Renal insufficiency, a frequent complication with age in oral-facial-digital syndrome type I.

The oral-facial-digital syndrome type I (OFD I) is characterized by multiple congenital malformations of the face, oral cavity and digits. A polycystic kidney disease (PKD) is found in about one-third of patients but long-term outcome and complications are not well described in the international literature. Renal findings have been retrospectively collected in a cohort of 34 females all carrying a pathogenic mutation in the OFD1 gene with ages ranging from 1 to 65 years. Twelve patients presented with PKD - 11/16 (69%) if only adults were considered -with a median age at diagnosis of 29 years [IQR (interquartile range) = (23.5-38)]. Among them, 10 also presented with renal impairment and 6 were grafted (median age = 38 years [IQR = (25-48)]. One grafted patient under immunosuppressive treatment died from a tumor originated from a native kidney. The probability to develop renal failure was estimated to be more than 50% after the age of 36 years. Besides, neither genotype-phenotype correlation nor clinical predictive association with renal failure could be evidenced. These data reveal an unsuspected high incidence rate of the renal impairment outcome in OFD I syndrome. A systematic ultrasound (US) and renal function follow-up is therefore highly recommended for all OFD I patients.

A new model of cancer cachexia: contribution of the ubiquitin-proteasome pathway.

A new model of cachexia is described in which muscle protein metabolism related to the ubiquitin-proteasome pathway was investigated. Cloning of the colon-26 tumor produced a cell line, termed R-1, which induced cytokine (noninterleukin-1beta, interleukin-6 and tumor necrosis factor-alpha)-independent cachexia. Implantation of R-1 cells in mice elicited significant (20-30%) weight loss and decreased blood glucose by 70%, and adipose tissue levels declined by 95% and muscle weights decreased by 20-25%. Food intake was unaffected. The decrease in muscle weight reflected a decline in insoluble, but not soluble, muscle protein that was associated with a significant increase in net protein degradation. The rate of ubiquitin conjugation of proteins was significantly elevated in muscles of cachectic mice. Furthermore, the proteasome inhibitor lactacystin blocked the increase in protein breakdown but had no significant effect on proteolysis. Several markers of the ubiquitin-proteasome pathway, E2(14k) mRNA and E2(14k) protein and ubiquitin-protein conjugates, were not elevated. Future investigations with this new model should gain further insights into the mechanisms of cachexia and provide a background to evaluate novel and more efficacious therapies.

Proteasome inhibitors: a novel class of potent and effective antitumor agents.

The ubiquitin-proteasome pathway plays a critical role in the regulated degradation of proteins involved in cell cycle control and tumor growth. Dysregulating the degradation of such proteins should have profound effects on tumor growth and cause cells to undergo apoptosis. To test this hypothesis, we developed a novel series of proteasome inhibitors, exemplified by PS-341, which we describe here. As determined by the National Cancer Institute in vitro screen, PS-341 has substantial cytotoxicity against a broad range of human tumor cells, including prostate cancer cell lines. The PC-3 prostate cell line was, therefore, chosen to further examine the antitumor activity of PS-341. In vitro, PS-341 elicits proteasome inhibition, leading to an increase in the intracellular levels of specific proteins, including the cyclin-dependent kinase inhibitor, p21. Moreover, exposure of such cells to PS-341 caused them to accumulate in the G2-M phase of the cell cycle and subsequently undergo apoptosis, as indicated by nuclear condensation and poly(ADP-ribose) polymerase cleavage. Following weekly i.v. treatment of PS-341 to mice bearing the PC-3 tumor, a significant decrease (60%) in tumor burden was observed in vivo. Direct injection of PS-341 into the tumor also caused a substantial (70%) decrease in tumor volume with 40% of the drug-treated mice having no detectable tumors at the end of the study. Studies also revealed that i.v. administration of PS-341 resulted in a rapid and widespread distribution of PS-341, with highest levels identified in the liver and gastrointestinal tract and lowest levels in the skin and muscle. Modest levels were found in the prostate, whereas there was no apparent penetration of the central nervous system. An assay to follow the biological activity of the PS-341 was established and used to determine temporal drug activity as well as its ability to penetrate tissues. As such, PS-341 was shown to penetrate PC-3 tumors and inhibit intracellular proteasome activity 1.0 h after i.v. dosing. These data illustrate that PS-341 not only reaches its biological target but has a direct effect on its biochemical target, the proteasome. Importantly, the data show that inhibition of this target site by PS-341 results in reduced tumor growth in murine tumor models. Together, the results highlight that the proteasome is a novel biochemical target and that inhibitors such as PS-341 represent a unique class of antitumor agents. PS-341 is currently under clinical evaluation for advanced cancers.

