We have previously described an inverse relationship between Cdx1 and Cdx2 mRNA levels and the extent of dysplasia and severity of clinical outcome in colorectal carcinoma, suggesting that altered expression of these genes was associated with colorectal carcinogenesis or tumor progression. To investigate further their involvement in the physiopathology of colorectal cancer, HT29 colon carcinoma cells that show very low Cdx expression were transfected with Cdx1 and/or Cdx2 cDNA to elicit their overexpression. Growth rate, tumorigenicity, resistance to apoptosis, and migration potential of the corresponding cells were analyzed. Growth rate of cells overexpressing Cdx2 decreased by half, whereas overexpression of Cdx1 had no effect. However, cells overexpressing both Cdxs had a growth rate reduced to 20% of control. In cells overexpressing Cdx1 or Cdx2, tumorigenicity and resistance to apoptosis induced by serum starvation, ceramide, or staurosporine were not changed compared with control cells; yet phorbol esterstimulated cell migration was decreased by 50%. In cells overexpressing both Cdx1 and Cdx2, tumorigenicity was decreased by 50%, resistance to apoptosis was significantly lowered, and stimulated cell migration was further decreased to 15% of control compared with cells expressing Cdx1 or Cdx2. Finally, cells overexpressing both Cdxs showed strongly decreased Bcl-2 expression, which could account for their increased sensitivity to apoptosis. These findings show that, in HT29 cells, both Cdx1 and Cdx2 genes must be expressed to reduce tumorigenic potential, to increase sensitivity to apoptosis, and to reduce cell migration, suggesting that the two genes control the normal phenotype by independent pathways. This may explain why loss of Cdx1 or Cdx2 expression is associated with tumor development and invasiveness in colorectal tumors.In an effort to characterize the mechanisms involved in colorectal cancer initiation and progression, we have developed a strategy based on the constitution of a large repertoire of transcripts from a colorectal tumor, all characterized by partial sequencing (1). Expression of these expressed sequence tags in normal and cancerous colon was compared, and those most differentially expressed were selected. Genes detected by these means may be causative or instrumental in tumor induction or/and progression. Looking for such genes, we found that the Cdx1 and Cdx2 homeotic genes were concomitantly down-regulated in about 85% of colorectal cancers (2). Such low expression of Cdx1 or Cdx2 in colon carcinoma was verified by immunohistochemistry (3, 4) and by reverse transcription polymerase chain reaction (5) studies. Cdx1 and Cdx2 are interesting candidates that could play a role in colon cancer pathology because Chawengsaksophak et al. (6) recently reported the occurrence of multiple intestinal adenomatous polyps in the proximal colon of Cdx2 ϩ/Ϫ mice, suggesting that lowering Cdx2 levels in intestinal cells would suffice to induce intestinal tumors. Also, Suh and Traber showed that expre...

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Abstract. Metanephrogenesis has been a long-standing model to study cell-matrix interactions. A number of adhesion molecules, including matrix receptors (i.e., integrins), are believed to be involved in such interactions. The integrins contain a and ~ subunits and are present in various tissues in different heterodimeric forms. In this study, one of the members of the integrin superfamily, av, was characterized, and its relevance in murine nephrogenesis was investigated. Mouse embryonic renal eDNA libraries were prepared and screened for av, and multiple clones were isolated and sequenced. The deduced amino acid sequence of the et v cDNA clones and hydropathic analysis revealed that it has a typical signal sequence and extracellular, transmembrane, and cytoplasmic domains, with multiple Ca 2÷ binding sites. No A(U)nA mRNA instability motifs were present. Conformational analysis revealed no rigid long-range-ordered structure in murine O~v. The O~v was expressed in the embryonic kidney at day 13 of the gestation, with a transcript size of ~7 kb. Its expression increased progressively during the later gestational stages and in the neonatal period. It was distributed in the epithelial elements of developing nephrons and was absent in the uninduced mesenchyme. In mature metanephroi, the expression was relatively high in the glomeruli and blood vessels, as compared to the tubules. Various heterodimeric associations of av, i.e., with [31, [33, [35, and [36, were observed in metanephric tissues. Inclusion of av-antisense-oligodeoxynucleotide or -antibody in metanephric culture induced dysmorphogenesis of the kidney with reduced population of the nephrons, disorganization of the ureteric bud branches, and reduction of mRNA and protein expressions of etv. The expressions of integrin [33, [35, and 136 were unaltered. These findings suggest that the integrin av is developmentally regulated, has a distinct spatio-temporal expression, and is relevant in the mammalian organogenesis.URING development, the interactions between cell adhesion molecules and extraceUular matrix (ECM) 1 glycoproteins are believed to be essential for the morphogenesis of various organs (Hay, 1991;Humphries et al., 1991;Reichardt and Tomaselli, 1991;Ruoslahti, 1991;Toole, 1991;Hynes, 1992;Juliano and Haskill, 1993;Kramer et al., 1993;Gladson and Cheresh, 1994;Hemler et al., 1994;Quaranta et al., 1994). The maturation of the mammalian metanephros constitutes many of the steps which are prototypic of the events relevant to the development of other organ systems (Ekblom, 1993;Clapp and Abrahamson, 1994). The renal organogenesis ensues after an initial reciprocal induction between the metanephric Address all correspondence to Dr. Y.S. Kanwar, Department of Pathology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 60611. Tel.: (312) 503-0084. Fax: (312) 503-0627.1. Abbreviations used in this paper: ECM, extracellular matrix; ODN, oligodeoxynucleotide; RT, reverse transcriptase. mesenchyme and the ureteric bud, which leads to...

