The impact of outpatient systemic anti-cancer treatment on patient costs and work practices.

BACKGROUND: There is extensive focus on the rising costs of healthcare. However, for patients undergoing cancer treatment, there are additional personal costs, which are poorly characterised. AIM: To qualify indirect costs during anti-cancer therapy in a designated Irish cancer centre. METHODS: An anonymous questionnaire collected demographic data, current work practice, and personal expenditure on regular and non-regular indirect costs during treatment. Differences between groups of interest were compared using the Mann-Whitney U test. RESULTS: In total, there were 151 responders of median age 58 years; 60 % were female and 74 % were not working. Breast cancer (29 %) was the most frequent diagnosis. Indirect costs totalled a median of €1138 (range €21.60-€7089.84) per patient, with median monthly outgoings of €354. The greatest median monthly costs were hair accessories (€400), transportation (€65), and complementary therapies (€55). The majority (74 %) of patients used a car and median monthly fuel expenditure was €31 (range €1.44-€463.32). Women spent more money during treatment (€1617) than men (€974, p = 0.00128). In addition, median monthly expenditure was greater for those less than 50 years old (€1621 vs €1105; p = 0.04236), those who lived greater than 25 km away (€2015 vs €1078; p = 0.00008) and those without a medical card (€2023 vs €961; p = 0.00024). CONCLUSION: This study highlights the need for greater awareness of indirect expenditures associated with systemic anti-cancer therapy in Ireland.

Robust anti-obesity and metabolic effects of a dual GLP-1/glucagon receptor peptide agonist in rodents and non-human primates.

AIMS: To characterize the pharmacology of MEDI0382, a peptide dual agonist of glucagon-like peptide-1 (GLP-1) and glucagon receptors. MATERIALS AND METHODS: MEDI0382 was evaluated in vitro for its ability to stimulate cAMP accumulation in cell lines expressing transfected recombinant or endogenous GLP-1 or glucagon receptors, to potentiate glucose-stimulated insulin secretion (GSIS) in pancreatic ß-cell lines and stimulate hepatic glucose output (HGO) by primary hepatocytes. The ability of MEDI0382 to reduce body weight and improve energy balance (i.e. food intake and energy expenditure), as well as control blood glucose, was evaluated in mouse models of obesity and healthy cynomolgus monkeys following single and repeated daily subcutaneous administration for up to 2 months. RESULTS: MEDI0382 potently activated rodent, cynomolgus and human GLP-1 and glucagon receptors and exhibited a fivefold bias for activation of GLP-1 receptor versus the glucagon receptor. MEDI0382 produced superior weight loss and comparable glucose lowering to the GLP-1 peptide analogue liraglutide when administered daily at comparable doses in DIO mice. The additional fat mass reduction elicited by MEDI0382 probably results from a glucagon receptor-mediated increase in energy expenditure, whereas food intake suppression results from activation of the GLP-1 receptor. Notably, the significant weight loss elicited by MEDI0382 in DIO mice was recapitulated in cynomolgus monkeys. CONCLUSIONS: Repeated administration of MEDI0382 elicits profound weight loss in DIO mice and non-human primates, produces robust glucose control and reduces hepatic fat content and fasting insulin and glucose levels. The balance of activities at the GLP-1 and glucagon receptors is considered to be optimal for achieving weight and glucose control in overweight or obese Type 2 diabetic patients.

The Budget Impact of Hepatitis C Treatment in Ireland 2001-2012.

Chronic Hepatitis C (HCV) is estimated to infect 20,000 to 50,000 people in Ireland. National estimates of the number of patients who have been treated for HCV, their demographics and the cost associated with that treatment have not been published. Prescriptions for the treatment of HCV from 2000-2012 were established by interrogating the records of the High-Tech Drug Scheme and the pharmacy records of the Genitourinary Medicine and Infectious Diseases department of St. James Hospital. 2320 patients were initiated on treatment for HCV. Over €27 million was spent on HCV treatment. €25.5 million was spent on anti-viral therapy and €2 million was spent on haematological growth factor support for the management of adverse effects. The budget impact of HCV treatment has been significant in Ireland. New agents for HCV will have a greater budget impact but should require less spend on adverse event management.

Chronic glucokinase activator treatment at clinically translatable exposures gives durable glucose lowering in two animal models of type 2 diabetes.

