Different Concentrations Of Imatinib Research Articles

Drug-induced reactive oxygen species-mediated inhibitory effect on growth of Trypanosoma evansi in axenic culture system.

Trypanosoma evansi, an extracellular haemoflagellate, has a wide range of hosts receptive and susceptible to infection, in which it revealed highly inconsistent clinical effects. Drugs used for the treatment of trypanosomosis have been utilized for more than five decades and have several problems like local and systemic toxicity. In the present investigation, imatinib and sorafenib were selected as drugs as they are reported to have the potential to cause reactive oxygen species (ROS)-mediated effect in cancer cells. Both have also been reported to have potential against T. brucei, T. cruzi and Leishmania donovani. To date, imatinib and sorafenib have not evaluated for their growth inhibitory effect against T. evansi. Imatinib and sorafenib showed significant (p < 0.001) inhibition on parasite growth and multiplication with IC50 (50% inhibitory concentration) values 6.12μM and 0.33μM respectively against T. evansi. Both the drug molecules demonstrated for the generation of ROS in T. evansi and were found up to 65% increased level of ROS as compared with negative control in the axenic culture system. Furthermore, different concentrations of imatinib and sorafenib were found non-toxic on horse peripheral blood mononuclear cells and Vero cell lines. Also, in conclusion, our results demonstrated that imatinib- and sorafenib-induced generation of ROS contributed inhibitory effect on the growth of Trypanosoma evansi in an axenic culture system.

Study of therapeutic effect of different concentrations of imatinib on Balb/c model of cutaneous leishmaniasis.

Leishmaniasis, as a tropical and subtropical disease, is endemic in more than 90 countries around the world. Today, cutaneous leishmaniasis (CL) that affects more than 1.5 million people per year lacks a definitive treatment approach. Imatinib is an anticancer drug that inhibits the abnormal function of Bcr-Abl due to its tyrosine kinase inhibitor, and that was the reason why the drug was tested for CL treatment because protein kinases are essential enzymes in the Leishmania genus. In this study, the L. major CL model of Balb/c mice was produced by injection of the cultured metacyclic form of parasite at the base of the tail. The possible recovery of CL ulcers and determination of the optimum dose of imatinib against Leishmania amastigotes were evaluated. A significant decrease was observed in mice treated with amphotericin B (positive control group) as well as imatinib 50 mg/kg compared to the unreceived drug, negative control group (P<0.05). This study could be promising in gaining insight into the potential of imatinib as an effective treatment approach against CL.

Evaluation of Different Concentrations of Imatinib on the Viability of Leishmania major: An In Vitro Study.

Background:Leishmaniasis is an infectious disease caused by an intracellular parasite of Leishmania and is transmitted through the female sandflies bite and may lead to severe skin lesions. Although drugs such as antimony compounds are available, their side effects such as toxicity, low efficacy, and emergence of resistance have raised the importance of effective replacement. Imatinib, as an inhibitor of tyrosine kinase (TK) of Leishmania, stops abnormal function of TK such as Bcr-Abl through assembling into transmembrane pores in a sterol-dependent manner. Hence, the evaluation of killing effects of different concentrations of imatinib against Leishmania major amastigotes and promastigotes in vitro were the objectives of the present study.Materials and Methods:The killing effects of different concentrations of imatinib (25, 50, and 100 μg) and 25 μg amphotericin B (as positive control) were evaluated against RPMI 1640-cultured promastigotes and the amastigote/macrophage model by MTS cell proliferation assay kit (ab197010) and Giemsa staining method during 24, 48, and 72 h.Results:The results showed anti-Leishmania effect of imatinib in concentration and time-dependent manner. The lowest number of live promastigotes and amastigotes were obtained due to treat with 100 μg/ml imatinib at 72 h. Furthermore, 100 μg concentration of imatinib had the same effect as 25 μg amphotericin B on both L. major promastigotes and amastigotes (P < 0.001).Conclusion:The anti-Leishmania effect of imatinib was confirmed by MTS and direct microscopy. Further study is recommended for evaluating possible therapeutic effects of imatinib on leishmaniasis in vivo.

