Erik Berglund

Expression and association of TRPC subtypes with Orai1 and STIM1 in human parathyroid

The mechanism behind Ca(2)(+) entry into the parathyroid cells has been widely debated, and the molecular identities of the responsible ion channels have not been established yet. In this study, we show that the parathyroid cells lack voltage-operated Ca(2)(+) channels. Passive store depletion by thapsigargin, on the other hand, induces a large non-voltage-activated non-selective cation current. The increase in intracellular Ca(2)(+) caused by thapsigargin is attenuated by 2-aminoethoxydiphenyl borate, a blocker of store-operated Ca(2)(+) entry (SOCE). Candidate molecules for non-voltage-operated Ca(2)(+) signaling were investigated. These included members of the transient receptor potential canonical (TRPC) ion channel family, as well as Ca(2)(+) release-activated Ca(2)(+) modulator 1 (Orai1) and stromal interaction molecule 1 (STIM1) that are key proteins in the SOCE pathway. Using RT-PCR screening, quantitative real-time PCR, and western blot, we showed expression of TRPC1, TRPC4, and TRPC6; Orai1; and STIM1 genes and proteins in normal and adenomatous human parathyroid tissues. Furthermore, co-immunoprecipitation experiments demonstrated a ternary complex of TRPC1-Orai1-STIM1, supporting a physical interaction between these molecules in human parathyroid.

Functional role of the Ca2+-activated Cl- channel DOG1/TMEM16A in gastrointestinal stromal tumor cells

DOG1, a Ca(2+)-activated Cl(-) channel (CaCC), was identified in 2004 to be robustly expressed in gastrointestinal stromal tumors (GIST). It was rapidly included as a tumor marker in routine diagnostics, but the functional role remained unknown. CaCCs are important regulators of normal physiological functions, but also implicated in tumorigenesis, cancer progression, metastasis, cell migration, apoptosis, proliferation and viability in several malignancies. We therefore investigated whether DOG1 plays a role in the three latter in GIST by utilizing in vitro cell model systems. Confocal microscopy identified different subcellular localizations of DOG1 in imatinib-sensitive and imatinib-resistant cells. Electrophysiological studies confirmed that DOG1-specific pharmacological agents possess potent activating and inhibiting properties. Proliferation assays showed small effects up to 72 h, and flow cytometric analysis of adherent cells with 7-AAD/Annexin V detected no pharmacological effects on viable GIST cells. However, inhibition of DOG1 conveyed pro-apoptotic effects among early apoptotic imatinib-resistant cells. In conclusion, DOG1 generates Cl(-) currents in GIST that can be regulated pharmacologically, with small effects on cell viability and proliferation in vitro. Inhibition of DOG1 might act pro-apoptotic on some early apoptotic GIST cell populations. Further studies are warranted to fully illuminate the function of DOG1 and its potential as therapeutic target.

Intracellular concentration of the tyrosine kinase inhibitor imatinib in gastrointestinal stromal tumor cells.

Gastrointestinal stromal tumor (GIST) is the most common mesenchymal neoplasm in the gastrointestinal tract. In most GISTs, the underlying mechanism is a gain-of-function mutation in the KIT or the PDGFRA gene. Imatinib is a tyrosine kinase inhibitor that specifically blocks the intracellular ATP-binding sites of these receptors. A correlation exists between plasma levels of imatinib and progression-free survival, but it is not known whether the plasma concentration correlates with the intracellular drug concentration. We determined intracellular imatinib levels in two GIST cell lines: the imatinib-sensitive GIST882 and the imatinib-resistant GIST48. After exposing the GIST cells to imatinib, the intracellular concentrations were evaluated using LC-MS (TOF). The concentration of imatinib in clinical samples from three patients was also determined to assess the validity and reliability of the method in the clinical setting. Determination of imatinib uptake fits within detection levels and values are highly reproducible. The GIST48 cells showed significantly lower imatinib uptake compared with GIST882 in therapeutic doses, indicating a possible difference in uptake mechanisms. Furthermore, imatinib accumulated in the tumor tissues and showed intratumoral regional differences. These data show, for the first time, a feasible and reproducible technique to measure intracellular imatinib levels in experimental and clinical settings. The difference in the intracellular imatinib concentration between the cell lines and clinical samples indicates that drug transporters may contribute toward resistance mechanisms in GIST cells. This highlights the importance of further clinical studies to quantify drug transporter expression and measure intracellular imatinib levels in GIST patients.

