双组分核定位信号介导Apoptin定位于肿瘤细胞核

Apoptin是一种来源于鸡贫血病毒的小蛋白,在肿瘤细胞中定位于细胞核,而在正常细胞中主要分布于细胞质。根据预测,Apoptin分子中有2段序列(NLS1和NLS2)可能是单组分核定位信号。通过基因突变和缺失构建了Apoptin各种不同的核定位信号突变体和磷酸化突变体,利用增强型绿色荧光蛋白(EGFP)作标签,观察了其在肿瘤细胞中亚细胞定位的变化。结果表明,NLS1和NLS2单独均不是有效的单组分核定位信号。Apoptin的核定位信号是由NLS1和NLS2这2段序列共同组成的双组分核定位信号,缺少任何一段序列都会严重影响Apoptin在肿瘤细胞中的核定位。其中,NLS2对于Apoptin的核定位起主要作用。Apoptin的获得型磷酸化突变体并不能转位到正常细胞的细胞核中,而其磷酸化负突变体仍定位于肿瘤细胞的细胞核。另外,丝氨酸／苏氨酸蛋白激酶抑制剂H7也不影响Apoptin在肿瘤细胞中的核定位。很可能,Apoptin的磷酸化并不参与调控其核定位信号的功能。     

Role of mitochondrial cardiolipin peroxidation in apoptotic photokilling of 5-aminolevulinate-treated tumor cells

In 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT), ALA taken up by tumor cells is metabolized to protoporphyrin IX (PpIX), which sensitizes photodamage leading to apoptotic or necrotic cell death. Since lipophilic PpIX originates in mitochondria, we postulated that photoperoxidation of highly unsaturated cardiolipin (CL), which anchors cytochrome c (cyt c) to the inner membrane, is an early proapoptotic event. As initial evidence, PpIX-sensitized photooxidation of liposomal CL to hydroperoxide (CLOOH) species precluded cyt c binding, but this could be reinstated by GSH/selenoperoxidase (GPX4) treatment. Further support derived from site-specific effects observed using (i) a mitochondrial GPX4-overexpressing clone (7G4) of COH-BR1 tumor cells, and (ii) an ALA treatment protocol in which most cellular PpIX is either inside (Pr-1) or outside (Pr-2) mitochondria. Sensitized cells were exposed to a lethal light dose, and then analyzed for death mechanism and lipid hydroperoxide (LOOH) levels. Irradiated Pr-1 vector control (VC) cells died apoptotically following cyt c release and caspase-3 activation, whereas 7G4 cells were highly resistant. Irradiated Pr-2 VC and 7G4 cells showed negligible cyt c release or caspase-3 activation, and both types died via necrosis. CLOOH (detected long before cyt c release) accumulated approximately 70% slower in Pr-1 7G4 cells than in Pr-1 VC, and this slowdown exceeded that of all other LOOHs. These and related findings support the hypothesis that CL is a key upstream target in mitochondria-dependent ALA-PDT-induced apoptosis.     

重组抗体工程及其在肿瘤靶向及癌症治疗中的应用

在过去的十几年中,重组抗体工程在基础研究、医学和药物生产上已经成为最有希望的领域之一。重组抗体及其片段在正在进行诊断和治疗的临床试验中占所有生物蛋白的30％以上。研究集中在抗体作为理想的癌症靶向试剂方面,最近由于FDA批准使用第一个工程化治疗抗体而使热度达到极点。过去的几年中,在设计、筛选及生产新型工程化抗体方面已经取得了重大进展。改革的筛选方法已经能够分离出高亲和力的癌－靶向及抗病毒的抗体,后能够抑制病毒用于基因治疗。癌症诊断和治疗的另一个策略是将重组抗体片段与放射性同位素、药物、毒素、酶以及生物传感器表面进行融合。双特异性抗体及相关融合蛋白也已经生产出来用于癌症的免疫治疗,在抗癌疫苗以及T细胞补充策略上有效地增强了人免疫应答。     

ERV3 human endogenous provirus mRNAs are expressed in normal and malignant tissues and cells, but not in choriocarcinoma tumor cells

Messenger RNA expression of a human endogenous provirus, ERV3, has been characterized in 170 specimens of normal and malignant human tissues and cells. In contrast to the high expression in first-trimester and full-term placental chorionic villi, most other human tissues expressed ERV3 mRNAs at a level of 2-30% of placenta. However, ERV3 mRNAs were not detected in choriocarcinoma tumor cell lines. These studies suggest that the ERV3 provirus may have been preempted for a biological function and disruption of its mRNA expression results in choriocarcinoma.     

