技术资料

Throughout its history,chronic myeloid leukemia (CML) has set precedents for cancer research and therapy. These range from the identification of the first specific chromosomal abnormality associated with cancer to the development of imatinib as a specific,targeted therapy for the disease. The successful development of imatinib as a therapeutic agent for CML can be attributed directly to decades of scientific discoveries. These discoveries determined that the BCR-ABL tyrosine kinase is the critical pathogenetic event in CML and an ideal target for therapy. This was confirmed in clinical trials of imatinib,with imatinib significantly improving the long-term survival of patients with CML. Continuing in this tradition of scientific discoveries leading to improved therapies,the understanding of resistance to imatinib has rapidly led to strategies to circumvent resistance. Continued studies of hematologic malignancies will allow this paradigm of targeting molecular pathogenetic events to be applied to many additional hematologic cancers. View Publication

Neurotrophins (NTs) and their receptors play a key role in neurogenesis and survival. The TRK (tropomyosin-related kinase) receptor protein tyrosine kinases (TRKA,TRKB,TRKC) are high-affinity NT receptors that are expressed in a variety of human tissues. Their role in normal and malignant hematopoiesis is poorly understood. In a prospective study involving 94 adult patients we demonstrate for the first time cell-surface expression of the 3 TRKs and constitutive activation in blasts from patients with de novo or secondary acute leukemia. At least one TRK was expressed in 55% of the analyzed cases. We establish a clear correlation between the TRK expression pattern and FAB classification. Although only few point mutations were found in TRK sequences by reverse-transcriptase-polymerase chain reaction (RT-PCR),we observed coexpression of BDNF (ligand for TRKB) in more than 50% of TRKB(+) cases (16/30). Activation of TRKA or TRKB by NGF and BDNF,respectively,efficiently rescued murine myeloid cells from irradiation-induced apoptosis. Coexpression of TRKB/BDNF or TRKA/NGF in murine hematopoietic cells induced leukemia. Moreover,activation of TRKs was important for survival of both human and murine leukemic cells. Our findings suggest that TRKs play an important role in leukemogenesis and may serve as a new drug target. View Publication

Most mouse embryonic stem (ES) cells are derived from a 129 or C57BL/6 background,whereas the derivation efficiency of ES cells is extremely low on certain refractory types of background for which ES cells are highly desired. Here we report an optimized,highly efficient protocol by combining pluripotin,a small molecule,and leukemia inhibitory factor (LIF) for the derivation of mouse ES cells. With this method,we successfully isolated ES cell lines from five strains of mice,with an efficiency of 57% for NOD-scid,63% for SCID beige,80% for CD-1,and 100% for two F1 strains from C57BL/6xCD-1. By tracking the Oct4-positive cells in the Oct4-green fluorescent protein embryos in the process of ES cell isolation,we found that pluripotin combined with LIF improved the efficiency of ES cell isolation by selectively maintaining the Oct4-positive cells in the outgrowth. To our knowledge,this is the first report of ES cells being efficiently derived from immunodeficient mice on refractory backgrounds (NOD-scid on a NOD background and SCID beige on a BALB/c background). View Publication

BACKGROUND Efficient slow freezing protocols within serum-free and feeder-free culture systems are crucial for the clinical application of human embryonic stem (hES) cells. Frequently,however,hES cells must be cryopreserved as clumps when using conventional slow freezing protocols,leading to lower survival rates during freeze-thaw and limiting their recovery and growth efficiency after thawing,as well as limiting downstream applications that require single cell suspensions. We describe a novel method to increase freeze-thaw survival and proliferation rate of single hES cells in serum-free and feeder-free culture conditions. METHODS hES cells maintained on Matrigel-coated dishes were dissociated into single cells with Accutase and slow freezing. After thawing at 37 degrees C,cells were cultured in mTeSR medium supplemented with 10 microM of Rho-associated kinase inhibitor Y-27632 for 1 day. RESULTS The use of Y-27632 and Accutase significantly increases the survival of single hES cells after thawing compared with a control group (P textless 0.01). Furthermore,by treatment of hES cell aggregates with EGTA to disrupt cell-cell interaction,we show that Y-27632 treatment does not directly affect hES cell apoptosis. Even in the presence of Y-27632,hES cells deficient in cell-cell interaction undergo apoptosis. Y-27632-treated freeze-thawed hES cells retain typical morphology,stable karyotype,expression of pluripotency markers and the potential to differentiate into derivatives of all three germ layers after long-term culture. CONCLUSIONS The method described here allows for cryopreservation of single hES cells in serum-free and feeder-free conditions and therefore we believe this method will be ideal for current and future hES cell applications that are targeted towards a therapeutic end-point. View Publication

