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Unsupervised explorative data analysis of normal human leukocytes and BCR/ABL positive leukemic cells mid-infrared spectra† G. Bellisola,*a,b M. Bolomini Vittori,b G. Cinque,c P. Dumas,d Z. Fiorini,b C. Laudanna,b M. Mirenda,b C. Sandt,d G. Silvestri,b L. Tomasello,b M. Vezzalini,b K. Wehbec and C. Sorio*b We proved the ability of Fourier Transform Infrared microspectroscopy (microFTIR) complemented by Principal Component Analysis (PCA) to detect protein phosphorylation/de-phosphorylation in mammalian cells. We analyzed by microFTIR human polymorphonuclear neutrophil (PMNs) leukocytes, mouse-derived parental Ba/F3 cells (Ba/F3#PAR), Ba/F3 cells transfected with p210BCR/ABL (Ba/F3#WT) and expressing high levels of protein tyrosine kinase (PTK), and human-derived BCR/ABL positive K562 leukemic cell sub-clones engineered to differently express receptor-type tyrosine-protein phosphatase gamma (PTPRG). Synchrotron radiation (SR) and conventional (globar) IR sources were used to perform microFTIR respectively, on single cells and over several cells within the same sample. Ex vivo time-course experiments were run, inducing maximal protein phosphorylation in PMNs by 100 nM N-formylated tripeptide fMLP. Within the specific IR fingerprint 1800–850 cm−1 frequency domain, PCA identified two regions with maximal signal variance. These were used to model and test the robustness of PCA in representing the dynamics of protein phosphorylation/de-phosphorylation processes. An IR signal ratio marker reflecting the homeostatic control by protein kinases and phosphatases was identified in normal leukocytes. The models identified by microFTIR and PCA in normal leukocytes also distinguished BCR/ABL positive Ba/F3#WT from BCR/ABL negative Ba/F3#PAR cells as well as K562 cells exposed to functionally active protein tyrosine phosphatase recombinant protein ICD-Tat transduced in cells by HIV-1 Tat technology or cells treated with the PTK inhibitor imatinib mesylate (IMA) from cells exposed to phosphatase inactive (D1028A)ICD-Tat recombinant protein and untreated control cells, respectively. The IR signal marker correctly reflected the degrees of protein phosphorylation associated with abnormal PTK activity in BCR/ABL positive leukemic cells and in general was inversely related to the expression/activity of PTPRG in leukemic sub-clones.
Received 22nd January 2015, Accepted 27th April 2015
In conclusion, we have described a new, reliable and simple spectroscopic method to study the ex vivo
DOI: 10.1039/c5an00148j
protein phosphorylation/de-phosphorylation balance in cell models: it is suitable for biomedical and pharmacological research labs but it also needs further optimization and its evaluation on large cohorts of
www.rsc.org/analyst
patients to be proposed in the clinical setting of leukemia.
a Azienda Ospedaliera Universitaria Intergrata di Verona, Department of Pathology and Diagnostics - Unit of Immunology, Policinico G. Rossi, P.le L.A. Scuro 10, I-37134 Verona, Italy. E-mail: [email protected]; Fax: +39 045 802 7127; Tel: +39 045 802 7688 b University of Verona, Department of Pathology and Diagnostics - General Pathology, Strada Le Grazie, 8, I-37134 Verona, Italy. E-mail: [email protected], [email protected], [email protected], [email protected], [email protected], [email protected], [email protected], [email protected]; Fax: +39 045 8027127; Tel: +39 045 802 7688 c Diamond Light Source Ltd, Diamond House, Harwell Science and Innovation Campus, Chilton, Didcot, Oxfordshire, UK. E-mail: [email protected], [email protected]; Tel: +44 (0)1235778410 d Synchrotron Soleil, L’Orme des Merisiers, Saint-Aubin - BP48, 91192 GIF-sur-Yvette Cedex, France. E-mail: [email protected], [email protected] † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c5an00148j
This journal is © The Royal Society of Chemistry 2015
Introduction Protein phosphorylation is a reversible post-translational modification and a common mechanism to transmit signals and regulate protein and cell functions in leukocytes.1,2 It almost exclusively occurs at the side chains of three amino acids: serine (Ser), threonine (Thr) and tyrosine (Tyr) which represent 6.8%, 5.9%, and 3.2% total protein amino acids, respectively. The transfer of the terminal gamma phosphate group (γ-PO32−) of adenosine triphosphate (ATP) to the nucleophilic (–OH) group of the amino acid side chain is catalyzed by the specific kinases necessary to lower the large amount of free energy of the phosphate–phosphate bond in ATP. Phosphate addition modifies the physico-chemical properties of
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Ser, Thr, and Tyr amino acids as well as those of proteins. For instance, a phosphorylated enzyme can undergo conformational changes which can either activate or inhibit its catalytic activity and facilitate the recruitment of neighboring proteins with structurally conserved domains capable of recognizing and binding to phosphomotifs.3 This occurs in polymorphonuclear neutrophil (PMN) leukocytes where the interactions of bacterial peptides with N-formyl peptide receptor (FPR) on the plasma membrane trigger enzymes that catalyze the generation of intermediate second messengers, such as inositol phosphate and diacylglycerol responsible for the rapid mobilization of Ca2+ from intracellular stores and for the activation of protein kinases.4 In particular, activated protein kinase C (PKC) phosphorylates Ser and Thr amino acid residues of recruited downstream proteins necessary for chemotaxis and cell adhesion, degranulation, generation of superoxide anions and phagocytosis in leucocytes.5 In addition to Ser/Thr protein kinases, receptor and non-receptor protein tyrosine kinases (PTKs) transfer phosphate groups from ATP to Tyr residues.6 Protein Ser/Thr phosphatases (PSPs) and Tyr phosphatases (PTPs) reverse protein phosphorylation and, together with protein kinases, contribute to the temporal and spatial balance of phosphorylation and de-phosphorylation processes leading to pleiotropic effects on cell proliferation, differentiation and survival.7,8 The large family of protein tyrosine phosphatases (PTPs) comprises 8 transmembrane (R1–R8) receptor-type enzymes, 5 with two catalytic tandem phosphatase (D1 and D2) domains and 3 with a single catalytic phosphatase domain, respectively. In tandem-domain receptor-type protein tyrosine phosphatases (PTPRs) the D1 domain adjacent to the plasma membrane displays catalytic activity whereas the D2 domain located in the