Adriamycin resistance is characterized by ultrastructural changes in human leukaemic K562 cells in vitro.

Ultrastructural changes associated with adriamycin (ADM) resistance have been investigated in the human K562 leukaemic cell line: sensitive K562 cells, a resistant subline cultured in the continuous presence of ADM and resistant cells without ADM Transmission electron microscopy (TEM) study revealed that K562-resistant cells displayed ultrastructural modifications of the cell surface, chromatin and nucleolus conformation. Alterations were not directly related to the presence of adriamycin as deprivated cells exhibited modificated characters through a slow progressive recovery phenomenon.     

Interaction between radiation and drug damage in mammalian cells. IV. Radiation response of adriamycin-resistant V79 cells

Adriamycin-resistant variants derived from V79 Chinese hamster cells were examined for their radiation response properties. A stable resistant cell line (77A) demonstrated a significant reduction in the extrapolation number of the single-dose radiation survival curve. Second-step mutants from 77A cells exhibited a spectrum of radiation response states including decreased D0 values and large extrapolation numbers. A highly Adriamycin-resistant line (LZ) was found to be radiation sensitive with increased capacity for the accumulation of sublethal radiation injury. LZ cells are known to contain double-minute chromosomes and an amplified gene for the multidrug phenotype and to exhibit multidrug resistant properties. These cells require the presence of Adriamycin in their growth medium to maintain their pleiotropic characteristics. LZ cells became more resistant to radiation following reversion to an intermediate Adriamycin response as the consequence of growth in Adriamycin-free medium. Reverted cells also lost their large capacity for sublethal damage. It is suggested that detailed study of these mutants may provide insight into the identification of radiation-sensitive sites and their relationship to the genetic changes characterizing Adriamycin-resistant cell lines.     

Thioltransferase overexpression increases resistance of MCF-7 cells to adriamycin

Thioltransferase, a small redox protein with thiol-disulfide oxidoreductase and dehydroascorbate reductase activities, has been reported to be expressed at higher levels in Adriamycin-resistant MCF-7 human breast tumor cells (MCF-7 ADR(R)) when compared with Adriamycin sensitive MCF-7 WT (MCF-7 WT) cells. The present study examined the effects of stably transfecting MCF-7 WT cells with the cDNA for human thioltransferase and the effects of subsequent Adriamycin cytotoxicity in the MCF-7 WT transfected cells. All transfected cell lines overexpressing thioltransferase activity were more resistant to Adriamycin than untransfected MCF-7 WT cells, supporting the hypothesis that increases in thioltransferase expression are related to Adriamycin resistance. This resistance was independent of the ability of thioltransferase to catalyze reduction of dehydroascorbic acid to ascorbic acid, as the addition of an ascorbate generating derivative, L-ascorbic acid-2-phosphate, to the media did not additionally increase Adriamycin resistance.     

Nrf2/HO-1/HMGB1信号通路在白血病细胞K562/A02化疗耐药中的机制研究

目的:通过观察高迁移率族蛋白1(HMGB1)、转录因子NF-E2相关因子2(Nrf2)及血红素加氧酶1(HO-1)基因沉默对白血病化疗耐药细胞(K562/A02细胞株)的影响,探讨该信号通路在白血病化疗耐药中的作用及其可能机制。方法:将HMGB1基因、Nrf2基因及HO-1基因的特异性干扰RNA分别转染阿霉素耐药细胞株K562/A02,荧光实时定量(RT-PCR)方法检测HMGB1、Nrf2及HO-1的mRNA表达水平,Western blot方法检测HMGB1、Nrf2及HO-1的蛋白表达水平,免疫荧光方法检测Nrf2的蛋白表达,并使用CCK-8方法检测转染前后K562/A02细胞株的细胞活性。结果:HMGB1基因、Nrf2基因或HO-1基因沉默的K562/A02细胞活性皆显著低于对照组及空白组(P0.05),化疗敏感性恢复。结论:HMGB1高表达导致了白血病细胞株K562/A02对阿霉素的化疗耐药,Nrf2/HO-1信号通路参与了HMGB1诱导的K562/A02细胞的化疗耐药,其表达上调可恢复K562/A02细胞对阿霉素的敏感性。     

Evidence for sialidase activity in K 562 cells: inhibition by adriamycin treatment

Sialidase activity has been studied in the human erythroleukemia K 562 cell line grown in vitro. The total sialidase activity was determined using disialoganglioside GD1a and fetuin as exogenous substrates. The enzymatic activity was stimulated by 0.08% Triton X-100 and reached the highest level at pH 4.0. Results obtained showed that gangliosides are hydrolysed more extensively than glycoproteins by K 562 sialidases. This finding could suggest that endogenous gangliosides may be the main source of metabolically available sialic acid in K 562 cell line. After treatment of K 562 cells by Adriamycin (40 nM), a potent anticancer drug, sialidase activity decreased by 40% as compared to control cells. This decrease occurs early during the first day of incubation with Adriamycin. This inhibition of sialidase activity could explain previous results obtained in our laboratory which show an enhanced sialylation of the membrane glycoconjugates after Adriamycin treatment.     