Evidence for Ca2+-regulated ATP release in gastrointestinal stromal tumors

Gastrointestinal stromal tumors (GISTs) are thought to originate from the electrically active pacemaker cells of the gastrointestinal tract. Despite the presence of synaptic-like vesicles and proteins involved in cell secretion it remains unclear whether GIST cells possess regulated release mechanisms. The GIST tumor cell line GIST882 was used as a model cell system, and stimulus-release coupling was investigated by confocal microscopy of cytoplasmic free Ca(2+) concentration ([Ca(2+)]i), flow cytometry, and luminometric measurements of extracellular ATP. We demonstrate that GIST cells have an intact intracellular Ca(2+)-signaling pathway that regulates ATP release. Cell viability and cell membrane integrity was preserved, excluding ATP leakage due to cell death and suggesting active ATP release. The stimulus-secretion signal transduction is at least partly dependent on Ca(2+) influx since exclusion of extracellular Ca(2+) diminishes the ATP release. We conclude that measurements of ATP release in GISTs may be a useful tool for dissecting the signal transduction pathway, mapping exocytotic components, and possibly for the development and evaluation of drugs. Additionally, release of ATP from GISTs may have importance for tumor tissue homeostasis and immune surveillance escape.

Calmodulin and calmodulin-dependent protein kinase II inhibit hormone secretion in human parathyroid adenoma

Intracellular calcium ([Ca(2+)](i)) is the most relevant modulator of parathyroid hormone (PTH) secretion. Uniquely, an increase in [Ca(2+)](i) results in an inhibition of PTH secretion, and it probably exerts its function via calcium-binding protein pathways. The ubiquitous calcium-binding proteins, calmodulin and calmodulin-dependent protein kinase II (CaMKII), have well-established roles in regulated exocytosis in neurons and neuroendocrine cells. However, their roles in parathyroid cells and PTH secretion are still unclear. Using reverse transcription-PCR and western blot analysis, we have demonstrated the expression of calmodulin and CaMKII in human normal parathyroid and parathyroid chief cell adenomas. Blocking of calmodulin and CaMKII activity by the specific antagonists calmidazolium and KN-62 respectively caused a rise in PTH secretion from parathyroid adenoma cells in spite of increased [Ca(2+)](i). The inhibitory effect of Ca(2+) calmodulin on PTH secretion may be due to the absence of synaptotagmin 1 protein in parathyroid adenomas, as demonstrated by western blot analysis. An increased extracellular calcium level acutely lowered the amount of active phosphorylated CaMKII (pCaMKII) in adenoma cells in vitro, indicating the physiological importance of this pathway. Moreover, a negative correlation between the levels of pCaMKII in parathyroid adenomas and serum calcium was found in 20 patients with primary hyperparathyroidism. Taken together, these results show that calmodulin negatively contributes to the regulation of PTH secretion in parathyroid adenoma, at least partially via a CaMKII pathway.

Secretome protein signature of human gastrointestinal stromal tumor cells

Strategies for correct diagnosis, treatment evaluation and recurrence prediction are important for the prognosis and mortality rates among cancer patients. In spite of major improvements in clinical management, gastrointestinal stromal tumors (GISTs) can still be deadly due to metastasis and recurrences, which confirms the unmet need of reliable follow-up modalities. Tumor-specific secreted, shed or leaked proteins (collectively known as secretome) are considered promising sources for biomarkers, and suitable for detection in biofluids. Herein, we stimulated cell secretion in the imatinib-sensitive GIST882 cell line and profiled the secretome, collected as conditioned media, by using a shotgun proteomics approach. We identified 764 proteins from all conditions combined, 51.3% being predicted as classically/non-classically secreted. The protein subsets found were dependent on the stimulatory condition. The significant increase in protein release by the classical pathway was strongly associated with markers already found in other cancer types. Furthermore, most of the released proteins were non-classically released and overlapped to a high degree with proteins of exosomal origin. Imatinib pre-treatment radically changed these secretory patterns, which can have clinical implications when investigating biomarkers in imatinib-treated versus non-treated GIST patients. Our results show, for the first time, that GISTs contain a secretome signature. In the search for suitable biomarkers in the more complex GIST patient samples, this study aids in the understanding of basic GIST secretome characteristics.

