Chemokine receptor CXCR4 and early-stage non-small cell lung cancer: pattern of expression and correlation with outcome.

BACKGROUND: The expression of CXCR4 has been implicated in metastatic dissemination in different models of breast cancer and melanoma. In the present study, we evaluated CXCR4 expression in non-small-cell lung cancer (NSCLC) and the relationship between CXCR4 expression and the prognosis of stage I disease. PATIENTS AND METHODS: Using immunohistochemical analysis, we retrospectively analyzed CXCR4 expression in specimens from 61 patients with completely resected pathologically confirmed stage I NSCLC for whom clinical follow-up data were available. RESULTS: In the present study, we have shown that: CXCR4 is expressed by tumor cells in stage I NSCLC; CXCR4 is located in the nucleus and/or in the cytoplasm of tumor cells; strong nuclear staining was observed in 17 cases (29.8%); patients whose tumors had CXCR4-positive nuclear staining had a significantly longer duration of survival than patients whose tumors had no nuclear expression (P = 0.039, log-rank test). Interestingly, the 5-year metastasis rates were 23.5% and 34.1% in patients with CXCR4-positive and CXCR4-negative nuclear expression, respectively (P = 0.2). CONCLUSION: Strong CXCR4-positive nuclear staining was associated with a significantly better outcome in early-stage NSCLC. The mechanisms underlying this clinically and biologically important finding need to be further explored.

Probing ligand-induced conformational changes of human CD38.

The lymphoid surface antigen CD38 is basically a NAD+glycohydrolase, which is also involved in the metabolism of cyclic ADP-ribose. Besides, this ecto-enzyme has potential signalling roles in T- and B-cells. Such multiple functions prompted us to study the molecular dynamics of the CD38 protein and especially the relationship between its ecto-enzymatic active site and its epitope, i.e. the binding site of most known anti-CD38 monoclonal antibodies. Both epitopic and enzymatic sites were shown to be degraded by proteases, such as trypsin or chymotrypsin. This sensitivity was almost entirely suppressed in the presence of substrates or inhibitors. Both sites were also degraded in the presence of reducing agents, as dithiothreitol. Inhibitory ligands induced the same resistance of both sites against reducing attack. The binding of CD38 ligands to the active site triggers therefore conformational changes that shield some backbone bonds and disulfide bridges against, respectively, proteolytic cleavage or reduction. This transconformation was found moreover to irreversibly take place after incubation with substrates such as NAD+ in the presence of dithiothreitol. The epitope remained preserved, while the enzymatic activity was lost. This inactivation probably resulted from the covalent trapping of the catalytically reactive intermediate in the active site (i.e. paracatalytic inactivation). These data have major implications in the knowledge of the CD38 structure, especially with regard to the location of disulfide bridges and their accessibility. Potential consequences of the conformational plasticity of CD38 should also be considered in its physiological functions such as signalling.

Upregulation of CD38 gene expression in leukemic B cells by interferon types I and II.

The activation antigen CD38, which has NAD+ glycohydrolase activity in its extracellular domain, is expressed by a large variety of cell types. Few investigations into the regulation of CD38 expression by physiologic stimuli have been reported. As the CD38 promoter contains potential binding sites for interferon (IFN) regulatory factor-1 (IRF-1), we investigated the influence of IFN type I (alpha and beta) and type II (gamma) on CD38 gene expression of leukemic B cells. Using the IFN-responsive B cell line Eskol, we found by RT-PCR analysis a rapid time-dependent induction in CD38 mRNA (starting at 6 h) with each type of IFN. This induction was independent of protein synthesis, suggesting that CD38 gene activation does not require IRF-1 but is merely under direct transcriptional regulation by latent IFN-inducible factors. mRNA stimulation was followed within 24 h by induction of membrane CD38, which coincided with rises of CD38-specific ectoenzymatic activities, that is, NAD+ glycohydrolase, (A/G)DP-ribosyl cyclase, and cyclic ADP ribose hydrolase activities. IFN failed to induce or upregulate the other CD38-related ectoenzymes analyzed, that is, CD39, CD73, CD157, and PC-1. Similarly, treatment of leukemic cells of patients with B chronic lymphocytic leukemia (B-CLL) with IFN resulted in an increase in CD38 mRNA mirrored by plasma membrane upregulation of CD38 and NAD+ glycohydrolase activity. Further investigation in relation to CD38 gene activation and B-CLL behavior remains to be defined.

