Myeloid Leukemia Tumors Research Articles

Signal transduction blockade with imatinib mesylate prevents and treats autoimmune arthritis (B76)

Abstract For over two decades researchers have speculated on the potential use of kinase inhibitors to treat autoimmune diseases. To date, investigations of kinase inhibitors have been largely limited by low therapeutic indices that result in unacceptable toxicity profiles. We show that imatinib mesylate, a tyrosine kinase inhibitor FDA-approved to treat chronic myeloid leukemia and gastrointestinal stromal tumors, has the potential to provide benefit for autoimmune diseases such as rheumatoid arthritis (RA). We demonstrate that imatinib, at a dose equivalent to 200–400mg/day in humans, prevents murine autoimmune arthritis and treats established disease. We further show that submicromolar imatinib concentrations inhibit tyrosine kinases and cellular responses that are central to RA pathogenesis, including PDGF-mediated proliferation of fibroblast-like synoviocytes, c-Fms activation and macrophage production of TNF-alpha, c-Kit-mediated mast cell release of proinflammatory cytokines, and B cell immunoglobulin production. Inhibition of tyrosine kinase pathways by imatinib represents a potent and promising therapeutic approach to treat RA and other autoimmune diseases.

Imatinib-induced immune hepatitis: Case report and literature review

Imatinib is one of the most recent medications used for the treatment of chronic myeloid leukemia (CML) and gastrointestinal stromal tumor (GIST). It is an orally administered protein–tyrosine kinase inhibitor, an enzyme which is produced by BCR–ABL fusion which results from translocation of 9:22 chromosome (Philadelphia chromosome). Imatinib blocks proliferation and induces apoptosis of BCR–ABL-expression in CML. Many side effects produced by imatinib have been documented but its induction of hepatotoxcity has been rarely reported. Only a few cases so far have been reported in the literature and almost all were in females. We describe another case of hepatotoxicity due to imatinib in a 17-year old female with clinical, laboratory and histopathological changes. The case described here suggests that imatinib may also induce immune hepatitis, in some patients.

Synthesis and positron emission tomography studies of carbon-11-labeled imatinib (Gleevec).

Imatinib mesylate (Gleevec) is a well known drug for treating chronic myeloid leukemia and gastrointestinal stromal tumors. Its active ingredient, imatinib ([4-[(4-methyl-1-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-pyridyl)-2-pyrimidinyl]amino]phenyl]benzamide), blocks the activity of several tyrosine kinases. Here we labeled imatinib with carbon-11 as a tool for determining the drug distribution and pharmacokinetics of imatinib, and we carried out positron emission tomography (PET) studies in baboons. [N-(11)C-methyl]imatinib was synthesized from [(11)C]methyl iodide and norimatinib was synthesized by the demethylation of imatinib (isolated from Gleevec tablets) according to a patent procedure [Collins JM, Klecker RW Jr, Anderson LW. Imaging of drug accumulation as a guide to antitumor therapy. US Patent 20030198594A1, 2003]. Norimatinib was also synthesized from the corresponding amine and acid. PET studies were carried out in three baboons to measure pharmacokinetics in the brain and peripheral organs and to determine the effect of a therapeutic dose of imatinib. Log D and plasma protein binding were also measured. [N-(11)C-methyl]imatinib uptake in the brain is negligible (consistent with P-glycoprotein-mediated efflux); it peaks and clears rapidly from the heart, lungs and spleen. Peak uptake and clearance occur more slowly in the liver and kidneys, followed by accumulation in the gallbladder and urinary bladder. Pretreatment with imatinib did not change uptake in the heart, lungs, kidneys and spleen, and increased uptake in the liver and gallbladder. [N-(11)C-methyl]imatinib has potential for assessing the regional distribution and kinetics of imatinib in the human body to determine whether the drug targets tumors and to identify other organs to which the drug or its labeled metabolites distribute. Paired with tracers such as 2'deoxy-2'-[(18)F]fluoro-D-glucose ((18)FDG) and 3'deoxy-3'-[(18)F]fluorothymidine ((18)FLT), [N-(11)C-methyl]imatinib may be a useful radiotracer for planning chemotherapy, for monitoring response to treatment and for assessing the role of drug pharmacokinetics in drug resistance.

