Christof Ludescher

Detection of activity of P-glycoprotein in human tumour samples using rhodamine 123

Summary Based on the fluorescent properties of the dye rhodamine 123 (Rh123), which is transported by the membrane efflux pump P‐glycoprotein (P‐gp), we developed a functional flow cytometric assay for the detection of multidrug‐resistant (MDR) cells. Using drug sensitive cell lines (KB‐3–1) and MDR mutants (KB‐8–5, KB‐C1) experimental conditions were established that enabled demonstration of significant differences in Rhl23 efflux and accumulation. Subsequently we investigated the applicability of this functional assay for the prediction of MDR in human peripheral blood and bone marrow samples. Using two‐colour flow cytometry, the leukaemic blast cells of six patients suffering from acute myeloid leukaemia (AML) were analysed. In three cases the blast cells showed a rapid and marked Rh123 efflux. In the presence of MDR inhibitors these cells retained Rh123. To determine whether the efflux of Rhl23 was associated with P‐gp expression, the leukaemic cells were stained with the monoclonal antibody MRK‐16. In addition extracted RNA was analysed by polymerase chain reaction to evaluate the expression of mdr 1 mRNA. In all three Rh123+ cases mdr 1 mRNA was detectable whereas only one AML case expressed P‐gp. In comparing Rh123 with daunorubicin, which also allows the detection of MDR cells, accumulation studies proved Rh123 to be the more sensitive drug for flow cytometric MDR screening. Additionally, two‐colour flow cytometry was much easier to perform with Rh123 than with daunorubicin.

Differential activity of P-glycoprotein in normal blood lymphocyte subsets

To better understand the phenomenon of P‐glycoprotein (P‐170) expression we investigated lymphocyte subpopulations for P‐170 function in healthy volunteers. Studies were based on three‐colour flow cytometry including the fluorescent probe rhodamine 123 (Rh123), which is transported by P‐170. Marked Rh123 efflux was detected in CD8+ T lymphocytes with CD8+/CD45RA+ T cells (naive cells) showing significantly higher P‐170 activity as compared with CD8+/CD45RA− cells (P < 0.04). Vice versa, CD8+/CD45RO+ T cells (memory cells) demonstrated less P‐170 activity than CD8+/CD45RO− cells (P < 0.04). P‐170 function was less prominent in CD4+ T cells, however, Rh123 efflux was higher in the CD4+/CD45RA+ and CD4+/CD45RO− subpopulations (P < 0.025) corresponding to the CD8+ results. Dye efflux differed significantly between activated and non‐activated CD8+ and CD4+ as well as CD8+/CD11b+ and CD8+/CD11b− T lymphocytes. Since CD16+ natural killer cells (NK) expressed the highest level of P‐170, the NK cytotoxicity against 51Cr‐labelled K562 target cells was assayed in the presence or absence of P‐170 inhibitors. NK related cytotoxicity was significantly reduced in the presence of R‐verapamil and dexnigaldipine‐HCP in a dose‐dependent manner.  The differential expression of P‐170 activity in naive and memory T cells together with the reduced NK related cytotoxicity in the presence of MDR‐modulators suggest a physiological role of P‐170 in immunological functions of these lymphocyte subsets. Consequently, the addition of MDR modulators to conventional chemotherapy as a strategy to overcome drug resistance should consider possible adverse immunosuppressive effects.

Reversal of multidrug resistance by B859-35, a metabolite of B859-35, niguldipine, verapamil and nitrendipine.

