Biomedical subjects

O Fardel

Publications and source records attributed to O Fardel.

At least 55 records · Page 3Linked to original sources

PubMed1996

A comparison of the effect of inducers on the expression of glutathione-S-transferases in the liver of the intact rat and in hepatocytes in primary culture.

Recently, we used human hepatocytes in primary culture to study the effects of inducers of glutathione-S-transferases (GSTs) in the expectation that information obtained can be used to predict the value of particular inducers for use in the chemoprevention of cancer and other toxicities. However, in vitro human studies cannot readily be confirmed by studies in vivo. This problem does not arise in experimental animals. In the current studies, the response of male rat hepatocytes in primary culture to the following inducers of GST isoenzymes has been determined: 3-methylcholanthrene (MC); phenobarbital (PB); 1,2-dithiole-3-thione and its 5-(2-pyrazinyl)-4-methyl derivative, oltipraz (OPZ), and the results have been compared with induction obtained in livers of MC- and OPZ-treated rats. Each type of inducer was found to elicit a different response. In vitro, phenobarbital increased messenger RNA (mRNA) levels of subunits 1b and 3 after 12 and 72 hours, respectively; MC had a rapid effect on GST alpha class mRNAs (bringing about increase after only 2 hours of treatment), increased subunit 7 mRNA slightly, and had no effect on mu class mRNAs; dithiolethiones induced both subunit lb and 7 mRNAs after 4 hours and, to a much lower extent, subunit 3 mRNA after 72 hours. In vivo, MC induced significantly both subunit lb and 7 mRNAs whereas OPZ increased significantly subunits lb, 3 and 7 mRNA levels, and to a lower extent those of subunit 2, after 3 days and beyond to at least 5 days of treatment. Results obtained in mRNA studies were confirmed by high-pressure liquid chromatography (HPLC) analysis of GST subunits. HPLC also showed an induction of subunit 10 at the protein level of which the mRNA was not analyzed. Our results show that rat hepatocytes in primary culture prove to be a good model for the effect of inducers on both the expression of GST mRNA and protein levels in the rat liver in vivo. The demonstration of this good correlation in the rat with respect to increases gives support for the use of human hepatocytes for predictive studies of chemoprotection in human pharmacology.

Animals↗

PubMed1996

The P-glycoprotein multidrug transporter.

1. P-glycoprotein (P-gp) is a transmembrane protein involved in ATP-dependent efflux of various structurally unrelated anticancer drugs. Its overexpression in cancer cells decreases intracellular drug concentrations and, thus, confers a multidrug resistance phenotype. 2. P-gp is encoded by MDR genes, which constitute a small gene family comprising two genes in humans and three genes in rodents. Only the MDR1 gene in humans and mdr1 and mdr3 genes in rodents have been demonstrated to be involved in drug resistance. 3. P-gp encoded by the human MDR1 gene is a phosphorylated and glycosylated protein 1289 amino acids long, and consists of 2 halves that share a high degree of similarity. 4. A wide variety of cancers have been shown to express P-gp, including solid tumors and hematological malignancies. This P-gp positivity can be evidenced at the time of diagnosis prior to chemotherapy or at relapse after treatment, and has been correlated with treatment failure and poor prognosis in several types of cancer. In addition, P-gp is also expressed by some normal tissues, such as liver and kidney. 5. P-gp expression is regulated by various factors, including xenobiotics and hormones. 6. P-gp-mediated multidrug resistance can be reversed by various unrelated compounds called chemosensitizers or reversing agents. These drugs act through inhibition of P-gp function and have entered clinical trials.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1996

Differential regulation of mdr genes in response to 2-acetylaminofluorene treatment in cultured rat and human hepatocytes.

