Limited lifespan in somatic cell hybrids and cybrids

The transformed phenotype is believed to be dominant in fusions between limited lifespan cells and transformed cells, based on heterokaryon experiments and on the isolation of transformed hybrids from mass cultures of fused cells. A series of fusions has been performed between limited lifespan Lesch-Nyhan fibroblast cells and a permanent HeLa cell line with a complementary genetic marker. The growth of independently isolated hybrid clones was followed in parallel with Lesch-Nyhan cells. In fusions involving Lesch-Nyhan cells which had completed about 50% of their lifespan, all hybrids initially show fibroblastic properties. Thirty-five hybrids had a limited lifespan slightly longer than Lesch-Nyhan controls. Three other hybrid clones, and all mass cultures of hybrids, showed the appearance of transformed colonies at a rate of approx. one transformant in 2 × 10₅ hybrid cells. These transformed cells showed anchorage independence, low serum requirement, chromosome loss, and have been maintained in culture for 50–100 population doublings with no signs of senescence. Fusions involving enucleated HeLa cells did not show transformation. Fusions with senescent Lesch-Nyhan cells yielded hybrids which grew beyond the normal Lesch-Nyhan cell lifespan, but which again showed limited lifespan and low frequency transformation. It is concluded that limited lifespan is expressed in a dominant manner in these fusions, and that transformation or “escape from senescence” is a low frequency event requiring the presence of the HeLa nucleus.     

Measurement of cell proliferation in microculture using Hoechst 33342 for the rapid semiautomated microfluorimetric determination of chromatin DNA

We report the development and characterization of a semiautomated method for measurement of cell proliferation in microculture using Hoechst 33342, a non-toxic specific vital stain for DNA. In this assay, fluorescence resulting from interaction of cell chromatin DNA with Hoechst 33342 dye was measured by an instrument that automatically reads the fluorescence of each well of a 96-well microtiter plate within 1 min. Each cell line examined was shown to require different Hoechst 33342 concentrations and time of incubation with the dye to attain optimum fluorescence in the assay. In all cell lines, cell chromatin-enhanced Hoechst 33342 fluorescence was shown to be a linear function of the number of cells or cell nuclei per well when optimum assay conditions were employed. Because of this linear relation, equivalent cell doubling times were calculated from growth curves based on changes in cell counts or changes in Hoechst/DNA fluorescence and the fluorimetric assay was shown to be useful for the direct assay of the influence of growth factors on cell proliferation. The fluorimetric assay also provided a means for normalizing the incorporation of tritiated thymidine ( [3H] TdR) into DNA; normalized values of DPM per fluorescence unit closely paralleled values of percent 3H-labelled nuclei when DNA synthesis was studied as a function of the concentration of rat serum in the medium. In summary, the chromatin-enhanced Hoechst 33342 fluorimetric assay provides a rapid, simple, and reproducible means for estimating cell proliferation by direct measurement of changes in cell fluorescence or by measurement of changes in the normalized incorporation of thymidine into DNA.     

Effect of mitochondrial DNA composition on the cellular properties of interspecific hybrid cells

Four subclones with single species of mitochondria and three subclones with both parental mitochondria were isolated from a mouse-rat hybrid cell line H2. The effects of the coexistence of different species of mitochondria on cellular properties were examined in these clones. Growth properties were studied by comparing the plating efficiencies and doubling times. The numbers of growing colonies and the doubling times of all the subclones were found to be almost the same, indicating that these growth properties were not affected by the presence of both mouse and rat mitochondria within the cells. The correlation between the expression of chloramphenicol (CAP)-resistance and the relative contents of mtDNA of CAP-resistant (CAP^r) rat and CAP-sensitive (CAP^s) mouse parent cells in the subclones were also examined. The expression of CAP resistance was measured as the relative plating efficiency. Subclones with a high content of mtDNA from CAP^r rat parent cells showed high relative plating efficiency.     

