技术资料

Aldehyde dehydrogenase (ALDH) activity has been implicated in multiple biological and biochemical pathways and has been used to identify potential cancer stem cells. Our main hypothesis is that ALDH activity may be a lung cancer stem cell marker. Using flow cytometry,we sorted cells with bright (ALDH(br)) and dim (ALDH(lo)) ALDH activity found in H522 lung cancer cell line. We used in vitro proliferation and colony assays as well as a xenograft animal model to test our hypothesis. Cytogenetic analysis demonstrated that the ALDH(br) cells are indeed a different clone,but when left in normal culture conditions will give rise to ALDH(lo) cells. Furthermore,the ALDH(br) cells grow slower,have low clonal efficiency,and give rise to morphologically distinct colonies. The ability to form primary xenografts in NOD/SCID mice by ALDH(br) and ALDH(lo) cells was tested by injecting single cell suspension under the skin in each flank of same animal. Tumor size was calculated weekly. ALDH1A1 and ALDH3A1 immunohistochemistry (IHC) was performed on excised tumors. These tumors were also used to re-establish cell suspension,measure ALDH activity,and re-injection for secondary and tertiary transplants. The results indicate that both cell types can form tumors but the ones from ALDH(br) cells grew much slower in primary recipient mice. Histologically,there was no significant difference in the expression of ALDH in primary tumors originating from ALDH(br) or ALDH(lo) cells. Secondary and tertiary xenografts originating from ALDH(br) grew faster and bigger than those formed by ALDH(lo) cells. In conclusion,ALDH(br) cells may have some of the traditional features of stem cells in terms of being mostly dormant and slow to divide,but require support of other cells (ALDH(lo)) to sustain tumor growth. These observations and the known role of ALDH in drug resistance may have significant therapeutic implications in the treatment of lung cancer. View Publication

Oxidized low-density lipoprotein (OxLDL) is a well-known risk marker for cardiovascular diseases. OxLDL has shown a variety of proatherogenic properties in experiments performed in vitro. In addition,immunological studies using monoclonal antibodies have revealed the occurrence of OxLDL in vivo in atherosclerotic lesions and patients' plasma specimens. Resent clinical studies have indicated the prospective significance of plasma OxLDL measurements; however,the behavior and metabolism of OxLDL in vivo is poorly understood. The mechanism by which LDL is oxidized is not clear,and the modified structures of OxLDL are not yet fully understood,partly because OxLDL is a mixture of heterogeneously modified particles. Here,I discuss the recent studies on oxidative modifications in OxLDL and its clinical and pathological features. 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

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

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

All-trans-retinoic acid (ATRA) and its associated analogues are important mediators of cell differentiation and function during the development of the nervous system. It is well known that ATRA can induce the differentiation of neural tissues from human pluripotent stem cells. However,it is not always appreciated that ATRA is highly susceptible to isomerisation when in solution,which can influence the effective concentration of ATRA and subsequently its biological activity. To address this source of variability,synthetic retinoid analogues have been designed and synthesised that retain stability during use and maintain biological function in comparison to ATRA. It is also shown that subtle modifications to the structure of the synthetic retinoid compound impacts significantly on biological activity,as when exposed to cultured human pluripotent stem cells,synthetic retinoid 4-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-ylethynyl)benzoic acid,4a (para-isomer),induces neural differentiation similarly to ATRA. In contrast,stem cells exposed to synthetic retinoid 3-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-ylethynyl)benzoic acid,4b (meta-isomer),produce very few neurons and large numbers of epithelial-like cells. This type of structure-activity-relationship information for such synthetic retinoid compounds will further the ability to design more targeted systems capable of mediating robust and reproducible tissue differentiation. View Publication

Current sources of mesenchymal cells,including bone marrow,fat and muscle,all require invasive procurement procedures,and provide relatively low frequencies of progenitors. Here,we describe the non-invasive isolation,and characterization,of a rich source of mesenchymal progenitor cells,which we call human umbilical cord perivascular cells (HUCPVCs). HUCPVCs show a similar immunological phenotype to bone marrow-derived mesenchymal stromal cells (BM-MSCs),since they are non-alloreactive,exhibit immunosuppression,and significantly reduce lymphocyte activation,in vitro. They present a non-hematopoietic myofibroblastic mesenchymal phenotype (CD45-,CD34-,CD105+,CD73+,CD90+,CD44+,CD106+,3G5+,CD146+); with a 1:300 frequency at harvest,a short-doubling time,and a clonogenic frequency of textgreater1:3 in culture. Furthermore,in addition to robust quinti-potential differentiation capacity in vitro,HUCPVCs have been shown to contribute to both musculo-skeletal and dermal wound healing in vivo. View Publication

