The MDR1 P-glycoprotein (Pgp), a member of the ATP-binding cassette family of transporters, is a transmembrane ATPase efflux pump for various lipophilic compounds, including many anti-cancer drugs. mAb UIC2, reactive with the extracellular moiety of Pgp, inhibits Pgp-mediated efflux. UIC2 reactivity with Pgp was increased by the addition of several Pgp-transported compounds or ATP-depleting agents, and by mutational inactivation of both nucleotide-binding domains (NBDs) of Pgp. UIC2 binding to Pgp mutated in both NBDs was unaffected in the presence of Pgp transport substrates or in ATP-depleted cells, whereas the reactivities of the wild-type Pgp and Pgps mutated in a single NBD were increased by these treatments to the level of the double mutant. These results indicate the existence of different Pgp conformations associated with different stages of transport-associated ATP hydrolysis and suggest trapping in a transient conformation as a mechanism for antibody-mediated inhibition of Pgp.P-glycoprotein (Pgp), the product of the human MDR1 gene, acts as a broad specificity plasma membrane efflux pump for many hydrophobic compounds (1, 2) and recently was shown to function as a short chain lipid translocase (3). Pgp is a member of a superfamily of ATP-binding cassette (ABC) transporters, characterized by the presence of conserved ABC domains containing consensus nucleotide-binding domain (NBD) sequence motifs (4). ABC transporters of a subgroup that includes the MDR1 Pgp, a closely related MDR2 gene product that acts as a phospholipid translocase (5, 6), the yeast STE6 protein that transports the a pheromone (7), and the cystic fibrosis transmembrane conductance regulator (8), are characterized by a common architecture. These proteins are composed of two halves separated by a ''linker'' region; each half comprises a hydrophobic region with six predicted membrane-spanning segments and the ABC domain.Expression of the MDR1 Pgp in tumor cells is associated with a clinically important phenotype of crossresistance to many structurally diverse anti-cancer drugs, which are pumped out by Pgp. Pgp was shown to bind its transport substrates (9), an event that most probably occurs in the lipid bilayer of the plasma membrane (10), and to hydrolyze ATP (11). The ATPase activity of Pgp is strongly stimulated by the addition of Pgp transport substrates (12). The stoichiometry, temporal sequence, and structural transitions linking the binding and transport of a Pgp substrate with the binding and hydrolysis of ATP are as yet unknown.We previously have developed a mouse mAb UIC2, specific for the extracellular moiety of the human MDR1 Pgp (13). In contrast to several other mAbs that react with Pgp on the surface of intact cells, the addition of UIC2 to tissue culture media decreases the activity of Pgp toward all the tested Pgp transport substrates (13-16). The conformational epitope that is recognized by UIC2 is distinct from the epitopes of the other mAbs, because only UIC2 fails to react with a mutant Pgp that carries a d...

P-glycoprotein (Pgp), encoded by the MDRI gene, is an active eflux pump for many structurally diverse lipophilic compounds. Cellular expression of Pgp results in multidrug resistance (MDR) in vitro and is believed to be a clinically relevant mechanism for tumor resistance to chemotherapy. We have developed a mouse monoclonal antibody, UIC2, that recognizes an extracellular epitope of human Pgp. UIC2 inhibited the efflux ofPgp substrates from MDR cells and significanty increased the cytotoxicity of Pgp-transported drugs, under the conditions where no effect was detectable with other anti-Pgp antibodies. Potentiation of cytotoxicity by UIC2was observed with all the tested drugs associated with MDR (vinblastine, vincristine, colchicine, taxol, doxorubicin, etoposide, actinomycin D, puromycin, and gramicidin D) but not with any ofthe drugs to which MDR cells are not cross-resistant (methotrexate, 5-fluorouracil, cis-platinum, G418, and gentamicin). The inhibitory effect of UIC2 in vitro was as strong as that of verapamil (a widely used Pgp inhibitor) at its highest clinally achievable concentrations. Our results suggest that UIC2 or its derivatives provide an alternative or supplement to chemical agents for the reversal of MDR in clinical cancer.

