291:	Immunocytochemical localization of the anticancer compound camptothecin to the vacuole of mesophyll cells and epidermal idioblasts.

Authors:	Nolte, Beth, A.(A)Griffing, Lawrence, R.(B)Rumpho, Mary, E.(A)
Affiliations:	(A): Program in Molecular and Environmental Plant Sciences and Dept. of Horticultural Sciences, Texas A&M University (B): Program in Molecular and Environmental Plant Sciences and Dept. of Biology, Texas A&M University
Presenter:	Nolte, Beth A., bnolte@tamu.edu

Camptothecin (CPT) is a monoterpene alkaloid with anticancer properties and a product of Camptotheca acuminata, commonly known as the Chinese happy tree. Semi-synthetic analogs of CPT are currently marketed in the U.S. for cancer treatment due to their role as topoisomerase I inhibitors. The availability of these drugs for therapy and future research is dependent on a supply of CPT from its natural source. Information concerning secondary metabolite compartmentalization has proven useful for the production of important quinolizidine alkaloids in cell cultures and could also be significant in identifying factors limiting biosynthesis. This work investigates CPT localization at the tissue and subcellular levels utilizing light-level immunocytochemical techniques and the compound's natural fluorescence under UV light. Immunocytochemistry is a powerful method for localizing nondiffusable cellular components, but presents a number of problems in detecting small, diffusable molecules that may not be retained by the tissue during fixation. Preparatory studies pertaining to alkaloid retention indicate that approximately 80% of the initial CPT concentrations are maintained in tissues fixed for light microscopy by conventional methods. The use of polyclonal antibodies generated to keyhole limpet hemacyanin- conjugated CPT, fluorescence and immunogold labeling reveal that the remaining alkaloid resides in vacuoles of mesophyll cells and epidermal idioblasts. These findings lend overall support to earlier work that attributes the blue autofluorescence of C. acuminata vacuoles to the presence of CPT. This research was supported by a Faculty Research Development Grant from the Texas Agricultural Experiment Station and Xylomed Corp.