Insulin receptor substrate-1 (IRS-1) is the major substrate of insulin receptor and IGF-1 receptor tyrosine kinases; it has an apparent relative molecular mass of 160-190,000 (M(r), 160-190K) on SDS polyacrylamide gel. Tyrosine-phosphorylated IRS-1 binds the 85K subunit of phosphatidylinositol 3-kinase which may be involved in the translocation of glucose transporters and the abundant src homology protein (ASH)/Grb2 which may be involved in activation of p21ras and MAP kinase cascade. IRS-1 also has binding sites for Syp and Nck and other src homology 2 (SH2) signalling molecules. To clarify the physiological roles of IRS-1 in vivo, we made mice with a targeted disruption of the IRS-1 gene locus. Mice homozygous for targeted disruption of the IRS-1 gene were born alive but were retarded in embryonal and postnatal growth. They also had resistance to the glucose-lowering effects of insulin, IGF-1 and IGF-2. These data suggest the existence of both IRS-1-dependent and IRS-1-independent pathways for signal transduction of insulin and IGFs.

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A human fibroblast cDNA expression library was screened for cDNA clones giving rise to f lat colonies when transfected into v-Ki-ras-transformed NIH 3T3 cells. One such gene, RECK, encodes a membrane-anchored glycoprotein of about 110 kDa with multiple epidermal growth factor-like repeats and serine-protease inhibitor-like domains. While RECK mRNA is expressed in various human tissues and untransformed cells, it is undetectable in tumor-derived cell lines and oncogenically transformed cells. Restored expression of RECK in malignant cells resulted in suppression of invasive activity with concomitant decrease in the secretion of matrix metalloproteinase-9 (MMP-9), a key enzyme involved in tumor invasion and metastasis. Moreover, purified RECK protein was found to bind to, and inhibit the proteolytic activity of, MMP-9. Thus, RECK may link oncogenic signals to tumor invasion and metastasis.Mutations of ras protooncogenes are found in a large variety of human tumors (1). It has been well established that Ras proteins are essential components in various intracellular signaling pathways involved in regulating gene expression and several other aspects of cellular behavior (2). Therefore, it is now important to find targets for these signals relevant to the expression of the malignant phenotype to understand the mechanism of cell transformation and to develop means to cure or prevent cancers.To this end, we have been isolating and characterizing genes that induce flat morphology (or ''flat reversion'') when expressed in a v-Ki-ras-transformed NIH 3T3 cell line, DT (3). The Krev-1 gene (4), also known as rap1A, which encodes a Ras-related protein containing a region identical to the effector domain of Ras, was isolated in a previous study (5) by using a plasmid-based human fibroblast cDNA expression library. Using a similar approach, Cutler et al. (6) isolated another transformation suppressor gene, rsp-1, encoding a leucinerich-repeat protein. Recently, we performed a similar screen of a human fibroblast cDNA expression library constructed with a new phagemid shuttle vector and obtained two cDNA clones exhibiting significant biological activities. One of these, clone CT124, was found to encode a truncated form of the MSX-2 homeobox protein, which induces flat reversion through a dominant-negative mechanism over the endogenous MSX-2 protein (7).Here we describe some properties of the other reversioninducing gene named RECK (reversion-inducing-cysteine-rich protein with Kazal motifs) and its product. This reversioninducing gene is unique in that it encodes an extracellular protein with protease inhibitor-like domains and its expression is suppressed strongly in many tumors and cells transformed by various kinds of oncogenes. Restored expression of the RECK gene inhibits the invasive and metastatic activities of tumor cells. We also found that RECK negatively regulates matrix metalloproteinase-9 (MMP-9) (8) in two ways: suppression of MMP-9 secretion from the cells and direct inhibition of its enzymatic activity. T...

The p53 tumor suppressor gene, which is induced by DNA damage and/or stress stimuli, causes cells to undergo G1-arrest or apoptotic death; thus it plays an essential role in human carcinogenesis. We have searched for p53-related genes by using degenerate PCR, and have identified two cDNA fragments similar to but distinct from p53: one previously reported, p73, and the other new. We cloned two major splicing variants of the latter gene and named these p51A and p51B (a human homologue of rat Ket). The p51A gene encodes a 448-amino-acid protein with a molecular weight of 50.9 kDa; and p51B, a 641-amino-acid protein with a molecular weight of 71.9 kDa. In contrast with the ubiquitous expression of p53, expression of p51 mRNA was found in a limited number of tissues, including skeletal muscle, placenta, mammary gland, prostate, trachea, thymus, salivary gland, uterus, heart and lung. In p53-deficient cells, p51A induced growth-suppression and apoptosis, and upregulated p21waf-1 through p53 regulatory elements. Mutations in p51 were found in some human epidermal tumors.

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The c-mos proto-oncogene product, pp39mos, is present in unfertilized Xenopus eggs, and disappears on fertilization. Microinjection of synthetic mos RNA into two-cell embryos induces cleavage arrest at metaphase. By contrast, egg cytosol extracts, when immunodepleted of endogenous pp39mos, lose their cleavage-arresting activity in injected embryos. These results demonstrate that Mos protein is the cytostatic factor CSF, long known as an endogenous meiotic inhibitor in vertebrate eggs.

Tenascin, an extracellular matrix protein, is expressed in an unusually restricted pattern during embryogenesis and has been implicated in a variety of morphogenetic phenomena. To directly assess the function of tenascin in vivo, we generated mutant mice in which the tenascin gene was nully disrupted by replacing it with the lacZ gene. In mutant mice, lacZ was expressed in place of tenascin, and no tenascin product was detected. Homozygous mutant mice were, however, obtained in accordance with Mendelian laws, and both females and males produced offspring normally. No anatomical or histological abnormalities were detected in any tissues, and no major changes were observed in distribution of fibronectin, laminin, collagen, and proteoglycan. The existence of these mutant mice, lacking tenascin yet phenotypically normal, casts doubt on the theory that tenascin plays and essential role in normal development.

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