Using Drugs to Probe the Variability of Trans-Epithelial Airway Resistance

BACKGROUND:Precision medicine aims to combat the variability of the therapeutic response to a given medicine by delivering the right medicine to the right patient. However, the application of precision medicine is predicated on a prior quantitation of the variance of the reference range of normality. Airway pathophysiology provides a good example due to a very variable first line of defence against airborne assault. Humans differ in their susceptibility to inhaled pollutants and pathogens in part due to the magnitude of trans-epithelial resistance that determines the degree of epithelial penetration to the submucosal space. This initial 'set-point' may drive a sentinel event in airway disease pathogenesis. Epithelia differentiated in vitro from airway biopsies are commonly used to model trans-epithelial resistance but the 'reference range of normality' remains problematic. We investigated the range of electrophysiological characteristics of human airway epithelia grown at air-liquid interface in vitro from healthy volunteers focusing on the inter- and intra-subject variability both at baseline and after sequential exposure to drugs modulating ion transport. METHODOLOGY/PRINCIPAL FINDINGS:Brushed nasal airway epithelial cells were differentiated at air-liquid interface generating 137 pseudostratified ciliated epithelia from 18 donors. A positively-skewed baseline range exists for trans-epithelial resistance (Min/Max: 309/2963 Ω·cm2), trans-epithelial voltage (-62.3/-1.8 mV) and calculated equivalent current (-125.0/-3.2 μA/cm2; all non-normal, P<0.001). A minority of healthy humans manifest a dramatic amiloride sensitivity to voltage and trans-epithelial resistance that is further discriminated by prior modulation of cAMP-stimulated chloride transport. CONCLUSIONS/SIGNIFICANCE:Healthy epithelia show log-order differences in their ion transport characteristics, likely reflective of their initial set-points of basal trans-epithelial resistance and sodium transport. Our data may guide the choice of the background set point in subjects with airway diseases and frame the reference range for the future delivery of precision airway medicine

The strange case of protein kinase CK2: how a constitutively active enzyme can mediate signal transduction. Lo strano caso della proteinchinasi CK2: come un enzima costitutivamente attivo può mediare la trasduzione del segnale.

