C7-07: Presence of amphiregulin autocrine-loop predicts in vitro sensitivity of EGFR wild type NSCLC and HNSCC cell lines to gefitinib and cetuximab

Background: Cell migration is an important step involved in heart regeneration and many cardiovascular diseases. However, cell migration dynamics in vivo is poorly understood due to the challenges from mammal hearts, which are opaque and fast beating, and thus individual cardiac cells cannot be imaged or tracked. Aims: In this study, cell migration dynamics in the heart is recorded with a novel strategy, in which fluorescence protein-tagged collagen is secreted from cells and deposited into extracellular matrix, forming visible trails when cells are moving in tissues. As a proof-of-concept, transplanted migration dynamics of cardiac progenitor cells in mouse hearts were investaged. Methods: Stable cell lines expressing mCherry-tagged type I collagen were generated from isolated cardiac progenitor cells, ABCG2 + CD45 - CD31 - cells (side populations), or c-kit + CD45 - CD31 - cells (c-kit + CPCs). The cell migration dynamics were monitored and measured based on the cell trails after cell transplantation into mouse tissues. Results: The stable cell lines form red cell trails both in vitro and in vivo (Fig. 1A & 1B, Green: GFP; Red: mCherry-collagen I, Blue: DAPI, bar: 50 microns). In culture dishes, the cells form visible cell trails of fluorescence protein. The cell moving directions are random, with a speed of 288 +/- 79 microns/day (side populations, n=3) or 143 +/-37 microns/day (c-kit + CPCs, n=3). After transplantation into wild-type mouse hearts, the cells form highly tortuous trails along the gaps between the heart muscle fibers. Angle between a cell trail and a muscle fiber is 16+/-16 degree (n=3). Side populations migrate twice as fast as c-kit+ CPCs in the heart (16.0 +/-8.7 microns/day vs. 8.1+/-0.0 microns/day, n=3, respectively), 18 time slower than the respective speeds in vitro . Additionally, side populations migrate significantly faster in the heart than in the skeletal muscles (26.4+/-5.8 microns/day, n=3). The side populations move significantly faster in immunodeficient mouse hearts (36.7+/-13.3 microns/day, n=3, typically used for studying cell therapies) than in wild-type mouse hearts. Conclusion: For the first time, cell migration dynamics in living hearts is monitored and examined with genetically modified cell lines. This study may greatly advance the fields of cardiovascular biology.

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Serotonin 5-HT2C Receptor Cys23Ser Single Nucleotide Polymorphism Associates with Receptor Function and Localization In Vitro

Scientific Reports ◽

10.1038/s41598-019-53124-2 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Michelle A. Land ◽

Holly L. Chapman ◽

Brionna D. Davis-Reyes ◽

Daniel E. Felsing ◽

John A. Allen ◽

...

Keyword(s):

Single Nucleotide Polymorphism ◽

Subcellular Localization ◽

Cell Lines ◽

Intracellular Signaling ◽

Calcium Release ◽

Wild Type ◽

Nucleotide Polymorphism ◽

Single Nucleotide ◽

Recycling Pathway

Abstract A non-synonymous single nucleotide polymorphism of the human serotonin 5-HT2C receptor (5-HT2CR) gene that converts a cysteine to a serine at amino acid codon 23 (Cys23Ser) appears to impact 5-HT2CR pharmacology at a cellular and systems level. We hypothesized that the Cys23Ser alters 5-HT2CR intracellular signaling via changes in subcellular localization in vitro. Using cell lines stably expressing the wild-type Cys23 or the Ser23 variant, we show that 5-HT evokes intracellular calcium release with decreased potency and peak response in the Ser23 versus the Cys23 cell lines. Biochemical analyses demonstrated lower Ser23 5-HT2CR plasma membrane localization versus the Cys23 5-HT2CR. Subcellular localization studies demonstrated O-linked glycosylation of the Ser23 variant, but not the wild-type Cys23, may be a post-translational mechanism which alters its localization within the Golgi apparatus. Further, both the Cys23 and Ser23 5-HT2CR are present in the recycling pathway with the Ser23 variant having decreased colocalization with the early endosome versus the Cys23 allele. Agonism of the 5-HT2CR causes the Ser23 variant to exit the recycling pathway with no effect on the Cys23 allele. Taken together, the Ser23 variant exhibits a distinct pharmacological and subcellular localization profile versus the wild-type Cys23 allele, which could impact aspects of receptor pharmacology in individuals expressing the Cys23Ser SNP.

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The Selection of NFκB Inhibitors to Block Inflammation and Induce Sensitisation to FasL-Induced Apoptosis in HNSCC Cell Lines Is Critical for Their Use as a Prospective Cancer Therapy

International Journal of Molecular Sciences ◽

10.3390/ijms20061306 ◽

2019 ◽

Vol 20 (6) ◽

pp. 1306 ◽

Cited By ~ 5

Author(s):

Mario Scheurer ◽

Roman Brands ◽

Mohamed El-Mesery ◽

Stefan Hartmann ◽

Urs Müller-Richter ◽

...

