Statistical controversies in clinical research: statistical significance-too much of a good thing .

The use and interpretation of P values is a matter of debate in applied research. We argue that P values are useful as a pragmatic guide to interpret the results of a clinical trial, not as a strict binary boundary that separates real treatment effects from lack thereof. We illustrate our point using the result of BOLERO-1, a randomized, double-blind trial evaluating the efficacy and safety of adding everolimus to trastuzumab and paclitaxel as first-line therapy for HER2+ advanced breast cancer. In this trial, the benefit of everolimus was seen only in the predefined subset of patients with hormone receptor-negative breast cancer at baseline (progression-free survival hazard ratio = 0.66, P = 0.0049). A strict interpretation of this finding, based on complex 'alpha splitting' rules to assess statistical significance, led to the conclusion that the benefit of everolimus was not statistically significant either overall or in the subset. We contend that this interpretation does not do justice to the data, and we argue that the benefit of everolimus in hormone receptor-negative breast cancer is both statistically compelling and clinically relevant.

Role of the mitogen-activated protein kinases and tyrosine kinases during leukotriene B4-induced eosinophil activation.

Exposure of guinea-pig eosinophils to leukotriene B4 (LTB4; 1 microM) resulted in a rapid generation of H2O2 (index of NADPH oxidase activation), stimulated [3H]arachidonic acid (AA) release (index of phospholipase A2 activity), and promoted CD18-dependent homotypic aggregation. Under similar conditions, LTB4 (1 microM) induced a rapid activation of extracellular-regulated kinases-1 and 2 (ERK-1/2) but not c-jun N-terminal kinases 46 and 54 (JNK-46/54) or p38 mitogen-activated protein kinase (p38 MAP kinase). To examine the role of ERK-1/2 in the mechanism of eosinophil activation, a selective inhibitor of MAP kinase kinase-1/2 (MEK-1/2), PD098059, was employed. However, PD 098059 at concentrations that attenuated ERK-1/2 activation had no significant affect on eosinophil activation. In contrast, a role for tyrosine kinases in LTB4-induced eosinophil activation was suggested by studies with the tyrosine kinase inhibitors, herbimycin A and lavendustin A. However, the results of those experiments implied divergent pathways for the control of eosinophil responses because the inhibitors were more effective at attenuating H2O2 generation than [3H]AA release, and had little effect on homotypic aggregation.

The academic global virtual concept in clinical cancer research and its application to breast cancer: The Breast Cancer International Research Group.

In contrast to previous decades, the 1990s have witnessed an increase of new agents with significant activity in breast cancer, including chemotherapy, hormone therapy, and, more recently, biologic modifiers. All information appears to confirm that such a trend will persist and even accelerate in the coming decades. Unless clear strategies of development for new drugs are strictly followed, it will become difficult to adequately assess the many new agents with potentially important activity against breast cancer, and patient access may become a limiting key factor. The academic, global virtual concept is calling for the definition of a new relationship between the pharmaceutical industry and clinical researchers. The main aspect is related to the creation of partnerships with an academically controlled global strategy of development for promising new agents, in which the quality and independence of processes (adjuvant setting, for example) are critical. The means are based on the globalization of patient access (worldwide network) and the virtuality of the approach (modern means of communication as well as access to subgroups of patients). The Breast Cancer International Research Group is the first academic global virtual cooperative group in breast cancer and is making contributions in the development of new drugs, such as taxanes, new antiestrogens, and new cytokines.

The MAP kinase inhibitors, PD098059, UO126 and SB203580, inhibit IL-1beta-dependent PGE(2) release via mechanistically distinct processes.

