Anthracycline incorporation in human lymphocytes. Kinetics of uptake and nuclear concentration

Tarasiuk, Jolanta; Frézard, Frédéric; Garnier‐Suillerot, Arlette; Gattegno, Liliane

doi:10.1016/0167-4889(89)90038-4

Cited by 84 publications

(45 citation statements)

References 25 publications

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“…No quenching of cytoplasmic fluorescence has been assumed to occur (see Materials and methods; Tarasiuk et al, 1989). Recently, however, a possible concentrationdependent quenching of daunorubicin in denucleated cells has been reported (Slapak et al, 1992).…”

Section: Resultsmentioning

confidence: 99%

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Changes in subcellular doxorubicin distribution and cellular accumulation alone can largely account for doxorubicin resistance in SW-1573 lung cancer and MCF-7 breast cancer multidrug resistant tumour cells

Schuurhuis¹,

Heijningen²,

Cervantes³

et al. 1993

Br J Cancer

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Summary Doxorubicin accumulation defects in multidrug reistant tumour cells are generally small in comparison to the resistance factors. Therefore additional mechanisms must be operative. In this paper we show by a quantitative approach that doxorubicin resistance in several P-glycoprotein-positive non-small cell lung cancer and breast cancer multidrug resistant cell lines can be explained by a summation of accumulation defect and alterations in the efficacy of the drug once present in the cell. This alteration of efficacy was partly due to changes in intracellular drug localisation, characterised by decreased nuclear/cytoplasmic doxorubicin fluorescence ratios (N/C-ratios). N/C-ratios were 2.8-3.6 in sensitive cells, 0.1-0.4 in cells with high (>70-fold) levels of doxorubicin resistance and 1.2 and 1.9 in cells with low or intermediate (7.5 and 24-fold, respectively) levels of doxorubicin resistance. The change of drug efficacy was reflected by an increase in the total amount of doxorubicin present in the cell at equitoxic (IC50) concentrations. N/C ratios in highly resistant P-glycoprotein-containing cells could be increased with the resistance modifier verapamil to values of 1.3-2.7, a process that was paralleled by a decrease of the cellular doxorubicin amounts present at IC50. At the low to moderate residual levels of resistance, obtained with different concentrations of verapamil, a linear relationship between IC50 and cellular doxorubicin amounts determined at IC50 was found. This shows that at this stage of residual resistance, extra reversal by verapamil should be explained by further increase of drug efficacy rather than by increase of cellular drug accumulation. A similar relationship was found for Pglycoprotein-negative MDR cells with low levels of resistance. Since in these cells N/C ratios could not be altered, verapamil-induced decrease of IC50 must be due to increased drug efficacy by action on as yet unidentified targets. Although the IC50 of sensitive human cells cannot be reached with resistance modifiers, when using these relationships it can be shown by extrapolation that cellular and nuclear doxorubicin amounts at IC50 at complete reversal of resistance were the same as in sensitive cells. It is concluded that doxorubicin resistance factors for multidrug resistant cells can for a large part, and in the case of P-glycoprotein-containing cells probably fully, be accounted for by decreased amounts of drug at nuclear targets, which in turn is characterised by two processes only: decreased cellular accumulation and a shift in the ratio nuclear drug/cytoplasmic drug.

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Section: Resultsmentioning

confidence: 99%

“…of two independent experiments, each determined in triplo, for nuclei of both cell lines. No quenching of doxorubicin in non-nuclear cellular compartments was assumed to occur (Tarasiuk et al, 1989).…”

Section: Drug Cytotoxicitymentioning

confidence: 99%

Changes in subcellular doxorubicin distribution and cellular accumulation alone can largely account for doxorubicin resistance in SW-1573 lung cancer and MCF-7 breast cancer multidrug resistant tumour cells

Schuurhuis¹,

Heijningen²,

Cervantes³

et al. 1993

Br J Cancer

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“…P-glycoprotein, MRP1) are responsible for the active efflux of drugs out of resistant cells resulting in the decreased intracellular accumulation insufficient to inhibit resistant cell proliferation (Gottesman and Pastan, 1993;Loe et al, 1996). It was reported by several researchers that DOX enters into sensitive cells and binds especially to the nucleus (Tarasiuk et al, 1989;Tarasiuk and Garnier-Suillerot, 1992;Skladanowski and Konopa, 1994;Taatjes and Koch, 2001;Cutts et al, 2003). It has also been presented that in the case of nonactivated DOX, the kinetics of cellular uptake was comparable with the rate of the efflux by MDR exporting pumps (P-glycoprotein and MRP1).…”