Overexpression of SHP-1 Enhances the Sensitivity of K562 Cells to Imatinib

To explore the effect of overexpression of SH2-containing tyrosine phosphatase 1 (SHP-1) on sensitivity of chronic myelogenous 1eukemia (CML) K562 cell line to imatinib and its related mechamism. K562 cells were infected with the lentiviral plasmids containing the specified retroviral vector (pEX-SHP-1-puro-Lv105) or the mock vector (pEX-EGFP-puro-Lv105). The expression of SHP-1 in K562 cells treated with 0.2 µmol/L imatinib (IM) for 72 h was determined by Western blot. After transfection the CCK-8 assay was used to determine the proliferation of the tramfected K562 cells (K562(SHP-1) and K562(EGFP) cells) at 72 h after exposure to different doses of IM, the half inhibitary concentration (IC50) was calculated. The mechanisms of the overexpression effects of SHP-1 and IM on the proliferation in K562 cells was investigated, the BCR-ABL1 activity and the level of tyrosine phosphorylation of CrkL (pCrkL) was measured by flow cytometry; the Western blot was used to detect the expression and activity of these molecules controlling cell growth, including MAPK, AKT, STAT5 and JAK2. After exposure of K562 cells to 0.08 µmol/L IM for 72 h, there was no significant change of SHP-1 expression in K562 cells. After exposure to 0.2 µmol/L of IM for 72 h, the inhibitory rate of K562(SHP-1) group was higher than that of K562(EGFP) group (P < 0.05), indicating that overexpression of SHP-1 in K562 cells could enhance the proliferation inhtibition effect of IM on K562 cells. The IC50 of IM in K562(SHP-1) cells was the lower as compared with that of K562(EGFP) cells (P < 0.05) after exposure to different concentrations of IM for 72 h. The slope of K562(SHP-1) cells was the largest ranging 0.02 - 0.16 µmol/L of IM. Overexpression of SHP-1 and IM could inhibit the activity BCR-ABL1, MAPK, AKT, STAT5 and JAK2 signaling pathways in the K562 cell line and displayed a synergistic effect. SHP-1 inhibits BCR-ABL1, MAPK, AKT, STAT5 and JAK2 signaling pathways in K562 cells, the overexpression of SHP-1 can enhance the sensitivity of K562 cells to IM.

Modulation of imatinib cytotoxicity by selenite in HCT116 colorectal cancer cells.

Imatinib is a principal therapeutic agent for targeting colorectal tumours. However, mono-targeting by imatinib does not always achieve complete cancer eradication. Selenite, a well-known chemopreventive agent, is commonly used in cancer patients. In this study, we aimed to explore whether selenite can modulate imatinib cytotoxicity in colorectal cancer cells. HCT116 cells were treated with different concentrations of imatinib and/or selenite for 24, 48 and 72hr. Imatinib-selenite interaction was analysed using isobologram equation. As indicators of apoptosis, DNA fragmentation, caspase-3 activity, Bcl-2 expression were explored. Autophagic machinery was also checked by visualizing acidic vesicular organelles and measuring Beclin-1 expression. Furthermore, reactive oxygen and nitrogen species were also examined. This study demonstrated that selenite synergistically augmented imatinib cytotoxicity in HCT116 cells as demonstrated by combination and dose reduction indices. Supranutritional dose of selenite when combined with imatinib induced apoptotic machinery by decreasing Bcl-2 expression, increasing caspase-3 activity and subsequently fragmenting DNA and blunted cytoprotective autophagy by decreasing Beclin-1 expression and autophagosomes formation. Moreover, their combination induced cell cycle S-phase block, increased total thiol content and reduced nitric oxide levels. In conclusion, selenite synergizes imatinib cytotoxicity through multi-barrelled molecular targeting, providing a novel therapeutic approach for colorectal cancer.

