Protein expression of G-protein inwardly rectifying potassium channels (GIRK) in breast cancer cells

Background Previous data from our laboratory has indicated that a functional link exists between the G-protein-coupled inwardly rectifying potassium (GIRK) channel and the beta-adrenergic receptor pathway in breast cancer cell lines, and these pathways were involved in growth regulation of these cells. Alcohol is an established risk factor for breast cancer and has been found to open GIRK. In order to further investigate GIRK channels in breast cancer and possible alteration by ethanol, we identified GIRK channel protein expression in breast cancer cells. Results Cell pellets were collected and membrane protein was isolated to determine GIRK protein expression. GIRK protein was also analyzed by immuno-precipitation. GIRK protein was over-expressed in cells by transfection of GIRK plasmids. Gene expression studies were done by real-time RT-PCR. GIRK protein expression was identified in breast cancer cell lines. Expression of GIRK1 at the indicated molecular weight (MW) (62 kDa) was seen in cell lines MDA-MB-453 and ZR-75-1. In addition, GIRK1 expression was seen at a lower MW (40–42 kDa) in MDA-MB-361, MDA-MB-468, MCF-7, ZR-75-1, and MDA-MB-453 cell lines. To prove the lower MW protein was GIRK1, MDA-MB-453 cells were immuno-precipitated. GIRK2 expression was seen in MDA-MB-468, MCF-7, and ZR-75-1 and was variable in MDA-MB-453, while GIRK4 protein expression was seen in all six cell lines tested. This is the first report indicating GIRK protein expression in breast cancer cells. To determine functionality, MDA-MB-453 cells were stimulated with ethanol. Decreased GIRK1 protein expression levels were seen after treatment with 0.12% ethanol in MDA-MB-453 breast cancer cells. Serum-free media decreased GIRK protein expression, possibly due to lack of estrogen in the media. Transfection of GIRK1 or GIRK4 plasmids increased GIRK1 protein expression and decreased gene expression in MDA-MB-453 breast cancer cells. Conclusion Our data indicates that functional GIRK channels exist in breast cancer cells that are involved in cellular signaling

Effects of Alparazolam and Triazolam on Isolation-Induced Aggression in Rats

Author Institution: Department of Biology, Western Michigan UniversityThe efficacy of two new benzodiazepine tranquilizers, alprazolam and triazolam, in the reduction of aggression produced in rats by isolation was tested. Rats were isolated for at least three weeks. They were then placed in a cage with another rat, and aggressive and submissive behaviors that occurred were noted. Several of the behaviors were changed by either alprazolam or triazolam. Aggressive behaviors of thrust, attack, offensive upright, and offensive sideways were decreased; bite was increased. The submissive behavior of defensive sideways was decreased, whereas crouch and freeze were increased. Reduction in aggression seems consistent with results for other benzodiazepine tranquilizers such as diazepam and chlordiazepoxide

G-Protein Inwardly Rectifying Potassium Channel 1 (GIRK1) Knockdown Decreases Beta-Adrenergic, MAP Kinase and Akt Signaling in the MDA-MB-453 Breast Cancer Cell Line

Previous data from our laboratory have indicated that there is a functional link between the beta-adrenergic receptor signaling pathway and the G-protein inwardly rectifying potassium channel (GIRK1) in breast cancer cell lines and that these pathways are involved in growth regulation of these cells. To determine functionality, MDA-MB-453 breast cancer cells were stimulated with ethanol, known to open GIRK channels. Decreased GIRK1 protein levels were seen after treatment with 0.12% ethanol. In addition, serum-free media completely inhibited GIRK1 protein expression. This data indicates that there are functional GIRK channels in breast cancer cells and that these channels are involved in cellular signaling. In the present research, to further define the signaling pathways involved, we performed RNA interference (siRNA) studies. Three stealth siRNA constructs were made starting at bases 1104, 1315, and 1490 of the GIRK1 sequence. These constructs were transfected into MDA-MB-453 cells, and both RNA and protein were isolated. GIRK1, β2-adrenergic and 18S control levels were determined using real-time PCR 24 hours after transfection. All three constructs decreased GIRK1 mRNA levels. However, β2 mRNA levels were unchanged by the GIRK1 knockdown. GIRK1 protein levels were also reduced by the knockdown, and this knockdown led to decreases in beta-adrenergic, MAP kinase and Akt signaling

