Toward an Enzyme/Prodrug Strategy for Cancer Gene Therapy: Endogenous Activation of Carboxypeptidase A Mutants by the PACE/Furin Family of Propeptidases
- 20 January 1999
- journal article
- research article
- Published by Mary Ann Liebert Inc in Human Gene Therapy
- Vol. 10 (2) , 235-248
- https://doi.org/10.1089/10430349950019020
Abstract
In an effort to develop a gene-dependent enzyme/prodrug therapy (GDEPT) for tumor-specific delivery of methotrexate (MTX) we have chosen to construct mutant forms of carboxypeptidase A1 (CPA) that circumvent the requirement for trypsin-dependent activation. The basis of this strategy is that methotrexate-alpha-peptides are inefficient substrates for the reduced folate carrier (RFC) and hence cannot be internalized by cells. However, the blocking amino acid can be cleaved by CPA to liberate MTX, which is then internalized by the RFC, resulting in inhibition of dihydrofolate reductase and cytotoxicity. A battery of mutant CPAs was generated, in which the putative trypsin cleavage sites in the propeptide were mutated to the consensus recognition sequence for mammalian subtilisin-like propeptidases. These mutant forms of CPA were evaluated for expression, activation, and catalytic activity by transiently transfecting them into COS-1 cells both in the absence and in the presence of cotransfected propeptidases. CPA95 was identified as the most efficiently cleaved mutant, and further studies of this mutant indicated that the endogenously activated enzyme had kinetic parameters identical to those of the trypsin-activated wild-type protein. In addition, endogenously activated CPA95 could effectively sensitize cells to MTX-Phe in culture, decreasing the IC50 of MTX-Phe from 25- to 250-fold in squamous cell carcinoma cells expressing active CPA as compared with the parental lines.Keywords
This publication has 29 references indexed in Scilit:
- SPC4, SPC6, and the novel protease SPC7 are coexpressed with bone morphogenetic proteins at distinct sites during embryogenesis.The Journal of cell biology, 1996
- Role of the Prodomain in Folding and Secretion of Rat Pancreatic Carboxypeptidase A1Biochemistry, 1996
- Procarboxypeptidase in rat pancreas Overall characterization and comparison of the activation processesEuropean Journal of Biochemistry, 1994
- Protein processing within the secretory pathwayCurrent Opinion in Biotechnology, 1992
- Internal translation initiation in the design of improved expression vectorsCurrent Opinion in Biotechnology, 1992
- In Vivo Gene Transfer with Retroviral Vector-Producer Cells for Treatment of Experimental Brain TumorsScience, 1992
- Three-dimensional structure of porcine pancreatic procarboxypeptidase A: A comparison of the A and B zymogens and their determinants for inhibition and activationJournal of Molecular Biology, 1992
- New Colorimetric Cytotoxicity Assay for Anticancer-Drug ScreeningJNCI Journal of the National Cancer Institute, 1990
- Carboxypeptidase-mediated release of methotrexate from methotrexate .alpha.-peptidesBiochemistry, 1989
- The severed activation segment of porcine pancreatic procarboxypeptidase a is a powerful inhibitor of the active enzyme Isolation and characterisation of the activation peptideBiochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1982