Transforming potential of the c-fms proto-oncogene (CSF-1 receptor)

The c-fms proto-oncogene encodes a transmembrane glycoprotein that is probably identical to the receptor for the macrophage colony stimulating factor, CSF-1. Forty C-terminal amino acids of the normal receptor are replaced by 11 unrelated residues in the feline v-fms oncogene product, deleting a C-terminal tyrosine residue (Tyr969) whose phosphorylation might negatively regulate the receptor kinase activity. We show that the human c-fms gene stimulates growth of mouse NIH 3T3 cells in agar in response to human recombinant CSF-1, indicating that receptor transduction is sufficient to induce a CSF-1 responsive phenotype. Although cells transfected with c-fms genes containing either Tyr969 or Phe969 were not transformed, cotransfection of these genes with CSF-1 complementary DNA induced transformation, with c-fms(Phe969) showing significantly more activity than c-fms(Tyr969). In the absence of CSF-1, chimaeric v-fms/c-fms genes encoding the wild-type c-fms C terminus were poorly transforming, whereas chimaeras bearing Phe969 were as transforming as v-fms. Thus, the Phe969 mutation, although not in itself sufficient to induce transformation, activates the oncogenic potential of c-fms in association with an endogenous ligand or in conjunction with mutations elsewhere in the c-fms gene that confer ligand-independent signals for growth.     

Chromosomal localization of the human proto-oncogene c-ets

E26 is an acute leukaemia avian retrovirus which induces myeloblastosis and erythroblastosis in vivo and transforms erythroblasts and myeloblasts in vitro. It contains the oncogene v-myb (ref. 4), first described for avian myeloblastosis virus (AMV), as well as a second specific nucleotide sequence, v-ets located 3' to v-myb (refs 5,6). We have reported that v-ets has a cellular counterpart (c-ets) in chicken and human DNA. Now, using two independent methods--hybridization with human c-ets probe of sorted chromosomes and in situ hybridization--we report the localization of the ets locus on human chromosome 11 at bands q23-q24. This finding may be important, as specific breakpoints around this position have been reported for human malignancies such as acute monocytic leukaemia and Ewing's sarcoma.     

Expression of c-myc changes during differentiation of mouse erythroleukaemia cells

The transforming gene of avian myelocytomatosis virus MC29, v-myc, causes a variety of malignancies in chickens. A cellular homologue, c-myc, has been implicated in B-cell malignancies in mice and humans but is also expressed in many normal cell types and may be important in the control of normal cell proliferation. c-myc is highly conserved in vertebrates. We have been investigating the relationship between c-myc expression and the terminal differentiation of cultured mouse erythroleukaemia (MEL) cells. We find that the level of c-myc messenger RNA shows a rapid biphasic change in MEL cells induced to differentiate by dimethyl sulphoxide or hypoxanthine. The changes occur during the first few hours of the differentiation programme and require active protein synthesis. These data suggest that changes in c-myc expression may be important in the irreversible commitment of MEL cells to terminal erythroid differentiation.     

Expression of a candidate sex-determining gene during mouse testis differentiation

The development of a eutherian mammal as a male is a consequence of testis formation in the embryo, which is thought to be initiated by a gene on the Y chromosome. In the absence of this gene, ovaries are formed and female characteristics develop. Sex determination therefore hinges on the action of this testis-determining gene, known as Tdy in mice and TDF in humans. In the past, several genes proposed as candidates for Tdy/TDF have subsequently been dismissed on the grounds of inappropriate location or expression. We have recently described a candidate for Tdy, which maps to the minimum sex-determining region of the mouse Y chromosome. To examine further the involvement of this gene, Sry, in testis development, we have studied its expression in detail. Fetal expression of Sry is limited to the period in which testes begin to form. This expression is confined to gonadal tissue and does not require the presence of germ cells. Our observations strongly support a primary role for Sry in mouse sex determination.     

Expression of cell-surface HLA-DR, HLA-ABC and glycophorin during erythroid differentiation

The unexpected discovery that Ia-like (HLA-DR) antigens in humans were present on blast cells from acute myeloblastic leukaemia led to the finding that normal granulocytic progenitors, in contrast to their mature descendents, also expressed HLA-DR antigens. Thus, anti-Ia sera stain a proportion of myeloblasts in normal bone marrow, inhibit myeloid progenitor (CFU-GM) colony formation in the presence of complement and can be used to label and separate CFU-GM on a fluorescence-activated cell sorter (FACS). Winchester et al. subsequently reported that erythroid progenitors (BFU-E and CFU-E) were also inhibited or killed by anti-Ia (p28,37) and complement. These observations raised the possibility that HLA-DR (or presumptive I-region equivalent) products might have a regulatory role in early haematopoiesis. We have now analysed HLA-DR and HLA-ABC antigen expression on normal erythroid progenitors using monoclonal antibodies to non-polymorphic determinants and fluorescence-activated cell sorting. In parallel experiments, we tested a monoclonal antibody to glycophorin, a well defined erythroid-specific cell-surface membrane glycoprotein. We report that HLA-DR, HLA-ABC and glycophorin are all expressed at various stages during erythroid differentiation.     

