Paulina Podszywalow-Bartnicka

The PERK-eIF2α phosphorylation arm is a pro-survival pathway of BCR-ABL signaling and confers resistance to imatinib treatment in chronic myeloid leukemia cells

Activation of adaptive mechanisms plays a crucial role in cancer progression and drug resistance by allowing cell survival under stressful conditions. Therefore, inhibition of the adaptive response is considered as a prospective therapeutic strategy. The PERK-eIF2α phosphorylation pathway is an important arm of the unfolded protein response (UPR), which is induced under conditions of endoplasmic reticulum (ER) stress. Our previous work showed that ER stress is induced in chronic myeloid leukemia (CML) cells. Herein, we demonstrate that the PERK-eIF2α phosphorylation pathway is upregulated in CML cell lines and CD34+ cells from CML patients and is associated with CML progression and imatinib resistance. We also show that induction of apoptosis by imatinib results in the downregulation of the PERK-eIF2α phosphorylation arm. Furthermore, we demonstrate that inactivation of the PERK-eIF2α phosphorylation arm decreases the clonogenic and proliferative capacities of CML cells and sensitizes them to death by imatinib. These findings provide evidence for a pro-survival role of PERK-eIF2α phosphorylation arm that contributes to CML progression and development of imatinib resistance. Thus, the PERK-eIF2α phosphorylation arm may represent a suitable target for therapeutic intervention for CML disease.

Downregulation of BRCA1 protein in BCR-ABL1 leukemia cells depends on stress-triggered TIAR-mediated suppression of translation

BRCA1 tumor suppressor regulates crucial cellular processes involved in DNA damage repair and cell cycle control. We showed that expression of BCR-ABL1 correlates with decreased level of BRCA1 protein, which promoted aberrant mitoses and aneuploidy as well as altered DNA damage response. Using polysome profiling and luciferase-BRCA1 3’UTR reporter system here we demonstrate that downregulation of BRCA1 protein in CML is caused by inhibition of BRCA1 mRNA translation, but not by increased protein degradation or reduction of mRNA level and half-life. We investigated 2 mRNA-binding proteins – HuR and TIAR showing specificity to AU-Rich Element (ARE) sites in 3’UTR of mRNA. BCR-ABL1 promoted cytosolic localization of TIAR and HuR, their binding to BRCA1 mRNA and formation of the TIAR-HuR complex. HuR protein positively regulated BRCA1 mRNA stability and translation, conversely TIAR negatively regulated BRCA1 translation and was found localized predominantly in the cytosolic stress granules in CML cells. TIAR-dependent downregulation of BRCA1 protein level was a result of ER stress, which is activated in BCR-ABL1 expressing cells, as we previously shown. Silencing of TIAR in CML cells strongly elevated BRCA1 level. Altogether, we determined that TIAR-mediated repression of BRCA1 mRNA translation is responsible for downregulation of BRCA1 protein level in BCR-ABL1 –positive leukemia cells. This mechanism may contribute to genomic instability and provide justification for targeting PARP1 and/or RAD52 to induce synthetic lethality in “BRCAness” CML and BCR-ABL1 –positive ALL cells.

Role of Annexin A6 Isoforms in Catecholamine Secretion by PC12 Cells: Distinct Influence on Calcium Response

Noradrenaline and adrenaline are secreted by adrenal medulla chromaffin cells via exocytosis. Exocytosis of catecholamines occurs after cell stimulation with various endogenous activators such as nicotine or after depolarization of the plasma membrane and is regulated by calcium ions. Cytosolic [Ca2+] increases in response to cell excitation and triggers a signal‐initiated secretion. Annexins are known to participate in the regulation of membrane dynamics and are also considered to be involved in vesicular trafficking. Some experimental evidence suggests that annexins may participate in Ca2+‐regulated catecholamine secretion. In this report the effect of annexin A6 (AnxA6) isoforms 1 and 2 on catecholamine secretion has been described. Overexpression of AnxA6 isoforms and AnxA6 knock‐down in PC12 cells were accompanied by almost complete inhibition or a 20% enhancement of dopamine secretion, respectively. AnxA6‐1 and AnxA6‐2 overexpression reduced Δ[Ca2+]c upon depolarization by 32% and 58%, respectively, while AnxA6 knock‐down increased Δ[Ca2+]c by 44%. The mechanism of AnxA6 action on Ca2+ signalling is not well understood. Experimental evidence suggests that two AnxA6 isoforms interact with different targets engaged in regulation of calcium homeostasis in PC12 cells. J. Cell. Biochem. 111: 168–178, 2010.