BACKGROUND AND PURPOSE: Pharmacological activation of glucokinase (GK) lowers blood glucose in animal models and humans, confirming proof of concept for this mechanism. However, recent clinical evidence from chronic studies suggests that the glucose-lowering effects mediated by glucokinase activators (GKAs) are not maintained in patients with type 2 diabetes (T2D). Existing preclinical data with GKAs do not explain this loss of sustained glucose-lowering efficacy in patients. Here, we have assessed the effects of chronic (up to 11 months) treatment with two different GKAs in two models of T2D. EXPERIMENTAL APPROACH: Two validated animal models of T2D, insulin-resistant obese Zucker rats and hyperglycaemic gk(wt/del) mice, were treated with two different GKAs for 1 or 11 months respectively at exposures that translate to clinical exposures in humans. Blood glucose, cholesterol, triglycerides and insulin were measured. GKA pharmacokinetics were also determined. KEY RESULTS: Treatment with either GKA provided sustained lowering of blood glucose for up to 1 month in the Zucker rat and up to 11 months in hyperglycaemic gk(wt/del) mice, with maintained compound exposures. This efficacy was achieved without increases in plasma or hepatic triglycerides, accumulation of hepatic glycogen or impairment of glucose-stimulated insulin secretion. CONCLUSIONS AND IMPLICATIONS: Chronic treatment with two GKAs in two animal models of diabetes provided sustained lowering of blood glucose, in marked contrast to clinical findings. Therefore, either these animal models of T2D are not good predictors of responses in human T2D or we need a better understanding of the consequences of GK activation in humans.

Antibacterial activity of epidural infusions.

The incidence of epidural abscess following epidural catheterisation appears to be increasing, being recently reported as one in 1000 among surgical patients. This study was designed to investigate the antibacterial activity of various local anaesthetics and additives, used in epidural infusions, against a range of micro-organisms associated with epidural abscess. The aim was to determine which, if any, epidural infusion solution has the greatest antibacterial activity. Bupivacaine, ropivacaine and levobupivacaine crystals were dissolved and added to Mueller-Hinton Agar in concentrations of 0.06%, 0.125%, 0.2%, 0.25%, 0.5% and 1%. Fentanyl, adrenaline and clonidine were also mixed with agar in isolation and in combination with the local anaesthetics. Using a reference agar dilution method, the minimum inhibitory concentrations were determined for a range of bacteria. Bupivacaine showed antibacterial activity against Staphylococcus aureus, Enterococcus faecalis and Escherichia coli with minimum inhibitory concentrations between 0.125% and 0.25%. It did not inhibit the growth of Pseudomonas aeruginosa at any of the concentrations tested. Levobupivacaine and ropivacaine showed no activity against Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa, even at the highest concentrations tested, and minimal activity against Escherichia coli (minimum inhibitory concentrations 0.5% and 1% respectively). The presence of fentanyl, adrenaline and clonidine had no additional effect on the antibacterial activity of any of the local anaesthetic agents. The low concentrations of local anaesthetic usually used in epidural infusions have minimal antibacterial activity. While the clinical implications of this in vitro study are not known, consideration should be given to increasing the concentration of bupivacaine in an epidural infusion or to administering a daily bolus of 0.25% bupivacaine to reduce the risk of epidural bacterial growth.

Predictive blood glucose lowering efficacy by Glucokinase activators in high fat fed female Zucker rats.

BACKGROUND AND PURPOSE: Glucokinase (GK) is the rate-limiting enzyme of hepatic glucose metabolism and acts as a sensor for glucose-stimulated insulin release in beta-cells. Here we examine whether the lowering of blood glucose levels in the rat by small molecule glucokinase activators (GKAs) can be predicted from in vitro enzyme potencies and plasma compound exposure. EXPERIMENTAL APPROACH: We developed an insulin resistant and hyperinsulinemic animal model, the high fat fed female Zucker (fa/fa) rat (HFFZ), and measured the acute in vivo glucose-lowering efficacy of a number of GKAs in an oral glucose tolerance test. KEY RESULTS: Four GKAs (at 1 to 30 mg kg(-1)), with different in vitro enzyme potencies, dose-dependently improved oral glucose tolerance in HFFZ rats (10-40% decrease glucose area under the curve (AUC) from vehicle control). The extent of glucose lowering, or the pharmacodynamic (PD) effect, of a GKA was directly related to the total compound concentration in the plasma; the pharmacokinetic (PK) measurement. This PK-PD relationship was extended across a series of GKAs by accounting for differences in protein binding and in the in vitro potency. CONCLUSIONS AND IMPLICATIONS: When the unbound GKA compound level is greater than the in vitro enzyme potency there was significant blood glucose lowering in vivo. This latter relationship was upheld in non-diabetic Wistar rats orally dosed with a GKA. The robust and predictive nature of the PK-PD relationship for GKAs may prove of value in testing these agents in early human clinical studies.

