Summary The control of cell proliferation by gastrin has been investigated in a rat pancreatic tumour cell line, AR4-2J. Exogenous gastrin, 10-12 to 10-8 M, stimulated cell growth of thymidine-synchronised AR4-2J cells cultured over 48 h in serum-free medium. Cell lysates of AR4-2J cells contained an average of 4.5 and 3.5 pg gastrin per 106 cells, when grown in serum-supplemented or serum-free media, respectively, as revealed by radioimmunoassay. In serum-free medium, AR4-2J secrete 34ngl 10-6 cells of gastrin over 48h. Addition of an anti-gastrin immunoglobulin preparation, but not control immunoglobulins, caused a maximum 52% reduction in cell growth. These data are consistent with an autocrine role for gastrin in the control of AR4-2J cell growth. These results were supported by studies with gastrin/CCK receptor antagonists. Six non-peptide gastrin/CCK receptor antagonists inhibited AR4-2J cell growth in a concentration-related manner. The concentration required for 50% inhibition (IC50) of cell growth by the amino acid-derived antagonists proglumide (3.5 x 10-M), benzotript (1.8 x 10-3 M), loxiglumide (1.1 x 10-4 M) and lorglumide (6.7 x 10-5 M) were of the same order and significantly correlated with their IC50 for inhibition of 1251-gastrin binding to AR4-2J cells. Inhibition of cell growth by these antagonists was partially reversed by the addition of exogenous gastrin. In contrast, the ICm for inhibition of cell growth with two benzodiazepine-derived antagonists, the CCK-B receptor antagonist L-365,260 (4.6 x 10-5 M) and the CCK-A receptor antagonist devazepide (1.7 x 10-' M) were two-three orders of magnitude greater than those required to inhibit gastrin binding (10-8-10-7 M). The growth inhibitory effects of L-365,260 and devazepide were not reversed by exogenous gastrin suggesting these benzodiazepine-derived antagonists do not inhibit cell growth by interaction with gastrin receptors. The results are consistent with gastrin being an autocrine growth factor in AR4-2J cells, and that stimulation of cell growth is due to stimulation of the gastrin, rather than CCK-B, receptor sub-type. This study highlights that gastrin receptor antagonists warrant further investigation as agents to control growth of tumours, such as those from the gastrointestinal tract, which express gastrin receptors.

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The role of gastrin in the control of growth of renal G401 cells isolated from a human nephroblastoma (Wilms' tumour) was investigated. G401 cell growth was enhanced in the presence of exogenous gastrin. Addition of anti-gastrin antibodies to serum-free medium significantly inhibited the growth of G401 cells. G401 cells contained the equivalent of 4.3 pg/10(6) cells of gastrin, and serum-free medium collected over 48 hr from G401 cells contained the equivalent of 38 ng/10(6) cells of gastrin, as determined by radioimmunoassay. Growth of G401 cells was inhibited in a concentration-related way by a variety of gastrin/CCK receptor antagonists. Devazepide and proglumide were, respectively, the most and the least potent inhibitors of G401 cell growth (potency order devazepide > L-365,260 = lorglumide > loxiglumide > benzotript > proglumide). These gastrin/CCK receptor antagonists had similar growth-inhibitory activities in human colonic adenocarcinoma HCT-116 cells. Growth of HCT-116 cells was stimulated to a lesser extent, as compared with G401 cells, by exogenous gastrin, and endogenous gastrin was not detectable in HCT-116 cells. The results are consistent with a role for a gastrin-like peptide in the control of growth of a renal cell line. The data suggest that gastrin/CCK receptor antagonists warrant further investigation as therapeutic agents for the control of gastrin-responsive tumours derived from outside, as well as inside, the gastrointestinal tract, including tumours derived from the kidney.

1H NMR spectroscopic methods have been applied to compare the in vitro reactivity of the renal papillary nephrotoxin 2-bromoethanamine (BEA) with those of selected halide-substituted nephrotoxic analogues, 2-chloroethanamine (CEA), 2-fluoroethanamine (FEA), and 1-phenyl-2-iodoethanamine (PIEA). The primary 1H NMR-detectable transformation during a 24 h incubation of confluent Madin Darby canine kidney (MDCK) cells with BEA, CEA, and FEA (at concentrations up to the IC50 determined by neutral red uptake) was the appearance in cell culture media of 2-oxazolidone (OX). Additional novel signals assigned as FEA carbamate (N-carbamoyl-2-fluoroethanamine) were observed in media collected following incubation of cells with FEA. We propose that N-carbamate intermediates are formed from the spontaneous reaction of these haloalkylamines with HCO(3-)-buffered growth media and that OX is formed from the carbamate via elimination of the hydrogen halide. Further 1H NMR experiments, conducted for up to 8 h at 25 degrees C on 5 mM solutions of BEA, CEA, and FEA in 2H2O containing a 20-fold excess of HCO3- at pH 7.6, demonstrated a time-dependent decrease in the concentration of the free haloalkylamines accompanied by the production of N-carbamate intermediates and OX. Under these pseudo-first-order reaction conditions, the formation of OX from BEA was complete within approximately 6 h. In similar reaction conditions OX formation from CEA (24 h after initiation) had reached 54% of its final equilibrium concentration. Equivalent experiments demonstrated that PIEA was almost completely converted to 4-phenyl-2-oxazolidinone (PHOX) within 2 h. These observations reveal the strong disposition of this series of haloalkylamines toward reaction with HCO3- and indicate that the compounds in this family may exist only transiently as free amines in vivo, where there will virtually always be excess HCO3-. The physiological relevance of the in vitro findings is further indicated by the NMR-detectable conversion of BEA to OX and also an alkylating aziridine (AZ) moiety in rat plasma containing BEA. The ability to form carbamoylated species and OX (or PHOX) may mediate the toxicity of this series of haloalkylamines and hence is potentially of considerable significance.

The growth and differentiation characteristics of MAC 15 murine adenocarcinoma cells, derived from routine passage in vivo for growth in vitro on a plastic substrate (MAC15j cells), were compared under conditions in which the cells were seeded onto a substrate of type-I collagen which was either attached to plastic or was released to float free in medium. Cells grown on a plastic substrate consisted of a heterogeneous, largely anaplastic population with a putative enterocytic morphology but with no evidence of junctional complexes or cell polarity typical of an epithelial phenotype. MAC 15j cells from cultures grown on a plastic substrate reestablished a moderate to well-defined degree of differentiation when transplanted back into NMRI mice. When MAC 15j cells were seeded from plastic onto type-I collagen, either attached to plastic or free-floating, tight junctional complexes were formed and the cells began to attain a more recognizable, columnar and polarised epithelial morphology. Cells grown on a type-I collagen gel which was free-floating showed a selective expression of alkaline phosphatase at the apical surfaces of approximately 10% of the cells. This expression was detectable by electron microscope histochemistry but could not be detected biochemically. Treatment of MAC 15j cells grown on a released collagen matrix with tetramethyl-urea (20mM) accelerated the expression of alkaline phosphatase activity at the apical surface as detected by microscopy.