Resistance to oxidants associated with elevated catalase activity in HL-60 leukemia cells that overexpress multidrug-resistance protein does not contribute to the resistance to daunorubicin manifested by these cells

Purpose: It has been recognized that enhanced antioxidant defenses can contribute to the resistance of cancer cells displaying multidrug resistance (MDR) that arises in conjunction with the overexpression of P-glycoprotein (Pgp). The purpose of this study was to determine if the defenses against oxidant stress in MDR human leukemia cells (HL-60/AR) that overexpress multidrug-resistance-associated protein (MRP), but not Pgp, contribute to the mechanism of drug resistance in this cell line. Methods: HL-60/AR cells were evaluated in comparison with wild-type cells with respect to sensitivity to the oxidants hydrogen peroxide (H₂O₂) and tert-butyl hydroperoxide (t-BuOOH), the activities and amounts of the antioxidant enzymes catalase and glutathione peroxidase (GSH-Px), and the effects that manipulation of the activities of these enzymes may have on cellular sensitivity to the oxidants and to daunorubicin. We also evaluated the ability of the cells to generate daunorubicin semiquinone free radical as measured by electron spin resonance (ESR) spectroscopy. Results: HL-60/AR cells were >10-fold resistant to the cytotoxic effects of the H₂O₂ or t-BuOOH as compared with parental, drug-sensitive HL-60 cells. This phenomenon could be attributed largely to elevated activity and protein levels of catalase in HL-60/AR cells. Furthermore, inhibition of catalase by 3-amino-1,2,4-triazole (AT) diminished the resistance of HL-60/AR to these oxidants by >80% or >50%, respectively. Despite these findings, AT was incapable of causing sensitization of HL-60/AR cells to the cytotoxic effects of daunorubicin. We found that the activity and amount of selenium-dependent glutathione peroxidase (GSH-Px) was no greater in HL-60/AR cells than in HL-60 cells. Cultivation of cells in selenium-deficient medium caused a marked reduction in GSH-Px activity in HL-60/AR cells and a profound inhibition of GSH-redox cycling manifested by a decrease in baseline hexose monophosphate shunt activity (HMPS) and markedly blunted stimulation of the HMPS by the oxidant t-BuOOH in both wild-type and resistant cells. These variations in GSH-Px activity and GSH-redox cycling, however, were not associated with an alteration in cellular sensitivity to daunorubicin. The failure of catalase inhibition or selenium manipulation of GSH-Px activity to affect daunorubicin cytotoxicity was not due to the inability of these cells to produce free-radical species of daunorubicin, since ESR studies revealed that the generation of daunorubicin semiquinone free radical by HL-60/AR cells was equal to and, in fact, 3-fold that obtained with HL-60 cells. Conclusions: In comparison with parental HL-60 cells, MRP-overexpressing HL-60/AR cells have demonstrable alterations in antioxidant defenses that are manifested by cellular resistance to the cytotoxic effects of H₂O₂ and t-BuOOH and by elevated protein levels and activity of catalase. Whether these alterations are epiphenomena or are related to overexpression of MRP remains to be determined. However, it does appear that the enhanced antioxidant defenses observed in HL-60/AR cells do not contribute to the resistance to daunorubicin manifested by this cell line. Although HL-60/AR cells generate daunorubicin semiquinone free radical to an extent equal to or greater than that observed in HL-60 cells, the failure of alterations in GSH-Px activity or inhibition of catalase to change the sensitivity of HL-60/AR cells to daunorubicin suggests that the cytotoxicity of daunorubicin in these cells in not mediated through H₂O₂ or other peroxide species detoxified by these enzymes. Received: 11 March 1994/Accepted: 15 July 1994     

Membrane glycoprotein changes associated with anthracycline resistance in HL-60 cells

