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Gastrin: growth enhancing effects on human gastric and colonic tumour cells.

Two colorectal (HT29, LoVo) and one gastric (MKN45) human tumour cell lines were examined for their in vitro trophic response to human gastrin-17. MKN45 and HT29 responded by increased 75Se selenomethionine uptake to exogenous gastrin (139 +/- 5.5% and 123 +/- 3% of control values respectively) whereas LoVo showed no significant response to this hormone. When these same cell lines were grown as xenografts in nude mice, similar responses were seen to exogenously administered human gastrin-17 (10 micrograms mouse-1 day-1, subcutaneous injection). MKN45 xenografts showed a greater response to continuously administered gastrin (osmotic mini-pumps, (10 micrograms mouse-1 day-1) when compared to the same dose given via a subcutaneous bolus injection. The hormone-treated xenografts had a two-fold increase in tumour cross-sectional area and growth rate when compared to saline-treated controls. Dose-response studies revealed that 0.4 micrograms gastrin mouse-1 day-1 appeared to be the minimally effective dose. As gastric and colorectal tumour cells show a trophic response to gastrin, antagonists of the gastrin receptor may prevent this effect causing tumour stasis. The gastric tumour cell line, MKN45, is gastrin-responsive and would be an ideal model for screening potent receptor antagonists.

The hormonal control of breast and prostatic tumours has become well established (Carter et al., 1977;Higgins & Hodges, 1941) and clinically effective hormone antagonists such as tamoxifen are widely used.
Gastrointestinal (GI) tract tumours are among the commonest found in humans (UK data: colon and rectum, 25,000; stomach 11,000 cancer deaths per year). Radiotherapy and cytotoxic drug therapy have failed to produce significant therapeutic results. An investigation into the hormonal control of such tumours with a view to developing antagonists of hormone action may yield alternative modes of treatment for gut tumours.
Gastrin appears to be an important trophic hormone for both normal and malignant GI mucosal cells. Gastric and duodenal mucosal cells were shown to respond to pentagastrin when cultured in vitro Lichtenberger et al., 1973). Naturally occurring gastrins (G171, G1711, G3411) were several times more potent than pentagastrin in their trophic effects on normal mucosa (Johnson, 1977). More recently it was shown that the density of fundic mucosal endocrine cells was positively related to levels of serum gastrin (Borch et al., 1986). GI tract tumour cells have been shown to respond to gastrin. Both established colorectal cell lines (Kusyk et al., 1986;McRae et al., 1986) and gastric cell lines (Watson et al., 1988) respond to gastrin in vitro. The same has been shown with gastric and colorectal primary tumour cells (Moyer, 1983;Sirinek et al., 1985).
The need to produce gastrin (receptor) antagonists and test them in reliable in vitro and in vivo systems is fundamental to determining the possible role of such agents in the treatment of GI cancer. This paper compares the gastrin responsiveness of human gastric and colorectal cell lines in vitro and in vivo.

Materials and methods
Established cell lines Three established human gastric and colorectal cell lines were examined for gastrin-dependence: (i) MKN45 is a human adenocarcinoma cell line originally derived from a metastatic tumour of the liver from a 62-year-old Japanese woman with an undifferentiated adenocarcinoma of the stomach (Hojo, 1977); (ii) HT29 and LoVo are human colorectal adenocarcinoma cell lines.
The cells used for in vitro assays had previously been grown in immunocompromised (nude) mice (Olac, Oxfordshire) and had been passaged less than five times in vitro, as previously described (Watson et al., 1988). The cells were grown in vitro in Dulbecco's growth medium (DMEM) supplemented with 10% fetal calf serum (FCS, Gibco) at 37°C in the presence of 5% CO2.
In vitro assessment of cell growth in the presence of gastrin Cells were synchronised in the GI phase of cell growth by the addition of excess thymidine and cell growth was assessed by 75Se-selenomethionine incorporation as previously described (Watson et al., 1988). Human gastrin-17 (G17, Sigma, Dorset) concentrations between 0.02 and l.O,g1-l were used.
Growth of cell lines as xenografts in nude mice Xenografts were initiated by injection of cell lines as previously described (Watson et al., 1988). The resultant tumours were aseptically dissected, mechanically minced and 5 mm3 pieces of tumour tissue transplanted into the animals and randomised into experimental groups. The control groups of animals received phosphate-buffered saline (PBS) either by a subcutaneous bolus injection (0.2ml) daily or by continuous infusion with the use of 14 day osmotic minipumps (Alzet, London, model 2002). Gastrin-treated animals received G17 either subcutaneously (single dose, 10jgmouse-1 day-', 0.2ml) or by 14 day mini-pump (0.4, 2.0, 10.0pgmouseday-1).
Tumour growth was evident from between days 4 and 7. The tumours were measured two or three times a week, with the use of calipers, for 3 weeks and the measurements were made by an independent observer. The two largest perpendicular diameters of the tumours were measured and from these the cross-sectional areas were derived.
Measurements of serum gastrin levels after administration of gastrin Nude mice were starved overnight and then tail bled to measure fasting serum gastrin levels.
Aliquots of 200,u1 blood were removed per mouse at each tail bleed, which yielded 20p1 serum. The gastrin levels of the serum were then assayed using a Radioimmunoassay kit (CIS Labs, UK) with the detection limit being 15 pg ml -1.

