Construction of tumor-specific toxins using ubiquitin fusion technique.
The use of cytotoxic agents to eliminate cancer cells is limited because of their nonselective toxicity and unwanted side effects. One of the strategies to overcome these limitations is to use latent prodrugs that become toxic in situ after being enzymatically activated in target cells. In this work we describe a method for producing tumor-specific toxins by using a ubiquitin fusion technique. The method is illustrated by the production of recombinant toxins by in-frame fusion of ubiquitin to saporin, a toxin from the plant Saponaria officinalis. Ubiquitin-fused toxins were rapidly degraded via the ubiquitin-proteasome system, significantly reducing their nonspecific toxicity. The insertion of the protease-cleavage sequence between ubiquitin and saporin led to the removal of ubiquitin by the protease and resulted in protease-dependent stabilization of the toxin. We engineered toxins that can be stabilized by specific proteases such as deubiquitinating enzymes and prostate-specific antigen (PSA). Both constructs were activated in vitro and in cultured cells by the appropriate enzyme. Processing by the protease resulted in a greater than 10-fold increase in the toxicity of these constructs. Importantly, the PSA-cleavable toxin was able to kill specifically the PSA-producing prostate cancer cells. The ubiquitin fusion technique is thus a versatile and reliable method for obtaining selective cytotoxic agents and can easily be adapted for different kinds of toxins and activating proteases.
Tcherniuk
Sergey O
SO
Laboratoire de Biophysique Moléculaire, Institut de Biologie Structurale J. P. Ebel (CEA/CNRS/UJF), 41 rue Jules Horowitz, 38027 Grenoble, France.
Chroboczek
Jadwiga
J
Balakirev
Maxim Y
MY
D045744:Cell Line, Tumor; D049109:Cell Proliferation; D015972:Gene Expression Regulation, Neoplastic; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008297:Male; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D011471:Prostatic Neoplasms / Q000235:genetics / Q000473:pathology / Q000628:therapy*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012156:Reticulocytes / Q000378:metabolism; D054789:Ribosome Inactivating Proteins, Type 1; D029750:Saponaria / Q000235:genetics; D000076985:Saporins; D012680:Sensitivity and Specificity; D014118:Toxins, Biological / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism*
D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D014118:Toxins, Biological; D025801:Ubiquitin; D000076985:Saporins