BMC Cancer 2005, 5:104 http://www.biomedcentral.com/1471-2407/5/104 cLpFauirgclmckuinorooenfmaG7rayIsRaKd1eneoxcparrecsisniomn ains ofrresphulsmurognicaarlytsisqsuuaemsoaumsples of Lack of GIRK1 expression in fresh surgical tissue samples of pulmonary adenocarcinomas or pulmo- nary squamous carcinomas. Top panel: GIRK1 was not expressed in two pulmonary squamous carcinomas or three adenocarcinomas obtained from cancer patients. Bottom panel: Cyclophilin, used as a positive reaction control was seen in all samples. For all gene expression experiments, neg- ative control reactions were performed and found to be neg- ative. The bands on the agarose gels were consistent with the expected sizes: GIRK1-441 bp; cyclophilin-216 bp; M-100 bp. expression of other GIRK subunits [34]. Further studies are needed to determine the role of GIRK3 in lung cancer. Since protein expression would also be necessary for func- tional GIRK channels, we determined GIRK1 protein expression in cell lines that expressed GIRK1 mRNA. Expression of GIRK1 protein was seen in the three SCLC cell lines that express GIRK1 mRNA. This is the first report of GIRK1 protein expression in SCLC cell lines. We also determined GIRK1 protein expression in NSCLC cell lines. None of the NSCLC cell lines that expressed GIRK1 showed protein expression for GIRK1. We feel that this indicates that GIRK1 expression may be a larger factor in SCLC than in NSCLC. Further study is needed to elucidate these differences. In the present study, we demonstrated a small decrease in GIRK1 mRNA expression (1.5×) in the H69 SCLC cell line after treatment for one week with the β2 adrenergic antag- onist ICI daily for seven days. In contrast to this data, exposure of the breast cancer cell line MDA-MB453 for six FGmiIRgRNuKrA1epa8rsoatsesiensseexdprbeysswioenstienrSnCbLloCt caenlalslytshisat express GIRK1 GIRK1 protein expression in SCLC cells that express GIRK1 mRNA as assessed by western blot analysis. In the three SCLC cell lines that expressed GIRK1 mRNA (Fig- ure 1), membrane protein expression was determined. Top panel: All three SCLC cell lines (WBA, H69, H146) expressed GIRK1 membrane protein. Bottom panel: Actin was used as a control for equal sample loading. The bands are consistent with the expected size: GIRK1-62 kDa; actin- 42 kDa. Table 3: Changes in gene expression of GIRK1 after seven day treatment of H69 cells with the β2 adrenergic antagonist ICI 118, 551 (ICI) (100 µ M) and lack of changes after seven day treatment with β1 adrenergic antagonist atenolol (AT) (100 µ M). GIRK1 18S Control 21.186 ± 0.13 12.028 ± 0.17 ICI 22.040 ± 0.06a 12.326 ± 0.03 AT 21.238 ± 0.06 12.090 ± 0.11 Changes in gene expression were determined by real time quantitative PCR, and are expressed as threshold values (CT), N = 5. a p < 0.0003. Threshold/CT cycle values will be higher for samples with less mRNA expression. days to the β-adrenergic antagonist propranolol (1 µ M) increased the GIRK1 mRNA levels [10]. Treatment with the β1 antagonist, atenolol, for the same time period had no effect, indicating that the β2-adrenergic receptor is more important for changes in GIRK mRNA. GIRK cur- rents have been shown to be increased in cells stimulated with the β-adrenergic agonist Iso in rat atrial myocytes transfected with β1 or β2 receptors [8]. Heterologous facil- itation of GIRK currents by β-adrenergic stimulation was also seen in rat cardiomyocytes [7]. The differences in sig- naling pathways stimulated by β-adrenergic ligands in breast cancer and SCLC remain to be elucidated. Page 8 of 10 (page number not for citation purposes)