Imatinib mesylate (STI571) abrogates the resistance to doxorubicin in human K562 chronic myeloid leukemia cells by inhibition of BCR/ABL kinase-mediated DNA repair

Imatinib mesylate (STI571), a specific inhibitor of BCR/ABL tyrosine kinase, exhibits potent antileukemic effects in the treatment of chronic myelogenous leukemia (CML). However, the precise mechanism by which inhibition of BCR/ABL activity results in pharmacological responses remains unknown. BCR/ABL-positive human K562 CML cells resistant to doxorubicin (K562DoxR) and their sensitive counterparts (K562DoxS) were used to determine the mechanism by which the STI571 inhibitor may overcome drug resistance. K562 wild type cells and CCRF-CEM lymphoblastic leukemia cells without BCR/ABL were used as controls. The STI571 specificity was examined by use of murine pro-B lymphoid Baf3 cells with or without BCR/ABL kinase expression. We examined kinetics of DNA repair after cell treatment with doxorubicin in the presence or absence of STI571 by the alkaline comet assay. The MTT assay was used to estimate resistance against doxorubicin and Western blot analysis with Crk-L antibody was performed to evaluate BCR/ABL kinase inhibition by STI571. We provide evidence that treatment of CML-derived BCR/ABL-expressing leukemia K562 cells with STI571 results in the inhibition of DNA repair and abrogation of the resistance of these cells to doxorubicin. We found that doxorubicin-resistant K562DoxR cells exhibited accelerated kinetics of DNA repair compared with doxorubicin-sensitive K562DoxS cells. Inhibition of BCR/ABL kinase in K562DoxR cells with 1 microM STI571 decreased the kinetics of DNA repair and abrogated drug resistance. The results suggest that STI571-mediated inhibition of BCR/ABL kinase activity can affect the effectiveness of the DNA-repair pathways, which in turn may enhance drug sensitivity of leukemia cells.     

Erythropoietin promotes resistance against the Abl tyrosine kinase inhibitor imatinib (STI571) in K562 human leukemia cells

Chronic myeloid leukemia is characterized by the Philadelphia chromosome translocation that causes expression of Bcr-Abl, a deregulated tyrosine kinase. Imatinib mesylate (STI571, Gleevec), a therapeutically used inhibitor of Bcr-Abl, causes apoptosis of Bcr-Abl-positive cells. In the leukemia cell line K562, we observed spontaneous resistance to imatinib at very low frequencies when cells were exposed to the drug (1 micro M) for more than 4 weeks. Surprisingly, in the presence of erythropoietin (Epo), K562 cells were temporarily able to sustain proliferation in the presence of imatinib, and imatinib-resistant clones could be isolated with high frequencies. From such imatinib-resistant, Epo-dependent clones, sublines could be established that were resistant to imatinib in the absence of Epo. Mitogen-activated protein (MAP) kinase activity was inhibited by imatinib treatment but could be partially restored by Epo. Inhibition of MAP kinase or phosphatidylinositol 3-kinase blocked the protective effect of Epo. The data suggest that K562 cells acquire factor dependency under imatinib/Epo treatment, allowing them to escape from imatinib-induced, immediate cell death. This pool of cells provides the basis for the outgrowth of imatinib-resistant clones of unlimited proliferative capacity. Thus, Epo, an endogenous regulator of hematopoiesis, promotes the development of resistance to imatinib.     

Imatinib (STI571) induces DNA damage in BCR/ABL-expressing leukemic cells but not in normal lymphocytes

Imatinib (STI571) is a 2-phenylaminopyrimidine derivative used mostly in the treatment of chronic myeloid leukaemia. It targets the BCR/ABL oncogenic tyrosine kinase, inhibiting its activity. Using the alkaline comet assay we showed that STI571 at concentrations ranging from 0.2 to 2 microM induced DNA damage in human leukemic K562 and BV173 cells expressing the BCR/ABL oncogene, whereas it had no effect in normal human lymphocytes and leukemic CCRF-CEM cells without the expression of BCR/ABL. Imatinib did not induce DNA strand breaks in the direct interaction with DNA as examined by the circular plasmid relaxation assay. Because the extent of DNA damage observed in the neutral and pH 12.1 versions of the comet assay was much lesser than in the alkaline version, we concluded that the drug induced DNA alkali-labile sites rather than strand breaks. K562 cells were unable to repair H(2)O(2)-induced DNA damage during a 120-min incubation, if they had been preincubated with STI571, whereas normal lymphocytes did so within 60 min. Pre-treatment of K562 cells with Vitamins A, C and E reduced the extent of DNA damage evoked by STI571. Similar results brought experiments with the nitrone spin traps POBN and PBN, suggesting that free radicals may be involved in the formation of DNA lesions induced by STI571 in K562 cells. These cells exposed to imatinib and treated with endonuclease III, formamidopyrimidine-DNA glycosylase and 3-methyladenine-DNA glycosylase II, the enzymes recognizing oxidized and alkylated bases, displayed greater extent of DNA damage than those not treated with these enzymes. Therefore, the mechanism of the anti-leukemic action of STI571 may involve not only the inhibition of BCR/ABL, but also DNA damage in the cells expressing this fusion protein. DNA damage induced by STI571 may follow from oxidative and alkylative base modifications.     