Summary The glycoproteins on the surface of HL-60/S wild-type, drug-sensitive human leukemia cells and HL-60/AR anthracycline-resistant cells which do not overexpress the P-glycoprotein, were characterized by labeling with [³⁵S]-methionine, NaB[³H₄], phosphorus 32, or sodium iodide I 125. HL-60/S and HL-60/AR cell lysates and membrane fractions tagged with [³⁵S]-methionine or phosphorus 32 showed no significant differences in their protein patterns as analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and by autoradiography. HL-60/S cells labeled with NaB[³H₄] yielded glycoproteins that were smeared predominantly in the molecular-weight range of 210,000 and 160,000 Da, with pI values ranging between pH 4 and pH 4.4. In contrast, NaB[³H₄]-labeled HL-60/AR cells showed 7–8 discrete glycoproteins within a molecular-weight range of 170,000 and 140,000 Da, with pI values also ranging between pH 4 and pH 4.4. In addition, [³H]-glucosamine incorporation into HL-60/S and HL-60/AR cells revealed that the latter showed lower uptake of [³H]-glucosamine than did the former. Following treatment with tunicamycin, [³H]-glucosamine uptake in HL-60/S cells decreased, whereas that in HL-60/AR cells remained unchanged. Surface-membrane radioiodination of HL-60/S and HL-60/AR cells showed two distinct protein electrophoretic patterns, with differences being observed in both the high-(220–95 kDa) and low-molecular-weight ranges (21 kDa). Flow cytometric analysis of HL-60/S and HL-60/AR cells using myeloid and lymphoid antigen-specific antibodies demonstrated no antigenic differences between HL-60/S and HL-60/AR cells. HL-60/S cells incubated in the presence of tunicamycin, an inhibitor ofN-linked glycosylation, or the protein kinase C agonist phorbol 12-myristate 13-acetate (PMA) developed a glycoprotein pattern similar to that observed in HL-60/AR cells. In addition, tunicamycin treatment of HL-60/S cells decreased daunorubicin (DNR) retention and altered its intracellular distribution as compared with that in HL-60/AR cells. These data indicate that HL-60/AR cells do not possess either de novo or amplified high-molecular-weight surface-membrane proteins; instead, existing proteins are hypoglycosylated. These results also show that HL-60/AR cells exhibit the multidrug-resistant phenotype in association with altered membrane glycoproteins of both high (220–95 kDa) and low molecular weight (21 kDa), but without overexpression of the P-glycoprotein. Furthermore, in HL-60/S cells, the multidrug-resistant phenotype is partially inducible by inhibition ofN-linked glycosylation of cell-surfac proteins.Abbreviations HL-60/AR anthracycline-resistant cells - HL-60/S parental HL-60 cells - DNR daunorubicin - RA retinoic acid - PBS phosphatebuffered saline (Hanks' Balanced Salt Solution) - PMA phorbol 12-myristate, 13-acetate - DVFM digitized video fluorescence microscopy - DFP diisopropylfluorophosphate - SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis - MDR multidrug resistance - 6GT 6-thioguanine - NP-40 Nonidet-40 detergent Supported by grants ACS CH-357, CA-31 761, CA-42 450, CA-40 188, the, William J. Matheson Foundation, the Medical Research Council of Canada, and the National Cancer Institute of Canada     

Subcellular distribution of daunorubicin in P-glycoprotein-positive and -negative drug-resistant cell lines using laser-assisted confocal microscopy.

Four well defined multidrug-resistant cell lines and their drug-sensitive counterparts were examined for intracellular distribution of daunorubicin (DNR) by laser-assisted confocal fluorescence microscopy: P-glycoprotein-negative HL-60/AR cells, and P-glycoprotein-positive P388/ADR, KBV-1, and MCF-7/ADR cells. Both drug sensitive cell lines (HL-60/S, P388/S, KB3-1, and MCF-7/S) and drug-resistant cell lines (HL-60/AR, P388/ADR, KBV-1, and MCF-7/ADR) exposed to DNR showed a similar rapid distribution of drug from the plasma membrane to the perinuclear region within the first 2 min. From 2-10 min, the drug sensitive HL-60/S, P388/S, and MCF-7/S cells redistributed drug to the nucleus and to the cytoplasm in a diffuse pattern. In contrast, drug-resistant HL-60/AR, P388/ADR, and MCF-7/ADR redistributed DNR from the perinuclear region into vesicles distinct from nuclear structures, thereby assuming a "punctate" pattern. This latter redistribution could be inhibited by glucose deprivation (indicating energy dependence), or by lowering the temperature of the medium below 18 degrees C. The differences in distribution between sensitive and resistant cells did not appear to be a function of intracellular DNR content, nor the result of drug cytotoxicity. Drug-sensitive KB3-1 and -resistant KBV-1 cells did not fully follow this pattern in that they demonstrated an intracellular DNR distribution intermediate between HL-60/S and HL-60/AR cells with both "punctate" and nuclear/cytoplasmic uptake sometimes in the same cell. These data indicate that the intracellular distribution of DNR is an important determinant of drug resistance regardless of the overexpression of P-glycoprotein. The intracellular movement of drug requires the presence of glucose and a temperature above 18 degrees C, implicating energy-dependent processes and vesicle fusion in the distribution process. This intracellular transport of DNR away from the nucleus in multidrug-resistant cells may protect putative cell targets such as DNA against drug toxicity.     

