A two-step fluorinase enzyme mediated (18)F labelling of an RGD peptide for positron emission tomography.

A two-step radiolabelling protocol of a cancer relevant cRGD peptide is described where the fluorinase enzyme is used to catalyse a transhalogenation reaction to generate [(18)F]-5'-fluoro-5'-deoxy-2-ethynyladenosine, [(18)F]FDEA, followed by a 'click' reaction to an azide tethered cRGD peptide. This protocol offers efficient radiolabelling of a biologically relevant peptide construct in water at pH 7.8, 37 °C in 2 hours, which was metabolically stable in rats and retained high affinity for αVß3 integrin.

Imaging tumour hypoxia with positron emission tomography.

Hypoxia, a hallmark of most solid tumours, is a negative prognostic factor due to its association with an aggressive tumour phenotype and therapeutic resistance. Given its prominent role in oncology, accurate detection of hypoxia is important, as it impacts on prognosis and could influence treatment planning. A variety of approaches have been explored over the years for detecting and monitoring changes in hypoxia in tumours, including biological markers and noninvasive imaging techniques. Positron emission tomography (PET) is the preferred method for imaging tumour hypoxia due to its high specificity and sensitivity to probe physiological processes in vivo, as well as the ability to provide information about intracellular oxygenation levels. This review provides an overview of imaging hypoxia with PET, with an emphasis on the advantages and limitations of the currently available hypoxia radiotracers.

Combination of sapacitabine and HDAC inhibitors stimulates cell death in AML and other tumour types.

BACKGROUND: Alternative treatments are needed for elderly patients with acute myeloid leukaemia, as the disease prognosis is poor and the current treatment is unsuitable for many patients. METHODS: In this study, we investigated whether combining the nucleoside analogue sapacitabine with histone deacetylase (HDAC) inhibitors could be an effective treatment. Synergy and mode-of-action analysis were studied in cultured cell lines and the efficacy of the combination was confirmed in a xenograft model. RESULTS: CNDAC (1-(2-C-cyano-2-deoxy-ß-D-arabino-pentofuranosyl)-cytosine), the active component of sapacitabine, synergised with vorinostat in cell lines derived from a range of tumour types. Synergy was not dependent on a specific sequence of drug administration and was also observed when CNDAC was combined with an alternative HDAC inhibitor, valproate. Flow cytometry and western blot analysis confirmed that the combination induced a significant increase in apoptosis. Mode-of-action analysis detected changes in Bcl-xl, Mcl-1, Noxa, Bid and Bim, which are all regulators of the apoptotic process. The sapacitabine/vorinostat combination demonstrated significant benefit compared with the single-agent treatments in an MV4-11 xenograft, in the absence of any observed toxicity. CONCLUSION: Sapacitabine and HDAC inhibitors are an effective drug combination that is worthy of clinical exploration.

Regulation of the Rac1-specific exchange factor Tiam1 involves both phosphoinositide 3-kinase-dependent and -independent components.

The small GTPase Rac1 is involved in regulating membrane ruffling, gene transcription, cell-cycle progression and cell transformation, and some of these events are blocked by inhibitors of phosphoinositide 3-kinase (PI 3-kinase). Moreover, Rac1 can be activated by several guanine nucleotide exchange factors, which facilitate the release of GDP. We therefore investigated the ability of PI 3-kinase lipid products to regulate Tiam1, a Rac1-specific exchange factor. Tiam1 bound to polyphosphorylated inositol lipids in the rank order PtdIns(3,4,5)P(3)>PtdIns(3,4)P(2) >>PtdIns(4,5)P(2), and this binding could be attributed to the N-terminal pleckstrin-homology (N-PH) domain. Both PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2) enhanced Tiam1 guanine nucleotide exchange activity in vitro, but PtdIns(4,5)P(2) had no effect. Co-expression of a constitutively active PI 3-kinase with Tiam1 increased the amount of GTP-bound Rac1 in vivo, a response which required the N-PH domain of Tiam1. Ectopic expression of Tiam1 caused membrane ruffling in Swiss 3T3 cells that was characterized by wortmannin-sensitive and -insensitive components, which required the N-PH domain and the C-terminal PH domain of Tiam1 respectively. These results reveal novel facets of Tiam1-dependent regulation of Rac1 function.