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Janus-activated kinase 2 (JAK2) mutations are common in myeloproliferative disorders; however,although they are detected in virtually all polycythemia vera patients,they are found in approximately 50% of essential thrombocythemia (ET) patients,suggesting that converging pathways/abnormalities underlie the onset of ET. Recently,the chromosomal translocation 3;21,leading to the fusion gene AML1/MDS1/EVI1 (AME),was observed in an ET patient. After we forced the expression of AME in the bone marrow (BM) of C57BL/6J mice,all the reconstituted mice died of a disease with symptoms similar to ET with a latency of 8 to 16 months. Peripheral blood smears consistently showed an elevated number of dysplastic platelets with anisocytosis,degranulation,and giant size. Although the AME-positive mice did not harbor Jak2 mutations,the BM of most of them had significantly higher levels of activated Stat3 than the controls. With combined biochemical and biological assays we found that AME binds to the Stat3 promoter leading to its up-regulation. Signal transducers and activators of transcription 3 (STAT3) analysis of a small group of ET patients shows that in about half of the patients,there is STAT3 hyperactivation independently of JAK2 mutations,suggesting that the hyperactivation of STAT3 by JAK2 mutations or promoter activation may be a critical step in development of ET. View Publication

Human skin-migratory dendritic cells (DCs) have the ability to prime and bias Th1 and Th2 CD4+ T lymphocytes. However,whether human cutaneous DCs are capable of initiating proinflammatory Th17 responses remains undetermined. We report that skin-migratory DCs stimulate allogeneic naive CD4+ T cells that differentiate simultaneously into two distinct effector Th17 and Th1 populations capable of homing to the skin,where they induce severe cutaneous damage. Skin-migratory Langerhans cells (smiLCs) were the main cutaneous DC subset capable of inducing Th17 responses dependent on the combined effects of IL-15 and stabilized IL-6,which resulted in IL-6 trans-signaling of naive CD4+ T cells. Different from smiLCs,purified skin-migratory dermal DCs did not synthesize IL-15 and were unable to bias Th17 responses. Nevertheless,these dermal DCs were capable of differentiating Th17 cells in mixed leukocyte cultures supplemented with IL-15 and stabilized IL-6. Overall,our data demonstrate that human epidermal smiLCs induce Th17 responses by mechanisms different from those previously described and highlight the need to target clinical treatments based on these variations. View Publication

Fanconi anemia (FA) is a heterogeneous genetic disorder characterized by bone marrow failure and complex congenital anomalies. Although mutations in FA genes result in a characteristic phenotype in the hematopoietic stem/progenitor cells (HSPCs),little is known about the consequences of a nonfunctional FA pathway in other stem/progenitor cell compartments. Given the intense functional interactions between HSPCs and the mesenchymal microenvironment,we investigated the FA pathway on the cellular functions of murine mesenchymal stem/progenitor cells (MSPCs) and their interactions with HSPCs in vitro and in vivo. Here,we show that loss of the murine homologue of FANCG (Fancg) results in a defect in MSPC proliferation and in their ability to support the adhesion and engraftment of murine syngeneic HSPCs in vitro or in vivo. Transplantation of wild-type (WT) but not Fancg(-/-) MSPCs into the tibiae of Fancg(-/-) recipient mice enhances the HSPC engraftment kinetics,the BM cellularity,and the number of progenitors per tibia of WT HSPCs injected into lethally irradiated Fancg(-/-) recipients. Collectively,these data show that FA proteins are required in the BM microenvironment to maintain normal hematopoiesis and provide genetic and quantitative evidence that adoptive transfer of WT MSPCs enhances hematopoietic stem cell engraftment. View Publication

Protein phosphatase 2A (PP2A) is a major serine/threonine protein phosphatase in eukaryotic cells and is involved in many essential aspects of cell function. The catalytic subunit of the enzyme (PP2Ac),a part of the core enzyme,has two isoforms,alpha (PP2Ac alpha) and beta (PP2Ac beta),of which PP2Ac alpha is the major form expressed in vivo. Deregulation of PP2A expression has been linked to several diseases,but the mechanisms that control the expression of this enzyme are still unclear. We conducted experiments to decipher molecular mechanisms involved in the regulation of the PP2Ac alpha promoter in human primary T cells. After preparing serially truncated PP2Ac alpha promoter luciferase constructs,we found that the region stretching around 240 bases upstream from the translation initiation site was of functional significance and included a cAMP response element motif flanked by three GC boxes. Shift assays revealed that CREB/phosphorylated CREB and stable protein 1 could bind to the region. Furthermore,we demonstrated that methylation of deoxycytosine in the CpG islands limited binding of phosphorylated CREB and the activity of the PP2Ac alpha promoter. In contrast,the binding of stable protein 1 to a GC box within the core promoter region was not affected by DNA methylation. Primary T cells treated with 5-azacitidine,a DNA methyltransferase inhibitor,showed increased expression of PP2Ac alpha mRNA. We propose that conditions associated with hypomethylation of CpG islands,such as drug-induced lupus,permit increased PP2Ac expression. View Publication