scholarly journals Methotrexate Resistance in Datura innoxia (Uptake and Metabolism of Methotrexate in Wild-Type and Resistant Cell Lines)

1993 ◽  
Vol 101 (2) ◽  
pp. 477-483 ◽  
Author(s):  
K. Wu ◽  
I. J. Atkinson ◽  
E. A. Cossins ◽  
J. King
Keyword(s):  
Cell Lines ◽  
Resistant Cell ◽  
Datura Innoxia ◽  
Wild Type ◽  
Methotrexate Resistance ◽  
Uptake And Metabolism

scholarly journals Inhibition of MUS81 By siRNA Reverses Resistance to Cisplatin in Human Ovarian Cancer Cell Lines

2021 ◽  
Author(s):  
Emma C. Bourton ◽  
Sheba Adam-Zahir ◽  
Piers N. Plowman ◽  
Hussein Nahidh Al-Ali ◽  
Helen A. Foster ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Protein Expression ◽  
Cell Lines ◽  
Cancer Cell ◽  
Ovarian Cancer Cell ◽  
Cancer Cell Lines ◽  
Resistant Cell ◽  
Wild Type ◽  
Ovarian Cancer Cell Lines ◽  
Sirna Knockdown

Abstract Bacground: Drugs that induce DNA interstrand crosslinks form the mainstay of anticancer treatments for different cancers. These drugs are used to treat ovarian cancer which is the most prevalent gynaecological cancer. Five-year survival rates are approximately 40% and the development of drug resistant disease is an important factor in treatment failure. Methods: In this study a comprehensive evaluation of the expression and function of the site-specific endonuclease MUS81 was conducted. Using quantitative real time PCR analysis and imaging flow cytometry we determined the mRNA and protein expression of MUS81 in three ovarian cancer cell lines and two immortalised human fibroblast cell lines which had been made resistant to cisplatin by chronic exposure. siRNA knockdown of MUS81 was employed to determine the effect on overall cell survival which was assessed using clonogenic assays. Results: In the five cisplatin-resistant cell lines we observed increased MUS81 mRNA expression. In addition MUS81 protein expression in the form of discrete nuclear foci in cells was observed in all cell lines following cisplatin exposure, there being significantly more foci in cisplatin resistant cell lines. siRNA knockdown of MUS81 significantly reduced both mRNA and protein levels in two cell lines (SK-OV-3 and MRC5-SV1 – wild-type and resistant) and critically re-sensitised cisplatin resistant cells to wild-type level, determined by clonogenic assay.Conclusion: MUS81 is central to the development of cisplatin resistance in ovarian cancer cell lines. Inhibition of MUS81 restored drug sensitivity to the cells. MUS81 may be a useful therapeutic target to overcome drug resistance in ovarian and other cancers.

Chromosome-mediated gene transfer of hydroxyurea resistance and amplification of ribonucleotide reductase activity

1983 ◽  
Vol 3 (6) ◽  
pp. 1053-1061
Author(s):  
W H Lewis ◽  
P R Srinivasan
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Ribonucleotide Reductase ◽  
Chinese Hamster ◽  
Fold Increase ◽  
Resistant Cell ◽  
Plating Efficiency ◽  
Wild Type Cell ◽  
Wild Type ◽  
Metaphase Chromosomes

Metaphase chromosomes purified from a hydroxyurea-resistant Chinese hamster cell line were able to transform recipient wild-type cells to hydroxyurea resistance at a frequency of 10(-6). Approximately 60% of the resulting transformant clones gradually lost hydroxyurea resistance when cultivated for prolonged periods in the absence of drug. One transformant was subjected to serial selection in higher concentrations of hydroxyurea. The five cell lines generated exhibited increasing relative plating efficiency in the presence of the drug and a corresponding elevation in their cellular content of ribonucleotide reductase. The most resistant cell line had a 163-fold increase in relative plating efficiency and a 120-fold increase in enzyme activity when compared with the wild-type cell line. The highly hydroxyurea-resistant cell lines had strong electron paramagnetic resonance signals characteristic of an elevated level of the free radical present in the M2 subunit of ribonucleotide reductase. Two-dimensional electrophoresis of cell-free extracts from one of the resistant cell lines indicated that a 53,000-dalton protein was present in greatly elevated quantities when compared with the wild-type cell line. These data suggest that the hydroxyurea-resistant cell lines may contain an amplification of the gene for the M2 subunit of ribonucleotide reductase.