1. In common with human bronchial epithelial cells, pulmonary A549 cells release prostaglandin (PG) E(2) in response to pro-inflammatory cytokines. We have therefore used these cells to examine the effect of the selective mitogen activated protein (MAP) kinase inhibitors; PD098059, a mitogen activated and extracellular regulated kinase kinase (MEK) 1 inhibitor, UO126, a dual MEK1 & MEK2 inhibitor, and SB203580, a p38 MAP kinase inhibitor in the IL-1beta-dependent release of PGE(2). 2. Following IL-1beta treatment the extracellular regulated kinases (ERKs) and the p38 MAP kinases were rapidly phosphorylated. 3. PD09059, UO126 and SB203580 prevented IL-1beta-induced PGE(2) release at doses that correlated closely with published IC(50) values. Small or partial effects at the relevant doses were observed on induction of cyclo-oxygenase (COX) activity or COX-2 protein suggesting that the primary effects were at the level of arachidonate availability. 4. Neither PD098059 nor SB203580 showed any effect on IL-1beta-induced arachidonate release. We therefore speculate that the MEK1/ERK and p38 kinase cascades play a role in the functional coupling of arachidonate release to COX-2. 5. In contrast, UO126 was highly effective at inhibiting IL-1beta-dependent arachidonate release, implicating MEK2 in the activation of the PLA(2) that is involved in IL-1beta-dependent PGE(2) release. 6. We conclude that the MEK1, MEK2 and p38 MAP kinase inhibitors, PD098059, UO126 and SB203580, are highly potent in respect of inflammatory PG release. Finally, we conclude that these inhibitors act via mechanistically distinct processes, which may have anti-inflammatory benefits.

Extracellular signal-regulated kinase 1/2 control Ca(2+)-independent force development in histamine-stimulated bovine tracheal smooth muscle.

The role of extracellular signal-regulated kinase (ERK)-1 and ERK-2 in controlling histamine-induced tone in bovine trachealis was investigated. PD 098059, an inhibitor of mitogen-activated protein kinase kinase (MKK)-1, had no effect on the histamine concentration-response relationship that described contraction. However, in the presence of EGTA, PD 098059 produced a parallel 5 fold rightwards shift of the histamine concentration-response curve without reducing the maximum response. The beta(2)-adrenoceptor agonist, procaterol, also displaced the histamine-concentration response curve to the right but the effect was much greater than that evoked by PD 098059, non-competitive and seen in the absence and presence of EGTA. A low basal level of pERK-1 and pERK-2 was always detected in untreated trachealis, which was significantly higher in EGTA-treated tissues and inhibited by PD 098059 and procaterol. Histamine markedly enhanced the phosphorylation of ERK-1 and ERK-2 by a mechanism that was also enhanced by EGTA and significantly attenuated by procaterol and PD 098059. Neither cholera toxin nor SP:-8-Br-cAMPS mimicked the ability of procaterol to dephosphorylate ERK. Similarly, neither pertussis toxin (PTX) nor RP:-8-Br-cAMPS, an inhibitor of cyclic AMP-dependent protein kinase (PKA), affected basal pERK levels or antagonized the inhibitory effect of procaterol. These data implicate the MKK-1/ERK signalling cascade in Ca(2+)-independent, histamine-induced contraction of bovine trachealis. In addition, the ability of procaterol to dephosphorylate ERK in an RP:-8-Br-cAMPS- and PTX-insensitive manner suggests that this may contribute to the anti-spasmogenic activity of beta(2)-adrenoceptor agonists by activating a novel PKA-independent pathway.

Pharmacological comparison of LTB(4)-induced NADPH oxidase activation in adherent and non-adherent guinea-pig eosinophils.

