Flow cytometric assay of modulation of P-glycoprotein function in whole blood by the multidrug resistance inhibitor GG918

Galactose-1-phosphate uridyl transferase (GALT) deficiency causes classical galactosemia in humans. Mice deficient in this enzyme were created by gene targeting. GALT-deficient mice develop biochemical features similar to those seen in humans with GALT deficiency, but fail to develop the pattern of acute toxicity seen in newborns with classical galactosemia. This study suggests that alternative routes of galactose metabolism are important in the pathogenesis of galactosemia.     

Enhanced sensitivity of P-glycoprotein-positive multidrug resistant tumor cells to complement-mediated lysis

The interaction of KB-V1, a multidrug resistant (MDR) variant of the KB-3-1 human oral carcinoma, with human complement was investigated. KB-V1 cells were found to be more sensitive than KB-3-1 cells to complement-mediated lysis. Detailed analysis of the capacity of KB cells to activate human complement demonstrated that both C3b deposition and formation of the membrane attack complex (MAC) are higher on KB-V1 than on KB-3-1 cells. Furthermore, the MAC formed on KB-V1 cells, but not on KB-3-1 cells, was found to be resistant to trypsin treatment, i.e. more stably inserted into the plasma membrane. Immunofluorescence analysis by flow cytometry showed that KB-V1 cells express less decay-accelerating factor (DAF, CD55) than KB-3-1 cells. Two other complement regulatory proteins, membrane cofactor protein (MCP, CD46) and CD59 are expressed to a similar extent on both KB-V1 and KB-3-1 cells. Treatment of KB-V1 cells with neutralizing anti-P-glycoprotein (P-gp) monoclonal antibodies reduced their sensitivity to complement. In addition, KB-V1 revertants which cease to express P-gp become more resistant to complement. These results indicate that multiple factors, such as reduced expression of DAF, enhanced deposition of C3b and increased binding and stability of the MAC may contribute to the increased complement sensitivity of KB-V1 cells. It is suggested that P-gp is responsible for the complement-sensitive phenotype of KB-V1 cells.     

Metabolism of 6-mercaptopurine in human leukemic cells

The PRPP concentrations, PRPP formation, and phosphorylation of 6-mercaptopurine in leukocyte suspensions and homogenates prepared from leukemic patients were studied...     

Reduced drug accumulation and multidrug resistance in human breast cancer cells without associated P-glycoprotein or MRP overexpression

MCF-7 human breast cancer cells selected in Adriamycin in the presence of verapamil developed a multidrug resistant phenotype, which was characterized by as much as 100,000-fold resistance to mitoxantrone, 667-fold resistance to daunorubicin, and 600-fold resistance to doxorubicin. Immunoblot and PCR analyses demonstrated no increase in MDR-1 or MRP expression in resistant cells, relative to parental cells. This phenotype is similar to one previously described in mitoxantrone-selected cells. The cells, designated MCF-7 AdVp, displayed a slower growth rate without alteration in topoisomerase II alpha level or activity. Increased efflux and reduced accumulation of daunomycin and rhodamine were observed when compared to parental cells. Depletion of ATP resulted in complete abrogation of efflux of both daunomycin and rhodamine. No apparent alterations in subcellular daunorubicin distribution were observed by confocal microscopy. No differences were noted in intracellular pH. Molecular cloning studies using DNA differential display identified increased expression of the alpha subunit of the amiloride-sensitive sodium channel in resistant cells. Quantitative PCR studies demonstrated an eightfold overexpression of the alpha subunit of the Na+ channel in the resistant subline. This channel may be linked to the mechanism of drug resistance in the AdVp cells. The results presented here support the hypothesis that a novel energy-dependent protein is responsible for the efflux in the AdVp cells. Further identification awaits molecular cloning studies.     

