Is rhodamine 123 an appropriate fluorescent probe to assess P-glycoprotein mediated multidrug resistance in vinblastine-resistant CHO cells?

Cellular drug resistance, which involves several mechanisms such as P-glycoprotein (P-gp) overexpression, kinetic and metabolic quiescence, or the increase in the intracellular levels of glutathione, limits the effectiveness of cancer treatment. It has been reported that functional assessment of the cationic dye rhodamine 123 (Rho123) efflux reveals accurately the drug-resistant phenotype. To study cellular drug resistance, we have obtained a CHO-K1 derived cell line resistant to vinblastine by means of multistep selection. This cell line (CHOVBR) displays high reactivity with a monoclonal antibody (MAb) (C219) directed against an internal domain of P-gp, and an active Rho123 efflux, as shown by parallel flow cytometric and fluorometric assays. However, under similar experimental conditions, the drug-sensitive parental cell line CHO-K1 (as well as the myeloblastic KG1 and KG1a cell lines), was also able to pump Rho123 out. These parental CHO-K1 cells had a very low reactivity against the C219 Mab, as confirmed by Western blot analysis. Both vinblastine and verapamil inhibited Rho123 efflux in CHO-K1 cells, but had no effect on CHOVBR cultures. Also, deprivation of vinblastine for one month did not affect Rho123 efflux in these cells. Our results suggest that the activity of P-gp appears to be essential, but not sufficient to confer drug resistance, and that Rho123-based functional assays of drug resistance should be evaluated for each cellular experimental model

Is Rhodamine 123 an Appropriate Fluorescent Probe to Assess P-Glycoprotein Mediated Multidrug Resistance in Vinblastine-Resistant CHO Cells?

Cellular drug resistance, which involves several mechanisms such as P‐glycoprotein (P‐gp) overexpression, kinetic and metabolic quiescence, or the increase in the intracellular levels of glutathione, limits the effectiveness of cancer treatment. It has been reported that functional assessment of the cationic dye rhodamine 123 (Rho123) efflux reveals accurately the drug‐resistant phenotype. To study cellular drug resistance, we have obtained a CHO‐K1 derived cell line resistant to vinblastine by means of multistep selection. This cell line (CHOVBR) displays high reactivity with a monoclonal antibody (MAb) (C219) directed against an internal domain of P‐gp, and an active Rho123 efflux, as shown by parallel flow cytometric and fluorometric assays. However, under similar experimental conditions, the drug‐sensitive parental cell line CHO‐K1 (as well as the myeloblastic KG1 and KG1a cell lines), was also able to pump Rho123 out. These parental CHO‐K1 cells had a very low reactivity against the C219 Mab, as confirmed by Western blot analysis. Both vinblastine and verapamil inhibited Rho123 efflux in CHO‐K1 cells, but had no effect on CHOVBR cultures. Also, deprivation of vinblastine for one month did not affect Rho123 efflux in these cells. Our results suggest that the activity of P‐gp appears to be essential, but not sufficient to confer drug resistance, and that Rho123‐based functional assays of drug resistance should be evaluated for each cellular experimental model

Is rhodamine 123 an appropriate fluorescent probe to assess P-glycoprotein mediated multidrug resistance in vinblastine-resistant CHO cells?