Recombinant adenovirus encoding H-ras ribozyme induces apoptosis in laryngeal cancer cells through caspase- and mitochondria-dependent pathways

Previously, we designed a ribozyme that targets the H-ras oncogene at the 12th codon mutation site (Chang et al., 1997). Ribozymes have antisense molecule and site-specific ribonuclease potential. In this study, an adenoviral vector was used to transduce the H-ras ribozyme into laryngeal cancer cells (HEp-2). This served to downregulate the H-ras gene expression in which this ribozyme performed antisense activity due to HEp-2 cells containing wild-type alleles in the 12th H-ras codon. Together, our data demonstrated that the recombinant adenovirus encoding H-ras ribozyme can be broadly regarded as a cytotoxic gene therapy in laryngeal cancer cells regardless of containing wild-type or mutant ras gene. In addition, the mechanism through which the H-ras ribozyme inhibited tumor growth was apoptosis and involved both caspase- and mitochondria-mediated pathways. The activators caspase-8 and -9 as well as the effector caspase-3 in the induction phase of apoptosis and the substrate PARP of caspase-3 in the execution phase were activated 48h following the H-ras ribozyme treatment. Mitochondrial events characterized by the production of superoxide anion and the release of cytochrome c started at 24h. Mitochondrial transmembrane potential loss occurred 48h after the ribozyme treatment. However, Bcl-2 delayed cytochrome c release to the cytosol, but it could not protect the apoptosis effect, suggesting that cytochrome c release from mitochondria may not play a role in H-ras ribozyme-induced apoptosis.     

Inhibition of the PI3 kinase/Akt pathway enhances doxorubicin-induced apoptotic cell death in tumor cells in a p53-dependent manner

Constitutive activation of the PI3 kinase/Akt pathway is associated with the neoplastic phenotype of a large number of human tumor cells. As the anti-apoptotic role of the PI3 kinase/Akt pathway has been established, we have examined whether specific blockade of this pathway sensitizes tumor cells to DNA-damaging agent-induced cytotoxicity by enhancing apoptotic cell death. Although a PI3 kinase inhibitor, LY294002, by itself does not induce apoptotic cell death, LY294002 selectively and markedly enhances the apoptosis-inducing efficacy of doxorubicin: such an enhanced cell death is only detected in tumor cells in which the PI3 kinase/Akt pathway is constitutively activated, and it is totally dependent on the functional p53 pathway. These results suggest that the combination of a PI3 kinase/Akt pathway inhibitor and doxorubicin provides an efficient chemotherapeutic strategy for the treatment of tumor cells in which the PI3 kinase/Akt pathway is constitutively activated and the p53 pathway is functional.     

Ectopic expression of von Hippel-Lindau tumor suppressor induces apoptosis in 786-O renal cell carcinoma cells and regresses tumor growth of 786-O cells in nude mouse

The von Hippel-Lindau (VHL) is a known tumor suppressor that binds to alpha-subunits of hypoxia-inducible factors and induces ubiquitin-mediated degradation of the protein in an oxygen-dependent manner. VHL is also involved in the regulation of tumor angiogenesis, glycolysis, cell cycle regulation, and apoptosis. In the present study, we showed that ectopic expression of VHL induces apoptosis in renal cell carcinoma 786-O cells which contain only the mutant VHL, evidenced by TUNEL assay and DAPI staining. Furthermore, biochemical studies indicated that expression of VHL in 786-O cells results in both PARP and CPP32 cleavage, suggesting that VHL-induced apoptosis in 786-O cells is caspase dependent. Moreover, we also observed that apoptosis induced by ectopic VHL expression was associated with up-regulation of p27 as well as Bax, implicating the roles of these two proteins in VHL-induced apoptosis. The up-regulation of p27 and Bax by VHL was specific since we did not detect any changes in the level of other apoptotic factors including Fas and Bcl2 by the expression of VHL. We next examined the effect of VHL expression on the tumor growth of 786-O renal cell carcinoma cells in nude mouse. The results showed that injection of Ad.VHL adenovirus regresses the tumor growth of 786-O cells in nude mouse. The analysis by TUNEL assay as well as DAPI staining of 786-O tumors injected with Ad.VHL showed clear evidence of apoptosis. These results suggest that ectopic VHL expression induces apoptotic response in 786-O VHL mutant cells both in vitro and in vivo.     

