技术资料

Overcoming imatinib mesylate (IM) resistance and disease persistence in patients with chronic myeloid leukemia (CML) is of considerable importance to the issue of potential cure. Here we asked whether autocrine signaling contributes to survival of BCR/ABL+ cells in the presence of IM and nilotinib (NI; AMN107),a novel,more selective Abl inhibitor. Conditioned media (CM) of IM-resistant LAMA84 cell clones (R-CM) was found to substantially protect IM-naive LAMA cells and primary CML progenitors from IM- or NI-induced cell death. This was due to an increased secretion of the granulocyte-macrophage colony-stimulating factor (GM-CSF),which was identified as the causative factor mediating IM resistance in R-CM. GM-CSF elicited IM and NI drug resistance via a BCR/ABL-independent activation of the janus kinases 2 (JAK-2)/signal transducer and activator of transcription 5 (STAT-5) signaling pathway in GM-CSF receptor alpha receptor (CD116)-expressing cells,including primary CD34+/CD116+ GM progenitors (GMPs). Elevated mRNA and protein levels of GM-CSF were detected in IM-resistant patient samples,suggesting a contribution of GM-CSF secretion for IM and NI resistance in vivo. Importantly,inhibition of JAK-2 with AG490 abrogated GM-CSF-mediated STAT-5 phosphorylation and NI resistance in vitro. Together,adaptive autocrine secretion of GM-CSF mediates BCR/ABL-independent IM and NI resistance via activation of the antiapoptotic JAK-2/STAT-5 pathway. Inhibition of JAK-2 overcomes GM-CSF-induced IM and NI progenitor cell resistance,providing a rationale for the application of JAK-2 inhibitors to eradicate residual disease in CML. View Publication

Chronic myeloid leukemia (CML) is genetically characterized by the Philadelphia (Ph) chromosome,formed through a reciprocal translocation between chromosomes 9 and 22 and giving rise to the constitutively active tyrosine kinase P210 BCR/ABL1. Therapeutic strategies aiming for a cure of CML will require full eradication of Ph chromosome-positive (Ph(+)) CML stem cells. Here we used gene-expression profiling to identify IL-1 receptor accessory protein (IL1RAP) as up-regulated in CML CD34(+) cells and also in cord blood CD34(+) cells as a consequence of retroviral BCR/ABL1 expression. To test whether IL1RAP expression distinguishes normal (Ph(-)) and leukemic (Ph(+)) cells within the CML CD34(+)CD38(-) cell compartment,we established a unique protocol for conducting FISH on small numbers of sorted cells. By using this method,we sorted cells directly into drops on slides to investigate their Ph-chromosome status. Interestingly,we found that the CML CD34(+)CD38(-)IL1RAP(+) cells were Ph(+),whereas CML CD34(+)CD38(-)IL1RAP(-) cells were almost exclusively Ph(-). By performing long-term culture-initiating cell assays on the two cell populations,we found that Ph(+) and Ph(-) candidate CML stem cells could be prospectively separated. In addition,by generating an anti-IL1RAP antibody,we provide proof of concept that IL1RAP can be used as a target on CML CD34(+)CD38(-) cells to induce antibody-dependent cell-mediated cytotoxicity. This study thus identifies IL1RAP as a unique cell surface biomarker distinguishing Ph(+) from Ph(-) candidate CML stem cells and opens up a previously unexplored avenue for therapy of CML. View Publication

The farnesyltransferase inhibitor tipifarnib exhibits modest activity against acute myelogenous leukemia. To build on these results,we examined the effect of combining tipifarnib with other agents. Tipifarnib inhibited signaling downstream of the farnesylated small G protein Rheb and synergistically enhanced etoposide-induced antiproliferative effects in lymphohematopoietic cell lines and acute myelogenous leukemia isolates. We subsequently conducted a phase 1 trial of tipifarnib plus etoposide in adults over 70 years of age who were not candidates for conventional therapy. A total of 84 patients (median age,77 years) received 224 cycles of oral tipifarnib (300-600 mg twice daily for 14 or 21 days) plus oral etoposide (100-200 mg daily on days 1-3 and 8-10). Dose-limiting toxicities occurred with 21-day tipifarnib. Complete remissions were achieved in 16 of 54 (30%) receiving 14-day tipifarnib versus 5 of 30 (17%) receiving 21-day tipifarnib. Complete remissions occurred in 50% of two 14-day tipifarnib cohorts: 3A (tipifarnib 600,etoposide 100) and 8A (tipifarnib 400,etoposide 200). In vivo,tipifarnib plus etoposide decreased ribosomal S6 protein phosphorylation and increased histone H2AX phosphorylation and apoptosis. Tipifarnib plus etoposide is a promising orally bioavailable regimen that warrants further evaluation in elderly adults who are not candidates for conventional induction chemotherapy. These clinical studies are registered at www.clinicaltrials.gov as NCT00112853. View Publication

