Role of protein kinase C in phosphorylation of vinculin in adriamycin-resistant HL-60 leukemia cells

In response to phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA), HL-60 cells differentiate to macrophage-like cells and exhibit the ability to phosphorylate vinculin in vitro. Adriamycin-resistant HL-60 (HL-60/ADR) cells similarly demonstrate this characteristic without prior treatment with TPA. Since protein kinase C (PK-C) is a cellular TPA receptor, we have examined the role of this enzyme in the inherent ability of HL-60/ADR cells to phosphorylate vinculin. DEAE-cellulose chromatography of cell extracts revealed that HL-60/ADR cells contained 2-fold more PK-C than did the parental cell line. All PK-C activity was found in the cytosol of wild type HL-60 cells, whereas 85% of PK-C activity was cytosolic and 15% was membrane-bound in HL-60/ADR cells. After a 2-day treatment with 10 nM TPA, PK-C activity was reduced 80-90% in both cell lines regardless of its intracellular distribution. Immunoblotting of cell extracts from HL-60/ADR cells or HL-60 cells following treatment with TPA revealed increased levels of a 52-kDa species of similar mass to M-kinase. Coincident with these changes after TPA treatment was a reduction in Ca2+ and phospholipid-independent phosphorylation of vinculin in vitro in extracts from HL-60/ADR cells, whereas HL-60 cells exhibited an elevation of this phosphoprotein. The phosphorylation of vinculin in TPA-treated HL-60 cells or untreated HL-60/ADR cells was blocked by antibodies to protein kinase C. These results suggest that it is not the absolute level of protein kinase C but rather the proteolytic activation of PK-C to a Ca2+ and phospholipid-independent form which is associated with the utilization of vinculin as an endogenous substrate.     

Cell cycle dependence of drug initiated apoptosis in HL-60 cells

The extent- and cell cycle specificity of apoptotic cell death were studied in human leukemia HL-60 cells treated with 1.25-20 mu M etoposide, 0.125-2.0 mu g/ml dactinomycin, 12.5-200 mu M 3-deazaadenosine (c(3)Ado) and 10-50 Gy gamma-irradiation. Flow cytometry was used to measure the fraction of apoptotic cells and determining their cell cycle position. With all agents the extent of apoptotic cell death showed a clear dose- and time-dependency. Specific apoptosis of S-phase cells was found in cultures treated with 1.25-2.5 mu M etoposide. At 5-20 mu M etoposide, 0.125-2.0 mu g/ml dactinomycin and 50-200 mu M c(3)Ado cells from all cell cycle phases responded by apoptosis. Exposure of the cells to 10 Gy gamma-irradiation resulted in apoptosis of G(2)+M-phase cells. At 50 Gy cells from the S- and G(0)/G(1)-phases also entered apoptosis. Our findings indicate that the cell cycle specificity of an anti-cancer agent initiated apoptotic response depends upon the drug/irradiation dose and exposure time used. This demonstrates that the resistance of subpopulations of cells to apoptosis is relative.     

Isolation and characterization of HL-60 cell variants with different potentials for spontaneous differentiation

Although HL-60 cells, an in vitro established cell line derived from a patient with acute promyelocytic leukemia, are blocked at the promyelocytic stage of myeloid differentiation, certain chemicals can induce the cells to undergo terminal differentiation into either granulocytes or macrophages. Moreover, a small fraction of the cell population undergoes differentiation spontaneously without the addition of any inducing agent. In this paper it is demonstrated that this cell line is heterogeneous with respect to the ability of the cells to differentiate spontaneously: some clones (SD+) have a higher tendency to do so than others (SD-). In semi-solid medium, SD+ cells developed diffused colonies containing mature monocytes and macrophages, whereas SD- cells developed compact colonies of promyelocytes. Based on these morphological differences the various clones were isolated and analysed. Although only a small fraction of the population actually became differentiated at any particular time, practically all the cells in the SD+ clones had the potential to differentiate spontaneously. The clones also differ in their response to differentiation inducers; whereas some agents induced complete differentiation in both types of clones, others (e.g. actinomycin C and cytosine arabinoside) induced only SD+ clones, suggesting that differentiation induced by the latter agents is related to the ability of the cells to differentiate spontaneously. Thus the potential of leukemic cells to undergo spontaneous differentiation may be an important factor when considering differentiation-inducing therapy for leukemic patients.     