Activation of p53-dependent responses in tumor cells treated with a PARC-interacting peptide

We tested the activity of a p53 carboxy-terminal peptide containing the PARC-interacting region in cancer cells with wild type cytoplasmic p53. Peptide delivery was achieved by fusing it to the TAT transduction domain (TAT-p53-C-ter peptide). In a two-hybrid assay, the tetramerization domain (TD) of p53 was necessary and sufficient to bind PARC. The TAT-p53-C-ter peptide disrupted the PARC-p53 complex. Peptide treatment caused p53 nuclear relocation, p53-dependent changes in gene expression and enhancement of etoposide-induced apoptosis. These studies suggest that PARC-interacting peptides are promising candidates for the enhancement of p53-dependent apoptosis in tumors with wt cytoplasmic p53.     

Bystander cell killing spreading from endothelial to tumor cells in a three-dimensional multicellular nodule model after Escherichia coli nitroreductase gene delivery

Tumor cells are elusive targets for standard anticancer chemotherapy due to their heterogeneity and genetic instability. On the other hand, proliferating host endothelial cells (ECs) are genetically stable and have a low mutational rate. Thus, antiangiogenic therapy directed against tumor's ECs should, in principle, improve the efficacy of antitumor therapy by inducing little or no drug resistance. Here we present a gene-directed enzyme prodrug therapy (GDEPT) strategy for targeting the tumor vasculature, using the Escherichia coli nitroreductase (ntr) gene delivery associated with the treatment with the prodrug CB1954. In a first time we demonstrated the ability of the ntr/CB1954 system to induce an apoptotic-mediated cell death on monolayer cultures of human umbilical vein ECs (HUV-EC-C). Then, when ntr-transfected HUV-EC-C cells (HUV-EC-C/ntr(+)) were associated in a three-dimensional (3-D) multicellular nodule model with untransfected B16-F10 murine melanoma cell line, we observed a CB1954-mediated bystander cell killing effect from endothelial to neighboring melanoma cells. To our knowledge, this is the first report indicating that GDEPT-based antiangiogenic targeting may be an effective approach for cancer treatment relied on the spreading of the bystander effect from endothelial to tumor cells.     

CD4<Superscript>+</Superscript> T cells kill Id<Superscript>+</Superscript> B-lymphoma cells: FasLigand-Fas interaction is dominant in vitro but is redundant in vivo

B-lymphoma cells express a highly tumor-specific antigen, monoclonal Ig, which is a promising target for immunotherapy. Previous work has demonstrated that B-lymphoma cells spontaneously process their endogenous monoclonal Ig and present variable (V) region peptides (Id-peptides) on their MHC class II molecules to CD4⁺ T cells. Id-specific CD4⁺ T cells protect mice against B-lymphoma cells in the absence of anti-idiotypic antibodies. The molecular mechanism by which Id-specific CD4⁺ T cells kill B-lymphoma cells is hitherto unknown. We here demonstrate in an Id-specific T-cell receptor (TCR)–transgenic mouse model that Id-specific CD4⁺ T cells induce apoptosis of Fas⁺ B-lymphoma cells in vitro by FasLigand (FasL)–Fas interaction. Moreover, the rare B lymphomas that had escaped rejection in TCR-transgenic mice had down-regulated their sensitivity to Fas-mediated apoptosis. Although these results suggest that FasL-Fas interaction is important, Id-specific CD4⁺ T cells could eliminate Id⁺ B-lymphoma cells in vivo by other mechanisms, since three independent ways of blocking FasL-Fas–mediated killing failed to abrogate tumor protection in TCR-transgenic mice. These results suggest that there are several redundant pathways by which Id-specific CD4⁺ T cells eliminate Id⁺ B-lymphoma cells in vivo, of which FasL-Fas interaction is only one.Supported by grants from the Norwegian Cancer Society, the Research Council of Norway, and the Multiple Myeloma Research Foundation.     

