Constitutive Bcl-2 expression throughout the hematopoietic compartment affects multiple lineages and enhances progenitor cell survival

Bcl-2, which can both reduce apoptosis and retard cell cycle entry, is thought to have important roles in hematopoiesis. To evaluate the impact of its ubiquitous overexpression within this system, we targeted expression of the human bcl-2 gene in mice by using the promoter of the vav gene, which is active throughout this compartment but rarely outside it. The vav-bcl-2 transgene was expressed in essentially all nucleated cells of hematopoietic tissues but not notably in nonhematopoietic tissues. Presumably because of enhanced cell survival, the mice displayed increases in myeloid cells as well as a marked elevation in B and T lymphocytes. The spleen was enlarged and the lymphoid follicles expanded. Although total thymic cellularity was normal, T cell development was altered: cells at the very immature and most mature stages were increased, whereas those at the intermediate stage were decreased. Unexpectedly, blood platelets were reduced by half, suggesting that their production from megakaryocytes is regulated by the Bcl-2 family. Colony formation by myeloid progenitor cells in vitro remained cytokine dependent, and the frequency of most progenitor and preprogenitor cells was normal. Macrophage progenitors were less frequent and yielded smaller colonies, however, perhaps reflecting inhibitory effects of Bcl-2 on cell cycling in specific lineages. After irradiation or factor deprivation, Bcl-2 markedly enhanced clonogenic survival of all tested progenitor and preprogenitor cells. Thus, Bcl-2 has multiple effects on the hematopoietic system. These mice should help to further clarify the role of apoptosis in the development and homeostasis of this compartment.

Expression of DFak56, a Drosophila homolog of vertebrate focal adhesion kinase, supports a role in cell migration in vivo

Focal adhesion kinase (FAK) is a highly conserved, cytoplasmic tyrosine kinase that has been implicated in promoting cell migration and transmission of antiapoptotic signals in vertebrate cells. In cultured cells, integrin engagement with the extracellular matrix promotes the recruitment of FAK to focal contacts and increases in its phosphotyrosine content and kinase activity, suggesting FAK is an intracellular mediator of integrin signaling. We have identified a Drosophila FAK homolog, DFak56, that is 33% identical to vertebrate FAK, with the highest degree of homology in domains critical for FAK function, including the kinase and focal adhesion targeting domains, and several protein–protein interaction motifs. Furthermore, when expressed in NIH 3T3 cells, DFak56 both localizes to focal contacts and displays the characteristic elevation of phosphotyrosine content in response to plating the cells on fibronectin. During embryogenesis, DFak56 is broadly expressed, and it becomes elevated in the gut and central nervous system at later stages. Consistent with a role in cell migration, we also observe that DFak56 is abundant in the border cells of developing egg chambers before the onset of, and during, their migration.

Derivation of pluripotent stem cells from cultured human primordial germ cells

Human pluripotent stem cells would be invaluable for in vitro studies of aspects of human embryogenesis. With the goal of establishing pluripotent stem cell lines, gonadal ridges and mesenteries containing primordial germ cells (PGCs, 5–9 weeks postfertilization) were cultured on mouse STO fibroblast feeder layers in the presence of human recombinant leukemia inhibitory factor, human recombinant basic fibroblast growth factor, and forskolin. Initially, single PGCs in culture were visualized by alkaline phosphatase activity staining. Over a period of 7–21 days, PGCs gave rise to large multicellular colonies resembling those of mouse pluripotent stem cells termed embryonic stem and embryonic germ (EG) cells. Throughout the culture period most cells within the colonies continued to be alkaline phosphatase-positive and tested positive against a panel of five immunological markers (SSEA-1, SSEA-3, SSEA-4, TRA-1–60, and TRA-1–81) that have been used routinely to characterize embryonic stem and EG cells. The cultured cells have been continuously passaged and found to be karyotypically normal and stable. Both XX and XY cell cultures have been obtained. Immunohistochemical analysis of embryoid bodies collected from these cultures revealed a wide variety of differentiated cell types, including derivatives of all three embryonic germ layers. Based on their origin and demonstrated properties, these human PGC-derived cultures meet the criteria for pluripotent stem cells and most closely resemble EG cells.