Demonstration of the cell cycle positions of taxol-induced "asters" and "bundles" by sequential measurements of tubulin immunofluorescence, DNA content, and autoradiographic labeling of taxol-sensitive and -resistant cells

We used reliable and relatively inexpensive equipment to make sequential sets of measurements of antitubulin immunofluorescence, Feulgen staining, and autoradiography on the same cells. This was done to evaluate tubulin conformations, DNA content, and [3H]-thymidine incorporation in cell lines sensitive (HL60) and resistant (K562) to the novel anti-tubulin chemotherapeutic agent taxol. Numbers of cells with microtubule bundles have been found to correlate with sensitivity to taxol by clonogenic assay for several leukemic cell lines. We have found that cells with "asters" produced by taxol exposure are in mitosis and that cells with taxol-induced "bundles" are in G0/G1, S, and G2 phases. We further found that S-phase cells with microtubule bundles in both sensitive (HL60) and resistant (K562) cell lines were able to incorporate [3H]-thymidine after 4-hr exposure to taxol. As microtubule bundles and asters occur in cells of the same cell cycle phases in both lines, we conclude that the greater frequency of cells with microtubule bundles reported for sensitive cells after taxol treatment cannot result from drug exclusion nor from different effects of the drug on cell microtubules in these two leukemic lines.     

Downregulation of Bcr-Abl in K562 cells restores susceptibility to apoptosis: characterization of the apoptotic death

We examined the susceptibility of a variety of human leukemic cell lines to the induction of apoptosis. K562, a chronic myelogenous leukemic cell line which expresses the bcr-abl fusion gene, was found to be extremely resistant to apoptosis, irrespective of the inducing agent. This resistance can be attributed to the deregulated Abl kinase activity of bcr-abl, as downregulation of its expression using antisense oligodeoxynucleotides targeted to the beginning of the abl sequence in this chimeric gene rendered these cells susceptible to cytotoxic drug-induced apoptosis. Examination of the morphological and biochemical features of apoptosis in K562 cells revealed the typical membrane blebbing and chromatin condensation associated with this form of cell death. In situ TdT-mediated end labeling of the DNA revealed the presence of strand breaks in the treated cells and field inversion gel electrophoresis revealed the presence of large 10-50 kb fragments. However there was an absence of oligonucleosomal DNA fragmentation, whether or not Bcr-Abl was expressed. Thus, while inhibition of expression of Bcr-Abl renders K562 cells susceptible to apoptosis, the absence of oligonucleosomal DNA fragmentation in these cells is independent of the function of this molecule.     

Potential mechanisms of leukemia cell resistance to TRAIL-induced apopotosis

There are many factors contributing to the resistance to TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand)-induced apoptosis. However, it is not clear whether the mechanism of resistance to TRAIL is constitutive or inductive. Therefore, the purpose of this study was to investigate the resistant mechanisms to TRAIL at different levels in the apoptotic pathway. The human T-lymphoblastic leukemic CEM cell line showed more resistant to TRAIL-induced apoptosis compared with the human chronic myeloid leukemic K562 cell line. Lower level of constitutive caspase-8 expression in the CEM cell line led to a poor response to both TRAIL-induced activation of caspase-3 and reduction in the mitochondrial membrane potential (m). There was no significant difference in the constitutive levels of NF-B in CEM and K562 cell lines. However, CEM cells showed a faster response to TRAIL-induced NF-B activation than K562 cells. TRAIL-induced regulation of Bcl-2 family of proteins included an up-regulation in Bcl-2/Bcl-XL and a down-regulation in Bax. IAPs, such as XIAP, cIAP-1, cIAP-2 and Survivin were all up-regulated during the treatment with TRAIL. In summary, our data suggest that the leukemic cells resistance to TRAIL-induced apoptosis might be due to the deficiency in the constitutive caspase-8 expression. Development of potential resistance to apoptosis by TRAIL can occur in both TRAIL-resistant and TRAIL-sensitive leukemic cells.     

