Acetylcholine analogue stimulates DNA synthesis in brain-derived cells via specific muscarinic receptor subtypes

Little is known about the factors which regulate the growth and development of the mammalian brain. Although proliferation of neuronal cells ceases relatively early in development, certain types of glial cells proliferate and differentiate mainly perinatally. In the perinatal period, the ability of acetylcholine to stimulate phosphoinositide (PI) hydrolysis in brain reaches peak levels, and indeed the stable acetylcholine analogue carbachol can stimulate PI hydrolysis of primary neonatal astroglial cells. As PI hydrolysis is thought to be important in the regulation of cell proliferation, we investigated whether cellular DNA synthesis can be induced by carbachol. Our results show that carbachol stimulates DNA synthesis via muscarinic acetylcholine receptors (mAChRs), in primary astrocytes derived from perinatal rat brain, in an age-dependent fashion. Carbachol is also mitogenic in certain brain-derived astrocytoma and neuroblastoma cell lines, as well as in chinese hamster ovary (CHO) cells expressing recombinant muscarinic receptors. DNA synthesis is strongly activated by carbachol in those brain-derived cell lines and transfected CHO cells that express mAChR subtypes which activate PI hydrolysis efficiently, and poorly activated in cells expressing mAChR subtypes which only weakly activate PI hydrolysis. These results strongly support a role for acetylcholine in regulating astroglial cell growth in the developing brain, and indicate that the specificity of acetylcholine-induced cell proliferation may be determined by the expression of those mAChR subtypes which activate PI hydrolysis.     

Self-renewal and expansion of single transplanted muscle stem cells

Adult muscle satellite cells have a principal role in postnatal skeletal muscle growth and regeneration. Satellite cells reside as quiescent cells underneath the basal lamina that surrounds muscle fibres and respond to damage by giving rise to transient amplifying cells (progenitors) and myoblasts that fuse with myofibres. Recent experiments showed that, in contrast to cultured myoblasts, satellite cells freshly isolated or satellite cells derived from the transplantation of one intact myofibre contribute robustly to muscle repair. However, because satellite cells are known to be heterogeneous, clonal analysis is required to demonstrate stem cell function. Here we show that when a single luciferase-expressing muscle stem cell is transplanted into the muscle of mice it is capable of extensive proliferation, contributes to muscle fibres, and Pax7(+)luciferase(+) mononucleated cells can be readily re-isolated, providing evidence of muscle stem cell self-renewal. In addition, we show using in vivo bioluminescence imaging that the dynamics of muscle stem cell behaviour during muscle repair can be followed in a manner not possible using traditional retrospective histological analyses. By imaging luciferase activity, real-time quantitative and kinetic analyses show that donor-derived muscle stem cells proliferate and engraft rapidly after injection until homeostasis is reached. On injury, donor-derived mononucleated cells generate massive waves of cell proliferation. Together, these results show that the progeny of a single luciferase-expressing muscle stem cell can both self-renew and differentiate after transplantation in mice, providing new evidence at the clonal level that self-renewal is an autonomous property of a single adult muscle stem cell.     

PPARγ罗格列酮对HT-29的生长抑制作用

PPARγ配体在抗肿瘤中发挥着重要的作用,这些配体具有抗肿瘤、诱导分化和抗血管生成等效果.目的主要是评估PPARγ激动剂罗格列酮对结肠癌细胞株HT-29生长的抑制作用.结果显示,罗格列酮可以有效地抑制体外培养的结肠癌细胞株HT-29的生长及克隆形成,并能抑制HT-29裸鼠移植瘤的生长.与此同时,罗格列酮能够升高PPARγ, p- PPARγ的表达水平.以上结果初步证明罗格列酮在体内外均可抑制结肠癌细胞株HT-29的成长,提示罗格列酮有可能发展成为治疗结肠癌的有效药物.     

