Table 1.

RLIP76 protein and transport activity in malignant and nonmalignant cell lines

RLIP76 proteinTransport activity (pmol/min/mg)
μg/108 cellsTotal protein (%)DoxorubicinDNP-SG
Malignant
    B16 (mouse melanoma)71 ± 60.8443 ± 341,796 ± 145
    DG-1 (human melanoma)63 ± 50.8384 ± 281,562 ± 112
    OVCAR-3 (human ovary)53 ± 30.7298 ± 231,194 ± 122
    PC-3 (human prostate)46 ± 30.6211 ± 26893 ± 66
    H358 (human lung, NSCLC)36 ± 30.6180 ± 15695 ± 56
    H1618 (human lung, SCLC)32 ± 30.596 ± 12361 ± 41
    MCF-7 (human breast)15 ± 10.236 ± 3105 ± 7
    HepG2 (human liver)17 ± 10.355 ± 5167 ± 14
Nonmalignant
    HLMVEC (human lung endothelium)19 ± 20.340 ± 5136 ± 14
    HLBEC (human lung epithelium)22 ± 20.446 ± 4150 ± 20
    HAVSM (human aorta smooth muscle)15 ± 10.340 ± 6125 ± 10
    HUVEC (human umbilical endothelial)14 ± 10.236 ± 4113 ± 12
  • NOTE: Cell lines were cultured in respective medium as described in Materials and Methods, and homogenate was prepared from 1 × 108 cells. RLIP76 was purified from total membrane fraction by DNP-SG affinity chromatography ( 28, 35), and quantified by ELISA. Purification table and SDS-PAGE of purified RLIP76 from different cells are presented in the Supplemental Data (Table A and Fig. A). Total membrane proteins were quantified by dye-binding method ( 39). For transport studies, plasma membrane fraction obtained from 2 × 107 cells was enriched for IOVs by wheat germ agglutinin affinity chromatography ( 32). Transport activity was calculated from measurements of uptake of 14C-doxorubicin (specific activity, 8.2 × 104 cpm/nmol) or 3H-DNP-SG (specific activity, 3.6 × 103 cpm/nmol) into the IOVs in the absence or presence of 4 mmol/L ATP after 10 minutes of incubation at 37°C as previously described ( 32). Each transport study was done with three replicates in three independent experiments (n = 9).