Cerebral cortical astroglia from the trisomy 16 mouse, a model for Down syndrome, produce neuronal cholinergic deficits in cell culture

Trisomy 21 (Down syndrome) is associated with a high incidence of Alzheimer disease and with deficits in cholinergic function in humans. We used the trisomy 16 (Ts16) mouse model for Down syndrome to identify the cellular basis for the cholinergic dysfunction. Cholinergic neurons and cerebral cortical astroglia, obtained separately from Ts16 mouse fetuses and their euploid littermates, were cultured in various combinations. Choline acetyltransferase activity and cholinergic neuron number were both depressed in cultures in which both neurons and glia were derived from Ts16 fetuses. Cholinergic function of normal neurons was significantly down-regulated by coculture with Ts16 glia. Conversely, neurons from Ts16 animals could express normal cholinergic function when grown with normal glia. These observations indicate that astroglia may contribute strongly to the abnormal cholinergic function in the mouse Ts16 model for Down syndrome. The Ts16 glia could lack a cholinergic supporting factor present in normal glia or contain a factor that down-regulates cholinergic function.

Mice transgenic for human CD4 and CCR5 are susceptible to HIV infection

HIV entry into human cells is mediated by CD4 acting in concert with one of several members of the chemokine receptor superfamily. The resistance to HIV infection observed in individuals with defective CCR5 alleles indicated that this particular chemokine receptor plays a crucial role in the initiation of in vivo HIV infection. Expression of human CD4 transgene does not render mice susceptible to HIV infection because of structural differences between human and mouse CCR5. To ascertain whether expression of human CD4 and CCR5 is sufficient to make murine T lymphocytes susceptible to HIV infection, the lck promoter was used to direct the T cell-specific expression of human CD4 and CCR5 in transgenic mice. Peripheral blood mononuclear cells and splenocytes isolated from these mice expressed human CD4 and CCR5 and were infectible with selected M-tropic HIV isolates. After in vivo inoculation, HIV-infected cells were detected by DNA PCR in the spleen and lymph nodes of these transgenic mice, but HIV could not be cultured from these cells. This indicated that although transgenic expression of human CD4 and CCR5 permitted entry of HIV into the mouse cells, significant HIV infection was prevented by other blocks to HIV replication present in mouse cells. In addition to providing in vivo verification for the important role of CCR5 in T lymphocyte HIV infection, these transgenic mice represent a new in vivo model for understanding HIV pathogenesis by delineating species-specific cellular factors required for productive in vivo HIV infection. These mice should also prove useful for the assessment of potential therapeutic and preventative modalities, particularly vaccines.

Modulation of Cell Proliferation and Differentiation through Substrate-dependent Changes in Fibronectin Conformation

Integrin-mediated cell adhesion to extracellular matrices provides signals essential for cell cycle progression and differentiation. We demonstrate that substrate-dependent changes in the conformation of adsorbed fibronectin (Fn) modulated integrin binding and controlled switching between proliferation and differentiation. Adsorption of Fn onto bacterial polystyrene (B), tissue culture polystyrene (T), and collagen (C) resulted in differences in Fn conformation as indicated by antibody binding. Using a biochemical method to quantify bound integrins in cultured cells, we found that differences in Fn conformation altered the quantity of bound α5 and β1 integrin subunits but not αv or β3. C2C12 myoblasts grown on these Fn-coated substrates proliferated to different levels (B > T > C). Immunostaining for muscle-specific myosin revealed minimal differentiation on B, significant levels on T, and extensive differentiation on C. Differentiation required binding to the RGD cell binding site in Fn and was blocked by antibodies specific for this site. Switching between proliferation and differentiation was controlled by the levels of α5β1 integrin bound to Fn, and differentiation was inhibited by anti-α5, but not anti-αv, antibodies, suggesting distinct integrin-mediated signaling pathways. Control of cell proliferation and differentiation through conformational changes in extracellular matrix proteins represents a versatile mechanism to elicit specific cellular responses for biological and biotechnological applications.

