A recombinant CHSE-214 cell line expressing an Mx1 promoter–reporter system responds to both interferon type I and type II from salmonids and represents a versatile tool to study the IFN-system in teleost fish

Interferons (IFNs) are cytokines that have proinflammatory, antiviral, and immunomodulatory effects and play a central role during a host response to pathogens. The IFN family contains both type I and type II molecules. While there are a number of type I IFNs, there is only one type II IFN. Recently both type I and type II IFN genes have been cloned in salmonid fish and recombinant proteins produced showing IFN activity. We have stimulated an Atlantic salmon cell line (SHK-1) with both type I and type II recombinant salmonid IFNs and analyzed the transcriptional response by microarray analysis. Cells were exposed to recombinant IFNs for 6 or 24 h or left unexposed as controls. RNA was hybridized to an Atlantic salmon cDNA microarray (salmon 17K feature TRAITS/SGP array) in order to assess differential gene expression in response to IFN exposure. For IFN I and II, 47 and 72 genes were stimulated, respectively; most genes were stimulated by a single IFN type, but some were affected by both IFNs, indicating coregulation of the IFN response in fish. Real-time PCR analysis was employed to confirm the microarray results for selected differentially expressed genes in both a cell line and primary leukocyte cultures.

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Type II RAF inhibition predicts superior ERK suppression compared to type I RAF inhibition in different BRAF mutant types recurrently found in lung cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.15_suppl.e23154 ◽

2017 ◽

Vol 35 (15_suppl) ◽

pp. e23154-e23154

Author(s):

Philippe Giron ◽

Amir Noeparast ◽

Sylvia De Brakeleer ◽

Ulrike De Ridder ◽

Erik Teugels ◽

...

Keyword(s):

Lung Cancer ◽

Cell Line ◽

Single Agent ◽

Mek Inhibitor ◽

Expression Vectors ◽

Type I ◽

Type Ii ◽

Braf Mutations ◽

Braf Mutant ◽

Mutant Braf

e23154 Background: Somatic driver BRAF mutations account for 6-8% of lung cancers. As opposed to melanoma in which V600E mutant BRAF predominates, the majority of lung cancer-derived BRAF mutations are non-V600. Yet, the efficacy of RAF-inhibitors and the possible resistance mechanisms in non-V600 BRAF mutant cells remain to be uncovered. Recently, we have shown that non-V600 BRAF mutations recurrently found in lung cancer predict sensitivity to the combination of type I RAF inhibitor Dabrafenib and a MEK inhibitor Trametinib. As a single agent, Dabrafenib shows only weak suppression of mutant BRAF-induced ERK signaling; moreover it can induce ERK paradoxical activation in CRAF overexpressing cells. Methods: Several recombinant BRAF expression vectors were generated by performing site-directed mutagenesis. We compared the effects of Dabrafenib and a type II RAF inhibitor (AZD-628) at clinically relevant dose as single agents or in combination with MEK inhibitor Trametinib on ERK activity in HEK293T cells expressing several tumor-derived BRAF mutants and a non-V600 BRAF mutant lung cancer cell line (H1666). Viability and caspase3/7 activation assays were performed using the H1666 cell line model. Results: In contrast to Dabrafenib, AZD-628 does not induce paradoxical ERK activation in CRAF expressing cells. Increased CRAF expression desensitizes BRAF-mutant expressing cells to Dabrafenib but not to AZD-628. Notably, AZD-628 has superior ERK-inhibitory effect in HEK293T cells co-expressing several different BRAF-mutants with CRAF and in H1666 cells. Combination of Trametinib and AZD-628 has superior MEK-inhibitory and pro-apoptotic effect in H1666 cells compared to combined Trametinib/Dabrafenib. Moreover, upon down titration of the RAF inhibitors with a steady dose of Trametinib, AZD-628 resulted in overall stronger effect on viability compared to Dabrafenib. Conclusions: In our in vitro model, we obtained strong indications that at conventional doses, type II RAF-inhibitor AZD628 is superior to type I RAF-inhibitor Dabrafenib in combination with MEK inhibitor Trametinib for the treatment of non-V600 BRAF mutant lung cancer.

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Organization and genesis of dihydrofolate reductase amplicons in the genome of a methotrexate-resistant Chinese hamster ovary cell line

Molecular and Cellular Biology ◽

10.1128/mcb.8.6.2316-2327.1988 ◽

1988 ◽

Vol 8 (6) ◽

pp. 2316-2327

Author(s):

C Ma ◽

J E Looney ◽

T H Leu ◽

J L Hamlin

Keyword(s):

Cell Line ◽

Dihydrofolate Reductase ◽

Chinese Hamster Ovary ◽

Chinese Hamster ◽

Resistant Cell ◽

Pulse Field ◽

Ovary Cell ◽

Type I ◽

Type Ii ◽

Gradient Gel Electrophoresis

We have recently isolated overlapping recombinant cosmids that represent the equivalent of two complete dihydrofolate reductase (dhfr) amplicon types from the methotrexate-resistant Chinese hamster ovary (CHO) cell line CHOC 400. In the work described in this report, we used pulse-field gradient gel electrophoresis to analyze large SfiI restriction fragments arising from the amplified dhfr domains. The junction between the 260-kilobase type I amplicons (which are arranged in head-to-tail configurations in the genome) has been localized, allowing the construction of a linear map of the parental dhfr locus. We also show that the 220-kilobase type II amplicons are arranged as inverted repeat structures in the CHOC 400 genome and arose from the type I sequence relatively early in the amplification process. Our data indicate that there are a number of minor amplicon types in the CHOC 400 cell line that were not detected in previous studies; however, the type II amplicons represent ca. 75% of all the amplicons in the CHOC 400 genome. Both the type I and type II amplicons are shown to be composed entirely of sequences that were present in the parental dhfr locus. Studies of less resistant cell lines show that initial amplicons can be larger than those observed in CHOC 400. Once established, a given amplicon type appears to be relatively stable throughout subsequent amplification steps. We also present a modification of an in-gel renaturation method that gives a relatively complete picture of the size and variability of amplicons in the genome.