Activation of the Wnt/β-Catenin Pathway Mediates Lenalidomide Resistance in Multiple Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 113-113 ◽  
Author(s):  
Chad C. Bjorklund ◽  
Deborah J. Kuhn ◽  
Jairo A. Matthews ◽  
Michael Wang ◽  
Veerabhadran Baladandayuthapani ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Cell Lines ◽  
Expression Profiling ◽  
Fold Increase ◽  
Resistant Cell ◽  
Refractory Disease ◽  
Wild Type ◽  
Myeloma Cells ◽  
Resistant Cells

Abstract Abstract 113 Background: Novel drugs such as the immunomodulatory agent lenalidomide have revolutionized the treatment of multiple myeloma, as evidenced by an increasing overall survival for patients with both newly-diagnosed, and relapsed and/or refractory disease. Despite these improvements, myeloma remains incurable, and is still characterized by a trend for increasing chemoresistance at relapse, with a decreasing duration of benefit from each successive line of therapy. By understanding the mechanisms responsible for the emergence of drug resistance, which have so far not been well characterized in the case of lenalidomide, it may be possible to rationally design novel regimens that could either overcome this resistance, or possibly prevent its emergence altogether. Methods: To improve our understanding of the mechanisms responsible for lenalidomide resistance, we developed cell line models of interleukin (IL)-6-dependent (ANBL-6 and KAS-6/1) and –independent (U266 and MM1.S) lenalidomide-resistant multiple myeloma cells. Starting at a concentration that was 1/10 of the IC50 for lenalidomide's anti-proliferative effects in drug-naïve cells, increasing drug concentrations were used until all the cell lines could proliferate and maintain cell membrane integrity in the presence of 10 μM lenalidomide. These cell lines were then used as an in vitro model of lenalidomide-specific drug resistance, and subjected to further characterization, including with gene expression profiling. Results: Resistance to lenalidomide was evidenced by a dramatic, 100-1000-fold increase in the IC50 values of these myeloma cells. In the case of ANBL-6 cells, for example, drug-naïve cells showed an IC50 of 0.14 μM using tetrazolium dye-based viability assays, but this increased to >100 μM in the drug-resistant cells, as was the case in U266 and MM1.S cells. This resistance was a stable phenotype, since removal of lenalidomide for seven to ninety days from cell culture conditions did not re-sensitize them when 10 μM lenalidomide was reintroduced. Gene expression profiling followed by pathway analysis to examine changes at the transcript level between wild-type parental and lenalidomide-resistant cell lines identified the Wnt/β-catenin pathway as the most altered across all cell lines. Increased expression was seen in several members of the low-density-lipoprotein receptor related protein family, including LRP1 and 5; members of the wingless-type MMTV integrations site family, including WNT3 and 4; β-catenin; and downstream Wnt/β-catenin targets such as CD44. Similar changes were detected in primary samples from a patient who developed clinically lenalidomide-refractory disease. Reporter assays revealed an up to 5-fold increase in LEF/TCF-dependent transcription both in drug-naïve cells acutely exposed to lenalidomide, and in their chronically exposed, lenalidomide-resistant clones. Western blotting and flow cytometry confirmed that these lenalidomide-resistant cells had increased expression by 2-20 fold of β-catenin and CD44, as well as other LEF/TCF targets, including Cyclin D1 and c-Myc. Comparable changes occurred after lenalidomide exposure in myeloma cells grown in the context of bone marrow stroma. Notably, lenalidomide-resistant cells showed decreased expression of casein kinase 1 and increased phosphorylation of glycogen synthase kinase 3 at Ser21/9, both of which would reduce the phosphorylation of β-catenin needed for its later proteasome-mediated degradation. Stimulation of the Wnt/β-catenin pathway with recombinant human Wnt3a resulted in resistance to lenalidomide in wild-type, drug-naïve cells, as evidenced by a 10-fold increase in the IC50. Conversely, exposure of lenalidomide-resistant cell lines to quercetin, a known antagonist of the β-catenin/TCF interaction, induced a partial re-sensitization to lenalidomide. Conclusions: These data support the hypothesis that activation of the Wnt/β-catenin pathway represents a mechanism of both acute and chronic resistance to the anti-proliferative effects of lenalidomide in multiple myeloma. Moreover, they support the development of strategies aimed at suppressing Wnt/β-catenin activity to resensitize multiple myeloma to the effects of this immunomodulatory agent in vivo. Disclosures: No relevant conflicts of interest to declare.

