"DNA clearing" from non-covalently bound agents in mammalian cells as a new mechanism of drug resistance.

Earlier, we have described the process of active dissociation or "DNA clearing" from non-covalently bound agents in living mammalian cells. The vital fluorescent bisbenzimidazole dye Hoechst 33342, which binds DNA in the minor groove tightly but non-covalently, was used for studying the interaction of non-covalently binding agents with DNA. Multiple drug resistance (MDR) in tumour cells is related to the expression of transport proteins that alter the cellular drug transport and distribution. Three different groups of genes (mdr, MRP, and LRP) and their products are implicated in MDR (A. Krishan, C. M. Fitz, and I. Andritsch, Cytometry 29:279-285 (1997)). To obtain new cell lines characterized by enhanced process of active dissociation of non-covalently bound agents from DNA or "DNA clearing", we carried out step-by-step selection with increasing concentrations of Hoechst 33342. The rodent cell lines hyperresistant to Hoechst 33342 and selected from AA8 were named AA8Hoe-R-1-AA8Hoe-R-10, and the cell lines selected from L cells were called LHoe-R-1-LHoe-R-10. The most resistant of them, AA8Hoe-R-6 and AA8Hoe-R-7, were able to grow in the presence of 80 microm/ml of Hoechst 33342 in the cell culture medium. All mutants were analyzed with the flow cytometric technique and were divided into two different groups. We conclude that the drug resistance of the first group of cell lines was due to changes in transport proteins. The second group of the resistant cell lines was characterized by an enhanced dissociation of the bisbenzimidazole dye-DNA complex. As we believe, the enhanced level of "DNA clearing" was caused by the amplification of some genes, because the gradual increase of Hoechst resistance in the same cell line resulted from the increase in the ability to remove the dye from DNA. These lines were shown to be also resistant to netropsin.     

A new human breast carcinoma cell line resistant to DNA-damaging drugs

To investigate the phenomenon of active dissociation of the vital dye, Hoechst 33342 (Ho342), from DNA (DNA clearing), a new MCF7HoeR-7 human breast carcinoma cell line was isolated from parent MCF7 cells by step-wise selection with increasing concentrations of Ho342. This cell line possesses an enhanced ability for DNA clearing. The MCF7HoeR-7 line is characterised in detail and compared with the parental MCF7 line and a typical P-glycoprotein-mediated multidrug resistant (MDR) cell line, MCF7/Adr. MCF7HoeR-7 cells have an increased population growth rate, a lower DNA content and a reduced number of chromosomes. Enhanced DNA clearing in MCF7HoeR-7 cells is associated with the high resistance of the cells to the toxic effects of Ho342 and cross-resistance to etoposide, a topoisomerase II inhibitor in clinical use. The MCF7HoeR-7 and parent MCF7 cell lines have similar expression levels of transport proteins. The results obtained confirm that DNA clearing is an atypical MDR mechanism in tumour cells.     

Binding of gemini bisbenzimidazole drugs with human telomeric G-quadruplex dimers: effect of the spacer in the design of potent telomerase inhibitors

The study of anticancer agents that act via stabilization of telomeric G-quadruplex DNA (G4DNA) is important because such agents often inhibit telomerase activity. Several types of G4DNA binding ligands are known. In these studies, the target structures often involve a single G4 DNA unit formed by short DNA telomeric sequences. However, the 3'-terminal single-stranded human telomeric DNA can form higher-order structures by clustering consecutive quadruplex units (dimers or n-mers). Herein, we present new synthetic gemini (twin) bisbenzimidazole ligands, in which the oligo-oxyethylene spacers join the two bisbenzimidazole units for the recognition of both monomeric and dimeric G4DNA, derived from d(T2AG3)4 and d(T2AG3)8 human telomeric DNA, respectively. The spacer between the two bisbenzimidazoles in the geminis plays a critical role in the G4DNA stability. We report here (i) synthesis of new effective gemini anticancer agents that are selectively more toxic towards the cancer cells than the corresponding normal cells; (ii) formation and characterization of G4DNA dimers in solution as well as computational construction of the dimeric G4DNA structures. The gemini ligands direct the folding of the single-stranded DNA into an unusually stable parallel-stranded G4DNA when it was formed in presence of the ligands in KCl solution and the gemini ligands show spacer length dependent potent telomerase inhibition properties.     