Statistics
Statistical analysis on both in vitro and in vivo data was performed by using the paired Student's t test and the term '% of control' used to compare data is defined as c.p.m. in presence of gastrin c.p.m. in absence of gastrin In vivo data were also subjected to a one-way analysis of variance which was performed over the complete time scale of each experiment. Where replicates were performed, the standard error of the mean was calculated.

Results
The cell lines examined for their gastrin dependence had been passaged less than five times in vitro as the growth response of tumour cells to gastrin may be lost on prolonged in vitro passage (Watson et al., 1988). Figure 1 shows a typical experiment in which the gastric cell line, MKN45 and the colorectal cell line, HT29 both incorporated 75Se-selenomethionine to greater levels in the presence of G-17. Maximum responses of 139 + 5.5% (MKN45) and 123+3% (HT29) of control values were achieved at gastrin concentrations of 0.2 and 1.0 pg 1-1 respectively ( Figure 1). The colorectal cell line, LoVo, failed to achieve a significant response to G17.
The same cell lines were grown as xenografts in nude mice. In all xenograft experiments, an arbitrary tumour area of 1.5 cm2 has been designated to indicate satisfactory tumour growth from transplanted tissue. With MKN45 xenografts (Figure 2a) 1/9 of PBS-treated mice achieved a tumour cross-sectional area greater than 1.5 cm2 whereas 4/10 gastrin treated mice had tumours of such a size. The mean growth rates of PBS-treated and gastrin-treated xenografts were 54+8 mm2 day-1 and 68 +11 mm2 day-1 respectively. Statistical analysis revealed no significant difference between the two groups.
To try and develop a more gastrin-responsive xenograft line, the four MKN45 xenografts achieving an area of greater than 1.5cm2 in the gastrin-treated group (Figure 2a) were removed, the tissue was combined and retransplanted into two more groups of experimental animals. These were randomised into PBS and gastrin-treated (10pgmouse-1 day-1) groups. From Figure 3, it can be seen that 3/10 PBStreated mice had xenografts greater than 1.5cm2 compared to 7/10 gastrin-treated animals. This was found to be statistically significant (one way analysis of variance, P<0.01). The mean growth rate of PBS-treated xenografts was 68 + 9mm2 day -1 compared to 90 + 9mm2 day -1 in the gastrin treated tumours.
As human gastrin-17 has a short half-life in the circulation (6min; Walsh & Grossman, 1975), it was decided to administer the hormone by continuous infusion with the use of an osmotic mini-pump. Gastrin-17 was delivered over a 14 day period from the day of xenograft transplantation. Figure 4 shows the growth response of both MKN45 and HT29 xenografts to pumped gastrin. The growth rate of MKN45 xenografts in response to gastrin (10 pg mouse -'day-1) was 178+20mm2day-I compared to 94+9mm2day-I in the PBS treated controls (P<0.01, Student's t test, P<0.01, oneway analysis of variance over entire experiment). The growth rate of HT29 xenografts in response to constantly infused gastrin was 148 + 21 mm2 day-1 compared to 105 + 15mm2 day-1, in the PBS-treated controls. There was a trend to increased growth in the presence of gastrin which was not significant.
A titration was performed on the growth response of MKN45 xenografts to pumped gastrin. Gastrin was administered at decreasing concentrations of 10.0, 2.0 and 0.4 pg mouse-1 day-I and the growth of the corresponding xenografts was measured over a 24-day period ( Figure 5). For comparative purposes, the mean of the tumour measurements for each group was calculated.

