ARTICLE |
Correspondence to: Martyn E. Caplin, Gastroenterology and Hepatobiliary Medicine, Royal Free Hospital, Pond Street, London NW3 2QG, UK.
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Immunotherapy has considerable potential in the treatment of cancer. Here we report on the uptake of an antibody raised against the CCK-B/Gastrin receptor (CCK-BR) by liver embryonic and liver tumor cell lines. In all five cell lines studied, expression of CCK-BR and uptake of labeled anti-CCK-BR antibody was observed. The labeled anti-CCK-BR antibody was localized in both the cytoplasm and nucleus of cells. In addition, we found a coincidence between the uptake of the labeled antibody by cells and the occurrence of apoptosis (cell death). The results suggest that antibodies directed against CCK-BR have potential for targeting and possibly destroying tumor cells bearing the receptor.
(J Histochem Cytochem 50:12131217, 2002)
Key Words: gastrin, CCK-B receptor, endocytosis, apoptosis, hepatocyte, tumor
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The incidence of hepatocellular carcinoma (HCC) is increasing in the United States (
Adoptive immunotherapy has been shown to lengthen the time between resection and recurrence in patients with HCC (
Gastrin is a peptide hormone which, in addition to stimulating gastric acid secretion, is also now believed to play a role in tumor growth. In addition to normal gut mucosa and brain, the receptor for gastrin (CCK-B/gastrin) is expressed in tumor cells including hepatic, gastrointestinal, and pancreatic cancers (
We have previously demonstrated that exposure of AR42J (pancreatic acinar carcinoma) cells to a labeled antibody (anti-CCK-BR) raised against the aminoterminus of the CCK-B/gastrin receptor results in translocation of the antibody, first to the cytoplasm and then to the nucleus (
The aims of this study were to detect the CCK-B/gastrin receptor in hepatoma cells and to determine whether uptake of an anti-CCK-BR antibody was able to induce apoptosis in these cells.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell lines
HepG2 (human HCC), WRL68 (human liver embryonic), HTC (rat hepatocellular carcinoma), PLC/PRF/5 (human HCC), and MCA RH 7777 (rat HCC) cells were obtained from the European Collection of Cell Cultures (ECACC; Porton Down, UK). NIH3T3 cells were a gift from Professor T. Matsui (Kobe University; Kobe, Japan).
Anti-CCK-BR Antibody
The anti-CCK-BR antibody (Aphton Corporation; Woodland, CA) was raised as previously described (
Immunoblotting
Lysate corresponding to 10 µg protein was loaded onto 10% polyacrylamide minigels for SDS-PAGE. In addition to the liver cell lines, normal mouse fibroblast NIH3T3 cells were included as a negative control. After electrotransfer and overnight blocking, the membranes were incubated with 3 µg/ml anti-CCK-BR antibody. The antibody was used either untreated or had been preabsorbed for 48 hr with a molar excess of the immunizing peptide. After incubation with goat anti-rabbit/mouse biotin and streptavidin/biotin-peroxidase complex, labeling was revealed using diaminobenzidine (DAB) reagent.
Uptake and Apoptosis Studies
Anti-CCK-BR antibody was labeled with Alexa Fluor 488 fluorescent dye (Molecular Probes; Eugene, OR). Cells were cultured on chamber well slides and exposed to the labeled antibody (20 µg/ml) for 1 hr at 37C and fixed with buffered formaldehyde solution. Cells were then counterstained with a nuclear stain (propidium iodide) or for apoptosis using the TUNEL method.
For propidium iodide staining, slides were incubated with 100 µg/ml RNase for 90 min at 37C followed by 3 µg/ml propidium iodide (both in 2 x SSC) for 7 min.
Apoptosis staining used the TUNEL method (ApopTag Red in situ apoptosis detection kit; Intergen, Purchase, NY) which labels the 3'-OH ends of DNA fragments generated by apoptosis with chemically modified nucleotides. The latter are then detected using a secondary antibody conjugated to the fluorescent dye rhodamine. Images were obtained using either a confocal or fluorescence microscope with appropriate filters for the Alexa Fluor 488 and rhodamine/propidium iodide fluorescence.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In all hepatocyte cell lines except negative control NIH3T3 (normal mouse fibroblasts), anti-CCK-BR immunoreactivity was found by blotting. Specific bands were distinguished by the absence of a corresponding band in the blots incubated with the preabsorbed anti-CCK-BR antibody. A common band at 162 kD was seen in all hepatocyte lines. Additional bands were seen at
137 kD in WRL68, HepG2, and PLC/PRF/5 cells and at
79 kD in the MCA RH7777 and HTC cells. A
183-kD band was also observed with the MCA RH7777 cells. No specific bands were seen with the NIH3T3 cells (Fig 1).
|
Immunofluorescence microscopy demonstrated uptake of anti-CCK-BR in all five cell lines studied (Fig 2). In cells that displayed uptake, nuclear localization of antibody was observed in nearly 100% of PLC/PRF/5, WRL68, Hep G2, and HTC cells but could not be confirmed in MCA RH7777 cells. Using the immunofluorescent TUNEL assay, cells that showed anti-CCK-BR uptake were also found to be positive for apoptosis (Fig 3). No uptake was seen in any of the cell lines studied when exposed to Alexa Fluor 488-labeled non-immune rabbit antibody alone.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have demonstrated that immunodetectable CCK-B/gastrin receptor is present in hepatic tumor and embryonic liver cells but not in normal fibroblasts. We have also shown that anti-CCK-BR antibody is internalized by these cells and that there was an association with apoptosis.
