Factors affecting valrubicin response in patients with bacillus Calmette-Guérin-refractory bladder carcinoma in situ.

OBJECTIVE: Patients with bacillus Calmette-Guérin (BCG)-refractory carcinoma in situ (CIS) of the bladder are candidates for intravesical (IVe) valrubicin. This post-hoc analysis of data from the pivotal phase 3, prospective, open-label study of valrubicin evaluated the effects of patient characteristics and past treatments on the response to valrubicin. METHODS: Enrolled patients had non-muscle-invasive CIS with or without concurrent papillary disease stage Ta and/or T1 for which papillary tumors had been resected before treatment, and had previously received ≥ 2 courses of IVe therapy (≥ 1 BCG course). Patients received a course of valrubicin, which consisted of 6 weekly IVe treatments of valrubicin (800 mg). Complete response was defined as no evidence of disease by urine cytology, cystoscopy, and biopsy at 3 and 6 months posttreatment. Patient characteristics, baseline urinary symptoms, and number and type of previous treatment courses and instillations were compared for complete versus nonresponders (including partial responders) to valrubicin. RESULTS: Ninety patients enrolled; 87 patients with positive biopsy at initiation completed a valrubicin course and underwent the 3-month assessment. Five had missing data at 6 months. Of the remaining 82 patients, 18 demonstrated a complete response; 64 demonstrated partial or no response. For complete responders versus partial or nonresponders, differences in patient characteristics, baseline urinary symptoms, and number of previous courses or instillations of BCG or other types of treatment were not significant (P > 0.05). More complete responders had evidence of inflammation before or during valrubicin treatment (P = 0.005 vs nonresponders). CONCLUSIONS: In these patients with BCG-refractory CIS, complete responders to valrubicin did not differ significantly from partial or nonresponders in the number of prior courses or instillations. The results suggest that therapy with valrubicin may be considered in appropriate candidates who have not responded to prior therapies. Cystectomy should be reconsidered when valrubicin treatment fails.

PKC epsilon has an alcohol-binding site in its second cysteine-rich regulatory domain.

Alcohols regulate the expression and function of PKC (protein kinase C), and it has been proposed that an alcohol-binding site is present in PKC alpha in its C1 domain, which consists of two cysteine-rich subdomains, C1A and C1B. A PKC epsilon-knockout mouse showed a significant decrease in alcohol consumption compared with the wild-type. The aim of the present study was to investigate whether an alcohol-binding site could be present in PKC epsilon. Here we show that ethanol inhibited PKC epsilon activity in a concentration-dependent manner with an EC50 (equilibrium ligand concentration at half-maximum effect) of 43 mM. Ethanol, butanol and octanol increased the binding affinity of a fluorescent phorbol ester SAPD (sapintoxin-D) to PKC epsilon C1B in a concentration-dependent manner with EC50 values of 78 mM, 8 mM and 340 microM respectively, suggesting the presence of an allosteric alcohol-binding site in this subdomain. To identify this site, PKC epsilon C1B was photolabelled with 3-azibutanol and 3-azioctanol and analysed by MS. Whereas azibutanol preferentially labelled His236, Tyr238 was the preferred site for azioctanol. Inspection of the model structure of PKC epsilon C1B reveals that these residues are 3.46 A (1 A=0.1 nm) apart from each other and form a groove where His236 is surface-exposed and Tyr238 is buried inside. When these residues were replaced by alanine, it significantly decreased alcohol binding in terms of both photolabelling and alcohol-induced SAPD binding in the mutant H236A/Y238A. Whereas Tyr238 was labelled in mutant H236A, His236 was labelled in mutant Y238A. The present results provide direct evidence for the presence of an allosteric alcohol-binding site on protein kinase C epsilon and underscore the role of His236 and Tyr238 residues in alcohol binding.

Interaction of anesthetics with the Rho GTPase regulator Rho GDP dissociation inhibitor.