Apoptosis and resistance to daunorubicin in human leukemic cells

We compared test methods based on specific mechanisms of daunorubicin (DNR) resistance to more global procedures. Assessment of P-glycoprotein (P-gp) expression and function by means of immunocytochemistry, DNR accumulation, and modulation of resistance and accumulation by the P-gp inhibitor cyclosporin A (CsA) were selected as parameters for multidrug resistance (MDR). On the other hand, we used the MTT assay and measured apoptosis and proliferative activity (S- and G2M-phases of the cell cycle) by flow cytometry. Validation of test methods was achieved for four leukemic cell lines (HL-60, KG-1a, K562/WT, K562/ADM). This battery of tests was then applied to mononuclear cells (MNC) from 18 leukemic patients. Low proficiency of MNC to undergo apoptosis and low proliferative activity rather than P-gp-mediated MDR correlated with DNR resistance as measured by the MTT assay. Bell-shaped dose-response curves for apoptosis, however, which reflect a switch from the apoptotic to the necrotic death mode with increasing cellular damage tend to limit practicability in clinical testing, because appropriate dose range and time points need to be explored. Thus, measurement of apoptosis by flow cytometry may be less convenient than the MTT assay for determination of chemosensitivity, if clinical samples with unknown patterns of responsiveness are to be tested. Spontaneous apoptosis in untreated MNC following 24 h incubation in vitro correlated significantly with DNR sensitivity in the MTT assay. A lack of essential viability factors (eg growth factors or cytokines) in vitro which are known to prevent apoptosis may contribute to DNR sensitivity.     

Dimethyl sulfoxide-induced apoptosis in human leukemic U937 cells

The effects of two oral contraceptives, containing gestodene and either 20 micrograms or 30 micrograms ethinylestradiol, on hemostatic parameters was investigated in a six-month randomized study involving a total of 40 healthy women between the ages of 18 and 30 years. A large number of hemostatic parameters were measured, which were categorized as either pro-coagulatory, anti-coagulatory, profibrinolytic, anti-fibrinolytic or indicative of fibrin turnover. Additionally, tissue plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI-1) were measured before and after venous occlusion and delta and ratio values calculated. Pro-coagulatory factors as well as reaction products reflecting in vivo coagulatory activity (thrombin-antithrombin III complex, prothrombin fragment 1 + 2) were found to increase. Among the anti-coagulatory parameters, only protein S concentration and protein S activity decreased, most notably in the 30 micrograms EE group. There was a corresponding increase in fibrinolytic activity reflected by reaction products of in vivo fibrinolysis (plasmin-antiplasmin 2-complex, fibrin-degradation products). Measurement of t-PA and PAI-1, before and after venous occlusion, revealed that the fibrinolytic response was more pronounced in the 20 micrograms EE group. There was also an increase in the threshold of fibrinolytic inhibition (ratio PAI-1) in both groups, which was less pronounced in the 20 micrograms EE group. Apart from isolated measurements, all parameters remained within their normal ranges and values returned to baseline in the follow-up cycle. It is concluded that both preparations had a balanced effect on the hemostatic system stimulating both pro-coagulant and fibrinolytic activity. No statistically significant differences were observed between the two groups; however, there was a trend towards greater fibrinolytic capacity in the 20 micrograms EE group.     

Effect of the phosphodiesterase inhibitor Pentoxyfylline on human sperm motility

Tyrode fluid and Tyrode fluid plus Pentoxifylline were individually added to aliquots of semen samples obtained from 6 normal men and 6 infertile patients considered to have idiopathic normogonadotropic oligoasthenozoospermia. Pentoxifylline was added to final concentrations of 0.15, 0.30 and 0.60 mM. One aliquot with no addition served as control. Samples were incubated in 37 degrees C and observed by light microscopy at 30 minutes and at 1, 2 and 4 hours after obtaining the material. At observation time, semen quality was evaluated by determining the percentages of forwardly progressive spermatozoa, slowly progressive spermatozoa, "in situ" motile spermatozoa, live and non-motile spermatozoa and dead spermatozoa. Results reported included only the first and last category. Tyrode fluid did not affect significantly the motility and the duration of activity of spermatozoa. Ejaculated human spermatozoa both from normal and asthenozoospermic men added the Pentoxifylline at 0.30 and 0.60 mM showed a longer lasting activity than those of control semen and semen added only with Tyrode fluid.     

