The Grb2 binding domain of mSos1 is not required for downstream signal transduction.

Cellular Ras proteins are activated primarily by specific guanine-nucleotide releasing factors such as the Son of Sevenless (Sos) proteins. This activation event is thought to occur in response to plasma membrane localization of a complex containing Sos and a small adapter protein Grb2. We have isolated a dominant mutant allele of mSos1 which transforms Rat1 cells, yet is no longer able to bind Grb2. Biochemical experiments reveal that the subcellular distribution of this truncated Sos protein is not altered with respect to the wild type Sos protein. These data argue against a role for Grb2 in the direct recruitment of Sos proteins to the plasma membrane and suggest that Grb2 may function to overcome negative regulation of Sos by its C terminus.

Fringe boundaries coincide with Notch-dependent patterning centres in mammals and alter Notch-dependent development in Drosophila.

In both vertebrate and invertebrate development, cells are often programmed to adopt fates distinct from their neighbors. Genetic analyses in Drosophila melanogaster have highlighted the importance of cell surface and secreted proteins in these cell fate decisions. Homologues of these proteins have been identified and shown to play similar roles in vertebrate development. Fringe, a novel signalling protein, has been shown to induce wing margin formation in Drosophila. Fringe shares significant sequence homology and predicted secondary structure similarity with bacterial glycosyltransferases. Thus fringe may control wing development by altering glycosylation of cell surface and/or secreted molecules. Recently, two fringe genes were isolated from Xenopus laevis. We report here the cloning and characterization of three murine fringe genes (lunatic fringe, manic fringe and radical fringe). We find in several tissues that fringe expression boundaries coincide with Notch-dependent patterning centres and with Notch-ligand expression boundaries. Ectopic expression of murine manic fringe or radical fringe in Drosophila results in phenotypes that resemble those seen in Notch mutants.

Transformation by oncogenes encoding protein kinases induces the metastatic phenotype.

Oncogenes encoding serine/threonine or tyrosine kinases were introduced into the established rodent fibroblast cell line NIH 3T3 and tested for tumorigenic and metastatic behavior in T cell-deficient nude mice. Transforming oncogenes of the ras family were capable of converting fibroblast cell lines to fully metastatic tumors. Cell lines transformed by the kinase oncogenes mos, raf, src, fes, and fms formed experimental metastases and (in some cases) these genes were more efficient at metastatic conversion than a mutant ras gene. In contrast, cells transformed by either of two nuclear oncogenes, myc or p53, were tumorigenic when injected subcutaneously but were virtually nonmetastatic after intravenous injection. These data demonstrate that, in addition to ras, a structurally divergent group of kinase oncogenes can induce the metastatic phenotype.

Mutant p53 tumor suppressor alleles release ras-induced cell cycle growth arrest.

Overexpression of an activated ras gene in the rat embryo fibroblast line REF52 results in growth arrest at either the G1/S or G2/M boundary of the cell cycle. Both the DNA tumor virus proteins simian virus 40 large T antigen and adenovirus 5 E1a are able to rescue ras induced lethality and cooperate with ras to fully transform REF52 cells. In this report, we present evidence that the wild-type activity of the tumor suppressor gene p53 is involved in the negative growth regulation of this model system. p53 genes encoding either a p53Val-135 or p53Pro-193 mutation express a highly stable p53 protein with a conformation-dependent loss of wild-type activity and the ability to eliminate any endogenous wild-type p53 activity in a dominant negative manner. In cotransfection assays, these mutant p53 genes are able to rescue REF52 cells from ras-induced growth arrest, resulting in established cell lines which express elevated levels of the ras oncoprotein and show morphological transformation. Full transformation, as assayed by tumor formation in nude mice, is found only in the p53Pro-193-plus-ras transfectants. These cells express higher levels of the ras protein than do the p53Val-135-plus-ras-transfected cells. Transfection of REF52 cells with ras alone or a full-length genomic wild-type p53 plus ras results in growth arrest and lethality. Therefore, the selective event for p53 inactivation or loss during tumor progression may be to overcome a cell cycle restriction induced by oncogene overexpression (ras). These results suggest that a normal function of p53 may be to mediate negative growth regulation in response to ras or other proliferative inducing signals.