carboxy-terminal part of the molecule can be either inactive or with negligible catalytic activity.9 In addition to D1–D2 intracellular domains (ICD) the receptor-type tyrosine-protein phosphatase gamma (PTPRG) has a carbonic anhydrase-like domain, a fibronectin 3-like domain in its extracellular part and a short transmembrane domain.10 In Chronic Myelogenous Leukemia (CML), a hematological malignancy, the balance between protein phosphorylation and de-phosphorylation regulated by protein kinases and protein phosphatases is completely subverted due to the presence of abnormal Bcr/Abl fusion oncogene.11 This codifies for a chimeric BCR/ABL protein with constitutively increased protein tyrosine kinase (PTK) activity. Downstream phosphorylation of CRKL, a SH2 and SH3 (src homology) domain containing protein, which is the main substrate of BCR/ABL PTK, activates RAS and JUN kinase signaling pathways allowing leukemic cells to become insensitive to pro-apoptotic stimuli and undergo sustained cell proliferation independently of the presence of growth factors. It has been observed that some BCR/ ABL positive K562 cell sub-clones with low expression/activity of PTPRG have a more aggressive phenotype than K562 subclones transfected with human PTPRG cDNA and with increased expression/activity of this PTP. Not only is the total
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amount of phosphorylated tyrosine reduced by the coexpression of PTPRG but also its specific targets such as BCR/ ABL and CRKL proteins are less phosphorylated.12 Different techniques are currently applied to detect leukemic cells and to monitor anti-leukemic therapy. Routine peripheral blood count associated with the examination of cell morphology in blood smear and bone marrow biopsy by light microscopy allows the formulation of diagnosis in almost cases. The flow cytometric analysis of cells stained with immunfluorescent probes directed towards different marker antigens is useful for establishing the lineage of the neoplastic cells and for assessing their maturation. Karyotyping and/or fluorescence in situ hybridization (FISH) analysis in bone marrow cells can detect chromosomal changes, for instance BCR/ABL. Molecular testing by real time quantitative polymerase chain reaction (RT-qPCR) of mRNA transcripts is the most sensitive routine approach for monitoring the presence of BCR/ABL fusion gene and therefore the response to therapy of patients with chronic myeloid leukaemia. Genome-wide scanning approaches, including gene expression profiling, comparative genomic hybridization (CGH), and single-nucleotide polymorphism (SNP), can measure the associations of specific gene polymorphisms with the phenotype and they are useful to predict the response of CML patients to therapy with tyrosine kinase inhibitors (TKIs).13 However, none of these approaches directly measure the effectiveness of the drug or drug combinations, a phenomenon associated with specific metabolic and biochemical changes which are potentially detectable by infrared analysis. We investigated whether a molecular-sensitive spectroscopic analytical technique such as Fourier Transform Infrared microspectroscopy (microFTIR) and unsupervised explorative multivariate data analyses could represent a rapid and objective method to snapshot the balance between protein phosphorylation and de-phosphorylation in cells as well as a useful method to monitor the effects of anti-leukemic drugs targeting intracellular phosphonetworks. In microFTIR, IR spectroscopy and optical microscopy techniques are combined, respectively.14,15 The first measures the absorbance bands due to IR active modes specific to molecules present in the specimen, the second locates spatially these signal changes at the micron scale over the sample area. The mid-IR spectrum of a single cell reflects thousands of detectable vibrations from chemical groups characterizing different molecules as well as their relative abundance and their interactions. Biochemical, structural, and dynamical information can be achieved by microFTIR at relatively high spatial resolution (down to subcellular resolution with the use of a synchrotron radiation source) allowing the exploration, for instance of structure–function related diseases or the testing and prediction of the sensitivity of leukemic sub-clones to drugs.16–18 A huge amount of useful information can be obtained by performing multivariate data analyses by dimension reductionbased methods such as Principal Component Analysis (PCA). The central idea of PCA is to reduce the dimensionality of a dataset consisting of a large number of interrelated variables,
This journal is © The Royal Society of Chemistry 2015
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while retaining as much as possible the variation present in the dataset.19 The final result is that from a large set of confusing data PCA identifies a few relevant variables that summarize the most relevant information necessary, for instance, classification of groups of cells with common biochemical profiles or identification of disease biomarkers.20 Direct evidence has been already provided that protein phosphorylation induced by external stimulation can be measured by SR microFTIR in cervical epithelial carcinomaderived A431 cells overexpressing the transmembrane epidermal growth factor receptor21 and more recently in individual living PC12 cells exposed to nerve growth factor.22 We extended the study of phosphate balance to normal leukocytes and to leukemic cell lines. By first round unsupervised explorative analysis with PCA we identified spectral regions with maximal variation induced by physiological stimulation of the FRP receptor in normal PMNs. Then, to cross-validate the models of PCA identified in normal leukocytes we tested the consistency of this unsupervised approach also in BCR/ABL negative and positive leukemic cell lines engineered to reproduce drug-resistance or tyrosine phosphatase imbalance and exposed ex vivo to drugs interfering with intracellular phoshonetworks, respectively. Although following different experimental protocols, other groups have independently shown that the FTIR microprobe might represent a valuable analytic tool suitable for monitoring drug resistance/efficacy in leukemic cell models.23–27 Associated with unsupervised explorative Principal Component Analysis (PCA), the IR spectroscopic approach might represent a powerful tool to explore ex vivo the efficacy of anti-leukemic drugs in leukemic blasts directly obtained from CML patients.