Identification of protein kinase C subspecies in wild-type and multidrug-resistant cell lines

1993 ◽  
Vol 21 (4) ◽  
pp. 378S-378S
Author(s):  
James A. L. Fenton ◽  
Alex Paton ◽  
Nigel Groome ◽  
J. Roger Warr ◽  
Martin G. Rumsby
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cell Lines ◽  
Multidrug Resistant ◽  
Resistant Cell ◽  
Wild Type ◽  
Kinase C

scholarly journals A phospho-proteomic study of cetuximab resistance in KRAS/NRAS/BRAFV600 wild-type colorectal cancer

2021 ◽  
Author(s):  
Alexandros Georgiou ◽  
Adam Stewart ◽  
Georgios Vlachogiannis ◽  
Lisa Pickard ◽  
Nicola Valeri ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Signal Transduction ◽  
Drug Resistance ◽  
Cell Lines ◽  
Ex Vivo ◽  
Pi3k Pathway ◽  
Resistant Cell ◽  
Wild Type ◽  
Proteomic Study

Abstract Purpose We hypothesised that plasticity in signal transduction may be a mechanism of drug resistance and tested this hypothesis in the setting of cetuximab resistance in patients with KRAS/NRAS/BRAFV600 wild-type colorectal cancer (CRC). Methods A multiplex antibody-based platform was used to study simultaneous changes in signal transduction of 55 phospho-proteins in 12 KRAS/NRAS/BRAFV600 wild-type CRC cell lines (6 cetuximab sensitive versus 6 cetuximab resistant) following 1 and 4 h in vitro cetuximab exposure. We validated our results in CRC patient samples (n = 4) using ex vivo exposure to cetuximab in KRAS/NRAS/BRAFV600 cells that were immunomagnetically separated from the serous effusions of patients with known cetuximab resistance. Results Differences in levels of phospho-proteins in cetuximab sensitive and resistant cell lines included reductions in phospho-RPS6 and phospho-PRAS40 in cetuximab sensitive, but not cetuximab resistant cell lines at 1 and 4 h, respectively. In addition, phospho-AKT levels were found to be elevated in 3/4 patient samples following ex vivo incubation with cetuximab for 1 h. We further explored these findings by studying the effects of combinations of cetuximab and two PI3K pathway inhibitors in 3 cetuximab resistant cell lines. The addition of PI3K pathway inhibitors to cetuximab led to a significantly higher reduction in colony formation capacity compared to cetuximab alone. Conclusion Our findings suggest activation of the PI3K pathway as a mechanism of cetuximab resistance in KRAS/NRAS/BRAFV600 wild-type CRC.