Keyword(s):

Cancer Therapy ◽

Cell Lines ◽

In Vitro Study ◽

Keratinocyte Cell Line Hacat ◽

Keratinocyte Cell ◽

Nfκb Pathway ◽

Induced Apoptosis ◽

Selection Of ◽

Hnscc Cell

Inflammation is a central aspect of tumour biology and can contribute significantly to both the origination and progression of tumours. The NFκB pathway is one of the most important signal transduction pathways in inflammation and is, therefore, an excellent target for cancer therapy. In this work, we examined the influence of four NFκB inhibitors—Cortisol, MLN4924, QNZ and TPCA1—on proliferation, inflammation and sensitisation to apoptosis mediated by the death ligand FasL in the HNSCC cell lines PCI1, PCI9, PCI13, PCI52 and SCC25 and in the human dermal keratinocyte cell line HaCaT. We found that the selection of the inhibitor is critical to ensure that cells do not respond by inducing counteracting activities in the context of cancer therapy, e.g., the extreme IL-8 induction mediated by MLN4924 or FasL resistance mediated by Cortisol. However, TPCA1 was qualified by this in vitro study as an excellent therapeutic mediator in HNSCC by four positive qualities: (1) proliferation was inhibited at low μM-range concentrations; (2) TNFα-induced IL-8 secretion was blocked; (3) HNSCC cells were sensitized to TNFα-induced cell death; and (4) FasL-mediated apoptosis was not disrupted.

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Interleukin-11 acts as an autocrine growth factor for human megakaryoblastic cell lines

Blood ◽

10.1182/blood.v81.4.889.889 ◽

1993 ◽

Vol 81 (4) ◽

pp. 889-893 ◽

Cited By ~ 74

Author(s):

S Kobayashi ◽

M Teramura ◽

I Sugawara ◽

K Oshimi ◽

H Mizoguchi

Keyword(s):

Growth Factor ◽

Cell Lines ◽

Antisense Oligonucleotides ◽

Leukemia Cell ◽

Dependent Manner ◽

Autocrine Growth ◽

Autocrine Loop ◽

Autocrine Growth Factor ◽

Interleukin 11

Abstract The cytokine interleukin-11 (IL-11) promotes normal human megakaryocytopoiesis in vitro. However, its role in abnormal megakaryocytopoiesis is not well known. Accordingly, we studied its effects on human megakaryoblastic cell lines CMK and Meg-J. IL-11 stimulated the proliferation of CMK and Meg-J in a dose-dependent manner with maximal growth being achieved at the concentration of 50 and 500 ng/mL, respectively. The growth of the cells was inhibited by anti-IL-11 antibody and IL-11 antisense oligonucleotides. IL-11 transcripts were detected in these two cell lines using a reverse transcriptase-polymerase chain reaction assay. These findings indicate that IL-11 might be an autocrine growth factor for megakaryoblastic cells. IL-11 transcripts also existed in other leukemia cell lines: HL- 60, U937, and K562. However, the growth of these cells was not stimulated by IL-11, and was not inhibited by IL-11 antisense oligonucleotides. These results suggested that IL-11 might regulate malignant cells of the megakaryocytic lineage, in part by an autocrine loop.

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Unstable Spinocerebellar Ataxia Type 10 (ATTCT)·(AGAAT) Repeats Are Associated with Aberrant Replication at the ATX10 Locus and Replication Origin-Dependent Expansion at an Ectopic Site in Human Cells

Molecular and Cellular Biology ◽

10.1128/mcb.01276-07 ◽

2007 ◽

Vol 27 (22) ◽

pp. 7828-7838 ◽

Cited By ~ 35

Author(s):

Guoqi Liu ◽

John J. Bissler ◽

Richard R. Sinden ◽

Michael Leffak

Keyword(s):

Molecular Mechanism ◽

Cell Lines ◽

Spinocerebellar Ataxia ◽

Replication Origin ◽

Spinocerebellar Ataxia Type ◽

Dna Unwinding ◽

Wild Type ◽

Normal Length ◽

Population Doublings

ABSTRACT Spinocerebellar ataxia type 10 (SCA10) is associated with expansion of (ATTCT) n repeats (where n is the number of repeats) within the ataxin 10 (ATX10/E46L) gene. The demonstration that (ATTCT) n tracts can act as DNA unwinding elements (DUEs) in vitro has suggested that aberrant replication origin activity occurs at expanded (ATTCT) n tracts and may lead to their instability. Here, we confirm these predictions. The wild-type ATX10 locus displays inefficient origin activity, but origin activity is elevated at the expanded ATX10 loci in patient-derived cells. To test whether (ATTCT) n tracts can potentiate origin activity, cell lines were constructed that contain ectopic copies of the c-myc replicator in which the essential DUE was replaced by ATX10 DUEs with (ATTCT) n . ATX10 DUEs containing (ATTCT)27 or (ATTCT)48, but not (ATTCT)8 or (ATTCT)13, could substitute functionally for the c-myc DUE, but (ATTCT)48 could not act as an autonomous replicator. Significantly, chimeric c-myc replicators containing ATX10 DUEs displayed length-dependent (ATTCT) n instability. By 250 population doublings, dramatic two- and fourfold length expansions were observed for (ATTCT)27 and (ATTCT)48 but not for (ATTCT)8 or (ATTCT)13. These results implicate replication origin activity as one molecular mechanism associated with the instability of (ATTCT) n tracts that are longer than normal length.