We developed a real-time copy number polymerase chain reaction assay for deletions on chromosome 20q (del20q),screened peripheral blood granulocytes from 664 patients with myeloproliferative disorders,and identified 19 patients with del20q (2.9%),of which 14 (74%) were also positive for JAK2-V617F. To examine the temporal relationship between the occurrence of del20q and JAK2-V617F,we performed colony assays in methylcellulose,picked individual burst-forming units-erythroid (BFU-E) and colony-forming units-granulocyte (CFU-G) colonies,and genotyped each colony individually for del20q and JAK2-V617F. In 2 of 9 patients,we found that some colonies with del20q carried only wild-type JAK2,whereas other del20q colonies were JAK2-V617F positive,indicating that del20q occurred before the acquisition of JAK2-V617F. However,in colonies from 3 of 9 patients,we observed the opposite order of events. The lack of a strict temporal order of occurrence makes it doubtful that del20q represents a predisposing event for JAK2-V617F. In 2 patients with JAK2-V617F and 1 patient with MPL-W515L,microsatellite analysis revealed that del20q affected chromosomes of different parental origin and/or 9pLOH occurred at least twice. The fact that rare somatic events,such as del20q or 9pLOH,occurred more than once in subclones from the same patients suggests that the myeloproliferative disorder clone carries a predisposition to acquiring such genetic alterations. View Publication

Defects in apoptosis contribute to poor outcome in pediatric acute lymphoblastic leukemia (ALL),calling for novel strategies that counter apoptosis resistance. Here,we demonstrate for the first time that small molecule inhibitors of the antiapoptotic protein XIAP cooperate with TRAIL to induce apoptosis in childhood acute leukemia cells. XIAP inhibitors at subtoxic concentrations,but not a structurally related control compound,synergize with TRAIL to trigger apoptosis and to inhibit clonogenic survival of acute leukemia cells,whereas they do not affect viability of normal peripheral blood lymphocytes,suggesting some tumor selectivity. Analysis of signaling pathways reveals that XIAP inhibitors enhance TRAIL-induced activation of caspases,loss of mitochondrial membrane potential,and cytochrome c release in a caspase-dependent manner,indicating that they promote a caspase-dependent feedback mitochondrial amplification loop. Of note,XIAP inhibitors even overcome Bcl-2-mediated resistance to TRAIL by enhancing Bcl-2 cleavage and Bak conformational change. Importantly,XIAP inhibitors kill leukemic blasts from children with ALL ex vivo and cooperate with TRAIL to induce apoptosis. In vivo,they significantly reduce leukemic burden in a mouse model of pediatric ALL engrafted in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. Thus,XIAP inhibitors present a promising novel approach for apoptosis-based therapy of childhood ALL. View Publication

Previous studies have demonstrated that normal mouse mammary tissue contains a rare subset of mammary stem cells. We now describe a method for detecting an analogous subpopulation in normal human mammary tissue. Dissociated cells are suspended with fibroblasts in collagen gels,which are then implanted under the kidney capsule of hormone-treated immunodeficient mice. After 2-8 weeks,the gels contain bilayered mammary epithelial structures,including luminal and myoepithelial cells,their in vitro clonogenic progenitors and cells that produce similar structures in secondary transplants. The regenerated clonogenic progenitors provide an objective indicator of input mammary stem cell activity and allow the frequency and phenotype of these human mammary stem cells to be determined by limiting-dilution analysis. This new assay procedure sets the stage for investigations of mechanisms regulating normal human mammary stem cells (and possibly stem cells in other tissues) and their relationship to human cancer stem cell populations. View Publication

Earlier reports have suggested that the BCR/ABL oncogene,associated with chronic myeloid leukemia,induces a mutator phenotype; however,it is unclear whether this leads to long-term changes in chromosomes and whether the phenotype is found in primary chronic myelogeneous leukemia (CML) cells. We have addressed both these issues. BCR/ABL-expressing cell lines show an increase in DNA breaks after treatment with etoposide as compared to control cells. However,although BCR/ABL-expressing cell lines have an equivalent cell survival,they have an increase in chromosomal translocations after DNA repair as compared to control cells. This demonstrates that BCR/ABL expression decreases the fidelity of DNA repair. To see whether this is true in primary CML samples,normal CD34+ progenitor cells and CML progenitor cells were treated with etoposide. CML progenitor cells have equivalent survival but have an increase in DNA double-strand breaks (DSBs). Spectral karyotyping demonstrates new chromosomal translocations in CML cells,but not normal progenitor cells,consistent with error-prone DNA repair. Taken together,these data demonstrate that BCR/ABL enhances the accumulation of DSBs and alters the apoptotic threshold in CML leading to error-prone DNA repair. View Publication

