Article Figures & Data
Figures
- Fig. 1.
Determination of the half-life of MDR1 mRNA in tet-repressible cells. A, demonstration of the linearity of the quantitative RT-PCR assay for the probes MDR1. Cp = −3.9 log10 (copy number) + 49.08. R2 = 0.9951. B, effect of tetracycline on MDR1 transcript of HeLa P-gpon cells. MDR primers match both endogenous mRNA and plasmid mRNA. MDR-3′UTR primers match only endogenous MDR1 transcripts. The time course was performed after addition of 2 μg/ml tetracycline. Decay of MDR1 was calculated at the steady state, from 4 h to 24 h. ΔCpt − ΔCpt = 0 = −0.3071.t +0.1746 R2 = 0.9116.
- Fig. 2.
Determination of time needed to return P-gp expression on tet-repressible cells to basal levels. A, to assay P-gp function, the ability of P-gp to pump out rhodamine 123 was studied. FACS was used to measure accumulation of rhodamine 123. HeLa P-gpon cells were treated up to 4 days in 2 μg/ml tetracycline (+T). Controls used were cells grown in colchicine (colch) and cells grown in the absence of drugs (−T). B, P-gp expression was also measured in a FACS assay with MRK16 (a mAb against human P-gp). C, a Western blot was used to verify the decrease of P-gp expression observed in the FACS assay. C219, another mAb against P-gp, was used to detect P-gp on blots. As a control for loading, an antibody against GAPDH was used. Lanes 1–4 are cells treated with 2 μg/ml tetracycline for 1, 2, 3, and 4 days, respectively; bars, ±SD.
- Fig. 3.
Tet-repressible cells are MDR as determined by colony forming assays. HeLa Tet-Off, the parental cells, (•) and HeLa MDR-Off cells with different treatments were assayed for resistance to increasing amounts of (A) colchicine, (B) doxorubicin, and (C) vinblastine. HeLa P-gpcol (▴) were cultured in colchicine, HeLa P-gpoff (□) were cultured in 2 μg/ml tetracycline for the 8–10-day cloning assay, and HeLa P-gpon (♦) were cultured in the absence of all drugs 4 days before the cloning assay. HeLa P-gpoff-t (▪) cells were cultured in 2 μg/ml tetracycline before the cloning assay; for the duration of the cloning assay, tetracycline was removed; bars, ±SD.
- Fig. 4.
Membrane potential measured with oxonol. Membrane potential of P-gp-expressing, drug-selected cells, KB-V1 (Lane 2) and KB-A1 (Lane 4), tet-repressible cells, 77.1 P-gpon (Lane 6) and HeLa P-gpon (Lane 8), and the control cells not expressing P-gp, KB-3–1 (Lane 1), KB-A1rev (Lane 3), 77.1 P-gpoff (Lane 5), and HeLa P-gpoff (Lane 7). Membrane potential was measured by flow cytometry, using oxonol (150 nm) as a membrane potential sensing dye, as detailed in “Materials and Methods.” Results show typical fluorescence intensities of a series of measurements with the cells (5 × 105 cells/ml). Several measurements with cells grown separately gave similar fluorescence intensity relations and relative membrane potentials (n = 3).
- Fig. 5.
Determination of motional freedom of ESR probe 5-doxyl-SA in tet-repressible and drug-selected cells. Motional freedom, as expressed by the order parameter, S, determined with the ESR probe 5-doxyl-SA inserted into plasma membranes of cells. Experimental details, and calculation of the order parameter are described in “Materials and Methods.” KB-3-1 cells (Lane 1), KB-A1rev (Lane 5), HeLa P-gpoff (Lane 7), and 77.1 P-gpoff (Lane 9) are P-gp nonexpressing cells, whereas KB-8–5 (Lane 2), KB-C1 (Lane 3), KB-V1 (Lane 4), and KB-A1 (Lane 6) are drug-selected cells that express P-gp in increasing amounts. Tet-repressible cells expressing P-gp are HeLa P-gpon (Lane 8) and 77.1 P-gpon (Lane 10). Mean order parameters were calculated from results obtained with separately grown cell-cultures (n = 3–4). t tests indicate P < 0.05 (∗) for the KB-8–5 and KB-C1 pair (∗); KB-3-1 and KB-V1 (∗); KB-A1 and KB-A1rev (∗). There were no significant differences between HeLa and 77.1 P-gpoff versus P-gpon; bars, ±SD.
- Fig. 6.
Polarization of TMA-DPH and AOSA probes in cells. A, polarization of the TMA-DPH fluorescence probe at the surface of the plasma membrane of cells. KB-3-1 (Lane 1), KB-A1rev (Lane 3), HeLa P-gpoff (Lane 5), and 77.1 P-gpoff (Lane 7) cells are P-gp-nonexpressing cells. KB-V1 (Lane 2) and KB-A1 (Lane 4) are drug-selected, and HeLa P-gpon (Lane 6) and 77.1 P-gpon (Lane 8) are the tet-repressible P-gp-expressing cells. Mean polarizations were calculated from results obtained with separately grown cells (n = 2) and several measurements with cells from the same culture (n = 4–6). Statistical analysis indicates significant differences, P < 0.05 between KB-3-1 and KB-V-1 cells, and between KB-A1rev and KB-A1 cells. However, there is no significant difference between 77.1 P-gpon and 77.1 P-gpoff cells, and between HeLa P-gpon and HeLa P-gpoff cells. B, polarization of the plasma membranes was determined by using AOSA probe that inserts in the membranes. KB-3-1 and KB-A1rev cells (Lanes 1 and 5) are P-gp nonexpressing, and KB-8-5, KB-C1, and KB-V1 (Lanes 2, 3, and 4, respectively) are P-gp-expressing cells. Mean polarization numbers were obtained from two independently grown cell cultures, and several measurements were made with each cultured cell population (n = 3–4). Statistical evaluation indicates that there is significant difference, P < 0.05, between KB-3-1 or KB-A1rev and KB-V-1 cells. Statistical differences between other cells are not significant at the level of P < 0.05. Experimental details and calculation of polarization, P, for both A and B are described in “Materials and Methods;” bars, ±SD.
- Fig. 7.
Membrane lipid packing as expressed by fluorescence intensity of Merocyanin 540 stained cells. Experimental details are given in “Materials and Methods.” KB-3-1 (Lane 1) is a P-gp-nonexpressing parental cell, and KB-8-5, KB-C1, and KB-V1 (Lanes 2, 3, and 4, respectively) are P-gp-expressing drug-selected cells. One typical result of several, obtained with separately grown cells, is shown (n = 3); bars, ±SD.
Volume 63, Issue 12