Introduction to the Kinases in Diabetes Biochemical Society Focused Meeting: are protein kinases good targets for antidiabetic drugs?

Insulin regulates whole-body glucose homoeostasis by modulating the activities of protein kinases in its target tissues: muscle, liver and fat. Defects in insulin's ability to modulate protein kinase activity lead to 'insulin resistance' or impaired insulin action. Insulin resistance in combination with defective insulin secretion from the pancreas results in the elevated blood glucose levels that are characteristic of diabetes mellitus. Pharmacological agents that selectively modulate protein kinase activities in insulin-resistant tissues may act either as insulin-sensitizing or insulin-mimetic drugs. Consistent with this, small molecule modulators of a number of protein kinases have demonstrated efficacy in animal models of insulin resistance and diabetes. Moreover, emerging data in humans suggest that marketed anti-diabetic agents may also act in part through modulating protein kinase activities. This meeting was convened to consider the potential to treat insulin resistance and Type II diabetes by modulating protein kinase activity.

Small molecule glucokinase activators as novel anti-diabetic agents.

The monomeric enzyme GK (glucokinase) has a low affinity for glucose and, quantitatively, is largely expressed in the liver and pancreatic beta-cells, playing a key 'glucose sensing' role to regulate hepatic glucose balance and insulin secretion. Mutations of GK in man can be inactivating, to cause a form of diabetes mellitus, or activating, to lower blood glucose levels. Recently, models of GK protein structure have helped to elucidate the role of inactivating and activating mutations, with the latter revealing an allosteric binding site, possibly for an unknown physiological activator. However, this discovery was pre-dated by Drug Discovery projects that have identified small organic molecules that activate pancreatic and liver GK enzyme activity. These compounds stimulate insulin secretion in islets and glucose metabolism in hepatocytes. The profile of these GK activators, both in vitro and in vivo and the potential role that GK activators play in lowering blood glucose levels in Type II diabetes mellitus will be discussed.

In vivo evaluation of the potency and bladder-vascular selectivity of the ATP-sensitive potassium channel openers (-)-cromakalim, ZD6169 and WAY-133537 in rats.

OBJECTIVE: To compare in vivo the potency and bladder-vascular selectivity of ATP-sensitive potassium channel openers (KCOs) (-)-cromakalim, WAY-133537 and ZD6169 and a muscarinic antagonist, tolterodine in rats. MATERIALS AND METHODS: Bladder and arterial pressures were monitored simultaneously, before and after increasing intravenous doses of compounds, in each of two urethane-anaesthetized rat bladder hyperactivity models: spontaneous non-voiding myogenic contractions secondary to partial outlet obstruction and volume-induced neurogenic contractions. RESULTS: (-)-Cromakalim, WAY-133537 and ZD6169 caused a dose-dependent suppression of spontaneous contractions in the obstructed model, with a 50% inhibition of the contraction area under the curve at doses of 0.06, 0.14 and 2.4 micro mol/kg (intravenous), respectively. Corresponding decreases in mean arterial pressure at these effective doses were 24%, 15% and 15%, respectively. The KCO potency rank order was the same and their relative potency highly comparable in the neurogenic model. There was complete inhibition of spontaneous contractions in obstructed rats at doses corresponding to approximately 50% inhibition of the neurogenic contractions. While tolterodine caused a dose-dependent inhibition of contractions in the neurogenic model, it was ineffective at inhibiting non-voiding contractions in obstructed rats. CONCLUSIONS: All KCOs tested caused significant decreases in arterial pressure at doses effective on the bladder in the model of obstructive instability, suggesting a lack of bladder-vascular selectivity. Similar KCO potency in both assays suggests no appreciable changes in KATP channel function as a result of partial outlet obstruction.

Nonsteroidal selective glucocorticoid modulators: the effect of C-5 alkyl substitution on the transcriptional activation/repression profile of 2,5-dihydro-10-methoxy-2,2,4-trimethyl-1H-[1]benzopyrano[3,4-f]quinolines.

