A New Protocol for Efficient and Non-Toxic Transfection of siRNA Into Leukemic Cell Lines and Primary Cells – a Direct Comparison Between Nucleofection and Accell Delivery Using Stem Cell Related Antigens As Validation Tools

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4802-4802
Author(s):  
Hanne Østergaard Larsen ◽  
Anne Stidsholt Roug ◽  
Claus Svane Sondergaard ◽  
Peter Hokland
Keyword(s):  
Cell Lines ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Transfection Efficiency ◽  
Leukemic Cell ◽  
Housekeeping Gene ◽  
Target Cells ◽  
Primary Cells ◽  
Cyclophilin B

Abstract Abstract 4802 BACKGROUND: Transient downregulation of genes in vitro by use of siRNA is a time-honored approach to study gene function. This can readily be applied in haematological malignancies, where many lesions derive from altered RNA activity arising e.g. from balanced translocations. PROBLEM FORMULATION: A crucial requirement to obtain this downregulation is an efficient and non-toxic delivery of the siRNA into the target cells, which has proven difficult to accomplish with some protocols; in particular, cells in suspension, such as leukemic blasts. HYPOTHESIS: Directly comparing the range of new transfection methodologies to unravel protocols suitable for cells in suspension. RESULTS: From pilot data, where a range of other methods were excluded due to either low transfection efficiencies or low viability of transfected cells, we compared the gold standard Nucleofection methodology to the novel non-viral based delivery method Accell in both suspension cell lines as well as primary patient AML and CML cells. As control the housekeeping gene Cyclophilin B was used, while hMICL and CD96, recently suggested to be associated with the putative leukemic stem cells, were chosen as target genes. Comparison of the two transfection methods yielded superior results using Accell methodology, as this technique not only yielded higher transfection rates but also retained superior cell viabilities for both cell lines and primary cells. Three AML and two CML samples were included in this study for the validation of transfection efficiency in primary cell employing the programs T-20 and U-15 for Nucleofection. Here, there was a large span in viabilities pre-experimentation among the five patient samples included in this study, probably reflecting the conditions of the patients. However, following transfection there was a significantly lower viability after Nucleofection (mean 37%, range 23%-65%) compared to Accell (mean 82%, range 57%-95%), in the two groups (p<0.001). Corroborating the cell line data transfection efficiencies were clearly superior after Accell transfection of primary patient cells (mean 85%, range 71%-97%) compared to Nucleofection transfection (mean 38%, range 23%-65%), p<0.01. Transfection efficiencies in primary cells after Accell delivery was 90–97% compared to 25–30% using Nucleofection for siRNA delivery. As a functional correlate to these data we performed CFU-GM assays on selected hMICL+ AML patients exposed to anti-hMICL siRNAs. While the AML blast growth of some patients were unaffected by the siRNA treatment others showed a clear inhibition of blast colony formation, suggesting a differential role of hMICL between AML patients, which needs to be further elucidated. CONCLUSION: Accell delivery is thus the method of choice for non-viral transfection of cells in suspension including primary AML and CML cells. These data should provide a platform for further studies of genes involved in early leukemogenesis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Preclinical Antileukemia Activity of Tramesan: A Newly Identified Bioactive Fungal Metabolite

2017 ◽  
Vol 2017 ◽  
pp. 1-8 ◽  
Author(s):  
M. R. Ricciardi ◽  
R. Licchetta ◽  
S. Mirabilii ◽  
M. Scarpari ◽  
A. Parroni ◽  
...  
Keyword(s):  
Cell Lines ◽  
Molecular Mechanisms ◽  
Mononuclear Cells ◽  
Leukemic Cell ◽  
Bioactive Compound ◽  
Primary Cells ◽  
Peripheral Blood Mononuclear ◽  
Normal Peripheral Blood ◽  
Leukemic Cell Lines

Despite improvements that occurred in the last decades in the acute myeloid leukemia (AML) treatment, clinical results are still unsatisfactory. More effective therapies are required, and innovative approaches are ongoing, including the discovery of novel antileukemia natural compounds. Several studies have described the activity of extracts from mushrooms which produce compounds that exhibited immunological and antitumor activities. The latter has been demonstrated to be promoted in vitro by mushroom polysaccharides via induction of apoptosis. However, the antileukemia activity of these compounds on primary cells is still not reported. In the present study, we examined the in vitro effects of Tramesan (TR), a bioactive compound extracted from Trametes versicolor, on leukemic cell lines and primary cells. Our results demonstrated that TR induced a marked growth inhibition of leukemic cell lines and primary cells from AML patients. The antiproliferative effects of TR were associated in primary AML cells with a significant increase of apoptosis. No significant cytotoxic effects were observed in normal peripheral blood mononuclear cells (MNC) from healthy donors. Our data demonstrated a cytotoxic activity of TR on leukemia cells prompting further translational applications. Ongoing studies are elucidating the molecular mechanisms underlying its antileukemic activity.