Single-cell analysis of K562 cells: An imatinib-resistant subpopulation is adherent and has upregulated expression of BCR-ABL mRNA and protein

In chronic myeloid leukemia (CML) cells from different stages of maturation may have differential expression of BCR-ABL at both messenger RNA (mRNA) and protein level. However, the significance of such differential expression to clinical disease behavior is unknown. Using the CML-derived, BCR-ABL expressing cell line, K562, distinct plastic-adherent (K562/Adh) and nonadherent (K562/NonAdh) subpopulations were established and then analyzed both as single cells and as bulk cell populations. BCR-ABL mRNA was upregulated in K562/Adh compared with K562/NonAdh cells in both single cell and bulk population analyses (p < 0.0001). Similarly, phosphorylation of BCR protein was upregulated in K562/Adh, compared with K562/NonAdh cells (63.42% vs. 23.1%; p = 0.007), and these two K562 subpopulations were found to express significantly different microRNA species. Furthermore, treatment with the BCR-ABL tyrosine kinase inhibitor, imatinib, reduced cell viability more rapidly in K562/NonAdh compared with K562/Adh cells (p < 0.005) both at single and bulk cell levels. This discovery of an adherent subpopulation of K562 cells with increased BCR-ABL mRNA, increased phosphorylated BCR protein expression, differential microRNA expression, and increased imatinib resistance suggests that a similar subpopulation of cells can also mediate clinical resistance to imatinib during treatment of patients with CML.

Effect of up-regulated expression of tumor suppressor gene p14(ARF) on apoptosis of chronic myeloid leukemia cells

To investigate the effect of up-regulated expression of tumor suppressor gene p14(ARF) on apoptosis of chronic myeloid leukemia (CML) cells and its interaction with imatinib. Tumor suppressor gene p14(ARF) was transduced into K562 (K562-p14(ARF)) and 4 blast crisis primary CML cells (CML-BC 1-4) using vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviral vector with cells transduced by empty vector as control. Fluorescence microscopy and flow cytometry were applied to measure transduction efficiency, and Western blotting assay was used to detect p14(ARF) protein of K562 cells. WST-8 method was used to determine cell growth inhibition rate of K562 cells transduced by the target gene under different concentrations of imatinib (0, 0.015, 0.062, 0.125, 0.25, 0.5, 1.0, 2.0 μmol/L). Cell apoptosis and leukemic cellular colony-forming ability were detected by Annexin V-FITC/PI dyeing using flow cytometry (FCM) and semi-solid culture method respectively. Fluorescence microscopy and FCM showed that transduction efficiency (GFP positive cells) of K562-p14(ARF), K562-VSV and CML-BC1 cells were close to 100%, and CML-BC 2-4 cells were 80% to 90% on average. Results of Western blotting showed that the levels of ARF protein expression of K562 cells transduced by p14(ARF) were significantly higher than of untransduced cells; the apoptosis rate of K562-p14(ARF) was 20%; the mean apoptosis rate of 4 primary leukemic cells transduced by the p14(ARF) [(71.1±22.4)%] was significantly higher than of control group [(12.4±6.2)%] (P<0.05). Imatinib significantly inhibited the proliferation of K562-p14(ARF) cells in a dose-dependent manner. The mean leukemic cellular colony-forming unit of 4 primary leukemic cells transduced by the p14(ARF) (41.5±13.2) was significantly lower than of the control group (88.5±7.9) (P<0.05). Increased p14(ARF) gene expression could induce apoptosis of CML cells; Moreover, it could enhance inhibitory effect on cell proliferation when combined with imatinib.

Inhibition by Imatinib of Expression of O-glycan-related Glycosyltransferases and Tumor-associated Carbohydrate Antigens in the K562 Human Leukemia Cell Line

To study changes of tumor associated carbohydrate antigen (TACAs) expression and mRNA levels for tumor associated glycosyltransferases, and assess subcellular localizations of N-acetyl galactosyltransferases (GalNAc-Ts) in the K562 leukemia cell line after imatinib treatment. RT-PCR was performed to analyze the expression of glycosyltransferases which synthesize O-glycan in tumor-associated carbohydrate antigens (TCTAs). The expression of Tn antigen, T antigen and sialyl T antigen on K562 cell membranes was measured by flow cytometry after treatment with different concentrations of imatinib. Co-localization of GalNAc-Ts and ER (endoplasmic reticulum) was determined by confocal laser scanning microcopy. Transcript expression levels of several glycosyltransferases related to TCTAs were decreased after imatinib (0-0.3μM) treatment. Expression of Tn antigen and T antigen was increased while that of sialyl T antigen was decreased. Co-localization of GalNAc-Ts and ER was reduced by 0.2μM of imatinib. Imatinib inhibited the expression of O-glycan related TACAs and several related glycosyltransferases, while decreasing the co-localization of GalNAc-Ts and ER and normalizing O-glycosylation in the K562 human leukemia cell.

