Reversal of the resistance to STI571 in human chronic myelogenous leukemia K562 cells

STI571, an Abl-specific tyrosine kinase inhibitor, selectively kills Bcr-Abl-containing cells in vitro and in vivo . However, some chronic myelogenous leukemia (CML) cell lines are resistant to STI571. We evaluated whether STI571 interacts with P-glycopro-tein (P-gp) and multidrug resistance protein 1 (MRP1), and examined the effect of agents that reverse multidrug resistance (MDR) on the resistance to SI571 in MDR cells. STI571 inhibited the [¹²⁵l]azidoagosterol A-photolabeling of P-gp, but not that of MRP1. K562/MDR cells that overexpress P-gp were 3.67 times more resistant to STI571 than the parental Philadelphia-chromosome-positive (Ph+) CML K562 cells, and this resistance was most effectively reversed by cepharanthine among the tested reversing agents. The concentration of STI571 required to completely inhibit tyrosine phosphorylation in K562/MDR cells was about 3 times higher than that in K562 cells, and cepharanthine abolished the difference. In KB-G2 cells that overexpress P-gp, but not Bcr-Abl, 2.5 μM STI571 partly reversed the resistance to vincristine (VCR), paclitaxel, etoposide (VP-16) and actinomycin D (ACD) but not to Adriamycin (ADM) or colchicine. STI571 increased the accumulation of VCR, but not that of ADM in KB-G2 cells. STI571 did not reverse resistance to any agent in KB/MRP cells that overexpress MRP1. These findings suggest that STI571 is a substrate for P-gp, but is less efficiently transported by P-gp than VCR, and STI571 is not a substrate for MRP1. Among the tested reversing agents that interact with P-gp, cepharanthine was the most effective agent for the reversal of the resistance to STI571 in K562/ MDR cells. Furthermore, STI571 itself was a potent reversing agent for MDR in P-gp-expressing KB-G2 cells.     

STI571在慢性髓细胞白血病急变期造血干细胞移植治疗中的作用

目的：探讨STI571在慢性髓细胞白血病(CML)急变期外周血干细胞移植中的应用价值。方法：CML急变期患者接受STI571治疗,然后进行异基因造血干细胞移植,同时进行临床观察。结果：3例CML急变期患者均获得临床血液学缓解,STI571对异基因造血干细胞移植后的造血恢复无影响。结论：STI571对CML急变期有治疗作用,可应用于造血干细胞移植前治疗,二者结合有可能使患者生存期延长。     

Mechanisms of resistance to STI571 (Imatinib) in Philadelphia-chromosome positive acute lymphoblastic leukemia

Resistance against STI571 (Imatinib) appears to be multifactorial, but the most likely mechanisms can be broadly categorized as interference with the pharmacologic activity of STI571 or genetic changes which alter the biologic behaviour of the leukemic cells. In Ph+ ALL, responses to STI571 are not sustained, and in the overwhelming majority of patients development of resistance is rapid. Clinically, two types of resistance to STI571 can be distinguished: 'primary resistance', corresponding to a failure to achieve fewer than 5% blasts in the bone marrow, and 'secondary resistance' in patients with STI571-induced complete remission who relapse despite continued STI571 treatment. Attempts to identify mechanisms by which Ph+ ALL acquire resistance to STI571 have already been successful. Mutations in the ATP binding site of ABL are frequent events which counteract the antileukemic effect of STI571. Gene expression profiling has been shown to discriminate between resistant and sensitive leukemic cells. Application of this technique has also generated several hypotheses regarding the ability of leukemic cells to bypass the BCR-ABL signal transduction pathway. This may result in the proliferation of Ph+leukemic cells even in the presence of STI571.     

