Ингибирование активности каспазы-2 в клетках Т-клеточной лимфомы человека Jurkat при помощи переключающего сплайсинг олигонуклеотида к её пре-мРНК

Caspase-2 is a key enzyme thinvolved in induction of apoptosis. The caspase-2 level is regulated by alternative splicing (AS) of its mRNA. The aim of this work was to determine the ability of an oligonucleotide complementary to Casp-2 pre-mRNA to induce AS. This oligonucleotide blocked the binding of splicing-regulating proteins to their sites at the end of exon 9 of Casp-2 pre-mRNA, leading to induction of AS of Casp-2 mRNA. The decrease in expression of full-size active splice-variant (Casp-2L) and the increase the expression of a shortened variant (Casp-2S) was demonstrated in human T-cell lymphoma Jurkat cell line. The expression level of total Casp-2 remained unchanged. Disproportion of splice variants of Casp-2 led to inhibition of enzymatic activity of caspase-2.Каспаза-2 является ферментом, участвующим в индукции апоптоза. Количество активного фермента каспазы-2 регулируется альтернативным сплайсингом (АС) её мРНК. Целью данной работы было определение способности олигонуклеотида, комплементарного пре-мРНК Casp-2, индуцировать АС. Данный олигонуклеотид блокировал связывание регулирующих сплайсинг белков со своими сайтами на конце экзона 9 пре-мРНК Casp-2, что приводило к индукции АС мРНК Casp-2: понижению экспрессии полноразмерного активного сплайс-варианта Casp-2L и повышению экспрессии укороченного варанта Casp-2S в клетках Т-клеточной лимфомы человека линии Jurkat. При этом уровень экспрессии общей Casp-2 не изменялся. Нарушуение пропорции сплайс-вариантов Casp-2 приводил к ингибированию ферментативной активности каспазы-2

Increased suppressor activity of transformed ex vivo regulatory T-cells in comparison with unstimulated cells of the same donor [Povyshennaia supressornaia aktivnost' transformirovannykh ex vivo reguliatornykh T-kletok v sravnenii s nestimulirovannymi kletkami togo zhe donora]

Regulatory T-cells CD4⁺CD25⁺FoxP3⁺CD127low (Tregs) play a key role in the maintenance of tolerance to auto antigens, inhibit function of effector T and B lymphocytes, and provide a balance between effector and regulatory arms of immunity. Patients with autoimmune diseases have decreased Treg numbers and impaired suppressive activity. Transformed ex vivo autologous Tregs could restore destroyed balance of the immune system. We developed a method for Treg precursor cell cultivation. Following the method, we were able to grown up 300-400 million of Tregs cells from 50 ml of peripheral blood during a week. Transformed ex vivo Tregs are 90-95% CD4⁺CD25⁺FoxP3⁺CD127low and have increased expression of transcription genes FoxP3 and Helios. Transformed ex vivo Tregs have increased demethylation of FoxP3 promoter and activated genes of proliferation markers Cycline B1, Ki67 and LGALS 1. Transformed ex vivo Tregs have increased suppressive activity and up to 80-90% these cells secrete cytokines TNFα и IFNγ. Our data suggest transformed ex vivo autologous Tregs have genetic, immunophenotypic and functional characteristics for regulatory T-cells and further can be used for adoptive immunotherapy autoimmune diseases and inhibition of transplantation immunity.Reguliatornye T-kletki CD4⁺CD25⁺FoxP3⁺SD127low (Treg) igraiut kliuchevuiu rol' v podderzhanii tolerantnosti k autoantigenam, podavliaiut funktsiiu éffektornykh T- i V-limfotsitov i obespechivaiut balans mezhdu éffektornym i reguliatornym zvenom immuniteta. U bol'nykh s autoimmunnymi zabolevaniiami snizheno soderzhanie Treg i narushena funktsiia étikh kletok. Zamestitel'naia terapiia autologichnymi kletkami patsienta, vyrashchennymi ex vivo, mozhet vosstanovit' narushennyĭ balans immunnoĭ sistemy. Nami razrabotana metodika kul'tivirovaniia kletok-predshestvennikov Treg vne organizma cheloveka, kotoraia pozvoliaet iz 50 ml perifericheskoĭ krovi vyrastit' 300-400 mln Treg v techenie odnoĭ nedeli. V nastoiashchem issledovanii pokazano, chto po sravneniiu s Treg perifericheskoĭ krovi, vyrashchennye ex vivo kletki na 90-95% imeiut fenotip CD4⁺CD25⁺FoxP3⁺SD127low Treg i povyshennuiu ékspressiiu kliuchevykh genov transkriptsii FoxP3 i Helios. V transformirovannykh Treg povyshena stepen' demetilirovaniia v promotornom uchastke gena FoxP3 i aktivirovany geny-markery proliferatsii tsiklina V1, Ki67 i LGALS 1. Transformirovannye ex vivo Treg obladaiut povyshennoĭ supressornoĭ aktivnost'iu, i do 80-90% kletok v populiatsii sekretiruiut tsitokiny TNFα i IFNγ. Nashi dannye pokazyvaiut, chto vyrashchennye ex vivo autologichnye Treg kletki imeiut geneticheskie, markernye i funktsional'nye kharakteristiki reguliatornykh T-kletok, kotorye v budushchem mogut byt' ispol'zovany dlia adaptivnoĭ immunoterapii bol'nykh s autoimmunnymi zabolevaniiami, a takzhe dlia podavleniia transplantatsionnogo immuniteta, v chastnosti, reaktsii transplantata protiv khoziaina

