BMC Cancer 2005, 5:104 http://www.biomedcentral.com/1471-2407/5/104 RT-PCR RNA from cell cultures, adult hamster brain, PNEC and from fresh surgical tissue samples was isolated by guani- dine isothiocyanate/cesium chloride ultracentrifugation [29] or by an Absolutely RNA kit (Stratagene, La Jolla, CA). RT-PCR was done as previously described [10]. The GIRK3 primers are forward 5'-gtgaccagcttcctccagac-3' and reverse 5'-gctaccatcttcccatccaa-3' which amplifies a 317 bp fragment (bases 1421–1737, Genbank Acession # NM_004983). PCR conditions are 94°C, 30 sec; 55°C, 30 sec; 72°C, 45 sec for 40 cycles. Cyclophilin primers were used an internal control (Ambion, Austin, TX). Real-time PCR Real-time PCR was done as previously described [10]. GIRK1 primers-forward 5'-ctctcggacctcttcaccac-3' and reverse 5'-gccacggtgtaggtgagaat-3' (bases 398–477, Gen- bank Acession # NM002239) and the internal TaqMan probe is 6-FAM-tcaagtggcgctggaacctc-TAMRA (bases 429– 449), annealing 62°. GIRK2 primers-forward 5'-gacct- gccaagacacatcag-3' and reverse 5'-cggtcaggtagcgataggtc-3' (bases 766–886, Genbank Acession # U52153) and the internal TaqMan probe is 6-FAM-gtgcaatgttcatcacggcaac- TAMRA (bases 837–859), annealing 56°. GIRK4 primers- forward 5'-agcgctacatggagaagagc-3' and reverse 5'-aagtt- gaagcgccacttgag-3' (bases 241–358, Genbank Acession # L47208) and the internal TaqMan probe is 6-FAM-accgg- tacctgagtgacctcttca-TAMRA (bases 301–324), annealing 62°. Reactions were run on a Cepheid SmartCycler (Sun- nyvale, CA). Reaction conditions are 200 µ M dNTPs, 0.3 µ M gene specific primers, 0.2 µ M TaqMan probe, 4 mM (GIRK1) or 6 mM (GIRK2or4) magnesium acetate, 2 µ l cDNA and 1.5 U MasterTaq (Eppendorf, Westbury, NY) and MasterTaq buffer in a final volume of 25 µ l. In some experiments, 18S primers were used an internal control (Qiagen, Valencia, CA). Proliferation assay Cells were counted and plated in RPMI without serum or phenol red at a density of 50,000 cells/well with appropri- ate treatments in a volume of 500 µ l. Cells were allowed to grow for 48 hours, and proliferation was measured by a cell proliferation assay for 5-bromo-2'-deoxyuridine (BrdU) according to the manufacturer's instructions (Roche, Indianapolis, IN), N = 5 for each treatment group. Western blots Cell pellets were collected and membrane protein was iso- lated with the ReadyPrep protein extraction kit (signal) (Biorad, Hercules, CA). Protein levels were determined using the RCDC kit (Biorad). Aliquots of 20–30 µ g pro- tein were boiled in 3× loading buffer (New England Biolabs, Beverly, MA) for two minutes, then loaded onto 12% Tris-glycine-polyacrylamide gels (Cambrex, Rock- land, ME), and transferred electrophoretically to nitrocel- lulose membranes. Membranes were incubated with the primary antibody (GIRK1; Upstate Biotechnology, Lake Placid, NY). In all western blots, membranes were addi- tionally probed with an antibody for actin (Sigma) to ensure equal loading of protein between samples. The membranes were then incubated with appropriate sec- ondary antibodies (Rockland, Gilbertsville, PA or Molec- ular Probes, Eugene OR). The antibody-protein complexes were detected by the LiCor Odyssey infrared imaging system (Lincoln, NE). Results In order to investigate possible expression similarities between breast cancer and lung cancer, we wanted to determine if GIRK channels are expressed in human lung cancers. Expression of mRNA for the GIRK1 channel was seen in three of six SCLC cell lines. GIRK 1 was expressed in the SCLC cell lines H69, H146 and WBA (Figure 1). Since GIRK1 cannot form channels alone, it must assem- ble with GIRK2, 3 or 4 [reviewed in [30]]. GIRK2 & 4 expression was determined in the six SCLC cell lines using real-time PCR. GIRK 4 expression was found in all six SCLC cell lines, and GIRK2 was found in all six SCLC cell lines with the exception of H146 and H187 (Table 1). In these experiments, real-time PCR was used as a second method of determining gene expression. Since gene expression was determined by only one sample (similar to gene expression studies by RT-PCR) no comparisons can be made between CT values for the samples. We wished to compare GIRK1 expression in SCLC to normal primary cells. GIRK1 was not expressed in the normal human SAE cells (Figure 1). Normal SAE cells express GIRK4 but do not express GIRK2 (data not shown). SCLC shares pheno- typic and functional features with pulmonary neuroendo- crine cells (PNEC), one of the possible origins of this cancer type [31]. Neither GIRK1 nor GIRK2 mRNA expres- sion was found in normal tissue from PNEC cells isolated from prenatal hamster lungs using the primers designed for human GIRK1 and 2 (Figure 2). In order to determine if this result was due to species differences in GIRK1 and 2 between humans and hamsters, tissue was isolated from hamster brain. In both cases, mRNA from GIRK1 and 2 were found to be expressed in hamster brain (Figure 2). However, our GIRK4 primers were found not to work in hamster tissue (data not shown). We wanted to determine if GIRK channels were also expressed in human non-SCLC (NSCLC) cancers. GIRK1 was not expressed in two adenocarcinoma cell lines of Clara cell phenotype, H322 and H441 (Figure 1). How- ever, three GIRK channels (1,2,4) were expressed in both the human adenocarcinoma cell line with alveolar type II cell phenotype (A549) and human carcinoid cell line expressing neuroendocrine features (H727) (Figure 3). GIRK1 and 4 are expressed in three squamous cell lines, Page 3 of 10 (page number not for citation purposes)