Treatment of Chronic Myeloid Leukemia Cells with Imatinib (STI571) Impairs p53 Accumulation in Response to DNA Damage

Chronic myelogenous leukaemia (CML) is induced by the Bcr-Abl fusion protein. Inhibition of Bcr-Abl by STI571 is widely used to treat CML patients. Unlike in most cancer types, the frequency of p53 mutations in CML is low. Here, we investigated the effect of STI571 treatment of CML cells on p53 regulation. Exposure of CML cells, including established cell lines and freshly isolated cells from patients, to STI571 reduced p53 protein levels, and severely impaired its accumulation in response to DNA damage. This may be explained by the status of p53 serine 20 phosphorylation. In non-stressed CML cells, serine 20 of p53 is constitutively phosphorylated by Chk1, and is inhibited by STI571. In response to DNA damage, however, this phosphorylation is mediated by Chk1 and Chk2, and is only partially inhibited by STI571. CML cells expressing wild-type p53 are more resistant to treatment with STI571, but moderately more sensitive to DNA damage, than CML cells lacking p53. An enhanced induction of apoptosis by STI571 and DNA damage is observed in CML cells bearing wild-type p53, but not in cells lacking functional p53. This implies that the status of p53 may affect the response of CML cells to this combined treatment.     

The tyrosine kinase inhibitor STI571 induces cellular clearance of PrPSc in prion-infected cells

The conversion of the cellular prion protein (PrP(c)) into pathologic PrP(Sc) and the accumulation of aggregated PrP(Sc) are hallmarks of prion diseases. A variety of experimental approaches to interfere with prion conversion have been reported. Our interest was whether interference with intracellular signaling events has an impact on this conversion process. We screened approximately 50 prototype inhibitors of specific signaling pathways in prion-infected cells for their capacity to affect prion conversion. The tyrosine kinase inhibitor STI571 was highly effective against PrP(Sc) propagation, with an IC(50) of < or =1 microM. STI571 cleared prion-infected cells in a time- and dose-dependent manner from PrP(Sc) without influencing biogenesis, localization, or biochemical features of PrP(c). Interestingly, this compound did not interfere with the de novo formation of PrP(Sc) but activated the lysosomal degradation of pre-existing PrP(Sc), lowering the half-life of PrP(Sc) from > or =24 h to <9 h. Our data indicate that among the kinases known to be inhibited by STI571, c-Abl is likely responsible for the observed anti-prion effect. Taken together, we demonstrate that treatment with STI571 strongly activates the lysosomal degradation of PrP(Sc) and that substances specifically interfering with cellular signaling pathways might represent a novel class of anti-prion compounds.     

Effects of the tyrosine kinase inhibitor imatinib mesylate (STI571) on bone marrow features in patients with chronic myelogenous leukemia

Preliminary data are available about bone marrow (BM) changes in patients with chronic myeloid leukemia (CML) who received the molecularly targeted and highly effective tyrosine kinase inhibitor Imatinib mesylate (STI571). This review is focused on a systematic assessment of BM features detectable at different stages of CML (stable, accelerated, blastic) following long-term (more than 10 months) treatment. By applying enzyme- and immunohistochemistry including monoclonal antibodies visualizing proliferating cell nuclear antigen (PCNA) and apoptosis (anti-apostatin), a more elaborate insight into alterations affecting hematopoiesis and the stroma compartment was gained. In patients with stable-phase CML therapy resulted in a significant reduction in cellularity, neutrophil granulopoiesis and number of megakaryocytes, accompanied by a retrieval of erythroid precursors. In patients with Imatinib as the only treatment morphometric analysis of CD61+ megakaryopoiesis was in keeping with a significant decrease in maturation defects implying a lesser amount of atypical micromegakaryocytes almost consistent with normalization. Moreover, a reduction of the initially enhanced (CD34+) microvessel density was detectable associated with a decrease in luminal distension. Regression of marked to moderate myelofibrosis was recognizable in about 70% of patients especially in the accelerated and blastic phases. The amount of myeloblasts, CD34+ progenitor cells and lysozyme-expressing immature myelomonocytic cells declined with treatment, but recurred in about 19% of patients that developed a leukemic relapse after 21+/-6 months of therapy. Data on proliferative activity and apoptosis in general supported in vitro findings concerning the inhibitory effect of this agent on growth associated with a tendency for stimulated apoptosis, at least in responding patients.     

