Insulin levels control female germline stem cell maintenance via the niche in Drosophila

Stem cell maintenance depends on local signals provided by specialized microenvironments, or niches, in which they reside. The potential role of systemic factors in stem cell maintenance, however, has remained largely unexplored. Here, we show that insulin signaling integrates the effects of diet and age on germline stem cell (GSC) maintenance through the dual regulation of cap cell number (via Notch signaling) and cap cell–GSC interaction (via E-cadherin) and that the normal process of GSC and niche cell loss that occurs with age can be suppressed by increased levels of insulin-like peptides. These results underscore the importance of systemic factors for the regulation of stem cell niches and, thereby, of stem cell numbers.     

Notch signaling directly controls cell proliferation in the Drosophila wing disc

Notch signaling is involved in cell differentiation and patterning during morphogenesis. In the Drosophila wing, Notch activity regulates the expression of several genes at the dorsal/ventral boundary, and this is thought to elicit wing-cell proliferation. In this work, we show the effect of clones of cells expressing different forms of several members of the Notch signaling pathway, which result in an alteration of Notch activity. The ectopic expression in clones of activated forms of Notch or of its ligands (Delta or Serrate) in the wing causes outgrowths associated with the appearance of ectopic wing margins. These outgrowths consist of mutant territories and of surrounding wild-type cells. However, the ectopic expression of Delta, at low levels in ventral clones, causes large outgrowths that are associated neither with the generation of wing margin structures nor with the expression of genes characteristic of the dorsal/ventral boundary. These results suggest that Notch activity is directly involved in cell proliferation, independently of its role in the formation of the dorsal/ventral boundary. We propose that the nonautonomous effects (induction of extraproliferation and vein differentiation in the surrounding wild-type cells) result from pattern accommodation to positional values caused by the ectopic expression of Notch.     

Loss of lysine-specific demethylase 1 nonautonomously causes stem cell tumors in the Drosophila ovary

Specialized microenvironments called niches keep stem cells in an undifferentiated and self-renewing state. Dedicated stromal cells form niches by producing a variety of factors that act directly on stem cells. The size and signaling output of niches must be finely tuned to ensure proper tissue homeostasis. Although advances have been made in identifying factors that promote niche cell fate, the mechanisms that restrict niche cell formation during development and limit niche signaling output in adults remain poorly understood. Here, we show that the histone lysine-specific demethylase 1 (Lsd1) regulates the size of the germline stem cell (GSC) niche in Drosophila ovaries. GSC maintenance depends on bone morphogenetic protein (BMP) signals produced by a small cluster of cap cells located at the anterior tip of the germarium. Lsd1 null mutant ovaries carry small germline tumors containing an expanded number of GSC-like cells with round fusomes that display ectopic BMP signal responsiveness away from the normal niche. Clonal analysis and cell type-specific rescue experiments demonstrate that Lsd1 functions within the escort cells (ECs) that reside immediately adjacent to cap cells and prevents them from ectopically producing niche-specific signals. Temporally restricted gene knockdown experiments suggest that Lsd1 functions both during development, to specify EC fate, and in adulthood, to prevent ECs from forming ectopic niches independent of changes in cell fate. Further analysis shows that Lsd1 functions to repress decapentaplegic (dpp) expression in adult germaria. The role of Lsd1 in regulating niche-specific signals may have important implications for understanding how disruption of its mammalian homolog contributes to cancer and metastasis.     

