CELL LINES

MGLVA1 is an ascitic variant of the human gastric cell line, MKN45G.18 ST16 is a human gastric cell line originally derived in the Cancer Research Campaign Labs, University of Nottingham, UK from a patient specimen. In vitro, the cell lines were maintained in RPMI 1640 medium (Gibco, Irvine, Scotland) supplemented with 10% heat inactivated foetal calf serum (FCS, Sigma, Poole, Dorset) and grown at 37°C in 5% CO₂ and humidified conditions.

REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION (RT-PCR) FOR DETECTION OF GASTRIN mRNA

Total RNA was extracted from cell lines using RNAzol-B (Biogenesis, Poole, Dorset) according to the manufacturer's instructions. Cells were lysed in 1 ml RNAzol-B with 110 ml chloroform (Sigma). An aqueous phase was separated by a 1300g spin at 4°C for 30 minutes and eluted. RNA from the eluate was precipitated in ice cold isopropanol and washed twice in 75% ethanol. The RNA pellet was then resuspended in 50 μl sterile diethyl pyrocarbonate (DEPC) treated water.

Reverse transcription was performed as described previously.19 Total RNA preparations were divided into two halves and incubated at 70°C for 10 minutes with random hexamer primers (pd(N)₆; GibcoBRL) and then cooled immediately on ice. One half was converted into cDNA while the other half acted as a control and was incubated in a reaction mix without the reverse transcriptase enzyme. Reaction mixes consisted of a 50 μl volume containing 30 μmol dNTPs (Pharmacia, St Albans, Herts), 0.01 M DTT (GibcoBRL), and 200 units Superscript reverse transcriptase which was buffered in 1st Strand Buffer (GibcoBRL). Reactions were carried out at 25°C for 10 minutes and 37°C for one hour with a denaturing stage of 95°C for five minutes.

A gastrin PCR reaction (50 μl volume) was performed using 2.5 units of Taq polymerase (Qiagen, UK), 40 μmol dNTPs (Pharmacia), and 10 μmol of each primer in 1× PCR buffer (Qiagen). Samples were separated on 1.5% (wt/vol) agarose gels and subjected to Southern blotting. The blots were hybridised at 52°C with digoxygenin (DIG) labelled oligonucleotide probes and washed at 47°C in 2× saline sodium citrate (SSC)/1% sodium dodecyl sulphate (SDS), and 0.5× SSC, both twice for five minutes. Any signal was detected using anti-DIG-AP antibodies and the CDP-Star chemiluminescent substrate (Boehringer Mannheim), both used according to the manufacturer's instructions. A 15 minute exposure to an x ray film was used to detect the chemiluminescence.

ENZYME LINKED OLIGONUCLEOTIDE LINKED CHEMILUMINESCENT ASSAY (ELOCA)

RNA extraction and reverse transcription were both performed as above. PCR was performed using a similar protocol to that described previously19 using 1 μl of cDNA. Reactions were prepared in a 50 μl volume containing 1.0 unit Dynazyme (Flowgen) in 1× PCR buffer (Flowgen) with 40 μmol dNTPs (Pharmacia) and 10 μmol of each primer. One drop of mineral oil was layered on top of the reaction mix which was incubated at 94°C for four minutes before cycles of 94°C for 60 seconds, 54°C for 90 seconds, and 72°C for 60 seconds. These cycles were repeated 23 times for GAPDH and 30 times for gastrin. A final incubation of 72°C for 600 seconds was performed immediately afterwards to convert all PCR products to double stranded DNA. The PCR reactions were dot blotted in duplicate onto Hybond-N⁺membrane (Amersham International) according to the ELOCA method.19 It was found that 1 μl of GAPDH and 10 μl of gastrin PCR reactions were optimal for semiquantitative detection. Calibration curves, containing known quantities of purified PCR product for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and gastrin were also blotted. The blots were hybridised at 37°C using 2× SSC/1% SDS and 0.5× SSC, twice each for five minutes. Blots were then incubated with anti-DIG-AP and CDP-Star as described above. After incubation with the chemiluminescent substrate, semiquantitation was performed by placing the blots in a Top Count (Hewlett Packard) and counting the chemiluminescence associated with each dot. The calibration curve was plotted on a log-log graph from which sample values were determined. A gastrin/GAPDH ratio was calculated in order to standardise for sample size.

