Sethi T, Herget T, Wu SV, Walsh JH, Rozengurt E.. CCKA and CCKB receptors are expressed in small cell lung cancer lines and mediate Ca2+ mobilization and clonal growth. Cancer Res 53: 5208-5213 (original) (raw)
Castrili, cholecystokinin (CCK), and CCK-related peptides comprise a Imimonili family characterized by an identical carboxy-terminal amino acid sequence, a domain critical for receptor binding. The addition of gastrin to small cell lung cancer (SCLC) cells causes a rapid and transient increase in the intracellular concentration of calcium i|(';r'|,i. Further more, gastrin acts as a direct growth factor through CCKn/gastrin recep tors. We report here that the expression of the mRNA coding for CCKu/ gastrin receptors correlates with the responsiveness of SCLC cells to gastrin in terms of (';r ' mobilization and stimulation of clonal growth in semisolid medium. The GLC19 SCLC cell line had no detectable expres sion of CCKn/gastrin receptor mRNA. Accordingly, gastrin (1-100 IIMIdid not cause any measurable increase in |('¡i '' |¡-In contrast, the addition of cholecystokinin residues 26â€"33(CCK-8) caused a rapid and transient increase in |< '¡r'1 ], in this cell line. CCK-8 mobilized « ;i '' in a dosedependent manner in the nanomolar range (half-maximal stimulatory concentration = 12 nvi). Furthermore, the selective CCKA antagonist CAM-1481 inhibited the increase in |(':i-'-|, induced by CCK-8 (halfmaximal inhibitory concentration = 3 n>i) in GLC19 but not in H510 cells. The selective CCKn/gastrin antagonist blocked the increase in |(;r ' |, induced by CCK-8 (half-maximal inhibitory concentration = 80 pM) in H510 but not in GLC19 cells. Thus, the effects of CCK-8 are mediated through CCKA receptors in GLC19 cells and via CCKB/gastrin receptors in H510 cells. CCK-8 markedly stimulated colony formation in GLC19 cells in a dose-dependent manner in the nanomolar range, whereas over the same concentration range, gastrin had no effect on clonal growth. CAM-1481 inhibited the CCK-stimulated colony formation in GLC19 but not in H510 cells. Our results show, for the first time, that CCKA receptors can mediate (a'' mobilization and growth in SCLC cells and that SCLC cells express two distinct functional CCK receptor subtypes.
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Annals of the New York Academy of Sciences, 1994
Thc incidence of carcinoma of the lung in the western world has been increasing at a dramatic rate during the last 50 years and it has become the principal cause of cancer deaths. Small cell lung cancer (SCLC), which constitutes 25% of all pulmonary cancers, follows a very aggressive clinical course despite initial sensitivity to chemotherapy and radi0therapy.l Novel therapeutic approaches are needed and they will arise, most likely, from a better understanding of the factors and intracellular events that are responsible for stimulating the rapid growth of SCLC cells.
Cancer research, 1993
Gastrin, cholecystokinin (CCK), and CCK-related peptides comprise a hormonal family characterized by an identical carboxy-terminal amino acid sequence, a domain critical for receptor binding. The addition of gastrin to small cell lung cancer (SCLC) cells causes a rapid and transient increase in the intracellular concentration of calcium ([Ca2+]i). Furthermore, gastrin acts as a direct growth factor through CCKB/gastrin receptors. We report here that the expression of the mRNA coding for CCKB/gastrin receptors correlates with the responsiveness of SCLC cells to gastrin in terms of Ca2+ mobilization and stimulation of clonal growth in semisolid medium. The GLC19 SCLC cell line had no detectable expression of CCKB/gastrin receptor mRNA. Accordingly, gastrin (1-100 nM) did not cause any measurable increase in [Ca2+]i. In contrast, the addition of cholecystokinin residues 26-33 (CCK-8) caused a rapid and transient increase in [Ca2+]i in this cell line. CCK-8 mobilized Ca2+ in a dose-depe...