The Relationship Between the Regulation of TOB1 Gene with Cell Proliferation, Apoptosis and Cell Cycle in BCR-ABL Positive Cells

AbstractAbstract 5125The TOB1 gene is a transcription factor responsible for the transduction of the gene ERBB2. It is a member of a family of cell suppressor proliferation proteins called TOB/BTG1 family; also, this gene operates on the inhibition of neoplastic transformation. The TOB1 gene presents a decreased expression in several types of cancer such as lung, breast, thyroid and stomach cancer. However, the function of this gene in chronic myeloid leukemia (CML) remains unknown. Aiming to evaluate the inhibition of gene TOB1 into BCR-ABL positive cells and trying to elucidate the molecular mechanisms associated with the inhibition of this gene in the CML we proceed to a more detailed study of this gene. The inhibition of this gene in K562 cells was performed using specific lentivirus. The effect of silencing TOB1 in the proliferation of K562 cells was assessed by the MTT assay after 48 hours of culture; in shTOB1 the proliferation was increased in comparison with shControl cells. To evaluate the synergistic effect between the inhibition of kinase tyrosine activity of BCR-ABL and the inhibition of TOB1 we performed a treatment with different concentrations of imatinib (0. 1, 0. 5 and 1μM), but we observed the decrease in cell proliferation of shTOB1 cells to similar levels of shControl cells only at the 1μM concentration. Therefore, the TOB1 silencing increased the proliferation of K562 cells without an additional effect of a treatment with Imatinib. To analyze the clonogenicity, we performed a formation of colonies assay, in methylcellulose, to determine whether silencing TOB1 could cause a change in the clonal growth of positive BCR-ABL cells. There was no significant change in the number of colonies that grew in cell culture shTOB1 compared to shControl cells. These results suggest that silencing TOB1 in K562 cells may not change the clonogenicity. In the assessment of cell cycle, the flow cytometry analysis revealed a significant accumulation of K562 cells in S phase, with consequent reduction of cells in the G2 phase of the cell cycle in cells shTOB1 compared to cells shControl. The TOB1 gene silencing in K562 cells kept the cells in the S phase and prevented the entry of cells in the G2 phase showing that the inhibition of gene TOB1 induced an increase in proliferation of K562 BCR-ABL cells. The level of apoptosis was assessed by flow cytometry after labeling the cells with anexin-V/PI. The Imatinib treatment presented dose-response in the induction of apoptosis as expected. However, a cumulative effect with TOB1 silencing was not observed. Furthermore, the apoptosis was also assessed by assays of caspases 3, 8 and 9, which showed an increase of the caspase activity of shControl cells in relation of the shTOB1 cells, showing that inhibition of this gene also changes the level of apoptosis. These results corroborate the literature data that report the relationship of this tumour suppressor gene in signalling pathways related to angiogenesis, carcinogenesis, apoptosis and metastasis. When we relate the results obtained with the LMC, we can consider the possibility of TOB1 regulation changes be related to modification of important signalling pathways such as AKT, PI3K, STAT3 and STAT5, among others. Furthermore, the inhibition of TOB1 may be related with an increase on the number of BCR-ABL positive cells and subsequent disease progression. In conclusion, this study confirmed literature data showing that TOB1 gene works as a tumour suppressor protein in cells of many types of cancer. From this work we can infer that in CML the expression of this gene is transformed, resulting in changing of the capacity of induction of apoptosis, decrease tumour necrosis and increase cell proliferation. This work was supported by FAPESP and INCT. Disclosures:No relevant conflicts of interest to declare.