Inhibition of NF-kappa B activation in vitro and in vivo: role of 26S proteasome.

It is becoming increasingly apparent that NF-kappa B plays a critical role in regulating the inflammatory response. Data obtained from studies in our laboratories demonstrate that the proteasome plays an important role in the inflammatory cascade by regulating the activation of NF-kappa B. Indeed, the availability of selective and orally active proteasome inhibitors should prove useful in delineating the roles of the proteasome and NF-kappa B in other pathophysiological conditions such as cancer and heart disease.

Role of the proteasome and NF-kappaB in streptococcal cell wall-induced polyarthritis.

The transcription factor NF-kappaB activates a number of genes whose protein products are proinflammatory. In quiescent cells, NF-kappaB exists in a latent form and is activated via a signal-dependent proteolytic mechanism in which the inhibitory protein IkappaB is degraded by the ubiquitin-proteasome pathway. Consequently, inhibition of the proteasome suppresses activation of NF-kappaB. This suppression should therefore decrease transcription of many genes encoding proinflammatory proteins and should ultimately have an anti-inflammatory effect. To this end, a series of peptide boronic acid inhibitors of the proteasome, exemplified herein by PS-341, were developed. The proteasome is the large multimeric protease that catalyzes the final proteolytic step of the ubiquitin-proteasome pathway. PS-341, a potent, competitive inhibitor of the proteasome, readily entered cells and inhibited the activation of NF-kappaB and the subsequent transcription of genes that are regulated by NF-kappaB. Significantly, PS-341 displayed similar effects in vivo. Oral administration of PS-341 had anti-inflammatory effects in a model of Streptococcal cell wall-induced polyarthritis and liver inflammation in rats. The attenuation of inflammation in this model was associated with an inhibition of IkappaBalpha degradation and NF-kappaB-dependent gene expression. These experiments clearly demonstrate that the ubiquitin-proteasome pathway and NF-kappaB play important roles in regulating chronic inflammation and that, as predicted, proteasome inhibition has an anti-inflammatory effect.

Mechanistic studies on the inactivation of the proteasome by lactacystin in cultured cells.

The natural product lactacystin exerts its cellular antiproliferative effects through a mechanism involving acylation and inhibition of the proteasome, a cytosolic proteinase complex that is an essential component of the ubiquitin-proteasome pathway for intracellular protein degradation. In vitro, lactacystin does not react with the proteasome; rather, it undergoes a spontaneous conversion (lactonization) to the active proteasome inhibitor, clasto-lactacystin beta-lactone. We show here that when the beta-lactone is added to mammalian cells in culture, it rapidly enters the cells, where it can react with the sulfhydryl of glutathione to form a thioester adduct that is both structurally and functionally analogous to lactacystin. We call this adduct lactathione, and like lactacystin, it does not react with the proteasome, but can undergo lactonization to yield back the active beta-lactone. We have studied the kinetics of this reaction under appropriate in vitro conditions as well as the kinetics of lactathione accumulation and proteasome inhibition in cells treated with lactacystin or beta-lactone. The results indicate that only the beta-lactone (not lactacystin) can enter cells and suggest that the formation of lactathione serves to concentrate the inhibitor inside cells, providing a reservoir for prolonged release of the active beta-lactone.

Generation of hybrid genes and proteins by vaccinia virus-mediated recombination: application to human immunodeficiency virus type 1 env.

The ability of poxviruses to undergo intramolecular recombination within tandemly arranged homologous sequences can be used to generate chimeric genes and proteins. Genes containing regions of nucleotide homology will recombine to yield a single sequence composed of portions of both original genes. A recombinant virus containing two genes with a number of conserved regions will yield a population of recombinant viruses containing a spectrum of hybrid sequences derived by recombination between the original genes. This scheme has been used to generate hybrid human immunodeficiency virus type 1 env genes. Recombinant vaccinia viruses that contain two divergent env genes in tandem array have been constructed. In the absence of selective pressure to maintain both genes, recombination between conserved homologous regions in these genes generated a wide range of progeny, each of which expressed a novel variant polypeptide encoded by the newly created hybrid env gene. Poxvirus-mediated recombination may be applied to map type-specific epitopes, to create novel pharmaceuticals such as hybrid interferons, to study receptor-binding or enzyme substrate specificities, or to mimic the antigenic diversity found in numerous pathogens.

Effective tumor immunotherapy directed against an oncogene-encoded product using a vaccinia virus vector.

