NUCLEAR IMMUNOLOCALIZATION OF P-GLYCOPROTEIN IN MULTIDRUG-RESISTANT CELL-LINES SHOWING SIMILAR MECHANISMS OF DOXORUBICIN DISTRIBUTION

The MDR1 gene product P-glycoprotein is a plasma membrane efflux pump which is responsible for multiple drug resistance of cancer cells. Alt hough the ability of multidrug-resistant cells to exclude drugs from t he nucleus is a distinctive and possibly the main mechanism for resist ance against a number of drugs, including doxorubicin, this phenomenon is not entirely understood, In this paper, the relationship between d oxorubicin subcellular distribution and P-glycoprotein activity at dif ferent cell sites has been investigated by different techniques, Cytof luorometry and confocal microscopy were used to study doxorubicin subc ellular distribution in U-2 OS human osteosarcoma cells and in the mul tidrug-resistant variant U-2 OS/DX(580), Stable levels of doxorubicin accumulation were found in the nuclei of sensitive cells, whereas the absence of detectable levels of drug in the nuclei of resistant cells could be attributed to an energy-dependent mechanism. Moreover, in res istant cells, inhibition of P-glycoprotein activity was able to induce drug accumulation in the nuclei of resistant cells and to achieve cyt otoxic effects comparable to those observed in sensitive cells, Simila r results were also found in isolated nuclei from U-2 OS/DX(580) cells . The expression of P-glycoprotein in U-2 OS/DX(580) and in two other multidrug-resistant cell lines (SW948-R-300 and Lo Vo-R-100) was inves tigated by confocal microscopy and immunoelectron microscopy, by using a panel of monoclonal antibodies directed against this protein, Highe r levels of P-glycoprotein expression, not only in the plasma membrane and inside tbe cytoplasm, but also in the nucleus, were found in U-2 OS/DX(580) and in Lo Vo-R-100 multidrug-resistant cells compared to th eir corresponding sensitive cells, SW948-R-300 cells, featuring increa sed amounts of MDR1 mRNA but lacking P-glycoprotein expression at the cell surface, showed a higher P-glycoprotein immunolabeling only in th e nucleus and in the cytoplasm, The localization of P-glycoprotein in the nucleus of multidrug-resistant cells was confirmed also by studies on isolated nuclei and nuclear matrices, and by Western blot analysis on total cell and isolated nuclear extracts. These findings, suggesti ng the possible involvement of nuclear P-glycoprotein in the regulatio n of subcellular doxorubicin distribution in multidrug-resistant cells , open new insights on the mechanisms of P-glyeoprotein-mediated resis tance to anticancer drugs.