Imatinib mesylate (STI-571) reduces Bcr-Abl-mediated vascular endothelial growth factor secretion in chronic myelogenous leukemia

A large and diverse spectrum of oncogenes has been implicated as a contributor to angiogenesis in solid tumors based, in part, on its ability to induce proangiogenic growth factors such as vascular endothelial growth factor (VEGF), and the fact that various anti-oncogenic signaling inhibitor drugs have been shown to reverse such proangiogenic effects both in vitro and in vivo. Because leukemias are now also considered to be angiogenesis-dependent malignancies, we asked whether a similar paradigm might exist for the BCR-ABL oncogene and the Bcr-Abl targeting drug, STI-571 (imatinib mesylate), in the context of chronic myelogenous leukemia (CML) cells. We found that levels of VEGF expression in BCR-ABL-positive K562 cells were reduced in vitro by treatment with STI-571 in a dose-dependent fashion. Transfection of BCR-ABL into murine myeloid 32D and human megakaryocyte MO7e hematopoietic cells resulted in enhanced VEGF expression, which could be further elevated by the exposure to cytokines such as interleukin 3 and granulocyte macrophage colony-stimulating factor. We also found that conditioned media taken from 32D-p210-transfected cells could stimulate human umbilical vein endothelial cells by increasing phosphorylation of VEGF-R2/KDR and the downstream serine/threonine kinase PKB/Akt, an important regulator of endothelial cell survival. Moreover, amplification of BCR-ABL in STI-571-resistant cells was associated with elevated VEGF expression levels which could be reversed by treatment with higher concentrations of STI-571. Taken together, our results implicate BCR-ABL as a possible regulator of CML angiogenesis and raise the possibility that STI-571 could mediate some of its anti-CML properties in vivo through an angiogenesis-dependent mechanism.     

Cisplatin-evoked DNA fragmentation in normal and cancer cells and its modulation by free radical scavengers and the tyrosine kinase inhibitor STI571

Cis-diamminedichloroplatinum(II) (cisplatin, cis-DDP) is well studied anticancer drug, whose activity can be attributed to its ability to form adducts with DNA, but this drug can also form DNA-damaging free radicals, however this mechanism of cisplatin action is far less explored. Using the comet assay we studied cisplatin-induced DNA damage in the presence of spin traps: DMPO and PBN, Vitamins A, C and E as well as the tyrosine kinases inhibitor STI571 in normal human lymphocytes and leukemic K562 cells. The latter cells express the BCR/ABL fusion protein, which can be a target of the tyrosine kinase inhibitor STI571. A 20 h incubation with cisplatin at 1-10 microM induced DNA cross-links and DNA fragmentation in normal and cancer cells. Cisplatin could induce intra- and interstrand DNA-DNA cross-links as well as DNA-protein cross-links. DNA damage in K562 cells was more pronounced than in normal lymphocytes. In the presence of spin traps and vitamins we noticed a decrease in the DNA fragmentation in both cell types. Co-treatment of the lymphocytes with cisplatin at 10 microM and STI571 at 0.25 microg/ml caused an increase of DNA fragmentation in comparison with DNA fragmentation induced by cisplatin alone. In the case of K562 cells, an increase of DNA fragmentation was observed after treatment with cisplatin at 1 microM. Our results indicate that the free radicals scavengers could decrease DNA fragmentation induced by cisplatin in the normal and cancer cells, but probably they have no effect on DNA cross-linking induced by the drug. The results obtained with the BCR/ABL inhibitor suggest that K562 cells could be more sensitive towards co-treatment of cisplatin and STI571. Our results suggest also that aside from the BCR/ABL other factors such as p53 level, signal transduction pathways and DNA repair processes can be responsible for the increased sensitivity of K562 cells to cisplatin compared with normal lymphocytes.     