Rapid detection, cloning and molecular cytogenetic characterisation of sequences from an MRP-encoding amplicon by chromosome microdissection.

Chromosome microdissection was utilised for the analysis of cytogenetic markers of gene amplification [homogeneously staining regions (hsrs) and double minutes (dmins)] in two doxorubicin-resistant cell lines, fibrosarcoma HT1080/DR4 and small-cell lung cancer H69AR. Microdissection products from the hsr(7)(p12p15) of HT1080/DR4 were amplified and used for fluorescent in situ hybridisation (micro-FISH) analysis of drug-sensitive HT1080, resistant HT1080/DR4 and normal lymphocytes. The results demonstrated that the hsr contains a domain of DNA amplification of complex origin including sequences derived from 16p11.2-16p13.1, 2q11.2, 7q32-7q34 and 10q22. The amplification was confirmed by converting the micro-dissected probe into a microclone library for probing HT1080 and HT1080/DR4 Southerns. A micro-FISH probe from normal band region 16p11-16p13 further demonstrated amplification of 16p sequences in both HT1080/DR4 and H69AR. During the course of this analysis, Cole et al. (1992) (Science, 258, 1650-1653) published the amplification of the MRP gene in H69AR cells, which maps to chromosome 16p13.1. Our results corroborate the finding of MRP amplification in these doxorubicin-resistant cell lines, but, importantly, they provide information on the composition of the complex amplicon contributions from four different chromosomes. This study demonstrates the potential utility of chromosome microdissection for the rapid recovery of sequences from amplified regions in drug-resistant cells.     

Coamplification of 3-hydroxy-3-methylglutaryl coenzyme A reductase genes in methotrexate-resistant human leukemia cell lines

Methotrexate (MTX)-resistant K562 human myelocytic leukemia sublines with 20- and 200-fold amplified dihydrofolate reductase (DHFR) genes localized to homogeneously staining regions (HSRs) on the long arms of chromosomes 5, 6, and 19 were used to examine whether other genes mapping close to the DHFR genes were coamplified. The gene for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, located on chromosome 5q13.3-14, was coamplified 4-14-fold, corresponding to the levels of resistance exhibited by these cells. Similar observations were made with a MTX-resistant subline of the promyelocytic leukemia cell line, HL-60R, with 200 gene copies of DHFR. These observations indicate a tight linkage of DHFR and HMG-CoA genes on chromosome 5q.     

槲皮素恢复柔红霉素在白血病耐药细胞K562/ADM和HL-60/ADM中的分布

背景与目的：槲皮素是一种天然黄酮类中药成分,儿有多种生理活性,最近发现其有逆转白血病细胞耐药的作用,本研究旨在探讨槲皮素恢复柔红霉素在白血病耐药细胞的分布从而达到逆转耐药的机制：方法：通过MTT体外药敏法检测槲皮素对柔红霉素的增敏作用并确定逆转的浓度范围,作用于K562／ADM、HL-60／ADM细胞及相应敏感株K562和HL-60,借助激光共聚焦显微镜观察槲皮素怍用前后柔红霉素在亚细胞水平的分布变化：结果：20～40μmol／L槲皮素在体外能日月显提高柔红霉素对K562／ADM和HL-60／ADM的敏感性,恢复柔红霉素在亚细胞水平的分布,使其回归细胞核内,从而逆转多药耐药。结论：黄酬类中药槲皮素能够成为蒽环类药物治疗白血病中有效的化疗增敏剂。     