The physiological effects of anesthetics have been ascribed to their interaction with hydrophobic sites within functionally relevant CNS proteins. Studies have shown that volatile anesthetics compete for luciferin binding to the hydrophobic substrate binding site within firefly luciferase and inhibit its activity (Franks, N. P., and Lieb, W. R. (1984) Nature 310, 599-601). To assess whether anesthetics also compete for ligand binding to a mammalian signal transduction protein, we investigated the interaction of the volatile anesthetic, halothane, with the Rho GDP dissociation inhibitor (RhoGDIalpha), which binds the geranylgeranyl moiety of GDP-bound Rho GTPases. Consistent with the existence of a discrete halothane binding site, the intrinsic tryptophan fluorescence of RhoGDIalpha was quenched by halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) in a saturable, concentration-dependent manner. Bromine quenching of tryptophan fluorescence is short-range and W192 and W194 of the RhoGDIalpha are located within the geranylgeranyl binding pocket, suggesting that halothane binds within this region. Supporting this, N-acetyl-geranylgeranyl cysteine reversed tryptophan quenching by halothane. Short chain n-alcohols ( n < 6) also reversed tryptophan quenching, suggesting that RhoGDIalpha may also bind n-alkanols. Consistent with this, E193 was photolabeled by 3-azibutanol. This residue is located in the vicinity of, but outside, the geranylgeranyl chain binding pocket, suggesting that the alcohol binding site is distinct from that occupied by halothane. Supporting this, N-acetyl-geranylgeranyl cysteine enhanced E193 photolabeling by 3-azibutanol. Overall, the results suggest that halothane binds to a site within the geranylgeranyl chain binding pocket of RhoGDIalpha, whereas alcohols bind to a distal site that interacts allosterically with this pocket.

Competitive binding of protein kinase Calpha to membranes and Rho GTPases.

Previously, we have shown that protein kinase C (PKC) forms a direct high-affinity, isozyme-specific and membrane lipid-independent interaction with Rho GTPases [Slater, S. J., Seiz, J. L., Stagliano, B. A., and Stubbs, C. D. (2001) Biochemistry 40, 4437-4445]. Since the cellular activation of PKCalpha involves an initial translocation from cytosolic to membrane compartments, the present study investigates the interdependence between the direct protein-protein interaction of PKCalpha with the Rho GTPase, Cdc42, and the protein-lipid interactions of PKCalpha with membranes. It was hypothesized that the interaction of PKCalpha with membrane-bound Cdc42 would contribute to the overall membrane-binding affinity of the kinase by providing an additional anchor. However, it was found that the incorporation of isoprenylated Cdc42 into membranes resulted in an apparent decrease in the membrane-binding affinity of PKCalpha, whereas the association of PKCbetaI, PKCdelta, PKCepsilon, and PKCzeta was each unaffected. The presence of membrane-bound Cdc42 resulted in a rightward shift in both the PS- and Ca2+-concentration response curves for PKCalpha membrane association and for the ensuing activation, whereas the maximal levels of binding and activation attained at saturating PS and Ca2+ concentrations were in each case unaffected. Overall, these findings suggest that PKCalpha undergoes a isozyme-specific interaction with membrane-bound Cdc42 to form a PKCalpha-Cdc42 complex, which possesses a membrane-binding affinity that is reduced relative to that of the individual components due to competition between Cdc42 and PS/Ca2+ for binding to PKCalpha. Consistent with this, it was found that the interaction of PKCalpha with membrane-bound Cdc42 was accompanied by the physical dissociation of the PKCalpha-Cdc42 complex from membranes. Thus, the study provides a novel mechanism by which the membrane association and activation of PKCalpha and Cdc42 may be regulated by competing protein-protein and protein-lipid interactions.

The use of time-resolved fluorescence imaging in the study of protein kinase C localisation in cells.