Ca2+/calmodulin-dependent protein kinase II regulates Tiam1 by reversible protein phosphorylation.

A number of guanine nucleotide exchange factors have been identified that activate Rho family GTPases, by promoting the binding of GTP to these proteins. We have recently demonstrated that lysophosphatidic acid and several other agonists stimulate phosphorylation of the Rac1-specific exchange factor Tiam1 in Swiss 3T3 fibroblasts, and that protein kinase C is involved in Tiam1 phosphorylation (Fleming, I. N., Elliott, C. M., Collard, J. G., and Exton, J. H. (1997) J. Biol. Chem. 272, 33105-33110). We now show, through manipulation of intracellular [Ca2+] and the use of protein kinase inhibitors, that both protein kinase Calpha and Ca2+/calmodulin-dependent protein kinase II are involved in the phosphorylation of Tiam1 in vivo. Furthermore, we show that Ca2+/calmodulin-dependent protein kinase II phosphorylates Tiam1 in vitro, producing an electrophoretic retardation on SDS-polyacrylamide gel electrophoresis. Significantly, phosphorylation of Tiam1 by Ca2+/calmodulin-dependent protein kinase II, but not by protein kinase C, enhanced its nucleotide exchange activity toward Rac1, by approximately 2-fold. Furthermore, Tiam1 was preferentially dephosphorylated by protein phosphatase 1 in vitro, and treatment with this phosphatase abolished the Ca2+/calmodulin-dependent protein kinase II activation of Tiam1. These data demonstrate that protein kinase Calpha and Ca2+/calmodulin-dependent protein kinase II phosphorylate Tiam1 in vivo, and that the latter kinase plays a key role in regulating the activity of this exchange factor in vitro.

Phospholipase C-gamma, protein kinase C and Ca2+/calmodulin-dependent protein kinase II are involved in platelet-derived growth factor-induced phosphorylation of Tiam1.

In Swiss 3T3 fibroblasts, the Rac1-specific guanine nucleotide exchange factor Tiam1 is phosphorylated by several different agonists. We show here that PDGF induces threonine phosphorylation of Tiam1 in a time- and dose-dependent manner. Tiam1 phosphorylation was significantly reduced by the selective protein kinase C inhibitor Ro-31-8220 and by KN93, an inhibitor of Ca2+/calmodulin-dependent protein kinase II. The Ca2+ chelator BAPTA/AM totally abrogated Tiam1 phosphorylation, indicating that Ca2+ is essential for this phosphorylation. Moreover, PDGF-stimulated Tiam1 phosphorylation was markedly reduced by 72 +/- 10% in PLC-gamma1 deficient mouse fibroblasts, compared to wild-type cells, indicating that phosphoinositide phospholipase C is involved.

Determination of interaction sites on the small G protein RhoA for phospholipase D.

Phospholipase D (PLD) has been identified as a target of small G proteins of the Rho family. The present study was directed at defining the interaction sites of RhoA with rat brain PLD in vitro using chimeric proteins between RhoA and Ha-Ras or Cdc42Hs and point mutations. The switch I region of RhoA, which is the common effector domain of Ras-like G proteins, was a crucial interaction site for PLD. Mutations in conserved amino acids (Tyr34, Thr37, Phe39) totally abolished PLD activation, while mutations in Val38 or Tyr42 caused partial loss. Two additional sites were responsible for the differential PLD activation ability between RhoA and Cdc42Hs. Changing Asp76 in the switch II region of RhoA to the corresponding amino acid in Cdc42Hs led to partial loss of PLD activation. A chimeric protein with the N-terminal third of Cdc42Hs changed to RhoA showed enhanced PLD activation. Analysis of other Rho/Ha-Ras chimeric proteins and mutations indicated that Gln52 adjacent to the switch II region is responsible for this gain of function. In conclusion, the present study shows that conserved amino acids in the switch I region of RhoA are major PLD interaction sites and that residues in the switch II and internal regions are responsible for the differential activation of PLD by RhoA and Cdc42Hs.