Purpose:To investigate the association between parameters obtained from dynamic contrast enhanced MRI (DCE-MRI) of breast cancer using different analysis approaches, as well as their correlation with angiogenesis biomarkers (vascular endothelial growth factor and vessel density).Materials and Methods: DCE-MRI results were obtained from 105 patients with breast cancer (108 lesions). Three analysis methods were applied: 1) whole tumor analysis, 2) regional hot-spot analysis, and 3) intratumor pixel-by-pixel analysis. Early enhancement intensities and fitted pharmacokinetic parameters were studied. Paraffin blocks of 71 surgically resected specimens were analyzed by immunohistochemical staining to measure microvessel counts (with CD31) and vascular endothelial growth factor (VEGF) expression levels.Results: MRI parameters obtained from the three analysis methods showed significant correlations (P Ͻ 0.0001), but a substantial dispersion from the linear regression line was noted (r ϭ 0.72-0.97). The entire region of interest (ROI) vs. pixel population analyses had a significantly higher association compared to the entire ROI vs. hot-spot analyses. Cancer specimens with high VEGF expression had significantly higher CD31 microvessel densities than did specimens with low VEGF levels (P Ͻ 0.005). No significant association was found between MRI parameters obtained from the three analysis strategies and IHC based measurements of angiogenesis. Conclusion:A consistent analysis strategy was important in the DCE-MRI study. In this series, none of these strategies yielded results for MRI based quantitation of tumor vascularity that were associated with IHC based measurements. Therefore, different analyses could not account for the lack of association.

We have developed a safe, simple, and efficient method for boron determination by means of direct-current plasma atomic emission spectroscopy. Tissues were solubilized by using concentrated sulfuric acid and 70% hydrogen peroxide to digest the samples without the need of high temperatures and pressures. Boron cluster compounds could be measured with sensitivity, precision, and accuracy similar to those of boric acid standards. Results obtained with [(C2H5)3NH]2B12H12, Cs2B12H11SH.H2O, and C15H32B10O6 show that this analytical method is applicable to a variety of compounds with different chemical structures. A sensitivity of 0.1 ppm has been obtained with known standards alone and in a variety of tissue matrices including tumor, blood, liver, skin, and cell suspensions. The measurement of total boron by direct-current plasma atomic emission spectroscopy (DCP-AES) has been achieved with as little as 50 mg of tissue or as few as 5 x 10(7) cells. The procedure is applicable to the analysis of boron in the ppm range with a high degree of precision and accuracy.

To formally test the hypothesis that the granulocyte/macrophage colony-forming unit (GM-CFU) cells can contribute to early hematopoietic reconstitution immediately after transplant, the frequency of genetically modified GM-CFU after retroviral vector transduction was measured by a quantitative in situ polymerase chain reaction (PCR), which is specific for the multidrug resistance-1 (MDR-1) vector, and by a quantitative GM-CFU methylcellulose plating assay. The results of this analysis showed no difference between the transduction frequency in the products of two different transduction protocols: “suspension transduction” and “stromal growth factor transduction.” However, when an analysis of the frequency of cells positive for the retroviral MDR-1 vector posttransplantation was carried out, 0 of 10 patients transplanted with cells transduced by the suspension method were positive for the vector MDR-1 posttransplant, whereas 5 of 8 patients transplanted with the cells transduced by the stromal growth factor method were positive for the MDR-1 vector transcription unit by in situ or in solution PCR assay (a difference that is significant at the P = 0.0065 level by the Fisher exact test). These data suggest that only very small subsets of the GM-CFU fraction of myeloid cells, if any, contribute to the repopulation of the hematopoietic tissues that occurs following intensive systemic therapy and transplantation of autologous hematopoietic cells.

P-glycoprotein (P-gp), the product of the MDR1 (multidrug resistance) gene, is a transmembrane efflux pump for different lipophilic compounds, including many anticancer drugs and fluorescent dyes. We have previously reported that the efflux of fluorescent dyes from lymphoid cells of human bone marrow was directly correlated with the cellular P-gp content. In the present study, we show that human peripheral blood lymphocytes (PBL) also express P-gp, and that P-gp expression correlates with the efflux of fluorescent dyes from PBL. This efflux was suppressed not only by chemical inhibitors of P-gp but also by a P-gp-specific monoclonal antibody UIC2, thus providing direct evidence that it was mediated by P-gp. We have also characterized dye efflux and UIC2 reactivity in specific PBL subsets. P-gp was expressed in the majority of CD56+, CD8+, and CD20+ lymphocytes, but in less than one half of CD4+ cells. P-gp-mediated dye efflux was highly heterogeneous relative to the expression of CD56RA, CD56RO, Leu-8, and HLA-DR antigens. No significant P-gp activity was detectable in CD14+ monocytes. MDR1 expression in normal lymphocytes may be a determinant of multidrug resistance in the corresponding malignancies.

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S U M M A R YWe describe an improved immunohistochemical procedure for detecting regions of hypoxia in normal organs and tumors in mice. The method employs a primary fluorescein-conjugated mouse monoclonal antibody directed against pimonidazole protein adducts that are created in hypoxic tissues and a secondary mouse anti-fluorescein antibody that is conjugated to horseradish peroxidase. Using these reagents, we clearly visualized the regions of relative hypoxia in implanted tumors in mice as well as in normal organs such as liver and kidney. Significantly, the resulting tissue sections were remarkably free of the background staining that is characteristically observed when rodent antibodies are used to detect antigens in rodent tissues.