BACKGROUND: Two-photon-excitation fluorescence lifetime imaging (2P-FLIM) was used to investigate the association of protein kinase C alpha (PKCalpha) with caveolin in CHO cells. PKCalpha is found widely in the cytoplasm and nucleus in most cells. Upon activation, as a result of increased intracellular Ca2+ and production of DAG, through G-protein coupled-phospholipase C signalling, PKC translocates to a variety of regions in the cell where it phosphorylates and interacts with many signalling pathways. Due to its wide distribution, discerning a particular interaction from others within the cell is extremely difficult. RESULTS: Fluorescence energy transfer (FRET), between GFP-PKCalpha and DsRed-caveolin, was used to investigate the interaction between caveolin and PKC, an aspect of signalling that is poorly understood. Using 2P-FLIM measurements, the lifetime of GFP was found to decrease (quench) in certain regions of the cell from approximately 2.2 ns to approximately 1.5 ns when the GFP and DsRed were sufficiently close for FRET to occur. This only occurred when intracellular Ca2+ increased or in the presence of phorbol ester, and was an indication of PKC and caveolin co-localisation under these conditions. In the case of phorbol ester stimulated PKC translocation, as commonly used to model PKC activation, three PKC areas could be delineated. These included PKCalpha that was not associated with caveolin in the nucleus and cytoplasm, PKCalpha associated with caveolin in the cytoplasm/perinuclear regions and probably in endosomes, and PKC in the peripheral regions of the cell, possibly indirectly interacting with caveolin. CONCLUSION: Based on the extent of lifetime quenching observed, the results are consistent with a direct interaction between PKCalpha and caveolin in the endosomes, and possibly an indirect interaction in the peripheral regions of the cell. The results show that 2P-FLIM-FRET imaging offers an approach that can provide information not only confirming the occurrence of specific protein-protein interactions but where they occur within the cell.

The nature of the hydrophobic n-alkanol binding site within the C1 domains of protein kinase Calpha.

The activator-binding sites within the C1 domains of protein kinase C (PKC) are also able to bind alcohols and anesthetics. In this study, the nature of the interaction of these agents with the hydrophobic region within the C1 domains was investigated and a structure-activity relationship for the alcohol effects was obtained. The effects of a series of n-alkanols on PKCalpha activity, determined using an in vitro assay system that lacked lipids, were found to be a nonlinear function of the chain length. In the absence of phorbol ester or diacylglycerol, 1-octanol potently activated PKCalpha in a concentration-dependent manner, while 1-heptanol was completely without effect, despite differing by one methylene unit. The minimal structural requirement for the activating effect corresponded to R-CH(OH)-(CH(2))(n)-CH(3), where R = H or an alkyl group and n >or= 6. Consistent with this, 2-octanol, for which n = 5, was without effect on the activity, even though this alcohol is only marginally less hydrophobic than 1-octanol, whereas 2-nonanol, for which n = 6, was able to produce activity. Importantly, it was found that PKCalpha was activated to a greater extent by R-2-nonanol than by the S enantiomer. The potentiation of phorbol ester-induced, membrane-associated PKCalpha activity by long-chain n-alkanols reported previously (Slater, S. J., Kelly, M. B., Larkin, J. D., Ho, C, Mazurek, A, Taddeo, F. J., Yeager, M. D., Stubbs, C. D. (1997) J. Biol. Chem. 272, 6167-6173), was also found here for nonmembrane associated PKC, indicating that this effect is an intrinsic property of the enzyme rather than a result of membrane perturbation. Overall, the results suggest that the alcohol-binding sites within the C1 domains of PKCalpha contain spatially distinct hydrophilic and hydrophobic regions that impose a high degree of structural specificity on the interactions of alcohols and other anesthetic compounds, as well as diacylglycerols and phorbol esters.

Effects of ethanol on protein kinase C alpha activity induced by association with Rho GTPases.