Expression of CCKB/gastrin receptor isoforms in gastro‐intestinal tumour cells
International Journal of Cancer, 1998
Anti-serum raised against the human cholecystokinin B (CCKB)/gastrin receptor was used in Western blotting to differentiate the cellular locations of receptor isoforms expressed by human gastro-intestinal (GI) tumour cell lines. Using anti-serum directed against the amino-terminal extracellular tail of the CCKB/gastrin receptor, 8/9 cell lines were shown to express immunoreactive proteins of either m.w. 70 or 40 kDa, or both. Both isoforms were found to be associated with intracellular, non-nuclear membranes, whereas only the 70 kDa protein was expressed in the plasma membrane. Receptor expression was related to gastrin production and secretion. Both progastrin and glycine-extended gastrin-17 were produced and secreted by the tumour cell lines; however, carboxy amidated gastrin was not detected by radioimmunoassay. A CCKB/gastrin receptor transfectant NIH3T3 cell line did not produce detectable gastrin and showed exclusive expression of the 70 kDa receptor on the plasma membrane. One cell line had Ͻ50 pg/ml cell-associated progastrin and no detectable receptor form. Cell lines expressing 50-150 pg/ml had both 40 and 70 kDa receptor forms. Those expressing Ͼ150 pg/ml progastrin had only the 40 kDa isoform, which was shown to be exclusively expressed on intracellular, non-nuclear membranes, in one of the cell lines. Of the 4 cell lines exclusively expressing the lower m.w. receptor, 3 had gastrin present within the cell, which was not secreted. Thus, if cell-associated gastrin induces a proliferative effect, it may be by an intracrine pathway. Our study has identified the presence of CCKB/gastrin receptor isoforms in different cellular locations and may help toward understanding the complex autocrine and intracrine pathways mediated by gastrin peptides. Int.
Regulatory Peptides, 2001
Introduction: Gastrin acts to stimulate gastric acid secretion and is an acknowledged growth factor for human gastrointestinal GI cancer. The identity of the exact receptor type mediating the growth promoting effects of gastrin in tumours is uncertain. However, the Ž. best-characterised gastrin receptor is the CCK receptor type B CCKB rgastrin receptor. The anti-GRE1 antibody is a polyclonal, affinity-purified antibody raised against GRE1, a synthetic 21 amino acid peptide homologous to part of the extracellular, N-terminal tail of the CCKB receptor. We have recently proven that GRE1 antiserum specifically localises CCKB receptors on CCKB receptor transfected NIH3T3 cells and human gastrointestinal tumour cells by Western blotting and immunocytochemistry. GRE1 antiserum also inhibits liver invasion in the C170HM colorectal liver-metastasis model. 2 Aim: To relate the ability of GRE1 antiserum to displace G17 from CCKB receptors with its impact on cellular transduction effects. Methods: Radioligand binding studies were performed with 125 IG17 and Calcium mobilisation studies by use of the fluorescent dye Fura 2-am. Results: GRE1 antiserum competitively displaced 50% radiolabelled gastrin-17 from whole cell NIH3T3 CCKB transfectants at a protein concentration of 250 mg ml y1. GRE1 antiserum did not stimulate calcium ion influx in the transfectant NIH3T3 cells when used at a range of protein concentrations. Pre-incubation with GRE1 antiserum was required to inhibit gastrin-stimulated calcium ion influx. This was found to be concentration-dependent, with inhibition shown at 30 and 5 mg ml y1 but not at 500 ng ml y1 or below. Conclusion: The GRE1 antiserum is specific for the CCKB receptor and may act to inhibit gastrin-stimulated signalling in tumour cells.