STI571 reduces NER activity in BCR/ABL-expressing cells

Nucleotide-excision repair (NER) is the most versatile mechanism of DNA repair, recognizing and dealing with a variety of helix-distorting lesions, such as the UV-induced photoproducts cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) photoproducts. We investigated the influence of an anticancer drug, STI571, on the efficacy of NER in removing UV-induced DNA damage. STI571 is used mostly in the treatment of chronic myeloid leukemia and inhibits activity of the BCR/ABL oncogenic tyrosine kinase, which is a hallmark of this disease. NER activity was examined in the BCR/ABL-expressing cell lines K562 and BV173 of myeloid and lymphoid origin, respectively, as well as in CCRF-CEM cells, which do not express BCR/ABL. A murine myeloid parental 32D cell line and its counterpart transfected with the BCR/ABL gene were also tested. NER activity was assessed in the cell extracts by use of an UV-irradiated plasmid as a substrate and by a modified single-cell gel electrophoresis (comet) assay on UV-treated nucleoids. Additionally, quantitative PCR was performed to evaluate the efficacy of the removal of UV-induced lesions from the p53 gene by intact cells. Results obtained from these experiments indicate that STI571 decreases the efficacy of NER in leukemic cells expressing BCR/ABL. Therefore, STI571 may overcome the drug resistance associated with increased DNA repair in BCR/ABL-positive leukemias.     

Imatinib mesylate (STI-571) attenuates liver fibrosis development in rats

It is widely recognized that activated hepatic stellate cells (HSC) play a pivotal role in development of liver fibrosis. A platelet-derived growth factor (PDGF) is the most potent mitogen for HSC. The aim of this study was to examine the effect of imatinib mesylate (STI-571, Gleevec), a clinically used PDGF receptor (PDGFR) tyrosine kinase inhibitor, on development of experimental liver fibrosis. The rat model of pig serum-induced hepatic fibrosis was used to assess the effect of daily oral administration of STI-571 on the indexes of fibrosis. STI-571 markedly attenuated development of liver fibrosis and hepatic hydroxyproline and serum fibrosis markers. The number of alpha-smooth muscle actin-positive cells and mRNA expression of alpha2-(I)-procollagen, tissue inhibitor of metalloproteinases-1, and transforming growth factor-beta were also significantly suppressed by STI-571. Our in vitro study showed that STI-571 markedly attenuated PDGF-BB-induced proliferation and migration and alpha-SMA and alpha2-(I)-procollagen mRNA of activated HSC in a dose-dependent manner. STI-571 also significantly attenuated PDGF-BB-induced phosphorylation of PDGFR-beta, MEK1/2, and Akt in activated HSC. Because STI-571 is widely used in clinical practice, it may provide an effective new strategy for antifibrosis therapy.     

Increased sensitivity to the platelet-derived growth factor (PDGF) receptor inhibitor STI571 in chemoresistant glioma cells is associated with enhanced PDGF-BB-mediated signaling and STI571-induced Akt inactivation

The platelet-derived growth factor receptor (PDGFR) is a tyrosine kinase, implicated in the development and progression of different tumors, including gliomas. Chemoresistance is a common feature of malignant gliomas. Since receptor tyrosine kinases contribute to chemoresistance in tumors, we addressed whether PDGFR signaling might confer selective growth advantage to chemoresistant cells. The effects of the PDGFR inhibitor STI571 on proliferation and PDGFR signaling were compared in chemosensitive and cisplatin-selected, chemoresistant sublines derived from glioma and from two other PDGFR-expressing tumors (ovarian carcinoma and neuroblastoma). The chemoresistant glioma U87/Pt cells were twofold more sensitive to STI571 growth-inhibitory effects than the chemosensitive U87 cells, and two- to threefold more sensitive than five unrelated glioma cell lines. The other two paired cell lines were equally responsive. Sensitization of U87/Pt cells correlated with upregulation of the PDGF-B isoform and with PDGF-BB-induced Akt overactivation, which was prevented by STI571. STI571 specifically inhibited PDGF-BB-, but not PDGF-AA- or stem cell factor-mediated signaling. In serum-containing medium, STI571 decreased phospho-Akt in U87/Pt cells, but not in U87, while activating extracellular signal-regulated kinase (Erk) in both. STI571 antiproliferative effects were partially reverted by constitutively active Akt. Cotreatment with inhibitors of phosphatidylinositol 3'-kinase (PI3K) or mitogen-activated protein kinase kinase (MEK) resulted in enhanced growth inhibition in glioma cells. Our results suggest that increased PDGF-BB signaling may sensitize chemoresistant glioma cells to STI571, suggesting a therapeutic potential for STI571 in patients with malignant gliomas refractory to chemotherapy. Simultaneous blockade of PDGFR and PI3K or Erk pathway may enhance therapeutic targeting in gliomas.     