Materials and methods Human peripheral blood polymorphonuclear neutrophil (PMN) leukocytes Human peripheral blood samples were collected from healthy donors according to the protocol approved by local ethical committee. Polymorphonuclear neutrophils (PMNs) were separated from other blood components by centrifugation of peripheral blood over Ficoll-Paque (Amersham Pharmacia Biotech, Upsala, Sweden) as previously described.28 To eliminate any contaminant erythrocytes, isolated PMNs were incubated at room temperature for 1 min in a hypotonic saline solution. Pure PMNs (>95% purity) were obtained by re-suspending the pellet of PMNs (3 min spinning at 200g) in isotonic phosphate buffered saline (PBS, pH 7.4). BCR/ABL positive cell line models BCR/ABL positive human-derived K562 leukemia cells (from American Type Culture Collection) had been previously subcloned to obtain K562 cells expressing undetectable levels of PTPRG.29 The transfection of these cells with the cDNA of functionally active human full-length PTPRG, or with the D1028A mutated and functionally PTP-inactive full-length
This journal is © The Royal Society of Chemistry 2015
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PTPRG, or with the empty plasmid ( pCR®3.1, Invitrogen, Palo Alto, CA, containing the G418 resistance gene for selection) allowed the generation of three K562 cell sub-clones: K562#G1, K562#DA and CTRL K562#MK, respectively.12 Replicate samples of these three K562 cell sub-clones were probed with microFTIR. A second leukemic cell model was derived from BCR/ABL negative mouse pro-B lymphocytes Ba/F3 cells (Ba/ F3#PAR). These Ba/F3 cells transfected with the human wildtype p210BCR/ABL cDNA (Ba/F3#WT) generated a stable cell clone with abnormal PTK activity that still retained sensitivity to the tyrosine kinase inhibitor imatinib mesylate (IMA).30 Cells were seeded in sterile T-25 culture flasks, maintained at 37 °C under a humidified atmosphere with 5% CO2 and 95% air and were expanded in RPMI 1640 medium with 10% Fetal Bovine Serum (FBS), 2 mM glutamine and without antibiotic. A WEHI-B3 conditioned supernatant was added to Ba/F3#PAR cells needing IL-3 as the growth factor.31 RT-PCR The expression of both BCR/ABL and PTPRG was crosschecked in Ba/F3 cells and in K562 sub-clones by real time polymerase chain reaction (RT-PCR). Total RNA was extracted from 1 × 107 cells using TRIzol® (Life Technologies, Rockville, MD) and according to the manufacturer’s instructions. The cDNA was obtained by reverse transcription of 450 ng polyAmRNA (isolated with Oligotex mRNA Spin Column, Qiagen) with random primers. Polymerase chain reaction (PCR) was carried out with specific primers available in the ESI (Table 1†). Primers were added to a volume (25 µL) of mastermix (Kapa Hifi, Kapa Biosystems, Wilmington, MA, USA) containing 25 ng cDNA. The reaction was carried out in a GeneAmp PCR System 9700 (Life Technologies; Milano, Italy) for 35 cycles (30 s denaturation at 94 °C, 30 s annealing at 60 °C, 30 s elongation at 72 °C with a single step of hot start activation carried out at 94 °C for 5 minutes). The amplified cDNAs were separated by electrophoresis on 1.5–3% agarose gel. Cloning, expression, and purification of recombinant intracellular domains (ICDs) of human PTPRG The cDNAs of PTP-active D1 and D2 intra-cellular domains (ICDs) and of aspartic acid to alanine-mutated and PTP-inactive ICD (D1028A)ICD of human PTPRG were separately cloned into a pRSET A vector bearing HIV-1Tat cDNA sequence and His tag sequence allowing high-level protein expression in Escherichia coli BL21(DE3)pLysS, and purification on nickel or cobalt columns.32 The expression of ICD- and (D1028A) ICD-Tat recombinant proteins was induced by the addition of 1.0 mM isopropyl-L-thio-β-D galactopyranoside (IPTG) to 2 L of each bacterial culture maintained for 18 hours under stirring (225 rpm) at 16 °C and at 26 °C, respectively. After sonication in 20 mM Tris buffer ( pH 7.8), containing 500 mM NaCl, 20 mM imidazole, and EDTA-free protease inhibitors (binding buffer), the clarified supernatant (10 min centrifugation at 12 500g) was loaded (flow rate 1 mL min−1) in a Prepacked HisTrap™ HP (1 mL) with pre-charged Ni Sepharose™ High