Genome-Wide Analysis Reveals Conserved and Divergent Features of Notch1/RBPJ Binding in Human and Murine T Lymphoblastic Leukemia Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5236-5236
Author(s):  
Jon C Aster ◽  
Hongfang Wang ◽  
James Zou ◽  
Yumi Yashiro-Ohtani ◽  
Bo Zhao ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Cell Lines ◽  
Binding Sites ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Human T Cell ◽  
Two Factors

Abstract Abstract 5236 Activated Notch1 regulates gene expression by associating with the DNA-binding factor RBPJ and is an important oncoprotein in murine and human T cell acute lymphoblastic leukemia/lymphoma (T-ALL), yet the interplay between Notch1 and other factors that regulate the transcriptional output of T-ALL cells is poorly understood. Using ChIP-Seq and starting with Notch1-dependent human and murine T-ALL cell lines, we find that Notch1 binds preferentially to promoters, to RBPJ binding sites, and near sites for ZNF143, as well as Ets and Runx factors. By ChIP-Seq, ZNF143 binds to ∼40% of Notch1 sites, whereas Ets1 binding is observed within 100 basepairs of ∼70% of genomic Notch1 binding sites. Notch1/ZNF143 “co-sites” have high Notch1 and ZNF143 signals, frequent co-binding of RBPJ to sites embedded within ZNF143 motifs, strong promoter bias, and low mean levels of “activated” chromatin marks. RBPJ and ZNF143 binding to DNA is mutually exclusive in vitro, suggesting RBPJ/Notch1 and ZNF143 complexes exchange on these sites in T-ALL cell lines. In contrast, Ets1 binding sites flank RBPJ/Notch1 binding sites and are associated with high levels of activated chromatin marks, whereas Runx sites are predominantly intergenic. Although Notch1 predominantly binds promoters, ∼75% of direct Notch1 target genes lack promoter binding and appear to be regulated by enhancers, which were identified near MYC, DTX1, IGF1R, IL7R and the GIMAP gene cluster. Both Ets1 and Notch1 binding to an intronic enhancer located in DTX1 were required for expression of this well characterized Notch1 target gene, suggesting that these two factors coordinately regulate DTX1 expression. Although the association of Notch1 binding with ZNF143, Ets, and Runx sites was highly conserved, binding near certain important genes showed substantial divergence. For example, in human T-ALL lines Notch1/RBPJ bind a 3' enhancer near the IL7R gene, whereas in murine T-ALL lines no binding was observed near Il7r. Similarly, in human T-ALL lines Notch1/RBPJ bound an enhancer located ∼565 kb 5' of MYC, whereas in murine T-ALL cells Notch1/RBPJ bound an enhancer located ∼1 Mb 3' of Myc. Human and murine T-ALL genomes also have many sites that bind only RBPJ. Murine RBPJ “only” sites are highly enriched for imputed sites for the corepressor REST, whereas human RPBJ “only” sites lack REST motifs and are more highly enriched for imputed CREB binding sites. Thus, there is a conserved network of cis-regulatory factors that interacts with Notch1 to regulate gene expression in T-ALL cell lines, as well as novel classes of divergent RBPJ “only” sites that also likely regulate transcription. To extend these findings to normal and pathophysiologic tissues, ChIP-Seq was used to identify RBPJ/Notch1 binding sites in primary murine thymocytes and primary murine T-ALL associated with Notch1 gain-of-function mutations. Early findings appear to indicate that primary T-ALLs closely resemble normal DN3a thymocytes in terms of the distribution of Notch1 binding sites and associated chromatin marks. These data suggest that Notch1-driven T-ALLs epigenetically resemble the DN3a stage of T cell development, during which Notch1 signaling is high and cells are rapidly proliferating. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Single and Dual Targeting Chimeric Antigen Receptor T-Cell Therapy in Acute Myeloid Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 25-25
Author(s):  
Sneha Chitre ◽  
Joop Gaken ◽  
Andrea Venuso ◽  
Ghulam J. Mufti
Keyword(s):  
T Cell ◽  
Cell Lines ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Target Cells ◽  
High Affinity ◽  
Leukemic Cell Lines ◽  
Dual Targeting ◽  
Car T

