Development of a Double-Antigen Microsphere Immunoassay for Simultaneous Group and Serotype Detection of Bluetongue Virus Antibodies.

Bluetongue viruses (BTV) are arboviruses responsible for infections in ruminants. The confirmation of BTV infections is based on rapid serological tests such as enzyme-linked immunosorbent assays (ELISAs) using the BTV viral protein 7 (VP7) as antigen. The determination of the BTV serotype by serological analyses could be only performed by neutralization tests (VNT) which are time-consuming and require BSL3 facilities. VP2 protein is considered the major serotype-defining protein of BTV. To improve the serological characterization of BTV infections, the recombinant VP7 and BTV serotype 8 (BTV-8) VP2 were synthesized using insect cells expression system. The purified antigens were covalently bound to fluorescent beads and then assayed with 822 characterized ruminant sera from BTV vaccinations or infections in a duplex microsphere immunoassay (MIA). The revelation step of this serological duplex assay was performed with biotinylated antigens instead of antispecies conjugates to use it on different ruminant species. The results demonstrated that MIA detected the anti-VP7 antibodies with a high specificity as well as a competitive ELISA approved for BTV diagnosis, with a better efficiency for the early detection of the anti-VP7 antibodies. The VP2 MIA results showed that this technology is also an alternative to VNT for BTV diagnosis. Comparisons between the VP2 MIA and VNT results showed that VNT detects the anti-VP2 antibodies in an early stage and that the VP2 MIA is as specific as VNT. This novel immunoassay provides a platform for developing multiplex assays, in which the presence of antibodies against multiple BTV serotypes can be detected simultaneously.

BRCA1 haplotype and clinical benefit of trabectedin in soft-tissue sarcoma patients.

BACKGROUND: This study aimed to determine whether the BRCA1 haplotype was associated with trabectedin efficacy in soft-tissue sarcoma (STS) patients. METHODS: We analysed BRCA1 single-nucleotide polymorphisms (SNPs) in tumour specimens from 135 advanced STS patients enrolled in published phase 2 trials or in a compassionate-use programme of trabectedin. Forty-four advanced STS patients treated with doxorubicin and 85 patients with localised STS served as controls. The 6-month nonprogression rate and overall survival (OS) were analysed according to BRCA1 haplotype using log-rank tests. RESULTS: A favourable BRCA1 haplotype (presence of at least one AAAG allele) was significantly associated with an improved 6-month nonprogression rate. It was the only variable significantly associated with OS. No correlations were found between outcomes for patients with localised or advanced STS treated with doxorubicin. CONCLUSIONS: The BRCA1 haplotype represents a potential DNA repair biomarker that can be used for the prediction of response to trabectedin in STS patients.

Antiproliferative activity of ecteinascidin 743 is dependent upon transcription-coupled nucleotide-excision repair.

While investigating the novel anticancer drug ecteinascidin 743 (Et743), a natural marine product isolated from the Caribbean sea squirt, we discovered a new cell-killing mechanism mediated by DNA nucleotide excision repair (NER). A cancer cell line selected for resistance to Et743 had chromosome alterations in a region that included the gene implicated in the hereditary disease xeroderma pigmentosum (XPG, also known as Ercc5). Complementation with wild-type XPG restored the drug sensitivity. Xeroderma pigmentosum cells deficient in the NER genes XPG, XPA, XPD or XPF were resistant to Et743, and sensitivity was restored by complementation with wild-type genes. Moreover, studies of cells deficient in XPC or in the genes implicated in Cockayne syndrome (CSA and CSB) indicated that the drug sensitivity is specifically dependent on the transcription-coupled pathway of NER. We found that Et743 interacts with the transcription-coupled NER machinery to induce lethal DNA strand breaks.

Characterization of a novel topoisomerase I mutation from a camptothecin-resistant human prostate cancer cell line.