Human CD38 is an authentic NAD(P)+ glycohydrolase.

The leucoyte surface antigen CD38 has been shown to be an ecto-enzyme with multiple catalytic activities. It is principally a NAD+ glycohydrolase that transforms NAD+ into ADP-ribose and nicotinamide. CD38 is also able to produce small amounts of cyclic ADP-ribose (ADP-ribosyl cyclase activity) and to hydrolyse this cyclic metabolite into ADP-ribose (cyclic ADP-ribose hydrolase activity). To classify CD38 among the enzymes that transfer the ADP-ribosyl moiety of NAD+ to a variety of acceptors, we have investigated its substrate specificity and some characteristics of its kinetic and molecular mechanisms. We find that CD38-catalysed cleavage of the nicotinamide-ribose bond results in the formation of an E.ADP-ribosyl intermediary complex, which is common to all reaction pathways; this intermediate reacts (1) with acceptors such as water (hydrolysis), methanol (methanolysis) or pyridine (transglycosidation), and (2) intramolecularly, yielding cyclic ADP-ribose with a low efficiency. This reaction scheme is also followed when using nicotinamide guanine dinucleotide as an alternative substrate; in this case, however, the cyclization process is highly favoured. The results obtained here are not compatible with the prevailing model for the mode of action of CD38, according to which this enzyme produces first cyclic ADP-ribose which is then immediately hydrolysed into ADP-ribose (i.e. sequential ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase activities). We show instead that the cyclic metabolite was a reaction product of CD38 rather than an obligatory reaction intermediate during the glycohydrolase activity. Altogether our results lead to the conclusion that CD38 is an authentic 'classical' NAD(P)+ glycohydrolase (EC 3.2.2.6).

Ecto-phosphodiesterase/pyrophosphatase of lymphocytes and non-lymphoid cells: structure and function of the PC-1 family.

Many developmentally regulated membrane proteins of lymphocytes are ecto-enzymes, with their active sites on the external surface of the cell. These enzymes commonly have peptidase, phosphodiesterase or nucleotidase activity. Their biological roles are just beginning to be discovered. Although their expression is usually associated with particular stages of lymphoid differentiation, the same gene products are often expressed on the surface of certain non-lymphoid cell types outside the immune system, indicating that their functions cannot be unique to lymphocytes, nor can they be ubiquitous. The plasma cell membrane protein PC-1 (phosphodiesterase I; EC 3.1.4.1/nucleotide pyrophosphatase; EC 3.6.1.9), which was one of the first serological markers for lymphocyte subsets to be discovered, is a typical example. Within the immune system, PC-1 is confined to plasma cells, which represent about 0.1% of lymphocytes. However, PC-1 is also expressed on cells of the distal convoluted tubule of the kidney, chondrocytes, osteoblasts, epididymis and hepatocytes. Recent work has shown that PC-1 is a member of a multigene family of ecto-phosphodiesterases that currently has two other members, PD-1 alpha (autotaxin) and PD-1 beta (B10). Within this family, the extracellular domains are highly conserved, especially around the active site. In contrast, the transmembrane and cytoplasmic domains are highly divergent. Individual members of the eco-phosphodiesterase family have distinct patterns of distribution in different cell types, and even within the same cell. For example, PC-1 is present only on the basolateral surface of hepatocytes, while B10 (PD-1 beta) is confined to the apical surface. Analysis of conservation and differences in the sequence of their cytoplasmic tails may illuminate intracellular targetting signals. Ecto-phosphodiesterases may play a part in diverse activities in different tissues, including recycling of nucleotides. They may also regulate the concentration of pharmacologically active extracellular compounds such as adenosine or its derivatives and cell motility. Some members may modulate local concentrations of pyrophosphate, and hence influence calcification in bone and cartilage.

