DLBCL cells with acquired resistance to venetoclax are not sensitized to BIRD-2 but can be resensitized to venetoclax through Bcl-XL inhibition

Anti-apoptotic Bcl-2-family members are frequently dysregulated in both blood and solid cancers, contributing to their survival despite ongoing oncogenic stress. Yet, such cancer cells often are highly dependent on Bcl-2 for their survival, a feature that is exploited by so-called BH3-mimetic drugs. Venetoclax (ABT-199) is a selective BH3-mimetic Bcl-2 antagonist that is currently used in the clinic for treatment of chronic lymphocytic leukemia patients. Unfortunately, venetoclax resistance has already emerged in patients, limiting the therapeutic success. Here, we examined strategies to overcome venetoclax resistance. Therefore, we used two diffuse large B-cell lymphoma (DLBCL) cell lines, Riva WT and venetoclax-resistant Riva (VR). The latter was obtained by prolonged culturing in the presence of venetoclax. We report that Riva VR cells did not become more sensitive to BIRD-2, a peptide targeting the Bcl-2 BH4 domain, and established cross-resistance towards BDA-366, a putative BH4-domain antagonist of Bcl-2. However, we found that Bcl-XL, another Bcl-2-family protein, is upregulated in Riva VR, while Mcl-1 expression levels are not different in comparison with Riva WT, hinting towards an increased dependence of Riva VR cells to Bcl-XL. Indeed, Riva VR cells could be resensitized to venetoclax by A-1155463, a selective BH3 mimetic Bcl-XL inhibitor. This is underpinned by siRNA experiments, demonstrating that lowering Bcl-XL-expression levels also augmented the sensitivity of Riva VR cells to venetoclax. Overall, this work demonstrates that Bcl-XL upregulation contributes to acquired resistance of DLBCL cancer cells towards venetoclax and that antagonizing Bcl-XL can resensitize such cells towards venetoclax

Driver mutations of pancreatic cancer affect Ca2+Ca^{2+} signaling and ATP production

Glandular pancreatic epithelia of the acinar or ductal phenotype may seem terminally differentiated, but they are characterized by remarkable cell plasticity. Stress-induced trans-differentiation of these cells has been implicated in the mechanisms of carcinogenesis. Current consensus links pancreatic ductal adenocarcinoma with onco-transformation of ductal epithelia, but under the presence of driver mutations in Kras and Trp53, also with trans-differentiation of pancreatic acini. However, we do not know when, in the course of cancer progression, physiological functions are lost by mutant acinar cells, nor can we assess their capacity for the production of pancreatic juice components. Here, we investigated whether two mutations—$Kras^{G12D}$ and $Trp53^{R172H}$—present simultaneously in acinar cells of KPC mice (model of oncogenesis) influence cytosolic Ca2+ signals. Since $Ca^{2+}$ signals control the cellular handling of digestive hydrolases, any changes that affect intracellular signaling events and cell bioenergetics might have an impact on the physiology of the pancreas. Our results showed that physiological doses of acetylcholine evoked less regular $Ca^{2+}$ oscillations in KPC acinar cells compared to the control, whereas responses to supramaximal concentrations were markedly reduced. Menadione elicited $Ca^{2+}$ signals of different frequencies in KPC cells compared to control cells. Finally, $Ca^{2+}$ extrusion rates were significantly inhibited in KPC cells, likely due to the lower basal respiration and ATP production. Cumulatively, these findings suggest that driver mutations affect the signaling capacity of pancreatic acinar cells even before the changes in the epithelial cell morphology become apparent

Activation of pancreatic stellate cells attenuates intracellular Ca2+ signals due to downregulation of TRPA1 and protects against cell death induced by alcohol metabolites

Alcohol abuse, an increasing problem in developed societies, is one of the leading causes of acute and chronic pancreatitis. Alcoholic pancreatitis is often associated with fibrosis mediated by activated pancreatic stellate cells (PSCs). Alcohol toxicity predominantly depends on its non-oxidative metabolites, fatty acid ethyl esters, generated from ethanol and fatty acids. Although the role of non-oxidative alcohol metabolites and dysregulated Ca2+ signalling in enzyme-storing pancreatic acinar cells is well established as the core mechanism of pancreatitis, signals in PSCs that trigger fibrogenesis are less clear. Here, we investigate real-time Ca2+ signalling, changes in mitochondrial potential and cell death induced by ethanol metabolites in quiescent vs TGF-β-activated PSCs, compare the expression of Ca2+ channels and pumps between the two phenotypes and the consequences these differences have on the pathogenesis of alcoholic pancreatitis. The extent of PSC activation in the pancreatitis of different aetiologies has been investigated in three animal models. Unlike biliary pancreatitis, alcohol-induced pancreatitis results in the activation of PSCs throughout the entire tissue. Ethanol and palmitoleic acid (POA) or palmitoleic acid ethyl ester (POAEE) act directly on quiescent PSCs, inducing cytosolic Ca2+ overload, disrupting mitochondrial functions, and inducing cell death. However, activated PSCs acquire remarkable resistance against ethanol metabolites via enhanced Ca2+-handling capacity, predominantly due to the downregulation of the TRPA1 channel. Inhibition or knockdown of TRPA1 reduces EtOH/POA-induced cytosolic Ca2+ overload and protects quiescent PSCs from cell death, similarly to the activated phenotype. Our results lead us to review current dogmas on alcoholic pancreatitis. While acinar cells and quiescent PSCs are prone to cell death caused by ethanol metabolites, activated PSCs can withstand noxious signals and, despite ongoing inflammation, deposit extracellular matrix components. Modulation of Ca2+ signals in PSCs by TRPA1 agonists/antagonists could become a strategy to shift the balance of tissue PSCs towards quiescent cells, thus limiting pancreatic fibrosis