神经酰胺诱导药物敏感型和耐药型细胞凋亡机制的探讨

以药物敏感型细胞株Ｋ５６２／Ｓ和耐药型细胞株Ｋ５６２／Ａ０２为对象．观察原癌基因Ｂｃｌ－２的表达量在两种细胞中的差异,以及神经酰胺作为一个新的脂质第二信使诱导细胞凋亡的能力,并利用酪氨酸激酶抑制剂ｇｅｎｉｓｔｅｉｎ,酪氨酸磷酸酯酶抑制剂ｖａｎａｄａｔｅ,观察酪氨酸可逆磷酸化与细胞凋亡间的关系．结果显示：在Ｋ５６２／Ａ０２中Ｂｃｌ－２的表达量明显高于Ｋ５６２／Ｓ;外源性神经酰胺能成功地诱导Ｋ５６２／Ｓ,Ｋ５６２／Ａ０２细胞凋亡,凋亡细胞具有典型的形态学改变和ＤＮＡ“Ｌａｄｄｅｒ”形成,ＦＣＭ检测出现凋亡细胞峰,但在同样的诱导条件下,Ｋ５６２／Ｓ细胞凋亡明显高于Ｋ５６２／Ａ０２细胞．ＦＣＭ检测ｇｅｎｉｓｔｅｉｎ能显著改变这两种细胞生长周期,但细胞阻滞于Ｇ２／Ｍ期,便对神经酰胺诱导的细胞凋亡无明显作用,ｖａｎａｄａｔｅ单独对细胞地明显作用,但与神经酰胺共同作用能明显提高细胞凋亡率．以上结果表明在药物诱导的细胞调亡中Ｂｃｌ－２基因起重要作用,神经酰胺能诱导Ｋ５６２／Ｓ和Ｋ５６２／Ａ０２细胞调亡．     

Involvement of miR-21 in resistance to daunorubicin by regulating PTEN expression in the leukaemia K562 cell line

Recent studies have shown microRNA-21 (miR-21) is overexpressed in several types of cancer and contributes to tumor resistance to chemotherapy. In this study, we investigated whether miR-21 mediated resistance of the leukaemia cell line K562 to the chemotherapeutic agent daunorubicin (DNR). miR-21 expression was upregulated in the DNR resistant cell line K562/DNR compared to its parental line K562. Stable transfection of miR-21 induced drug resistance in K562, while suppression of miR-21 in K562/DNR led to enhanced DNR cytotoxicity. Additional experiments indicate that the mechanism of miR-21 drug resistance involves the PI3K/Akt pathway and changes following PTEN protein expression. This study provides a novel mechanism for understanding leukaemia drug resistance.     

Protoplast-mediated gene transfer into human leukemia (K562) cells

We have examined the usefulness of a protoplast fusion technique as a tool to transfer cloned genes into hematopoietic cells. Protoplasts carrying cloned plasmids, which would express specific markers when successfully transfected into human cells, were prepared and fused with human leukemic cell line K562 cells using polyethylene glycol as a fusogenic factor. As a result, K562 cells fused with protoplasts containing a plasmid pSV2-cat constructed to code for chloramphenicol acetyltransferase (CAT) expressed CAT activity efficiently. K562 cells were also readily transformed to geneticin-(G418) resistant cells following fusion with protoplasts carrying a plasmid pSV2-neo-SV-gpt, which confers the resistance of mammalian cells to G418 and mycophenolic acid. It was also demonstrated that the plasmid genome was stably integrated into the chromosomal DNA of G418-resistant K562 cells. Our results proved that protoplast fusion could be used to study the specific expression and the biologic activities of cloned genes in human hematopoietic cells.     

Different mechanisms account for the relative resistance of KG-1 and HL-60 cell lines to retrovirus infection.

I infected three different human leukemic cell lines (K562, KG-1, and HL-60) with an amphotropic retrovirus vector (designated PA317/N2) which confers G418 resistance and contains the Moloney murine leukemia virus long terminal repeat. Compared with K562 cells, both KG-1 and HL-60 cells were relatively resistant to infection with this retrovirus vector. In HL-60 cells, this resistance appeared to result from diminished viral DNA synthesis, while in KG-1 cells there was a block to the genomic integration of the viral DNA.     