The influence of compound ITEL1296 on telomerase activity and the growth of cancer cells

Telomerase is a ribonueleoprotein that synthesizes telomeric repeats and identified as a promising target for anticancer therapy. Here we describe a new compound aITEL 1296 as a potent telomcrase inhibitor. Its inhibitory activity was a bit higher (IC50 = 0,19±0,02 ng/ml) than that of BIBR1532, one of the most potent telomerase inhibitors known to date. Besides telomerase inhibition aITEL 1296 activated apoptotic mechanisms and effectively suppressed proliferation of tumor cell lines (GI50 = 5,0±0,2 ng/ml for most sensitive cell line LnCap) but not normal fibroblast cell line

The influence of compound aITEL1296 on telomerase activity and growth of cancer cells

Telomerase is a ribonucleoprotein complex, which synthesizes telomeric repeats and which has been identified as a promising target for anticancer therapy. Here we have investigated the effect of a new compound aITEL1296 on telomerase activity. aITEL1296 effectively inhibited telomerase activity; its inhibitory activity was a bit higher (IC 50 = 0.19 ± 0.02 ng/mL) than that of BIBR1532, one of the most potent telomerase inhibitors known to date. In addition to telomerase inhibition aITEL1296 activated apoptotic mechanisms and effectively suppressed proliferation of tumor cell lines (GI 50 = 5.0 ± 0.2 ng/mL for most sensitive cell line LnCap) but not normal fibroblast cell line. © Pleiades Publishing, Ltd., 2012

The influence of compound ITEL1296 on telomerase activity and the growth of cancer cells

Telomerase is a ribonueleoprotein that synthesizes telomeric repeats and identified as a promising target for anticancer therapy. Here we describe a new compound aITEL 1296 as a potent telomcrase inhibitor. Its inhibitory activity was a bit higher (IC50 = 0,19±0,02 ng/ml) than that of BIBR1532, one of the most potent telomerase inhibitors known to date. Besides telomerase inhibition aITEL 1296 activated apoptotic mechanisms and effectively suppressed proliferation of tumor cell lines (GI50 = 5,0±0,2 ng/ml for most sensitive cell line LnCap) but not normal fibroblast cell line

