Hypo-osmotic Stress Induces ATP Release via Volume-regulated Anion Channels in Undifferentiated Mammary Cells

The high interstitial ATP concentration in the cancer microenvironment is a major source of adenosine, which acts as a strong immune suppressor. However, the source of ATP release has not been elucidated. We measured the ATP release during hypotonic stress using a real-time ATP luminescence imaging system in primary cultured mammary cells and in breast cell lines. In primary cultured cells, ATP was intermittently released with transient-sharp peaks, while in breast cell lines ATP was released with a slowly rising diffuse pattern. The diffuse ATP release pattern was changed to a transient-sharp pattern by cholera toxin treatment and the reverse change was induced by transforming growth factor (TGF) β treatment. DCPIB, an inhibitor of volume-regulated anion channels (VRACs), only suppressed the diffuse pattern. The inflammatory mediator sphingosine-1-phosphate (S1P) induced a diffuse ATP release pattern isovolumetrically. The knockdown of A isoform of leucine-rich repeat-containing protein 8 (LRRC8A), the essential molecular entity of VRACs, using shRNA suppressed the diffuse pattern. These results suggest that abundantly expressed VRACs are a conduit of ATP release in undifferentiated cells, including cancer cells.

Download Full-text

Sphingosine-1-Phosphate Induces ATP Release via Volume-Regulated Anion Channels in Breast Cell Lines

Life ◽

10.3390/life11080851 ◽

2021 ◽

Vol 11 (8) ◽

pp. 851 ◽

Cited By ~ 1

Author(s):

Kishio Furuya ◽

Hiroaki Hirata ◽

Takeshi Kobayashi ◽

Masahiro Sokabe

Keyword(s):

Cell Lines ◽

Cultured Cells ◽

Imaging System ◽

Atp Release ◽

Cancer Microenvironment ◽

Anion Channels ◽

Breast Cell Lines ◽

Undifferentiated Cells ◽

Sphingosine 1 Phosphate ◽

Dose Dependent

High interstitial level of ATP and its lysate adenosine in the cancer microenvironment are considered a halo mark of cancer. Adenosine acts as a strong immune suppressor. However, the source of ATP release is unclear. We clarified the release of ATP via volume-regulated anion channels (VRACs) in breast cell lines using an ATP luminescence imaging system. We detected a slowly rising diffuse pattern of ATP release that was only observed in undifferentiated cells, not in differentiated primary cultured cells. This was confirmed by suppression with DCPIB, a blocker of VRACs, and shRNA for LRRC8A, an indispensable subunit of VRACs. We herein demonstrated that the inflammatory mediator sphingosine-1-phosphate (S1P), which exists abundantly in the cancer microenvironment, induced a diffuse pattern of ATP release isovolumetrically. The response was dose-dependent and suppressed by the knock-down of LRRC8A. It was also suppressed by blockers of S1P receptor 1 and 2 (W146 and JTE013, respectively). RTqPCR demonstrated the prominent presence of S1PR1 and S1PR2 mRNAs. We discussed the roles of S1P-induced ATP release in the cancer microenvironment.

Download Full-text

Establishment of adult mouse Sertoli cell lines by using the starvation method

Reproduction ◽

10.1530/rep-12-0086 ◽

2013 ◽

Vol 145 (5) ◽

pp. 505-516 ◽

Cited By ~ 11

Author(s):

Yoko Sato ◽

Kaoru Yoshida ◽

Shiari Nozawa ◽

Miki Yoshiike ◽

Michiko Arai ◽

...

Keyword(s):

Growth Factor ◽

Sertoli Cell ◽

Cell Lines ◽

Germ Cells ◽

Sertoli Cells ◽

Transforming Growth Factor ◽

Cultured Cells ◽

Transforming Growth Factor Β1 ◽

Leukaemia Inhibitory Factor ◽

Specific Gene

Sertoli cells were isolated from the testes of 6-week-old mice and stable Sertoli cell lines with higher proliferation rates were subcloned after starvation of primary cultured cells. After two rounds of this subcloning, 33 subcloned lines were selected on the basis of their proliferation rates. In addition, these subclones were screened according to their phagocytic activity and the characteristics of mature Sertoli cells, such as the expression of androgen receptors (ARs) and progesterone receptors, by using western blotting and immunocytochemical analysis, in addition to their morphology and proliferation rates. After the third round of subcloning, 12 subclones were selected for the final selection using RT-PCR for identification of genes specifically expressed by various testicular cells. Three clones were selected that expressed Sertoli-cell-specific genes, i.e. stem cell factor, clusterin, AR, α-inhibin, transferrin, Wilms' tumour-1, Müllerian inhibitory substance, sex-determining region Y-box 9, FSH receptor (Fshr) and occludin; however, these clones did not express globulin transcription factor 1, steroidogenic factor or androgen-binding protein. These clones also expressed growth and differentiation factors that act on germ cells, such as leukaemia inhibitory factor, transforming growth factor β1 and basic fibroblast growth factor 2, but did not express c-kit (specific for germ cells), LH receptor and 3β-hydroxyl-dehydrogenase (specific for Leydig cells). Immunocytochemical data confirmed the expression of clusterin in these clones. Furthermore, the Bromodeoxyuridine incorporation assay confirmed the proliferation activity of these clones throughFshrafter treatment with FSH. These clones are considered to be valuable tools for the study of Sertoli cell-specific gene expression and function.