A GPI-Anchored Co-Receptor for TFPI Controls Its Intracellular Trafficking and Endothelial Surface Expression.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1927-1927
Author(s):  
Anna C. Cunningham ◽  
Charles M. Mansbach ◽  
Alan E. Mast
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Tissue Factor ◽  
Cell Lines ◽  
Surface Expression ◽  
Resistant Cell ◽  
Wild Type ◽  
Endothelial Surface ◽  
Cellular Processing ◽  
Resistant Cells

Abstract Tissue factor pathway inhibitor (TFPI) is the major endogenous inhibitor of tissue factor initiated blood coagulation and a is key regulator of the development of intravascular thrombosis. TFPI indirectly binds to the endothelial surface through tight association with a GPI-anchored co-receptor. The location of recombinant TFPI expression in mammalian cells varies depending on the cell line used. In cell lines that do not produce endogenous TFPI, CHO and HEK293 cells, recombinant TFPI is secreted into the culture media. However, in a cell line that produces endogenous surface TFPI, EaHy926 cells, recombinant TFPI is expressed on the cell surface. These data suggest that TFPI is expressed on the surface of cells that produce the GPI-anchored co-receptor and is secreted by cells that do not. To further investigate the function of the co-receptor in TFPI cellular trafficking we developed aerolysin resistant ECV304 and EaHy926 cells lines. Both of these cell lines produce endogenous, surface associated TFPI. The cell lines were mutated with ethyl methanesulfonate and selected with aerolysin. Mutant cells lacking surface GPI-anchored proteins are resistant to the toxic effects of aerolysin and survive. The morphology and growth rate of the two aerolysin resistant cell lines are identical to that of the wild-type cells. They were first characterized to rule out the presence of a mutation that could directly alter cellular metabolism of TFPI. Sequencing of TFPI cDNA indicates that no mutations are present in the TFPI exons. Analysis of mRNA by real time PCR demonstrates that the aerolysin resistant cells make similar amounts of TFPI mRNA as their wild-type counterparts. Thus, transcription and translation of TFPI appear identical to the wild-type cells. In addition, the two independently derived cell lines have very similar phenotypes, as described below, indicating that the aerolysin resistant cell lines have a defect in GPI-anchor biosynthesis but not additional random mutations that could alter cellular processing of TFPI. Characterization of protein expression by flow cytometry indicates that the aerolysin resistant cell lines do not express GPI-anchored proteins (CD59, uPAR) or TFPI on the cell surface but do have wild-type surface expression of transmembrane proteins (CD9, tissue factor). Interestingly, instead of being secreted, western blot analysis of cellular lysates indicates that TFPI is degraded within the aerolysin resistant cells in a manner similar to that observed for GPI-anchored proteins. Intracellular degradation of TFPI is prevented by brefeldin A indicating that degradation takes place in a post endoplasmic reticulum compartment. Pepstatin A, but not MG-132, also prevents degradation, indicating that degradation is lysosomal rather than proteosomal. It appears that binding of TFPI to its co-receptor occurs early in cellular processing, likely within the endoplasmic reticulum. Cellular trafficking of TFPI is controlled by its co-receptor, which has not yet been identified. The co-receptor directs TFPI to the cell surface in wild-type endothelial cells or to be degraded in aerolysin resistant cells. In the absence if its co-receptor TFPI is secreted. Therefore, regulation of co-receptor expression provides a mechanism for the production of cell associated TFPI, as occurs in endothelial cells, versus soluble TFPI, as may occur in megakaryocytes/platelets.

Interferon-γ-Induced Upregulation of Immunoproteasome Subunit Assembly Overcomes Bortezomib Resistance of Leukemia Cell Lines Harbouring Bortezomib-Induced Mutations in Constitutive PSMB5

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1346-1346 ◽  
Author(s):  
Denise Niewerth ◽  
Niels Franke ◽  
Gerrit Jansen ◽  
Yehuda Assaraf ◽  
Johan van Meerloo ◽  
...  
Keyword(s):  
Cell Lines ◽  
Acquired Resistance ◽  
Hematologic Malignancies ◽  
Interferon Γ ◽  
Resistant Cell ◽  
Proteasome Activity ◽  
Mrna Levels ◽  
Wild Type ◽  
Ic50 Values ◽  
Ifn Γ