Expression of P-glycoprotein (P-gp), the mdr gene product, was investigated in primary cultures of rat and human hepatocytes exposed to 2-acetylaminofluorene (2-AAF). Increased levels of mdr1 mRNAs were evident in 2-AAF-treated rat hepatocytes by Northern blot analysis using rat mdr gene-specific probes, while transcripts of the mdr2 and mdr3 genes were decreased and unaffected respectively. Rat hepatocytes exposed to 2-AAF were also found to accumulate doxorubicin, an anticancer drug known to be transported by P-gp, poorly, thereby demonstrating that 2-AAF-mediated mdr1 induction resulted in increased P-gp activity. In contrast to their rat counterparts, human hepatocytes obtained from 10 individuals exhibited no change in both MDR1 and MDR2 mRNA levels, as well as in doxorubicin intracellular retention, in response to 2-AAF treatment, while cytochromes P-450 CYP1A1 and CYP1A2 were induced in both human and rat hepatocyte cultures. These data provide strong evidence that regulation of expression of mdr genes in liver cells in response to carcinogens such as 2-AAF is gene- and species-specific.

2-Acetylaminofluorene↗

PubMed1996

Inhibition of P-glycoprotein activity in human leukemic cells by mifepristone.

The antiprogestatin drug mifepristone has previously been shown to potentiate anti-cancer drug activity in rodent multidrug-resistant cell lines through inhibition of P-glycoprotein (P-gp) function. In order to characterize P-gp-mifepristone interactions in human tumoral cells, we have studied the effect of the antiprogestatin agent on P-gp activity in human CD34+ leukemic cells known to display high levels of P-gp-related drug efflux. P-gp-mediated transport of the fluorescent dye rhodamine 123 occurring in the CD34+ KG1a myeloid leukemia cell line was found to be strongly inhibited by mifepristone in a dose-dependent manner. Similarly to verapamil, a well-known chemosensitizer agent, the antiprogestatin drug increased doxorubicin cytotoxicity in KG1a cells. Mifepristone, when used at a 10 microM concentration thought to be achievable in vivo without major toxicity, was also able to markedly decrease cellular rhodamine 123 efflux occurring in CD34+ blast cells isolated from six patients suffering from myeloid acute leukemias. These results thus indicate that mifepristone can strongly inhibit P-gp activity in human cells, including tumoral cells freshly isolated from patients, therefore suggesting that the clinical use of this compound may contribute to down-modulate P-gp-mediated drug resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

Rifampicin enhances anti-cancer drug accumulation and activity in multidrug-resistant cells.

Rifampicin, a semi-synthetic antibiotic used in the treatment of tuberculosis and belonging to the chemical class of rifamycins, was examined for its effect on anti-cancer drug accumulation and activity in multidrug resistant cells overexpressing P-glycoprotein (P-gp). Rifampicin was shown to strongly enhance vinblastine accumulation in both rat hepatoma RHC1 and human leukemia K562 R7 multidrug resistant cells, but had no effect in rat SDVI drug-sensitive liver cells. By contrast, two other rifamycins, rifamycins B and SV, had no or only minor effect on vinblastine accumulation in RHC1 cells. Efflux experiments revealed that rifampicin was able, like the well-known chemosensitizer agent verapamil, to decrease export of vinblastine out of resistant cells. Rifampicin, when used at a concentration close to plasma concentrations achievable in humans (25 microM), was able to increase sensitivity of RHC1 cells to both vinblastine and doxorubicin. Rifampicin was also demonstrated to inhibit P-gp radiolabeling by the photoactivable P-gp ligand azidopine, thereby suggesting that the antituberculosis compound can interfere directly with P-gp drug binding sites. These results thus indicate that rifampicin was able to down-modulate P-gp-associated resistance through inhibition of P-gp function.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

Multidrug resistance in B-cell chronic lymphocytic leukemia.

