技术资料

Small populations within an increasing array of solid tumors,labeled cancer stem cells (CSC) or tumor-initiating cells (TIC),have the ability to differentiate,self-renew,and replicate the original tumor in vivo. To date,these cells have been distinguished from the bulk-tumor population by the expression pattern of cell-surface proteins (e.g.,CD24,CD44,CD133) and cellular activities,such as the efflux of Hoechst dye or aldehyde dehydrogenase activity. Recent data have shown that these markers are inducible by exposure to anticancer agents; this finding highlights not only the potential fluidity of the CSC compartment,but also the functionality of these markers. The involvement of CD44 in invasion,adhesion,and metastasis,or the role of CD24 in modulation of src,FAK,and GLI1 are examples of these relevant roles. Instead of looking solely at the marker expression in these populations,we hope to clarify the biologically significant roles these markers and activities play in tumor progression,metastases,and as possible targets for therapy. View Publication

Human solid tumors frequently have pronounced heterogeneity of both neoplastic and normal cells on the histological,genetic,and gene expression levels. While current efforts are focused on understanding heterotypic interactions between tumor cells and surrounding normal cells,much less is known about the interactions between and among heterogeneous tumor cells within a neoplasm. In glioblastoma multiforme (GBM),epidermal growth factor receptor gene (EGFR) amplification and mutation (EGFRvIII/DeltaEGFR) are signature pathogenetic events that are invariably expressed in a heterogeneous manner. Strikingly,despite its greater biological activity than wild-type EGFR (wtEGFR),individual GBM tumors expressing both amplified receptors typically express wtEGFR in far greater abundance than the DeltaEGFR lesion. We hypothesized that the minor DeltaEGFR-expressing subpopulation enhances tumorigenicity of the entire tumor cell population,and thereby maintains heterogeneity of expression of the two receptor forms in different cells. Using mixtures of glioma cells as well as immortalized murine astrocytes,we demonstrate that a paracrine mechanism driven by DeltaEGFR is the primary means for recruiting wtEGFR-expressing cells into accelerated proliferation in vivo. We determined that human glioma tissues,glioma cell lines,glioma stem cells,and immortalized mouse Ink4a/Arf(-/-) astrocytes that express DeltaEGFR each also express IL-6 and/or leukemia inhibitory factor (LIF) cytokines. These cytokines activate gp130,which in turn activates wtEGFR in neighboring cells,leading to enhanced rates of tumor growth. Ablating IL-6,LIF,or gp130 uncouples this cellular cross-talk,and potently attenuates tumor growth enhancement. These findings support the view that a minor tumor cell population can potently drive accelerated growth of the entire tumor mass,and thereby actively maintain tumor cell heterogeneity within a tumor mass. Such interactions between genetically dissimilar cancer cells could provide novel points of therapeutic intervention. View Publication

MED1/TRAP220,a subunit of the transcriptional Mediator/TRAP complex,is crucial for various biological events through its interaction with distinct activators,such as nuclear receptors and GATA family activators. In hematopoiesis,MED1 plays a pivotal role in optimal nuclear receptor-mediated myelomonopoiesis and GATA-1-induced erythropoiesis. In this study,we present evidence that MED1 in stromal cells is involved in supporting hematopoietic stem and/or progenitor cells (HSPCs) through osteopontin (OPN) expression. We found that the proliferation of bone marrow (BM) cells cocultured with MED1 knockout (Med1(-/-)) mouse embryonic fibroblasts (MEFs) was significantly suppressed compared to the control. Furthermore,the number of long-term culture-initiating cells (LTC-ICs) was attenuated for BM cells cocultured with Med1(-/-) MEFs. The vitamin D receptor (VDR)- and Runx2-mediated expression of OPN,as well as Mediator recruitment to the Opn promoter,was specifically attenuated in the Med1(-/-) MEFs. Addition of OPN to these MEFs restored the growth of cocultured BM cells and the number of LTC-ICs,both of which were attenuated by the addition of the anti-OPN antibody to Med1(+/+) MEFs and to BM stromal cells. Consequently,MED1 in niche appears to play an important role in supporting HSPCs by upregulating VDR- and Runx2-mediated transcription on the Opn promoter. View Publication

