CORRELATION BETWEEN FUNCTIONAL AND MOLECULAR ANALYSIS OF MDR1 P-GLYCOPROTEIN IN HUMAN SOLID-TUMOR XENOGRAFTS

The contribution of P-glycoprotein (Pgp) to multidrug resistance in hu man solid tumors is generally estimated from bulk mRNA measurements or immunohistochemistry, while direct measurement of the effect of Pgp o n intracellular drug concentrations has not been pursued. We investiga ted the feasibility and sensitivity of a method for probing Pgp-mediat ed drug transport in cells isolated from solid tumors, using xenograft models. Human tumor xenografts (XG) were grown by s.c. injection of P gp-expressing cell lines 2780(AD), BRO/mdr1 and KB8-5. Tumor uptake of doxorubicin (DOX) after administration of DOX to the mice was determi ned. XG from untreated mice were enzymatically dissociated. The effect of the Pgp modulator bepridil on steady-state cellular daunorubicin ( DNR) and vincristine (VCR) accumulation and chemosensitivity of these XG cells was compared with its effects in the cell lines (CL). mdr1 mR NA and Pgp (by now cytometry) were measured. Also, the dependence on i ntracellular ATP concentration, [ATP](i), of the modulator effect was determined in intact KB8-5 cells. The results showed that i.v. adminis tration of DOX to the mice led to lower DOX levels in the Pgp-expressi ng XG than in the ''sensitive'' XG, suggesting the presence of an in v ivo functional Pgp in these XG tumor models. Dissociated, viable XG ce lls appeared to have ATP levels sufficient to sustain Pgp-ATPase-coupl ed drug transport. This was inferred from experiments using KB8-5 CL, which showed half-maximal inhibition of DNR transport at an [ATP](i) o f 1 to 2 mM. The effect of bepridil on DNR and VCR accumulation and ch emosensitivity in the XG cells was in accordance with the XG expressio n of mdr1/Pgp. In KB8-5 XG cells, Pgp function was hardly detectable, in accordance with decreased mdr1/Pgp expression in vivo. In conclusio n, Pgp activity can be determined in freshly dissociated XG human tumo r cells. The results obtained with the more necrotic KB8-5 XG may repr esent some of the interpretation problems arising when low levels of P gp expression occur within a heterogeneous cell population, such as ma y be expected in clinical human tumors. Also our results indicate that Pgp activity may be impaired in vivo at [ATP](i) below 2 mM, which ar e realistic values for human solid tumors. (C) 1995 Wiley-Liss, Inc.