Purification and characterization of an altered topoisomerase II from a drug-resistant Chinese hamster ovary cell line

The cytotoxicity and DNA damage induced by the epipodophyllotoxins and several intercalating agents appear to be mediated by DNA topoisomerase II. We have purified topoisomerase II to homogeneity both from an epipodophyllotoxin-resistant Chinese hamster ovary cell line, VpmR-5, and from the wild-type parental cell line. Immunoblots demonstrate similar topoisomerase II content in these two cell lines. The purified enzymes are dissimilar in that DNA cleavage by VpmR-5 topoisomerase II is not stimulated by VP-16 or m-AMSA. Furthermore, the VpmR-5 enzyme is unstable at 37 degrees C. Thus, the drug resistance of VpmR-5 cells appears to result from the presence of an altered topoisomerase II in these cells. Purified topoisomerase II from VPMR-5 and wild-type cells has the same monomeric molecular mass as well as equivalent catalytic activity with respect to decatenation of kinetoplast DNA. Etoposide (VP-16) inhibits the activity of both enzymes. Noncovalent DNA-enzyme complex formation, assayed by nitrocellulose filter binding, is also similar, as is protection from salt dissociation of this complex by ATP and VP-16. The data suggest a model in which the drug-resistant cell line, VpmR-5, has religation activity which is less affected by drug than that of the wild-type cells. Drug effect on DNA religation and catalytic activity are dissociated mechanistically. In addition, under certain circumstances, the "cleavable complex" observed following denaturation of a drug-stabilized DNA-enzyme complex may not adequately reflect the nature of the antecedent lesion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anticancer activity of some bisbenzimidazoles

The discovery of DNA topoisomerases has added a new dimension to the study of anticancer drugs. Bisbenzimidazole derivatives are important compounds known as DNA topoisomerase I inhibitors. In the present study, some symmetrical bisbenzimidazole derivatives were synthesized and investigated for their anticancer activity. Anticancer activity screening was applied on HT-29 (colon carcinoma) and MCF-7 (breast carcinoma) cell lines by investigation of cytotoxicity, analysis of DNA synthesis, and DNA fragmentation assays. One of the seven compounds tested showed significant cytotoxicity in both cell lines and caused DNA degradation in the HT-29 cell line.     

Gamma-ray and hydrogen peroxide induction of gene amplification in hamster cells deficient in DNA double strand break repair

To investigate the role of DNA double strand breaks (DSBs) and of their repair in gene amplification, we analyzed this process in the V3 Chinese hamster cell line and in the parental line AA8, after exposure to gamma-rays and to hydrogen peroxide (H2O2). V3 is defective in DSB repair because of a mutation in the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) gene, a gene involved in the non-homologous end-joining pathway. As a measure of gene amplification we used the frequency of colonies resistant to N-(phosphonacetyl)-L-aspartate (PALA), since in rodent cells PALA resistance is mainly achieved through the amplification of the CAD (carbamyl-P-synthetase, aspartate transcarbamylase, dihydro-orotase) gene. After treatment with different doses of gamma-rays and of H2O2, we found a dose related increase in the frequency of gene amplification and of chromosome aberrations. When the same doses of damaging agents were used, these increments were higher in V3 than in AA8. These results indicate that DSBs that are not efficiently repaired can be responsible for the induction of gene amplification. H2O2 stimulates gene amplification as well as gamma-rays, however, at similar levels of amplification induction, chromosome damage was about 50% lower. This suggests that gene amplification can be induced by H2O2 through pathways alternative to a direct DNA damage. Stimulation of gene amplification by H2O2, which is one of the products of the aerobic metabolism, supports the hypothesis that cellular metabolic products themselves can be a source of genome instability.     