DE-cadherin-mediated cell adhesion is essential for maintaining somatic stem cells in the Drosophila ovary

Evidence from many systems has shown that stem cells are maintained in "niches" or specific regulatory microenvironments formed by stromal cells. The question of how stem cells are maintained in their niches is important, and further studies will lead to a better understanding of stem cell regulation and enhance the future use of stem cells in regenerative medicine. Here we show that cadherin-mediated cell adhesion is required for anchoring somatic stem cells (SSCs) to their niches in the Drosophila ovary. DE-cadherin and Armadillo/beta-catenin accumulate in the junctions between SSCs and their neighboring cells, inner germarial sheath cells. Removal of DE-cadherin from SSCs results in stem cell loss in the adult ovary. Furthermore, the cadherin-mediated adhesion is also important for maintaining SSCs in their niches before adulthood. This study provides further support that SSCs are located in a niche formed by their neighboring cells. We have previously shown that DE-cadherin-mediated cell adhesion is essential for anchoring germ-line stem cells to their niches in the Drosophila ovary. This study further implicates cadherin-mediated cell adhesion as a general mechanism for anchoring stem cells to their niches in a variety of systems.     

Crystal structure of the yeast eIF4A-eIF4G complex: an RNA-helicase controlled by protein-protein interactions

Translation initiation factors eIF4A and eIF4G form, together with the cap-binding factor eIF4E, the eIF4F complex, which is crucial for recruiting the small ribosomal subunit to the mRNA 5' end and for subsequent scanning and searching for the start codon. eIF4A is an ATP-dependent RNA helicase whose activity is stimulated by binding to eIF4G. We report here the structure of the complex formed by yeast eIF4G's middle domain and full-length eIF4A at 2.6-A resolution. eIF4A shows an extended conformation where eIF4G holds its crucial DEAD-box sequence motifs in a productive conformation, thus explaining the stimulation of eIF4A's activity. A hitherto undescribed interaction involves the amino acid Trp-579 of eIF4G. Mutation to alanine results in decreased binding to eIF4A and a temperature-sensitive phenotype of yeast cells that carry a Trp579Ala mutation as its sole source for eIF4G. Conformational changes between eIF4A's closed and open state provide a model for its RNA-helicase activity.     

Reversing chemoresistance by small molecule inhibition of the translation initiation complex eIF4F

Deregulation of cap-dependent translation is associated with cancer initiation and progression. The rate-limiting step of protein synthesis is the loading of ribosomes onto mRNA templates stimulated by the heterotrimeric complex, eukaryotic initiation factor (eIF)4F. This step represents an attractive target for anticancer drug discovery because it resides at the nexus of the TOR signaling pathway. We have undertaken an ultra-high-throughput screen to identify inhibitors that prevent assembly of the eIF4F complex. One of the identified compounds blocks interaction between two subunits of eIF4F. As a consequence, cap-dependent translation is inhibited. This compound can reverse tumor chemoresistance in a genetically engineered lymphoma mouse model by sensitizing cells to the proapoptotic action of DNA damage. Molecular modeling experiments provide insight into the mechanism of action of this small molecule inhibitor. Our experiments validate targeting the eIF4F complex as a strategy for cancer therapy to modulate chemosensitivity.     

Phagocytosis genes nonautonomously promote developmental cell death in the Drosophila ovary