Apoptotic and necrotic cell death are both induced by electroporation in HL60 human promyeloid leukaemia cells

Cell death was induced by electroporation in HL60 cells, a human promyeloid leukaemia strain, in order to determine by both morphological and biochemical criteria whether necrotic or apoptotic processes occurred. Cells sampled at several times after electroporation were analyzed for the assessment of the following end-points: (i) chromosomal DNA fragmentation; (ii) cell viability; (iii) mono- and oligonucleosomes in the cytoplasmic fraction; (iv) apoptotic index; and (v) morphology of treated cells. The results indicate that about 50% of the cells killed by electroporation die through necrosis, while the remaining 50% of the cells undergo apoptosis. Chromosome damage was also studied by cytogenetic analysis at metaphase. The possibility of killing tumour cells by electroporation, as a variant of electrotherapy, constitutes, in our opinion, a promising procedure in cancer therapy, avoiding the undesirable side effects normally derived from treatment with cytotoxic drugs.     

Effects of karanjin on cell cycle arrest and apoptosis in human A549, HepG2 and HL-60 cancer cells

Background

We have investigated the potential anticancer effects of karanjin, a principal furanoflavonol constituent of the Chinese medicine Fordia cauliflora, using cytotoxic assay, cell cycle arrest, and induction of apoptosis in three human cancer cell lines (A549, HepG2 and HL-60 cells).

Results

MTT cytotoxic assay showed that karanjin could inhibit the proliferation and viability of all three cancer cells. The induction of cell cycle arrest was observed via a PI (propidium iodide)/RNase Staining Buffer detection kit and analyzed by flow cytometry: karanjin could dose-dependently induce cell cycle arrest at G2/M phase in the three cell lines. Cell apoptosis was assessed by Annexin V-FITC/PI staining: all three cancer cells treated with karanjin exhibited significantly increased apoptotic rates, especially in the percentage of late apoptosis cells.

Conclusion

Karanjin can induce cancer cell death through cell cycle arrest and enhance apoptosis. This compound may be effective clinically for cancer pharmacotherapy.     

Release of iC3b from apoptotic tumor cells induces tolerance by binding to immature dendritic cells in vitro and in vivo

Chemo- as well as immunotherapeutical approaches induce apoptosis in tumor cells. Apoptotic cells are known to activate homologous complement and to be opsonized with iC3b. Since maturation of dendritic cells (DC) can be inhibited by binding of iC3b to the complement receptor 3 (CR3, CD11b/CD18) and because immature DC induce tolerance, we investigated the induction of tolerance after pulsing DC with apoptotic cells in the presence or absence of native serum. Apoptosis in pancreatic carcinoma cells was induced either by heat-stress, chemotherapy or anti-Her2 antibody. Monocyte-derived DC were pulsed with apoptotic cells with or without native serum. DC were analyzed for the maturation state by flow cytometry and the cytotoxic activity was determined. Tolerance was prevented by addition of substances such as anti CD11b or N–acetyl-D-Glucosamine (NADG) which block iC3b binding to CR3. Furthermore, binding of iC3b from apoptotic cells to DC was blocked in a syngeneic pancreatic carcinoma mouse model. All of the former strategies for apoptosis induction resulted in iC3b release. Pulsing DC with apoptotic cells in the presence of serum prevents maturation of DC and induces finally tolerance. This tolerance could be prevented almost completely by blocking the interaction of iC3b with the CR3 receptor. This could be shown as well in an immunocompetent mouse model. Chemo- as well as immunotherapeutical approaches induce apoptosis in tumor cells. Release of iC3b from apoptotic tumor cells prevents fully maturation of DC and immature DC induce antigen-specific silencing or tolerance. Blocking of iC3b-binding could mostly prevent this effect.     

Apoptin® specifically causes apoptosis in tumor cells and after UV-treatment in untransformed cells from cancer-prone individuals: A review

Tumor formation is caused by an imbalance between cell replication and apoptosis, which is a physiological form of cell death. For instance, UV damage can result in tumor formation due to mutations of the tumor-suppressor gene p53, a major apoptosis-inducing protein. Over-expression of the proto-oncogene Bcl-2, due to chromosomal translocation, can also inhibit apoptosis resulting in, e.g., lymphomas and leukemias. Anti-tumor therapies are often based on induction of apoptosis mediated via p53 and/or inhibited by Bcl-2, which explains the frequently poor results of anti-tumor treatment. The avian-virus-derived protein ‘Apoptin’, induces apoptosis in a p53-independent way, is stimulated by Bcl-2 and is insensitive to BCR-ABL, another inhibitor of chemotherapeutic agents. Apoptin induces apoptosis in human transformed/tumorigenic cells but not in normal diploid cells. Co-synthesis of SV40 large T antigen and Apoptin results in induction of apoptosis, illustrating that the establishment of a stable transformed state is not required. UV-irradiation causes an aberrant SOS-response in primary diploid cells from cancer-prone individuals and renders such cells susceptible to Apoptin-induced apoptosis. All these features make Apoptin a potential candidate as a therapeutic and diagnostic tool in cancer treatment.     