Histone H2AX-dependent GABA(A) receptor regulation of stem cell proliferation

Stem cell self-renewal implies proliferation under continued maintenance of multipotency. Small changes in numbers of stem cells may lead to large differences in differentiated cell numbers, resulting in significant physiological consequences. Proliferation is typically regulated in the G1 phase, which is associated with differentiation and cell cycle arrest. However, embryonic stem (ES) cells may lack a G1 checkpoint. Regulation of proliferation in the 'DNA damage' S/G2 cell cycle checkpoint pathway is known for its role in the maintenance of chromatin structural integrity. Here we show that autocrine/paracrine gamma-aminobutyric acid (GABA) signalling by means of GABA(A) receptors negatively controls ES cell and peripheral neural crest stem (NCS) cell proliferation, preimplantation embryonic growth and proliferation in the boundary-cap stem cell niche, resulting in an attenuation of neuronal progenies from this stem cell niche. Activation of GABA(A) receptors leads to hyperpolarization, increased cell volume and accumulation of stem cells in S phase, thereby causing a rapid decrease in cell proliferation. GABA(A) receptors signal through S-phase checkpoint kinases of the phosphatidylinositol-3-OH kinase-related kinase family and the histone variant H2AX. This signalling pathway critically regulates proliferation independently of differentiation, apoptosis and overt damage to DNA. These results indicate the presence of a fundamentally different mechanism of proliferation control in these stem cells, in comparison with most somatic cells, involving proteins in the DNA damage checkpoint pathway.     

Point mutation in FGF receptor eliminates phosphatidylinositol hydrolysis without affecting mitogenesis.

Stimulation of growth factor receptors with tyrosine kinase activity is followed by rapid receptor dimerization, tyrosine autophosphorylation and phosphorylation of signalling molecules such as phospholipase C gamma (PLC gamma) and the ras GTPase-activating protein. PLC gamma and GTPase-activating protein bind to specific tyrosine-phosphorylated regions in growth factor receptors through their src-homologous SH2 domains. Growth factor-induced tyrosine phosphorylation of PLC gamma is essential for stimulation of phosphatidylinositol hydrolysis in vitro and in vivo. We have shown that a short phosphorylated peptide containing tyrosine at position 766 from a conserved region of the fibroblast growth factor (FGF) receptor is a binding site for the SH2 domain of PLC gamma (ref. 8). Here we show that an FGF receptor point mutant in which Tyr 766 is replaced by a phenylalanine residue (Y766F) is unable to associate with and tyrosine-phosphorylate PLC gamma or to stimulate hydrolysis of phosphatidylinositol. Nevertheless, the Y766F FGF receptor mutant can be autophosphorylated, and can phosphorylate several cellular proteins and stimulate DNA synthesis. Our data show that phosphorylation of the conserved Tyr 766 of the FGF receptor is essential for phosphorylation of PLC gamma and for hydrolysis of phosphatidylinositol, but that elimination of this hydrolysis does not affect FGF-induced mitogenesis.     

Capillary endothelial cells express basic fibroblast growth factor, a mitogen that promotes their own growth

Angiogenesis, the formation of new capillaries, which is observed in embryonic and injured tissue and is particularly prominent in the vicinity of solid tumours, involves the migration and proliferation of capillary endothelial cells. It is probably triggered by agents, such as basic fibroblast growth factor (bFGF), thought to be released from tissues adjacent to proliferating capillaries. As well as being a potent inducer of cell division in capillary endothelial cells in vitro, bFGF can act as an angiogenic agent in vivo. It is present in a wide variety of richly vascularized tissues including brain, pituitary, retina, adrenal gland, kidney, corpus luteum, placenta and various tumours. So far, however, the normal bFGF-producing cell species in these tissues have not been identified. We report here that capillary endothelial cells express the bFGF gene, that they produce and release bFGF and that bFGF derived from them can stimulate the proliferation of capillary endothelial cells. We conclude that bFGF can act as a self-stimulating growth factor for capillary endothelial cells, and that it is possible that the formation of new capillaries is induced by capillary endothelial cells themselves.     

