Identification of alternatively spliced TIMP-1 mRNA in cancer cell lines and colon cancer tissue

RT-PCR on cultured cell lines (A–C) and colon cancer tissue samples (D). The following primer combinations were used. Panel A: forward exon 1 and reverse exon 6; panel B and D: forward exon 1 and reverse exon 3; panel C: forward exon 4 and reverse exon 6. Full-length TIMP-1 mRNA was detected in all cell lines (A, upper band at 648bp), and all cell lines except EVSA-T and CAMA express more than one transcript, most abundantly a second transcript of 515/519bp (lower band in A). RT-PCR with primers located in exon 1 and 3 shows that all cell lines except EVSA-T and CAMA express del-2 variant (B, lower band at 134bp), and RT-PCR with primers located in exon 4 and 6 shows that MDA-MB-231-BAG, MDA-MD-435-BAG, EAHY-926 and PC-3 cell lines express del-5 variant (C, lower band at 127bp). Both full-length TIMP-1 and del-2 variant mRNA were detected in 11 of the 12 colon cancer tissue samples (D). L: 100bp DNA ladder.

Detection of TIMP-1 and alternative TIMP-1 products in cancer cell lines (A), lysates of tumor samples from CRC patients (B) and in plasma from CRC patients (C). Equal amounts of protein were separated by SDS-PAGE, and TIMP-1 was subsequently detected with a monoclonal anti-TIMP-1 antibody (VT7). Low control, platelet lysate (contains high TIMP-1 protein levels) and recombinant human (rh) TIMP-1 serve as controls. The plasma samples are representative of a total of 12 samples analysed. The experiment was repeated once with similar results.

TIMP-1 expressions in colon cancer. Serial sections from a colon adenocarcinoma were hybridized with antisense or sense probes for full-length TIMP-1 (A, C, D) or exon 2 splice variant of TIMP-1 (B). Full-length TIMP-1 mRNA is seen in fibroblast-like cells localized in the stroma (St) adjacent to the cancer cells (Ca) with similar hybridization patterns seen with two non-overlapping antisense probes (transcribed from pTIMP-1 01 and 02) (arrows in A and C). The exon 2 variant of TIMP-1 mRNA is detected in some of the same fibroblast-like cells seen in a parallel section (arrows in B). No signal is detected with a sense probe corresponding to the antisense probe shown in panel A (D). Scale bar: A–D=50μm.

Identification of TIMP-1 positive cells in colon cancer. Serial sections from a colon adenocarcinoma were processed for in situ hybridization with TIMP-1 antisense probe 01 (A), or immunostained with monoclonal antibodies against TIMP-1 (B) or α-SMA (C). TIMP-1 mRNA expressing cells (arrows in A) are seen the stroma (St) with no signal observed in the cancer cells (Ca), and immunoreactivity for TIMP-1 is detected in the same cells (arrows in B). TIMP-1 mRNA and protein co-localize with a sub-population of α-SMA positive cells, as seen on an adjacent section (arrows in C). No immunoreactivity is seen when the primary antibody is substituted with an irrelevant antibody against TNP (D). Scale bar: A–D=50μm.

Schematic representation of full-length and alternatively spliced TIMP-1. Exons are numbered from 1 to 6 and translated regions are shown in grey. White boxes indicate untranslated regions. Translation initiation sites are marked by black arrow and ATG. The corresponding translated proteins are shown underneath the mRNAs and the theoretical size is marked with number of amino acids (aa). The signal peptide sequences are shown as black boxes at the N-terminal end of full-length TIMP-1 and del-5 variant. The translation initiation site in del-2 TIMP-1 is shifted from exon 2 to exon 3, which results in a shorter protein lacking the signal peptide. A theoretical translation of the del-5 variant results in a protein consisting of 136 aa due a shift in reading frame of the exon 6 sequence.