Cellular Signalling, 2000
Depending upon experimental model, the CCK-B/gastrin receptor ligand CI-988 exhibits either agonist or antagonist activity. To confirm that CI-988 behaves as an antagonist toward gastrin-stimulated growth, its effects on cell proliferation were investigated in unsynchronized and synchronized AR42J rat pancreatic tumour cells. In unsynchronized cultures CI-988 alone had no effect, but inhibited gastrin-stimulated cell proliferation. In contrast, in synchronized cultures, CI-988 stimulated cell proliferation. Similarly, CI-988 inhibited gastrin-stimulated cAMP production in unsynchronized cells, but stimulated cAMP formation in synchronized cultures. Therefore, CI-988 stimulation of cAMP production and proliferation in AR42J cell cultures appears to be cell cycle-dependent. CI-988 inhibited gastrin-stimulated intracellular calcium ([Ca 2ϩ ] i) mobilization in both populations and thus acted as an antagonist toward this pathway. Because CCK receptor densities and affinities were similar in both cell populations, the data suggest that CI-988's divergent effects on cell proliferation are governed by postreceptor signalling events which vary with cell cycle.
High affinity binding of cholecystokinin to small cell lung cancer cells
Peptides, 1987
affinity binding of cholecystokinin to small cell lung cancer cells. PEPTIDES 8(1) 103-107, 1987.-The binding of '2'~I-Bolton Hunter-cholecystokinin octapeptide ('2'~I-BH-CCK-8) to small cell lung cancer cell lines was investigated. ~zSI-BH-CCK-8 bound with high affinity (Kd=2.4 nM) to an apparent single class of sites (1700/cell) using cell line NCI-H209. Binding was time dependent and the ratio of specific/nonspecific binding was 8/1. Pharmacology studies indicated that gastrin, caerulein, CCK-33 and nonsulfated CCK-8 were potent inhibitors of specific ~2~I-BH-CCK-8 binding whereas CCK-26-32-NH2 was not. Because CCK receptors are present on small cell lung cancer cells, CCK may function as a regulatory peptide in this disease. CCK CCK receptors Small cell lung cancer Neuroendocrine peptides
Gastroenterology, 2005
Background & Aims: The role of amidated gastrin 17 (G17) and the gastrin/CCKB/CCK2 receptor in colorectal carcinogenesis is still a controversial issue. Here, we investigated the effect of G17 on proliferation and apoptosis of CCK2 receptor-expressing human colon cancer cell lines in vitro and in vivo. Methods: Proliferation was determined by cell counting and cell cycle analysis. Apoptosis was analyzed by annexin V staining, TUNEL staining, caspase-3/7 assay, and JC1 (⌬⌿) assay. Signal-transduction pathways were analyzed by Western blotting and gelshift and luciferase assays. An in vivo tumor model with subcutaneously inoculated colon cancer cells in SCID mice was used, and systemic hypergastrinemia was induced by omeprazole. Results: In Colo320 cells stably transfected with the wild-type CCK2 receptor (Colo320wt) or in Lovo cells endogenously expressing CCK2 receptors, G17 treatment inhibited proliferation along with a G2/M cell cycle arrest. Furthermore, the administration of G17 significantly augmented apoptosis of CCK2 receptor-expressing cells. In contrast, G17 had no effect on proliferation and apoptosis in Colo320 cells stably transfected with a tumor-derived CCK2 receptor mutant (Colo320mut) or in cells lacking CCK2 receptor expression. Systemic hypergastrinemia in severe combined immunodeficiency (SCID) mice suppressed the growth of Colo320wt tumors accompanied by enhanced apoptosis as compared with untreated tumors. In contrast, omeprazole did not affect Colo320mut tumors reflecting a loss-offunction state of the CCK2(mut) receptor. This is supported by the observation that, in Colo320wt cells, but not in Colo320mut cells, G17 treatment induced the MAPK/ERK/AP-1 pathway and inhibited the activity of NF-B. Conclusions: G17 exerts an antiproliferative and proapoptotic effect on human colon cancer cells expressing the wild-type CCK2 receptor. This supports the view that amidated gastrin prevents rather than promotes colorectal carcinogenesis.
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