Hematological and nonhematological toxicities of imatinib mesylate in patients with chronic myeloid leukemia and gastrointestinal stromal tumor

<b>Objectives</b> : To determine the hematological and nonhematological toxicities of imatinib mesylate in patients with chronic myeloid leukemia (CML) and gastrointestinal stromal tumors (GIST) and to review the literature to compile a list of the etiologic agents responsible for these events. <br><b> Materials and Methods</b> : This was a prospective study conducted from May 2001 to February, 2007. Two hundred and thirty-two patients with CML and GIST treated with imatinib mesylate at the Aga Khan University Hospital were included in the study. Side effects were graded according to the common toxicity criteria of the National Cancer Institute version 3.0. <br><b> Results</b> : Ninety-seven patients experienced various side effects which, in decreasing order of frequency, were: generalized hypopigmentation, periorbital edema, nausea, and weight gain. Hematological toxicities included mainly grade I/II anemia and thrombocytopenia. Grade III/ IV hematological adverse events were rare in our group. The frequency of all events is equally distributed in all phases of CML and GIST. The side effects rarely lead to permanent discontinuation of therapy. <br><b>Conclusion</b> : Imatinib mesylate is a well-tolerated drug with some adverse events that are only rarely a permanent barrier to therapy.

Platelet-Derived Growth Factor as a Therapeutic Target for Systemic Autoimmune Diseases

Some systemic rheumatic diseases and disorders, especially fibrotic and vascular disorders, are often refractory to corticosteroid therapy. Recently, ever accumulating evidence suggests that platelet-derived growth factor (PDGF) is involved in those refractory diseases. Imatinib mesylate inhibits the activation of PDGF receptor as well as c-Abl, Bcr-Abl and c-Kit tyrosine kinases. It has therefore been widely used for the treatment of chronic myeloid leukemia and gastrointestinal stromal tumors. Imatinib effectively suppresses the activation and proliferation of fibroblasts, mesangial cells and smooth muscle cells both in vitro and in vivo. Additionally, it has recently been reported that some patients with rheumatoid arthritis or idiopathic pulmonary arterial hypertension demonstrated a good clinical response to imatinib therapy. Imatinib may therefore overcome the limitations of current therapeutic strategy with corticosteroids and immunosuppressive agents for refractory diseases, such as systemic sclerosis and interstitial lung diseases, without clinical intolerability.

Cancer Risks in Fanconi Anemia: Experience of the German Fanconi Anemia (GEFA) Registry.

OBJECTIVE: Acute Myeloid Leukemia (AML) and solid tumors (ST) occur frequently in Fanconi Anemia (FA). Our objective was to characterize the age of onset of cancer and identify any neoplasms occurring in excess.METHODS: We analyzed data from the German Fanconi Anemia (GEFA) Registry, a retrospective cohort. For competing adverse events of bone marrow failure (BMF), AML, and ST, we determined cause-specific hazards and cumulative incidence curves. We calculated the ratio of observed to expected cancers (O/E ratio) in GEFA compared to age- and sex-matched persons from the general North American population. We studied outcomes after bone marrow transplantation (BMT) using survival analysis.RESULTS: In GEFA, 182 patients contributed 2548 person-years of observation prior to BMT; 63 had BMF as the first adverse event, 15 had AML, and 10 had ST. The cumulative incidence by age 50 was 48% for BMF and 29% for ST. The cumulative incidence by age 20 was 9% for AML. The hazard of BMF peaked at 4%/y at age 10. The hazard of AML increased to 1.6%/y at age 20. The hazard of ST increased from 1%/y at age 20, to 5%/y at age 40, to ~10%/y at age 50. The O/E ratio was 45 for all cancers, 24 for all solid tumors, and 926 for AML; these increased risks were statistically significant. Significantly elevated O/E ratios were observed for esophagus (6346), vulva (2436), oral cavity and pharynx (121), breast (34), and brain (23) cancers. Forty-eight patients had BMT prior to cancer. Subsequently, there were 20 deaths and 3 malignancies in 216 person-years. The 3 malignancies (tongue, liver, and esophagus) occurred 2, 16, and 17 years after mismatched, matched, and matched transplant at ages 13, 23, and 34, respectively. The age-specific hazard of ST was 3.8-fold higher in transplanted versus untransplanted patients; this increased risk was not significant (P = 0.11). During 2000–2004, none of 5 patients with matched, and 3 of 18 patients with mismatched donors, died during the period from 0 – 6 months. In patients with matched donors, acute and chronic GVH were significant risk factors for death beyond 6 months.CONCLUSIONS: Absolute and relative risks of cancer in GEFA are quantitatively similar to previously reported estimates from the North American Survey. Outcomes after transplantation in GEFA are comparable to the Hôpital St Louis Cohort. Our prior observation that FA patients who survive BMF are at extraordinary risk of specific ST has been replicated.