Expression of H-ras correlates with metastatic potential: evidence for direct regulation of the metastatic phenotype in 10T1/2 and NIH 3T3 cells.

Using three independent approaches, we studied the effects of H-ras on metastasis formation. Analysis of five in vitro-ras-transfected 10T1/2 clones with either flat or refractile morphologies revealed a relationship between metastatic potential, H-ras expression, and anchorage-independent growth. Four metastatic variants derived from a poorly metastatic, low-H-ras-expressing line all expressed high levels of H-ras RNA and grew efficiently in soft agar. Activation of H-ras expression in the metastatic tumors had occurred through amplification and rearrangement of H-ras sequences. In addition, preinduction of p21 synthesis in NIH 3T3 line 433, which contains v-H-ras under transcriptional control of the glucocorticoid-sensitive mouse mammary tumor virus long terminal repeat, significantly increased metastatic efficiency. Glucocorticoid treatment of normal or pEJ-transformed NIH 3T3 cells did not affect metastatic potential. These data reveal a direct relationship between ras expression and metastasis formation and suggest that metastatic and transformed phenotypes may be coregulated in ras-transformed 10T1/2 and NIH 3T3 cells.

Natural killer cell regulation of implantation and early lung growth of H-ras-transformed 10T1/2 fibroblasts in mice.

We examined the relative role of the natural killer (NK) cell and H-ras gene in controlling metastasis formation using a novel assay for quantitating viable tumor cells entering and surviving in the lung for up to 13 days following i.v. tumor inoculation. This assay utilized the resistance to G418 sulfate conferred by transfection of the neoR gene into 10T1/2 fibroblasts along with activated H-ras. We had previously shown that the metastatic efficiency of T-24-H-ras-transformed 10T1/2 fibroblasts correlated with H-ras expression at the RNA level. In this paper we show that the NK cell could recognize H-ras-transformed fibroblasts in vivo and control experimental metastasis formation using NK-suppressed and -activated syngeneic C3H recipients. Evaluation of NK sensitivity in vitro of individual lines did not predict metastatic ability. However, NK susceptibility in vitro did inversely correlate with the ability of tumor cells to arrest and survive in the lung for the first 48 h after i.v. inoculation. Although the level of H-ras RNA correlated with the ultimate metastatic potential, it did not correlate with the initial rate of tumor cell pulmonary retention or clearing. Over the next 10 to 12 days, however, we detected a preferential survival and outgrowth of high H-ras-expressing variants, which correlated well with the ultimate metastatic ability but not NK susceptibility. These observations argue that the NK cell has its major effect early in the course of the disease, while subsequent tumor growth occurs preferentially in high H-ras-expressing cell lines.

RAS is required for epidermal growth factor-stimulated arachidonic acid release in rat-1 fibroblasts.

Previous studies have provided suggestive evidence for an interaction between ras activation and signalling pathways involved in agonist-stimulated arachidonic acid release in a variety of cell systems. In order to clarify this interaction, we have measured epidermal growth factor (EGF)-stimulated arachidonic acid release in rat-1 fibroblasts transfected with the N-17 dominant negative mutation of ras. Cells transfected with the N-17 ras mutant, display a markedly attenuated arachidonic acid-release response to EGF, compared to sham-transfected and non-transfected cells. In contrast, the response to phorbol myristate acetate (PMA) was not attenuated in the N-17-mutant expressing cells. No differences were detected between sham-transfected and N-17 mutant expressing cells in levels of immunodetectable EGF receptor, cytosolic phospholipase A2 or mitogen-activated protein (MAP) kinase. Attenuation of EGF-stimulated arachidonic acid release in the N-17 mutant expressing cells, was accompanied by a marked diminution in EGF-stimulated tyrosine phosphorylation of MAP kinase. We conclude that the signalling pathway involved in epidermal growth factor-stimulated arachidonic acid release is similar to the signalling pathway for mitogenic responses to epidermal growth factor and requires ras activation, likely followed by a downstream cascade of kinases eventuating in MAP kinase activation.

Cysteine proteinase cathepsin L expression correlates closely with the metastatic potential of H-ras-transformed murine fibroblasts.

