Abstract
The p53 tumor suppressor is mutated in over 50% of human cancers. Mutations resulting in amino acid changes within p53 result in a loss of activity and consequent changes in expression of genes that regulate DNA repair and cell cycle progression. Replacement of p53 using protein therapy would restore p53 function in p53-deficient tumor cells, with a consequence of tumor cell death and tumor regression. p53 functions in a tetrameric form in vivo. Here, we refolded a wild-type, full-length p53 from inclusion bodies expressed in Escherichia coli as a stable tetramer. The tetrameric p53 binds to p53-specific DNA and, when transformed into a p53-deficient cancer cell line, induced apoptosis of the transformed cells. Next, using the same expression and refolding technology, we produced a stable tetramer of recombinant gonadotropin-releasing hormone-p53 fusion protein (GnRH-p53), which traverses the plasma membrane, slows proliferation, and induces apoptosis in p53-deficient, GnRH-receptor-expressing cancer cell lines. In addition, we showed a time-dependent binding and internalization of GnRH-p53 to a receptor-expressing cell line. We conclude that the GnRH-p53 fusion strategy may provide a basis for constructing an effective cancer therapeutic for patients with tumors in GnRH-receptor-positive tissue types.
Highlights
The p53 tumor suppressor is a transcription factor that resides in the cytosol, and after activation by posttranslational modifications, it translocates to the nucleus, where it tetramerizes, binds DNA, and activates transcription of genes important in cell cycle regulation and DNA repair (1 – 3)
We describe the production of a wild-type tetrameric p53 that binds DNA and induces apoptosis when introduced into p53-deficient cancer cell lines
The overall aim of this study is to show the feasibility of using tetrameric p53 as a cancer therapeutic
Summary
The p53 tumor suppressor is a transcription factor that resides in the cytosol, and after activation by posttranslational modifications, it translocates to the nucleus, where it tetramerizes, binds DNA, and activates transcription of genes important in cell cycle regulation and DNA repair (1 – 3). Activation of p53 regulates over 100 cellular genes. Despite the fact that p53 is mutated in many human cancers and its role in tumor suppression is well characterized, there are no p53-based antineoplastic therapies available. Some of the current standard cancer therapies, including chemotherapy (e.g., cisplatin, carboplatin, and oxaliplatin) and radiation therapy, may partly depend on activation of p53-dependent pathways for induction of tumor cell death. In many tumors, p53 is inactivated by mutation, and these therapies are not as effective as cancer cells with wild-type p53. Only a modest sensitization of the target tissue to second-line chemotherapy has been elicited from this treatment
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