Development and characterization of a human myelogenous leukemia cell line resistant to 4'-(9-acridinylamino)-3-methanesulfon-m-anisidide

The human myelogenous leukemia cell line HL-60 was made resistant to amsacrine (m-AMSA) by repeated exposure in vitro to increasingly large doses of the drug. Resistance to m-AMSA developed in a triphasic process and was accompanied by a slightly slower growth rate and cloning efficiency and a more differentiated morphological phenotype. Extensive chromosomal rearrangement also took place. Among other chromosomal aberrations, one of the No. 6 homologues showed an added segment on the long arm in the form of an homogeneously staining region. One of the homologues of chromosome 14 in every cell showed a deletion of the distal end of the long arm that was replaced by an unidentified homogeneously staining segment. Membrane-associated 170 kd glycoprotein was not overexpressed in the resistant cells, which together with an absence of cross-resistance to Vinca alkaloids and anthracyclines points toward a mechanism of resistance different from multidrug resistance. The ability of resistant cells to respond to differentiation-inducing agents was not significantly changed as compared with that of the parental line. Growth of resistant cells in the absence of m-AMSA for over 200 population doublings within a period of more than 1.5 years did not result in reversion of the resistance, suggesting a stable genomic change. Resistance was not due to a decrease in the bioavailability of the drug. Uptake of [14C]m-AMSA by either whole cells or isolated nuclei of resistant cells exceeded that of the parental cell line, and outward transport of the drug was not more active; thus there were higher levels of intracellularly bound drug. The cell line represents an excellent model for studies of the mechanisms of resistance to m-AMSA and its modulation in human myelogenous leukemia.     

Allelotyping of acute myelogenous leukemia: loss of heterozygosity at 7q31.1 (D7S486) and q33-34 (D7S498, D7S505)

Loss of a whole chromosome 7(-7) or a deletion of the long arm of chromosome 7 del(7q) occurs frequently in many types of primary cancers including cases of acute myelogenous leukemia (AML). We analyzed for loss of heterozygosity (LOH) of chromosome arm 7q in 26 AML cases using a set of 15 microsatellite markers in order to begin to determine the location of putative tumor suppressor genes (TSG) important to this disease. Seven samples (27%) showed LOH at one or more loci on chromosome 7q. We identified the smallest commonly deleted regions to be at 7q31.1 (D7S486) and 7q33-34 (D7S498, D7S505) suggesting that alterations of a TSG in each region have an important role in de novo AML.     

Chromosomal organization of amplified genes in multidrug-resistant Chinese hamster cells

Six independently derived multidrug-resistant Chinese hamster cell lines selected with vincristine, daunorubicin, actinomycin D, or colchicine were probed by in situ hybridization techniques with the cloned cDNA, p5L-18, to chromosomally localize known or presumed amplified P-glycoprotein genes. One or two clusters of amplified genes were demonstrable in all of the highly resistant sublines and were localized to homogeneously staining regions and/or abnormally banding regions, gene amplification-associated cytogenetic abnormalities, on eight different chromosomes. Analysis of trypsin-Giemsa banded karyotypes revealed additional, multiple chromosomal rearrangements that were apparently nonspecific. Mapping studies localized the native P-glycoprotein gene(s) to the region q23 to 31 (most probably band 26) on the long arm of chromosome 1 of normal Chinese hamster bone marrow fibroblasts and normal chromosome 1 homologues in resistant cells. Southern blot analysis of restriction endonuclease fragments indicated the amplification of one or both of (at least) two wild-type nonallelic genes in four of the lines and the presence in one line (DC-3F/DMM XX) of a unique 5.0-kilobase BamHI fragment resulting from a recombinational event during amplification. Comparison with the cytogenetic data indicated no correlation between restriction patterns generated by EcoRI, HindIII, PstI, or BamHI and chromosomal location of amplified genes. However, the only sublines in which the homogeneously staining region or abnormally banding region is positioned at 1q26 (at or near the site of the native gene) exhibit either alterations in gene structure (DC-3F/DM XX) or in regulation of gene expression (DC-3F/AD X), suggesting a process more complex than simply amplification of the gene in loco.     