Increased Levels of Myeloid-Derived Suppressor Cells (MDSCs) in Chronic Myeloid Leukemia and the Effect of Tyrosine Kinase Inhibitors on MDSCs in Vitro

Abstract 2744 BackgroundMyeloid-derived suppressor cells (MDSCs) are cells of myeloid origin that are able to inhibit immune cells such as T cells. MDSCs have been found in many different solid tumors and the role of these cells in cancer is currently in focus since they hamper anti-tumor immune responses and thereby lead to a worse prognosis for the patients. The presence and function of MDSCs in hematological malignancies such as chronic myeloid leukemia (CML) has so far not been extensively studied. Since patients with CML have malignant immature myeloid cells in blood and bone marrow it is of interest to investigate if these cells are myeloid suppressor cells. Furthermore, the level of MDSCs in solid tumors decreases after treatment with the tyrosine kinase inhibitor (TKI) sunitinib. CML patients are treated with other TKIs such as imatinib and dasatinib and it is of interest to investigate if these have the same effect on MDSCs. Imatinib and dasatinib are intended to block bcr-abl in the tumor cells. In addition to this, these TKIs have been shown to interact with other tyrosine kinases present in immune cells. AimThe aim of the study was to investigate the presence of MDSCs and the MDSC-associated molecule Arginiase-1 in CML patients and cell lines as well as to investigate the effect of the TKIs on MDSCs in vitro. MethodsCryopreserved leukapheresis samples from newly diagnosed patients in chronic phase (n=18) from high (n=11) and low (n=7) Sokal score risk group CML patients and buffy coats from healthy controls (HC, n=21) as well as CML cell lines BV173, K562 and CML-T1 were investigated for the presence of MDSCs (CD11b+CD14-CD33+ cells) by flow cytometry. Peripheral blood mononuclear cells (PBMCs) from healthy donors were cultured in different concentrations of imatinib or dasatinib and the level of MDSCs in cultures was assessed after 2 and 4 days of culture by flow cytometry. The level of the MDSC-associated molecule Arginase-1 in cells from CML patients (n=5), controls (n=5) and CML cell lines as well as in healthy PBMCs after culture with imatinib or dasatinib was assessed by real time PCR. ResultsCML patients had significantly higher levels of MDSCs compared to health controls (medians 2,6% in CML and 0,8% in HC). When CML patients were divided into Sokal high and low risk groups the high risk group had higher MDSC level (median 3,6%) compared to the low risk group (median 0,8%). The level of MDSC in Sokal low risk group did not differ from the level in HCs. Arginase-1 is a molecule linked to the immune inhibitory effects of MDSCs. The mRNA expression level of Arginase-1 was higher in CML patients compared to HCs as assessed by real time PCR. When healthy PBMCs were cultured with imatinib or dasatinib for 2 to 4 days, the level of MDSCs increased in a dose dependent manner. In accordance with this, the Arginase-1 mRNA expression also increased with treatment in a dose dependent manner. All cell lines investigated had cells with the MDSC phenotype, however not all cells exhibited that phenotype. Only K562 expressed Arginase-1 and the expression increased when the cells were treated with imatinib or dasatinib. ConclusionsCML have higher MDSC levels compared to healthy individuals and the level correlates to Sokal score. The level of MDSCs also correlates with mRNA expression of Arginase-1. Interestingly, treatment of healthy donor PBMCs with the TKIs imatinib or dasatinib increases the level of MDSCs in contrast to results shown with sunitinib. Disclosures:Simonsson:Novartis, BMS, Merck, Pfizer: Consultancy, Honoraria.

Influence of imatinib combined with ABT-737 on apoptosis of gastrointestinal stromal tumor cells

Objective To observe the effect of imatinib (IM) combined with ABT-737 on apoptosis of gastrointestinal stromal tumor cells.Methods We treated the GIST-882 with ABT-737 alone or ABT737 combined with imatinib.The effect of different concentrations of ABT-737 at different time points on the cell viability was determined by methyl thiazol tetrazolium (MTT).The expression of bcl-2 in GIST882 after IM treatment alone was analyzed by Western blotting.The activation form of Caspase-3 after IM or ABT-737 treatment alone and combination of IM with ABT-737 was analyzed by Western blotting.The effect of combination of IM with ABT-737 on the cell viability was determined by MTT.Results After treatment of GIST-882 cells with ABT-737 alone at a concentration of 10 μmol/L,the 24-,48- and 72-h survival rate of tumor cells was respectively 70％,55％ and 38％.At the concentration of 20 μmol/LABT-737,the 24-,48- and 72-h survival rate was less than 20％.After treatment with IM at a concentration of 0.1 μmol/L combined with ABT-737 at a concentration of 0.1 μmol/L,the survival rate of GIST-882 cells was less than 20％.The combined use of IM with ABT-737 both at the concentration of 10 μmol/L could reduce the survival rate of GIST-882 cells to ＜ 10％.IM treatment alone did not have any effect on the expression of bcl-2.The activation form of Caspase-3 was upregulated by the treatment of combination of IM with ABT-737.Different concentrations of IM combined with ABT-737 could decline the cells viability.Conclusion Combination of ABT-737 with IM could enhance the cell apoptosis. Key words: Imatinib; ABT-737 ; Gastrointestinal stromal tumor; Apoptosis