The extrinsic and intrinsic apoptotic pathways are involved in manganese toxicity in rat astrocytoma C6 cells

Manganese (Mn) is a trace element known to be essential for maintaining the proper function and regulation of many biochemical and cellular reactions. However, chronic exposure to high levels of Mn in occupational or environmental settings can lead to its accumulation in the brain resulting in a degenerative brain disorder referred to as Manganism. Astrocytes are the main Mn store in the central nervous system and several lines of evidence implicate these cells as major players in the role of Manganism development. In the present study, we employed rat astrocytoma C6 cells as a sensitive experimental model for investigating molecular mechanisms involved in Mn neurotoxicity. Our results show that C6 cells undergo reactive oxygen species-mediated apoptotic cell death involving caspase-8 and mitochondrial-mediated pathways in response to Mn. Exposed cells exhibit typical apoptotic features, such as chromatin condensation, cell shrinkage, membrane blebbing, caspase-3 activation and caspase-specific cleavage of the endogenous substrate poly (ADP-ribose) polymerase. Participation of the caspase-8 dependent pathway was assessed by increased levels of FasL, caspase-8 activation and Bid cleavage. The involvement of the mitochondrial pathway was demonstrated by the disruption of the mitochondrial membrane potential, the opening of the mitochondrial permeability transition pore, cytochrome c release, caspase-9 activation and the increased mitochondrial levels of the pro-apoptotic Bcl-2 family proteins. In addition, our data also shows for the first time that mitochondrial fragmentation plays a relevant role in Mn-induced apoptosis. Taking together, these findings contribute to a deeper elucidation of the molecular signaling mechanisms underlying Mn-induced apoptosis.     

Melatonin effect on the ultrastructure of Ehrlich ascites tumor cells,lifetime and histopathology in Swiss mice

Aims

One of the models used for studying cancer is the Ehrlich ascites tumor (EAT) due to its ability to grow in liquid suspension, allowing a standard number of cells to be inoculated, growth quantification and regression of tumor mass. Among the oncostatic substances, melatonin has shown effectiveness in limiting the tumor cell proliferation. However, studies have shown contradictory effects of melatonin on the EAT. This study has investigated the melatonin effect on tumor growth, time and survival percentage, ultrastructure and metastasis of EAT cells in mice submitted or not to pinealectomy.

Main methods

Animals were inoculated with 5 × 10⁶ cells/mL and treated or not with exogenous melatonin with doses of at 150 and 300 μg/30 g animal weight for 12 days. Melatonin significantly reduced the abdominal circumference, volume of ascites liquid and EAT-cell viability, raising rates of time and mice survival percentage.

Key findings

Ultrastructurally, the melatonin treatment revealed changes in the shape of cells, the cell surface showed numerous projections, some bifurcated, cytoplasmic vacuolation, mitochondrial degeneration and nuclear fragmentation, peculiar characteristics of apoptosis. Histopathology revealed no metastasis in the liver, small intestine and large intestine in any of the animals in the experimental groups; however this process was evident in the lungs and kidneys, being inhibited by melatonin administration.

Significance

Thus, we can conclude that doses of 150 and 300 μg/30 g of melatonin for 12 consecutive days have a very effective oncostatic and cytotoxic activity on EAT cells in mice.     

