Search

Two-dimensional gel electrophoresis of intermediate-sized filament-enriched cytoskeletons of epithelial monkey kidney TC7 cells has shown that they are composed of at least two keratins (isoelectric focusing 36, Mr = 48,500; IEF 46, Mr = 43,500; HeLa protein catalogue number) and vimentin. Indirect immunofluorescence as well as immunoelectron microscopy using antibodies directed against specific polypeptides sometimes revealed a discontinuous staining of keratin-containing filaments. Indirect immunofluorescence analysis of cells stained with keratin or vimentin antibodies also revealed a bright perinuclear staining in 58% of the cells in interphase. Of particular interest were focal centers from which filaments radiated. Double-label immunofluorescence using tubulin and keratin antibodies showed that these centers codistributed with focal arrays of microtubules (most likely centrosomes) in interphase cells but were not colocalized with centrioles in mitosis or, in many cases, with the microtubule organizing centers seen after release from nocodazole treatment. Treatment of TC7 cells with demecolcine (10 micrograms/ml, 20 hr) resulted in a drastic rearrangement of the keratin and vimentin filaments. Likewise, treatment with cytochalasin B (10 micrograms/ml, 1 hr) produced a star-like arrangement of the keratin and vimentin filaments and, in most cases, these codistributed with patches of actin. The results provide evidence for the interaction of intermediate filaments (keratins and vimentin) with both microtubules and microfilaments.

A cell population highly enriched in human epidermal basal cells has been obtained and characterized by using antibodies specific for various cell types in the epidermis. Quantitative two-dimensional gel electrophoretic analysis (isoelectric focusing) of [35S]methionine-labeled polypeptides from basal cells and simian virus 40-transformed keratinocytes showed that the basal cells synthesize very low amounts (less than 0.02% of the total protein) of the nuclear, transformation-sensitive protein cyclin as compared to the transformed cells, which synthesize this protein constitutively (0.15% of the total protein). Very low levels of cyclin were observed in total human epidermis, and preliminary studies of two basaliomas have shown a significant synthesis of this protein in these tumors. Immunofluorescence studies using antibodies to proliferating cell nuclear antigen that immunoprecipitate cyclin confirmed the above observations at least in the case of the cultured cells. Taken together, these results support the notion that cyclin may be a central component of the pathway(s) that controls cell proliferation.

Studies of the polypeptides synthesized by normal and transformed human cultured cells under a variety of physiological conditions have revealed a basic 54-kDa protein NEPHGE 10a, whose rate of synthesis is sensitive to changes in the rate of cell proliferation. This nuclear phosphoprotein, which we have termed "dividin" (present only in populations of cells committed to divide), is synthesized almost exclusively during the S phase of the cell cycle of transformed human amnion cells (AMA). Dividin synthesis is first detected late in G1 near the G1/S transition border, reaches a maximum late in S phase, and declines thereafter. As expected for an S phase-specific protein, no detectable synthesis of dividin was observed in growth-arrested normal human cultured cells of epithelial and fibroblast origin. These findings suggest a role for this protein in events leading to DNA replication and cell division.

The Drosophila Notch gene product is a transmembrane protein that functions as a receptor of intercellular signals in several Drosophila developmental processes. Two other transmembrane proteins, encoded by the genes Delta and Serrate, genetically and molecularly behave as Notch ligands. All these proteins share the presence of epidermal growth factor (EGF)-like repeats in their extracellular domain. The Notch protein has 36 EGF-like repeats, 2 of which, numbers 11 and 12, are required for the interaction with the Delta and Serrate ligands. We have isolated and molecularly characterized a Notch mutation in its Delta- and Serrate-binding domain that behaves genetically as both a Notch antimorphic and a loss-of-function mutation. This mutation, NM1, carries a Glu-->Val substitution in the Notch EGF repeat 12. The NM1 allele interacts with other Notch alleles such as Abruptex and split and with mutations in the Notch-ligand genes Delta and Serrate. The basis for the genetic antimorphism of NM1 seems to reside in the titration of Notch wild-type products into NM1/N+ nonfunctional dimers and/or the titration of Delta products into nonfunctional ligand-receptor complexes.