STI571对恶性多发性神经胶质母细胞瘤(GBM)的体外抑瘤作用

目的:研究酪氨酸激酶抑制剂(STI571)对恶性胶质瘤细胞的肿瘤生物学调节作用.方法:用免疫组化法探查血小板源生长因子(PDGF)及其受体在T98G,U87MG,LNZ308,RG四种胶质瘤细胞的表达;MTT法用于检查STI571/TMZ单独或联合应用对胶质瘤细胞的增殖抑制作用,STI571诱导的细胞凋亡途径由流式细胞仪检测,免疫印迹杂交法用来探查STI571抵抗细胞的蛋白激酶网络变化.结果:所用胶质瘤细胞均不同程度的表达PDGF及受体.STI571与TMZ相互协同作用于胶质瘤细胞株T98G;10μmol/L STI571可部分抑制所测胶质瘤细胞增殖、可完全抑制其形成集落;高于10μmol/L浓度的STI571可快速诱导胶质瘤细胞凋亡或坏死;胶质瘤细胞株T98G与STI571长期共培养后,瘤细胞蛋白激酶网络有较大改变.结论:研究表明,PDGF受体酪氨酸激酶抑制剂STI571有较强的抑制恶性胶质瘤细胞(GBM)增殖的作用并可能与其他化疗药物协同抗肿瘤.     

Role of alpha1 acid glycoprotein in the in vivo resistance of human BCR-ABL(+) leukemic cells to the abl inhibitor STI571

BACKGROUND: Chronic myeloid leukemia is caused by a chromosomal translocation that results in an oncogenic fusion protein, Bcr-Abl. Bcr-Abl is a tyrosine kinase whose activity is inhibited by the antineoplastic drug STI571. This drug can cure mice given an injection of human leukemic cells, but treatment ultimately fails in animals that have large tumors when treatment is initiated. We created a mouse model to explore the mechanism of resistance in vivo. METHODS Nude mice were injected with KU812 Bcr-Abl(+) human leukemic cells. After 1 day (no evident tumors), 8 days, or 15 days (tumors >1 g), mice were treated with STI571 (160 mg/kg every 8 hours). Cells recovered from relapsing animals were used for in vitro experiments. Statistical tests were two-sided. RESULTS: Tumors regressed initially in all STI571-treated mice, but all mice treated 15 days after injection of tumor cells eventually relapsed. Relapsed animals did not respond to further STI571 treatment, and their Bcr-Abl kinase activity in vivo was not inhibited by STI571, despite high plasma concentrations of the drug. However, tumor cells from resistant animals were sensitive to STI571 in vitro, suggesting that a molecule in the plasma of relapsed animals may inactivate the drug. The plasma protein alpha1 acid glycoprotein (AGP) bound STI571 at physiologic concentrations in vitro and blocked the ability of STI571 to inhibit Bcr-Abl kinase activity in a dose-dependent manner. Plasma AGP concentrations were strongly associated with tumor load. Erythromycin competed with STI571 for AGP binding. When animals bearing large tumors were treated with STI571 alone or with a combination of STI571 and erythromycin, greater tumor reductions and better long-term tumor-free survival (10 of 12 versus one of 13 at day 180; P:<.001) were observed after the combination treatment. CONCLUSION: AGP in the plasma of relapsed animals binds to STI571, preventing this compound from inhibiting the Bcr/Abl tyrosine kinase. Molecules such as erythromycin that compete with STI571 for binding to AGP may enhance the therapeutic potential of this drug.     

Phosphatidylinositol-3 kinase inhibitors enhance the anti-leukemia effect of STI571

BCR/ABL fusion tyrosine kinase is responsible for the initiation and maintenance of the Philadelphia chromosome (Ph(1))-positive chronic myelogenous leukemia (CML) and a cohort of acute lymphocytic leukemias (ALL). STI571 (Gleevec), a novel anti-leukemia drug targeting BCR/ABL kinase can induce remissions of the Ph(1)-positive leukemias. STI571 was recently combined with the standard cytostatic drugs to achieve better therapeutic results and to overcome emerging drug resistance mechanisms. We decided to search for a more specific partner compound for STI571. Our previous studies showed that a signaling protein phosphatidylinositol-3 kinase (PI-3k) is essential for the growth of CML cells, but not of normal hematopoietic cells (Blood, 86:726,1995). Therefore the anti- Ph(1)-leukemia effect of the combination of BCR/ABL kinase inhibitor STI571 and PI-3k inhibitor wortmannin (WT) or LY294002 (LY) was tested. We showed that STI571+WT exerted a synergistic effect against the Ph(1)-positive cell lines, but did not affect the growth of Ph(1)-negative cell line. Moreover, the combinations of STI571+WT or STI571+LY were effective in the inhibition of clonogenic growth of CML-chronic phase and CML-blast crisis patient cells, while sparing normal bone marrow cells. Single colony RT-PCR assay showed that colonies arising from the mixture of CML cells and normal bone marrow cells after treatment with STI571+WT were selectively depleted of BCR/ABL-positive cells. Biochemical analysis of the CML cells after the treatment revealed that combination of STI571+WT caused a more pronounced activation of caspase-3 and induced massive apoptosis, in comparison to STI571 and WT alone. In conclusion, combination of STI571+WT or STI571+LY may represent a novel approach against the Ph(1)-positive leukemias.     