In this study, we characterized the structure and function of topoisomerase I (top1) protein in the camptothecin (CPT)-resistant prostate cancer cell lines, DU-145/RC0.1 and DU-145/RC1 (RC0.1 and RC1, respectively). Both of the cell lines were previously selected by continuous exposure to 9-nitro-CPT. The RC0.1 and RC1 cells have high cross-resistance to CPT derivatives including SN-38 and topotecan, but are not cross-resistant to the non-top1 inhibitors etoposide, doxorubicin, and vincristine. Although the top1 protein levels were not decreased in the resistant cells compared with the parental cells, CPT-induced DNA cleavage was markedly reduced in the RC0.1 and RC1 nuclear extracts. The resistant-cell-line nuclear extracts also demonstrated top1 catalytic activity and resistance to CPT, in in vitro assays. Reverse transcription-PCR products from the resistant cell lines were sequenced, and revealed a point mutation resulting in a R364H mutation in the top1 of both RC0.1 and RC1. No wild-type top1 RNA or genomic DNA was detected in the resistant cell lines. Using a purified recombinant R364H top1, we found that the R364H mutant top1 was CPT resistant and fully active. In the published top1 crystal structure, the R364H mutation is close to the catalytic tyrosine and other well-known mutations leading to CPT resistance.

Topoisomerase I-mediated cytotoxicity of N-methyl-N'-nitro-N-nitrosoguanidine: trapping of topoisomerase I by the O6-methylguanine.

Alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) are known to covalently link alkyl groups at the position 6 of guanines (O6MG) in DNA. O6-alkylguanine-DNA alkyltransferase (AGT) specifically removes the methyl group of the O6MG. Using purified human topoisomerase I (Top1), we found an 8-10-fold enhancement of Top1 cleavage complexes when O6MG is incorporated in oligonucleotides at the +1 position relative to a unique Top1 cleavage site. Top1 poisoning by O6MG is attributable to a decrease of the Top1-mediated DNA religation as well as an increase in the enzyme cleavage step. Increased cleavage is probably linked to a change in the hydrogen bonding pattern, such as in the case of the 8-oxoguanine, whereas inhibition of religation could be attributed to altered base pairing, such as abasic sites or base mismatches, because incorporation of a 6-thioguanine did not affect Top1 activity. Top1-DNA covalent complexes are also induced in MNNG-treated CHO cells constitutively lacking the AGT enzyme. Conversely, no increase could be detected in CHO cells transfected with the wild-type human AGT. Moreover, we show that yeasts overexpressing the human Top1 are more sensitive to MNNG, whereas knock-out Top1 strain cells display some resistance to the drug. Altogether, these results suggest a role for Top1 poisoning by alkylated bases in the antiproliferative activity of alkylating agents as well as in the DNA lesions resulting from endogenous and carcinogenic DNA modifications.

Topoisomerase I-mediated DNA damage.

Topoisomerase I is a ubiquitous and essential enzyme in multicellular organisms. It is involved in multiple DNA transactions including DNA replication, transcription, chromosome condensation and decondensation, and probably DNA recombination. Besides its activity of DNA relaxation necessary to eliminate torsional stresses associated with these processes, topoisomerase I may have other functions related to its interaction with other cellular proteins. Topoisomerase I is the target of the novel anticancer drugs, the camptothecins. Recently a broad range of physiological and environmentally-induced DNA modifications have also been shown to poison topoisomerases. This review summarizes the various factors that enhance or suppress top1 cleavage complexes and discusses the significance of such effects. We also review the different mechanisms that have been proposed for the repair of topoisomerase I-mediated DNA lesions.

Substitutions of Asn-726 in the active site of yeast DNA topoisomerase I define novel mechanisms of stabilizing the covalent enzyme-DNA intermediate.

Eukaryotic DNA topoisomerase I (Top1p) catalyzes changes in DNA topology and is the cellular target of camptothecin. Recent reports of enzyme structure highlight the importance of conserved amino acids N-terminal to the active site tyrosine and the involvement of Asn-726 in mediating Top1p sensitivity to camptothecin. To investigate the contribution of this residue to enzyme catalysis, we evaluated the effect of substituting His, Asp, or Ser for Asn-726 on yeast Top1p. Top1N726S and Top1N726D mutant proteins were resistant to camptothecin, although the Ser mutant was distinguished by a lack of detectable changes in activity. Thus, a basic residue immediately N-terminal to the active site tyrosine is required for camptothecin cytotoxicity. However, replacing Asn-726 with Asp or His interfered with distinct aspects of the catalytic cycle, resulting in cell lethality. In contrast to camptothecin, which inhibits enzyme-catalyzed religation of DNA, the His substituent enhanced the rate of DNA scission, whereas the Asp mutation diminished the enzyme binding of DNA. Yet, these effects on enzyme catalysis were not mutually exclusive as the His mutant was hypersensitive to camptothecin. These results suggest distinct mechanisms of poisoning DNA topoisomerase I may be explored in the development of antitumor agents capable of targeting different aspects of the Top1p catalytic cycle.

