Molecular Signaling Pathways of Recombinant Human Cripto-1

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Human oncofetal protein Cripto-1 (CR-1) is considered as a potential biomarker and target for cancer therapy. CR-1 is overexpressed in various types of cancers, including colorectal, breast, nasopharyngeal and gastric cancer. It has been shown that Cripto-1 activates several processes linked with neoplasia, including cell proliferation. The mitogenic effect of CR-1 has been observed in various experimental systems. However, the finer molecular details of CR-1 mediated mitogenic signaling and the controls of those signaling pathways require further investigations. It is now well established that many growth factors can have contextual dichotomous effects. Epidermal growth factor (EGF), a well known mitogen, inhibits cellular proliferation and induces apoptosis, depending upon cell type, expression level of receptors and dosage. Similarly, members of TGF-b superfamily, including TGF-b itself, function in a context dependent fashion. CR-1 is a modulator of signaling by TGF-b ligands and has a modified EGF-like domain. Therefore, one can expect that CR-1 may also have such dichotomous effects. Here, in this thesis, we investigate differential effects of recombinant human CR-1 on cell proliferation in different cellular systems and identify a previously uncharacterized anti-proliferative pathway induced by CR-1. We have expressed human CR-1 in both mammalian as well as in bacterial expression systems and have used recombinant CR-1 for our investigations. CR-1 interacts with cell surface Glypican-1 to induce the pro-proliferative pathways. We have used two different cell lines, human cervical adenocarcinoma cell line HeLa and human glioblastoma cell line U-87 MG, for most of our investigations. The expression of Glypican-1 is very low in Hela cells in comparison to U-87 MG. As expected, recombinant CR-1 activates proproliferative pathways and induces proliferation of U-87 MG cells. However, we have observed that treatment with recombinant CR-1 leads to decrease in proliferation of HeLa cells in a dose dependent fashion. This anti-proliferative effect is measured using different biochemical assays and is confirmed in two other cell lines. Using a series of experiments, we have proved that recombinant CR-1 reduces cell proliferation of HeLa cells by increasing the doubling time, without arresting cell cycle or inducing cell death..
Supervisor: Biplob Bose