Adhesion properties of hematopoietic stem cells (HSCs) in the bone marrow (BM) niches control their migration and affect their cell-cycle dynamics. The serum response factor (Srf) regulates growth factor-inducible genes and genes controlling cytoskeleton structures involved in cell spreading,adhesion,and migration. We identified a role for Srf in HSC adhesion and steady-state hematopoiesis. Conditional deletion of Srf in BM cells resulted in a 3-fold expansion of the long- and short-term HSCs and multipotent progenitors (MPPs),which occurs without long-term modification of cell-cycle dynamics. Early differentiation steps to myeloid and lymphoid lineages were normal,but Srf loss results in alterations in mature-cell production and severe thrombocytopenia. Srf-null BM cells also displayed compromised engraftment properties in transplantation assays. Gene expression analysis identified Srf target genes expressed in HSCs,including a network of genes associated with cell migration and adhesion. Srf-null stem cells and MPPs displayed impair expression of the integrin network and decreased adherence in vitro. In addition,Srf-null mice showed increase numbers of circulating stem and progenitor cells,which likely reflect their reduced retention in the BM. Altogether,our results demonstrate that Srf is an essential regulator of stem cells and MPP adhesion,and suggest that Srf acts mainly through cell-matrix interactions and integrin signaling. View Publication

Neutrophil apoptosis is essential for the resolution of inflammation but is delayed by several inflammatory mediators. In such terminally differentiated cells it has been uncertain whether these agents can inhibit apoptosis through transcriptional regulation of anti-death (Bcl-X(L),Mcl-1,Bcl2A1) or BH3-only (Bim,Bid,Puma) Bcl2-family proteins. We report that granulocyte/macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor (TNF)-α prevent the normal time-dependent loss of Mcl-1 and Bcl2A1 in neutrophils,and we demonstrate that they cause an NF-κB-dependent increase in Bcl-X(L) transcription/translation. We show that GM-CSF and TNF-α increase and/or maintain mRNA levels for the pro-apoptotic BH3-only protein Bid and that GM-CSF has a similar NF-κB-dependent effect on Bim transcription and BimEL expression. The in-vivo relevance of these findings was indicated by demonstrating that GM-CSF is the dominant neutrophil survival factor in lung lavage from patients with ventilator-associated pneumonia,confirming an increase in lung neutrophil Bim mRNA. Finally GM-CSF caused mitochondrial location of Bim and a switch in phenotype to a cell that displays accelerated caspase-9-dependent apoptosis. This study demonstrates the capacity of neutrophil survival agents to induce a paradoxical increase in the pro-apoptotic proteins Bid and Bim and suggests that this may function to facilitate rapid apoptosis at the termination of the inflammatory cycle. View Publication

The bisdichloroacetyldiamine WIN 18,446 reversibly inhibits spermatogenesis in many species,including humans; however,the mechanism by which WIN 18,446 functions is unknown. As retinoic acid is essential for spermatogenesis,we hypothesized that WIN 18,446 might inhibit retinoic acid biosynthesis from retinol (vitamin A) within the testes by inhibiting the enzyme aldehyde dehydrogenase 1a2 (ALDH1a2). We studied the effect of WIN 18,446 on ALDH1a2 enzyme activity in vitro,and on spermatogenesis and fertility in vivo,in mature male rabbits for 16 weeks. WIN 18,446 markedly inhibited ALDH1a2 enzyme activity in vitro with an IC(50) of 0.3 μM. In vivo,the oral administration of 200 mg/kg WIN 18,446 to male rabbits for 16 weeks significantly reduced intratesticular concentrations of retinoic acid,severely impaired spermatogenesis,and caused infertility. Reduced concentrations of intratesticular retinoic acid were apparent after only 4 weeks of treatment and preceded the decrease in sperm counts and the loss of mature germ cells in tissue samples. Sperm counts and fertility recovered after treatment was discontinued. These findings demonstrate that bisdichloroacetyldiamines such as WIN 18,446 reversibly suppress spermatogenesis via inhibition of testicular retinoic acid biosynthesis by ALDH1a2. These findings suggest that ALDH1a2 is a promising target for the development of a reversible,nonhormonal male contraceptive. View Publication

