Ascorbate free radical stimulates the growth of a human promyelocytic leukemia cell line

Ascorbate free radical stimulates the growth of human promyelocytic leukemia cells (HL-60) in the presence of a limited amount of serum (1%) when added to the cells under conditions where it is impermeable. Maximum growth stimulation occurs at concentrations from 5 x 10(-9) to 2 x 10(-8) M. Ascorbate mimicks the stimulation effect of its free radical but stimulates at higher concentrations. Autoxidation of ascorbate by oxygen produces its free radical, which apparently causes growth stimulation. Ascorbate could be regenerated by intact cells in vitro, since prevention of autoxidation of ascorbate in the presence of cells is observed. Neither dehydroascorbate nor isoascorbate increases HL-60 cell growth. Short term incubation of cells in the presence of ascorbate free radical induced intracellular NADH oxidation. We propose that the stimulation of growth of HL-60 cells shown here could be caused by activation of the transplasma membrane electron transport system by the ascorbate free radical.     

Multiple mechanisms confer drug resistance to mitoxantrone in the human 8226 myeloma cell line

Selection for in vitro drug resistance can result in a complex phenotype with more than one mechanism of resistance emerging concurrently or sequentially. We examined emerging mechanisms of drug resistance during selection with mitoxantrone in the human myeloma cell line 8226. A novel transport mechanism appeared early in the selection process that was associated with a 10-fold resistance to mitoxantrone in the 8226/MR4 cell line. The reduction in intracellular drug concentration was ATP-dependent and ouabain-insensitive. The 8226/MR4 cell line was 34-fold cross-resistant to the fluorescent aza-anthrapyrazole BBR 3390. The resistance to BBR 3390 coincided with a 50% reduction in intracellular drug concentration. Confocal microscopy using BBR 3390 revealed a 64% decrease in the nuclear:cytoplasmic ratio in the drug-resistant cell line. The reduction in intracellular drug concentration of both mitoxantrone and BBR 3390 was reversed by a novel chemosensitizing agent, fumitremorgin C. In contrast, fumitremorgin C had no effect on resistance to mitoxantrone or BBR 3390 in the P-glycoprotein-positive 8226/DOX6 cell line. Increasing the degree of resistance to mitoxantrone in the 8226 cell line from 10 to 37 times (8226/MR20) did not further reduce the intracellular drug concentration. However, the 8226/MR20 cell line exhibited 88 and 70% reductions in topoisomerase II beta and alpha expression, respectively, compared with the parental drug sensitive cell line. This decrease in topoisomerase expression and activity was not observed in the low-level drug-resistant, 8226/MR4 cell line. These data demonstrate that low-level mitoxantrone resistance is due to the presence of a novel, energy-dependent drug efflux pump similar to P-glycoprotein and multidrug resistance-associated protein. Reversal of resistance by blocking drug efflux with fumitremorgin C should allow for functional analysis of this novel transporter in cancer cell lines or clinical tumor samples. Increased resistance to mitoxantrone may result from reduced intracellular drug accumulation, altered nuclear/cytoplasmic drug distribution, and alterations in topoisomerase II activity.     

Amiloride potentiation of differentiation of human promyelocytic cell line HL-60

Cells of the human acute promyelocytic cell line HL-60 undergo differentiation when exposed to dimethyl sulfoxide (DMSO); in this report, amiloride, an inhibitor of passive intracellular Na+ flux, potentiated the DMSO-induced differentiation of HL-60 cells. This effect was seen at several concentrations of DMSO. Amiloride alone did not affect HL-60 differentiation. Various analogues of amiloride were tested for their ability to potentiate differentiation of HL-60 cells. The synergistic induction of myeloid differentiation by a membrane solvent (DMSO) and an Na+ transport inhibitor (amiloride) suggested membrane cation flux as being important in initiating differentiation.     

Radiation resistance in a doxorubicin-resistant human fibrosarcoma cell line.

In clinical practice, cancers refractory to chemotherapy can appear relatively radioresistant. Recent work in multidrug-resistant cell lines has yielded conflicting results concerning the relationship between drug resistance and radiation resistance. The current study examines the radiation response of a human fibrosarcoma (HT1080) and a doxorubicin-resistant subline (HT1080/DR4). Using soft-agar colony formation after graded doses of x-rays as an endpoint, HT1080/DR4 had an increased D0 (D0 = 2.1 Gy) and a broader initial shoulder (n = 2.7, Dq = 2.1 Gy) than the parental HT1080 line (D0 = 0.7, Gy, n = 1.2, Dq = 0.3 Gy), suggesting that HT1080/DR4 has an increased capacity to repair radiation-induced DNA damage. This possibility was tested by comparing the cell lines' ability to accumulate sublethal damage. In split-dose recovery experiments, HT1080/DR4 demonstrated increased ability to repair sublethal radiation damage following fractionated irradiation, compared with the HT1080 parental line. The mechanism for this radiation resistance is not clear, but a variety of cellular alterations seen in drug-resistant cell lines are discussed with reference to areas of further study.     