Programmed cell death (PCD) is usually considered a cell-autonomous suicide program, synonymous with apoptosis. Recent research has revealed that PCD is complex, with at least a dozen cell death modalities. Here, we demonstrate that the large-scale nonapoptotic developmental PCD in the Drosophila ovary occurs by an alternative cell death program where the surrounding follicle cells nonautonomously promote death of the germ line. The phagocytic machinery of the follicle cells, including Draper, cell death abnormality (Ced)-12, and c-Jun N-terminal kinase (JNK), is essential for the death and removal of germ-line–derived nurse cells during late oogenesis. Cell death events including acidification, nuclear envelope permeabilization, and DNA fragmentation of the nurse cells are impaired when phagocytosis is inhibited. Moreover, elimination of a small subset of follicle cells prevents nurse cell death and cytoplasmic dumping. Developmental PCD in the Drosophila ovary is an intriguing example of nonapoptotic, nonautonomous PCD, providing insight on the diversity of cell death mechanisms.Programmed cell death (PCD) is the genetically controlled elimination of cells that occurs during organismal development and homeostasis. Cells are considered dead when they have undergone irreversible plasma membrane permeabilization or have become completely fragmented (1). Apoptosis is the most well-characterized form of PCD, however there are at least a dozen cell death modalities that are morphologically, biochemically, and genetically distinct (2, 3). Two examples of nonapoptotic cell death are autophagic cell death and necrosis, but there are several alternative cell death mechanisms that are less well understood.Nonapoptotic PCD occurs on a large scale in the Drosophila ovary. Drosophila females can produce hundreds of eggs during their lifetime, and for every egg that is formed, developmental PCD of supporting nurse cells (NCs) occurs. However, the mechanisms of developmental PCD in the Drosophila ovary are poorly understood. Each egg forms from a 16-cell germ-line cyst, comprised of the single oocyte and 15 NCs that support the oocyte throughout 14 stages of oogenesis (4, 5). Hundreds of somatically derived follicle cells (FCs) surround the germ-line cyst, forming an egg chamber. At stage 11 of oogenesis, NCs rapidly transfer (“dump”) their cytoplasm into the oocyte. Concurrently, the NCs asynchronously undergo developmental PCD, resulting in mature stage 14 egg chambers that no longer contain any NCs (4–6). Interestingly, caspases, proteases associated with apoptosis, play only a minor role in the death of the NCs in late oogenesis (7–9). Furthermore, combined inhibition of caspases and autophagy does not significantly block NC death during late oogenesis (10). To date, defining the major mechanism of developmental PCD in the Drosophila ovary has remained elusive.An intriguing possibility is that the somatic FCs non–cell-autonomously promote developmental PCD of the NCs during late oogenesis. Non–cell-autonomous regulation of PCD occurs when a cell or group of cells extrinsically initiates or promotes the death of another cell. This concept challenges the idea that PCD is largely a self-regulated, autonomous suicide program in which a cell controls its own demise. One well-characterized example of non–cell-autonomous control of PCD is apoptosis induced by the death ligands Fas or TNF (11, 12).Another type of non–cell-autonomous PCD is phagoptosis (or primary phagocytosis), in which engulfing cells directly cause the death of other cells via “murder” or “assisted suicide.” Phagoptosis is distinct from the engulfment of cell corpses, as the engulfing cell plays an active role in the death of a cell, rather than simply degrading a cell that died via another mechanism. The defining characteristic of phagoptosis is that inhibition of phagocytosis leads to a failure in cell death (13, 14). Phagoptosis has been demonstrated in activated microglia that phagocytose viable neurons, resulting in their destruction (13–15). Entosis is another example of non–cell-autonomous PCD, often referred to as “cell cannibalism,” in which a viable cell invades another cell, where it is degraded by lysosomes. Entosis is distinct from phagoptosis, as the inhibition of phagocytosis genes does not prevent entosis (16). Phagocytosis has also been shown to promote PCD in Caenorhabditis elegans, although this is an example of assisted suicide, as dying cells also require apoptotic machinery (17, 18).Genetic studies in C. elegans have identified two partially redundant signaling pathways that control phagocytosis: the cell death abnormality (CED)-1, 6, 7 and CED-2, 5, 12 pathways (19–21). The CED-1, 6, 7 and CED-2, 5, 12 pathways act in parallel to promote the activation of CED-10, a Rac GTPase responsible for cytoskeletal rearrangements that allow for internalization of the cell corpse. In Drosophila, the roles of the Ced-1, 6, 7 and Ced-2, 5, 12 pathways appear to be conserved. The CED-1 ortholog, Draper, is a transmembrane protein that localizes to the surface of the engulfing cell and acts as a receptor to recognize dying cells. Draper was first shown to be required for engulfment of apoptotic neurons in the embryonic central nervous system with mutants displaying lingering cell corpses (22). Additionally, Draper has been shown to be important in several other contexts including the engulfment of severed axons, bacteria, imaginal disc cells, hemocytes, and apoptotic NCs in midoogenesis (23–27). In addition to Draper, other Drosophila engulfment receptors include Croquemort (28) and integrins (29–31). Croquemort is related to CD36, a scavenger receptor involved in engulfment in mammals (32), and integrins also act as engulfment receptors in C. elegans and mammals (33, 34). The upstream activators of the Ced-2, 5, 12 pathway are largely unknown, although integrins may activate the pathway (34). As in C. elegans, it appears that Ced-12 and draper function in separate pathways in Drosophila. Ced-12 and draper have been shown to function in distinct steps in axon clearance (35). In macrophages, Ced-12 has been shown to function in a separate pathway from simu, a bridging molecule that acts upstream of draper (36). A number of other engulfment genes have been identified in Drosophila, and their molecular interactions are under active investigation (36–39).Given the minor role for apoptosis and autophagic cell death during developmental PCD in the Drosophila ovary, we investigated the possibility that the FCs non–cell-autonomously promote NC death. Previously we showed that FCs of the Drosophila ovary are capable of phagoptosis in midoogenesis when phagocytosis genes are overexpressed (27), and we questioned whether phagocytosis genes might normally function to control cell death in late oogenesis. Indeed, we found that the phagocytosis genes draper and Ced-12/ELMO are required in the FCs for NC removal in late oogenesis and that they function partly in parallel. We also show that the FCs non–cell-autonomously control events associated with the death of the NCs, including nuclear envelope permeabilization, acidification, and DNA fragmentation. Furthermore, the genetic ablation of stretch FCs disrupted all cellular changes associated with developmental PCD of the NCs. Therefore, PCD of the NCs is a unique model of a naturally occurring developmental cell death program that is nonapoptotic and non–cell-autonomously controlled.     