Download Full-text

Maxi-anion channel and pannexin 1 hemichannel constitute separate pathways for swelling-induced ATP release in murine L929 fibrosarcoma cells

AJP Cell Physiology ◽

10.1152/ajpcell.00459.2011 ◽

2012 ◽

Vol 303 (9) ◽

pp. C924-C935 ◽

Cited By ~ 28

Author(s):

Md. Rafiqul Islam ◽

Hiromi Uramoto ◽

Toshiaki Okada ◽

Ravshan Z. Sabirov ◽

Yasunobu Okada

Keyword(s):

Connexin 43 ◽

Regulatory Volume Decrease ◽

Atp Release ◽

Anion Channel ◽

Molecular Entity ◽

Channel Blockers ◽

Channel Activity ◽

Anion Channels ◽

Pannexin 1 ◽

Fibrosarcoma Cells

The maxi-anion channel plays a classically recognized role in controlling the membrane potential through the chloride conductance. It also has novel functions as a regulated pathway for the release of the anionic signaling molecules ATP and excitatory amino acids from cells subjected to osmotic perturbation, ischemia, or hypoxia. Because hemichannels formed by pannexins and connexins have been reported to mediate ATP release from a number of cell types, these hemichannels may represent the molecular correlate of the maxi-anion channel. Here, we found that L929 fibrosarcoma cells express functional maxi-anion channels which mediate a major portion of swelling-induced ATP release, and that ATP released via maxi-anion channels facilitates the regulatory volume decrease after osmotic swelling. Also, it was found that the cells express the mRNA for pannexin 1, pannexin 2, and connexin 43. Hypotonicity-induced ATP release was partially suppressed not only by known blockers of the maxi-anion channel but also by several blockers of pannexins including the pannexin 1-specific blocking peptide 10Panx1 and small interfering (si)RNA against pannexin 1 but not pannexin 2. The inhibitory effects of maxi-anion channel blockers and pannexin 1 antagonists were additive. In contrast, maxi-anion channel activity was not affected by pannexin 1 antagonists and siRNAs against pannexins 1 and 2. Although a connexin 43-specific blocking peptide, Gap27, slightly suppressed hypotonicity-induced ATP release, maxi-anion channel activity was not affected by Gap27 or connexin 43-specific siRNA. Thus, it is concluded that the maxi-anion channel is a molecular entity distinct from pannexin 1, pannexin 2, and connexin 43, and that the maxi-anion channel and the hemichannels constitute separate pathways for swelling-induced ATP release in L929 cells.

Download Full-text

Direct protein delivery into intact plant cells using polyhistidine peptides

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbab055 ◽

2021 ◽

Author(s):

Yoshino Tanaka ◽

Yoshihiko Nanasato ◽

Kousei Omura ◽

Keita Endoh ◽

Tsuyoshi Kawano ◽

...

Keyword(s):

Cell Lines ◽

Fusion Proteins ◽

Protein Delivery ◽

Fluorescent Protein ◽

Cultured Cells ◽

Plant Cells ◽

Adenosine Monophosphate ◽

Cell Penetrating Peptides ◽

Intracellular Space ◽

Intracellular Fluorescence

Abstract Polyhistidine peptides (PHPs), sequences comprising only histidine residues (>His8), are effective cell-penetrating peptides for plant cells. Using PHP-fusion proteins, we aimed to deliver proteins into cultured plant cells from Nicotiana tabacum, Oryza sativa, and Cryptomeria japonica. Co-cultivation of cultured cells with fusion proteins combining maltose-binding protein (MBP), red fluorescent protein (RFP), and various PHPs (MBP-RFP-His8–His20) in one polypeptide showed the cellular uptake of fusion proteins in all plant cell lines. Maximum intracellular fluorescence was shown in MBP-RFP-His20. Further, adenylate cyclase (CyaA), a synthase of cyclic adenosine monophosphate (cAMP) activated by cytosolic calmodulin, was used as a reporter for protein delivery in living cells. A fusion protein combining MBP, RFP, CyaA, and His20 (MBP-RFP-CyaA-His20) was delivered into plant cells and increased intracellular fluorescence and cAMP production in all cell lines. The present study demonstrates that PHPs are effective carriers of proteins into the intracellular space of various cultured plant cells.