Summary Protein kinase CK2 is a Ser/Thr protein kinase composed of two catalytic (alpha and/or alpha’) and two regulatory (beta) subunits. It is ubiquitously expressed, constitutively active, and highly pleiotropic, with more than 300 protein substrates known so far (Meggio and Pinna, 2003); consequently, CK2 plays a key role in several physiological and pathological processes (Pinna, 2002; Ahmed et al., 2002). Unlike the majority of the other protein kinases that are activated in response to specific stimuli by second messengers, phosphorylation events or association with regulatory molecules, CK2 activity is not regulated, thus understanding the mechanism by which it controls cellular events is challenging. Many of the efforts to understand its involvement in signaling pathways are directed to identify cellular substrates that are responsible for mediating its action in cells. This last consideration, together with the fact that the CK2 function in cells is well recognized to be pro-survival and anti-apoptotic, thus related to neoplastic transformation (Ruzzene and Pinna, 2010), has rendered CK2 a reasonable “druggable target” in many different pathologies in which it is involved (Guerra and Issinger, 2008), giving rise to the development of many specific inhibitors, useful not only for investigating its physiological role, but, possibly, also for therapy (Sarno and Pinna, 2008; Pierre et al., 2011). The main issue which gave rise to this work is therefore how can a constitutively active kinase mediate external stimuli, which need to be transient? With this premise, we examined the CK2 involvement in different signaling pathways and in different contexts: I. The role of protein kinase CK2 in the differentiation process of acute promyelocytic leukemia (APL) cells induced by retinoic acid (RA) treatment; II. The role of CK2 in mediating plant response to salicylic acid (SA); III. The CK2-dependent regulation of multimolecular chaperone complexes, under survival/apoptotic conditions, in normal and multidrug resistant (MDR) cells. I. Role of protein kinase CK2 in the differentiation process of APL cells: With this work we demonstrate that the CK2 activity is required for RA-induced APL cells differentiation since CK2 inhibitors block this response. Moreover, being the CK2 catalytic activity unchanged in response to RA, we focused on changes in the substrate phosphorylation pattern. A major change in phosphorylation revealed to concern beta-actin, that was not known as a CK2 substrate before. By means of in vitro kinase assays, we confirmed that actin is phosphorylated by CK2. Moreover, we found that RA induces an increase of beta-actin expression level detectable both in the cytosol and in the nucleus; interestingly, the CK2 inhibition markedly reduced the nuclear beta-actin increase in response to RA, suggesting a role of the CK2-dependent phosphorylation in actin nuclear translocation and its possible involvement in mediating the RA-induced APL cells differentiation. II. Role of protein kinase CK2 in plant response to SA treatment: SA treatment in plants is known to induce a complex cellular response, accompained by the production of nitric oxide (NO), which is prevented by inhibition of CK2 (Zottini et al., 2007). We demonstrated that a major protein phosphorylated in Arabidopsis in response to SA treatment is the homologous of the human co-chaperone protein p23, known to be involved with Hsp90 in the cell chaperone machinery. Our experiments also showed that CK2 (from human and maize) phosphorylates Arabidopsis p23 in vitro with favourable kinetic parameters, and that endogenous Arabidopsis CK2 is the major kinase responsible for the phosphorylation of this protein. Moreover, we demonstrate that CK2 physically associates with p23 in vitro and that the two proteins co-localize in vivo. Although at present we do not know the exact function and effect of p23 phosphorylation. All together our results are consistent with a role of this protein in the requirement of CK2 for plant response to SA. III. The CK2-dependent regulation of multimolecular chaperone complexes, under survival/apoptotic conditions, in normal and multidrug resistant (MDR) cells: For this work we take advantage of a cell model, the CEM cells, which are available in two variants: the S-CEM, normally sensitive to drug-induced apoptosis, and the R-CEM, which are multidrug resistant (Dupuis et al., 2003). This model is particularly interesting since we have previously demonstrated that R-CEM express a higher level of CK2 alpha respect to S-CEM. In these cells, we analysed a specific substrate of CK2, the co-chaperone protein Cdc37, a protein essential in the Hsp90 chaperone machinery committed to protein kinases folding and activation. We found that in the two cell variants, two different isoforms of Cdc37 are expressed: the protein in S-CEM is shorter at the C-terminus than that expressed in R-CEM. These two isoforms display different features: we found that Cdc37 of S-CEM is rapidly degraded in apoptosis, while the isoform of R-CEM is more stable, and, during apoptosis, undergoes the only C-terminal cleavage. Surprisingly, despite the higher amount of CK2 exhibited by R-CEM , the CK2-dependent phosphorylation of Cdc37 Ser13 was not significantly different in the two cell lines. This suggested to us the possibility that, in dependence of the of the isoform expressed, Ser13 is differently accessible to the kinase and/or the phosphatase responsible for its modification. In agreement with this hypothesis, we found that during apoptosis, when the C-terminal part of Cdc37 in R-CEM is cleaved, a dephosphorylation of Ser13 also occurs. Moreover, the two Cdc37 isoforms seems to participate to different multimolecular complexes, as judge by their migration upon density gradient ultracentrifugation, and these complexes are differently susceptible to disruption by chaperone inhibitors. In summary, in apoptosis resistant cells, a more fuctional chaperone machinery is observed, and CK2, expressed at particularly high levels in these cells, might exploit this substrate as a powerful tool to exert its anti-apoptotic function.Riassunto La proteinchinasi CK2 è un Ser/Thr chinasi composta da due subunità catalitiche (alfa e/o alfa') e due subunità regolatorie (beta). E' ubiquitaria, costitutivamente attiva e altamente pleiotropica con più di 300 substrati noti sino ad oggi (Meggio e Pinna, 2003); proprio per queste sue caratteristiche, CK2 gioca un ruolo chiave in molti processi fisiologici e patologici (Pinna, 2002; Ahmed et al., 2002). Diversamente dalla maggior parte delle altre chinasi, che in risposta a stimoli specifici sono attivate da secondi messaggeri, fosforilazioni o associazione con molecole regolatorie, l’attività di CK2 non è regolata, e questo rende particolarmente impegnativa la comprensione dei meccanismi tramite i quali essa controlla i diversi processi cellulari. Molti degli sforzi al riguardo sono diretti all’identificazione di substrati cellulari responsabili del suo intervento in tali processi. Queste considerazioni ed il fatto che è largamente riconosciuto il ruolo anti-apoptotico e pro-sopravvivenza che CK2 riveste nelle cellule, quindi legato alla trasformazione neoplastica (Ruzzene e Pinna, 2010), hanno reso CK2 un interessante bersaglio farmacologico nelle varie patologie nelle quali è stata riconosciuta avere un ruolo (Guerra e Issinger, 2008). Sono stati infatti sviluppati molti inibitori specifici per CK2, non solo allo scopo di studiare il suo ruolo fisiologico, ma anche, potenzialmente, per uso terapeutico (Sarno e Pinna, 2008; Pierre et al., 2011). La domanda principale che ha dato origine a questo lavoro di tesi è stata quindi: come può una chinasi costitutivamente attiva mediare stimoli esterni, che, per loro nature, devono essere transitori? Con queste premesse, abbiamo quindi studiato il coinvolgimento di CK2 in differenti contesti e vie di segnalazione. I tre principali argomenti descritti in questa tesi sono: I. Ruolo di CK2 nel differenziamento di cellule di leucemia promielocitica acuta (APL) in risposta ad acido retinoico (RA); II. Ruolo di CK2 nella risposta delle piante al trattamento con acido salicilico (SA); III. Regolazione da parte di CK2 di complessi chaperone multimolecolari, in condizioni di sopravvivenza ed apoptosi, in cellule normali e farmacoresistenti (MDR) I. Ruolo di CK2 nel differenziamento di cellule di leucemia promielocitica acuta (APL) in risposta ad acido retinoico (RA): Con questo lavoro, abbiamo dimostrato che l’attività di CK2 è necessaria per il differenziamento indotto da RA delle cellule di leucemia promielocitica acuta, infatti l’inibizione di CK2 blocca tale risposta cellulare. Inoltre, abbiamo dimostrato che l’attività catalitica di CK2 non è influenzata dal trattamento; abbiamo