Oocyte-somatic cell communication and microRNA function in the ovary

An enormous amount of knowledge about the ovary has been generated over the last two decades, due in part to the development of strategies to genetically manipulate the mouse using embryonic stem cell technology. Our group and others have identified multiple factors that are important and essential at all stages of ovarian folliculogenesis from formation of the primordial factor to ovulation. It is obvious that an oocyte, the key cargo of the ovary, and the surrounding granulosa cells, the support cells of the follicle, entertain a dialog that is key for granulosa growth and differentiation and oocyte growth, maturation, and fertilization. In addition to the involvement of genes in these processes, small non-coding RNAs including microRNAs and siRNAs have been implicated as key regulators, especially in the oocyte. These studies have direct implications for human fertility control in the assisted reproductive technology (ART) laboratory.     

Steroid receptor coactivator-3, a homolog of Taiman that controls cell migration in the Drosophila ovary, regulates migration of human ovarian cancer cells

Border cell migration is a process that occurs during Drosophila ovarian development in which cells derived from a simple epithelium migrate and invade neighboring tissue. This process resembles the behavior of cancerous cells that derive from the simple epithelium of the human ovary. One important regulator of border cell migration is Taiman, a homolog of steroid receptor coactivator-3 (SRC-3). Because increasing evidence indicates that similarities exist between the molecular control of migration of border cells and of cancer cells, we investigated whether SRC-3 controls ovarian cancer cell migration. Little or no SRC-3 expression was detected in normal ovarian surface epithelium, ovarian cysts and borderline ovarian tumors that lack stromal invasion. In contrast, SRC-3 was abundantly expressed in high-grade ovarian carcinomas. Inhibiting SRC-3 expression in ovarian cancer cells markedly reduced cell spreading and migration, and altered intracellular localization of focal adhesion kinase. This inhibitory effect on cell migration was independent of the estrogen receptor (ER) status of the cells. These studies reveal a novel role for SRC-3 in ovarian cancer progression by promoting cell migration, independently of its role in estrogen receptor signaling.     