Cellular drug resistance, which involves several mechanisms such as P-glycoprotein (P-gp) overexpression, kinetic and metabolic quiescence, or the increase in the intracellular levels of glutathione, limits the effectiveness of cancer treatment. It has been reported that functional assessment of the cationic dye rhodamine 123 (Rho123) efflux reveals accurately the drug-resistant phenotype. To study cellular drug resistance, we have obtained a CHO-K1 derived cell line resistant to vinblastine by means of multistep selection. This cell line (CHOVBR) displays high reactivity with a monoclonal antibody (MAb) (C219) directed against an internal domain of P-gp, and an active Rho123 efflux, as shown by parallel flow cytometric and fluorometric assays. However, under similar experimental conditions, the drug-sensitive parental cell line CHO-K1 (as well as the myeloblastic KG1 and KG1a cell lines), was also able to pump Rho123 out. These parental CHO-K1 cells had a very low reactivity against the C219 Mab, as confirmed by Western blot analysis. Both vinblastine and verapamil inhibited Rho123 efflux in CHO-K1 cells, but had no effect on CHOVBR cultures. Also, deprivation of vinblastine for one month did not affect Rho123 efflux in these cells. Our results suggest that the activity of P-gp appears to be essential, but not sufficient to confer drug resistance, and that Rho123-based functional assays of drug resistance should be evaluated for each cellular experimental model

Impact of small molecules immunosuppressants on P-glycoprotein activity and T-cell function

Purpose. P-glycoprotein (Pgp) is a member of the ABC-transporter family that transports substances across cellular membranes acting as an efflux pump extruding drugs out of the cells. Pgp plays a key role on the pharmacokinetics of several dr ugs. Herein, we have studied the effects of immunosuppressants on Pgp function, assessing rhodamine-123 (Rho123) uptake and efflux in different T- cell subsets. Methods. Different immunosuppressants such as Cyclosporine (CsA), Rapamycin (Rapa) and Tacrolimus (Tac) were used to assess the in vitro effect on Pgp function of main T-cell subsets among healthy volunteers. We measured Rho123 upta ke, efflux and kinetic of extrusion in CD4 + and CD8 + subsets by flow cytometry. Antigen-specific memory T-ce ll responses were assessed by measuring T-cell proliferation and cytokine secretion using an allogeneic mixed lymphocyte reaction. Results. Rho123 uptake in groups treated with CsA and CsA+Rapa was signif icantly decreased compared to non-treated group and the other immunosupressants in both T cells subsets. Pgp activity was also reduced in CsA and CsA+Rapa compared to the other immunosupressants but it was only significant in the CsA group for CD8 + subset. Kinetic extrusion of Rho123 by Pgp in all groups was faster in CD8 + T cells. All immunosuppressants and the specific Pgp inhibitor PSC833 diminished antigen-primed T-cell proliferation, especially CD8 + T-cell subset. Conclusions. Our data indicate that small molecules immunosuppressants, especially CsA, inhibit Pgp activity and T-cell function being the CD8 + T cells more susceptible to this effect. These findings support the importance of Pgp when designing combined immunosuppressive regimens

Transgene Expression Levels Determine the Immunogenicity of Transduced Hematopoietic Grafts in Partially Myeloablated Mice

We investigated whether transgene expression levels influence the immunogenicity of transduced hematopoietic grafts upon transplantation into partially myeloablated mice. To this aim, bone marrow cells (BMCs) transduced with retroviral vectors driving green fluorescent protein (GFP) expression either at high (high-EGFP) or low levels (low-EGFP) were transplanted into congenic recipients conditioned with sublethal doses of total body irradiation (TBI) or busulfan. Virtually all recipients showed evidence of donor engraftment 4 weeks after transplantation. However, as opposed to recipients receiving low-EGFP transduced grafts, the risk of rejecting the EGFP+ cells by 30 days after transplantation was significantly higher in mice conditioned with busulfan and receiving high-EGFP transduced grafts. Anti-EGFP cellular immune responses were demonstrated in high-EGFP-treated mice conditioned with busulfan by interferon-γ (IFN-γ), enzyme-linked immunospot assay (ELISPOT), and cytotoxic T lymphocyte (CTL) assays, in contrast to that observed in mice transplanted with low-EGFP BMC. These results show for the first time that transgene expression levels can be critical for the immunogenicity of gene-modified hematopoietic grafts, especially in immunocompetent or in partially immunosuppressed recipients. These results have profound implications in vector choice and in the design of gene therapy (GT) protocols