A set of H-ras-transfected mouse C3H 10T1/2 cell lines that vary in their experimental and spontaneous metastatic potential has been analysed for the level of expression of the cysteine proteinase cathepsin L, also known as the major excreted protein (MEP). We found a good positive correlation between the extent of ras expression, the metastatic potential, and the amount of the secreted protease in nine independently isolated lines. There was an increased abundance of the MEP mRNA in cells with increased ras expression, suggesting that this gene is under ras control. Expression of glyceraldehyde phosphate dehydrogenase mRNA was also elevated.

Evidence for synergistic interactions between ras, myc and a mutant form of p53 in cellular transformation and tumor dissemination.

Mouse 10T1/2 cells were transfected with combinations of T24 H-ras, human c-myc and the proline 193 mutant form of p53. The three-gene ras/myc/p53 combination was significantly more efficient than single genes or double gene combinations in inducing transformed foci in vitro. An analysis of cell lines isolated after transfections with ras, ras/myc, ras/p53 and ras/myc/p53 indicated that the last combination contained significantly higher levels of ras protein than the other combinations, produced tumors in syngeneic mice with a shorter latency period, and exhibited an increased ability to form lung tumors in an in vivo experimental metastasis assay. Synergistic interactions between ras, myc and mutant p53 genes were observed in focus formation and metastasis assays, suggesting that the action of the three oncogenes in malignant transformation occurs along separate but interactive pathways. These results support a working model of oncogene cooperativity in which alterations in myc and p53 permit elevated expression of ras, which is important in a mechanism affecting both cellular transformation in vitro and tumor dissemination in vivo.

Expression of notch receptors, notch ligands, and fringe genes in hematopoiesis.

OBJECTIVE: Hematopoiesis is the process by which mature blood cell types are generated from a small population of pluripotent hematopoietic stem cells. How these cells undergo fate selection, however, is not fully understood. The Notch signaling system is known to mediate cell fate decisions of multipotent precursors in a wide range of complex animals throughout development. As Notch signaling involves cell-cell interactions, we sought to determine the expression of Notch receptors, ligands, and regulators in individual cell populations along the hematopoietic differentiation pathway. MATERIALS: Described here is a single cell RT-PCR analysis of Notch1, Notch3, Notch4, Notch ligands (Dll1 and Jagged1), and Fringe gene expression in cells of the blood system. As previously described, single cell globally amplified cDNA was generated by RT-PCR from various hematopoietic precursor cells whose potential was known from sibling analysis. A precursor hierarchy slot blot was created containing these cDNAs as well as samples from maturing blood cell populations and two fibroblast cell lines. The precursor slot blot was screened with probes for each of the candidate genes. RESULTS: Macrophage precursors expressed high levels of Notch1 transcript, while maturing macrophages expressed high levels of both Notch1 and Notch4. The Jagged 1 ligand transcript was highly expressed in terminally maturing cells including mast cells and megakaryocytes. In contrast, the Manic Fringe gene was highly expressed in uncommitted bi- and tri-potential precursors as well as in committed neutrophil and macrophage precursors. CONCLUSIONS: Distinct expression patterns of Jagged1 and Manic Fringe suggest that their corresponding proteins could regulate cell fate choices during hematopoiesis and may be responsible for regulating communication between lineage compartments during hematopoietic development.

Molecular determinants of metastatic transformation.

In recent years, experimental systems have developed to analyze genetic and epigenetic regulation of the metastatic phenotype. Numerous studies have uncovered a potent role for transforming oncogenes in metastatic conversion. In addition, it has been shown that oncoprotein products operate in a dose-dependent fashion. The continued expression of oncoproteins is required to induce and regulate metastatic dissemination of tumor cells and, consequently, many of the signal transduction pathways that are controlled by the oncogene products can regulate metastasis. Exogenous growth factors that act through these same pathways also alter metastatic potential. Some primary and immortalized cells can be transformed by oncogenes but remain completely benign and nonmetastatic. Malignant transformation can be achieved in these cells through the cooperative interaction of specific oncogenes or loss of active suppression regulated by recessive genetic determinants. Therefore, it is likely that tumor cells acquire the metastatic phenotype through the cooperative interaction of dominant and recessive genetic alterations. This model is consistent with the correlative data accumulating in studies of human tumor specimens where more malignant carcinomas often contain both activating mutations in oncogenes and either inactivating mutations or loss of tumor-suppressor genes.