Megakaryocytopoiesis gives rise to platelets by proliferation and differentiation of lineage-specific progenitors,identified in vitro as Colony Forming Unit-Megakaryocytes (CFU-Mk). The aim of this study was to refine and optimize the in vitro Standard Operating Procedure (SOP) of the CFU-Mk assay for detecting drug-induced thrombocytopenia and to prevalidate a model for predicting the acute exposure levels that cause maximum tolerated decreases in the platelets count,based on the correlation with the maximal plasma concentrations (C max) in vivo. The assay was linear under the SOP conditions,and the in vitro endpoints (percentage of colonies growing) were reproducible within and across laboratories. The protocol performance phase was carried out testing 10 drugs (selected on the base of their recognised or potential in vivo haematotoxicity,according to the literature). Results showed that a relationship can be established between the maximal concentration in plasma (C max) and the in vitro concentrations that inhibited the 10-50-90 percent of colonies growth (ICs). When C max is lower than IC10,it is possible to predict that the chemicals have no direct toxicity effect on CFU-Mk and could not induce thrombocytopenia due to bone marrow damage. When the C max is higher than IC90 and/or IC50,thrombocytopenia can occur due to direct toxicity of chemicals on CFU-Mk progenitors. View Publication

Protein geranylgeranyltransferase type I (GGTase-I) is responsible for the posttranslational lipidation of CAAX proteins such as RHOA,RAC1,and cell division cycle 42 (CDC42). Inhibition of GGTase-I has been suggested as a strategy to treat cancer and a host of other diseases. Although several GGTase-I inhibitors (GGTIs) have been synthesized,they have very different properties,and the effects of GGTIs and GGTase-I deficiency are unclear. One concern is that inhibiting GGTase-I might lead to severe toxicity. In this study,we determined the effects of GGTase-I deficiency on cell viability and K-RAS-induced cancer development in mice. Inactivating the gene for the critical beta subunit of GGTase-I eliminated GGTase-I activity,disrupted the actin cytoskeleton,reduced cell migration,and blocked the proliferation of fibroblasts expressing oncogenic K-RAS. Moreover,the absence of GGTase-I activity reduced lung tumor formation,eliminated myeloproliferative phenotypes,and increased survival of mice in which expression of oncogenic K-RAS was switched on in lung cells and myeloid cells. Interestingly,several cell types remained viable in the absence of GGTase-I,and myelopoiesis appeared to function normally. These findings suggest that inhibiting GGTase-I may be a useful strategy to treat K-RAS-induced malignancies. View Publication

In a previous study of prevalidation,a standard operating procedure (SOP) for two independent in vitro tests (human and mouse) had been developed,to evaluate the potential hematotoxicity of xenobiotics from their direct and the adverse effects on granulocyte-macrophages (CFU-GM). A predictive model to calculate the human maximum tolerated dose (MTD) was set up,by adjusting a mouse-derived MTD for the differential interspecies sensitivity. In this paper,we describe an international blind trial designed to apply this model to the clinical neutropenia,by testing 20 drugs,including 14 antineoplastics (Cytosar-U,5-Fluorouracil,Myleran,Thioguanine,Fludarabine,Bleomycin,Methotrexate,Gemcitabine,Carmustine,Etoposide,Teniposide,Cytoxan,Taxol,Adriamycin); two antivirals (Retrovir,Zovirax,); three drugs for other therapeutic indications (Cyclosporin,Thorazine,Indocin); and one pesticide (Lindane). The results confirmed that the SOP developed generates reproducible IC90 values with both human and murine GM-CFU. For 10 drugs (Adriamycin,Bleomycin,Etoposide,Fludarabine,5-Fluorouracil,Myleran,Taxol,Teniposide,Thioguanine,and Thorazine),IC90 values were found within the range of the actual drug doses tested (defined as the actual IC90). For the other 10 drugs (Carmustine,Cyclosporin,Cytosar-U,Cytoxan,Gemcitabine,Indocin,Lindane,Methotrexate,Retrovir,and Zovirax) extrapolation on the regression curve out of the range of the actual doses tested was required to derive IC90 values (extrapolated IC90). The model correctly predicted the human MTD for 10 drugs out of 10 that had actual IC90 values" and 7 drugs out of 10 for those having only an extrapolated IC90. Two of the incorrect predictions (Gemcitabine and Zovirax) were within 6-fold of the correct MTD� View Publication