1. Leukotriene B(4) (LTB(4)) stimulation of guinea-pig peritoneal eosinophils, induced a biphasic activation of the NADPH oxidase composed of a rapid (<3 min) phase mediated by non-adherent cells and a sustained (3 - 120 min) phase mediated by CD11b/CD18 adherent eosinophils. Studies were undertaken to compare the intracellular mechanism that mediate these responses. 2. SB 203580 and PP1, inhibitors of p38 mitogen-activated protein (MAP) kinase and the src-family protein tyrosine kinases, respectively caused concentration-dependent attenuation of both the rapid (SB203580: pD(2)=-6.31; PP1: pD(2)=-5.50) and sustained (SB203580: pD(2)=-6.50; PP1: pD(2)=-5.73) phases. Similarly, the MAP kinase kinase-1 inhibitor, PD098059 produced partial inhibition of the both phases of superoxide generation. 3. The protein kinase C (PKC) inhibitors Ro-31 8220, GF 109203X and Gö 6976 attenuated the rapid NADPH oxidase response (pD(2)s=-6.10, -6.72, -6.15 respectively) and, to a lesser extent, (pD(2)s=-5.54, -6.02, -6.51 respectively) the sustained phase. 4. An inhibitor of phosphatidylinositol 3-kinase (PtdIns 3-kinase), wortmannin caused concentration dependent attenuation of the sustained (pD(2)=-8.68) but not rapid phase of superoxide generation. In contrast, the syk kinase inhibitor, piceatannol abolished the rapid (pD(2)=-6.43) but not sustained respiratory responses. 5. This study demonstrates that LTB(4)-induced superoxide generation from adherent and non-adherent eosinophils is mediated via both common (p38 MAP kinase, MEK-1, PKC and the src kinases) and divergent intracellular pathways (syk kinases and PtdIns 3-kinase). This suggests the possibility of therapeutic intervention to selective attenuate activation of adherent tissue eosinophils.

'Outside-in' signalling mechanisms underlying CD11b/CD18-mediated NADPH oxidase activation in human adherent blood eosinophils.

1 Incubation of human eosinophils in BSA-coated tissue culture plates resulted in time-dependent adhesion and attendant activation of the NADPH oxidase that were both inhibited (by >85%) by blocking antibodies raised against CD11b and CD18. 2 SB 203580, an inhibitor of p38 mitogen-activated protein (MAP) kinase, did not influence adhesion but inhibited superoxide anion generation (pIC50=-6.57). 3 PP1, an inhibitor of the src-family of protein tyrosine kinases, inhibited adhesion and CD11b/CD18-mediated superoxide anion generation with similar potencies (pEC50s=-5.53 and -5.99 respectively) suggesting that inhibition of the NADPH oxidase was a direct consequence of blocking adhesion. 4 The protein kinase C (PKC) inhibitors Ro-31 8220 (broad spectrum inhibitor), GF 109203X (inhibitor of conventional and novel isoforms) and Gö 6976 (inhibitor of conventional isoforms) suppressed adhesion-dependent NADPH oxidase activation (pIC50s=-6.61, -6.05 and -4.89 respectively) without affecting adhesion. Based upon the selectivity of these drugs PKCdelta and PKCepsilon are implicated in the suppression of oxidant production. 5 Wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PtdIns 3-kinase), abolished superoxide anion production in adherent eosinophils (pEC50=-9.06). Similarly, CD11b/CD18-dependent adhesion was suppressed with the same potency (pEC50=-9.29) although the maximum effect did not exceed 50% implying that wortmannin also had an affect on those processes that govern adhesion-driven oxidase activation. 6 PD 098059 and piceatannol, inhibitors of MAP kinase kinase-1 and the syk tyrosine kinase respectively, had no effect on CD11b/CD18-mediated adhesion or NADPH oxidase activation. 7 The results of this study demonstrate that human eosinophils adhere to BSA-coated plastic by a CD11b/CD18-dependent mechanism, which is responsible for activation of the NADPH oxidase. Although the signalling pathway(s) utilized by CD11b/CD18 is still to be elucidated, the data presented herein implicate p38 MAP kinase, novel PKCs and PtdIns 3-kinase.

p38 MAP kinase and MKK-1 co-operate in the generation of GM-CSF from LPS-stimulated human monocytes by an NF-kappa B-independent mechanism.

