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PDF file - 62K, Gpr19 siRNA-induced increase in the relative number of DMS 53 cells with G2/M DNA content

PDF file - 51K, Detailed cell line information for human NSCLC, SCLC, and normal lung cell lines and HEK-293 cells

PDF file - 337K, Reduced cellular proliferation upon Gpr19 siRNA treatment. Thumbnail visualization of confluence measurement for NCI-H1703 cells and phase contrast microscopy pictures of NCI-H1703 and DMS 53 cells subject to siRNA transfection

PDF file - 45K, Information about TaqMan assays used in RT-qPCR

PDF file - 88K, Gpr19 siRNA-induced increase in binucleated and polylobed nuclei

PDF file - 74K, Binding of E2F1-4 to the promoter of Gpr19. The result of chromatin immunoprecipitation is shown (full size agarose gel).

PDF file - 253K, Detailed patient information for NSCLC, SCLC and normal lung RNA samples from Origene Technologies

It has long been known that G protein-coupled receptors (GPCR) are subject to illegitimate expression in tumor cells. Presumably, hijacking the normal physiologic functions of GPCRs contributes to all biologic capabilities acquired during tumorigenesis. Here, we searched for GPCRs that were expressed in lung cancer: the mRNA encoding orphan G protein-coupled receptor 19 (GPR19) was found frequently overexpressed in tissue samples obtained from patients with small cell lung cancer. Several observations indicate that overexpression of Gpr19 confers a specific advantage to lung cancer cells by accelerating transition through the cell-cycle. (i) Knockdown of Gpr19 mRNA by RNA interference reduced cell growth of human lung cancer cell lines. (ii) Cell-cycle progression through G2–M-phase was impaired in cells transfected with siRNAs directed against Gpr19 and this was associated with increased protein levels of cyclin B1 and phosphorylated histone H3. (iii) The expression levels of Gpr19 mRNA varied along the cell-cycle with a peak observed in S-phase. (iv) The putative control of Gpr19 expression by E2F transcription factors was verified by chromatin immunoprecipitation: antibodies directed against E2F-1 to -4 allowed for the recovery of the Gpr19 promoter. (v) Removal of E2F binding sites in the Gpr19 promoter diminished the expression of a luciferase reporter. (vi) E2f and Gpr19 expression correlated in lung cancer patient samples. To the best of knowledge, this is the first example of a GPCR showing cell-cycle-specific mRNA expression. Our data also validate GPR19 as a candidate target when overexpressed in lung cancer. Mol Cancer Res; 10(10); 1343–58. ©2012 AACR.

Selective serotonin reuptake inhibitors (SSRIs) have been observed to drive programmed cell death in Burkitt lymphoma cells. Further studies, however, showed that SSRIs induce apoptosis with little, if any, appreciable selectivity. Actually, the selectivity appears to be so low that SSRIs can kill protozoa such as Trichomonas vaginalis. Although the serotonin transporter SERT was initially considered as SSRI target in inducing programmed cell death, this concept has been rejected repeatedly. N-acetylated versions of SSRIs, which are not capable of inhibiting serotonin reuptake anymore, were shown to kill cells in concentration ranges comparable to those of their non-acetylated original versions. Our working hypothesis is that SSRIs induce cell death via activation of the endoplasmic reticulum stress response/unfolded protein response (UPR). To address this hypothesis, we performed a luciferase reporter assay (in HEK293 and HeLa cells), in which the firefly luciferase gene is under the control of the promoter for glucose-regulated protein of 78 kD (GRP78), an endoplasmic reticulum (ER) chaperone mainly expressed during the UPR. Several SSRIs (paroxetine, fluvoxamine, fluoxetine and citalopram) and their N-acetylated versions induce GRP78 expression with steep concentration-response curves, comparable to those obtained for killing cells. SSRIs also trigger activation of caspase 3/7, as observed with a caspase 3/7-dependent fluorescent substrate, and expression of the endoplasmic reticulum stress protein C/EBP-homologous protein (CHOP-10). Our results suggest that SSRIs induce cell death via the UPR, and further experiments are designed to strengthen our hypothesis. We will also investigate, if this effect is conserved throughout species (e.g. in Trichomonas vaginalis).

Background Selective serotonin reuptake inhibitors (SSRIs) have been observed to drive programmed cell death in Burkitt lymphoma cells. Accordingly, they were proposed as a new modality in the treatment of Burkitt lymphoma. However, they induce apoptosis with little if any appreciable selectivity. Actually, the selectivity appears to be so low that SSRIs can also kill protozoa (such as Trichomonas vaginalis). Acetylated versions of SSRIs, which do not inhibit serotonin reuptake, kill cells in concentration ranges comparable to those of their non-acetylated original versions. Thus, the obvious SSRI target, SERT, is not required for cell killing.