Isolation and characterization of intraspecific cybrids : Effect of mitochondrial DNA on their cellular properties

Cybrid clones were obtained by fusing whole cells of rat glioma C6BU-1, resistant to 5-bromodeoxyuridine (BrdU), with cytoplasts of embryonic rat 3Y1CAP cells, resistant to chloramphenicol (CAP), in selective medium with BrdU and CAP. The clones resistant to BrdU and CAP were confirmed to be cybrids by chromosome and mtDNA analyses. More than half the mtDNA of all the cybrid clones was from the 3Y1CAP cells. After cultivation of a cybrid clone Y22 for 3 months in the absence of CAP, subclones were isolated. One subclone Y22-22 contained predominantly mitochondrial DNA (mtDNA) from the 3Y1CAP cells. Using this subclone, the effects of the mitochondrial genome on cellular properties were examined. The growth patterns, expression of glioma-specific beta-adrenergic receptor, and composition of the major proteins of C6BU-1 cells were not affected by transmitted mtDNA from the 3Y1CAP cells. This procedure for isolating cells containing predominantly foreign mtDNA will be useful in studies on the interaction between genomes of the mitochondria and nucleus.     

Size maturation process of nascent DNA intermediates into chromosomal-sized DNA in Tetrahymena pyriformis macronuclear DNA replication

An analysis was made of the size maturation process of nascent DNA intermediates in macronuclear DNA replication of Tetrahymena pyriformis. The first discrete size class of nascent intermediates larger than Okazaki fragments were replicon-sized DNA (about 2 X 10(7) D single-stranded (ss) DNA) and accumulated in cells treated with cycloheximide. On removal of cycloheximide, the replicon-sized intermediates were converted to middle-sized intermediates (about 10 X 10(7) D ssDNA) and then merged into chromosomal-sized DNA. As indicated by either aphidicolin inhibition or the technique of the photolysis of bromodeoxyuridine (BrdU)-substituted DNA with long-wave ultraviolet light, four to eight replicon-sized intermediates were joined together to form a middle-sized intermediate after rapid sealing by DNA synthesis of the late-replicating regions located between adjacent replicon-sized intermediates. The late-replicating regions may represent the short gaps or terminal regions where DNA synthesis was retarded by cycloheximide, since the size of late-replicating regions was suggested to be shorter than the replicon size by DNA fiber autoradiography. Therefore, it is probable that four to eight completed replicons are joined as a group such as a replicon cluster, as has been reported in DNA replication of other eukaryotic cells.     

Cell division cycle in mammalian cells : VIII. Mapping of G1 into six segments using temperature-sensitive cell cycle mutants

The G1 blocks in three temperature-sensitive (ts) Syrian hamster cell-cycle mutants have been mapped in relation to other G1 landmarks. Two mutants reported here, ts-559 and ts-694, show defective progression only in G1. When shifted from the permissive temperature of 33 degrees C to the non-permissive temperature of 39 degrees C, G1 cells of these two mutants show no further cell cycle progression, while cells in S, G2 and mitosis progress through the cell cycle but become blocked after entering G1. The two mutants complement each other, and also complement the previously reported mutant ts-550C with blocks in both G1 and G2 of the cell cycle. The locations of the G1 blocks in both ts-559 and ts-694 are before the hydroxyurea arrest point. The G1 ts point in ts-694 is prior to the isoleucine deprivation and serum starvation points, while the G1 block in ts-559 is after the serum starvation point but before the isoleucine block. Other G1 block points which have been reported are in mutants of different species and isolated in different laboratories, causing difficulties for relative positioning of the blocks in G1. The mutants for mapping in this study have been isolated from the same cell line. The G1 ts arrest points of ts-559 and ts-694, and that found in ts-550C, together with nutritional deprivations and metabolic inhibitors, provide seven reference points which divide G1 into six segments, each of which is bracketed by two adjacent points: mitosis, ts-694 block, serum starvation arrest point, ts-559 block, isoleucine deprivation arrest point, ts-550C block, hydroxyurea or excess-thymidine arrest segment.     

