Mitogen-activated protein (MAP) kinase kinase (MAPKK) activates MAP kinase in a signal transduction pathway that mediates cellular responses to growth and differentiation factors. Oncogenes such as ras, src, raf, and mos have been proposed to transform cells by prolonging the activated state of MAPKK and of components downstream in the signaling pathway. To test this hypothesis, constitutively active MAPKK mutants were designed that had basal activities up to 400 times greater than that of the unphosphorylated wild-type kinase. Expression of these mutants in mammalian cells activated AP-1-regulated transcription. The cells formed transformed foci, grew efficiently in soft agar, and were highly tumorigenic in nude mice. These findings indicate that constitutive activation of MAPKK is sufficient to promote cell transformation.

The met protooncogene product, Met, is the tyrosine kinase growth factor receptor for hepatocyte growth factor/scatter factor (HGF/SF). NIH 3T3 cells express HGF/SF endogenously and become tumorigenic in nude mice via an autocrine mechanism when murine Met is expressed ectopically (Metmu cells) or when human Met and human HGF/SF are coexpressed (HMH cells). Here, we show that Metmu and HMH cells are invasive in vitro and display enhanced protease activity necessary for the invasive phenotype. In experimental and spontaneous metastasis assays, Metmu or HMH cells metastasize to the lung, but lower numbers of subcutaneously injected Metmu and HMH cells produced invasive tumors in the heart, diaphragm, salivary gland, and retroperitoneum. It has been reported elsewhere that Met expression increased with tumor passage in athymic nude mice, and these tumor explants show enhanced activity in the metastasis assays. Autocrine-mediated Met-HGF/SF signal transduction in NIH 3T3 mesenchymal cells may provide an important system for understanding the biological process of metastasis.

Hepatocyte growth factor/scatter factor (HGF/SF) is a pleiotropic effector of cells expressing the Met tyrosine kinase receptor. Although HGF/SF is synthesized by mesenchymal cells and acts predominantly on epithelial cells, we have recently demonstrated that human sarcoma cell lines often inappropriately express high levels of Met and respond mitogenically to HGF/SF. In the present report we show that HGF/SF-Met signalling in the human leiomyosarcoma cell line SK-LMS-1 enhances its in vivo tumorigenicity, an effect for which the mitogenicity of this signalling pathway is likely to play a role. In addition, we found that HGF/SF-Met signalling dramatically induces the in vitro invasiveness and in vivo metastatic potential of these cells. We have studied the molecular basis by which HGF/SF-Met signalling mediates the invasive phenotype. A strong correlation has previously been demonstrated between the activation of the urokinase plasminogen activator (uPA) proteolysis network and the acquisition of the invasive-metastatic phenotype, and we show here that HGF/SF-Met signalling significantly increases the protein levels of both uPA and its cellular receptor in SK-LMS-1 cells. This results in elevated levels of cell-associated uPA and enhanced plasmin-generating ability by these cells. These studies couple HGF/SF-Met signalling to the activation of proteases that mediate dissolution of the extracellular matrix-basement membrane, an important property for cellular invasionmetastasis.

The met proto-oncogene is the tyrosine kinase growth factor receptor for hepatocyte growth factor/scatter factor (HGF/SF). It was previously shown that, like the oncogenic tpr-met, the mouse met proto-oncogene transforms NIH 3T3 cells. We have established NIH 3T3 cells stably expressing both human (Methu) and mouse (Metmu) met proto-oncogene products. The protein products are properly processed and appear on the cell surface. NIH 3T3 cells express endogenous mouse HGF/SF mRNA, suggesting an autocrine activation mechanism for transformation by Metmu. However, the tumor-forming activity of Methu in NIH 3T3 cells is very low compared with that of Metmu, but efficient tumorigenesis occurs when Methu and HGF/SFhu are coexpressed. These results are consistent with an autocrine transformation mechanism and suggest further that the endogenous murine factor inefficiently activates the tumorigenic potential of Methu. The tumorigenicity observed with reciprocal chimeric human and mouse receptors that exchange external ligand-binding domains supports this conclusion. We also show that HGF/SFhu expressed in NIH 3T3 cells produces tumors in nude mice.

