Cyclin: a nuclear protein whose level correlates directly with the proliferative state of normal as well as transformed cells.
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Biological Transport↗
Biomedical subjects
R Bravo
Publications and source records attributed to R Bravo.
Epidermal growth factor inhibits the synthesis of the nuclear protein cyclin in A431 human carcinoma cells.
The effect of epidermal growth factor (EGF) on the synthesis of the nuclear protein cyclin and its relationship with cell proliferation has been studied in human carcinoma A431 cells. Quantitative two-dimensional gel electrophoretic analysis of [35S]methionine-labeled polypeptides from EGF-treated and untreated A431 cells showed that the synthesis of cyclin (0.07% of total labeled protein) could be decreased to 20% (less than 0.01% of total labeled protein) by EGF at a concentration of 100 ng/ml. A good correlation between the effect of different concentrations of EGF on cyclin synthesis and A431 cell proliferation was found. A431-derived cells resistant to the growth-inhibitory effect of EGF showed no significant changes in cyclin synthesis after EGF treatment. Taken together, these results support the idea that cyclin may be an important protein that is involved in the control of cell proliferation.
Carcinoma↗
Transcytosis of the G protein of vesicular stomatitis virus after implantation into the apical membrane of Madin-Darby canine kidney cells. II. Involvement of the Golgi complex.
In the preceding paper (Pesonen M., W. Ansorge, and K. Simons, 1984, J. Cell Biol., 99:796-802), we have shown that transcellular transport of the membrane glycoprotein G of vesicular stomatitis virus implanted into the apical membrane of Madin-Darby canine kidney cells is transcytosed through the endosomal compartment to the basolateral plasma membrane. To determine whether the Golgi complex was involved in this process, G protein lacking sialic acid or all of the terminal sugars was implanted into the apical membrane and allowed to move to the basolateral membrane. Using the criteria of endoglycosidase H sensitivity, binding to Ricinus communis agglutinin and two-dimensional gel electrophoresis, the sugars on the transcytosed G protein were found to be the same as in the starting material. The absence of any involvement of the Golgi complex in transcytosis was supported by subcellular fractionation studies in which transcytosing G protein was never found in fractions containing galactosyl transferase.
Animals↗
Differential immunological crossreactivity of HeLa keratin antibodies with human epidermal keratins.
HeLa cells contain four keratin-like proteins having molecular weights of 50,000 (IEF 31), 48,500 (IEF 36), 44,000 (IEF 44), and 43,500 (IEF 46), respectively. Mouse polyclonal antibodies prepared against two of these keratins (IEF 31 and 46) have been used in this study to identify human epidermal keratins with common antigenic determinants. Using a sensitive immunoprecipitation procedure we show that the IEF 31 antibody crossreacts with three human acidic epidermal keratins, termed K1, K2, and K3, having molecular weights of 44,000, 47,500, and 54,000, respectively. One of these keratins (K1) comigrated with HeLa keratin IEF 44 and exhibited an identical one-dimensional peptide map. This protein is also abundant in basaliomas. In contrast to these results, the IEF 46 antibody showed no crossreactivity with any of the human acidic or basic [35S]methionine-labeled epidermal proteins. The lack of crossreactivity of this antibody was further confirmed by indirect immunofluorescence staining of cryostat sections from human split skin. These results emphasize both the similarity and diversity of antigenic determinants among HeLa and epidermal keratins.
Antibody Specificity↗
Proteins IEF (isoelectric focusing) 31 and IEF 46 are keratin-type components of the intermediate-sized filaments: keratins of various human cultured epithelial cells.