UV mutagenesis, cytotoxicity and split-dose recovery in a human-CHO cell hybrid having intermediate (6-4) photoproduct repair

Somatic cell hybrids constructed between UV-hypersensitive Chinese hamster ovary cell line UV20 and human lymphocytes were used to examine the influence of a human DNA repair gene, ERCC1, on UV photoproduct repair, mutability at several drug-resistance loci, UV cytotoxicity and UV split-dose recovery. In hybrid cell line 20HL21-4, which contains human chromosome 19, UV-induced mutagenesis at the APRT, HPRT and Na+/K+-ATPase loci was comparable to that in repair-proficient CHO AA8 cells, whereas cell line 20HL21-7, a reduced human-CHO hybrid not containing human chromosome 19, exhibited a hypermutable phenotype at all 3 loci indistinguishable from that of UV20 cells. The response of 20HL21-4 cells to UV cytotoxicity reflected substantial but incomplete restoration of wild-type UV cytotoxic response, whereas responses of UV20 and 20HL21-7 cell lines to UV cytotoxicity were essentially the same, reflecting several-fold UV hypersensitivity. Repair of UV-induced (5-6) cyclobutane dimers and (6-4) photoproducts was examined by radioimmunoassay; (6-4) photoproduct repair was deficient in UV20 and 20HL21-7 cell lines, and intermediate in 20HL21-4 cells relative to wild-type CHO AA8 cells. UV split-dose recovery in 20HL21-4 cells was also intermediate relative to AA8 cells. These results show that the human ERCC1 gene on chromosome 19 is responsible for substantial restoration of UV survival and mutation responses in repair-deficient UV20 cells, but only partially restores (6-4) UV photoproduct repair and UV split-dose recovery.     

A simple,rapid, and sensitive DNA assay procedure

A simple and rapid assay for quantitative determinations of DNA in crude homogenates is described. The method is based on the enhancement of fluorescence seen when bisbenzimidazole (Hoechst 33258) binds to DNA. Crude homogenates in which chromatin has been dissociated with high salt buffer can be assayed directly and reliably in a few minutes. The dissociation of chromatin is critical to accurate determinations of DNA in biological materials using this method. The assay can detect as little as 10 ng of DNA with rather unsophisticated instrumentation.     

The relationship between sister-chromatid exchange and perturbations in DNA replication in mutant EM9 and normal CHO cells

The majority of the high (12-fold elevated) baseline sister-chromatid exchanges (SCEs) that occur in the CHO mutant line EM9 appear to be a consequence of incorporated BrdUrd, and they arise during replication of DNA containing BrdUrd in a template strand. In normal CHO cells the alkaline elution patterns of DNA newly replicated on a BrdUrd-containing template are significantly altered compared with those seen during the replication on an unsubstituted template. The nascent DNA synthesized on such an altered template is delayed in reaching mature size, possibly because replication forks are temporarily blocked at sites occurring randomly along the template. Transient blockage of replication forks may be a prerequisite for SCE. The delay in replication on BrdUrd-substituted templates was greater in EM9 cells than in parental AA8 cells and was also greater in AA8 cells treated with benzamide, an inhibitor of poly(ADPR) polymerase, than in untreated AA8 cells. Under these conditions, treatment with benzamide also produced a 7-fold increase in SCEs in AA8. An EM9-derived revertant line that has a low baseline SCE frequency showed less delay in replication on BrdUrd-substituted templates than did EM9. However, under conditions where the template strand contained CldUrd, which was shown to produce 4-fold more SCEs than BrdUrd in AA8 cells, the replication delay in AA8 was not any greater in the CldUrd-substituted cells. Thus, other factors besides the delay appear to be involved in the production of SCEs by the template lesions resulting from incorporation of the halogen-substituted pyrimidine molecules.     