Induction of topoisomerase I cleavage complexes by 1-beta -D-arabinofuranosylcytosine (ara-C) in vitro and in ara-C-treated cells.

1-beta-d-Arabinofuranosylcytosine (Ara-C) is a nucleoside analog commonly used in the treatment of leukemias. Ara-C inhibits DNA polymerases and can be incorporated into DNA. Its mechanism of cytotoxicity is not fully understood. Using oligonucleotides and purified human topoisomerase I (top1), we found a 4- to 6-fold enhancement of top1 cleavage complexes when ara-C was incorporated at the +1 position (immediately 3') relative to a unique top1 cleavage site. This enhancement was primarily due to a reversible inhibition of top1-mediated DNA religation. Because ara-C incorporation is known to alter base stacking and sugar puckering at the misincorporation site and at the neighboring base pairs, the observed inhibition of religation at the ara-C site suggests the importance of the alignment of the 5'-hydroxyl end for religation with the phosphate group of the top1 phosphotyrosine bond. This study also demonstrates that ara-C treatment and DNA incorporation trap top1 cleavage complexes in human leukemia cells. Finally, we report that camptothecin-resistant mouse P388/CPT45 cells with no detectable top1 are crossresistant to ara-C, which suggests that top1 poisoning is a potential mechanism for ara-C cytotoxicity.

Benzo[a]pyrene diol epoxide adducts in DNA are potent suppressors of a normal topoisomerase I cleavage site and powerful inducers of other topoisomerase I cleavages.

The catalytic intermediates of DNA topoisomerase I (top1) are cleavage complexes that can relax DNA supercoiling (intramolecular reaction) or mediate recombinations (intermolecular religation). We report here that DNA adducts formed from benzo[a]pyrene bay-region diol epoxides can markedly affect top1 activity. Four oligonucleotide 22-mers of the same sequence were synthesized, each of which contained a stereoisomerically unique benzo[a]pyrene 7, 8-diol 9,10-epoxide adduct at the 2-amino group of a central 2'-deoxyguanosine residue. These four adducts correspond to either cis or trans opening at C-10 of the (+)-(7R, 8S, 9S, 10R)- or (-)-(7S, 8R, 9R, 10S)-7,8-diol 9,10-epoxides. Their solution conformations in duplex DNA (intercalated and minor-groove bound for the cis and trans opened adducts respectively) can be deduced from previous NMR studies. All four adducts completely suppress top1 cleavage activity at the alkylation site and induce the formation of new top1cleavage complexes on both strands of the DNA 3-6 bases away from the alkylation site. The trans opened adduct from the highly carcinogenic (+)-diol epoxide is the most active in inducing top1 cleavage independently of camptothecin, demonstrating that minor groove alkylation can efficiently poison top1. We also found that this isomer of the diol epoxide induces the formation of top1-DNA complexes in mammalian cells, which suggests a possible relationship between induction of top1 cleavage complexes and carcinogenic activity of benzo[a]pyrene diol epoxides.

DNA protein cross-links produced by NSC 652287, a novel thiophene derivative active against human renal cancer cells.

2, 5-bis(5-Hydroxymethyl-2-thienyl)furan (NSC 652287), is a representative of a series of thiophene derivatives that exhibit potent and selective antitumor activity against several tumor cell lines in the National Cancer Institute Anticancer Drug Screen. NSC 652287 has noticeable activity for the renal cell lines and produces cures in certain corresponding xenografts. The cellular mechanisms of action of NSC 652287 were therefore investigated in this study in greater detail. The most sensitive renal carcinoma cell line, A498, exhibited cell cycle arrest in G(0)-G(1) and G(2)-M at 10 nM NSC 652287, with increased p53 and p21(WAF1) protein. At higher concentrations, NSC 652287 still induced p53 elevation but with p21(WAF1) reduction and massive apoptosis. These results collectively suggested that NSC 652287 induced DNA damage. Using alkaline elution techniques, we found that NSC 652287 induced both DNA-protein and DNA-DNA cross-links with no detectable DNA single-strand breaks. These DNA-protein cross-links (DPC) persisted for at least 12 h after drug removal and their frequency was correlated with cytotoxicity in the renal cell lines studied. The most sensitive cells (A498) produced the highest DPC followed by the cell line with intermediate sensitivity (TK-10). DPC were minimal in the two resistant cell lines, ACHN and UO-31. Nonetheless, a similar degree of DPC occurred at doses imparting equitoxic effects. These results indicate that DNA is a primary target for the novel and potent anticancer thiophene derivative, NSC 652287. NSC 652287 did not cross-link purified DNA or mammalian topoisomerase I suggesting the importance of active metabolite(s) for the cross-linking activity.