Multidrug resistance (MDR) was investigated in peripheral blood cells isolated from 40 patients with B cell chronic lymphocytic leukemia (B-CLL) and from 7 healthy volunteers, using a flow cytometric assay that detects cellular efflux of the fluorescent dye rhodamine 123 (Rh 123), which has been demonstrated to be transported from the cell by the P-glycoprotein pump. The proportion of B leukemic cells effluxing Rh 123 and thus displaying MDR was low (14 +/- 17%) in B-CLL and in only 4 cases did the contingent of B leukemic cells showing MDR represent more than 30% of the total leukemic cells. In contrast, a higher proportion of cells effluxing Rh 123 (44 +/- 13%) was demonstrated in normal B lymphocytes. No statistical correlation was found between the number of leukemic B cells displaying MDR and clinical parameters or previous treatment. These results clearly suggest that MDR activity is usually low in B-CLL.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

Multi-drug resistance (MDR) activity in acute leukemia determined by rhodamine 123 efflux assay.

We prospectively analyzed MDR functional activity by the Rh123 efflux assay in 84 de novo acute leukemias. Thirty of the 60 AML cases (50%) showed a positive dye efflux (in more than 10% of blast cells). In 19 cases, the dye efflux was superior to 30%. Twenty-four of the 30 efflux positive cases were CD34+ and could be studied in double staining. The mean percentage of effluxing CD34+ blast cells was 54%. There was a high correlation between CD34 expression and MDR activity (P < 10(-4)), MDR activity and PgP expression (P < 10(-6)). All the efflux negative samples were PgP negative. Nine efflux positive cases were PgP negative. Five of the 24 ALL were efflux positive. MDR activity did not correlate with FAB subtype (with the exception of AML3: 1/6 was efflux positive), age, white blood cell count or LDH level. Forty-seven AML patients were treated with conventional chemotherapy including cytarabine and an anthracycline. Thirty-one (66%) entered complete remission (CR). CR rate was statistically lower for efflux positive as compared to efflux negative patients, 46 vs 87% (P = 0.003), for PgP+ as compared to PgP- patients, 40 vs 78% (P = 0.01), for CD34+ as compared to CD34- patients, 45 vs 84% (P = 0.005). There was no correlation between P110 expression (32 AML cases studied) and FAB subtype, MDR status and clinical outcome. Two years survival was 20% for efflux positive patients as compared to 54% for efflux negative patients (P < 0.07), 15% for PgP+ vs 54% for PgP- patients (P < 0.04). The finding of efflux+/PgP- cases suggests the existence of other membrane efflux pumps. Rh123 efflux assay is straightforward in routine and could be included in MDR screening because of its potential interest in clinical outcome in AML.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

The antiprogestatin drug RU 486 potentiates doxorubicin cytotoxicity in multidrug resistant cells through inhibition of P-glycoprotein function.

The antiprogestatin drug RU 486 was examined for its effect on doxorubicin cellular retention and cytotoxicity in multidrug resistant cells overexpressing P-glycoprotein (P-gp). RU 486 was shown to strongly enhance intracellular accumulation of doxorubicin in both rat hepatoma RHC1 and human leukemia K562 R7 drug-resistant cells but had no action in SDVI drug-sensitive liver cells. The antiprogestatin drug when used at 10 microM, a concentration close to plasma concentrations achievable in humans, was able to hugely increase the sensitivity of RHC1 cells to doxorubicin. RU 486 appeared to prevent the P-gp-mediated doxorubicin efflux out of RHC1 cells and was demonstrated to interfere directly with P-gp drug binding sites since it blocked P-gp labelling by the photoactivable P-gp ligand azidopine. These results thus demonstrate that RU 486 can downmodulate anticancer drug resistance through inhibition of P-gp function.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

Constitutive expression of functional P-glycoprotein in rat hepatoma cells.