Discussion
As radiotherapy and chemotherapy of GI tumours have failed to produce beneficial therapeutic results an alternative mode of treatment has been sought. This study investigates the hormonal control of such tumours with a view to developing antagonists, if tumour growth shows hormonal dependence.
Early results showed that GI tract tumour cells have a low growth response to gastrin after long-term in vitro growth (Watson et al., 1988). However, if the same cells were grown as xenografts in nude mice and re-examined after short-term in vitro growth, they showed an elevated response.
In the present study, MKN45 and HT29 cells incorporated elevated levels of 75Se-selenomethionine in the presence of human G-17 whereas LoVo cells failed to respond. Time (days) Figure 5 The dose-response of MKN45 xenografts to gastrin administered via an osmotic mini-pump. Mean tumour measurements (n=5), PBS control LI, gastrin (pgmouse' day'1) 0.4~, 2.0 EL 10.0 S. These same cell lines, grown as xenografts in nude mice, did not respond significantly to daily subcutaneous injections of human G-17. This is not surprising as the tumour cells will only be exposed to the hormone for a short time, as the serum half-life of G-17 is 6min (Walsh & Grossman, 1975). However, when MKN45 xenografts were exposed to continuously administered gastrin it induced a two-fold increase in cross-sectional area and mean rate of tumour growth when compared to saline-treated controls. HT29 xenografts showed a trend to increased growth in the presence of continuously infused gastrin that was not statistically significant.
In the MKN45 xenograft model, pumped gastrin which induced up to three fold increases in circulating fasting serum gastrin levels (when compared to PBS-treated animals) appeared to increase the growth of the tumours in the shortterm. In accord with this, studies in humans have revealed that fasting serum gastrin levels are elevated 8.5-fold in patients with colorectal cancer (Smith et al., 1987) and 3.5fold in patients with carcinoma of the stomach (McGuigan & Trudeau, 1973).
A similar study was performed with xenotransplanted human gastric and colon carcinomas (Sumiyoshi et al., (1984). A human gastric carcinoma, SC-6-JCK, responded trophically to gastrin whereas a second human gastric carcinoma, ST15, and a colonic adenocarcinoma, Co3, failed to respond. However, the response with SC-6-JCK was obtained with pentagastrin (10 pg mouse-Iday-1) which is known to be less potent at promoting a trophic response with normal cells than gastrin-17 (Johnson, 1977) and has been shown to have a lower binding affinity for the gastrin receptor when compared to gastrin-17 (P. Singh, personal communication). The pentagastrin was also administered via a single subcutaneous injection. It is possible therefore that SC-6-JCK may have greater sensitivity to the action of gastrin than MKN45.
A recent study has shown that a mouse colon adenocarcinoma cell line, MC26, responded to pentagastrin (3.25-15.00 pgmouse-1 day-1) administered by intraperitoneal injections (Winsett et al., 1986). It has also been found that both exogenous gastrin (pentagastrin, 2 mg kg-1, subcutaneous injection) and endogenous hypergastrinaemia (induced by antral exclusion) exerted trophic effects on chemically induced colorectal neoplasms in the rat (McGregor et al., 1982). The lack of gastrin response seen in the human colorectal tumour cells examined in the present study may be dose related and further experiments will be performed to investigate this.
In conclusion, we have demonstrated that a human gastric adenocarcinoma cell line, MKN45, when initiated as a xenograft in nude mice, almost doubles in size upon continuous administration of human G-17. Together with the cell line's gastrin responsiveness in vitro it is an ideal candidate for screening gastrin (receptor) antagonists with a view to examining them as potential therapeutic agents.