Mouse polyclonal antibodies have been raised against two human proteins (IEF [isoelectric focusing] 31, Mr = 50,000; IEF 46, Mr = 43,500) that have previously been shown to be present in HeLa cytoskeletons enriched in intermediate-sized filaments. Immunoprecipitation studies show that both proteins share common antigenic determinants with each other and with the putative human keratins IEF 36 and 44, also present in HeLa cytoskeletons. Indirect immunofluorescence studies showed that both antibodies revealed similar filamentous networks in various cultured epithelial cells of human origin. These included AMA (transformed amnion), HeLa (cervical carcinoma), normal amnion cells, Fl-amnion (transformed amnion), WISH-amnion (transformed amnion), Chang liver (liver), and Detroid-98 (sternal marrow). Human cells that did not react with both antibodies included skin fibroblasts, lung fibroblasts (WI-38), SV40-transformed lung fibroblasts, Molt 4 (leukemia), lymphocytes, and monocytes. These results were in complete agreement with the presence or absence of both proteins in two-dimensional gels of the different cell types. Exposure of AMA cells to demecolcine (24 h; 10 micrograms/ml) caused the total collapse of vimentin filaments but, as seen by indirect immunofluorescence, caused only a partial redistribution of the IEF 31 and 46 filaments. These results are taken to suggest that both proteins are components of the intermediate-sized filaments of the "keratin" type. The antibodies could be clearly differentiated by staining human bladder carcinoma EJ 19 cells, as only the IEF 46 antibody stained a filamentous network in these cells The occurrence of keratins IEF 31, 36, 44, and 46 in different cultured human epithelial cells has been studied using two-dimensional gel electrophoresis.
Antibody Specificity↗
Synthesis and conversion of the renin precursor in vitro.
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Animals↗
Architecture and polypeptide composition of HeLa cytoskeletons. Modification of cytoarchitectural polypeptides during mitosis.
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Cytoskeleton↗
Putative association of mitochondria with a subpopulation of intermediate-sized filaments in cultured human skin fibroblasts.
Cultured cell Triton cytoskeletons enriched in intermediate filaments by extraction in high and low salt show, in addition to known intermediate-filament proteins and actin, a few other tenaciously bound polypeptides. An antibody raised against one of these polypeptides (IEF 24) is shown to react specifically with mitochondria of different cell types. With methanol-acetone fixation, required for the antibody reaction, the characteristically long mitochondria of human skin fibroblasts fragment into strings of beads, as identified by antibody staining in the light microscope and whole-mount electron microscopy. From the colinear organization of these beads, their codistribution with the intermediate-filament network and their retention in salt-extracted cytoskeletons, it is suggested that a subpopulation of the intermediate filaments can serve as an anchorage site for mitochondria.
Amnion↗
Changes in the relative proportion of transformation-sensitive polypeptides in giant HeLa cells produced by irradiation with lethal doses of x-rays.
Irradiation of HeLa cells with 1,100 rads (1 rad = 0.01 J/kg = 0.01 Gy) of x-rays yielded a pure population of giant cells 5-7 days after irradiation. These cells do not divide but go through an intermittent DNA synthetic phase. The population of giant cells in S phase (8%) is considerably lower than that of control asynchronous HeLa cells (30%), but 80% of the giant cells go through S phase as determined by 48-hr labeling with [3H]thymidine. Previous studies with high-resolution two-dimensional gel electrophoresis identified 58 [35S]methionine-labeled polypeptides common to human epithelial amnion cells and lung fibroblasts, whose rate of synthesis is sensitive to neoplastic transformation [Bravo, R. & Celis, J. (1982) Clin. Chem. (Winston-Salem, NC) 28, 949-955]. These polypeptides also have been identified in HeLa cells and other transformed human cells such as Detroit 98, Chang liver, Fl-amnion, and WISH-amnion [Bravo, R. & Celis, J. (1982) Clin. Chem. (Winston-Salem, NC) 28, 949-955]. After irradiation of HeLa cells and giant cell formation, the relative proportions of most of the transformation-sensitive polypeptides (43 of 47) reverted to levels similar to those observed in non-tumorigenic cells. This suggests that their relative proportions are dependent on the growth properties of the cells. In particular, the relative proportions of three polypeptides (designated 12g and 60d1 in isoelectric focusing and 27b in nonequilibrium pH gradient electrophoresis) were not affected, indicating that their reduced amounts in transformed cells could reflect a fundamental change that develops during transformation.