P-glycoprotein is a plasma-membrane glycoprotein involved in multidrug resistance. P-glycoprotein overexpression has been demonstrated to occur in tumor cells after cytotoxic drug exposure, but also in some cancers including hepatocellular carcinomas before any chemotherapeutic treatment. In order to better analyze this constitutive type of tumoral drug resistance, we have investigated P-glycoprotein expression and function in rat liver tumors induced experimentally by administration of diethylnitrosamine and in two cell clones derived from one of these tumors designated as RHC1 and RHC2. High levels of P-glycoprotein mRNAs were found in both liver tumor samples and the two hepatoma cell clones as assessed by Northern blotting; both RHC1 and RHC2 cells displayed altered liver functions commonly observed in rat hepatoma cells, particularly the decreased expression of albumin and overexpression of the fetal glutathione S-transferase 7-7. The use of specific multidrug resistance (mdr) probes revealed a major induction of the mdr1 gene in liver tumor samples while RHC1 and RHC2 cells expressed both mdr1 and mdr3 genes without displaying a major alteration in the number of mdr gene copies as assessed by Southern blotting. High amounts of P-glycoprotein were also demonstrated in RHC1 and RHC2 cells by Western blotting. These cells were strongly resistant to doxorubicin and vinblastine, two anticancer drugs transported by P-glycoprotein. Doxorubicin intracellular retention was low in RHC1 and RHC2 cells, but was strongly enhanced in the presence of verapamil, a known modulator agent of P-glycoprotein; low retention appeared to occur via a drug efflux mechanism, indicating that P-glycoprotein was fully active. These results show that rat hepatoma cells can display elevated levels of functional P-glycoprotein without any prior cytotoxic drug selection and suggest that these cells represent a useful model for analyzing P-glycoprotein regulation in intrinsically clinical drug-resistant cancers.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

Transport and metabolism of polyamines in wild and multidrug resistant human leukemia (K 562) cells.

Multidrug resistance (MDR) can be defined as the resistance of cancer cells not just to chemotherapeutic agents to which they have been exposed but also to other apparently unrelated compounds. This MDR phenotype is commonly associated with the high expression of levels of 170 kDa P-glycoprotein, encoded by MDR genes. In the present study, the uptake kinetics of polyamines and their biosynthesis were studied in wild and multidrug resistant (MDR) K 562 cells in culture. The rate (Vmax) of polyamine uptake was significantly lower in MDR cells than that in wild type cells, whereas the Km for the uptake was not significantly different in these cells, suggesting that polyamine transporter is not modified in MDR cells, though their different physiological state influences the uptake process. In a 32 h chase, the transported radioactive polyamines were gradually interconverted. [14C]putrescine was converted into [14C]spermidine following between 15 min and 32 h of culture, and into [14C]-spermine after 16 h of culture, in both the cell types; however, the levels of interconverted radioactive polyamines were always lower in MDR cells as compared with wild type cells. Similarly, internalized [14C]spermidine was converted into [14C]spermine, but not into [14C]putrescine in both the cells types. [14C]spermidine is metabolized into [14C]spermine after 4 h of culture in wild type cells, whereas in MDR cells the interconversion of [14C]spermidine into [14C]spermine is seen only after 16 h of culture. Blocking of the transmembrane drug efflux pump, expressed in the MDR cells, by preincubation in the presence of verapamil, did not influence the uptake of either of the two polyamines (putrescine and spermidine) by MDR cells. On the contrary, this kind of preincubation of wild type cells in the presence of verapamil significantly increased the uptake of these two polyamines. The levels of intracellular polyamine contents in MDR cells were always lower than those in the parental cell line. These results demonstrate that MDR cells are defective in both the uptake of polyamines and their biosynthesis as compared with wild type cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1993

Use of human hepatocyte cultures for drug metabolism studies.

Among the in vitro models developed to investigate drug metabolism isolated hepatocytes have become the most powerful model. Human hepatocytes can be prepared from whole livers and surgical wedge biopsies. When placed in culture they retain their specific drug metabolizing activities including inducible cytochrome P450 enzymes for several days. Primary human hepatocyte cultures are now increasingly used for studying drug behavior during preclinical development, e.g. drug interactions, stereoselective drug metabolism and drug metabolic profiles. As a rule there is a good in vivo/in vitro correlation in drug biotransformation activity. The main metabolites found in vivo are recovered in vitro. However, quantitative differences are frequently observed.

Animals↗

PubMed1993

Regulation by dexamethasone of P-glycoprotein expression in cultured rat hepatocytes.