Identification of plectin in different human cell types and immunolocalization at epithelial basal cell surface membranes

The occurrence of plectin in various human tissues and cell lines was investigated using immunofluorescence microscopy and antibody gel overlay/immunoblotting techniques. Plectin was identified in all tissues and cell lines tested, namely placenta, kidney, cornea, foreskin and eyelid skin, skin fibroblasts, monocytes, keratinocytes and HeLa cells. In frozen sections of cornea and skin, plectin was found to be enriched at epithelial basal cell surface membranes. Consequently, antibodies to plectin could serve as a tool in the classification of mechanobullous diseases.     

Co-dominant markers for the selection of somatic dihybrids and polyhybrids

Resistance to ouabain and puromycin are shown to represent very useful co-dominant characters for the selection of somatic hybrids between mammalian cells, after fusion with polyethylene glycol. We therefore used, with success, a number of Chinese hamster and mouse cell lines carrying these markers in association with thymidine kinase and hypoxanthine-guanine-phosphoribosyl transferase deficiency for selection of hybrids of triparental origin.     

Differential effects of pure human alpha and gamma interferons on fibroblast cell growth and the cell cycle

Pure human alpha and recombinant gamma interferons had differential effects on two strains of fetal lung fibroblasts in vitro. Alpha interferon had little effect on long-term cell growth, whereas gamma interferons, both glycosylated and non-glycosylated, were cytotoxic. However, when synchronized cells were studied, alpha interferon prolonged both G1 and S + G2 phases of the cell cycle, whereas gamma interferon only affected the G1 phase.     

In situ hybridization of DNA sequences in human metaphase chromosomes visualized by an indirect fluorescent immunocytochemical procedure

In situ hybridization and immunocytochemical procedures are described which allow identification and localization of specific DNA sequences in human chromosomes by fluorescence microscopy. With this method the genes coding for 18S and 28S ribosomal RNA (rRNA) were localized on human metaphase chromosomes by in situ hybridization of 18S or 28S rRNA followed by an immunocytochemical incubation with specific anti-RNA-DNA hybrid antiserum. Visualization of the immunocytochemically localized RNA-DNA hybrids was achieved by indirect immuno-fluorescence. The antiserum against RNA-DNA hybrid molecules was raised in a rabbit injected with poly(rA)-poly(dT). The specificity of the sera was determined using a model system of Sephadex beads to which various nucleic acids had been coupled. To obtain optimal specific fluorescence and very low aspecific background staining, several modifications of the in situ hybridization and the immunocytochemical procedures were investigated. The use of aminoalkylsilane-treated glass slides, removal of unbound fluorochrome molecules from the fluorochromelabelled antibody solutions and application of a proteinase K treatment during the hybridization procedure and the immunocytochemical procedure proved to be essential for optimal results.     

Expression of transformation-associated traits in the myogenic cell lines L6 and L8

The presence of cellular alterations, usually associated with transformation, has been studied in two permanent myogenic cell lines, L6 and L8, that retain the ability to differentiate in vitro. We present evidence that, beside being immortal, both cell lines are anchorage-independent for proliferation, a feature not found in primary muscle cells. L6 secretes constitutively high levels of plasminogen activator. L8 is able to undergo multinucleation in the presence of cytochalasin B (cytB) and is tumorigenic in vivo. Single anchorage-independent clones were shown to possess differentiative potentials similar to those of the parental line. Moreover, cell fusion could be directly observed in L8 while still growing as colonies in soft agar. We discuss our data with respect to (i) the reported differences in the regulation of differentiation between primary myogenic cells and continuous cell lines; (ii) the relationship between transformation and differentiation in muscle cells.     

Reinitiation of host DNA synthesis in senescent human diploid cells by infection with simian virus 40

Human diploid fibroblasts, TIG-1, cease to proliferate at about 60-62 population doubling level. In their senescent state used in this study, the percentage of nuclei labeled by [3H]thymidine for 48 h was around 1-2% in fresh medium containing 5-40% fetal bovine serum. The percentage of labelled nuclei increased up to 10-fold after infection with SV40. This increase reflects stimulation of cell DNA synthesis because: 1. The increase also occurred when ts A900 was used for infection at the non-permissive temperature, under these conditions viral DNA synthesis is inhibited; 2, the increase paralleled the stimulation of [3H]thymidine incorporation into DNA in a Hirt-precipitate fraction from SV40-infected cells. UV-irradiated SV40 had reduced ability to induce DNA synthesis. A viable deletion mutant of SV40, d1940, had almost the same activity to induce cell DNA synthesis as did wild-type SV40. Equilibrium density gradient centrifugation analysis of DNA labelled with 5-bromodeoxyuridine (BrdU) supported semiconservative replication rather than repair synthesis. We conclude that a considerable fraction of human diploid cells in a senescent population initiate host DNA replication by infection with SV40, although these cells cannot be stimulated with fetal bovine serum.     