Downregulation of Receptor Activator of Nuclear Factor kB Ligand (RANKL) in Chronic Myeloid Leukemia Patients Receiving Imatinib Mesylate Is Dose-Dependent.

The receptor activator of the nuclear factor kB ligand (RANKL) is a key molecule in the pathogenesis of malignant osteolytic lesions in multiple myeloma or bone metastases in prostate and breast cancer. Inhibition of RANKL is therefore a promising new therapy for controlling bone loss and pain in bone tumors and associated osteolysis. Changes in bone and mineral metabolism developing in a proportion of patients taking imatinib for either chronic myeloid leukemia (CML) or gastrointestinal stromal tumors have previously been described. The aim of our study was to verify in CML patients the effect of imatinib on RANKL bone marrow molecular expression.The study included 13 CML patients (7 males, 6 females, median age 37 yrs). Six were treated with imatinib at standard dosage (400 mg) and the remaining 7 at higher doses (> 400 mg). The median duration of imatinib therapy was 28 months (range 13 – 44 months).The RANKL gene expression level was determined by Real-Time PCR using 1X Platinum SYBR Green qPCR SuperMix-UDG. Variations in RANKL gene expression level were estimated by comparing the values of 2−Δ Δ Ct obtained in CML patients at diagnosis and during imatinib treatment. For each of the tested cases the sample at diagnosis was utilized as calibrator and 28S rRNA was used as reference gene.Eight (62%) patients showed a downregulation of RANKL gene expression, the fold decrease ranging from 0.01 to 0.47 as compared to the value at diagnosis; among these 8 patients, 6 (75%) were treated with imatinib at standard dosage. Instead, 5 (38%) patients showed an upregulation of RANKL, the gene expression fold increase ranging from 1.43 to 4.94 as compared to the gene level observed at CML onset; all these 5 patients were treated with high doses of imatinib. There was no association between RANKL expression and the duration of imatinib treatment.These findings suggest that imatinib mesylate could control RANKL expression. At standard dosage, imatinib can downregulate RANKL production. The RANKL upregulation observed in CML patients treated with high doses of imatinib could be explained by the possible activation of the immune system cells, such as T and B cells, induced by the tyrosine kinases inhibitor. Further studies are needed to verify the minimal effective dose of imatinib on RANKL expression, with the aim of administering it as an antiosteolytic agent in such diseases as osteoporosis, metastatic bone disease, and multiple myeloma.

Thiopurine methyltransferase Genotype Is Not a Risk Factor for Secondary Malignant Neoplasias after Treatment for Childhood Acute Lymphoblastic Leukemia on Berlin-Frankfurt-Muenster Protocols.

The thiopurines mercaptopurine and thioguanine are important components of contemporary polychemotherapeutic treatment protocols for acute leukemias. Thiopurines are prodrugs that undergo intestinal and hepatic metabolism. Activation occurs via a multistep pathway to form thioguanine nucleotides, which are thought to be the major cytotoxic compound through triggering cell cycle arrest and apoptosis. This process is in competition with direct inactivation of thiopurines or their metabolites by thiopurine S-methyltransferase (TPMT). TPMT is a cytosolic enzyme that is ubiquitously expressed in the human body and catalyzes the S-methylation of thiopurines. The TPMT locus is subject to genetic polymorphism, with heterozygous individuals (5 to 11% of Caucasians) having intermediate TPMT activity, and homozygous individuals (0.3 to 0.5% of Caucasians) having low TPMT activity. At least 20 variant TPMT alleles (*2 to *18) have been described so far that confer decreased enzyme activities compared to the TPMT*1 wild-type allele. TPMT genotype is highly concordant with TPMT phenotype. With regard to long-term adverse effects, patients who have diminished TPMT activity were shown to be at increased risk of developing chemotherapy-induced acute myeloid leukemia and radiation-induced second brain tumors tumors after exposure towards mercaptopurine during therapy for childhood acute lymphoblastic leukemia (ALL). To investigate if such an association is generalizable to other entities of secondary malignant neoplasms (SMN) and different treatment approaches for ALL, we collected specimens of 72 patients who developed a SMN after ALL treatment on Berlin-Frankfurt-Muenster (BFM) protocols, analyzed their TPMT genotype and compared genotype frequencies to these in the general ALL patient population. The 72 patient cohort consisted of 49 hematological SMN (half of them received cranial irradiation), 12 brain tumors, and 11 other solid SMN. Neither in the entire patient group nor in subgroup analyses, differences in allele frequencies of TPMT variants conferring diminished enzyme activity were detectable when comparing SMN patients to the overall ALL patient population. Thus, low TPMT activity does not seem to play a major role in the etiology of SMN after treatment for childhood ALL according to BFM treatment strategies. Factors potentially helpful for the explanation of the previously described relationsship of TPMT activity with SMN will be presented.