Monoamine neurotransmitters play major roles in regulating a range of brain functions in adults and increasing evidence suggests roles for monoamines in brain development. Here we show that mice lacking the monoamine metabolic enzymes MAO A and MAO B (MAO AB-deficient mice) exhibit diminished proliferation of neural stem cells (NSC) in the developing telencephalon beginning in late gestation [embryonic day (E) 17.5],a deficit that persists in neonatal and adult mice. These mice showed significantly increased monoamine levels and anxiety-like behaviors as adults. Assessments of markers of intermediate progenitor cells (IPC) and mitosis showed that NSC in the subventricular zone (SVZ),but not in the ventricular zone,are reduced in MAO AB-deficient mice. A developmental time course of monoamines in frontal cortical tissues revealed increased serotonin levels as early as E14.5,and a further large increase was found between E17.5 and postnatal day 2. Administration of an inhibitor of serotonin synthesis (parachlorophenylalanine) between E14.5 and E19.5 restored the IPC numbers and SVZ thickness,suggesting the role of serotonin in the suppression of IPC proliferation. Studies of neurosphere cultures prepared from the telencephalon at different embryonic and postnatal ages showed that serotonin stimulates proliferation in wild-type,but not in MAO AB-deficient,NSC. Together,these results suggest that a MAO-dependent long-lasting alteration in the proliferation capacity of NSC occurs late in embryonic development and is mediated by serotonin. Our findings reveal novel roles for MAOs and serotonin in the regulation of IPC proliferation in the developing brain. View Publication

The contribution of mammary epithelial cells (MEC) to human immunodeficiency virus type 1 (HIV-1) in breast milk remains largely unknown. While breast milk contains CD4(+) cells throughout the breast-feeding period,it is not known whether MEC directly support HIV-1 infection or facilitate infection of CD4(+) cells in the breast compartment. This study evaluated primary human MEC for direct infection with HIV-1 and for indirect transfer of infection to CD4(+) target cells. Primary human MEC were isolated and assessed for expression of HIV-1 receptors. MEC were exposed to CCR5-,CXCR4- and dual-tropic strains of HIV-1 and evaluated for viral reverse transcription and integration and productive viral infection. MEC were also tested for the ability to transfer HIV to CD4(+) target cells and to activate resting CD4(+) T cells. Our results demonstrate that MEC express HIV-1 receptor proteins CD4,CCR5,CXCR4,and galactosyl ceramide (GalCer). While no evidence for direct infection of MEC was found,HIV-1 virions were observed in MEC endosomal compartments. Coculture of HIV-exposed MEC resulted in productive infection of activated CD4(+) T cells. In addition,MEC secretions increased HIV-1 replication and proliferation of infected target cells. Overall,our results indicate that MEC are capable of endosomal uptake of HIV-1 and can facilitate virus infection and replication in CD4(+) target cells. These findings suggest that MEC may serve as a viral reservoir for HIV-1 and may enhance infection of CD4(+) T lymphocytes in vivo. View Publication

This study evaluated the hypothesis that smoke from harm reduction cigarettes impedes attachment and proliferation of H9 human embryonic stem cells (hESCs). Smoke from three harm reduction brands was compared with smoke from a conventional brand. Doses of smoke were measured in puff equivalents (PE) (1 PE = the amount of smoke in one puff that dissolves in 1 ml of medium). Cytotoxic doses were determined using morphological criteria and trypan blue staining,and apoptosis was confirmed using Magic Red staining. Attachment and proliferation of hESC were followed at a noncytotoxic dose in time-lapse videos collected using BioStation technology. Data were mined from videos either manually or using video bioinformatics subroutines developed with CL-Quant software. Mainstream (MS) and sidestream (SS) smoke from conventional and harm reduction cigarettes induced apoptosis in hESC colonies at 1 PE. At 0.1 PE (noncytotoxic),SS smoke from all brands inhibited attachment of hESC colonies to Matrigel with the strongest inhibition occurring in harm reduction brands. At 0.1 PE,SS smoke,but not MS smoke,from all brands inhibited hESC growth,and two harm reduction brands were more potent than the conventional brand. In general,hESC appeared more sensitive to smoke than their mouse ESC counterparts. Although harm reduction cigarettes are often marketed as safer than conventional brands,our assays show that SS smoke from harm reduction cigarettes was at least as potent or in some cases more potent than smoke from a conventional brand and that SS smoke was more inhibitory than MS smoke in all assays. View Publication