We have examined P-glycoprotein (P-gp) expression and function in cultured rat hepatocytes in response to dexamethasone (DEX), which is known to modulate various liver functions. Northern blot analyses revealed high levels of P-gp mRNAs in cultured untreated liver cells in comparison to those found in freshly isolated hepatocytes, while DEX-treated hepatocytes also displayed elevated, although weaker, P-gp levels. Similarly, Western blotting analysis indicated high levels of P-gp in liver cells maintained in the absence of DEX. The use of mdr gene-specific probes allowed us to show that DEX-modulated P-gp induction in cultured hepatocytes involved mostly, if not specifically, mdr1 gene regulation. Doxorubicin P-gp-mediated efflux analyses revealed lower intracellular doxorubicin accumulation in DEX-untreated liver cells than in DEX-treated cells, thus indicating that over-expressed P-gp was functional. These data clearly show that DEX treatment strongly modulates P-gp expression in primary rat hepatocyte cultures through a specific effect on the mdr1 gene.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1993

Expression of the multidrug resistance-associated P-glycoprotein (P-170) in 59 cases of de novo acute lymphoblastic leukemia: prognostic implications.

Immunocytochemical detection of the multidrug resistance (MDR)-associated membrane protein (P-170) was performed at time of diagnosis in a series of 36 children and 23 adults with acute lymphoblastic leukemia (ALL) using two monoclonal antibodies JSB1 and C219. Immunophenotypes were obtained in all cases and karyotypes were analyzed in 37 cases. Detection with JSB1 or with C219 led to similar results in terms of positive cells and cases, but the intensity of staining was higher with JSB1. In the populations studied, the rate of first complete remission differed between MDR-positive and MDR-negative in adult patients only (56% v 93%, respectively, P = .05). Of the 16 MDR-positive patients who had presented a first complete remission, 13 (81%) relapsed, compared with 13 of 35 (37%) MDR-negative (P = .008) patients. A higher rate of relapse among MDR-positive compared with MDR-negative patients was observed in adults and in children taken separately (adults 100% v 46%; children 73% v 32%, respectively). The survival rates (Kaplan-Meier method) were significantly higher in MDR-negative compared with MDR-positive populations as a whole (P = .002) and among children (P = .05) and adults (P = .03) taken separately. Event-free survival curves followed this trend. The percentage of second complete remission was very low in the MDR-positive group (15%) compared with 38% for the MDR-negative group. These results were shown by multivariate analysis to be independent of age, immunophenotypes, and karyotypes and clearly show the importance of MDR phenotype detection in ALL.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1993

Preferential increase of glutathione S-transferase class alpha transcripts in cultured human hepatocytes by phenobarbital, 3-methylcholanthrene, and dithiolethiones.

In rodents, a diversity of compounds are able to protect against acute and chronic toxicities of various xenobiotics including carcinogens, at least in part through induction of drug-metabolizing enzymes including glutathione S-transferase (GST) enzymes. We have posed the question as to whether or not these compounds also induce GSTs in human liver. Primary human hepatocyte cultures were exposed to phenobarbital, 3-methylcholanthrene, and two dithiolethiones [1,2-dithiole-3-thione and its 5-(2-pyrazinyl)-4-methyl derivative, oltipraz], and steady-state mRNA levels of GST classes alpha, mu, and pi were determined by Northern blot analysis. After 3 daily treatments, the two dithiolethiones were the most potent inducers; phenobarbital was also effective but to a lesser extent and 3-methylcholanthrene increased GST mRNA in only 2 of the 6 samples, although it stimulated cytochrome P-450 1A2 mRNA in all cell preparations. Whatever the compound only GSTA1 and/or A2 transcripts were induced. GST M1 mRNAs were not responsive or only slightly responsive, and GST P1 mRNAs, which were mostly undetectable in control cells, were not affected by treatment with any of the four chemicals. Large individual variations were observed in the level of induction of GST A1 and/or A2 mRNAs, and no sex difference could be demonstrated. These results clearly indicate that phenobarbital, 3-methylcholanthrene, and dithiolethiones are able to markedly increase mRNA levels of GST in human hepatocytes and that the GST alpha class is preferentially involved.