Lysophosphatidic acid induces threonine phosphorylation of Tiam1 in Swiss 3T3 fibroblasts via activation of protein kinase C.

The Rho family of GTPases plays an important role in the control of cell shape, adhesion, movement, and growth. Several guanine nucleotide exchange factors have been identified that activate Rho family GTPases by promoting the binding of GTP to these proteins. However, little is known concerning the regulation of these GDP/GTP exchange factors. In this study, we demonstrate that lysophosphatidic acid (LPA) induces a rapid, sustainable phosphorylation of the Rac1-specific nucleotide exchange factor Tiam1 in Swiss 3T3 fibroblasts. LPA stimulated Tiam1 phosphorylation in a dose-dependent manner, and the protein was phosphorylated on threonine, but not tyrosine or serine. Tiam1 phosphorylation was also induced by platelet-derived growth factor, endothelin-1, bombesin, and bradykinin but not by epidermal growth factor. Significantly, pretreatment of Swiss 3T3 fibroblasts with 1 microM phorbol 12-myristate 13-acetate for 24 h, or with the selective protein kinase C inhibitor Ro-31-8220, reduced LPA-stimulated phosphorylation of Tiam1 by approximately 75%. Moreover, acute stimulation with 100 nM phorbol 12-myristate 13-acetate was sufficient to induce Tiam1 phosphorylation in vivo, and protein kinase C could phosphorylate purified Tiam1 on threonine residues in vitro. These data indicate that agonist-induced phosphorylation of Tiam1 is a general mechanism and suggest that it is likely to be important in its regulation. Protein kinase C appears to play a key role in phosphorylation of Tiam1.

Differential translocation of rho family GTPases by lysophosphatidic acid, endothelin-1, and platelet-derived growth factor.

The small GTPases of the Rho family play a key role in a number of signaling pathways activated by lysophosphatidic acid (LPA). However, little is known concerning the mechanism of regulation of these proteins. In this study we demonstrate that in Swiss 3T3 fibroblasts, LPA induces a sustained, time-dependent relocalization of RhoA to the Triton X-100-soluble low speed membrane fraction, which can be reversed by removal of LPA from the medium. Translocation was only observed with micromolar concentrations of LPA and was inhibited by pretreating the cells with pertussis toxin but not with tyrosine kinase inhibitors. LPA also induced translocation of CDC42Hs to the membranes but had no effect on the distribution of Rac1, RhoB, or Rho-GDI. Translocation of RhoA was also induced by endothelin-1. Conversely, platelet-derived growth factor did not cause the translocation of RhoA to any membrane fraction but stimulated relocalization of Rac1 to the high speed membrane fraction. Significantly, incubation of cell lysates with guanosine 5'-O-(thiotriphosphate) was sufficient to translocate RhoA, Rac1, and CDC42Hs from the cytosol to the membranes, whereas incubation with GDP had the opposite effect. These data suggest that the translocation of the Rho family proteins to the membrane fraction is controlled by their activation state and that agonists show selectivity in inducing the activation/translocation of these proteins.

Purification and characterization of N-ethylmaleimide-insensitive phosphatidic acid phosphohydrolase (PAP2) from rat liver.

