Cellular aging studied by the reconstruction of replicating cells from nuclei and cytoplasms isolated from normal human diploid cells

Cytochalasin B (CB) was used to enucleate cells (cytoplasts) and to obtain karyoplasts (nuclei) from the human diploid fetal lung fibroblast strain WI-38. Fusion of cytoplasts and nuclei from young and old cells was accomplished with the aid of inactivated Sendai virus. Viable nuclei may be obtained from the karyoplast pellet after passage through a layer of bovine albumin which retains any contamination cytoplasts. The majority of successful fusions forming “whole cells” occurred when cytoplast from “old” cultures (PDL 40–51) and karyoplasts from “young” cultures were used (PDL 12–22), but almost always resulted in limited division of the viable reconstructed cells. When successful fusion occurred between “young” cytoplasts and “young” karyoplasts the number of cell divisions obtained was comparable to control cells kept under similar conditions.     

Synthesis of viral components in hybrids of differentially permissive cells infected with adenovirus type 12

Sendai virus induced heterokaryocytes of non-permissive BHK21 cells and permissive HEp2 cells were infected with adenovirus Type 12. Neoantigens, virus structural antigens and viral DNA were detected in the BHK21 nuclei as well as the HEp2 nuclei of heterokaryocytes, thus demonstrating positive control of these viral functions by the permissive cell component in the heterokaryocyte.     

Expression of malignancy in hybrids between normal and malignant cells.

Somatic cell hybrids between either normal human fibroblasts, phenotypically normal mouse fibroblasts or mouse peritoneal macrophages and HT1080 human diploid fibrosarcoma cells were studied for their ability to form tumors in nude mice. The results of this study indicate that tumorigenic behavior is expressed as a dominant trait in both human-human and mouse-human hybrid cells.     

Fusion-mediated microinjection of active amine and diamine oxidases into cultured cells: effect on protein and DNA synthesis in chick embryo fibroblasts and in glioma cells

Serum amine oxidase and/or porcine kidney diamine oxidase were trapped within reconstituted Sendai virus envelopes, and retained their activity. The trapped enzymes that were detected by radioimmunoblots were microinjected into cultured cells by fusion. When diamine oxidase was microinjected into cultured fibroblasts of chick or rat embryos, a temporary arrest in protein and DNA synthesis was observed. The inhibitory effect was more significant when both serum amine oxidase and kidney diamine oxidase were microinjected into those cultured cells. Fibroblasts of either chick or rat embryos transformed by Rous sarcoma virus were more susceptible to the injected enzymes than the normal cultures, showing a complete arrest in protein and DNA synthesis within 4 hours. Similar results were obtained by microinjecting diamine oxidase into cultured glioma cells. The injected enzyme catalyzed the oxidation of intracellular polyamines. The resulting oxidation product (hydrogen peroxide and aminoaldehydes) apparently caused the arrest in the synthesis of macromolecules.     

Intercellular exchange of lysosomal hydrolases between mutant human fibroblasts and other cell types

Human fibroblasts with a genetic deficiency of a single lysosomal enzyme and fibroblasts from a patient with ‘I-cell’ disease with a multiple deficiency of lysosomal hydrolases were used as recipient cells in studies on recognition and uptake of β-N-acetylhexosaminidase (hexosaminidase), β-glucuronidase and β-galactosidase. Normal human fibroblasts, and fibroblasts, hepatocytes and hepatoma cells from the rat were used as donor cells. The release of hexosaminidase was found to be similar among these different cell types, but the extracellular activities of β-glucuronidase and β-galactosidase were much higher in the rat cell cultures than in cultures of normal human fibroblasts. The enzymes released by rat fibroblasts were ingested by deficient human fibroblasts; enzyme from normal human fibroblasts was shown to be taken up by rat fibroblasts by means of electrophoresis. This indicates that reciprocal transfer of lysosomal hydrolases occurs between human and rat fibroblasts. Rat hepatocytes released hydrolases that were poorly taken up by human recipient fibroblasts and uptake of human fibroblast enzyme was not detected in the hepatocytes. Rat hepatoma cells, on the other hand, released lysosomal enzymes that were taken up by human deficient cells with a higher efficiency than those from fibroblasts. The uptake was subject to competitive inhibition by mannose 6-phosphate, the kinetics of which were comparable with those reported for ‘high-uptake’ forms of lysosomal enzymes [1–2]. Electrophoretic studies showed that rat hepatoma cells were not only capable of ingesting hexosaminidase from normal human fibroblasts, but also defectively processed enzyme [4–5] released by ‘I-cells’. These findings make rat hepatoma cells a useful model for the study of recognition and uptake of lysosomal enzymes.     