正常人骨髓成纤维样基质细胞对HL-60/VCR耐药细胞增殖的影响

目的观察正常人骨髓成纤维样细胞系HFCL对白血病多药耐药细胞HL-60/VCR增殖和分化的影响.方法采用四唑氮蓝(MTT)法进行HL-60/VCR细胞药物敏感实验.建立HL-60/VCR细胞和HFCL细胞共培养体系,苔盼蓝拒染法测定生长曲线;硝基四氮唑蓝(NBT)确定细胞分化;流式细胞仪检测细胞周期和CD11b、CD13、CD14、CD33细胞表面抗原进一步鉴定细胞分化;Western blot检测增殖细胞核抗原(Proliferating Cell Nuclear Antigen,PCNA)和P糖蛋白(P-glycoprotein,P-gp).结果HL-60/VCR细胞对多种药物耐药.与HFCL细胞共培养后,HL-60/VCR细胞生长受抑,且与HFCL细胞直接接触组的抑制作用＞用transwell组.同时发现HL-60/VCR细胞与HFCL细胞共培养后,G1期细胞增多,S期细胞减少;同时CD11b和CD14表达增高,CD13和CD33变化不大;且NBT阳性细胞轻度增多.Western blot检测结果显示,PCNA表达下调,以直接接触组为甚.但是P-gp表达无变化.结论正常人骨髓成纤维样细胞HFCL能抑制白血病MDR细胞HL-60/VCR的增殖,抑制PCNA的表达,出现G1期阻滞,并部分向单核细胞分化.     

Allelic loss on chromosome 17 in human ovarian cancer

In order to identify a common region of deletion on chromosome 17 potentially containing a tumor-suppressor gene, 27 ovarian carcinomas and 3 ovarian tumors of low malignant potential (LMP) were examined for loss of heterozygosity (LOH) at 6 p arm and 10 q arm loci. Ninety percent of all tumors had deletions at one or more loci. On the p arm, there was a single near-common region of deletion on 17p 13.3 (D/7S30/ pYNZ22.1; 86% LOH), an intervening locus with a low LOH rate, and a more proximal locus on 17p11.2 (D/7S58/pEW301; 82% LOH) with a high LOH rate. In less aggressive tumors, LOH at Df 7S30 was not accompanied by LOH at p53. The q arm had a common region of deletion for high-stage carcinoma at D/7S579 (Mfd 188; 74% LOH) on q21, a locus tightly linked to the familial breast-ovarian-cancer syndrome (BRCAI) locus. D/7S579 was lost in all informative high-stage carcinomas and retained in all low-stage carcinomas and tumors of LMP. There may be at least 2 tumor-suppressor genes, an early-acting gene on the p arm and a gene on the q arm involved in tumor progression and metastasis.     

Indoloquinoxaline compounds that selectively antagonize P-glycoprotein.

Tumor cells often develop drug resistance through overexpression of membrane transport proteins that effectively efflux anticancer agents. The pharmacologies of the two best-studied transporters, P-glycoprotein (Pgp) and MRP1, are partially overlapping but distinct. To improve the therapeutic potential of drug resistance reversing agents, we have developed a program to identify compounds with selectivity for Pgp or MRP1. Screening of a commercial library of compounds identified indoloquinoxaline compounds with transporter selectivity, and certain examples were synthesized and further evaluated. 1,4-Dibutoxy-6H-indolo[2,3-b]quinoxaline and 4,7-dibutoxy-2,3-dihydrobenzimidazole-2-spiro-3-indolin-2-one were synthesized by condensation of 3,6-dibutoxy-1,4-diaminobenzene and isatin. Neither compound was cytotoxic to MCF-7 cells, nor did either one affect the sensitivity of MCF-7/VP or HL-60/ADR cells at doses up to at least 20 microM, indicating that they do not antagonize MRP1. In contrast, each compound, at doses as low as 0.25 microM, sensitized NCI/ADR cells to vinblastine, actinomycin D, Taxol, and doxorubicin, indicating that they effectively reverse Pgp-mediated multidrug resistance (MDR). Furthermore, the compounds sensitized two additional cell lines that overexpress Pgp to this panel of anticancer drugs. However, these compounds did not affect the sensitivities of MCF-7 or T24 cells to these cytotoxic drugs, and did not alter the sensitivities of any of the tested cell lines to cisplatin or 5-fluorouracil. Both compounds enhanced the intracellular accumulation of [3H]vinblastine by NCI/ADR cells, but did not inhibit photoaffinity labeling of Pgp by [3H]azidopine at concentrations up to at least 100 microM. Therefore, these novel nontoxic indoloquinoxalines selectively sensitize Pgp-overexpressing cells to drugs that are subject to transport by this protein, without modulating the sensitivities of MRP1-overexpressing or non-Pgp cells to cytotoxic drugs. Because of this transporter selectivity, we predict that these compounds will be effective MDR modulators in vivo.     