Topoisomerase poisoning activity of novel disaccharide anthracyclines.

Doxorubicin and idarubicin are very effective anticancer drugs in the treatment of human hematological malignancies and solid tumors. These agents are well known topoisomerase II poisons; however, some anthracycline analogs recently have been shown to poison topoisomerase I. In the present work, we assayed novel disaccharide analogs and the parent drug, idarubicin, for their poisoning effects of human topoisomerase I and topoisomerases IIalpha and IIbeta. Drugs were evaluated with a DNA cleavage assay in vitro and with a yeast system to test whether the agents were able to poison the enzymes in vivo. We have found that the test agents are potent poisons of both topoisomerases IIalpha and IIbeta. The axial orientation of the second sugar relative to the first one of the novel disaccharide analogs was shown to be required for poisoning activity and cytotoxicity. Interestingly, idarubicin and the new analogs stimulated topoisomerase I-mediated DNA cleavage at low levels in vitro. As expected, the cytotoxic level of the drug was highly affected by the content of topoisomerase II; nevertheless, the test agents had a yeast cell-killing activity that also was weakly dependent on cellular topoisomerase I content. The results are relevant for the full understanding of the molecular mechanism of topoisomerase poisoning by anticancer drugs, and they define structural determinants of anthracyclines that may help in the rational design of new compounds directed against topoisomerase I.

Poisoning of human DNA topoisomerase I by ecteinascidin 743, an anticancer drug that selectively alkylates DNA in the minor groove.

Ecteinascidin 743 (Et743, National Service Center 648766) is a potent antitumor agent from the Caribbean tunicate Ecteinascidia turbinata. Although Et743 is presently in clinical trials for human cancers, the mechanisms of antitumor activity of Et743 have not been elucidated. Et743 can alkylate selectively guanine N2 from the DNA minor groove, and this alkylation is reversed by DNA denaturation. Thus, Et743 differs from other DNA alkylating agents presently in the clinic (by both its biochemical activities and its profile of antitumor activity in preclinical models). In this study, we investigated cellular proteins that can bind to DNA alkylated by Et743. By using an oligonucleotide containing high-affinity Et743 binding sites and nuclear extracts from human leukemia CEM cells, we purified a 100-kDa protein as a cellular target of Et743 and identified it as topoisomerase I (top1). Purified top1 was then tested and found to produce cleavage complexes in the presence of Et743, whereas topoisomerase II had no effect. DNA alkylation was essential for the formation of top1-mediated cleavage complexes by Et743, and the distribution of the drug-induced top1 sites was different for Et743 and camptothecin. top1-DNA complexes were also detected in Et743-treated CEM cells by using cesium chloride gradient centrifugation followed by top1 immunoblotting. These data indicate that DNA minor groove alkylation by Et743 induces top1-mediated protein-linked DNA breaks and that top1 is a target for Et743 in vitro and in vivo.

Human DNA topoisomerase I-mediated cleavage and recombination of duck hepatitis B virus DNA in vitro.

In this study, we report that eukaryotic topoisomerase I (top1) can linearize the open circular DNA of duck hepatitis B virus (DHBV). Using synthetic oligonucleotides mimicking the three-strand flap DR1 region of the DHBV genome, we found that top1 cleaves the DNA plus strand in a suicidal manner, which mimics the linearization of the virion DNA. We also report that top1 can cleave the DNA minus strand at specific sites and can linearize the minus strand via a non-homologous recombination reaction. These results are consistent with the possibility that top1 can act as a DNA endo-nuclease and strand transferase and play a role in the circularization, linearization and possibly integration of viral replication intermediates.