Abstract Abstract 1346 Acquired resistance to the proteasome inhibitor (PI) bortezomib (BTZ) is an emerging factor limiting its efficacy in the treatment of hematologic malignancies. The clinical impact of acquired resistance has been shown in Multiple Myeloma (MM) patients who were re-treated with BTZ. Although BTZ-retreatment was found to be effective, the response rate as well as the duration of response were less as compared to initial treatment, indicating the development of BTZ-resistance in a subgroup of patients. In line with that, we previously found increased expression of constitutive proteasome (cP) subunit ß5 harbouring a mutation in the BTZ-binding pocket and a decreased expression of non-mutated immunoproteasome subunits in BTZ-resistant cell lines of hematologic malignancies (Franke and Niewerth et al, Leukemia 2012). We here explore whether upregulation of immunoproteasome (iP) expression could restore sensitivity in BTZ-resistant leukemia cells towards BTZ and two epxoyketone-based irreversible PIs; carfilzomib (CFZ) and the ß5i-targeted ONX 0914. BTZ-resistant cell lines were of multiple myeloma (8226), T-cell (CEM) and myelomonocytic (THP1) origin and displayed resistance towards cell growth inhibition in the presence of 7–200 nM BTZ. Induction of iP in wild type (WT) and BTZ-resistant 8226, CCRF-CEM and THP1 cells was achieved by exposure to 100U/ml Interferon- γ (IFN-γ) for 6–72 h. IFN-γ transiently increased (maximum between 24–48 hours) mRNA levels of β5i, β1i, and β2i up to 8-fold, 30-fold and 4-fold, respectively. These findings were corroborated at the β5i, β1i and β2i protein expression level using Western blot analysis. Following IFN-γ exposure, chymotrypsin-like proteasome activity increased up to 2.5-fold compared to unstimulated controls, trypsin-like activity increased up to 1.5-fold, whereas caspase-like activity was slightly decreased. Consistent with increased proteasome activity, there was also an increased expression of cell surface HLA Class I molecules. The impact of IFN-γ induced upregulation of iPs on the sensitivity to the PI BTZ, CFZ, and ONX 0914, defined by the decrease in IC50, is summarized in Table 1. 8226/BTZ100 cells became 4-fold more sensitive towards BTZ after IFN-γ exposure, whereas THP1/BTZ200 and CEM/BTZ200 cells displayed nearly 2-fold increased sensitivity. For CFZ, a modest level of sensitization was observed in all cell lines with high level BTZ resistance. Interestingly, for the immunoproteasome inhibitor ONX 0914, IC50 values were markedly decreased (7-fold for 8226/BTZ100 and 3-fold for THP1/BTZ200 and CEM/BTZ200 cells). Additionally, in 8226 cells with low levels of BTZ resistance (8226/BTZ7), IFN-γ restored parental cell sensitivity to ONX 0914. Restoration of PI sensitivity after IFN-γ exposure was further confirmed by activation of PARP cleavage and accumulation of ubiquitinated proteins, pointing to restoration of BTZ activity under proteasome inhibition and consequent induction of apoptosis. Finally, to provide evidence that upregulation of β5i and or β1i by IFN-γ was responsible for the observed sensitization, siRNA downregulation of β5i and β1i was applied prior to exposure to IFN-γ. Under these conditions, mRNA levels and proteasome activity of β5i remained suppressed, even after exposure to IFN-γ. Moreover, after β5i silencing, PI sensitization and apoptosis were attenuated. Silencing of β1i expression had no effect on PI-sensitization. In conclusion, down-regulation of β5i subunit expression is a major determinant of BTZ-resistance and increasing its proteasomal assembly after IFN-γ exposure facilitates restoration of sensitivity in BTZ-resistant leukemia cells towards cP inhibitors and in particular iP inhibitors. Table 1. IC50 values of PIs ± IFN-γ pre-incubation (48 hr) of wild type and BTZ-resistant hematologic cell lines Cell lines BTZ BTZ + IFNy SF ONX 0914 ONX 0914 + IFNy SF CFZ CFZ + IFNy SF 8226/wt 2.6 1.8 1.4 54 46 1.5 0.4 0.4 1 8226/BTZ7 13.5 5.8 2.3 99 47 2.1 0.9 0.8 1.1 8226/BTZ100 208 57 3.6 1837 249 7.4 28 13 2.2 CEM/wt 4.1 3.9 1.1 75 65 1.2 0.4 0.3 1.3 CEM/BTZ200 416 223 1.9 1763 566 3.1 42 26 1.6 THP1/wt 6.2 5.1 1.2 52 19 2.7 0.9 1.3 0.7 THP1/BTZ200 641 347 1.8 4236 1376 3.1 49 34 1.4 50% inhibitory concentration compared to untreated controls (IC50 nM) as determined in a 4 days growth inhibition assay (MTT). Results depicted are means of at least 3 separate experiments. SF: sensitization factor: IC50 control/IC50 with IFN-g. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Chitosan-Based Polyelectrolyte Complexes for Doxorubicin and Zoledronic Acid Combined Therapy to Overcome Multidrug Resistance