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Performance (GE Healthcare, Milano, Italy) for affinity chromatography purification. Recombinant Tat proteins were eluted with a linear gradient of imidazole (flow rate 1 mL min−1 from 10 to 300 mM imidazole) and their absorbance was continuously checked at 280 nm ( peak around 180 mM imidazole). The presence of pure recombinant protein was checked in fractions by gel electrophoresis on 10% acrylamide/bisacrylamide gel and Coomassie blue staining. Fractions were then pooled, subjected to buffer exchange (20 mM Tris, 200 mM Arginine and 150 mM NaCl buffer ( pH 7.8)) and concentrated by centrifugation (Amicon® Pro Purification System with 50 kDa Amicon® Ultra-0.5 Device, Millipore).33 PMN stimulation Replicate samples of 5 × 106 PMNs mL−1 suspended in phosphate buffered saline (PBS) containing 1.0 mM CaCl2 and 1.0 mM MgCl2 were maintained at 37 °C under stirring and exposed for 1, 5 and 10 minutes to the action of 100 nM formyl-methionyl-leucyl-phenylalanine (fMLP) tripeptide (Sigma, St Louis, MO, USA), respectively. Some replicates were pre-incubated for 30 min with 100 nM wortmannin (WT, from Alexis Pharmaceuticals, San Diego, CA) before fMLP stimulation. Wortmannin (WT) is a fungal metabolite that inhibits the activity of phosphatidylinositol 3-kinases (PI3Ks) specifically and irreversibly and prevents downstream propagation of the stimulus triggered by the activation of fMLP receptor.34–36 Cell stimulation was ceased by the addition of 1 volume of ice cold 4.0% buffered paraformaldehyde (PFA) solution in PBS prepared without methanol to three volumes of sample.17 Appropriate non-stimulated control samples (CTRL) were collected. Perturbation of K562 cell sub-clones Aliquots (1.5 mL) of K562#MK cells plated at a cell density of 1 × 106 cells per mL were separately exposed for 4 and 8 hours to 0.2 µM PTP-active ICD-Tat, 0.2 µM PTP-inactive (D1028) ICD-Tat recombinant proteins, or 5.0 µM imatinib mesylate (4-[(4-methylpiperazin-1-yl)methyl]-N-[4-methyl-3-[(4-pyridin3-ylpyrimidin-2yl)amino]-phenyl]-benzamide, IMA from Novartis Pharma AG, Basel, CH), respectively. IMA competing for the binding of ATP to the active kinase domain of BCR/ABL inhibits the associated abnormal PTK activity. In 24 hour experiments K562 cell sub-clones were separately exposed to 1.0 µM IMA or to 2.0 µM ICD- or (D1028A)ICD-Tat recombinant proteins, respectively.
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Cell fixation preserves lipid, phosphate, and protein components from degradation while inducing very poor distortion in the IR spectrum of cells.37 microFTIR measurements All the spectra were acquired in transmission mode. The FTIR absorbance spectra of single K562 cells were acquired in samples using the instrumentation available at the SMIS (Spectroscopy and Microscopy in the Infrared using Synchrotron) beamline of synchrotron SOLEIL (FR). Exploiting the high brilliance of the infrared synchrotron source (SR) and the confocal operational mode of the microscope (Continuum XL, Thermo Fisher Scientific Inc., USA), several individual cells (>50 per replicate) were analyzed for each sample. The projected size of the aperture onto the sample was set at 12 × 12 μm2, fitting the dimensions of a cell (diameter around 12 μm). The microscope coupled to a FTIR spectrometer (Nexus 5700) used a pair of 32× magnification objective/condenser (N.A. 0.63) and was equipped with a liquid nitrogen cooled single MCT detector of 50 microns sizes. To obtain spectra with acceptable signal to noise ratio (S/N) values within the interval of wavenumbers between 4000 cm−1 and 600 cm−1, 128 co-added interferograms were recorded (scanner velocity 20 kHz, spectral resolution 4 cm−1) both for the sample and for the background (ZnSe). The sample compartment was continuously fluxed with nitrogen to maintain stable environmental conditions during the acquisition of spectra. OMNIC software suite (version 7.3, Thermo Scientific, Madison, WI, USA) was used for the data acquisition. PMNs and Ba/F3 cell samples were analyzed at the Multimode InfraRed Microspectroscopy And Imaging (MIRIAM) beamline B22 of Diamond Light Source (UK). SR was used as the external IR source for single cell analysis via a Hyperion 3000 microscope equipped with a pair of 36× objective/condenser and a 50 × 50 μm2 active area high sensitivity MCT detector for the mid-IR region. For single cell analysis slits were set as specified before. A conventional IR source (globar) was used to analyze simultaneously several cells by delimiting areas ≥30 × 30 µm2 via microscope slits and using a 20× objective/condenser set up. The IR microscope was connected with an in vacuum Bruker Vertex 80 V Fourier Transform IR interferometer. A number of 128 scans (40 kHz scanner velocity and 4 cm−1 spectral resolution, respectively) was accumulated to obtain necessary quality spectra (S/N) within the 4000–700 cm−1 interval. Opus software (release 7.0 from Bruker Optics, Ettlingen, Germany) was used to manage spectra.
