A single-step FACS sorting strategy in conjunction with fluorescent vital dye imaging efficiently assures clonality of biopharmaceutical production cell lines

2017 ◽  
Vol 12 (6) ◽  
pp. 1700002 ◽  
Author(s):  
Jürgen Fieder ◽  
Patrick Schulz ◽  
Ingo Gorr ◽  
Harald Bradl ◽  
Till Wenger
Keyword(s):  
Cell Lines ◽  
Single Step ◽  
Vital Dye ◽  
Biopharmaceutical Production ◽  
Production Cell ◽  
Facs Sorting

Breaking limitations of complex culture media: Functional non-viral miRNA delivery into pharmaceutical production cell lines

2013 ◽  
Vol 168 (4) ◽  
pp. 589-600 ◽  
Author(s):  
Simon Fischer ◽  
Andreas Wagner ◽  
Aron Kos ◽  
Armaz Aschrafi ◽  
René Handrick ◽  
...  
Keyword(s):  
Cell Lines ◽  
Culture Media ◽  
Complex Culture ◽  
Pharmaceutical Production ◽  
Production Cell

scholarly journals Synthesis, Anticancer Activity, and Molecular Modeling of New Halogenated Spiro[pyrrolidine-thiazolo-oxindoles] Derivatives

2020 ◽  
Vol 10 (6) ◽  
pp. 2170 ◽  
Author(s):  
Mohammad Shahidul Islam ◽  
Abdullah Mohammed Al-Majid ◽  
Fardous F. El-Senduny ◽  
Farid A. Badria ◽  
A. F. M. Motiur Rahman ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
Cancer Cell Lines ◽  
Single Step ◽  
One Pot ◽  
Antiproliferative Effects ◽  
Physiochemical Parameters ◽  
Hct 116 ◽  
Mcf 7

A one-pot, single-step, and an atom-economical process towards the synthesis of highly functionalized spirooxindoles analogues was efficiently conducted to produce a satisfactory chemical yields (70–93%) with excellent relative diastereo-, and regio-selectivity. An in vitro antiproliferative assay was carried out on different cancer cell lines to evaluate the biological activity of the synthesized tetrahydro-1’H-spiro[indoline-3,5’-pyrrolo[1,2-c]thiazol]-2-one 5a–n. The prepared hybrids were then tested in vitro for their antiproliferative effects against three cancer cell lines, namely, HepG2 (liver cancer), MCF-7 (breast cancer), and HCT-116 (colon cancer). The spirooxindole analogue 5g exhibited a broad activity against HepG2, MCF-7, and HCT-116 cell lines of liver, breast, and colorectal cancers when compared to cisplatin. Modeling studies including shape similarity, lipophilicity scores, and physicochemical parameters were calculated. The results of this study indicated that spirooxindole analogue 5g retained a good physiochemical parameters with acceptable lipophilicity scores.

Microwave-assisted Single Step Cinnamic Acid Derivatization and Evaluation for Cytotoxic Potential

2020 ◽  
Vol 21 (3) ◽  
pp. 236-243
Author(s):  
Sonali Mishra ◽  
Shilpi Singh ◽  
Arif Ali ◽  
Amit C. Gupta ◽  
Karuna Shanker ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cinnamic Acid ◽  
Human Cancer ◽  
Single Step ◽  
Breast Adenocarcinoma ◽  
Microwave Assisted Synthesis ◽  
Hek 293 ◽  
Anticancer Properties ◽  
Microwave Assisted

Background: Phenylpropylene biosynthesis pathway plays a crucial role in the vanillin and their derivative(s) production in the plants. The intermediate of vanillin synthesis i.e. cinnamic acid (CA) is converted into 2-Hydroxy 4-MethoxyBenzaldehyde (HMB) in Decalepis arayalpathra having a number of therapeutic value. Objective : Microwave-assisted modifications in cinnamic acid were planned for potential anticancer properties with better yield and efficiency. The present study also confirms the presence of HMB and its precursor i.e. cinnamic acid in D. arayalpathra tubers. Methods: We used a single step Microwave Assisted Synthesis (MAS) to modify cinnamic acid, and then examined the synthetic and natural cinnamic acid derivatives anticancer potential against six human cancer (K-562, WRL-68, A549, A431, MCF-7, and COLO-201) and two normal (L-132 and HEK-293) cell lines at 2, 10 and 50 µg/ml concentrations. Results: β-bromostyrene and β -nitrostyrene have shown inhibition with IC50 values ranging 0.10-21 µM and 0.03-0.06 µM, respectively to the cancer cell lines. β-bromostyrene was the most potent anticancer derivative of CA with better cellular safety and biocompatibility. Conclusions: The present study of microwave-assisted synthesis demonstrates a single-step modification in cinnamic acid. MAS is a fast, reliable, and robust method. The resultant compounds have shown in-vitro anticancer activity against human lung carcinoma and breast adenocarcinoma.