The preparation and characterization of a series of selective glucocorticoid receptor modulators are described. The preliminary structure-activity relationship of nonaromatic C-5 substitution on the tetracyclic quinoline core showed a preference for small lipophilic side chains. Proper substitution at this position maintained the transcriptional repression of proinflammatory transcription factors while diminishing the transcriptional activation activity of the ligand/glucocorticoid receptor complex. The optimal compounds described in this study were the allyl analogue 18 and cyclopentyl analogue 32. These candidates showed slightly less potent, highly efficacious E-selectin repression with significantly reduced levels of glucocorticoid response element activation in reporter gene assays vs prednisolone. Allyl analogue 18 was evaluated in vivo. An oral dose of 18 showed an ED(50) = 1.7 mg/kg as compared to 1.2 mg/kg for prednisolone in the Sephadex-induced pulmonary eosinophilia model and an ED(50) = 15 mg/kg vs 4 mg/kg for prednisolone in the carrageenan-induced paw edema model.

Symptomatic lactic acidosis in hospitalized antiretroviral-treated patients with human immunodeficiency virus infection: a report of 12 cases.

We retrospectively investigated the clinical and histopathologic features of hospitalized patients infected with human immunodeficiency virus who had symptomatic lactic acidosis syndrome at a university teaching hospital during 1995-2000. Twelve patients were identified, 11 during 1998-2000; of these, 5 died with rapid progression to otherwise unexplained multiple-organ failure. All had extensive prior exposure to nucleoside analog reverse-transcriptase inhibitors (NRTIs). At presentation, the most commonly identified NRTI component of antiretroviral regimens was stavudine plus didanosine. Eleven patients presented with abdominal pain, nausea, and/or emesis. Eight patients had prior acute weight loss (mean [+/-SD], 12+/-5.3 kg). Median venous plasma lactate levels were > or =2-fold greater than the upper limit of normal (2.1 mmol/L). Serum transaminase levels were near normal limits at presentation. Histopathologic studies confirmed hepatic macrovesicular and microvesicular steatosis in 6 patients. Concurrent chemical pancreatitis was identified in 6 patients. The increasing number of cases identified during the study period suggests that physicians better recognize symptomatic lactic acidosis and/or that cumulative NRTI exposure may increase the risk for this syndrome.

Validation of FLIPR membrane potential dye for high throughput screening of potassium channel modulators.

A fluorescence-based assay using the FLIPR Membrane Potential Assay Kit (FMP) was evaluated for functional characterization and high throughput screening (HTS) of potassium channel (ATP-sensitive K+ channel; K(ATP)) modulators. The FMP dye permits a more sensitive evaluation of changes in membrane potential with a more rapid response time relative to DiBAC4(3). The time course of responses is comparable to ligand-evoked activation of the channel measured by patch-clamp studies. The pharmacological profile of the K+ channel evaluated by using reference K(ATP) channel openers is in good agreement with that derived previously by DiBAC4(3)-based FLIPR assays. Improved sensitivity of responses together with the diminished susceptibility to artifacts such as those evoked by fluorescent compounds or quenching agents makes the FMP dye an alternative choice for HTS screening of potassium channel modulators.

Synthesis and characterization of non-steroidal ligands for the glucocorticoid receptor: selective quinoline derivatives with prednisolone-equivalent functional activity.

A novel class of functional ligands for the human glucocorticoid receptor is described. Substituents in the C-10 position of the tetracyclic core are essential for glucocorticoid receptor (GR) selectivity versus other steroid receptors. The C-5 position is derivatized with meta-substituted aromatic groups, resulting in analogues with a high affinity for GR (K(i) = 2.4-9.3 nM) and functional activity comparable to prednisolone in reporter gene assays of glucocorticoid-mediated gene transcription. The biological activity of these novel quinolines was also prednisolone-equivalent in whole cell assays of glucocorticoid function, and compound 13 was similar to prednisolone (po ED(50) = 2.8 mpk for 13 vs ED(50) = 1.2 mpk for prednisolone) in a rodent model of asthma (sephadex-induced eosinophil influx).

Inhibition of GSK-3 selectively reduces glucose-6-phosphatase and phosphatase and phosphoenolypyruvate carboxykinase gene expression.