N-Ethylmaleimide-insensitive phosphatidic acid phosphohydrolase (PAP; EC 3.1.3.4) was purified 5900-fold from rat liver. The enzyme was solubilized from membranes with octylglucoside, fractionated with (NH4)2SO4, and purified in the presence of Triton X-100 by chromatography on Sephacryl S300, hydroxyapatite, heparin-Sepharose and Affi-Gel Blue. Silver-stained SDS/PAGE indicated that the enzyme was an 83 kDa polypeptide. Sephacryl S-300 gel filtration also produced a second peak of enzyme activity, which was eluted from all of the chromatography columns at a different position from the purified enzyme. SDS/PAGE indicated that it contained three polypeptides (83 kDa, 54 kDa and 34 kDa), and gel filtration suggested that it was not an aggregate of the purified enzyme. Both forms were sensitive to inhibition by amphiphilic amines, Mn2+ and Zn2+, but not by N-ethylmaleimide. Purified PAP required detergent for activity, but was not activated by Mg2+, fatty acids or phospholipids. The enzyme was able to dephosphorylate lysophosphatidic acid or phosphatidic acid, and was inhibited by diacylglycerol and monoacylglycerol. No evidence was obtained for regulation of PAP by reversible phosphorylation.

Subcellular distribution of N-ethylmaleimide-sensitive and -insensitive phosphatidic acid phosphohydrolase in rat brain.

The dephosphorylation of phosphatidic acid by phosphatidic acid phosphohydrolase (PAP) is important in both cell-signalling and in glycerolipid metabolism. However, these roles are apparently performed by two different enzymes, which can be distinguished by their sensitivity in vitro to N-ethylmaleimide (NEM). Both of these enzymes are present in rat brain as well as a wide range of other rat tissues. However, the quantity and specific activity of each enzyme varies considerably between different tissues, as does the ratio of the two enzymes in each tissue. Tissues rich in glycerolipids are abundant in NEM-sensitive PAP, whereas there is no obvious pattern to the distribution of the NEM-insensitive enzyme in the different tissues tested. Studies on brain cortex, which is relatively rich in both forms of PAP, indicate that the NEM-insensitive PAP is located in the synaptosomes, and the NEM-sensitive enzyme present in the cytosol and microsomes. The NEM-sensitive PAP can also be translocated from the cytosol to the microsomes by oleate. When assayed against a range of phosphatidic acids, NEM-sensitive PAP showed a preference for phosphatidic acids with short acyl chains and for those containing arachidonate, whereas NEM-insensitive PAP had a preference for short and unsaturated acyl chains. The two isozymes also had different activity profiles against these substrates suggesting that they are in fact different enzymes. The implications for these results on the putative roles of the two forms of PAP are discussed.

The activity of the metabolic form of hepatic phosphatidate phosphohydrolase correlates with the severity of alcoholic fatty liver in human beings.

Increased esterification of fatty acids to triglyceride is common to most of the mechanisms proposed to explain the causation of alcoholic fatty liver. However, it is unclear whether this is caused by increased substrate supply or whether direct stimulation of the enzymes of the esterification pathway occurs after excessive alcohol intake. The rate-limiting step in triglyceride synthesis is catalyzed by the enzyme phosphatidate phosphohydrolase, which is present in the cytosol and microsomes and is sensitive to inhibition by N-ethylmaleimide. This enzyme is physically distinct from a second form of phosphatidate phosphohydrolase that is located predominantly in the plasma membrane, is insensitive to N-ethylmaleimide inhibition and has a putative role in cell-signaling. We have investigated whether the activity of the N-ethylmaleimide-sensitive ("metabolic") form of phosphatidate phosphohydrolase is increased in patients with alcoholic liver disease and whether any increased activity correlates with the severity of steatosis. N-ethylmaleimide-sensitive and -insensitive phosphatidate phosphohydrolase activities were measured in needle liver biopsy specimens from 42 alcoholic patients and 6 patients with primary biliary cirrhosis and in wedge biopsy specimens from 6 normal patients undergoing routine cholecystectomy. Steatosis was "scored" on coded slides from 0 to 3. N-ethylmaleimide-sensitive activity was higher in alcoholic biopsy specimens scoring 3 (3.25 +/- 0.4 units/mg protein, n = 10) than in those scoring either 0 (1.21 +/- 0.2, n = 14) or 1 to 2 (1.58 +/- 0.2, n = 18), and it was also higher than in biopsy specimens from normal and primary biliary cirrhosis patients (1.65 +/- 0.3, n = 12; p < 0.0001, analysis of variance).(ABSTRACT TRUNCATED AT 250 WORDS)