Coordinated regulation by iron of the synthesis of two transformation-sensitive membrane proteins in NRK cells

Studies were designed to characterize the plasma membrane polypeptide composition of normal rat kidney (NRK) and virus-transformed NRK cells as a function of time following iron deprivation. Using this approach we found that rapid depletion of iron from the cells increases the amounts of two membrane-associated glycoproteins of apparent MW 160,000 (160 K) and 130,000 (130 K) in NRK cells. In contrast, virus-transformed NRK cells subjected to iron deprivation showed an altered induction phenomenon manifested by reduced levels of both 160 K and 130 K and altered time-sequence of the induction. Possible relationship of the 160 K and 130 K membrane-associated glycoproteins described in this study to growth control and viral transformation are discussed.     

Identification of a transformation-sensitive 110-kDa plasma membrane glycoprotein of rat hepatocytes

Monoclonal antibodies were used to define cell surface antigens which are present on rat hepatocytes but are absent from hepatoma cells. One monoclonal antibody, referred to as Be 9.2, recognizes a major component of purified rat liver plasma membranes with a Mr of 110 000. This antigen (gp110) was not found in the transplantable Morris hepatoma 9121 and 7777 nor on two cultured hepatoma cell lines. Isoelectric focussing showed that gp110 is a very acidic membrane component with an isoelectric point of 3.6 to 3.8. Treatment with neuraminidase reduced the Mr to 95 000. Gp110 while bound to the membrane was resistant to trypsin, but sensitive to papain. The tissue distribution of gp110 was examined by indirect immunofluorescence in frozen sections. The antigen was found on the bile canalicular domain of hepatocytes, the microvillous zone of enterocytes of the small intestinal villi, the luminal plasma membrane of acinar cells in the submaxillary and extraorbital gland and of epithelial cells of the vesicular gland. Gp110 could not be detected in the stomach, pancreas, large intestine, kidney, thymus, spleen, heart, lung, muscle cells and fibers and in the brain. Identical results were obtained by the use of an antiserum raised against purified gp110. They confirm the transformation-sensitive character of this glycoprotein. A possible identity with dipeptidyl peptidase IV and aminopeptidase M, which have similar molecular weights and are also present in rat liver on the bile canalicular domains, could be excluded. The results suggest that the loss of gp110 might be regarded as a marker for transformation or dedifferentiation of hepatocytes.     

Induction of motility and alteration of surface membrane polypeptides in lymphocytes by contact with autologous and allogeneic fibroblasts

Contact with cultured fibroblasts induced and maintained motile behavior in autologous and allogeneic human lymphocytes. After 3 h of contact with fibroblasts, 50 +/- 19% of the autologous lymphocytes were motile and after 24 h the corresponding figure was 49 +/- 18%. On a plastic surface the number of motile lymphocytes in the same individuals generally persisted below 15%. SDS-PAGE of iodine-labeled lymphocytes indicated that contact with fibroblasts but not with plastic for a 3-h period caused the appearance of a 300-kda band and the disappearance of several bands of lower molecular weight. During the course of T-lymphocyte activation by concanavalin A or allogeneic cells on a plastic surface, the number of motile forms did not reach a maximum (30 to 50% in separate individuals) until after 2 to 4 days in culture. Thus, in terms of both rate of development and number of motile forms, the fibroblast-dependent motility mechanism was more effective than conventional lymphocyte activation to blast transformation. Conditioned medium from fibroblasts did not induce motile behavior in the lymphocytes and did not provoke alteration of surface membrane polypeptides as revealed by iodination. Fibroblasts also triggered lymphocyte locomotion in serum-free medium, but their triggering effect was enhanced markedly by serum. The development and maintenance of lymphocyte motility required protein synthesis. These data suggest that during contact with fibroblasts lymphocytes acquire locomotor capacity by a mechanism different from activation to blast transformation.     

Enhancement of UDPgalactose: glycoprotein galactosyltransferase in cultured human skin fibroblasts by cationic polypeptides

UDPgalactose : glycoprotein galactosyltransferase in normal human skin fibroblast homogenates has been assayed using ovalbumin as an acceptor. The activity in the homogenate fraction sedimenting between 51 300 X g and 105 000 X g was enhanced by the addition of a number of catonic polypeptides of L-configuration but not by those of D-configuration. In contrast to the enhancing effect of poly(L-lysine), poly(L-glutamic acid) inhibited the activity. Poly(D-glutamic acid) had no effect. Cationic or anionic amino acid derivatives, spermine or spermidine had no effect on activity. The enhancement of transferase activity by poly(L-arginine) is probably due to an increase in V for UDPgalactose and ovalbumin. The implication of these results for the regulation of glycoprotein synthesis in cultivated skin fibroblasts and for the pathogenesis of cystic fibrosis is discussed.     

