CELL CULTURE

Cells were grown in RPMI media (Gibco BRL, Paisley, UK) containing 10% heat inactivated newborn calf serum (Sigma, Poole, Dorset, UK) at 37°C and in 5% CO₂.

RNA EXTRACTION

Total RNA was extracted from 10⁶ cells using RNAzol-B (Biogenesis, Poole, Dorset, UK). Cells were lysed in RNAzol-B (1 ml) with chloroform (110 μl; Sigma). An aqueous phase was separated by a 13 000 g spin and eluted. RNA from the eluate was precipitated using isopropanol and washed twice in 75% ethanol. The RNA pellet was then resuspended in diethylpyrocarbonate (DEPC; Sigma) treated sterile water (50 μl).

REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION

Reverse transcription was performed as previously described.10 Total RNA preparations were divided into two halves (25 μl) and randomly primed. Both halves were incubated at 25°C for 10 minutes, 37°C for one hour, and 95°C for 10 minutes in reverse transcription reaction buffer (1× Dynazyme PCR buffer (Flowgen, Sittingbourne, Kent, UK), 30 μmol dNTPs (Pharmacia, St Albans, Herts, UK), 0.01 M dithiothreitol (Gibco BRL), 200 U Superscript reverse transciptase (Gibco BRL, UK)). One half was reverse transcribed into cDNA while the other half was used as a negative control and the reverse transcriptase was replaced with water. PCR was performed using a similar protocol to that described previously10 except that different cDNA volumes were utilised for individual reactions. Reactions (50 μl) were prepared in 1.0 U Dynazyme (Flowgen) in 1× Dynazyme PCR buffer (Flowgen) with 40 μmol dNTPs (Pharmacia) and 10 μmol of upper and lower primers. The different volumes of cDNA preparations used for each PCR were as follows: GAPDH, 1 μl; gastrin, 5 μl; CCK-B and ΔCCK-B, 10 μl. One drop of mineral oil was layered on top of the reaction mixture which was incubated in a thermal cycler (Hybaid, Teddington, Middlesex, UK) at 95°C for five minutes before 40 cycles of 95°C for 45 seconds, 60°C for 90 seconds, and 72°C for 90 seconds. This was followed by a single stage of 60°C for 120 seconds and 72°C for 180 seconds.

SOUTHERN BLOTTING

Polymerase chain reaction products (10–20 μl) were electrophoresed on 2% (wt/vol) agarose gels and transferred on to Hybond-N nylon membrane (Amersham International, Aylesbury, Buckinghamshire, UK) by capillary blotting using an alkaline transfer buffer (0.5 M NaOH, 0.6 M NaCl). Blots were neutralised in 2× saline sodium citrate (2×SSC; Sigma) and ultraviolet fixed.

PROBE LABELLING AND PRIMER SEQUENCES

Primers (H11 and H33) and the probe (H34) for ΔCCK-B are identical to the sequences published by Miyake.9 All other primers and probes were designed on the OLIGO program (Medprobe a/s, Oslo, Norway) based on cDNA sequences taken from Seqnet (Daresbury Laboratory, UK). The gastrin probe was synthesised and 5′-digoxygenin (DIG) labelled by R&D Systems (Abingdon, Oxon, UK). The CCK-B and ΔCCK-B probes were synthesised as unlabelled oligos and 3′ tailed with poly dATP and multiple DIG labelled dUTP using a DIG tailing kit (Boehringer Mannheim). All primers and the CCK-B and ΔCCK-B probes were synthesised by the Biopolymer Synthesis Unit (Department of Biochemistry, University of Nottingham). Table 1 and figure 1 present all sequences.

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Figure 1

The “total CCK-B” RT-PCR assay using primers GR1 and GR2 detects both isoforms, while primers H33 and H11 specifically detect ΔCCK-B, the truncated CCK-B receptor.

