Location of T-cell leukaemia cells in a model rat system by means of a fluorescent probe

Abstract

Departments of' IBiocheimistry' anCd 2Pharniacolo!gu, Stop! l-rd This study demonstrates the use of a fluorescent probe for the active centre of a cell surface protease (Steven et al., 1985) to locate the malignant cells of a rat T-cell leukaemia, (Diblev et a!., 1975;Jackson et al., 1984) in the host tissues. The enzyme, guanidinobenzoatasc, degrades fibronectin and has been shown to be associated with the surface of cells capable of migration (Steven et al., 1985). Using the fluorescent probe leukaemic cells can readily be demonstrated in sections of kidney, liver, testis, and epididymis in this experimental model; even individual leukaemia cells can be so located. We illustrate the application of the fluorescent probes in both wax and resin embedded sections, but frozen tissue sections can also be used.Fluorescent labelling is achieved by treating sections with aqueous 9-aminoacridine which is known to be a competitive inhibitor at the active centre of the protease. Cells possessing this protease 'stack' 9-aminoacridine and exhibit a yellow surface fluorescence on a blue background. A second staining procedure using an aqueous solution of propidium iodide following the 9-aminoacridine staining, enhances the colour contrast for photography. This combined staining procedure relies on the fact that both 9-amino acridine and propidium iodide are planar molecules which are capable of intercalating in DNA. If a cell surface possesses guanidinobenzoatase, then it is possible to stack 9-aminoacridine and subsequently co-stack propidium iodide on the cell surface (Steven et al., 1986). The co-stacking of propidium iodide leads to a change in fluorescent emission from yellow to pink. The structures of these fluorescent molecules are shown diagrammatically in Figure 1. Materials and methodsAnaial.s T-Leukaemia cell suspensions were prepared for intramuscular transmission of the disease in the inbred hooded Oxford strain of rats following the procedure previously described (Jackson et al., 1984). Chenmic als 9-Aminoacridine, propidium iodide and N-tosyl-lysyl-chloromethylketone were purchased from Sigma Chemical Company, St Louis, Mo., USA.Flulorescent staining Dewaxed sections (5 pm) were placed in an aqueous solution containing 9-aminoacridine (10 3 M) and N-tosyl-lysy-lchloromethylketone (10 -M) for 2min. Excess reagent was removed by 2 min washing in each of a series of 3 tanks containing isotonic sodium chloride. Resin sections (1I im) were stained in the same mixture for 5 min then washed with isotonic saline for 30 sec prior to microscopic examination. The combined staining procedure involved placing the 9-aminoacridine-stained slide in an aqueous solution of propidium iodide (6 x 10-I M) for 1 min followed by washing with water for 10 sec prior to microscopic analysis.The combined staining had 3 results; (a) nuclei of all cellsshowed an overall red fluorescence as would be expected (DNA reaction) (b) the plasma membranes of those cells possessing guanidinobenzoatase now appeared pink and (c) mast cells exhibite...