Age-related alterations in plasma membrane glycoprotein content and scheduled or unscheduled DNA synthesis.

WI-38 cells of various ages and SV40-transformed WI-38 cells were examined for differences in plasma membrane composition of glycoproteins and DNA synthesis. Sialic acid per milligram of protein content of the membranes of WI-38 cells decreased with passage of time in culture. Other glycoprotein fractions and alkaline phosphatase activity disappeared in the WI-38 cells with passage of time in culture (Phase III). Studies of DNA repair correlated with changes observed in the plasma membrane glycoprotein content of WI-38 cells over a passage of time in culture were also reported. Both the extent and rate of ultraviolet-induced unscheduled DNA synthesis remained relatively constant during the passage of the WI-38 cells until late phase III. At that time the extent of unscheduled DNA synthesis was measurably reduced. The number of cells in a population of phase III cells able to perform semiconservative DNA synthesis diminished with age in culture but not to an extent capable of explaining the observed changes seen in membrane composition of semiconservative DNA synthesis during passage of the cells in culture. Cells with an extended lifespan SV40-transformed WI-38 (VA 13.2 RA) cells, did not vary in membrane composition, semiconservative DNA synthesis, or unscheduled DNA synthesis over 200 serial subpassages of the cells in culture.     

The 21-kDa protein is a transformation-sensitive metalloproteinase inhibitor of chicken fibroblasts.

We report the electrophoretic purification and characterization of the 21-kDa protein, an extracellular matrix component synthesized during the early stages of transformation of chicken embryo fibroblasts infected with Rous sarcoma virus (Blenis, J., and Hawkes, S. P. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 770-774; Blenis, J., and Hawkes, S. P. (1984) J. Biol. Chem. 259, 11563-11570). The NH2-terminal amino acid sequence of the protein is greater than 60% identical to a consensus sequence of mammalian tissue inhibitor of metalloproteinases (TIMP). It shares several biochemical properties with other metalloproteinase inhibitors, including evidence of intrachain disulfide bonds and resistance to cleavage by trypsin. An electrophoretic assay employing a metal ion-dependent gelatinase from conditioned cell culture medium demonstrates inhibitor activity for purified 21-kDa protein. The 21-kDa protein is the major inhibitor in the extracellular matrix and appears unique in solubility properties among inhibitors with a TIMP-like sequence. Statistical analysis of amino acid composition data for these inhibitors defines two distinct groups (TIMP and TIMP-2) and supports a close relationship for the 21-kDa protein with the TIMP group. However, the apparent size and lack of glycosylation align it more closely with the TIMP-2 group of proteins. Therefore, it is possible that the 21-kDa protein is a variant of TIMP or, alternatively, represents a third protein within the metalloproteinase inhibitor family. This report provides the first evidence that avian metalloproteinase inhibitors are similar in sequence to their mammalian counterparts.     

Intracellular synthesis of mouse mammary tumor virus polypeptides: indication of a precursor glycoprotein.

Mouse mammary tumor virus polypeptides were detected in the cytoplasm of mouse mammary tumor cell cultures using immunological precipitation techniques. The anti-mouse mammary tumor virus serum precipitated the major virion glycoproteins gp49 and gp37.5/33.5 and a viral-related nonvirion glycoprotein of 76,000 daltons. Subcellular fractionation studies revelaed that the cell-associated virion glycoproteins were present in the membrane fraction. Pulsechase experiments indicated that a viral-related nonvirion glycoprotein of 76,000 daltons may be a precursor to one or more of the virion glycoproteins.     

Carbohydrate-deficient glycoprotein syndrome type 1: correction of the glycosylation defect by deprivation of glucose or supplementation of mannose

In the carbohydrate deficient glycoprotein syndrome (CDGS) type 1 glycoproteins with less and shorter N-linked oligosaccharides are synthesized due to a deficiency of phosphomannomutase. Glucose deprivation or mannose addition are shown to partially or fully correct the size of oligosaccharides incorporated into lipid linked oligosaccharides and nascent glycoproteins in skin fibroblasts from CDGS type 1 patients with a phosphomannomutase defect. The corrective effect is ascribed to regulatory mechanisms and/or metabolic pathways that bypass phosphomannomutase.     