Ceramide promotes the death of human cervical tumor cells in the absence of biochemical and morphological markers of apoptosis

C8-ceramide, a synthetic cell-permeable analog of endogenous ceramides, interfered with cell proliferation, and was cytotoxic to papilloma virus-containing human cervix carcinoma cells, CALO, INBL, and HeLa, that match two clinical stages of tumor progression. C8-ceramide (3 microM) markedly reduced the tumor cell number after 48 h of treatment, an effect that endured even after the removal of C8-ceramide. The carcinoma cells showed morphologic changes, characteristic of necrosis and released lactate dehydrogenase (LDH). A biologically inactive analog C8-dihydro-ceramide had no effect on cell viability in any of the cell lines tested. Seventy-two hours after C8-ceramide treatment none of the biochemical and morphological markers characteristic of apoptosis: (a) nuclear chromatin condensation, (b) DNA fragmentation, (c) proteolysis of the caspase-3 substrate poly-(ADP-ribose)-polymerase (PARP), and (d) appearance of phosphatidylserine on the external cell membrane, were observed. C8-ceramide had no effect on human cervix fibroblasts and induced a mild reduction (30%) in the proliferation of normal human cervix epithelia and HeLa cells (IV-B metastatic stage). The cytotoxicity of C8-ceramide was restricted to CALO (early II-B) and INBL (IV-A non-metastatic) carcinoma cells. The possible application of ceramide in the treatment of early stages of cervical cancer is discussed.     

番茄红素对骨肉瘤大鼠后肢移植瘤及移植瘤细胞凋亡的影响

目的探讨番茄红素对骨肉瘤大鼠后肢移植瘤及移植瘤细胞凋亡的影响。方法将30日龄1级Wistar大鼠20只,于大鼠后肢股骨远端的骨髓腔中注射骨肉瘤细胞,建立大鼠后肢移植瘤及移植瘤动物模型,并分为对照组和实验组。两组均常规喂养,实验组按100mg／kg食物添加番茄红素,观察番茄红素对大鼠健康状况、体重变化及移植瘤变化的影响,并用TUNEI。方法检测其对移植瘤细胞凋亡的影响。结果番茄红素对大鼠健康状况及体重变化无明显影响（P〉0．05）;对移植瘤的生长有抑制作用,肿瘤抑制率为74．5％（P〈0．05）;对移植瘤细胞的凋亡有促进作用（P〈0．05）。结论番茄红素可能通过促进肿瘤细胞凋亡等途径对骨肉瘤产生抑制作用,成为骨肉瘤化学预防及治疗的辅助手段。     

Lupulone, a hop bitter acid, activates different death pathways involving apoptotic TRAIL-receptors, in human colon tumor cells and in their derived metastatic cells

Our study aimed to compare death signalling pathways triggered by lupulone in TRAIL-sensitive human colon cancer cells (SW480) and in their derived TRAIL-resistant metastatic cells (SW620). Lupulone (40 μg/ml) up-regulated expression of TRAIL DR4/DR5 death receptors at the cell surface of both cell lines, even in the absence of exogenous TRAIL ligand. Cell death induced by lupulone was inhibited in SW480 and SW620 cells exposed to blocking anti-DR4/DR5 antibodies. In SW480 cells, lupulone triggered cell death through a cross-talk between TRAIL-DR4/DR5 and the mitochondrial (intrinsic) pathways involving caspase-8 activation and Bid protein cleavage. As a consequence mitochondrial cytochrome c was released into the cytosol and activation of caspases-9 and -3 was observed. In the metastatic SW620 cells, lupulone restored the sensibility of these cells to TRAIL ligand and activated the extrinsic apoptotic pathway via DR4/DR5 death receptors and the involvement of the caspase-8/caspase-3 cascade. The demonstration that lupulone is able to activate TRAIL-death signalling pathways even in TRAIL resistant cancer cells highlights the potential of this natural compound for cancer prevention and therapy.     

Changes in the free-radical state and the level of free iron during the development of drug resistance in tumor cells

The free-radical state of K562 human erythroleukemia cells changes during the development of resistance to doxorubicin, an antitumor antibiotic with prooxidant action widely used in oncology. It was found that the level of superoxide anion in the resistant cells decreased. The addition of doxorubicin to the culture medium induced a much smaller increase in O ₂ ^? generation in the resistant cells than in the sensitive cells. Again, the semiquinone-type EPR signal with a g-factor of 2.006 considerably decreased in the resistant cells grown without doxorubicin as compared with the sensitive cells under the same conditions. The EPR study has shown that the level of paramagnetic nitrosyl complexes of nonheme iron in the resistant cells decreased, which indicates that the content of free nonheme iron declined in development of drug resistance. In addition, we have found with the use of RT-PCR that the level of mRNA of the transferrin receptor decreased in the resistant cells. The data suggest that the suppression of free-radical processes during the development of resistance of K562 cells to doxorubicin is a coordinated redox-dependent adaptive response.