Coordinated regulation in human T cells of nucleotide-hydrolyzing ecto-enzymatic activities, including CD38 and PC-1. Possible role in the recycling of nicotinamide adenine dinucleotide metabolites.

The human leukocyte surface Ag CD38 was recently identified as a nicotinamide adenine dinucleotide (NAD)(+)-glycohydrolase ecto-enzyme, degrading NAD into nicotinamide and ADP-ribose. We show here that expression of CD38 is increased in the Jurkat T cell line after treatment with agents that augment intracellular cAMP, with the permeant cAMP analogue dibutyryl-cAMP (db-cAMP), and also with PMA, which activates protein kinase C. Treatment of human PBL T cells with db-cAMP or submitogenic doses of PMA also increased CD38 expression. Two other nucleotide-hydrolyzing activities were induced on the T cell surface concomitantly with CD38: the human PC-1 molecule, a nucleotide phosphodiesterase/pyrophosphatase that produces AMP from NAD or ADP-ribose, and a nucleotidase that produces adenosine from AMP, but which may be distinct from the CD73 5'-nucleotidase. All three enzymes were up-regulated after stimulation of human peripheral blood T cells with PHA. The coordinated regulation of these ecto-enzymes suggested that, besides a possible signaling function, they may recycle extracellular NAD by degrading it to adenosine and nicotinamide, which can be taken up by cells. In support of this hypothesis, db-cAMP-treated Jurkat cells could degrade extracellular NAD for de novo synthesis of purines, while untreated cells could not. Activated lymphocytes are often located in tissues in which cell death is common. It is suggested that the coordinated expression of these enzymes may allow activated T cells to re-use NAD and nucleotides from dead cells.

Activation of phosphodiesterase by transducin in bovine rod outer segments: characteristics of the successive binding of two transducins.

In bovine retinal rods, transducin loaded with GTP or GTP gamma S (T*) activates a cGMP phosphodiesterase (PDE) by forming a tightly membrane-bound complex with it [Catty, P., et al. (1992) J. Biol. Chem. 267, 19489-19493]. Up to two T*s are able to bind to PDE [Clerc, A., & Bennett, N. (1992) J. Biol. Chem. 267, 6620-6627]. We analyze here PDE activation by two successive bindings of T*. In the mathematical model used, we took into account that the membrane concentration determines the amount of PDE able to interact efficiently with T* through the attachment of PDE itself to the membrane. We therefore fitted the data obtained over a wide range of membrane and PDE concentrations. We found that the binding of the first T* to PDE elicits 80-100% of the maximal activity of PDE, whereas the binding of the second T* to PDE elicits little or no additional activation of PDE. This finding profoundly differs from previous conclusions. The carefully controlled conditions of our experiments permit one to understand these discrepancies. In the physiological situation, PDE would be nearly maximally activated through its interaction with only one T*. The efficient binding of the second T* to those complexes would then ensure a rapid deactivation of T* through the enhancement of the rate of GTP hydrolysis in T* bound to PDE [Pagès, F., et al. (1992) J. Biol. Chem. 267, 22018-22021; Pagès, F., et al. (1993) J. Biol. Chem. 268, 26358-26364].

Cell calcium signaling via GM1 cell surface gangliosides in the human Jurkat T cell line.