Effect of ultraviolet light on thymidine incorporation, DNA chain elongation and replicon initiation in wild-type and excision-deficient Chinese hamster ovary cells

Wild-type Chinese hamster ovary cells (AA8) and five excision-deficient clones derived from the AA8 line (UV-4, UV-5, UV-20, UV-24 and UV-41) were exposed to ultraviolet light and then analyzed for their ability to incorporate [3H]thymidine and to initiate as well as elongate replicon-sized DNA fragments. After exposure to ultraviolet light, all cell lines exhibited a depression in the rate of thymidine incorporation. For exposures of 4.0 J/m2 or higher the wild-type cells recovered normal rates of thymidine incorporation within a few hours, while none of the excision-deficient lines exhibited complete recovery. For fluences below 4.0 J/m2 all but the UV-5 line exhibited at least some recovery. The ability to elongate DNA chains appeared to correlate with the thymidine incorporation data, with the UV-5 line exhibiting the strongest blockage of DNA chain elongation, the AA8 line exhibiting the least blockage, and the UV-20 line exhibiting an intermediate response. All cell lines exhibited a decrease in the distance between replication origins, thus supporting models which propose that exposure to ultraviolet light results in the use of alternative sites for the initiation of replication.     

Topoisomerase II in multiple drug resistance

Topoisomerase II is a target of alkaloid, anthracycline and related antitumor agents. Two types of multiple drug resistance are associated with these enzymes. In classical (typical) multidrug resistance, inhibitors are actively effluxed from cells by P-glycoprotein. In atypical multidrug resistance, topoisomerase II is either reduced in cellular content or mutated to a form that does not interact with inhibitors. Because cytotoxicity of most antineoplastic topoisomerase II inhibitors is directly related to the number of active topoisomerase II molecules, a reduction in this number leads to resistance. In the topoisomerase II mechanism, through which the DNA linking number is altered, DNA double strands are cleaved, and the termini transiently bound covalently (5) or noncovalently (3) to the enzyme while a second double strand is passed through the break in the first. This transition state complex then decays to enzyme and DNA of altered linking number. Most cytotoxic topoisomerase II inhibitors stabilize these reaction intermediates as ternary complexes, which are converted to lethal lesions when cells attempt to utilize the damaged DNA as templates. Toxicity is related to topoisomerase II content as well as to drug concentration. Thus, multidrug resistance results from either 1) decreasing cellular content of the inhibitor by P-glycoprotein (typical) or 2) decreasing cellular content and/or activity of the target, topoisomerase II, as, for example, when its content or activity is modulated downward by decreased expression, deactivation, or by mutations to the TopII gene, producing an enzyme that reacts poorly with inhibitors (atypical). Mixed types,i.e., both typical and atypical, are known. Attempts to abrogate or prevent both typical and atypical multidrug resistance to topoisomerase II inhibitors have been described.Abbreviations atMDR atypical multidrug resistance - kDa kilodaltons - MDR multidrug resistance - Pgp P-glycoprotein - TOPO II topoisomerase II     

Induction and repair of DNA single-strand breaks in EM9 mutant CHO cells treated with hydrogen peroxide

In this study we investigated the induction and rejoining of DNA single-strand breaks (SSBs) produced by H2O2 in the repair-deficient EM9 mutant Chinese hamster ovary (CHO) cell line. The effect of the poly(ADP-ribose)-transferase inhibitor 3-aminobenzamide (3-ABA) on SSB-rejoining and on cell killing was also evaluated. Results were compared with those obtained previously with the parent cell line (AA8). Cells were treated with H2O2 on ice for 1 h, after which they were either harvested or allowed to repair their damage at 37 degrees C either in the presence or absence of 3-ABA (5 mM). The cells were then assayed either for survival using a colony-forming assay or for their level of DNA SSBs using alkaline elution. EM9 cells were somewhat more sensitive than AA8 cells to the cytotoxic effects of H2O2. However, because the repair mutant showed slightly lower levels of DNA SSBs than did its parental cell line, this sensitivity could not be explained on the basis of alterations in initial damage. The rejoining of the H2O2-induced DNA SSBs followed exponential kinetics in both cell lines; however, EM9 cells rejoined these breaks at a slower rate (t1/2 of 10 min) than did AA8 cells (t1/2 of 5 min). The increased sensitivity of the EM9 cells therefore appears to correlate with a reduced ability to remove these lesions from their DNA. As previously demonstrated for the AA8 cells, 3-ABA treatment resulted in both a retardation of the removal of H2O2-induced DNA SSBs and potentiation of cytotoxicity in the EM9 cells. However, the degree of these effects were similar for both AA8 and EM9 cells. These data provide further evidence that the cytotoxic effects of low concentrations of H2O2 are mediated by damage to DNA, and suggest that the rate at which DNA SSBs are rejoined is important for cell survival.     