1. The extent to which the p38 mitogen-activated protein (MAP) kinase and MAP kinase kinase (MKK)-1-signalling pathways regulate the expression of granulocyte/macrophage colony-stimulating factor (GM-CSF) from LPS-stimulated human monocytes has been investigated and compared to the well studied cytokine tumour necrosis factor-alpha (TNF alpha). 2. Lipopolysaccharide (LPS) evoked a concentration-dependent generation of GM-CSF from human monocytes. Temporally, this effect was preceded by an increase in GM-CSF mRNA transcripts and abolished by actinomycin D and cycloheximide. 3. LPS-induced GM-CSF release and mRNA expression were associated with a rapid and time-dependent activation of p38 MAP kinase, ERK-1 and ERK-2. 4. The respective MKK-1 and p38 MAP kinase inhibitors, PD 098059 and SB 203580, maximally suppressed LPS-induced GM-CSF generation by >90%, indicating that both of these signalling cascades co-operate in the generation of this cytokine. 5. Electrophoretic mobility shift assays demonstrated that LPS increased nuclear factor kappa B (NF-kappa B) : DNA binding. SN50, an inhibitor of NF-kappa B translocation, abolished LPS-induced NF-kappaB : DNA binding and the elaboration of TNFalpha, a cytokine known to be regulated by NF-kappaB in monocytes. In contrast, SN50 failed to affect the release of GM-CSF from the same monocyte cultures. 6. Collectively, these results suggest that the generation of GM-CSF by LPS-stimulated human monocytes is regulated in a co-operative fashion by p38 MAP kinase- and MKK-1-dependent signalling pathways independently of the activation of NF-kappa B.

Identification of the protein kinase C isoenzymes in human lung and airways smooth muscle at the protein and mRNA level.

The protein kinase C (PKC) isoenzymes expressed by human peripheral lung and tracheal smooth muscle resected from individuals undergoing heart-lung transplantation were identified at the protein and mRNA level. Western immunoblot analyses of human lung identified multiple PKC isoenzymes that were differentially distributed between the soluble and particulate fraction. Thus, PKC alpha, PKC betaII, PKC epsilon, and PKC zeta were recovered predominantly in the soluble fraction whereas the eta isoform was membrane-associated together with trace amounts of PKC alpha and PKC epsilon. PKC beta1-like immunoreactivity was occasionally seen although the intensity of the band was uniformly weak. Immunoreactive bands corresponding to PKCs gamma, delta, or theta were never detected. Reverse transcription-polymerase chain reaction (RT-PCR) of RNA extracted from human lung using oligonucleotide primer pairs that recognise unique sequences in each of the PKC genes amplified cDNA fragments that corresponded to the predicted sizes of PKC alpha, PKC betaI, PKC betaII, PKC epsilon, PKC zeta, and PKC eta (consistent with the expression of PKC isoenzyme protein) and, in addition, mRNA for PKC delta; PCR fragments of the expected size for the supposedly muscle-specific isoform, PKC theta, or the atypical isoenzyme, PKC lambda, were never obtained. The complement and distribution of PKC isoforms in human trachealis were similar, but not identical, to human lung. Thus, immunoreactive bands corresponding to the alpha, betaI, betaII, epsilon, and zeta isoenzymes of PKC were routinely labelled in the cytosolic fraction. In the particulate material PKC alpha, PKC epsilon, PKC alpha, PKC eta, and PKC mu were detected by immunoblotting. With the exception of PKC zeta, RT-PCR analyses confirmed the expression of the PKC isoforms detected at the protein level and, in addition, identified mRNA for PKC delta. Collectively, these data clearly demonstrate the expression of multiple PKC isoenzymes in human lung and tracheal smooth muscle, suggesting that they subserve diverse multifunctional roles in these tissues.

Role of arachidonic acid in leukotriene B(4)-induced guinea-pig eosinophil homotypic aggregation.