RADIOIMMUNOASSAY OF CELL ASSOCIATED AND SECRETED GASTRIN LEVELS

Cells were seeded into T75 (Costar) flasks containing serum free medium composed of RPMI 1640 medium in a 1:1 ratio with HAMS F12 (Gibco) containing 0.5% bovine serum albumin (BSA, Sigma). When the cell monolayers had reached confluence, they were harvested, pelleted in 1 ml of sterile phosphate buffered saline, pH 7.2 (PBS, Oxoid, Bucks), and heated in a boiling water bath. Spent media from the cell lines were centrifuged to remove cell debris and dialysed overnight in PBS. The levels of progastrin, glycine extended gastrin, and carboxy amidated gastrin were measured by radioimmunoassay (RIA) using the specific antisera L289, 109-21, and L2, respectively which have been described previously.20

IN VITRO ASSESSMENT OF CELL GROWTH RATES AND EFFECT OF TREATMENT WITH ANTISERUM RAISED AGAINST GASTRIMMUNE

Cells were seeded into 96 flat bottomed well plates (Costar) at a concentration of 1 × 10⁴ per well in a 100 μl volume in the serum free medium. After 72 hours, cell proliferation was assessed by the methylthiazoyl tetrazolium (MTT) assay.21

Protein A purified immunoglobulins (IgG) raised against Gastrimmune (test serum) and normal rabbit control IgG were normalised to a standard protein concentration of 0.5 mg/ml. Cells were seeded into the 96 well plates described above at a concentration of 1 × 10⁴ per well in a 100 μl volume in serum free medium. IgG dilutions from 1/20 to 1/1000 were prepared in serum free medium (relating to antigen binding capacities (ABCs) ranging from 5.5 × 10^–8 to 1.1 × 10^–9 M for amidated G17 (affinity 0.15 nM) and 2 × 10^–7 to 4 × 10^–9 M for GlyG17 (affinity 0.47 nM)) and added to the cells in 100 μl volumes per well. Each condition was performed with five replicates. Cell proliferation was assessed after 72 hours by the MTT assay.

INITIATION OF IN VIVO ASCITES MODELS

Severe combined immunodeficient (SCID) mice, aged 6–10 weeks (Cancer Studies Unit, University of Nottingham) were used for the in vivo studies. The mice were fed and watered ad libitum.

Cells were harvested from semiconfluent monolayers and suspended at the required concentration in sterile PBS. A 1 ml volume was then injected into the peritoneal cavity of the experimental mice, randomised such that animal weights were equalised across the experimental groups, which were composed of 8–16 animals of mixed sex.

Animals were inspected daily and, at the onset of morbidity (cachexia, weight loss, and abdominal swelling due to ascites formation), termination was carried out by an experienced independent observer who was blind to the treatment groups. Initial validation studies showed that variation in solid tumour burden (diffusely spread throughout the peritoneal cavity), ascites volume, and cell density between animals, at onset of morbidity, were within 15%. Thus the model allows the assessment of time taken to achieve a definitive tumour burden.

IN VIVO ADMINISTRATION OF ANTIBODIES RAISED BY GASTRIMMUNE

Antisera were raised in rabbits by immunisation with Gastrimmune as described previously22 and denoted rabbit anti-G17 (9):DT. Normal rabbit antiserum was used as the vehicle control. Antiserum was administered from day 1 following tumour cell injection, intravenously, once daily, at a concentration generating a stable serum ABC of 7.5 × 10^–9 M for amidated G17 and 3 × 10^–8 M for GlyG17.17

All animal experimentation was performed according to the UK Coordinating Committee for Cancer Research (UKCCCR) guidelines.

STATISTICS

Statistical evaluation of in vitro data was performed by a one way analysis of variance, and survival analysis was assessed using a log rank test, both on the SPSS statistical package.

mRNA LEVELS IN MGLVA1 AND ST16 AS DETERMINED BY THE ELOCA ASSAY

The gastrin/GAPDH mRNA levels were determined for MGLVA1 and ST16 cells growing in vitro in both serum containing and serum free media. In serum free medium the gastrin: GAPDH ratio was 0.0909 for MGLVA1 and 0.0052 for ST16 cells, showing a 17.5 fold increase with MGLVA1 cells. When the cells were grown in serum containing medium, the ratio was reduced by 76% to 0.0217 in MGLVA1 cells and to 0.0015 in ST16 cells (71% reduction). This experiment has been repeated twice with similar results (fig 1 shows a representative Southern blot).

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Figure 1

A Southern blot showing gastrin mRNA expression of MGLVA1 and ST16 cells grown in serum free conditions in vitro. The standard curve is generated with PCR products from a gastrin positive control (human G cells) of known DNA content.

