BMC Cancer 2005, 5:104 http://www.biomedcentral.com/1471-2407/5/104 EFxigpurersesi5on of GIRK3 in SCLC cell lines Expression of GIRK3 in SCLC cell lines. Top panel: GIRK3 expression was seen in all six SCLC cell lines, in WBA, H69, H146, H187, H209, and H526. Bottom panel: Cyclophilin, used as a positive reaction control was seen in all samples. For all gene expression experiments, negative control reactions were performed and found to be negative. The bands on the agarose gels were consistent with the expected sizes: GIRK3-317 bp; cyclophilin-216 bp; M-100 bp. FGiIgRuKr3ee6xpression in NSCLC cell lines and in normal cells GIRK3 expression in NSCLC cell lines and in normal cells. Top panel: GIRK3 was expressed in normal human SAEC and hamster PNEC as well as being expressed in all NSCLC cell lines tested. These NSCLC cell lines are: A549, H727, H226, H520, and H2170. Bottom panel: Cyclophilin, used as a positive reaction control was seen in all samples. For all gene expression experiments, negative control reac- tions were performed and found to be negative. The bands on the agarose gels were consistent with the expected sizes: GIRK3-317 bp; cyclophilin-216 bp; M-100 bp. collected from patients at the UT Graduate School of Medicine's Cancer Center. None of these tissue samples expressed GIRK1 (Figure 7). For functional GIRK channels in lung cells, protein expres- sion is needed as well as gene expression. We determined GIRK1 protein expression in cell lines that express GIRK1 mRNA. Isolating enriched membrane protein using a Bio- rad kit, we found GIRK1 protein expression in the three SCLC cell lines that expressed GIRK1 mRNA, H69, H146 and WBA (Figure 8). In addition, in the NSCLC cell lines that express GIRK1 mRNA, GIRK1 protein expression was determined. GIRK1 protein expression was not seen in A549, H727, H2170, H226, and H520 (data not shown). Since β-adrenergic ligands affected both gene expression of GIRK1 and cell proliferation for breast cancer cell lines in our laboratory [10], we wanted to determine if β-adren- ergic agonists and antagonists had effects on either gene expression or cell proliferation in SCLC cells. H69 cells were used for these experiments because it is one of the two cell lines that expressed mRNA for all four GIRK chan- nels as well as GIRK1 protein. The second cell line, WBA, is not as well characterized in the literature. Exposure of MDA-MB-453 breast cancer cells for six days to the β- adrenergic antagonist propranolol increased the GIRK1 mRNA levels [10]. In the present experiments, we used two specific β-adrenergic antagonists to possibly induce changes in GIRK1 mRNA levels. The specific β1 antagonist atenolol (100 µ M) had no effect on gene expression when H69 cells were treated for seven days (Table 3). However, when H69 cells were treated with 100 µ M of the β2 adren- ergic antagonist ICI daily for seven days, differences in gene expression were seen as determined by real-time quantitative PCR (Table 3). Calculating differences in gene expression using the 2-∆ CT Formula [32], ICI treat- ment caused a 1.47× decrease in GIRK1 gene expression p < 0.0003 (Table 3). H69 cells express mRNA for both β1 and β2 (data not shown). As indicated above, we wanted to determine if β-adrenergic ligands altered proliferation in H69 cells. We determined the effects of the β-adrenergic agonist Iso and the GIRK channel inhibitor U5 [28] on proliferation in H69 cells. Iso (10 µ M) inhibited prolifer- ation (p < 0.001) (Figure 9). The GIRK inhibitor U5 (2 µ M) also inhibited proliferation (p < 0.001), and this decrease was potentiated by Iso (p < 0.001) (Figure 9). Discussion This is the first report that describes the expression of mRNA for G-Protein Inwardly Rectifying Potassium Channels (GIRKs) in lung cancer cell lines. Previous reports have indicated that voltage gated K+, Na+ and Ca2+ channels have been shown in SCLC cell lines [20], and these voltage activated K+ channels have a role in modu- lating cell proliferation [21]. Classical inwardly rectifying potassium channels not linked with G-proteins have been shown in a few SCLC cell lines [22,23]. Since GIRK1 can- not form functional channels by itself, other GIRK chan- nels are needed [30]. All six SCLC cell lines tested express mRNA for either GIRK2 or GIRK4 indicating that func- tional GIRK potassium channels are possible in these SCLC cancer cell lines. GIRK1 has been shown in tissue samples from approximately 40% of primary human Page 6 of 10 (page number not for citation purposes)