Microinjection of arginase into enzyme-deficient cells with the isolated glycoproteins of Sendai virus as fusogen

A method of introducing enzymes into the cytoplasm of fibroblasts in culture is described. Erythrocytes obtained from normal and arginase-deficient individuals were loaded with arginase in vitro and fused to arginase-deficient mouse and human fibroblasts. Erythrocyte ghost-fibroblast fusion was quantified by a 14C-radioactive assay for arginase in solubilized fibroblasts. Fusion was successfully induced by Sendai virus and also by the isolated glycoproteins of Sendai virus. After fusion the arginase activity associated with the Fibroblasts was 700--1500 U of arginase/mg of cell protein; this enzyme activity was 5- to 10-times higher than that normally found in the fibroblasts. The enrichment in arginase activity indicated that between four and ten ghosts had fused per fibroblast. The use of isolated viral proteins to mediate the transfer of enzymes into cells in vivo might alleviate clinical complications inherent in the use of whole virions. The enzyme replacement technique described in this report for a hyperargininemic model cell system should be applicable to the group of inborn errors of metabolism characterized by deficiency of an enzyme normally localized in the cytoplasmic compartment of cells.     

Enzyme activity during the growth and aging of human cells in vitro

Acid phosphatase, alkaline phosphatase, and lactic dehydrogenase activities have been compared in normal human diploid cell strains and in SV₄₀-transformed heteroploid cell lines derived from them. A higher level of acid phosphatase activity was observed in diploid cultures derived from adult lung than in cultures derived from fetal lung of similar passage levels. The alkaline phosphatase activity of normal diploid fibroblasts was significantly higher than that of SV₄₀-transformed cell lines derived from them. Generally, the lactic dehydrogenase activities of all these cell cultures were similar. Human diploid cells in culture “age,” in the sense that their ability to proliferate decreases with time during serial subcultivation. Evaluation of the activities of these three enzymes during the “aging” process showed that, although alkaline phosphatase and lactic dehydrogenase activities were similar in “young” and “senescent” cells, acid phosphatase showed a small but significant increase in the senescent cells.     

Variation in S phase in synchronous human cell lines.

Growth parameters of diploid and trisomic human fibroblasts were determined. The rate of growth of both classes of cells was examined in asynchronous cultures, and diploid and trisomic cells had similar growth rates. Synchronous cultures were developed using simple mitotic selection. The patterns and length of the DNA synthetic period (S phase) were found to be altered in trisomy 21 cells when compared to diploid human or to heteroploid HeLa cells. Early S-phase synthesis was absent or reduced and the overall length of the S phase was extended. However, the trisomic cells have apparently normal rates of DNA chain elongation and normal replicon sizes.     

Repair DNA synthesis in heterokaryons during reactivation of chick erythrocytes fused with human diploid fibroblasts or HeLa cells

Suppression of unscheduled DNA synthesis (UDS) after exposure to ultraviolet (UV) light in the human nuclei results when diploid human fibroblasts are fused with chick erythrocytes. The suppression is positively correlated with the number of erythrocyte nuclei in the heterokaryons, with a maximal effect at 36 h after fusion. Evidence is presented that this suppression is due to lowered levels of the enzymes involved in UDS as a result of inhibition of the RNA synthesis by chick components. No suppression of UDS is detected in the human nuclei of the HeLa-chick erythrocyte heterokaryons. In HeLa cells the rate of RNA synthesis is about 10 times higher than the rate in the normal diploid fibroblasts, and the relatively small inhibitory influence of the chick components will therefore not lead to a limitation of the enzymes involved in UDS in the HeLa-chick erythrocyte heterokaryons.     

Cytological identification of colony formation of intergeneric somatic hybrid cells

Summary Colony formation of intergeneric somatic hybrid cells was obtained by culturing highly viable heterokaryocytes. The high viability of theAtropa belladonna × Petunia hybrida,A. belladonna × Nicotiana tabacum, andDaucus carota × hybrida, D. carota × N. tabacum heterokaryocytes was due to the use of a refined polyethylene glycol (PEG) treatment. The heterokaryocytes underwent continuous division and formed colonies when placed in contact with a nutrient medium which stimulated growth of the parental cells. To confirm the original observations which were made using vacuolar anthocyanin pigments and the presence of chloroplasts as phenotypic markers, the formation of colonies was investigated by using cytological procedures. Nuclear fusion was frequently observed during synchronous mitosis. Completion of the mitotic cycle led to the formation of real hybrid daughter cells, which in the course of further division formed hybrid colonies. The high mitotic activity of the cells during the first 10–14 days of culture enabled the identification of hybrid colonies up to the 20–30 cell stage. Random tests with macroscopically visible calli indicated that up to this stage the hybrid character is retained. All calli obtained from PEG treatedA. belladonna andP. hybrida protoplasts were exposed to environmental conditions which induced differentiation. 240 plants, which subsequently flowered have been regenerated, 235 of these so far were identified asP. hybrida plants by analysis of the karyogram of the root tip cells. In 53 instances theP. hybrida plants were tetraploid. Five tetraploidA. belladonna plants were also obtained. In addition, 33 shoots were regenerated which grow rather slowly and do not show phenotypic markers characteristic of eitherA. belladonna orP. hybrida plants.     

mRNA sequence diversity during early embryogenesis in Drosophila melanogaster.

