Bacterial cellulose films for L-asparaginase delivery to melanoma cells.

L-asparaginase (L-ASNase) is an enzyme that catalyzes the hydrolysis of L-asparagine to L-aspartic acid and ammonia and is used to treat acute lymphoblastic leukemia. It is also toxic to the cells of some solid tumors, including melanoma cells. Immobilization of this enzyme can improve its activity against melanoma tumor cells. In this work, the properties of bacterial cellulose (BC) and feasibility of BC films as a new carrier for immobilized L-ASNase were investigated. Different values of growth time were used to obtain BC films with different thicknesses and porosities, which determine the water content and the ability to adsorb and release L-ASNase. Fourier transform infrared spectroscopy confirmed the adsorption of the enzyme on the BC films. The total activity of adsorbed L-ASNase and its release were investigated for films grown for 48, 72 or 96 h. BC films grown for 96 h showed the most pronounced release as described by zero-order and Korsmayer-Peppas models. The release was characterized by controlled diffusion where the drug was released at a constant rate. BC films with immobilized L-ASNase could induce cytotoxicity in A875 human melanoma cells. With further development, immobilization of L-ASNase on BC may become a potent strategy for anticancer drug delivery to superficial tumors.

Induction of FoxP3 Pre-mRNA Alternative Splicing to Enhance the Suppressive Activity of Regulatory T Cells from Amyotrophic Lateral Sclerosis Patients.

Forkhead box protein 3 (FoxP3) is a key transcription factor responsible for the development, maturation, and function of regulatory T cells (Tregs). The FoxP3 pre-mRNA is subject to alternative splicing, resulting in the translation of multiple splice variants. We have shown that Tregs from patients with amyotrophic lateral sclerosis (ALS) have reduced expression of full-length (FL) FoxP3, while other truncated splice variants are expressed predominantly. A correlation was observed between the reduced number of Tregs in the peripheral blood of ALS patients, reduced total FoxP3 mRNA, and reduced mRNA of its FL splice variant. Induction of FL FoxP3 was achieved using splice-switching oligonucleotides capable of base pairing with FoxP3 pre-mRNA and selectively modulating the inclusion of exons 2 and 7 in the mature mRNA. Selective expression of FL FoxP3 resulted in the induction of CD127low, CD152, and Helios-positive cells, while the cell markers CD4 and CD25 were not altered. Such Tregs had an increased proliferative activity and a higher frequency of cell divisions per day. The increased suppressive activity of Tregs with the induced FL FoxP3 splice variant was associated with the increased synthesis of the pro-apoptotic granzymes A and B, and perforin, IL-10, and IL-35, which are responsible for contact-independent suppression, and with the increased ability to suppress telomerase in target cells. The upregulation of Treg suppressive and proliferative activity using splice-switching oligonucleotides to induce the predominant expression of the FoxP3 FL variant is a promising approach for regenerative cell therapy in Treg-associated diseases.

Electrochemical approach for the analysis of DNA degradation in native DNA and apoptotic cells.

The aim of this work was to develop an electrochemical approach for the analysis of DNA degradation and fragmentation in apoptotic cells. DNA damage is considered one of the major causes of human diseases. We analyzed the cleavage processes of the circular plasmid pTagGFP2-N and calf thymus DNA, which were exposed to restriction endonucleases (the restriction endonucleases BstMC I and AluB I and the nonspecific endonuclease I). Genomic DNA from the leukemia K562 cell line was used as a marker of the early and late (mature) stages of apoptosis. Registration of direct electrochemical oxidation of nucleobases of DNA molecules subjected to restriction endonuclease or apoptosis processes was proposed for the detection of these biochemical events. Label-free differential pulse voltammetry (DPV) has been used to measure endonuclease activities and DNA damage using carbon nanotube-modified electrodes. The present DPV technique provides a promising platform for high-throughput screening of DNA hydrolases and for registering the efficiency of apoptotic processes. DPV comparative analysis of the circular plasmid pTagGFP2-N in its native supercoiled state and plasmids restricted to 4 and 23 parts revealed significant differences in their electrochemical behavior. Electrochemical analysis was fully confirmed by means of traditional methods of DNA analysis and registration of apoptotic process, such as gel electrophoresis and flow cytometry.

Engineering and Expression Strategies for Optimization of L-Asparaginase Development and Production.

