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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.

Hance MW, Dhar MS, Plummer HK - Breast Cancer (Auckl) (2008)

Bottom Line: All three constructs decreased GIRK1 mRNA levels.However, β(2) 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.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cancer Analysis Laboratory, Department of Pathobiology, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996-4542, U.S.A.

ABSTRACT
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, β(2)-adrenergic and 18S control levels were determined using real-time PCR 24 hours after transfection. All three constructs decreased GIRK1 mRNA levels. However, β(2) 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.

No MeSH data available.


Related in: MedlinePlus

GIRK1 protein expression of MDA-MB-453 cells transfected with GIRK1 stealth siRNA. MDA-MB-453 breast cancer cells were transfected with 3 stealth siRNA constructs (Invitrogen). Enriched membrane protein was isolated 1, 3, and 5 days after transfection by the ReadyPrep protein extraction kit (signal) (Bio-Rad, Richmond, CA, U.S.A). Western blotting was performed, and the membranes were probed with antibodies to GIRK1 and GAPDH. (A) Western blots indicating GIRK1 and GAPDH expression of MDA-MB-453 cells transfected with three GIRK1 siRNA constructs (1104, 1315, 1490). Samples were collected at 24 hours, 3 days and 5 days. (B) Graphs indicate densitometry of the Western blots. The densitometry of GIRK1 was divided by the densitometry of the control GAPDH (N = 3). All three siRNA constructs reduced GIRK1 protein levels 24 hours after transfection. These reductions were reversed by 3–5 days, and in the case of one construct, increased over control levels. The bands are consistent with the expected size: GIRK1 (40–42 kDa; Dhar and Plummer 2006); GAPDH (37 kDa). These are gels representative of two separate experiments.
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f2-bcbcr-2008-025: GIRK1 protein expression of MDA-MB-453 cells transfected with GIRK1 stealth siRNA. MDA-MB-453 breast cancer cells were transfected with 3 stealth siRNA constructs (Invitrogen). Enriched membrane protein was isolated 1, 3, and 5 days after transfection by the ReadyPrep protein extraction kit (signal) (Bio-Rad, Richmond, CA, U.S.A). Western blotting was performed, and the membranes were probed with antibodies to GIRK1 and GAPDH. (A) Western blots indicating GIRK1 and GAPDH expression of MDA-MB-453 cells transfected with three GIRK1 siRNA constructs (1104, 1315, 1490). Samples were collected at 24 hours, 3 days and 5 days. (B) Graphs indicate densitometry of the Western blots. The densitometry of GIRK1 was divided by the densitometry of the control GAPDH (N = 3). All three siRNA constructs reduced GIRK1 protein levels 24 hours after transfection. These reductions were reversed by 3–5 days, and in the case of one construct, increased over control levels. The bands are consistent with the expected size: GIRK1 (40–42 kDa; Dhar and Plummer 2006); GAPDH (37 kDa). These are gels representative of two separate experiments.

Mentions: These three siRNA constructs were also transfected into additional MDA-MB-453 cells, and protein was isolated after 24 hours, 3 days and 5 days after transfection. Enriched membrane protein was isolated, Western blotting was performed, and the membranes were probed with antibodies to GIRK1. All three siRNA constructs reduced GIRK1 protein levels 24 hours after transfection (Fig. 2). At 24 hours, the GIRK1 protein levels were 57.1% of control levels for the 1104 construct, 41.2% of control levels for the 1315 construct and 52.1% of control levels for the 1490 construct. These reductions were reversed by 3–5 days, and in the case of one construct, increased over control levels (Fig. 2). At 3 days, the GIRK1 protein levels were 109.7% of control levels for the 1104 construct, 267.8% of control levels for the 1315 construct and 78.1% of control levels for the 1490 construct. At 5 days, the GIRK1 protein levels were 99.0% of control levels for the 1104 construct, 81.1% of control levels for the 1315 construct and 178.8% of control levels for the 1490 construct. When the low GC RNAi negative control (Invitrogen) for the GIRK1 stealth siRNA was used, there were no effects on GIRK1 protein expression at either one, three or five days (data not shown).