急性早幼粒细胞白血病耐药机制的研究进展

全反式维甲酸(ATRA)与三氧化二砷(As2O3)作为一线药物治疗急性早幼粒细胞白血病(APL)以来,APL患者的治愈率得到显著提高.但ATRA和(或)As2O3耐药的出现成为一个严重问题.现就近年来新提出的一些耐药机制如融合基因突变、细胞信号通路异常、染色质重构复合物异常、凋亡调控异常、骨髓微环境介导耐药等方面进行综述.     

HL-60/VCR多药耐药细胞株耐药机制的研究

背景与目的：白血病细胞对化疗药物的耐药是白血病治疗失败的主要原因,多药耐药细胞株为白血病多药耐药机制和逆转多药耐药性的研究提供了良好的模型。为探讨药物诱导产生多药耐药机制,我们对HL-60/VCR细胞的耐药机制进行了研究。方法：应用流式细胞术和一组抗体,对药物敏感细胞株HL-60和多药耐药细胞株HL-60/VCR细胞的耐药相关蛋白P-gp、MRP、LRP、BCRP、GST-π,以及凋亡调节蛋白bcl-2、bax、bcl-x、bad的表达进行分析。结果：在HL-60/VCR细胞株中,耐药相关蛋白P-gp、MRP、BCRP、GST-π分别是其在HL-60细胞株中的18.62、1.19、1.50、1.32倍,而LRP无变化。凋亡抑制蛋白bcl-2、bcl-x分别是其在HL-60细胞株中的2.48、1.25倍,凋亡调节蛋白bad是HL-60细胞株中的1.08倍;而凋亡诱导蛋白bax反而降低,是HL-60细胞株中的0.88倍。结论：多种机制参与HL-60/VCR的多药耐药,涉及耐药蛋白P-gp、MRP、BCRP和GST-π的表达增强,而且凋亡调节蛋白bcl-2、bax、bcl-x、bad均可能参与其耐药机制的形成。     

Multidrug resistance in a human leukemic cell line selected for resistance to trimetrexate

Trimetrexate (TMQ) is a lipophilic antifolate shown to have antitumor activity in humans. TMQ-resistant sublines of the MOLT-3 human acute lymphoblastic leukemia cell line were developed and were designated as MOLT-3/TMQ200, MOLT-3/TMQ800, and MOLT-3/TMQ2500 based on degrees of resistance to TMQ. The TMQ resistance was accompanied by 5- to 7-fold increases in dihydrofolate reductase activity and markedly reduced cellular TMQ accumulation. Methotrexate accumulation was not impaired in TMQ-resistant cells. TMQ retention (efflux) was unchanged in these TMQ-resistant cells. Verapamil enhanced the TMQ accumulation in the resistant cells to the level seen in the parent cells but had no effects on the TMQ retention. These sublines were cross-resistant not only to methotrexate but also to vincristine, doxorubicin, daunorubicin, and mitoxantrone. There was no cross-resistance to bleomycin or cisplatin. Resistance to vincristine, doxorubicin, daunorubicin, and mitoxantrone was reversed by verapamil. TMQ resistance was only minimally reversed by verapamil and methotrexate resistance not affected at all. Both cellular accumulation and retention of vincristine and daunorubicin in the TMQ-resistant cells were markedly decreased. Verapamil enhanced both accumulation and retention of the drug. Plasma membrane fractions of the TMQ-resistant cells analyzed by urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by staining with Coomassie Blue revealed the presence of a distinct band with a molecular weight of 170,000. Immunoblot analysis with 125I-labeled monoclonal antibody raised against P-glycoprotein of multidrug-resistant Chinese hamster ovary cells (C219) cross-reacted with the Mr 170,000 protein of the TMQ-resistant cells. These results show that the TMQ-resistant cells displayed not only decreased TMQ uptake and increased dihydrofolate reductase but also characteristics associated with a classical multidrug-resistant phenotype. Multidrug resistance includes lipophilic antifolate.     