The cell surface ganglioside GM1 is the specific receptor for the B subunit of cholera toxin. We show here that in the human Jurkat T cell line an increase in intracellular free Ca2+ concentration can be elicited by using B subunits to ligate GM1 molecules. This Ca2+ signaling effect is clearly mediated through GM1 because it can be observed after direct insertion of exogenous GM1 in a Jurkat cell variant deficient in GM1 expression. The observed Ca2+ response clearly involves both the release of Ca2+ from intracellular stores and a Ca2+ influx from extracellular spaces. It is sustained in the presence of 1 mM extracellular Ca2+, whereas it becomes transient in Ca(2+)-free medium. We show that the GM1-mediated stimulation partially empties the CD3-dependent and inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ pool suggesting a dependence of the Ca2+ response from activation of phospholipase C (PLC) metabolism. Accordingly, tyrosine phosphorylation of PLC gamma-1 can be evidenced but only in Jurkat cells highly expressing GM1. GM1 stimulation results in an IL-2 production comparable to that obtained after CD3 activation. Finally, the GM1-linked cell Ca2+ activation pathway is also observed in a Jurkat cell clone lacking Ag-specific receptor expression suggesting that the presence of functional CD3/TCR molecules is not essential for GM1-induced cell Ca2+ response. Altogether, these data show that cell surface gangliosides GM1 may act as a signaling molecule in Jurkat T cells possibly by a new pathway, a finding of importance when considering a possible function for ubiquitous membrane carbohydrate structures in T cell recognition systems.

Enhancement by phosphodiesterase subunits of the rate of GTP hydrolysis by transducin in bovine retinal rods. Essential role of the phosphodiesterase catalytic core.

Phosphodiesterase (PDE) in bovine retinal rod outer segments is activated when it forms a membrane-bound complex with the alpha-subunit of transducin loaded with GTP (T alpha*). At maximal activation, this complex contains two T alpha* and all the subunits of native PDE (PDE alpha, PDE beta, and two inhibitory PDE gamma). We observed previously (Pagès, F., Deterre, P., and Pfister, C. (1992) J. Biol. Chem. 267, 22018-22021) that the rate of GTP hydrolysis by transducin in a rod outer segment suspension is enhanced when T alpha* is bound to native PDE (PDE alpha beta gamma 2). In this article, we compare the effects of PDE species with different PDE gamma contents. We show that T alpha* hydrolyzes its GTP faster not only when bound to PDE alpha beta gamma 2, but also when bound to PDE alpha beta gamma or PDE alpha beta. Moreover, trypsin-treated PDE (PDE gamma-deprived soluble PDE) also induces an acceleration of GTP hydrolysis. On the contrary, addition of isolated PDE gamma alone does not accelerate GTP hydrolysis. The interaction between T alpha* and PDE gamma, which is essential for the activation of PDE by T alpha*, is apparently not responsible of the feedback of PDE on T alpha*. The interaction of primary importance for the acceleration of GTP hydrolysis would be that existing between T alpha* and PDE alpha beta.

The lymphocyte surface antigen CD38 acts as a nicotinamide adenine dinucleotide glycohydrolase in human T lymphocytes.

The extracellular domain of the lymphocyte surface antigen CD38 has been recently shown to share a high sequence homology with a nicotinamide adenine dinucleotide (NAD+)-specific hydrolyzing enzyme cloned from the ovotestis of the gastropod Aplysia (E. States, D.J., Walseth, T.F., Lee, H. C., Trends Biochem. Sci. 1992. 17:495). In agreement with this finding, we present here evidence that CD38-overexpressing T cells, such as human thymocytes and cells from the human HPB-ALL T cell line, exhibit a NAD(+)-hydrolyzing enzymatic activity present on the outer surface of the cell membrane. In contrast, T lymphocytes with relatively low levels of CD38 marker, such as the human Jurkat cell line, display a lower activity. This suggests a relationship between ecto-NAD+ glycohydrolase activity and CD38 expression, as confirmed here when comparing wild-type Jurkat cells and a Jurkat cell variant overexpressing the CD38 molecule. Moreover, CD38 immunoprecipitates from thymocytes behave as an authentic NAD+ glycohydrolase enzyme: it transforms NAD+ stoichiometrically into nicotinamide plus adenosine 5'-diphosphoribose. Altogether these results strongly support the assumption that CD38 is actually a lymphocyte-specific NAD(+)-hydrolyzing enzyme, a finding that give new prospects to understand the in vivo function of this cell membrane protein.