The isolation of an antimycin A-resistant human cell line

An antimycin A-resistant derivative of the human cell line, D98, has been obtained by selective mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. The derivative, designed MA65, is capable of continuous growth in 15 microM antimycin and the resistant phenotype is stable in the absence of selection. MA65 is not cross-resistant to chloramphenicol or triethyl tin. Crude membrane preparations from MA65 after propagation in medium containing antimycin have normal succinate-cytochrome c oxidoreductase activity and the respiratory activity of whole cells continues in the presence of the drug. The mitochondrially synthesized proteins of D98 and MA65 are similar when compared on sodium dodecyl sulphate (SDS), or isoelectric focusing gels, but there is a reproducible difference in the extent of labelling of one band detected by isoelectric focusing. Genetic analysis is consistent with the existence of a cytoplasmically localized determinant conferring resistance.     

Analysis of a Chinese hamster temperature-sensitive cell cycle mutant arrested in early S phase

E36 ts24 is a temperature-sensitive cell cycle mutant which has been derived from the Chinese hamster lung cell line E36. This mutant is arrested in phase S when incubated at the restrictive temperature (40.3 degrees C) for growth. At this temperature, proliferation of the mutant cells ceases after 10 h. About 2 h earlier, DNA synthesis is arrested. These kinetic studies indicate that the execution point of the mutant cells is in early S phase well beyond the G1/S boundary. The pattern of replication bands in E36 ts24 cell grown for 9 h at 40.3 degrees C strengthen the kinetic studies and map the execution point to early S phase. The exact point of arrest of the mutant cells in phase S was mapped in early S phase near the execution point. At the point of arrest the cells continue to synthesize DNA at at a high rate but practically all of the newly synthesized DNA is degraded. This high rate of DNA degradation is limited to nascent DNA at the point of arrest. In the presence of 5-bromodeoxyuridine (5-BudR), the last E36 ts24 cells which reach mitosis at the restrictive temperature for growth show asymmetric replication bands which illustrate DNA degradation and resynthesis occurring in these cells at 40.3 degrees C.     

Decrease in the average size of replicons in a Werner syndrome cell line by Simian Virus 40 infection

We have measured the distance between replicon initiation sites as well as the rate of DNA chain elongation in Simian Virus 40 (SV40)-infected and uninfected Werner syndrome (WS) and normal cell lines by DNA fiber-autoradiography. There was no difference in the rate of chain elongation among these cell lines. On the other hand, the replicon center-to-center distance was clearly longer in WS fibroblasts than that in normal fibroblasts. SV40 infection changed the center-to-center distance in WS cells toward that in normal cells.     

Reversible and irreversible stages in the transition of cell surface marker during the differentiation of pluripotent teratocarcinoma cell induced with retinoic acid

Treatment of a pluripotent teratocarcinoma cell line with retinoic acid (RA) results in the disappearance of peanut agglutinin (PNA) receptors, accompanied with the decrease in F9 antigens and the enhanced secretion of plasminogen activator. However, this type of differentiation was inhibited by feeder cells. Furthermore, the transition of PNA receptor was reversible on the cells treated with RA for 2 days and became irreversible by an additional 2-day treatment with RA. Thus, two stages of teratocarcinoma cell differentiation—reversible and irreversible—were demonstrated.     