Neural Hedgehog signaling maintains stem cell renewal in the sensory touch dome epithelium

The touch dome is a highly patterned mechanosensory structure in the epidermis composed of specialized keratinocytes in juxtaposition with innervated Merkel cells. The touch dome epithelium is maintained by tissue-specific stem cells, but the signals that regulate the touch dome are not known. We identify touch dome stem cells that are unique among epidermal cells in their activated Hedgehog signaling and ability to maintain the touch dome as a distinct lineage compartment. Skin denervation reveals that renewal of touch dome stem cells requires a perineural microenvironment, and deleting Sonic hedgehog (Shh) in neurons or Smoothened in the epidermis demonstrates that Shh is an essential niche factor that maintains touch dome stem cells. Up-regulation of Hedgehog signaling results in neoplastic expansion of touch dome keratinocytes but no Merkel cell neoplasia. These findings demonstrate that nerve-derived Shh is a critical regulator of lineage-specific stem cells that maintain specialized sensory compartments in the epidermis.The skin is one of several tissues in which the epithelium is organized into specialized regions, and each compartment is maintained by lineage-specific stem cells (1). Epithelial–mesenchymal interactions are classically regarded as critical signals in establishing and maintaining epidermal lineage compartments (2). The importance of adjacent dermal mesenchyme in regulating epidermal lineages has been demonstrated in the cycling hair follicle (3) and the interscale epidermis in mouse tail (4). However, other tissues influence epidermal stem cell biology, including subcutaneous fat (5), differentiated keratinocytes (6), and nerves (7). The importance of sensory nerves in sustaining epithelia is suggested by the fact that nerve damage can result in degeneration of the innervated tissue, including the corneal epithelium (8), lingual taste buds (9), and cutaneous Merkel cells (10). In this study, we investigate how neural signals maintain Merkel cell progenitors and sustain the mammalian touch dome lineage in skin.The touch dome (Haarscheibe) is a specialized epidermal structure that mediates slow-adapting touch sensation (11). In mice, touch domes form in association with guard hair follicles and are found adjacent to adult guard hairs (12). The touch dome is thicker than the surrounding epidermis and has morphologically distinct columnar keratinocytes in the basal layer. Arrayed among the touch dome basal keratinocytes are neuroendocrine Merkel cells that form synapse-like associations with myelinated sensory nerve endings (13). Merkel cells are epithelial cells that express keratin 8 (K8) (14), and originate from keratin 14 (K14)-positive progenitors in the epidermis (15). Touch dome keratinocytes have a distinct molecular profile compared with the surrounding epidermis, including expression of keratin 17 (K17) in basal cells (16). There is ongoing cellular turnover in touch domes, with constant production of stratified epithelial keratinocytes and cyclical expansion of terminally differentiated Merkel cells that coincides with anagen regeneration in the hair cycle (17). Genetic fate mapping has shown that the progenitors maintaining these two cell types are found among the K17⁺ cells in the touch dome (18), with unipotent atonal homolog 1 (Atoh1)-positive progenitors maintaining the Merkel cells (19). However, the signaling pathways involved in touch dome and Merkel cell homeostasis remain unclear.Here we use genetic fate mapping to confirm the touch dome is a distinct lineage within the epidermis and show that Gli1⁺ stem cells maintain touch dome Merkel cells and keratinocytes. Surgical denervation demonstrates that the entire touch dome lineage is dependent on a perineural microenvironment. Notably, a specific requirement for nerve-derived Sonic hedgehog (Shh) in long-term touch dome maintenance is shown via conditional knockout of Shh from dorsal root ganglion (DRG) neurons or of Smoothened (Smo) from the epidermis. Although Shh is necessary for touch dome stem cell renewal, overactivation of Hedgehog (Hh) signaling fails to induce proliferation of Merkel cells. Thus, by signaling to lineage-specific touch dome stem cells with Shh, neurons maintain the specialized sensory epithelium they innervate and sustain functional regionalization of the skin epithelium.     

