[Frontiers in Bioscience 2, a18-25, July 15, 1997]

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Siamak Tabibzadeh, Ravi Kothapalli , Ibrahim Buyuksal

Dept of Pathology, H. Lee Moffitt Cancer Center at the University of South Florida, College of Medicine, 12902 Magnolia Drive, Tampa, FL 33612

Received 7/10/97 Accepted 7/15/97 *: Patent pending


In the present report, we defined the pattern of expression of TGFB4 (ebaf) mRNA in normal tissues. In addition to the endometrium, the 2.1 and 2.5 kb TGFB4 (ebaf) mRNAs were expressed in the pancreas and the expression of the 2.1 kb TGFB4 (ebaf) mRNA in the rectum and testis was weak and was rarely noted in the ovary. The weak to lack of expression of TGFB4 (ebaf) mRNA may be due to the proportionally low number of cells that express TGFB4 (ebaf) in these tissues. However, in contrast to the endometrium that primarily exhibits the 1.5 kb, 2.1 kb and 2.5 kb TGFB4 (ebaf) mRNAs, the major TGFB4 (ebaf) mRNA species expressed in these tissues was the 2.1 kb in size. Sequencing of the TGFB4 (ebaf) cDNAs obtained from normal endometria, revealed that the underlying basis for the difference in the sizes of TGFB4 (ebaf) mRNAs were attributable to the deletion of parts of the coding sequence of TGFB4 (ebaf) (unpublished data). Therefore, TGFB4 (ebaf) mRNA seems to exist as spliced variants. In addition, TGFB4 (ebaf) mRNA seems to have a distinct tissue distribution pattern. In situ hybridization revealed that in endometrium, TGFB4 (ebaf) mRNA expression was primarily confined to the endometrial stroma (15). Few, if any, endometrial glands expressed TGFB4 (ebaf) mRNA and the endometrial endothelial cells did not express TGFB4 (ebaf) mRNA (15). The expression of lefty, the mouse homolog of the TGFB4 (ebaf), was also limited to the mesenchymal cells in the mouse embryos (16). This expression was polarized and during embryogenesis was seen only on the left side of the body (16). Furthermore, the expression of lefty mRNA quickly disappeared postnatally. These findings show that TGFB4 (ebaf) mRNA has distinct tissue and cell specific expression in the embryo and in the adult tissues. It is conceivable that different splice variants of TGFB4 (ebaf) mRNA may exist in different cell types and lineages.

Northern blot analysis of tumors derived from different cell lineages confirmed the specific tissue distribution of the TGFB4 (ebaf) mRNA. TGFB4 (ebaf) mRNA was not expressed in tumors derived from lymphoid cells such as T and B cell lymphomas, and Hodgkin’s disease. Tumors derived from endothelial cells, smooth muscle, bone, cartilage, synovium and melanocytes also did not express TGFB4 (ebaf) mRNA. The TGFB4 (ebaf) mRNA was expressed in adenocarcinomas of colon, ovary and testis. The type of the tumor in the colon and ovary that expressed the gene was derived from glandular structures of these tissues, namely adenocarcinomas. In situ hybridization showed that the TGFB4 (ebaf) mRNA was expressed primarily in the neoplastic glands rather than the tumor stroma. Histologic evaulation of adenocarcinomas expressing the TGFB4 (ebaf) mRNA showed that these tumors exhibit mucinous differentiation. In the testis, both the seminomas and tumors that contained an embryonal carcinoma component exhibited TGFB4 (ebaf) mRNA expression. However, the TGFB4 (ebaf) mRNA that was expressed in the seminomas was different in size from that expressed in the embryonal carcinomas. This difference may be attributable to the cell lineage of these tumors. Taken together, these findings show that TGFB4 (ebaf) mRNA is expressed in tumors of specific types and cell lineages. The difference in the expression of the 2.5 versus the 2.1 kb of TGFB4 (ebaf) mRNA may be dependent on the cell type that expresses the gene.

The predicted protein sequence of TGFB4 (ebaf) showed homology with and structural features of the members of TGF-b superfamily (15). The members of the TGF-b superfamily are synthesized as prepro-proteins which are cleaved at RXXR site to release the mature form of the protein. The predicted protein of TGFB4 (ebaf) exhibits two such RXXR sites which are located respectively at amino acid residues of 73-76 and 131-134 (15). The deduced amino acid sequence of lefty also contained two potential cleavage sites at amino acid residues of 74-77 and 132-135 (15). Therefore, TGFB4 (ebaf) gene products may be secreted by the tumor cells and may be released into the peripheral circulation. The confined expression of TGFB4 (ebaf) to tumors of distinct phenotype, therefore, will make detection of TGFB4 (ebaf) mRNA or its protein a useful tumor marker. Tumor markers in the blood include glycoproteins secreted by the solid tumors as well as those expressed on the cell surface. The most successful markers for the diagnosis of solid tissue cancers have been a-fetoprotein and prostate specific antigen (14). The serum level of markers such as CEA and a number of carbohydrate epitopes, e.g., CA 15.3, CA 19.9, CA 50, CA 242, and mucin epitopes, such as MCA, CA 125, and DU-PAN-2 are now being used to determine the prognosis of a variety of cancers and to monitor their response to therapy (13-14). If secreted, presence of TGFB4 (ebaf) in the peripheral circulation may be potentially used for screening, diagnosis, prognosis, and monitoring of the treatment, or the detection of relapse in tumors expressing the gene.

In summary, in the present report, we defined the expression of TGFB4 (ebaf) mRNA in normal and neoplastic tissues. In normal tissues, the expression of TGFB4 (ebaf) was limited to the endometrium, and only a weak expression of TGFB4 (ebaf) mRNA was detectable in the rectum, ovary and testis. In the pancreas, the TGFB4 (ebaf) mRNA was detectable. In the neoplastic tissues, the expression of the TGFB4 (ebaf) mRNA was restricted to tumors of colon, ovary and testis and tumors of lymphoid, melanocytic, and mesenchymal origins did not express the TGFB4 (ebaf) mRNA. These findings show that TGFB4 (ebaf) mRNA has a narrow and distinct tissue distribution in both normal and neoplastic tissues.