Cells, Cultured↗

PubMed1993

Detection of P glycoprotein activity on normal and leukemic CD34+ cells.

Rhodamine 123 is transported by the transmembrane efflux pump P glycoprotein (Pgp). We used this fluorescent dye to study multidrug resistance (MDR) activity in normal and leukemic CD34+ cells. These immature cells had a high degree of MDR activity. Among leukemic cells, CD34+ leukemias had significantly higher MDR activity as compared to CD34- leukemias. Heterogeneous results in cell subpopulations, however, indicate that prognosis should be interpreted in the light of MDR analysis.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1993

P-glycoprotein expression in human, mouse, hamster and rat hepatocytes in primary culture.

P-Glycoprotein (P-gp), the multidrug resistance (MDR) gene product, has previously been shown to be functional and overexpressed in adult rat hepatocytes during primary culture. In the present study, we examined P-gp expression in human, mouse and hamster hepatocytes cultured in conditions similar to those used for rat liver cells. Northern blotting and doxorubicin P-gp-mediated efflux analyses revealed that liver parenchymal cells from these three species exhibited only limited increased MDR mRNA levels with no intracellular decreased drug retention, thus suggesting that the marked functional P-gp induction observed in rat hepatocytes could reflect a species-specific response of these cells to an unfamiliar environment. An 8 h exposure to cycloheximide, which strongly induced MDR mRNAs in rat liver cells, also increased, although to a far lesser extent, MDR mRNA levels in human, mouse and hamster hepatocytes, indicating that P-gp expression in normal liver parenchymal cells could be at least partly negatively regulated by a labile protein factor.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1992

Modulation of multidrug resistance gene expression in rat hepatocytes maintained under various culture conditions.

P-glycoprotein (P-gp), the multidrug resistance gene product, is overexpressed in normal adult rat hepatocytes under standard culture conditions. We have studied the modulation of P-gp expression in this in vitro model in the presence of both epidermal growth factor and pyruvate, which favor hepatocyte growth, as well as in the presence of either dimethyl sulfoxide (DMSO) or nicotinamide, which favors maintenance of differentiated functions. P-gp overexpression, estimated by northern blotting and doxorubicin-mediated drug efflux analyses, was similarly observed during culture in both standard and proliferating conditions, while it was delayed, but not inhibited, in the presence of DMSO or nicotinamide. These results suggest that the functional P-gp overexpression occurring in rat hepatocytes when exposed to an unfamiliar environment is at least partly not related to cell proliferation or the degree of cell differentiation in vitro.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1992

Overexpression of the multidrug resistance gene product in adult rat hepatocytes during primary culture.

Expression of P-glycoprotein (P-gp), the product of multidrug resistance gene(s), was investigated in primary cultures of normal adult rat hepatocytes. Levels of P-gp mRNAs determined by Northern blotting and of P-gp measured by immunoblotting increased in parallel with time in culture. As in normal liver, P-gp was found to be localized on the membrane of bile canaliculus-like structures. This increased expression of P-gp was associated with decreased intracellular retention of doxorubicin, which could be restored by compounds such as verapamil and cyclosporin; doxorubicin (and also vincristine) was more cytotoxic to early than to late cultures. As in preneoplastic and neoplastic liver, overexpression of P-gp in cultured hepatocytes was associated with differential changes in drug-metabolizing enzymes, including increased glutathione S-transferase 7-7. Functional P-gp over-expression was observed in the absence of xenobiotic exposure or cell division; it could be linked to cellular stress occurring during cell isolation and plating. Increased expression of P-gp was blocked by actinomycin D, indicating its dependence on increased transcription, while cycloheximide led to a superinduction suggesting negative regulation by a protein factor.

ATP Binding Cassette Transporter, Subfamily B, Mem↗