The anticancer effect of L-asparaginases (L-ASNases) is attributable to their ability to hydrolyze L-asparagine in the bloodstream and cancer cell microenvironment.
(RrA) has dual mechanism of action ...and plays a role in the suppression of telomerase activity. The aim of this work was to investigate the possible mechanism of RrA penetration into human cancer cells. Labeling of widely used L-ASNases by fluorescein isothiocyanate followed by flow cytometry and fluorescent microscopy demonstrated that only RrA can interact with cell membranes. The screening of inhibitors of receptor-mediated endocytosis demonstrated the involvement of clathrin receptors in RrA penetration into cells. Confocal microscopy confirmed the cytoplasmic and nuclear localization of RrA in human breast cancer SKBR3 cells. Two predicted nuclear localization motifs allow RrA to penetrate into the cell nucleus and inhibit telomerase. Chromatin relaxation promoted by different agents can increase the ability of RrA to suppress the expression of telomerase main catalytic subunit. Our study demonstrated for the first time the ability of RrA to penetrate into human cancer cells and the involvement of clathrin receptors in this process.
Complex heterogeneous systems, such as micelles or blood plasma, represent a particularly challenging environment to measure the catalytic parameters of some enzymes, including l-asparaginase. ...Existing methods are strongly interfered by the presence of plasma proteins, amino acids, as well as other components of plasma. Here we show that FTIR spectroscopy enables continuous real-time measurement of catalytic activity of l-asparaginase, in native and in PEG-chitosan conjugated form, in aqueous solutions as well as in heterogeneous non-transparent multicomponent systems, including colloidal systems or blood plasma, with minimal or no sample preparation. The approach developed is potentially applicable to other enzymatic reactions where the spectroscopic properties of substrate and product do not allow direct measurement with absorption or fluorescence spectroscopy.
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•New method is developed to determine catalytic parameters of l-asparaginase using FTIR spectroscopy.•The new method works well in complex heterogeneous systems, including blood plasma and reversed micelles.•Using the new method we performed the first ever direct measurement of dimeric L-asparaginase activity.
Human (h) telomerase (TL; EC 2.7.7.49) plays a key role in sustaining cancer cells by means of elongating telomeric repeats at the 3′ ends of chromosomes. Since TL-inhibitor (TI) stand-alone cancer ...therapy has been proven to be remarkably challenging, a polypharmacological approach represents a valid alternative. Here we consider a series of compounds able to inhibit both hTL and the tumor-associated carbonic anhydrases (CAs; EC 4.2.1.1) IX and XII. Compounds 7 and 9 suppressed hTL activity in both cell lysates and human colon cancer cell lines, and prolonged incubation with either 7 or 9 resulted in telomere shortening, cell cycle arrest, replicative senescence, and apoptosis. Enzyme kinetics showed that 7 and 9 are mixed-type inhibitors of the binding of DNA primers and deoxynucleoside triphosphate (dNTP) to the TL catalytic subunit hTERT, which is in agreement with docking experiments. Compound 9 showed antitumor activity in Colo-205 mouse xenografts and suppressed telomerase activity by telomere reduction.
Telomerase activity is regulated at the mRNA level by alternative splicing (AS) of its catalytic subunit hTERT. The aim of this study was to define the ability of splice-switching oligonucleotides ...(SSOs) that pair with hTERT pre-mRNA to induce AS and inhibit telomerase activity in human CD4+ T lymphocytes. SSOs that blocked the binding of a single splicing regulatory protein, SRp20 or SRp40, to its site within intron 8 of hTERT pre-mRNA demonstrated rather moderate capacities to induce AS and inhibit telomerase. However, a SSO that blocked the interaction of both SRp20 and SRp40 proteins with pre-mRNA was the most active. Cultivation of lymphocytes with spliced hTERT and inhibited telomerase resulted in the reduction of proliferative activity without significant induction of cell death. These results should facilitate further investigation of telomerase activity regulation, and antitelomerase SSOs could become promising agents for antiproliferative cell therapy.
•Splice-switching oligonucleotides inhibit telomerase activity.•Blocking of SRp20 and SRp40 proteins induces alternative splicing of hTERT.•Telomerase activity inhibition affects proliferation of human lymphocytes.
The nuclease activity of deoxyribonuclease 1 (DNase I) is regulated by alternative splicing (AS) of its mRNA. The aim of this study was to define the ability of a splice-switching oligonucleotide ...(SSO) that base-paired with DNase I pre-mRNA to induce AS and inhibit nuclease activity in human T, B and NK lymphocytes. The SSO for DNase I could significantly downregulate the expression of full-length active DNase I and upregulate a truncated splice variant with a deleted exon 4. Such an induction of AS resulted in inhibition of nuclease activity and slowed apoptosis progression in anti-CD95/FAS stimulated lymphocytes. These results should facilitate further investigations of apoptosis regulation in lymphocytes and demonstrate that SSOs for DNase I are promising cytoprotective agents.
•A splice-switching oligonucleotide inhibits the nuclease activity of DNase I.•Blocking SC35 and SRp40 proteins induces alternative splicing of DNase I.•Inhibition of nuclease activity slows apoptosis progression in human lymphocytes.
•Human Tregs induce target lymphocytes death upon contact-independent co-cultivation.•Tregs induces alternative splicing of telomerase mRNA in target lymphocytes.•Alternative splicing is associated ...with endonuclease G up-regulation.•Alternative splicing leads to telomerase inhibition and replicative senescence.•Target lymphocytes escape death after Tregs removal from co-cultivation.