Enhanced GTPase activity of transducin when bound to cGMP phosphodiesterase in bovine retinal rods.

The generation of the physiological response of a retinal rod cell to an incident photon involves activation of a cGMP phosphodiesterase (PDE) by a GTP-binding protein, transducin (T). This activation has been shown to occur by formation of a membrane-bound T alpha GTP-PDE complex (Clerc, A., and Bennett, N. (1992) J. Biol. Chem. 267, 6620-6627; Catty, P., Pfister, C., Bruckert, F., and Deterre, P. (1992) J. Biol. Chem 267, 19489-19493). The recovery of the response involves turning-off of T by its intrinsic GTPase activity. We show here that the formation of the membrane-bound T alpha GTP-PDE complex correlates with an enhanced rate of GTP hydrolysis. In vivo, this would provide an appropriate mechanism for fast turn-off of cGMP hydrolysis.

The cGMP phosphodiesterase-transducin complex of retinal rods. Membrane binding and subunits interactions.

cGMP-specific phosphodiesterase (PDE) of vertebrate retinal rod outer segments (ROS) is composed of two catalytic subunits (PDE alpha and PDE beta) and two identical inhibitory subunits (PDE gamma). Native PDE alpha beta gamma 2 is peripherally bound to the membranes of ROS discs. We studied quantitatively its partition between soluble and membrane-bound fractions in ROS homogenates. In the presence of its activator, the alpha-subunit of transducin loaded with a triphosphate guanine nucleotide (T alpha*), PDE displayed a greatly enhanced membrane binding. Neither the purified PDE gamma.T alpha* complex, nor the PDE alpha beta and PDE alpha beta gamma forms of active PDE, showed a membrane binding comparable to that of PDE alpha beta gamma 2 in the presence of T alpha*. The T alpha*-activated PDE is therefore an undissociated complex tightly bound to the ROS membranes. Using limited proteolysis, we showed that the membrane anchoring of the whole complex implies not only PDE (mainly by the C terminus of PDE beta) but also both termini of T alpha*. The membrane binding of the purified PDE alpha beta species was also enhanced in the presence of T alpha*; a direct link would therefore exist between the activator and the catalytic subunits. From this work emerges a plausible structural model of the T alpha*-activated PDE, with its internal interactions and its sites of anchoring into the ROS membrane.

Role of G protein beta gamma subunits in the regulation of the plasma membrane Ca2+ pump.

In Zajdela hepatoma cells (ZHC) the plasma membrane Ca2+ pump displayed no sensitivity to glucagon (19-29) (mini-glucagon), whereas in hepatocyte this metabolite of glucagon evoked a biphasic regulation of the Ca2+ pump system via a cholera toxin-sensitive G protein. Analysis of G protein subunits in ZHC membranes indicated the presence of cholera toxin-sensitive Gs alpha and G beta gamma proteins, whose functionality was manifested by GTP and NaF stimulation of adenylylcyclase activity, and pertussis toxin-catalyzed ADP-ribosylation of Gi alpha, respectively. However, immunoblotting experiments suggested a lower content in beta gamma subunits in ZHC as compared with hepatocyte plasma membranes. Complementation of ZHC or hepatocyte plasma membranes with purified beta gamma subunits from transducin (T beta gamma) caused inhibition of the basal activity of the Ca2+ pump at 10 and 300 ng/ml, respectively, and revealed (in ZHC) or increased (in hepatocytes) sensitivity of the system to mini-glucagon. After cholera toxin treatment of ZHC, T beta gamma no longer reconstituted the response of the Ca2+ pump to mini-glucagon, suggesting that the mechanism of beta gamma action is dependent on an association with the alpha subunit of a cholera toxin-sensitive G protein. It is concluded that G beta gamma subunits control both the basal activity of the plasma membrane Ca2+ pump and its inhibition by mini-glucagon.