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.

Hance MW, Dhar MS, Plummer HK - Breast Cancer (Auckl) (2008)

GIRK1 protein expression of MDA-MB-453 cells transfected with GIRK1 stealth siRNA. MDA-MB-453 breast cancer cells were transfected with 3 stealth siRNA constructs (Invitrogen). Enriched membrane protein was isolated 1, 3, and 5 days after transfection by the ReadyPrep protein extraction kit (signal) (Bio-Rad, Richmond, CA, U.S.A). Western blotting was performed, and the membranes were probed with antibodies to GIRK1 and GAPDH. (A) Western blots indicating GIRK1 and GAPDH expression of MDA-MB-453 cells transfected with three GIRK1 siRNA constructs (1104, 1315, 1490). Samples were collected at 24 hours, 3 days and 5 days. (B) Graphs indicate densitometry of the Western blots. The densitometry of GIRK1 was divided by the densitometry of the control GAPDH (N = 3). All three siRNA constructs reduced GIRK1 protein levels 24 hours after transfection. These reductions were reversed by 3–5 days, and in the case of one construct, increased over control levels. The bands are consistent with the expected size: GIRK1 (40–42 kDa; Dhar and Plummer 2006); GAPDH (37 kDa). These are gels representative of two separate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3091401&req=5

f2-bcbcr-2008-025: GIRK1 protein expression of MDA-MB-453 cells transfected with GIRK1 stealth siRNA. MDA-MB-453 breast cancer cells were transfected with 3 stealth siRNA constructs (Invitrogen). Enriched membrane protein was isolated 1, 3, and 5 days after transfection by the ReadyPrep protein extraction kit (signal) (Bio-Rad, Richmond, CA, U.S.A). Western blotting was performed, and the membranes were probed with antibodies to GIRK1 and GAPDH. (A) Western blots indicating GIRK1 and GAPDH expression of MDA-MB-453 cells transfected with three GIRK1 siRNA constructs (1104, 1315, 1490). Samples were collected at 24 hours, 3 days and 5 days. (B) Graphs indicate densitometry of the Western blots. The densitometry of GIRK1 was divided by the densitometry of the control GAPDH (N = 3). All three siRNA constructs reduced GIRK1 protein levels 24 hours after transfection. These reductions were reversed by 3–5 days, and in the case of one construct, increased over control levels. The bands are consistent with the expected size: GIRK1 (40–42 kDa; Dhar and Plummer 2006); GAPDH (37 kDa). These are gels representative of two separate experiments.
Mentions: These three siRNA constructs were also transfected into additional MDA-MB-453 cells, and protein was isolated after 24 hours, 3 days and 5 days after transfection. Enriched membrane protein was isolated, Western blotting was performed, and the membranes were probed with antibodies to GIRK1. All three siRNA constructs reduced GIRK1 protein levels 24 hours after transfection (Fig. 2). At 24 hours, the GIRK1 protein levels were 57.1% of control levels for the 1104 construct, 41.2% of control levels for the 1315 construct and 52.1% of control levels for the 1490 construct. These reductions were reversed by 3–5 days, and in the case of one construct, increased over control levels (Fig. 2). At 3 days, the GIRK1 protein levels were 109.7% of control levels for the 1104 construct, 267.8% of control levels for the 1315 construct and 78.1% of control levels for the 1490 construct. At 5 days, the GIRK1 protein levels were 99.0% of control levels for the 1104 construct, 81.1% of control levels for the 1315 construct and 178.8% of control levels for the 1490 construct. When the low GC RNAi negative control (Invitrogen) for the GIRK1 stealth siRNA was used, there were no effects on GIRK1 protein expression at either one, three or five days (data not shown).

Bottom Line: All three constructs decreased GIRK1 mRNA levels.However, β(2) 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.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cancer Analysis Laboratory, Department of Pathobiology, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996-4542, U.S.A.

ABSTRACT
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, β(2)-adrenergic and 18S control levels were determined using real-time PCR 24 hours after transfection. All three constructs decreased GIRK1 mRNA levels. However, β(2) 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.

No MeSH data available.


Related in: MedlinePlus