Helminth products bypass the need for TSLP in Th2 immune responses by directly modulating dendritic cell function

Thymic stromal lymphopoietin (TSLP) is an interleukin (IL)-7-like cytokine, mainly expressed by epithelial cells, and key to the development of allergic responses. The well-documented involvement of TSLP in allergy has led to the conviction that TSLP promotes the development of inflammatory Th2 cell responses. However, we now report that the interaction of TSLP with its receptor (TSLPR) has no functional impact on the development of protective Th2 immune responses after infection with 2 helminth pathogens, Heligmosomoides polygyrus and Nippostrongylus brasiliensis. Mice deficient in the TSLP binding chain of the TSLPR (TSLPR^−/−) exhibited normal Th2 cell differentiation, protective immunity and memory responses against these two distinct rodent helminths. In contrast TSLP was found to be necessary for the development of protective Th2 responses upon infection with the helminth Trichuris muris (T. muris). TSLP inhibited IL-12p40 production in response to T. muris infection, and treatment of TSLPR^−/− animals with neutralizing anti-IL-12p40 monoclonal antibody (mAb) was able to reverse susceptibility and attenuate IFN-γ production. We additionally demonstrated that excretory-secretory (ES) products from H. polygyrus and N. brasiliensis, but not T. muris, were capable of directly suppressing dendritic cell (DC) production of IL-12p40, thus bypassing the need for TSLP. Taken together, our data show that the primary function of TSLP is to directly suppress IL-12 secretion, thus supporting Th2 immune responses.CD4⁺ T cells can differentiate into Th1, Th2, Th17, and Treg subsets, whose different immunological functions are associated with the production of particular cytokines (1). Of these subsets, Th2 cells play a pivotal role in immunity against helminth infection and are responsible for the pathology associated with allergic disorders (2, 3). Th2 cells typically produce IL-4, IL-5, IL-13, and IL-9 resulting in antibody-isotype switching to IgE, eosinophilia, basophilia, mucin production, and smooth muscle cell hyperreactivity (1).Extensive work has highlighted the key polarizing factors underlying the development of Th1, Th2, Th17, and Treg cells. IL-4 acts as both an effector cytokine and a Th2-polarizing factor (4, 5), although the early signals influencing T cell IL-4 production remain unclear. TSLP activates human DC to up-regulate costimulatory molecules, produce Th2 cell-attracting chemokines, and to promote the production of IL-4, IL-5, IL-13, and TNF-α, but not IL-10, by naïve CD4⁺ T cells (6, 7). TSLP simultaneously fails to activate DC IL-12 secretion (6, 7) and can even inhibit LPS-induced IL-12 production by murine DC in vitro (8, 9). More recently, TSLP has been reported to act directly on naïve mouse CD4⁺ T cells to promote IL-4 production in vitro (10), and to promote Th2 cytokine production by skin-infiltrating effector T cells in vivo (11). Consistent with its role in Th2 differentiation and effector function, TSLPR^−/− mice exhibit strongly attenuated allergic airway (12, 13) and skin inflammation (11), whereas over-expression of TSLP in lung epithelial cells or keratinocytes causes spontaneous allergic inflammation within the respective tissues (13, 14).In the current study, we investigated the impact of TSLP-TSLPR signaling on protective Th2 immune responses against the helminths H. polygyrus and N. brasiliensis, of the Trichostrongyloidea superfamily and T. muris, of the Trichineloidea superfamily. Protective immunity against all 3 helminths requires Th2 immune responses and is abrogated in the absence of Stat6-mediated IL-4/IL-13 signals (15–18). H. polygyrus and N. brasiliensis elicit strong Th2 immune responses in most mouse strains investigated (19). In contrast, the response elicited against T. muris is highly dependent on the genetic background of the host, with resistance or susceptibility tightly correlating with the generation of a Th2 and Th1 immune response, respectively (20, 21). Surprisingly, we found that TSLPR signaling had no detectable impact on H. polygyrus-induced Th2 polarization and only a minor impact on N. brasiliensis-induced CD4⁺ T cell cytokine production. In addition, TSLPR signaling had no impact on Th2 memory responses and did not alter the ability of mice to expel either helminth after secondary infection. In contrast, TSLPR signaling was necessary to prevent IL-12p40 production after T. muris infection, and lack of TSLPR signaling led to impaired protective Th2 responses. Secreted products from H. polygyrus and N. brasiliensis, but not T. muris, were found to modulate DC function in vitro, such that these cells were refractory to LPS-induced production of the proinflammatory cytokine IL-12p40. These data indicate that particular helminths can directly modulate host DC to suppress IL-12p40 production and thus render TSLP redundant for the development of Th2 immune responses.     