Section: Discussionmentioning

confidence: 99%

“…The data obtained also evidenced that it was (they were) (a) relatively long-lived specie(s) because the increasing effect in cytotoxic activity of DOX was conserved even when the drug was added 10 min after the preincubation step with NADPH and CPR. Identification of this (these) reactive metabolite(s) as well as investigating its (their) rate of cellular accumulation, level of nuclear retention and rate of active export by MDR exporting pumps (P-glycoprotein, MRP1) Tarasiuk et al, 1989Tarasiuk et al, , 2004Tkaczyk-Gobis et al, 2001) is needed in order to clarify the role of reductive activation of DOX by cellular oxidoreductases in the mode of action of this drug in regard to resistant tumour cells. These studies are in progress in our laboratory.…”

Section: Discussionmentioning

confidence: 99%

The role of bioreductive activation of doxorubicin in cytotoxic activity against leukaemia HL60-sensitive cell line and its multidrug-resistant sublines

et al. 2005

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Clinical usefulness of doxorubicin (DOX) is limited by the occurrence of multidrug resistance (MDR) associated with the presence of membrane transporters (e.g. P-glycoprotein, MRP1) responsible for the active efflux of drugs out of resistant cells. Doxorubicin is a well-known bioreductive antitumour drug. Its ability to undergo a one-electron reduction by cellular oxidoreductases is related to the formation of an unstable semiquionone radical and followed by the production of reactive oxygen species. There is an increasing body of evidence that the activation of bioreductive drugs could result in the alkylation or crosslinking binding of DNA and lead to the significant increase in the cytotoxic activity against tumour cells. The aim of this study was to examine the role of reductive activation of DOX by the human liver NADPH cytochrome P450 reductase (CPR) in increasing its cytotoxic activity especially in regard to MDR tumour cells. It has been evidenced that, upon CPR catalysis, DOX underwent only the redox cycling (at low NADPH concentration) or a multistage chemical transformation (at high NADPH concentration). It was also found, using superoxide dismutase (SOD), that the first stage undergoing reductive activation according to the mechanism of the redox cycling had the key importance for the metabolic conversion of DOX. In the second part of this work, the ability of DOX to inhibit the growth of human promyelocytic-sensitive leukaemia HL60 cell line as well as its MDR sublines exhibiting two different phenotypes of MDR related to the overexpression of P-glycoprotein (HL60/VINC) or MRP1 (HL60/DOX) was studied in the presence of exogenously added CPR. Our assays showed that the presence of CPR catalysing only the redox cycling of DOX had no effect in increasing its cytotoxicity against sensitive and MDR tumour cells. In contrast, an important increase in cytotoxic activity of DOX after its reductive conversion by CPR was observed against HL60 as well as HL60/VINC and HL60/DOX cells.

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“…These ratios as such do not reflect the actual concentrations of doxorubicin in each compartment, since they do not take into account the quenching of doxorubicin fluorescence at several different localisations of the drug: quenching of doxorubicin fluorescence due to DNA intercalation has been estimated at 95% (Chaires et al, 1982), while cytoplasmic fluorescence may be largely unaffected (Budge & Tritton, 1985;Tarasiuk et al, 1989). We have used an independent technique to show that N/C ratio changes reflect changes in the actual compartmental amounts of doxorubicin.…”

Section: Resultsmentioning

confidence: 99%

Early multidrug resistance, defined by changes in intracellular doxorubicin distribution, independent of P-glycoprotein

Schuurhuis¹,

Broxterman²,

Lange³

et al. 1991

Br J Cancer

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Summary Resistance to multiple antitumour drugs, mostly antibiotics or alkaloids, has been associated with a cellular plasma membrane P-glycoprotein (Pgp), causing energy-dependent transport of drugs out of cells. However, in many common chemotherapy resistant human cancers there is no overexpression of Pgp, which could explain drug resistance. In order to characterise early steps in multidrug resistance we have derived a series of P-glycoprotein-positive (Pgp/+) and P-glycoprotein-negative (Pgp/-) multidrug resistant cell lines, from a human non-small cell lung cancer cell line, SW-1573, by stepwise selection with increasing concentrations of doxorubicin. These cells were exposed to doxorubicin and its fluorescence in nucleus (N) and cytoplasm (C) was quantified with laserscan microscopy and image analysis. The fluorescence N/C ratio in parent cells was 3.8 and decreased both in Pgp/+ and Pgp/-cells with increasing selection pressure to

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Anthracycline incorporation in human lymphocytes. Kinetics of uptake and nuclear concentration

Cited by 84 publications

References 25 publications

Changes in subcellular doxorubicin distribution and cellular accumulation alone can largely account for doxorubicin resistance in SW-1573 lung cancer and MCF-7 breast cancer multidrug resistant tumour cells

Changes in subcellular doxorubicin distribution and cellular accumulation alone can largely account for doxorubicin resistance in SW-1573 lung cancer and MCF-7 breast cancer multidrug resistant tumour cells

The role of bioreductive activation of doxorubicin in cytotoxic activity against leukaemia HL60-sensitive cell line and its multidrug-resistant sublines

Early multidrug resistance, defined by changes in intracellular doxorubicin distribution, independent of P-glycoprotein

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