SummaryIt has been shown previously that verapamil and other calcium antagonists and calmodulin inhibitors can reverse multidrug resistance. We compared the potency of the dihydropyridine derivatives (4R)-3-[3-(4,4-diphenyl-l-piperadinyl)-propyl]-5-methyl-1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-pyridine-3,5-dicarboxylate-hydrochloride (B859-35), a metabolite of B859-35, niguldipine and (R)-nitrendipine to that of (RS)-verapamil in reversing multidrug resistance. The accumulation of the fluorescent dye rhodamine 123, which is transported by the P-glycoprotein, was determined by a flow cytometer. Multidrug-resistant human HeLa KB-8-5 and Walker rat carcinoma cells were incubated in the presence and in absence of the drugs indicated above. We found that 0.1 μM B859-35 increases the accumulation of rhodamine 123 in multidrug-resistant KB-8-5 and Walker cells more effectively than 1 μM (RS)-verapamil. In sensitive KB-3-1 cells addition of the drugs had no significant influence on the accumulation of rhodamine 123. In KB-8-5 cells, 10 nM Adriamycin caused a reduction of cell growth to 85% compared to untreated controls (=100%). If 1 μM B859-35, B859-35 metabolite, niguldipine, verapamil or (R)-nitrendipine was added to 10 nM Adriamycin, growth reduction compared with untreated controls increased to 12%, 11%, 23%, 63%, and 82% respectively. The effect of 0.1 μM B859-35 was a reduction in proliferation to 38%, that of 0.1 μM verapamil to 72%. These data illustrate that B859-35, a compound with antitumor activity in several tumors, is at least ten times more potent than racemic verapamil in reversing multidrug resistance.

Multidrug resistance in acute leukemia: a comparison of different diagnostic methods.

Accurate measurement of P-glycoprotein (P-170) expression in clinical samples still remains a controversial issue. In this study tumor cell P-170 expression was assessed in 29 patients suffering from acute leukemia (17 acute myeloid leukemia (AML) and 12 acute lymphoblastic leukemia (ALL)) using three different techniques: flow cytometry measuring rhodamine 123 (Rh123) efflux (functional level), immunocytochemistry (protein level) and RT-PCR (mRNA level). Rh123 efflux was detectable in 10/29 (34%) of all cases, in 9/17 (53%) of AML and in 1/12 (8%) of ALL samples. In AML patients a significant association of CD34 expression and P-170 activity was observed (P < 0.02). all aml patients with the fab subtype m5 were rh123 negative (P < 0.007). cytospin preparations were analyzed for staining with monoclonal antibodies jsb1 and mm4.17. eight of 16 (50%) aml and 0/9 (0%) all cases expressed the multidrug resistance (mdr) protein assessed by jsb1. with mm4.17 87% of aml and 50% of all patients were scored positive. agreement between both antibodies was found in only 13/23 (57%) samples. extracted rna from 12 patients was analyzed by rt-pcr to evaluate the expression of mdr1 and multidrug resistance-associated protein (mrp) mrna. an increased level of mdr1 mrna was detectable in 4/7 aml and 0/5 all cases. mrp expression was found in 3/7 aml and 0/5 all patients. comparison of rh123 assay and immunocytochemistry revealed a very good correlation when using moab jsb1 (P < 0.004) but not with mm4.17 (not significant (ns)). jsb1 also showed a much better association with the pcr results (P < 0.05) than mm4.17 (ns). finally, we compared the results of the functional rh123 assay and rt-pcr and observed a high correlation for rh123/mdr1 (r = 0.819, P < 0.001) but low for rh123/mrp (r = 0.562, NS). We conclude that measurement of Rh123 efflux and immunocytochemical staining of cytospin preparations with JSB1 allows the accurate monitoring of P-170 expression in acute leukemia. The simplicity of these two MDR assays suggests their use for routine MDR screening.