Potentiation of adriamycin accumulation and effectiveness in adriamycin-resistant cells by aclacinomycin A

Variants of Friend leukemia cells (FLC) selected for resistance to either adriamycin (ADM), daunorubicin (DNR) or aclacinomycin A (ACM) by step-wise exposure to each drug, were found to be cross-resistant to ADM and DNR but not to ACM. In addition, an epithelial cell line isolated from normal monkey kidney (CV-1) was found to be intrinsically resistant to ADM and DNR but not to ACM. In contrast, a human breast carcinoma cell line (MCF-7) was found to be sensitive to all three compounds. In these latter cell lines as well as in the FLC variants, lowered intracellular amounts of ADM and DNR correlated with resistance, but ACM levels were the same in sensitive and resistant cells. When cells with either acquired or intrinsic resistance were treated with ACM in combination with ADM or DNR, significant increases in the intracellular amounts of these latter compounds were found. Increased drug accumulation in resistant cells treated this way was accompanied by increased cytotoxicity. When resistant cells were exposed to ACM in combination with other anthracyclines, similar results were obtained. In comparison, these phenomena were not observed when either one of the sensitive cell types (parental FLC and MCF-7) were treated similarly. Since ADM and DNR resistant cells are sensitive to ACM and their resistance circumvented by ACM, this drug may have important clinical applications when used in combination with other anthracyclines.     

Detection and characterization of membrane protein changes in multidrug resistant HL-60 cells.

Antisera were prepared against fractionated membrane proteins of HL-60 cells isolated for resistance to adriamycin. Analysis of these antisera revealed that one (GSBl) was capable of detecting major protein changes in three independent isolates selected for anthracycline resistance. Thus, in studies using western blot analysis, the antiserum was found to be reactive with two proteins of 130 and 150 kDa which are present in plasma membranes of resistant but not sensitive cells. The antibody also reacted with a plasma membrane protein of 180 kDa that is present in sensitive cells but is increased in resistant isolates. Additional studies showed that P180 was greatly increased in both plasma membranes and endoplasmic reticulum in sensitive cells induced to differentiate in the presence of 12-O-tetradecanoylphorbol13-acetate (TPA). Resistant cells treated under identical conditions showed only a slight increase in the levels of P180. TPA had no effect on the levels of P150 or P130. In contrast, differentiation of HL-60 cells in the presence of dimethylsulfoxide (DMSO) resulted in the induction of P150 expression with major levels of protein contained in plasma membranes. DMSO has essentially no effect on the levels of plasma membrane P180, P150, or P130 in HL-60/Adr cells. These results therefore demonstrate a strong correlation between the development of resistance and the overexpression of proteins reactive with the GSBl antiserum. The results also show that development of anthracycline resistance in HL-60 cells results in the overexpression of P150, a protein associated with the differentiation of myeloid cells to granulocytes.     

Involvement of intracellular pH elevation in the effect of 1,25-dihydroxyvitamin D3 on HL-60 cells

Besides its effect in inhibiting proliferation and inducing differentiation of HL-60 to monocyte-like cells, 1,25-dihydroxyvitamin D3 also causes a rise in intracellular pH (pHi) from 7.17 +/- 0.02 to 7.3 +/- 0.05, as measured by the fluorescence of 2',7'-bis(carboxyethyl)-5-(6)-carboxy-fluorescein. The effect of 1,25-dihydroxyvitamin D3 on pHi is dose dependent in a parallel manner to its effect on the proliferation and differentiation processes. The elevation of pHi by 1,25-dihydroxyvitamin D3 is gradual but precedes the differentiation process. A significant increase in pHi was obtained after 16 h of incubation with the hormone and reached its maximum level after 48 h. pHi then dropped back to its initial level, which is also similar to that of peripheral normal blood monocytes. A rise in pHi was not observed during incubation of HL-60 cells with 24,25-dihydroxyvitamin D3, a metabolite which does not promote their differentiation. In contrast, other agents such as phorbol 12-myristate 13-acetate known to induce differentiation in this cell line do cause an increase in pHi. Moreover, alkalinization of HL-60 cells by NH4Cl causes induction of differentiation to monocytes. The results suggest that a rise in pHi plays a role in regulating the molecular mechanism of the inhibition of proliferation and the induction of differentiation in HL-60 cells.     

