The in vivo effect of the administration of resistance-modulating agents on rhodamine 123 distribution in mice thymus and lymph nodes.

P-glycoprotein (Pgp) has been widely associated with the multidrug resistance phenotype. Nevertheless, this protein has been detected in many normal tissues and cells, including liver, kidney, endothelial cells that constitute the hematological barrier of the brain and testes, and cells from the immune system. Many in vitro models have been used to study drugs that modulate Pgp activity and the multidrug resistance phenomenon. In the present work, we investigate the in vivo effects of resistance-modulating agents on lymphoid organs. Rhodamine 123 (Rho123), a well-known Pgp substrate, was administered to mice, and the fluorescence level in thymus and lymph node cells measured. The fluorescence level on these organs showed a dose-dependent response. Cyclosporin A (CSA), Verapamil (VP) and Trifluoperazine (TFP), three resistance-modulating agents, were administered to mice 1 h prior to 1 mg/kg Rho123 administration. Surprisingly, VP (10 mg/kg) and TFP (750 microg/kg) did not modulate Rho123 retention by thymus and lymph node cells. CSA (50 mg/kg) was the only drug that increased the fluorescence level in both organs. These results point out to the need of a wider study on the in vivo effects of resistance-modulating agents in different organs and systems.

Comparison between anthracyclines and rhodamine-123 accumulation in chronic lymphoid leukemia: effect of cyclosporin A and verapamil.

Multidrug resistance in leukemic cells is associated with decreased drug accumulation. A resistant cell line and cells from 11 patients with chronic lymphoid leukemia B were used for the evaluation of intracellular accumulation of daunorubicin (DNR), idarubicin (IDA), epirubicin (EPI) and rhodamine-123 (Rh-123). Cyclosporin A (CSA) and verapamil were used to test their modulatory effects on anthracyclines and the fluorescent dye. In leukemic samples there was a tendency for a lower accumulation index in samples tested with Rh-123 as compared to anthracyclines. IDA was a poorer substrate to P-glycoprotein (Pgp) than two of its analogues, e.g. DNR and EPI. A good correlation (80%) was found between Rh-123 accumulation and Pgp expression by phosphatase-anti-alkaline phosphatase. A strict correlation (100%) was found between modulation by CSA of Rh-123 accumulation and immunoreactivity to Pgp. Two discordant results were seen suggesting that other mechanisms of resistance could be present. The Rh-123 accumulation test seems to give a better indication than anthracyclines, however, it is not selective and may allow the detection of other drug-transport pumps.

Ouabain effects on activated lymphocytes: augmentation of CD25 expression on TPA-stimulated cells and of CD69 on PHA-and TPA-stimulated cells.

Ouabain (OUA) was capable of inhibiting peripheral blood lymphocyte (PBL) proliferation induced by phyothaemagglutinin (PHA) of phorbol ester (TPA), as measured by thymidine incorporation or cell cycle analysis. In this latter case it was possible to detect a block in the progression from G1 to S phase. This inhibition could not be reversed by interleukin (IL)-2 and was not due to an effect on CD 25 expression, as this molecule was only reduced in PHA cultures treated with OUA. Conversely, cultures activated by TPA and OUA showed an increased expression of CD25. The activation antigen CD69 was increased in both situation, suggesting that despite the absence of proliferative response the cells were being activated. The possibility that these cells were being deviated to the activation pathway leading to apoptosis is now under investigation. This study also suggested that CD25 induction may occur via different pathways, and that the selective effect of OUA for PHA-activated cells may become a useful tool for the understanding of the process.

Phenotypic analysis of PBMCs from uterine cervix cancer patients with high natural killer activity.

Phenotypic analysis of peripheral blood mononuclear cells from uterine cervix cancer patients, with increased natural killer cell activity, treated with radiation therapy was carried out. An increase in the percentage of CD56+ cells was observed in 5 out of 7 patients. When the expression of CD69, a phenotypic marker of cellular activation, was analyzed in 6 patients, an increase was observed in 4 of them. No direct correlation between cytolytic activity and the levels of CD56+ or CD69+ cells were observed. After 72 hr, an increased expression of CD56 was observed in 3 patients and a similar picture was seen at the same time following activation with IL-2 or IFN.

Sensitivity of vincristine-sensitive K562 and vincristine-resistant K562-Lucena 1 cells to anthracyclines and reversal of multidrug resistance.

The phenomenon of multidrug resistance (MDR), that involves the efflux pump P-glycoprotein, can be reversed by a number of substances known as MDR modulators or reversing agents. In the present study we investigated the action of three anthracyclines, mitoxantrone and vincristine on short-term (72 h) cultures using 2 methods ([3H] incorporation and MTT (3-[4,5-dimethylthiasol-2-yl]-2,5-diphenyltetrazolium bromide)), on 2 cell lines: K562, a human erythroleukemia, and a vincristine-resistant subline K562-Lucena 1. Using the same culture methods plus flow cytometry analysis, the reversing potentials of cyclosporin A and verapamil were studied in both cell lines. There were differences in the sensitivity and resistance profiles of the two lines to the various drugs but daunorubicin (5 micrograms/ml) and idarubicin (0.035 micrograms/ml) were the most effective when each was used in high concentration. Cyclosporine at 200 ng/ml and verapamil at 5 micrograms/ml reversed MDR in the resistant line, and had a synergistic action with chemotherapeutic agents on the sensitive line. Again differences were demonstrable between combinations of the various drugs and reversal was only clearly shown with the method measuring cell proliferation ([3H] incorporation) but not by the method measuring metabolic activity (MTT). The efflux of rhodamine-123 mimics the functional activity of the pump and cyclosporine was a better reversing agent by this criteria. These data show that the results obtained in in vitro studies attempting to identify treatments for different types of leukemias depend to a large extent on the methods used to measure cell response.