Cell Division↗
More than one-third of the discernible mouse polypeptides are not expressed in a Chinese hamster-mouse embryo fibroblast hybrid that retains all mouse chromosomes.
Gene expression at the level of polypeptide synthesis has been investigated in a somatic cell hybrid (20 BW-4) isolated after fusion of spontaneously transformed, tumorigenic Chinese hamster lung fibroblasts with mouse embryo fibroblasts. This hybrid exhibited suppression of tumorigenicity and retained--in addition to the parental Chinese hamster genome--copies of all mouse chromosomes as demonstrated by direct karyotype analysis and confirmed for 18 different mouse chromosomes by analysis of 18 different mouse isozymes. Two-dimensional gel electrophoresis of [35S]methionine-labeled polypeptides from hybrid 20 BW-4 showed that the overall polypeptide pattern corresponded to that of the hamster parent. All polypeptides detected in the hamster parental cells were also expressed in the hybrid although some of them were expressed in altered amounts. Of approximately 1200 labeled polypeptides revealed in the parental cells, 115 mouse polypeptides could be clearly distinguished from the hamster polypeptides due to their different electrophoretic mobilities. Forty-two of these (i.e., 37%) were not expressed in the hybrid 20 BW-4. These observations were confirmed by analysis of another independently isolated hybrid (2W 23) of the same parental cells that also exhibited suppression of malignancy and that retained copies of all mouse chromosomes except no. 5. The results suggest that the genome of the tumorigenic cell after hybridization can suppress expression of more than one-third of the normal parental genome. The suppressed mouse genetic information is probably located on many, if not all, different mouse chromosomes. Even if the level of genetic suppression is high the mouse genome is able to reduce the tumorigenicity of the hamster parental cell.
Animals↗
In vitro studies on the conversion of the biosynthetic precursor of renin.
The initial translation product of mouse submaxillary gland mRNA has a molecular weight of about 50,000. We have now translated renin mRNA in frog oocytes which are known to be able to remove the so-called "pre"- or "signal"- sequence from products of injected mRNAs. From these injected oocytes, we could precipitate with antirenin a 48,000 dalton polypeptide. Treatment with kallikrein or trypsin of the oocyte product resulted in a decrease in amount of this 48,000 dalton protein and a corresponding increase in a 45,000 dalton protein, precipitable with antirenin. We conclude that the initial translation product of renin mRNA is a preprorenin of molecular weight 50,000, which in oocytes is enzymatically cleaved to render 48,000 dalton prorenin. Although kallikrein may be a physiological activator of renin, it appears to be not the only one since no mature, 40,000 dalton renin was formed.
Animals↗
Human proteins sensitive to neoplastic transformation in cultured epithelial and fibroblast cells.
We resolved the [35S]methionine polypeptides synthesized by normal [lung fibroblasts (WI-38), amnion cells] and transformed [SV40 transformed WI-38, AMA (spontaneously transformed amnion cells)] cultured human cells, using two-dimensional gel electrophoresis under conditions in which about 1300 polypeptides could be reproducibly separated. These studies demonstrated important changes in the relative proportions of several polypeptides that are present both in normal and transformed cells. Of a total of 400 common polypeptides that we quantitated for each cell type, 53 (22 basic and 31 acidic) varied by 40% of more in both cell pairs. Among these, we have identified vimentin (IEF 26), cyclin (IEF 49), and a tropomyosin-related polypeptide (IEF 52). No new major polypeptide was detected in the transformed cells, at least at the level of resolution currently achieved by this technique. Similar but qualitative studies of [32P]orthophosphate-labeled proteins revealed that, of 250 analyzed, only seven common phosphoproteins, including phosphovimentin (IEF 26e), varied consistently in both cell pairs. These results strengthen our previous conclusion that transformation results in changes in the relative proportions of polypeptides synthesized in normal and transformed cells rather than in the appearance of new polypeptides in transformed cells.
Amnion↗
Up-dated catalogue of HeLa cell proteins: percentages and characteristics of the major cell polypeptides labeled with a mixture of 16 14C-labeled amino acids.