Apoptotic response of HL-60 human leukemia cells to the antitumor drug TAS-103

TAS-103 is a DNA intercalating indeno-quinoline derivative that stimulates DNA cleavage by topoisomerases. This synthetic drug has a broad spectrum of antitumor activity against many human solid tumor xenografts and is currently undergoing clinical trials. We investigated the induction of apoptosis in human promyelocytic leukemia cells treated with TAS-103. The treatment of proliferating human leukemia cells for 24 h with various concentrations of the drug induces significant variations in the mitochondrial transmembrane potential (delta(psi)mt) measured by flow cytometry using the fluorochromes 3,3-dihexyloxacarbocyanine iodide, Mitotracker Red, and tetrachloro-tetraethylbenzimidazolcarbocyanine iodide. The collapse of delta(psi)mt is accompanied by a marked decrease of the intracellular pH. Cleavage experiments with the substrates N-acetyl-Asp-Glu-Val-Asp-pNA, poly(ADP-ribose) polymerase, and pro-caspase-3 reveal unambiguously that caspase-3 is a key mediator of the apoptotic pathway induced by TAS-103. Caspase-8 is also cleaved, and the bcl-2 oncoprotein is underexpressed. Drug-induced internucleosomal DNA fragmentation and the externalization of phosphatidylserine residues in the outer leaflet of the plasma membrane were also characterized. The cell cycle perturbations produced by TAS-103 can be connected with the changes in deltapsi(mt). At low concentrations (2-25 nM), the drug induces a marked G2 arrest and concomitantly provokes an increase in the potential of mitochondrial membranes. In contrast, treatment of the HL-60 cells with higher drug concentrations (50 nM to 1 microM) triggers massive apoptosis and a collapse of deltaP(mt) that is a signature for the opening of the mitochondrial permeability transition pores. The discovery of a correlation between the G2 arrest and changes in mitochondrial membrane potential provides an important mechanistic insight into the action of TAS-103.     

DNA adduct formation by the anticancer drug ellipticine in human leukemia HL-60 and CCRF-CEM cells

Ellipticine induces formation of two DNA adducts in leukemia HL-60 and CCRF-CEM cells, identical with deoxyguanosine adducts generated by ellipticine metabolites 13-hydroxyellipticine and 12-hydroxyellipticine in vitro and in vivo. The ellipticine cytotoxicity to HL-60 (IC(50)=0.64microM) and CCRF-CEM cells (IC(50)=4.7microM) correlates with levels of DNA adducts. The different expressions of enzymes activating ellipticine in cells explain this finding. While cytochrome P450 1A1 and cyclooxygenase-1 are expressed in both cells, HL-60 cells express also high levels of another activator, myeloperoxidase. The results suggest the adduct formation as a new mode of antitumor action of ellipticine for leukemia.     

ISOLATION AND CHARACTERIZATION OF AN ADRIAMYCIN－RESISTANT SUBLINE OF THE HUMAN GASTRIC ADENOCARCINOMA CELLLINE

ＩＳＯＬＡＴＩＯＮＡＮＤＣＨＡＲＡＣＴＥＲＩＺＡＴＩＯＮＯＦＡＮＡＤＲＩＡＭＹＣＩＮ－ＲＥＳＩＳＴＡＮＴＳＵＢＬＩＮＥＯＦＴＨＥＨＵＭＡＮＧＡＳＴＲＩＣＡＤＥＮＯＣＡＲＣＩＮＯＭＡＣＥＬＬＬＩＮＥＷａｎｇＹａｎｐｉｎｇ；王艳萍；ＸｕＧａｎｇ；徐刚（Ｉ...     

P-glycoprotein expression does not change the apoptotic pathway induced by curcumin in HL-60 cells

Purpose One of the mechanisms responsible for the multidrug resistance (MDR) phenotype of cancer cells is overexpression of so-called ATP-dependent drug efflux proteins: the 170-kDa P-glycoprotein (P-gp) encoded by the MDR1 gene and the 190-kDa multidrug resistance-associated protein 1 encoded by the MRP1 gene. The purpose of the present study was to verify the hypothesis postulating that P-gp expression, apart from enabling drug efflux, confers on the cells resistance to apoptosis by inhibiting caspase-8 and caspase-3.Materials and methods Human HL-60 cells, either drug-sensitive or with the MDR phenotype caused by overexpression of P-gp (HL-60/Vinc) or MRP1 (HL-60/Adr), were treated with the natural dye curcumin at 50 M or with UVC to induce apoptosis. Symptoms of cell death were assessed by morphological observation after Hoechst staining, DNA fragmentation was measured by flow cytometry and the TUNEL method, and caspase-8 and caspase-3 activation and cytochrome c release from mitochondria were measured by Western blotting.Results Curcumin induced cell death in HL-60 cells, both sensitive and with the MDR phenotype, which could be classified as caspase-3-dependent apoptosis, together with cytochrome c release, activation of caspase-3 and oligonucleosomal DNA fragmentation. No active caspase-8 was detected. Also UVC caused caspase-3 activation in both the sensitive and the MDR HL-60 cells.Conclusions Our findings show that there was no correlation between P-gp expression and resistance to caspase-3-dependent apoptosis induced by curcumin and UVC, at least in HL-60 cells. However, we cannot exclude the possibility of parallel P-gp expression and caspase-3 inhibition in some other cell lines, as cancer cells can acquire many different apoptosis-resistance mechanisms.     