Genetic regulation of metastatic progression.

Metastasis is a complex process which results in the growth of tumor cells at sites distant from the primary neoplasm. It is likely that many of the large number of biological changes associated with metastatic ability are controlled through alterations in the expression of a relatively small number of key genes usually referred to as cellular oncogenes and tumor suppressor genes. These genes are normally required for the regulation of growth-related processes in the cell, and alterations through mutations and/or expression are important in establishing metastatic properties of malignant cells. In this article, we review evidence indicating that oncogenes play an important role in metastatic spread, and we have placed emphasis on studies with the ras oncogenes. Metastatic progression is dependent upon an accumulation of multiple genetic changes in malignant cells. Therefore, we have also briefly addressed the related questions of altered growth factor regulation, and the cooperative interactions observed between dominantly acting oncogenes and between these genes, and tumor suppressor genes.

Growth factor modulation of metastatic lung colonization.

Altered production and response to growth factors is often involved in neoplasia but little is known about their effect on the dissemination of tumors. Therefore, we have examined the effect of growth factors on metastatic lung colonization. Autocrine induction of the metastatic phenotype was demonstrated in NIH-3T3 cells transformed by a signal peptide-bFGF gene but not bFGF in the absence of the signal peptide. In addition, exogenous growth factor regulation of metastasis was demonstrated. Treatment of ras transformed C3H-10T 1/2 cells and ras or src transformed NIH-3T3 cells with bFGF prior to intravenous injection resulted in potent inhibition of metastatic potential. Stimulation of v-fms transformed cells by the natural fms-ligand, CSF-1, resulted in potent stimulation of metastatic behavior in freshly plated or refed cells, whereas following autocrine conditioning of the medium, the metastatic properties of these cells were sensitive to inhibitory effects of CSF-1. These observations indicate that specific growth factors can regulate the metastatic phenotype and, depending on the oncogenes responsible for cell transformation and autocrine conditioning, these effects can be either stimulatory or inhibitory.

Recovery of DNA for forensic analysis from lip cosmetics.

To obtain a reference DNA profile from a missing person, we analyzed a variety of personal effects, including two lip cosmetics, both of which gave full DNA profiles. Further investigations were undertaken to explore this previously unreported source of DNA. We have tested a range of brands and types of lip cosmetics. Our studies have revealed that lip cosmetics are an excellent source of DNA, with almost 80% of samples giving a result. However, artifacts are frequently observed in the DNA profiles when Chelex is used for the DNA extraction and additional DNA purification procedures are required to ensure that an accurate DNA profile is obtained.

Oncogenes and metastatic progression.

It is now established that ras oncogenes can induce metastatic characteristics in primary diploid fibroblasts, nonsenescing fibroblasts and nonmetastasizing tumors. The issue of whether ras is directly involved in maintaining the metastatic phenotype through the expression and action of its gene product has been examined by analyzing the relationship to ras expression and to the production of the p21 ras-GTP complex, which is thought to mediate ras-transforming activity. While these expression and mutation studies support the idea that p21 ras directly regulates metastasis formation, it is also evident that there are many examples of human and murine cancers which show no differences in ras expression in primary and metastatic tumor cells. This may be partially explained by the ability of protein kinase-encoding oncogenes to also induce metastatic potential. In addition, the ability of ras to induce metastasis may be dependent on the regulation of its activity by other genes. Furthermore, transformation does not occur as an isolated genetic event, but is rather the result of interaction of two or more oncogenes. We suggest that the nature of these gene interactions will ultimately determine whether a cell is a benign transformant or a malignant and metastatic cancer.

A complex of Grb2 adaptor protein, Sos exchange factor, and a 36-kDa membrane-bound tyrosine phosphoprotein is implicated in ras activation in T cells.

