Xiu F. Hu

Altered expression of the IGF-1 receptor in a tamoxifen-resistant human breast cancer cell line

The relationship between oestrogen (E2) and insulin-like growth factor-one (IGF-1) was examined in both tamoxifen-sensitive (MCF 7/5–21) and tamoxifen-resistant (MCF 7/5–23) subclones of the MCF 7 cell line. Both subclones were grown in defined, serum-free (SF) medium over a period of 7 days with the addition of E2or IGF-1 or a combination of both agents. Growth of both MCF 7/5–21 and 7/5–23 cells was stimulated (245% and 350%, respectively) by E2. However, only the growth of MCF 7/5–23 cells was stimulated (266%) by IGF-1. A combination of E2and IGF-1 significantly enhanced MCF 7/5–21 and 7/5–23 cell growth (581% and 695%, respectively). E2-induced IGF-1 receptor (IGF-1R) levels (as measured by 125I-IGF-1 binding and Northern analyses) in only MCF 7/5–23 cells. This effect was partially inhibited by tamoxifen. In medium containing serum, the growth of only the MCF 7/5–23 cells was significantly inhibited by the IGF-1R monoclonal antibody, αIR-3. The detection of E2-induced expression of IGF-2 using RT-PCR was demonstrated in the MCF 7/5–23 cells. These experiments indicate that E2may sensitize tamoxifen-resistant MCF 7/5–23 cells to the growth stimulatory actions of IGF-2 via up-regulation of the IGF-1R and describes a cell-survival mechanism that may manifest itself as tamoxifen resistance.

MRP1 not MDR1 gene expression is the predominant mechanism of acquired multidrug resistance in two prostate carcinoma cell lines

Multidrug resistant prostate cancer cell lines DU 0.03 and PC 0.03 were established from the parental prostate cancer cell lines DU145 and PC-3 respectively by stepwise selection in doxorubicin (DOX) from 0.001 to 0.03 μg/ml. As cells adapted to each concentration of DOX. the drug concentration was increased by 0.001 μg/ml. The chemosensitivity of each line was determined by growth inhibition assay. The DU 0.03 and PC 0.03 lines exhibit a 5–10-fold and 1.3–2.8-fold increase in resistance to anthracyclines, vinblastine (VLB) and mitozantrone (Mito), respectively. Verapamil (5 μM) partially reversed the resistance to the anthracycline and completely reversed the resistance to VLB and Mito. Drug kinetic studies measured by intracellular accumulation of 3H-daunorubicin demonstrated a 3 fold decrease in the level of intracellular 3H-daunorubicin in the PC 0.03 and DU 0.03 resistant lines compared with their respective parental line. This effect was partially reversed by 5 μM verapamil. The expression of MDR1 and MRP genes was analysed by Northern blotting and RT-PCR. P-glycoprotein (Pgp) and MRP protein were tested by immunocytochemistry staining using the monoclonal antibodies J-SB1. C219 and MRK16 (Pgp) and MRPm6 and MRPr1 (MRP). Neither Northern blot analysis nor the more sensitive RT-PCR demonstrated detectable MDR1 transcripts in any of the prostate cancer cell lines and the three Pgp monoclonal antibodies failed to reveal expression of Pgp. A 2–4-fold increase in MRP1 mRNA levels in the drug resistant DU 0.03 and PC 0.03 lines were demonstrated by both Northern blotting and RT-PCR consistent with the findings observed after staining by the two specific monoclonal antibodies, MRPm6 and MRPr1. Southern blot analysis demonstrated a 2-fold increase in the MRP1 gene copy number in the PC 0.03 line but not in the DU 0.03 line, suggesting that the overexpression of the MRP gene was regulated at the level of transcription in the latter line. We conclude that MRP1 not MDR1 overexpression. contributes to acquired drug resistance in these two prostate cancer cell lines.

Induction of MDR1 gene expression by anthracycline analogues in a human drug resistant leukaemia cell line