HL-60 cells isolated for resistance to vincristine are defective in 12-O-tetradecanoylphorbol-13-acetate induced differentiation and the formation of a functional AP-1 complex.

HL-60 cells isolated for resistance to vincristine are multidrug resistant and defective in the cellular accumulation of drug. Further studies demonstrate that these cells are also highly defective in 12-O-tetradecanoylphorbol-13-acetate (TPA) induced differentiation to macrophages. Analysis of this system demonstrates that certain protooncogenes which may contribute to differentiation are expressed at similar levels in sensitive and resistant cells. Thus, treatment of cells with TPA results in a reduction in the levels of c-myb and c-myc mRNA, while the expression of c-fos, c-jun, and junB is greatly enhanced. Immunoprecipitation experiments also demonstrate a TPA induced increase in the c-jun protein in both sensitive and resistant cells. Gel mobility shift assays show that TPA induces AP-1 formation in sensitive cells, whereas in parallel experiments with the HL-60/Vinc isolate, AP-1 is essentially absent. It has been found, however, that in resistant cells which have reverted to drug sensitivity, the levels of TPA inducible AP-1 is essentially identical to that of sensitive cells. Revertant and sensitive cells differentiate at similar levels in the presence of TPA. These studies therefore demonstrate that HL-60/Vinc cells are defective in the TPA induction of a functional AP-1 complex and that this may account for the inability of these cells to differentiate to macrophages. The molecular basis of the finding that AP-1 is not formed in resistant cells remains to be determined.     

Biochemical characterization of tyrosine kinase and phosphotyrosine phosphatase activities of HL-60 leukemia cells

The cellular phosphotyrosine content of the HL-60 promyelocytic leukemia markedly decreased during the induced granulocytic and monocytic maturation of these cells. This occurs in the face of major increases in tyrosine kinase and protein phosphotyrosine phosphatase activities (D. A. Frank and A. C. Sartorelli, Biochem. Biophys. Res. Commun., 140: 440-447, 1986). In the present work, these two activities were characterized in the particulate fraction of HL-60 cells, since both enzymes are membrane bound. The tyrosine kinase activity utilized ATP as a phosphate donor, although GTP and other nucleotides were competitive with ATP. The enzyme was temperature sensitive, had a pH optimum of 6.5, and required Mg2+ or Mn2+ for activity, with additional stimulation of activity being produced by Zn2+. Agents such as epidermal growth factor and insulin, which stimulate other tyrosine kinase enzymes, were without effect on the tyrosine kinase activity of HL-60 cells. Enzyme activity was stimulated, however, by non-ionic detergents and was inhibited by quercetin. The protein phosphotyrosine phosphatase activity was paralleled by that of p-nitrophenyl phosphatase, was inhibited by VO3-4, Zn2+ and F-, and was maximally active at a pH of 7 to 8. The characteristics of the tyrosine kinase and the protein phosphotyrosine phosphatase activities were distinct from those of other known proteins of these classes. Tyrosine kinase activity was predominantly located on the plasma membrane, while the protein phosphotyrosine phosphatase activity was concentrated on internal membranes. The activities of both enzymes present on the plasma membrane appeared to exist on the cytoplasmic face of this membrane. Further characterization of the activities of these enzyme systems and their contribution to the regulation of tyrosine phosphorylation would appear to be important to an understanding of the control of cellular proliferation and differentiation.     