Structure and organization of the human Ki-ras proto-oncogene and a related processed pseudogene

Analysis of the organization and nucleotide sequence of two human loci related to the transforming gene of Kirsten murine sarcoma virus establishes one as a functional gene and the other as a processed pseudogene. The two final coding exons of the functional gene seem to have arisen by duplication. Differentially spliced mRNAs incorporating one or other of the duplicated exons probably served as the intermediates by which the viral transforming gene and the pseudogene were generated. This suggests that the functional gene may specify either of two related polypeptides depending on the pattern of RNA splicing.     

Expression of a transfected human c-myc oncogene inhibits differentiation of a mouse erythroleukaemia cell line

The Friend-virus-derived mouse erythroleukaemia (MEL) cell lines represent transformed early erythroid precursors that can be induced to differentiate into more mature erythroid cells by a variety of agents including dimethyl sulphoxide (DMSO). There is a latent period of 12 hours after inducer is added, when 80-90% of the cells become irreversibly committed to the differentiation programme, undergoing several rounds of cell division before permanently ceasing to replicate. After DMSO induction, a biphasic decline in steady-state levels of c-myc and c-myb messenger RNAs occurs. Following the initial decrease in c-myc mRNA expression, the subsequent increase occurs in, and is restricted to, the G1 phase of the cell cycle. We sought to determine whether the down-regulation is a necessary step in chemically induced differentiation. Experiments reported here indicate that expression in MEL cells of a transfected human c-myc gene inhibits the terminal differentiation process.     

Molecular characterization of human reticulon 3 as a potential marker during the differentiation of human neuroblastoma SH-SY5Y cells

Neuroendocrine-specific protein (NSP) -reticulons are endoplasmic reticulum-associated protein complexes, which are localized in the endoplasmic reticulum (ER) and identified as markers for neuroendocrine differentiation. In the present study, human reticulon 3 gene (hRTN3) was cloned and its expression pattern in a variety of tissues was investigated. Truncated hRTN3s corresponding to the C-terminal (hRTN3-C) domain were expressed and purified. hRTN3 mRNA was down-regulated during the differentiation of human neuroblastoma cell line SH-SY5Y induced by all-trans-retinoic acid (RA), which suggests that, like other members of the reticulon family, hRTN3 is a potential marker for neuroendocrine differentiation.     

Activated human monocytes express the c-sis proto-oncogene and release a mediator showing PDGF-like activity

Current ideas about the mechanism of wound healing and the pathogenesis of atherosclerosis, pulmonary fibrosis and hepatic fibrosis suggest a central role for the mononuclear phagocyte in attracting and/or stimulating the proliferation of mesenchymal cells. We demonstrate here that activated human blood monocytes, but not resting monocytes, release a mediator that attracts smooth muscle cells and cooperates with other mediators to stimulate fibroblast proliferation. This mediator is very similar to platelet-derived growth factor (PDGF): its chromatographic properties and chemical stability are similar to those of PDGF, it competes with 125I-PDGF for binding to fibroblasts and it immunoprecipitates with anti-PDGF antibodies. In parallel, stimulated monocytes, but not resting monocytes, express the c-sis proto-oncogene, a gene coding for one of the PDGF chains, consistent with the concept that expression of the c-sis proto-oncogene may be involved in the ability of mononuclear phagocytes to modulate the accumulation of mesenchymal cells.     

A yeast gene encoding a protein homologous to the human c-has/bas proto-oncogene product

Organisms amenable to easy genetic analysis should prove helpful in assessing the function of at least those proto-oncogene products which are highly conserved in different eukaryotic cells. One obvious possibility is to pursue the matter in Drosophila melanogaster DNA, which has sequences homologous to several vertebrate oncogenes. Another is to turn to the yeast Saccharomyces cerevisiae, if it contains proto-oncogene sequences. Here we report the identification of a gene in S. cerevisiae which codes for a 206 amino acid protein (YP2) that exhibits striking homology to the p21 products of the human c-has/bas proto-oncogenes and the transforming p21 proteins of the Harvey (v-rasH) and Kirsten (v-rasK) murine sarcoma viral oncogenes. The YP2 gene is located between the actin and the tubulin gene on chromosome VI and is expressed in growing cells. The protein it encodes might share the nucleotide-binding capacity of p21 proteins.     