Deciding to exit pluripotency and undergo differentiation is of singular importance for pluripotent cells,including embryonic stem cells (ESCs). The molecular mechanisms for these decisions to differentiate,as well as reversing those decisions during induced pluripotency (iPS),have focused largely on transcriptomic controls. Here,we explore the role of translational control for the maintenance of pluripotency and the decisions to differentiate. Global protein translation is significantly reduced in hESCs compared to their differentiated progeny. Furthermore,p70 S6K activation is restricted in hESCs compared to differentiated fibroblast-like cells. Disruption of p70 S6K-mediated translation by rapamycin or siRNA knockdown in undifferentiated hESCs does not alter cell viability or expression of the pluripotency markers Oct4 and Nanog. However,expression of constitutively active p70 S6K,but not wild-type p70 S6K,induces differentiation. Additionally,hESCs exhibit high levels of the mTORC1/p70 S6K inhibitory complex TSC1/TSC2 and preferentially express more rapamycin insensitive mTORC2 compared to differentiated cells. siRNA-mediated knockdown of both TSC2 and Rictor elevates p70 S6K activation and induces differentiation of hESCs. These results suggest that hESCs tightly regulate mTORC1/p70 S6K-mediated protein translation to maintain a pluripotent state as well as implicate a novel role for protein synthesis as a driving force behind hESC differentiation. View Publication

One of the factors that can impact human embryonic stem cell expansion in stirred microcarrier culture reactors is mechanical stress caused by agitation. Therefore,we have investigated the effects of agitation on human embryonic stem cell growth and expression of pluripotent markers. Agitation of HES-2 cell line in microcarrier cultures in stirred spinner and agitated six-well plates did not affect expression of pluripotent markers,cell viability,and cell doubling times even after seven passages. However,HES-3 cell line was found to be shear sensitive,showing downregulation of three pluripotent markers Oct-4,mAb 84,and Tra-1-60,and lower cell densities in agitated as compared with static cultures,even after one passage. Cell viability was unaffected. The HES-3-agitated cultures showed increased expression of genes and proteins of the three germ layers. We were unable to prevent loss of pluripotent markers or restore doubling times in agitated HES-3 microcarrier cultures by addition of five different known cell protective polymers. In addition,the human induced pluripotent cell line IMR90 was also shown to differentiate in agitated conditions. These results indicate that the effect of agitation on cell growth and differentiation is cell line specific. We assume that the changes in the growth and differentiation of the agitation-sensitive (HES-3) cell line do not result from the effect of shear stress directly on cell viability,but rather by signaling effects that influence the cells to differentiate resulting in slower growth. View Publication

The importance of cancer metabolism has been appreciated for many years,but the intricacies of how metabolic pathways interconnect with oncogenic signaling are not fully understood. With a clear understanding of how metabolism contributes to tumorigenesis,we will be better able to integrate the targeting of these fundamental biochemical pathways into patient care. The mevalonate (MVA) pathway,paced by its rate-limiting enzyme,hydroxymethylglutaryl coenzyme A reductase (HMGCR),is required for the generation of several fundamental end-products including cholesterol and isoprenoids. Despite years of extensive research from the perspective of cardiovascular disease,the contribution of a dysregulated MVA pathway to human cancer remains largely unexplored. We address this issue directly by showing that dysregulation of the MVA pathway,achieved by ectopic expression of either full-length HMGCR or its novel splice variant,promotes transformation. Ectopic HMGCR accentuates growth of transformed and nontransformed cells under anchorage-independent conditions or as xenografts in immunocompromised mice and,importantly,cooperates with RAS to drive the transformation of primary mouse embryonic fibroblasts cells. We further explore whether the MVA pathway may play a role in the etiology of human cancers and show that high mRNA levels of HMGCR and additional MVA pathway genes correlate with poor prognosis in a meta-analysis of six microarray datasets of primary breast cancer. Taken together,our results suggest that HMGCR is a candidate metabolic oncogene and provide a molecular rationale for further exploring the statin family of HMGCR inhibitors as anticancer agents. View Publication