Anti-proliferative effect of the abl tyrosine kinase inhibitor STI571 on the P-glycoprotein positive K562/ADM cell line

STI571, an abl tyrosine kinase inhibitor, is less effective in chronic myelogenous leukemia (CML) patients in the accelerated phase and in blastic crisis. We addressed whether STI571 is effective for the CML blastic crisis cell line K562 and the P-glycoprotein (P-gp) positive, multidrug resistance cell line K562/ADM. The present results demonstrate that P-gp positive K562/ADM cells were more resistant than K562 cells to the anti-proliferative and apoptotic effect of STI571, but the co-addition of a P-gp modulator augmented the sensitivity of K562/ADM cells to STI571. For patients in CML blastic crisis, simultaneous use of a P-gp modulator may increase the efficacy of STI571.     

Pivanex, a histone deacetylase inhibitor, induces changes in BCR-ABL expression and when combined with STI571, acts synergistically in a chronic myelocytic leukemia cell line

Chronic myelogenous leukemia (CML) is associated with the high TK activity chimeric protein BCR-ABL, known to contribute to cell tumorogenicity, resistance to apoptosis and differentiation. STI571, the TK inhibitor, is the current treatment for CML. One possible approach to overcome STI571 resistance appearing in some cases, involves the combination of histone deacetylase inhibitors (HDI) and STI571. We demonstrated that in K562, the CML cell line, pivaloyloxymethyl butyrate (Pivanex)-induced apoptosis, differentiation and reduced BCR-ABL protein levels and that the combination of Pivanex with STI571 acted synergistically. These data suggest the possible benefit of combining this HDI with STI571 for treatment of CML.     

STI571诱导K562细胞红系分化及其机制的初步研究

目的 :探讨 STI5 71诱导 K5 6 2细胞向红系分化的可能机制。方法 :单用 STI5 71及联用信号传导阻滞剂处理 K5 6 2细胞 ,采用联苯胺染色法检测 K5 6 2细胞向红系分化比例变化 ,流式细胞术检测细胞周期改变 ,Western blot检测 Rb、p- Rb、Erk、p- Erk、p2 7蛋白改变 ,RT- PCR检测 GATA1、GATA2、p2 7m RNA水平变化。结果 :STI5 71诱导 K5 6 2细胞向红系分化 ,呈时间剂量依赖性改变。K5 6 2细胞经 STI5 71处理后 1 2 h即可发生 G1 期阻滞 ,随时间延长趋于明显 ,伴随 p2 7,p- Rb水平下降。p- Erk水平升高。STI5 71与信号传导阻滞剂 U0 1 2 6联合应用后 ,K5 6 2细胞联苯胺阳性率明显下降 (P<0 .0 5 )。STI5 71作用后 ,GATA1、GATA2、m RNA水平未见明显变化。结论 :STI5 71通过上调p2 7,降低 p- Rb水平 ,诱导 K5 6 2细胞发生 G1 期阻滞 ,活化 Erk促使 K5 6 2细胞向红系分化     

Synergistic activity of the new ABL-specific tyrosine kinase inhibitor STI571 and chemotherapeutic drugs on BCR-ABL-positive chronic myelogenous leukemia cells.