BACKGROUND: Discovery of chimeric antigen receptors (CARs) specific for tumour-associated antigens are emerging to be an effective form of immunotherapy for cancer treatment in recent years. However, the lack of a compelling acute myeloid leukemia (AML)-specific cell surface antigen and the safety concerns for myeloid-directed CAR T therapies causing prolonged myeloablation/aplasia which necessitate bone marrow transplantation make it challenging to develop CARTs for AML. In spite of these challenges, the high relapse rate of the disease i.e. 43% and 18% patients never attaining clinical remission (CR) with front-line induction treatments highlight an unmet need for developing improved CAR T vectors with enhanced specificity towards leukemic blasts in refractory/resistant cases with poor cytogenetics. Therefore, we aim to develop improved CAR T vectors for AML that produce safe and consistent responses in patients with high-risk disease. Herein, we demonstrate the in-vitro data for (a) efficacy of a second-generation CAR expressing six single-chain variable fragments (scFv) with different affinities for CD123 [interleukin three receptor alpha (IL3RA; CD123), a molecule over expressed on AML blasts and leukemic stem cells (LSC)] and (b) evaluate the cytotoxic effects of a dual targeting CARCD123/CD33 (against CD123 and CD33; an important myeloid marker specifically expressed on bulk AML disease) to enhance specificity towards leukemic cells therefore reducing "on-target off-organ effects". METHOD: Six lentiviral vectors with CAR against CD123 were constructed i.e. two high affinity (4nM kD & 4nM kD with a point mutation resulting in amino acid change K136Q), two moderate (56nM kD & 56nM kD with mutation at A105G) and two low affinity vectors (101nM kD & 101nM kD with mutation at V24G). To improve the specificity of the single targeting CARCD123, the high affinity (4nM kD, K136Q) vector was utilized to generate two dual targeting (CARCD123/CD33) constructs containing the activation domain (CD3ζ) directed against CD33 and the costimulatory domain (either CD28 or 41BB) directed against CD123. All constructs were transduced (MOI 1:5) into peripheral blood mononuclear cells (PBMCs) from healthy donors or AML patients and their cytotoxicity was examined by flowcytometry on leukemic cell lines; Kg1, U937, K562 [Fig:1a], Ramos wild type (CD19+, CD123-), artificially engineered Ramos cells (transduced by lentiviral vectors with CD123 and/or CD33 cDNA) i.e. Ramos 123 (CD123+), Ramos 33 (CD33+), Ramos 123/33 (CD123+ and CD33+) and AML mononucelar cells (MNCs). RESULTS: Flowcytometric analysis confirmed the expansion of T cells from PBMCs and cytotoxicity of the eight CAR constructs against target cells in in-vitro co-culture assay. High affinity CARCD123 (4nM kD & 4nM kD K136Q) T cells demonstrated enhanced cytotoxicity [Fig 1a] compared to moderate (56nM kD, 56nM kD A105G) CARCD123 in all leukemic cell lines while the low affinity (101nM kD, 101nM kD V24G) vectors had no effect. Efficacy of the high affinity CARCD123 constructs was validated on Kg1 [Fig 1b] and Ramos 123+ target cells by the increasing effector: target ratios (1:2, 1:4 & 1:10). Similar cytotoxic effects were also consistently observed against autologous AML MNCs (target cells) [n=4] and allogenic (effector cells are PBMCs from healthy donors) AML MNCs [Fig 1c] [n=3]. T cell activation was confirmed by ELISA and showed increased IFN-γ (500-2000 fold) and TNF-α (150-200 fold) levels after 24hr co-culture. Furthermore, we also elucidated the exclusive cytotoxicity of the two dual targeting CARCD123/CD33 in Ramos 123+/33+ cells [Fig 1d] with no effect being observed on Ramos 123+, Ramos 33+ and Ramos wild type (123-/33-) cell lines. This confirmed the absence of non-specific targeting, validated the improved specificity of the CARs towards leukemic cells and demonstrated a potential to reduce deleterious "on-target but off organ effects". CONCLUSION: In summary, we illustrate in-vitro data establishing the importance of scFv on CAR T cell cytotoxicity and exemplify for the first time an improved specificity of CARTs by targeting two antigens simultaneously in AML. Future work will involve examining the in-vivo dynamics of CAR CD123 and CAR CD123 CD33 on the hematopoietic system and on disease pathogenesis with an aim to proceed to phase I clinical trial. Figure 1 Disclosures Mufti: BMS, Novartis: Research Funding; Abbvie, Novartis: Consultancy.