Expression of inwardly rectifying potassium channels (GIRKs) and beta-adrenergic regulation of breast cancer cell lines

BACKGROUND: Previous research has indicated that at various organ sites there is a subset of adenocarcinomas that is regulated by beta-adrenergic and arachidonic acid-mediated signal transduction pathways. We wished to determine if this regulation exists in breast adenocarcinomas. Expression of mRNA that encodes a G-protein coupled inwardly rectifying potassium channel (GIRK1) has been shown in tissue samples from approximately 40% of primary human breast cancers. Previously, GIRK channels have been associated with beta-adrenergic signaling. METHODS: Breast cancer cell lines were screened for GIRK channels by RT-PCR. Cell cultures of breast cancer cells were treated with beta-adrenergic agonists and antagonists, and changes in gene expression were determined by both relative competitive and real time PCR. Potassium flux was determined by flow cytometry and cell signaling was determined by western blotting. RESULTS: Breast cancer cell lines MCF-7, MDA-MB-361 MDA-MB 453, and ZR-75-1 expressed mRNA for the GIRK1 channel, while MDA-MB-468 and MDA-MB-435S did not. GIRK4 was expressed in all six breast cancer cell lines, and GIRK2 was expressed in all but ZR-75-1 and MDA-MB-435. Exposure of MDA-MB-453 cells for 6 days to the beta-blocker propranolol (1 μM) increased the GIRK1 mRNA levels and decreased beta(2)-adrenergic mRNA levels, while treatment for 30 minutes daily for 7 days had no effect. Exposure to a beta-adrenergic agonist and antagonist for 24 hours had no effect on gene expression. The beta adrenergic agonist, formoterol hemifumarate, led to increases in K(+ )flux into MDA-MB-453 cells, and this increase was inhibited by the GIRK channel inhibitor clozapine. The tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a high affinity agonist for beta-adrenergic receptors stimulated activation of Erk 1/2 in MDA-MB-453 cells. CONCLUSIONS: Our data suggests β-adrenergic receptors and GIRK channels may play a role in breast cancer

Expression of G-protein inwardly rectifying potassium channels (GIRKs) in lung cancer cell lines

BACKGROUND: Previous data from our laboratory has indicated that there is a functional link between the β-adrenergic receptor signaling pathway and the G-protein inwardly rectifying potassium channel (GIRK1) in human breast cancer cell lines. We wanted to determine if GIRK channels were expressed in lung cancers and if a similar link exists in lung cancer. METHODS: GIRK1-4 expression and levels were determined by reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR. GIRK protein levels were determined by western blots and cell proliferation was determined by a 5-bromo-2'-deoxyuridine (BrdU) assay. RESULTS: GIRK1 mRNA was expressed in three of six small cell lung cancer (SCLC) cell lines, and either GIRK2, 3 or 4 mRNA expression was detected in all six SCLC cell lines. Treatment of NCI-H69 with β(2)-adrenergic antagonist ICI 118,551 (100 μM) daily for seven days led to slight decreases of GIRK1 mRNA expression levels. Treatment of NCI-H69 with the β-adrenergic agonist isoproterenol (10 μM) decreased growth rates in these cells. The GIRK inhibitor U50488H (2 μM) also inhibited proliferation, and this decrease was potentiated by isoproterenol. In the SCLC cell lines that demonstrated GIRK1 mRNA expression, we also saw GIRK1 protein expression. We feel these may be important regulatory pathways since no expression of mRNA of the GIRK channels (1 & 2) was found in hamster pulmonary neuroendocrine cells, a suggested cell of origin for SCLC, nor was GIRK1 or 2 expression found in human small airway epithelial cells. GIRK (1,2,3,4) mRNA expression was also seen in A549 adenocarcinoma and NCI-H727 carcinoid cell lines. GIRK1 mRNA expression was not found in tissue samples from adenocarcinoma or squamous cancer patients, nor was it found in NCI-H322 or NCI-H441 adenocarcinoma cell lines. GIRK (1,3,4) mRNA expression was seen in three squamous cell lines, GIRK2 was only expressed in one squamous cell line. However, GIRK1 protein expression was not seen in any non-SCLC cells. CONCLUSION: We feel that this data may indicate that stimulation of GIRK1 or GIRK2 channels may be important in lung cancer. Stimulation of GIRK channels and β-adrenergic signaling may activate similar signaling pathways in both SCLC and breast cancer, but lead to different results