The ABL-specific tyrosine kinase inhibitor STI571 (formerly CGP57148B) induced cytogenetic remissions in 33% of chronic myelogenous leukemia (CML) patients in a phase I trial (Druker et al 1999). Combination therapy may increase this proportion. We tested whether combinations of STI571 and cytarabine or other chemotherapeutic agents such as hydroxyurea, mafosfamide or etoposide would display synergistic activity in BCR-ABL-positive chronic myelogenous leukemia (CML) cell lines derived from patients in blast crisis. In addition, the toxicity of these combinations on BCR-ABL-negative cells was investigated. A tetrazolium-based MTT assay was used to quantity growth inhibition after 48 h of exposure to cytotoxic agents alone and in simultaneous combination with STI571. The drug interactions were analyzed using the median-effect method of Chou and Talalay. The combination index (CI) was calculated according to the classic isobologram equation. At growth inhibition levels of over 50%, STI571 + cytarabine as well as STI571 + etoposide were significantly synergistic (CI < 1, P < 0.05) in the BCR-ABL-positive cell lines evaluated. At 60% inhibition or higher, a similar synergistic pattern became apparent for STI571 + mafosfamide (P < 0.05), while STI571 + hydroxyurea showed ambiguous, cell line-dependent synergism (BV173), additivity (EM-3) or antagonism (K562) in CML cell lines. Furthermore, the BCR-ABL-negative HL-60, KG1a and normal CD34+ progenitor cells were not affected by 0.8 microM STI571, a concentration which produced more than 50% growth inhibition in all BCR-ABL-positive cells tested, and no potentiation of growth inhibition was observed in these BCR-ABL-negative cells when STI571 was combined with chemotherapeutic agents. Our in vitro data with CML blast crisis cell lines strongly suggest that combinations of STI571 with cytarabine or etoposide be rapidly considered for clinical testing.     

MTT assays cannot be utilized to study the effects of STI571/Gleevec on the viability of solid tumor cell lines

Purpose  This study will determine whether MTT assays accurately assess the effect of STI571 (Gleevec; Abl kinase inhibitor) on the viability of cancer cells containing highly active Abl kinases. Methods  Growth kinetics, tritiated thymidine, fluorescent caspase, MTT, and Cell Titer Glo (CTG) assays were used to determine the effect of STI571 on growth, proliferation, apoptosis, and viability of melanoma and breast cancer cells. Results  STI571 inhibited growth and proliferation, and increased apoptosis. However, MTT assays indicated that STI571 increased cell viability. In contrast, STI571 induced a dose-dependent decrease in viability using CTG assays. Conclusions  Doses of STI571 (1–10 μM) required to inhibit endogenous Abl kinases interfere with the MTT assay, and therefore MTT cannot be used to determine the effect of STI571 on viability using these doses. Additionally, caution should be utilized when interpreting the results of MTT assays used to screen kinase inhibitors for anti-cancer activity, as drug effectiveness may be minimized.     

Growth inhibition and modulation of kinase pathways of small cell lung cancer cell lines by the novel tyrosine kinase inhibitor STI 571

Small cell lung cancer (SCLC) is an aggressive cancer characterized by several autocrine growth mechanisms including stem cell factor and its receptor c-Kit. In order to arrive at potentially new and novel therapy for SCLC, we have investigated the effects of the tyrosine kinase inhibitor, STI 571, on SCLC cell lines. It has been previously reported that STI 571 does not only inhibit cellular Abl tyrosine kinase activity but also the PDGF receptor and c-Kit tyrosine kinases at similar concentrations (approximately 0.1 microM). There is no expression of the PDGF-receptor, and the Abl kinase is not activated by SCLC, but over 70% of SCLC contain the c-Kit receptor. Utilizing this preliminary data, we have determined that three (NCI-H69, NCI-H146 and NCI-H209) of five (including NCI-H82 and NCI-H249) SCLC cell lines had detectable c-Kit receptors and were inhibited in growth and viability at concentrations 1 - 5 microM of STI 571 after 48 h of treatment. The SCLC cell lines, NCI-H69, NCI-H146 and NCI-H209, showed a dose-response (tested between 0.1 - 10 microM) inhibition of tyrosine phosphorylation of c-Kit as well as in vitro kinase activity (at 5 microM) of c-Kit in response to STI 571. STI 571 inhibited cell motility, as assessed by time-lapsed video microscopy, within 6 h of STI 571 treatment (5 microM). STI 571 also decreased intracellular levels of reactive oxygen species (ROS) by at least 60%, at a concentration (5 microM) that also inhibited cell growth. Cell cycle analysis of STI 571 responsive cells showed that cells were generally slowed in G2/M phase, but there was no arrest at G1/S. A downstream phosphorylation target of c-Kit, Akt, was not phosphorylated in response to stem cell factor in the presence of STI 571. These data imply that STI 571 inhibits growth of SCLC cells through a mechanism that involves inactivation of the tyrosine kinase c-Kit. The effectiveness of STI 571 in this study suggests this drug may be useful in a clinical trial, for patients with SCLC. Oncogene (2000) 19, 3521 - 3528     