Determination of the DNA content of a six-gene amplicon in the multidrug-resistant Chinese hamster cell line CHRB3 by flow karyotyping

Acquired multidrug resistance in cultured cells is often due to amplification of pgp genes, which gives rise to overproduction of P-glycoproteins that confer resistance by reducing the intracellular drug accumulation. The size of these amplicons varies between multidrug resistant cell lines and is often much larger than the gene selected for. Amplicons of the multidrug resistant Chinese hamster ovary cell line CHRC5 and its progenitor CHRB3, for example, span at least five different genes besides the pgp genes. Linkage of these gene classes with pgp had been shown by in situ hybridization and by long distance mapping using pulsed field gradient gel electrophoresis. Because the boundaries of the larger amplicons could not be determined, the size of such amplicons is not yet known, even though the six genes span at least 1500 kilobases. In the present study we have determined the amplicon size in B3+, a subclone of CHRB3 with a homogeneously staining region on chromosome 7q+ that harbors the amplified genes. We estimated the amplicon size in revertant clones by correlating the decreased DNA content of the 7q+ homogeneously staining region with the number of lost amplicons. The reduction of the homogeneously staining region DNA that accompanied reversion was determined by flow cytometry of propidium iodide stained chromosome suspensions of the various cell lines. We found that about 107 megabase pairs were lost together with 11-24 P-glycoprotein gene copies, suggesting that the mean amplicon size is in the range of 4.5-10.1 megabase pairs.     

Identification of two distinct regions of allelic imbalance on chromosome 18Q in metastatic prostate cancer

Like most cancers, prostate cancer (CaP) is believed to be the result of the accumulation of genetic alterations within cells. Previous studies have implicated numerous chromosomal regions with elevated rates of allelic imbalance (AI), using mostly primary CaPs with an unknown disease outcome. These regions of AI are proposed sites for tumor suppressor genes. One of the regions previously implicated as coding for at least one tumor suppressor gene is the long arm of chromosome 18 (18q). To confirm this observation, as well as to narrow the critical region for this putative tumor suppressor, we analyzed 32 metastatic CaP specimens for AI on chromosome 18q. Thirty-one of these 32 specimens (96.8%) exhibited AI at one or more loci on chromosome 18q. Our analysis using 17 polymorphic markers revealed statistically significant AI on chromosome 18q at 3 markers, D18S35, D18S64 and D18S461. Using these markers as a guide, we have been able to identify 2 distinct minimum regions of AI on 18q. The first region is between the genetic markers D18S1119 and D18S64. The second region lies more distal on the long arm of the chromosome and is between the genetic markers D18S848 and D18S58. To determine if 18q loss is a late event in the progression of CaP, we also examined prostatic intraepithelial neoplasia (PIN) and primary prostate tumors from 17 patients for AI with a subset of 18q markers. We found significantly higher AI in the metastatic samples. Our results are consistent with 18q losses occurring late in CaP progression.     