Circumvention of confluence-dependent resistance in a human multi-drug-resistant colon-cancer cell line

Colorectal adenocarcinomas are inherently resistant to anthracyclines and other topoisomerase-II inhibitors. Resistance to doxorubicin of colon cancer cells (Caco2) depends on 2 main mechanisms. The first is typical multi-drug resistance, characterized by the mdr1 gene and its product the P170 membrane glycoprotein. P170 effluxes anthracyclines out of cancer cells and is antagonized in vitro by verapamil. The second mechanism, which develops when cell-culture density increases, we have designated confluence-dependent resistance. Confluence-dependent resistance depends on the reduced topoisomerase II content of the G₀/G₁,-phase cells which accumulate in the confluent population. We show here that short treatments of confluent Caco2 cells with slightly toxic concentrations of DNA-damaging agents (cisplatin, melphalan or mitomycin C) produced a transient accumulation of cells in S- and G₂/M-phases of the cell cycle. Concomitantly with the increase in the S-phase population, the topoisomerase II cellular level and the sensitivity of cells to doxorubicin were greatly enhanced. Over-coming confluence-dependent resistance through S-phase accumulation and inhibition of multi-drug resistance by verapamil were fully additive, and a nearly complete reversal of confluent Caco2 cells' resistance to doxorubicin was obtained when both strategies were combined. © 1995 Wiley-Liss, Inc.     

Multidrug resistance in a human small cell lung cancer cell line selected in adriamycin

A multidrug resistant variant (H69AR) of the human small cell lung cancer cell line NCI-H69 was obtained by culturing these cells in gradually increasing doses of Adriamycin up to 0.8 microM after a total of 14 months. H69AR expresses the multidrug resistant phenotype because it is cross-resistant to anthracycline analogues including daunomycin, epirubicin, menogaril, and mitoxantrone as well as to acivicin, etoposide, gramicidin D, colchicine, and the Vinca alkaloids, vincristine and vinblastine. H69AR is also similar to other multidrug resistant cell lines in that it displays little or no cross-resistance to bleomycin, 5-fluorouracil, and carboplatin. It has a slight collateral sensitivity to 1-dehydrotestosterone and lidocaine. H69AR has increased cell-cell adhesiveness compared to H69, but a similar growth rate in vitro and tumorigenicity in nude mice. When cultured in the absence of Adriamycin, there is a 40% decrease in resistance by 35 days of culture, compared to cells in continuous culture in drug, but no further decrease in resistance up to 181 days. Monoclonal antibodies to P-glycoprotein have no detectable reactivity with H69AR cells as determined by enzyme-linked immunosorbent assay and immunoblotting techniques. Thus, unlike most multidrug resistant cell lines, H69AR does not appear to express enhanced levels of P-glycoprotein. H69AR will provide a useful model for the study of multidrug resistance in human small cell lung cancer.     

c-Ha-ras-I oncogene-induced differentiation and natural killer cell resistance in a human colorectal carcinoma cell line

Natural killer (NK) activity is primarily a peripheral blood function of a lymphocyte population capable of spontaneous lysis of many transformed and metastatic targets. However, NK-susceptible targets tend to be relatively poorly differentiated. We have previously shown that poorly differentiated human colorectal carcinoma are lysed by NK cells. Well-differentiated and chemically differentiated colorectal carcinomas are insensitive to NK lysis. The present study demonstrates that transfection of the c-Ha-ras-I oncogene into a poorly differentiated colorectal carcinoma cell line also renders it NK resistant. This resistance is accompanied by a more differentiated colorectal carcinoma phenotype. Two ras-transfected lines (Clone-A-5 and Clone-A-4) showed a 30-66% decrease in susceptibility to NK lysis as compared to the parental line in standard cytotoxicity assays. The resistance of these transfectants was strictly dependent on expression of the activated p21, the H-ras protein product. Studies to assess the integrity of the initial binding step in NK lysis showed a significant decrease in the ability of these transfectants to form conjugates with fresh NK cells. It is likely that transfection with c-Ha-ras-I has selectively modulated critical NK target recognition structures.     

Molecular mechanisms behind the resistance of cisplatin in germ cell tumours

Cisplatin has been one of the principal chemotherapy agents for the last 30 years and is still used widely in the treatment of testicular, ovarian, lung, head and neck, bladder and several other tumours. Resistance to chemotherapeutic agents is a major obstacle for successful treatment. Treatment effect on germ cell tumours (GCTs) is more successful than in adults suffering from almost any other solid tumour, but resistance still appears in 20% of patients with metastatic disease. However, because of the young age of patients and few data regarding the process of becoming resistant, this situation is still a challenge. In this review we are going to analyse the published literature on cisplatin resistance in GCTs and explain the initiatives that the Spanish Germ Cell Cancer Group (GG) is taking to try to elucidate the molecular mechanisms behind this process.     