Although we observed an association of uptake of labeled anti-CCK-BR antibody and apoptosis in all five hepatocyte cell lines examined, we did not observe nuclear uptake in the MCA RH7777 cells. This suggests that nuclear uptake of the antibody may not be essential for inducing apoptosis.
We do not know if the translocation to the nucleus involves the entire receptor or a fragment of the receptor. Ligand-induced internalization of the CCK-B receptor was demonstrated by
Crosslinking of cell surface Fas molecules mediated by binding of either Fas ligand or anti-Fas antibody leads to apoptosis (
In summary, this study has demonstrated CCK-B/gastrin receptor expression in hepatocyte cell lines and that binding and uptake of anti-CCK-BR antibody is associated with apoptosis. The therapeutic options for hepatocellular carcinoma are very limited and further studies are ongoing to determine whether the findings of this study can be applied in vivo and subsequently in human subjects.
![]() |
Acknowledgments |
---|
Supported by a research grant from Aphton Corporation.
Received for publication November 26, 2001; accepted March 22, 2002.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Caplin ME, Clarke P, Grimes S, Dhillon AP, Khan K, Savage K, Lewin J et al. (1999a) Demonstration of new sites of expression of the CCK-B/gastrin receptor in pancreatic acinar AR42J cells using immunoelectron microscopy. Regul Peptides 84:81-89[Medline]
Caplin ME, Khan K, Grimes S, Micheali D, Savage K, Pounder RE, Dhillon AP (2001) Effect of gastrin and anti-gastrin antibodies on proliferation of hepatocyte cell lines. Dig Dis Sci 46:1356-1366[Medline]
Caplin ME, Khan K, Savage K, Rode J, Varro A, Micheali D, Grimes S et al. (1999b) Expression of gastrin in hepatocellular carcinoma, fibrolamellar carcinoma and cholangiocarcinoma. J Hepatol 30:519-526[Medline]
Dockray GJ (2000) Cholecystokinin(CCK)-A and CCK-B/gastrin receptors in human tumors. Gut 47:747-748
Matsumoto M, Park J, Yamada T (1987) Gastrin receptor characterization: affinity cross-linking of the gastrin receptor on canine gastric parietal cells. Am J Physiol 252:G143-147
Mehlen P, Bredesen DE (2000) Les récepteurs à dépendance: interface entre apoptose, développement du système nerveux et contrôle de la tumorigenèse. Bull Cancer 87:537-541[Medline]
Morisset J, Wong H, Walsh JH, Laine J, Bourassa J (2000) Pancreatic CCK(B) receptors: their potential roles in somatostatin release and delta-cell proliferation. Am J Physiol 279:G148-156
Nagata S, Golstein P (1995) The Fas death factor. Science 267:1449-1456[Medline]
Nair S, Shivakumar KS, Thuluvath PJ (2002) Mortality from hepatocellular and biliary cancers: changing epidemiological trends. Am J Gastroenterol 97:167-171[Medline]
Pohl M, SilventePoirot S, Pisegna JR, Tarasova NI, Wank SA (1997) Ligand-induced internalization of cholecystokinin receptors. Demonstration of the importance of the carboxyl terminus for ligand-induced internalization of the rat cholecystokinin type B receptor but not the type A receptor. J Biol Chem 29:18179-18184
Reubi JC, Schaer J-C, Waser B (1997) Cholecystokinin(CCK)-A and CCK-B/gastrin receptors in human tumors. Cancer Res 57:1377-1386[Abstract]
Savage K, Khan K, Dhillon AP, Grimes S, Micheali D, Brett B, Pounder RE et al. (1999) Trafficking of CCK-B/gastrin receptor in tumour cells using confocal microscopy. Gastroenterology 116:G2801
Takayama T, Sekine T, Makuuchi M, Yamasaki S, Kosuge T, Yamamoto J, Shimada K et al. (2000) Adoptive immunotherapy to lower postsurgical recurrence rates of hepatocellular carcinoma: a randomised trial. Lancet 356:802-807[Medline]
Wallach D, Varfolomeev EE, Malinin NL, Goltsev YV, Kovalenko AV, Boldin MP (1999) Tumor necrosis factor receptor and Fas signaling mechanisms. Annu Rev Immunol 17:331-367[Medline]
Watson SA, Clarke PA, Morris TM, Caplin ME (2000) Antiserum raised against an epitope of the cholecystokinin B/gastrin receptor inhibits hepatic invasion of a human colon tumor. Cancer Res 60:5902-5907
Watson SA, Clarke P, Smith A, Varro A, Micheali D, Grimes S, Caplin ME et al. (1998) Expression of CCKB/gastrin isoforms in gastro-intestinal tumour cells. In J Cancer 77:572-577
Yao CZ, Bold RJ, Ishizuka J, Townsend CM, Jr, Thompson JC (1995) Growth of mouse hepatocytes is stimulated by gastrin. J Cell Physiol 163:532-537[Medline]