The induction of DNA-protein crosslinks in hypoxic cells and their possible contribution to cell lethality

The induction of single-strand breaks (SSBs) in the DNA of Chinese hamster ovary cells by X rays under different irradiation conditions was measured by the alkaline elution technique. The oxygen enhancement ratio (OER) for SSB induction determined for cells irradiated in air versus irradiation of cells made hypoxic by metabolic depletion of O2 was 9.7. However, when proteinase K was included in the cell lysis solution the OER was reduced to 4.2. The proteinase affected the elution rate only of the cells irradiated under hypoxic conditions, suggesting that DNA-protein crosslinks (DPCs) are preferentially produced in hypoxic cells by radiation. The ability to repair these DPCs was compared in two cell lines: the wild-type AA8 line and an excision-repair-deficient mutant line, UV-41. The AA8 line removed about 80% of the DPCs induced by radiation under hypoxic conditions within a 24-h repair incubation. The UV-41 line, on the other hand, removed only about 20% of the DPCs in the same time. The OERs for cell survival of these two lines are 3.1 for AA8 but only 1.9 for UV-41, suggesting that the DPCs preferentially induced in the DNA of cells irradiated under hypoxic conditions may contribute to cell killing when the normal DNA-repair mechanisms are compromised.     

The DRAG test: an assay for detection of genotoxic damage

A high throughput assay (the DRAG test) is described, which could be a useful tool for the detection of repairable DNA adducts, and which is based on the inhibition of the growth of DNA repair-deficient Chinese hamster ovary (CHO) cells. The cytotoxicity of a test substance towards DNA repair-deficient CHO cell lines is compared with the corresponding cytotoxicity in the parental wild-type CHO cell line (AA8). A more pronounced toxicity toward a DNA repair-deficient cell line is interpreted as being the consequence of its inability to repair the DNA adduct induced by the compound. (+)-7beta,8alpha-Dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, camptothecin, ethyl methanesulphonate and mitomycin C were used as reference substances, and the overall results indicate that the DRAG test could be useful in the screening of compounds for the production of repairable DNA adducts. The main advantages with the DRAG test are that it provides a relevant endpoint, it is rapid, it requires small amounts of the test item, and it permits a large number of compounds to be tested.     

The high rate of endoreduplication in the repair deficient CHO mutant EM9 parallels a reduced level of methylated deoxycytidine in DNA

It has been recently proposed that hypomethylation of DNA induced by 5-azacytidine (5-azaC) leads to reduced chromatid decatenation that ends up in endoreduplication, most likely due to a failure in topo II function [S. Mateos, I. Domínguez, N. Pastor, G. Cantero, F. Cortés, The DNA demethylating 5-azaC induces endoreduplication in cultured Chinese hamster cells, Mutat. Res. 578 (2005) 33-42]. The Chinese hamster mutant cell line EM9 has a high spontaneous frequency of endoreduplication as compared to its parental line AA8. In order to see if this is related to the degree of DNA methylation, we have investigated the basal levels of both endpoints in AA8 and EM9, as well as the effect of extensive 5-azaC-induced demethylation on the production of endoreduplication. Based on the correlation between the levels of DNA methylation and indices of endoreduplication we propose that genomic DNA hypomethylation in EM9 cell line is probably an important factor that bears significance in relation to the high basal level of endoreduplication observed in this cell line.     