Electrical short-circuit in β-cells from a patient with non-insulinoma pancreatogenous hypoglycemic syndrome (NIPHS): a case report

IntroductionNon-insulinoma pancreatogenous hypoglycemic syndrome is a rare disorder among adults, and, to our knowledge, only about 40 cases have been reported in the literature.Case presentationThe patient is a previously healthy 35-year-old Caucasian man. His symptoms began four years ago when he suddenly felt weakness in his legs and started sweating for unknown reasons. The symptoms worsened, and laboratory tests revealed hypoglycemia and hyperinsulinemia at the time of the symptoms. All diagnostics attempts using magnetic resonance imaging, computed tomography, and endoscopic ultrasound did not reveal any abnormalities. At this stage, surgical intervention was planned, and a distal 80% pancreatectomy was performed. The histopathologic and immunohistochemical investigations of the pancreas showed an increased number of islets of different sizes, more or less evenly distributed in the gland, but no insulinoma. Patch-clamp recordings from isolated pancreatic β-cells showed that, even at a low glucose concentration (3 mmol/L), the β-cell membrane was depolarized, and action potentials were seen. Surprisingly, in patch-clamp experiments, the addition of diazoxide had a marked effect on K-ATP channel activity and membrane potential, but no effect on insulin levels in vivo before surgery.ConclusionThis case report adds new information on the pathogenesis of non-insulinoma pancreatogenous hypoglycemic syndrome, as we performed an electrophysiologic characterization of isolated islet cells. We show, for the first time, that β-cells isolated from a non-insulinoma pancreatogenous hypoglycemic syndrome patient are constantly depolarized, even at low glucose levels, but display normal K-ATP channel physiology.

miR-125a-5p regulation increases phosphorylation of FAK that contributes to imatinib resistance in gastrointestinal stromal tumors

ABSTRACT The use of imatinib mesylate has greatly improved the clinical outcome for gastrointestinal stromal tumor (GIST) patients. However, imatinib resistance is still a major clinical challenge, and the molecular mechanisms are not fully understood. We have previously shown that miR‐125a‐5p and its mRNA target PTPN18 modulate imatinib response in GIST cells. Herein, we evaluated phosphorylated FAK (pFAK) as a candidate downstream target of PTPN18 and the possible association of this regulation with imatinib resistance in GIST. FAK and pFAK expressions were evaluated in GIST882 cells transfected with short hairpin RNA or short interfering RNA targeting PTPN18 or miR‐125a‐5p mimic, imatinib‐resistant GIST882R subclones and clinical samples using Western blot analyses. FAK phosphorylation was blocked using the FAK inhibitor 14 (FAKi) and the effects on cell viability and apoptosis were evaluated using WST‐1 assay and cleaved PARP expression. Clinical associations of FAK and pFAK expression with imatinib resistance, KIT mutation and patient outcome were assessed by Fishers exact test or log‐rank test. Over‐expression of miR‐125a‐5p and silencing of PTPN18 increased pFAK, but not FAK, expression in GIST cells. Higher pFAK expression was observed in the GIST882R subclones with acquired imatinib resistance compared to their imatinib‐sensitive parental cells. Treatment with FAKi in imatinib‐resistant GIST882R cells reduced cell viability and increased apoptosis upon imatinib treatment. Additionally, FAKi could rescue the imatinib resistance effect mediated by miR‐125a‐5p over‐expression. In clinical samples, high FAK and pFAK expressions were associated with KIT mutation status, and high FAK expression was also associated with metastasis in GIST. Higher pFAK was found in cases with shorter overall survival. Our findings highlight an important role for miR‐125a‐5p regulation and its downstream target pFAK for imatinib resistance in GIST. pFAK and FAK may have prognostic values in GIST. Graphical abstract Figure. No caption available. HighlightsPhosphorylation of FAK is regulated by PTPN18 and miR‐125a‐5p.pFAK level is higher in imatinib‐resistant than imatinib‐sensitive GIST882 cells.FAK inihibitor treatment in imatinib‐resistant cells increases imatinib‐induced apoptosis.Blocking FAK phosphorylation rescues miR‐125a‐5p mediated imatinib resistance.