Activation and solubilization of the retinal cGMP-specific phosphodiesterase by limited proteolysis. Role of the C-terminal domain of the beta-subunit.

The cGMP-specific phosphodiesterase (PDE) of vertebrate retinal rod outer segments (ROS) is a peripheral enzyme activated in vivo by transducin. In vitro artificial activation can be achieved using trypsin. This was described as resulting from degradation of the inhibitory gamma subunit (2 copies/PDE molecule), leaving intact the alpha beta catalytic core. It was, however, observed that trypsin could induce the release of PDE (or solubilization) from the ROS membranes before its activation [Wensel, T. G. & Stryer, L. (1986) Proteins Struct. Funct. Genet. 1, 90-99]. Studying the time course of this solubilization, we were able to purify a trypsin-solubilized PDE still completely inhibited (i.e. with its two gamma subunits bound). The tryptic solubilization of PDE is therefore complete before any functional degradation of the gamma subunits occurs. It was recently suggested that this solubilization could coincide with the cleavage of a C-terminal fragment of the alpha subunit, which can be labeled by methylation of a terminal cysteine residue [Ong, O. C., Ota, I. M., Clarke, S. & Fung, B. K. K. (1989) Proc. Natl Acad. Sci. USA 86, 9238-9242]. We present the following evidence indicating that the C-terminus of the PDE beta subunit is mainly responsible for PDE anchorage to the ROS membrane. (a) The trypsin-solubilized PDE alpha beta gamma 2 has intact blocked N-termini. (b) It is still methylated on PDE alpha. (c) The C-terminus of PDE beta can also be labeled by methylation and its tryptic cleavage coincides well with the PDE solubilization. (d) Sequential cleavage of the alpha and beta polypeptides can also be detected by high-resolution gel electrophoresis: the first cleavage appears on the beta subunit and is completed when cleavage of the alpha subunit begins. The time course for cleavage of the gamma subunits appears to be slower than for the beta subunit and comparable to that of the alpha subunit. Upon longer trypsinization, a 70-kDa polypeptide appears which seems to be a degradation product of PDE beta. Gel-filtration analysis, however, shows that this 70-kDa fragment does not dissociate from the catalytic core.

cGMP phosphodiesterase of retinal rods is regulated by two inhibitory subunits.

The cGMP phosphodiesterase (PDE) of cattle retinal rod outer segments comprises three types of subunits: the two heavy catalytic ones, PDE alpha and PDE beta, each around 85 kDa, and the light inhibitory one, PDE gamma or I (11 kDa). The relative stoichiometry is usually assumed to be 1:1:1. PDE activation in the visual transduction cascade results from removal of the inhibitor by the alpha subunit of transducin (T alpha). The stoichiometric complex T alpha-I, separated from activated PDE, has been isolated and characterized. Analyzing now the activated PDE, we find that it still contains some inhibitor and is resolvable into two species, one with 50% of the inhibitor content of the native enzyme and the other totally devoid of it. The same two species are observed upon activation of PDE by very short tryptic proteolysis, which specifically degrades the inhibitor. This leads us to conclude that the composition of the native enzyme is PDE alpha beta-I2. The two inhibitory subunits are differentially bound, sequentially removable, and exchangeable between the native complex PDE alpha beta-I2 and the fully active PDE alpha beta. The possibility of this exchange precludes as yet an unambiguous estimate of the actual activity of the intermediate complex PDE alpha beta-I. The differential binding and the exchangeability of the inhibitors raises the possibility of a fast, diffusion controlled, switch-off mechanism of PDE activity after a flash, which would shortcut the inactivation resulting from the slow GTPase rate of transducin.