scholarly journals 2-Sulfonylpyrimidines: Mild alkylating agents with anticancer activity toward p53-compromised cells

2016 ◽  
Vol 113 (36) ◽  
pp. E5271-E5280 ◽  
Author(s):  
Matthias R. Bauer ◽  
Andreas C. Joerger ◽  
Alan R. Fersht
Keyword(s):  
Reactive Oxygen Species ◽  
Anticancer Activity ◽  
Cell Lines ◽  
Target Genes ◽  
Alkylating Agents ◽  
Reactive Oxygen ◽  
Glutathione Depletion ◽  
Target Cells ◽  
Oxygen Species

The tumor suppressor p53 has the most frequently mutated gene in human cancers. Many of p53’s oncogenic mutants are just destabilized and rapidly aggregate, and are targets for stabilization by drugs. We found certain 2-sulfonylpyrimidines, including one named PK11007, to be mild thiol alkylators with anticancer activity in several cell lines, especially those with mutationally compromised p53. PK11007 acted by two routes: p53 dependent and p53 independent. PK11007 stabilized p53 in vitro via selective alkylation of two surface-exposed cysteines without compromising its DNA binding activity. Unstable p53 was reactivated by PK11007 in some cancer cell lines, leading to up-regulation of p53 target genes such as p21 and PUMA. More generally, there was cell death that was independent of p53 but dependent on glutathione depletion and associated with highly elevated levels of reactive oxygen species and induction of endoplasmic reticulum (ER) stress, as also found for the anticancer agent PRIMA-1MET(APR-246). PK11007 may be a lead for anticancer drugs that target cells with nonfunctional p53 or impaired reactive oxygen species (ROS) detoxification in a wide variety of mutant p53 cells.

Downregulation of MiR-449a Is Essential for the Survival of EVI1 Positive Leukemic Cells through Modulation of NOTCH1 and BCL2.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 361-361
Author(s):  
An De Weer ◽  
Pieter Mestdagh ◽  
Katleen De Preter ◽  
Joni Van der Meulen ◽  
Pieter Van Vlierberghe ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Lines ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Chromosomal Rearrangements ◽  
Ectopic Expression ◽  
Leukemic Cell ◽  
Model Systems ◽  
Leukemic Cells

Abstract Abstract 361 Chromosomal rearrangements involving the EVI1 gene are a recurrent finding in malignant myeloid disorders. These translocations or inversions contribute to ectopic expression or to the formation of fusion genes involving the EVI1 gene. EVI1 transcriptional activation has been reported in up to 10% of acute myeloid leukemia (AML) and is a prognostic marker of poor outcome. MicroRNA (miRNA) deregulation was recently identified as a major contributor to cancer initiation and progression. As miRNA genes were shown to be directly regulated by activated proto-oncogenes, we aimed to identify miRNAs under direct or indirect control of EVI1. To this purpose, we analyzed the expression of 366 miRNAs in 38 EVI1 rearranged/overexpressing patient samples, 6 normal bone marrow controls and 2 EVI1 knockdown model systems (siRNA mediated EVI1 knockdown in the EVI1 rearranged/overexpressing cell lines Kasumi-3 and UCSD-AML1). In total, 24 upregulated and 25 downregulated miRNAs (p<0.05) were shown to be related to the EVI1 expression status. Amongst these, miR-449a was selected for further study based on its homology to the known cancer associated miRNA miR-34a. Downregulation of miR-449a by EVI1 was further confirmed in the leukemic cell line U937 with tetracycline controllabel (tet-off) EVI1 overexpression. Next, direct transcriptional regulation of miR-449a expression by EVI1 was demonstrated by chromatin immunoprecipitation (ChIP). To test the functional consequences of downregulation of miR-449a in AML cells, reconstitution of the expression of miR-449a in the Kasumi-3 and UCSD-AML1 cell lines was performed, which resulted in significantly decreased cell viability, increased apoptosis and differentiation towards the megakaryocytic and monocytic lineages. Interestingly, siRNA mediated knockdown of EVI1 expression in Kasumi-3 or UCSD-AML1 almost completely abrogated the miR-449a induced reduction in cell viability, while electroporation of both cell lines with EVI1 siRNAs alone had essentially no effect on cell viability. These data strongly suggest that repression of miR-449a expression is essential for the survival and growth of EVI1 overexpressing cells and that this requirement is specifically imposed by EVI1 itself. We next demonstrated that the predicted miR-449a targets NOTCH1 and BCL2 were bona fide miR-449a targets using promoter reporter assays. To asses the contribution of these target genes to the observed phenotype upon miR-449a upregulation, knockdown of NOTCH1 and BCL2 was performed, revealing similar effects on cell viability and apoptosis. These results indicated that the effects seen upon treatment of cells with a precursor miR-449a are at least partly mediated through NOTCH1 and BCL2. In conclusion, we provided for the first time evidence that EVI1 mediated downregulation of miR-449a leads to NOTCH1 and BCL2 upregulation and is required for sustained proliferation and survival of EVI1 overexpressing cells. These data also open new perspectives for therapeutic intervention through modulation of miR-449a and/or its target genes. Disclosures: No relevant conflicts of interest to declare.