Screening and cloning of multi-drug resistant genes in HL-60/MDR cells

ObjectiveTo screen and clone multi-drug resistance (MDR) related genes in MDR acute myeloid leukemia cells (HL-60/MDR).MethodsHL-60/MDR was established using All-Trans Retinoic Acid. With the HL-60 cells as “tester” and HL60/MDR as “driver”, the cDNA library of HL-60/MDR was established by suppression subtractive hybridization. Then 12 of the resulting subtracted cDNA clones were selected for DNA sequencing and homology analysis. The obtained expressed sequence tags (ESTs) were analyzed with the GenBank BLASTN program to identify sequence homologies to known genes.ResultsThe HL-60/MDR cells had different multi-drug resistance to six kinds of chemotherapeutic drugs. The 211 positive gene clones in differential cDNA library of HL-60/MDR cells were amplified with PCR and 46 gene clones exhibited differential expression (ratio >3). Twelve gene clones with significant differential expression (ratio >5) were screened out to homology analysis. Of these, 11 matched known genes and the rest 1 showed no significant homology to human or non-human known sequences. It was named as gene clone HA117.ConclusionsThis effort provides the partial list of genes differential expressed in HL-60/MDR cells and a novel gene HA117 was found to be related to MDR. Identification of these genes contributes to our understanding of MDR development, and potentially provides candidate target genes to overcome MDR.     

Intracellular distribution and pharmacokinetics of daunorubicin in anthracycline-sensitive and -resistant HL-60 cells

Anthracycline-sensitive (HL-60) and -resistant (HL-60/AR) cells, which do not overexpress the P-glycoprotein, each transport and distribute daunorubicin (DNR) into distinct intracellular locations, as visualized by digitized video fluorescence microscopy. At pH 7.4, the fluorescence of DNR in HL-60 cells appears distributed diffusely in both the nucleus and cytoplasm. In contrast, HL-60/AR cells show much less fluorescence in the nucleus and cytoplasm; most of the fluorescence localizes first to the Golgi apparatus and is then gradually shifted to the lysosomes and/or mitochondria. In pharmacokinetic studies, HL-60/AR cells exposed to different extracellular concentrations of [14C]DNR consistently accumulated less radioactive drug than the parent HL-60 cells. Incubation of HL-60/AR cells with sodium azide and deoxyglucose blocked the efflux of [14C]DNR and also prevented the shift of DNR fluorescence from the Golgi apparatus to the lysosomes/mitochondria. The efflux and the intracellular shift of DNR could also be inhibited by lowering the temperature to 18 degrees C, which stops endosomal membrane fusion. When DNR was allowed to accumulate in HL-60 or HL-60/AR cells at pH 5 there was an increase in the proportion of drug fluorescence in the membranes of both HL-60 and HL-60/AR cells; a decrease in the amount of drug retained by HL-60, but not by HL-60/AR cells; and a decrease in the cytostatic effects of DNR on both HL-60 and HL-60/AR cells. These data suggest that DNR resistance is associated with a failure of DNR to pass through membranes and to bind to cytoplasmic and nuclear structures. Instead, most of the drug is taken up by the Golgi apparatus from which it is then shifted to the lysosomes or to mitochondria, or out of the cell.     

Genistein modulates the decreased drug accumulation in non-P-glycoprotein mediated multidrug resistant tumour cells.

In tumour cells the pharmacological basis for multidrug resistance (MDR) often appears to be a reduced cellular cytostatic drug accumulation caused by the drug efflux protein, P-glycoprotein (Pgp MDR), or by other drug transporters (non-Pgp MDR). Here we report the reversal of the decreased daunorubicin (DNR) accumulation in five non-Pgp MDR cell lines (GLC4/ADR, SW-1573/2R120, HT1080/DR4, MCF7/Mitox and HL60/ADR) by genistein. Genistein inhibited the enhanced DNR efflux in the GLC4/ADR cells. In these cells the decreased VP-16 accumulation was also reversed by genistein. Three other (iso)flavonoids biochanin A, apigenin and quercetin also increased the DNR accumulation in the GLC4/ADR cells. In contrast to the effects on non-Pgp MDR cells, 200 microM genistein did not increase the reduced DNR accumulation in three Pgp MDR cell lines (SW-1573/2R160, MCF7/DOX40 and KB8-5) or in the parental cell lines. In conclusion the use of genistein provides a means to probe non-Pgp related drug accumulation defects.     

Loss of heterozygosity at 7q22 and mutation analysis of the CDP gene in human epithelial ovarian tumors

Many tumor types including that of the ovary show loss of heterozygosity (LOH) on chromosome arm 7q, which suggests the existence of at least one tumor suppressor gene (TSG) on this chromosome arm. We have studied the region surrounding the putative tumor suppressor gene CUTL1 at 7q22 in 127 epithelial ovarian tumors. LOH was found across 7q22 in 31% of malignant and 14% of benign ovarian tumors. In 16% of the tumors the LOH appeared to be centered on the CUTL1 gene. This gene has been implicated previously as a TSG in both uterine leiomyomas and breast carcinoma. However, mutation analysis of the CUTL1 gene in 47 tumors with 7q22 LOH failed to identify any somatic alterations in the coding regions. This finding suggests that CUTL1 may not be the target of the 7q22 LOH in ovarian cancers.     