Methyl-β-cyclodextrin in HL-60 parental and multidrug-resistant cancer cell lines: effect on the cytotoxic activity and intracellular accumulation of doxorubicin

The purpose of this work was to determine the role of methyl-β-cyclodextrin (MEBCD) in combination with doxorubicin (DOX) on the cellular proliferation of a sensitive parental and a multidrug-resistant human cancer cell line (HL-60 S and HL-60 R) and to study the effect of MEBCD on DOX intracellular accumulation. The cytotoxicity of DOX at five concentrations (50–50,000 nM ) was evaluated with or without the coadministration of four fixed noncytotoxic concentrations of MEBCD (100, 200, 500, and 1,000 μM ). Intracellular DOX concentrations were determined by a high-performance liquid chromatography (HPLC) method with fluorescence detection. MEBCD applied at 500 and 1000 μM in combination with doxorubicin (DOX) significantly potentiated the activity of DOX used alone on both sensitive and multidrug-resistant cell lines; 50% growth-inhibitory (IC₅₀) ratios (IC₅₀ MEBCD-DOX/IC₅₀ DOX ) were about 3:4 and 1.6:4 for HL-60 S and HL-60 R, respectively. Moreover, intracellular DOX accumulation, determined by HPLC during 6 h of drug exposure, was about 2–4 times higher for cells treated with MEBCD in combination with DOX than in those treated with DOX alone. Similar results were obtained using other paired MCF 7 sensitive and resistant cell lines. Correlation between these results and an MEBCD-cell membrane interaction was discussed. These initial data provide a basis for the potential therapeutic application of MEBCD in cancer therapy. Received: 13 July 1996 / Accepted: 25 March 1997     

水杨酸钠诱导HL-60细胞凋亡及其与胞内游离Ca~(2+)浓度的关系

 目的　目的研究水杨酸钠对HL 6 0细胞的促凋亡活性及其与胞内游离Ca2 + 浓度的关系。方法Annexin V结合分析检测靶细胞磷脂酰丝氨酸外化 ;琼脂糖凝胶电泳检测断裂DNA ;激光共聚焦显微镜分析细胞内游离Ca2 + 浓度变化。结果　HL 6 0细胞经 5mmol/L(IC50 )水杨酸钠分别作用 10h、16h后 ,靶细胞的磷脂酰丝氨酸外化率显著高于对照组 (0 .2 4vs 0 .0 3) ,其基因组DNA在琼脂糖凝胶电泳中呈典型的梯状带型 ;此间 ,HL 6 0细胞内游离Ca2 + 浓度进行性升高。结论　水杨酸钠触发的HL 6 0细胞凋亡与胞内游离Ca2 + 浓度升高密切相关。     

HL-60及其耐药细胞之间线粒体膜及线粒体中Bid差异研究

目的研究线粒体膜及线粒体中Bid参与肿瘤耐药的机制.方法采用紫外分光光度仪和流式细胞仪检测Ca2 诱导下HL-60及其耐药细胞HL-60/E6线粒体膜通透改变孔道开放和线粒体膜电位的变化,Western blot检测线粒体中Bid表达.结果Ca2 诱导下HL-60/E6细胞线粒体膜电位下降幅度和膜通透改变孔道开放程度明显低于HL-60细胞.药物AMD作用后线粒体中Bid表达呈时间依赖性,而且在不同时间,HL-60/E6细胞线粒体中Bid表达均明显低于HL-60细胞.结论HL-60/E6细胞耐药与Ca2 诱导下线粒体膜电位下降幅度及膜通透改变孔道开放程度的降低有关,还与线粒体上Bid表达降低有关.     

Correlation between the molecular weight and potency of polar compounds which induce the differentiation of HL-60 human promyelocytic leukemia cells

Structure-activity studies of a series of polar organic compounds, including N,N-dimethylformamide, N-methylformamide, and related ureas and acetamides, were performed with regard to their ability to promote the terminal differentiation of the human promyelocytic leukemia cell line HL-60 to granulocyte-like cells. Functional and morphological criteria were used to assess the percentage of differentiated cells which arose from their continuous incubation with different concentrations of each agent over a period of 96 h. All of the alkylformamides, alkylacetamides, and alkylureas tested were found to induce differentiation, regardless of structure. Inspection of the results showed that there was a linear relationship (r = -0.937) between the molecular weight of each compound and the logarithm of the concentration which was required to bring about the differentiation of the greatest number of cells, while viability was generally maintained at greater than 85%. Once established, this relationship was used to predict the potency of several polar solvents which were structurally unrelated to the formamides. For example, methanol, ethanol, and acetone were all inducers of differentiation with a potency predictable from their molecular weight alone. The terminal differentiation induced by all of the compounds was only accomplished by cells which were capable of replication prior to differentiation. At concentrations which prevented a single replication and brought about a fall in cell viability over 96 h, no differentiation was observed. A correlation was observed between the molecular weight of each compound and the logarithm of its concentration to bring about cytotoxicity without differentiation (r = -0.935), and the line was almost parallel to that defining the concentration required for optimal differentiation (slope values of -0.02126 and -0.02288). A poorer (r = -0.6654) correlation was found between the logarithm of the octanol-water partition coefficient and the logarithm of the concentration required for optimal differentiation, when the data for 12 of the polar organic compounds were analyzed. The results suggest that no special structural requirements are necessary for the alkylformamides, -acetamides, -ureas, and related compounds to induce the terminal differentiation of HL-60 cells to granulocyte-like cells, but that the activity of each compound could be predicted from their molecular weight. The concentrations required to induce differentiation were marginally lower than those which were cytostatic or cytotoxic, which suggested that a toxic threat to the cells was sufficient to induce differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)     

