scholarly journals Biochemical studies on cell fusion. I. Lipid composition of fusion-resistant cells.

1985 ◽  
Vol 101 (4) ◽  
pp. 1578-1590 ◽  
Author(s):  
D S Roos ◽  
P W Choppin
Keyword(s):  
Lipid Composition ◽  
Neutral Lipids ◽  
Mutant Cell ◽  
Ether Lipid ◽  
Resistant Cell ◽  
Fatty Acyl ◽  
Resistant Cell Line ◽  
Parental Line ◽  
Acyl Chains ◽  
Resistant Cells

A series of stable cell mutants of mouse fibroblasts were previously isolated (Roos, D. S. and R. L. Davidson, 1980, Somatic Cell Genet., 6:381-390) that exhibit varying degrees of resistance to the fusion-inducing effect of polyethylene glycol (PEG), but are morphologically similar to the parental cells from which they were derived. Biochemical analysis of these mutant cell lines has revealed differences in whole cell lipid composition which are directly correlated with their susceptibility to fusion. Fusion-resistant cells contain elevated levels of neutral lipids, particularly triglycerides and an unusual ether-linked lipid, O-alkyl, diacylglycerol. This ether lipid is increased approximately 35-fold over parental cells in the most highly PEG-resistant cell line. Fusion-resistant cells also contain more highly saturated fatty acyl chains (ratio of saturated to polyunsaturated fatty acids [S/P ratio] approximately 4:1) than the parental line (S/P ratio approximately 1:1). Cells which are intermediate in their resistance to PEG have ether lipid and fatty acid composition which is intermediate between the parental cells and the most fusion-resistant mutants. In a related communication (Roos, D. S. and P. W. Choppin, 1985, J. Cell. Biol., 100:1591-1598) evidence is presented that alteration of lipid content can predictably control the fusion response of these cells.

Avian Sarcoma Virus Transformed Hamster Cells made Resistant to Ethidium Bromide

1974 ◽  
Vol 16 (3) ◽  
pp. 603-621
Author(s):  
C. ALTANER ◽  
J. MATOSKA
Keyword(s):  
Cell Lines ◽  
Ethidium Bromide ◽  
Virus Genome ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Avian Sarcoma Virus ◽  
Sarcoma Virus ◽  
Original Cell ◽  
Avian Sarcoma ◽  
Resistant Cells

Hamster cells transformed with the Schmidt-Ruppin strain of avian sarcoma virus were selected for resistance to ethidium bromide (EB). The resistant cell lines proliferated in the presence of up to 30 µg/ml EB. From avian sarcoma virus-transformed hamster cells already resistant to bromodeoxy-uridine (BrdU), ethidium bromide-resistant cells which were able to grow in 10 µg/ml EB were also prepared. These cells remain deficient in thymidine kinase activity and are suitable for selective preparation of hybrid cells. The EB resistance was genetically stable. The EB-resistant cell lines, and doubly resistant cells (BrdU, EB) showed no differences in mitochondrial ultrastructure compared with the original cell lines. Thymidine incorporation into mitochondrial DNA was not influenced by EB resistance. All resistant cell lines, including the doubly resistant cell line, contained the avian sarcoma virus genome. The number of cells needed for positive rescue experiments for avian sarcoma virus genome by cell fusion with permissive chicken embryo cells was the same as with the original cell lines. The single EB-resistant cell lines contained R-type virus-like particles, while in BrdU-resistant and doubly resistant cells the R-type particles were absent. The possible nature of EB resistance is discussed.

scholarly journals Generating Paclitaxel-Resistant in Cervical Cancer HeLa Cell Line

2020 ◽  
Vol 11 (2) ◽  
pp. 90
Author(s):  
Muhammad Hasan Bashari ◽  
Fachreza Aryo Damara ◽  
Isna Nisrina Hardani ◽  
Gita Widya Pradini ◽  
Tenny Putri ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Cell Line ◽  
Hela Cells ◽  
Molecular Mechanisms ◽  
Inhibitory Concentration ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Cancer Drug ◽  
Drug Resistant ◽  
Resistant Cells

