Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas

Although cancer arises from a combination of mutations in oncogenes and tumour suppressor genes, the extent to which tumour suppressor gene loss is required for maintaining established tumours is poorly understood. p53 is an important tumour suppressor that acts to restrict proliferation in response to DNA damage or deregulation of mitogenic oncogenes, by leading to the induction of various cell cycle checkpoints, apoptosis or cellular senescence. Consequently, p53 mutations increase cell proliferation and survival, and in some settings promote genomic instability and resistance to certain chemotherapies. To determine the consequences of reactivating the p53 pathway in tumours, we used RNA interference (RNAi) to conditionally regulate endogenous p53 expression in a mosaic mouse model of liver carcinoma. We show that even brief reactivation of endogenous p53 in p53-deficient tumours can produce complete tumour regressions. The primary response to p53 was not apoptosis, but instead involved the induction of a cellular senescence program that was associated with differentiation and the upregulation of inflammatory cytokines. This program, although producing only cell cycle arrest in vitro, also triggered an innate immune response that targeted the tumour cells in vivo, thereby contributing to tumour clearance. Our study indicates that p53 loss can be required for the maintenance of aggressive carcinomas, and illustrates how the cellular senescence program can act together with the innate immune system to potently limit tumour growth.     

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).     

Abrogation of IL-3 and IL-2 dependence by recombinant murine retroviruses expressing v-myc oncogenes

Several oncogenes are thought to cause transformation by affecting the signal transmission pathway of growth factors. One example is the induction of c-myc, the cellular homologue of the avian transforming oncogene v-myc, by platelet-derived growth factor (PDGF) among a set of genes associated with competence induction in fibroblasts. Another of the competence genes, r-fos, has been shown to be related to v-fos, the transforming gene of the FBJ sarcoma virus. In addition, PDGF induces c-fos, the cellular homologue of v-fos. The importance of c-myc induction is suggested by the observation that c-myc, under the control of a glucocorticoid regulator, can partially relieve the requirement of fibroblasts for PDGF. We have examined the effects of oncogenes on haematopoietic/lymphoid cell differentiation, immortalization and factor dependence for growth. Here we report the effects of recombinant murine retroviruses capable of expressing the avian v-myc. With interleukin-3 (IL-3)- or interleukin-2 (IL-2)-dependent cells, the viruses abrogated the requirement for growth factors and suppressed c-myc expression.     

Retinoids specifically enhance the number of epidermal growth factor receptors

Retinoids elicit many biological and biochemical responses from cells in vitro. One widely used criterion for the responsiveness of cells to retinoids is inhibition of growth; retinoids reduce the saturation density and/or growth rate of many normal and tumorigenic cell lines. Propagation of eukaryotic cells has been demonstrated to be dependent on the presence of macromolecular growth factors such as epidermal growth factor (EGF), which can stimulate proliferation of epithelial and fibroblastic cell lines. We now describe the effect of retinoids on the binding of EGF to its receptor. Retinoic acid enhances binding of 125I-labelled EGF to various fibroblastic and epidermal cell lines. It has no marked effect on the affinity of this growth factor for its receptor, but increases the number of EGF receptor sites. Retinoic acid has little effect on the binding of concanavalin A (Con A) and insulin, indicating the specific nature of the action of retinoids on cell-surface glycoproteins. Treatment of cells with the phorbol ester 12-o-tetradecanoyl phorbol-13-acetate (TPA) and retinoic acid shows poor antagonism between these compounds on EGF binding. It has been previously shown that retinoids induce or stimulate differentiation of embryonal carcinoma cells. EGF binding can be used as a marker to monitor differentiation of these cells.     

The Hardy-Zuckerman 2-FeSV, a new feline retrovirus with oncogene homology to Abelson-MuLV

There is substantial evidence that oncogenes (v-onc) of acute transforming retroviruses have been acquired by transduction of cellular genes (c-onc) with retroviruses. Feline leukaemia virus (FeLV)-associated feline fibrosarcomas have proven to be extremely useful for the isolation of acute transforming retroviruses of a mammalian species. Three different v-onc genes have been identified in five acute transforming feline retroviruses. The Susan McDonough feline sarcoma virus (SMFeSV) contains the oncogene fms (ref. 4). The Snyder-Theilen (ST) and Gardner-Arnstein (GA) FeSVs contain the oncogene fes (ref. 4), which is homologous to the oncogene fps of the avian sarcoma viruses FSV, RRCII, PRCIV and 16L (refs 7, 8). The v-onc sequences of the Parodi-Irgens (PI) FeSV have recently been found to be homologous with the v-sis sequences of the simian sarcoma virus. We report here the isolation of another acute transforming feline retrovirus from a naturally occurring feline fibrosarcoma, designated the Hardy-Zuckerman 2 feline sarcoma virus (HZ2-FeSV) and demonstrate that the HZ2-FeSV and Abelson murine leukaemia virus (A-MuLV) have homologous oncogenes.     