Liposome-mediated modulation of multidrug resistance in human HL-60 leukemia cells.

BACKGROUND: Multidrug resistance (MDR) is a major obstacle in cancer treatment. Resistance of cultured tumor cells to major classes of cytotoxic drugs is frequently due to expression of a plasma membrane P-glycoprotein encoded by MDR genes. We have demonstrated that liposome-encapsulated doxorubicin is more toxic than the free drug and that it modulates MDR in Chinese hamster LZ cells and human colon cancer cells. PURPOSE: To investigate further the association between expression of P-glycoprotein and modulation of MDR by liposome-encapsulated doxorubicin, we studied vincristine-resistant HL-60/VCR leukemia cells, which express P-glycoprotein, and doxorubicin-resistant HL-60/ADR leukemia cells, which do not. METHODS: Cells were exposed to various concentrations of free doxorubicin and liposome-encapsulated doxorubicin. The cellular content of doxorubicin was determined by fluorescence analysis, and cytotoxicity was determined by cell growth inhibition. Photoaffinity-labeling studies of P-glycoprotein binding were performed on HL-60/VCR and HL-60/ADR cells and KB-GSV2 cells transfected with the MDR1 gene (also known as PGY1). RESULTS: The concentrations that caused 50% inhibition of growth (IC50) for free doxorubicin in HL-60, HL-60/ADR, and HL-60/VCR cells were 30 nM, 9 microM, and 0.9 microM, respectively. The values for liposome-encapsulated doxorubicin in parental HL-60 cells and HL-60/ADR cells were 20 nM and 9 microM, respectively, indicating little or no sensitization. In contrast, HL-60/VCR cells were fivefold more sensitive to liposome-encapsulated doxorubicin than to free doxorubicin, and IC50 was reduced to 0.17 microM. In HL-60 cells exposed to liposome-encapsulated doxorubicin, intracellular doxorubicin accumulation was less than that seen with free drug. In contrast, in HL-60/VCR cells, accumulation was twofold to threefold higher than that with free doxorubicin. Liposome-encapsulated doxorubicin completely inhibited the photoaffinity labeling of P-glycoprotein by azidopine in membrane vesicles of HL-60/VCR cells, with a potency comparable to that of azidopine, suggesting that circumvention of MDR by liposomes is related to their specific interaction with P-glycoprotein. The studies with KB-GSV2 cells indicated that blank liposomes can directly inhibit photoaffinity labeling of P-glycoprotein. CONCLUSIONS: These results demonstrate the effectiveness of liposome-encapsulated doxorubicin in overcoming resistance in the multidrug-resistant phenotype of HL-60/VCR cells by direct interaction with P-glycoprotein. Furthermore, they indicate that liposome-encapsulated doxorubicin may be an effective treatment for human cancers.     

槲皮素逆转白血病细胞株HL-60/ADM多药耐药的研究

目的探讨槲皮素(Que)逆转白血病细胞多药耐药在膜转运蛋白方面的机制。方法通过四唑蓝体外药敏法,检测Que对柔红霉素(DNR)的增敏作用,并以不同浓度作用于HL-60／ADM耐药株及相应敏感株HL-60;运用逆转录多聚酶链式反应和流式细胞术,检测HL-60／ADM和HL-60细胞株多药耐药相关基因1(MRP1)及其膜蛋白产物MRP1蛋白的表达情况;借助激光共聚焦显微镜,观察DNR在亚细胞水平的分布变化。结果20～40μmol／L终浓度的Que在体外能明显提高DNR对HL-60／ADM耐药株的敏感性,并能下凋MRP1基因及其膜蛋白产物MRP1的表达,使DNR回归于细胞核内,从而逆转多药耐药,而且对细胞本身无明显毒性作用。结论Que有可能成为蒽环类药物治疗白血病的有效且低毒的化疗增敏剂。