scholarly journals Herpesvirus Entry Mediator HVEM Mediates Cell-Cell Spread in BHK(TK−) Cell Clones

1998 ◽  
Vol 72 (2) ◽  
pp. 1411-1417 ◽  
Author(s):  
Richard J. Roller ◽  
Daniel Rauch
Keyword(s):  
Cell Lines ◽  
Pseudorabies Virus ◽  
Cell Types ◽  
Single Step ◽  
Cell Clones ◽  
Step Growth ◽  
Simplex Virus ◽  
Cell Spread ◽  
Hsv 1 ◽  
Cell Cell

ABSTRACT 95-19 and US11cl19.3 are BHK(TK−)-derived cell lines that are highly resistant to postattachment entry of herpes simplex virus type 1 (HSV-1) and HSV-2 but not to later steps in single-step replication. The resistance properties of these two cell types are not identical. US11cl19.3 cells are fully susceptible to pseudorabies virus (PRV), as shown by single-step growth experiments, whereas 95-19 cells are resistant to entry of free PRV but not to entry by cell-cell spread. We have tested the ability of HVEM to overcome the block to infection in both cell lines following transient and stable transfection. HVEM was able to mediate entry of free HSV-1 into both cell lines, as shown by an increase in the number of β-galactosidase-expressing cells in cultures transiently transfected with an HVEM expression plasmid and infected withlacZ-expressing HSV-1. In stably transfected 95-19 cells, HVEM enhanced infection by free HSV-1, as shown by an increase in the number of infectious centers obtained following infection. In both cell types, HVEM strongly enhanced entry of HSV-1 and HSV-2 by cell-cell spread, suggesting that HVEM can function as an entry mediator both in entry of free virus and in entry by cell-cell spread.

A Sensitive Molecular Method for Detection of Disseminating Hematopoietic Tumor Cells by Using Specific Epigenetic DNA Methylation Biomarkers

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4850-4850
Author(s):  
Michael X Wang ◽  
Xiaohui Zhao ◽  
Srilatha Nalluri ◽  
Huan-You Wang ◽  
Kristen H. Taylor ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bone Marrow ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Genomic Dna ◽  
Cpg Island ◽  
Lymphoblastic Leukemia ◽  
Single Step ◽  
Tumor Cell Lines

Abstract Background: Most cancer deaths are caused by hematogenous spread and subsequent metastasis. Emerging data indicates that the presence of circulating tumor cells in peripheral blood (CTC) and disseminating tumor cells in bone marrow (DTC) is an early event in tumorigenesis. These circulating or disseminating rare tumor cells may represent a distinct clone with cancer stem cell properties (metastatic stem cells). Clinically, the presence of CTC and DTC is relevant to overt metastases. Early detection and characterization of these rare biologically distinct tumor cells with sensitive and specific methods provide vital information for cancer biology and early clinical intervention and thus improve patients’ survival. Method Design: Genomic DNA was extracted from patient blood, bone marrow aspirate specimens and cancer cell lines prior to digestion with 4 methylation sensitive restriction enzymes. Hypermethylated CpG island regions in tumors, in contrast to their counterpart in normal cells which are completely digested, remain resistant to digestion and therefore can be differentially amplified by PCR. Thus, the specific PCR products on the gel represent the specific methylation loci in the tumor cells in the patient specimens. Results: First, we tested a total of 26 cancer cell lines including 17 hematopoietic tumors and 9 solid tumors. The hematopoietic tumor cell lines represent a spectrum of B-cell malignancies, T-cell lymphoblastic leukemia and acute myeloid leukemia. The solid tumor cell lines represent the major anatomic sites of human carcinoma including lung, colon, breast, prostate, ovarian and skin (melanoma). We found multiple DNA methylation markers specific for lymphoid, myeloid and solid tumors. By using DLC-1 (deleted in liver cancer-1), a tumor suppressor gene, as a single marker, CpG island methylation was detected in 13 out of 17 hematopoietic tumor cell lines (76%) and 6 out of 9 solid tumor cell lines (67%). We then used B-cell acute lymphoblastic leukemia (B-ALL) as a testing case to verify the method in a total of 135 clinical specimens. DLC-1 methylation was detected in 64% and 54% of diagnostic bone marrow aspirates and peripheral blood specimens, respectively, but none was detected in normal or non-leukemic bone marrow or blood control samples. By adding two additional methylation markers PCDHGA12 and CDH1, greater than 90% of B-ALL patients can be detected. We also traced 4 B-ALL cases and 4 follicular lymphoma cases up to 10 years retrospectively and found that the DLC-1 methylation is exactly correlated with patient clinical status. Lastly, by mixing normal and leukemic cell genomic DNA, analytic sensitivity was determined as 0.1% or 10−3 in a single-step PCR and 0.00001% or 10−6 in a nested PCR suggesting that this method is capable of detecting as few as 5 leukemic cells in a single-step PCR. Conclusion: By utilizing tumor specific DNA methylation marker(s), we have developed a simple, highly sensitive and specific gel-based PCR assay, namely Multiple Methylation Sensitive Enzyme Restriction PCR (MSR-PCR) for detection of CTC and DTC from blood and bone marrow of majority of hematopoietic malignancies. The method can potentially be used for early diagnosis and molecular monitoring in vast majority of clinical cancer patients.