The structure and metabolism of membrane glycoprotein carbohydrate units were studied in three clonal strains of contact-deficient mouse L cells as compared with several contact-competent rodent fibroblasts. The L cells appeared deficient in the formation of stable adhesive and communicating contacts. Formation of these contacts could not be induced by artificial adhesion induced by Sendai virus. The absence of functional contacts in L cells was associated with synthesis of incomplete, dialysable fucosyl glycopeptides exposed at the cell surface. Somatic cell hybrids between communication incompetent L cells and mouse leukemia cells synthesized membrane carbohydrates of near-normal size distribution, but these cells remained deficient in functional contacts. However, in hybrids of L cells and normal human fibroblasts or lymphocytes, glycoprotein synthesis and formation of functional contacts were concomitantly restored.     

Inducers of DNA synthesis: levels higher in transformed cells than in normal cells

The objective of this study was to determine whether transformed cells have greater DNA synthesis-inducing ability (DSIA) than normal cells when fused with G1 phase cells. HeLa cells synchronized in G1 phase, prelabeled with large latex beads, were fused separately with (a) quiescent human diploid fibroblasts (HDF), (b) HDF partially synchronized in late G1, and random populations of (c) HeLa, (d) WI-38, (e) SV-40 transformed WI-38, (f) CHO, (g) chemically transformed mouse cells (AKR-MCA), and (h) T98G human glioblastoma cells (all prelabeled with small latex beads) using UV-inactivated Sendai virus. The fusion mixture was incubated with [3H] thymidine, sampled at regular intervals, and processed for radioautography. Among the heterodikaryons, the frequency of those with a labeled and an unlabeled nuclei (L/U) were scored as a function of time after fusion. The faster the induction of DNA synthesis in HeLa G1, the steeper the drop in the L/U class and hence the higher DSIA in the S phase cells. The DSIA, which is indicative of the intracellular levels of the inducers of DNA synthesis, was the highest in HeLa and virally transformed WI-38 cells and the lowest in normal human diploid fibroblasts (HDF) while those of chemically and spontaneously transformed cells are intermediate between these two extremes. Higher level of DNA synthesis inducers appears to be one of the pleotropic effects of transformation by DNA tumor viruses. These studies also revealed that initiation of DNA synthesis per se is regulated by the presence of inducers and not by inhibitors.     

Thymidine Kinase from Normal, Simian Virus 40-Transformed and Simian Virus 40-Lytically Infected Cells

Simian virus 40 (SV40) infection of human diploid cells failed to cause an enhanced production of thymidine kinase during the first 10 days after infection. Thymidine kinase activities from extracts of SV40-transformed cultures (human or simian) were considerably higher than the activity levels in extracts from the normal cells of origin. In addition, whereas the kinase activities obtained for human diploid cultures decreased as the cell sheet became confluent, the kinase activities for SV40-transformed human cells remained high after confluence was reached. Antisera obtained from hamsters bearing SV40 or adeno-7-SV40 hybrid virus tumors selectively inhibited enzyme from transformed sources (human or simian). Also, the antisera selectively inhibited enzyme extracted from SV40-lytically infected monkey cells. Sera from normal animals or from hamsters bearing polyoma tumors failed to inhibit enzymes from normal, SV40-transformed, or SV40-lytically infected cells. The Michaelis constant of partially purified enzyme from SV40-transformed cells was two to five times as high as that obtained for partially purified enzyme from human diploid cell cultures.     

Complementation in heterokaryons derived from a thymidine kinase deficient cell line and senescent human diploid cells.

Incorporation of [3H]thymidine was observed in both parental nuclei in heterokaryons resulting from the fusion of post-mitotic, "senescent" human diploid cells and a thymidine kinase-deficient murine cell line (3T3der-4E). The senescent nuclei displayed a sudden increase of activity approximately 4--6 hours after fusion. A moderate increase of thymidine incorporation was observed in 3T3der-4E cells cocultivated with but not fused to senescent cells, consistent with metabolic cooperation. Chromosome preparations of cultures fixed approximately 24 hr after fusion revealed the presence of hybrid metaphase cells containing almost the entire human complement. All of the identifiable human chromosomes were bi-armed, suggesting that the senescent nuclei were stimulated to reinitiate replicative DNA synthesis rather than repair synthesis in these heterokaryons.     