Ex Vivo Generation of Donor Antigen-Specific Immunomodulatory Cells: A Comparison Study of Anti-CD80/86 mAbs and CTLA4-lg Costimulatory Blockade

Adoptive transfer of alloantigen-specific immunomodulatory cells generated ex vivo with anti-CD80/CD86 mAbs (2D10.4/IT2.2) holds promise for operational tolerance after transplantation. However, good manufacturing practice is required to allow widespread clinical application. Belatacept, a clinically approved cytotoxic T-lymphocyte antigen 4-immunoglobulin that also binds CD80/CD86, could be an alternative agent for 2D10.4/IT2.2. With the goal of generating an optimal cell treatment with clinically approved reagents, we evaluated the donor-specific immunomodulatory effects of belatacept- and 2D10.4/IT2.2-generated immunomodulatory cells. Immunomodulatory cells were generated by coculturing responder human peripheral blood mononuclear cells (PBMCs) (50 × 106 cells) with irradiated donor PBMCs (20 × 106 cells) from eight human leukocyte antigen-mismatched responder–donor pairs in the presence of either 2D10.4/IT2.2 (3 μg/106 cells) or belatacept (40 μg/106 cells). After 14 days of coculture, the frequencies of CD4+ T cells, CD8+ T cells, and natural killer cells as well as interferon gamma (IFN-γ) production in the 2D10.4/IT2.2- and belatacept-treated groups were lower than those in the control group. The percentage of CD19+ B cells was higher in the 2D10.4/IT2.2- and belatacept-treated groups than in the control group. The frequency of CD4+CD25+CD127lowFOXP3+ T cells increased from 4.1±1.0% (preculture) to 7.1±2.6% and 7.3±2.6% (day 14) in the 2D10.4/IT2.2- and belatacept-treated groups, respectively (p<0.05). Concurrently, delta-2 FOXP3 mRNA expression increased significantly. Compared with cells derived from the no-antibody treated control group, cells generated from both the 2D10.4/IT2.2- and belatacept-treated groups produced lower IFN-γ and higher interleukin-10 levels in response to donor-antigens, as detected by enzyme-linked immunospot. Most importantly, 2D10.4/IT2.2- and belatacept-generated cells effectively impeded the proliferative responses of freshly isolated responder PBMCs against donor-antigens. Our results indicate that belatacept-generated donor-specific immunomodulatory cells possess comparable phenotypes and immunomodulatory efficacies to those generated with 2D10.4/IT2.2. We suggest that belatacept could be used for ex vivo generation of clinical grade alloantigen-specific immunomodulatory cells for tolerance induction after transplantation.

Evidence for intracellular calcium-regulated secretion in gastrointestinal stromal tumor.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Gastrointestinal stromal tumors (GISTs) are considered to originate from the electrically active pacemaker cells of the gut. Despite the presence of synaptic like vesicles and proteins involved in exocytosis and secretory response in other cell types, it still remains unclear if GIST cells have a regulated secretion. A GIST tumor cell line (GIST882) was used as a model cell system, and stimulus-secretion coupling was investigated performing measurements of intracellular Ca2+, confocal microscopy, flow cytometry and an ATP-based assay to measure secretory response. We demonstrate that GIST cells have an intracellular signaling pathway downstream regulating cell secretion observed as an ATP release. Cell membrane integrity was preserved showing that ATP is released and not related to cell membrane rupture or cell death. The stimulus-secretion signal transduction is, at least partly, dependent on Ca2+ influx, since exclusion of extracellular Ca2+ diminishes the secretory response. We conclude that measurements of the secretory response in GIST by using ATP as a marker may be a useful tool in dissecting the signal transduction pathway, identification of secretory substances and in the development of drugs. Citation Format: Erik Berglund, David Berglund, Pinar Akcakaya, Mehran Ghaderi, Elisabetta Dare, Per-Olof Berggren, Craig A. Aspinwall, Weng-Onn Lui, Jan Zedenius, Catharina Larsson, Robert Branstrom. Evidence for intracellular calcium-regulated secretion in gastrointestinal stromal tumor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5317. doi:10.1158/1538-7445.AM2013-5317 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.