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STL-based Analysis of TRAIL-induced Apoptosis Challenges the Notion of Type I/Type II Cell Line Classification

PLoS Computational Biology ◽

10.1371/journal.pcbi.1003056 ◽

2013 ◽

Vol 9 (5) ◽

pp. e1003056 ◽

Cited By ~ 15

Author(s):

Szymon Stoma ◽

Alexandre Donzé ◽

François Bertaux ◽

Oded Maler ◽

Gregory Batt

Keyword(s):

Cell Line ◽

Type I ◽

Type Ii ◽

Type Ii Cell ◽

Induced Apoptosis ◽

Line Classification

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Activin and inhibin have antagonistic effects on ligand-dependent heteromerization of the type I and type II activin receptors and human erythroid differentiation.

Molecular and Cellular Biology ◽

10.1128/mcb.17.3.1682 ◽

1997 ◽

Vol 17 (3) ◽

pp. 1682-1691 ◽

Cited By ~ 123

Author(s):

J J Lebrun ◽

W W Vale

Keyword(s):

Cell Line ◽

Transforming Growth Factor ◽

Erythroid Differentiation ◽

Stable Cell Line ◽

Type I ◽

Dependent Manner ◽

Type Ii ◽

Tgf Beta ◽

Activin Receptor ◽

Antagonistic Effects

Activins and inhibins belong to the transforming growth factor beta (TGF-beta)-like superfamily and exert their effects on a broad range of cellular targets by modulating cell differentiation and proliferation. Members of this family interact with two structurally related classes of receptors (type I and type II), both containing a serine/threonine kinase domain. When expressed alone, the type II but not the type I activin receptor can bind activin. However, the presence of a type I receptor is required for signaling. For TGF-beta1, ligand binding to the type II receptor results in the recruitment and transphosphorylation of the type I receptor. Transient overexpression of the two types of activin receptor results in ligand-independent receptor heteromerization and activation. Nevertheless, activin addition to the transfected cells increased complex formation between the two receptors, suggesting a mechanism of action similar to that observed for the TGF-beta receptor. In the present study, we generated a stable cell line, overexpressing the two types of activin receptor upon induction, in the human erythroleukemia cell line K562. We demonstrate here that activin specifically induces heteromer formation between the type I and type II receptors in a time-dependent manner. Using this stable line, we analyzed the effects of activin and inhibin on human erythroid differentiation. Our results indicate that activin signal transduction mediated through its type I and type II receptors results in an increase in the hemoglobin content of the cells and limits their proliferation. Finally, using cell lines that can be induced to overexpress ActRII and ActRIB or ActRIB only, we show that the inhibin antagonistic effects on activin-induced biological responses are mediated through a competition for the type II activin receptor but also require the presence of an inhibin-specific binding component.

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Organization and genesis of dihydrofolate reductase amplicons in the genome of a methotrexate-resistant Chinese hamster ovary cell line.

Molecular and Cellular Biology ◽

10.1128/mcb.8.6.2316 ◽

1988 ◽

Vol 8 (6) ◽

pp. 2316-2327 ◽

Cited By ~ 33

Author(s):

C Ma ◽

J E Looney ◽

T H Leu ◽

J L Hamlin

Keyword(s):

Cell Line ◽

Dihydrofolate Reductase ◽

Chinese Hamster Ovary ◽

Chinese Hamster ◽

Resistant Cell ◽

Pulse Field ◽

Ovary Cell ◽

Type I ◽

Type Ii ◽

Gradient Gel Electrophoresis

We have recently isolated overlapping recombinant cosmids that represent the equivalent of two complete dihydrofolate reductase (dhfr) amplicon types from the methotrexate-resistant Chinese hamster ovary (CHO) cell line CHOC 400. In the work described in this report, we used pulse-field gradient gel electrophoresis to analyze large SfiI restriction fragments arising from the amplified dhfr domains. The junction between the 260-kilobase type I amplicons (which are arranged in head-to-tail configurations in the genome) has been localized, allowing the construction of a linear map of the parental dhfr locus. We also show that the 220-kilobase type II amplicons are arranged as inverted repeat structures in the CHOC 400 genome and arose from the type I sequence relatively early in the amplification process. Our data indicate that there are a number of minor amplicon types in the CHOC 400 cell line that were not detected in previous studies; however, the type II amplicons represent ca. 75% of all the amplicons in the CHOC 400 genome. Both the type I and type II amplicons are shown to be composed entirely of sequences that were present in the parental dhfr locus. Studies of less resistant cell lines show that initial amplicons can be larger than those observed in CHOC 400. Once established, a given amplicon type appears to be relatively stable throughout subsequent amplification steps. We also present a modification of an in-gel renaturation method that gives a relatively complete picture of the size and variability of amplicons in the genome.

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