual interval < 45 days). Oligomenorrheic PCOS patients (intermenstrual interval>> 45 days) did not demonstrate temporal coupling between kisspeptin and LH secretory peaks.

The study of the endogenous kisspeptin and LH pulsatile release revealed the temporal coupling of kisspeptin with LH secretory pulses only in eumenorrheic. This data supports the hypothesis that neuroendocrine impairments in PCOS affect the coupling of kisspeptin with LH pulses and potentially worsen as the disease progresses, becoming unequivocally evident in oligomenorrheic PCOS patients.

Vascular calcification is associated with increased cardiovascular morbidity and mortality in patients with atherosclerosis, diabetes and chronic kidney disease. However, no viable treatments for this condition have been identified. This study aimed to determine whether farnesyl transferase inhibitors (FTIs) can reduce vascular calcification and the mechanism by which this reduction occurs.

We demonstrate that FTI-277 significantly inhibits phosphate-induced mineral deposition by vascular smooth muscle cells (VSMC) in vitro, prevents VSMC osteogenic differentiation, and increases mRNA expression of matrix Gla protein (MGP), an inhibitor of mineralization. FTI-277 increases Akt signaling in VSMC in short-term serum-stimulation assays and in long-term mineralization assays. In contrast, manumycin A has no effect on Akt signaling or mineralization. Co-incubation of VSMC with FTI-277 and SH6 (an Akt inhibitor) significantly reduces the inhibitory effect of FTI-277 on mineralization, demonstrating that FTI-277 inhibits calcification by activating Akt signaling. Over-expression of the constitutively active p110 sub-unit of PI3K in VSMC using adenovirus activates Akt, inhibits mineralization, suppresses VSMC differentiation and significantly enhances MGP mRNA expression. FTI-277 also inhibits phosphate-induced activation of caspase 3 and apoptosis of VSMC, and these effects are negated by co-incubation with SH6. Finally, using an ex vivo model of vascular calcification, we demonstrate that FTI-277 inhibits high phosphate-induced mineralization in aortic rings derived from rats with end-stage renal failure.

Together, these results demonstrate that FTI-277 inhibits VSMC mineral deposition by up-regulating PI3K/Akt signaling and preventing apoptosis, suggesting that targeting farnesylation, or Akt specifically, may have therapeutic potential for the prevention of vascular calcification.