All-trans-retinoic acid increases cytosine arabinoside cytotoxicity in HL-60 human leukemia cells in spite of decreased cellular ara-CTP accumulation

Background: Accumulation of the cytosine arabinoside (ara-C) metabolite ara-C-triphosphate (ara-CTP) in leukemic blast cells is considered to be the main determinant of ara-C cytotoxicity in vitro and in vivo. Retinoids such as all-trans-retinoic acid (ATRA) have been shown to increase the sensitivity of acute myelogenous leukemic (AML) blast cells to ara-C. To investigate the mechanism of this sensitisation, the hypothesis was tested that ATRA augments cellular ara-CTP levels in human-derived myelogenous leukemia HL-60 cells.Materials and methods: The effect of ATRA and 13-cis-retinoic acid on ara-CTP accumulation and ara-C-induced apoptosis was studied. Ara-CTP levels were measured by high-performance liquid chromatography (HPLC), cytotoxicity by the tetrazolium (MTT) assay, and apoptosis by occurrence of DNA fragmentation (gel electrophoresis), cell shrinkage and DNA loss (flow cytometry).Results: Pretreatment of HL-60 cells with ATRA (0.01–1 µM) caused a significant decrease in intracellular ara-CTP levels; e.g., incubation for 72 hours with ATRA 1 µM prior to one hour ara-C 10 µM reduced ara-CTP levels to 41% ± 4% of control. Similar results were obtained after preincubation with 13-cis-retinoic acid. In spite of decreased ara-CTP levels, the cytotoxicity of the combination was supraadditive and ATRA augmented ara-C-induced apoptosis.Conclusions: At therapeutically relevant concentrations ATRA increased ara-C cytotoxicity and ara-C induced apoptosis but this augmentation is not the corollary of elevated ara-CTP levels. The feasibility of ara-C treatment optimisation via strategies other than those involving elevation of ara-CTP levels should be investigated further.     

Impairment of Fas-antigen expression in adriamycin-resistant but not TNF-resistant MCF7 tumor cells

Anti-cancer drugs and cytotoxic cytokines such as members of the TNF/Fas-ligand family play a predominant role in apoptosis induction in tumor cells, and are critical in cancer therapy. In this study we used the human breast-carcinoma cell line MCF7, its derivatives MCF7Adr (resistant to adriamycin) and R-A1 (resistant to TNF), to determine the impact of acquired drug and cytokine resistance on susceptibility to Fas-induced cytotoxicity and Fas-antigen expression. While MCF7 and R-A1 cells were killed by anti-Fas in the presence of IFN-γ, MCF7Adr was found to be resistant to Fas-mediated apoptosis. This resistance was correlated with a loss of surface Fas-protein expression. Fas-gene transfer in MCF7Adr resulted in high sensitivity to Fas-mediated cytotoxicity, indicating that the Fas signalling pathway is virtually intact in this cell line. Over-expression of the MDRI gene in MCF7 following gene transfer did not affect Fas expression and anti-Fas sensitivity, suggesting that the P-gp-mediated multidrug-resistance phenotype is not directly involved in the loss of Fas expression, contrary to what has been observed by others in T-cell lines. Furthermore, the down-regulation of Fas expression and subsequent resistance to anti-Fas were observed in drug-resistant human ovarian-carcinoma IGR-OVI/VCR cells and leukemic lymphoblast CEM/VLB cells, suggesting that the alteration of Fas expression following drug-resistance selection is not restricted to one cell type. © 1996 Wiley-Liss, Inc.