A Specific Light Chain of Kinesin Associates with Mitochondria in Cultured Cells

The motor protein kinesin is implicated in the intracellular transport of organelles along microtubules. Kinesin light chains (KLCs) have been suggested to mediate the selective binding of kinesin to its cargo. To test this hypothesis, we isolated KLC cDNA clones from a CHO-K1 expression library. Using sequence analysis, they were found to encode five distinct isoforms of KLCs. The primary region of variability lies at the carboxyl termini, which were identical or highly homologous to carboxyl-terminal regions of rat KLC B and C, human KLCs, sea urchin KLC isoforms 1–3, and squid KLCs. To examine whether the KLC isoforms associate with different cytoplasmic organelles, we made an antibody specific for a 10-amino acid sequence unique to B and C isoforms. In an indirect immunofluorescence assay, this antibody specifically labeled mitochondria in cultured CV-1 cells and human skin fibroblasts. On Western blots of total cell homogenates, it recognized a single KLC isoform, which copurified with mitochondria. Taken together, these data indicate a specific association of a particular KLC (B type) with mitochondria, revealing that different KLC isoforms can target kinesin to different cargoes.

Electric Field–directed Cell Motility Involves Up-regulated Expression and Asymmetric Redistribution of the Epidermal Growth Factor Receptors and Is Enhanced by Fibronectin and Laminin

Wounding corneal epithelium establishes a laterally oriented, DC electric field (EF). Corneal epithelial cells (CECs) cultured in similar physiological EFs migrate cathodally, but this requires serum growth factors. Migration depends also on the substrate. On fibronectin (FN) or laminin (LAM) substrates in EF, cells migrated faster and more directly cathodally. This also was serum dependent. Epidermal growth factor (EGF) restored cathodal-directed migration in serum-free medium. Therefore, the hypothesis that EGF is a serum constituent underlying both field-directed migration and enhanced migration on ECM molecules was tested. We used immunofluorescence, flow cytometry, and confocal microscopy and report that 1) EF exposure up-regulated the EGF receptor (EGFR); so also did growing cells on substrates of FN or LAM; and 2) EGFRs and actin accumulated in the cathodal-directed half of CECs, within 10 min in EF. The cathodal asymmetry of EGFR and actin staining was correlated, being most marked at the cell–substrate interface and showing similar patterns of asymmetry at various levels through a cell. At the cell–substrate interface, EGFRs and actin frequently colocalized as interdigitated, punctate spots resembling tank tracks. Cathodal accumulation of EGFR and actin did not occur in the absence of serum but were restored by adding ligand to serum-free medium. Inhibition of MAPK, one second messenger engaged by EGF, significantly reduced EF-directed cell migration. Transforming growth factor β and fibroblast growth factor also restored cathodal-directed cell migration in serum-free medium. However, longer EF exposure was needed to show clear asymmetric distribution of the receptors for transforming growth factor β and fibroblast growth factor. We propose that up-regulated expression and redistribution of EGFRs underlie cathodal-directed migration of CECs and directed migration induced by EF on FN and LAM.

An Apical-Type Trafficking Pathway Is Present in Cultured Oligodendrocytes but the Sphingolipid-enriched Myelin Membrane Is the Target of a Basolateral-Type Pathway

Myelin sheets originate from distinct areas at the oligodendrocyte (OLG) plasma membrane and, as opposed to the latter, myelin membranes are relatively enriched in glycosphingolipids and cholesterol. The OLG plasma membrane can therefore be considered to consist of different membrane domains, as in polarized cells; the myelin sheet is reminiscent of an apical membrane domain and the OLG plasma membrane resembles the basolateral membrane. To reveal the potentially polarized membrane nature of OLG, the trafficking and sorting of two typical markers for apical and basolateral membranes, the viral proteins influenza virus–hemagglutinin (HA) and vesicular stomatitis virus–G protein (VSVG), respectively, were examined. We demonstrate that in OLG, HA and VSVG are differently sorted, which presumably occurs upon their trafficking through the Golgi. HA can be recovered in a Triton X-100-insoluble fraction, indicating an apical raft type of trafficking, whereas VSVG was only present in a Triton X-100-soluble fraction, consistent with its basolateral sorting. Hence, both an apical and a basolateral sorting mechanism appear to operate in OLG. Surprisingly, however, VSVG was found within the myelin sheets surrounding the cells, whereas HA was excluded from this domain. Therefore, despite its raft-like transport, HA does not reach a membrane that shows features typical of an apical membrane. This finding indicates either the uniqueness of the myelin membrane or the requirement of additional regulatory factors, absent in OLG, for apical delivery. These remarkable results emphasize that polarity and regulation of membrane transport in cultured OLG display features that are quite different from those in polarized cells.

