Dov Michaeli

Expression and processing of gastrin in hepatocellular carcinoma, fibrolamellar carcinoma and cholangiocarcinoma.

BACKGROUND/AIMS Gastrin is a trophic factor within the normal gastrointestinal tract and is also a mitogen for a number of gastrointestinal and non-gastrointestinal tumours. Precursor forms of gastrin including progastrin (proG) and glycine-extended gastrin (G-gly) as well as the fully processed amidated gastrin (G-NH2) are expressed by tumours. There has been little study of the role of gastrin in either normal liver or liver tumours. The aim of this study was to identify the expression of CCK-B/gastrin receptor (CCK-BR), proG, G-gly and G-NH2 in normal liver and liver tumours. METHODS Tissue sections from patients with hepatocellular carcinoma, fibrolamellar carcinoma, cholangiocarcinoma as well as normal liver biopsies were assessed for expression of CCK-BR and gastrin isoforms. RESULTS Most liver tumours express CCK-BR and are able to process gastrin as far as proG and G-gly, although not as far as the amidated form. There appears to be little expression of the receptor and no expression of precursor forms of gastrin in normal liver. CONCLUSIONS Liver tumours express the CCK-BR and precursor forms of gastrin. This expression may be associated with tumour proliferation.

Expression of CCKB/gastrin receptor isoforms in gastro‐intestinal tumour cells

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 <50 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 >150 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. J. Cancer 77:572–577, 1998.

Antibodies raised by gastrimmune inhibit the spontaneous metastasis of a human colorectal tumour, AP5LV.

Both precursor forms of gastrin and mature amidated gastrin peptides can enhance proliferation of colorectal tumours and may regulate growth in an autocrine manner. The purpose of this study was to evaluate the effect of neutralization of precursor and amidated gastrin on primary and secondary in vivo growth of a human colorectal tumour. The human colorectal cell line, AP5LV, when injected into the muscle layer of the abdominal wall of severe combined immunodeficient (SCID) mice, grows as a well-vascularized primary tumour and metastasis to the lung. AP5LV expressed the precursor gastrin forms; progastrin and glycine-extended gastrin and gastrin/CCKB receptors, as assessed by immunocytochemistry. Gastrimmune is a gastrin immunogen in which the amino terminus of the gastrin-17 molecule is linked to diphtheria toxoid and induces antibodies which neutralise the amidated and glycine-extended forms of gastrin-17. Rabbit antiserum, raised against Gastrimmune, was administered intravenously into SCID mice bearing AP5LV tumours. Control animals were treated with antiserum raised against diphtheria toxoid only. Antibodies raised against Gastrimmune significantly limited the growth of primary AP5LV tumours, as assessed by median cross-sectional area (controls = 244 mm2; antibody-treated = 179 mm2; P = 0.033). In addition Gastrimmune-induced antiserum limited the growth of lung metastasis as assessed by nodule number (controls = 3.5; antibody-treated = 1.0; P = 0.0001) and nodule cross-sectional as assessed by image analysis (controls = 11.9 mm2; antibody-treated = 3.75 mm2; P = 0.0064). In conclusion in vivo neutralization of gastrin forms, which may potentially be fueling growth by an autocrine pathway, inhibited both primary growth and, to a greater degree, lung metastasis of a human colorectal tumour cell line. Immunization against tumour-associated gastrin forms may provide an effective therapy for advanced colorectal cancer.

Effect of Gastrin and Anti-Gastrin Antibodies on Proliferation of Hepatocyte Cell Lines

Gastrin (G-17) and its precursor glycine-extended gastrin (G-17-gly) have been shown to be trophic to some gastrointestinal tumors. This in vitro study assessed the effect of G-17, G-17-gly, anti-gastrin antibodies (anti-G-17), and the CCK-B receptor antagonist PD135,158 on three hepatoma cell lines (PLC/PRF/5, HepG2 and MCA-RH7777) and an embryonic liver cell line (WRL68). The pancreatic adenocarcinoma cell line AR42J was used as a positive control. G-17 and G-17-gly caused significant proliferation of AR42J and WRL68 cell lines. G-17-gly but not G-17 induced significant proliferation of the PLC/PRF/5 cell line. Anti-G-17 and PD135,158 significantly inhibited unstimulated AR42J and WRL68 cell lines. Anti-G-17 also inhibited the proliferative effects of G-17 and G-17-gly on AR42J, WRL68, and PLC/PRF/5 cell lines, whereas PD135,158 inhibited the proliferative effect of G-17 only. G-17 and G-17-gly as well as anti-G-17 and PD135,158 had no effect on HepG2 and MCA-RH77777 cell lines. It is concluded that G-17-stimulated proliferation is mediated via the CCK-B receptor and G-17-gly via a separate, as yet uncharacterized, receptor. There may therefore be a role for gastrin in embryonic hepatocellular proliferation and perhaps also in the proliferation of some hepatocellular tumors.