Cervical cancer is one of the most leading causes of women death. Currently, paclitaxel is still one of the main therapeutic regimens for cervical cancer patients. However, some patients developed to be paclitaxel-resistant. Hence, studies to find out the novel strategies to resolve this problem are important. Generating resistant cancer cell lines can be utilized as the potent tool to evaluate the efficacy of any therapeutic agent toward cancer drug-resistant problems. Current studies describing the methods to establish chemoresistance are lacking. Moreover, study in Indonesia conducting chemoresistance in cell line is limited. This study was aimed to elaborate the characteristics of HeLa cells during generation of paclitaxel-resistant cervical cancer cells. The parental HeLa cells were exposed to an escalating concentration of paclitaxel for a long time period. Subsequently, cells were divided into two groups for the evaluation of resistance characteristics. The values of inhibitory concentration 50 (IC50) and inhibitory concentration 90 (IC90) were analyzed using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Our data showed that the longer exposing periods of paclitaxel, the higher IC50 and IC90 values of HeLa cells are. IC90 of paclitaxel in HeLa Pac RB was increased from 69 pM, 440 pM, 2,561 pM and 10,337 pM on 0th, 1st, 2nd, 3rd and 4th months, respectively. Interestingly, the resistant cells were recovered to be paclitaxel-sensitive when they were not being continuously exposed to paclitaxel. In addition, the paclitaxel resistant cells become less sensitive against 5-FU but not doxorubicin, cisplatin and etoposide. We were able to generate cervical cancer HeLa paclitaxel-resistant cell line. These cell line could potentially be utilized for further studies in order to understand the molecular mechanisms of drug resistance in cervical cancer and as a tool for cancer drug discovery.Keywords: cervical cancer, drug resistant cell line, paclitaxel resistant cells, stepwise escalating concentration.

scholarly journals Radiosensitization by Kinase Inhibition Revealed by Phosphoproteomic Analysis of Pancreatic Cancer Cells

2020 ◽  
Vol 19 (10) ◽  
pp. 1649-1663
Author(s):  
Svenja Wiechmann ◽  
Elena Saupp ◽  
Daniela Schilling ◽  
Stephanie Heinzlmeir ◽  
Günter Schneider ◽  
...  
Keyword(s):  
Cell Lines ◽  
Molecular Mechanisms ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Actin Dynamics ◽  
Ductal Adenocarcinoma ◽  
Pancreatic Cancer Cells ◽  
Intrinsic Radiosensitivity ◽  
Resistant Cells

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers and known for its extensive genetic heterogeneity, high therapeutic resistance, and strong variation in intrinsic radiosensitivity. To understand the molecular mechanisms underlying radioresistance, we screened the phenotypic response of 38 PDAC cell lines to ionizing radiation. Subsequent phosphoproteomic analysis of two representative sensitive and resistant lines led to the reproducible identification of 7,800 proteins and 13,000 phosphorylation sites (p-sites). Approximately 700 p-sites on 400 proteins showed abundance changes after radiation in all cell lines regardless of their phenotypic sensitivity. Apart from recapitulating known radiation response phosphorylation markers such as on proteins involved in DNA damage repair, the analysis uncovered many novel members of a radiation-responsive signaling network that was apparent only at the level of protein phosphorylation. These regulated p-sites were enriched in potential ATM substrates and in vitro kinase assays corroborated 10 of these. Comparing the proteomes and phosphoproteomes of radiosensitive and -resistant cells pointed to additional tractable radioresistance mechanisms involving apoptotic proteins. For instance, elevated NADPH quinine oxidoreductase 1 (NQO1) expression in radioresistant cells may aid in clearing harmful reactive oxygen species. Resistant cells also showed elevated phosphorylation levels of proteins involved in cytoskeleton organization including actin dynamics and focal adhesion kinase (FAK) activity and one resistant cell line showed a strong migration phenotype. Pharmacological inhibition of the kinases FAK by Defactinib and of CHEK1 by Rabusertib showed a statistically significant sensitization to radiation in radioresistant PDAC cells. Together, the presented data map a comprehensive molecular network of radiation-induced signaling, improves the understanding of radioresistance and provides avenues for developing radiotherapeutic strategies.

Evaluation of possible mechanisms of phorbol ester stimulation of phosphatidylcholine synthesis in HeLa cells

1985 ◽  
Vol 63 (2) ◽  
pp. 145-151 ◽  
Author(s):  
H. W. Cook ◽  
D. E. Vance
Keyword(s):  
Fatty Acids ◽  
Phorbol Ester ◽  
Hela Cells ◽  
Phorbol Esters ◽  
Neutral Lipids ◽  
Cellular Fatty Acid ◽  
Fatty Acyl ◽  
Ctp:Phosphocholine Cytidylyltransferase ◽  
Intracellular Release ◽  
Acyl Chains