Sample preparation for IR analysis
Pre-processing and data analysis
Cells were fixed for 30 min by adding one volume of 4% buffered-PFA solution in PBS prepared without methanol to 3 volumes of sample, washed twice in distilled H2O, pelleted by centrifugation (80g for 2 min at room temperature), and resuspended in H2O. A drop (5 μL) of each cell suspension was deposited and dried on a 50 × 25 × 2 mm ZnSe IR transparent substrate to obtain an array of sample spots.17
The spectra were inspected to identify the number, position and shape of marker peaks. Signal-to-noise ratio (S/N) value was calculated within the 2100–1900 cm−1 interval and spectra with values
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Unsupervised explorative data analysis of normal human leukocytes and BCR/ABL positive leukemic cells mid-infrared spectra† G. Bellisola,*a,b M. Bolomini Vittori,b G. Cinque,c P. Dumas,d Z. Fiorini,b C. Laudanna,b M. Mirenda,b C. Sandt,d G. Silvestri,b L. Tomasello,b M. Vezzalini,b K. Wehbec and C. Sorio*b We proved the ability of Fourier Transform Infrared microspectroscopy (microFTIR) complemented by Principal Component Analysis (PCA) to detect protein phosphorylation/de-phosphorylation in mammalian cells. We analyzed by microFTIR human polymorphonuclear neutrophil (PMNs) leukocytes, mouse-derived parental Ba/F3 cells (Ba/F3#PAR), Ba/F3 cells transfected with p210BCR/ABL (Ba/F3#WT) and expressing high levels of protein tyrosine kinase (PTK), and human-derived BCR/ABL positive K562 leukemic cell sub-clones engineered to differently express receptor-type tyrosine-protein phosphatase gamma (PTPRG). Synchrotron radiation (SR) and conventional (globar) IR sources were used to perform microFTIR respectively, on single cells and over several cells within the same sample. Ex vivo time-course experiments were run, inducing maximal protein phosphorylation in PMNs by 100 nM N-formylated tripeptide fMLP. Within the specific IR fingerprint 1800–850 cm−1 frequency domain, PCA identified two regions with maximal signal variance. These were used to model and test the robustness of PCA in representing the dynamics of protein phosphorylation/de-phosphorylation processes. An IR signal ratio marker reflecting the homeostatic control by protein kinases and phosphatases was identified in normal leukocytes. The models identified by microFTIR and PCA in normal leukocytes also distinguished BCR/ABL positive Ba/F3#WT from BCR/ABL negative Ba/F3#PAR cells as well as K562 cells exposed to functionally active protein tyrosine phosphatase recombinant protein ICD-Tat transduced in cells by HIV-1 Tat technology or cells treated with the PTK inhibitor imatinib mesylate (IMA) from cells exposed to phosphatase inactive (D1028A)ICD-Tat recombinant protein and untreated control cells, respectively. The IR signal marker correctly reflected the degrees of protein phosphorylation associated with abnormal PTK activity in BCR/ABL positive leukemic cells and in general was inversely related to the expression/activity of PTPRG in leukemic sub-clones.
Received 22nd January 2015, Accepted 27th April 2015
In conclusion, we have described a new, reliable and simple spectroscopic method to study the ex vivo
DOI: 10.1039/c5an00148j
protein phosphorylation/de-phosphorylation balance in cell models: it is suitable for biomedical and pharmacological research labs but it also needs further optimization and its evaluation on large cohorts of
www.rsc.org/analyst
patients to be proposed in the clinical setting of leukemia.
a Azienda Ospedaliera Universitaria Intergrata di Verona, Department of Pathology and Diagnostics - Unit of Immunology, Policinico G. Rossi, P.le L.A. Scuro 10, I-37134 Verona, Italy. E-mail: [email protected]; Fax: +39 045 802 7127; Tel: +39 045 802 7688 b University of Verona, Department of Pathology and Diagnostics - General Pathology, Strada Le Grazie, 8, I-37134 Verona, Italy. E-mail: [email protected], [email protected], [email protected], [email protected], [email protected], [email protected], [email protected], [email protected]; Fax: +39 045 8027127; Tel: +39 045 802 7688 c Diamond Light Source Ltd, Diamond House, Harwell Science and Innovation Campus, Chilton, Didcot, Oxfordshire, UK. E-mail: [email protected], [email protected]; Tel: +44 (0)1235778410 d Synchrotron Soleil, L’Orme des Merisiers, Saint-Aubin - BP48, 91192 GIF-sur-Yvette Cedex, France. E-mail: [email protected], [email protected] † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c5an00148j
This journal is © The Royal Society of Chemistry 2015
Introduction Protein phosphorylation is a reversible post-translational modification and a common mechanism to transmit signals and regulate protein and cell functions in leukocytes.1,2 It almost exclusively occurs at the side chains of three amino acids: serine (Ser), threonine (Thr) and tyrosine (Tyr) which represent 6.8%, 5.9%, and 3.2% total protein amino acids, respectively. The transfer of the terminal gamma phosphate group (γ-PO32−) of adenosine triphosphate (ATP) to the nucleophilic (–OH) group of the amino acid side chain is catalyzed by the specific kinases necessary to lower the large amount of free energy of the phosphate–phosphate bond in ATP. Phosphate addition modifies the physico-chemical properties of
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Ser, Thr, and Tyr amino acids as well as those of proteins. For instance, a phosphorylated enzyme can undergo conformational changes which can either activate or inhibit its catalytic activity and facilitate the recruitment of neighboring proteins with structurally conserved domains capable of recognizing and binding to phosphomotifs.3 This occurs in polymorphonuclear neutrophil (PMN) leukocytes where the interactions of bacterial peptides with N-formyl peptide receptor (FPR) on the plasma membrane trigger enzymes that catalyze the generation of intermediate second messengers, such as inositol phosphate and diacylglycerol responsible for the rapid mobilization of Ca2+ from intracellular stores and for the activation of protein kinases.4 In particular, activated protein kinase C (PKC) phosphorylates Ser and Thr amino acid residues of recruited downstream proteins necessary for chemotaxis and cell adhesion, degranulation, generation of superoxide anions and phagocytosis in leucocytes.5 In addition to Ser/Thr protein kinases, receptor and non-receptor protein tyrosine kinases (PTKs) transfer phosphate groups from ATP to Tyr residues.6 Protein Ser/Thr phosphatases (PSPs) and Tyr phosphatases (PTPs) reverse protein phosphorylation and, together with protein kinases, contribute to the temporal and spatial balance of phosphorylation and de-phosphorylation processes leading to pleiotropic effects on cell proliferation, differentiation and survival.7,8 The large family of protein tyrosine phosphatases (PTPs) comprises 8 transmembrane (R1–R8) receptor-type enzymes, 5 with two catalytic tandem phosphatase (D1 and D2) domains and 3 with a single catalytic phosphatase domain, respectively. In tandem-domain receptor-type protein tyrosine phosphatases (PTPRs) the D1 domain adjacent to the plasma membrane displays catalytic activity whereas the D2 domain located in the carboxy-terminal part of the molecule can be either inactive or with negligible catalytic activity.9 In addition to D1–D2 intracellular domains (ICD) the receptor-type tyrosine-protein phosphatase gamma (PTPRG) has a carbonic anhydrase-like domain, a fibronectin 3-like domain in its extracellular part and a short transmembrane domain.10 In Chronic Myelogenous Leukemia (CML), a hematological malignancy, the balance between protein phosphorylation and de-phosphorylation regulated by protein kinases and protein phosphatases is completely subverted due to the presence of abnormal Bcr/Abl fusion oncogene.11 This codifies for a chimeric BCR/ABL protein with constitutively increased protein tyrosine kinase (PTK) activity. Downstream phosphorylation of CRKL, a SH2 and SH3 (src homology) domain containing protein, which is the main substrate of BCR/ABL PTK, activates RAS and JUN kinase signaling pathways allowing leukemic cells to become insensitive to pro-apoptotic stimuli and undergo sustained cell proliferation independently of the presence of growth factors. It has been observed that some BCR/ ABL positive K562 cell sub-clones with low expression/activity of PTPRG have a more aggressive phenotype than K562 subclones transfected with human PTPRG cDNA and with increased expression/activity of this PTP. Not only is the total
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amount of phosphorylated tyrosine reduced by the coexpression of PTPRG but also its specific targets such as BCR/ ABL and CRKL proteins are less phosphorylated.12 Different techniques are currently applied to detect leukemic cells and to monitor anti-leukemic therapy. Routine peripheral blood count associated with the examination of cell morphology in blood smear and bone marrow biopsy by light microscopy allows the formulation of diagnosis in almost cases. The flow cytometric analysis of cells stained with immunfluorescent probes directed towards different marker antigens is useful for establishing the lineage of the neoplastic cells and for assessing their maturation. Karyotyping and/or fluorescence in situ hybridization (FISH) analysis in bone marrow cells can detect chromosomal changes, for instance BCR/ABL. Molecular testing by real time quantitative polymerase chain reaction (RT-qPCR) of mRNA transcripts is the most sensitive routine approach for monitoring the presence of BCR/ABL fusion gene and therefore the response to therapy of patients with chronic myeloid leukaemia. Genome-wide scanning approaches, including gene expression profiling, comparative genomic hybridization (CGH), and single-nucleotide polymorphism (SNP), can measure the associations of specific gene polymorphisms with the phenotype and they are useful to predict the response of CML patients to therapy with tyrosine kinase inhibitors (TKIs).13 However, none of these approaches directly measure the effectiveness of the drug or drug combinations, a phenomenon associated with specific metabolic and biochemical changes which are potentially detectable by infrared analysis. We investigated whether a molecular-sensitive spectroscopic analytical technique such as Fourier Transform Infrared microspectroscopy (microFTIR) and unsupervised explorative multivariate data analyses could represent a rapid and objective method to snapshot the balance between protein phosphorylation and de-phosphorylation in cells as well as a useful method to monitor the effects of anti-leukemic drugs targeting intracellular phosphonetworks. In microFTIR, IR spectroscopy and optical microscopy techniques are combined, respectively.14,15 The first measures the absorbance bands due to IR active modes specific to molecules present in the specimen, the second locates spatially these signal changes at the micron scale over the sample area. The mid-IR spectrum of a single cell reflects thousands of detectable vibrations from chemical groups characterizing different molecules as well as their relative abundance and their interactions. Biochemical, structural, and dynamical information can be achieved by microFTIR at relatively high spatial resolution (down to subcellular resolution with the use of a synchrotron radiation source) allowing the exploration, for instance of structure–function related diseases or the testing and prediction of the sensitivity of leukemic sub-clones to drugs.16–18 A huge amount of useful information can be obtained by performing multivariate data analyses by dimension reductionbased methods such as Principal Component Analysis (PCA). The central idea of PCA is to reduce the dimensionality of a dataset consisting of a large number of interrelated variables,
This journal is © The Royal Society of Chemistry 2015
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while retaining as much as possible the variation present in the dataset.19 The final result is that from a large set of confusing data PCA identifies a few relevant variables that summarize the most relevant information necessary, for instance, classification of groups of cells with common biochemical profiles or identification of disease biomarkers.20 Direct evidence has been already provided that protein phosphorylation induced by external stimulation can be measured by SR microFTIR in cervical epithelial carcinomaderived A431 cells overexpressing the transmembrane epidermal growth factor receptor21 and more recently in individual living PC12 cells exposed to nerve growth factor.22 We extended the study of phosphate balance to normal leukocytes and to leukemic cell lines. By first round unsupervised explorative analysis with PCA we identified spectral regions with maximal variation induced by physiological stimulation of the FRP receptor in normal PMNs. Then, to cross-validate the models of PCA identified in normal leukocytes we tested the consistency of this unsupervised approach also in BCR/ABL negative and positive leukemic cell lines engineered to reproduce drug-resistance or tyrosine phosphatase imbalance and exposed ex vivo to drugs interfering with intracellular phoshonetworks, respectively. Although following different experimental protocols, other groups have independently shown that the FTIR microprobe might represent a valuable analytic tool suitable for monitoring drug resistance/efficacy in leukemic cell models.23–27 Associated with unsupervised explorative Principal Component Analysis (PCA), the IR spectroscopic approach might represent a powerful tool to explore ex vivo the efficacy of anti-leukemic drugs in leukemic blasts directly obtained from CML patients.