Reinitiation of DNA synthesis in senescent human fibroblasts upon fusion with cells of unlimited growth potential

Postreplicative, "senescent" human fibroblasts were fused to HeLa or to SV-40 transformed human fibroblasts with Sendai virus. DNA synthesis was reinitiated in senescent nuclei in a high proportion of the heterodikaryons. The [3H]thymidine labeling index of senescent fibroblast nuclei in heteropolykaryons was a function of the ratio of HeLa to senescent nuclei.     

Replication of simian virus 40 DNA in normal human fibroblasts and in fibroblasts from xeroderma pigmentosum.

Simian virus 40 infection of semipermissive human diploid fibroblasts (HF), at early passage in cell culture, was compared with that of permissive established monkey cell lines. Viral DNA can be readily detected at 24 to 48 h postinfection at 37 degrees C with a high multiplicity of infection, approaching 10% of that of monkey cells (TC7). The length of time necessary for replication of an average molecule of viral DNA was found to be indistinguishable in HF and TC7 cells. Strand elongation plus termination were assessed by following the accumulation of DNA I at 40 degrees C from replicative intermediates of tsA30 prelabeled at 33 degrees C, obviating isotope pool problems. Combined initiation and elongation of wild-type viral DNA was measured by density shift experiments involving a 5-bromodeoxyuridine chase of prelabeled [3H]thymidine-labeled viral DNA. Determination of accumulation of viral T and V antigens supports the conclusion that the most likely basis for the reduced virus yield in HF cells results from the inefficiency of an early stage in virus infection, before or during uncoating. Similar results were obtained in fibroblasts derived from patients with xeroderma pigmentosum, suggesting that enzymes of UV repair are not required in unirradiated simian virus 40 DNA synthesis.     

Presence of two active X chromosomes in hybrids between normal human and SV40-transformed fibroblasts from patients with the Lesch-Nyhan syndrome

Skin fibroblasts (LNSV) derived from a hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficient patient with the Lesch-Nyhan syndrome, who has glucose-6-phosphate dehydrogenase (G6PD) type A, were transformed with SV40 and hybridized with WI38 human diploid fibroblasts derived from a female embryo which have normal HGPRT and G6PD type B activities. The hybrid clones selected in hypoxanthine, aminopterin and thymidine (HAT) medium, were essentially tetraploid and contained three X and one Y chromosomes. These hybrids contained HGPRT, types A and B and the AB heteropolymeric form of G6PD enzymes which were indicative that in these cells X linked genes of both parental cells were fully active. Hybrids back-selected in medium containing 8-azaguanine (8-AG) contained only two X chromosomes. They had no HGPRT activity and they contained only G6PD type A enzyme. It is concluded that the hybrid cells which grew in the presence of 8-AG retained the X chromosome of the LNSV parental cell and apparently the inactive X of the WI 38 cell.     

Aging in vitro : Growth of cultured cells from the Galapagos tortoise

Skin biopsies from two growing and two fully-grown Galapagos tortoises, a species that lives twice as long as man, were explanted in vitro. Cellular outgrowth was more vigorous from explants of young tortoises but epithelial cells stopped dividing early in the history of all cultures, even at the optimum temperature of 30 °C. Serially subcultured fibroblasts from young tortoises divided more rapidly and achieved longer lifespans than fibroblasts from old tortoises in terms of mean population doublings and to a lesser extent, calendar time. For all cultures the lifespans exceeded those reported for human diploid fibroblasts. The results indicate that a proportionality exists between the potential (remaining) lifespan in vivo and the mitotic capacity of cultured diploid cells in vitro.     

Human cytomegalovirus: demonstration of permissive epithelial cells and nonpermissive fibroblastic cells in a survey of human cell lines.

To more clearly define the characteristics which render a cell permissive for human cytomegalovirus (HCMV), we screened a panel of human cell lines differing in morphology, ploidy, and extent of differentiation for the ability to sustain productive HCMV replication. Cells were exposed to HCMV at 5 to 20 PFU per cell and examined at 4 to 14 days postinfection to detect the production of infectious virus by a plaque assay and the assembly of progeny virions by electron microscopy. By these criteria, high-titer HCMV replication (10(6) to 10(7) PFU/ml) occurred in a well-differentiated, diploid, epithelial cell line, HCMC, which had been derived from normal human colonic mucosa. In contrast, all aneuploid human cell types proved to be nonpermissive, including a fibroblastic cell line designated HT-144. These results indicate that HCMV replication in cultures is not strictly limited to fibroblasts and conversely that not all human fibroblastic cells are permissive for HCMV. Nonpermissive cell types were further investigated by attempts to chemically induce HCMV replication. Treatment of nonpermissive cell types with 25 to 500 micrograms of 5-iodo-2'-deoxyuridine per ml prior to infection did not convert them to the permissive state. The implications of these findings for the possible mechanisms maintaining the nonpermissive state are discussed.