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Subcellular Distribution of Plasminogen Activator in Cultured Human Fibrosarcoma Cells¹

Division of Hematology-Oncology, Childrens Hospital of Los Angeles, USC School of Medicine, Los Angeles, California [W. E. L.] and Research Institute Wander, a Sandoz Research Unit, Wander Ltd., Berne, Switzerland [B. D., J. S., M. B.]

The subcellular distribution of plasminogen activator (PA) was studied in the human fibrosarcoma cell line HT 1080. The cells were homogenized and the cytoplasmic extract (postnuclear supernatant) was fractionated by rate sedimentation and isopyknic equilibration in continuous sucrose density gradients. The distribution of PA was compared with that of a number of subcellular marker enzymes. PA was found to be associated with light, slowly sedimenting particles. It was clearly resolved from the lysosomal marker enzymes ß-glucuronidase and N-acetyl-ß-glucosaminidase and from catalase used as a marker for peroxisomes. In both fractionation systems, the distribution profile for PA was intermediate between that of the putative plasmalemmal marker enzymes alkaline phosphodiesterase I and leucyl-ß-naphthylamidase and that of esterase, a marker of the endoplasmic reticulum. PA-containing structures were resolved to a similar degree from plasmalemma and endoplasmic reticulum fragments by isopyknic equilibration in discontinuous sucrose gradients. Furthermore, following treatment of the postnuclear supernatant with digitonin, the shift in modal equilibrium density of the PA distribution profile was smaller than that of the plasmalemmal markers and larger than that of esterase.

The consistent dissociation of PA from alkaline phosphodiesterase I and leucyl-ß-naphthylamidase suggests that PA is not primarily a constituent of the plasmalemma of transformed cells. It is most likely associated with the membrane of Golgiderived secretory or carrier vesicles and could this way become incorporated into the plasmalemma and subsequently gain access to the extracellular environment.

The subcellular distribution of PA was also studied in clones of the fibrosarcoma cell line HT 1080 with high or low secretory activity and found to be very similar. This suggests that PA secretion in transformed cells does not depend on a particular subcellular distribution of this proteinase and that low secretory activity is not related to increased intracellular storage.

¹ This work was supported in part by a contract from National Cancer Institute (NO1-CP-55641) and by grants from the National Heart, Lung and Blood Institute (HL 23500) and the American Cancer Society (CD-15).

² Recipient of a Junior Faculty Clinical Fellowship of the American Cancer Society. To whom requests for reprints should be addressed, at Division of Hematology-Oncology, Childrens Hospital of Los Angeles, 4650 Sunset Boulevard, Los Angeles, California 90027.

Received 4/20/82. Accepted 10/ 6/82.