BACKGROUND: 1,25-Dihydroxyvitamin D(3) inhibits growth of several types of human cancer cells in vitro,but its therapeutic use is hampered because it causes hypercalcemia. 19-nor-1,25-Dihydroxyvitamin D(2) (paricalcitol) is a noncalcemic vitamin D analog that is approved by the Food and Drug Administration for the treatment of secondary hyperparathyroidism. We investigated the antitumor activity and mechanism of action of paricalcitol in vitro and in vivo. METHODS: Effects of paricalcitol on proliferation,the cell cycle,differentiation,and apoptosis were examined in cancer cell lines. Effects on tumor growth were examined with colon cancer cell xenografts in nude mice (five in the experimental group and five in the control group). The interaction of paricalcitol with the vitamin D receptor (VDR) in mononuclear spleen cells and myeloid stem cells from wild-type and VDR knockout mice was examined. All statistical tests were two-sided. RESULTS: Paricalcitol inhibited the proliferation of myeloid leukemia cell lines HL-60,NB-4,and THP-1 cells at an effective dose that inhibited growth 50% (ED(50)) of 2.4-5.8 x 10(-9) M by inducing cell cycle arrest and differentiation. Paricalcitol inhibited the proliferation of NCI-H929 myeloma cells at an ED(50) of 2.0 x 10(-10) M by inducing cell cycle arrest and apoptosis. Paricalcitol also inhibited the proliferation of colon cancer cell lines HT-29 (ED(50) = 1.7 x 10(-8) M) and SW837 (ED(50) = 3.2 x 10(-8) M). HT-29 colon cancer xenografts in paricalcitol-treated nude mice were smaller (1044 mm(3) and 1752 mm(3),difference = 708 mm(3),95% confidence interval = 311 to 1104 mm(3); P =.03) and weighed less (1487 mg and 4162 mg,difference = 2675 mg,95% confidence interval = 2103 to 3248 mg; Ptextless.001) than those in vehicle-treated mice. Paricalcitol induced committed myeloid hematopoietic stem cells from wild-type but not from VDR knockout mice to differentiate as macrophages. CONCLUSION: Paricalcitol has anticancer activity against myeloid leukemia,myeloma,and colon cancer cells that may be mediated through the VDR. Because it has been approved by the Food and Drug Administration,clinical trials of this agent in certain cancers are reasonable. View Publication

Because of the wide use of pesticides for domestic and industrial purposes,the evaluation of their potential effects is of major concern for public health. The myelotoxicity of the herbicide propanil (3,4-dichloroproprioanilide) and its metabolite 3,4-dichloroaniline (DCA) is well documented in mice,but evidence that pesticides may severely compromise hematopoiesis in humans is lacking. In this study,an interspecies comparison of in vitro toxicity of these two compounds on murine and human burst- and colony-forming unit-erythrocyte (BFU-E,CFU-E) and colony-forming unit-granulocyte/macrophage (CFU-GM) progenitors,has been carried out. Murine bone marrow progenitors and human cord blood cells were exposed to propanil or DCA in doses ranging from 10 micro M to 1000 micro M,and the toxic effect was detected by a clonogenic assay with continuous exposure to the compounds. The results on murine cells indicate that the erythrocytic lineage is the most sensitive target for propanil and DCA. On the other hand,human progenitors seem to be less sensitive to the toxic effects of both compounds than murine progenitors at the same concentrations (IC(50) values are 305.2 +/- 22.6 micro M [total erythroid colonies] and textgreater500 micro M [CFU-GM] for propanil). Propanil was significantly more toxic to human erythroid progenitors than to human CFU-GM progenitors,as was found for the murine cells,emphasizing the role of the heme pathway as the target for propanil. These data confirm the evidence that the compounds investigated interfere with erythroid colony formation at different stages of the differentiation pathway and have different effects according to the dose. View Publication

Imatinib is a novel tyrosine kinase inhibitor used for the treatment of Philadelphia chromosome-positive leukemias and other malignancies. Side effects are mostly moderate; however,a dose-dependent hematologic toxicity affecting all hematopoietic lineages is observed clinically. The aim of this study was to investigate the effect of imatinib on normal hematopoietic stem and progenitor cells in vitro. A dose-dependent decrease in proliferation potential was found when CD34+ cells were expanded in serum-free medium supplemented with 6 growth factors and imatinib. Functionally,a decrease in colony-forming capacity was observed under increasing doses of imatinib. However,no such effect on more primitive cobblestone area-forming cells was detectable. Both withdrawal of stem cell factor from our expansion cultures or functional inhibition of c-kit led to a similar degree of inhibition of expansion,whereas the effect of imatinib was substantially greater at all dose levels tested. These data suggest a significant inhibitory effect of imatinib on normal CD34+ progenitor (but not stem) cells that is largely independent of c-kit signaling. View Publication