Previous studies have shown that n-alkanols have biphasic chain length-dependent effects on protein kinase C (PKC) activity induced by association with membranes or with filamentous actin [Slater, S. J., et al. (1997) J. Biol. Chem. 272, 6167-6173; Slater, S. J., et al. (2001) Biochim. Biophys. Acta 1544, 207-216]. Recently, we showed that PKCalpha is also activated by a direct membrane lipid-independent interaction with Rho GTPases. Here, the effects of ethanol and 1-hexanol on Rho GTPase-induced activity were investigated using an in vitro assay system to provide further insight into the mechanism of the effects of n-alkanols on PKC activity. Both ethanol and 1-hexanol were found to have two competing concentration-dependent effects on the Ca(2+)- and phorbol ester- or diacylglycerol-dependent activities of PKCalpha associated with either RhoA or Cdc42, consisting of a potentiation at low alcohol levels and an attenuation of activity at higher levels. Measurements of the Ca(2+), phorbol ester, and diacylglycerol concentration-response curves for Cdc42-induced activation indicated that the activating effect corresponded to a shift in the midpoints of each of the curves to lower activator concentrations, while the attenuating effect corresponded to a decrease in the level of activity induced by maximal activator levels. The presence of ethanol enhanced the interaction of PKCalpha with Cdc42 within a concentration range corresponding to the potentiating effect, whereas the level of binding was unaffected by higher ethanol levels that were found to attenuate activity. Thus, ethanol may either enhance activation of PKCalpha by Rho GTPases by enhancing the interaction between the two proteins or attenuate the level of activity of Rho GTPase-associated PKCalpha by inhibiting the ensuing activating conformational change. The results also suggest that the effects of ethanol on Rho GTPase-induced activity may switch between an activation and inhibition depending on the concentration of Ca(2+) and other activators.

A novel interaction between perlecan protein core and progranulin: potential effects on tumor growth.

In an in vivo search of novel partners for perlecan, a major heparan sulfate proteoglycan of basement membranes and cell surfaces, we identified progranulin, a secreted growth factor, as a strong interacting protein. Unambiguous interaction, first observed with the yeast two-hybrid system, was corroborated by co-immunoprecipitation studies using cell-free transcription/translation and transient cell transfection assays. The interaction of progranulin with perlecan domain V involved the first two laminin- and epidermal growth factor-like repeats. Within progranulin, the subdomains interacting most with perlecan harbored granulins F and B. Kinetics analysis of the interaction using surface plasmon resonance showed a saturable binding of relative low affinity (KD approximately 1 microM). These results were supported by significant expression overlap of these two proteins in a series of ovarian tumor tissue microarrays. Progranulin was present within proliferating blood vessels of ovarian carcinomas and perivascular matrices, with a distribution similar to perlecan. Notably, both progranulin and domain V stimulated the growth of adrenal carcinoma cells. However, when used together in equimolar amounts, the two proteins counteracted each other's activity. Thus, progranulin/perlecan interaction could contribute to a fine regulation of tumor angiogenesis and could ultimately affect cancer growth.

Angiogenesis in collagen I requires alpha2beta1 ligation of a GFP*GER sequence and possibly p38 MAPK activation and focal adhesion disassembly.

Angiogenesis depends on proper collagen biosynthesis and cross-linking, and type I collagen is an ideal angiogenic scaffold, although its mechanism is unknown. We examined angiogenesis using an assay wherein confluent monolayers of human umbilical vein endothelial cells were overlain with collagen in a serum-free defined medium. Small spaces formed in the cell layer by 2 h, and cells formed net-like arrays by 6-8 h and capillary-like lumens by 24 h. Blocking of alpha2beta1, but not alpha1 or alpha(v)beta3 integrin function halted morphogenesis. We found that a triple-helical, homotrimeric peptide mimetic of a putative alpha2beta1 binding site: alpha1(I)496-507 GARGERGFP*GER (where single-letter amino acid nomenclature is used, P* = hydroxyproline) inhibited tube formation, whereas a peptide carrying another putative site: alpha1(I)127-138 GLP*GERGRP*GAP* or control peptides did not. A chemical inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), SB202190, blocked tube formation, and p38 MAPK activity was increased in collagen-treated cultures, whereas targeting MAPK kinase (MEK), focal adhesion kinase (FAK), or phosphatidylinositol 3-kinase (PI3K) had little effect. Collagen-treated cells had fewer focal adhesions and 3- to 5-fold less activated FAK. Thus capillary morphogenesis requires endothelial alpha2beta1 integrin engagement of a single type I collagen integrin-binding site, possibly signaling via p38 MAPK and focal adhesion disassembly/FAK inactivation.

Inhibition of protein kinase C by resveratrol.