IMBES pre-conference: using insight from research to improve education

In September 2016, the International Mind, Brain, and Education Society (IMBES) biennial conference took place in Toronto, Canada. The pre-conference (organized by the Wellcome Trust) was designed to share new findings, lessons learned, and inspirational innovation within the field, and to encourage the community to not only identify current challenges, but to start planning potential ways to address them. This article provides a brief summary of the discussions from the day, and suggests steps that members of the community, conference organizers, and funders can take in helping to move the field forward. Finally, it outlines new work Wellcome has commissioned since the conference in response to some of the challenges defined on the day

Prevalence of deleterious variants in MC3R in patients with constitutional delay of growth and puberty.

CONTEXT: The melanocortin 3 receptor (MC3R) has recently emerged as a critical regulator of pubertal timing, linear growth and the acquisition of lean mass in humans and mice. In population-based studies, heterozygous carriers of deleterious variants in MC3R report a later onset of puberty than non-carriers. However, the frequency of such variants in patients who present with clinical disorders of pubertal development is currently unknown. OBJECTIVE: To determine whether deleterious MC3R variants are more frequently found in patients clinically presenting with constitutional delay of growth and puberty (CDGP) or normosmic idiopathic hypogonadotropic hypogonadism (nIHH). DESIGN, SETTING AND PARTICIPANTS: We examined the sequence of MC3R in 362 adolescents with a clinical diagnosis of CDGP and 657 patients with nIHH, experimentally characterised the signalling properties of all non-synonymous variants found and compared their frequency to that in 5774 controls from a population-based cohort. Additionally, we established the relative frequency of predicted deleterious variants in individuals with self-reported delayed vs normally timed menarche/voice breaking in the UK Biobank cohort. RESULTS: MC3R loss-of-function variants were infrequent but overrepresented in patients with CDGP (8/362 (2.2%), OR = 4.17, p = 0.001). There was no strong evidence of overrepresentation in patients with nIHH (4/657 (0.6%), OR = 1.15, p = 0.779). In 246,328 women from UK Biobank, predicted deleterious variants were more frequently found in those self-reporting delayed (≥16 years) vs normal age at menarche (OR = 1.66, p = 3.90E-07). CONCLUSIONS: We have found evidence that functionally damaging variants in MC3R are overrepresented in individuals with CDGP but are not a common cause of this phenotype

Prior mucosal exposure to heterologous cells alters the pathogenesis of cell-associated mucosal feline immunodeficiency virus challenge

<p>Abstract</p> <p>Background</p> <p>Several lines of research suggest that exposure to cellular material can alter the susceptibility to infection by HIV-1. Because sexual contact often includes exposure to cellular material, we hypothesized that repeated mucosal exposure to heterologous cells would induce an immune response that would alter the susceptibility to mucosal infection. Using the feline immunodeficiency virus (FIV) model of HIV-1 mucosal transmission, the cervicovaginal mucosa was exposed once weekly for 12 weeks to 5,000 heterologous cells or media (control) and then cats were vaginally challenged with cell-associated or cell-free FIV.</p> <p>Results</p> <p>Exposure to heterologous cells decreased the percentage of lymphocytes in the mucosal and systemic lymph nodes (LN) expressing L-selectin as well as the percentage of CD4+ CD25+ T cells. These shifts were associated with enhanced ex-vivo proliferative responses to heterologous cells. Following mucosal challenge with cell-associated, but not cell-free, FIV, proviral burden was reduced by 64% in cats previously exposed to heterologous cells as compared to media exposed controls.</p> <p>Conclusions</p> <p>The pathogenesis and/or the threshold for mucosal infection by infected cells (but not cell-free virus) can be modulated by mucosal exposure to uninfected heterologous cells.</p