Reprogramming the Cell Cycle for Endoreduplication in Rodent Trophoblast Cells

Differentiation of trophoblast giant cells in the rodent placenta is accompanied by exit from the mitotic cell cycle and onset of endoreduplication. Commitment to giant cell differentiation is under developmental control, involving down-regulation of Id1 and Id2, concomitant with up-regulation of the basic helix-loop-helix factor Hxt and acquisition of increased adhesiveness. Endoreduplication disrupts the alternation of DNA synthesis and mitosis that maintains euploid DNA content during proliferation. To determine how the mammalian endocycle is regulated, we examined the expression of the cyclins and cyclin-dependent kinases during the transition from replication to endoreduplication in the Rcho-1 rat choriocarcinoma cell line. We cultured these cells under conditions that gave relatively synchronous endoreduplication. This allowed us to study the events that occur during the transition from the mitotic cycle to the first endocycle. With giant cell differentiation, the cells switched cyclin D isoform expression from D3 to D1 and altered several checkpoint functions, acquiring a relative insensitivity to DNA-damaging agents and a coincident serum independence. The initiation of S phase during endocycles appeared to involve cycles of synthesis of cyclins E and A, and termination of S was associated with abrupt loss of cyclin A and E. Both cyclins were absent from gap phase cells, suggesting that their degradation may be necessary to allow reinitiation of the endocycle. The arrest of the mitotic cycle at the onset of endoreduplication was associated with a failure to assemble cyclin B/p34cdk1 complexes during the first endocycle. In subsequent endocycles, cyclin B expression was suppressed. Together these data suggest several points at which cell cycle regulation could be targeted to shift cells from a mitotic to an endoreduplicative cycle.

Fibronectin Regulates Assembly of Actin Filaments and Focal Contacts in Cultured Cells via the Heparin-binding Site in Repeat III13

Fibroblasts, when plated on the extracellular matrix protein fibronectin (FN), rapidly spread and form an organized actin cytoskeleton. This process is known to involve both the central α5β1 integrin-binding and the C-terminal heparin-binding regions of FN. We found that within the heparin-binding region, the information necessary for inducing organization of stress fibers and focal contacts was located in a 29–amino acid segment of FN type III module 13 (III13). We did not find a cytoskeleton-organizing role for repeat III14, which had previously been implicated in this process. Within III13, the same five basic amino acids known to be most important for heparin binding were also necessary for actin organization. A substrate of III13 alone was only weakly adhesive but strongly induced formation of filopodia and lamellipodia. Stress fiber formation required a combination of III13 and III7–11 (which contains the integrin α5β1 recognition site), either as a single fusion protein or as separate polypeptides, and the relative amounts of the two binding sites appeared to determine whether stress fibers or filopodia and lamellipodia were the predominant actin structures formed. We propose that a balance of signals from III13 and from integrins regulates the type of actin structures assembled by the cell.

Inhibition of Chromosomal Separation Provides Insights into Cleavage Furrow Stimulation in Cultured Epithelial Cells

While astral microtubules are believed to be primarily responsible for the stimulation of cytokinesis in Echinoderm embryos, it has been suggested that a signal emanating from the chromosomal region and mediated by the interzonal microtubules stimulates cytokinesis in cultured mammalian cells. To test this hypothesis, we examined cytokinesis in normal rat kidney cells treated with an inhibitor of topoisomerase II, (+)-1,2-bis(3,5-dioxopiperaz-inyl-1-yl)propane, which prevents the separation of sister chromatids and the formation of a spindle interzone. The majority of treated cells showed various degrees of abnormality in cytokinesis. Furrows frequently deviated from the equatorial plane, twisting daughter cells into irregular shapes. Some cells developed furrows in regions outside the equator or far away from the spindle. In addition, F-actin and myosin II accumulated at the lateral ingressing margins but did not form a continuous band along the equator as in control cells. Imaging of microinjected 5- (and 6-) carboxymtetramethylrhodamine-tubulin revealed that a unique set of microtubules projected out from the chromosomal vicinity upon anaphase onset. These microtubules emanated toward the lateral cortex, where they delineated sites of microtubule bundle formation, cortical ingression, and F-actin and myosin II accumulation. As centrosome integrity and astral microtubules appeared unperturbed by (+)-1,2-bis(3,5-dioxopiperaz-inyl-1-yl)propane treatment, the present observations cannot be easily explained by the conventional model involving astral microtubules. We suggest that in cultured epithelial cells the organization of the chromosomes dictates the organization of midzone microtubules, which in turn determines and maintains the cleavage activity.