Pharmaceutics ◽  
2018 ◽  
Vol 10 (4) ◽  
pp. 180 ◽  
Author(s):  
Simona Giarra ◽  
Silvia Zappavigna ◽  
Virginia Campani ◽  
Marianna Abate ◽  
Alessia Cossu ◽  
...  
Keyword(s):  
Zoledronic Acid ◽  
Cell Lines ◽  
Combined Therapy ◽  
Polymer Concentration ◽  
Resistant Cell ◽  
Wild Type ◽  
Polyelectrolyte Complexes ◽  
Ionotropic Gelation ◽  
Experimental Parameters

This study aimed to develop nanovectors co-encapsulating doxorubicin (Doxo) and zoledronic acid (Zol) for a combined therapy against Doxo-resistant tumors. Chitosan (CHI)-based polyelectrolyte complexes (PECs) prepared by ionotropic gelation technique were proposed. The influence of some experimental parameters was evaluated in order to optimize the PECs in terms of size and polydispersity index (PI). PEC stability was studied by monitoring size and zeta potential over time. In vitro studies were carried out on wild-type and Doxo-resistant cell lines, to assess both the synergism between Doxo and Zol, as well as the restoring of Doxo sensitivity. Polymer concentration, incubation time, and use of a surfactant were found to be crucial to achieving small size and monodisperse PECs. Doxo and Zol, only when encapsulated in PECs, showed a synergistic antiproliferative effect in all the tested cell lines. Importantly, the incubation of Doxo-resistant cell lines with Doxo/Zol co-encapsulating PECs resulted in the restoration of Doxo sensitivity.

scholarly journals Genetic Transformation of LoHDZ2 and Analysis of its Function to Enhance Stress Resistance in Larch

2021 ◽  
Author(s):  
Peiqi An ◽  
Ruofan Qin ◽  
Qingrong Zhao ◽  
Xuefeng Li ◽  
Chen Wang ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Differentially Expressed Genes ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Wild Type Cell ◽  
Differentially Expressed ◽  
Wild Type ◽  
Gus Staining ◽  
Transgenic Cell