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Pore mutants of HERG and KvLQT1 downregulate the reciprocal currents in stable cell lines

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00479.2009 ◽

2010 ◽

Vol 299 (5) ◽

pp. H1525-H1534 ◽

Cited By ~ 18

Author(s):

Xiao-Qin Ren ◽

Gong Xin Liu ◽

Louise E. Organ-Darling ◽

Renjian Zheng ◽

Karim Roder ◽

...

Keyword(s):

Cell Lines ◽

Rabbit Model ◽

Chinese Hamster ◽

Expression Vectors ◽

Delayed Rectifier ◽

Wild Type ◽

Cho Cell ◽

Surface Plasmon Resonance Analysis ◽

Transgenic Rabbit

We previously reported a transgenic rabbit model of long QT syndrome based on overexpression of pore mutants of repolarizing K+ channels KvLQT1 (LQT1) and HERG (LQT2).The transgenes in these rabbits eliminated the slow and fast components of the delayed rectifier K+ current ( IKs and IKr, respectively), as expected. Interestingly, the expressed pore mutants of HERG and KvLQT1 downregulated the remaining reciprocal repolarizing currents, IKs and IKr, without affecting the steady-state levels of the native polypeptides. Here, we sought to further explore the functional interactions between HERG and KvLQT1 in heterologous expression systems. Stable Chinese hamster ovary (CHO) cell lines expressing KvLQT1-minK or HERG were transiently transfected with expression vectors coding for mutant or wild-type HERG or KvLQT1. Transiently expressed pore mutant or wild-type KvLQT1 downregulated IKr in HERG stable CHO cell lines by 70% and 44%, respectively. Immunostaining revealed a severalfold lower surface expression of HERG, which could account for the reduction in IKr upon KvLQT1 expression. Deletion of the KvLQT1 NH2-terminus did not abolish the downregulation, suggesting that the interactions between the two channels are mediated through their COOH-termini. Similarly, transiently expressed HERG reduced IKs in KvLQT1-minK stable cells. Coimmunoprecipitations indicated a direct interaction between HERG and KvLQT1, and surface plasmon resonance analysis demonstrated a specific, physical association between the COOH-termini of KvLQT1 and HERG. Here, we present an in vitro model system consistent with the in vivo reciprocal downregulation of repolarizing currents seen in transgenic rabbit models, illustrating the importance of the transfection method when studying heterologous ion channel expression and trafficking. Moreover, our data suggest that interactions between KvLQT1 and HERG are mediated through COOH-termini.

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Monitoring and Tracking Changes in Sensitivity to Azoxystrobin Fungicide in Alternaria solani in Wisconsin

Plant Disease ◽

10.1094/pdis-92-4-0555 ◽

2008 ◽

Vol 92 (4) ◽

pp. 555-560 ◽

Cited By ~ 29

Author(s):

N. Rosenzweig ◽

G. Olaya ◽

Z. K. Atallah ◽

S. Cleere ◽

C. Stanger ◽

...

Keyword(s):

Management Practices ◽

Alternaria Solani ◽

Wild Type ◽

In Vitro Sensitivity ◽

Commercial Potato ◽

Repeated Applications ◽

Polymerase Chain ◽

Sensitivity Changes ◽

Sensitivity Decrease

Azoxystrobin is a common fungicide used by farmers of Solanaceous crops against Alternaria solani, but there was growing concern about decreased sensitivity with repeated applications. In 2002 and 2003, monitoring of A. solani from commercial potato fields in Wisconsin indicated increased frequency and a statewide distribution of isolates with decreased in vitro sensitivity to azoxystrobin. Mean effective concentration in inhibiting spore germination by 50% values gathered in 2002 and 2003 were approximately 20-fold higher than baseline isolates of A. solani collected in 1998 from fields that had never been treated with azoxystrobin. This sensitivity decrease was correlated with site-specific mutations in the cytochrome b detected by quantitative real-time polymerase chain reaction. The F129L and the G143A substitution have been shown to cause a reduction in sensitivity or resistance, respectively, to quinone outside inhibitors. All of the recovered A. solani isolates collected in 2002 and 2003 were wild type at position 143. However, all three mutations responsible for the F129L substitution (TTA, CTC, and TTG) were detected in our samples. In addition, the frequency of this amino acid substitution in A. solani isolates was statistically different across sampling sites and years, indicating that sensitivity changes depended on specific disease management practices.

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