A balance between the diap1 death inhibitor and reaper and hid death inducers controls steroid-triggered cell death in Drosophila

The steroid hormone ecdysone directs the massive destruction of obsolete larval tissues during Drosophila metamorphosis, providing a model system for defining the molecular mechanisms of steroid-regulated programmed cell death. Although earlier studies have identified an ecdysone triggered genetic cascade that immediately precedes larval tissue cell death, no death regulatory genes have been functionally linked to this death response. We show here that ecdysone-induced expression of the death activator genes reaper (rpr) and head involution defective (hid) is required for destruction of the larval midgut and salivary glands during metamorphosis, with hid playing a primary role in the salivary glands and rpr and hid acting in a redundant manner in the midguts. We also identify the Drosophila inhibitor of apoptosis 1 as a survival factor in the larval cell death pathway, delaying death until its inhibitory effect is overcome by rpr and hid. This study reveals functional interactions between rpr and hid in Drosophila cell death responses and provides evidence that the precise timing of larval tissue cell death during metamorphosis is achieved through a steroid-triggered shift in the balance between the Drosophila inhibitor of apoptosis 1 and the rpr and hid death activators.     

Lessons from border cell migration in the Drosophila ovary: A role for myosin VI in dissemination of human ovarian cancer

Dissemination of ovarian cancer is a major clinical challenge and is poorly understood at the molecular level due to a lack of suitable experimental models. During normal development of the Drosophila ovary, a dynamic process called border cell migration occurs that resembles the migratory behavior of human ovarian cancer cells. In this study, we found that myosin VI, a motor protein that regulates border cell migration, is abundantly expressed in high-grade ovarian carcinomas but not in normal ovary and ovarian cancers that behave indolently. Inhibiting myosin VI expression in high-grade ovarian carcinoma cells impeded cell spreading and migration in vitro. Optical imaging and histopathologic studies revealed that inhibiting myosin VI expression reduces tumor dissemination in nude mice. Therefore, using genetic analysis of border cell migration in Drosophila is a powerful approach to identify novel molecules that promote ovarian cancer dissemination and represent potential therapeutic targets.     

Mimicking the functional hematopoietic stem cell niche in vitro: recapitulation of marrow physiology by hydrogel-based three-dimensional cultures of mesenchymal stromal cells

Background

A culture system that closely recapitulates marrow physiology is essential to study the niche-mediated regulation of hematopoietic stem cell fate at a molecular level. We investigated the key features that play a crucial role in the formation of a functional niche in vitro.

Design and Methods

Hydrogel-based cultures of human placenta-derived mesenchymal stromal cells were established to recapitulate the fibrous three-dimensional architecture of the marrow. Plastic-adherent mesenchymal stromal cells were used as controls. Human bone marrow-derived CD34⁺ cells were co-cultured with them. The output hematopoietic cells were characterized by various stem cell-specific phenotypic and functional parameters.

Results

The hydrogel-cultures harbored a large pool of primitive hematopoietic stem cells with superior phenotypic and functional attributes. Most importantly, like the situation in vivo, a significant fraction of these cells remained quiescent in the face of a robust multi-lineage hematopoiesis. The retention of a high percentage of primitive stem cells by the hydrogel-cultures was attributed to the presence of CXCR4-SDF1α axis and integrin beta1-mediated adhesive interactions. The hydrogel-grown mesenchymal stromal cells expressed high levels of several molecules that are known to support the maintenance of hematopoietic stem cells. Yet another physiologically relevant property exhibited by the hydrogel cultures was the formation of hypoxia-gradient. Destruction of hypoxia-gradient by incubating these cultures in a hypoxia chamber destroyed their specialized niche properties.