The influence of compound aITEL1296 on telomerase activity and growth of cancer cells

Telomerase is a ribonucleoprotein complex, which synthesizes telomeric repeats and which has been identified as a promising target for anticancer therapy. Here we have investigated the effect of a new compound aITEL1296 on telomerase activity. aITEL1296 effectively inhibited telomerase activity; its inhibitory activity was a bit higher (IC 50 = 0.19 ± 0.02 ng/mL) than that of BIBR1532, one of the most potent telomerase inhibitors known to date. In addition to telomerase inhibition aITEL1296 activated apoptotic mechanisms and effectively suppressed proliferation of tumor cell lines (GI 50 = 5.0 ± 0.2 ng/mL for most sensitive cell line LnCap) but not normal fibroblast cell line. © Pleiades Publishing, Ltd., 2012

D-amino acids in nature, agriculture and biomedicine

Information accumulated over the past decades on the physiological functions and metabolic pathways of biosynthesis and degradation of D-amino acids has led to a renewed interest in their study. These isomers are known to form both in nature and during the chemical synthesis of L-amino acids for feeding and pharmacological purposes, as well as in the industrial processing of some raw materials. This article discusses the positive and negative effects of D-amino acids on the human body, animals and the environment. In addition, the scientific data concerning the mechanisms of cytotoxic action of D-amino acids and their industrial and biomedical potential are summarized. © 2019, © 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group

Phenotypical and functional characteristics of human regulatory t cells during ex vivo maturation from cd4+ t lymphocytes

Regulatory T cells (Tregs) participate in the negative regulation of inflammatory reactions by suppressing effector cells. In a number of autoimmune disorders, the suppressive function and/or the number of Tregs is compromised. The lack of active functioning Tregs can be restored with adoptive transfer of expanded ex vivo autologous Tregs. In our study, we traced the differentiation and maturation of Tregs CD4+CD25+FoxP3+CD127low over 7 days of cultivation from initial CD4+ T cells under ex vivo conditions. The resulting ex vivo expanded cell population (eTregs) demonstrated the immune profile of Tregs with an increased capacity to suppress the proliferation of target effector cells. The expression of the FoxP3 gene was upregulated within the time of expansion and was associated with gradual demethylation in the promotor region of the T cell-specific demethylation region. Real-time RT-PCR analysis revealed changes in the expression profile of genes involved in cell cycle regulation. In addition to FOXP3, the cells displayed elevated mRNA levels of Ikaros zinc finger transcription factors and the main telomerase catalytic subunit hTERT. Alternative splicing of FoxP3, hTERT and IKZF family members was demonstrated to be involved in eTreg maturation. Our data indicate that expanded ex vivo eTregs develop a Treg-specific phenotype and functional suppressive activity. We suggest that eTregs are not just expanded but transformed cells with enhanced capacities of immune suppression. Our findings may influence further development of cell immunosuppressive therapy based on regulatory T cells. © 2021 by the authors. Licensee MDPI, Basel, Switzerland

L-lysine α-oxidase: Enzyme with anticancer properties

L-lysine α-oxidase (LO), one of L-amino acid oxidases, deaminates L-lysine with the yield of H2 O2, ammonia, and α-keto-ε-aminocaproate. Multiple in vitro and in vivo studies have reported cytotoxic, antitumor, antimetastatic, and antitumor activity of LO. Unlike asparaginase, LO has a dual mechanism of action: depletion of L-lysine and formation of H2 O2, both targeting tumor growth. Prominent results were obtained on murine and human tumor models, including human colon cancer xenografts HCT 116, LS174T, and T47D with maximum T/C 12, 37, and 36%, respectively. The data obtained from human cancer xenografts in immunodeficient mice confirm the potential of LO as an agent for colon cancer treatment. In this review, we discuss recently discovered molecular mechanisms of biological action and the potential of LO as anticancer enzyme. © 2021 by the authors. Licensee MDPI, Basel, Switzerland