The activation of eosinophils with the lipid mediator, leukotriene B(4), induces their homotypic aggregation. Upon activation with leukotriene B(4), eosinophils release a significant amount of arachidonic acid, a process dependent on the activation of phospholipase A(2). Here, we have evaluated whether arachidonic acid could induce aggregation of eosinophils and whether the release of arachidonic acid mediated the aggregation induced by leukotriene B(4). The exogenous administration of arachidonic acid induced a concentration-dependent eosinophil homotypic aggregation. Pretreatment of eosinophils with a 5-lipoxygenase inhibitor or a leukotriene B(4) receptor antagonist abrogated arachidonic-acid-induced aggregation. Arachidonic acid induced a significant increase in leukotriene B(4) levels and desensitised leukotriene B(4)-induced aggregation in a dose-dependent manner. Moreover, this desensitisation was effectively reversed by a 5-lipoxygenase inhibitor. However, arachidonic acid failed to induce a rise in intracellular Ca(2+) in eosinophils and failed to desensitise these cells to rises in intracellular Ca(2+) induced by leukotriene B(4). Pretreatment of eosinophils with the phospholipase A(2) inhibitor, mepacrine, inhibited the aggregation responses induced by 1 nM leukotriene B(4) by approximately 50% but had no significant effect on the other concentrations of leukotriene B(4) tested (0.1 to 100 nM). In conclusion, arachidonic acid stimulates eosinophil aggregation indirectly via the release of leukotriene B(4). Although a significant amount of arachidonic acid is released in response to activation of eosinophils with leukotriene B(4), the arachidonic acid released does appear to play a major role in mediating leukotriene B(4)-induced eosinophil aggregation.

Transglutaminase involvement in the secretion of insulin from electropermeabilised rat islets of Langerhans.

Ca(2+)-Induced insulin release from electropermeabilised islets is inhibited by the transglutaminase inhibitors monodansylcadaverine, glycine methylester, methylamine and cystamine but not by the control compounds dimethyl monodansylcadaverine and sarcosine methylester which lack the primary amine group. Neither monodansylcadaverine nor glycine methylester inhibited insulin secretion induced by either cAMP or the phorbol ester PMA at basal levels (10 nM) of Ca2+. These data provide further evidence for the involvement of transglutaminase in Ca2+ induced insulin secretion, they also suggest that insulin secretion induced by either cAMP or PMA may act in part by a mechanism independent of that induced by Ca2+.

Chemotherapy of breast cancer: are the taxanes going to change the natural history of breast cancer?

Among the novel chemotherapeutic drugs introduced in the last decade, taxanes have emerged as the most powerful compounds and results available to date suggest that they will be remembered in the future as the breast cancer chemotherapy of the 1990s. The two taxanes (paclitaxel, Taxol, Bristol-Myers Squibb and docetaxel, Taxotere, Rhône-Poulenc Rorer) share some characteristics, but are also significantly different both in preclinical profile and, most importantly, in clinical characteristics. The main clinical differences are related to their different efficacy-toxicity ratio in relation to dose and schedule; the differing integrability of paclitaxel and docetaxel in anthracycline-taxane containing regimens, secondary to major differences in pharmacokinetic interactions between each taxane and the anthracyclines, and; the potential differences in level of synergism between each taxane and herceptin (HeR2Neu antibody/trastuzumab, Genentech/Roche). In clinical practice, the taxanes are now standard therapy in metastatic breast cancer after prior chemotherapy, in particular anthracyclines, has failed. Their role in combination with anthracyclines in first-line therapy of advanced breast cancer is emerging and sheds new light on the potential role of taxanes in the adjuvant setting. However, the impact of taxanes on the natural history of breast cancer is yet to be defined, despite the trend of results suggesting that these agents have the potential for significant improvements in advanced and, most importantly, adjuvant therapy of breast cancer. The results of all completed or ongoing Phase III trials in first-line metastatic and the adjuvant setting will help determine if taxanes will further improve the outcome of breast cancer or not.

Human eosinophil isolation and the measurement of apoptosis.

Eosinophils have been implicated in allergic diseases, such as bronchial asthma, which is characterized by elevated eosinophil numbers in the bronchoalveolar lavage fluid and peripheral blood. Their accumulation and activation within the airway mucosa is thought to cause tissue injury, contraction of airway smooth muscle, and increased bronchial responsiveness (1-3). The balance between cell maturation and death is of great importance in determining the number of eosinophils in the blood and tissues (4-6). Following in vitro culture in the absence of cytokines, eosinophils undergo apoptosis, or programmed cell death (7,8), a process that can be inhibited by cytokines such as interleukin-3 and -5 and granulocyte-macrophage colony-stimulating factor (GM-CSF), and accelerated by such factors as corticosteroids and Fas (7-11).