The effect of HER-2/neu overexpression on chemotherapeutic drug sensitivity in human breast and ovarian cancer cells

INTRODUCTION: Determination of the optimal electrode configuration during implantable cardioverter defibrillator (ICD) implantation remains largely an empirical process. This study investigated the feasibility of using a finite element model of the thorax to predict clinical defibrillation metrics for internal defibrillation in humans. Computed defibrillation metrics from simulations of three common electrode configurations with a monophasic waveform were compared to pooled metrics for similar electrode and waveform configurations reported in humans. METHODS AND RESULTS: A three-dimensional finite element model was constructed from CT cross-sections of a human thorax. Myocardial current density distributions for three electrode configurations (epicardial patches, right ventricular [RV] coil/superior vena cava [SVC] coil, RV coil/SVC coil/subcutaneous patch) and a truncated monophasic pulse with a 65% tilt were simulated. Assuming an inexcitability threshold of 25 mA/cm2 (10 V/cm) and a 75% critical mass criterion for successful defibrillation, defibrillation metrics (interelectrode impedance, defibrillation threshold current, voltage, and energy) were calculated for each electrode simulation. Values of these metrics were within 1 SD of sample-size weighted means for the corresponding metrics determined for similar electrode configurations and waveforms reported in human clinical studies. Simulated myocardial current density distributions suggest that variations in current distribution and uniformity partially explain differences in defibrillation energy requirements between electrode configurations. CONCLUSION: Anatomically realistic three-dimensional finite element modeling can closely simulate internal defibrillation in humans. This may prove useful for characterizing patient-specific factors that influence clinically relevant properties of current density distributions and defibrillation energy requirements of various ICD electrode configurations.     

Effect of the antitumor drug lonidamine on glucose metabolism of adriamycin-sensitive and -resistant human breast cancer cells

The efficacies of direct percutaneous transluminal coronary angioplasty (PTCA) and thrombolysis for the treatment of acute myocardial infarction were investigated in 80 patients treated within 12 hours of the onset of myocardial infarction by either PTCA (39 patients) or thrombolytic therapy (41 patients) followed by conservative care. The therapeutic approach was selected according to the treatment strategy at each of the 16 participating centers before the admission of the patients. The two treatment groups were closely matched in clinical characteristics except for the history of hypertension which occurred more in the thrombolysis group (22/39 vs 12/41, p = 0.026). The mean time before starting reperfusion therapy from the onset of symptoms was shorter in the thrombolysis group (2.3 +/- 1.5 vs 5.3 +/- 5.7 hours, p = 0.0001). Chest pain resolved more quickly in the PTCA group. Serial changes in the mean numbers of abnormal Q waves and mean values of the sum of elevated ST-segments on the electrocardiograms were similar in both groups. Serial changes of wall motion abnormality index on echocardiograms were similar in both groups. Coronary angiography after 4 weeks showed the thrombolysis group had greater residual luminal stenosis in the infarct-related artery. Left ventriculography after 4 weeks showed the PTCA group had better mean ejection fraction (68.1 +/- 11.2% vs 58.7 +/- 14.2%, p = 0.0263). Death (3/39 vs 1/41) and cardiac events (6/39 vs 6/41) after 4 weeks were similar in both groups. There was no significant difference in death and cardiac events between these two groups. However, the PTCA group had less severe residual luminal stenosis in the infarct-related artery and better left ventricular function after 4 weeks than the thrombolysis group.     