Immunofluorescence analysis of synchronously growing transformed human amnion cells (AMA) using autoantibodies specific for cyclin has revealed dramatic changes in the nuclear distribution of this protein during the S phase of the cell cycle. Cells in G1, G2, and mitosis exhibit weak staining with the antibody, while S-phase cells show variable patterns of staining in terms of both intensity and distribution of the antigen. Early in S phase, cyclin is localized throughout the nucleoplasm with the exception of the nucleoli. A similar, but stronger, staining pattern is observed as the cells progress through the S phase. At a later stage, before maximum DNA synthesis, cyclin redistributes to reveal a punctuated pattern with foci of staining throughout the nucleus. This pattern precedes a major change in the distribution of this protein, which is then detected in the nucleolus. At this stage, DNA synthesis is at or near a maximum. Thereafter, there are further changes in the distribution of this protein, with the pattern becoming punctuated and of decreasing intensity. All these staining patterns have also been detected in asynchronously growing normal human amnion cells (AF type), suggesting that the distribution of this protein is not a consequence of transformation. Analysis of cultured cells from several vertebrate species also revealed similar staining patterns. These results are consistent with the idea that cyclin is a central component of the pathway(s) leading to DNA replication and cell division.

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.

tRNAs from yeast (tRNAPhe and 4S RNA) and Escherichia coli (suIII+ tRNAITyr) have been transferred to mouse cells by means of a two-step transfer procedure [Loyter, Zakai, and Kulka (1975) J. Cell Biol. 66, 292-304; Schlegel and Rechsteiner (1975) Cell 5, 371-379]. In the first stage of the transfer tRNAs were incorporated into rabbit red blood cells (RBCs). Thereafter, the loaded erythrocytes were fused with recipient mouse cells by means of Sendai virus. At least 0.3-0.4% of the total input of tRNA used to load the RBCs could be transferred to mouse cells. Of the tRNA incorporated in the mouse cells, at least 50% could be recovered in the form of intact tRNA molecules when yeast 4S RNA was used. With E. coli suIII+ tRNAITyr a rather smaller proportion of the tRNA remained intact (33%). Although the loading of tRNA into RBCs is not essential for its uptake, we find that the transfer is four times more efficient with RBCs as a vehicle for the injection. Significantly, a fraction (2%) of the recipient cells possessed much more incorporated tRNA than the average cell when Sendai virus and loaded RBCs were used. Such cells were not found in control experiments in which tRNA uptake was induced by Sendai virus alone.

The gene Notch (N) codes for a transmembrane protein with an extracellular domain that has homologies to epidermal growth factors and an intracellular domain that could be involved in signal transduction. N null alleles cause the transformation of most epidermal cells into neuroblasts in central and peripheral nervous systems. Alleles of the same gene, called Abruptex (Ax), that map to the extracellular domain of N protein cause the absence of adult sensory organs. Both types of alleles show cell autonomy in mosaic analysis carried out in the last stages of the formation of adult sensory organs. The phenotypes are different: cells lacking N gene products differentiate as sensory organ mother cells early and as its neural sublineage later, whereas in the homozygous Ax condition epidermal cells do not enter the sensory organ mother cell pathway. The results indicate that N gene products act internally in the cell, probably as receptors of intercellular signals both in sensory organ mother cell singularization and in fate specification of its daughter cells. Ax mutations behave as an excess of N+ function in this signal transduction process. N proteins modified by these mutations act as constitutively activated.

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.

Elongation factor 2 was identified in the two-dimensional gel patterns of asynchronous human amnion cells (AMA) by comigration with purified rabbit reticulocyte elongation factor 2 and by two-dimensional gel immunoblot analysis using a specific rabbit polyclonal antibody. In all, four polypeptides were identified corresponding to isoelectric focusing polypeptides 2713 (95.0 kDa), 2714 (94.8 kDa), 3727 (94.8 kDa), and 3702 (93.6 kDa) (listed in order of decreasing pI values) in the computerized comprehensive two-dimensional gel data base of human AMA proteins. The relative proportion of two of these variants (isoelectric focusing polypeptides 3727 and 3702), which are phosphorylated, increased dramatically during mitosis. Since phosphorylation is known to render elongation factor 2 inactive in translation, this observation may partly explain the decline in the rate of protein synthesis observed during cell division.

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.