Loop Regulation Between microRNAs and Epigenetics Underlie microRNA Dysregulation in Multiple Myeloma and Is Associated with the Disease Progression

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3013-3013 ◽  
Author(s):  
Kei Kimura ◽  
Yuko Kuroda ◽  
Yuta Masuda ◽  
Arito Yamane ◽  
Hikaru Hattori ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Multiple Myeloma ◽  
Cell Lines ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Methylation Status ◽  
Expression Level ◽  
Expression Levels ◽  
Control Subjects

Abstract Background: MicroRNAs (miRs) are small non-coding RNAs of 19-25 bases in length that have the ability to modulate gene expression. Some miRs are involved in carcinogenesis and act as tumor suppressor genes (TSG). It has been shown that epigenetics and miRs play an important role in multiple myeloma (MM) progression; however, precise mechanism underlying miR dysregulation has not yet been fully elucidated. Transcriptional silencing of TSG in cancer cells is often associated with DNA methylation and carried out by DNMTs. miR-29 family directly targets DNMTs and promoters of miR-34 family are also methylated in cancers. In this study, we attempted to clarify the interaction between miR and epigenetics focusing on the miR-29 and miR-34 families and their associated genes to understand mechanism of miR dysregulation in MM. Methods: Bone marrow plasma cells from 123 MM patients, 57 MGUS patients, 20 control subjects and 9 MM cell lines were analyzed. This study was approved by the IRB of Gunma University and all patients provided their informed consent prior to enrollment. MiRs and their target gene mRNA values were determined by RQ-PCR. DNA methylation status was determined by methylation-specific PCR. Decitabine, nutlin-3, c-myc inhibitor 10058-F4, and miRNA-mimicTM were used. Result: We found a significantly reduced expression of miR-29a, -29b, -29c, -34a, -34b, and -34c in MM patients compared with MGUS patients and the control subjects (all: p<0.001). DNMT1, -3A and -3B were elevated in MM patients compared with MGUS patients and the control subjects (p<0.001, p<0.001, p=0.01, respectively). DNMT1 was inversely correlated with miR-29a and miR-29b (r=-0.419, p=0.005, r=-0.407, p=0.006, respectively). DNMT3A was inversely correlated with miR-29a, miR-29b and miR-29c (r=-0.315, p=0.04; r=-0.371, p=0.01; r=-0.315, p=0.04, respectively). DNMT3B was inversely correlated with miR-29a and miR-29b (r=-0.353, p=0.02; r=-0.358, p=0.02, respectively). The promoter regions of miR-34a and miR-34b/c were methylated in the MM cell lines, and the rate of methylation of these miRs were higher in MM patients (45.4%, 70.2%, respectively) compared with MGUS patients (15.8%, 26.3%, respectively) (p<0.001). There were significant positive correlations among the miRs expression levels: 29a-34a r=0.448, p<0.001; 29a-34b r=0.309, p=0.001; 29b-34a r=0.500, p<0.001; 29b-34b r=0.297, p=0.002). The expression level of TP53 and its downstream target p21 was higher in MM patients (p=0.004, p<0.001) compared with MGUS patients and the control subjects. The expression level of TP53 was positively correlated with p21 (r=0.33, p<0.001), but not with miR-34a, -b or -c, which were presumed to be upregulated by TP53. In the MM cell lines, nutlin-3, which accumulates TP53 protein, did not increase the expression of miR-34a, -b or -c; however, decitabine increased the expression of pri-miR-34a by 1.2-7.3 fold, pri-miR-34b by 3.3-7.1 fold and increased miR-34a and miR-34b/c by 1.3-2.3 fold and 1.2-5.4 fold, respectively, suggesting that miR-34 family transcription was suppressed by methylation. The transfection of miR-29a or -29b reduced DNMT3A and 3B expression by 0.5 fold and increased pri-miR-34 expression by 2-8 fold. Treatment with a c-myc inhibitor increased the expression of pri-miR-29a/b-1 by 6-8 fold, and miR-29b by 4-7 fold in the MM cell lines. Moreover, the c-myc inhibitor increased the expression of pri-miR-34a by 20-192 fold and miR-34a by 3-6 fold. The transfection of miR-34 reduced the expression of target genes CDK6, SIRT1 and c-myc by 0.4 to 0.6 fold. The expression levels of CDK6, SIRT1 and c-myc were significantly higher in MM patients compared with MGUS patients and the control subjects (p=0.04, p<0.0001, p<0.0001, respectively). Conclusion: We found a significant reduction in the miRs expression levels in MM patients and cell lines, which was partly associated with methylation. Correlations between miRs and related transcripts in patients and an in vitro study demonstrate a negative regulation loop where c-myc suppresses the expression of the miR-29 family, the miR-29 family suppresses DNMT, DNMT suppresses the miR-34 family through promoter methylation, and the miR-34 family suppresses c-myc. This mechanism might underlie the dysregulation of miRs in MM and the disruption of this loop mechanism may be a novel target for MM treatment. Disclosures No relevant conflicts of interest to declare.