Hyperglycaemia modifies energy metabolism and reactive oxygen species formation in endothelial cells in vitro

There is significant evidence for an involvement of reactive oxygen species (ROS) in the pathogenesis of diabetic vascular complications through many metabolic and structural derangements. However, despite the advanced knowledge on the crucial role of ROS in cardiovascular damage, their intracellular source in endothelial cells exposed to high concentrations of glucose has not been precisely defined. Moreover, the molecular mechanism of action of elevated glucose on mitochondria has not been fully elucidated. The main aim of this study was to describe changes in the mitochondrial metabolism of human umbilical vein endothelial cells (HUVECs) treated with high glucose concentrations and to indicate the actual source of ROS in these cells. HUVECs exposed to 30 mM glucose exhibited an increased content of vascular adhesive molecule-1 (VCAM-1) and an excessive ROS production. Faster oxygen consumption and increased abundance of selected respiratory complexes coexist with slightly declined mitochondrial membrane potential and substantially elevated amount of uncoupling protein-2 (UCP2). Inhibition of NADPH oxidase (NOX) and modification of mitochondrial ROS generation with a mitochondrial uncoupler or respiratory chain inhibitors allowed concluding that the major source of ROS in HUVECs exposed to hyperglycaemic conditions is NOX. The mitochondrial respiratory chain seems not to participate in this phenomenon.

Interaction of plasma membrane Ca 2+ -ATPase isoform 4 with calcineurin A: Implications for catecholamine secretion by PC12 cells

PMCA1-4 isoforms have been recently recognised as regulators of various signalling pathways in mammalian cells. PMCAs were found to interact with calcineurin A in an isoform specific manner. In this study we focus on the interaction of calcineurin A with PMCA4 and its effect on catecholamine secretion in PC12 cells with reduced PMCA2 or PMCA3 content. Reduction of synthesis of PMCA2 or PMCA3 led to upregulation of PMCA4 manifested by preferential interaction of PMCA4 with calcineurin A. On the other hand, we observed a significant reduction of dopamine secretion, which did not correspond with an increased [Ca(2+)](c). This result indicates that the interaction of PMCA4 with calcineurin A plays a regulatory role in the signalling during catecholamine secretion.

Expression of Oncogenic Kinase Bcr-Abl Impairs Mitotic Checkpoint and Promotes Aberrant Divisions and Resistance to Microtubule-Targeting Agents

Recent findings showed that BRCA1, in addition to its role in DNA damage response, acts as an upstream regulator of genes involved in the mitotic checkpoint regulation, thus protecting against promotion of aberrant divisions and aneuploidy. Moreover, there is also an indication that the BRCA1 protein is downregulated in chronic myeloid leukemia (CML) patients. We have investigated a possible functional relationship between BRCA1 and mitotic checkpoint competence in cells with the same genetic background expressing different levels of Bcr-Abl, an oncogene responsible for CML. Herein, we show that Bcr-Abl strongly downregulates the BRCA1 protein level, which is partially reversed on treatment with imatinib, an inhibitor of Bcr-Abl tyrosine kinase. Bcr-Abl leads to decreased expression of genes involved in the mitotic checkpoint activation—Mad2, Bub1, Bub3, and BubR1, resulting in mitosis perturbances, weakened mitotic checkpoint function, and mitotic slippage after nocodazole treatment. Furthermore, high Bcr-Abl–expressing cells showed also postmitotic checkpoint dysfunctions and inability to effectively arrest in the 4NG1 phase of the cell cycle, which was associated with limited p21 induction. These observations had significant biological consequences, as we found a high level of improper divisions, chromosomal missegregation, and generation of polyploid cells on mitotic checkpoint prolonged activation. Additionally, Bcr-Abl–expressing cells showed resistance to death activated by spindle defects, reversed by imatinib. Our study presents new facts and supports the hypothesis concerning the mutator nature of Bcr-Abl itself. The functional interaction between Bcr-Abl and mitosis dysfunctions, due to compromised mitotic checkpoints, may have important implications for the generation of aneuploidy and CML progression. Mol Cancer Ther; 9(5); 1328–38. ©2010 AACR.

Calcium‐ and pH‐dependent localization of annexin A6 isoforms in Balb/3T3 fibroblasts reflecting their potential participation in vesicular transport

Annexin A6 (AnxA6), calcium‐ and membrane‐binding protein, is involved in membrane dynamics. It exists in the cell in two isoforms, AnxA6‐1 and AnxA6‐2, varying only by the VAAEIL sequence. In most cells, AnxA6‐1 predominates. A limited number of observations suggests that both isoforms differ from each other functionally. The EGF‐dependent Ca2+ influx in A431 cells is inhibited only by AnxA6‐1. Moreover, AnxA6‐2 was found to exhibit higher affinity for Ca2+. In this report we addressed the potential significance of the VAAEIL deletion in AnxA6‐2. For this purpose, we expressed AnxA6 isoform cDNAs in bacteria or mouse Balb/3T3 fibroblasts. The recombinant AnxA6‐2 was characterized by a less extended molecular shape than that of AnxA6‐1 and required a narrower [Ca2+] range to bind liposomes. Upon lowering pH in the presence of EGTA recombinant AnxA6‐2 became less hydrophobic than AnxA6‐1 as revealed by the Triton X‐114 partition. Furthermore, AnxA6‐2 revealed stronger F‐actin binding than that of AnxA6‐1. Immunofluorescence microscopy showed that the EGFP‐tagged AnxA6 isoforms expressed in Balb/3T3 fibroblasts relocate in a Ca2+‐ and H+‐sensitive manner to the vesicular structures in a perinuclear region or in cytosol. Cell fractionation showed that in resting conditions AnxA6‐1 is associated with early endosomes and AnxA6‐2 with late endosomes, and an increase in [Ca2+] and/or [H+] induced their opposite distribution. These findings suggest a potentially independent regulation, localization, and function of AnxA6 isoforms in Balb/3T3 fibroblasts. More generally, our findings suggest distinct functions of AnxA6 isoforms in membrane dynamics. J. Cell. Biochem. 104: 418–434, 2008.