pertanto focalizzato la nostra attenzione su possibili variazioni di fosforilazione dei suoi substrati. Il principale cambiamento èstato osservato nel grado di fosforilazione della beta-actina, una proteina mai annoverata sino ad ora tra i substrati di CK2. Attraverso saggi di fosforilazione in vitro, abbiamo confermato che l’actina è effettivamente un substrato di CK2. Abbiamo potuto inoltre appurare che il trattamento con RA induce un aumento dell’espressione della beta-actina, individuabile sia a livello citosolico che nucleare; particolarmente interessante è risultato il fatto che l’inibizione di CK2 riduce fortemente tale incremento a livello nucleare, suggerendo un ruolo della fosforilazione di CK2 nella traslocazione della beta-actina nel nucleo e, di conseguenza, la sua possibile importanza nel mediare l’effetto di RA sul differenziamento delle cellule APL. II. Ruolo di CK2 nella risposta delle piante al trattamento con acido salicilico (SA); Il trattamento con SA, nelle piante, è noto indurre una risposta di varia natura, accompagnata dalla produzione di ossido nitrico (NO), che viene però a mancare qualora CK2 sia inibita (Zottini et al., 2007). Con questo studio in Arabidopsis, abbiamo dimostrato che la principale proteina che viene fosforilata in risposta al trattamento con SA, è p23, una proteina omologa alla p23 umana che è un co-chaperone noto per essere coinvolto, con Hsp90, nel macchinario delle “chaperonine”. I nostri risultati hanno dimostrato che CK2 (umana e di mais) fosforilano p23 di Arabidopsis in vitro con parametri cinetici favorevoli, e inoltre, che la CK2 endogena di Arabidosis è la principale chinasi responsabile della fosforilazione di questa proteina. Abbiamo anche dimostrato che CK2 e p23 possono interagire fisicamente in vitro e che hanno, in vivo, la stessa localizzazione subcellulare. Al momento non conosciamo la funzione di p23 e l’effetto della sua fosforilazione, ma, nell’insieme, i nostri dati sono coerenti con un suo ruolo nel mediare l’azione di CK2, in risposta all’SA nelle piante. III. Regolazione da parte di CK2 di complessi chaperone multimolecolari, in condizioni di sopravvivenza ed apoptosi, in cellule normali e farmacoresistenti (MDR): In questo lavoro ci siamo serviti del modello cellulare costituito dalle cellule CEM, che avevamo disponibili in due varianti: le S-CEM, che sono sensibili all’apoptosi indotta da farmaci, e le R-CEM, che sono invece farmacoresistenti (Dupuis et al., 2003). Questo modello è particolarmente interessante, in quanto abbiamo dimostrato in precedenza che le cellule R-CEM esprimono un livello più elevato della subunità catalitica di CK2 rispetto alle S-CEM. In queste cellule, abbiamo analizzato un substrato specifico di CK2, la proteina co-chaperone Cdc37, che è essenziale nel macchinario chaperone di Hsp90 diretto al folding ed all’attivazione di proteinchinasi. Abbiamo visto che le due linee cellulari esprimono due isoforme diverse di Cdc37: le S-CEM esprimono una isoforma più corta al C-terminale rispetto all’isoforma presente nelle R-CEM. Le due isoforme dimostrano inoltre di avere caratteristiche diverse: quella delle S-CEM è rapidamente degradata in apoptosi, mentre l’isoforma delle R-CEM è più stabile e, in apoptosi, è sottoposta al taglio del solo C-terminale. Sorprendentemente però, nonostante l’elevata espressione di CK2, nelle R-CEM, il livello della fosfo-Ser13, che è il residuo fosforilato in maniera specifica da CK2 in Cdc37, non è significativamente diverso nelle due linee cellulari. Questo ci ha suggerito l’ipotesi che, a seconda dell’isoforma espressa, la Ser13 sia diversamente accessibile alla chinasi e/o alla fosfatasi responsabili della sua modificazione. In accordo con ciò, abbiamo infatti osservato che in apoptosi, quando il C-terminale di Cdc37 delle R-CEM viene tagliato, si assiste anche alla defosforilazione della Ser13. Inoltre, le due isoforme sembrano partecipare a complessi multimolecolari diversi, come appurato tramite centrifugazione in gradiente di densità, che mostrano anche una diversa sensibilità alla disgregazione indotta da inibitori degli chaperone. Nel complesso, i dati ottenuti suggeriscono che CK2, nelle cellule farmacoresistenti, dove è espressa a livelli particolarmente elevati, può servirsi di Cdc37, che in queste cellule sembra essere più funzionale, come un potente strumento per esplicare la sua funzione anti-apoptotica

