Reversible switching of primary cells between normal and malignant state by oncogenic virus KSHV and CRISPR/Cas9‐mediated targeting of a major viral latent protein

It has been proposed that gonadotropin-releasing hormone (GnRH) stimulates Ca2+ entry by activation of voltage-independent, receptor-mediated Ca2+ channels in the rat gonadotroph. Little work has been done on the role of calcium in GnRH-induced luteinizing hormone (LH) release in species other than the rat. Therefore, this study was done to compare the effects of agents that alter Ca2+ or Na+ entry on LH release from calf anterior pituitary primary cells in culture. GnRH (100 ng/ml), Ca2+ ionophore A23187 (2.5 microM), and the depolarizing agent ouabain (0.1-10 microM) all produced significant increases (P less than 0.05) in LH release; these effects were significantly reduced when the cells were preincubated with the organic Ca2+ channel blockers nifedipine (1-10 microM) and verapamil (1-10 microM) and with Co2+ (0.01-1 mM). The effect of ouabain was inhibited by tetrodotoxin (TTX; 1-10 nM) as well as by nifedipine at 0.1-10 microM. In contrast to its effect on rat pituitary LH release, TTX significantly inhibited GnRH-stimulated LH release at 1-100 nM. These results suggest that GnRH-induced LH release may employ Ca2+ as a second messenger in bovine gonadotrophs and support recent speculation that GnRH-induced Ca2+ mobilization may in part be voltage dependent.

Download Full-text

Gender-Dependent Phenotype in Polycystic Kidney Disease Is Determined by Differential Intracellular Ca2+ Signals

International Journal of Molecular Sciences ◽

10.3390/ijms22116019 ◽

2021 ◽

Vol 22 (11) ◽

pp. 6019

Author(s):

Khaoula Talbi ◽

Inês Cabrita ◽

Rainer Schreiber ◽

Karl Kunzelmann

Keyword(s):

Cell Proliferation ◽

Kidney Disease ◽

Polycystic Kidney Disease ◽

Collecting Duct ◽

Primary Cells ◽

Loss Of Function ◽

Polycystic Kidney ◽

Chloride Currents ◽

Duct Cells ◽

Collecting Duct Cells

Autosomal dominant polycystic kidney disease (ADPKD) is caused by loss of function of PKD1 (polycystin 1) or PKD2 (polycystin 2). The Ca2+-activated Cl− channel TMEM16A has a central role in ADPKD. Expression and function of TMEM16A is upregulated in ADPKD which causes enhanced intracellular Ca2+ signaling, cell proliferation, and ion secretion. We analyzed kidneys from Pkd1 knockout mice and found a more pronounced phenotype in males compared to females, despite similar levels of expression for renal tubular TMEM16A. Cell proliferation, which is known to be enhanced with loss of Pkd1−/−, was larger in male when compared to female Pkd1−/− cells. This was paralleled by higher basal intracellular Ca2+ concentrations in primary renal epithelial cells isolated from Pkd1−/− males. The results suggest enhanced intracellular Ca2+ levels contributing to augmented cell proliferation and cyst development in male kidneys. Enhanced resting Ca2+ also caused larger basal chloride currents in male primary cells, as detected in patch clamp recordings. Incubation of mouse primary cells, mCCDcl1 collecting duct cells or M1 collecting duct cells with dihydrotestosterone (DHT) enhanced basal Ca2+ levels and increased basal and ATP-stimulated TMEM16A chloride currents. Taken together, the more severe cystic phenotype in males is likely to be caused by enhanced cell proliferation, possibly due to enhanced basal and ATP-induced intracellular Ca2+ levels, leading to enhanced TMEM16A currents. Augmented Ca2+ signaling is possibly due to enhanced expression of Ca2+ transporting/regulating proteins.

Download Full-text

Cyclopenta[b]indole Derivative Inhibits Aurora B in Primary Cells

ACS Omega ◽

10.1021/acsomega.0c05491 ◽

2020 ◽

Vol 5 (51) ◽

pp. 33455-33460

Author(s):

Andreas Ekebergh ◽

Jerker Mårtensson ◽

Christine Lingblom Ekebergh

Keyword(s):

Indole Derivative ◽

Aurora B ◽

Primary Cells

Download Full-text

Identification of Novel FNIN2 and FNIN3 Fibronectin-Derived Peptides That Promote Cell Adhesion, Proliferation and Differentiation in Primary Cells and Stem Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22063042 ◽

2021 ◽

Vol 22 (6) ◽

pp. 3042

Author(s):

Eun Ju Lee ◽

Khurshid Ahmad ◽

Shiva Pathak ◽

SunJu Lee ◽

Mohammad Hassan Baig ◽

...

Keyword(s):

Stem Cells ◽

Cell Culture ◽

Cell Adhesion ◽

3D Culture ◽

Human Mesenchymal Stem Cells ◽

Cell Types ◽

Primary Cells ◽

Cell Culture Techniques ◽

Culture Techniques ◽

Promote Cell Adhesion

In recent years, a major rise in the demand for biotherapeutic drugs has centered on enhancing the quality and efficacy of cell culture and developing new cell culture techniques. Here, we report fibronectin (FN) derived, novel peptides fibronectin-based intergrin binding peptide (FNIN)2 (18-mer) and FNIN3 (20-mer) which promote cell adhesion proliferation, and the differentiation of primary cells and stem cells. FNIN2 and 3 were designed based on the in silico interaction studies between FN and its receptors (integrin α5β1, αvβ3, and αIIbβ3). Analysis of the proliferation of seventeen-cell types showed that the effects of FNINs depend on their concentration and the existence of expressed integrins. Significant rhodamine-labeled FNIN2 fluorescence on the membranes of HeLa, HepG2, A498, and Du145 cells confirmed physical binding. Double coating with FNIN2 or 3 after polymerized dopamine (pDa) or polymerized tannic acid (pTA) precoating increased HBEpIC cell proliferation by 30–40 percent, suggesting FNINs potently affect primary cells. Furthermore, the proliferation of C2C12 myoblasts and human mesenchymal stem cells (MSCs) treated with FNINs was significantly increased in 2D/3D culture. FNINs also promoted MSC differentiation into osteoblasts. The results of this study offer a new approach to the production of core materials (e.g., cell culture medium components, scaffolds) for cell culture.

Download Full-text

New insights on CRISPR/Cas9-based therapy for breast Cancer

Genes and Environment ◽

10.1186/s41021-021-00188-0 ◽

2021 ◽

Vol 43 (1) ◽

Author(s):

Hussein Sabit ◽

Shaimaa Abdel-Ghany ◽

Huseyin Tombuloglu ◽

Emre Cevik ◽

Amany Alqosaibi ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Research ◽

Animal Models ◽

Human Cancer ◽

Step Process ◽

Cancer Initiation ◽

Malignant State ◽

Treat Breast Cancer

AbstractCRISPR/Cas9 has revolutionized genome-editing techniques in various biological fields including human cancer research. Cancer is a multi-step process that encompasses the accumulation of mutations that result in the hallmark of the malignant state. The goal of cancer research is to identify these mutations and correlate them with the underlying tumorigenic process. Using CRISPR/Cas9 tool, specific mutations responsible for cancer initiation and/or progression could be corrected at least in animal models as a first step towards translational applications. In the present article, we review various novel strategies that employed CRISPR/Cas9 to treat breast cancer in both in vitro and in vivo systems.

Download Full-text