Phorbol esters, including 12-O-tetradecanoylphorbol-13-acetate (TPA) and 12,13-dibutyrylphorbol acetate, markedly stimulate the synthesis of phosphatidylcholine (PC) and the activity of CTP:phosphocholine cytidylyltransferase (CT) in cultured HeLa cells. Two possible mechanisms whereby the phorbol esters stimulated PC biosynthesis were investigated. One consideration was that phorbol esters may induce the release of fatty acyl chains from endogenous complex lipids; increased fatty acids or fatty acyl-CoAs could cause the translocation of CT from cytosol to microsomes and thereby increase the activity of the rate-limiting enzyme in PC synthesis. In HeLa cells prelabeled with [3H]oleate or [3H]arachidonate, phorbol ester treatment increased the redistribution of arachidonate in phospholipids and neutral lipids and release of label to the medium, but there was little effect on the cellular fatty acid pools with either of the labeled fatty acids or of the phorbol esters. A second possibility was that protein kinase C (PKC), a receptor for phorbol esters, might be involved in activation of CT activity. TPA stimulated the phosphorylation of cytosolic proteins of HeLa cells more than twofold during a 10- or 60-min incubation with 32Pi. However, an approximate sixfold purified preparation of PKC from rat brain did not stimulate the activity of partially purified (12- to 15-fold) CT; a slight inhibition, dependent on ATP but independent of Ca2+ and diolein, was observed. Our results suggest that intracellular release of free fatty acids or direct phosphorylation of CT by PKC probably do not account for the observed levels of stimulation by phorbol esters. Other possible mechanisms are discussed.

scholarly journals Discrete human dihydrofolate reductase gene transcripts present in polysomal RNA map with their 5' ends several hundred nucleotides upstream of the main mRNA start site.

1985 ◽  
Vol 5 (3) ◽  
pp. 493-500 ◽  
Author(s):  
J N Masters ◽  
G Attardi
Keyword(s):  
Cell Lines ◽  
Dihydrofolate Reductase ◽  
Transcription Initiation ◽  
Initiation Site ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Parental Line ◽  
Dhfr Gene ◽  
Reading Frame ◽  
Polyadenylic Acid

The 5' ends of dihydrofolate reductase (DHFR)-specific transcripts have been mapped in the 5'-flanking region of the amplified DHFR gene of the human methotrexate-resistant cell line 6A3 by primer extension and S1 protection experiments. The main 5' end, at position -71 relative to the first nucleotide of the DHFR reading frame, corresponds to the recently identified main transcription initiation site for the DHFR gene and pertains to transcripts representing approximately 99% of the DHFR-specific polysomal polyadenylic acid-containing RNA, and including the previously described DHFR mRNAs with sizes of 3.8, 1.0, and 0.8 kilobases. At least six other minor 5' ends have been mapped to nucleotide positions -449 to -480 upstream of the DHFR gene and pertain to approximately 1% of the DHFR-specific polysomal polyadenylic acid-containing RNA. These upstream initiating transcripts appear to include five major discrete species with sizes of 4.3, 3.8, 3.1, 2.1, and 1.0 kilobases and four minor ones with sizes of 7.3, 5.0, 1.4, and 0.8 kilobases. These species, with the exception of those of 3.1- and 2.1-kilobase sizes, also have been found in VA2-B cells, the parental line of 6A3, and in HeLa cells. The upstream initiating transcripts present in all three cell lines are increased in amount in 6A3 cells as compared with the other cell lines, in about the same proportion as the three identified DHFR mRNAs.

Tyrosine Kinase Proteins profiling of Nilotinib Resistant Chronic Myelogenous Leukemia Cells Unravels a Tyrosine Kinase-Mediated Bypass.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2175-2175
Author(s):  
Jean-Max Pasquet ◽  
Romain Gioia ◽  
Claire Drullion ◽  
Valerie Lagarde ◽  
Cedric Leroy ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Cell Line ◽  
Tyrosine Kinases ◽  
Kinase Activity ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Tyrosine Kinase Activity ◽  
Tyrosine Residues ◽  
Lyn Kinase ◽  
Resistant Cells