Accumulation of a heat shock-like protein during differentiation of human erythroid cell line K562

The human erythroid cell line K562 provides a model system for studying erythroid differentiation and eukaryotic gene regulation. These cells express glycophorin A, spectrin and i antigen. They accumulate embryonic and fetal haemoglobins on induction of erythroid differentiation with haemin, sodium butyrate or hydroxyurea. In the present study, the protein composition of K562 cells during haemin-mediated induction of erythroid maturation was analysed by two-dimensional gel electrophoresis. Under conditions in which haemin did not effect cell viability and proliferation, a protein of approximately 70,000 molecular weight (MW) accumulated in the differentiated K562 cells. The accumulation appears to be due to an increase in the rate of RNA synthesis for this protein. The protein is related in sequence to a 70,000-MW heat shock protein. An antigenically related protein was also demonstrated in human bone marrow and accumulates at particular stages of human erythroid maturation.     

Fibronectin inhibits the terminal differentiation of human keratinocytes

In the epidermis proliferation of keratinocytes is restricted to the basal layer, which is in contact with the basement membrane, and cells undergo terminal differentiation as they move upwards through the suprabasal layers. In stratified cultures of human keratinocytes, upward migration is a consequence, not a cause, of terminal differentiation and occurs because keratinocytes become less adhesive to their substratum and to one another. Most keratinocytes can be induced to differentiate to completion by placing them in suspension in methylcellulose: within 12 h DNA synthesis is irreversibly inhibited and by 24 h most cells express involucrin (ref 4; P. A. Hall, J.C.A. and F.M.W., unpublished observations). Here we report that when fibronectin is added to the methylcellulose, keratinocytes still withdraw from the cell cycle, but induction of involucrin expression is largely inhibited. The effect of fibronectin is concentration- and time-dependent and is mediated by a receptor of the integrin family. These results provide an explanation for why overt terminal differentiation is normally restricted to suprabasal cells, whereas cell-cycle withdrawal occurs within the basal layer; they also have important implications for the mechanism of epidermal wound healing. Furthermore, our data show that the binding of an extracellular matrix protein to its receptor can regulate differentiated gene expression in the absence of changes in cell shape.     

Requirement for ras proto-oncogene function during serum-stimulated growth of NIH 3T3 cells

Human tumours often contain DNA sequences not found in normal tissues which are able to transform cultured NIH 3T3 cells. In some tumours the gene responsible for this transformation belongs to the cellular ras gene family. A specific type of mutation is responsible for converting the cellular proto-oncogene into a ras oncogene capable of inducing transformation. In a study of the function of a cellular ras gene, its protein product (produced in a bacterial cell) was microinjected into NIH 3T3 cells; the recipient cells became morphologically transformed and were induced to initiate DNA synthesis in the absence of added serum, but only when cellular ras protein was injected at much higher concentrations than required with protein of the transforming ras gene. To further analyse the function of the cellular ras gene, we have now injected monoclonal antibodies against ras proteins into NIH 3T3 cells. We report here that NIH 3T3 cells induced to divide by adding serum to the culture medium are unable to enter the S phase of the cell cycle after microinjection of anti-ras antibody, showing that the protein product of the ras proto-oncogene is required for initiation of the S-phase in NIH 3T3 cells.     

生长分化因子-9在大鼠卵巢中的表达

目的：探讨生长分化因子-9(GDF-9)在大鼠卵巢中的表达部位。方法：选择不同发育时期的大鼠卵巢,采用免疫组化和Western blot技术检测大鼠卵泡各发育阶段GDF-9蛋白表达情况及分布规律。结果：免疫组化方法显示,出生0～2d卵巢中未见GDF-9表达,出生3d卵巢中见部分原始卵泡卵母细胞GDF-9表达阳性,以后从初级卵泡到发育成熟的卵泡卵母细胞均明显可见GDF-9蛋白表达。Western blot技术进一步提示,出生0-2d卵巢中无GDF-9蛋白形成,第3d开始有GDF-9蛋白产生,以后在卵巢发育的各时期均有GDF-9蛋白分泌。结论：GDF-9在卵巢发育的早期即开始产生,以后在各阶段卵巢中持续表达,提示卵母细胞通过产生GDF-9等因子对卵泡的发育进行调节。