BMC Cancer 2005, 5:104 http://www.biomedcentral.com/1471-2407/5/104 Background Recent studies in human cancer cell lines or in animal models have shown that the growth of adenocarcinomas of the lungs, pancreas and colon are under β-adrenergic control [1-5]. The tobacco carcinogenic nitrosamine 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) has recently been identified as a high affinity β-adrenergic agonist that stimulated the growth of pulmonary and pan- creatic adenocarcinomas in vitro and in animal models [1,3,5]. 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 tested [6], and this expression of GIRK1 was associated with a more aggres- sive clinical behavior. Increases in GIRK currents by β- adrenergic stimulation have been reported in adult rat car- diomyocytes and in Xenopus laevis oocytes coexpressing β2-adrenergic receptors and GIRK1/GIRK4 subunits [7]. In addition, in rat atrial myocytes transiently transfected with β1 or β2 adrenergic receptors, the β-adrenergic agonist isoproterenol (Iso) stimulated GIRK currents, whereas this stimulation was not seen in non-transfected cells [8]. Previous data from our laboratory has indicated that there is a functional link between the β-adrenergic receptor pathway and the G-protein inwardly rectifying potassium channel (GIRK1) in breast cancer cell lines and these path- ways were involved in growth regulation of these cells [9,10]. Additional data from our laboratory has also indi- cated that the normal breast epithelial cell line MCF 10A lacks GIRK1 expression [10]. Established risk factors for breast cancer include age, increased hormone exposure, alcohol consumption and family history as well as many other factors [11]. Smoking is a controversial risk factor for the development of this malignancy [12-14]. However, increases in pulmonary metastatic disease have been seen in smokers with breast cancer [15]. In addition, a study of 141,000 women showed a significantly increased risk of developing lung cancer for breast cancer patients, possibly due to interac- tions between radiotherapy and smoking [16]. An eight- fold increase in breast cancer risk has been seen in women who were smokeless tobacco users [17]. Smokeless tobacco has higher levels of nicotine and the tobacco car- cinogen NNK than cigarette smoke [18]. However, many types of lung cancer are smoking related [19]. In order to investigate possible similarities between breast cancer and lung cancer, we wanted to determine if GIRK channels are expressed in human lung cancers and if β- adrenergic signaling is also involved in lung cancer. Volt- age gated K+, Na+ and Ca2+ channels have been shown in the small cell lung cancer (SCLC) cell lines NCI-H128, NCI-H69 and NCI-H146 by measurement of currents [20]. These voltage activated K+ channels in the SCLC cell lines have a role in modulating cell proliferation [21]. A classical inwardly rectifying potassium channel not linked with G-proteins has been shown in a subclone of NCI- H69, H69AR, a subline with overexpressed multidrug resistance associated protein, but these currents were not seen in the original H69 cell line [22]. Inwardly rectifying potassium currents have also been measured in RERF-LC- MA SCLC cells, and these cells express the Kir2.1 mRNA and protein [23]. GIRK1 expression has been shown in tis- sue specimens from patients with non-small lung cancer and this expression was associated with lymph node metastasis [24]. Prior to this study, GIRK channels have not been identified in lung cancer cell lines or SCLC cell lines and clinical samples. In the present research we have screened small cell and non-small cell lung cancer cell lines and several normal lung cell types for GIRK mRNA and protein expression. Methods Cell culture The human SCLC cell lines NCI-H69 (H69), NCI-H146 (H146), NCI-H187 (H187), NCI-H209 (H209), and NCI- H526 (H526), the human adenocarcinoma cell lines NCI- H322 (H322), NCI-H441 (H441), and A549, the carci- noid cell line NCI-H727 (H727), and the squamous cell lines NCI-H226 (H226), NCI-H2170 (H2170), and NCI- H520 (H520) were purchased from the American Type Culture Collection (Manassas, VA). The human SCLC cell line WBA [25] was a gift of Dr. G. Krystal, Medical College of Virginia. All cancer cell lines except A549 were main- tained in RPMI medium supplemented with fetal bovine serum (10% v/v), L-glutamine (2 mM), penicillin (100 U/ ml) and streptomycin (100 µ g/ml) at 37°C in an atmos- phere of 5% CO2. A549 cells were grown in Hams F12 media with supplements as above. Human small airway epithelia cells (SAEC) were purchased from Clonetics/ BioWhittaker (Walkersville, MD). These primary cells were maintained in SAEC basal medium with supple- ments (Clonetics) at 37°C in an atmosphere of 5% CO2. Fresh surgical tissue samples were collected from patients at the University of Tennessee (UT) Graduate School of Medicine's Cancer Center and processed for reverse tran- scription polymerase chain reaction (RT-PCR). The collec- tion of tissue was approved by the UT Institutional Review Board, and the authors have been certified by the NIH Office of Human Subjects Research. Cultures of fetal ham- ster pulmonary neuroendocrine cells (PNEC) were estab- lished from fetal lung periphery harvested on day 15 of gestation as previously described [26,27]. Studies with fetal hamster cells were approved by the UT Institutional Animal Care and Use Committee. Exposure of cells to iso- proterenol (Iso), the GIRK inhibitor U50488H (U5) [28] (Sigma, St. Louis, MO), or ICI 118,551 (ICI) (Tocris, Ball- win, MO) for experiments was as detailed in the figure leg- ends or results. Page 2 of 10 (page number not for citation purposes)