FLOW CYTOMETRIC ASSAY OF PINOCYTOSIS - CORRELATION WITH MEMBRANE RUFFLING AND METASTATIC POTENTIAL IN THE DUNNING R-3327 RAT PROSTATIC ADENOCARCINOMA MODEL

Membrane ruffling has been associated with neoplastic transformation, Harvey ras expression, and metastatic capability. In the Dunning R-332 7 rat prostatic adenocarcinoma model, membrane ruffling graded visuall y upon live cultured cells filmed by time-lapse video-microscopy has d istinguished sublines of high and low metastatic potential. Fluid-phas e pinocytosis is a constitutive, noninducible internalization of mediu m by cell membrane. Fluid phase pinocytosis may be measured flow cytom etrically by cellular uptake of fluorescein-labelled medium constituen ts. The optimum conditions for a flow cytometric assay of pinocytosis were determined using AT-2 subline that has an intermediate degree of membrane ruffling. The optimum dextran concentration was selected from the midpoint of the linear portion of the dose-response (0.01-10.00 m g/ml) curve, whereas the optimum incubation time was determined from a time course (1-405 min.) curve study. Cultured cells from 6 Dunning s ublines incubated with 1.0 mg/ml of fluorescein-labelled dextran for 9 0 min were washed, fixed, and the fluorescence of 10,000 cells studied by flow cytometry. For each subline, dextran fluorescence was measure d in four independent experiments. Pinocytosis failed to distinguish s ublines of high (AT-3 63.5 +/- standard error 4.1 mean channel number, MAT-LyLu 63.2 +/- 6.3, MAT-Lu 64.3 +/-5.6) and low (G 33.5 +/- 1.2, A T- 1 63.5 +/- 4. 1, AT-2 58.4 +/- 3.6) (rank p = 0.38) metastatic pote ntial but correlated strongly with visually graded membrane ruffling ( r = 0.95, p = 0.003). Pinocytosis assayed by flow cytometry reflects m embrane ruffling observed visually and thus flow cytometric assays may facilitate study of membrane activity. (C) 1993 Wiley-Liss, Inc.