Materials and methods Human peripheral blood polymorphonuclear neutrophil (PMN) leukocytes Human peripheral blood samples were collected from healthy donors according to the protocol approved by local ethical committee. Polymorphonuclear neutrophils (PMNs) were separated from other blood components by centrifugation of peripheral blood over Ficoll-Paque (Amersham Pharmacia Biotech, Upsala, Sweden) as previously described.28 To eliminate any contaminant erythrocytes, isolated PMNs were incubated at room temperature for 1 min in a hypotonic saline solution. Pure PMNs (>95% purity) were obtained by re-suspending the pellet of PMNs (3 min spinning at 200g) in isotonic phosphate buffered saline (PBS, pH 7.4). BCR/ABL positive cell line models BCR/ABL positive human-derived K562 leukemia cells (from American Type Culture Collection) had been previously subcloned to obtain K562 cells expressing undetectable levels of PTPRG.29 The transfection of these cells with the cDNA of functionally active human full-length PTPRG, or with the D1028A mutated and functionally PTP-inactive full-length
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PTPRG, or with the empty plasmid ( pCR®3.1, Invitrogen, Palo Alto, CA, containing the G418 resistance gene for selection) allowed the generation of three K562 cell sub-clones: K562#G1, K562#DA and CTRL K562#MK, respectively.12 Replicate samples of these three K562 cell sub-clones were probed with microFTIR. A second leukemic cell model was derived from BCR/ABL negative mouse pro-B lymphocytes Ba/F3 cells (Ba/ F3#PAR). These Ba/F3 cells transfected with the human wildtype p210BCR/ABL cDNA (Ba/F3#WT) generated a stable cell clone with abnormal PTK activity that still retained sensitivity to the tyrosine kinase inhibitor imatinib mesylate (IMA).30 Cells were seeded in sterile T-25 culture flasks, maintained at 37 °C under a humidified atmosphere with 5% CO2 and 95% air and were expanded in RPMI 1640 medium with 10% Fetal Bovine Serum (FBS), 2 mM glutamine and without antibiotic. A WEHI-B3 conditioned supernatant was added to Ba/F3#PAR cells needing IL-3 as the growth factor.31 RT-PCR The expression of both BCR/ABL and PTPRG was crosschecked in Ba/F3 cells and in K562 sub-clones by real time polymerase chain reaction (RT-PCR). Total RNA was extracted from 1 × 107 cells using TRIzol® (Life Technologies, Rockville, MD) and according to the manufacturer’s instructions. The cDNA was obtained by reverse transcription of 450 ng polyAmRNA (isolated with Oligotex mRNA Spin Column, Qiagen) with random primers. Polymerase chain reaction (PCR) was carried out with specific primers available in the ESI (Table 1†). Primers were added to a volume (25 µL) of mastermix (Kapa Hifi, Kapa Biosystems, Wilmington, MA, USA) containing 25 ng cDNA. The reaction was carried out in a GeneAmp PCR System 9700 (Life Technologies; Milano, Italy) for 35 cycles (30 s denaturation at 94 °C, 30 s annealing at 60 °C, 30 s elongation at 72 °C with a single step of hot start activation carried out at 94 °C for 5 minutes). The amplified cDNAs were separated by electrophoresis on 1.5–3% agarose gel. Cloning, expression, and purification of recombinant intracellular domains (ICDs) of human PTPRG The cDNAs of PTP-active D1 and D2 intra-cellular domains (ICDs) and of aspartic acid to alanine-mutated and PTP-inactive ICD (D1028A)ICD of human PTPRG were separately cloned into a pRSET A vector bearing HIV-1Tat cDNA sequence and His tag sequence allowing high-level protein expression in Escherichia coli BL21(DE3)pLysS, and purification on nickel or cobalt columns.32 The expression of ICD- and (D1028A) ICD-Tat recombinant proteins was induced by the addition of 1.0 mM isopropyl-L-thio-β-D galactopyranoside (IPTG) to 2 L of each bacterial culture maintained for 18 hours under stirring (225 rpm) at 16 °C and at 26 °C, respectively. After sonication in 20 mM Tris buffer ( pH 7.8), containing 500 mM NaCl, 20 mM imidazole, and EDTA-free protease inhibitors (binding buffer), the clarified supernatant (10 min centrifugation at 12 500g) was loaded (flow rate 1 mL min−1) in a Prepacked HisTrap™ HP (1 mL) with pre-charged Ni Sepharose™ High
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Performance (GE Healthcare, Milano, Italy) for affinity chromatography purification. Recombinant Tat proteins were eluted with a linear gradient of imidazole (flow rate 1 mL min−1 from 10 to 300 mM imidazole) and their absorbance was continuously checked at 280 nm ( peak around 180 mM imidazole). The presence of pure recombinant protein was checked in fractions by gel electrophoresis on 10% acrylamide/bisacrylamide gel and Coomassie blue staining. Fractions were then pooled, subjected to buffer exchange (20 mM Tris, 200 mM Arginine and 150 mM NaCl buffer ( pH 7.8)) and concentrated by centrifugation (Amicon® Pro Purification System with 50 kDa Amicon® Ultra-0.5 Device, Millipore).33 PMN stimulation Replicate