A total of 1357 polypeptides [946 acidic (isoelectric focusing) and 411 basic (nonequilibrium pH-gradient electrophoresis)] from human HeLa cells have been separated and catalogued with use of high-resolution two-dimensional gel electrophoresis. Of these polypeptides, 1266 were detected by labeling cells with [35S]methionine, while the rest were revealed by silver staining or by labeling with a mixture of 16 14C-labeled amino acids. For convenience, all these polypeptides have been numbered and are indicated in a large fold-out protein map. The percentages of some of the major 14C-labeled proteins have been determined, and for some we list a few characteristics such as: variation during the cell cycle; cellular distribution in cytoplasts and karyoplasts; presence in Triton- and salt-extracted cytoskeletons; and phosphorylation and sensitivity to neoplastic transformation.
Autoradiography↗
Identification of a nuclear polypeptide ("cyclin") whose relative proportion is sensitive to changes in the rate of cell proliferation and to transformation.
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Animals↗
Identification of a nuclear and of a cytoplasmic polypeptide whose relative proportions are sensitive to changes in the rate of cell proliferation.
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Animals↗
Coexistence of three major isoactins in a single sarcoma 180 cell.
Actin is transformed sarcoma 180 cells is composed of the nonmuscle beta and gamma species and of a third, more acidic stable variant termed zeta. Two-dimensional peptide analysis shows that zeta is similar to beta actin, differing in the mobility of only one tryptic peptide. Several lines of evidence indicate that zeta is not a modified beta-actin species. This third actin species comprises 20% of the total labeled actin, has the same molecular weight as the beta and gamma actins and has a different mobility in isoelectric focusing gels from that of the known alpha actins from skeletal, cardiac and vascular smooth muscle. Like beta and gamma actin, zeta can be extracted with the actin depolymerizing factor from slime mold. Two-dimensional gel electrophoresis (isoelectric focusing) of the 35S-methionine-labeled polypeptides synthesized by a single sarcoma 180 cell showed that all three major actin species coexist within the same cell. This analysis also showed for the first time the coexistence and alpha and beta tubulin, vimentin, alpha actinin and three other polypeptides present in intermediate-filament-enriched cytoplast cytoskeletons (spots 12, 24 and 31). Determination of the ratio of gamma plus beta to zeta actin in different cytoskeletal preparations of intact and enucleated sarcoma 180 cells indicated that this actin species is not localized specifically to any of the major actin-containing structures preserved in the cytoskeletons.
Actinin↗
[35S]-methionine labelled polypeptides from HELA cells. Coordinates and percentage of some major polypeptides.
A total of 1169 [35S]-methionine labelled polypeptides (855-acidic (IEF) and 314 basic (NEPHGE) from 100 asynchronous human HeLa cells have been separated using high resolution two dimensional gel electrophoresis. Beside numbering these spots, we give for the major polypeptides their coordinates (M.W. and relative mobility respect to beta-actin (IEF) or NEPHGE), and percentage of total protein label. A few major polypeptides have been identified.
Electrophoresis, Polyacrylamide Gel↗
Distribution of HeLa cells polypeptides in cytoplasts and karyoplasts.
The polypeptide composition of cytoplasts and karyoplasts prepared form HeLa cells prelabelled with [35S]-methionine and enucleated with Cytochalasin B has been analyzed using high resolution two dimensional gel electrophoresis (IEF and NEPHGE). Of the 259 major proteins followed in this study we have identified 73 polypeptides (30 acidic(IEF) and 43 basic (NEPHGE)) that are present mainly in karyoplasts. One of these polypeptides (IEF 49) has previously been shown to be a polypeptide marker for cycling cells. A total of 59 polypeptides (27 acidic and 32 basic) were found to be present mainly in cytoplasts. Many polypeptides (109 acidic and 18 basic) including Y and beta-actin (60% in cytoplasts), beta-tubulin (60% in cytoplasts), vimentin (75% in cytoplasts) and alpha-actinin (65% in cytoplasts) were found to be present in both cellular fragments. These results could be of value in assigning the cellular distribution of potential regulatory proteins.
Cell Nucleus↗