Alkylphosphocholines: Effects on human leukemic cell lines and normal bone marrow cells

The anti-leukemic activity of a series of alkylphosphocholines (APCs) was studied against a panel of human leukemic cell lines (HL-60, K-562, Reh, MOLT-4, Jurkat, Ramos and Raji). Cytotoxic efficacy was measured by the MTT cell survival assay. All cell lines were found to be sensitive, except the multipotential CML-derived K-562 cell line. Flow cytometry of HL-60 cells showed a significant decrease of cells in S phase and the formation of a sub-G fraction. DNA fragmentation typical for programmed cell death was detected by DNA gel electrophoresis in these cells but not in any of the other leukemic lines. At concentrations below the cytotoxic range, mitogenic effects were seen in HL-60 cells after 14-hr exposure. Colony formation by K-562 cells revealed an augmented clonogenicity after exposure to APC with a short alkyl chain. In contrast, cells of lymphoid origin did not undergo DNA fragmentation or show mitogenic stimulation after exposure to APC. Normal bone marrow cells were also investigated for mitogenic and genotoxic effects. No decrease was found in the number of hematopoietic progenitors in long-term bone marrow cell cultures after exposure to APC. On the contrary, a significant increase was found after short exposure. Dodecylphosphocholine, hexadecylphosphocholine (HPC) and (octadecyl-[2-(N-methylpiperidino)-ethyl]phosphate exhibited a mild clastogenicity at equimolar high doses on murine bone marrow cells in vivo, which is unusual for the majority of classical DNA-interacting anti-cancer drugs. In conclusion, APCs are agents with a broad spectrum of in vitro anti-leukemic effects, which lack hematological toxicity.     

Radioligand-binding assay employing P-glycoprotein-overexpressing cells: testing drug affinities to the secretory intestinal multidrug transporter

We report a perceptual phenomenon that originates from a nonlinear operation during the visual process, and we use these observations to study the functional organization of the responsible nonlinearity; the regulation of visual sensitivity to light. When the contrast of a high frequency grating was modulated while its spatial and temporal average luminance was kept constant, observers saw brightness changes or desaturation in the field. If the contrast was modulated periodically between zero and a peak value, observers saw vivid flicker (contrast-modulation flicker), and this flicker could be seen even when the grating was too fine to be visually resolved as a pattern. This uniform-field flicker can be nulled by a modulation of space-average luminance at the contrast-modulation frequency, with appropriate phase and modulation depth. Contrast-modulation flicker is still measurable with gratings at 100 cycles/deg. The dynamics of contrast-modulation flicker suggest that it results from an early sensitivity-controlling mechanism, acting very rapidly (within about 20 msec). Its dependence on stimulus spatial frequency implies a strictly local luminance nonlinearity, one that either resides within individual photoreceptors or operates on signals from individual receptors.     

A leukotriene receptor antagonist, ONO-1078, modulates drug sensitivity and leukotriene C4 efflux in lung cancer cells expressing multidrug resistance protein

In this preliminary observation, a group of seven mentally and physically handicapped persons of chronological ages ranging from 15.4 yr. to 26.8 yr. experienced 15 sec. of physical rocking. For the further analysis, the poststimulus periods were classified into either those when the subjects' spontaneous head, mouth, and body movements had increased from the prestimulus period or those decreased. The median heart rates recorded in the poststimulus period were not significantly different from those in the prestimulus period on trials on which there was an observable increase in the rates of spontaneous head, mouth, and body movements; however, the median heart rates decreased during those trials on which a decrease in the rates of the movements occurred. Since it is said that rocking heightens arousal of persons with mental and physical handicaps, it is suggested that spontaneously emitted, aimless head, mouth, and body movements attributed to low arousal were reduced by heightened arousal rather than by a decline in participants' activities.     

c-Cbl tyrosine phosphorylation and subcellular localization in human primary leukemic cells