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Several integrin alpha subunits undergo post-translational endoproteolytic processing at pairs of basic amino acids that is mediated by the proprotein convertase furin. Here we ask whether other convertase family members can participate in these processing events. We therefore examined the endoproteolysis rate of the integrin subunits pro-alpha5, alpha6 and alphav by recombinant furin, proprotein convertase (PC)5A, paired basic amino acid converting enzyme (PACE)4, PC1, PC2 and PC7 in vitro and/or ex vivo after overexpression in LoVo cells that were deficient in furin activity. We found that 60-fold more PC1 than furin was needed to produce 50% cleavage of pro-alpha subunit substrates in vitro; the defective pro-alpha chain endoproteolysis in LoVo cells was not rescued by overexpression of PC1 or PC2. No endoproteolysis occurred with PC7 either in vitro or ex vivo, although similar primary sequences of the cleavage site are found in integrins and in proteins efficiently processed by PC7, which suggests that a particular conformation of the cleavage site is required for optimal convertase-substrate interactions. In vitro, 50% cleavage of pro-alpha subunits was obtained with one-third of the amount of PC5A and PACE4 than of furin. In LoVo cells, PC5A remained more active than furin, PACE4 activity was quite low, and PC5B, which differs from PC5A by a C-terminal extension containing a transmembrane domain, was very inefficient in processing integrin alpha-subunit precursors. In conclusion, these results indicate that integrin alpha-subunit endoproteolytic processing involves the redundant function of furin and PC5A and to a smaller extent PACE4, but not of PC1, PC2, PC5B or PC7.

In the present report the biosynthesis of the integrin alpha-chains endowed with constitutive endoproteolytic cleavage was evaluated in LoVo cells where furin, a subtilisin-like convertase involved in post-translational endoproteolytic processing, is not functional. It was found that cell-surface alpha 3, alpha 6 and alpha v subunits were not processed endoproteolytically into heavy and light chains as they were in HT29-D4 cells, a furin-competent cell line. Complete removal of N-linked oligosaccharides and pulse-chase experiments confirmed that the cleavage of the alpha 6 integrin subunit occurring 45 min after translation in HT29 cells did not take place in LoVo cells. Apart from cleavage deficiency, alpha 6 subunit glycosylation, association with beta 4 subunits and targeting to the plasma membrane seemed comparable in LoVo and HT29 cells. The pro-alpha 6 and the pro-alpha 3 subunits immunopurified from LoVo cells were highly sensitive to endoproteolysis by recombinant furin. Furin cleavage was calcium dependent and resulted in the conversion of the 140 kDa pro-alpha 6 into a 120 kDa heavy chain. These results suggest strongly that furin is involved in the endoproteolytic processing of cleavable integrin alpha subunits.

Integrins are transmembrane glycoproteins, composed of noncovalently associated ␣ and ␤ subunits, that are involved in cell-extracellular matrix (ECM) 1 and cell-cell interactions (1). Many integrin ␣ chains undergo a post-translational endoproteolytic cleavage. The ␣ 3 , ␣ 5 , ␣ 6 , ␣ 7 , ␣ 8 , ␣ 9 , ␣ v , and ␣ IIb subunits are cleaved in the membrane-proximal extracellular region, resulting in a heavy chain that is disulfide-linked to a membrane spanning light chain (2). The ␣ 4 and ␣ E subunits can also be cleaved, but at unusual positions, near the middle and in the N-terminal region of the molecule, respectively (3, 4). Endoproteolytic cleavage of integrin ␣ subunits occurs at specific sites comprising pairs of basic amino acids.Post-translational proteolysis is a common mechanism required for the synthesis of biologically active proteins in bacteria, fungi, yeast, invertebrates, and mammals (5). However, the role of endoproteolytic cleavage of integrin ␣ subunits is not clear. The cleavage is conserved, not only in different ␣ chains but also across species, suggesting that it might be of functional importance. It has been established, by site-directed mutagenesis of cleavage sites, that uncleaved ␣ IIb ␤ 3 and ␣ 4

In the tumor microenvironment, autocrine/paracrine loops of insulin-like growth factors (IGFs) contribute to cancer cell survival. However, we report here that IGF-I can send contradictory signals that interfere with cell death induced by different ligands of the tumor necrosis factor (TNF) superfamily. IGF-I protected human colon carcinoma cells from TNF-alpha-induced apoptosis, but it enhanced the apoptotic response to anti-Fas antibody and TNF-related apoptosis inducing ligand stimulation. This proapoptotic effect of IGF-I, observed in several but not all tested colon cancer cell lines, was mediated via the phosphatidylinositol 3'-kinase (PI3K)/Akt pathway. Furthermore, IGF-I receptors (IGF-IR) were located in and out of membrane lipid rafts and were tyrosine autophosphorylated in response to IGF-I. However, disruption of rafts by acute cholesterol depletion shifted IGF-IR to non-raft domains, abolished the IGF-I-mediated proapoptotic effect, and inhibited the IGF-I-dependent IRS-1 and Akt recruitment into and phosphorylation/activation within lipid rafts. Replenishing cell membranes with cholesterol reversed these effects. Activation of extracellular-regulated kinase-1/2 and p38 mitogen-activated protein kinase, which convey the IGF-I anti-apoptotic effect, occurred independently of lipid rafts. Thus, we propose that segregation of IGF-IR in and out of lipid rafts may dynamically regulate the pro- and anti-apoptotic effects of IGF-I on apoptosis induced by TNF superfamily members.