A comparison of an anti-gastrin antibody and cytotoxic drugs in the therapy of human gastric ascites in SCID mice.

The therapeutic effect of antibodies raised by the immunogen Gastrimmune was compared with both a CCKB/gastrin receptor antagonist, CI‐988, and 5‐Fluorouracil/leucovorin in a gastric cancer model. The human gastric ascites cell line, MGLVA1asc, produced and secreted progastrin and glycine‐extended gastrin as determined by radioimmunoassay and immunocytochemistry. Cells were also stained with an antiserum directed against the human CCKB/gastrin receptor. MGLVA1asc cells were injected i.p. into SCID mice. Antibodies raised by Gastrimmune immunization of rabbits (affinity for G17 of 0.15 nM and GlyG17 of 0.47 nM) were passively infused i.p. and significantly enhanced survival by up to 5 days (p=0.0024 from vehicle controls). The enhancement in survival was not significantly different from that achieved by treatment with 5‐Fluorouracil and leucovorin. A CCKB/gastrin receptor antagonist, CI‐988, did not affect survival with cells injected at 7.5×105 cells/mouse but significantly increased the survival of mice injected with a lower cell innoculum of 5×105 cells/mouse from 30 to 35 days (p=0.0186). At this lower innoculum antibodies raised by Gastrimmune induced complete survival in 2 animals with the remaining dead by day 36 (p=0.0022). Thus, both endocrine and autocrine pathways mediated by precursor and mature gastrin molecules may be jointly operational in the gastric cancer scenario and may be important targets for therapeutic agents. Int. J. Cancer 81:248–254, 1999.

Demonstration of new sites of expression of the CCK-B/gastrin receptor in pancreatic acinar AR42J cells using immunoelectron microscopy

The CCK-B/gastrin receptor has been characterised in both normal and tumour tissues. Endocytosis of the CCK-B/gastrin receptor has recently been demonstrated and this has similarly been described for other peptide receptors. In addition, ligand and ligand-receptor translocation to the nucleus has been demonstrated for other peptides. The aim of this study was to identify the sites of expression of the CCK-B/gastrin receptor in the known CCK-B/gastrin receptor bearing pancreatic acinar AR42J cells. The specificity of the CCK-B/gastrin receptor antibody (alpha-CCKBR-Ser antibody) was demonstrated by inhibition ELISA studies, radioligand inhibition studies and immunofluorescence binding studies on AR42J cells. Western blotting and immunogold electron microscopy techniques were used to identify the receptor in AR42J cell preparations. The affinity purified alpha-CCKBR-Ser antibody was shown to be specific for the CCK-B/gastrin receptor. The receptor was expressed on the cell membrane, in the cytoplasm and within the nucleus. Isoforms of the receptor protein identified in extra-nuclear and nuclear extracts ranged in molecular weight from 58 to 66 kDa. We conclude that the CCK-B/gastrin receptor is not only expressed on the cell membrane, but also in the cytoplasm and nucleus of AR42J pancreatic acinar cells.

Transforming growth factor‐α–mediated growth pathways in human gastro‐intestinal cell lines in relation to the gastrin autocrine pathway