Induction of glycolytic enzyme synthesis in proliferating fibroblasts. Study of phosphofructokinase, glucose phosphate isomerase and pyruvate kinase.

Specific activity of phosphofructokinase is 7-8-fold higher in exponentially growing human fibroblasts than in quiescent cells, but the difference is considerably less pronounced for two other glycolytic enzymes, glucose phosphate isomerase and pyruvate kinase. The ratio of the F-type to L-type phosphofructokinase subunits is essentially the same in growing and resting cells, 4:1. F-type-phosphofructokinase-related antigen concentration is decreased in resting cells as compared with proliferating fibroblasts, but relatively less than the enzyme activity; the ratio of the enzyme activity to the antigen concentration (immunological specific activity) is therefore lower in resting than in growing fibroblasts. Synthesis of phosphofructokinase, as a percentage of the total protein synthesis, is about 30-fold greater during the proliferative phase than in quiescent cells, but this difference is only 3-4-fold for glucose phosphate isomerase and pyruvate kinase. Modulation of the synthesis of phosphofructokinase therefore seems to be responsible for the changes of its specific activity in function of cell proliferation. The appearance of some inactive cross-reacting material in quiescent cells is probably due to post-translational alteration of the pre-synthesized molecules. Compared with other glycolytic enzymes, such as glucose phosphate isomerase and pyruvate kinase, phosphofructokinase seems to be the (or one of the) preferential target of glycolytic induction in proliferating cells.     

Phase-specific protein synthesis in human fibroblasts after synchronization by a double thymidine block

Kinetics of acid-insoluble non-histone protein synthesis during S and G2 cell cycle phases of diploid human fibroblasts and heteroploid transformed cells was investigated. Two distinct groups of protein with different kinetic pattern depending on the cell culture type were revealed. Export of one group of protein and turnover of the other group of acid-insoluble non-histone protein is arrested in heteroploid transformed cells.     

Inhibition of Tamm-Horsfall glycoprotein induction of alkaline phosphatase in cystic fibrosis fibroblasts by medium conditioned by normal cells.

It is confirmed that the level of alkaline phosphatase in fibroblasts derived from cystic fibrosis patients can be induced many-fold by growing the cells in the presence of Tamm-Horsfall glycoprotein. It is further shown that normal fibroblasts produce a "CF corrective factor" which markedly inhibits this phenomenon. These observations support a previous hypothesis on the nature of the metabolic defect in cystic fibrosis.     

Cloning and characterization of a cDNA encoding transformation-sensitive tropomyosin isoform 3 from tumorigenic human fibroblasts.

We isolated a cDNA clone from the tumorigenic human fibroblast cell line HuT-14 that contains the entire protein coding region of tropomyosin isoform 3 (Tm3) and 781 base pairs of 5'- and 3'-untranslated sequences. Tm3, despite its apparent smaller molecular weight than Tm1 in two-dimensional gels, has the same peptide length as Tm1 (284 amino acids) and shares 83% homology with Tm1. Tm3 cDNA hybridized to an abundant mRNA of 1.3 kilobases in fetal muscle and cardiac muscle, suggesting that Tm3 is related to an alpha fast-tropomyosin. The first 188 amino acids of Tm3 are identical to those of rat or rabbit skeletal muscle alpha-tropomyosin, and the last 71 amino acids differ from those of rat smooth muscle alpha-tropomyosin by only 1 residue. Tm3 therefore appears to be encoded by the same gene that encodes the fast skeletal muscle alpha-tropomyosin and the smooth muscle alpha-tropomyosin via an alternative RNA-splicing mechanism. In contrast to Tm4 and Tm5, Tm3 has a small gene family, with, at best, only one pseudogene.     

Restoration of normal morphology, adhesion and cytoskeleton in transformed cells by addition of a transformation-sensitive surface protein.