Morphogenetic Effects of Neuregulin (Neu Differentiation Factor) in Cultured Epithelial Cells

Neuregulin, or neu differentiation factor, induces cell proliferation or differentiation through interaction with members of the ErbB family of receptor tyrosine kinases. We report that neuregulin can also induce profound morphogenic responses in cultured epithelial cells of different origins. These effects include scattering of small epithelial islands and rearrangement of larger cell islands into ordered ring-shaped arrays with internal lumens. The ring-forming cells are interconnected by cadherin- and β-catenin-containing adherens junctions. In confluent cultures, neuregulin treatment induces formation of circular lumenlike gaps in the monolayer. Both cell scattering and ring formation are accompanied by a marked increase in cell motility that is independent of hepatocyte growth factor/scatter factor and its receptor (c-Met). Affinity-labeling experiments implied that a combination of ErbB-2 with ErbB-3 mediates the morphogenic signal of neuregulin in gastric cells. Indeed, a similar morphogenic effect could be reconstituted in nonresponsive cells by coexpression of ErbB-2 and -3. We conclude that a heterodimer between the kinase-defective neuregulin receptor, ErbB-3, and the coreceptor, ErbB-2, mediates the morphogenetic action of neuregulin.

Gradual Phenotypic Conversion Associated with Immortalization of Cultured Human Mammary Epithelial Cells

Examination of the process of immortal transformation in early passages of two human mammary epithelial cell (HMEC) lines suggests the involvement of an epigenetic step. These lines, 184A1 and 184B5, arose after in vitro exposure of finite lifespan 184 HMEC to a chemical carcinogen, and both are clonally derived. Although early-passage mass cultures of 184A1 and 184B5 maintained continuous slow growth, most individual cells lost proliferative ability. Uniform good growth did not occur until 20–30 passages after the lines first appeared. Early-passage cultures expressed little or no telomerase activity and telomeres continued to shorten with increasing passage. Telomerase activity was first detected when the telomeres became critically short, and activity levels gradually increased thereafter. Early-passage cultures had little or no ability to maintain growth in transforming growth factor-β (TGFβ); however, both mass cultures and clonal isolates showed a very gradual increase in the number of cells displaying progressively increased ability to maintain growth in TGFβ. A strong correlation between capacity to maintain growth in the presence of TGFβ and expression of telomerase activity was observed. We have used the term “conversion” to describe this process of gradual acquisition of increased growth capacity in the absence or presence of TGFβ and reactivation of telomerase. We speculate that the development of extremely short telomeres may result in gradual, epigenetic-based changes in gene expression. Understanding the underlying mechanisms of HMEC conversion in vitro may provide new insight into the process of carcinogenic progression in vivo and offer novel modes for therapeutic intervention.

Initiation of Assembly of the Cell Envelope Barrier Structure of Stratified Squamous Epithelia

The cell envelope (CE) is a specialized structure that is important for barrier function in terminally differentiated stratified squamous epithelia. The CE is formed inside the plasma membrane and becomes insoluble as a result of cross-linking of constituent proteins by isopeptide bonds formed by transglutaminases. To investigate the earliest stages of assembly of the CE, we have studied human epidermal keratinocytes induced to terminally differentiate in submerged liquid culture as a model system for epithelia in general. CEs were harvested from 2-, 3-, 5-, or 7-d cultured cells and examined by 1) immunogold electron microscopy using antibodies to known CE or other junctional proteins and 2) amino acid sequencing of cross-linked peptides derived by proteolysis of CEs. Our data document that CE assembly is initiated along the plasma membrane between desmosomes by head-to-tail and head-to-head cross-linking of involucrin to itself and to envoplakin and perhaps periplakin. Essentially only one lysine and two glutamine residues of involucrin and two glutamines of envoplakin were used initially. In CEs of 3-d cultured cells, involucrin, envoplakin, and small proline-rich proteins were physically located at desmosomes and had become cross-linked to desmoplakin, and in 5-d CEs, these three proteins had formed a continuous layer extending uniformly along the cell periphery. By this time >15 residues of involucrin were used for cross-linking. The CEs of 7-d cells contain significant amounts of the protein loricrin, typically expressed at a later stage of CE assembly. Together, these data stress the importance of juxtaposition of membranes, transglutaminases, and involucrin and envoplakin in the initiation of CE assembly of stratified squamous epithelia.