STI571 (Gleevec) improves tumor growth delay and survival in irradiated mouse models of glioblastoma

PURPOSE: Glioblastoma multiforme (GBM) is a devastating brain neoplasm that is essentially incurable. Although radiation therapy prolongs survival, GBMs progress within areas of irradiation. Recent studies in invertebrates have shown that STI571 (Gleevec; Novartis, East Hanover, NJ) enhances the cytotoxicity of ionizing radiation. In the present study, the effectiveness of STI571 in combination with radiation was studied in mouse models of GBM. METHODS AND MATERIALS: Murine GL261 and human D54 GBM cell lines formed tumors in brains and hind limbs of C57BL6 and nude mice, respectively. GL261 and D54 cells were treated with 5 micromol/L of STI571 for 1 h and/or irradiated with 3 Gy. Protein was analyzed by Western immunoblots probed with antibodies to caspase 3, cleaved caspase 3, phospho-Akt, Akt, and platelet-derived growth factor receptor (PDGFR) alpha and beta. Tumor volumes were assessed in mice bearing GL261 or D54 tumors treated with 21 Gy administered in seven fractionated doses. Histologic sections from STI571-treated mice were stained with phospho-Akt and phospho-PDGFR beta antibodies. Kaplan-Meier survival curves were used to study the response of mice bearing intracranial implants of GL261. RESULTS: STI571 penetrated the blood-brain barrier, which resulted in a reduction in phospho-PDGFR in GBM. STI571-induced apoptosis in GBM was significantly enhanced by irradiation. STI571 combined with irradiation induced caspase 3 cleavage in GBM cells. Glioblastoma multiforme response to therapy correlated with an increase in tumor growth delay and survival when STI571 was administered in conjunction with daily irradiation. CONCLUSION: These findings suggest that STI571 has the potential to augment radiotherapy and thereby improve median survival.     

Inhibition of platelet-derived growth factor receptor signaling restricts the growth of human breast cancer in the bone of nude mice.

PURPOSE: Bone is a common site for breast cancer metastasis. Platelet-derived growth factor (PDGF) and PDGF receptors (PDGFR) are involved in the regulation of bone resorption. This study examined the effects of STI571 (imatinib mesylate), which inhibits PDGFR tyrosine kinase signaling, on the growth of human breast cancer cells in the bone of nude mice with consequent osteolysis. EXPERIMENTAL DESIGN: Human breast cancer MDA-MB-435 cells were injected into the tibia of female nude mice. Two weeks later the mice were treated with p.o. and injected water (control), daily p.o. STI571, weekly injection of paclitaxel, or daily STI571, plus weekly paclitaxel, for up to 8 weeks. Growth of tumors in bones and osteolysis were monitored by digital radiography and tumors were collected for histochemical analysis. RESULTS: Mice treated with STI571 or STI571 plus paclitaxel had smaller bone tumors with less lytic bone destruction than did mice treated with water or paclitaxel alone. The results of treatment with paclitaxel plus STI571 did not differ from those with STI571 alone. Immunohistochemistry showed that PDGF-A, PDGF-B, PDGFRalpha, and PDGFRbeta were expressed in the bone tumors. STI571 treatment inhibited PDGFR phosphorylation in tumor cells and tumor-associated endothelial cells, coincident with increased apoptosis, reduced proliferation, and lower microvessel density in the tumors. CONCLUSIONS: Activated PDGFRs are expressed by endothelial and tumor cells in breast cancer tumors growing in the bone of nude mice. Interfering with PDGFR signaling may be an approach to control the progressive growth of breast cancer cells and thus reduce bone lysis.     