Cytotoxicity and genotoxicity evaluation of antidote oxime HI-6 tested on eight cell lines of human and rodent origin

Oxime HI-6 is an efficient reactivator of the acetylcholinesterase inhibited by organophosphorous nerve agents. In this study we have estimated cytotoxicity of HI-6 by the colony forming assay and genotoxicity by the comet assay on human and rodent cell lines. IC50 of HI-6 assessed by the colony forming capacity was 3.59 mM for HeLa cells and 5.18 mM for a mouse cell line L929. Small difference in cytotoxicity was found among other cell lines tested: IC50 was 1.61 mM for human A549 cells, 1.14 mM for UROtse line, 1.96 mM and 1.71 mM for Chinese hamster cells AA8 and UV-20, respectively. The A549 cell viability measured with the MTT test was 5 times decreased comparing 2 and 24 hours of HI-6 oxime treatment. The 5 mM HI-6 concentration reduced the viability within 2 hours to 95% only, however, it induced a significant number of DNA breaks in mouse cells L929, and also in human UROtse and HepG2 cells. 1-β-D-arabinofuranosylcytosine (10(-4) M) and hydroxyurea (10(-2) M), supplemented to the cultivation medium, did not cause any significant accumulation of DNA breaks during treatment, which indicated that the nucleotide excision repair was not acting on the induced DNA damage.     

Induction of chromosomal aberrations in the CHO mutant EM9 and its parental line AA8 by EcoRI restriction endonuclease: electroporation experiments

EcoRI restriction endonuclease (RE), which produces cohesive-ended double-strand breaks (dsb) in DNA, was tested in the ethyl methanesulfonate- and X-ray-sensitive CHO mutant EM9 and its parental cell strain AA8 for its chromosomal aberration-inducing effect. The RE was efficiently introduced by electroporation into AA8 cells, while the mutant cells showed a very high sensitivity to electroporation, which consistently resulted in cell death. Nevertheless, the incubation of EM9 cells in the presence of EcoRI, without electroporation, was sufficient to induce about three times the chromosome damage observed in the electroporated parental cell line AA8 for any given dose of the RE.     

Transfection of a human gene for the repair of X-ray- and EMS-induced DNA damage

EM9 cells are a line of Chinese hamster ovary cells that are sensitive to killing by ethylmethanesulfonate (EMS) and X ray, since they are unable to repair the DNA damage inflicted by these agents. Through DNA-mediated gene transfer, human DNA and a selectable marker gene, pSV2neo, were transfected into EM9 cells. Resistant clones of transfected cells were selected for by growth in EMS and G418 (an antibiotic lethal to mammalian cells not containing the transfected neo gene). One primary clone (APEX1) and one secondary clone (TEMS2) were shown to contain both marker and human DNA sequences by Southern blot. In cell survival studies, APEX1 was shown to be as resistant to EMS and X ray as the parental cell type AA8 (CHO cells). TEMS2 cells were found to be partially resistant to EMS and X ray, displaying an intermediate phenotype more sensitive than AA8 cells but more resistant than EM9 cells. Alkaline elution was used to assess the DNA strand-break rejoining ability of these cells at 23 degrees C. APEX1 cells showed DNA repair capacity equal to that of AA8 cells; 75% of the strand breaks were repaired with a rejoining T 1/2 of 3 min. TEMS2 showed similar levels of repair but a T 1/2 for repair of 9 min. EM9 cells repaired only 25% of the breaks and showed a T 1/2 for repair of 16 min. The DNA repair data are consistent with the survival data in that the more resistant cell lines showed a greater capacity for DNA repair. The data support the conclusion that APEX1 and TEMS2 cells contain a human DNA repair gene.     