SummaryThe effects of 4-demethoxydaunorubicin (idarubicin, IDA) and MX2, a new morpholino-anthracycline, on up-regulation of the MDR1 gene in the low-level multidrug resistant (MDR) cell line CEM/A7R were compared at similar concentrations (IC10, IC50and IC90) over a short time exposure (4 and 24 h). The chemosensitivity of each drug was determined by a 3-day cell growth inhibition assay. Compared with epirubicin (EPI), IDA and MX2 were 17- and eightfold more effective in the CEM/A7R line respectively. No cross-resistance to 5-FU was seen in the CEM/A7R line. Verapamil (5 μM) and PSC 833 (1 μM), which dramatically reversed resistance to EPI in the CEM/A7R line, had no sensitizing effect on the resistance of this line to MX2, but slightly decreased resistance to IDA. The sensitivity to 5-FU was unchanged by these modulators. The induction of MDR1 mRNA expression by IDA, MX2 and 5-FU was analysed by Northern blotting and semiquantitatively assessed by scanning Northern blots on a phosphorimager. The relative level of MDR1 expression was expressed as a ratio of MDR1 mRNA to the internal RNA control glyceraldehyde-3-phosphate dehydrogenase (GAPDH). IDA, MX2 and 5-FU differentially up-regulated MDR1 mRNA in the CEM/A7R line in a dose-dependent manner. Both IDA and MX2 induced MDR1 expression within 4 h. 5-FU up-regulated MDR1 expression only when drug exposure was prolonged to 24 h. Based on MRK 16 binding, flow cytometric analysis of P-glycoprotein (Pgp) expression paralleled the increase in MDR1 mRNA levels. For the three anthracyclines, the increase in MDR1 expression was stable in cells grown in the absence of drug for more than 3 weeks after drug treatment. The induction of MDR1 expression by 5-FU was transient, associated with a rapid decrease in the increased Pgp levels which returned to baseline 72 h after the removal of 5-FU. This study demonstrates that MDR1 expression can be induced by analogues of anthracyclies not pumped by Pgp, and that this induction appears to be stable despite a 3-week drug-free period.

Induction of insulin-like growth factor binding protein expression by ICI 182,780 in a tamoxifen-resistant human breast cancer cell line

Earlier studies in our laboratory demonstrated that the steroidal antiestrogen ICI 182,780 is very effective in abolishing the tamoxifen‐resistant proliferation of MCF 7/5‐23 cells [1]. In addition, preliminary binding studies showed that ICI 182,780 increased the binding of insulin‐like growth factor (IGF)‐I to the MCF 7/5‐23 cells, although this finding was not the result of an increase in the expression of the insulin‐like growth factor‐I receptor (IGF‐IR). Hence, we reasoned that the inhibition of tamoxifen‐resistant cell growth by ICI 182,780 might have been due to increased expression of insulin‐like growth factor binding proteins (IGFBPs).We observed the up‐regulation of non‐insulin‐suppressible IGF‐I binding in both the tamoxifen‐sensitive MCF 7/5‐21 cell line (1.5‐fold) and the tamoxifen‐resistant MCF 7/5‐23 cell line (2.5‐fold) after 5 days of treatment with ICI 182,780 (10−7 M) in serum‐free medium, suggesting a role for cell‐associated IGFBPs. Affinity cross‐linking experiments confirmed the presence of an IGF‐I:IGFBP complex of approximately 38‐kDa in tamoxifen or ICI 182,780‐treated cells. Western ligand blots showed higher levels of a soluble 30‐kDa IGFBP in media conditioned by either of the subclones that had been treated with ICI 182,780, an effect consistently opposed by estrogen (E2:10−9 M). RT‐PCR showed higher levels of IGFBP‐5 mRNA than any of the other known IGFBPs, suggesting that this was the major IGFBP subtype. The protein was subsequently identified by Western immunoblotting as IGFBP‐5. In conclusion, we postulate that this may be a mechanism contributing to the greater potency of ICI 182,780 in the growth inhibition of the MCF 7/5‐23, tamoxifen‐resistant cell line.

Effect of cyclosporin and verapamil on the cellular kinetics of daunorubicin

Both cyclosporin and verapamil modulate the multidrug-resistant (MDR) phenotype in the classical MDR cell lines, CEM/VLB100 and CEM/VLB1000. Initial studies demonstrated a significant reduction in daunorubicin accumulation in the two resistant lines compared with the drug-sensitive parent line CEM/CCRF. Both cyclosporin and verapamil increased drug accumulation in the resistant lines. This effect was dose-dependent although a plateau occurred in CEM/VLB100 cells at concentrations of cyclosporin exceeding 4.2 mumol/l. Cyclosporin 4.2 mumol/l and verapamil 10 mumol/l significantly increased daunorubicin uptake and reduced drug efflux in the CEM/VLB100 and CEM/VLB1000 lines. At low clinical concentrations of cyclosporin (0.8-1.6 mumol/l and verapamil (1-2 mumol/l), there was a synergistic increase in drug accumulation in the two resistant cell lines (P less than 0.007). These data suggest that cyclosporin modulates the classical MDR phenotype by altering the cellular kinetics of daunorubicin. The in vitro synergistic action of cyclosporin and verapamil could be interesting clinically.

Clinical application of a rapid, functional assay for multidrug resistance based on accumulation of the fluorescent dye, fluo-3

A rapid and simple functional assay for P-glycoprotein (Pgp) using flow cytometry to measure the accumulation of the flurophore fluo-3 has been applied to samples from patients with B-cell chronic lymphocytic leukaemia (B-CLL). Peripheral blood lymphocytes from 37 patients with B-CLL were studied for Pgp. Pgp expression, using MRK-16, a monoclonal antibody recognising an external surface epitope of Pgp, was detected in 92% of patients with B-CLL. The functional assays for Pgp expression were positive in 78 and 59% of patients using the fluo-3 and doxorubicin (dox) assays, respectively. When compared with the MRK-16 assay, the fluo-3 assay had a sensitivity of 82% compared to a sensitivity of 56% for the dox assay (P = 0.004). The specificity of the fluo-3 and dox assays could not be evaluated because of the low number of MRK-16 negative CLL cells.