A major action of insulin is to regulate the transcription rate of specific genes. The expression of these genes is dramatically altered in type 2 diabetes. For example, the expression of two hepatic genes, glucose-6-phosphatase and PEPCK, is normally inhibited by insulin, but in type 2 diabetes, their expression is insensitive to insulin. An agent that mimics the effect of insulin on the expression of these genes would reduce gluconeogenesis and hepatic glucose output, even in the presence of insulin resistance. The repressive actions of insulin on these genes are dependent on phosphatidylinositol (PI) 3-kinase. However, the molecules that lie between this lipid kinase and the two gene promoters are unknown. Glycogen synthase kinase-3 (GSK-3) is inhibited following activation of PI 3-kinase and protein kinase B. In hepatoma cells, we find that selectively reducing GSK-3 activity strongly reduces the expression of both gluconeogenic genes. The effect is at the level of transcription and is observed with induced or basal gene expression. In addition, GSK-3 inhibition does not result in the subsequent activation of protein kinase B or inhibition of the transcription factor FKHR, which are candidate regulatory molecules for these promoters. Thus, GSK-3 activity is required for basal activity of each promoter. Inhibitors of GSK-3 should therefore reduce hepatic glucose output, as well as increase the synthesis of glycogen from L-glucose. These findings indicate that GSK-3 inhibitors may have greater therapeutic potential for lowering blood glucose levels and treating type 2 diabetes than previously realized.

Functional implication of spare ATP-sensitive K(+) channels in bladder smooth muscle cells.

ATP-sensitive K(+) (K(ATP)) channels play important roles in the regulation of excitability in urinary bladder smooth muscle cells. Patch-clamp studies revealed that the current density was about 9-fold higher in the pig bladder smooth muscle cells, compared with guinea pig, although the rank order of potencies for suppression of electrical field-stimulated contraction of bladder strips by K(ATP) channel openers (KCOs) showed a nearly 1:1 correlation between pig and guinea pig. To investigate the existence of spare K(ATP) channels, P1075-evoked current and membrane potential responses were studied in bladder smooth muscle cells. During a 10-min exposure to P1075 (10 microM), K(ATP) currents ran down by approximately 30.5%, whereas membrane hyperpolarization remained constant. P1075 evoked membrane hyperpolarization with an EC(50) value of 0.20 +/- 0.02 microM, comparable to that required for smooth muscle relaxation (EC(50) = 0.11 +/- 0.01 microM). However, these potencies are 6-fold higher than those required for current activation (EC(50) = 0.73 +/- 0.4 microM). These findings demonstrate that the reduction in membrane excitability by KCOs is associated with membrane hyperpolarization, and that a low amount of K(ATP) channel opening is sufficient to suppress bladder smooth muscle contraction.

Potassium channels: molecular defects, diseases, and therapeutic opportunities.

Potassium channels play important roles in vital cellular signaling processes in both excitable and nonexcitable cells. Over 50 human genes encoding various K(+) channels have been cloned during the past decade, and precise biophysical properties, subunit stoichiometry, channel assembly, and modulation by second messenger and ligands have been elucidated to a large extent. Recent advances in genetic linkage analysis have greatly facilitated the identification of many disease-producing loci, and naturally occurring mutations in various K(+) channels have been identified in diseases such as long-QT syndromes, episodic ataxia/myokymia, familial convulsions, hearing and vestibular diseases, Bartter's syndrome, and familial persistent hyperinsulinemic hypoglycemia of infancy. In addition, changes in K(+) channel function have been associated with cardiac hypertrophy and failure, apoptosis and oncogenesis, and various neurodegenerative and neuromuscular disorders. This review aims to 1) provide an understanding of K(+) channel function at the molecular level in the context of disease processes and 2) discuss the progress, hurdles, challenges, and opportunities in the exploitation of K(+) channels as therapeutic targets by pharmacological and emerging genetic approaches.

Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription.