Activity of Dasatinib Against Novel KIT-N822I Mutation In Familial Cutaneous Mastocytosis

AbstractAbstract 4088 Background:In contrast to mastocytosis associated with somatic KIT mutations, the accounts of familial forms of mastocytosis with KIT germline mutations are extremely rare. We report the family that met the WHO criteria for cutaneous mastocytosis, which was diagnosed in the father and two children. Patients and Methods:The clinical follow-up of mastocytosis in the father included bone marrow histopatological/cytological examinations and flow cytometry, and histopatological examination of the skin. In children tryptase measurement and skin histopatological examination was performed. The father and children presented with urticaria pigmentosa as the only manifestation of the disease. Blood, urine and buccal swabs specimens were collected from the family members. Molecular analysis of the KIT coding sequence revealed a novel missense mutation (p.N822I) in the affected members of the family.Ba/F3 cell lines expressing KIT-N822I, KIT-D816V and KIT-V559D mutants were treated with different concentrations of imatinib and dasatinib. The effect of treatment on proliferation, survival, and signaling was determined. In addition, Cos-7 cells were transiently transfected with plasmids expressing KIT-WT and KIT-N882I. Results:By in vitro assays, both imatinib and dasatinib exhibited a high efficacy toward the control, imatinib-sensitive KIT-V559D mutant. In contrast, only dasatinib potently inhibited the KIT-N822I and control, imatinib-resistant KIT-D816V isoform with an IC50 value of 68 nmol/L and 77nmol/L, respectively. Finally, experiments with Cos-7 cells showed that on the contrary to KIT-WT the KIT-N822I mutation constitutively activated KIT tyrosine phosphorylation. Conclusion:In summary, we proved that novel germline KIT mutation (p.N822I) has transforming potential and cause a constitutive activation of KIT. Moreover, we demonstrated that treatment with dasatinib may provide a therapeutic alternative for patients with mastocytosis whose carry imatinib-resistant KIT mutant isoforms. Disclosures:No relevant conflicts of interest to declare.

IFN-Alpha and gm-CSF Can Reverse Immunomodulatory Effects of Imatinib

Presently, Imatinib (IM) is the first line therapy for chronic myeloid leukaemia (CML), but in most patients CML recurs after discontinuation of IM. In contrast, allogeneic stem cell transplantation (ASCT) is considered to be the only curative treatment for this malignancy curing by the immune effects of donor T-cells against CML progenitor cells. Recent reports have described that IM has inhibitory effects on both the function of T-cells and antigen presenting cells (APCs) like dendritic cells. In the present study we further characterized the immunomodulatory effects of IM on T-cells and APCs and analysed whether IFN-alpha (interferon alpha, IFN-a) alone or in combination with GM-CSF (granulocyte-macrophage colony-stimulating factor) can influence the negative IM effects in vitro. Patient derived CML cells were examined to stimulate allogeneic HLA-mismatched T-cells without and with Imatinib (1, 2 or 5 micro M). The activation profile (CD25) and the expression of the TCR-alpha (T-cell receptor) on the T-cells was determined by FACS after 5 days in presence of IM as well as the proliferative T-cell response which was evaluated using CFSE (Carboxy Fluoroscein Succinimidyl Ester). In [51Cr]-release assays the cytotoxicity of CD8+ T-cells was assessed. The cytokine profile of the culture supernatants was detected by CBA (Cytokine bead array, Beckton Dickinson, USA). We also characterized the antigen profile (HLA-DR, CD40, CD54, CD58, CD80 and CD86) of the CML cells as APCs over a 5 day culture with and without IM. The rescue of the CML cells was initiated by IFN-a or IFN-a/GM-CSF after 3 days incubation with IM. On day 6 the APC profile of the CML cells was obtained and cells were used as stimulators in terms of T-cell proliferation which was determined by CFSE. We could show that the proliferation of allogeneic T-cell was inhibited in the presence of IM in a dose-dependent manner. In addition, the T-cell activation marker CD25 and the TCR-alpha expression were significantly downregulated by different concentrations of IM. Moreover, IM impaired the cytotoxic function of allogeneic T-cells in a dose-dependent manner. Interestingly, after stimulation with CML-cells cytotoxic T-cell activity could only be induced in those cases that did not secrete IL-6. Down-regulation of the APC profile on CML-cells (adhesion/costimulatory molecules) by increasing concentrations of IM could be in part reversed by the addition of IFN-a and completely restored by the combination of IFN-a and GM-CSF. Although, the proportion of proliferating T-cells could not be increased further by the combination of IFN-a ± GM-CSF, the number of T-cells was increased in the presence of IFN-a + GM-CSF. In conclusion, the down-modulating effects of IM could be almost completely reversed by the addition of IFN-a and GM-CSF. It remains to be seen whether these findings can be successfully applied to the treatment of CML-patients.