Conclusions

Our data show that hydrogel-based cultures of mesenchymal stromal cells form a functional in vitro niche by mimicking key features of marrow physiology.     

甲状腺结节伴微钙化患者5年内发生恶变的临床研究

目的:探讨甲状腺结节伴钙化患者甲状腺激素与5年内发生恶变的相关性。方法:采用多普勒超声检查仪对427例甲状腺结节伴钙化患者(观察组)和1147例单纯甲状腺结节患者(对照组)定期随访约5年,观察2组患者甲状腺结节恶变情况,并比较2组患者甲状腺激素水平。结果:观察组中有51例(11.94%)患者发生恶变,对照组中有58例(5.06%)患者发生恶变,观察组恶变发生率明显高于对照组(P0.01);观察组患者结节恶变发生在随访后的25.0～75.0个月,恶变中位时间48.5个月,对照组发生在随访后40.0～81.5个月,中位时间66.5个月。观察组恶变时间明显短于对照组(P0.01)。在观察组51例发生恶变的患者中,有48例为微钙化,占94.12%,剩下3例为粗大钙化。2组中,恶变与未恶变患者,在随访前后甲状腺激素(FT_3、FT_4和TSH)水平均无显著差异(均P0.05),患者甲状腺激素与恶变无明显相关性(r=0.217,P0.05)。结论:健康人群中甲状腺结节伴钙化恶变发生率要高于无钙化的单纯甲状腺结节患者,绝大部分为微钙化,且恶变的发生与甲状腺激素水平无关。     

慢病毒靶向溴样结构域蛋白4通过抑制SHH通路对非小细胞肺癌细胞A549能量代谢水平的影响

目的:探究溴样结构域蛋白4(BRD4)siRNA通过SHH通路对非小细胞肺癌细胞A549能量水平的影响。方法:RT-qPCR分析非小细胞肺癌细胞A549、HLF-1细胞内BRD4表达水平;将BRD4 siRNA转入A549细胞内,细胞计数试剂盒-8(CCK-8)和细胞克隆形成实验检测BRD4 siRNA对A549生长增殖的影响;2-[N-(7-硝基苯并-2-氧-1,3-二唑-4-基)氨基]-2-脱氧葡萄糖法(2-NBDG)法和乳酸检测试剂盒检测BRD4 siRNA对A549细胞葡萄糖摄取率和乳酸生成量的影响;RT-qPCR和Western blot检测BRD4 siRNA对基因SHH、胶质瘤相关癌基因同源物1(GLI1)的mRNA和蛋白表达水平的影响。结果:BRD4基因在A549细胞中上调表达(P0.05);BRD4 siRNA对A549细胞的增殖、克隆形成、葡萄糖摄入和乳酸生成具有抑制作用(P0.05),且能够促进SHH和GLI1基因的mRNA和蛋白表达显著下调(均P0.05)。结论:慢病毒靶向BRD4基因能够通过抑制SHH通路降低非小细胞肺癌细胞A549的能量代谢水平。     

Induction of sertoli cell tumors in the rat ovary by N-alkyl-N-nitrosoureas

Summary Relatively high incidences of Sertoli cell tumors of the ovary were induced in Donryu rats given a 400 ppm N-ethyl-N-nitrosourea solution as drinking water for 4 weeks or a single dose of 200 mg/kg N-propyl-N-nitrosourea by stomach tube. Typical Sertoli cell tumors were composed of solid areas showing tubular formation. Tubules were lined by tall, columnar cells having abundant, faintly eosinophilic, often vacuolated cytoplasm, and basally oriented round nuclei. In some cases, Sertoli cell tumors were found to be mixed with granulosa cell tumors.