Interferon-resistant Daudi Cell Line with a Stat2 Defect Is Resistant to Apoptosis Induced by Chemotherapeutic Agents

Interferon-α (IFNα) has shown promise in the treatment of various cancers. However, the development of IFN resistance is a significant drawback. Using conditions that mimic in vivo selection of IFN-resistant cells, the RST2 IFN-resistant cell line was isolated from the highly IFN-sensitive Daudi human Burkitt lymphoma cell line. The RST2 cell line was resistant to the antiviral, antiproliferative, and gene-induction actions of IFNα. Although STAT2 mRNA was present, STAT2 protein expression was deficient in RST2 cells. A variant STAT2 mRNA, which resulted from alternative splicing within the intron between exon 19 and 20, was expressed in several human cell lines but at relatively high levels in RST2 cells. Most importantly, the RST2 line showed an intrinsic resistance to apoptosis induced by a number of chemotherapeutic agents (camptothecin, staurosporine, and doxorubicin). Expression of STAT2 in RST2 cells not only rescued their sensitivity to the biological activities of IFNs but also restored sensitivity to apoptosis induced by these chemotherapeutic agents. The intrinsic resistance of the RST2 cells to IFN as well as chemotherapeutic agents adds a new dimension to our knowledge of the role of STAT2 as it relates to not only biological actions of IFN but also resistance to chemotherapy-induced apoptosis.IFN²α/β regulates a number of cellular responses, such as proliferation, differentiation, and development (1). Although IFN triggers the death of some tumor cells by inducing proapoptotic proteins (tumor necrosis factor-related apoptosis-inducing ligand, PKR, etc.), IFN also promotes cell survival through a nuclear factor κB-dependent pathway (2–4). IFN is used to treat various human malignancies (chronic myeloid leukemia, non-Hodgkin lymphomas, Kaposi sarcoma, hairy cell leukemia, multiple myeloma, and malignant melanoma), viral infections, as well as various other diseases (5, 6). However, only a fraction of patients are responsive to IFN therapy, and many patients eventually develop resistance after chronic IFN exposure. The underlying mechanism for IFN resistance is still unclear, but it is reasonable to suggest that genetic variation and selection during prolonged IFN exposure may reflect IFN signaling defects.IFN binds to its cell surface receptor resulting in the activation of JAK1 and TYK2 nonreceptor protein-tyrosine kinases, which phosphorylate STAT proteins (7). Phosphorylated STAT1 and STAT2 in a complex with IRF9 bind to a conserved IFN stimulus-response element (ISRE) present in the promoters of hundreds of IFN-stimulated genes (ISGs) inducing their expression. Mutant cell lines with defined signaling defects have made significant contributions in elucidating the IFN-activated JAK/STAT signal transduction pathway (7, 8). Such IFN-resistant mutants were isolated after multiple rounds of chemical mutagenesis and selection of IFN-resistant mutants. However, this procedure does not mimic in vivo what happens to patients who are subjected to long term IFN treatment. Moreover, these IFN-resistant cells have undergone multiple mutational events, because complementation of a single defect rescues aspects of IFN signaling but not sensitivity to all of the biological actions of IFN (8, 9). As an alternative approach, our laboratory has resorted to long term IFN treatment of cells to isolate naturally arising IFN-resistant mutants, which more closely resemble what occurs in vivo (10, 11). Using this strategy, we previously identified a STAT3-defective IFN-resistant cell line (11).In this study, a mutant cell line (RST2) that was highly resistant to the antiviral, antiproliferative, and gene-inducing actions of IFN was isolated from the highly IFN-sensitive Daudi cell line by growth in the continuous presence of IFN. Sequencing of STAT2 mRNA identified an alternative splice site between exon 19 and 20 that is expressed in RST2 cells, causing translation termination at the beginning of the Src homology 2 domain of STAT2. Expression of STAT2 in RST2 cells rescued sensitivity to the antiviral, antiproliferative, and gene-inducing actions of IFN. Furthermore, although RST2 cells are intrinsically resistant to the induction of apoptosis by a variety of chemotherapeutic agents, reconstitution of STAT2 restored sensitivity to chemotherapy-induced apoptosis.