Immune Effects of Imatinib in Chronic Myelogenous Leukemia In Vitro.

Imatinib mesylate, a potent and selective inhibitor of the BCR-ABL tyrosine kinase, has been shown to induce durable haematological and major cytogenetic responses in a high percentage of CML patients. However in most patients the disease recurs, when imatinib is discontinued. In contrast, allogeneic stem cell transplantation (ASCT) is considered to be curative by the immune effect of donor T cells against CML progenitor cells. In this context, the role of imatinib is controversial; it may improve the results of ASCT by reducing the tumour load, it may also reduce the effect of donor lymphocyte transfusions (DLT) by impairing the function of T cells and the capacity of myeloid cells to present antigen. Patient derived CML-cells were studied for the stimulation of allogeneic HLA-matched and mismatched T-cells in the presence and absence of imatinib. In a 5 day culture the proliferative response of HLA-mismatched T cells was evaluated in presence of different concentrations of imatinib (0, 1, 2, or 5 micro M) and various responder-to-stimulator ratios. Thereby, proliferation was detected via a CFDA based assays and the activation profile (CD25, CD69) of the T-cells was determined by FACS. Cr51-release assays were performed after a 7 day culture of CML cells with HLA-matched T cells to test cytotoxicity of CD8+ T-cells. In addition, we characterized the antigen-presenting profile (CD14, CD33, HLA-DR, CD40, CD80, CD86, CD54, CD58) of the CML cells over a 5 day culture with and without imatinib. The presence of imatinib inhibited the proliferative capacity of allogeneic T-cells in a dose-dependent manner. Also, the expression of T cell activation markers was reduced in the presence of the different imatinib concentrations. Preincubation of CML cells with imatinib for 48 hours strengthened the effect on proliferation and activation of T cells. Moreover, imatinib impaired the cytotoxic function of T-cell (HLA-matched setting; CR51-release assay) also in a dose-dependent manner. Finally, the antigen-presenting profile of the myeloid leukemia cells was down regulated by increasing concentrations of imatinib. In summary, imatinib may interfere with the T cellular immune response and the antigen presenting profile on the CML cells in vitro. These results may have an impact on new strategies of treatment of CML with immunotherapy.

Immunomodulatory Effects of STI571 in Chronic Myeloid Leukaemia.