Simultaneous determination of AMN107 and Imatinib (Gleevec, Glivec, STI571) in cultured tumour cells using an isocratic high-performance liquid chromatography procedure with UV detection

A reversed phase high-performance liquid chromatographic (HPLC) method with UV detection was developed for the simultaneous determination of imatinib (Gleevec, Glivec, STI571) and AMN107 in cultured tumour cells, using clozapine as an internal standard. The compounds of interest were extracted by liquid-liquid extraction using TOXI-TUBES((R)) A extraction tubes. Chromatographic separation was performed on a Phenomenex Gemini C18 reversed phase column (150 mm x 2.0 mm, 5 microm particle size), using a mixture of 65% CH(3)OH (methanol) and 35% NH(4)Ac (Ammonium acetate) buffer (20mM, pH 10). Separation was achieved under isocratic conditions at a flow rate of 0.5 ml/min. Imatinib, clozapine and AMN107 are detected by UV detection at 260 nm. Calibration curves were linear from 50 to 7500 ng/ml with correlation coefficients (r(2)) better than 0.998. The limit of quantitation (LOD) was 50 ng/ml. The method has been successfully applied to a cellular kinetics study.     

D-ribose inhibits DNA repair synthesis in human lymphocytes

D-ribose is cytotoxic for quiescent human lymphocytes and severely inhibits their PHA-induced proliferation at concentrations (25-50 mM) at which other simple sugars are ineffective. In order to explain these effects, DNA repair synthesis was evaluated in PHA-stimulated human lymphocytes treated with hydroxyurea and irradiated. D-ribose, in contrast to other reducing sugars, did not induce repair synthesis and therefore did not apparently damage DNA in a direct way, although it markedly inhibited gamma ray-induced repair. Taking into account that lymphocytes must rejoin physiologically-formed DNA strand breaks in order to enter the cell cycle, we suggest that D-ribose exerts its cytotoxic activity by interfering with metabolic pathways critical for the repair of DNA breaks.     

Cadmium inhibits human DNA mismatch repair in vivo

The heavy metal cadmium (Cd) is a human carcinogen that inhibits DNA repair activities. We show that DNA mismatch repair (MMR)-mediated cell cycle arrest after alkylation damage is suppressed by exposure to Cd and that this effect is reversed by preincubation with excess of zinc (Zn). We show that Cd-mediated inactivation of MMR activity is not caused by disruption of complex formation between the MMR proteins hEXO1-hMutS alpha and hEXO1-hMutL alpha nor does Cd inhibit 5'-exonuclease activity of hEXO1 in vitro. Thus, our studies show that exposure of human cells to Cd suppresses MMR activity, a repair activity known to play an important role in colon cancer and that this effect can be reversed by Zn treatment.     

Aphidicolin inhibits repair of DNA in UV-irradiated human fibroblasts

Aphidicolin, a specific inhibitor of DNA polymerase α, is shown to inhibit DNA repair in human diploid fibroblasts. Although aphidicolin has no apparent effect on the DNA of unirradiated cells, it causes a large number of strand breaks to accumulate in UV-irradiated cellular DNA. The number of breaks is the same as the number observed following a similar dose of ultraviolet light when cells are treated with arabinofuranosyl cytosine (araC) and hydroxyurea (HU), known inhibitors of repair. Moreover, two-dimensional paper chromatography shows that aphidicolin completely blocks removal of pyrimidine dimers. These observations are discussed in light of the proposed roles of DNA polymerases α β in DNA replication and repair and the action of aphidicolin on polymerase α.     

Tetrahydrocannabinol inhibits adenyl cyclase in human leukemia cells

Delta 9-tetrahydrocannabinol has been shown to induce incomplete maturation in ML2 human leukemia cell lines. We extend the observation of its induction of morphologic maturation to HL60 cells and of its induction of growth restriction to HL60 and K562 cells. We show that tetrahydrocannabinol reduces the cyclic AMP content of ML2 cells. Finally we demonstrate that this agent inhibits adenyl cyclase activity in ML2 cell membrane-enriched fractions. This finding in myeloid cells is compatible with one hypothesis of cannabinoid action in neuronal cells.