Repositioning of Difluorinated Propanediones as Inhibitors of Histone Methyltransferases and their Biological Evaluation in Human Leukemic Cell Lines

2019 ◽  
Vol 18 (13) ◽  
pp. 1892-1899 ◽  
Author(s):  
Tanushree Pal ◽  
Asmita Sharda ◽  
Bharat Khade ◽  
C. Sinha Ramaa ◽  
Sanjay Gupta
Keyword(s):  
Cell Lines ◽  
Physiological Role ◽  
Leukemic Cell ◽  
Docking Studies ◽  
Biological Evaluation ◽  
Leukemic Cell Lines ◽  
Histone Lysine ◽  
Histone Lysine Methyltransferase ◽  
Subsequent Decrease

Background: At present, ‘pharmaco-epigenomics’ constitutes the hope in cancer treatment owing to epigenetic deregulation- a reversible process and playing a role in malignancy. Objective: Chemotherapy has many limitations like host-tissue toxicity, drug resistance. Hence, it is imperative to unearth targets to better treat cancer. Here, we intend to repurpose a set of our previously synthesized difluorinated Propanediones (PR) as Histone lysine Methyltransferase inhibitors (HMTi). Methods: The cell lines of leukemic origin viz. histiocytic lymphoma (U937) and acute T-cell leukemia (JURKAT) were treated with PR-1 to 7 after docking studies with active pocket of HMT. The cell cycle analysis, in vitro methylation and cell proliferation assays were carried out to delineate their physiological role. Results: A small molecule PR-4, at 1 and 10µM, has shown to alter the methylation of histone H3 and H4 in both cell lines. Also, treatment shows an increase in G2/M population and a subsequent decrease in the G0/G1 population in U937. In JURKAT, an increase in both G2/M and S phase population was observed. The sub-G1 population showed a steady rise with increase in dose and prolonged time intervals in U937 and JURKAT cell lines. In SRB assay, the PR showed a cell growth of 42.6 and 53.4% comparable to adriamycin; 44.5 and 53.2% in U937 and JURKAT, respectively. The study suggests that PR-4 could emerge as a potential HMT inhibitor. Conclusion: The molecule PR-4 could be a lead in developing more histone lysine methyltransferases inhibitors with potential to be pro-apoptotic agents.