Imatinib mesylate selectively impairs expansion of memory cytotoxic T cells without affecting the control of primary viral infections

AbstractImatinib mesylate (imatinib) is a potent inhibitor of defined tyrosine kinases (TKs) and is effective in the treatment of malignancies characterized by constitutive activation of these TKs such as chronic myeloid leukemia and gastrointestinal stromal tumors. TKs also play an important role in T-cell receptor (TCR) signal transduction. Inhibitory as well as stimulating effects of imatinib on T cells and dendritic cells have been described. Here, we analyzed the effects of imatinib treatment on antiviral immune responses in vivo. Primary cytotoxic T-cell (CTL) responses were not impaired in imatinib-treated mice after infection with lymphocytic choriomeningitis virus (LCMV) or after immunization with a tumor cell line expressing LCMV glycoprotein (LCMV-GP). Similarly, neutralizing antibody responses to vesicular stomatitis virus (VSV) were not affected. In contrast, secondary expansion of LCMV-specific memory CTLs was reduced in vitro and in vivo, resulting in impaired protection against reinfection. In addition, imatinib treatment delayed the onset of diabetes in a CTL-induced diabetes model. In summary, imatinib treatment in vivo selectively inhibits the expansion of antigen-experienced memory CTLs without affecting primary T- or B-cell responses. Therefore, imatinib may be efficacious in the suppression of CTL-mediated immunopathology in autoimmune diseases without the risk of acquiring viral infections.

Phase-2 study of imatinib in metastasized medullary thyroid carcinoma

15500 Background: Medullary thyroid carcinoma (MTC) is a relatively rare tumor that metastasizes to regional lymph nodes, lung, liver and bone. When surgery is no longer a curative treatment option, no effective systemic therapy is currently available. In most cases a (somatic or germline) mutation in the RET (REarranged during Transfection) gene is considered to be an essential step in the pathogenesis of MTC. Imatinib is an oral inhibitor of several protein-tyrosine kinases including RET kinase. It has shown to be an effective drug in chronic myeloid leukemia and gastrointestinal stromal tumors. Methods: A two-step open label phase-2 study was initiated using 600 mg imatinib daily with a scheduled dose increase to 800 mg in case of minimal toxicity. Standard RECIST criteria were used for response evaluation using CT/MRI scans every 2 months. The two-step design allowed no further inclusion in the absence of objective responses in 15 patients. Results: 15 patients with metastasized MTC were included. No objective responses were observed but long term stable disease over 18 months was noted in 4 patients. However, toxicity was considerable. Emergency tracheotomy had to be performed in 2 cases due to mucosal swelling in patients with laryngeal nerve palsy and a narrow vocal cleft. In 3 cases the dose of Imatinib had to be decreased because of unacceptable toxicity (malaise, mucosal swelling (2) and fatigue). In 7 patients with a history of a thyreoidectomy, the dose of thyroxine had to be increased considerably to avoid serious hypothyroidism due to an incompletely understood interaction. Conclusion: Treatment with Imatinib yielded no objective responses and considerable toxicity in patients with MTC. Long-term stabilization of the disease was observed in only a minority of the patients. Patients with hypothyroidism and with laryngeal nerve palsy are at risk for serious adverse events and need special attention when treated with Imatinib. No significant financial relationships to disclose.

