Abstract
Using a doxorubicin-resistant subline (K562/ADM) of human leukaemia K562 cells (Tsuruo et al, 1986), the effect of immunoliposomes that targeted a cellular transferrin receptor (TFR) was examined by neutralization of doxorubicin (DOX) resistance. OKT9-CIL, prepared by conjugation of DOX-encapsulated liposome with an anti-TFR monoclonal antibody, OKT9 (Aisenberg and Wilkes, 1980), showed similar binding to both K562 and K562/ADM. Although an 80-fold higher sensitivity to free DOX on cell growth inhibition in K562 than in K562/ADM was found, the difference was clearly diminished after OKT9-CIL treatment through the increased sensitivity of K562/ADM. The cellular DOX level 30 min after the exposure of free DOX was 45-fold lower in K562/ADM than in K562, whereas nearly equivalent DOX levels were detected in K562 and K562/ADM after OKT9-CIL treatment. In addition, DOX in K562/ADM in the free DOX treatment was efficiently excreted by 54% within 120 min of incubation, whereas almost all DOX supplied by OKT9-CIL remained uncleared. Fluorescence microscopic observation showed that OKT9-CIL was internalized into juxtanuclear vesicles in K562/ADM cells. These results suggest that OKT9-CIL has a potency to accumulate DOX, resulting in augmentation of DOX cytotoxicity in DOX-resistant tumour cells.
Highlights
The levels of cellsurface OKT9-CIL were decreased during incubation at 37°C in both cells, but the rates of the decrease were higher in K562 (68% at 120 min) than in K562/ADM (37% at 120 min)
The decrease in total cellular DOX level was, within 5% of the initial value in both cells. This process did not occur in fixed cells and was suppressed in the presence of some endocytosis inhibitors (Berinstein et al, 1987; Collins et al, 1989), such as sodium azide, ammonium chloride, chloroquin and colchicine
In K562 cells treated with free DOX for 2 h at 37°C, bright DOX fluorescence was observed in both the nucleus and cytosol (Figure 3A)
Summary
Human myelogeneous leukaemia K562 and its DOX-resistant subline K562/ADM (Tsuruo et al, 1986) were generously provided by Dr Tsuruo, Institute of Molecular and Cellular Biosciences, University of Tokyo. These cell lines were maintained in Dulbecco's modified Eagle minimal essential medium (Nissui Pharmaceutical, Tokyo), 2 mM L-glutamine, 1 gM sodium pyruvate, 10 mM Hepes, kanamycin at 60 gg ml-', pH 7.4 (standard medium) containing 10% heat-inactivated fetal calf serum (FCS) (MA Bioproducts, Walkersville, MD, USA) and with 0.3 jg ml-' DOX only in the case of K562/ADM. Leucinefree medium was prepared from RPMI-1640 select amine kit (Gibco, NY, USA). L-[4,5-3H]leucine ([3H]leucine) was obtained from Amersham Lab., Buckinghamshire, UK
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