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.     

Haemonectin, a bone marrow adhesion protein specific for cells of granulocyte lineage

There is substantial evidence that the haematopoietic microenvironment is crucial to the growth and differentiation of haematopoietic cells. This microenvironment is composed of stromal cells, soluble factors and extracellular matrix (ECM). We have shown that a complex extract of bone marrow ECM can stimulate the growth and differentiation of haematopoietic cells in vitro. Furthermore, the use of inhibitors or stimulators of ECM synthesis in long-term marrow culture affects cell proliferation. On a molecular level, however, the interactions between ECM and haematopoietic cells are not well understood. We have investigated the adhesion between specific bone marrow ECM components and haematopoietic cells, and found a protein, 'haemonectin', of relative molecular mass 60,000 in bone marrow ECM which is a lineage- and organ-specific attachment molecule for cells of granulocyte lineage. This specificity distinguishes haemonectin from previously described adhesion proteins which have a wider tissue distribution and cell type specificity.     

Induction by E1A oncogene expression of cellular susceptibility to lysis by TNF

Tumour necrosis factor alpha (ref. 1), synthesized primarily by monocytes in response to various invasive agents, induces a wide variety of biological effects relevant to regulating cell growth and differentiation, including the selective killing of some tumour cells and the growth stimulation of some normal fibroblasts. As tumour necrosis factor (TNF) appears to kill tumour cells preferentially, we asked whether TNF sensitivity correlates with the expression of specific oncogene(s). If so, by examining the cellular target(s) of the oncogene product, it might be possible to identify specific factor(s) which mediate TNF action. By using an in vitro cytotoxicity assay with NIH 3T3 and Fisher BRK-derived cells expressing exogenously introduced oncogenes, we found that adenovirus E1A proteins induce susceptibility to TNF killing.     

Granulocyte- and granulocyte-macrophage-colony stimulating factors induce human endothelial cells to migrate and proliferate

Granulocyte-colony stimulating factor (G-CSF) and granulocyte-macrophage-colony stimulating factor (GM-CSF) belong to a family of glycoprotidic growth factors required for the survival, growth and differentiation of haematopoietic precursors and which affect the function of circulating mature cells. They are produced by resting or stimulated stromal cells of the haematopoietic microenvironment (fibroblasts and endothelium) and by immunocompetent cells (T cells and monocytes/macrophages). The action of these CSF molecules was thought to be restricted to cells of haematopoietic origin. Here, we report that G-CSF and GM-CSF influence the migration and proliferation of human endothelial cells suggesting that these molecules may act as regulatory signals outside the haematopoietic system.     

Mature murine B lymphocytes immortalized by Kirsten sarcoma virus

Clonal, antigen-specific, functionally responsive cell populations have proved critical for the analysis of the activation and regulation of lymphocytes. Such studies with B lymphocytes, the precursors of antibody-secreting cells, are hampered by the difficulty in generating phenotypically mature, antigen-reactive lines from defined cell populations. One method is to use acutely transforming retroviruses, which can transform B-lineage lymphocytes in vitro. However, Abelson murine leukaemia virus (A-MuLV) infection of murine bone marrow cells in vitro yields mostly immature B-cell lines, and infection of murine bone marrow cells with murine sarcoma viruses carrying ras related genes produces only immature lymphoid cell lines. Retroviruses which contain ras can immortalize nonlymphoid cells without causing loss of mature phenotypic characteristics. We used ras-containing Kirsten sarcoma virus (KiSV) pseudotyped with an amphotropic MuLV helper virus, to infect a purified population of mature, hapten-binding murine splenic B lymphocytes, aiming to generate mature B-cell lines to use as models for the study of B-cell growth and differentiation physiology. Immortalized B-cell lines which retain the same mature phenotype as the starting population, including hapten-specific binding, were produced. This is the first demonstration of a method for immortalizing selected antigen-binding B lymphocytes, and the first example of immortalization of mature B cells in vitro with an acutely transforming ras-containing retrovirus.     