The role of MCPIP1 in activation of NF-κB in cellular model of non-alcoholic fatty liver disease

Niealkoholowa stłuszczeniowa choroba wątroby (NAFLD) jest jedną z najczęściej występujących chorób w krajach zachodnich. Główną przyczyną NAFLD jest zwiększona akumulacja trójglicerydów w hapatocytach, co może prowadzić do odpowiedzi zapalnej, marskości wątroby a nawet do nowotworu. Wykazano, że rozwój NAFLD wiąże się z nadekspresją białka MCPIP1, które odgrywa kluczową rolę w regulacji stanu zapalnego. Celem badań było wykazanie czy białko MCPIP1 wpływa na aktywację czynnika transkrypcyjnego NF-κB w komórkowym modelu NAFLD, który jest głównym regulatorem stanu zapalnego i apoptozy. Poziom aktywności NF-κB został sprawdzony w teście opartym o dwa geny reporterowe, w którym komórki były transfekowane dwoma plazmidami zawierającymi sekwencje kodujące lucyferazę pochodzącą z dwóch różnych organizmów: świetlika oraz renilli. W promotorze genu dla lucyferazy w plazmidzie eksperymentalnym była obecna sekwencja rozpoznawana przez czynnik transkrypcyjny z rodziny NF-κB, co umożliwiło pomiar poziomu aktywności tego białka w komórce. Podczas doświadczeń, komórki linii HepG2 stymulowane były 0.5 mM oraz 1 mM oleinianem sodu w celu wygenerowania komórkowego modelu choroby NAFLD. Pokazano, że poziom aktywności NF-κB w dzikich komórkach nie zmienia się po stymulacji tłuszczem, podczas gdy w komórkach ‘MCPIP1’ ulega obniżeniu. Te wyniki świadczą o tym, że białko MCPIP1 obniża aktywność czynnika transkrypcyjnego NF-κB w NAFLD i oba białka mogą mieć wpływ na progresję choroby.Nonalcoholic fatty liver disease (NAFLD) is one of the most prevalent liver illness that affects people, especially in Western nations. The main cause of NAFLD is increased accumulation of triglycerides in hepatocytes, which may result in inflammatory response, cirrhosis and liver cancer. It was shown that progression of NAFLD is connected with overexpression of MCPIP1 which is one of the most significant protein engaged in regulation of inflammation. The aim of this research was to determine whether MCPIP1 influences activation of transcription factor NF-κB in cellular model of NAFLD, which is a major regulator of inflammation and cell death. The level of activation of NF-κB was examined in Dual-Luciferase Reporter Assay System in which cells were transfected with two plasmids. Experimental plasmid consists of sequence encoding luciferase from a firefly, whereas control plasmid has sequence for luciferase from a sea pansy. In the promoter of luciferase gene in expermiental plasmid was present the sequence for NF-κB binding what made it possible to check the level of activation of this transcription factor. During the research, HepG2 cells were treated 0.5 mM and 1 mM sodium oleate to generate cellular model of disease. It was shown that the level of activation of NF-κB in wild-type cells had not changed after stimulation, while in ‘MCPIP1’ cells had dropped after sodium oleate treatment. This results indicate that MCPIP1 decreases the level of activation of NF-κB and both proteins may have impact on progression of NAFLD

"Figuring the chaos out" : organization of the Krakow Film Music Festival from the perspective of project manager and stage manager

The article was written on the basis of ethnographic research of the Krakow Film Music Festival in 2019. The research in the form of observation and interviews was held by a team of ten researchers belonging to the "Konfraternia Eksploratorów Kultury" ("Confraternity of Culture Explorers") scientific circle operating at the Institute of Culture of the Jagiellonian University. The article presents how the organization of the festival looks like in the perspective of two people managing different spheres of it - the project manager and the stage manager. The organization itself can be reduced to the common denominator of "figuring the chaos out". The festival is a complex collection of spontaneous and often unexpected situations. The managers and the physical space in which the organizers operate and the audience stays, have therefore tasks that introduce order, so the tasks of "figuring out". The article is then a story about ordering the festival reality

Pancreatic Cancer and Its Microenvironment—Recent Advances and Current Controversies

Pancreatic ductal adenocarcinoma (PDAC) causes annually well over 400,000 deaths world-wide and remains one of the major unresolved health problems. This exocrine pancreatic cancer originates from the mutated epithelial cells: acinar and ductal cells. However, the epithelia-derived cancer component forms only a relatively small fraction of the tumor mass. The majority of the tumor consists of acellular fibrous stroma and diverse populations of the non-neoplastic cancer-associated cells. Importantly, the tumor microenvironment is maintained by dynamic cell-cell and cell-matrix interactions. In this article, we aim to review the most common drivers of PDAC. Then we summarize the current knowledge on PDAC microenvironment, particularly in relation to pancreatic cancer therapy. The focus is placed on the acellular stroma as well as cell populations that inhabit the matrix. We also describe the altered metabolism of PDAC and characterize cellular signaling in this cancer