ITCC phase II study of imatinib mesylate in children with solid tumors expressing imatinib-sensitive tyrosine kinase receptors

9003 Background: Imatinib mesylate inhibits selectively specific activations of the platelet-derived growth factor receptor (PDGFR), c-KIT and BCR/ABL tyrosine kinases and is approved for the treatment of chronic myeloid leukemia and gastro-intestinal stromal tumors (GIST). This study evaluated efficacy of imatinib in solid childhood tumors. Methods: Phase II study of imatinib as single agent in children and adolescents with refractory or relapsing solid tumor expressing at least one of the receptors. Patients were to be treated at 340 mg/m2, a dose escalation allowed to 440 mg/m2 after 2 months in case of insignificant improvement. C-KIT, PDGFRα and β expression was determined on archive tissue sections by immunohistochemistry prior to study entry. Gene mutations, pharmacokinetics, pharmacogenetics, and positron emission tomography imaging were assessed. Results: 36 patients, 21 boys, median age 13.7 years (2.2–22.5 y), 12 with brain tumors, 6 fibromatosis, 8 mesenchymal/bone tumors, and 10 other solid tumors, including 1 GIST and 3 chordoma, were treated at 340 mg/m2 daily during a total of 168 months (median 1.9 month/patient, range 0.5–19). 18/36 expressed c-KIT, 10 PDGFRα, 21 PDGRβ; 12 expressed more than one receptor. Ten patients were escalated to 440 mg/m2 due to lack of efficacy. During the 1st month, 17 patients experienced mild toxicity (grade 1 and 2) related to study treatment: gastro-intestinal (n=22), face edema (n=7), asthenia (n=5), tumor induration (n=2), skin toxicity (n=2), thrombocytopenia (n=1). No partial or complete response was observed; 5 patients (2 fibromatosis, 1 GIST, 1 medulloblastoma, 1 pseudo-inflammatory tumor) experiencing durable stable disease have been under treatment for more than 12 months. Interesting tumor stabilization during 10 and 7 months, respectively, was achieved in a brain stem glioma and a renal carcinoma. Glucose uptake on 18FDG PET scan was reduced in a chordoma, although the child progressed and died due to disease. Pharmacokinetic and genetic data are currently evaluated. Conclusions: Imatinib as single agent was well tolerated, but—as used in our study —failed to show measurable anti-tumor effects according the standard criteria in the pediatric malignancies studied. No significant financial relationships to disclose.

Targeted Therapies in Gynecologic Cancers

With the rapid development of high-throughput techniques for identifying novel specific molecular targets in human cancer over the past few years, attention to targeted cancer therapy has dramatically increased. The term "targeted cancer therapy" refers to a new generation of drugs designed to interfere with a specific molecular target that is believed to play a critical role in tumor growth or progression, is not expressed significantly in normal cells, and is correlated with clinical outcome. There has been a rapid increase in the identification of targets that have potential therapeutic application. The clinical success of the small-molecule kinase inhibitor imatinib mesylate in chronic myeloid leukemia and gastrointestinal stromal tumors has accelerated the development of a new era of molecular targeted cancer therapy. The number of agents under preclinical and clinical investigation has grown accordingly. This emphasis on molecular biology and genetics has also resulted in significant changes in the treatment of gynecologic cancers. Several promising drugs targeting tyrosine kinases (EGFR and Her-2/Neu), mTOR, Raf kinase, proteasome, and histone deacetylases, as well as drugs affecting apoptosis and mitosis, are under development for clinical application. However, some clinical trials of p53 gene therapies and farnesyl transferase inhibitors have had limited success. In this review, we will focus on potential novel targets in gynecologic cancer and the development of targeted therapy and its clinical applications in gynecologic cancer.

STI571 (Glivec) induces cell death in the gastrointestinal stromal tumor cell line, GIST-T1, via endoplasmic reticulum stress response

STI571 is a specific inhibitor of tyrosine kinases, such as BCR-ABL, platelet-derived growth factor receptor, and c-KIT, and has recently been approved for the treatment of chronic myeloid leukemia and gastrointestinal stromal tumors (GISTs). This study demonstrated that STI571 induces cell death in the gastrointestinal stromal tumor cell line, GIST-T1. In these cells, STI571 induced pro-caspase-12 or pro-caspase-7 cleavage and it affected caspase-3 activity and induced the endoplasmic reticulum (ER)-resident chaperone, glucose-regulated protein 78. The STI571-induced cell death was blocked by the protein synthesis inhibitor, cycloheximide. Together, these results suggest that STI571 induces cell death in GIST-T1 cells, at least in part, via the ER stress response.