Several studies indicate that a number of signal-transducing molecules involved in the proliferation, differentiation, and functional activation of normal hemopoietic cells may be constitutively activated in primary leukemic cells and play a role in the outcome or in the progression of these neoplastic disorders. In this study we show that the product of the proto-oncogene c-Cbl, whose function is still unknown, is constitutively tyrosine phosphorylated not only in cells from chronic myelogenous leukemias (CMLs) in the blast phase, but also in cells from acute myeloblastic leukemias (AMLs), Ph-negative acute T-lymphoblastic leukemias (T-ALLs), and Ph-negative pre-B lymphoblastic leukemias (pre-B ALL). Moreover, in acute leukemia cells, c-Cbl was not stably complexed with the tyrosine-phosphorylated adaptor protein CrkL. The analysis of Grb2/c-Cbl interaction demonstrated that, in both acute leukemia and CML blasts, c-Cbl was stably complexed with the N-terminal Src homology (SH) 3 domain of Grb2 and, in blasts from ALL patients, with the Grb2 SH2 domain. The analysis of c-Cbl subcellular distribution showed that in all cases of leukemia tested, as well as in growth factor-stimulated M-07e cells, c-Cbl was present in the cytosolic, in the membrane, and in the detergent-insoluble fractions. Finally, in polymorphonuclear neutrophils (PMNs) from CML patients, c-Cbl was found stably associated with the detergent-insoluble fraction, whereas in PMNs from normal donors, it was detected only in the cytosolic fraction. Our findings that c-Cbl is constitutively tyrosine phosphorylated and associated with the detergent-insoluble fraction in AML and ALL blasts and in PMNs from CML patients suggest that this event represents a common step in the neoplastic transformation of both myeloid and lymphoid progenitor cells.     

Glucocorticoid-induced apoptosis and regulation of NF-kappaB activity in human leukemic T cells

Glucocorticoid-induced apoptosis was investigated in glucocorticoid-sensitive 6TG1.1 and resistant ICR27TK.3 human leukemic T cells. Following glucocorticoid treatment of 6TG1.1 cells, chromatin fragmentation was observed after a delay of 24 h. Fragmentation was not observed in ICR27TK.3 cells containing mutant glucocorticoid receptors (L753F) that are activation-deficient but retain the ability to repress AP-1 activity. Nor was fragmentation observed after treatment with RU38486, indicating that repression of AP-1 activity is not involved. As described in other systems, fragmentation required ongoing protein synthesis. However, inhibition of protein synthesis with cycloheximide anytime during the first 18 h of steroid treatment was as effective in blocking chromatin fragmentation as inhibition for the entire period, suggesting that synthesis of a component with a rapid turnover rate is required. Dexamethasone treatment completely blocked 12-O-tetradecanoylphorbol 13-acetate induction of nuclear factor-kappaB (NF-kappaB) activity and elicited an increase in the amount of immunoreactive IkappaB alpha in sensitive 6TG1.1 cells but not in resistant ICR27TK.3 cells. In addition, mild detergent treatment of cell extracts indicated that a substantial amount of cytoplasmic NF-kappaB is complexed with IkappaB alpha or some other inhibitory factor. These results suggest that induction of a labile inhibitory factor such as IkappaB alpha may contribute to glucocorticoid-induced apoptosis.     

A multidrug resistance transporter from human MCF-7 breast cancer cells

MCF-7/AdrVp is a multidrug-resistant human breast cancer subline that displays an ATP-dependent reduction in the intracellular accumulation of anthracycline anticancer drugs in the absence of overexpression of known multidrug resistance transporters such as P glycoprotein or the multidrug resistance protein. RNA fingerprinting led to the identification of a 2.4-kb mRNA that is overexpressed in MCF-7/AdrVp cells relative to parental MCF-7 cells. The mRNA encodes a 655-aa [corrected] member of the ATP-binding cassette superfamily of transporters that we term breast cancer resistance protein (BCRP). Enforced expression of the full-length BCRP cDNA in MCF-7 breast cancer cells confers resistance to mitoxantrone, doxorubicin, and daunorubicin, reduces daunorubicin accumulation and retention, and causes an ATP-dependent enhancement of the efflux of rhodamine 123 in the cloned transfected cells. BCRP is a xenobiotic transporter that appears to play a major role in the multidrug resistance phenotype of MCF-7/AdrVp human breast cancer cells.     