Stimulation of multipotential, erythroid and other murine haematopoietic progenitor cells by adherent cell lines in the absence of detectable multi-CSF (IL-3)

It is well established that murine multipotential and committed erythroid progenitor cells require the presence of a glycoprotein, termed multi-CSF (multi-colony-stimulating factor, IL-3) for clonal proliferation and differentiation in vitro. The initial proliferation of these cells can also be stimulated by two other glycoproteins, granulocyte-macrophage CSF (GM-CSF) and granulocyte CSF (G-CSF), although continued proliferation and differentiation requires the subsequent presence of multi-CSF. Here we report the stimulation of multipotential, erythroid and other haematopoietic progenitor cells by a number of adherent cell lines including a cloned bone marrow cell line (B.Ad). The positive cell lines, as feeder layers, exhibit colony-stimulating, erythropoietin-like and burst-promoting (BPA) activities. Optimal erythropoietic stimulation by the B.Ad line requires close cell-cell contact. The cell lines also support the in vitro clonal growth of multipotential colony-forming cells and progenitors of six other haematopoietic lineages. The biological activities observed seem not to be mediated by known multipotential or erythroid colony-stimulating factors (multi-CSF, IL-3, MCGF, HCGF, PSF, BPA).     

Structure, expression and function of a schwannoma-derived growth factor

During the development of the nervous system, cells require growth factors that regulate their division and survival. To identify new growth factors, serum-free growth-conditioned media from many clonal cell lines were screened for the presence of mitogens for central nervous system glial cells. A cell line secreting a potent glial mitogen was established from a tumour (or 'schwannoma') derived from the sheath of the sciatic nerve. The cells of the tumour, named JS1 cells, were adapted to clonal culture and identified as Schwann cells. Schwann cells secrete an autocrine mitogen and human schwannoma extracts have mitogenic activity on glial cells. Until now, neither mitogen has been purified. Here we report the purification and characterization of a mitogenic molecule, designated schwannoma-derived growth factor (SDGF), from the growth-conditioned medium of the JS1 Schwann cell line. SDGF belongs to the epidermal growth factor family, and is an autocrine growth factor as well as a mitogen for astrocytes, Schwann cells and fibroblasts.     

Bmi1 is essential for cerebellar development and is overexpressed in human medulloblastomas

Overexpression of the polycomb group gene Bmi1 promotes cell proliferation and induces leukaemia through repression of Cdkn2a (also known as ink4a/Arf) tumour suppressors. Conversely, loss of Bmi1 leads to haematological defects and severe progressive neurological abnormalities in which de-repression of the ink4a/Arf locus is critically implicated. Here, we show that Bmi1 is strongly expressed in proliferating cerebellar precursor cells in mice and humans. Using Bmi1-null mice we demonstrate a crucial role for Bmi1 in clonal expansion of granule cell precursors both in vivo and in vitro. Deregulated proliferation of these progenitor cells, by activation of the sonic hedgehog (Shh) pathway, leads to medulloblastoma development. We also demonstrate linked overexpression of BMI1 and patched (PTCH), suggestive of SHH pathway activation, in a substantial fraction of primary human medulloblastomas. Together with the rapid induction of Bmi1 expression on addition of Shh or on overexpression of the Shh target Gli1 in cerebellar granule cell cultures, these findings implicate BMI1 overexpression as an alternative or additive mechanism in the pathogenesis of medulloblastomas, and highlight a role for Bmi1-containing polycomb complexes in proliferation of cerebellar precursor cells.     

Negative regulation of lymphocyte activation and autoimmunity by the molecular adaptor Cbl-b

The signalling thresholds of antigen receptors and co-stimulatory receptors determine immunity or tolerance to self molecules. Changes in co-stimulatory pathways can lead to enhanced activation of lymphocytes and autoimmunity, or the induction of clonal anergy. The molecular mechanisms that maintain immunotolerance in vivo and integrate co-stimulatory signals with antigen receptor signals in T and B lymphocytes are poorly understood. Members of the Cbl/Sli family of molecular adaptors function downstream from growth factor and antigen receptors. Here we show that gene-targeted mice lacking the adaptor Cbl-b develop spontaneous autoimmunity characterized by auto-antibody production, infiltration of activated T and B lymphocytes into multiple organs, and parenchymal damage. Resting cbl-b(-/-) lymphocytes hyperproliferate upon antigen receptor stimulation, and cbl-b(-/-) T cells display specific hyperproduction of the T-cell growth factor interleukin-2, but not interferon-gamma or tumour necrosis factor-alpha. Mutation of Cbl-b uncouples T-cell proliferation, interleukin-2 production and phosphorylation of the GDP/GTP exchange factor Vav1 from the requirement for CD28 co-stimulation. Cbl-b is thus a key regulator of activation thresholds in mature lymphocytes and immunological tolerance and autoimmunity.     