Assessment of CK2 Constitutive Activity in Cancer Cells

At variance with the great majority of protein kinases that become active only in response to specific stimuli and whose implication in tumors is caused by genetic alterations conferring to them unscheduled activity, the highly pleiotropic Ser/Thr-specific protein kinase CK2 is constitutively active even under normal conditions and no gain-of-function CK2 mutants are known. Nevertheless, CK2 level is abnormally high in cancer cells where it is believed to generate an environment favorable to the development of malignancy, through a mechanism denoted as "non-oncogene addiction." This makes CK2 not only an appealing target to counteract different kinds of tumors but also a valuable marker of cells predisposed to undergo neoplastic transformation owing to the presence in them of CK2 level exceeding a critical threshold. Such a prognostic exploitation of CK2 would imply the availability of methods suitable for the reliable, sensitive, and specific quantification of its activity in biological samples and in living cells. The aim of this chapter is to describe a number of procedures applicable to the quantitative determination of CK2 activity and to provide experimental details designed for rendering these assays as sensitive and selective as possible even in the presence of many other protein kinases. The procedures described roughly fall in three categories: (i) in vitro quantification of CK2 activity in crude biological samples and cell lysates; (ii) in-cell assay of endogenous CK2 activity based on the phosphorylation of reporter substrates; (iii) identification of CK2 targets in malignant and normal cell

Protein kinase CK2 accumulation in "oncophilic" cells: causes and effects

none5At variance with protein kinases expressed by oncogenes, CK2 is endowed with constitutive activity under normal conditions, and no CK2 gain-of-function mutants are known. Its amount, however, is abnormally high in malignant cells where it appears to be implicated in many of the cell biology phenomena associated with cancer. These observations can be reconciled assuming that tumor cells develop an overdue reliance ("non-oncogene addiction") on abnormally high CK2 level. While the potential of this latter to generate an environment favorable to neoplasia is consistent with the global antiapoptotic and prosurvival role played by CK2, it is not clear what is determining accumulation of CK2 in cells "predisposed" to become malignant. Exploiting the apoptosis sensitive (S) or resistant (R) CEM cell model, characterized by sharply different CK2 levels, we have now correlated the level and degradation rate of CK2 to those of the chaperone proteins Hsp90 and Cdc37. We show in particular that persistence of high CK2 level in R-CEM, as opposed to S-CEM, is accompanied by the presence of an immunospecific form of Cdc37 not detectable in S-CEM and refractory to staurosporine-induced degradation.noneM. RUZZENE; Tosoni K; Zanin S; Cesaro L; Pinna LARuzzene, Maria; Tosoni, Kendra; Zanin, Sofia; Cesaro, Luca; Pinna, Lorenz

Pharmacological inhibition of protein kinase CK2 reverts the multidrug resistance phenotype of a CEM cell line characterized by high CK2 level.

Protein kinase CK2 is an ubiquitous and constitutively active kinase, which phosphorylates many cellular proteins and is implicated in the regulation of cell survival, proliferation and transformation. We investigated its possible involvement in the multidrug resistance phenotype (MDR) by analysing its level in two variants of CEM cells, namely S-CEM and R-CEM, normally sensitive or resistant to chemical apoptosis, respectively. We found that, while the CK2 regulatory subunit beta was equally expressed in the two cell variants, CK2alpha catalytic subunit was higher in R-CEM and this was accompanied by a higher phosphorylation of endogenous protein substrates. Pharmacological downregulation of CK2 activity by a panel of specific inhibitors, or knockdown of CK2alpha expression by RNA interference, were able to induce cell death in R-CEM. CK2 inhibitors could promote an increased uptake of chemotherapeutic drugs inside the cells and sensitize them to drug-induced apoptosis in a co-operative manner. CK2 blockade was also effective in inducing cell death of a different MDR line (U2OS). We therefore conclude that inhibition of CK2 can be considered as a promising tool to revert the MDR phenotype

Comparative analysis of CK2 expression and function in tumor cell lines displaying sensitivity vs. resistance to chemical induced apoptosis

CK2 is a pleiotropic protein kinase, which phosphorylates many substrates and has a global role in promoting cell survival and preventing apoptosis. In this study, we investigated its involvement in the phenomenon of the drug resistance, by which tumor cells frequently become unresponsive to chemical apoptosis. By comparing the expression of CK2 subunits in four different pairs of sensitive (S) and resistant (R) cancer cell lines, we found that in three cases the resistant phenotype is accompanied by the overexpression of the CK2 catalytic alpha subunit, either alone or in combination with the regulatory beta subunit. The degree of CK2 expression correlates with the CK2 catalytic activity, when measured toward endogenous protein substrates. All the tested R cell lines, including the one with no CK2 overexpression, can be induced to undergo death by treatment with CK2 inhibitors. We therefore conclude that, although CK2 overexpression is not an absolute requirement for the resistant phenotype, its activity is essential for cell survival and contributes to a high degree of resistance. We also found that CK2 inhibition increases the accumulation of cytotoxic drugs inside the R cells, presumably by impairing the functionality of the extrusion pump P-gp. We therefore propose that CK2 should be considered a target to counteract the pharmaco-resistant phenotype