Abstract Abstract 2175 Poster Board II-152 Targeting the tyrosine kinase activity of Bcr-Abl is an attractive therapeutic strategy in Chronic Myeloid Leukemia (CML) and in Bcr-Abl positive Acute Lymphoblastic Leukemia. Whereas imatinib, a selective inhibitor of Bcr-Abl tyrosine kinase, is now used in frontline therapy for CML, second generation inhibitors such as nilotinib or dasatinib have been developed for the treatment of imatinib-resistant or –intolerant disease. We have shown that one of the mechanisms of resistance to nilotinib is an increasing expression of the p53/56 Lyn kinase, both at mRNA and protein level in cell lines. This result was confirmed in vivo in nilotinib-resistant CML patients (Mahon et al. Cancer Res., 2008, 68(23):9809-16.). To elucidate Lyn mediated-nilotinib resistance, a phosphoproteomic study was performed by Stable Isotope Labelling with Amino acid in Cell culture (SILAC) which highlights the potential role of downstream tyrosine kinases. Among different candidate proteinsThe Spleen tyrosine kinase Syk and the UFO family receptor tyrosine kinase Axl were the most relevant in the nilotinib resistant cell line as compared to the sensitive counterpart. Syk hyperphosphorylation was confirmed in the nilotinib resistant cell line using western blot at least on tyrosine residues Y323 and Y525/526, two critical tyrosine residues respectively involved in Lyn-mediated Syk phosphorylation and autophosphorylation-associated Syk activation. Lyn interacts with Syk as detected in Syk immunoprecipitates in nilotinib resistant cells. Furthermore, Syk-Lyn interaction is inhibited by dasatinib suggesting the requirement of Lyn kinase activity and Syk phosphorylation. Targeting Syk expression in nilotinib resistant cells by siRNA or tyrosine kinase activity by pharmacological inhibitors leads respectively to a partial (35%) or to a full restoration of nilotinib sensitivity. Moreover, the identification of Axl by SILAC is correlated to a 9 fold increase of its level of expression in the resistant cell line and the inhibition of Axl tyrosine kinase activity decreases proliferation of both nilotinib sensitive and resistant CML cells. All together these results disclose a new pathway for tyrosine kinase inhibitors resistance in CML involving at least the two Lyn downstream tyrosine kinases Syk and Axl. Disclosures: Mahon: Amgen: Honoraria; Novartis Pharma: Consultancy, Honoraria, Research Funding; Alexion: Consultancy, Honoraria.

scholarly journals Selection of mammalian cells resistant to a chloramphenicol analog.

1975 ◽  
Vol 65 (2) ◽  
pp. 492-498 ◽  
Author(s):  
R B Wallace ◽  
K B Freeman
Keyword(s):  
Protein Synthesis ◽  
Mammalian Cells ◽  
Mitochondrial Protein ◽  
Specific Inhibitor ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Triton X ◽  
Resistant Cells

This study describes the selection and preliminary characterization of mammalian cells resistant to 100 mug Tevenel/ml. Tevenel, the sulfamoyl analog of chloramphenicol, is a specific inhibitor of mitochondrial protein synthesis. After growth in suspension culture for 5 days in 100 mug Tevenel/ml and subsequent plating in 100 mug Tevenel/ml, LMTK- cells yielded resistant clones. As a control, L cells treated identically yielded no clones. Three resistant clones were chosen for study. Each resistant cell line had an identical growth rate in the presence and absence of 100 mug Tevenel/ml. By plating efficiency analysis, the resistant cells were found to be cross-resistant to D-chloramphenicol. The change responsible for resistance was found to be stable for at least 100 generations in the absence of the drug. Protein synthesis by isolated mitochondria of resistant cells was found to be less inhibited by concentrations of both Tevenel and D-chloramphenicol up to 200 mug/ml than the protein synthesis by LMTK- mitochondria. This resistance in vitro was not changed by incubation of the mitochondria in 0.01% Triton X-100.

scholarly journals Misregulation of translation drives prostate cancer drug resistance

2021 ◽  
Author(s):  
Emeline I. J. Lelong ◽  
France Hélène Joncas ◽  
Pauline Adjibade ◽  
Valerie ST.-Sauveur Grenier ◽  
Jean-Philippe Lambert ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Drug Resistance ◽  
Cell Line ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Cancer Drug ◽  
Biological Processes ◽  
Drug Resistant ◽  
Cancer Resistance ◽  
Resistant Cells