T lymphocytes contain both Grb2, an SH2 and SH3 domain containing adaptor protein, and Sos, a guanine nucleotide exchange factor for Ras. Immunoprecipitates of Sos from the lysates of T cells contain a 36-kDa protein which is phosphorylated on tyrosine residues in response to T cell receptor/CD3 cross-linking. In vitro studies using different bacterially synthesized GST-Sos fusion proteins confirm the formation of complexes containing p36 and the proline-rich COOH-terminal domain of Sos. The use of mutant GST-Grb2 proteins in which both SH3 domains have been mutationally inactivated shows that Grb2 binds to tyrosine phosphorylated p36 via its SH2 domain. In Jurkat cells phosphorylated p36 is localized exclusively in the particulate fraction. In addition, another SH2 domain-containing protein, p52Shc is tyrosine phosphorylated upon TCR.CD3 cross-linking and associates with a 150-kDa phosphotyrosine containing protein. Taken together these data suggest that activation of Ras in T cells via the TCR.CD3 complex might be controlled, at least in part, by mechanisms similar to those found in fibroblasts, involving in this case formation of a complex of Grb2, Sos, and a membrane-bound tyrosine phosphoprotein of molecular mass 36-kDa.

Mapping GRB2, a signal transduction gene in the human and the mouse.

We have mapped GRB2, a signal transduction gene whose protein product is an essential component of the pathway between tyrosine kinases (such as the epidermal growth factor receptor) and downstream proteins (such as Ras and Sos). We assigned GRB2 to human chromosome 17 by hybridization to a somatic cell hybrid mapping panel. To position the locus at a much finer resolution, we have isolated the human GRB2 gene in three different cosmids, which we have mapped by fluorescence in situ hybridization to the long arm of human chromosome 17 (17q24-q25). We have hybridized a human GRB2 open reading frame probe to mouse DNAs from the European Interspecific Backcross. The segregation patterns reveal that the mouse Grb2 locus maps distally on chromosome 11, and an additional Grb2-related locus is present on chromosome 4 of one of the parental strains, Mus spretus/CRC.

Mapping the gene that encodes phosphatidylinositol-specific phospholipase C-gamma 2 in the human and the mouse.

We have mapped the PLCG2 gene, which encodes the enzyme phosphatidyl inositol-specific phospholipase C-gamma 2. This is one of the phospholipases responsible for catalyzing the hydrolysis of phosphatidyl inositol in response to a great many mitogenic stimuli. PL C-gamma 2 is an essential component of the signal transduction pathway between tyrosine kinases and downstream events such as protein kinase C activation and intracellular calcium release. We assigned PLCG2 to human chromosome 16 by amplification within a somatic cell hybrid mapping panel. To position the locus at a much finer resolution, PLCG2 sequences were amplified from a chromosome 16-specific somatic cell hybrid panel, which placed the gene on the long arm of the chromosome in band 16q24.1, a region that has few known genes. We have hybridized a mouse Plcg2 open reading frame probe to mouse DNAs from the European Interspecific Backcross. The segregation pattern reveals the mouse Plcg2 locus maps to distal chromosome 8.

Molecular characterization of the Entner-Doudoroff pathway in Escherichia coli: sequence analysis and localization of promoters for the edd-eda operon.

The nucleotide sequence of the entire Escherichia coli edd-eda region that encodes the enzymes of the Entner-Doudoroff pathway was determined. The edd structural gene begins 236 bases downstream of zwf. The eda structural gene begins 34 bases downstream of edd. The edd reading frame is 1,809 bases long and encodes the 602-amino-acid, 64,446-Da protein 6-phosphogluconate dehydratase. The deduced primary amino acid sequences of the E. coli and Zymomonas mobilis dehydratase enzymes are highly conserved. The eda reading frame is 642 bases long and encodes the 213-amino-acid, 22,283-Da protein 2-keto-3-deoxy-6-phosphogluconate aldolase. This enzyme had been previously purified and sequenced by others on the basis of its related enzyme activity, 2-keto-4-hydroxyglutarate aldolase. The data presented here provide proof that the two enzymes are identical. The primary amino acid sequences of the E. coli, Z. mobilis, and Pseudomonas putida aldolase enzymes are highly conserved. When E. coli is grown on gluconate, the edd and eda genes are cotranscribed. Four putative promoters within the edd-eda region were identified by transcript mapping and computer analysis. P1, located upstream of edd, appears to be the primary gluconate-responsive promoter of the edd-eda operon, responsible for induction of the Entner-Doudoroff pathway, as mediated by the gntR product. High basal expression of eda is explained by constitutive transcription from P2, P3, and/or P4 but not P1.