MAP Kinase Expression in Eosinophils.

The mitogen-activated protein kinases (MAPK) are an expanding family of proline-directed serine/threonine kinases that are activated, following their dual phosphorylation at conserved threonine and tyrosine residues, by a family of MAPK kinases (MEK). Presently, the MAPK family can be divided into three groups: the extracellular signal-regulated kinases (ERK), composed of ERK-1/2/3/4/5, the c-jun N-terminal kinases (JNK)-46/54; and the p38 MAP kinase (p38 MAPK) composed of p38/p38ß/p38γ/p38δ (1). Of these enzymes, the authors have detected ERK-1/2/3/5, JNK-46/54, and p38 in guinea pig peritoneal eosinophils by Western analysis: ERK-4 and p38ß were apparently absent.

Phospholipases and the activation and priming of neutrophils by peritoneal dialysis effluent.

OBJECTIVE: To investigate the role of phospholipase during the activation and priming of neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase by peritoneal dialysis effluent (PDE). DESIGN: Examine the action of 4-hour dwell PDE upon phospholipase activation in the circulating neutrophils obtained from healthy individuals. RESULTS: We have previously reported that PDE stimulated superoxide release by the NADPH oxidase of human neutrophils and primed the response to the bacterial peptide, fMLP (fMetLeuPhe). To elucidate the biochemical mechanisms underlying these observations, we have examined the roles of phospholipases (PL) C, D, and A2, whose activation causes the release of a range of intracellular secondary messengers. Following fMLP stimulation, we observed a rapid activation of both PLC and PLD as well as a small but nonsignificant increase in PLA2 activity. Peritoneal dialysis effluent alone failed to stimulate either PLC or PLD, while pre-incubation with PDE had no affect upon fMLP-induced PLC and PLD activation. However, PDE caused a small but nonsignificant increase in PLA2 activity (which was comparable to that observed with fMLP) and primed the fMLP-induced response. In common with a role for PLA2 and the subsequent release of arachidonic acid (AA), we have demonstrated dose-dependent inhibition of PDE-induced superoxide release by the PLA2 inhibitor mepacrine, as well as activation and priming of the fMLP-induced superoxide generation by AA. CONCLUSIONS: These results imply that PDE-induced NADPH-oxidase activation and priming in human neutrophils is mediated via a PLA2-dependent but PLC- and PLD-independent mechanism.

The perception of veterinary herd health management by Dutch dairy farmers and its current status in the Netherlands: a survey.

The importance of veterinary herd health management (VHHM) is increasing in both dairy farming and veterinary practice. Little is known, however, about how VHHM is perceived by farmers in terms of structure, content and satisfaction. In 2007 a questionnaire, containing questions about these three items was therefore sent to 800 Dutch dairy farmers. Farmers received two questionnaires, one for participants in VHHM and one for non-participants, allowing them to choose the appropriate one. Results were summarized and statistically analyzed. Farmers who were participating in VHHM had better farm performance. They were satisfied with the way VHHM was executed on their farm. However, there were some pressure points. Goal setting and evaluation was still not a regular part of VHHM, even though it is said to be effective in literature. Time spent on VHHM not visible to the farmer was often not charged or not clearly specified on the bill. The differences in opinions between participants and non-participants of VHHM indicated a lack of communication and/or product differentiation. Satisfaction with the way VHHM was executed on the farm had no significant influence on 305-day production. There was, however, some influence on calving interval and bulk milk somatic cell count (BMSCC).

Cell-penetrating-peptide-mediated siRNA lung delivery.