Regulatory T cells (Tregs) play a fundamental role in the maintenance of immunological tolerance by suppressing effector target T, B and NK lymphocytes. Contact-dependent suppression mechanisms have been well–studied, though contact-independent Treg activity is not fully understood. In the present study, we showed that human native Tregs, as well as induced ex vivo Tregs, can cause in vitro telomere-dependent senescence in target T, B and NK cells in a contact-independent manner. The co-cultivation of target cells with Tregs separated through porous membranes induced alternative splicing of the telomerase catalytic subunit hTERT (human Telomerase Reverse Transcriptase), which suppressed telomerase activity. Induction of the hTERT splicing variant was associated with increased expression of the apoptotic endonuclease EndoG, a splicing regulator. Inhibited telomerase in target cells co-cultivated with Tregs for a long period of time led to a decrease in their telomere lengths, cell cycle arrest, conversion of the target cells to replicative senescence and apoptotic death. Induced Tregs showed the ability to up-regulate EndoG expression, TERT alternative splicing and telomerase inhibition in mouse T, B and NK cells after in vivo administration. The results of the present study describe a novel mechanism of contact-independent Treg cell suppression that induces telomerase inhibition through the EndoG-provoked alternative splicing of hTERT and converts cells to senescence and apoptosis phenotypes.
•Tregs induce death of effector lymphocytes through a contact-independent mechanism.•The death of effector cells is promoted by telomere-dependent senescence.•Tregs induces alternative splicing of ...telomerase catalytic subunit in target cells.•Alternative splicing results in telomerase inhibition and telomere loss.•Tregs-driven EndoG translocation to cell nuclei induces alternative splicing.
Regulatory T cells (Tregs) suppress the activity of effector T, B and NK lymphocytes and sustain immunological tolerance, but the proliferative activity of suppressed cells remains unexplored. In the present study, we report that mouse Tregs can induce replicative senescence and the death of responder mouse CD4+CD25− T cells, CD8+ T cells, B cells and NK cells in vitro and in vivo. Contact-independent in vitro co-cultivation with Tregs up-regulated endonuclease G (EndoG) expression and its translocation to the nucleus in responder cells. EndoG localization in the nucleus induced alternative mRNA splicing of the telomerase catalytic subunit Tert and telomerase inhibition. The lack of telomerase activity in proliferating cells led to telomere loss followed by the development of senescence and cell death. Injection of Tregs into mice resulted in EndoG-associated alternative splicing of Tert, telomerase inhibition, telomere loss, senescence development and increased cell death in vivo. The present study describes a novel contact-independent mechanism by which Tregs specify effector cell fate and provides new insights into cellular crosstalk related to immune suppression.
Apoptotic endonucleases act cooperatively to fragment DNA and ensure the irreversibility of apoptosis. However, very little is known regarding the potential regulatory links between endonucleases. ...Deoxyribonuclease 1 (DNase I) inactivation is caused by alternative splicing (AS) of DNase I pre-mRNA skipping exon 4, which occurs in response to EndoG overexpression in cells. The current study aimed to determine the role of EndoG in the regulation of DNase I mRNA AS and the modulation of its enzymatic activity. A strong correlation was identified between the EndoG expression levels and DNase I splice variants in human lymphocytes. EndoG overexpression in CD4+ T cells down-regulated the mRNA levels of the active full-length DNase I variant and up-regulated the levels of the non-active spliced variant, which acts in a dominant-negative fashion. DNase I AS was induced by the translocation of EndoG from mitochondria into nuclei during the development of apoptosis. The DNase I spliced variant was induced by recombinant EndoG or by incubation with EndoG-digested cellular RNA in an in vitro system with isolated cell nuclei. Using antisense DNA oligonucleotides, we identified a 72-base segment that spans the adjacent segments of exon 4 and intron 4 and appears to be responsible for the AS. DNase I-positive CD4+ T cells overexpressing EndoG demonstrated decreased progression towards bleomycin-induced apoptosis. Therefore, EndoG is an endonuclease with the unique ability to inactivate another endonuclease, DNase I, and to modulate the development of apoptosis.
•EndoG expression correlates with DNase I splice variants in human lymphocytes.•The DNase I splice variant has dominant-negative activity.•EndoG induces the alternative splicing of DNase I mRNA through its RNase activity.•EndoG-produced oligonucleotide modulate the alternative splicing of DNase I.•The alternative splicing of DNase I mRNA is associated with the inhibition of apoptosis.
•Apoptotic endonuclease EndoG induces hTERT alternative splicing.•EndoG expression correlates with the expression of hTERT splice variants.•EndoG expression down-regulates telomerase activity in CD4+ ...T lymphocytes.•Telomerase inhibition results in results in telomere loss.•EndoG overexpression induces death of CD4+ T lymphocytes.
Telomerase activity is regulated by alternative splicing of its catalytic subunit human Telomerase Reverse Transcriptase (hTERT) mRNA. Induction of a non-active spliced hTERT leads to inhibition of telomerase activity. However, very little is known about the mechanism of hTERT mRNA alternative splicing. The aim of this study was to determine the role of the apoptotic endonuclease EndoG in alternative splicing of hTERT and telomerase activity. A strong correlation was identified between EndoG expression levels and hTERT splice variants in human CD4+ and CD8+ T lymphocytes. Overexpression of EndoG in CD4+ T cells down-regulated the expression of the active full-length hTERT variant and up-regulated expression of the non-active spliced variant. A reduction in full-length hTERT transcripts down-regulated telomerase activity. Long-term in vitro cultivation of EndoG-overexpressing CD4+ T cells led to dramatically shortened telomeres, conversion of cells into a replicative senescence state, and activation of the BCL2/BAX-associated apoptotic pathway finally leading to cell death. These data indicated the participation of EndoG in alternative mRNA splicing of the telomerase catalytic subunit hTERT, regulation of telomerase activity and determination of cell fate.
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