We have constructed a vaccinia virus recombinant that expresses the extracellular domain of the rat neu oncogene-encoded protein, a 185-kDa transmembrane glycoprotein termed p185. Strain NFS mice immunized with this recombinant virus developed a strong antibody response against the neu oncogene product and were fully protected against subsequent tumor challenge with neu-transformed NIH 3T3 cells. No tumor immunoprotection was found when recombinant virus-immunized mice were challenged with Ha-ras-transformed NIH 3T3 cells. These data indicate that immunization with a single oncogene-encoded antigen can fully and specifically protect animals against tumor cells bearing this antigen.

Fibronectin binds to some bacteria but does not promote their uptake by phagocytic cells.

The involvement of plasma fibronectin in phagocytosis of bacteria was investigated by testing the binding of fibronectin to several species of bacteria and by evaluating the ability of fibronectin to promote binding and endocytosis of two species of these bacteria by phagocytic cells. Fibronectin binds non-covalently to Gram-positive and Gram-negative bacteria and to yeast but did not appear to be necessary or sufficient for uptake of Staphylococcus aureus and Salmonella typhimurium by several different phagocytic cell types.

Similarities and differences between the fibronectins of normal and transformed hamster cells.

Fibronectins from normal and virally transformed hamster cells were compared by several criteria. The fibronectin from transformed cells was similar to that from normal cells in being an intact dimeric glycoprotein with the ability to bind to gelatin, activated thiol-Sepharose, and cells. No evidence was found for proteolytic cleavages or abnormalities in disulfide bonding of transformed cell fibronectin. This fibronectin was also shown to be active in promoting cell attachment, elongation, and alignment. Therefore, the fibronectin produced by transformed cells is not defective. However, it was shown that the transformed cells were partially deficient in their capacity to bind fibronectins from either normal or transformed cells. This deficiency has implications for the significance of the loss of fibronectin on oncogenic transformation. Partial proteolysis of the fibronectins from normal and transformed cells gave rise to the same fragments. However, the glycosylated fragments from transformed cell fibronectin appeared somewhat larger than those from normal cell fibronectin. Analysis of fibronectin glycopeptides showed that transformation leads both to more branches per core and to a higher sialylation of the asparagine-linked complex carbohydrate side chains.

Cell surface fibronectin and oncogenic transformation.

Fibronectin is a large glycoprotein at the cell surface of many different cell types; a related protein is present in plasma. Fibronectin is a dimer of 230,000-dalton subunits and also occurs in larger aggregates; it forms fibrillar networks at the cell surface, between cells and substrata and between adjacent cells, and it is not a typical membrane protein. Cell surface fibronectin is reduced in amount or absent on transformed cells and in many cases its loss correlates with acquisition of tumorigenicity and, in particular, metastatic ability. Exceptions to the correlations with transformation and tumorigenicity exist. Loss of fibronectin and the resulting reduced adhesion appear to be involved in pleiotropic alterations in cell behavior and may be responsible for several aspects of the transformed phenotype in vitro. Fibronectin interacts with other macromolecules (collagen/gelatin, fibrin/fibrinogen, proteoglycans) and is apparently connected to microfilaments inside the cell.

10 nm filaments in normal and transformed cells.

An antibody was raised against an electrophoretically homogeneous protein from cultured fibroblasts and shown to be directed against 10 nm filaments. The antiserum did not stain microtubules or actin microfilaments. The distribution of 10 nm filaments in normal cells was studied during growth, spreading, locomotion, mitosis, and after treatment with colchicine and cytochalasin B. The 58,000 dalton subunit protein is apparently all polymerized in the filaments which are insoluble in nonionic detergent. The distribution of 10 nm filaments is altered by colchicine treatments which disrupt microtubules. The organization of 10 nm filaments is altered in transformed cells.

LETS glycoprotein: arrangement and function at the cell surface.

LETS is a large surface glycoprotein that is found on normal fibroblasts, but is absent or exists in amounts on transformed cells. Immunofluorescent staining shows LETS protein fibrils arrayed around the cells, particularly concentrated beneath the cells and in the area between neighboring cells. LETS glycoprotein is disulfide-bonded at the cell surface into dimers and higher aggregates. Other surface proteins also appear to participate in disulfide bonding. Reduction of disulfide bonds leads to increased release of LETS protein from the cells, as does the addition of cytochalasin B. Purified LETS protein added to transformed cells binds to the cells in a fibrillar array similar to that seen on normal cells. Addition of LETS protein leads to increased attachment and spreading of cells and causes transformed cells to align like normal ones. It also causes the appearance of actin cables in transformed cells, which normally lack them. These effects are inhibited by specific antisera to LETS protein or by reduction of disulfide bonds in the protein and are blocked or reversed by proteolysis. The results suggest that LETS protein plays a role in adhesion of cells.