Anesthesia and Fanconi anemia: a case report and review of literature

Fanconi anemia (FA) is the most frequently reported of the rare inherited bone marrow failure syndromes. It is an autosomal recessive chromosomal instability disorder, which is characterized by congenital abnormalities, defective hemopoiesis and a high risk of developing acute myeloid leukemia and certain solid tumors (1). We report the anesthetic management for a Hickman catheter insertion in a 4.5-year-old girl with this condition. Possible anesthetic implications in the presence of FA are discussed.

Ultra-Sensitive Immunodetection of 5'Methyl Cytosine for DNA Methylation Analysis on Oligonucleotide Microarrays

For the determination of methylation levels in genomic regulatory DNA sequences a high-sensitive assay for detecting 5'methyl-cytosines (5'mC) in non-bisulfite-treated DNA has been established. The system is designed for the application of immunofluorescence using a monoclonal antibody that specifically recognizes 5'mC in single-stranded DNA hybridized to oligonucleotide microarrays. For assay readout an ultra-sensitive fluorescence scanner with submicrometer resolution was used. To minimize autofluorescence 150-microm thin glass slides with an aldehyde-functionalized surface were developed. These methodological improvements allowed the detection of 5'mC in synthetic oligonucleotides hybridized to microarrays with atto molar analytical sensitivity. Using enzymatic fragmented genomic DNA from myeloid leukemia tumor cell lines differences in the methylation status of gene regulatory sequences for E-cadherin, p15/CDKN2b and p16/CDKN2a were demonstrated. Thus, this novel technique can potentially be used for DNA methylation analysis in various scientific fields.

Cardiac Safety Alert for Users of Cancer Drug

The FDA has issued a safety alert about potential cardiotoxicity of imatinib mesylate (Gleevec), a drug used to treat chronic myeloid leukemia and gastrointestinal stromal tumors. In a new warning added to the drug label, the manufacturer notes that Gleevec users with heart disease or at risk for heart failure (HF) should be monitored carefully and that users who …

Squamous cell carcinoma of the tongue after bone marrow transplantation in a patient with Fanconi anemia

Fanconi anemia (FA) is an autosomal recessive disorder that might cause a variety of congenital and developmental abnormalities. The most important features of FA are progressive bone marrow failure and development of malignancies, particularly acute myeloid leukemia and solid tumors. This paper reports the case of a 12-year-old patient with FA assisted at the Stomatology and Bucomaxillofacial Cancer Prevention Service of São Lucas Hospital, Brazil, who had been submitted to bone marrow transplantation (BMT) at the age of 5 and exhibited oral lesions characteristic of chronic graft versus host disease (GVHD). The patient was treated and followed-up for the oral lesions. Eleven years after the BMT, he developed squamous cell carcinoma of the tongue with an aggressive behavior, which was considered an untreatable condition. The patient died few months later from asphyxia at the age of 16. The reasons for development of these malignant conditions are unknown. However, chromosomal instability typically observed in FA cases, BMT factors and GVHD have been considered. Systematic follow-up of these patients allows early and less invasive therapeutic approaches.

Thapsigargin Enhances Cell Death in the Gastrointestinal Stromal Tumor Cell Line, GIST-T1, by Treatment with Imatinib (Glivec)

Imatinib is a specific inhibitor of c-KIT that has recently been approved for the treatment of chronic myeloid leukemia and gastrointestinal stromal tumor (GIST). Thapsigargin is an inhibitor of calcium transport to the endoplasmic reticulum (ER) and can inhibit protein maturation. In this study, we evaluated the synergistic cytotoxic effect of thapsigargin and imatinib on the GIST cell line, GIST-T1. Cell viability and cell death were determined by 3,-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry, respectively. In addition, the amount and activation of c-KIT on the cell surface were measured by flow cytometry and western blot analysis, respectively. Thapsigargin alone (for 5 hr incubation) was shown to decrease the amount of c-KIT on the GIST-T1 cell surface and to slightly inhibit the associated tyrosine kinase activity. Interestingly, thapsigargin significantly enhanced the cell death induced by imatinib compared with the effect of imatinib alone. The results further suggested that thapsigargin induced the increased cell death in GIST-T1 cells co-treated with imatinib, at least in part, via disrupting the translocation of c-KIT to the cell surface, and decreasing the activated c-KIT molecules.

