No abstract

In a hope to isolate genes whose expression was elevated in colon cancer cells, we used a plus/minus screening of a rat colon carcinoma cDNA library. We were thus able to isolate a cDNA clone (B9) encoding the rat S13 ribosomal protein (1). The corresponding mRNA is-30-fold more abundant in the cancer cells than in normal colon, and 50-fold more important in lung metastasis than in normal lung. The expression level of this gene was found to be closely correlated with the growth rate of rat cell lines (1). Unfortunately, the B9 probe was short, and did not hybridize to RNA prepared from human tissues. In order to obtain longer clones encoding the rat S13 ribosomal protein we first rescreened the PROb cDNA library. A longer insert was then used to screen a cDNA library prepared from the T-84 colonic epithelial cell line constructed in the Uni-ZAP XR vector (Stratagene, La Jolla, CA). Inserts were excised with helper phage R408 to generate subclones in the pBluescript plasmid and sequenced by the dideoxy-chain termination method (2) using T7 DNA polymerase. The final sequences were determined from both strands. This cDNA contains 548 nucleotides and includes a 5' noncoding sequence of 32 nucleotides, an open reading frame of 456 nucleotides, a 3' noncoding sequence of 60 nucleotides, followed by a 19 bp polyA tail (Figure 1). The presumed polyadenylation signal, AATAAA, is located at bases 509-514. The nucleotide sequence in the coding region differs from the rat S13 ribosomal protein sequence (3) in the first position in 6 codons and in the third position in 52 codons. It encodes a sequence of 151 aminoacids which is identical to the rat protein. This highly conserved primary structure of ribosomal proteins across species has been observed for other ribosomal proteins (4, 5). Northern blot analysis revealed that the size of the human S13 ribosomal protein mRNA is approximately 600 bp. This probe hybridized to 8-10 genomic DNA fragments, probably representing one (or a few) functional S13 gene and a family of nonfunctional pseudogenes, as it has been shown for many other ribosomal protein genes (6, 7).

In order to define biological markers of aggressiveness, 2 rat colon-carcinoma cell lines differing by their tumorigenicity were used to clone genes over-expressed in colon carcinoma as compared with normal epithelial cells. A progressive rat colon-carcinoma clone (PROb) cDNA library was hybridized with 32P-cDNA synthesized from mRNA prepared from these PROb cells, or from regressive cells (REGb) derived from the same tumor. Several clones were isolated after the initial screening. The specificity of each clone was confirmed by RNA blotting. One of these (B9) was found to hybridize to an mRNA 30-fold more abundant in PROb cells than in normal adult rat colon, and was therefore selected for further study. No gene amplification was detected by Southern blot analysis, indicating that the difference in mRNA content was most likely due to an increased transcription of this gene. Sequencing of the cDNA revealed approximately 98% homology with the rat S13 ribosomal protein. The expression level of this gene was determined in a series of rat cell lines with different growth rates. A good correlation was found between these 2 parameters. Our data suggest that the S13 ribosomal-protein gene can be used to evaluate the growth rate of tumor cells, which might be correlated with their aggressiveness. In an initial trial experiment, S13 ribosomal-protein mRNA was detected in a series of human colorectal tumors by in situ hybridization. A strong signal was seen in the 4 tumors analyzed.

No abstract

Germline mutations of the APC (adenomatous polyposis coli) gene lead to multiple intestinal tumors in familial adenomatous polyposis patients and in Min (multiple intestinal neoplasia) mice. Consequently, these mice provide an excellent model for familial colon cancer. We have identified an Mr approx. 66 kDa glycoprotein which is preferentially expressed at the cell surface of cell lines established from chemically induced rat colon carcinomas. Cloning of the corresponding Tage4 cDNA has revealed that this protein contains the conserved amino acids characteristic of members of the immunoglobulin gene superfamily. Here, we analyze expression of the mouse Tage4 gene in Min mouse intestinal adenomas. RT-PCR analysis allowed us to detect expression of this gene in all the mouse adenomas tested. In contrast, lower levels of Tage4 mRNA were found in the intestinal tract and barely detectable levels in other tissues of normal mice. Furthermore, Tage4 mRNA was detected in a series of mouse intestinal adenomas by in situ hybridization. A strong signal was seen in the samples analyzed.