Evidence is emerging that resveratrol (RV), a polyphenolic phytoaxelin present in dietary sources including red wine, may protect against atherosclerosis and cardiovascular disease by enhancing the integrity of the endothelium. In this study, the possibility that such beneficial effects of RV may arise from a modulation of protein kinase C (PKC)-mediated signaling was investigated by determining the effects of RV on the in vitro activities of PKC isozymes. It was found that the Ca(2+)-dependent activities of membrane-associated PKCalpha induced by either phorbol ester or diacylglycerol were potently inhibited by RV, each with an IC(50) of approximately 2 microM. The inhibitory effect of RV was also observed for conventional PKCbetaI, whereas the activities of novel PKC epsilon and atypical PKCzeta were each unaffected. The inhibition of PKCalpha activity was found to be competitive with respect to phorbol ester concentration but noncompetitive with respect to Ca(2+) and phosphatidylserine concentrations, suggesting that the RV may compete for phorbol ester-binding to the C1 domains. Supporting this, it was found that RV bound to a fusion peptide containing the C1A and C1B domains of PKCalpha. Similar to the effects of diacylglycerol and phorbol ester, the interaction of RV with the C1 domains induced the association of PKCalpha with membrane lipid vesicles, although this did not result in activation. Overall, the results suggest that the inhibitory effect of RV on PKC activity, and therefore on the associated signaling networks, may, in part, underlie the mechanism(s) by which this agent exerts its beneficial effects on endothelial and cardiovascular function. Furthermore, the effects of RV on these signaling networks are predicted to differ according to the cellular localization and the regulating PKC isozyme.

The use of fluorescent phorbol esters in studies of protein kinase C-membrane interactions.

The family of protein kinase C (PKC) isozymes belongs to a growing class of proteins that become active by associating with membranes containing anionic phospholipids, such as phosphatidylserine. Depending on the particular PKC isoform, this process is mediated by Ca(2+)-binding to a C2 domain and interaction of activators such as 1,2-diacyl-sn-glycerol or phorbol esters with tandem C1 domains. This cooperation between the C1 and C2 domains in inducing the association of PKC with lipid membranes provides the energy for a conformational change that consists of the release of a pseudosubstrate sequence from the active site, culminating in activation. Thus, the properties of the interactions of the C1 and C2 domains with membranes, both as isolated domains, and as modules in the full length PKC isoforms, have been the subject of intense scrutiny. Here, we review the findings of studies in which fluorescent phorbol esters have been utilized to probe the properties of the C1 domains of PKC with respect to the interaction with activators, the subsequent interaction with membranes, and the role of the activating conformational change that leads to activation.

Regulation of PKC alpha activity by C1-C2 domain interactions.

In this study, the role of interdomain interactions involving the C1 and C2 domains in the mechanism of activation of PKC was investigated. Using an in vitro assay containing only purified recombinant proteins and the phorbol ester, 4 beta-12-O-tetradecanoylphorbol-13-acetate (TPA), but lacking lipids, it was found that PKC alpha bound specifically, and with high affinity, to a alpha C1A-C1B fusion protein of the same isozyme. The alpha C1A-C1B domain also potently activated the isozyme in a phorbol ester- and diacylglycerol-dependent manner. The level of this activity was comparable with that resulting from membrane association induced under maximally activating conditions. Furthermore, it was found that alpha C1A-C1B bound to a peptide containing the C2 domain of PKC alpha. The alpha C1A-C1B domain also activated conventional PKC beta I, -beta II, and -gamma isoforms, but not novel PKC delta or -epsilon. PKC delta and -epsilon were each activated by their own C1 domains, whereas PKC alpha, -beta I, -beta II, or -gamma activities were unaffected by the C1 domain of PKC delta and only slightly activated by that of PKC epsilon. PKC zeta activity was unaffected by its own C1 domain and those of the other PKC isozymes. Based on these findings, it is proposed that the activating conformational change in PKC alpha results from the dissociation of intra-molecular interactions between the alpha C1A-C1B domain and the C2 domain. Furthermore, it is shown that PKC alpha forms dimers via inter-molecular interactions between the C1 and C2 domains of two neighboring molecules. These mechanisms may also apply for the activation of the other conventional and novel PKC isozymes.