Proliferation and differentiation of neuronal stem cells regulated by nerve growth factor

Nerve growth factor plays an important part in neuron-target interactions in the late embryonic and adult brain. We now report that this growth factor controls the proliferation of neuronal precursors in a defined culture system of cells derived from the early embryonic brain. Neuronal precursor cells were identified by expression of the intermediate filament protein nestin. These cells proliferate in response to nerve growth factor but only after they have been exposed to basic fibroblast growth factor. On withdrawal of nerve growth factor, the proliferative cells differentiate into neurons. Thus, in combination with other growth factors, nerve growth factor regulates the proliferation and terminal differentiation of neuroepithelial stem cells.     

Ornithine decarboxylase activity is critical for cell transformation.

The enzyme ornithine decarboxylase is the key regulator of the synthesis of polyamines which are essential for cell proliferation. Expression of this enzyme is transiently increased upon stimulation by growth factors, but becomes constitutively activated during cell transformation induced by carcinogens, viruses or oncogenes. To test whether ornithine decarboxylase could be a common mediator of transformation and oncogenic itself, we transfected NIH3T3 cells with expression vectors carrying the complementary DNA encoding human ornithine decarboxylase in sense and antisense orientations. The increased expression of the enzyme (50-100-times endogenous levels) induced not only cell transformation, but also anchorage-independent growth in soft agar and increased tyrosine phosphorylation of a protein of M(r) 130K. Expression of ornithine decarboxylase antisense RNA was associated with an epithelioid morphology and reduced cell proliferation. Moreover, blocking the endogenous enzyme using specific inhibitor or synthesizing antisense RNA prevented transformation of rat fibroblasts by temperature-sensitive v-src oncogene. Our results imply that the gene encoding ornithine decarboxylase is a proto-oncogene central for regulation of cell growth and transformation.     

Cellular immortalization by a cDNA clone encoding the transformation-associated phosphoprotein p53

Malignant transformation of primary cells requires at least two distinct and characteristic alterations in cellular behaviour. The first, cellular immortality, can be induced by chemical carcinogens or by cloned oncogenes such as polyoma large T (ref. 4), adenovirus early region 1A (E1A) or the oncogene from avian (MC29) myelocytomatosis virus, v-myc. Cells whose in vitro life-span has been extended by these procedures can be fully transformed by transfection with oncogenes belonging to a different complementation group, including genes of the ras family, adenovirus E1b and polyoma virus middle T (refs 4, 5). The unstable cellular phosphoprotein p53 is frequently present at elevated levels in transformed cells and is stabilized by the formation of complexes with simian virus 40 (SV40) large T or adenovirus E1b 57K protein. Although several reports have associated p53 with cell proliferation, its role remains obscure. We have cloned complementary DNA sequences encoding murine p53 and report here that transfection of p53 expression constructs into cells of finite lifespan in vitro results in cellular immortality and susceptibility to transformation by a ras oncogene.     

Sarcoma viruses carrying ras oncogenes induce differentiation-associated properties in a neuronal cell line

The growth-promoting and/or differentiation-blocking activities of Kirsten (Ki-MSV) or Harvey murine sarcoma virus (Ha-MSV) on various types of cells in vitro are well documented. Here we report an unexpected effect of these viruses on a rat phaeochromocytoma cell line, PC12. PC12 cells, which multiply indefinitely in growth medium, are known to respond to nerve growth factor (NGF) by cessation of cell division and expression of several properties resembling those of differentiated sympathetic neurones. We have found that Ki- and Ha-MSV mimic some, if not all, of the activities of NGF in PC12 cells, and there is evidence that the viral oncogenes, v-Ki-ras and v-Ha-ras, are responsible for this phenomenon. This system may be of value for studying the mechanism of action of the v-ras genes as well as the regulatory mechanism of growth and differentiation in neuronal cells.     

Effect of Steel factor and leukaemia inhibitory factor on murine primordial germ cells in culture.