Effect of DNA topoisomerase I inhibitor on the transduction efficiency of an deno-associated virus vector in human airway epithelial cells

We tested the effects of a DNA topoisomerase inhibitor (camptothecin; CPT) on the transduction efficiency of AAV vectors in cultured human airway epithelial cells. The cells were treated with CPT for 24 hours, then exposed to AAV-CMV-LacZ for 1 hour at different multiplicities of infection (moi). Transduction efficiency of AAV vectors was assessed using X-gal staining as the percentage of LacZ-expressing cells. The transduction efficiency was approximately 1.5 to 10 fold increased by treatment with CPT prior to AAV vector exposure. However, treatment with CPT after AAV vector infection did not enhance the transduction efficiency of the vectors. These results suggest that pre-treatment with CPT increases the transduction efficiency of AAV vectors, probably by nodulating cellular function.     

Pirarubicin nuclear uptake does not correlate with its induced cell death effect during reversal of multidrug resistance by quinine in human K562 and CEM leukemic cells

A number of small and lipophilic cations are able to reverse in vitro the resistance to anthracyclines and other natural products through their interaction with P-glycoprotein or P-gp. However, some modulators do not interact with P-gp. We have demonstrated in a previous a work, using confocal laser microspectrofluorometry, that quinine does not increase nuclear anthracycline uptake in multidrug-resistant Chinese hamster ovary LR73 cells. In this case the LR73 cells were transfected with the mdr1 gene. Moreover, quinine induced in these cells an increase of mdr1 gene expression. In the present study, we investigated verapamil and quinine for their ability to increase nuclear pirarubicin uptake in multidrug-resistant K562R and CEMR human leukemic cell lines. These two cell lines resist, respectively, to doxorubicin and vinblastine and both overexpress the P-gp. Verapamil was able to restore nuclear pirarubicin in both cell lines. On the other hand, quinine was unable to significantly increase nuclear pirarubicin uptake. Both modulators were able to restore pirarubicin sensitivity in both resistant cell lines. After treatment with quinine, mdr1 gene and P-gp expression was not significantly altered as observed previously in the LR73 cells. This suggest that the effect of quinine on mdr1 gene expression is dependent on the cell line studied. These data suggest that quinine could modify the molecular environment of anthracyclines and/or its binding to a possible cytoplasmic target, and that the mechanisms by which anthracyclines induce cell death, and ways by which chemotherapy fails in multidrug-resistant leukemic cells remain complex and are related to more than one target.     

The influence of 1-beta-D-arabinofuranosylcytosine on the metabolism of phosphatidylcholine in human leukemic HL 60 and Raji cells

We report that high-dose 1-beta-D-arabinofuranosylcytosine (Ara-C) treatment leads to substantial changes of membrane lipid composition in human leukemic cell lines. HL 60 cells are at least 10- to 20-fold more sensitive to Ara-C than Raji cells. After 4 h incubation with 50 microM Ara-C, both cells show deviations in their phosphatidylcholine (PC) and triglyceride (TG) contents, starting as early as 8 h after treatment. After 24 h, the Ara-C-induced changes in lipid metabolism are accompanied by a severe loss of viability in HL 60 cells but not in Raji cells. At this time point the HL 60 cells show a 20% depletion of PC with a concomitant increase in TG of 25%, whereas in Raji cells both PC and TG are increased 20 and 22%, respectively. The addition of lysophosphatidylcholine (lysoPC) antagonizes Ara-C-induced cell death in various leukemic cell lines and primary AML blasts from patients. Since lysoPC is a direct precursor for PC and increases the PC content of the membrane, we assume that the loss of PC in the sensitive cell line HL 60 and in other cells plays a role in Ara-C-induced toxicity. Further evidence for this mechanism is presented by the observation that hexadecylphosphocholine, an inhibitor of PC synthesis shows synergistic antiproliferative effects with Ara-C. We conclude that the rapid cell lysis described during high-dose Ara-C treatment seems to be mediated by reduction of cell membrane PC content.