Inappropriate Expression of CSF-1 in CSF-1R-Expressing Cells in Mice Leads to Osteoporosis, Macrophage Activation and Early Death.

The CSF-1R is selectively expressed on mononuclear phagocytes, osteoclasts and cells of the female reproductive tract. CSF-1 is normally expressed in a wide variety of cell types, excluding those expressing the CSF-1 receptor (CSF-1R). Autocrine/paracrine regulation by CSF-1 plays an important role in the development of disease in mouse models of acute myeloid leukemia and mammary tumor progression to metastasis. To determine the consequences of autocrine regulation by CSF-1 in the absence of other interventions, we generated transgenic mice that expressed CSF-1 in CSF-1R bearing cells. In contrast to mice that expressed the CSF-1R in CSF-1-expressing cells, which had no gross phenotype at 1 year of age, mice expressing CSF-1 under the control of mouse CSF-1R promoter and second intron (TgRC mice), had a short lifetime, starting to lose weight and mobility after 3 weeks of age. CSF-1 mRNA was expressed in peritoneal macrophages (PM) and sequence analysis of the RT-PCR products verified the expression of a chimeric mRNA of the CSF-1R exon 2, 3 and CSF-1 mRNAs under control of the CSF-1R promoter. Quantitative real-time RT-PCR analysis revealed that CSF-1 mRNA expression was increased by 19-fold in PM and 980-fold in bone marrow derived macrophages (BMM) from a TgRC founder mouse, compared to these populations from non-transgenic mice. Consistent with autocrine regulation by CSF-1, in contrast to BMM from non-transgenic mice, TgRC BMM proliferated in the absence of CSF-1. However, there was no elevation of the circulating CSF-1 in TgRC mice, indicating that transgene-regulated expression of CSF-1 only affected local CSF-1 concentrations. X-ray and histological analysis demonstrated that TgRC mice developed osteoporosis with increased numbers of femoral TRAP-positive osteoclasts. Macrophage densities were significantly increased in bone marrow, liver, spleen and brain. In several tissues, there were local regions of increased macrophage density, consistent with effects of locally produced CSF-1 on macrophage survival and/or proliferation. Also, macrophages in several tissues had a more dendritic appearance than in non-transgenic mice, suggesting that they may be activated. TgRC PM, cultured in the presence of CSF-1 exhibited a highly dendritic morphology with multiple protruding pseudopods also suggesting an activated state. Studies of the macrophage cytokine expression profile by the real-time quantitative RT-PCR revealed elevated levels of mRNA for the proinflammatory cytokines, IL-1β (33-fold) (in BMM) and IL-10 (10-fold) and IL-6 (2.6-fold) (in PM), consistent with the priming effect of CSF-1 on proinflammatory cytokine release by macrophages. These studies indicate that inappropriate local expression of CSF-1 by or near CSF-1R-expressing cells can result in activation of macrophages and osteoclasts and severe disease. Supported by NIH grant CA32551 (ERS) and an ASH Fellow Scholar Award and Leukemia and Lymphoma Society Special Fellow Award (X-MD).

Molecular interactions in imatinib–DPPC liposomes

Imatinib (Gleevec ®) is a novel chemotherapeutic agent against Bcr-Abl protein tyrozine kinase, playing a crucial role in the therapy of chronic myeloid leukemia (CML) and gastrointestinal stromal tumors (GIST). Our study aimed at designing a liposomal imatinib formulation and investigating molecular interactions between lipid and imatinib, within the liposomal membrane. Multilamellar (MLV) and small unilamellar (SUV) vesicles were prepared from α- l-dipalmitoyl-phosphatidylcholine (DPPC). The effect of imatinib on the DPPC membrane was studied by electron paramagnetic resonance (EPR) spectroscopy and differential scanning calorimetry (DSC), at pH 5.2 and 9.0, where imatinib is in monocationic and neutral form, respectively. Our results indicate that imatinib interacts mainly with the DPPC head groups, leading to a slight increase in the mobility of the polar headgroups in case of MLVs. Contrary to that, imatinib causes a significant decrease in the fluidity of SUVs, which can be the result of a pH-dependent fusion/fission effect. The size distribution and morphology of liposomes were checked by dynamic light scattering and freeze-fracture electron microscopy. Our results direct attention to investigate the interactions of imatinib with artificial/biological membranes.