The therapeutic application of siRNA (short interfering RNA) shows promise as an alternative approach to small-molecule inhibitors for the treatment of human disease. However, the major obstacle to its use has been the difficulty in delivering these large anionic molecules in vivo. A potential approach to solving this problem is the chemical conjugation of siRNA to the cationic CPPs (cell-penetrating peptides), Tat-(48-60) (transactivator of transcription) and penetratin, which have been shown previously to mediate protein and peptide delivery in a host of animal models. In this transaction, we review recent studies on the utility of siRNA for the investigation of protein function in the airways/lung. We show that, despite previous studies showing the utility of cationic CPPs in vitro, conjugation of siRNA to Tat-(48-60) and penetratin failed to increase residual siRNA-mediated knockdown of p38 MAPK (mitogen-activated protein kinase) (MAPK14) mRNA in mouse lung in vivo. Significantly, we will also discuss potential non-specific actions and the induction of immunological responses by CPPs and their conjugates and how this might limit their application for siRNA-mediated delivery in vivo.

Signal transduction and activation of the NADPH oxidase in eosinophils.

Activation of eosinophil NADPH oxidase and the subsequent release of toxic oxygen radicals has been implicated in the mechanism of parasite killing and inflammation. At present, little is known of the signal transduction pathway that govern agonist-induced activation of the respiratory burst and is the subject of this review. In particular, we focus on the ability of leukotrine B4 to activate the NADPH oxidase in guinea-pig peritoneal eosinophils which can be obtained in sufficient number and purity for detailed biochemical experiments to be performed.

Mammalian Sterile20-like kinase 1 and the regulation of apoptosis.

Mammalian Sterile20-like kinase 1 (Mst1) is a ubiquitously expressed serine/threonine kinase which represents a member of the rapidly expanding family of enzymes related to the yeast Sterile20 kinase. Although the physiological function of Mst1 and its role in intracellular signalling is still unclear, reports to date suggest that Mst1, similar to its yeast homologue, operates in the MAPK (mitogen-activated protein kinase) pathway and, in this capacity, may represent a putative MAPK kinase kinase kinase. Moreover, there is abundant evidence for a role of this enzyme in apoptosis, where not only is it a target for caspases, but may also serve as an activator of these proteases to amplify the apoptotic signalling pathway. This paper reviews the investigations that have led to our current understanding of the mechanisms by which Mst1 may be activated and thereby contribute to apoptosis.

Regulation of m2 muscarinic receptor gene expression by platelet-derived growth factor: involvement of extracellular signal-regulated protein kinases in the down-regulation process.

To study the role of mitogen-activated protein kinase in the regulation of M2 receptors, we studied the effect of platelet-derived growth factor (PDGF) on M2 receptor gene expression. PDGF (4 ng/ml) caused a time-dependent decrease in M2 receptor number and in m2 receptor mRNA levels in HEL 299 cells. The PDGF-induced loss in m2 mRNA required de novo protein synthesis and occurred through a decrease in the rate of transcription of the m2 receptor gene. The down-regulation of M2 receptors was not accompanied by an uncoupling of the remaining receptors, indicating a large receptor reserve in these cells. Preincubations with the phosphatidylinositol 3-kinase inhibitor wortmannin, the protein kinase C inhibitor GF 109203X and the cAMP-dependent protein kinase inhibitor H-8 did not attenuate PDGF-induced down-regulation, indicating a lack of involvement of these enzymes in the down-regulation process. Activation of the extracellular signal-regulated protein kinase (ERK) 1 and 2 proteins was measured by an "in gel" phosphorylation assay. Carbachol did not activate ERK1 or 2, whereas PDGF and 4 beta-phorbol 13,14-dibutyrate resulted in a large increase in ERK1 and 2 activity along with a decrease in m2 mRNA. Preincubation with PD 098059, an inhibitor of mitogen-activated protein kinase kinase, inhibited PDGF- and 4 beta-phorbol 13,14-dibutyrate-mediated activation of ERK 1 and 2 in a concentration-dependent manner. The inhibitory action of PD 098059 was reflected at the mRNA level attenuating both PDGF- and 4 beta-phorbol 13,14-dibutyrate-mediated decreases in m2 mRNA. These results suggest a role of ERK1 and 2 in the regulation of muscarinic m2 receptor gene expression.