辛伐他汀对NB4细胞分化与凋亡的影响

 目的　探讨磁性纳米Fe3O4颗粒（Fe3O4-MNP）联合多柔比星（ADM）对Raji细胞的影响。方法　采用MTT法检测细胞的增殖,锥虫蓝染色计数细胞的活性,流式细胞术检测细胞凋亡情况,Western blot检测p53和NF-κB的表达水平。结果　ADM及Fe3O4-MNP-ADM对Raji细胞的生长抑制率与药物浓度、作用时间呈正相关（r＝0.412,P＝0.027;r＝0.523,P＝0.014）;12、24、48 h细胞凋亡率二者分别为8.76 % 比 14.85 %、35.08 %比 44.50 %、44 %比69.4 %,差异有统计学意义（P＝0.012、0.041、0.024）;Western blot检测示ADM组与Fe3O4-MNP-ADM组NF-κB蛋白的灰度条带与内参比较分别为4.22±0.32、3.31±0.28,p53蛋白的条带灰度值分别为1.042±0.114、1.270±0.091,差异具有统计学意义（t＝-54.416,P＝0.035;t＝33.963,P＝0.047）。结论　Fe3O4-MNP联合ADM能够有效抑制Raji细胞增殖并诱导其凋亡,作用机制可能与增强p53蛋白活化、抑制NF -κB通路的激活有关。     

Isolation and characterization of a deoxycytidine kinase-deficient human promyelocytic leukemic cell line highly resistant to 1-beta-D- arabinofuranosylcytosine

A deoxycytidine kinase-deficient variant of a human promyelocytic leukemic cell line (HL-60/ara-C) has been isolated and characterized. These cells are capable of proliferating in the presence of 10(-6) M 1-beta-D-arabinofuranosylcytosine (ara-C), a level achieved in the plasma of leukemic patients undergoing conventional-dose ara-C therapy. The cells share numerous biological and biochemical features with the parent line, including: morphology; rate of growth; cloning characteristics; karyotype; rates of DNA, RNA, and protein synthesis; and ability to undergo terminal differentiation in the presence of agents such as 12-O-tetradecanoylphorbol acetate and dimethyl sulfoxide. In contrast, these cells display a great reduction in the total intracellular accumulation of ara-C following a 4-hr exposure to 10(-6) M ara-C (2.4 versus 99.0 pmol ara-C/10(6) cells). Resistant cells exposed to 10(-6) M ara-C for 1 hr also exhibited a reduction in the generation [1.2 versus 31.9 pmol 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP)/10(6) cells] and the 4-hr retention (0.30 versus 3.87 pmol ara-CTP/10(6) cells) of ara-CTP, the lethal ara-C metabolite, in comparison to parent cells. Incorporation of ara-C into resistant HL-60 cell DNA was also profoundly decreased. These biochemical alterations were associated with a 1000-fold decrease in the sensitivity of clonogenic cells to continuously administered ara-C (ara-C 50% inhibitory concentration: 1.8 X 10(-6) M for HL-60/ara-C; 3.0 X 10(-9) M for HL-60). A variety of antagonists of de novo pyrimidine synthesis inhibited the growth of ara-C-sensitive and -resistant cells to a similar extent. When HL-60 cells were exposed to a lethal concentration of thymidine (5 X 10(-3) M), coadministration of 5 X 10(-6) M deoxycytidine restored 90 +/- 4% (S.D.) of colony-forming capacity. Normal human bone marrow progenitor cells were protected to a similar degree by 3 X 10(-3) M deoxycytidine. In contrast, deoxycytidine concentrations as high as 5 X 10(-3) M were unable to confer any protection to HL-60/ara-C cells under identical conditions. These studies suggest that an enzymatic perturbation rendering human leukemic cells highly resistant to ara-C may be exploited to achieve a selective in vitro chemotherapeutic effect.     

Molecular mechanisms of cisplatin resistance

Platinum-based drugs, and in particular cis-diamminedichloroplatinum(II) (best known as cisplatin), are employed for the treatment of a wide array of solid malignancies, including testicular, ovarian, head and neck, colorectal, bladder and lung cancers. Cisplatin exerts anticancer effects via multiple mechanisms, yet its most prominent (and best understood) mode of action involves the generation of DNA lesions followed by the activation of the DNA damage response and the induction of mitochondrial apoptosis. Despite a consistent rate of initial responses, cisplatin treatment often results in the development of chemoresistance, leading to therapeutic failure. An intense research has been conducted during the past 30 years and several mechanisms that account for the cisplatin-resistant phenotype of tumor cells have been described. Here, we provide a systematic discussion of these mechanism by classifying them in alterations (1) that involve steps preceding the binding of cisplatin to DNA (pre-target resistance), (2) that directly relate to DNA-cisplatin adducts (on-target resistance), (3) concerning the lethal signaling pathway(s) elicited by cisplatin-mediated DNA damage (post-target resistance) and (4) affecting molecular circuitries that do not present obvious links with cisplatin-elicited signals (off-target resistance). As in some clinical settings cisplatin constitutes the major therapeutic option, the development of chemosensitization strategies constitute a goal with important clinical implications.