Trifluoperazine reduces the expression of CD69 in phytohemagglutinin-activated lymphocytes.

Trifluoperazine (TFP) is a phenothiazine capable of inhibiting lymphocyte proliferation as well as natural killer cells (NK) and lymphokine-activated killer cells (LAK) cytotoxic activity. CD69 is a surface molecule induced by various mechanisms of cellular activation. In the present work the modulation of CD69 expression by TFP was investigated on PHA-stimulated peripheral blood mononuclear cells and compared to that of CD25 (IL-2 receptor) expression. Determination of surface molecules was performed in an indirect immunofluorescence assay using anti-CD69 or anti-CD25 monoclonal antibodies, and analyzed by flow cytometry. The time course of the expression of these two molecules differed: CD69 expression was already declining at 48 h, whereas CD25 was still increasing at 72 h after stimulation. TFP (10 microM) reduced CD69 expression by 71.8% at 24 h, 68.4% at 48 h and 24% at 72 h following activation. In contrast, the same dose of TFP did not significantly affect CD25 expression at 24 h but showed an inhibitory effect at later times. These results suggest that different activation pathways are involved in the expression of CD25 and CD69.

Sensitivity of vincristine-sensitive K562 and vincristine-resistant K562-Lucena 1 cells to anthracyclines and reversal of multidrug resistance

The phenomenon of multidrug resistance (MDR), that involves the efflux pump P-glycoprotein, can be reversed by a number of substances known as MDR modulators or reversing agents. In the present study we investigated the action of three anthracyclines, mitoxantrone and vincristine on short-term (72 h) cultures using 2 methods ([3H] incorporation and MTT (3-[4,5-dimethylthiasol-2-yl]-2,5-diphenyltetrazolium bromide)), on 2 cell lines: K562, a human erythroleukemia, and a vincristine-resistant subline K562-Lucena 1. Using the same culture methods plus flow cytometry analysis, the reversing potentials of cyclospotin A and verapamil were studied in both cell lines. There were differences in the sensitivity and resistance profiles of the two lines to the various drugs but daunorubicin (5 mug/ml) and idarubicin (0.035 mug/ml) were the most effective when each was used in high concentration. Cyclosporine at 200 mug/ml and verapamil at 5 mug/ml reversed MDR in the resistant line, and had a synergistic action with chemotherapeutic agents on the sensitive line. Again differences were demonstrable between combinations of the various drugs and reversal was only clearly shown with the method measuring cell proliferation ([3H] incorporation) but not by the method measuring metabolic activity (MIT). The efflux of rhodamine-123 mimics the functional activity of the pump and cyclosporine was a better reversing agent by this criteria. These data show that the results obtained in in vitro studies attempting to identify treatments for different types of leukemias depend to a large extent on the methods used to measure cell response.

Trifluoperazine reduces the expression of CD69 in phytohemagglutinin-activated lymphocytes

Trifluoperazine (TFP) is a phenothiazine capable of inhibiting lymphocyte proliferation as well as natural killer cells (NK) and lymphokine-activated killer cells (LAK) cytotoxic activity. CD69 is a surface molecule induced by various mechanisms of cellular activation. In the present work the modulation of CD69 expression by TFP was investigated on PHA-stimulated peripheral blood mononuclear cells and compared to that of CD25 (IL-2 receptor) expression. Determination of surface molecules was performed in an indirect immunofluorescence assay using anti-CD69 or anti-CD25 monoclonal antibodies, and analyzed by flow cytometry. The time course of the expression of these two molecules differed: CD69 expression was already declining at 48 h, whereas CD25 was still increasing at 72 h after stimulation. TFP (10 muM) reduced CD69 expression by 71.8 per cent at 24 h, 68.4 per cent at 48 h and 24 per cent at 72 h following activation. In contrast, the same dose of TFP did not significantly affect CD25 expression at 24 h but showed an inhibitory effect at later times. These results suggest that different activation pathways are involved in the expression of CD25 and CD69.

Increase of chondroitin 4-sulfate concentration in the endochondral ossification cartilage of normal dogs.

The absolute concentrations of chondroitin 4- and 6-sulfate are compared in articular and endochondral ossification cartilage from normal dogs. In newborn dogs, the absolute concentration of chondroitin 4-sulfate decreases nearly 3-fold from the deeper endochondral ossification cartilage to the articular surface, whereas that of chondroitin 6-sulfate does not change. In cartilage from the articular surface of the epiphysis in adults, where the ossification process is complete, the concentration of chondroitin 4-sulfate is also low. These observations suggest that chondroitin 4-sulfate may be important in the ossification process. The pathogenesis of heritable disorders involving the sulfation of chondroitin sulfate is discussed in view of these findings.