samples of 5 × 106 PMNs mL−1 suspended in phosphate buffered saline (PBS) containing 1.0 mM CaCl2 and 1.0 mM MgCl2 were maintained at 37 °C under stirring and exposed for 1, 5 and 10 minutes to the action of 100 nM formyl-methionyl-leucyl-phenylalanine (fMLP) tripeptide (Sigma, St Louis, MO, USA), respectively. Some replicates were pre-incubated for 30 min with 100 nM wortmannin (WT, from Alexis Pharmaceuticals, San Diego, CA) before fMLP stimulation. Wortmannin (WT) is a fungal metabolite that inhibits the activity of phosphatidylinositol 3-kinases (PI3Ks) specifically and irreversibly and prevents downstream propagation of the stimulus triggered by the activation of fMLP receptor.34–36 Cell stimulation was ceased by the addition of 1 volume of ice cold 4.0% buffered paraformaldehyde (PFA) solution in PBS prepared without methanol to three volumes of sample.17 Appropriate non-stimulated control samples (CTRL) were collected. Perturbation of K562 cell sub-clones Aliquots (1.5 mL) of K562#MK cells plated at a cell density of 1 × 106 cells per mL were separately exposed for 4 and 8 hours to 0.2 µM PTP-active ICD-Tat, 0.2 µM PTP-inactive (D1028) ICD-Tat recombinant proteins, or 5.0 µM imatinib mesylate (4-[(4-methylpiperazin-1-yl)methyl]-N-[4-methyl-3-[(4-pyridin3-ylpyrimidin-2yl)amino]-phenyl]-benzamide, IMA from Novartis Pharma AG, Basel, CH), respectively. IMA competing for the binding of ATP to the active kinase domain of BCR/ABL inhibits the associated abnormal PTK activity. In 24 hour experiments K562 cell sub-clones were separately exposed to 1.0 µM IMA or to 2.0 µM ICD- or (D1028A)ICD-Tat recombinant proteins, respectively.
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Cell fixation preserves lipid, phosphate, and protein components from degradation while inducing very poor distortion in the IR spectrum of cells.37 microFTIR measurements All the spectra were acquired in transmission mode. The FTIR absorbance spectra of single K562 cells were acquired in samples using the instrumentation available at the SMIS (Spectroscopy and Microscopy in the Infrared using Synchrotron) beamline of synchrotron SOLEIL (FR). Exploiting the high brilliance of the infrared synchrotron source (SR) and the confocal operational mode of the microscope (Continuum XL, Thermo Fisher Scientific Inc., USA), several individual cells (>50 per replicate) were analyzed for each sample. The projected size of the aperture onto the sample was set at 12 × 12 μm2, fitting the dimensions of a cell (diameter around 12 μm). The microscope coupled to a FTIR spectrometer (Nexus 5700) used a pair of 32× magnification objective/condenser (N.A. 0.63) and was equipped with a liquid nitrogen cooled single MCT detector of 50 microns sizes. To obtain spectra with acceptable signal to noise ratio (S/N) values within the interval of wavenumbers between 4000 cm−1 and 600 cm−1, 128 co-added interferograms were recorded (scanner velocity 20 kHz, spectral resolution 4 cm−1) both for the sample and for the background (ZnSe). The sample compartment was continuously fluxed with nitrogen to maintain stable environmental conditions during the acquisition of spectra. OMNIC software suite (version 7.3, Thermo Scientific, Madison, WI, USA) was used for the data acquisition. PMNs and Ba/F3 cell samples were analyzed at the Multimode InfraRed Microspectroscopy And Imaging (MIRIAM) beamline B22 of Diamond Light Source (UK). SR was used as the external IR source for single cell analysis via a Hyperion 3000 microscope equipped with a pair of 36× objective/condenser and a 50 × 50 μm2 active area high sensitivity MCT detector for the mid-IR region. For single cell analysis slits were set as specified before. A conventional IR source (globar) was used to analyze simultaneously several cells by delimiting areas ≥30 × 30 µm2 via microscope slits and using a 20× objective/condenser set up. The IR microscope was connected with an in vacuum Bruker Vertex 80 V Fourier Transform IR interferometer. A number of 128 scans (40 kHz scanner velocity and 4 cm−1 spectral resolution, respectively) was accumulated to obtain necessary quality spectra (S/N) within the 4000–700 cm−1 interval. Opus software (release 7.0 from Bruker Optics, Ettlingen, Germany) was used to manage spectra.
Sample preparation for IR analysis
Pre-processing and data analysis
Cells were fixed for 30 min by adding one volume of 4% buffered-PFA solution in PBS prepared without methanol to 3 volumes of sample, washed twice in distilled H2O, pelleted by centrifugation (80g for 2 min at room temperature), and resuspended in H2O. A drop (5 μL) of each cell suspension was deposited and dried on a 50 × 25 × 2 mm ZnSe IR transparent substrate to obtain an array of sample spots.17
The spectra were inspected to identify the number, position and shape of marker peaks. Signal-to-noise ratio (S/N) value was calculated within the 2100–1900 cm−1 interval and spectra with values