Extensive disulfide bonding at the mammalian cell surface.

Cell surface proteins of cultured cells are disulfide bonded to a greater degree than are total cellular proteins. In particular, the "large external transformation-sensitive" (LETS) protein, a major surface protein, is present almost exclusively in disulfide-bonded complexes including homodimers and also higher aggregates held together by disulfide bonds or concovalent interactions. Other cell surface proteins also appear to be involved in disulfide bonding, both intramolecular and intermolecular. In virally transformed cells, LETS protein and its disulfide complexes are absent and certain other disulfide-bonded proteins are also not observed.

Synthesis, secretion, and attachment of LETS glycoprotein in normal and transformed cells.

LETS glycoprotein is a surface glycoprotein which is absent or greatly diminished on the surfaces of transformed cells. Normal cells secrete large amounts of this protein into the medium; transformed cell medium contains much less. The difference is not due to degradation of the soluble LETS protein. Biosynthesis of LETS protein can be studied by analysis of cell extracts by detergent extraction and immune precipitation and appears to proceed in transformed cells at a reduced rate compared with normal cells. Addition of inhibitors of protein synthesis to transformed cell cultures causes the small amount of LETS protein in the medium to attach to the cells. Addition of normal conditioned medium, which contains LETS protein, to transformed cells alters their morphology towards normal. Addition of purified LETS protein to transformed cells causes the cells to attach, spread, align with one another, and regain actin cables. The results indicate that LETS protein can exchange between cell surface and medium and that it affects cellular adhesion, morphology, and cytoskeleton.

Spatial organization at the cell surface.

Approaches are described for analysis of spatial organization of cell surface structure. Extraction of cells with nonionic and ionic detergents, chelating and chaotropic agents, salts, and reducing agents results in selective solubilization of surface proteins. Bisimidate disulfide-containing crosslinking reagents produce complexes containing surface proteins which can be analyzed by subsequent dissociation of the complexes. Disulfide-bonded complexes are also found without addition of crosslinkers, and reducing agents aid in extracting surface proteins. These results suggest a possible role for disulfide bonds in cell surface organization. Immunofluorescent staining of cells with antisera to LETS protein and to actin reveals fibrillar structures which survive NP40 extraction. These results indicate a complex organization at the cell surface which is amenable to analysis by permutations of the methods described.

Chromosomally depleted interspecific hybrid cell clones selected with cytotoxic antisera: utilization in the study of control of murine leukemia virus host-range.

A chromosomally stable mouse-Chinese hamster hybrid cell line was subjected to five rounds of selection with cytotoxic antisera raised in rabbits against either the parental mouse 3T3 cells or the parental Chinese hamster Wg-1 cells. Routine karyological analysis of clones isolated at each stage of serum selection revealed that treatment with either serum resulted in a limited loss of chromosomes (compared to the untreated hybrid cell cultured in parallel) and that the pattern of chromosome loss could not be correlated with the particular antiserum used for selection. However, more detailed analysis with the SSC-formamide C-banding technique, which identifies chromosomes containing a mouse centromere region, demonstrated that while large-scale chromosome loss was not achieved as a result of antiserum selection, the limited loss of chromosomes did, in fact, reflect a specific depletion of chromosomes in response to treatment with cytotoxic antiserum. Specific chromosomal elimination was shown to occur as early as the first round of antiserum treatment. Antigenic analysis of the serum-selected clones revealed a quantitative decrease in the expression of the species-specific surface antigens selected against, but no qualitative loss of antigens was detected. The results suggest that treatment with cytotoxic antiserum may select for clones that have lost specific chromosomes bearing genes regulating the expression of species-specific surface antigens, rather than for those demonstrating large-scale depletion of chromosomes bearing the corresponding structural genes. Some of these chromosomally depleted hybrid cell clones have been used (along with pseudotype viruses containing the genome of vesicular stomatitis virus within the envelope of murine leukemia virus, VSV [MuLV]), to study the mechanisms regulating MuLV replication in Chinese hamster cells. The results indicate that the restriction of MuLV replication in Chinese hamster cells operates at two levels: (a) an inability to adsorb to or penetrate Chinese hamster cells; and (b) an additional intracellular block which is dominant in the mouse-Chinese hamster hybrid cell clones examined. This latter block is presently under study.