Antiproliferative effect of STI571 on cultured human cutaneous melanoma-derived cell lines

Standard antineoplastic treatment for metastatic melanoma is ineffective in the large majority of patients. Therefore, alternative approaches need to be investigated. STI571 is a new antineoplastic compound, which selectively inhibits the tyrosine kinase activity of ABL, c-Kit and platelet-derived growth factor receptor (PDGFR). Melanoma may express all of these proteins. The aim of this study was to investigate whether STI571 inhibits the in-vitro growth of melanoma cells. Nineteen cell lines were obtained from four primary and 15 metastatic melanomas of cutaneous origin. The percentages of positive cells for the putative targets of STI571 were as follows: ABL, 41-100%; c-Kit, 8-97%; PDGFR-alpha, 41-98%; PDGFR-beta, 51-99%. 3-(4,5-Dimethylthiazol-yl)-2,5-diphenyltetrazolium (MTT) and viability assays showed that STI571 clearly inhibits the proliferation of eight of the 19 (42.1%) cell lines. No relationship could be established between the expression of c-Kit, ABL, PDGFR-alpha or PDGFR-beta and the response of cell lines to STI571. Our study shows, for the first time, an antiproliferative effect of STI571 on human melanoma cell lines of cutaneous origin, raising the possibility of the future clinical use of STI571. The identification of the target of STI571 in human cutaneous melanoma cells would allow the selection of patients who could benefit from this treatment.     

Chemosensitization by STI571 targeting the platelet-derived growth factor/platelet-derived growth factor receptor-signaling pathway in the tumor progression and angiogenesis of gastric carcinoma

BACKGROUND: Autocrine and paracrine growth mediated by the platelet-derived growth factor (PDGF)/PDGF receptor (PDGFR)-signaling pathway plays an important role in the progression of solid tumors. The authors assessed the effect of STI571 on the tumor growth of gastric carcinoma in combination with 5-fluorouracil (5-FU) or paclitaxel targeting the PDGF/PDGFR-signaling pathway. METHODS: In MKN-45 gastric carcinoma cells, the cytotoxic effect was evaluated by 3-(4,5 dimethiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, and the in vivo antitumor effect was evaluated in a nude mouse xenograft. Both STI571 and an antitumor drug were administered intraperitoneally. Gene expression was assessed by Western blot analysis and immunohistochemical staining. Apoptotic cell death was evaluated by the terminal deoxyuridine triphosphate-biotin nick-end labeling assay, and tumor angiogenesis was evaluated by microvessel density analysis. RESULTS: Treatment with STI571 alone was not effective in vitro, as assessed by a 50% inhibitory concentration value of 24.3 microM. Combination treatment with STI571 and 5-FU or paclitaxel enhanced the cytotoxic effect somewhat when the concentration of STI571 was increased to 10 microM. Combination treatment with STI571 and 5-FU or paclitaxel enhanced the antitumor effect of the antitumor drug significantly in vivo. The enhanced antitumor effect was associated with increased apoptotic cell death and inhibition of tumor angiogenesis. Treatment with STI571 down-regulated the expression of PDGF-BB and PDGFR-beta in tumor cells and decreased the production of phosphorylated PDGFR-beta and phosphorylated Akt. Furthermore, treatment with STI571 inhibited the expression of PDGFR-beta in stromal cells. CONCLUSIONS: STI571 was an effective chemosensitizer of antitumor drugs, such as 5-FU and paclitaxel for gastric carcinoma, targeting the PDGF/PDGFR-signaling pathway of tumor cells and stromal cells in disease progression and angiogenesis.     

The influence of imatinib mesylate (STI571) used alone or in combination with purine nucleoside analogues on the normal and chronic myelogenous leukaemia progenitor cells in vitro

Imatinib mesylate (STI571, Glivec), a signal transduction inhibitor used as a single agent demonstrates significant activity in patients with chronic myelogenous leukaemia (CML). Nevertheless, the interaction between STI571 and other antileukaemic drugs such as hydroxyurea, interferon alpha or cytarabine have also been investigated in order to further improve its effectiveness. In this study we have tried to answer the question if the combination of STI571 with purine nucleoside analogues (PNAs)- cladribine (2-CdA) and fludarabine (F-ara-A) intensifies the antiproliferative effect on granulocyte-macrophage progenitor cells (CFU-GM) from patients with CML as well as from normal persons. Our studies were based on the method of semisolid CFU-GM cultures in vitro. We added STI571 or PNAs singly to the culture, each of the drugs at three concentrations, as well as in combinations of the concentrations used. We showed that STI571 (0.5, 1.0 and 2.0 microM) used alone inhibited the colony growth of CML CFU-GM, as compared to CFU-GM derived from normal donors (p = 0.03; p = 0.0004; p = 0.0001). We also observed that STI571 used together with 2-CdA (5,10 and 20 microM) or F-ara-A (0.2, 0.4 and 0.8 microM) at all the combinations significantly inhibited the colony growth of CML CFU-GM, as compared either to the control or to STI571 used alone (p < 0.05). In addition, the differences between CML and normal CFU-GM colony growth inhibition after the use of the combination of the highest concentrations of STI571 either with 2-CdA or F-ara-A were statistically significant (p = 0.03 and p = 0.01, respectively). In conclusion, STI571 used together with both the PNAs had an additive effect on CML CFU-GM cells. However, further experimental and clinical studies concerning the usefulness of these combinations in the treatment of CML patients seem warranted.     

