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.

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.

Intricate combinatorial patterns of exon splicing generate multiple regulated troponin T isoforms from a single gene.

Mechanisms of alternative RNA splicing, important in the generation of protein diversity, are common but incompletely understood. Among the contractile proteins, troponin T exists in several isoforms, shown to be derived in part from a novel pattern of differential RNA splicing in the 3' region of the rat skeletal fast troponin T gene. In fact, this gene has a previously unsuspected capacity to encode multiple isoforms. The isolation of four distinct but related cDNAs from this gene, which share discontinuous subsegments of sequence identity in their 5' regions, and the determination of the genomic sequence, demonstrate that small exons with characteristic split codon structure are differentially spliced in intricate combinatorial patterns to generate a minimum of 10, and potentially 64, distinct troponin T mRNAs, encoding different isoforms, in a developmentally regulated and tissue-specific manner. At least two of these mRNAs are spliced from structurally identical primary transcripts, necessitating control by trans-acting factors.

A novel mechanism of alternative RNA splicing for the developmentally regulated generation of troponin T isoforms from a single gene.

Troponin T (TnT) is a major regulatory protein of the striated muscle that exhibits developmental and tissue-specific structural heterogeneity. The molecular basis for this heterogeneity was studied at the level of TnT structural gene organization and RNA expression. Two tissue-specific and developmentally regulated TnT mRNAs, alpha and beta, are derived from a single fast skeletal muscle TnT gene. Although otherwise structurally identical from amino acid 70 to the end of the 3' untranslated region, the alpha and beta TnT mRNAs differ by a small internal oligonucleotide coding for amino acids 229 to 242. These isoform-specific oligopeptides, both spanning the same internal portion of the TnT protein, are encoded by two distinct and adjacent miniexons in the TnT gene. Alternative and mutually exclusive splicing of these two miniexons results in the incorporation of either exon into the mature TnT mRNA and argues persuasively against a processive scanning model of RNA splice site selection.

Complex regulation of fibronectin synthesis by cells in culture.

We have studied the biosynthesis of fibronectin by NIL8 hamster embryo cells in various stages of growth. Pulse labeling with [35S]methionine, immunoprecipitation, electrophoresis, and autoradiography were employed to compare fibronectin synthesis by cells in subconfluent monolayers, confluent monolayers, and "aged" postquiescent monolayers. We have determined that fibronectin synthesis is proportionally low while cells are subconfluent, rises to maximal levels at confluence but just prior to quiescence, and then declines with increasing culture age in quiescent cultures. Furthermore, when cells are stimulated to grow, the effects on rates of fibronectin synthesis depend on the prior history of the cells; freshly confluent cells stimulated to grow show reductions in rate of fibronectin synthesis, whereas aged postquiescent cultures show increases in this rate. These results are in contrast to those on the rates of synthesis of other proteins (including the precursor to the C3 component of complement), which strictly correlate with quiescence and do not decline significantly with culture age postconfluence. We have also determined that the microheterogeneity of pulse-labeled fibronectin differs between exponentially growing and quiescent cells and that it becomes less heterogeneous once cells are quiescent. We conclude that the microheterogeneity of pulse-labeled fibronectin and the proportionate amount of total fibronectin synthesized both depend on growth state prior to the entry of cells into quiescence and that they depend on culture age thereafter.

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.

Relationships between fibronectin (LETS protein) and actin.

Double label immunofluorescence was used to study the distribution of fibronectin (LETS protein), actin and intermediate filaments in cultured cells. No relationship was observed between fibronectin and intermediated filaments, but fibronectin and actin showed coincident staining in a large proportion of cells during spreading or when fully spread. The distributions of actin and fibronectin staining during the course of cell spreading progressed through a series of patterns. Certain actin patterns correlated with certain fibronectin patterns. When fibrillar patterns developed, there was correspondence between the two fibrillar arrays in 80--100% of the cells. These results suggest a transmembrane relationship between microfilament bundles and fibronectin. We propose that fibronectin may participate in the formation of attachment plaques and discuss the interrelationship between plaques, microfilament bundles and fibronectin in cell-substratum and cell-cell contacts.

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.

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.

Selective depletion of chromosomes in a stable mouse-Chinese hamster hybrid cell line using antisera directed against species-specific cell surface antigens.

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 asthe 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.