The p23 co-chaperone protein is a novel substrate of CK2 in Arabidopsis

The ubiquitous Ser/Thr protein kinase CK2, which phosphorylates hundreds of substrates and is essential for cell life, plays important roles also in plants; however, only few plant substrates have been identified so far. During a study aimed at identifying proteins targeted by CK2 in plant response to salicylic acid (SA), we found that the Arabidopsis co-chaperone protein p23 is a CK2 target, readily phosphorylated in vitro by human and maize CK2, being also a substrate for an endogenous casein kinase activity present in Arabidopsis extracts, which displays distinctive characteristics of protein kinase CK2. We also demonstrated that p23 and the catalytic subunit of CK2 interact in vitro and possibly in Arabidopsis mesophyll protoplasts, where they colocalize in the cytosol and in the nucleus. Although its exact function is presently unknown, p23 is considered a co-chaperone because of its ability to associate to the chaperone protein Hsp90; therefore, an involvement of p23 in plant signal transduction pathways, such as SA signaling, is highly conceivable, and its phosphorylation may represent a fine mechanism for the regulation of cellular responses

Electrophysiological values on an individual donor basis (A-R).

<p>Box plots per donor highlighting minimum, maximum (whiskers) and 25^th/75^th percentile and median (box). Donors arranged in ascending order ranked by mean values of TER in (B) with number of ALIs per donor above the boxes. These are compared with either (A) voltage, (C) [I_eq] and (D) time at ALI.</p

Relationship between baseline voltage and response of TER after amiloride addition.

<p>Logarithm of baseline V plotted against ratio of TER upon amiloride addition in drug regime I, applying the initial baseline (A-I), or the rolling baseline approach (B-I). In drug regime II (A-II), grouping produced a significant difference between the two groups (LOW, n = 57; HIGH, n = 24). For both drug regimes, grouping was performed as in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149550#pone.0149550.g005" target="_blank">5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149550#pone.0149550.g006" target="_blank">6</a>. Additional regression and statistical analysis data are shown in Table D, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149550#pone.0149550.s008" target="_blank">S1 File</a>.</p

Drug induced effect on TER with respect to the rolling baseline.

<p>TER values at each stage of the drug regime chronologically are considered as baseline. The hypotenuse of the shaded triangular area represents the line of identity while magnification is shown in the box inserts. (A-I) The initial baseline TER values (closed grey circles) are plotted against the values obtained after addition of forskolin (n = 44). (B-I) TER values +FSK are plotted against values after addition of CFTR_Inh172 (n = 44). (C-I) Values of TER with CFTR_Inh172 plotted against values +AMI (n = 44); line of regression: dashed line +99% CI (grey vertical bars). In D-I same data as in C-I showing two distinct populations of amiloride responders designated as LOW (closed grey circles-black dashed regression line +99% CI vertical bars, n = 35) and HIGH (open squares, black regression line +99% CI dotted line, n = 9). (A-II) The initial baseline TER values are plotted against the values obtained after addition of amiloride showing two distinct populations designated as LOW (n = 57) and HIGH (n = 24) amiloride responders. (B-II) Amiloride values plotted against values +FSK (LOW n = 56, HIGH n = 24). (C-II) Values after addition of forskolin plotted against values +CFTR_Inh172 (LOW n = 56, HIGH n = 21). Additional regression and statistical analysis data are shown in Table C in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149550#pone.0149550.s008" target="_blank">S1 File</a>.</p