Genetic engineering for heterologous expression has advanced in recent years. Model systems such as Escherichia coli, Bacillus subtilis and Pichia pastoris are often used as host microorganisms for the enzymatic production of L-asparaginase, an enzyme widely used in the clinic for the treatment of leukemia and in bakeries for the reduction of acrylamide. Newly developed recombinant L-asparaginase (L-ASNase) may have a low affinity for asparagine, reduced catalytic activity, low stability, and increased glutaminase activity or immunogenicity. Some successful commercial preparations of L-ASNase are now available. Therefore, obtaining novel L-ASNases with improved properties suitable for food or clinical applications remains a challenge. The combination of rational design and/or directed evolution and heterologous expression has been used to create enzymes with desired characteristics. Computer design, combined with other methods, could make it possible to generate mutant libraries of novel L-ASNases without costly and time-consuming efforts. In this review, we summarize the strategies and approaches for obtaining and developing L-ASNase with improved properties.

Modulation of Suppressive Activity and Proliferation of Human Regulatory T Cells by Splice-Switching Oligonucleotides Targeting FoxP3 Pre-mRNA.

The maturation, development, and function of regulatory T cells (Tregs) are under the control of the crucial transcription factor Forkhead Box Protein 3 (FoxP3). Through alternative splicing, the human FoxP3 gene produces four different splice variants: a full-length variant (FL) and truncated variants with deletions of each of exons 2 (∆2 variant) or 7 (∆7 variant) or a deletion of both exons (∆2∆7 variant). Their involvement in the biology of Tregs as well as their association with autoimmune diseases remains to be clarified. The aim of this work was to induce a single FoxP3 splice variant in human Tregs by splice switching oligonucleotides and to monitor their phenotype and proliferative and suppressive activity. We demonstrated that Tregs from peripheral blood from patients with multiple sclerosis preferentially expressed truncated splice variants, while the FL variant was the major variant in healthy donors. Tregs with induced expression of truncated FoxP3 splice variants demonstrated lower suppressive activity than those expressing FL variants. Reduced suppression was associated with the decreased expression of Treg-associated suppressive surface molecules and the production of cytokines. The deletion of exons 2 and/or 7 also reduced the cell proliferation rate. The results of this study show an association between FoxP3 splice variants and Treg function and proliferation. The modulation of Treg suppressive activity by the induction of the FoxP3 FL variant can become a promising strategy for regenerative immunotherapy.

New Genetic Bomb Trigger: Design, Synthesis, Molecular Dynamics Simulation, and Biological Evaluation of Novel BIBR1532-Related Analogs Targeting Telomerase against Non-Small Cell Lung Cancer.

Telomeres serve a critical function in cell replication and proliferation at every stage of the cell cycle. Telomerase is a ribonucleoprotein, responsible for maintaining the telomere length and chromosomal integrity of frequently dividing cells. Although it is silenced in most human somatic cells, telomere restoration occurs in cancer cells because of telomerase activation or alternative telomere lengthening. The telomerase enzyme is a universal anticancer target that is expressed in 85-95% of cancers. BIBR1532 is a selective non-nucleoside potent telomerase inhibitor that acts by direct noncompetitive inhibition. Relying on its structural features, three different series were designed, and 30 novel compounds were synthesized and biologically evaluated as telomerase inhibitors using a telomeric repeat amplification protocol (TRAP) assay. Target compounds 29a, 36b, and 39b reported the greatest inhibitory effect on telomerase enzyme with IC50 values of 1.7, 0.3, and 2.0 µM, respectively, while BIBR1532 displayed IC50 = 0.2 µM. Compounds 29a, 36b, and 39b were subsequently tested using a living-cell TRAP assay and were able to penetrate the cell membrane and inhibit telomerase inside living cancer cells. Compound 36b was tested for cytotoxicity against 60 cancer cell lines using the NCI (USA) procedure, and the % growth was minimally impacted, indicating telomerase enzyme selectivity. To investigate the interaction of compound 36b with the telomerase allosteric binding site, molecular docking and molecular dynamics simulations were used.

Cytoprotective Activity of Polyamines Is Associated with the Alternative Splicing of RAD51A Pre-mRNA in Normal Human CD4+ T Lymphocytes.