Association of ganglioside-protein conjugates into cell and Sendai virus. Requirement for the HN subunit in viral fusion

We have prepared several electron and light microscopic labels of epidermal growth factor (EGF) to analyse the morphologic features of its binding and internalization by cultured cells. These include a ferritin conjugate of EGF, a covalent conjugate of EGF and horseradish peroxidase (EGF-HRP), a colloidal gold marker system using EGF-HRP as a primary antigen, and a covalent complex of EGF with rhodamine-labelled lactalbumin. All of the light and electron microscopic labels showed similar patterns of binding. EGF initially bound to diffusely distributed cell surface receptors at 4 degrees C. The EGF-receptor complexes clustered into clathrin-coated pits on the cell surface only when the temperature was raised to 37 degrees C. In KB and Swiss 3T3 cells, this was followed by rapid internationalization into receptosomes, compartmentalization into the Golgi system, clustering in the clathrin-coated regions of the Golgi, and finally delivery into lysosomes from the Golgi. This general pathway was seen in Swiss 3T3 cells which have a low number of EGF receptors, KB cells which have a moderate number of receptors and A431 cells that have a high number of receptors. However, the ruffling activity induced in A431 cells by EGF produced some internalization through macropinosomes, making the pathway of entry more difficult to evaluate. Double label experiments showed that EGF is internalized together with alpha 2-macroglobulin and adenovirus particles. These data clarify the route of entry of EGF in different cell types using multiple labels, and shows that it enters cells through the same coated pit entry pathway as most other ligands previously examined.     

Resolution of mitochondrial DNA structures in the large yeast Wickerhamia fluorescens

DAPI (4′6 Diamidino-2-phenylindole) staining of the large yeast Wickerhamia fluorescens revealed extensive cytoplasmic staining material attributable to mitochondrial DNA (mtDNA), often present in large network-like structures. The maintenance of this mtDNA appears to be insensitive to a variety of mitochondrial specific mutagens, suggesting that W. fluorescens may be classified as a petite negative yeast. Restriction enzyme analysis generated a unit genome size of 42(10⁶) D for this mtDNA which, together with determinations of the average mtDNA per cell, allowed an estimate of the cellular copy number of mitochondrial genomes. A physical map of this mtDNA was also derived. These experiments suggested models which might reflect the cytological structures resolved by DAPI staining in W. fluorescens relative to other yeasts.     

Formation of vinculin plaques precedes other cytoskeletal changes during retinoic acid-induced teratocarcinoma cell differentiation

Immunofluorescence and immunoblotting techniques were used to study the presence and distribution of vimentin and keratin type intermediate filaments, actin, and vinculin (130 kD protein) during retinoic acid (RA)-induced differentiation of F9 embryonal carcinoma (EC) cells. The undifferentiated F9 cells regularly expressed vimentin, usually concentrated close to the nucleus, but not keratin. Actin appeared as short intracellular filaments and as spikes at the edges of the colonies, together with some diffuse cytoplasmic staining. F9 cells also showed a weak, diffuse cytoplasmic vinculin-specific fluorescence in addition to occasional small focal vinculin patches at the edges of the cell colonies. RA treatment led into a series of changes in the cytoskeletal organization of F9 cells. These changes were initiated by the appearance of distinct vinculin plaques and followed by formation of actin stress fibers and by profound changes in the organization of vimentin in the flattening cells. RA treatment finally led to the appearance and co-expression of keratin fibrils in many of the vimentin-containing F9 cells. This sequence of changes suggests that the vinculin-containing adhesion plaques may be important in the mechanism of RA-induced differentiation of EC cells.     

Identification of an Indian muntjac DNA fragment preferentially hybridizing to the X-chromosome

Upon digestion of DNA from male and female Indian muntjac (Muntiacus muntjak) fibroblasts with the restriction enzyme Hae III or Alu I, a prominent fragment of DNA (greater than 20 kb in length) was observed. This excluded DNA (ex-DNA) appeared not to contain sequences recognized by a variety of restriction enzymes and constituted about 0.6% of the total DNA in the female genome. For equal amounts of DNA digested, female DNA contained more of this material. In situ hybridization indeed revealed strong hybridization of the ex-DNA to the entire X chromosome with a few less intense sites of hybridization on other chromosomes. Hybridization studies against total muntjac DNA indicated the presence of repetitive sequences in the ex-DNA. These repetitive sequences did not cross-hybridize with human or mouse DNA.