Background: The tyrosine kinase inhibitor imatinib (imatinib, STI571, Glivec, and Gleevec) is highly effective in the treatment of chronic myeloid leukaemia (CML) and has already been shown to be effective in the setting of allogeneic stem cell transplantation. But until now, less is known with respect to its immunomodulating effects.Objective: In the present survey we investigated, whether imatinib could modify the antigen-presenting capacity of myeloid cells and in turn affects the cellular immune response.Method: For this purpose, patient derived chronic myeloid cells were incubated with different concentrations of imatinib (0, 1, 2, or 5microM), characterized for their antigen-presenting profile and their stimulatory capacity in the context of HLA-matched and mismatched T-cells. After 5 days, the proliferative immune response was evaluated in presence or absence of different concentrations of imatinib and altered effector-to-target ratios. Thereby, proliferation was detected via a CFDA, SE (5,6-carboxyfluorescein diacetate succinimidyl ester) based assay.Result: The proliferative capacity of the T- cells (allogeneic, HLA-mismatched) was inhibited by imatinib in a dose-dependent manner. Also, the expression of the activation markers was reduced in the presence of the different STI571 concentrations. Moreover, myeloid blasts were sensitized for T cell mediated effector functions by pre-treatment with increasing concentrations of imatinib.Conclusion: Taken together, imatinib can interfere with the T cellular immune response in vitro, and its impact on graft-versus-leukemia (GvL) and graft-versus-host (GvH) reactions will be further investigated.

Transcriptional Regulation of the Bach2 B-Cell Differentiation and Apoptotic Factor by the Bcr-Abl Oncoprotein.

Transformation by the Bcr-Abl oncoprotein of CML is mediated by the activation of a variety of signalling pathways, leading to transcriptional regulation of genes conferring the malignant phenotype of increased proliferation, altered adhesion and inhibition of apoptosis. We previously reported that expression of the BACH2 gene is downregulated by Bcr-Abl. Bach2 is a B-lymphoid specific transcription factor, which regulates somatic hypermutation and class switch recombination of Ig genes. It is also a pro-apoptotic factor, coupling oxidative stress to transcription repression. It is possible that in an environment of increased genomic instability, Bcr-Abl transformed cells may repress pro-apoptotic signals by suppressing BACH2 transcription. To determine the direct association between Bcr-Abl and decreased BACH2 transcription, we infected human B-lymphoid cells with a retroviral vector expressing both p210Bcr-Abl and eGFP genes. Infected cells were treated with imatinib, an Abl tyrosine kinase inhibitor, prior to quantification of BACH2 transcripts by Real Time RT/PCR. Ectopic expression of BCR-ABL significantly decreased BACH2 mRNA levels, and this effect was completely abolished by imatinib. To investigate whether this regulation was exerted at the transcriptional level, we identified the BACH2 transcription initiation site (TIS), and then cloned and characterised a 3.9 Kb genomic DNA fragment including the BACH2 promoter region. By generating luciferase reporter constructs of various lengths of the BACH2 promoter we found that a region of 725 bp upstream the TIS conferred maximum promoter activity in human B-lymphoid cells. The effect of Bcr-Abl on promoter activity was demonstrated by co-transfection of the reporter and p210Bcr-Abl constructs. BACH2-promoter activity was reduced up to 60% in the presence of Bcr-Abl. Furthermore, when co-transfected cells were incubated with different concentrations of imatinib, the Bcr-Abl-mediated promoter repression was abrogated in a dose dependent manner, confirming the dependence of the effect on the tyrosine kinase activity of the oncoprotein. In support of these data, no effect on promoter activity was seen when the BACH2 promoter was co-transfected with a kinase-dead BCR-ABL construct. Moreover, treatment with imatinib of the BCR-ABL+ cell line BV173 transfected with the reporter induced a nearly 2-fold upregulation in its activity. Bioinformatics inspection of the promoter sequence revealed potential sites for the Pax5 B-cell differentiation factor and the Foxo3a transcription factor, a regulator of pro-apoptotic genes. In co-transfection experiments of either factor with the BACH2 promoter, both demonstrated a significant inducing effect on its activity. Gel shift and chromatin immunoprecipitation showed direct binding of Pax5 within the BACH2 promoter in vitro and in vivo. Moreover, Western analysis showed elevated Pax5 levels in BCR-ABL+ cell lines after imatinib treatment, indicating that inhibition of Bach2 expression by Bcr-Abl is mediated at least in part by Pax5. As to Foxo3a, it has been reported to be constitutively phosphorylated and inactivated in BCR-ABL+ cells, processes which prevent its translocation to the cell nucleus. Altogether, our data suggest that Bcr-Abl transcriptional repression of Bach2 via Pax5 could lead to a differentiation arrest in transformed B-cells, and that Foxo3a may induce imatinib-mediated apoptosis through up-regulation of the Bach2 apoptotic function.