scholarly journals Griseofulvin resistance mutation of Chinese hamster ovary cells that affects the apparent molecular weight of a congruent to 200,000-dalton protein.

1984 ◽  
Vol 4 (9) ◽  
pp. 1761-1768 ◽  
Author(s):  
R S Gupta
Keyword(s):  
Molecular Weight ◽  
Cell Lines ◽  
Peptide Mapping ◽  
Chinese Hamster Ovary Cells ◽  
Resistance Mutation ◽  
Chinese Hamster ◽  
Apparent Molecular Weight ◽  
Resistant Mutant ◽  
Single Step ◽  
Cross Resistance

A single-step griseofulvin-resistant mutant (GrsR-4) of CHO cells which exhibit very specific cross-resistance towards certain microtubule inhibitors showed the absence of a protein of molecular weight congruent to 200,000 (designated P5) and the concomitant presence of a new protein spot, M5, of lower molecular weight (Mr congruent to 180,000) which is not present in other cell lines. Peptide mapping studies showed that proteins P5 and M5 are related to each other and that M5 may be missing a peptide fragment present in P5. In GrsR-4 X GrsS cell hybrids, both P5 and M5 were present in equal amounts, which provided evidence against post-translation mechanisms in the origin of M5 and indicated that the GrsR-4 mutant most likely contains a nonsense mutation in the structural gene for protein P5, which causes its premature termination and leads to the formation of M5. Our studies also showed that in different Chinese hamster cell lines the two alleles of the protein P5 are nonidentical and make protein products which differ from each other in isoelectric points. It is suggested that protein P5 and its isoelectric variant P6 may constitute microtubule-associated proteins.

scholarly journals Properties of single-step mutants of Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate.

1983 ◽  
Vol 3 (11) ◽  
pp. 2089-2098 ◽  
Author(s):  
J Zieg ◽  
C E Clayton ◽  
F Ardeshir ◽  
E Giulotto ◽  
E A Swyryd ◽  
...  
Keyword(s):  
Cell Lines ◽  
Syrian Hamster ◽  
Resistant Cell ◽  
Single Step ◽  
Similar Degree ◽  
Fluctuation Analysis ◽  
Aspartate Transcarbamylase ◽  
Resistant Lines ◽  
Cad Gene

Eleven independent lines of Syrian hamster cells were selected by using very low levels of N-(phosphonacetyl)-L-aspartate (PALA), an inhibitor of aspartate transcarbamylase. The protocol employed insured that each resistant cell arose during one of the last divisions before selection was applied. Cells of each mutant line contained an amplification of the structural gene for CAD, a trifunctional protein which includes aspartate transcarbamylase and two other enzymes of UMP biosynthesis. Strikingly, despite the minimal selection employed, the degree of amplification of the CAD gene was 6 to 10 times the normal diploid number in all 11 cases. In situ hybridization indicated that the amplified CAD genes were almost always present at a single chromosomal site in each line. Therefore, one of the two alleles was amplified 11- to 19-fold. The rates at which cells became resistant to PALA, determined by fluctuation analysis, were 100 times less dependent on drug concentration than were the frequencies of resistant cells in steady-state populations. The relatively shallow dependence of this rate upon PALA concentration is consistent with our independent observation that most events gave rise to a similar degree of amplification. In six of six cell lines examined, the levels of CAD mRNA and aspartate transcarbamylase activity were elevated two- to fourfold. These lines were resistant to PALA concentrations 20- to 80-fold higher than the ones used for selection. The organization of amplified DNA was examined by hybridizing Southern blots with cloned DNA fragments containing amplified sequences, previously isolated from two cell lines resistant to high levels of PALA. A contiguous region of DNA approximately 44 kilobases long which included the CAD gene was amplified in five of five single-step mutants examined. Outside this region, these mutants shared amplified sequences with only one of the two highly resistant lines.

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