Gene expression and mutation-guided synthetic lethality eradicates proliferating and quiescent leukemia cells

Quiescent and proliferating leukemia cells accumulate highly lethal DNA double-strand breaks that are repaired by 2 major mechanisms: BRCA-dependent homologous recombination and DNA-dependent protein kinase–mediated (DNA-PK–mediated) nonhomologous end-joining, whereas DNA repair pathways mediated by poly(ADP)ribose polymerase 1 (PARP1) serve as backups. Here we have designed a personalized medicine approach called gene expression and mutation analysis (GEMA) to identify BRCA- and DNA-PK–deficient leukemias either directly, using reverse transcription-quantitative PCR, microarrays, and flow cytometry, or indirectly, by the presence of oncogenes such as BCR-ABL1. DNA-PK–deficient quiescent leukemia cells and BRCA/DNA-PK–deficient proliferating leukemia cells were sensitive to PARP1 inhibitors that were administered alone or in combination with current antileukemic drugs. In conclusion, GEMA-guided targeting of PARP1 resulted in dual cellular synthetic lethality in quiescent and proliferating immature leukemia cells, and is thus a potential approach to eradicate leukemia stem and progenitor cells that are responsible for initiation and manifestation of the disease. Further, an analysis of The Cancer Genome Atlas database indicated that this personalized medicine approach could also be applied to treat numerous solid tumors from individual patients.

NFAT1 and NFAT3 Cooperate with HDAC4 during Regulation of Alternative Splicing of PMCA Isoforms in PC12 Cells

Background The bulk of human genes undergo alternative splicing (AS) upon response to physiological stimuli. AS is a great source of protein diversity and biological processes and is associated with the development of many diseases. Pheochromocytoma is a neuroendocrine tumor, characterized by an excessive Ca2+-dependent secretion of catecholamines. This underlines the importance of balanced control of calcium transport via regulation of gene expression pattern, including different calcium transport systems, such as plasma membrane Ca2+-ATPases (PMCAs), abundantly expressed in pheochromocytoma chromaffin cells (PC12 cells). PMCAs are encoded by four genes (Atp2b1, Atp2b2, Atp2b3, Atp2b4), whose transcript products undergo alternative splicing giving almost 30 variants. Results In this scientific report, we propose a novel mechanism of regulation of PMCA alternative splicing in PC12 cells through cooperation of the nuclear factor of activated T-cells (NFAT) and histone deacetylases (HDACs). Luciferase assays showed increased activity of NFAT in PC12 cells, which was associated with altered expression of PMCA. RT-PCR experiments suggested that inhibition of the transcriptional activity of NFAT might result in the rearrangement of PMCA splicing variants in PC12 cells. NFAT inhibition led to dominant expression of 2x/c, 3x/a and 4x/a PMCA variants, while in untreated cells the 2w,z/b, 3z,x/b,c,e,f, and 4x/b variants were found as well. Furthermore, chromatin immunoprecipitation experiments showed that NFAT1-HDAC4 or NFAT3-HDAC4 complexes might be involved in regulation of PMCA2x splicing variant generation. Conclusions We suggest that the influence of NFAT/HDAC on PMCA isoform composition might be important for altered dopamine secretion by PC12 cells.

Inhibitors of SRC kinases impair antitumor activity of anti-CD20 monoclonal antibodies

Clinical trials with SRC family kinases (SFKs) inhibitors used alone or in a combination with anti-CD20 monoclonal antibodies (mAbs) are currently underway in the treatment of B-cell tumors. However, molecular interactions between these therapeutics have not been studied so far. A transcriptional profiling of tumor cells incubated with SFKs inhibitors revealed strong downregulation of MS4A1 gene encoding CD20 antigen. In a panel of primary and established B-cell tumors we observed that SFKs inhibitors strongly affect CD20 expression at the transcriptional level, leading to inhibition of anti-CD20 mAbs binding and increased resistance of tumor cells to complement-dependent cytotoxicity. Activation of the AKT signaling pathway significantly protected cells from dasatinib-triggered CD20 downregulation. Additionally, SFKs inhibitors suppressed antibody-dependent cell-mediated cytotoxicity by direct inhibition of natural killer cells. Abrogation of antitumor activity of rituximab was also observed in vivo in a mouse model. Noteworthy, the effects of SFKs inhibitors on NK cell function are largely reversible. The results of our studies indicate that development of optimal combinations of novel treatment modalities with anti-CD20 mAbs should be preceded by detailed preclinical evaluation of their effects on target cells.