Decreased potency of MDR‐modulators under serum conditions determined by a functional assay

Summary. A variety of agents are capable of overcoming P‐glycoprotein‐mediated multidrug resistance (MDR) in vitro. However, the clinical potential of these compounds is often limited due to high plasma protein binding. We compared the efficacy of several MDR‐reversing compounds in serum‐free culture medium and under serum conditions by means of a functional assay. Using flow cytometry the efflux of the fluorescent dye rhodamine 123 (Rhl23) was measured from normal peripheral blood CD8+ T‐lymphocytes which express low levels of P‐glycoprotein. Inhibition of Rhl23 efflux by R‐verapamil, dexnigludipine‐HCl, cyclosporin A, SDZ PSC833 and the protein kinase C (PKC) inhibitor CGP 41251 was determined in serum‐free medium and in serum at concentrations from 0.1 to 50μmol/l. With the exception of SDZ PSC833 all MDR modulators showed an insufficient or suboptimal modulation of P‐glycoprotein under serum conditions at concentrations achievable in vivo. The highest potency under serum conditions demonstrated SDZ PSC833: even at a concentration of 0.5μxmol/1 a sufficient inhibitory effect was observed. Subsequently this approach was applied to patients suffering from B‐cell chronic lymphocytic leukaemia (B‐CLL; n= 3) and acute myeloid leukaemia (AML; n= 2) which were positive in the Rh l23 efflux assay. As for normal CD8+ T‐lymphocytes, much higher drug concentrations were required under serum conditions to effectively inhibit Rhl23 efflux from the leukaemic cells. Thus the interpretation of results of clinical‘modulator’trials should consider the decreased bioavailability of MDR‐reversing agents.

Phenotypic and Functional Lymphocyte Recovery After CD34+-Enriched Versus Non-T Cell-Depleted Autologous Peripheral Blood Stem Cell Transplantation

To determine the effect of CD34+ selection on immune recovery after high-dose chemo/radiotherapy in the setting of autologous stem cell transplantation (ASCT), we analyzed quantitative and qualitative lymphocyte reconstitution for up to 1 year post-transplantation in 27 consecutive adult patients receiving either CD34+-enriched or unmanipulated autologous stem cell (SC) grafts. Pretransplant immunological parameters were identical for both treatment groups. Total lymphocyte counts as well as CD3+ T cells provided a similar course of recovery in both cohorts, returning to baseline values within the first 3 months. There were no significant differences in the reconstitution kinetics of CD4+, CD8+, CD45RA+, and CD45RO+ T cells. CD4+ and CD45RA+ T cells between the two groups were significantly decreased within the first 6 months, returning to pretransplant baseline values by 1 year. Although within the first 3 months the majority of CD3+ cells were activated as demonstrated by expression of HLA-DR, we observed a significant loss of CD25+ T cells in both groups within the first 6 months. B cell numbers returned to baseline values within 3 months but in vivo B cell function measured by serum immunoglobulin M (IgM) and IgA levels did not recover as early as 6 months post-transplantation. T cell function measured by proliferation in response to the lectins phytohemagglutinin (PHA) and Concanavalin A (ConA) and to alloantigens in the mixed lymphocyte reaction (MLR) was significantly impaired, but tended to return to pretransplant baseline values by 1 year. Although preliminary, our results provide strong evidence that T cell depletion (TCD) by CD34+ enrichment using the CellPro device does not result in delayed phenotypic immune reconstitution after autologous peripheral blood stem cell transplantation (PB-SCT). Even in the absence of a high thymic T cell regenerative capacity in adults, T cell numbers and subset distributions were restored within the time frame studied. T and B cell function, however, remained significantly impaired for a prolonged period of time (>6 months after SCT) with a more profound defect in patients autografted with CD34+-enriched SC.

Reversal of multidrug resistance by the staurosporine derivatives CGP 41251 and CGP 42700