Download Full-text

Structure-guided selection of puromycin N-acetyltransferase mutants with enhanced selection stringency for deriving mammalian cell lines expressing recombinant proteins

Scientific Reports ◽

10.1038/s41598-021-84551-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Alessandro T. Caputo ◽

Oliver M. Eder ◽

Hana Bereznakova ◽

Heleen Pothuis ◽

Albert Ardevol ◽

...

Keyword(s):

Cell Lines ◽

Mammalian Cell ◽

Recombinant Proteins ◽

Cultured Cells ◽

Hek293 Cells ◽

Reaction Products ◽

Enzyme Form ◽

X Ray Crystallography ◽

Mammalian Cell Lines ◽

Apparent Loss

AbstractPuromycin and the Streptomyces alboniger-derived puromycin N-acetyltransferase (PAC) enzyme form a commonly used system for selecting stably transfected cultured cells. The crystal structure of PAC has been solved using X-ray crystallography, revealing it to be a member of the GCN5-related N-acetyltransferase (GNAT) family of acetyltransferases. Based on structures in complex with acetyl-CoA or the reaction products CoA and acetylated puromycin, four classes of mutations in and around the catalytic site were designed and tested for activity. Single-residue mutations were identified that displayed a range of enzymatic activities, from complete ablation to enhanced activity relative to wild-type (WT) PAC. Cell pools of stably transfected HEK293 cells derived using two PAC mutants with attenuated activity, Y30F and A142D, were found to secrete up to three-fold higher levels of a soluble, recombinant target protein than corresponding pools derived with the WT enzyme. A third mutant, Y171F, appeared to stabilise the intracellular turnover of PAC, resulting in an apparent loss of selection stringency. Our results indicate that the structure-guided manipulation of PAC function can be utilised to enhance selection stringency for the derivation of mammalian cell lines secreting elevated levels of recombinant proteins.

Download Full-text

Growth inhibition and induction of phenotypic alterations by tiazofurin: Differential effects on MCF-7 breast cancer and HBL-100 breast cell lines

European Journal of Cancer and Clinical Oncology ◽

10.1016/0277-5379(89)90136-3 ◽

1989 ◽

Vol 25 (5) ◽

pp. 883-889 ◽

Cited By ~ 10

Author(s):

Y. Sidi ◽

E. Beery ◽

C. Panet ◽

L. Wasserman ◽

A. Novogrodsky ◽

...

Keyword(s):

Breast Cancer ◽

Growth Inhibition ◽

Cell Lines ◽

Differential Effects ◽

Breast Cell Lines ◽

Mcf 7

Download Full-text

Heparan sulfates of cultured cells. II. Acid-soluble and -precipitable species of different cell lines

Biochemistry ◽

10.1021/bi00784a027 ◽

1971 ◽

Vol 10 (8) ◽

pp. 1445-1451 ◽

Cited By ~ 139

Author(s):

Paul M. Kraemer

Keyword(s):

Cell Lines ◽

Cultured Cells ◽

Heparan Sulfates

Download Full-text

Establishment of Cell Lines from Both Myeloma Bone Marrow and Plasmacytoma: SNU_MM1393_BM and SNU_MM1393_SC from a Single Patient

BioMed Research International ◽

10.1155/2014/510408 ◽

2014 ◽

Vol 2014 ◽

pp. 1-8 ◽

Cited By ~ 1

Author(s):

Youngil Koh ◽

Woo-June Jung ◽

Kwang-Sung Ahn ◽

Sung-Soo Yoon

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Cell Lines ◽

Mononuclear Cells ◽

Cultured Cells ◽

Clonal Evolution ◽

Myeloma Cell ◽

Mouse Bone Marrow ◽

Single Patient ◽

U266 Cell

Purpose.We tried to establish clinically relevant human myeloma cell lines that can contribute to the understanding of multiple myeloma (MM).Materials and Methods.Mononuclear cells obtained from MM patient’s bone marrow were injected via tail vein in an NRG/SCID mouse. Fourteen weeks after the injection, tumor developed at subcutis of the mouse. The engraftment of MM cells into mouse bone marrow (BM) was also observed. We separated and cultured cells from subcutis and BM.Results.After the separation and culture of cells from subcutis and BM, we established two cell lines originating from a single patient (SNU_MM1393_BM and SNU_MM1393_SC). Karyotype of the two newly established MM cell lines showed tetraploidy which is different from the karyotype of the patient (diploidy) indicating clonal evolution. In contrast to SNU_MM1393_BM, cell proliferation of SNU_MM1393_SC was IL-6 independent. SNU_MM1393_BM and SNU_MM1393_SC showed high degree of resistance against bortezomib compared to U266 cell line. SNU_MM1393_BM had the greater lethality compared to SNU_MM1393_SC.Conclusion.Two cell lines harboring different site tropisms established from a single patient showed differences in cytokine response and lethality. Our newly established cell lines could be used as a tool to understand the biology of multiple myeloma.

Download Full-text