P-糖蛋白在多药耐药的K562细胞转运苯妥英纳与卡马西平中的作用

研究证实,多药转运体与难治性癫痫耐药机制密切相关,P-糖蛋白在其中起重要作用．主要研究P-糖蛋白拮抗剂维拉帕米对P-糖蛋白过表达的K562细胞耐药性及细胞内苯妥英纳与卡马西平浓度的影响．首先建立了P-糖蛋白高表达的K562/Dox(阿霉素诱导)耐药细胞株,比较耐药细胞株和P-糖蛋白表达阴性的K562细胞株对苯妥英纳和卡马西平的耐药性,并观察给予维拉帕米后,耐药细胞内抗癫痫药物的浓度变化．结果发现,苯妥英纳和卡马西平对K562/Dox细胞株的半数抑制浓度(IC₅₀)明显高于K562细胞株,加入维拉帕米后,苯妥英纳和卡马西平对K562/Dox 细胞的IC₅₀明显下降,逆转倍数分别为2.5和1.5．进一步研究发现,K562/Dox细胞内苯妥英纳和卡马西平的浓度均显著少于其药敏K562细胞,仅分别为正常K562细胞的23.6%和32.2%．当加入维拉帕米后,K562/Dox细胞内抗癫痫药物浓度明显升高(P < 0.05)．由此证明,高表达的P-糖蛋白参与了细胞的药物转运,在难治性癫痫的耐药机制中扮演重要角色．     

Early human decidual cells exhibit NK activity against the K562 cell line but not against first trimester trophoblast

The susceptibility of cultured first trimester human trophoblast cells to lysis by NK cells has been studied. Trophoblast cells are resistant to lysis by NK cells from both peripheral blood and decidua. However, decidual cells extracted by enzymatic disaggregation do exhibit cytotoxicity against the NK-sensitive cell line K562. The relevance of these findings to successful implantation of the blastocyst is discussed.     

Apaf-1XL is an inactive isoform compared with Apaf-1L

Apaf-1 plays a crucial role in the cytochrome c/dATP-dependent activation of caspase-9 and -3. We found that the human myeloid leukemic K562 cells were more resistant to cytochrome c-induced activation of caspase-9 and -3 in a cell-free system compared with the human T-lymphoblastic subclone CEM/VLB(100) cells. Apaf-1 cDNA sequencing revealed an additional insert of 11 aa between the CARD and CED-4 (ATPase) domains in K562 cells, which was identical to the sequence of Apaf-1XL. Immunoprecipitation of Apaf-1 with caspase-9 after a cell-free reaction demonstrated that Apaf-1XL in the K562 cell line showed a lower binding ability to caspase-9 compared with Apaf-1L protein. The resistance of K562 cells to cytochrome c-dependent apoptosis may be partly due to this Apaf-1XL form. These results suggest that the additional insert between CARD and CED-4 domains might affect Apaf-1 recruitment of caspase-9 during apoptosis.     

cDNA microarray analysis of altered gene expression in Ara-C-treated leukemia cells

The acute lymphoblastic leukemia cell line CCRF-CEM is sensitive to Ara-C and undergoes apoptosis. In contrast, the chronic myelogenous leukemia (CML) cell line K562 is highly resistant to Ara-C, which causes the cells to differentiate into erythrocytes before undergoing apoptosis. We used cDNA microarrays to monitor the alterations in gene expression in these two cell lines under conditions leading to apoptosis or differentiation. Ara-C-treated CCRF-CEM cells were characterized by a cluster of down-regulated chaperone genes, whereas Ara-C-treated K562 cells were characterized by a cluster of up-regulated hemoglobin genes. In K562 cells, Ara-C treatment induced significant down-regulation of the asparagine synthetase gene, which is involved in resistance to L-asparaginase. Sequential treatment with Ara-C and L-asparaginase had a synergistic effect on the inhibition of K562 cell growth, and combination therapy with these two anticancer agents may prove effective in the treatment of CML, which cannot be cured by either drug alone.     

Assessment of multidrug resistance reversal using dielectrophoresis and flow cytometry

In cancer, multidrug resistance (MDR) is the simultaneous resistance of tumor cells to different natural product anticancer drugs that have no common structure. This is an impediment to the successful treatment of many human cancers. A common correlate of MDR is the overexpression of a membrane protein, P-glycoprotein. Many studies have shown that MDR can be reversed after the use of substrate analogs, called MDR modulators. However, our understanding of MDR modulation is incomplete. In this article, we examine the electrical properties of the human leukemic cells (K562) and its MDR counterpart (K562AR) using dielectrophoresis and flow cytometry (with a membrane potential sensitive dye, DIOC5), both before and after treatment with XR9576 (a P-glycoprotein-specific MDR-reversal agent). The results show significant differences in the cytoplasmic conductivity between the cell lines themselves, but indicate no significant changes after modulation therapy. We conclude that the process of MDR modulation is not associated with changes in the electrical properties of cancer cells. Moreover, the results demonstrate that using the flow cytometry method alone, with MDR cells, may produce artifactual results--whereas in combination with dielectrophoresis, the results show the role of MDR modulators in preventing drug efflux in MDR cells.     