Epidermal growth factor (EGF) and transforming growth factor‐α (TGF‐α) increase transcription of the gastrin gene, and the gastrin peptide may be phosphorylated by EGF‐stimulated tyrosine kinase. Our aims were to compare EGF/TGF‐α interactions in 2 human gastro‐intestinal cell lines: MGLVA1, with a strong gastrin autocrine pathway, and C170HM2, with a weak pathway. Both cell lines expressed the TGF‐α gene. MGLVA1 expressed TGF‐α protein as determined by immuno‐cytochemistry, which was absent in C170HM2. Both cell lines expressed the same level of EGF receptors, as assessed by flow cytometry; however, MGLVA1 did not have enhanced in vitro proliferation in response to EGF or TGF‐α, unlike C170HM2. The basal growth of MGLVA1 was inhibited by anti‐sera against TGF‐α, the EGF receptor and G17. C170HM2 was not inhibited by any of the anti‐sera. Neutralisation of TGF‐α resulted in undetectable cell‐associated progastrin levels in MGLVA1 (untreated had 391.7 fmol/5 × 106 cells). The progastrin level of C170HM2 remained unaffected. Tyrosine kinase activity, as assessed by phosphopeptide concentration, of unstimulated MGLVA1 was 2.6 times higher than that of C170HM2 in the cell membrane fraction (0.097 compared to 0.037 μg/mg protein, p < 0.001) and 4.8 times higher in the cytosolic fraction (0.269 compared to 0.056 μg/mg protein, p < 0.05). Following treatment with EGF, the phosphopeptide concentration increased in both the membrane and cytosolic fractions of both cell lines. Tyrphostin B42, which inhibits autophosphorylation of the EGF receptor, inhibited the basal growth of MGLVA1 (IC50 1.3 μM) and C170HM2 (9.5 μM, p < 0.05 from MGLVA1). Herbimycin, which inhibits pp60c‐src kinase, reduced the basal growth of MGLVA1 (0.67 μM) but not C170HM2. Immunofluorescence studies confirmed the presence of tyrosine‐phosphorylated proteins and pp60c‐src within the cytoplasm of unstimulated MGLVA1 cells. There was no specific immunofluorescence for either parameter in C170HM2 cells until after treatment with EGF. Int. J. Cancer 87:20–28, 2000.

A phase II study of gastrimmune in advanced pancreatic cancer

A PHASE II STUDY OF GASTRIMMUNE IN ADVANCED PANCREATIC CANCER. Bernard T. Brett, Stephen C. Smith, Catherine V. Bouvier, Dov Michaeli, Daniel Hochhauser, Brian R. Davidson, Tom R. Kurzawinski, Tony Watkinson, Neil Van-Someren, Roy E. Pounder, Martyn E. Caplin, The Middlesex Hosp, London, United Kingdom; Royal Free and Univ Coli Med Sch, London, United Kingdom; Aphton Corp, Woodlands, CA; Royal Free Hosp, London, United Kingdom; Chase Farm Hosp, London, United Kingdom; Dept of Gastroenterology, Royal Free Hosp, London, United Kingdom.

Expression of CCK-B/gastrin receptor and endocytosis of anti-CCK-B/gastrin receptor antibody

Introduction: We have previously reported endocytosis of anti-CCK-BI gastrin receptor antibody (GR#l) by tumour cell lines. Aim: To demonstrate expression of CCK-B/gastrin receptor using RT-PCR, and confirm that uptake of anti-CCK-B/gastrin receptor antibody is dependent on expression of CCK-B/gastrin receptor. Materials and methods: GR#l is an antibody to the amino terminal end of the human CCK-BR. It was labelled with either Alexa-546 (red) or Alexa-488 (green) fluorescent marker. GR#l was added to live cells from the AR42J pancreatic adenocarcinoma cell line, as well as to fibroblast NIH3T3 cells transfected with human CCKB/gastrin receptor gene. GR#l was also added to non-transfected NIH3T3 cells as a negative control. Expression of CCK-B/gastrin receptor was demonstrated by RT-PCR. Total RNA was isolated from I . 3 X 106 cells using the Promega SV isolation system, according to the manufacturers instructions. RT-PCR was performed using l-2!Lg total RNA and the Access RT-PCR system (Promega). Specific primers were used for transcription and amplification of gastrin receptor and f3 actin as a positive control; a double round of PCR was used. Results were analysed using agarose gel electrophoresis. Results. Uptake of anti gastrin receptor antibody was seen in AR42J cells and in NIH3T3 cells transfected with gastrin receptor. No uptake was seen in non-transfected NIH3T3 cells. Using RT-PCR, mRNA for f3 actin was detected in all cell lines: mRNA for gastrin receptor was detected in AR42J cells and in transfected NIH3T3 cells, but not in non-transfected NIH3T3 cells. Conclusions. Anti gastrin receptor antibody was only taken up by cells expressing CCK-BR. This specific uptake confirms that this antibody may be a potential targeting agent in tumours expressing CCK-BR.