BACKGROUND: Glycogen synthase kinase-3 (GSK-3) is a serine/threonine protein kinase, the activity of which is inhibited by a variety of extracellular stimuli including insulin, growth factors, cell specification factors and cell adhesion. Consequently, inhibition of GSK-3 activity has been proposed to play a role in the regulation of numerous signalling pathways that elicit pleiotropic cellular responses. This report describes the identification and characterisation of potent and selective small molecule inhibitors of GSK-3. RESULTS: SB-216763 and SB-415286 are structurally distinct maleimides that inhibit GSK-3alpha in vitro, with K(i)s of 9 nM and 31 nM respectively, in an ATP competitive manner. These compounds inhibited GSK-3beta with similar potency. However, neither compound significantly inhibited any member of a panel of 24 other protein kinases. Furthermore, treatment of cells with either compound stimulated responses characteristic of extracellular stimuli that are known to inhibit GSK-3 activity. Thus, SB-216763 and SB-415286 stimulated glycogen synthesis in human liver cells and induced expression of a beta-catenin-LEF/TCF regulated reporter gene in HEK293 cells. In both cases, compound treatment was demonstrated to inhibit cellular GSK-3 activity as assessed by activation of glycogen synthase, which is a direct target of this kinase. CONCLUSIONS: SB-216763 and SB-415286 are novel, potent and selective cell permeable inhibitors of GSK-3. Therefore, these compounds represent valuable pharmacological tools with which the role of GSK-3 in cellular signalling can be further elucidated. Furthermore, development of similar compounds may be of use therapeutically in disease states associated with elevated GSK-3 activity such as non-insulin dependent diabetes mellitus and neurodegenerative disease.

Atypical protein kinases Clambda and -zeta associate with the GTP-binding protein Cdc42 and mediate stress fiber loss.

Both the Rho family of low-molecular-weight GTP-binding proteins and protein kinases C (PKCs) mediate responses to a variety of extracellular and intracellular signals. They share many downstream targets, including remodeling of the actin cytoskeleton, activation of p70(S6) kinase and c-jun N-terminal kinase (JNK), and regulation of transcription and cell proliferation. We therefore investigated whether Rho family GTP-binding proteins bind to PKCs. We found that Cdc42 associates with atypical PKCs (aPKCs) PKCzeta and -lambda in a GTP-dependent manner. The regulatory domain of the aPKCs mediates the interaction. Expression of activated Cdc42 results in the translocation of PKClambda from the nucleus into the cytosol, and Cdc42 and PKClambda colocalize at the plasma membrane and in the cytoplasm. Expression of activated Cdc42 leads to a loss of stress fibers, as does overexpression of either the wild type or an activated form of PKClambda. Kinase-dead PKClambda and -zeta constructs acted as dominant negatives and restored stress fibers in cells expressing the activated V12 Cdc42 mutant, indicating that Cdc42-dependent loss of stress fibers requires aPKCs. Kinase-dead PKClambda and -zeta and dominant-negative N17 Cdc42 also blocked Ras-induced loss of stress fibers, suggesting that this pathway may also be important for Ras-dependent cytoskeletal changes. N17 Rac did not block Ras-induced loss of stress fibers, nor did kinase-dead PKClambda block V12 Rac-stimulated loss of stress fibers. These results indicate that Cdc42 and Rac use different pathways to regulate stress fibers.

c-Jun is a JNK-independent coactivator of the PU.1 transcription factor.

The ETS domain transcription factor PU.1 is necessary for the development of monocytes and regulates, in particular, the expression of the monocyte-specific macrophage colony-stimulating factor (M-CSF) receptor, which is critical for monocytic cell survival, proliferation, and differentiation. The bZIP transcription factor c-Jun, which is part of the AP-1 transcription factor complex, is also important for monocytic differentiation, but the monocyte-specific M-CSF receptor promoter has no AP-1 consensus binding sites. We asked the question of whether c-Jun could promote the induction of the M-CSF receptor by collaborating with PU.1. We demonstrate that c-Jun enhances the ability of PU.1 to transactivate the M-CSF receptor promoter as well as a minimal thymidine kinase promoter containing only PU.1 DNA binding sites. c-Jun does not directly bind to the M-CSF receptor promoter but associates via its basic domain with the ETS domain of PU.1. Consistent with our observation that AP-1 binding does not contribute to c-Jun coactivation is the observation that the activation of PU.1 by c-Jun is blocked by overexpression of c-Fos. Phosphorylation of c-Jun by c-Jun NH2-terminal kinase on Ser-63 and -73 does not alter the ability of c-Jun to enhance PU.1 transactivation. Activated Ras enhances the transcriptional activity of PU.1 by up-regulating c-Jun expression without changing the phosphorylation pattern of PU.1. The activation of PU.1 by Ras is blocked by a mutant c-Jun protein lacking the basic domain. The expression of this mutant form of c-Jun also completely blocks 12-O-tetradecanoylphorbol-13-acetate-induced M-CSF receptor promoter activity during monocytic differentiation. We propose therefore that c-Jun acts as a c-Jun NH2-terminal kinase-independent coactivator of PU.1, resulting in M-CSF receptor expression and development of the monocytic lineage.