Non-Viral Vectors for Gene Delivery

2018 ◽  
Vol 9 (1) ◽  
pp. 4-11 ◽  
Author(s):  
Aparna Bansal ◽  
Himanshu
Keyword(s):  
Gene Therapy ◽  
Viral Vectors ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Expression System ◽  
Target Cells ◽  
Specific Expression ◽  
Naked Dna

Introduction: Gene therapy has emerged out as a promising therapeutic pave for the treatment of genetic and acquired diseases. Gene transfection into target cells using naked DNA is a simple and safe approach which has been further improved by combining vectors or gene carriers. Both viral and non-viral approaches have achieved a milestone to establish this technique, but non-viral approaches have attained a significant attention because of their favourable properties like less immunotoxicity and biosafety, easy to produce with versatile surface modifications, etc. Literature is rich in evidences which revealed that undoubtedly, non–viral vectors have acquired a unique place in gene therapy but still there are number of challenges which are to be overcome to increase their effectiveness and prove them ideal gene vectors. Conclusion: To date, tissue specific expression, long lasting gene expression system, enhanced gene transfection efficiency has been achieved with improvement in delivery methods using non-viral vectors. This review mainly summarizes the various physical and chemical methods for gene transfer in vitro and in vivo.

scholarly journals Efficient genome editing in primary cells and in vivo using viral-derived “Nanoblades” loaded with Cas9/sgRNA ribonucleoproteins

2017 ◽  
Author(s):  
Philippe E. Mangeot ◽  
Valérie Risson ◽  
Floriane Fusil ◽  
Aline Marnef ◽  
Emilie Laurent ◽  
...  
Keyword(s):  
Stem Cells ◽  
Genome Editing ◽  
Target Genes ◽  
Bone Marrow Cells ◽  
Leukemia Virus ◽  
Target Cells ◽  
Mouse Bone Marrow ◽  
Primary Cells ◽  
Hematopoietic Stem

AbstractProgrammable nucleases have enabled rapid and accessible genome engineering in eukaryotic cells and living organisms. However, their delivery into target cells can be technically challenging when working with primary cells or in vivo. Using engineered murine leukemia virus-like particles loaded with Cas9/sgRNA ribonucleoproteins (“Nanoblades”), we were able to induce efficient genome-editing in cell lines and primary cells including human induced pluripotent stem cells, human hematopoietic stem cells and mouse bone-marrow cells. Transgene-free Nanoblades were also capable of in vivo genome-editing in mouse embryos and in the liver of injected mice. Nanoblades can be complexed with donor DNA for “all-in-one” homology-directed repair or programmed with modified Cas9 variants to mediate transcriptional up-regulation of target genes. Nanoblades preparation process is simple, relatively inexpensive and can be easily implemented in any laboratory equipped for cellular biology.

scholarly journals Comparative In Vitro Sensitivities of Human Immune Cell Lines, Vaginal and Cervical Epithelial Cell Lines, and Primary Cells to Candidate Microbicides Nonoxynol 9, C31G, and Sodium Dodecyl Sulfate

2002 ◽  
Vol 46 (7) ◽  
pp. 2292-2298 ◽  
Author(s):  
Fred C. Krebs ◽  
Shendra R. Miller ◽  
Bradley J. Catalone ◽  
Raina Fichorova ◽  
Deborah Anderson ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Sodium Dodecyl Sulfate ◽  
Cell Viability ◽  
Cell Lines ◽  
Sodium Dodecyl ◽  
Primary Cells ◽  
Cytokine Activation ◽  
Dodecyl Sulfate ◽  
Human Immune

ABSTRACT In experiments to assess the in vitro impact of the candidate microbicides nonoxynol 9 (N-9), C31G, and sodium dodecyl sulfate (SDS) on human immune and epithelial cell viability, cell lines and primary cell populations of lymphocytic and monocytic origin were generally shown to be equally sensitive to exposures ranging from 10 min to 48 h. However, U-937 cells were more sensitive to N-9 and C31G after 48 h than were primary monocyte-derived macrophages. Cytokine activation of monocytes and lymphocytes had no effect on cell viability following exposure to these microbicidal compounds. Primary and passaged vaginal epithelial cultures and cell lines differed in sensitivity to N-9 and C31G but not SDS. These studies provide a foundation for in vitro experiments in which cell lines of human immune and epithelial origin can be used as suitable surrogates for primary cells to further investigate the effects of microbicides on cell metabolism, membrane composition, and integrity and the effects of cell type, proliferation, and differentiation on microbicide sensitivity.

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