Molecular Oncology



Our interests in Molecular Oncology touch several fields:




Trefoil peptides involved in carcinogenesis of the digestive tract

The molecular events leading to carcinogenesis of the upper digestive tract are poorly understood, and, in analogy to the colon adenoma-carcinoma multistep models, would be a prerequisite for diagnosis and eventually therapy (Goett and Blin 1996 ). A group of genes presumably involved in this process belong to the family of trefoil peptides. These are small secretory proteins of about 50 aa characterized by three intrachain disulfide bonds forming the trefoil-domain. They are abundantly expressed at mucosal surfaces, especially of the gastrointestinal tract. The expression of trefoil peptides in certain pathological conditions like ulcera, meta- and neoplasia of mucosa is disturbed. In chronic ulcerative diseases as well as in many gastrointestinal tumors, the transcription of the corresponding genes is enhanced, whereas some tumors of the stomach have ceased expression. Trefoil peptides promote the maintainance of the mucosal integrity, possibly by interaction with mucins. Conversely, knock-out mice, lacking the stomach specific trefoil peptide pS2, develop gastric adenomas and carcinomas.

Genomic organization of the three human trefoil peptide genes
We recently showed the three genes coding for human trefoil peptides pS2(TFF1/BCEI), spasmolytic protein SP (TFF2/SML1) and intestinal trefoil factor (TFF3/ITF) to be clustered on 21q22.3 (Schmitt et al. 1996a ) and established a genomic contig using the bacterial arteficial chromosome (BAC) recombinants (Beck et al. 1996 ). The three genes are transcribed in tandem orientation within a region of 50 kb distance. This structural clustering may reflect a prerequisite for coordinated, interrelated gene expression (Goett et al. 1996 ). On an evolutionary level, these genes may have evolved by exon shuffling. TFF2, located in the middle of the cluster, contains two trefoil-domains located on exons 2 and 3. Interestingly, a tandem repeat sequence is found in between, which may have arisen in mammalian evolution.

Transcriptional regulation of trefoil peptide genes
The expression pattern of all three trefoil peptides regarding cell and tissue specificity is regulated by yet to be determined factors. We have cloned the 5┤-flanking regions of the corresponding three human genes for characterization. Homology searches define short elements with almost identical sequence and spacing. Two motifs with identical sequence and positions are shared by TFF1 and TFF2 thus presenting possible targets for stomach specific gene regulation. Two other motifs are shared within all known human and rat trefoil genes suggesting a coordinated regulation and/or a common locus controlling region. Using luciferase reporter gene technology we are currently investigating the function of regulatory sequences in transiently transfected cell lines. Transcription factors involved in regulation will be determined by gel shift assays and will be further characterized.

Expression of trefoil peptides and mucins in meta- and neoplasia of human esophagus and stomach
Using semiquantitative RT-PCR, a change in the pattern of trefoil gene expression is detected in tissue samples of normal gastric mucosa, in hyperplastic polyps, gastric cancer, and gastric cancer cell lines, respectively. In gastric carcinomas pS2 expression was observed in 66 % of cases, being significantly higher in diffuse (89%) than in intestinal type of carcinoma (Machado et al 1996 ). By using immunohistochemistry, normal gastric mucosa, chronic atrophic gastritis with hyperplastic polyps and adenomatous polyps were analysed. Major changes in pS2 expression can be used to differentiate at least two seperate pathways of malignant transformation of gastric mucosa. Biopsy material from esophageal carcinoma and Barretts esophagus is being analysed by RT-PCR and immunohistochemistry for changes in trefoil peptide expression. Expression of mucin genes will be included for integration of more detailed pathways of malignant transformation.

Transgenic expression of trefoil peptides in cell lines
Studies using exogenous addition of trefoil peptides to cell lines indicate a role in cell migration and wound healing. To establish the physiological function of pS2, a gastric tumor cell line was transfected with a pS2-antisense vector to shut down endogenous pS2 expression. Influence of pS2 on morphological changes, growth and migration behaviour are the main parameters in current studies. Additional gastrointestinal cell lines will be transfected by antisense vectors. Transgenic cell lines expressing pS2 via a inducible promoter will help in fine tuning the physiological level of pS2.

Suicide gene therapy by means of trefoil gene promoter constructs
Since TFF3 is expressed in tumors of the upper digestive tract and on the other hand some colorectal tumors express TFF1, we are investigating this abberrant regulation pattern on the level of transcriptional initiation. Tissue-and tumor specific promoter boxes are currently analysed by reporter gene constructs in gastrointestinal tumor lines, and will be cloned in front of suicide genes, which can alter a prodrug to a cytotoxic metabolite (e.g. 5-fluorocytosine). This will eventually lead to a gene therapeutic approach for gastrointestinal cancer.



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Last updated: 23 January 1997

This page is maintained by Holger Schmitt and Jose Carlos Machado
For comments or suggestions please e-mail Mail Holger(holger.schmitt@uni-tuebingen.de)