It has been shown previously that the staurosporine derivative CGP 41251, a specific inhibitor of protein kinase C (IC50 = 50 nM), exhibits antitumor activity and reverses mdr1‐mediated multidrug resistance. At present, the compound is evaluated as an anticancer drug in clinical phase I trials. We compared the effects of CGP 41251 with CGP 42700, another staurosporine derivative, which exhibits low protein kinase C inhibiting activity (IC50 = >100 μM). We found that in contrast to CGP 41251, CGP 42700 does not show antiproliferative activity in HeLa and KB cells in tissue culture (up to a concentration of 10 μM). We compared both compounds for their ability to reverse mdr1‐mediated resistance in KB‐C1 and in HeLa‐MDR1 cells (transfected with the mdr1 gene). CGP 42700 is able to reverse mdr1‐mediated resistance to a similar extent as CGP 41251. The intracellular accumulation of rhodamine 123 in KB‐C1 cells following pretreatment with CGP 41251 for 30 min was higher than that following treatment with CGP 42700 if determined in medium without serum. However, quantitation of rhodamine efflux in an ex vivo assay using human CD8+ cells in serum showed that CGP 42700 is more effective in inhibiting the efflux of rhodamine 123 than CGP 41251. We conclude from our results that (1) CGP 42700 is more effective in reversal of multidrug resistance in serum than CGP 41251, indicating that the compound may be useful for treatment of patients, and (2) CGP 42700 does not inhibit protein kinase C and cell proliferation and, therefore, may be less toxic and elicit less side effects in humans than other chemosensitizers. Int. J. Cancer 77:64–69, 1998.© 1998 Wiley‐Liss, Inc.

CYTOKINE PRIMING OF THE GRANULOCYTE RESPIRATORY BURST IN MYELODYSPLASTIC SYNDROMES

Increased susceptibility to infections in patients with myelodysplastic syndromes (MDS) is thought to be due to neutropenia as well as functional abnormalities of neutrophils. In the present study we examined the effect of two different stimulants (fMLP, PMA) and three cytokines (alphaTNF, G-CSF and GM-CSF), both singly and in combination on granulocyte (RB) in 25 MDS patients compared to seven healthy controls. Single fMLP and PMA-stimulation showed similar results for both groups. Preincubation with cytokines enhanced fMLP-stimulated RB in most MDS patients and controls, but in patients to a significantly lesser extent when compared to the control group (p < or = 0,05). Combinations of alphaTNF + GM-CSF and alphaTNF + G-CSF were highly synergistic in priming fMLP-stimulated burst in both groups. But again, as with the single cytokine priming this effect was markedly reduced in MDS patients compared to controls (p < or = 0,05). A specific priming defect for one of the cytokines or a cytokine combination could not be demonstrated. Serum alphaTNF levels were measured in 18 and neutrophil alkaline phosphatase (NAP) index in 23 patients. Results did not correlate with variations of the RB in MDS patients. We conclude that reduced alphaTNF, GM-CSF and G-CSF priming of granulocyte RB is a frequent finding in MDS and may contribute to the enhanced susceptibility to bacterial infections.

Osteosclerotic myeloma with polyneuropathy and hypocalcemia

SummaryA case is presented of a 46-year-old man with multifocal osteosclerotic bone lesions, peripheral polyneuropathy and hypocalcemia. Histologic examination of a bone marrow biopsy disclosed a multiple myeloma. Immunoelectrophoresis revealed a small M-component identified as IgG-lambda. Osteosclerotic myeloma lacking any osteolytic lesions seems to be very rare and shows several different features as compared with classical myeloma. A review of the current literature suggests that multiple myeloma is not a uniform disease but rather a group of clinical syndromes characterized by the special properties of their proliferating plasma cell clones.

Treatment of relapsed and refractory acute myelogenous leukaemia with aclacinomycin A (ACA) and etoposide (VP-16)

Ten patients with refractory and relapsed acute myelogenous leukaemia (AML) and one patient with CML in blast crisis were treated with aclacinomycin A (ACA, 60 mg/m2/day for 5 days) and etoposide (VP-16, 100 mg/m2/day for 5 days) and analysed retrospectively. Of 11 patients, seven (63%) achieved an objective response including four CRs (36%). Most impressively, two patients experienced consecutive CRs (second, third, and fourth) following relapses. Only two patients (18%) were primary resistant with persisting leukaemia. In conclusion, the combination of ACA and VP-16 is an active regimen in refractory and relapsed AML with a toxicity comparable with other salvage regimens.