Physiological polyamines are ubiquitous polycations with pleiotropic biochemical activities, including regulation of gene expression and cell proliferation as well as modulation of cell signaling. They can also decrease DNA damage and promote cell survival. In the present study, we demonstrated that polyamines have cytoprotective effects on normal human CD4+ T lymphocytes but not on cancer Jurkat or K562 cells. Pretreatment of lymphocytes with polyamines resulted in a significant reduction in cells with DNA damage induced by doxorubicin, cisplatin, or irinotecan, leading to an increase in cell survival and viability. The induction of RAD51A expression was in response to DNA damage in both cancer and normal cells. However, in normal cells, putrescin pretreatment resulted in alternative splicing of RAD51A and the switch of the predominant expression from the splice variant with the deletion of exon 4 to the full-length variant. Induction of RAD51A alternative splicing by splice-switching oligonucleotides resulted in a decrease in DNA damage and cell protection against cisplatin-induced apoptosis. The results of this study suggest that the cytoprotective activity of polyamines is associated with the alternative splicing of RAD51A pre-mRNA in normal human CD4+ T lymphocytes. The difference in the sensitivity of normal and cancer cells to polyamines may become the basis for the use of these compounds to protect normal lymphocytes during lymphoblastic chemotherapy.

Mechanisms of the Antiproliferative and Antitumor Activity of Novel Telomerase-Carbonic Anhydrase Dual-Hybrid Inhibitors.

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.

Inhibition of nuclease activity by a splice-switching oligonucleotide targeting deoxyribonuclease 1 mRNA prevents apoptosis progression and prolong viability of normal human CD4+ T-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.

Inhibition of telomerase activity by splice-switching oligonucleotides targeting the mRNA of the telomerase catalytic subunit affects proliferation of human CD4+ T lymphocytes.

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.

Endonuclease G modulates the alternative splicing of deoxyribonuclease 1 mRNA in human CD4+ T lymphocytes and prevents the progression of apoptosis.

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.

Contact-independent suppressive activity of regulatory T cells is associated with telomerase inhibition, telomere shortening and target lymphocyte apoptosis.

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.

Murine regulatory T cells induce death of effector T, B, and NK lymphocytes through a contact-independent mechanism involving telomerase suppression and telomere-associated senescence.

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.

Inhibition of telomerase activity and induction of apoptosis by Rhodospirillum rubrum L-asparaginase in cancer Jurkat cell line and normal human CD4+ T lymphocytes.

Rhodospirillum rubrum L-asparaginase mutant E149R, V150P, F151T (RrA) down-regulates telomerase activity due to its ability to inhibit the expression of telomerase catalytic subunit hTERT. The aim of this study was to define the effect of short-term and long-term RrA exposure on proliferation of cancer Jurkat cell line and normal human CD4+ T lymphocytes. RrA could inhibit telomerase activity in dose- and time-dependent manner in both Jurkat and normal CD4+ T cells. Continuous RrA exposure of these cells resulted in shortening of telomeres followed by cell cycle inhibition, replicative senescence, and development of apoptosis. Complete death of Jurkat cells was observed at the day 25 of RrA exposure while normal CD4+ T cells died at the day 50 due to the initial longer length of telomeres. Removal of RrA from senescent cells led to a reactivation of hTERT expression, restoration telomerase activity, re-elongation of telomeres after 48 h of cultivation, and survival of cells. These findings demonstrate that proliferation of cancer and normal telomerase-positive cells can be limited by continuous telomerase inhibition with RrA. Longer telomeres of normal CD4+ T lymphocytes make such cells more sustainable to RrA exposure that could give them an advantage during anti-telomerase therapy. These results should facilitate further investigations of RrA as a potent anti-telomerase therapeutic protein.

Rhodospirillum rubruml-asparaginase targets tumor growth by a dual mechanism involving telomerase inhibition.

Rhodospirillum rubruml-asparaginase mutant RrA E149R, V150P, F151T (RrA) was previously identified to down-regulate telomerase activity along with catalyzing the hydrolysis of l-asparagine. The aim of this study was to define the effect of prolonged RrA exposure on telomerase activity, maintenance of telomeres and proliferation of cancer cells in vitro and in vivo. RrA could inhibit telomerase activity in SCOV-3, SkBr-3 and A549 human cancer cell lines due to its ability to down-regulate the expression of telomerase catalytic subunit hTERT. Telomerase activity in treated cells did not exceeded 29.63 ± 12.3% of control cells. Continuous RrA exposure of these cells resulted in shortening of telomeres followed by cell death in vitro. Using real time PCR we showed that length of telomeres in SCOV-3 cells has been gradually decreasing from 10105 ± 2530 b.p. to 1233 ± 636 b.p. after 35 days of cultivation. RrA treatment of xenograft models in vivo showed slight inhibition of tumor growth accompanied with 49.5-53.3% of decrease in hTERT expression in the all tumors. However down-regulation of hTERT expression, inhibition of telomerase activity and the loss of telomeres was significant in response to RrA administration in xenograft models. These results should facilitate further investigations of RrA as a potent therapeutic protein.