One gene fragment of approximately 80 bp was expressed in the 4 normal tissue samples but not, or to a much lesser degree, in 24 colorectal tumors in the mRNA DD-PCR experiment. The band was cut and reamplified using the same primers, and cloned. Clones were then selected by size, and by using inverse northern hybridization it was found that all clones represented the same DNA sequence. Sequencing and subsequent search in the genome database showed that this differentially expressed gene fragment corresponded to the sequence of aquaporin 8 (AQP8).
The result was confirmed with RT-PCR on newly synthesized cDNA and northern blot hybridization (Fig. ) on the original samples from the mRNA DD-PCR experiment. The number of samples used for northern blot was small depending on limited amounts of RNA from the samples. We extended the experiment and included besides the 28 samples used in the mRNA DD-PCR, another 7 sporadic colorectal cancers, 3 samples from normal colonic mucosa, 5 sporadic colonic adenomas and 3 HNPCC tumors from patients with known germline mutations in hMLH1. Using a semiquantitative RT-PCR it was found that samples from adenomas, rectal cancers, and colonic cancers, including those from HNPCC patients, expressed the AQP8 to various degrees, but generally to a much lesser extent than normal colonic tissue (Fig. ). The expression of AQP8 in some tumors could be explained by a normal cell contamination in those samples.
Figure 1 Northern blot demonstrating AQP8 in normal colonic tissue, and not in adenomas, or carcinomas. Symbols first in sample identification relate to type; N, normal, H, HNPCC carcinoma, C, sporadic colonic carcinoma, R, sporadic rectal carcinoma, P, adenomatous (more ...)
RT-PCR comparing expression of GADPH2 and AQP8 in normal colonic tissue (N), adenomas (P), colon (C) and rectal (R) carcinomas and carcinomas from HNPCC-patients (H). GADPH2, 25 cycles of PCR amplifications and AQP8, 35 cycles of amplification.
To study what cells expressed the AQP8 mRNA, in situ hybridization was used. In samples from 2 normal colonic mucosa there was a staining of mRNA AQP8 expression in the columnar surface epithelium only (Fig. ). No expression of AQP8 was detected in any of one sporadic rectal carcinoma, one sporadic colon carcinoma or one HNPCC carcinoma tested (the HNPCC carcinoma is shown)(Fig. ). The RNA in situ experiment suggested that only normal mucosa expressed AQP8.
Figure 3 A) Antisens, RNA-in situ demonstrates AQP8 in normal colonic columnar epithelium from one patient. B) Sens, RNA-in situ demonstrate no signal. C) Antisens, HNPCC carcinoma demonstrates that there is no AQP8 expression in the tumor. Epithelial cells of (more ...)
In total 15 colon cancer cell lines, six microsatellite unstable (SW48, LOVO, HT116, RKO, LS174T, DDL1) and 9 microsatellite stable (SW480, SW620, HT29, CACO2, Crl7184, Crl7456, Crl2405, 238CCL, 233CCL), were tested for expression of AQP8 using RT-PCR. None of them demonstrated expression of the AQP8 transcript, supporting the hypothesis that tumor cells do not express AQP8 (Fig. ).
RT-PCR demonstrating that there is no expression of AQP8 in any of the tested colorectal cancer cell lines. Line one, normal mucosa demonstrate the band corresponding to AQP8 expression.