HYBRIDISATION

Blots were prehybridised in 5×SSC (Sigma), 1% (wt/vol) Blocking Reagent (Boehringer Mannheim, Lewes, East Sussex, UK), 0.1% (wt/vol) N-lauroyl sarcosine (Sigma), and 0.02% (wt/vol) sodium dodecyl sulphate (SDS; Sigma) for 30 minutes and then hybridised for 60 minutes with specific oligo probes. The hybridisation temperatures varied depending on the probes used. For gastrin, hybridisation proceeded at 34°C whereas the CCK-B and ΔCCK-B probes were used at 55°C with the inclusion of poly(A) (Boehringer Mannheim) to block non-specific binding. Post-hybridisation washes were performed at room temperature for gastrin and 50°C for CCK-B and ΔCCK-B. First washes were with 2×SSC + 1% (wt/vol) SDS for 10 minutes twice. Second washes were with 2×SSC for gastrin for 10 minutes twice or 0.5×SSC for CCK-B and ΔCCK-B for 10 minutes twice. Probe detection and chemiluminescence was carried out using anti-DIG-AP conjugate and CSPD (Boehringer Mannheim) following the manufacturer’s instructions. After a 15 minute incubation at 37°C, the blots were exposed to a light sensitive x ray film (X-OMAT; Sigma) for between 45 and 90 minutes.

SEQUENCING

Polymerase chain reaction products were excised from agarose gels and purified using the Bandprep Kit (Pharmacia Biotech). Agarose gel slices were melted and DNA allowed to bind to Sephaglas (Pharmacia Biotech). Samples were washed and allowed to dry before resuspension in sterile water prior to sequencing. Sequencing was performed on an A373A sequencing machine (Advanced Biotechnologies, Warrington, UK) using the upper primer of each product, by the Biopolymer Synthesis Unit (Department of Biochemistry, University of Nottingham).

GAPDH PCR PRODUCTS

All cDNA preparations gave an expected PCR product for GAPDH, verifiying successful RNA extraction and reverse transcription.

GASTRIN PCR PRODUCTS

Normal human gastric antrum cDNA samples gave an expected gastrin PCR product of 215 bp which was detected on Southern blots by a specific oligo probe. The PCR product was sequenced and was found to correspond to the published gastrin cDNA sequence.11 Table2 presents results of RT-PCR. Gastrin was expressed in 5/5 and 7/8 human gastric and colorectal cell lines respectively and was also detected in lymphoblastic leukaemia, fibrosarcoma, epidermoid, and osteosarcoma derived cell lines. Gastrin PCR products from tumour cell line samples showed a characteristic two band pattern (fig 2A) as reported previously in colon tumour cells.12 ,13 The expected product, derived from a mature mRNA, of 215 bp was coexpressed with a higher molecular weight band of 345 bp. Negative control RT-PCR lanes showed no hybridisation on Southern blots, verifiying that the 345 bp band was derived from a gastrin RNA species. The gastrin PCR primers bind to regions on exons 2 and 3 of the cDNA and so the extra band is derived from retention of intron 2 as previously reported.12 ,13

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Figure 2

(A) Southern blot of gastrin RT-PCR products from cell lines showing 215 bp and 345 bp bands. From left to right: AGS, A431, HT29(L), Colo205, 791T. (B) Southern blot of ΔCCK-B RT-PCR products showing a 281 bp band in those cell lines where it was detected. From left to right: HCT116, LIM1215, C170HM2, MCF-7B, AGS, AGS, MKN45, C170HM2, LoVo. (C) Southern blot of CCK-B RT-PCR products. From left to right: AGS, MCF-7B, HCT116, ST16 ascites.

EXPRESSION OF ΔCCK-B AND TOTAL CCK-B

The total CCK-B RT-PCR assay detected a region common to both ΔCCK-B and non-truncated CCK-B. Expected total CCK-B RT-PCR products of 169 bp to 184 bp were detected on agarose gels and comigrated and specifically hybridised with an oligo probe on Southern blots (fig 2C). LoVo cells were used as a positive control for a specific ΔCCK-B RT-PCR and a product of the expected molecular weight, 281 bp, was observed on agarose gels and specifically hybridised on Southern blots (fig 2B).

The assay for total CCK-B receptor detected expression in 4/5 and 6/8 human gastric and colorectal cell lines respectively (table 2), as well as the breast (MCF7) and lymphoblastic leukaemia (Molt 4) derived cell lines. The truncated isoform, ΔCCK-B, was expressed in 3/5 gastric and 5/8 colorectal cell lines (table 2) and the one lymphoblastic leukaemia derived cell line (Molt 4). This is the first study showing widespread expression of ΔCCK-B in GI tract tumour cell lines. The GI cell lines RD19 and LIM 1215 as well as the breast line MCF7 gave PCR products for total CCK-B but not ΔCCK-B. They therefore exclusively express non-truncated CCK-B receptor molecules.