The CD31(+) subset of human naive CD4(+) T cells is thought to contain the population of cells that have recently emigrated from the thymus,while their CD31(-) counterparts have been proposed to originate from CD31(+) cells after homeostatic cell division. Naive T-cell maintenance is known to involve homeostatic cytokines such as interleukin-7 (IL-7). It remains to be investigated what role this cytokine has in the homeostasis of naive CD4(+) T-cell subsets defined by CD31 expression. We provide evidence that IL-7 exerts a preferential proliferative effect on CD31(+) naive CD4(+) T cells from adult peripheral blood compared with the CD31(-) subset. IL-7-driven proliferation did not result in loss of CD31 expression,suggesting that CD31(+) naive CD4(+) T cells can undergo cytokine-driven homeostatic proliferation while preserving CD31. Furthermore,IL-7 sustained or increased CD31 expression even in nonproliferating cells. Both proliferation and CD31 maintenance were dependent on the activation of phosphoinositide 3-kinase (PI3K) signaling. Taken together,our data suggest that during adulthood CD31(+) naive CD4(+) T cells are maintained by IL-7 and that IL-7-based therapies may exert a preferential effect on this population. View Publication

Recently it has been demonstrated that CD30 expression was rather specific for transformed than for normal human ES cells and therefore CD30 maybe suggested as a potential marker for human ES cells bearing chromosomal abnormalities. Using immunohistochemistry and RT-PCR analysis we examined ?¡D30 expression in 10 hESCs lines with normal and abberant karyotypes. All hESC lines expressed CD30 antigen and RNA in undifferentiated state whether cell line beared chromosomal abnormalities or not. In contrast to previous notions our data demonstrate that CD30 could be considered as marker of undifferentiated hESCs without respect to karyotype changes. View Publication

The mechanisms underlying the decision of a stem or progenitor cell to either self-renew or differentiate are incompletely understood. To address the role of Myc in this process,we expressed different forms of the proto-oncogene Myc in multipotent neural progenitor cells (NPCs) using retroviral transduction. Expression of Myc in neurospheres increased the proportion of self-renewing cells fivefold,and 1% of the Myc-overexpressing cells,but none of the control cells,retained self-renewal capacity even under differentiation-inducing conditions. A Myc mutant (MycV394D) deficient in binding to Miz-1,did not increase the percentage of self-renewing cells but was able to stimulate proliferation of NPCs as efficiently as wild-type Myc,indicating that these two cellular phenomena are regulated by at least partially different pathways. Our results suggest that Myc,through Miz-1,enhances self-renewal of NPCs and influences the way progenitor cells react to the environmental cues that normally dictate the cellular identity of tissues containing self-renewing cells. View Publication

We recently reported that the antineoplastic thiodioxopiperazine natural product chaetocin potently induces cellular oxidative stress,thus selectively killing cancer cells. In pursuit of underlying molecular mechanisms,we now report that chaetocin is a competitive and selective substrate for the oxidative stress mitigation enzyme thioredoxin reductase-1 (TrxR1) with lower K(m) than the TrxR1 native substrate thioredoxin (Trx; chaetocin K(m) = 4.6 +/- 0.6 microM,Trx K(m) = 104.7 +/- 26 microM),thereby attenuating reduction of the critical downstream ROS remediation substrate Trx at achieved intracellular concentrations. Consistent with a role for TrxR1 targeting in the anticancer effects of chaetocin,overexpression of the TrxR1 downstream effector Trx in HeLa cells conferred resistance to chaetocin-induced,but not to doxorubicin-induced,cytotoxicity. As the TrxR/Trx pathway is of central importance in limiting cellular reactive oxygen species (ROS)--and as chaetocin exerts its selective anticancer effects via ROS imposition--the inhibition of TrxR1 by chaetocin has potential to explain its selective anticancer effects. These observations have important implications not just with regard to the mechanism of action and clinical development of chaetocin and related thiodioxopiperazines,but also with regard to the utility of molecular targets within the thioredoxin reductase/thioredoxin pathway in the development of novel candidate antineoplastic agents. View Publication