Murine leukaemogenesis: monoclonal antibodies to T-cell determinants arrest T-lymphoma cell proliferation

We have proposed a receptor-mediated leukaemogenesis hypothesis wherein T lymphomas would be clones of T cells bearing mitogen-linked surface receptors specific for the envelope determinants of the inducing MuLV. A prediction of the hypothesis is that T-lymphoma proliferation is dependent on continued presentation of MuLV envelope determinants to these cell-surface receptors, and that substances which interfere with receptor-virus interactions should inhibit T-lymphoma proliferation. Rat monoclonal antibodies were raised to the AKR mouse T lymphoma KKT-2, and these antibodies were screened independently for blockade of virus-binding and for cytostatic activity on KKT-2 cells. We report here that those monoclonal antibodies which block virus binding inhibit growth of KKT-2 cells in vitro, whereas monoclonal antibodies which bind to these cells but do not block virus binding are not cytostatic. Three of the four cytostatic antibodies detect determinants on the Thy-1 molecule, while none of the other (noncytostatic) antibodies detect Thy-1. Antibody inhibition of KKT-2 cell growth is precluded by saturation of KKT-2 virus receptors with the inducing leukaemia virus.     

Autocrine growth factors and cancer

The ability of cancer cells to produce and to respond to their own growth factors (autocrine secretion) has become a central concept linking oncogene and growth factor research. Oncogenes confer growth factor autonomy on cells not only by coding directly for autocrine peptide growth factors or their receptors, but also by amplifying the mitogenic signals generated by a growth factor at its receptor. Antagonists of positive autocrine growth factors can inhibit growth of cancer cells in experimental animals. Recently identified negative autocrine growth factors might themselves control aberrant cell growth.     

Loss of heterozygosity of chromosome 3p markers in small-cell lung cancer

Specific chromosomal deletions sometimes associated with tumours such as retinoblastoma (chromosome 13q14) and Wilm's tumour (chromosome 11p13) have led to the hypothesis that recessive genes may be involved in tumorigenesis. This hypothesis is supported by demonstration of allele loss specific for these regions using polymorphic DNA markers and by the isolation of a complementary DNA clone for the retinoblastoma gene. A cytogenetic deletion in chromosome 3 (p14-p23) was reported in small-cell lung cancer (SCLC) by Whang-Peng et al. At least one homologue of chromosome 3 was affected in the majority of SCLC tumours; however, the multiple chromosomal changes seen presented the possibility that chromosome 3 was rearranged, not deleted. We used polymorphic DNA probes for chromosome 3p and compared tumour and constitutional genotypes of nine SCLC patients. Our data show loss of alleles of chromosome 3p markers in tumour DNA of all nine patients supporting the hypothesis that this region contributes to tumorigenesis in SCLC.     

Raf-1 protein kinase is required for growth of induced NIH/3T3 cells

Many growth factors regulate the cytoplasmic Raf-1 protein kinase, consistent with its having a central role in transduction of growth signals. The kinase is ubiquitously expressed and can promote proliferation, presumably in a manner dependent on growth-factor receptors and membrane-associated oncogenes. We have now examined the dependence of serum- and TPA (12-O-tetradecanoylphorbol-13-acetate)-regulated NIH/3T3 cell growth on RAF-1 kinase to determine whether Raf-1 is essential for receptor signalling. We inhibited Raf-1 function by expressing c-raf-1 antisense RNA or kinase-defective c-raf-1 mutants. Antisense RNA for c-raf-1 interferes with proliferation of normal NIH/3T3 cells and reverts raf-transformed cells. In revertant cells, DNA replication induced by serum or TPA was eliminated or reduced proportionately to the reduction in Raf protein levels. Expression of a kinase-defective Raf-1 mutant (craf301) or a regulatory domain fragment (HCR) inhibited serum-induced NIH/3T3-cell proliferation and raf transformation even more efficiently. Inhibition by antisense RNA or craf301 blocked proliferation and transformation by Ki- and Ha-ras oncogenes. We conclude that raf functions as an essential signal transducer downstream of serum growth factor receptors, protein kinase C and ras.     