ABSTRACTEmerging evidence associates translation factors and regulators to tumorigenesis. Recent advances in our ability to perform global translatome analyses indicate that our understanding of translational changes in cancer resistance is still limited. Here, we characterize global translational changes that occur during the acquisition of prostate cancer (PCa) drug resistance. We generated a patient derived xenograft (PDX) model created from PCa cells to recapitulate key features of resistant PCa progression. From an enzalutamide-sensitive patient derived cell line (VCaP), we generated a castration resistant cell line (VCaPCRPC) and an enzalutamide resistant cell line (VCaPER). We performed Total and polyribosome-bound RNA sequencing and mass spectroscopy from both VCaPCRPC and VCaPER to reveal their respective translatomes. We found that in drug-resistant cells, RNAs associated to ribosomes were enriched for nuclear RNA and DNA binding related biological processes, whereas RNAs that are less associated showed enrichment for processes such as cell membrane and cell-cell junction related biological processes. These results were corroborated by mass spectrometry and suggest that translation is indeed affected during drug resistance. Furthermore, our analysis revealed enrichment of long non-coding RNAs associated to ribosomes, which may suggest aberrant translation or translation of novel peptides that can be considered as new biomarkers. Our findings thus point towards novel therapeutic avenues that may target drug-resistant cells.

scholarly journals Discrete human dihydrofolate reductase gene transcripts present in polysomal RNA map with their 5' ends several hundred nucleotides upstream of the main mRNA start site

1985 ◽  
Vol 5 (3) ◽  
pp. 493-500
Author(s):  
J N Masters ◽  
G Attardi
Keyword(s):  
Cell Lines ◽  
Dihydrofolate Reductase ◽  
Transcription Initiation ◽  
Initiation Site ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Parental Line ◽  
Dhfr Gene ◽  
Reading Frame ◽  
Polyadenylic Acid

The 5' ends of dihydrofolate reductase (DHFR)-specific transcripts have been mapped in the 5'-flanking region of the amplified DHFR gene of the human methotrexate-resistant cell line 6A3 by primer extension and S1 protection experiments. The main 5' end, at position -71 relative to the first nucleotide of the DHFR reading frame, corresponds to the recently identified main transcription initiation site for the DHFR gene and pertains to transcripts representing approximately 99% of the DHFR-specific polysomal polyadenylic acid-containing RNA, and including the previously described DHFR mRNAs with sizes of 3.8, 1.0, and 0.8 kilobases. At least six other minor 5' ends have been mapped to nucleotide positions -449 to -480 upstream of the DHFR gene and pertain to approximately 1% of the DHFR-specific polysomal polyadenylic acid-containing RNA. These upstream initiating transcripts appear to include five major discrete species with sizes of 4.3, 3.8, 3.1, 2.1, and 1.0 kilobases and four minor ones with sizes of 7.3, 5.0, 1.4, and 0.8 kilobases. These species, with the exception of those of 3.1- and 2.1-kilobase sizes, also have been found in VA2-B cells, the parental line of 6A3, and in HeLa cells. The upstream initiating transcripts present in all three cell lines are increased in amount in 6A3 cells as compared with the other cell lines, in about the same proportion as the three identified DHFR mRNAs.

scholarly journals Genomic Characterization of Cisplatin Response Uncovers Priming of Cisplatin-Induced Genes in a Resistant Cell Line

2021 ◽  
Vol 22 (11) ◽  
pp. 5814
Author(s):  
Hadar Golan Berman ◽  
Pooja Chauhan ◽  
Shira Shalev ◽  
Hiba Hassanain ◽  
Avital Parnas ◽  
...  
Keyword(s):  
Cell Line ◽  
Nucleotide Excision Repair ◽  
Cisplatin Resistance ◽  
Excision Repair ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Nucleotide Excision ◽  
Genomic Characterization ◽  
Resistant Cells

Cisplatin is a chemotherapy drug that kills cancer cells by damaging their DNA. In human cells, this damage is repaired primarily by nucleotide excision repair. While cisplatin is generally effective, many cancers exhibit initial or acquired resistance to it. Here, we studied cisplatin resistance in a defined cell line system. We conducted a comprehensive genomic characterization of the cisplatin-sensitive A2780 ovarian cancer cell line compared to A2780cis, its resistant derivative. The resistant cells acquired less damage, but had similar repair kinetics. Genome-wide mapping of nucleotide excision repair showed a shift in the resistant cells from global genome towards transcription-coupled repair. By mapping gene expression changes following cisplatin treatment, we identified 56 upregulated genes that have higher basal expression in the resistant cell line, suggesting they are primed for a cisplatin response. More than half of these genes are novel to cisplatin- or damage-response. Six out of seven primed genes tested were upregulated in response to cisplatin in additional cell lines, making them attractive candidates for future investigation. These novel candidates for cisplatin resistance could prove to be important prognostic markers or targets for tailored combined therapy in the future.

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