Both cell lines have previously been shown to express mRNA for the truncated and classical CCKBR8 as well as the receptor protein as characterised by western blotting.9

CELL ASSOCIATED AND SECRETED GASTRIN LEVELS AS DETERMINED BY RADIOIMMUNOASSAY

Table 1 presents the results. MGLVA1 has previously been shown to secrete cell associated progastrin and glycine extended gastrin but not carboxy amidated gastrin.23 ST16 had cell associated progastrin but no cell associated glycine extended or carboxy amidated gastrin, and no detectable secreted gastrin species. Growth of both of the cell lines in serum containing growth medium, resulted in a lack of detection of gastrin peptides by the RIA.

IN VITRO GROWTH LEVELS IN SERUM FREE MEDIUM

MTT uptake was used to measure in vitro proliferation which assesses the oxidative ability of the cell to convert a soluble tetrazolium compound into an insoluble coloured crystalline product which can be dissolved and assessed colorimetrically. This has previously been shown to correlate with direct cell counts of human G1 cell lines.21 The mean basal MTT uptake of ST16 from three experiments was 0.513 with a standard deviation of 0.08 compared with a mean of 0.906 for MGLVA1 with a standard deviation of 0.03 (p<0.001, Student's t test). ST16 has previously been shown to respond proliferatively to exogenously administered G17 in vitro,24 whereas MGLVA1 showed no proliferative response.18 Figure 2 shows the MTT uptake of MGLVA1 and ST16 in the presence of increasing dilutions of rabbit anti-G17 (9):DT (calculated as a percentage of normal rabbit IgG control at each antiserum dilution). In two experiments, the growth of MGLVA1 was significantly reduced by treatment with rabbit anti-G17 (1–9):DT IgG to 30–40% of control at a 1/20 dilution, relating to an ABC of 5 × 10^–7 M (fig 2A), and modestly inhibited at dilutions of 1/100 and 1/200 (p<0.05, one way analysis of variance). The growth of ST16 was not significantly reduced in the two experiments performed (fig 2B).

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Figure 2

Effect of rabbit anti-G17 (9):DT IgG on the in vitro growth of (A) MGLVA1, and (B) ST16 in serum free growth conditions. *p<0.05. MTT, methylthiazoyl tetrazolium.

IN VIVO EFFECT OF ANTISERUM RAISED AGAINST GASTRIMMUNE

Figure 3 shows the effect of rabbit anti-G17 (9):DT treatment on mice injected with increasing concentrations (5 × 10⁵ to 1 × 10⁶ cells per mouse) of MGLVA1 cells. With initial cell concentrations of 1 × 10⁶ there was a significant effect on survival by treatment with rabbit anti-G17 (9):DT when compared with normal rabbit (p=0.0124, log rank test) with 50% median survival increasing by 21% from 19 days to 23 days (fig 3A). A significant effect on survival, following treatment with rabbit anti-G17 (9):DT, was also seen when the cell concentration was reduced to 7.5 × 10⁵ cells (p=0.0023 when compared with normal rabbit control) with the 50% median survival increasing by 26.3% from 19 days to 24 days (fig 3B). When the seeding concentration was lowered to 5 × 10⁵ cells/mouse, treatment with rabbit anti-G17 (9):DT increased the 50% median survival by 25% from 20 days to 25 days (p=0.0146, fig 3C).

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Figure 3

Effect of Gastrimmune induced antiserum on the in vivo growth of MGLVA1 cells at three different seeding concentrations: (A) 1 × 10⁶; (B) 7.5 × 10⁵; (C) 5 × 10⁵.

Figure 4 shows the effect of rabbit anti-G17 (9):DT treatment on mice injected with increasing concentrations of ST16 cells. With initial cell innocula of 1 × 10⁶ cells, no significant effect on survival was achieved (fig 4A). When mice were injected with cell concentrations of 7.5 × 10⁵ cells, the 50% median survival was increased by 72% from 25 to 43 days in the rabbit anti-G17 (9):DT treated group (p<0.0001, fig 4B). When mice were injected with 5 × 10⁵ cells, the median 50% survival was 27 days following normal rabbit IgG treatment and 47 days following treatment with rabbit anti-G17 (9):DT (fig 4C, p<0.0001, 74% increase). Tumour burden, ascites volume, and density were consistent in all animals at onset of morbidity. However, at day 60, there was no evidence of tumour within the peritoneal cavity of rabbit anti-G17 (9):DT antiserum treated mice.

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Figure 4

Effect of Gastrimmune induced antiserum on the in vivo growth of ST16 cells at three different seeding concentrations: (A) 1 × 10⁶; (B) 7.5 × 10⁵; (C) 5 × 10⁵.