A chemical approach to stem-cell biology and regenerative medicine

An improved understanding of stem-cell and regenerative biology, as well as a better control of stem-cell fate, is likely to produce treatments for many devastating diseases and injuries. Chemical approaches are starting to have an increasingly important role in this young field. Attention has focused on chemical approaches that allow the precise manipulation of cells in vitro to obtain homogeneous cell types for cell-based therapies. Another promising approach is the development of conventional chemical and biological therapeutics to stimulate endogenous cells to regenerate. Such therapeutics can act on target cells or their niches in vivo to promote cell survival, proliferation, differentiation, reprogramming and homing.     

Requirement of hormone for thermal conversion of the glucocorticoid receptor to a DNA-binding state

A central question arising from the model of eukaryotic gene regulation by steroid hormone receptors is whether or not proteins represent pre-existing gene regulatory proteins that are activated on exposure to the extracellular signal. It has been generally believed that the ligand-binding of steroid hormone receptors triggers an allosteric change in receptor structure, manifested by an increased affinity of the receptor for DNA in vitro and nuclear target elements in vivo, as monitored by nuclear translocation. But this model has been challenged by recent reports indicating that glucocorticoid and progesterone receptors bind specifically in vitro to target DNA sequences even in the absence of hormone. On the other hand, it appears that the hormone induces protection in vivo of the glucocorticoid response element of the tyrosine amino transferase gene. Here we show that under conditions permitting minimal in vitro manipulation, the steroid-free glucocorticoid receptor in crude cytosol associates with the hsp90 heat shock protein (relative molecular mass Mr approximately equal to 90,000) to form a large 300K complex, rather than the 94K liganded receptor monomer. More importantly, we have developed an assay to demonstrate the requirement of hormone to dissociate the 300K complex by heat treatment. Specific DNA-binding activity of the receptor becomes apparent in this process, showing that DNA binding occurs but is inhibited in the large heteromeric complex. We propose a model in which receptor function is repressed by association of the receptor with hsp90. Dissociation of this complex is induced by the binding of steroid and is apparently an irreversible process.     

聚苯乙烯薄膜三维培养肝细胞的实验研究

采用三维聚苯乙烯薄膜作为细胞培养支架,在体外进行大鼠肝细胞的三维培养,并与常规二维肝细胞培养进行比较.分析了细胞的生长状态及肝细胞功能,包括细胞存活率、白蛋白分泌功能、尿素合成功能及葡萄糖消耗功能.结果显示:三维细胞增殖活力均始终明显高于二维;在显微镜下观察,可看到三维支架表面黏附生长的肝细胞逐渐增多,有肝细胞黏附成团,且肝细胞保持着良好的形态学结构.研究表明,三维培养的肝细胞其增殖生长和代谢功能都优于同样条件下的二维培养,三维聚苯乙烯薄膜可以作为体外肝细胞培养支架,在生物人工肝培养体系中具有应用前景.     

Cytokine-induced pseudopodial protrusion is coupled to tumour cell migration

Pseudopodia protrusion is a prominent feature of actively motile cells in vitro and invading tumour cells in vivo; however, the function and regulation of pseudopodia are poorly understood. Tumour autocrine motility factor (AMF) represents a new class of cytokines which are secreted by tumour cells and embryonic cells and induce random motility in the producer cells or in heterologous cells with appropriate receptors. Here we report that a major effect of this factor is to induce the extension of cell pseudopodia before cell translocation. Using a new method to quantify and isolate pseudopodia, we find that human breast carcinoma cell AMF (at concentrations of 1 nM or below) stimulates random pseudopodia formation in a dose-dependent and time-dependent manner. Anti-AMF antibodies inhibit pseudopodia protrusion and cell motility, showing the importance of pseudopodia formation during locomotion. AMF-stimulated motility and pseudopodia formation occur on a wide variety of adhesive substrata which suggests that certain intrinsic motility events are independent of the attachment mechanism. Induced pseudopodia show a prominent axial actin network in the electron microscope. The number of laminin receptor and fibronectin RGD recognition sites is increased by a factor of 20 in the induced pseudopodia when compared to the average distribution in unstimulated cells. Exploratory pseudopodia regulated by cell-derived motility factors contain receptors for matrix proteins and could serve as 'senseorgans' essential to the process of cell locomotion.