Gadd45, a p53-responsive stress protein, modifies DNA accessibility on damaged chromatin.

This report demonstrates that Gadd45, a p53-responsive stress protein, can facilitate topoisomerase relaxing and cleavage activity in the presence of core histones. A correlation between reduced expression of Gadd45 and increased resistance to topoisomerase I and topoisomerase II inhibitors in a variety of human cell lines was also found. Gadd45 could potentially mediate this effect by destabilizing histone-DNA interactions since it was found to interact directly with the four core histones. To evaluate this possibility, we investigated the effect of Gadd45 on preassembled mononucleosomes. Our data indicate that Gadd45 directly associates with mononucleosomes that have been altered by histone acetylation or UV radiation. This interaction resulted in increased DNase I accessibility on hyperacetylated mononucleosomes and substantial reduction of T4 endonuclease V accessibility to cyclobutane pyrimidine dimers on UV-irradiated mononucleosomes but not on naked DNA. Both histone acetylation and UV radiation are thought to destabilize the nucleosomal structure. Hence, these results imply that Gadd45 can recognize an altered chromatin state and modulate DNA accessibility to cellular proteins.

Topoisomerase I and II Activity Assays.

DNA topoisomerases I and II (top1 and top2, respectively) are ubiquitous enzymes that play an essential role in transcription, replication, chromosome segregation, and DNA repair. The basic enzymatic reaction of topoisomerases, namely reversible DNA nicking, is a transesterification reaction where a DNA phosphodiester bond is transferred to a specific enzyme tyrosine residue. Eukaryotic top1 and top2 exhibit major differences concerning their mechanism of action. Top1 acts as a monomer and forms a covalent bond with the 3'-terminus of a DNA single-strand break (1-3) whereas top2 acts as an homodimer and forms a covalent bond with the 5'-terminus of the DNA double-strand break with a four base-pairs overhang (Fig. 1) (1-4). No energy cofactor is required for top1 activity, whereas top2 hydrolyzes adenosine triphosphate (ATP) during its catalytic cycle. Fig. 1. Top1- and top2-cleavage complexes. (A) Top1 acts as a monomer, makes a single-strand break and covalently binds to the 3'-end of the break, leaving a 5'-hydroxyl end. (B) Top2 acts as a dimer, and generally makes a double-strand break. Each strand is cleaved by one monomer, with a 4-base overhang. Each monomer covalently binds to the 5'-end of the break and leaves a 3'-hydroxyl end.

Induction of topoisomerase I cleavage complexes by the vinyl chloride adduct 1,N6-ethenoadenine.

We used purified mammalian topoisomerases I (top1) and oligonucleotides to study top1-mediated cleavage and religation in the presence of a potent carcinogenic adduct, 1,N6-ethenoadenosine (epsilonA) incorporated immediately downstream of a unique top1 cleavage site. We found tha epsilonA markedly enhanced top1 cleavage complexes when it was incorporated at the +1 position of the top1 cleavage. This enhancement was due to a reduction of the religation step of the top1 reaction. In addition, epsilonA reduced the top1-mediated cleavage and decreased binding of the enzyme to DNA. We also studied the effects of the epsilonA adduct on top1 trapping by camptothecin (CPT), a well known top1 inhibitor. CPT was inactive when epsilonA was present at the +1 position. Alkylation of the top1 cleavage complex by 7-chloromethyl-10,11-methylenedioxycamptothecin (7-ClMe-MDO-CPT) was also blocked by the epsilonA adduct. Altogether, these results demonstrate that the epsilonA carcinogenic adduct can efficiently trap human top1 and mimic CPT effects. Normal hydrogen bonding of the base pairs immediately downstream from the top1 cleavage site is probably essential for efficient DNA religation and binding of camptothecins in the top1 cleavage complex.

Mechanism of action of eukaryotic DNA topoisomerase I and drugs targeted to the enzyme.

DNA topoisomerase I is essential for cellular metabolism and survival. It is also the target of a novel class of anticancer drugs active against previously refractory solid tumors, the camptothecins. The present review describes the topoisomerase I catalytic mechanisms with particular emphasis on the cleavage complex that represents the enzyme's catalytic intermediate and the site of action for camptothecins. Roles of topoisomerase I in DNA replication, transcription and recombination are also reviewed. Because of the importance of topoisomerase I as a chemotherapeutic target, we review the mechanisms of action of camptothecins and the other topoisomerase I inhibitors identified to date.