Hepatocyte growth factor/scatter factor (HGF/SF) is a pleiotropic effector of cells expressing the Met tyrosine kinase receptor. While HGF/SF-Met signaling clearly plays a role in a variety of normal cellular process, this signaling pathway has also been implicated in the generation and metastatic spread of tumors. This review discusses in detail several model systems that have been developed to investigate the role of HGF/SF-Met signaling in malignancy and describes additional data regarding the expression of these molecules in human tumors. Collectively the findings support a role for this receptor-ligand pair in human malignancy.

Coexpression of the human Met receptor and its ligand, hepatocyte growth factor/scatter factor (HGF/SF), in NIH 3T3 fibroblasts causes the cells to become tumorigenic in nude mice. The resultant tumors display lumen-like morphology, contain carcinoma-like focal areas with intercellular junctions resembling desmosomes, and coexpress epithelial (cytokeratin) and mesenchymal (vimentin) cytoskeletal markers. The tumor cells also display enhanced expression of desmosomal and tight-junction proteins. The apparent mesenchymal to epithelial conversion of the tumor cells mimics the conversion that occurs during embryonic kidney development, suggesting that Met-HGF/SF signaling plays a role in this process as well as in tumors that express both epithelial and mesenchymal markers.

We cloned the wild-type allele of the spolID locus of Bacillus subtilis. This DNA region was shown to be transcribed beginning within an hour after the onset of sporulation. The amount of spoIID mRNA present in ceUs at 1 h after the end of growth was more than 50-fold greater than it was in growing cells; the pool of this mRNA decreased steadily after 1.5 h after the end of growth. spoIlD mRNA was present in stationary-phase cells of sporulation mutants with lesions in the spoOJ and spolIB genes but was absent in cells carrying spoOB, spoOH, spoIA, spollE, spoliG, or spollIA mutations. In vitro runoff transcription with the Ef55, Ec37, Ea32, and Eu29 forms of RNA polymerase indicated that only the Ea29 form was able to transcribe the spoliD gene.This result is consistent with results of studies with the Spo-mutants, because only mutants that produced Ea29 were able to produce spollD mRNA in vivo. In the course of this work, two additional transcription units were discovered in the DNA region neighboring the spolID gene. One of these was expressed during vegetative growth; the other was expressed early during sporulation and corresponded to an in vitro transcript produced by the Eu29 form of RNA polymerase.Temporal control of gene expression during sporulation of Bacillus subtilis is thought to occur by sequential replacement of the sigma factor component of RNA polymerase (17,18). This mechanism can account for simultaneous activation and silencing of large groups of genes, even if they are scattered around the chromosome. In the last several years, this hypothesis has received strong experimental support. At least five forms of RNA polymerase (Ea55, Ea7, EJ32, Er29, Eu28) that differ only in their sigma factors are present in vegetative or sporulating cells or both. Each enzyme has specific promoter recognition sequences which permit it to transcribe only a certain group of genes (15). These genes are transcribed in vivo at characteristic times during growth or during sporulation or both.In vegetative cells, most of the RNA polymerase holoenzyme contains a sigma subunit designated {J55 (apparent molecular weight, 55,000). In an in vitro system, the purified form of this enzyme transcribes genes expressed in vivo during growth, such as the tms and veg genes (16, 20). Ear37 and E&2 are minor forms of RNA polymerase in vegetative cells. In vitro, these forms of RNA polymerase transcribe genes that are expressed at the end of the logarithmic growth phase or very early during sporulation (e.g., spoVG, ctc, and the subtilisin E gene) (15,27,31). Another minor form of vegetative cell RNA polymerase, Ea28, transcribes a small number of growth genes (13). The Ea29 form of RNA polymerase is found only in sporulating cells, and its appearance is developmentally regulated (30). It is most abundant about 3 h after sporulation begins; when it appears it seems to replace all known vegetative cell sigma factors (14, 30). Ea29 directs transcription of the L gene (14), as well as ctc (27) and, very weakly, the veg gene (14), ...

Hepatocyte growth factor/scatter factor (HGF/SF) is a pleiotropic effector of cells expressing the Met tyrosine kinase receptor. While HGF/SF-Met signaling clearly plays a role in a variety of normal cellular process, this signaling pathway has also been implicated in the generation and metastatic spread of tumors. This review discusses in detail several model systems that have been developed to investigate the role of HGF/SF-Met signaling in malignancy and describes additional data regarding the expression of these molecules in human tumors. Collectively the findings support a role for this receptor-ligand pair in human malignancy.