Progenitor cells from patients with advanced phase chronic myeloid leukaemia respond to STI571 in vitro and in vivo

STI571 targets p210(BCR-ABL) in chronic myeloid leukaemia (CML). In vitro, STI571 reduces self-replication (replating ability) by chronic-phase CML CFU-GM. Here, we studied CFU-GM in advanced-phase (accelerated and blast crisis) CML. The numbers and self-replication of CFU-GM in advanced phase were greater than in the chronic phase. Self-replication by CFU-GM from advanced phase patients was reduced by STI571 or IFN alfa to the same extent as in the chronic phase. The reduced replating ability induced by STI571 correlated with that induced by IFN alpha (r=0.73). STI571 treatment in vivo also reduced replating ability and the numbers of CFU-GM/ml of blood.     

Inhibition of cell survival and invasive potential of colorectal carcinoma cells by the tyrosine kinase inhibitor STI571

Inhibiting tyrosine kinases has recently emerged as a therapeutic modality in several forms of neoplasia. The tyrosine kinase inhibitor STI571 (IMATINIB MESYLATE; GLEEVEC; GLIVEC) is a case in point as it has shown promise in the treatment of malignancies expressing the BCR/ABL fusion protein. In addition to BCR/ABL, STI571 inhibits the tyrosine kinase moieties of several cell surface receptors including the platelet-derived growth factor (PDGF) receptors and c-Kit. Previous work demonstrated that c-Kit activation supports migration, invasion and, survival of certain colorectal carcinoma cells including DLD-1. Here we describe that blocking c-Kit with STI571 inhibits these malignant traits not only in DLD-1 cells but also in two early passage colorectal carcinoma cell strains. Specifically, STI571 inhibited anchorage-independent colony formation and cell scattering in semi-solid medium. Furthermore, it enhanced apoptosis susceptibility and abrogated invasion of DLD-1 cells through Matrigel. In addition, STI571 treatment affected the balance of the Bcl-2 family of apoptosis regulators on favor of a pro-apoptotic phenotype. Specifically, STI571 treatment of DLD-1 cells was associated with lower levels of Bcl-2 expression accompanied by de novo expression of Bcl-xS. Finally, STI571 acted as a chemosensitizing agent in DLD-1 cells when used in combination with 5-fluorouracil.     

STI571 sensitizes nasopharyngeal carcinoma cells to cisplatin: sustained activation of ERK with improved growth inhibition

Previously, we demonstrated that c-kit and stem cell factors (SCF) commonly co-expressed in primary and metastatic nasopharyngeal carcinomas (NPC), and in HONE-1 NPC cells with tyrosine autophosphorylation of c-kit. These findings suggest that the SCF/c-kit signaling may contribute to pathogenesis of NPC. Therefore, the efficacy of STI571 treatment alone and when combined with cisplatin on HONE-1 cells were evaluated. STI571 induced growth inhibition at the IC50 concentration (14.9 microM). When the concentration was at or higher than 30 microM, the induction of cell apoptosis was observed. The effects of STI571 were shown to be mediated by the sustained activation of ERK but did not involve the inhibition of c-kit signal activity. When the STI571 (5 microM) and cisplatin (5 microg/ml) treatments were combined, there were further inductions of ERK activation resulting in obviously enhanced growth inhibition and induction of cell apoptosis. In a xenograft model, STI571 (50 mg/kg/day) showed only a limited ability to inhibit HONE-1 cell growth, but when combined with cisplatin (3 mg/kg/twice a week) treatment, there was a significant improvement in growth inhibition compared with STI571 or cisplatin treatment alone. Our results provide experimental support for the advanced NPC therapeutic trials using the combined STI571 and cisplatin treatment.