Transformed cells lack a large, external, transformation-sensitive (LETS) glycoprotein which is a major surface component of their normal counterparts. Addition of LETS glycoprotein isolated from normal cells to transfomed cells restores certain morphological features and adhesive properties characteristic of normal cells. LETS protein is detected on the cell surface both by iodination using lactoperoxidase and by immunofluorescent staining. The surface distribution pattern detected by immunofluorescence is strikingly similar to that of normal cells. After addition of LETS protein, transformed cells also exhibit well defined actin cables which are not seen in untreated, transformed cells. All these alterations can be blocked by treating LETS protein with specific antisera or by subjecting it to mild trypsinization prior to addition to transformed cells. The effects are rapidly reversible by mild trypsinization, which removes the added LETS protein. The high rate of uptake of 2-deoxyglucose, characteristic of transformed cells, is not affected by LETS protein. These results suggest that LETS protein may have a role in cell attachment and spreading, and affect the organization of cytoskeleton.     

Induction of proline-rich glycoprotein synthesis in mouse salivary glands by isoproterenol and by tannins

Glycoproteins which contain about 45 mol% proline were dramatically induced in mouse parotid and submandibular glands by isoproterenol treatment, but these unusual proteins were not detected in control animals. These acid-soluble substances were obtained by extracting tissues with 10% trichloroacetic acid, as reported previously for isolating proline-rich proteins from rat submandibular glands (Mehansho, H., and Carlson, D.M. (1983) J. Biol. Chem. 258, 6616-6620). Three major proline-rich glycoproteins were induced in parotid glands with apparent molecular weights of 66,000 (GP-66p), 45,000 (GP-45p), and 27,000 (GP-27p), whereas only one such protein was expressed by the submandibular glands (66,000 (GP-66sm]. Both GP-66p and GP-66sm contained about 19% carbohydrate with the following molar ratios, respectively; GalNAc, 1.0, 1.0; Gal, 1.6, 2.3; GlcNAc, 0.8, 1.1; sialic acid, 0.9, 1.9. The peptide chains of GP-66p and GP-66sm appear to be identical by amino acid compositions, glycopeptide analysis, and preliminary amino acid sequencing data. Northern blot analysis of RNAs from parotid glands of normal and isoproterenol-treated rats, probed with a 32P-labeled proline-rich protein cDNA, confirmed that control animals were devoid of mRNAs encoding these proteins and that isoproterenol treatment dramatically induced expression of these genes. Feeding sorghum high in tannins caused changes in the parotid glands similar to those observed upon isoproterenol treatment, as noted earlier with rats (Mehansho, H., Hagerman, A., Clements, S., Butler, L., Rogler, J., and Carlson, D.M. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 3948-3952). These glycoproteins have high affinities for tannins as demonstrated by competitive binding curves.     

Quantitation of a transformation-sensitive, adhesive cell surface glycoprotein. Decrease of several untransformed permanent cell lines

We have quantitated the transformation-sensitive, cell surface LETS glycoprotein on many untransformed cell types. By SDS-polyacrylamide gel electrophoresis, this trypsin-sensitive iodinatable glycoprotein comprises 1-3% of total cellular protein of the seven early passage cell types tested. In contrast, it constitutes less than 0.15% of the protein in four of six continuous cell lines. This decrease is reflected in alterations both in [14C]glucosamine labeling and in the immunofluorescent staining of early passage vs. these four permanent cell lines. These results help to clarify previous experiments in which CSP, a purified LETS protein, partially restored a fibroblastic phenotype to cells transformed by tumor viruses. These findings also indicate that a major decrease in this cell surface glycoprotein can occur in the establishment of a continuous cell line without resulting in cellular transformation.     

Induction of chromosome damage by Neurospora endonuclease in repair-inhibited quiescent normal human fibroblasts

The purpose of these experiments was to determine the role of double-strand breaks in chromosome aberration formations. Quiescent normal human fibroblasts were treated with 3 μM nitrogen mustard and then allowed to repair their DNA damage for 24 h prior to cell fusion and induction of premature chromosome condensation. The extent of chromosome damage was determined in the G1 prematurely condensed chromosomes (G1 PCC). The presence of cytosine arabinoside and hydroxyurea during the repair period in order to accumulate single-strand DNA breaks resulted in an increase in the chromosome-break frequency. Treatment of these repair-inhibited cells with single-strand-specific neurospora endonuclease during fusion to change single-strand lesions into double-strand breajs resulted in a doubling of the aberration frequency. These results support the notion that double-strand breaks are important in chromosome-aberration formation.