Despite the importance of germ cells to the survival of species, surprisingly little is known about their embryological origin, proliferation, migration and entry into mitotic arrest or meiosis. Mutations in the murine Dominant White Spotting (W) and Steel genes, which respectively encode the c-kit tyrosine kinase receptor and the c-kit ligand (or Steel factor), impair the development of primordial germ cells (PGCs) in vivo, as well as haematopoietic stem cells and neural crest-derived melanoblasts. Here we use a monoclonal antibody against c-kit tyrosine kinase receptor and recombinant Steel factor to study the c-kit receptor-ligand system in cultured PGCs. In addition, we show that leukaemia inhibitory factor (also known as differentiation inhibitory activity), a factor secreted by STO fibroblasts, can stimulate proliferation of primordial germ cells in vitro.     

人脂肪组织来源干细胞生物学特性研究

为进一步研究脂肪组织来源的干细胞增殖和多向分化潜能,用改进的方法分离脂肪组织来源的干细胞,通过cck-8检测细胞的增殖能力、流式细胞仪检测干细胞相关表面标记的表达、RT-PCR对干细胞相关基因的表达进行分析;并对分离得到的细胞向脂肪、软骨、骨及心肌细胞诱导,观察其多向分化能力.结果显示:采用改进的方法,从400～600 mg脂肪组织可收获约5×105个脂肪组织来源的干细胞,并且细胞可以重叠生长1个月以上,期间细胞表现出几个对数增殖期;所有增殖的细胞其干细胞相关表面标记都呈阳性表达;转录因子Nanog、Oct-4、Sox-2和Rex-1也呈强阳性表达;ADSCs向脂肪、骨、软骨和心肌细胞诱导分化后能够分别表达脂滴、碱性磷酸酶和矿化结节、富含黏多糖的软骨细胞外基质,以及少量心肌特异性连接蛋白Connexin-43,表明ADSCs具有向多个胚层细胞分化的能力.此外,为获得更多具有强增殖能力的细胞,根据生长曲线,对细胞进行每14 d传代而非传统的5 d传代,发现所得到的细胞仍保持强的增殖能力、干细胞表型以及更强的多向分化潜能.     

Serine- and threonine-specific protein kinase activities of purified gag-mil and gag-raf proteins

Retroviruses carry cell-derived oncogenes (v-onc) that have the potential to transform cells in culture and induce tumours in vivo. One of the few carcinoma-inducing viruses is the acutely transforming retrovirus MH2, which carries the putative oncogene v-mil and the known oncogene v-myc. Recently, a high degree of homology was discovered between v-mil and v-raf, the transforming gene of the murine retrovirus 3611 murine sarcoma virus (MSV), whereas homology to v-src is low. Both viruses express their oncogenes as the gag-fusion polyproteins p100gag-mil and p75gag-raf (of respective relative molecular mass (Mr) 100,000 and 75,000), while the myc oncogene of MH2 is expressed by means of a subgenomic messenger RNA. We have recently demonstrated that p100gag-mil is not a nuclear protein. Here we report that purified p100gag-mil and p75gag-raf exhibit protein kinase activities in vitro which, in contrast to the src-related p130gag-fps of Fujinami sarcoma virus (FSV) and all other characterized oncogene-encoded protein kinases, phosphorylate serine and threonine but not tyrosine. Both types of protein kinases phosphorylate lipids in vitro.     

Bombesin-like peptides can function as autocrine growth factors in human small-cell lung cancer

The autocrine hypothesis proposes that a cell produces and secretes a hormone-like substance that can interact with specific membrane receptors on its surface to induce effects such as proliferation. Thus, a cancer cell could act to stimulate its own growth. Bombesin and bombesin-like peptides (BLPs) such as gastrin-releasing peptide (GRP) cause various physiological responses in mammals, including stimulation of proliferation of 3T3 mouse fibroblasts and normal human bronchial epithelial cells in vitro and induction of gastrin cell hyperplasia and increased pancreatic DNA content in vivo in rats. Human small-cell lung cancer (SCLC) cell lines produce and secrete BLPs and can express a single class of high-affinity receptors for BLPs. Exogenously added BLPs can also stimulate the clonal growth and DNA synthesis of SCLC in vitro. These findings suggest that BLPs function as autocrine growth factors for this tumour. One way to test this hypothesis is to interrupt the function of the endogenously produced BLPs. Here, we demonstrate that a monoclonal antibody to bombesin binds to the C-terminal region of BLPs, blocks the binding of the hormone to cellular receptors and inhibits the clonal growth of SCLC in vitro and the growth of SCLC xenografts in vivo. These results demonstrate that BLPs can function as autocrine growth factors for human SCLC.