Circumvention of doxorubicin resistance in multi-drug resistant human leukaemia and lung cancer cells by the pure antioestrogen ICI 164384

ICI 164384, a new steroidal antioestrogen, entirely devoid of oestrogenic activity, modulates doxorubicin resistance in vitro. At non-cytotoxic concentrations, ICI 164384 potentiated the cytotoxicity of doxorubicin in a dose-dependent manner in both the classical multi-drug resistant (MDR) human leukaemia cell lines CEM/VLB 100 and CEM/VLB 1000 and the human small cell lung cancer cell line H69 LX4. ICI 164384 had no effect on the two respective parental cell lines, CEM/CCRF and H69 P. None of these cell lines expressed the oestrogen receptor. In comparative studies at concentrations ranging from 1.25 to 10 mumols/l, ICI 164384 was significantly more effective (1.2-6-fold) than tamoxifen in reducing the IC50 of doxorubicin in the CEM/VLB 100 line. In resistant cells, ICI 164384 increased 3H-daunomycin accumulation in a dose-dependent manner and was significantly more effective than tamoxifen at concentrations ranging from 2.5 to 10 mumol/l. ICI 164384 reduced the efflux of daunomycin from resistant cells more effectively than tamoxifen. These studies suggest that ICI 164384 is an effective modulator of MDR.

Differential effects of estrogen, tamoxifen and the pure antiestrogen ICI 182,780 in human drug-resistant leukemia cell lines

ICI 182,780, a potent, new steroidal antiestrogen without apparent agonist activity, appears to be a potent modulator of the classic multidrug resistance (MDR) phenotype in the CEM/A7, CEM/VLB100 and K562/VIN100 MDR cell lines. This reagent had no effect on the respective parental CCRF-CEM and K562 cell lines. The use of 1.25 μM ICI 182,780 resulted in a 6- to 7-fold decrease in doxorubicin resistance in the CEM/A7 and CEM/VLB100 cell lines. A dose-response effect was observed at ICI 182,780 concentrations of up to 5 μM. As compared with tamoxifen (TAM), ICI 182,780 was 2 and 4 times more effective in the K562/VIN100 and CEM/A7 cell lines, respectively. ICI 182,780 at 0.625 μM increased [3H]-daunomycin uptake (P<0.0001) as effectively as 5 μM TAM in the resistant CEM/A7 line. Drug-efflux studies showed that 5 μM ICI 182,780 significantly decreased drug efflux as compared with 5 μM TAM (P<0.0001). Estradiol (EST) at 10 μM increased doxorubicin resistance by 1.2–1.3 times in the CEM/A7 and CEM/VLB100 cell lines and significantly decreased drug accumulation (P=0.002) and retention (P<0.001) in the CEM/A7 cell line. However, the addition of 10 μM EST to 1–2 μM ICI 182,780 did not inhibit the ability of ICI 182,780 to modulate doxorubicin resistance in the two resistant cell lines. Using reverse-phase high-performance liquid chromatography (HPLC) to measure lipophilicity, we found no apparent association between the ability of ICI 182,780, TAM or EST to modulate resistance and their relative hydrophobicity.

Inheritance of chromosome 7 is associated with a drug-resistant phenotype in somatic cell hybrids.

A major form of drug resistance in tumour cells known as classical multidrug resistance (MDR) is associated with the overexpression of the mdr1 gene product, the membrane protein P-glycoprotein (P-gp), which acts as an energy-dependent drug efflux pump. In this study the inheritance of P-gp expression was examined using hybrids formed after somatic cell fusion between a drug-sensitive human T-cell leukaemia cell line, CEM/CCRF, and a drug-resistant derivative, CEM/A7, which is characterized by a clonal chromosomal duplication dup(7)(q11.23q31.2). Fourteen hybrids, chosen at random, were analysed by reverse transcriptase-polymerase chain reaction (RT-PCR) and by binding studies involving the monoclonal antibody MRK16, which recognises an external P-gp epitope. Only two hybrids were positive for both MRK16 antibody labelling and mdr1 mRNA. Partial karyotypic analysis of all hybrids revealed that only the MRK16-positive hybrids contained the duplication in chromosome 7 seen in the CEM/A7 parental MDR line. Therefore, P-gp overexpression in the MRK16-positive hybrids may be linked to the inheritance of chromosome 7 from CEM/A7 and possibly associated with the chromosome 7 abnormality.