Abstract To study the function of LoHDZ2 in larch, we first constructed a p1302-LoHDZ2::GUS overexpression vector. Through Agrobacterium-mediated infection, the expression vector was transferred into a larch embryogenic cell line. A stable resistant cell line was subsequently screened, and mature embryos were induced to grow until they developed into seedlings. Antagonistic cell lines were identified at both the DNA and RNA levels. The transgenic cell lines were then subjected to GUS staining, and transgenic cell lines were ultimately identified and obtained. These transgenic cell lines were sequenced to identify differentially expressed genes, and a cluster analysis was performed. The resistant cell lines were cultured under stress conditions involving 20% PEG6000 and 200 mM NaCl proliferation media (1/10-BM). After the stress treatment, the contents of peroxidase (POD), malondialdehyde (MDA) and superoxide dismutase (SOD) in both wild-type and transgenic cell lines were measured.The results are summarized below.1. When the specific fragment of the target gene in the genome of the resistant cell line was amplified, at the RNA level, the expression of the fragment in four resistant lines increased. In addition, GUS staining showed a blue reaction, indicating that LoHDZ2 was successfully integrated into the larch embryonic cell lines.2. To verify the accuracy and reliability of the transcriptome data, 10 differentially expressed genes (5 upregulated and 5 downregulated ones) were subjected to qRT–PCR verification. The results showed that the expression trend of the 10 differentially expressed genes was the same as that revealed by RNA-seq, indicating that the transcriptome data were reliable.3. The transcriptome sequencing showed that 176 genes were upregulated and that 140 genes were downregulated. Through GO enrichment analysis and KEGG metabolic pathway analysis, the screened differentially expressed genes were related to biological processes such as larch metabolism and response to stimuli, indicating that these genes may be closely involved in the regulation of the larch response to external stimuli, including heat stress, drought stress, metal ion stress and bacterial infection, and may participate in the growth process.4. After PEG6000 treatment, the POD enzyme activity of the transgenic cell line was greater than that of the wild-type; this activity could effectively remove the amount of peroxide produced. The MDA content of the transgenic cell lines was lower than that of the wild-type cell lines, and the accumulation degree of harmful substances was low, indicating that the degree of oxidative damage of the transgenic cell lines was lower than that of the wild-type cell lines. The SOD content of the transgenic cell lines was lower than that of the wild-type cell lines, indicating that the drought resistance of the transgenic cell lines was enhanced. After 200 mM NaCl treatment, although the increase in SOD content was not obvious, the same trend was detected, indicating that the resistance of the transgenic cell lines was indeed stronger than that of the wild-type cell lines. According to the results of previous experiments, after this gene was overexpressed in tobacco, the transformed plants showed obvious dwarfing, which may indicate that the stress resistance of the plant was enhanced.In conclusion, a transgenic larch cell line was successfully obtained, and transgenic larch seedlings were successfully induced. LoHDZ2, which is a member of the HD-ZipII subfamily, of Larix olgensis may participate in the response of plants to the external environment and may participate in the growth and development of Larix olgensis by affecting plant metabolic pathways.

scholarly journals Inhibition of MUS81 by siRNA Reverses Resistance to Cisplatin in Human Ovarian Cancer Cell Lines

2021 ◽  
Author(s):  
Emma Bourton ◽  
Sheba Adam Zahir ◽  
Piers Plowman ◽  
Hussein Al-Ali ◽  
Helen Foster ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Protein Expression ◽  
Cell Lines ◽  
Cancer Cell ◽  
Ovarian Cancer Cell ◽  
Cancer Cell Lines ◽  
Resistant Cell ◽  
Wild Type ◽  
Ovarian Cancer Cell Lines ◽  
Sirna Knockdown

Abstract Purpose: Drugs that induce DNA interstrand crosslinks form the mainstay of anticancer treatments for different cancers. These drugs are used to treat ovarian cancer which is the most prevalent gynaecological cancer. Five-year survival rates are approximately 40% and the development of drug resistant disease is an important factor in treatment failure. Methods: In this study a comprehensive evaluation of the expression and function of the site-specific endonuclease MUS81 was conducted. Using quantitative real time PCR analysis and imaging flow cytometry we determined the mRNA and protein expression of MUS81 in three ovarian cancer cell lines and two immortalised human fibroblast cell lines which had been made resistant to cisplatin by chronic exposure. siRNA knockdown of MUS81 was employed to determine the effect on overall cell survival which was assessed using clonogenic assays. Results: In the five cisplatin-resistant cell lines we observed increased MUS81 mRNA expression. In addition MUS81 protein expression in the form of discrete nuclear foci in cells was observed in all cell lines following cisplatin exposure, there being significantly more foci in cisplatin resistant cell lines. siRNA knockdown of MUS81 significantly reduced both mRNA and protein levels in two cell lines (SK-OV-3 and MRC5-SV1 – wild-type and resistant) and critically re-sensitised cisplatin resistant cells to wild-type level, determined by clonogenic assay. Conclusion: MUS81 is central to the development of cisplatin resistance in ovarian cancer cell lines. Inhibition of MUS81 restored drug sensitivity to the cells. MUS81 may be a useful therapeutic target to overcome drug resistance in ovarian and other cancers.

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