Rescue of K562 cells from MDM2-modulated p53-dependent apoptosis by growth factor-induced differentiation

The wild-type human MDM2 protooncogene was tested for its ability to modulate apoptotic activity of the de novo expressed p53 tumor suppressor gene in K562 cells. We also studied the role of some cytokines in this phenomenon. K562, a human myeloid leukemia cell line, does not express p53 at the mRNA or protein level. In this study, we stably transfected K562 with eukaryotic vectors containing either normal p53 cDNA (pC53-SN3) or mutated p53 (143Val-->Ala) cDNA (pC53-SCX3). Transfectants expressing WT p53 or those expressing mutant p53 are called K562 SN and K562 SM respectively. Many leukemic cell lines undergo apoptosis when de novo WT p53 is expressed alone. In contrast, while the resulting clones (K562 SN and K562 SM) expressed p53, they did not undergo apoptosis. However, when treated with MDM2 mRNA antisense (MDM2 AS) oligodeoxynucleotides (ODNs), K562 SN demonstrated apoptotic features at both molecular and morphological levels. No change was observed when the other clones (K562 and K562 SM) were treated with MDM2 AS. Apoptosis induced in this manner was associated with a relatively small increase in intracellular calcium [Ca2+]i. Cells cultured in medium previously supplemented with recombinant human (rh) interleukin (IL)-3 and rh-erythropoietin (Epo) did not undergo apoptosis. Moreover, K562 SN cells were induced to differentiate. This differentiation was evaluated by measuring hemoglobin (Hb) level in cellular extracted proteins and by analyzing erythroid colony number and morphology. High Hb synthesis was obtained when K562 SN cells were cultured with cytokines (IL-3 + Epo) combined with MDM2 AS. Our results are consistent with the hypothesis that the function of the proto-oncogene MDM2 is to provide a 'feedback' mechanism for the p53-dependent pathway of apoptosis that could be shunted toward differentiation.     

Inhibitory effect of cinnamoyl compounds against human malignant cell line

In the present study, anti-proliferative effects of dietary polyphenolic compounds have been observed and demonstrated the strong anticancer efficacy of curcumin (CMN), an active constituent of dietary spice (turmeric) using human leukemia cancer cell line. CMN inhibited the proliferation of K562 leukemic cells by induction of apoptosis. The current study demonstrated synergy with combination of drug therapy, and suggested that combination of ferulic acid and cisplatin synergistically inhibited cellular proliferation. Cytotoxic synergy was observed independent of the sequence of addition of two drugs to cultured cells. The synergized growth inhibitory effect with cisplatin was probably associated with G2-M arrest in cell cycle progression. These findings suggested that among the cinnamoyl compounds, CMN was most potent and FER appeared to be a better modulating agent on human malignant cell line.     

Response of chemosensitive and chemoresistant leukemic cell lines to drug therapy: simultaneous assessment of proliferation, apoptosis, and necrosis

BACKGROUND: The balance between cell proliferation and drug-induced cell death by apoptosis or necrosis plays a major role in determining response to chemotherapy. Commonly-used DNA analysis methods cannot study both parameters simultaneously. A new approach described here combines a green fluorescent membrane-intercalating dye (PKH67) with Hoechst 33342 or annexin V and propidium iodide, to allow simultaneous assessment of cell division, cell cycle status, apoptosis, and necrosis, respectively. METHODS: To test this approach, we used cultured K562 leukemic cell lines which are drug-sensitive (K562S) or drug-resistant (K562R) by virtue of whether they lack or exhibit expression, respectively, of the gp-170 (PGP) glycoprotein pump involved in multidrug resistance. RESULTS: We found that: 1) PKH67 fluorescence intensity decreases proportionately to number of cell divisions, 2) labeling with PKH67 does not alter either cell cycle distribution, as assessed by vital DNA staining with Hoechst 33342, or cell growth, and 3) using a simple threshold analysis method suitable for real-time sorting decisions, subpopulations of proliferating cells present at initial levels of >/= 10% can readily be detected after two cell division times, based on decreased PKH67 intensity. Finally, we demonstrated that after treatment of an admixture of K562S and K562R with vincristine, triple-labeling with PKH67, annexin V, and propidium iodide can be used to identify and sort those cells which remain not only viable (nonnecrotic, nonapoptotic) but actively dividing (decreased PKH67 intensity) in the presence of drug. CONCLUSIONS: Although the studies described here were carried out in a model system using cells having known drug resistance phenotypes, we expect that the methods described will be useful in ex vivo studies of clinical leukemic specimens designed to identify the role played by specific chemoresistance proteins and mechanisms in therapeutic outcomes for individual patients.