Doxorubicin-induced alterations of c-myc and c-jun gene expression in rat glioblastoma cells: role of c-jun in drug resistance and cell death.

We studied the effect of doxorubicin on the expression of c-myc and c-jun in the rat glioblastoma cell line C6 and its doxorubicin-resistant variant C6 0.5, at equitoxic exposures. For quantitation, the mRNA levels of these oncogenes were related to those of two domestic genes, beta-actin and glyceraldehyde phosphate dehydrogenase. After a transient overexpression of the genes during the first hour of incubation, there was a selective, dose-dependent down-regulation of both genes by doxorubicin in the sensitive cells. In the resistant cell line, c-myc expression was also decreased in response to doxorubicin incubation, but the expression of c-jun remained unchanged over the whole range of concentrations. In contrast, vincristine had no effect on the amounts of c-myc and c-jun mRNAs in either line. The effect of doxorubicin on the mRNA levels of c-jun was also observed on the JUN proteins by immunoblotting, but the MYC protein levels remained unchanged upon doxorubicin treatment. There was a significant correlation between the levels of c-myc and c-jun gene expression and the degree of growth inhibition induced by doxorubicin. In addition, doxorubicin induced a fragmentation of DNA in sensitive cells, but not in resistant cells, thus revealing a resistance to apoptosis in this line. Doxorubicin-induced cell death did not appear to be mediated by p53 in either cell line.

Purification and characterization of a Mg2+-dependent endonuclease (AN34) from etoposide-treated human leukemia HL-60 cells undergoing apoptosis.

An important biochemical hallmark of apoptosis is the cleavage of chromatin into oligonucleosomal fragments. Here, we purified a Mg2+-dependent endonuclease from etoposide-treated HL-60 cells undergoing apoptosis. High levels of Mg2+-dependent endonuclease activity were detected in etoposide-treated HL-60 cells, and this activity increased in a time-dependent manner following etoposide treatment. Such an activity could not be detected in untreated cells or in cells treated with etoposide in the presence of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-(OMe)-fluoromethyl ketone (zVAD-fmk) or the serine protease inhibitor tosyl-L-phenylalanine chloromethyl ketone (TPCK). This Mg2+-dependent endonuclease was purified by a series of chromatographic procedures. The enzyme preparation showed a single major protein band with Mr 34,000, determined by SDS-PAGE. The presence of the Mr 34,000 Mg2+-dependent endonuclease was also confirmed by activity gel analysis. The enzyme required only Mg2+ for full activity. pH optimum was in the range of 6.5-7.5. This enzyme introduced single- and double-strand breaks into SV40 DNA and produced internucleosomal DNA cleavage in isolated nuclei from untreated cells. The DNA breaks were terminated with 3'-OH, consistent with characteristic products of apoptotic chromatin fragmentation. We propose to designate this Mr 34,000 Mg2+-dependent endonuclease AN34 (apoptotic nuclease Mr 34,000).

Transcriptional down-regulation of c-myc expression in an erythroleukemic cell line, K562, and its doxorubicin-resistant variant by two topoisomerase II inhibitors, doxorubicin and amsacrine.

We have evaluated the effect of two topoisomerase II (Topo II) poisons, amsacrine and doxorubicin, on the expression of the c-myc oncogene, both at the mRNA and protein levels, in the leukemia cell line, K562, and its doxorubicin-resistant counterpart, K562 DoxR. We report in this study a concentration-dependent decrease in c-myc mRNA levels upon exposure of both cell lines to amsacrine and doxorubicin, with a more pronounced effect for amsacrine in the resistant line. In either case, c-myc down-regulation closely paralleled the drug-induced growth inhibition. We have also used the technique of PCR stop-assay to detect the occurrence of DNA breaks within the P2 promoter of the c-myc gene. We have shown that Topo II-mediated breaks induced by amsacrine are probably responsible for the down-regulation of c-myc in the resistant line. In addition, amsacrine induced apoptosis only in the resistant line while doxorubicin did not induce apoptosis in any cell line. These results suggest that c-myc is not involved in the resistance of K562 DoxR cells, but can induce the apoptosis pathway in these cells, while no drug-induced apoptosis could be detected in the sensitive line.